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Novel functions of MCL-1 in ATM signalling, homologous recombination, and heterochromatin dynamics Pesarchuk, Eric 2016

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NOVEL FUNCTIONS OF MCL-1 IN ATM SIGNALLING, HOMOLOGOUS RECOMBINATION, AND HETEROCHROMATIN DYNAMICS by  Eric Pesarchuk  B.Sc., The University of Guelph, 2009  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  June 2016  © Eric Pesarchuk, 2016 ii  Abstract  MCL-1 is a pro-survival member of the BCL-2 family of proteins that is over-expressed in a wide variety of human cancers. Beyond its canonical role regulating apoptosis, our lab was the first to describe an additional function of MCL-1 in the nucleus where it is chromatin-bound at sites of DNA double strand breaks and potentiates DNA damage response signalling. This thesis further characterizes the nuclear functions of MCL-1 and shows that it has a shorter protein half life in the nucleus compared to the cytosol. In response to low dose etoposide, MCL-1-/- MEFs had diminished γH2AX and pS824 KAP-1, while pS1981 ATM and pT68 Chk2 were lower across a 100-fold range of etoposide concentrations. Analysis of a panel of pathway-specific U2OS reporter cell lines determined that MCL-1 participates in homologous recombination. Analysis of γH2AX and RPA foci by immunofluorescence revealed that MCL-1-depleted cells had elevated levels of RPA at 8 hours post-ionizing radiation but exhibited no difference in γH2AX kinetics.  A LC-MS/MS screen of endogenous MCL-1 immunoprecipitations from etoposide-treated HeLa-S3 cells identified HP1α and p150CAF-1 as putative binding partners, which were validated by immunoblot and suggested a role for MCL-1 in heterochromatin dynamics. MCL-1 was found to bind HP1α and p150CAF-1 in the presence or absence of exogenous DNA damage, and was dispensable for the established interaction of HP1α and p150CAF-1. MCL-1 was also not required for the localization of HP1α, p150CAF-1, or KAP-1 to sites of DNA damage. MCL-1 nevertheless had a functional role in promoting heterochromatin compaction as iii  determined using the U2OS 2-6-3 cell line that has a quantifiable heterochromatin array. Furthermore, cells lacking MCL-1 had reduced basal levels of H3K9me2/3. Taken together, I propose that MCL-1 promotes heterochromatin compaction and H3K9me3 via novel interactions with HP1α and p150CAF-1. Other reports indicate that H3Kme3 leads to Tip60-mediated ATM activation following DNA damage, and that heterochromatic factors enhance downstream processes in homologous recombination. Therefore, MCL-1 is capable of regulating numerous double strand break repair processes by influencing chromatin architecture.   iv  Preface  The work in this thesis is currently unpublished but manuscripts encompassing all of the work are in preparation. I was responsible for all of the optimization and execution of experimental procedures, and all data analysis. Experimental design and interpretation of results was done with the help of Dr. Vince Duronio, my PhD supervisor, and Dr. Aaron Goodarzi, a collaborator at the University of Calgary with whom I worked during the summer of 2015. Advice for mass spectrometry experiments was given by Drs. Juergen Kast, Gregg Morin, Cordula Klockenbusch, Shujun Lin, Jason Rogalski, and Grace Cheng. All studies were performed according to Biosafety Approval B14-0167. v  Table of Contents  Abstract .......................................................................................................................................... ii!Preface ........................................................................................................................................... iv!Table of Contents ...........................................................................................................................v!List of Tables ..................................................................................................................................x!List of Figures ............................................................................................................................... xi!List of Abbreviations ................................................................................................................. xiii!Acknowledgements .................................................................................................................... xvi!Dedication ................................................................................................................................. xviii!Chapter 1: Introduction ................................................................................................................1!1.1! DNA damage response – a focus on double strand breaks ............................................. 2!1.1.1! Cell cycle checkpoints ............................................................................................ 3!1.1.2! Double strand break repair pathways ...................................................................... 3!! Non-homologous end joining ............................................................................. 4!! Homologous recombination ................................................................................ 4!! Additional double strand break repair pathways ................................................ 8!! Experimental generation of double strand breaks ............................................... 9!1.1.3! Chromatin response to double strand breaks ........................................................ 10!1.2! The BCL-2 family of proteins regulates apoptosis ....................................................... 13!1.2.1! Linking the DNA damage response to apoptosis signaling .................................. 15!1.3! Focusing on MCL-1: a BCL-2 family member with many unique characteristics ....... 15!1.3.1! MCL-1 protein structure ....................................................................................... 16!vi  1.3.2! MCL-1 protein turnover ........................................................................................ 20!1.3.3! Fluctuations of MCL-1 protein levels during the cell cycle ................................. 22!1.3.4! Nuclear translocation of MCL-1 ........................................................................... 23!1.3.5! MCL-1 has an active role in the DNA damage response ...................................... 24!1.3.6! Additional nuclear interactions of MCL-1 ............................................................ 25!! Cyclin dependent kinase 1 ................................................................................ 25!! Proliferating cell nuclear antigen ...................................................................... 26!! Translationally controlled tumor protein .......................................................... 26!1.3.7! Functions of additional pro-survival BCL-2 family members in double strand break repair and G2/M arrest ................................................................................................ 27!! BCL-2 inhibits double strand break repair ........................................................ 27!! BCL-XL potentiates G2/M arrest in response to etoposide ............................... 28!1.4! Research aims and chapter summary ............................................................................ 28!Chapter 2: Methods .....................................................................................................................32!2.1! Cell culture .................................................................................................................... 32!2.2! Chemicals ...................................................................................................................... 32!2.3! siRNA and plasmid transfections .................................................................................. 32!2.4! Antibodies ..................................................................................................................... 33!2.5! Immunoblotting............................................................................................................. 35!2.6! Nuclear fractionation .................................................................................................... 35!2.7! Cell cycle analysis ......................................................................................................... 36!2.8! Double strand break repair reporter assays ................................................................... 36!2.9! Immunofluorescence ..................................................................................................... 37!vii  2.10! Confocal microscopy .................................................................................................... 37!2.11! Ionizing radiation-induced foci quantification of G2 cells ........................................... 37!2.12! Laser microirradiation ................................................................................................... 38!2.13! Targeted LacO array heterochromatin relaxation assay ............................................... 38!2.14! Targeted LacO array heterochromatin double strand break-induction assay ............... 39!2.15! Co-immunoprecipitation ............................................................................................... 39!2.15.1! Antibody-agarose bead conjugation ..................................................................... 39!2.15.2! Fractionated lysate preparation ............................................................................. 40!! Nuclear lysate preparation ............................................................................ 40!! Chromatin-bound protein complex enrichment ............................................ 40!2.15.3! Co-immunoprecipitation procedure ...................................................................... 40!2.16! LC-MS/MS methods ..................................................................................................... 41!2.16.1! Cell culture ............................................................................................................ 41!2.16.2! In-gel digestion ..................................................................................................... 41!2.16.3! LC-MS/MS analysis ............................................................................................. 42!! Qstar XL ........................................................................................................ 42!! LTQ-FT-ICR ................................................................................................. 42!2.16.4! Mass spectrometry data analysis ........................................................................... 43!Chapter 3: Identifying signaling and double strand break repair pathways requiring MCL-1 ...........................................................................................................................................44!3.1! Nuclear MCL-1 has decreased protein stability compared to cytosolic MCL-1. ......... 44!3.2! Cells lacking MCL-1 have impaired phosphorylation of ATM, Chk2, KAP-1, and γH2AX in response to etoposide. .............................................................................................. 47!viii  3.3! MCL-1 promotes double strand break repair efficiency via homologous recombination, as determined by pathway-specific reporter assays. ................................................................. 49!3.4! MCL-1 knockdown prevents RPA foci resolution in G2 with no impact on γH2AX kinetics. ..................................................................................................................................... 52!3.5! Discussion ..................................................................................................................... 55!Chapter 4: LC-MS/MS analysis of MCL-1 coimmunoprecipitations to identify novel protein interactions ......................................................................................................................59!4.1! LC-MS/MS data sets ..................................................................................................... 61!4.1.1! Nuclear lysate preparation and analysis on Qstar ................................................. 61!4.1.2! Chromatin-bound complex enrichment and analysis on the LTQ-FT-ICR .......... 66!4.2! Validating novel binding partners of MCL-1 by immunoblot ...................................... 73!4.2.1! Rationale for characterizing HP1α and p150CAF-1 interaction with MCL-1 ..... 73!4.2.2! MCL-1 coimmunoprecipitation from adherent HeLa cells confirms HP1α and p150CAF-1 as novel interactions by immunoblot. ............................................................... 77!4.3! Discussion ..................................................................................................................... 80!Chapter 5: Investigating a role for MCL-1 in heterochromatic double strand break repair85!5.1! MCL-1 is not required for recruitment of HP1α, p150CAF-1, or KAP-1 to DNA damage. ..................................................................................................................................... 85!5.2! MCL-1 is not required for retention of pS824 KAP-1 at late-repairing double strand breaks or with PML nuclear bodies .......................................................................................... 88!5.3! MCL-1 promotes heterochromatin compaction ............................................................ 92!5.4! Discussion ..................................................................................................................... 95!Chapter 6: Discussion ..................................................................................................................99!ix  6.1! MCL-1 potentiation of ATM activity may be linked to H3K9me3-mediated Tip60 activation ................................................................................................................................. 101!6.2! MCL-1 may propagate H3K9me3 through interactions with HP1α and p150CAF-1 104!6.3! MCL-1 may facilitate downstream processes in HR as a HC promoting factor ........ 106!6.4! Experimental considerations when affecting basal chromatin architecture and analyzing DSB repair .............................................................................................................. 110!6.5! Future directions ......................................................................................................... 112!References ...................................................................................................................................117! x  List of Tables  Table 2-1: Cell lines ...................................................................................................................... 32!Table 2-2: Primary antibodies ....................................................................................................... 33!Table 2-3: Secondary antibodies ................................................................................................... 34!Table 4-1: Putative MCL-1 interactions identified from standard nuclear fractionation protocol and Qstar LC-MS/MS analysis, organized by ion score. .............................................................. 63!Table 4-2: Putative MCL-1 interactions identified from chromatin complex enrichment protocol and LTQ-FT-ICR LC-MS/MS analysis, organized by ion score. ................................................. 68!Table 4-3: MCL-1 and HP1α/CBX5 protein identifications from a preliminary MCL-1 IP using nuclear lysate preparation with a lysis buffer containing 250mM NaCl and 2.5% Triton X-100, and Qstar LC-MS/MS analysis. .................................................................................................... 76! xi  List of Figures  Figure 1-1: Summary of the Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) DNA double strand break (DSB) repair pathways. ...................................... 7!Figure 1-2: Structural alignments of MCL-1 and other BCL-2 family members. ........................ 20!Figure 1-3: Proposed model for the involvement of MCL-1 in heterochromatin dynamics, ATM signalling, and homologous recombination. ................................................................................. 31!Figure 3-1: MCL-1 has decreased protein stability in the nucleus compared to cytoplasm. ........ 46!Figure 3-2: Activation of ATM, Chk2, KAP-1, and γH2AX is impaired in MCL-1-/- MEFs. ..... 48!Figure 3-3: MCL-1 promotes DSB repair efficiency in HR specifically. ..................................... 51!Figure 3-4: MCL-1 knockdown prevents RPA foci resolution in G2 cells with no effect on γH2AX kinetics. ............................................................................................................................ 54!Figure 4-1: CoIP samples from nuclear lysates analyzed on Qstar LC-MS/MS. ......................... 62!Figure 4-2: CoIP samples from chromatin complex enriched samples analyzed on LTQ-FT-ICR LC-MS/MS. .................................................................................................................................. 67!Figure 4-3: MCL-1 CoIPs in HeLa cells validate HP1α and p150CAF-1 as novel binding partners by immunoblot. ............................................................................................................... 79!Figure 4-4: MCL-1 is not required for HP1α/p150CAF-1 interaction. ........................................ 80!Figure 5-1: MCL-1 is not required for recruitment of HP1α or KAP-1 to laser track DNA damage. ......................................................................................................................................... 87!Figure 5-2: MCL-1 is not required for recruitment of p150CAF-1 or KAP-1 to heterochromatic DSBs. ............................................................................................................................................ 88!xii  Figure 5-3: MCL-1 is not required for retention of pS824 KAP-1 with γH2AX foci or PML bodies 24 hours post irradiation. ................................................................................................... 91!Figure 5-4: MCL-1 promotes compaction of the LacO heterochromatic array. ........................... 94!Figure 5-5: MCL-1 depletion decreases basal Histone H3K9 methylation. ................................. 95!Figure 6-1: Proposed model of how MCL-1 can impact multiple stages of the DNA damage response by compacting chromatin. ............................................................................................ 100! xiii  List of Abbreviations 4-OHT   4-hydroxytamoxifen 53BP1   p53-binding protein 1 Alt-EJ   Alternative end joining ATM   Ataxia telangiectasia mutated ATR   Ataxia telangiectasia mutated and Rad3-related BCL-2   B-cell lymphoma 2 BCL-XL  B-cell lymphoma-extra large BAD   BCL-2-associated death promoter Bak   BCL-2 agonist killer BH1/2/3  BCL-2 homology 1/2/3 BRCA1/2  Breast cancer type 1/2 susceptibility protein BRCT   BRCA1 C terminus BrdU   Bromodeoxyuridine BSA   Bovine serum albumin CAF-1   Chromatin assembly factor 1 Cdc20/25  Cell division cycle protein 20/25 homolog CDK1   Cyclin-dependent kinase 1 CENPF  Centromere protein F CHD3   Chromodomain-helicase-DNA-binding prtein 3 Chk1/2  Checkpoint kinase 1/2  CHX   Cycloheximide  CoIP   Co-immunoprecipitation CRISPR  Clustered regularly-interspaced short palindromic repeats CtIP   CtBP-interacting protein Ctrl   Control DAPI   4’,6’-diamidino-2-phenylindole DD   Destabilization domain DDR   DNA damage response DDX41  DEAD box protein 41 DMSO   Dimethyl sulfoxide DNA-PKcs  DNA protein kinase catalytic subunit DSB   DNA double strand break DTT   Dithiothritol EDTA   Ethylenediaminetetraacetic acid ER   Estrogen receptor Exo1   Exonuclease 1 FATC   FAT-C-terminal FBS   Fetal bovine serum FHA   Forkhead-associated FT-ICR  Fourier transform ion cyclotron resonance GFP   Green fluorescent protein H3K9   Histone H3 lysine 9 H3K9me2/3  Histone H3 lysine 9 di-/tri-methylated xiv  HC   Heterochromatin HDAC   Histone deacetylase HEK   Human embryonic kidney HEPES  4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HP1α/β/γ  Heterochromatin protein 1 α/β/γ  HPLC   High performance liquid chromatography HR   Homologous recombination hTERT  Human telomerase reverse transcriptase IEX-1   Immediate early response 3 IF   Immunofluorescence  IgG   Immunoglobulin G IR   Ionizing radiation IP   Immunoprecipitation KAP-1   KRAB-associated protein 1 LacO   Lac operon LacR   Lac repressor LC-MS/MS  Liquid chromatography tandem mass spectrometry MCL-1  Myeloid cell leukemia 1 MCM 2-7  Minichromosome maintainence 2-7 MEF   Mouse embryonic fibroblast Mre11   Meiotic recombination 11 homolog 1 MNase   Micrococcal nuclease MRN   Mre11-Rad50-NBS1 NBS1   Nijmegen Breakage Syndrome 1 NCS   Neocarzinostatin NHEJ   Non-homologous end joining NLS   Nuclear localization sequence NMR   Nuclear magnetic resonance p14ARF   Alternate reading frame p14 p84-ZFN  Zinc fingerp84 PARP1  Poly [ADP-ribose] polymerase 1 PBS   Phosphate-buffered saline PCNA   Proliferating cell nuclear antigen PCR   Polymerase chain reaction PFA   Paraformaldehyde PI   Propidium iodide PML   Promyelocytic leukemia PML NB  Promyelocytic leukemia nuclear body PMSF   Phenylmethylsulfonyl fluoride PPP1R12C  Protein phosphatase 1 regulatory subunit 12C PUMA   p53 up-regulated modulator of apoptosis RPA   Replication protein A RPM   Revolutions per minute SCE   Sister chromatid exchange Scr   Scrambled xv  SDS-PAGE  Sodium dodecyl sulfate polyacrylamide gel electrophoresis SETDB1  SET domain bifurcated 1 SILAC   Stable isotope labeling by amino acids in cell culture siRNA   Small interfering ribonucleic acid SSA   Single strand annealing ssDNA  Single-stranded DNA STAGE-tip  Stop and go extraction tip TCTP   Translationally-controlled tumor protein Tip60   60 kDa Tat-interactive protein UV   Ultraviolet  WT   Wildtype XLF   XRCC4-like factor XRCC4  X-ray repair cross-complementing protein 4  xvi  Acknowledgements  First and foremost, I want to thank my supervisor, Dr. Vince Duronio. Thank you for taking a chance on an eager 22-year old kid who thought the BCL-2 family was interesting enough to hold his attention for 6 years. This project took a lot of twists and turns, but your calm and persistent optimism guided my curiosity and helped develop me into a critical, lighthearted, and independent scientist. Thank you for trusting me to pursue my ideas and always dropping whatever you were doing to engage with my ramblings about them.   I am also very grateful to Dr. Aaron Goodarzi at the University of Calgary. I never would have thought that randomly requesting a protocol from a publication’s corresponding author would lead to ongoing dialogue resulting in a working visit in your lab. The accelerated pace of discovery at the end of my PhD was initiated by my summer with you and I cannot overstate my appreciation for your whole team’s welcome and insights.  I must also thank my supervisory committee – Drs. Chris Maxwell, Michel Roberge, Gregg Morin, and Juergen Kast – for their help in shaping the direction of my project over the years. The latter two’s labs were incredibly helpful in troubleshooting and interpreting my LC-MS/MS experiments and Dr. Kast in particular was very generous in allowing me many hours of instrument time. I also received valuable theoretical input from Dr. Peter Stirling and members of the Genome Instability Group at the BC Cancer Agency. David Ko and Wenbo Xu were incredibly helpful with my flow cytometry experiments. And without Dr. Yemin Wang I would not have been able to do any IR experiments, and I greatly appreciate his willingness to help. xvii  Many thanks also to Dr. Horacio Bach for teaching me numerous techniques, acquiring industrial side projects for me, and having a joie de vivre that permeates the 4th floor at JBRC.   I want to thank Dr. Sarwat Jamil for the critical findings that laid the groundwork for my project, and Payman Hojabrpour for keeping the lab energetic and running smoothly. There are also so many fellow students that it was a joy to navigate this journey with, especially Sylvia Cheung, Alice Lau, Sneha Thomas, Peng Zhang, Rouhollah Mousavizadeh, and Malihe Pourmasjedi. And collectively to all students in Experimental Medicine, it was an honour to be twice elected as your Student Representative, which was a formative experience during my PhD.   I am grateful to the agencies/institutions that provided operational funds and salary support to make this project possible – CIHR, UBC, the Experimental Medicine program, the BC Proteomics Network, and MITACS.  Lastly, my ongoing gratitude and admiration for my friends and family. I moved to Vancouver without knowing anyone, in search of a change and a challenge. My friends in Ontario have never waned in their support nor have they let time or geography impede our relationships. I was fortunate to immediately collide with brilliant, creative, and engaging people that have become the cornerstone of the life and community I’ve developed in this amazing city. To Ren – my partner, my love – every day you inspire me to illuminate additional dimensions of the man I want to be, and strengthen me to become him. And to my family, your love, support, insights, and financial help have been foundational in getting me to this point. Thank you, and I love you! xviii  Dedication     For Ren, my parents, my sister, Dido, and Atis. 1  Chapter 1:!Introduction DNA is under constant assault from numerous damaging agents; each cell in the body encounters tens of thousands of lesions per day (Lindahl and Barnes, 2000). Damage can be from endogenous sources such as reactive oxygen species that are natural by-products of metabolism or collapse of stalled replication forks (Allen et al., 2011; De Bont and van Larebeke, 2004). Genotoxic stress can also arise from external sources such as radiation, chemical exposure, or viral infection (Valko et al., 2006; Ward, 1988; Xiaofei and Kowalik, 2014). The resulting damage can modify the nucleotide sugar and base moeities, create covalent DNA adducts, crosslink DNA strands, or cleave the phosphate backbone to create single-strand or double-strand breaks (DSBs) (Marnett and Plastaras, 2001). Cells possess numerous DNA repair pathways to recognize and replace modified bases, or process and re-ligate DNA strand breaks.  The accumulation of unrepaired or mis-repaired DNA damage can yield a mutational landscape that drives cancer initiation, progression, and metastasis. As such, the development of genomic instability is a hallmark of cancer (Hanahan and Weinberg, 2000; Hanahan and Weinberg, 2011). Therefore, cells have evolved a highly orchestrated DNA damage response (DDR) to maintain genomic integrity (Harper and Elledge, 2007; Jackson and Bartek, 2009; Rouse and Jackson, 2002). The function of the DDR is to recognize that damage has occurred, pause the cell cycle, activate repair mechanisms appropriate for the specific lesion, remodel chromatin to facilitate repair, and if unrepairable, initiate apoptosis. DSBs in particular can be highly lethal as they may result in breakage, loss, or translocation of chromosomes to yield genomic instability (Khanna and Jackson, 2001; Wyman and Kanaar, 2006).  2  1.1! DNA damage response – a focus on double strand breaks The study of DSB-induced signalling began with the discovery of a kinase that bound and was activated by DNA (Carter et al., 1990; Jackson et al., 1990; Lees-Miller et al., 1990), now called DNA protein kinase catalytic subunit DNA-PKcs. Through sequence similarity, this kinase was found to be related to Ataxia-telangiectasia mutated (ATM), a gene whose mutation is responsible for a rare autosomal recessive disorder exhibiting neurodegeneration, chromosomal instability, and a predisposition to cancer (Hartley et al., 1995; Savitsky et al., 1995). DNA-PKcs and ATM are each activated by DSBs while a related kinase, Ataxia-telangiectasia mutated and Rad3-related (ATR) (Cimprich et al., 1996), requires exposure to single stranded DNA (ssDNA) for activation (Cortez et al., 2001; Zou and Elledge, 2003). These three kinases are part of the Phosphatidylinositol 3-kinase-related kinase family and together with the downstream checkpoint kinases, Chk1 and Chk2, are responsible for activating a plethora of effectors for cell cycle arrest, DNA repair, and apoptosis (Bensimon et al., 2011; Blasius et al., 2011; Mu et al., 2007; Shiloh and Ziv, 2013; Yang et al., 2003).  The factors needed for DSB repair are constitutively expressed. Once activated, the DDR initiates a myriad of post-translational modifications to coordinate the spatiotemporal organization and functional activation of protein complexes at sites of damage (Polo and Jackson, 2011). The best example of this is phosphorylation of the histone variant H2AX on S139 (then referred to as γH2AX) by each of the DDR kinases within seconds of damage (Podhorecka et al., 2010; Rogakou et al., 1998). γH2AX quickly propagates in both directions for up to megabases away from DSBs (Liu et al., 2005; Zhang et al., 2002c). This provides a scaffold to assemble large protein complexes that contain effectors for chromatin remodeling and 3  DSB repair. In the same fashion, many DDR factors do not possess enzymatic function and exert their role by facilitating protein-protein interactions. The focal accumulation of these proteins can be visualized by immunofluorescence and the analysis of DSB repair protein foci kinetics is a mainstay experimental method in the field of DNA repair research.  1.1.1! Cell cycle checkpoints In response to DNA damage cells arrest the cell cycle to prevent division and allow time for repair. There are three checkpoints that can be activated by the DDR kinases: two at the transitions of G1/S and G2/M, and one that is intra-S-phase. There are two main strategies through which phosphorylation events by DDR kinases activate cell cycle arrest. First, activation of the Wee1 kinase and inhibition of the cdc25 phosphatase maintains the tyrosine phosphorylation that inhibits cyclin dependent kinases (CDKs) (Donzelli and Draetta, 2003; Perry and Kornbluth, 2007). Secondly, DDR kinase phosphorylation of p53 precludes its interaction with the MDM2 ubiquitin ligase to rapidly increase its protein levels (Banin et al., 1998; Lees-Miller et al., 1992; Loughery et al., 2014; Ou et al., 2005; Saito et al., 2002; Shieh et al., 1997; Tibbetts et al., 1999). p53 then transcriptionally upregulates the p21 cell cycle inhibitor that represses the cyclin-CDK complexes responsible for progression from G1 through mitosis (Bunz et al., 1998; Waldman et al., 1995).  1.1.2! Double strand break repair pathways There are two predominant strategies through which DSBs are repaired: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). While their repair mechanism and 4  required factors differ, they share a sequential hierarchy of DSB recognition, processing, and ligation. The NHEJ and HR pathways are described below and outlined in Figure 1-1.! Non-homologous end joining NHEJ is a rapid repair mechanism that can occur at any stage in the cell cycle but can result in small base pair additions/deletions leading to mutations (Lees-Miller, 1996; Meek et al., 2004; Weterings and Chen, 2008). NHEJ is also important for V(D)J recombination during immune development (Malu et al., 2012). In response to DSBs, the broken termini are quickly bound by the high-abundance Ku70/80 heterodimer, which then recruits DNA-PKcs to form the catalytically active DNA-PK holoenzyme (Dvir et al., 1992; Gottlieb and Jackson, 1993; Suwa et al., 1994). DSB ends are then processed by various factors, including the Artemis and Mre11 nucleases (Gu et al., 2010; Moshous et al., 2001; Xie et al., 2009; Yannone et al., 2008), to make the termini amenable for ligation. The final ligation step is carried out by a complex containing DNA Ligase IV, XRCC4, and XLF (Ahnesorg et al., 2006; Buck et al., 2006; Grawunder et al., 1997; Grawunder et al., 1998; Li et al., 1995; Modesti et al., 1999). While these fundamental molecular determinants of NHEJ have been studied for 10 – 20 years, new critical factors continue to be discovered (Ochi et al., 2015).! Homologous recombination HR is a high fidelity process because it uses homology within the sister chromatid as a template for repair (Moynahan and Jasin, 2010). Accordingly, HR is limited to S and G2 phases of the cell cycle, but represents a major repair pathway within mammalian cells (Liang et al., 1998). The process begins as the constitutively bound Mre11-Rad50-NBS1 (MRN) complex is recruited to 5  DSBs (D'Amours and Jackson, 2002; Lamarche et al., 2010). Mre11 binds broken DNA termini (de Jager et al., 2001a; Furuse et al., 1998; Usui et al., 1998), while Rad50 functions as a molecular hook to hold the ends in proximity (Anderson et al., 2001; de Jager et al., 2001b; Hopfner et al., 2000), and NBS1 facilitates protein interactions with other repair factors (Hari et al., 2010). The MRN complex recruits inactive ATM dimers within seconds of damage (Paull and Lee, 2005; Uziel et al., 2003). The robust activation of ATM kinase activity that follows DNA damage requires Tip60-mediated acetylation of ATM (Sun et al., 2005; Sun et al., 2007), followed by autophosphorylation on S1981, which then releases active ATM monomers (Bakkenist and Kastan, 2003; Kozlov et al., 2006; Shiloh and Ziv, 2013)  HR requires 3’ ssDNA overhangs for strand invasion and homology searching in the sister chromatid, which are generated through end resection by nucleases away from the DSB. The intrinsic nuclease activity of Mre11, in conjunction with that of CtIP, initiate 5’ – 3’ end resection to reveal ~20bp of overhang (Sartori et al., 2007). Once a DSB is resected, NHEJ is no longer efficient at repair, therefore end resection plays a critical role in the choice of DNA repair pathways (Grabarz et al., 2012; Huertas, 2010). Furthermore, the initiation of CtIP-mediated end resection is regulated by CDKs to limit HR to S, G2, and mitosis, when sister chromatid are present (Huertas et al., 2008; Huertas and Jackson, 2009; Peterson et al., 2011). Resected DNA is extended through the nuclease activities of Dna2 and Exo1, and helicase activity of BLM, to reveal longer sequences for homologous pairing (Nimonkar et al., 2008). ATM activity not only facilitates processes that promote resection, but it directly regulates the enzymes that initiate and extend resection (Ababou et al., 2000; Bolderson et al., 2010; Kijas et al., 2015; Limbo et al., 2011; Peterson et al., 2013). 6  The exposed ssDNA is quickly bound by replication protein A (RPA), a heterotrimeric complex composed of p70, p32, and p14 subunits. RPA binds ssDNA with high affinity to protect against nucleases and dissolve the secondary nucleic acid structure (de Laat et al., 1998; Fanning et al., 2006; Iftode and Borowiec, 2000). A series of protein complexes are recruited to RPA-coated ssDNA leading to ATR activation (Cimprich and Cortez, 2008; Shiotani and Zou, 2009). The exposed ssDNA also serves as the template for homology searching in the sister chromatid but the catalysis of strand invasion requires the formation of nucleoprotein filament with Rad51 (Baumann et al., 1996; Krejci et al., 2012; Li and Heyer, 2008; Sung, 1994). Breast cancer type 2 susceptibility protein (BRCA2) is the key effector in the replacement of RPA for Rad51 on ssDNA and its mutation is associated with hereditary breast and ovarian cancer (Fackenthal and Olopade, 2007; Holloman, 2011; Moynahan and Jasin, 2010). Rad51-coated ssDNA forms a D-loop structure as it invades the sister chromatid and then produces a double Holliday junction as DNA polymerases perform extensions. The final step of HR involves resolving the Holliday junction in a process that can result in strand crossovers (Matos and West, 2014; Swuec and Costa, 2014). 7   Figure 1-1: Summary of the Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) DNA double strand break (DSB) repair pathways. NHEJ begins as the Ku70/80 heterodimer binds DSB ends and recruits DNA-PKCS to form the DNA-PK holoenzyme. Processing factors (including Artemis, and Mre11 of the MRN complex) then make the DNA termini amenable for ligation by a complex containing DNA Ligase IV, XRCC4, and XLF. In HR, DSB ends are bound by the MRN complex that acts to both recruit ATM and initiate end resection. The exposed ssDNA is bound by RPA, which is then replaced by Rad51 in a process requiring BRCA2. The Rad51 nucleoprotein filament invades the sister chromatid for homology searching and the Holliday junction is resolved to complete repair. This figure is copied from Lans et al. (Lans et al., 2012), published by the open-access BioMed Central. 8! Additional double strand break repair pathways There are two additional repair pathways also initiated by end resection: single strand annealing (SSA) and microhomology-mediated end joining, also known as alternative end joining (Alt-EJ). Their mechanisms are distinct from NHEJ and HR (Bennardo et al., 2008; Howard et al., 2015), but our understanding of them is still limited and requires further elucidation. SSA and Alt-EJ are highly mutagenic and do not involve strand invasion with the sister chromatid. Instead, they rely on sequence homology to be present within the same DNA molecule during resection from a DSB.  SSA has been best studied in yeast (Haber and Leung, 1996; Ivanov et al., 1996; Sugawara et al., 2000). Following extensive resection, sequence homologies of ~30 bp can be revealed and bound by Rad52 (Symington, 2002). The large intervening sequence is then removed by the ERCC1 endonuclease (Sargent et al., 2000; Sargent et al., 1997) to yield sequence deletions. Mammalian cells have also shown the same dependency of these factors for SSA fidelity (Bennardo et al., 2008).  Alt-EJ occurs after limited resection reveals regions of microhomology (~10 bp) flanking the DSB and results in short base pair deletions/insertions (Ceccaldi et al., 2016; Truong et al., 2013). The critical factors currently known to be involved in Alt-EJ are PARP-1, DNA Ligase I/III, XRCC1, and DNA polymerase θ (Arakawa et al., 2012; Audebert et al., 2004; Ceccaldi et al., 2015; Liang et al., 2008; Mansour et al., 2010; Mateos-Gomez et al., 2015; Paul et al., 2013; Wang et al., 2005). Inhibition of Alt-EJ with PARP inhibitors has shown great therapeutic 9  benefit for cancers with BRCA1/2 deficiencies. These tumors have defects in downstream processes of HR and are therefore more reliant on Alt-EJ for repair. Thus, treating these cancers with a PARP inhibitor produces synthetic lethality (Bryant et al., 2005; Farmer et al., 2005). In December 2014, the FDA approved Lynparza™ (Olaparib), a PARP inhibitor, as a monotherapy for ovarian cancer patients possessing germline BRCA1/2 mutations. This was a milestone for therapeutic targeting of the DDR and personalized medicine.! Experimental generation of double strand breaks There are several methods to induce DSBs experimentally and for cancer therapy, and each works by one of two general principles. First, there are chemical compounds that inhibit the function of Topoisomerase II. This enzyme works in conjunction with DNA replication and transcription to relieve the torsional strain imposed by unwinding DNA. Topoisomerase II has a two-step process whereby it creates an enzymatic DSB that allows the DNA strands to uncoil, followed by a religation step. Etoposide is a derivative of podophyllotoxin isolated from the wild mandrake (Podophyllum peltatum) that covalently stabilizes the intermediate structure of Topoisomerase II preventing the religation step and leaving the enzymatic DSB (Baldwin and Osheroff, 2005). The other method of DSB generation is through the action of free radicals that cause a variety of DNA lesions (Cooke et al., 2003; Valko et al., 2006; Ward, 1988). These can be produced by ionizing radiation (IR), including X-rays and γ-rays, which contain enough energy to strip electrons off atoms. There are also radiomimetic compounds (e.g. neocarzinostatin and bleomycin) whose chemistry generates free radicals. DSBs can have greater complexity depending on the source of damage (etoposide < X-ray < carbon ion), which 10  corresponds to slower repair kinetics and a propensity towards the HR pathway (Shibata et al., 2011).  1.1.3! Chromatin response to double strand breaks It is conceptually important to remember that DDR signalling and DNA repair take place in the context of chromatin and higher order DNA structures that influence pathway choice and repair kinetics. This notion was appreciated long before the mechanisms began to be elucidated (Smerdon, 1991). The basic unit of chromatin is the nucleosome in which 147 bp of DNA are wound around a (H2A-H2B-H3-H4)2 histone octamer (Luger et al., 1997; Richmond and Davey, 2003). This structure is assembled into newly synthesized DNA during replication and repair through association of the PCNA sliding clamp with the Chromatin Assembly Factor-1 (CAF-1) complex (Gaillard et al., 1996; Gerard et al., 2006; Green and Almouzni, 2003; Moggs et al., 2000). Nucleosomes are then packed into higher order structures. Euchromatin exists in a more open configuration and localizes transcriptionally active genes within the nuclear interior (Harnicarova et al., 2006; Williams et al., 2006; Zink et al., 2004). The term heterochromatin (HC) was first used in cytological descriptions of deeply stained chromatin regions that remain condensed throughout the cell cycle (Heitz, 1928). HC consists of gene-poor regions that localize to the nuclear periphery with a highly condensed architecture (Croft et al., 1999). DSBs within euchromatin are fixed with rapid kinetics (< 4 hours) and preferentially utilize the NHEJ pathway (Lorat et al., 2012; Shibata et al., 2011). Whereas heterochromatic structure is refractory to repair and DSBs within it require chromatin remodeling – repair occurs with slow kinetics (4 – 48 hours), primarily via HR (Goodarzi et al., 2008; Kakarougkas et al., 2013; Noon et al., 2010; Shibata et al., 2011). 11   HC is molecularly characterized by the presence of heterochromatin protein 1 (HP1) isoforms, and trimethylation on lysine 9 of histone H3 (H3K9me3), each of which is needed for the nucleation, maintenance, and propagation of HC structure (Hall et al., 2002; Litt et al., 2001; Nakayama et al., 2001; Noma et al., 2001; Yamada et al., 2005). The H3K9me3 mark is evolutionarily conserved in yeast and fungi up to mammals (Du et al., 2015), and is generated by the SETDB1 and SUV39H1/2 histone methyltransferases (Bakkenist and Kastan, 2015; Becker et al., 2016; Du et al., 2015; Kim and Kim, 2012). HP1 proteins were first identified in Drosophila and are found in many organisms from S. pombe to mammals (Grewal and Jia, 2007; Huisinga et al., 2006). Their amino terminal chromodomain binds H3K9me3 (Bannister et al., 2001; Jacobs and Khorasanizadeh, 2002; Lachner et al., 2001; Nielsen et al., 2002) while their chromoshadow domain facilitates protein-protein interactions with other factors that promote HC compaction (Lechner et al., 2005; Murzina et al., 1999; Nishibuchi and Nakayama, 2014; Smothers and Henikoff, 2000; Thiru et al., 2004; Zhang et al., 2002b).   One such binding partner of the HP1 chromoshadow domain is KRAB domain-associated protein 1 (KAP-1), which was originally discovered as a transcriptional repressor (Friedman et al., 1996; Kim et al., 1996; Le Douarin et al., 1996; Moosmann et al., 1996). KAP-1 is known to interact with several HC promoting factors, including nucleosome remodelers, histone deacetylases (HDACs), and the histone methyltransferases that generate H3K9me3 (Fritsch et al., 2010; Ivanov et al., 2007; Lechner et al., 2000; Li et al., 2010b; Nielsen et al., 1999). Following DNA damage, ATM-mediated phosphorylation of KAP-1 on S824 disrupts these complexes and results in pan-nuclear HC relaxation to facilitate repair (Ayrapetov et al., 2014; 12  Goodarzi et al., 2011; Goodarzi et al., 2008; Ziv et al., 2006). This phosphosite is constitutively targeted by phosphatases and localized ATM activity must be retained to maintain the open configuration required for HC DSB repair (Lee et al., 2012; Li et al., 2010a; Liu et al., 2012; Noon et al., 2010).  While it is appreciated that HC poses a barrier to DNA repair that must be overcome (Goodarzi et al., 2010; Goodarzi and Jeggo, 2012; Price and D'Andrea, 2013), there is also significant evidence that HC promoting factors (including HP1 isoforms, histone methyltransferases, CAF-1 subunits, and KAP-1) are immediately and transiently recruited to various types of DNA damage (Alagoz et al., 2015; Ayoub et al., 2008; Ayrapetov et al., 2014; Baldeyron et al., 2011; Green and Almouzni, 2003; Luijsterburg et al., 2009; Martini et al., 1998; Moggs et al., 2000; Soria and Almouzni, 2013; Zarebski et al., 2009). Furthermore, a recent study revealed that much like γH2AX, H3K9me3 is a histone modification that propagates for hundreds of kilobases away from euchromatic DSBs (Ayrapetov et al., 2014). This may reflect the need to immobilize broken DNA termini and/or repress transcription at sites of damage. The spread of H3K9me3 in response to damage is also likely a key regulator of DDR initiation since it activates the acetyltransferase activity of Tip60, which in turn initiates robust ATM activation and DDR signaling (Kaidi and Jackson, 2013; Sun et al., 2009). Chromatin compaction has also been shown to promote processes of HR downstream of end resection, potentially by holding sister chromatid together during homology searching (Alagoz et al., 2015; Geuting et al., 2013).  13  1.2! The BCL-2 family of proteins regulates apoptosis The initiation of apoptosis from intrinsically derived stimuli (e.g. DNA damage) is under control of the BCL-2 family of proteins. Release of cytochrome c from mitochondria marks the point of no return in apoptosis signalling since it combines with Apaf-1 to form the apoptosome complex that initiates the caspase cascade responsible for the orchestrated dismantling of a cell (Ola et al., 2011; Reubold and Eschenburg, 2012.; Tait and Green, 2013) The BCL-2 family proteins are subdivided based on their apoptosis promoting/inhibiting function, and composition of BCL-2 homology (BH) domains. There are multi-domain pro-apoptotic members (Bax and Bak) and multi-domain pro-survival members (BCL-2, BCL-XL, MCL-1, BCL-w), which each have BH1, BH2, and BH3 domains. There are also BH3-only containing proteins (including Bid, Bim, Bad, Puma, and Noxa) that respond to toxic stimuli and regulate the transition from pro-life to pro-death signals within a cell.   BH domains 1 – 3 form a hydrophobic groove that mediates interaction with the BH3 domain of other BCL-2 family members. Bax and Bak are thought to form the pore through which cytochrome c is released from mitochondria and each undergo extensive conformational changes to execute this step (Bogner et al., 2010; Leber et al., 2007). The dynamics of how the other BCL-2 family members regulate Bax and Bak continues to be revealed and there are competing models that are likely not mutually exclusive. To summarize, the key observations regarding BCL-2 family interactions are: 1) Bax and Bak can be activated directly by certain BH3-only proteins; 2) Bax and Bak can be held inactive by multi-domain pro-survival proteins and these interactions can be de-repressed by BH3-only proteins, which thus have pro-apoptotic activity; 3) the potency with which a BH3-only protein initiates apoptosis relates to the subset of multi-14  domain BCL-2 family members it can bind. (Brahmbhatt et al., 2015; Brunelle and Letai, 2009; Chipuk et al., 2010; Green and Llambi, 2015; Llambi and Green, 2011; Luna-Vargas and Chipuk, 2015; Moldoveanu et al., 2014; Shamas-Din et al., 2013; Zheng et al., 2015). It is important to highlight that none of the BCL-2 family members possess enzymatic function and instead regulate apoptosis by protein-protein interactions and conformational changes in their binding partners.  A hallmark of cancer is the evasion of apoptosis signals and many tumors have elevated levels of pro-survival BCL-2 family proteins to achieve this (Hanahan and Weinberg, 2000; Hanahan and Weinberg, 2011; Kelly and Strasser, 2011; Yip and Reed, 2008). Cancer cells have been described as “primed for death” and can be sensitized for apoptosis by repressing the pro-survival proteins they are reliant on (Certo et al., 2006). As such, there is interest to produce compounds that mimic BH3 domain interactions with the hydrophobic groove of pro-survival BCL-2 family members (Besbes et al., 2015; Delbridge and Strasser, 2015; Nemec and Khaled, 2008; Opferman, 2015; Roy et al., 2014). The first BH3-mimetic was ABT-737, modeled on the BCL-2/BAD interaction, which showed high binding affinity for BCL-2, BCL-XL, and BCL-w (Park et al., 2006; Petros et al., 2006). This was followed by an orally-available derivative, ABT-263 (Navitoclax) (Tse et al., 2008), and more recently ABT-199 (Venetoclax), which solely inhibits BCL-2 (Souers et al., 2013). The BH3-mimetics Navitoclax and Venetoclax are currently undergoing a number of Phase II and III clinical trials for a wide range of human cancers.  15  1.2.1! Linking the DNA damage response to apoptosis signaling The most defined mechanism that links DDR signalling to apoptosis is through stabilization of p53 by the DDR kinases as previously described. p53 then upregulates the pro-apoptotic BCL-2 family members Bax, Noxa, and Puma (Kurata et al., 2008; Lee et al., 2007; Miyashita et al., 1994; Yu et al., 2001). p53 also has transcription-independent mechanisms that can promote apoptosis by directly binding BCL-XL, Bax, and Bak at mitochondria, thus behaving somewhat like a BH3-only protein (Chi, 2014; Chipuk et al., 2005; Chipuk et al., 2004; Leu et al., 2004; Mihara et al., 2003; Moll et al., 2005).   Additionally, ATM and ATR have been shown to directly phosphorylate a nuclear pool of Bid leading to efficient S phase arrest in response to DNA damage (Kamer et al., 2005; Liu et al., 2011; Maryanovich et al., 2012; Oberkovitz et al., 2007; Zinkel et al., 2005). There is also evidence that Bid functions in DSB repair and leukemia development, separate from its canonical BCL-2 family function (Biswas et al., 2013).  1.3! Focusing on MCL-1: a BCL-2 family member with many unique characteristics MCL-1 is a multi-domain pro-survival member of the BCL-2 family that was first identified as a key regulator of monocyte and macrophage differentiation (Kozopas et al., 1993), and later characterized as an early response gene induced by DNA damage (Zhan et al., 1997). MCL-1 is required for the development and maintenance of several cell lineages including hematopoetic stem cells (Opferman et al., 2005), lymphocytes (Dzhagalov et al., 2008; Opferman et al., 2003), neutrophils (Dzhagalov et al., 2007; Steimer et al., 2009), plasma cells (Peperzak et al., 2013), neurons (Arbour et al., 2008), cardiomyocytes (Wang et al., 2013), and oocytes (Omari et al., 16  2015). MCL-1 is also critical for development since knockout embryos died prior to implantation; however, there was no detectable increase in apoptosis, which suggests that this protein has additional essential functions (Rinkenberger et al., 2000). MCL-1 is also critical for the mother’s biology during embryo implantation where it functions as a transcriptional co-activator in the nucleus of uterine stromal cells to promote the necessary mesenchymal-epithelial transition (Renjini et al., 2014). Additional nuclear functions of MCL-1 are discussed below and there is also growing evidence that MCL-1 regulates mitochondrial physiology and fission/fusion dynamics (Morciano et al., 2016; Omari et al., 2015; Perciavalle et al., 2012; Varadarajan et al., 2013a; Wang et al., 2013).  Given its pro-survival function, MCL-1 is known to be overexpressed and/or facilitate therapeutic resistance in a wide range of hematological and solid tumors, including: myelomas (Derenne et al., 2002; Wuilleme-Toumi et al., 2005; Zhang et al., 2002a), leukemias (Aichberger et al., 2005; Glaser et al., 2012), lymphoma (Khoury et al., 2003), and cancers of the  breast (Ding et al., 2007b; Long et al., 2015), liver (Fleischer et al., 2006; Sieghart et al., 2006), pancreas (Wei et al., 2008), lung (Song et al., 2005; Zhang et al., 2011), stomach (Akagi et al., 2013; Wacheck et al., 2006), and skin (Boisvert-Adamo et al., 2009; Skoda et al., 2008; Skvara et al., 2005). In fact, the genetic locus of MCL-1 located at chromosome 1q21 is one of the most highly amplified regions in all human cancer genomes (Beroukhim et al., 2010).  1.3.1! MCL-1 protein structure The MCL-1 gene contains 3 exons and yields a full length protein of 350 amino acids. The C-terminal transmembrane domain localizes MCL-1 primarily to mitochondria (Germain and 17  Duronio, 2007; Yang, 1995) while the BH1/2/3 domains form the hydrophobic groove that allows MCL-1 to perform its canonical BCL-2 family function (Clohessy et al., 2006). MCL-1 is structurally unique within the BCL-2 family as it also has an extended N-terminus of 170 amino acids that bears no similarity to other BCL-2 family members. This portion of the protein lacks any discernable structural motifs and has not been included in any of the NMR or crystal structures of MCL-1. The N-terminus does contain two PEST domains rich in proline (P), glutamate (E), serine (S), and threonine (T) residues. Structural diagrams of MCL-1 and other BCL-2 family members are depicted in Figure 1-2.  There also exist short and extra short splice variants named MCL-1S and MCL-1ES, respectively. MCL-1S arises from the skipping of exon 2 to generate a 271 amino acid protein that only contains the PEST and BH3 domains (Bae et al., 2000; Bingle et al., 2000). The MCL-1ES variant results from splicing within exon1 to yield a 197 amino acid protein that only lacks the PEST regions (Kim and Bae, 2013; Kim et al., 2009). Both of these shorter variants are expressed at low levels in normal cells and exhibit the opposite function, being pro-apoptotic, and show binding exclusivity towards full length MCL-1, compared to other BCL-2 family members (Bae et al., 2000; Bingle et al., 2000; Kim and Bae, 2013; Kim et al., 2009). Another BCL-2 family gene, Bcl-X, similarly undergoes alternatively splicing whereby BCL-XL is the most predominant variant and is pro-survival, while BCL-XS antagonizes this function (Boise et al., 1993; Fang et al., 1994; Heermeier et al., 1996; Michels et al., 2013; Willimott et al., 2011). There is evidence that alternative splicing of MCL-1 is differentially regulated throughout the cell cycle (Moore et al., 2010) and that increasing the ratio of MCL-1S with antisense oligonucleotides can induce apoptosis in cancer cells (Kim et al., 2011; Morciano et al., 2016; Shieh et al., 2009).  18   However, studying the endogenous proteins of these shorter splice variants by immunoblotting is confounded by the fact that most MCL-1 antibodies also detect proteolytic fragments of full length MCL-1 that have altered protein stability and subcellular localization (De Biasio et al., 2007; Fan et al., 2014; Huang and Yang-Yen, 2010; Jamil et al., 2005; Perciavalle et al., 2012; Stewart et al., 2010a). Furthermore, in our lab’s experience, full length MCL-1 can be degraded during sample preparation, post-lysis, to generate an immunoblot band at the same size as MCL-1S. This can be minimized by using broad spectrum protease inhibition, including PMSF, and working quickly with samples on ice (unpublished data). It is necessary to highlight that all investigations of MCL-1 in this thesis were with the full length protein.    The hydrophobic groove of MCL-1 is structurally unique within the BCL-2 family multi-domain pro-survival proteins (Dutta et al., 2010; Yang and Wang, 2012) and exhibits a unique binding profile with BH3-only proteins – it does not bind Bad and is the only family member that interacts with Noxa (Gelinas and White, 2005; Luna-Vargas and Chipuk, 2015; Thomas et al., 2010). Interestingly, the BH3 domain of MCL-1 itself shows potent and exclusive binding to full length MCL-1 (Stewart et al., 2010b), which may explain the pro-apoptotic effect of the shorter splice variants. Because of these structural differences in its hydrophobic groove, MCL-1 has low affinity for each of the BH3-mimetic drugs currently available (ABT-737, ABT-263, ABT-199) and its high protein expression in cancer cells is associated with resistance to each compound (Chen et al., 2011; Chen et al., 2007a; Choudhary et al., 2015; Konopleva et al., 2006; Lin et al., 2007; Tahir et al., 2007; van Delft et al., 2006.) Accordingly, there are a number of groups trying to develop potent and selective MCL-1 inhibitors (Abulwerdi et al., 2014; 19  Bernardo et al., 2010; Doi et al., 2012; Friberg et al., 2013; Petros et al., 2014; Richard et al., 2013). An effective BH3-mimetic drug should have a high affinity (Kd < 100 nM) to outcompete endogenous protein interactions, and elicit hallmarks of apoptosis signaling within 2 – 4 hours (Souers et al., 2013; Tao et al., 2014; Tse et al., 2008). Thus, the specificity of many MCL-1 inhibitors has been called into question due to the need for high concentrations and their induction of apoptosis via other toxic mechanisms (Albershardt et al., 2011; Eichhorn et al., 2013; Varadarajan et al., 2013a; Varadarajan et al., 2013b). Recently though, a high-throughput screen of compounds that disrupt the MCL-1/Bim interaction yielded a potent (Kd = 0.454 nM) and selective BH3-mimetic for MCL-1 that induced apoptosis in a MCL-1-dependent cancer cell line and lowered the lethal dose of ABT-263 in others (Bruncko et al., 2015; Leverson et al., 2015).  20   Figure 1-2: Structural alignments of MCL-1 and other BCL-2 family members. Multi-domain members have BH1-3 domains that fold into a hydrophobic groove for interactions with the BH3 domain of other family members. MCL-1 is structurally unique within the BCL-2 family as it contains an extended N-terminus of 170 amino acids that bears no similarity to other members, but does contain two PEST domains.   1.3.2! MCL-1 protein turnover One of the most interesting properties of MCL-1 is its short half-life, ranging from 30 minutes to 21  4 hours depending on the cell type (Chao et al., 1998; Cuconati et al., 2003; Ding et al., 2007a; Jamil et al., 2010b; Liu et al., 2005; Maurer et al., 2006; Nijhawan et al., 2003; Schubert and Duronio, 2001; Stewart et al., 2010a). The value of this measurement appears to depend on whether experiments were done by pulse-chase or with cycloheximide, suggesting that protein turnover of newly synthesized MCL-1 may occur faster than that of more mature forms. MCL-1 protein levels are under dynamic regulation by transcriptional, translational, and post-translational mechanisms (Opferman, 2006; Thomas et al., 2010; Warr and Shore, 2008). The PEST domains contain several sites of phosphorylation, ubiquitination, and caspase cleavage, and were originally thought to dictate protein stability (Rechsteiner and Rogers, 1996). However, there is accumulating evidence that deletion of the PEST domains has no effect on MCL-1 protein turnover (Akgul et al., 2000; Clohessy et al., 2004; Xiao et al., 2014).  To date, five ubiquitin E3 ligases have been identified that target MCL-1: βTrCP, Fbw7, Trim17, MULE, and cdc20. Factors affecting MCL-1 ubiquitination, including key phosphorylation sites, have been extensively reviewed (Mojsa et al., 2014; Opferman, 2006; Thomas et al., 2010; Warr and Shore, 2008) but specific points related to nuclear functions of MCL-1 will be addressed in following sections. It is important to highlight that a non-ubiquitinatable mutant of MCL-1 in which all Lys residues were mutated to Arg had the same protein stability and resistance to etoposide and staurosporine treatments as the wildtype protein (Stewart et al., 2010a). Additionally, in a cell-free system with unmodified in vitro synthesized proteins, the 20S proteasome was able to directly degrade MCL-1 (Stewart et al., 2010a). There is evidence that intrinsically unstructured proteins can undergo ubiquitin-independent proteasomal degradation (Ben-Nissan and Sharon, 2014; Jariel-Encontre et al., 2008) and the N-terminus of MCL-1 may 22  fit into this paradigm. However, a recent study identified the VTLISFG sequence at the C-terminal end of the BH1 domain as the minimum requirement for MCL-1 degradation (Xiao et al., 2014). This motif neither contains Lys residues for ubiquitination nor is located in an unstructured area of the protein and further reinforces our lack of understanding of how MCL-1 protein stability may be regulated. While polyubiquitination appears dispensable for baseline MCL-1 turnover, it does still add a layer of refined regulation in response to various stimuli and stresses.   1.3.3! Fluctuations of MCL-1 protein levels during the cell cycle MCL-1 protein dynamics are regulated throughout the normal unperturbed cell cycle. Immunofluorescence of asynchronous cells shows that only certain cells in a population possess nuclear MCL-1 (Liu et al., 2005). More specifically, releasing U2OS cells from double thymidine block showed that MCL-1 levels rise in S and G2, peak in M, and return to baseline in the following G1 (Harley et al., 2010). In hematopoetic cells, a proteolytic fragment of MCL-1 showed nuclear localization only during S and G2 (Jamil et al., 2005).  Overexpression of MCL-1 leads to a decrease in cell proliferation (Fujise et al., 2000; Jamil et al., 2005) but the precise effects on cell cycle arrest are less clear. Some studies saw that MCL-1 overexpression resulted in G2/M arrest (Jamil et al., 2008; Renjini et al., 2014; Yi et al., 2012), while another saw no effect (Jamil et al., 2005). Conversely, there is also evidence that MCL-1 knockdown lead to G2/M arrest (Yi et al., 2012). These conflicting results may reflect lineage-specific roles for MCL-1 or may highlight that the level of MCL-1 expression in a cell needs to 23  be held within a critical window for proper cell cycle progression. The latter is certainly achievable with a rapidly turned over protein.  1.3.4! Nuclear translocation of MCL-1 There is a growing body of evidence for diverse roles of MCL-1 in the nucleus, yet the protein contains neither a conventional nuclear localization sequence (NLS) nor nuclear export sequence, and the mechanisms of its nuclear translocation are poorly understood. Despite the canonical mitochondrial function and localization of MCL-1, in U2OS cells its predominant subcellular localization is in the nucleus (Fujise et al., 2000; Zhang et al., 2002c). However, in most cell types MCL-1 is primarily cytosolic and shuttled into the nucleus in response to genotoxic stress (Jamil et al., 2008; Pawlikowska et al., 2010; Wang et al., 2014a), or other stimuli (Renjini et al., 2014). Even in unperturbed cells though, a nuclear pool is maintained and MCL-1 shows rapid redistribution into the nucleus during fluorescent recovery after photobleaching (Thomas et al., 2012). Adding to the complexity is the observation that MCL-1 is primarily mitochondrial the day after seeding cells in culture, but can be seen in the nucleus after two days and beyond (Liu et al., 2005). Analysis of a human tissue microarray revealed that nuclear staining of MCL-1 is correlated with later stages of prostate cancer (Reiner et al., 2015).  Post-translational modifications of MCL-1 can affect its nuclear localization. Ser62 phosphorylation appears to play a key role in this process as S62A MCL-1 was preferentially located in the nucleus, but excluded from nucleoli. This construct differed from wildtype canonical function, with higher protein turnover and it was unable to protect cells from apoptosis induced by Bak overexpression (Thomas et al., 2012). Different proteolytic fragments of MCL-1 24  also showed nuclear accumulation and reduced cellular proliferation (Fan et al., 2014; Jamil et al., 2005).  The nuclear translocation of MCL-1 likely depends on protein binding partners. Like MCL-1, IEX-1 is also an early response gene following DNA damage that is shuttled into the nucleus. IEX-1 interacts with the transmembrane domain of MCL-1 (but does not bind BCL-2 or BCL-XL) in an ATM-dependent manner following IR treatment (Pawlikowska et al., 2010). Mutation of the NLS in IEX-1 still permitted binding to MCL-1 but abolished the nuclear localization of each (Pawlikowska et al., 2010). To date this is the only protein interaction identified that regulates MCL-1 nuclear trafficking but it is possible that other proteins may have a similar function.  1.3.5! MCL-1 has an active role in the DNA damage response Our lab was the first to demonstrate that nuclear MCL-1 facilitates DDR signaling, showing that MCL-1-null MEFs have diminished Chk1 activation in response to DNA damage (Jamil et al., 2008). This was later confirmed by an independent group (Pawlikowska et al., 2010). A follow up study revealed that MCL-1 is chromatin-bound directly at sites of DNA damage and interacts with the NBS1 subunit of the MRN complex (Jamil et al., 2010a). Cells lacking MCL-1 also have decreased DNA repair kinetics and accumulated a greater number of chromosomal aberrations (Jamil et al., 2010a; Pawlikowska et al., 2010; Reiner et al., 2015). Recently, it was also observed that Ku70 binds MCL-1 in response to DNA damage and can directly deubiquitinate K48 linkages to prolong protein half-life of MCL-1 (Wang et al., 2014a).  25  1.3.6! Additional nuclear interactions of MCL-1 Additional nuclear proteins have been identified as MCL-1 binding partners through yeast 2-hybrid screens and CoIPs, and are discussed below. This adds to the evidence that MCL-1 has functions in the nucleus separate from its canonical role within the BCL-2 family pathway.! Cyclin dependent kinase 1 CDK1 is a critical cell cycle regulatory protein that promotes the progression and transition through G2 into mitosis via interactions with cyclin A and B, respectively. MCL-1 and CDK1 have reciprocal regulatory functions on each other that implicates MCL-1 in the process of mitotic arrest. The two proteins are direct binding partners and MCL-1 inhibits CDK1 kinase activity (Jamil et al., 2005; Yi et al., 2012). Conversely, the cyclin B1-CDK1 complex phosphorylates Thr92 MCL-1 to engage the anaphase promoting complex leading to the MCL-1 degradation that occurs in response to mitotic arrest from microtubule inhibitors (Harley et al., 2010; Sakurikar et al., 2012; Wertz et al., 2011). During prolonged mitotic arrest, there is caspase-dependent activation of DDR effectors (ATM, Chk1, Chk2, γH2AX) to prime cells for apoptosis if they incorrectly slip through the checkpoint (Ganem and Pellman, 2012; Heijink et al., 2013; Orth et al., 2012). In response to DSBs, MCL-1 potentiates DDR signaling (Jamil et al., 2008; Jamil et al., 2010a; Pawlikowska et al., 2010) but when treated with microtubule inhibitors, the overexpression of MCL-1 actually inhibits activation of DDR kinases (Colin et al., 2015).  26! Proliferating cell nuclear antigen PCNA is a sliding DNA clamp at sites of synthesis that acts as a scaffold to recruit a vast array of proteins required for DNA replication and repair, chromatin assembly, and epigenetic programming (Alabert et al., 2014; Naryzhny, 2008). A yeast 2-hybrid screen of PCNA identified MCL-1 as a protein binding partner, but not BCL-XL, Bax, or Bak (Fujise et al., 2000). This interaction was validated by coimmunoprecipitation (CoIP) in human cell lines and shown to be dependent on amino acids 221-228 of MCL-1, but was unaffected by DNA polymerase or microtubule inhibitors (Fujise et al., 2000; Jamil et al., 2005; Yi et al., 2012). However, NMR structural analysis of PCNA interactions using purified recombinant proteins failed to detect an association with MCL-1, suggesting that the interaction is weak or requires additional factors (De Biasio et al., 2012).! Translationally controlled tumor protein TCTP is a protein that bears no sequence homology to any known protein but is highly expressed in eukaryotes and is critical for development since knockout mice are embryonic lethal (Chen et al., 2007b). TCTP is a multifunctional chaperone protein that is upregulated in many cancers and has regulatory roles in apoptosis, cell cycle, proliferation, and cytoskeletal dynamics (Acunzo et al., 2014; Bommer and Thiele, 2004). In response to IR, TCTP makes chromatin associations in conjunction with ATM and γH2AX, and potentiates the DNA binding affinity of the Ku70/80 heterodimer (Zhang et al., 2012). MCL-1 and TCTP were identified as binding partners in separate yeast 2-hybrid screens using each as the bait, and they reciprocally increased the post-translational protein stability of each other (Liu et al., 2005; Zhang et al., 2002c).   27  1.3.7! Functions of additional pro-survival BCL-2 family members in double strand break repair and G2/M arrest Failure to properly repair DNA damage may result in genomic instability that is potentially oncogenic and cells initiate apoptosis to avoid propagating damaged genomes. Besides the aforementioned roles of MCL-1, other pro-survival BCL-2 family members have active roles in the nucleus regulating DSB repair and cell cycle arrest. Thus, there is accumulating evidence for a paradigm wherein BCL-2 family members have dual functions at the crossroads of DDR and apoptosis regulation.! BCL-2 inhibits double strand break repair There are several interspersed studies implicating BCL-2 in DSB repair, but with disparate mechanisms of action. A repeated observation is that BCL-2 overexpression inhibits the fidelity of HR when assessed using reporter assays (Dumay et al., 2006; Laulier et al., 2011; Saintigny et al., 2001). In one of these studies, BCL-2 overexpression had no effect on Rad51 foci kinetics but did suggest differences in Rad51 post-translational modification using 2D electrophoresis (Saintigny et al., 2001). In another, the transmembrane domain of BCL-2 was shown to bind BRCA1 and sequester it to mitochondria and the endoplasmic reticulum to inhibit HR function (Laulier et al., 2011).  BCL-2 translocation into the nucleus is dependent on its binding partner, FKBP38 (Portier and Taglialatela, 2006). In response to IR, BCL-2 interacts with Ku70/Ku80, independent of canonical BH1-3 hydrophobic groove associations, to prevent their binding to DSB ends (Dutta et al., 2012; Wang et al., 2008). Conversely, an inhibitory association of BCL-2 on PARP1 is 28  relieved by treatment with BH3 mimetics (Dutta et al., 2012). While a nuclear role of BCL-2 is clear, there is still much to be learned about its mechanism of action.! BCL-XL potentiates G2/M arrest in response to etoposide The Bertrand lab at the Université de Montréal did a robust screen of BCL-XL phosphosite mutants and identified two key phosphorylation events that oscillate reciprocally during normal mitotic progression: pS49 peaks during G2, telophase, and cytokinesis, while pS62 is strongest during metaphase and anaphase (Wang et al., 2011; Wang et al., 2014b). Each of these phosphosites contributes to BCL- XL -mediated G2/M arrest in response to etoposide by inhibiting CDK1, but through different mechanisms. pS49 BCL- XL directly inhibits CDK1 activity (Wang et al., 2011), while pS62 BCL- XL sequesters CDK1 in nucleoli, away from its targets, without affecting its kinase activity (Wang et al., 2014b). The exact role of BCL- XL in DSB repair may depend on cell type since HR efficiency was increased by overexpression in human B lymphoblasts (Wiese et al., 2002) but decreased by overexpression in human fibroblasts (Dumay et al., 2006) and mouse L cells (Saintigny et al., 2001).  1.4! Rationale, hypothesis, and chapter summary It is clear that MCL-1 has functions in the nucleus beyond its canonical interactions with BCL-2 family members to regulate apoptosis. And the fact that MCL-1 knockout mice experience embryonic lethality prior to implantation, with no apparent increase in apoptosis, suggests additional essential functions unrelated to its traditional roles of the BCL-2 family pathway (Rinkenberger et al., 2000). Therefore, we hypothesized that MCL-1 has an essential role in the nucleus to facilitate DSB repair and maintain genome stability. My approach to investigating this 29  hypothesis was two-fold: 1) identify additional DDR signaling events and processes requiring MCL-1; 2) perform a proteomic screen of putative MCL-1 protein interactions and validate a mechanism through which MCL-1 promotes DNA repair.  Chapter 3: reveals that cells lacking MCL-1 have a robust impairment of ATM and Chk2 activation, decreased efficiency of the HR pathway, and elevated RPA foci > 4 hours following IR. In addition, the nuclear pool of nuclear MCL-1 protein has a shorter half-life than that of the cytosol. Chapter 4: describes the results of a proteomic screen for MCL-1 protein interacting partners using two separate CoIP techniques, which discovered HP1α and p150CAF-1 as novel MCL-1 binding partners. These interactions suggested a role for MCL-1 as a HC protein; together with the diminished KAP-1 phosphorylation and defective RPA kinetics of MCL-1-depleted cells that I identified in Chapter 3:, I hypothesized that MCL-1 potentiates repair of DSBs specifically within HC. However, Chapter 5: demonstrates that key phenotypes associated with HC DSB repair do not require MCL-1. Instead, MCL-1 plays an active role in HC compaction and maintains basal levels of H3K9me2/3, all in the absence of exogenous DNA damage.  There are numerous lines of evidence that HC compacting factors are immediately and transiently recruited to DNA damage (Alagoz et al., 2015; Ayoub et al., 2008; Ayrapetov et al., 2014; Baldeyron et al., 2011; Green and Almouzni, 2003; Luijsterburg et al., 2009; Martini et al., 1998; Moggs et al., 2000; Soria and Almouzni, 2013; Zarebski et al., 2009). The heterochromatic H3K9me3 mark propagates away from DSBs (Ayrapetov et al., 2014) and increases the acetyltransferase activity of Tip60 needed for robust ATM activation (Chailleux et al., 2010; 30  Ikura et al., 2015; Jiang et al., 2006; Kaidi and Jackson, 2013; Sun et al., 2005; Sun et al., 2010; Sun et al., 2009; Sun et al., 2007; Xu et al., 2012b). Recent studies also have revealed that downstream processes of HR are enhanced by chromatin compaction, possibly by keeping entangled sister chromatid stable during homology searching (Alagoz et al., 2015; Geuting et al., 2013). Thus, by promoting chromatin compaction and H3K9me2/3, MCL-1 is capable of participating in multiple phases of the DDR and HR, and a proposed model summarizing this concept is outlined in Figure 1-3.  31   Figure 1-3: Proposed model for the involvement of MCL-1 in heterochromatin dynamics, ATM signalling, and homologous recombination. The novel protein complex of MCL-1 with HP1α and p150CAF-1 recruits histone methyltransferases to generate heterochromatin structure, characterized by H3K9me3. In response to DNA damage, H3K9me3 activates the acetyltransferase activity of Tip60, which in turn activates robust ATM kinase activity. Downstream targets of ATM initiate cell cycle arrest and homologous recombination (HR). ATM phosphorylation of KAP-1 acts as a negative feedback to limit the amplification of H3K9me3 away from the DSB. Heterochromatin factors also indirectly enhance the latter stages of HR by keeping sister chromatid in close proximity during homology searching.    32  Chapter 2:!Methods 2.1! Cell culture All cells lines and culture media used in this work are outlined in Table 2-1.  Table 2-1: Cell lines  2.2! Chemicals Cycloheximide (Calbiochem Cat #239763) was used at 100µg/mL. Etoposide (Calbiochem Cat #341205) was used at the indicated concentrations between 1 – 100µM. Aphidicolin (Calbiochem Cat #178273) was used 1µg/mL. Each of these chemicals was prepared in DMSO stocks and stored at -20°C.   2.3! siRNA and plasmid transfections All transfections were performed with Metafectene Pro (Biontex Cat #T040). Transfection complexes were mixed in PBS and added directly to complete media, according to Cell Line Description Media HeLa Human cervical adenocarcinoma DMEM, 10% FBS, L-Glu, P/S MEF WT and MCL-L∆/- were obtained from Joe Opferman (Opferman et al., 2003). MCL-1∆/- henceforth referred to as MCL-1-/- HEK 293T Human embryonic kidney U2OS Human osteocarcinoma DMEM (no pyruvate), 10% FBS, L-Glu, P/S A549 Human lung adenocarcinoma RPMI, 10% FBS, L-Glu, P/S LNCaP Human prostate carcinoma 1BR3 Human dermal fibroblasts, normal, hTERT-immortalized MEM, 15% FBS, L-Glu, P/S PromoCell  C-12302 Primary human dermal fibroblasts, normal Commercial from PromoCell: C-23020 basal media; C39325 supplements 33  manufacturer’s instructions. siRNA for HeLa cells was added at 50nM and 1:2 Metafectene Pro (µg nucleic acid:µL transfection reagent), while all other cell lines were treated with 25nM siRNA at 1:5 Metafectene Pro. For plasmids, all cell lines were transfected with 0.5 or 1µg at 1:4 Metafectene Pro.   Scrambled siRNA was either ON-TARGETplus Non-targeting Control Pool (Dharmacon Cat #D-001810-10) or Stealth siRNA sequence 5’-UACUCUUUGCGUACUCUUGAUGCCG-3’ (ThermoFisher). MCL-1 siRNA was ON-TARGETplus Human MCL-1 SMARTpool (Dharmacon Cat #L-004501-00) and CtIP siRNA was siGENOME Human RBBP8 (Dharmacon Cat #D-011376-01).  2.4! Antibodies All primary antibodies are listed in Table 2-2, which specifies the particular species reactivity and applications utilized in this body of work. All secondary antibodies used for immunoblotting and immunofluorescence are outlined in Table 2-3  Table 2-2: Primary antibodies Target Manufacturer Catalogue # Host Species, Clonality Species Reactivity Application Dilution ATM Novus Biologicals NB100-104 Rabbit Polyclonal Human, Mouse WB 1:500 pS1981 ATM Novus Biologicals NB100-306 Mouse Monoclonal Human, Mouse WB 1:500 p150CAF-1 Santa Cruz sc10205 Goat Polyclonal Human, Mouse WB, IP, IF 1:250 p150CAF-1 Santa Cruz sc10206 Goat Polyclonal Mouse WB, IF 1:250 Chk2 Novus Biologicals NB100-56546 Mouse Monoclonal Human, Mouse WB 1:500 pT68 Chk2 Novus Biologicals NB100-92502 Rabbit Polyclonal Human, Mouse WB 1:500 Fibrillarin Abcam ab5821 Rabbit Polyclonal Human IF 1:1000 34  Target Manufacturer Catalogue # Host Species, Clonality Species Reactivity Application Dilution γH2AX ABM YO11268 Rabbit Polyclonal Human, Mouse WB 1:1000 γH2AX Millipore 05-636 Mouse Monoclonal Human, Mouse IF 1:1000 H3 Millipore 06-755 Rabbit Polyclonal Human, Mouse WB 1:500 pS10 H3 Abcam ab47297 Rabbit Polyclonal Human IF 1:500 H3K9me2 Abcam ab1220 Mouse Monoclonal Human, Mouse WB 1:1000 H3K9me3 Abcam ab8898 Rabbit PolyClonal Human, Mouse WB 1:1000 HP1α/β Cell Signaling 2623 Rabbit Monoclonal Human IP 1:50 HP1α Cell Signaling 2616 Rabbit Polyclonal Human, Mouse WB, IF 1:500 KAP-1 Novus Biologicals NB500-158 Rabbit Polyclonal Human, Mouse WB, IF 1:1000 pS824 KAP-1 Bethyl A300-767A Rabbit Polyclonal Human, Mouse WB 1:1000 pS824 KAP-1 Jeggo Lab Acquired from Goodarzi Lab Rabbit Polyclonal Human IF 1:200 Lamin B1 Abcam ab16048 Rabbit Polyclonal Human, Mouse WB 1:1000 MCL-1 Rockland 600-401-394 Rabbit Polyclonal Mouse WB 1:1000 MCL-1 Santa Cruz sc819 Rabbit Polyclonal Human WB, IP 1:500 PML Santa Cruz sc966 Mouse Monoclonal Human IF 1:1000 RPA Calbiochem NA18 Mouse Monoclonal Human IF 1:500 Vinculin Sigma V9131 Mouse Monoclonal Human, Mouse WB 1:5000   Table 2-3: Secondary antibodies Species Reactivity Excitation Wavelength Manufacturer Catalogue # Host Species Application Dilution Rabbit 680nm Li-cor 928-68023 Donkey WB 1:5000 Goat 680nm Li-cor 926-68024 Donkey WB 1:5000 Mouse 800nm Li-cor 926-32212 Donkey WB 1:5000 Mouse 488nm Thermo Fischer A11029 Goat IF 1:400 Mouse 594nm Thermo Fischer A11005 Goat IF 1:200 Rabbit 488nm Thermo Fischer A11034 Goat IF 1:400 Rabbit 594nm Thermo Fischer A11037 Goat IF 1:200   35  2.5! Immunoblotting Cells were washed twice with PBS and lysed in a buffer containing 50mM Tris-HCl pH 7.7, 100mM NaCl, 1% Triton X-100, 10% glycerol, 2.5mM EDTA, 10mM sodium fluoride, 0.2mM sodium orthovanadate, and 1mM sodium molybdate, supplemented with a protease inhibitor cocktail (Roche Cat #11836170001). Samples were sonicated on ice, cleared by centrifugation at 13,200 RPM for 5 minutes at 4°C, and resuspended in Laemmli sample buffer. Protein lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 1% casein in PBS (BioRad Cat #161-0783) then probed with primary antibodies (Table 2-2) in Tris-buffered saline containing 0.05% Tween-20, 0.5% BSA, and 5mM NaF ≥ 2 hours and fluorescent secondary antibodies (Table 2-3) in 1% casein in PBS (BioRad Cat #161-0783) + 0.05% Tween-20 + 5mM NaF for 1 hour, all at room temperature. Blots were scanned on the Licor Odyssey CLx (Model 9140) imager; data acquisition and band quantification were performed with Image Studio software.   2.6! Nuclear fractionation Cells were trypsinized and washed twice with PBS, centrifuging 5 minutes at 1400 RPM. Cells were then swelled in a hypotonic buffer (10mM HEPES pH 7.9, 10mM KCl, 1.5mM MgCl2, 1mM DTT, 1mM PMSF, 10% glycerol) on ice for 10 minutes. The cytoplasmic membrane was ruptured by addition of 0.1% Triton X-100 and gentle flicking of the microfuge tube. Nuclei were pelleted by centrifugation at 3500 RPM for 5 minutes. Supernatant was taken as cytoplasmic sample. Nuclei were washed once with hypotonic buffer then lysed as per the immunoblot lysate preparation above (Section 2.5).   36  2.7! Cell cycle analysis U2OS cells were transfected with siRNA for 24 hours, then trypsinized and fixed in cold 70% ethanol for 1 hour at 4°C. Cells were washed once with PI buffer [PBS + 0.1% Triton X-100 (vol/vol) + 0.1mM EDTA]. Cells were incubated with PI buffer + 0.1mg/mL RNase A (ThermoFisher Cat #EN0531) for 30 minutes at room temperature, then propidium iodide (Thermo Fischer Cat #P3566) was added to a final concentration of 50µg/mL and incubated 20 minutes at room temperature. Flow cytometry was performed on a BD FACS Canto II and data acquired using the BD FACSDiva software. Single cells were selected by hierarchical gating of the center populations of FSC vs SSC, then PI-W vs PI-A.   2.8! Double strand break repair reporter assays Four DSB repair pathway-specific U2OS reporter cell lines were obtained from Dr. Jeremy Stark: DR-GFP (HR), SA-GFP (SSA), EJ-2-GFP (Alt-EJ), and EJ-5-GFP (Distal EJ) (Gunn et al., 2011; Gunn and Stark, 2012). Cells were transfected with siRNA at 40% confluency for 24 hours, followed by plasmid transfection of pCBASce-1 (Addgene plasmid #26477) for 48 hours. Cells were trypsinized and washed twice in FACS buffer [PBS + 2% FBS (vol/vol) + 0.1mM EDTA], spinning 5 minutes at 1400 RPM to pellet cells. Final samples were suspended in FACS buffer + 1:10,000 Hoechst 33342 (ThermoFischer Cat #H3570). Flow cytometry was performed on a BD FACS Canto II and data acquired using the BD FACSDiva software. Live cells were selected by hierarchical gating of the center population of FSC vs SSC, then the Hoechst 33342-negative population. GFP positive cells were quantified against the APC-Cy7 axis to mitigate the effects of autofluorescence.   37  2.9! Immunofluorescence Cells were grown on glass cover slips or in 8-well chamber slides (Thermo Cat #154534). Samples were washed twice with PBS and fixed with 3% (wt/vol) PFA + 2% sucrose (wt/vol) in PBS for 10 minutes, then permeabilized with 0.2% Triton X-100 (vol/vol) in PBS for 3 minutes. For RPA staining, cells were pre-extracted with 0.2% Triton X-100 (vol/vol) for 30 seconds prior to fixation. Samples were immunostained with primary antibodies (Table 2-2) for 1 hour, washed three times with PBS, and incubated with secondary antibodies (Table 2-3) for 30 minutes, all in 5% BSA (wt/vol) at room temperature. Cover slips were mounted in ProLong Gold Antifade Mountant with DAPI (ThermoFisher Cat #P-36931).  2.10! Confocal microscopy Cells were imaged on a LSM 780, AxioObserver confocal microscope (Carl Zeiss) with a Plan-Apochromat 40X1.4 Oil DIC M27 objective. Images were acquired and analyzed with Zen Black and Zen Blue software (Carl Zeiss), respectively.  2.11! Ionizing radiation-induced foci quantification of G2 cells HeLa cells were transfected with siRNA for 24 hours, then treated with 1µg/mL aphidicolin just prior to receiving 2Gy X-Ray delivered with a 300 keV Pantak Siefert XRAD320 irradiation system at 5.2Gy/min with filtration through a 0.5mm copper filter. Cells were fixed and stained for immunofluorescence of pS10 H3 and one of γH2AX or RPA. Z stacks were acquired at an interval of 0.5µm by confocal microscopy and deconvoluted using the Maximum Intensity Projection function. Foci enumeration within G2 cells was performed as previously described (Beucher et al., 2009; Geuting et al., 2013; Shibata et al., 2011). Briefly, G2 cells were identified 38  by positive pS10 H3 staining and foci were counted for at least 100 nuclei across three biological replicates.  2.12! Laser microirradiation WT and MCL-/- MEFs were incubated with 10 µM BrdU for 16 - 24 hours prior to laser microirradiation. DSB tracks were generated using a 355 nm, 5 mW self-aligning solid-state diode laser at 30% power projected through an EC Plan Neofuor 100×/1.3 NA oil immersion objective, using a laser microdissection module (PALM MicroBeam; Carl Zeiss) on an Axio Observer Z1 platorm. This is equivalent to 8 – 10 Gy delivered along the laser track (Bekker-Jensen et al., 2006). Laser irradiation was controlled by RoboSoftware 4.5 (Carl Zeiss) and images were acquired with Zen Pro software (Carl Zeiss) using an AxioCam MRm Rev.3 camera. Cells were fixed within 10 minutes of damage and stained for IF.  2.13! Targeted LacO array heterochromatin relaxation assay Human MCL-1 was amplified from pBabe-Flag hMCL-1 (Addgene plasmid #25371), introducing HindIII and BamH1 restriction sites at the 5’ and 3’ ends, respectively, with the forward primer 5’-ATAAAGCTTTTTGGCCTCAAAAGAAACG-3’ and reverse primer 5’-ATAGGATCCCTATCTTATTAGATATGCCAAA-3’. PCR amplification required 8% DMSO and a 60°C annealing temperature. The MCL-1 gene was inserted into mCherry-LacR-C1 (Coppotelli et al., 2013) with a CMV promoter and pmCherry-C1 as a backbone (Addgene plasmid #632524).  39  U2OS 2-6-3 cells possessing a heterochromatic array with 256 sequential copies of LacO (Janicki et al., 2004) were obtained with permission from Dr. Susan Janicki. These cells were either 1) transfected with LacR-mCherry or LacR-mCherry-MCL-1 plasmids for 48 hours or 2) transfected with Scr vs MCL-1 siRNA for 24 hours followed by LacR-mCherry plasmid for 48 hours. Cells were fixed and stained for immunofluorescence, and imaged by confocal microscopy.  2.14! Targeted LacO array heterochromatin double strand break-induction assay U2OS 2-6-5 cells, a derivative of the U2OS 2-6-3 heterochromatic LacO array cell line also stably expressing ER-Fok1-mCherry-LacR-DD (Tang et al., 2013), were obtained with permission from Dr. Roger Greenberg. These cells were treated for 5 hours with 1µM Shield-1 (Clontech Cat #631037) and 1µM 4-OHT (Sigma Cat #H7904) to induce stabilization and nuclear translocation of the Fok1 construct, respectively. Once targeted to the LacO array, Fok1 can induce ≥50,000 DSBs locally. Cells were fixed and stained for immunofluorescence, and imaged by confocal microscopy.  2.15! Co-immunoprecipitation 2.15.1! Antibody-agarose bead conjugation A mixture of CoIP antibody and Protein G agarose beads (Roche Cat #11243233001) were rotated in PBS for 1 hour at room temperature then washed twice with 10 volumes of 200mM sodium borate pH 9.0, centrifuging 1 minute in a bench top centrifuge after each wash. Beads were resuspended in 10 volumes of 200mM sodium borate pH 9.0 and covalently linked to the antibody by addition of 20mM dimethylpimelimidate and rotating 30 minutes at room 40  temperature. The reaction was stopped by washing once with 10 volumes of 200mM ethanolamine pH 8.0, then beads were rotated 2 hours at room temperature in 10 volumes of 200mM ethanolamine pH 8.0. Antibody not covalently attached to beads was removed by 3 washes with 10 volumes of 100mM glycine pH 2.0, followed by several PBS washes to neutralize, and stored at 4°C in PBS + 0.01% sodium azide.  2.15.2! Fractionated lysate preparation! Nuclear lysate preparation Cell nuclei were fractionated as per Section 2.6. For HeLa lysates, the protease inhibitor tablet used in the lysis buffer was Roche Ultra (Cat #5892791001). The same buffer was also used for post-IP washes.! Chromatin-bound protein complex enrichment The Active Motif Nuclear Complex CoIP Kit (Cat #54001) was used to enhance CoIP of nuclear protein complexes assembled on DNA. Cells were trypsinized and washed twice with PBS, centrifuging 5 minutes at 1400 RPM. Nuclear fractionation and lysate preparation was performed as per the kit’s reagents and instructions. Incubation with the Enzymatic Shearing Cocktail was 10 minutes at 37°C. Samples were diluted in the High Stringency Wash Buffer without added DTT or BSA, which was also used for post-IP washes.  2.15.3! Co-immunoprecipitation procedure Lysates were pre-cleared with Protein G agarose beads (Roche Cat #11243233001) by rotating for 1 hour at 4°C. Samples were spun 5 minutes at 13,200 RPM. An aliquot of supernatant was 41  taken as the Input sample for immunoblotting and the remainder was added to antibody-conjugated beads and rotated 2 hours at 4°C. Post-IP, samples were washed 5 times using 10 bead volumes of lysis buffer, spinning 1 minute in a bench top centrifuge. A small hole was made in the top and bottom of the microfuge tube using a 28G needle and remaining wash buffer was drained by 1-minute centrifugation at 3500 RPM in a swinging bucket rotor. Bound protein complexes were eluted with 3 sequential 500mM formic acid elutions, draining and pooling after each. The formic acid was evaporated by centrifugation under vacuum at 45°C. Samples were resuspended in Laemmli buffer and neutralized as necessary with ammonium hydroxide vapors.   2.16! LC-MS/MS methods 2.16.1! Cell culture Suspension-adapted HeLa-S3 cells were grown at multi-liter quantities in Joklik’s modified MEM + 5% FBS by the National Cell Culture Center (Minneapolis, MN). ~0.65 x 106 cells/mL were treated with 15µM etoposide (Calbiochem Cat #341205) for 3 hours, then washed with PBS + 1mM PMSF, pelleted into multiple equivalent fractions, snap frozen in liquid nitrogen, and shipped overnight on dry ice.  2.16.2! In-gel digestion CoIP protein samples were separated by SDS-PAGE and stained with PageBlue coomassie (ThermoFisher Cat #24620). Bands were excised across the length of the whole lane and chopped into ~1 x 1mm cubes. Gel pieces were shrunk in acetonitrile for 10 minutes between each of the following steps: reduction with 10mM DTT in 100mM ammonium bicarbonate for 30 minutes at 56°C, followed by alkylation with 55mM iodoacetamide in 100mM ammonium 42  bicarbonate for 45 minutes at room temperature in the dark. Gel pieces were washed with 1:1 100mM ammonium bicarbonate:acetonitrile to remove residual coomassie stain. Gel pieces were rehydrated with sequencing grade trypsin (Roche Cat #11418475001) in 50mM ammonium bicarbonate + 5mM CaCl2 and incubated overnight at 37°C with shaking at 600rpm. Digests were extracted with two sequential acetonitrile shrinkage steps, rehydrating in 5% formic acid before the second. Samples were dried in a vacuum centrifuge at 45°C and stored at -20°C until analyzed.  2.16.3! LC-MS/MS analysis! Qstar XL Peptides were reconstituted in 6 µl of 0.1 % formic acid and analyzed on a Qstar XL LC-MS/MS (Applied Biosystems). A PepMap C18, 3µm particle size and 100Å pore size column (LC Packings) was used for peptide separation. Solvents A and B contained 5 % and 80 % acetonitrile, respectively, in water with 0.1 % formic acid. LC conditions started at 2 % solvent B with a gradient to 30 % B over 60 min, to 80 % B at 63 min, which was held for ten minutes before returning to 2 % B.! LTQ-FT-ICR STAGE-tip protocol was utilized for peptide desalting upstream of LC-MS/MS; solvent A was 0.5% acetic acid in water, and solvent B was 0.5% acetic acid + 80% acetonitrile in water. Empore C18 filters (3M Cat #14-386) were conditioned with 20µL methanol and washed with solvent A. Digested samples were reconstituted in 5% formic acid and passed through the filter, 43  then washed with solvent A and eluted with solvent B. Samples were dried in a vacuum centrifuge at 45°C.  Peptides were resuspended in 6µL solvent A and analyzed using a LTQ-FT-ICR (Thermo Fisher Scientific). Peptide mixtures were separated on a PicoTip column (o.d. = 360, I.d. = 75, tip = 15 ± 1 µm, New Objective) packed with reverse-phase C18 material (15 cm, C18 magic, 100 Å, 3 µm, Michrom Bioresources). A linear gradient of 6% to 80% solvent B over 30 min at a flow rate of 0.6 µl / min was applied via an Agilent 1100 nano HPLC pump (Agilent).  2.16.4! Mass spectrometry data analysis Proteins were identified by searching the MS and MS/MS spectra against the Uniprot-Swissprot Homo sapiens database (15/11/2012) using Mascot (version 2.4.1). Search parameters included one missed cleavage by trypsin, fixed carbamidylmethyl modification on cysteine, and variable modification of methionine oxidation. The mass tolerance for precursor ions were set to 0.15 Da for QSTAR and 20 ppm for LTQ-FT-ICR. The mass tolerance for product ions was 0.5 Da for both QSTAR and LTQ-FTICR. Protein identifications were filtered by only considering peptides with an ion score ≥ 20, and proteins with ≥ 2 peptides identified. Putative MCL-1 interactions were filtered by removing all proteins identified in the parallel IgG control IP.     44  Chapter 3:!Identifying signaling and double strand break repair pathways requiring MCL-1 While the canonical BCL-2 family function of MCL-1 occurs in the cytosol and at mitochondria, there is evidence that a substantial nuclear pool of MCL-1 is dynamically maintained (Fujise et al., 2000; Liu et al., 2005; Thomas et al., 2012; Zhang et al., 2002c). MCL-1 is also translocated into the nucleus in response to DNA damage (Jamil et al., 2008; Pawlikowska et al., 2010; Wang et al., 2014a). Our lab was the first to show, and others have confirmed, that MCL-1 null MEFs have impaired Chk1 and γH2AX activation following DNA damage, and slower DSB repair kinetics (Jamil et al., 2008; Jamil et al., 2010a; Pawlikowska et al., 2010). However, a mechanistic view of how MCL-1 exerts these functions is largely unknown. Therefore, the aim of this chapter was to assess the characteristics of nuclear MCL-1, and identify additional DDR signaling events and specific DSB repair pathways potentiated by MCL-1.  3.1! Nuclear MCL-1 has decreased protein stability compared to cytosolic MCL-1. One of the most interesting properties of MCL-1 is its short half-life, ranging from 30 minutes to 4 hours depending on the cell type (Chao et al., 1998; Cuconati et al., 2003; Ding et al., 2007a; Jamil et al., 2010b; Liu et al., 2005; Maurer et al., 2006; Nijhawan et al., 2003; Schubert and Duronio, 2001; Stewart et al., 2010a). This characteristic is unique within the BCL-2 family and is facilitated by both ubiquitin-dependent and –independent proteasomal degradation (Stewart et al., 2010a). Suppression of protein turnover allows for rapid expansion of MCL-1 protein levels in response to external stimuli or differentiation programs to provide cells with transient pro-survival windows in times of stress (Craig, 2002; Kozopas et al., 1993; Sitailo et al., 2009). 45  Conversely, enhanced protein turnover of MCL-1 promotes a pro-apoptotic environment when cells encounter heightened toxicity (Mojsa et al., 2014; Nijhawan et al., 2003; Opferman, 2006; Thomas et al., 2010).  Our lab’s previous work revealed additional functions of MCL-1 in the nucleus and I was curious whether its protein stability was altered by subcellular localization. Since basal MCL-1 protein levels and subcellular localization are known to fluctuate during the cell cycle (Harley et al., 2010; Jamil et al., 2005), I chose to investigate growth arrested human fibroblasts to mitigate these effects. In response to cycloheximide (CHX) treatment, which inhibits de novo protein synthesis, MCL-1 levels dropped more quickly in the nucleus compared to the cytoplasm (Figure 3-1). The cytosolic amount of MCL-1 only dropped by 30% during the 4 hour CHX treatment, which is less dramatic than what is seen in other reports. To our knowledge, this is the first investigation of MCL-1 half-life in the 1BR3 cell line, and the first time specifically inducing growth arrest to avoid cell cycle regulatory effects, which could account for the observed stability of MCL-1 in the cytosol. These results indicate that cells have mechanisms by which the nuclear pool of MCL-1 is regulated differently than that of the cytosol.  46   Figure 3-1: MCL-1 has decreased protein stability in the nucleus compared to cytoplasm. Normal human hTERT-immortalized fibroblasts (1BR3) were grown to confluence and maintained for 3 days to induce growth arrest. Cells were treated with 100µg/mL cycloheximide (CHX) for the indicated times and fractionated into cytosolic and nuclear lysates. (A) Representative immunoblots probing for MCL-1, and Vinculin and Lamin B1 as cytosolic and nuclear loading controls, respectively. (B) Quantification of MCL-1 protein levels normalized to the loading control from the same lane, and then the untreated (UT) sample of the same cellular compartment. Data represents mean + SEM from at least 3 biological replicates. Statistical analysis by Student’s t-test comparing cytosolic and nuclear MCL-1 levels at each time point.  47  3.2! Cells lacking MCL-1 have impaired phosphorylation of ATM, Chk2, KAP-1, and γH2AX in response to etoposide. Previous results from our lab and another demonstrated that MCL-1-/- MEFs have delayed Chk1 activation and diminished γH2AX levels at low doses of etoposide (Jamil et al., 2010a; Pawlikowska et al., 2010). This led me to investigate additional DDR signalling events requiring MCL-1. The ATM kinase is a master DDR regulator known to be critical for DSB repair in each stage of the cell cycle (Shiloh and Ziv, 2013). ATM is recruited to DSBs as an inactive dimer, but autophosphorylation at S1981 releases active monomers that then activate the Chk2 kinase by T68 phosphorylation – these two kinases each target key effectors of cell cycle arrest (Jackson, 2001; Rouse and Jackson, 2002; Smith et al., 2010). ATM also promotes DSB repair by phosphorylating γH2AX at sites flanking the damage to recruit numerous repair factors (Khanna and Jackson, 2001; Polo and Jackson, 2011), and by phosphorylating KAP-1 at S824 to induce the heterochromatin relaxation that precedes several DNA repair pathways (Goodarzi et al., 2010; Goodarzi and Jeggo, 2012; Lemaitre and Soutoglou, 2014; Soria et al., 2012; Ziv et al., 2006).  Comparing WT and MCL-1-/- MEFs treated with etoposide at 1, 10, and 100µM for 30 minutes and 3 hours, MCL-1 was shown to be required for activation of ATM and Chk2 across all doses and time points (Figure 3-2). Conversely, the activation of γH2AX and KAP-1 in MCL-1-/- MEFs was most pronounced at 1µM, or 30 minutes of 10µM etoposide. This may be due to functional redundancy of other DDR kinases capable of these phosphorylation events. Alternatively, it could mean that MCL-1 works by amplifying DDR signalling as opposed to functioning directly at the DSB. As more DSBs are generated with increasing etoposide concentrations, signaling 48  from the DSB recognition complexes may be sufficient to elicit DDR activation of γH2AX and KAP-1 in MCL-1-/- MEFs comparable to WT. This data expands the repertoire of DDR events potentiated by MCL-1 and implicates the protein as a potential co-activator of the ATM – Chk2 axis in response to etoposide.   Figure 3-2: Activation of ATM, Chk2, KAP-1, and γH2AX is impaired in MCL-1-/- MEFs. WT and MCL-1-/- MEFs were untreated (UT) or treated with etoposide at the indicated concentrations and time points. Whole cell lysates were prepared and immunoblotted for the indicated targets, with Vinculin serving as a loading control. Blots are representative of the results observed in at least 3 biological replicates.    49  3.3! MCL-1 promotes double strand break repair efficiency via homologous recombination, as determined by pathway-specific reporter assays.  A number of I-Sce1 endonuclease-based reporter assay systems have been developed that allow quantification of DNA repair efficiency in a specific DSB pathway. In each case, the parental cell line is selected for stable integration of a single copy of a DNA cassette designed in such a way that upon transfection with I-Sce1, repair of the induced DSB by the desired pathway will cause the cell to express an active copy of GFP. I-Sce1 is widely used in DSB repair studies because it is a rare-cutting endonuclease with a 18 bp recognition sequence that is not present in the human genome (Jasin, 1996).  For our purposes, we obtained validated and widely-used U20S reporter systems from Dr. Jeremy Stark to investigate homologous recombination (HR), single strand annealing (SSA), alternative end joining (Alt-EJ), and distal EJ of tandem DSBs (Bennardo et al., 2008; Bennardo et al., 2009; Gunn et al., 2011; Gunn and Stark, 2012; Howard et al., 2015). The rationale behind each DNA repair cassette is outlined in Figure 3-3a and explained below:  DR-GFP (HR): I-Sce1 site and two STOP codons are integrated into the upstream GFP gene. If this cut site is repaired by HR using the downstream truncated and inactive copy of GFP (iGFP) for homology the cell becomes GFP+.  SA-GFP (SSA): A downstream 3’ GFP segment contains a I-Sce1 cut site and shares 266 nucleotide homology with an upstream 5’ GFP fragment. This homology can bridge the cut site to generate active GFP if the intervening 2.7 kilobases are resected.  50   EJ-2-GFP (Alt-EJ): I-Sce1 cut site is followed by a STOP codon, and these together are flanked by 8 nucleotides of microhomology. Alt-EJ (microhomology-mediated EJ) results in a 35 nucleotide deletion that yields GFP expression.  EJ5-GFP (Distal EJ): Tandem I-Sce1 cut sites flank a puromycin resistance cassette. Numerous EJ processes can join the distal sites, deleting the intervening sequence and placing the GFP directly downstream of the promoter. This system is not specific for NHEJ as demonstrated by KU70-/- having minimal impact on GFP expression (Bennardo et al., 2008).   Because MCL-1 levels are known to rise in S and G2 in U2OS cells (Harley et al., 2010), I adopted an experimental design to ensure cells were actively dividing throughout the duration of the experiment. Cells at 40% confluency were transfected with siRNA for 24 hours, followed by transfection with an I-Sce1 plasmid for an additional 48 hours. The 24 hour siRNA transfection resulted in efficient MCL-1 knockdown (Figure 3-3b) and had negligible effects on cell cycle distribution (Figure 3-3c). Representative flow cytometry dot plots from the DR-GFP reporter cell line are shown in Figure 3-3d.  CtIP knockdown was used as a control and results were in accordance with previous studies showing the most pronounced effects in pathways dependent on DSB end resection (Bennardo et al., 2008; Howard et al., 2015). MCL-1 knockdown resulted in reduced repair efficiencies for each of the pathways tested, but only achieved statistical significance with HR, showing 65.4% fidelity (Figure 3-3e). The magnitude of the differences diminished if cells were transfected at a 51  higher confluency with fewer cells actively cycling (data not shown). This does suggest that the function of MCL-1 in these repair pathways may fluctuate during the cell cycle and thus, results of the asynchronous population in Figure 3-3e would underestimate the role of MCL-1 in S or G2 specifically.   Figure 3-3: MCL-1 promotes DSB repair efficiency in HR specifically. Established I-Sce1 endonuclease-based U2OS reporter cell lines were used to assess pathway-specific DSB repair 52  efficiencies. The designs of the repair cassettes are outlined in (A) for DR-GFP (HR), SA-GFP (SSA), EJ2-GFP (Alt-EJ), and EJ-5-GFP (Distal EJ). Panel A was copied with permission of Springer from Gunn et al. (Gunn and Stark, 2012). Each cell line was transfected at 40% confluency with siRNA for 24 hours to achieve efficient MCL-1 knockdown (B) with negligible effects on cell cycle profile, as measured by Propidium Iodide (PI) staining (C). Cells were subsequently transfected with an I-Sce1 plasmid for 48 hours and analyzed by flow cytometry. After gating for live cells, the GFP positive population was quantified against APC-Cy7 to negate autofluorescence. Representative flow cytometry dot plots for DR-GFP are shown in (D). (E) Data represents mean + SEM for 3 independent experiments. Statistical analysis of each reporter cell line by one-way ANOVA with Dunnett’s multiple comparison post-test. Significance reported compared to Ctrl.   3.4! MCL-1 knockdown prevents RPA foci resolution in G2 with no impact on γH2AX kinetics. The results in Figure 3-3e led me to investigate ionizing radiation-induced foci associated with HR. Since this pathway requires the presence of a sister chromatid for homology searching, it is limited to S and G2 phases of the cell cycle, with each phase having unique regulatory features for HR (Branzei and Foiani, 2008; Kocher et al., 2012; Mathiasen and Lisby, 2014). Since MCL-1 reaches maximal protein levels in G2 (Harley et al., 2010) I adopted an established technique of quantifying foci within G2 cells whereby aphidicolin (a DNA polymerase inhibitor) is added to cells just prior to IR to prevent S phase cells from slipping into G2 during the course of the experiment. Samples were then prepared for immunofluorescence by co-staining the target of interest with a G2 marker (pS10-H3, CENPF, etc.) (Alagoz et al., 2015; Beucher et al., 2009; Geuting et al., 2013; Shibata et al., 2011).  Using this technique I investigated overall DSB repair kinetics by γH2AX foci, and resection by RPA foci, for up to 8 hours following 2Gy X-Ray treatment of HeLa cells. As seen in Figure 3-4b G1 cells were easily identifiable as pS10-H3-negative, with γH2AX foci and no RPA foci; 53  S phase cells were pS10-H3-negative, with pan-nuclear γH2AX and RPA foci; G2 cells had punctate pS10-H3 staining with both γH2AX and RPA foci.  The control cells in Figure 3-4c showed the same foci kinetics as observed in other studies  - γH2AX and RPA foci reached maxima immediately and at 2 hours, respectively, and gradually declined as DSBs were repaired (Alagoz et al., 2015; Bakr et al., 2015; Beucher et al., 2009; Geuting et al., 2013; Kocher et al., 2012; Shibata et al., 2011). The RPA foci of MCL-1-depleted cells revealed that resection initiation was unaffected, but levels plateaued and remained elevated out to 8 hours. This pattern has been observed with BRCA2-deficient cells, which also exhibited a downstream defect in Rad51 loading (Beucher et al., 2009; Geuting et al., 2013; Shibata et al., 2011). Surprisingly, there was no difference in γH2AX foci between control and MCL-1 siRNA. This may indicate that another DSB repair pathway is capable of compensating for an HR defect in MCL-1-depleted cells. Interestingly, at the 8 hour time point in the MCL-1 knockdown the number of γH2AX and RPA foci were equal, implying that all remaining DSBs were associated with resected ends.  54   Figure 3-4: MCL-1 knockdown prevents RPA foci resolution in G2 cells with no effect on γH2AX kinetics. HeLa cells were transfected with siRNA for 24 hours, then treated with 1µg/mL aphidicolin just prior to receiving 2Gy X-Ray. Cells were fixed at 30 minutes, and 1, 2, 4, and 8 hours (with 30 second 0.2% Triton X-100 pre-extraction for RPA foci samples), then stained for immunofluorescence of the indicated proteins. G2 cells were identified by punctate pS10-H3 staining and within those cells γH2AX and RPA foci were quantified for at least 100 nuclei across 3 independent experiments. (A) Efficient MCL-1 knockdown achieved in HeLa cells with 24-hour siRNA transfection. (B) Representative images from the 8-hour time point. (C) Data represents mean ± SEM across 3 replicates. Statistical analysis by Student’s t-test at each time point. 55   3.5! Discussion This chapter has expanded the understanding of DNA repair processes that require MCL-1. I have also shown that the amount of MCL-1 protein in the nucleus decreased faster than in the cytosol following CHX treatment (Figure 3-1). Whether this is from higher protein turnover or nuclear import/export dynamics is a question that can be addressed in future experiments. For example, the same experimental setup as Figure 3-1 could also incorporate treatment with a proteasome inhibitor (e.g. MG132, bortezomib) to investigate a role of ubiquitin-mediated proteasomal degradation. If this co-treatment prevents a decrease in nuclear MCL-1 levels, follow up studies could look at the role of individual E3 ubiquitin ligases or phosphorylation sites associated with MCL-1 protein turnover (Mojsa et al., 2014; Thomas et al., 2010; Warr and Shore, 2008). Alternatively, the nuclear pool of MCL-1 that is actively retained (Thomas et al., 2012) may depend on continual nuclear translocation. To date, only one binding partner has been shown to regulate MCL-1 nuclear trafficking, IEX-1, which binds the transmembrane domain of MCL-1 (Pawlikowska et al., 2010). However, there are several other MCL-1 protein interactions that each exhibit both cytoplasmic and nuclear functions, and may similarly regulate translocation, including PCNA (Bouayad et al., 2012; Fujise et al., 2000; Jamil et al., 2005; Yi et al., 2012), CDK1 (Gavet and Pines, 2010; Jamil et al., 2005; Yi et al., 2012), TCTP (Liu et al., 2005; Ma and Zhu, 2012; Zhang et al., 2002c), and Tankyrase (Bae et al., 2003; De Rycker et al., 2003). MCL-1 mutant constructs that preclude these interactions may reveal if any are required for maintaining basal nuclear MCL-1 levels.  56  I next assessed a number of phosphorylation events associated with DDR activation. MCL-1 appears to be a potent activator of ATM since pS1981 ATM and pT68 Chk2 levels were impaired across a 100-fold range of etoposide concentrations in MCL-1-/- MEFs at all time points tested (Figure 3-2). Other studies have shown that these same cells had diminished Chk1 activation in response to DSBs (Jamil et al., 2010a; Pawlikowska et al., 2010). ATM-deficient human fibroblasts have also been shown to exhibit impaired Chk1 activation following ionizing radiation, likely from delayed exposure of RPA-coated ssDNA (Kocher et al., 2012). Thus, all of these findings fit with a model of MCL-1 as an ATM activator. Upstream activation of ATM requires acetylation by Tip60 (Kaidi and Jackson, 2013; Sun et al., 2005; Sun et al., 2009; Sun et al., 2007) and the potential involvement of MCL-1 in this process is discussed in Section 6.1, incorporating findings from Chapter 5:. It should be highlighted that ATM and ATR phosphorylate a [S/T]-Q consensus sequence and proteomic analyses revealed 900+ protein targets of these kinases in response to DNA damage (Matsuoka et al., 2007; Mu et al., 2007). MCL-1 contains several phosphorylation sites (Mojsa et al., 2014; Thomas et al., 2010) but does not possess this ATM/ATR consensus sequence.  To identify which DSB repair pathway(s) involved MCL-1 I utilized a panel of U2OS reporter cell lines and found that only HR showed a statistically significant reduction in MCL-1-depleted cells (Figure 3-3). This finding required that cells be actively cycling throughout the duration of the experiment, which fits with a previous finding showing that basal MCL-1 levels rise during S and G2 in U2OS cells (Harley et al., 2010). If the role of MCL-1 is cell cycle-specific, analysis of asynchronous populations would underestimate its effects and the reduced fidelity seen in other pathways may in fact be statistically significant if cells could be stratified based on cell 57  cycle stage. To our knowledge there has never been a study that integrates cell cycle specificity with a I-Sce1 reporter system. Many protocols utilize a 72-hour siRNA knockdown followed by 48-hour plasmid transfection. But my approach of transfecting at low confluency and truncating the overall duration of the experiment to 3 days may tease out cell cycle-specific effects in an asynchronous population. This phenomenon may explain the large discrepancy seen with HP1γ knockdown on HR efficiency using the U2OS DR-GFP reporter system. Lee et al. saw a ~90% reduction in HR efficiency while knocking down HP1γ in a scheme similar to my own (Lee et al., 2013); while Soria and Almouzni report a ~30% increase in HR efficiency with HP1γ knockdown using a longer overall experimental duration (Soria and Almouzni, 2013).  Future DSB repair pathway reporter experiments could make use of the pDSRed-I-Sce1-Glucocorticoid Receptor construct in conjunction with cell synchronization protocols to look at pathway defects in specific stages of the cell cycle. Triamcinolone binds the glucocorticoid receptor to translocate the construct into the nucleus. Conditions could be optimized to activate the I-Sce1 endonuclease at different time points after release from cell synchronization to have site specific DSBs generated in S or G2 cells. Incorporating the necessary upstream siRNA knockdown may prove difficult with this technique and it has also been observed that cell synchronization effects basal levels of DNA damage in cells (Shibata et al., 2011).  Lastly, to gain more insights about the mechanism of MCL-1 in the HR pathway I investigated the repair kinetics of γH2AX and RPA foci. Resection initiation in MCL-1-depleted HeLa cells was unaffected since RPA reached its normal peak at 2 hours, however, this level remained at the same level out to 8 hours (Figure 3-4c). BRCA2-deficient cells exhibit this same RPA profile 58  due to a resolution defect and inability to properly load Rad51 onto ssDNA (Beucher et al., 2009; Geuting et al., 2013; Shibata et al., 2011). However, these studies also had elevated numbers of γH2AX foci ≥ 4 hours, which was not observed in Figure 3-4c. This may reflect the ability of a separate DSB repair pathway to compensate for the HR defect in MCL-1 knockdown cells, which has been described in BRCA2-deficient cells with impaired HC structure (Geuting et al., 2013). Another interpretation of Figure 3-4c is that MCL-1 affects pathway choice, since the number of DSBs is unchanged but the proportion of them undergoing resection is increased. The arguments for each of these explanations and proposed experiments to delineate them are further discussed in Section 6.3, which incorporates additional results from Chapter 5:  In this chapter I have broadened the characterization of nuclear MCL-1 and DDR events it is involved in. I also provided evidence that MCL-1 functions in HR, but a robust understanding of its mechanism will be aided by studying the nuclear complexes containing MCL-1. 59  Chapter 4:!LC-MS/MS analysis of MCL-1 complexes to identify novel protein interactions There is a breadth of accumulating evidence that MCL-1 has roles in the nucleus separate from its canonical pro-survival BCL-2 family function in the cytosol and at mitochondria. Three nuclear proteins have been identified as novel MCL-1 binding partners using yeast 2-hybrid screens -  PCNA (Fujise et al., 2000), TCTP (Liu et al., 2005; Zhang et al., 2002c), and Tankyrase (Bae et al., 2003). Our lab and others have discovered additional nuclear proteins that MCL-1 interacts with by CoIP, including Chk1 (Jamil et al., 2008), NBS1 (Jamil et al., 2010a), Ku70 (Wang et al., 2014a), and CDK1 (Jamil et al., 2005; Yi et al., 2012).  MCL-1 is known to shuttle from the cytosol to the nucleus in response to DNA damage and other stimuli, and potentiate DDR signalling (Fan et al., 2014; Jamil et al., 2008; Jamil et al., 2010a; Pawlikowska et al., 2010; Renjini et al., 2014; Wang et al., 2014a). However, the precise mechanism through which MCL-1 is exerting these fundamental nuclear functions remains elusive and is fertile ground for discovery. Therefore, I sought to screen putative nuclear interactions of MCL-1 and research those that would shed light on its mechanism of action in DSB repair.  It is important to remember that MCL-1 does not have an enzymatic function and potentially acts as a protein scaffold for the spatiotemporal organization of effectors. MCL-1 also contains neither a conventional nuclear localization sequence (NLS) nor nuclear export sequence and likely depends on protein interactions for its nuclear translocation. Furthermore, MCL-1 does not 60  have orthologs in species below vertebrates meaning that screens in model organisms like yeast or Drosophila would not reveal functions for MCL-1.  The strategy for my screen was to perform CoIP of endogenous MCL-1 from large-scale nuclear fractionated lysates. I used etoposide-treated HeLa cell cultures because I was interested in isolating DNA repair complexes. Collected proteins were then analyzed by LC-MS/MS to generate lists of potential MCL-1 binding partners to be validated by additional methods and investigated for functional consequences in the context of DSB repair.  Preliminary experiments with only MCL-1 IPs were performed on nuclear lysates in a buffer containing 250mM NaCl and 2.5% Triton X-100 in an attempt to minimize non-specific interactions. This was later changed to 150mM NaCl to be more physiologically relevant, and 0.5% Triton X-100 to be less harsh. Under these conditions, MCL-1 and control IgG IPs were performed from nuclear lysates and the LC-MS/MS protein identifications (n=1) are presented in  Section 4.1.1. In additional experiments, chromatin-bound protein complexes were enriched using the Active Motif Nuclear Complex CoIP Kit (Cat #54001) as per Section; similarly, MCL-1 and IgG control IPs were performed in parallel and the putative MCL-1 interactions (n=1) are presented in Section 4.1.2. All proteins presented as putative interactions met the exclusion criteria (Section 2.16.4) and were not observed in the parallel IgG IP.  61  4.1! LC-MS/MS data sets 4.1.1! Nuclear lysate preparation and analysis on Qstar For this replicate, ~3.25 x 108 etoposide treated HeLa-S3 cells were fractionated to isolate nuclei as per Section The lysis buffer contained 150mM NaCl and 0.5% Triton X-100. Prior to pre-clearing, the lysate was split in half for parallel IgG and MCL-1 IPs. The IgG IP sample was pre-cleared with blank Protein G beads while the MCL-1 IP was pre-cleared with beads conjugated to rabbit IgG. CoIP was performed as per Section 2.15.3 with the following exceptions: post-IP, the last two washes were with 500mM NaCl; and bound proteins were eluted with 100mM glycine pH 2.0, neutralized with Tris, and concentrated by acetone precipitation. Proteins were separated by SDS-PAGE in a 7.5% gel, in-gel digested, and peptides were analyzed on the Qstar.  Aliquots of the IgG and MCL-1 IP samples, input lysates, and equivalent post-IP lysate volumes were analyzed by immunoblot to assess the specificity and efficiency of the IPs (Figure 4-1a). The intense negative exposure of MCL-1 in its IP lane indicates an abundant enrichment. The remainder of the IP samples were run in a separate SDS-PAGE for LC-MS/MS analysis; this coomassie stained gel is shown in Figure 4-1b. Putative MCL-1 interactions that met the exclusion criteria (Section 2.16.4) are listed in Table 4-1 with MCL-1 outlined in red. MCL-1 was identified with 19.7% coverage and an ion score of 317.8. CDK1 (also called cdc2) is an established nuclear binding partner of MCL-1 (Jamil et al., 2005; Yi et al., 2012) observed in this replicate and is highlighted in blue in Table 4-1 with 6.1% coverage and an ion score of 90.5. 62   Figure 4-1: CoIP samples from nuclear lysates analyzed on Qstar LC-MS/MS. 3% of the IgG and MCL-1 IP samples, and 1.5% of the input/post-IP lysates were analyzed by immunoblot for MCL-1 (A). MCL-1 was not observed in the IgG IP but was highly abundant and overexposed in the MCL-1 IP. Probing also revealed the heavy chain of the IP antibodies in the IP lanes running at ~55kDa. The remainder of the IP samples were separated by SDS-PAGE and coomassie stained (B) for band excision, in-gel digestion, and LC-MS/MS analysis.   63  Table 4-1: Putative MCL-1 interactions identified from standard nuclear fractionation protocol and Qstar LC-MS/MS analysis, organized by ion score. Identification of MCL-1 is highlighted in red. Cdc2, an estabilished MCL-1 nuclear binding partner is highlighted in blue. These proteins were observed in the MCL-1 IP but not the parallel IgG IP. Description Accession  Ion Score  Total Peptides Unique Peptides % Coverage Mass (kDa) Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 SV=1 ACTB_HUMAN  1215.5 171 23 48 42.1 DNA replication licensing factor MCM7 OS=Homo sapiens GN=MCM7 PE=1 SV=4 MCM7_HUMAN  463.5 11 11 16.6 81.9 Histone H2B type 1-M OS=Homo sapiens GN=HIST1H2BM PE=1 SV=3 H2B1M_HUMAN  449.9 24 9 53.2 14 Histone H2B type 1-B OS=Homo sapiens GN=HIST1H2BB PE=1 SV=2 H2B1B_HUMAN  424.6 22 8 48.4 13.9 Myc box-dependent-interacting protein 1 OS=Homo sapiens GN=BIN1 PE=1 SV=1 BIN1_HUMAN  411 19 9 13.7 64.9 Keratin, type II cytoskeletal 4 OS=Homo sapiens GN=KRT4 PE=1 SV=4 K2C4_HUMAN  346.1 41 7 12.4 57.6 Induced myeloid leukemia cell differentiation protein MCL-1 OS=Homo sapiens GN=MCL1 PE=1 SV=3 MCL1_HUMAN  317.8 13 8 19.7 37.4 Kinesin-like protein KIFC1 OS=Homo sapiens GN=KIFC1 PE=1 SV=2 KIFC1_HUMAN  279.1 7 7 11.1 74.3 DNA replication licensing factor MCM6 OS=Homo sapiens GN=MCM6 PE=1 SV=1 MCM6_HUMAN  251.9 6 6 7.2 93.8 Nucleolin OS=Homo sapiens GN=NCL PE=1 SV=3 NUCL_HUMAN  233.3 6 6 6.8 76.6 Polyadenylate-binding protein 1 OS=Homo sapiens GN=PABPC1 PE=1 SV=2 PABP1_HUMAN  180.5 6 5 8.2 70.9 Ubiquitin OS=Homo sapiens GN=RPS27A PE=1 SV=1 UBIQ_HUMAN  155.3 5 4 52.6 8.6 Glial fibrillary acidic protein OS=Homo sapiens GN=GFAP PE=1 SV=1 GFAP_HUMAN  153.9 26 3 5.3 49.9 ATP synthase subunit g, mitochondrial OS=Homo sapiens GN=ATP5L PE=1 SV=3 ATP5L_HUMAN  153.1 3 3 35.9 11.4 Alpha-actinin-1 OS=Homo sapiens GN=ACTN1 PE=1 SV=2 ACTN1_HUMAN  141 3 3 2.9 103.6 64  Description Accession  Ion Score  Total Peptides Unique Peptides % Coverage Mass (kDa) Erlin-2 OS=Homo sapiens GN=ERLIN2 PE=1 SV=1 ERLN2_HUMAN  136.3 3 3 8.8 38 DNA replication licensing factor MCM4 OS=Homo sapiens GN=MCM4 PE=1 SV=5 MCM4_HUMAN  126.7 3 3 2.8 97.1 NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial OS=Homo sapiens GN=NDUFS3 PE=1 SV=1 NDUS3_HUMAN  120.3 2 2 9.8 30.3 40S ribosomal protein S28 OS=Homo sapiens GN=RPS28 PE=1 SV=1 RS28_HUMAN  112.3 2 2 21.7 7.9 60S acidic ribosomal protein P2 OS=Homo sapiens GN=RPLP2 PE=1 SV=1 RLA2_HUMAN  110.6 2 2 24.3 11.7 Cytochrome b-c1 complex subunit 8 OS=Homo sapiens GN=UQCRQ PE=1 SV=4 QCR8_HUMAN  91.7 3 3 29.3 9.9 Cell division control protein 2 homolog OS=Homo sapiens GN=CDC2 PE=1 SV=1 CDC2_HUMAN  90.5 3 2 6.1 34.1 ATP synthase subunit f, mitochondrial OS=Homo sapiens GN=ATP5J2 PE=1 SV=3 ATPK_HUMAN  88.8 2 2 17 11 Translocon-associated protein subunit alpha OS=Homo sapiens GN=SSR1 PE=1 SV=3 SSRA_HUMAN  88.3 3 2 6.6 32.2 Protein transport protein Sec31A OS=Homo sapiens GN=SEC31A PE=1 SV=3 SC31A_HUMAN  85.8 6 3 1.1 133.9 Plectin-1 OS=Homo sapiens GN=PLEC1 PE=1 SV=3 PLEC1_HUMAN  84.3 4 3 0.6 533.5 Lysozyme C OS=Homo sapiens GN=LYZ PE=1 SV=1 LYSC_HUMAN  81.4 4 2 14.2 17 UPF0568 protein C14orf166 OS=Homo sapiens GN=C14orf166 PE=1 SV=1 CN166_HUMAN  79.3 2 2 6.1 28.2 Sodium channel protein type 1 subunit alpha OS=Homo sapiens GN=SCN1A PE=1 SV=2 SCN1A_HUMAN  77.3 3 3 1 230.9 Ran GTPase-activating protein 1 OS=Homo sapiens GN=RANGAP1 PE=1 SV=1 RAGP1_HUMAN  76.1 4 2 3.2 64 Protein S100-A9 OS=Homo sapiens GN=S100A9 PE=1 SV=1 S10A9_HUMAN  73.8 2 2 24.6 13.3 Transmembrane and coiled-coil domain-containing protein 1 OS=Homo sapiens GN=TMCO1 PE=1 SV=1 TMCO1_HUMAN  66.5 2 2 9 21.4 65  Description Accession  Ion Score  Total Peptides Unique Peptides % Coverage Mass (kDa) 14-3-3 protein beta/alpha OS=Homo sapiens GN=YWHAB PE=1 SV=3 1433B_HUMAN  64.8 3 2 6.5 28.2 60S ribosomal protein L10-like OS=Homo sapiens GN=RPL10L PE=1 SV=3 RL10L_HUMAN  64 2 2 9.3 25 Elongation factor Tu, mitochondrial OS=Homo sapiens GN=TUFM PE=1 SV=2 EFTU_HUMAN  60.6 2 2 4.2 49.9 Serine/threonine-protein phosphatase 1 regulatory subunit 10 OS=Homo sapiens GN=PPP1R10 PE=1 SV=1 PP1RA_HUMAN  60.5 2 2 2.1 99.3 60S ribosomal protein L37a OS=Homo sapiens GN=RPL37A PE=1 SV=2 RL37A_HUMAN  57 2 2 15.2 10.5 Obscurin OS=Homo sapiens GN=OBSCN PE=1 SV=3 OBSCN_HUMAN  55.3 2 2 0.2 879.6 Pumilio homolog 1 OS=Homo sapiens GN=PUM1 PE=1 SV=3 PUM1_HUMAN  54 4 2 1 127.1 Fibrinogen alpha chain OS=Homo sapiens GN=FGA PE=1 SV=2 FIBA_HUMAN  51.8 3 2 2.5 95.7 SAP30-binding protein OS=Homo sapiens GN=SAP30BP PE=1 SV=1 S30BP_HUMAN  45.7 2 2 7.5 34   66   4.1.2! Chromatin-bound complex enrichment and analysis on the LTQ-FT-ICR The Active Motif Nuclear Complex CoIP kit (Cat #54001) was used in this replicate to enrich for chromatin-bound protein complexes. ~1.07 x 109 etoposide treated HeLa-S3 cells were fractionated to obtain nuclei as described in Section, but using the kit’s Low Stringency Wash buffer. CoIP was as per Section 2.15.3, but with the addition of a second pre-clearing step using rabbit IgG-conjugated beads, after which the lysate was split in half for parallel IgG and MCL-1 IPs.   In all previous replicates the CoIP was optimized in 1.5mL microfuge tubes and for the LC-MS/MS sample preparation, volumes were scaled up and carried out in larger tubes. However, the efficiency of MCL-1 recovery was always reduced at these larger scales. Therefore, in this replicate multiple equivalent IPs were performed simultaneously for each of the IgG and MCL-1 conditions. Eluted samples were pooled during the vacuum centrifuge evaporation step to yield single IgG and MCL-1 IP samples. Proteins were separated in a 9% SDS-PAGE gel and excised bands were in-gel trypsin digested for analysis on the LTQ-FT-ICR.  Aliquots of the IgG and MCL-1 IP samples, input lysates, and equivalent volumes of each individual post-IP lysate were analyzed by immunoblot (Figure 4-2a). The remainder of the IP samples were run in a separate SDS-PAGE for LC-MS/MS analysis; this coomassie stained gel is shown in Figure 4-2b. Putative MCL-1 interactions that met the exclusion criteria (Section 2.16.4) are listed in Table 4-2 with MCL-1 outlined in red. MCL-1 was identified with an ion score of 289.4 and 37.4% coverage. The proteins chosen for further characterization in the 67  following sections are highlighted in green and the rationale behind their selection is outlined in Section 4.2.1.    Figure 4-2: CoIP samples from chromatin complex enriched samples analyzed on LTQ-FT-ICR LC-MS/MS. 3% of the IgG and MCL-1 IP samples, 1.5% of the input lysates, and equivalent amounts of each post-IP lysates were analyzed by immunoblot (A). The last lane of each blot are the IP samples; MCL-1 was not detected in the IgG IP but was strongly identified in the MCL-1 IP, denoted by the red boxes. The remainder of the IP samples were separated by SDS-PAGE and coomassie stained (B) for band excision, in-gel digestion, and LC-MS/MS analysis.  68  Table 4-2: Putative MCL-1 interactions identified from chromatin complex enrichment protocol and LTQ-FT-ICR LC-MS/MS analysis, organized by ion score. Identification of MCL-1 is highlighted in red, and proteins further characterized in the following sections are outlined in green. These proteins were observed in the MCL-1 IP but not the parallel IgG IP. Description Accession  Ion Score  Total Peptides Unique Peptides % Coverage Mass (kDa) U4/U6.U5 tri-snRNP-associated protein 1 OS=Homo sapiens GN=SART1 PE=1 SV=1 O43290|SNUT1_HUMAN  950.5 20 18 23.6 90.4 Putative ribosomal RNA methyltransferase NOP2 OS=Homo sapiens GN=NOP2 PE=1 SV=2 P46087|NOP2_HUMAN  814.3 18 14 22.4 89.6 Probable ATP-dependent RNA helicase DDX17 OS=Homo sapiens GN=DDX17 PE=1 SV=2 Q92841|DDX17_HUMAN  639.3 15 12 21.5 80.9 Heterogeneous nuclear ribonucleoprotein H OS=Homo sapiens GN=HNRNPH1 PE=1 SV=4 P31943|HNRH1_HUMAN  579.5 20 12 32.1 49.5 Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2 Q86YZ3|HORN_HUMAN  428.8 33 8 5.1 283.1 Pre-mRNA-splicing factor ISY1 homolog OS=Homo sapiens GN=ISY1 PE=1 SV=3 Q9ULR0|ISY1_HUMAN  405.6 8 7 22.1 33 ELAV-like protein 1 OS=Homo sapiens GN=ELAVL1 PE=1 SV=2 Q15717|ELAV1_HUMAN  395.4 6 6 26.4 36.2 Probable ATP-dependent RNA helicase DDX10 OS=Homo sapiens GN=DDX10 PE=1 SV=2 Q13206|DDX10_HUMAN  366.7 7 7 10.3 101.2 Nucleophosmin OS=Homo sapiens GN=NPM1 PE=1 SV=2 P06748|NPM_HUMAN  363.4 7 7 24.1 32.7 Splicing factor, proline- and glutamine-rich OS=Homo sapiens GN=SFPQ PE=1 SV=2 P23246|SFPQ_HUMAN  356.2 9 8 12.3 76.2 Probable rRNA-processing protein EBP2 OS=Homo sapiens GN=EBNA1BP2 PE=1 SV=2 Q99848|EBP2_HUMAN  333.1 6 5 22.5 34.9 Protein Red OS=Homo sapiens GN=IK PE=1 SV=3 Q13123|RED_HUMAN  310.1 8 8 16.9 65.7 Heterogeneous nuclear ribonucleoprotein U-like protein 2 OS=Homo sapiens GN=HNRNPUL2 PE=1 SV=1 Q1KMD3|HNRL2_HUMAN  308.4 6 5 7.4 85.6 Lamin-B2 OS=Homo sapiens GN=LMNB2 PE=1 SV=3 Q03252|LMNB2_HUMAN  305.7 5 4 8.5 67.8 Induced myeloid leukemia cell differentiation protein MCL-1 OS=Homo sapiens GN=MCL1 PE=1 SV=3 Q07820|MCL1_HUMAN  289.4 4 4 22.9 37.4 Pre-mRNA-splicing factor SYF1 OS=Homo sapiens GN=XAB2 PE=1 SV=2 Q9HCS7|SYF1_HUMAN  282 7 7 9.4 100.7 69  Description Accession  Ion Score  Total Peptides Unique Peptides % Coverage Mass (kDa) Protein Smaug homolog 2 OS=Homo sapiens GN=SAMD4B PE=1 SV=1 Q5PRF9|SMAG2_HUMAN  248.5 4 4 8.6 75.9 U3 small nucleolar RNA-associated protein 14 homolog A OS=Homo sapiens GN=UTP14A PE=1 SV=1 Q9BVJ6|UT14A_HUMAN  236.8 5 5 6.2 88.1 WD repeat-containing protein 74 OS=Homo sapiens GN=WDR74 PE=1 SV=1 Q6RFH5|WDR74_HUMAN  229.2 6 6 15.1 43 PHD finger-like domain-containing protein 5A OS=Homo sapiens GN=PHF5A PE=1 SV=1 Q7RTV0|PHF5A_HUMAN  219.8 4 3 33.6 13.1 Splicing factor U2AF 65 kDa subunit OS=Homo sapiens GN=U2AF2 PE=1 SV=4 P26368|U2AF2_HUMAN  214.9 3 3 13.5 53.8 Importin subunit alpha-2 OS=Homo sapiens GN=KPNA2 PE=1 SV=1 P52292|IMA2_HUMAN  210.2 4 4 7.9 58.2 Myb-binding protein 1A OS=Homo sapiens GN=MYBBP1A PE=1 SV=2 Q9BQG0|MBB1A_HUMAN  210 5 5 3.6 149.7 Chromobox protein homolog 5 OS=Homo sapiens GN=CBX5 PE=1 SV=1 P45973|CBX5_HUMAN  204 4 3 16.2 22.4 RNA-binding protein 28 OS=Homo sapiens GN=RBM28 PE=1 SV=3 Q9NW13|RBM28_HUMAN  200.1 5 4 5.3 86.2 Transcription elongation factor SPT6 OS=Homo sapiens GN=SUPT6H PE=1 SV=2 Q7KZ85|SPT6H_HUMAN  188.1 3 3 1.9 200.2 Pre-mRNA 3'-end-processing factor FIP1 OS=Homo sapiens GN=FIP1L1 PE=1 SV=1 Q6UN15|FIP1_HUMAN  187.8 3 3 7.9 66.6 Nucleolar GTP-binding protein 1 OS=Homo sapiens GN=GTPBP4 PE=1 SV=3 Q9BZE4|NOG1_HUMAN  178.6 4 4 7.6 74.3 Probable ATP-dependent RNA helicase DDX41 OS=Homo sapiens GN=DDX41 PE=1 SV=2 Q9UJV9|DDX41_HUMAN  177.9 4 4 7.6 70.5 Chromatin assembly factor 1 subunit A OS=Homo sapiens GN=CHAF1A PE=1 SV=2 Q13111|CAF1A_HUMAN  176.5 6 5 4.7 108.1 Synemin OS=Homo sapiens GN=SYNM PE=1 SV=2 O15061|SYNEM_HUMAN  170.5 3 3 2.4 173 70  Description Accession  Ion Score  Total Peptides Unique Peptides % Coverage Mass (kDa) DBIRD complex subunit ZNF326 OS=Homo sapiens GN=ZNF326 PE=1 SV=2 Q5BKZ1|ZN326_HUMAN  161.1 2 2 4.6 66 Nuclear pore complex protein Nup98-Nup96 OS=Homo sapiens GN=NUP98 PE=1 SV=4 P52948|NUP98_HUMAN  156.6 4 4 2.2 198.6 Trifunctional enzyme subunit alpha, mitochondrial OS=Homo sapiens GN=HADHA PE=1 SV=2 P40939|ECHA_HUMAN  152.4 3 3 6 83.7 Cytochrome c oxidase subunit 5A, mitochondrial OS=Homo sapiens GN=COX5A PE=1 SV=2 P20674|COX5A_HUMAN  146 6 3 20 16.9 Nuclear valosin-containing protein-like OS=Homo sapiens GN=NVL PE=1 SV=1 O15381|NVL_HUMAN  134.6 2 2 3 96 Eukaryotic translation initiation factor 6 OS=Homo sapiens GN=EIF6 PE=1 SV=1 P56537|IF6_HUMAN  133.6 4 3 14.7 27.1 Chromatin assembly factor 1 subunit B OS=Homo sapiens GN=CHAF1B PE=1 SV=1 Q13112|CAF1B_HUMAN  130.7 2 2 4.1 61.9 PRKC apoptosis WT1 regulator protein OS=Homo sapiens GN=PAWR PE=1 SV=1 Q96IZ0|PAWR_HUMAN  128.7 3 3 7.4 36.7 Nuclear mitotic apparatus protein 1 OS=Homo sapiens GN=NUMA1 PE=1 SV=2 Q14980|NUMA1_HUMAN  127 2 2 1.1 239.2 Rho guanine nucleotide exchange factor 12 OS=Homo sapiens GN=ARHGEF12 PE=1 SV=1 Q9NZN5|ARHGC_HUMAN  122 3 3 2.5 174.1 WW domain-binding protein 11 OS=Homo sapiens GN=WBP11 PE=1 SV=1 Q9Y2W2|WBP11_HUMAN  120.1 2 2 3.4 70 Polymerase delta-interacting protein 3 OS=Homo sapiens GN=POLDIP3 PE=1 SV=2 Q9BY77|PDIP3_HUMAN  111.9 3 3 7.8 46.3 WD repeat-containing protein 43 OS=Homo sapiens GN=WDR43 PE=1 SV=3 Q15061|WDR43_HUMAN  111.2 2 2 3.2 75.8 Proline-, glutamic acid- and leucine-rich protein 1 OS=Homo sapiens GN=PELP1 PE=1 SV=2 Q8IZL8|PELP1_HUMAN  111 2 2 1.8 120.9 Ig lambda-6 chain C region OS=Homo sapiens GN=IGLC6 PE=4 SV=1 P0CF74|LAC6_HUMAN  109.9 2 2 28.3 11.4 71  Description Accession  Ion Score  Total Peptides Unique Peptides % Coverage Mass (kDa) Probable ATP-dependent RNA helicase DDX27 OS=Homo sapiens GN=DDX27 PE=1 SV=2 Q96GQ7|DDX27_HUMAN  105.8 3 3 4.3 90.3 Nucleolar protein 6 OS=Homo sapiens GN=NOL6 PE=1 SV=2 Q9H6R4|NOL6_HUMAN  96.9 2 2 1.9 128.4 Zinc finger CCCH domain-containing protein 11A OS=Homo sapiens GN=ZC3H11A PE=1 SV=3 O75152|ZC11A_HUMAN  94.2 7 2 3.5 89.9 Cleavage and polyadenylation specificity factor subunit 2 OS=Homo sapiens GN=CPSF2 PE=1 SV=2 Q9P2I0|CPSF2_HUMAN  93.6 2 2 6 89.3 Antigen KI-67 OS=Homo sapiens GN=MKI67 PE=1 SV=2 P46013|KI67_HUMAN  93.6 2 2 0.7 360.7 U3 small nucleolar ribonucleoprotein protein MPP10 OS=Homo sapiens GN=MPHOSPH10 PE=1 SV=2 O00566|MPP10_HUMAN  92.6 3 3 5.7 78.9 Nucleolar protein 7 OS=Homo sapiens GN=NOL7 PE=1 SV=2 Q9UMY1|NOL7_HUMAN  90.9 4 3 12.5 29.5 EF-hand domain-containing protein D2 OS=Homo sapiens GN=EFHD2 PE=1 SV=1 Q96C19|EFHD2_HUMAN  90.8 2 2 11.7 26.8 Neuroguidin OS=Homo sapiens GN=NGDN PE=1 SV=1 Q8NEJ9|NGDN_HUMAN  89.9 2 2 7.6 35.9 Pre-mRNA-splicing factor SLU7 OS=Homo sapiens GN=SLU7 PE=1 SV=2 O95391|SLU7_HUMAN  89.3 3 2 5.6 68.8 Putative 40S ribosomal protein S26-like 1 OS=Homo sapiens GN=RPS26P11 PE=5 SV=1 Q5JNZ5|RS26L_HUMAN  80.8 2 2 20.9 13.3 Nucleolar complex protein 2 homolog OS=Homo sapiens GN=NOC2L PE=1 SV=4 Q9Y3T9|NOC2L_HUMAN  80.6 2 2 2.8 85.7 Ribosomal biogenesis protein LAS1L OS=Homo sapiens GN=LAS1L PE=1 SV=2 Q9Y4W2|LAS1L_HUMAN  79.4 2 2 3.1 84 Pre-mRNA-splicing factor SYF2 OS=Homo sapiens GN=SYF2 PE=1 SV=1 O95926|SYF2_HUMAN  78.7 2 2 17.3 28.8 Regulation of nuclear pre-mRNA domain-containing protein 2 OS=Homo sapiens GN=RPRD2 PE=1 SV=1 Q5VT52|RPRD2_HUMAN  76.7 2 2 2.6 156.4 Microtubule-associated protein 4 OS=Homo sapiens GN=MAP4 PE=1 SV=3 P27816|MAP4_HUMAN  69.3 2 2 1.5 121.4 40S ribosomal protein S2 OS=Homo sapiens GN=RPS2 PE=1 SV=2 P15880|RS2_HUMAN  68.5 2 2 7.8 31.6 72  Description Accession  Ion Score  Total Peptides Unique Peptides % Coverage Mass (kDa) Splicing factor 1 OS=Homo sapiens GN=SF1 PE=1 SV=4 Q15637|SF01_HUMAN  67.7 2 2 2.8 68.5 Unconventional myosin-Ig OS=Homo sapiens GN=MYO1G PE=1 SV=2 B0I1T2|MYO1G_HUMAN  62.3 2 2 1.9 117.4 Synaptopodin OS=Homo sapiens GN=SYNPO PE=1 SV=2 Q8N3V7|SYNPO_HUMAN  59.9 2 2 3.9 99.9   73  4.2! Validating novel binding partners of MCL-1 by immunoblot 4.2.1! Rationale for characterizing HP1α and p150CAF-1 interaction with MCL-1 From the list of putative interacting proteins generated by LC-MS/MS analysis of endogenous MCL-1 CoIPs I chose to follow up on two: Chromobox protein homolog 5 (CBX5), more commonly referred to as HP1α, is a non-histone component of heterochromatin that binds H3K9me3 (Bannister et al., 2001; Jacobs and Khorasanizadeh, 2002; Lachner et al., 2001; Nielsen et al., 2002); and Chromatin assembly factor 1 subunit A (CAF1A), also called p150CAF-1, which is part of a complex that assembles (H2A-H2B-H3-H4)2 histone octamers onto nascent newly synthesized DNA (Gaillard et al., 1996; Gerard et al., 2006; Green and Almouzni, 2003; Kaufman et al., 1995; Moggs et al., 2000). HP1α/CBX5 and CAF1A/p150CAF-1 were each identified using the chromatin complex enrichment sample preparation method and are highlighted in green in Table 4-2. HP1α/CBX5 was identified with 16.2% coverage and an ion score of 204, while CAF1A/p150CAF-1 had 4.7% coverage and an ion score of 176.5. It should be highlighted that other than MCL-1, no proteins were concomitant in both of the data sets presented in Section 4.1.  HP1α and p150CAF-1 are themselves established binding partners (Murzina et al., 1999; Quivy et al., 2008) that are each recruited to DNA damage sites (Baldeyron et al., 2011; Green and Almouzni, 2003; Luijsterburg et al., 2009; Martini et al., 1998; Moggs et al., 2000; Soria and Almouzni, 2013; Zarebski et al., 2009). Furthermore, the recruitment of HP1α to laser track DNA damage is dependent upon its interaction with p150CAF-1 (Baldeyron et al., 2011). Several studies also show that knockdown of HP1α reduces the fidelity of HR using reporter cell 74  lines like those in Figure 3-3 (Alagoz et al., 2015; Baldeyron et al., 2011; Lee et al., 2013; Soria and Almouzni, 2013). Taken together, the observed DNA damage response characteristics of HP1α and p150CAF-1, and the MCL-1 data from Chapter 3: provided a rationale to validate and functionally investigate the interaction of all three proteins.   The data sets presented in Section 4.1 only identified HP1α and p150CAF-1 in the MCL-1 IP when enriching for chromatin complexes using the Active Motif kit. An additional replicate under these conditions also identified p150CAF-1 with 7.8% coverage and an ion score of 282.8 (comparable to that seen in Table 4-2); however, the MCL-1 identification in this replicate only had one peptide with 5.7% coverage and an ion score of 64.8, which makes the efficiency of this IP questionable. A preliminary experiment using a nuclear lysate also had strong identification of HP1α. This replicate was performed with a lysis buffer containing 250mM NaCl and 2.5% Triton X-100 to minimize non-specific interactions; however, a control IgG IP was never performed under these conditions. 914 proteins were identified with ≥2 peptides of an ion score ≥20 in this replicate; the MCL-1 and HP1α peptides from this replicate are listed in Table 4-3.  HP1α is part of a family also containing HP1β and HP1γ. The latter two isoforms are also recruited to sites of DNA damage where HP1β promotes homologous recombination, while HP1γ suppresses the fidelity of DNA repair (Alagoz et al., 2015; Ayoub et al., 2008; Kalousi et al., 2015; Luijsterburg et al., 2009; Soria and Almouzni, 2013; Zarebski et al., 2009). Of the 3 family members, only HP1α was ever identified in the MCL-1 CoIP LC-MS/MS experiments and was the only isoform I chose to investigate by immunoblot. The Chromatin Assembly Factor 75  1 complex is heterotrimeric and consists of A (p150), B (p60), and C (p48) subunits. While p60CAF-1 was also seen in Table 4-2, I did not pursue validation and characterization because this subunit has been shown to be dispensable to the DNA repair function of the complex (Baldeyron et al., 2011).  76  Table 4-3: MCL-1 and HP1α/CBX5 protein identifications from a preliminary MCL-1 IP using nuclear lysate preparation with a lysis buffer containing 250mM NaCl and 2.5% Triton X-100, and Qstar LC-MS/MS analysis. Description Accession  Ion Score  Total Peptides Unique Peptides % Coverage Mass (kDa) Induced myeloid leukemia cell differentiation protein MCL-1 OS=Homo sapiens GN=MCL1 PE=1 SV=3 - [MCL1_HUMAN] Q07820 195.22 12 7 18.00 37.4 Chromobox protein homolog 5 OS=Homo sapiens GN=CBX5 PE=1 SV=1 - [CBX5_HUMAN] P45973 77.32 3 2 9.95 22.2   77  4.2.2! MCL-1 coimmunoprecipitation from adherent HeLa cells confirms HP1α and p150CAF-1 as novel interactions by immunoblot. The immunoblots in Figure 4-3 validate HP1α and p150CAF-1 as novel binding partners of MCL-1 in etoposide treated HeLa cells, and show the same pattern of associations dependent on lysate preparation method as was observed in the LC-MS/MS experiments. Both HP1α and p150CAF-1 were detected in MCL-1 CoIPs with lysates prepared using the Active Motif kit to enrich chromatin-bound complexes (Figure 4-3a), while only HP1α was observable when using nuclear lysates (Figure 4-3b). Detection of MCL-1 in reciprocal CoIPs of endogenous HP1α or p150CAF-1 was not observed when using chromatin complex enrichment (Figure 4-3a), but was seen with nuclear lysates (Figure 4-3b).  The methods used to generate Figure 4-3b also incorporated alterations in NaCl concentrations in an attempt to enhance CoIP of hydrophobic interactions. Nuclear fractionated lysates were prepared at 100mM NaCl then divided in half. One half received antibody-conjugated beads immediately and was analogous to the nuclear lysate preparation in Section The other half was spiked with NaCl to a final concentration of 800mM to dissociate ionic protein interactions and favour those that are hydrophobic (Jelesarov et al., 1998; Roy et al., 2015); the antibody-conjugated beads were then added and the sample was diluted to 50mM NaCl for CoIP. The only interaction that was affected by the NaCl manipulation was the detection of HP1α in the MCL-1 CoIP, which was less evident with the NaCl alteration (Figure 4-3b).  78  I next sought to determine if these novel MCL-1 interactions were enhanced by DNA damage. Figure 4-3c shows that MCL-1 is associated with p150CAF-1 in both DMSO and etoposide treated HeLa cells. Similar results were observed with HP1α in previous experiments. The replicate in Figure 4-3c had CoIP antibody present in the final sample and the light chain obfuscated HP1α detection since both are ~25kDa. The lower MCL-1 band in the input lanes is a degradation product known to arise during lysate incubation. Collectively, the immunoblots in Figure 4-3 validate HP1α and p150CAF-1 as novel binding partners of MCL-1.  I next investigated whether the established interaction of HP1α and p150CAF-1 is dependent upon MCL-1. For this purpose I performed p150CAF-1 IPs in WT and MCL-1-/- etoposide treated MEFs, since the p150CAF-1 antibody robustly CoIPed HP1α under all conditions tested in Figure 4-3. As seen in Figure 4-4, the interaction between HP1α and p150CAF-1 was unaffected by the absence of MCL-1.    79   Figure 4-3: MCL-1 CoIPs in HeLa cells validate HP1α and p150CAF-1 as novel binding partners by immunoblot. (A) HeLa cells were treated with 15µM etoposide for 3 hours and a chromatin complex-enriched lysate was prepared using the Active Motif Nuclear Complex CoIP Kit. The lysate was split in thirds for IPs of endogenous MCL-1, HP1α, and p150CAF-1. (B) HeLa cells were treated with 15µM etoposide for 3 hours and a nuclear fractionated lysate was prepared, then split in half. One half remained at 100mM NaCl, was split in thirds, and received antibody-conjugated beads immediately (100); the other half was spiked with NaCl to 800mM, split in thirds, given antibody conjugated beads, then diluted to a final NaCl concentration of 50mM (800/50). IPs of endogenous MCL-1, HP1α, and p150CAF-1 were performed under each lysate preparation method. (C) HeLa cells were treated with 15µM etoposide (or DMSO control) for 3 hours and a chromatin complex-enriched lysate was prepared using the Active Motif Nuclear Complex CoIP Kit. Each lysate was split in half and IPs of MCL-1 and control rabbit IgG was performed. The heavy chain of the IP antibodies is visible in the IP lanes running just above the MCL-1 band.   80   Figure 4-4: MCL-1 is not required for HP1α/p150CAF-1 interaction. WT and MCL-1-/- MEFs were treated with 10µM etoposide for 3 hours. Nuclear fractionated lysates were prepared using the Active Motif Nuclear Complex CoIP Kit and IPs of endogenous p150CAF-1 were performed to assess its interaction with HP1α.   4.3! Discussion In this chapter I employed a proteomic LC-MS/MS screen of MCL-1 IPs and validated HP1α and p150CAF-1 as novel protein interactions. These three proteins were found to bind each other in the presence or absence of exogenous DNA damage (Figure 4-3), and MCL-1 was dispensable for the established interaction between HP1α and p150CAF-1 (Figure 4-4). These latter results are not surprising given that a number of DSB repair complexes are constitutively bound and DNA damage instead influences their localization (Ayrapetov et al., 2014; Chailleux et al., 2010; Paull and Lee, 2005; Sun et al., 2009; Uziel et al., 2003). It is noteworthy that HP1α and p150CAF-1, which are both chromatin-bound proteins, were not readily identified in nuclear lysates of soluble proteins. This emphasizes the importance sample preparation techniques in the isolation of protein complexes.  81  It is striking that HP1α and p150CAF-1, which are themselves established binding partners (Murzina et al., 1999; Quivy et al., 2008), show opposite results with reciprocal CoIPs of each other in Figure 4-3a, b. Using p150CAF-1 as the bait resulted in robust CoIP of HP1α in all of the methods investigated, while none of the HP1α CoIPs had observable p150CAF-1. This same pattern has been observed in proteomic studies of tandem-affinity-purification tagged constructs of each protein, which were also performed in HeLa cells. Using p150CAF-1 as bait identified HP1α, HP1γ, and p60CAF-1 (Smith et al., 2014). With HP1β or HP1γ as bait, LC-MS/MS results contained each of the other HP1 isoforms, p150CAF-1 and p60CAF-1; however, with HP1α as the bait, none of these interactions were seen (Rosnoblet et al., 2011). Clearly the dynamics of these interactions can be affected by the CoIP procedure and may be sensitive to epitope masking by affinity tags or the CoIP antibodies themselves. MCL-1 was not identified in either of these p150CAF-1 or HP1α proteomic studies, but 5 of the 19 reported p150CAF-1 interactions (Smith et al., 2014) were also seen in my screen using the Active Motif kit (Table 4-2). This suggests that the interaction may not be direct and requires additional factors.  The putative MCL-1 interactions identified using a nuclear lysate, listed in Table 4-1, contains MCM4, 6, and 7, which could be validated in future experiments to implicate MCL-1 in DNA replication and/or repair at stalled replication forks. The high NaCl and high Triton X-100 preliminary replicate referenced in Table 4-3 also identified these proteins. The MCM2-7 complex is the DNA helicase holoenzyme that is fundamental to DNA replication and can stall at DNA lesions leading to replication fork collapse and genomic instability (Bochman and Schwacha, 2009; Das et al., 2014; Hills and Diffley, 2014; Vijayraghavan and Schwacha, 2012). 82  The three specific subunits identified in the nuclear lysate form the core of the complex and possess the helicase activity (Ishimi and Komamura-Kohno, 2001; Lee and Hurwitz, 2001; You et al., 1999). MCL-1 has also been shown to interact with PCNA, another key factor in replication fork progression (Fujise et al., 2000; Jamil et al., 2005; Yi et al., 2012); however, this interaction is weak or requires additional components as it was not detected with recombinant proteins in NMR structural analysis (De Biasio et al., 2012). This may account for why PCNA was not identified in my screen. The only previously reported nuclear binding partner of MCL-1 to be identified in my CoIP LC-MS/MS screen was CDK1/cdc2, which is highlighted in blue in Table 4-1 and was identified from a nuclear lysate.  There was a recent quantitative proteomic analysis, also in HeLa-S3 cells, of proteins associated with nascent newly replicated DNA. It revealed that all three HP1 isoforms, all three subunits of the CAF1 complex, MCM2-7, and numerous DNA repair proteins are all enriched on newly forming chromatin, however, MCL-1 was not identified in this study (Alabert et al., 2014). This design looked at unperturbed DNA replication and did not take exogenous damage into account. Another proteomic investigation examined MCM2 and MCM5 interactions in response to etoposide, but did not identify MCL-1 (Drissi et al., 2015). However, my screen identified MCM4, 6, and 7 as the putative interactions, which were not used as bait in the latter study. It is important to remember that a role for MCL-1 in DNA replication would be restricted to S phase and the work in Section 3.4 looked at G2 cells.   The proteins identified using chromatin complex enrichment (Table 4-2) contain 18 factors related to pre-mRNA splicing. These include RNA binding and processing proteins, splicing 83  factors, members of the DDX RNA helicase family, and binding partners of the core spliceosome proteins, the U small nuclear ribonucleoproteins. Each of these proteins are involved in the highly orchestrated processing and handoff of intermediate RNA-protein complexes that results in the joining of exon sequences (Abou Faycal et al., 2016; Fredericks et al., 2015; Liu and Cheng, 2015; Papasaikas and Valcarcel, 2016; Salton and Misteli, 2016). Searching all of the RNA processing proteins in Table 4-2 against the Spliceosome Database (Cvitkovic and Jurica, 2013) revealed that the greatest functional commonality was 5 proteins that are components of the C and P complexes of the spliceosome cycle: DDX41, ISY1, SYF1, SYF2, and SLU7. It is interesting to speculate that the protein product of MCL-1, a gene which undergoes alternative splicing, may itself regulate the overall process of alternative splicing. Before moving forward with this characterization it would be critical to treat lysates with a nuclease to destroy RNA (e.g. Benzonase) and ensure that the proteins in the CoIP are not due to a non-specific enrichment of RNA during sample preparation. It is also important to note that the Spliceosome Database, which curates a growing list of spliceosome proteomic studies, does not list MCL-1 as an effector of RNA splicing.   There are additional improvements that could be implemented in further proteomic analysis of MCL-1 interactions. First of all, the advent of CRISPR technology has changed the way parallel control IPs are optimally performed when using endogenous proteins as bait. Instead of splitting the lysate for a MCL-1 IP and parallel pre-immunized rabbit IgG control IP, it would be better to have two MCL-1 IPs from separate lysates, with one being from the same parental cell line validated to have a CRISPR-mediated MCL-1 knockout. This design would make it easier to remove non-specific interactions uniquely associated with the MCL-1 antibody. Tandem affinity 84  purification tagged constructs of MCL-1 could also be designed to minimize non-specific interactions in CoIP experiments with exogenous protein. Secondly, quantitative methods (e.g. SILAC) could be implemented to study the dynamics of MCL-1 interactions in response to genotoxic treatments, or in different stages of the cell cycle following release from cell synchronization. Thirdly, to enrich for chromatin-bound interactions the lysates could be treated with DNase and micrococcal nuclease (MNase), then formaldehyde-crosslinked to preserve protein complexes and minimize their pelleting during sample preparation. The MNase treatment cuts DNA in the linker regions between histone octamers, which generates a plethora of DSBs that can initiate DDR signaling post-lysis. Therefore, if phosphorylation sites are to be assessed it is necessary to use a lysis buffer with DDR kinase inhibitors (e.g. caffeine and/or Wortmanin) and phosphatase inhibitors to maintain proteins’ phosphorylation status for analysis (Klement et al., 2014; Noon et al., 2010). The Active Motif kit may use DNase and/or MNase but that proprietary information cannot be ascertained.   The most important factor to improve future proteomic analysis of the MCL-1 nuclear interactome is to have ≥3 replicates with parallel controls, all performed under the same conditions. Then a robust data set can be interrogated with strict exclusion criteria to yield the most confident identification of putative protein interactions.  85  Chapter 5:!Investigating a role for MCL-1 in heterochromatic double strand break repair The identification of HP1α and p150CAF-1 as novel MCL-1 interactions (Figure 4-3) implicates MCL-1 in heterochromatic structure. The compact nature of HC is refractory to DSB repair and this steric hindrance must be relaxed to allow repair. Several observations in Chapter 3: are consistent with those seen in HC DSB repair: MCL-1 knockdown yields diminished pS824 KAP-1 at low levels of damage when assessed by immunoblotting (Figure 3-2), as do the mediators of pS824 KAP-1 retention, MDC1 and 53BP1 (Noon et al., 2010); MCL-1 is a potent activator of ATM (Figure 3-2), which is needed to relax HC structure at DSBs (Ayrapetov et al., 2014; Goodarzi et al., 2011; Goodarzi et al., 2008; Ziv et al., 2006); MCL-1 knockdown decreases the fidelity of HR (Figure 3-3e), which is the predominant pathway for HC DSB repair (Kakarougkas et al., 2013); and the elevated RPA foci in MCL-1 depleted cells was most pronounced > 4 hours (Figure 3-4c), which corresponds with the kinetics of HC DSB repair (Goodarzi et al., 2008; Kakarougkas et al., 2013; Noon et al., 2010; Shibata et al., 2011). Therefore, my hypothesis for this chapter was that MCL-1 promotes DSB repair specifically within HC.  5.1! MCL-1 is not required for recruitment of HP1α, p150CAF-1, or KAP-1 to DNA damage. HP1α, p150CAF-1, and KAP-1 are each actively recruited to various forms of DNA damage (Alagoz et al., 2015; Ayoub et al., 2008; Ayrapetov et al., 2014; Baldeyron et al., 2011; Green and Almouzni, 2003; Luijsterburg et al., 2009; Martini et al., 1998; Moggs et al., 2000; Soria and 86  Almouzni, 2013; Zarebski et al., 2009). I therefore sought to determine if MCL-1 is required for these localizations. In the case of laser track DNA damage, the recruitment of HP1α or KAP-1 was previously found to be dependent on the PxVxL motif of p150CAF-1 (Baldeyron et al., 2011). However, using WT and MCL-1-/- MEFs for microirradiation experiments revealed that MCL-1 is dispensable for HP1α or KAP-1 recruitment to laser tracks (Figure 5-1). The localization of p150CAF-1 to laser tracks could not be determined due to a lack of the necessary secondary antibodies.  To look at DSB repair within HC specifically, we acquired U2OS 2-6-5 cells, with permission from Dr. Roger Greenberg. This cell line was derived from the U2OS 2-6-3 line, which contains 256 sequential repeats of the Lac operon (LacO); this repetitive sequence is heterochromatic and associates with H3K9me3 and all three HP1 isoforms (Janicki et al., 2004). The U2OS 2-6-5 cell line also stably expresses an Estrogen Receptor (ER) ligand-binding domain-Fok1 endonuclease-mCherry-LacR-Destabilization Domain (DD) construct (Tang et al., 2013). The DD targets fusion proteins for proteasomal degradation, which is blocked by treatment with the Shield-1 ligand. 4-hydroxytamoxifen (4-OHT) treatment causes nuclear translocation of the fusion protein by binding the ER. The LacR sequence specifically targets the construct to the heterochromatic LacO array where the non-specific Fok1 endonuclease can then induce potentially 50,000 DSBs that can be visualized by the mCherry tag. This LacR-Fok1 system has been used to visualize heterochromatic DSB recruitment of several proteins (Klement et al., 2014; Shanbhag et al., 2010; Tang et al., 2013).  87  Using the same MCL-1 knockdown conditions as the U2OS reporter cell lines in Figure 3-3b, the U2OS 2-6-5 cell line revealed that MCL-1 is not required for heterochromatic DSB recruitment of p150CAF-1 or KAP-1 (Figure 5-2). The HP1α antibody had poor quality staining in human cell lines and its colocalization with mCherry was not assessed.    Figure 5-1: MCL-1 is not required for recruitment of HP1α or KAP-1 to laser track DNA damage. WT and MCL-1-/- MEFs were sensitized for DNA damage with overnight BrdU pre-treatment before laser microirradiation. Laser tracks were visualized with γH2AX immunofluorescence and colocalized with either (A) HP1α or (B) KAP-1.  88   Figure 5-2: MCL-1 is not required for recruitment of p150CAF-1 or KAP-1 to heterochromatic DSBs. (A) Schematic diagram of the U2OS 2-6-5 site-specific heterochromatic DSB recruitment assay. These cells were treated with 1µM Shield-1 and 1µM 4-OHT for 5 hours to translocate active Fok1 endonuclease to the heterochromatic LacO array. Cells were then fixed and stained for immunofluorescence to detect colocalization of (B) p150CAF-1 or (C) KAP-1 with mCherry at the LacO array DSBs, denoted by the white arrows.    5.2! MCL-1 is not required for retention of pS824 KAP-1 at late-repairing double strand breaks or with PML nuclear bodies DSBs that persist > 4 hours are preferentially located within HC and require sustained ATM activity to maintain the focal accumulation of pS824 KAP-1 (Geuting et al., 2013; Goodarzi et 89  al., 2011; Goodarzi et al., 2008; Kakarougkas et al., 2013; Noon et al., 2010). The formation of pS824 KAP-1 foci is dependent on HP1 (White et al., 2012) and by 24 hours post-IR (≤10 Gy), all remaining γH2AX foci colocalize with pS824 KAP-1 in a process dependent on the DDR signaling mediator proteins MDC1, RNF8, and 53BP1 (Noon et al., 2010). These mediators were also shown to be required for total pS824 KAP-1 retention by immunoblotting, but only at low doses of IR (Noon et al., 2010). Given that MCL-1 is a novel binding partner of HP1α (Figure 4-3a,b) and immunoblotting revealed that MCL-1 potentiates KAP-1 S824 phosphorylation most strongly at low doses of etoposide (Figure 3-2), I hypothesized that MCL-1 would facilitate the retention of pS824 KAP-1 foci at late-repairing DSBs.  pS824 KAP-1 foci are best visualized in growth arrested primary fibroblasts since HC perturbations during the normal cell cycle can preclude the intensity of immunofluorescent detection (personal communication with Dr. Goodarzi). Therefore, I used a commercial primary human fibroblast cell line (PromoCell) that could yield efficient MCL-1 knockdown (Figure 5-3a) when fully confluent. Cells were analyzed 24 hours after 3Gy or 8Gy X-Ray since the effect of the signaling mediators is most evident at the lower dose, and all remaining γH2AX foci at this time point colocalize with pS824 KAP-1 (Noon et al., 2010). Figure 5-3b shows that the knockdown of MCL-1 did not affect the robust colocalization of pS824 KAP-1 and γH2AX foci in growth arrested human fibroblasts at either dose of IR 24 hours post irradiation.   Late-repairing γH2AX foci are also known to be associated with PML nuclear bodies (PML NBs), with immunofluorescence showing overlap at the periphery of the structures instead of 90  being a complete colocalization (Dellaire et al., 2006a; Munch et al., 2014). PML NBs are subnuclear compartments that contain numerous DNA repair factors and make contact with chromatin to navigate through nuclear space and arrive at sites of DNA damage (Dellaire and Bazett-Jones, 2007; Lallemand-Breitenbach and de The, 2010; Sleeman and Trinkle-Mulcahy, 2014). Structurally characterized by the PML protein, PML NBs normally replicate by a fission process during S phase (Dellaire et al., 2006b); they also exhibit an ATM-, Chk2-, and ATR-mediated increase in number following DNA damage, reaching a peak hours after most other DDR signaling events (Dellaire et al., 2006a; Kepkay et al., 2011). Furthermore, PML NBs interact with numerous HR proteins and promote the pathway’s fidelity (Carbone et al., 2002; Dellaire et al., 2006a; Dellaire et al., 2006b; Dellaire et al., 2009; Foltankova et al., 2013; Kepkay et al., 2011; Lallemand-Breitenbach and de The, 2010; Munch et al., 2014; Xu et al., 2003; Yeung et al., 2012).   Since late-repairing γH2AX foci are known to associate with both pS824 KAP-1 foci and PML I speculated that the latter two proteins would themselves show an association by immunofluorescence. I also wanted to test a potential role of MCL-1 in facilitating this interaction since MCL-1 and PML NBs are each associated with ATM/ATR signaling, HC contact, and HR effector foci defects at late time points. Again using growth-arrested primary human fibroblasts analyzed 24 hours after IR, Figure 5-3c provides (to our knowledge) the first visualization of pS824 KAP-1 foci association with PML; this interaction presents with the classic configuration of DNA damage foci overlapping with PML NB periphery. Once again though, MCL-1 knockdown did not affect this association at either of the IR doses tested.   91     Figure 5-3: MCL-1 is not required for retention of pS824 KAP-1 with γH2AX foci or PML bodies 24 hours post irradiation. Primary human fibroblasts (PromoCell) were grown to confluence and maintained for 2 days to induce growth arrest. They were then transfected with siRNA for 48 hours to achieve efficient MCL-1 knockdown (A). The cells were treated with 3Gy or 8Gy X-ray, fixed at 24 hours, and stained for immunofluorescence to detect association of pS824 KAP-1 foci with (B) γH2AX foci or (C) PML NBs.     92  5.3! MCL-1 promotes heterochromatin compaction I have demonstrated that MCL-1 potentiates ATM activity and promotes HR efficiency, and its knockdown results in elevated RPA levels > 4 hours. MCL-1 also has novel protein interactions with HP1α and p150CAF-1; however, MCL-1 binds both proteins in the presence or absence of exogenous DNA damage, is not required for their established interaction or recruitment to DNA damage, and does not affect KAP-1 localizations associated with heterochromatic DSB repair. Therefore, I next tested whether MCL-1 plays a functional role in the dynamic process of HC relaxation/compaction. I hypothesized that MCL-1 would facilitate HC relaxation based on its potentiation of KAP-1 S824 phosphorylation in response to etoposide (Figure 3-2).  With the permission of Dr. Susan Janicki, we acquired the U2OS 2-6-3 cell line that was previously described, which has a stably integrated heterochromatic array consisting of 256 sequential LacO repeats (Janicki et al., 2004). Protein constructs can be targeted to this array by fusion with LacR, and visualized by fusion with mCherry. This approach has been used to quantify the array area as a fraction of nuclear area to assess proteins’ functional roles in HC relaxation/compaction (Klement et al., 2014; Luijsterburg et al., 2012).  MCL-1 was cloned to generate a mCherry-LacR-NLS-MCL-1 construct, and a separate plasmid with only mCherry-LacR-NLS was used as a control to visualize the basal size of the LacO heterochromatic array. As seen in Figure 5-4b, overexpression of NLS-tagged MCL-1 yielded nucleolar accumulation. Attempts were made to validate this as a true functional and endogenous phenomenon but the nucleolar staining was deemed non-specific. Furthermore, another study showed that overexpression of a S162A MCL-1 construct, without a NLS tag, localized to the 93  nucleus and was excluded from nucleoli (Thomas et al., 2012). The nucleolar accumulation of mCherry-LacR-NLS-MCL-1 is likely an artifact that has been reported by others when overexpressing NLS-tagged constructs (Musinova et al., 2011). In Figure 5-4b, co-staining with the nucleloar protein Fibrillarin allowed discernment of which mCherry signal was the HC LacO array. Zen Blue software was used to quantify the array area as a percentage of total nuclear area and Figure 5-4c reveals that MCL-1 targeting to LacO compacted the structure by 21%. The basal array size was 0.50% of the nucleus (n=117, SEM=0.03), while targeting MCL-1 decreased it to 0.39% (n=111, SEM=0.02).   This result of MCL-1 promoting HC compaction was surprising and the opposite of my hypothesis. To confirm, I performed siRNA knockdown of MCL-1 (as per Figure 3-3b) followed by LacR-mCherry plasmid transfection to measure the basal LacO array area in the presence or absence of MCL-1. Figure 5-4e shows that MCL-1 knockdown increased the HC array by 30%, a magnitude comparable to the decrease due to the targeted overexpressed construct. With scrambled siRNA, the basal array area was 0.42% of the nucleus (n=106, SEM=0.03) and MCL-1 knockdown increased it to 0.55% (n=105, SEM=0.03). Taken together, these results demonstrate that MCL-1 plays a role in compacting HC structures.  To further investigate MCL-1 as a HC promoting factor I assessed levels H3K9 methylation. H3K9me2/3 is characteristic of HC and is generated by the SETDB1 and SUV39H1/2 histone methyltransferases (Bakkenist and Kastan, 2015; Becker et al., 2016; Du et al., 2015; Kim and Kim, 2012). Figure 5-5 reveals that the basal level of H3K9me2/3 was diminished in cells depleted for MCL-1, using a panel of human cell lines and WT vs MCL-1-/- MEFs. 94    Figure 5-4: MCL-1 promotes compaction of the LacO heterochromatic array. (A) Schematic diagram of U2OS 2-6-3 cells with integrated heterochromatic LacO array and targeting of LacR-mCherry constructs to quantify array area. U2OS 2-6-3 cells were transfected with LacR-mCherry or LacR-mCherry-MCL-1 plasmids for 48 hours, then fixed and stained for Fibrillarin to visualize nucleoli. (B) Representative images of constructs targeted to LacO array and (C) cumulative quantification of array area/nuclear area for at least 100 nuclei per condition across three replicates. In a separate experiment, U2OS 2-6-3 cells were transfected with Ctrl or 95  MCL-1 siRNA for 24 hours, followed by 48-hour transfection of LacR-mCherry plasmid, and then fixed. (D) Representative images of LacO array in Ctrl vs MCL-1 knockdown conditions and (E) cumulative quantification of relative array area (% nuclear volume) for at least 100 nuclei per condition across three replicates. All quantification performed using Zen Blue software and statistical analysis by Student’s t-test. Data presented is mean ± SD. LacO HC arrays are denoted by white arrows.       Figure 5-5: MCL-1 depletion decreases basal Histone H3K9 methylation. WT and MCL-/- MEFs, and four human cell lines transfected with MCL-1 siRNA for 48 hours were harvested for whole cell lysates without any exogenous treatment. Samples were immunoblotted for H3K9me3, with Vinculin and total histone H3 as loading controls.   5.4! Discussion My initial hypothesis for this chapter was that MCL-1 participates in DSB repair and chromatin relaxation within HC specifically. A hallmark of these repair events is that they occur at late time 96  points and require retention of pS824 KAP-1 to maintain an open chromatin structure (Goodarzi et al., 2011; Goodarzi et al., 2008; Kakarougkas et al., 2013; Lee et al., 2012; Noon et al., 2010; Woodbine et al., 2011; Ziv et al., 2006). However, in Figure 5-3b I demonstrated that MCL-1 is dispensable for the colocalization of pS824 KAP-1 and γH2AX at late-repairing DSBs. To our knowledge, I visualized for the first time an association between pS824 KAP-1 and PML nuclear bodies at late-repairing DSBs (Figure 5-3c). However, MCL-1 was also dispensable for this event. And contrary to my hypothesis that MCL-1 would promote HC relaxation, I found that it enhances chromatin compaction (Figure 5-4) and maintains basal levels of H3K9me2/3 (Figure 5-5). I therefore conclude that MCL-1 is not an essential factor for DSB repair within HC. The implications for the results in this chapter will be further discussed in Chapter 6:.  It is important to highlight that the evidence presented here for the involvement of MCL-1 in HC compaction was in untreated cells. De novo HC production during DNA replication requires HP1α and interaction between p150CAF-1 and PCNA (Dohke et al., 2008; Gerard et al., 2006; Rivera et al., 2014). It is reasonable to postulate a mechanistic link of MCL-1 with DNA replication given its interactions with HP1α, p150CAF-1 (Figure 4-3), PCNA (Fujise et al., 2000; Jamil et al., 2005; Yi et al., 2012), and putative interactions with MCM4, 6, and 7 identified in Table 4-1.   To investigate chromatin dynamics in response to DNA damage, further techniques will need to be utilized. Ayrapetov et al presented an elegant system to assess the propogation of factors away from euchromatic DSBs – intron 1 of the PPP1R12L gene is known to be euchromatic and expression of p84-ZNF induces a single DSB within it (Brunet et al., 2009; Xu et al., 2012a; Xu 97  et al., 2010). Chromatin IP was then used to detect proteins and histone modifications at specific distances from the DSB (Ayrapetov et al., 2014). The presence of MCL-1, or requirement of it for the presence of other factors, could be analyzed in this way. Such experiments would complement our lab’s previous data showing that MCL-1 is bound directly at I-Ppo1-induced DSBs (Jamil et al., 2010a).  Conversely, it is possible that the effect of MCL-1 on HC structure is different in response to DNA damage, and may in fact relax chromatin. The micrococcal nuclease (MNase) assay is a classic technique to assess HC relaxation in response to DNA damage. MNase treatment cleaves DNA in the linker regions separating histone octamers. The result is a ladder pattern of DNA fragments in multiples of ~147 base pairs (number of base pairs in one nucleosome) that can be visualized in an agarose gel. This technique requires high doses of DNA damage and the results are only observable at times < 2 hours after damage. There is also much inconsistency in the literature about the type of results expected in response to 30-minute treatment with 200ng/mL of the radiomimetic drug neocarzinostatin (NCS). Some studies show a ladder pattern only after DNA damage (Goodarzi et al., 2011), while others report an increase in the intensity of the lowest DNA bands following NCS (Klement et al., 2014; Ziv et al., 2006), or see no difference at all (Hamilton et al., 2011). Inconsistencies can also arise from differences in cell lines, confluency at time of treatment, siRNA transfection, and even the lot of MNase used (personal communication with Dr. Goodarzi). Multiple attempts and protocols were tried to investigate the effect of MCL-1 on HC structure using the MNase assay but no consistent results were seen between replicates.    98  Another method to investigate HC dynamics following DNA damage involves isolating chromatin from untreated or IR-treated cells followed by time course nuclease treatment and immunoblot detection of proteins and histone modifications (Goodarzi et al., 2008; Klement et al., 2014). HC factors require longer nuclease treatment in order to be released and this strategy could be used to detect MCL-1, or its requirement for the presence/absence of other components, in different digested chromatin fractions following DNA damage.  99  Chapter 6:!Discussion In this thesis I have expanded upon the Duronio lab’s previous work establishing a function of the pro-survival BCL-2 family member, MCL-1, in the nucleus regulating DNA repair (Jamil et al., 2008; Jamil et al., 2010a). MCL-1 is unique within the BCL-2 family as it has a short half life ranging from 0.5 – 4 hours (Chao et al., 1998; Cuconati et al., 2003; Ding et al., 2007a; Jamil et al., 2010b; Liu et al., 2005; Maurer et al., 2006; Nijhawan et al., 2003; Schubert and Duronio, 2001; Stewart et al., 2010a) and I demonstrated that its half life is shorter in the nucleus compared to the cytosol (Figure 3-1). I have also shown in Chapter 3: that MCL-1 is a potent activator of the ATM – Chk2 axis (Figure 3-2), potentiates HR efficiency (Figure 3-3e), and that its loss impacts RPA foci kinetics > 4 hours (Figure 3-4). In Chapter 4: I employed a LC-MS/MS proteomic screen of MCL-1 CoIPs to identify novel protein interactions that facilitate DNA repair. I identified HP1α and p150CAF-1 as novel MCL-1 binding partners and validated by immunoblot that the interactions are constitutive (Figure 4-3). In Chapter 5: I investigated the hypothesis that these interactions implicate MCL-1 in the repair of heterochromatic DSBs. However, the absence of MCL-1 had no impact on the established HP1α-p150CAF-1 interaction (Figure 4-4) or the recruitment of HP1α, p150CAF-1, or KAP-1 to sites of DNA damage (Figure 5-1,Figure 5-2). Furthermore, the retention of pS824 KAP-1 foci at late repairing HC DSBs, an event required for efficient repair (Goodarzi et al., 2011; Goodarzi et al., 2008; Kakarougkas et al., 2013; Lee et al., 2012; Noon et al., 2010), was not affected by MCL-1 knockdown (Figure 5-3b). To our knowledge I have also provided the first evidence that pS824 KAP-1 associates with PML nuclear bodies at HC DSBs but similarly, MCL-1 was dispensable for this event 100  (Figure 5-3c). Functional analysis revealed that MCL-1 promotes the compaction of HC (Figure 5-4) and maintains basal levels of H3K9me2/3 (Figure 5-5).  I propose a model whereby MCL-1-mediated HC formation regulates both the upstream activation of ATM kinase activity and key events downstream in the HR pathway. This model is depicted in Figure 6-1 and the details are discussed below.   Figure 6-1: Proposed model of how MCL-1 can impact multiple stages of the DNA damage response by compacting chromatin. MCL-1 promotes heterochromatin compaction and H3K9 trimethylation, potentially through its novel interactions with HP1α and p150CAF-1. In response to DNA damage, H3K9me3 is transiently propagated away from euchromatic DSBs, which activates the Tip60 acetyltransferase for downstream ATM activation. Heterochomatic DSBs require chromatin relaxation mediated by ATM phosphorylation of KAP-1. MCL-1 potentiates 101  early phosphorylation of ATM, Chk2, KAP-1, and γH2AX placing it upstream of ATM as well. Once resection has been initiated, chromatin compaction factors enhance the efficiency of homologous recombination and keep sister chromatid in close proximity. Thus, by compacting chromatin and generating H3K9me3, MCL-1 may promote the replacement of RPA for Rad51 to enhance homology searching and fidelity of the homologous recombination pathway.   6.1! MCL-1 potentiation of ATM activity may be linked to H3K9me3-mediated Tip60 activation Cells lacking MCL-1 have a dramatic reduction in ATM activation and downstream signalling to Chk2 at early time points across a 100-fold range of etoposide concentrations (Figure 3-2). Upstream of the damage-induced increase in ATM kinase activity and dissociation into active monomers is acetylation of ATM by Tip60 (Jiang et al., 2006; Kaidi and Jackson, 2013; Sun et al., 2005; Sun et al., 2010; Sun et al., 2009; Sun et al., 2007; Xu et al., 2012b). The acetylation site is Lys 3016 in the FATC domain of ATM, immediately adjacent to its kinase domain (Sun et al., 2007). The DSB recruitment of Tip60 is dependent on the MRN complex but the damage-induced increase of its acetyltransferase activity is dependent upon interaction with H3K9me3 via its chromodomain; this enhanced activity was not seen with unmodified H3 or methylated sites of other histone residues (Kaidi and Jackson, 2013; Sun et al., 2009). A kinase-dead mutant of ATM still interacts with and is acetylated by Tip60, and a ATM K3016R mutant failed to dissociate into a monomers, validating that this acetylation event is upstream of ATM activation (Sun et al., 2005; Sun et al., 2007).  Experiments that utilize site mutations to prevent the association of Tip60 with ATM or H3K9me3, or conditions that lower basal H3K9me3 levels, all show dramatically impaired 102  activation of pS1981 ATM and pT68 Chk2 ≤4 hours across a range of genotoxic stresses (Kaidi and Jackson, 2013; Sun et al., 2005; Sun et al., 2009; Sun et al., 2007). This matches the DDR signalling in MCL-1-/- MEFs (Figure 3-2) and provides a potential mechanistic link to the MCL-1-mediated maintenance of basal H3K9me2/3 (Figure 5-5), whereby cells lacking MCL-1 have decreased H3K9me3 that would reduce Tip60 activation and subsequent ATM activation. Furthermore, the recruitment of Tip60 to DSBs is dependent on Mre11 (Sun et al., 2009) and Tip60 CoIPs with each component of the MRN (Mre11-Rad50-NBS1) complex (Chailleux et al., 2010). The CoIP of MCL-1 and NBS1 (Jamil et al., 2010a) may provide an indirect interaction between MCL-1 and Tip60. Size exclusion chromatography of the Tip60-MRN complex revealed a size of ~1MDa (Chailleux et al., 2010) suggesting that there are uncharacterized proteins present, which may include MCL-1. It has also been observed that p14ARF binds and increases the protein half life of Tip60, with the overexpression of p14ARF leading to increased basal levels of pS1981 ATM, pT68 Chk2, pS345 Chk1, and induced G2/M arrest, all in the absence of exogenous DNA damage (Eymin et al., 2006). This matches previous work from our lab showing that MCL-1 overexpression alone induced pS345 Chk1 and G2/M arrest (Jamil et al., 2008). If MCL-1 is in a complex that increases the protein stability of Tip60 then it would be a similar effect to that seen on TCTP (Zhang et al., 2002c). However, given the short half life of MCL-1, it would have to be continually replaced in complexes to exert a stabilizing effect on other proteins, unless the increased stability is reciprocal.   Future studies could explore a direct effect of MCL-1 on the enzymatic activity of Tip60 and ATM. There are well established in vitro assays for Tip60 acetyltransferase activity on ATM or Histone H4, ATM kinase activity on p53, and detection of acetylated ATM following CoIP and 103  immunoblotting with a pan-acetyl Lysine antibody (Kaidi and Jackson, 2013; Sun et al., 2005; Sun et al., 2009; Sun et al., 2007).  Regardless of the mechanism, Figure 3-2 demonstrates that MCL-1 is a potent activator of the ATM - Chk2 axis. ATM-deficient cells are also known to have delayed ATR – Chk1 signaling, likely due to delayed exposure of RPA-coated ssDNA (Kocher et al., 2012). Therefore, our lab’s previous result showing that MCL-1-/- MEFs have delayed Chk1 activation at low doses of etoposide (Jamil et al., 2010a) is in agreement with the model of MCL-1 as an ATM activator. In Figure 3-2, the effect of MCL-1 on γH2AX and pS824 KAP-1 was most pronounced at the lowest doses of etoposide. This may reflect functional redundancy of other DDR kinases that can compensate for the diminished ATM activity. Alternatively, it may reveal MCL-1 is involved in DDR signal amplification spreading away from the DSB since the activity of DDR kinases localized directly at DSBs may be sufficient to elicit a robust DDR response as the number of DSBs increase with higher etoposide concentrations.   The reduced early KAP-1 S824 phosphorylation in MCL-1-/- MEFs is particularly interesting. KAP1 was originally discovered as a transcriptional corepressor (Friedman et al., 1996; Kim et al., 1996; Le Douarin et al., 1996; Moosmann et al., 1996). The pan-nuclear HC relaxation associated with early KAP-1 S824 phosphorylation (Goodarzi et al., 2011; Goodarzi et al., 2008; Ziv et al., 2006) leads to the upregulation of genes involved in cell cycle arrest and apoptosis, including the BCL-2 family members Bax, Noxa, and Puma (Kurata et al., 2008; Lee et al., 2007; Miyashita et al., 1994; Yu et al., 2001). While these genes are each transcriptionally upregulated by p53 (Nakano and Vousden, 2001; Oda et al., 2000; Shibue et al., 2003; Toshiyuki 104  and Reed, 1995), cells deficient for p53 still display upregulation and sensitivity to genotoxic stress (Broude et al., 2007; Li et al., 2007; Passalaris et al., 1999; Wang et al., 1996). This suggests that suppression of KAP-1 transcriptional repressor function may contribute to p53-independent upregulation of apoptotic genes. It is interesting to speculate that through such a mechanism the pro-survival BCL-2 family member, MCL-1, may regulate the expression of pro-apoptotic family members in response to genotoxic stress.  6.2! MCL-1 may propagate H3K9me3 through interactions with HP1α and p150CAF-1 The specific requirement of H3K9me3 for the activation of Tip60 acetyltransferase activity on ATM initially led to the hypothesis that this mechanism was limited to HC regions. Indeed, it was shown that HP1β dispersal following DNA damage made H3K9me3 available for Tip60 binding (Ayoub et al., 2008; Sun et al., 2009). However, recent evidence reveals that like γH2AX, DNA damage elicits the spread of de novo H3K9me2/3 up to 200bp away from euchromatic DSBs (Ayrapetov et al., 2014). This process was dependent on the immediate and transient recruitment of a previously identified complex containing HP1α/β, KAP-1, and the SUV39H1/2 histone methyltransferases that generates H3K9me2/3 (Ayrapetov et al., 2014; Fritsch et al., 2010; Ivanov et al., 2007; Lechner et al., 2000; Li et al., 2010b; Nielsen et al., 1999). This complex was recruited to DSBs dependent on HP1α/β interaction with H3K9me3, while the spread of H3K9me3 was dependent on the methyltransferase activity of SUV39H1/2, and the complex’s dispersal from DNA damage within 15 minutes was mediated by ATM phosphorylation of S824 KAP-1 (Ayrapetov et al., 2014). These properties may be specific to the HP1α/β isoforms since HP1γ has been shown to inhibit DNA repair processes (Kalousi et al., 105  2015; Soria and Almouzni, 2013). H3K9me3 is also produced by SETDB1, which similarly is transiently recruited to sites of DNA damage (Alagoz et al., 2015). This initial and transient burst of HC compaction at DSBs may act to inhibit transcription at sites surrounding DNA damage and/or immobilize DSB ends.   The HP1α/β-KAP-1-SUV39H1/2 and Tip60-ATM complexes exist in the presence or absence of exogenous DNA damage (Ayrapetov et al., 2014; Sun et al., 2005). Also, the transient DSB recruitment of HP1 isoforms, KAP-1, and histone methyltransferases requires the presence of each other, p150CAF-1, and ATM activity (Alagoz et al., 2015; Ayoub et al., 2008; Ayrapetov et al., 2014; Baldeyron et al., 2011; Luijsterburg et al., 2009; Soria and Almouzni, 2013; Zarebski et al., 2009). MCL-1 therefore fits this scheme as it constitutively binds HP1α and p150CAF-1 (Figure 4-3) and potentiates ATM signaling (Figure 3-2). However its role is more nuanced since the absence of MCL-1 does not affect the interaction of HP1α-p150CAF-1 (Figure 4-4) or recruitment of HP1α, p150CAF-1, or KAP-1 to sites of DNA damage (Figure 5-1,Figure 5-2). Nonetheless, MCL-1 has a clear functional role in promoting HC compaction by facilitating H3K9me2/3 methylation (Figure 5-5) and compacting the heterochromatic LacO array of U2OS 2-6-3 cells (Figure 5-4). The latter is not a trivial finding - targeting the HC-promoting CHD3 nucleosome remodeler to this array had no effect (Klement et al., 2014) but MCL-1 is able to further condense the already-compact LacO array. Future experiments could utilize the same U2OS 2-6-3 cells to see if LacR-MCL-1 is concomitant with the presence/absence of other HC factors at the array.  106  6.3! MCL-1 may facilitate downstream processes in HR as a HC promoting factor Using U2OS pathway-specific reporter systems I showed that MCL-1 functions in HR (Figure 3-3e). This is in agreement with my model since the knockdown of HP1α, p150CAF-1, Tip60, KAP-1, SUV39H1/2, or SETDB1 have all been shown to decrease HR efficiency in I-Sce1-based reporter assays (Alagoz et al., 2015; Ayrapetov et al., 2014; Baldeyron et al., 2011; Chailleux et al., 2010; Lee et al., 2013; Soria and Almouzni, 2013; Tang et al., 2013).  A limitation of I-Sce1 based reporter systems is that they do not account for HC structure. This is best represented by investigations of 53BP1, which has been widely described to inhibit DSB resection and antagonize BRCA1 foci formation and recruitment of HR effectors associated with it (Bouwman et al., 2010; Daley et al., 2015; Daley and Sung, 2014; Panier and Boulton, 2014; Zhou et al., 2014; Zimmermann and de Lange, 2014). Accordingly, there are examples where 53BP1 knockdown results in lower HR efficiency or has no effect when using I-Sce1-based reporter systems (Bunting et al., 2010; Tang et al., 2013; Ward et al., 2004). However, the true complexity of the these processes continues to be elucidated and high resolution microscopy reveals that as BRCA1 foci enlarge from 2 – 8 hours following damage they exist in partnership with 53BP1 that encapsulates the focal periphery (Alagoz et al., 2015; Chapman et al., 2012). And when assessing HR processes via microscopy instead of I-Sce1 reporter assays, 53BP1 presents as a typical HR factor with its knockdown resulting in fewer RPA foci, Rad51 foci, and sister chromatid exchanges (SCEs), with a γH2AX repair defect at 8 hours in G2 cells (Kakarougkas et al., 2013). As well, HR is the predominant repair pathway for HC DSB repair and 53BP1 is required for maintaining the ATM-mediated pS824 KAP-1 that enables this process (Kakarougkas et al., 2013; Noon et al., 2010). Thus, 53BP1 has a complex role in 107  regulating chromatin structure during HR that is not reflected in I-Sce1 assays. Similarly, the magnitude of diminished pathway efficiency by MCL-1 knockdown in these systems may under-represent the protein’s complete function involving chromatin packing. It should also be highlighted that my I-Sce1 assays were performed in U2OS parental cell lines, which exhibited a modest effect of MCL-1 knockdown on basal H3K9me2/3 levels in Figure 5-5. Performing these same assays in other cell lines (e.g. HEK 293T) may yield more dramatic results.  Besides the U2OS DR-GFP reporter results, MCL-1 is also implicated in HR since siRNA knockdown resulted in elevated RPA foci in G2 cells > 4 hours after IR (Figure 3-4c). Following DNA damage, the number of RPA foci in normal cells reaches a peak at 2 hours then gradually declines as it is exchanged for Rad51 and the DSBs are repaired. MCL-1 depleted cells achieve the same RPA peak but this level persists out to 8 hours, which is the same pattern observed with BRCA2-deficient cells (Beucher et al., 2009; Geuting et al., 2013; Shibata et al., 2011). BRCA2 is well characterized as promoting Rad51 nucleoprotein filament formation by directly binding with DNA and Rad51, and interacting with RPA through its binding partner, DSS1 (Jensen et al., 2010; Liu et al., 2010; San Filippo et al., 2006; Zhao et al., 2015). However, BRCA2-deficient G2 cells also exhibit a γH2AX repair defect at 8 hours post irradiation (Beucher et al., 2009; Geuting et al., 2013; Shibata et al., 2011), which was not seen with MCL-1 knockdown (Figure 3-4c). This could be because the cited papers use cell lines with functional p53, while I used HeLa cells. Alternatively, it could implicate the HC compaction functionality of MCL-1; the γH2AX repair defect of BRCA2-deficient cells could be eliminated via the Alt-EJ pathway, but only if there was global HC relaxation by KAP-1 co-depletion (Geuting et al., 2013). These authors concluded that once resection has commenced, HC compaction favours HR over Alt-EJ.   108   I hypothesize that MCL-1 promotes HR processes downstream of end resection, including the exchange of RPA for Rad51, by facilitating chromatin compaction. It is important to remember that resection, RPA/Rad51 loading, and homology searching are not linear stepwise processes. Instead, they exist as part of a continuum whereby a Rad51 nucleoprotein filament can be engaged in a sister chromatid while further downstream resection is taking place to expose a longer sequence for homology searching. These entangled DNA structures are likely very vulnerable to instability and a compact chromatin environment would keep the sister chromatid in close proximity to enhance the latter stages of HR.  In addition to Geuting et al. there is evidence that HC promoting factors potentiate downstream processes in HR. HP1α/β, SETDB1A/B, and SUV39H1/2 were dispensable for the initiation of resection but were epistatic with the proceeding wave of resection elongation and necessary for proper architecture of 53BP1/BRCA1 foci of G2 cells (Alagoz et al., 2015). Furthermore, the proximity of sister chromatids in G2 was dependent on HP1α/β, SETDB1A/B, and SUV39H1/2 whereas BRCA1 was dispensable (Alagoz et al., 2015). Another recent study revealed that ATM has functions during HR in G2 cells, distinct from its role in initiating resection. Chemical inhibition of ATM kinase activity after resection had initiated resulted in impaired Rad51 resolution and a γH2AX repair defect ≥10 hours, with ~50% HR efficiency by a I-Sce1 assay (Bakr et al., 2015). As well, Tip60 is the acetyltransferase component of the NuA4 complex that targets Histone H4 (Murr et al., 2006; Price and D'Andrea, 2013; Soria et al., 2012; Xu et al., 2012a) to promote the proper focal accumulation of 53BP1 and BRCA1 at sites of DNA damage (Tang et al., 2013; Xu et al., 2010). 109   My model is based on data implicating MCL-1 in HC compaction and ATM activation, with the hypothesis that Tip60 links these two processes. All of these factors potentiate key events downstream in HR processing and fit with a role for MCL-1 facilitating the switch of RPA for Rad51 by compacting chromatin. Thus, in the context of RPA foci, MCL-1 would be epistatic with BRCA2; but MCL-1 knockdown would mimic the co-depletion of BRCA2 and KAP-1 that resulted in no γH2AX repair defect due to Alt-EJ fidelity (Geuting et al., 2013), while still showing impaired HR efficiency (Figure 3-3e).   However, another interpretation of Figure 3-4c is that the MCL-1 knockdown does not prevent RPA resolution, but instead influences pathway choice, increasing resection and RPA foci formation at DSBs that would not normally undergo these processes. The number of DSBs was the same, but cells depleted of MCL-1 had more RPA foci per cell. There is growing evidence that chromatin architecture and histone methylation status can affect DSB repair pathway choice (Aymard et al., 2014; Clouaire and Legube, 2015; Pai et al., 2014; Pfister et al., 2014). A direct role for MCL-1 in pathway choice may be mediated by CDK1; resection initiation by CtIP is promoted by CDK phosphorylation during S and G2 (Huertas et al., 2008; Huertas and Jackson, 2009; Peterson et al., 2011) and our lab previously demonstrated that in hematopoetic cells, a proteolytic fragment of MCL-1 shows nuclear accumulation during S and G2 where it inhibits CDK1 activity (Jamil et al., 2005). Thus, MCL-1 may be part of a feedback mechanism to limit end resection and its knockdown would therefore shift DSB repair towards HR. How this would reconcile with the decreased HR efficiency in the U2OS DR-GFP reporter cells would need further mechanistic elucidation.  110   To delineate these two explanations of Figure 3-4c, additional experiments could be performed. Using the same experimental setup, Rad51 foci could be enumerated. Also in G2 cells with control vs MCL-1 siRNA, sister chromatid exchanges and BRCA1/53BP1 focal architecture could be assessed. If MCL-1 promotes the exchange of RPA for Rad51, then the downstream events in HR processing would be impaired in cells lacking MCL-1 at late time points following DNA damage. And if repair is able to proceed via Alt-EJ then the addition of a PARP inhibitor during the experiment would elicit a γH2AX repair defect in the MCL-1 knockdown. Conversely, if MCL-1 influences pathway choice and knockdown shifts cells towards HR, we would expect to see elevated levels of Rad51 foci and sister chromatid exchanges in the knockdown. It is noteworthy that in Figure 3-4c the number of RPA and γH2AX foci at 8 hours in the MCL-1 knockdown are equal, suggesting that all remaining DSBs are associated with resected HR sites. Including longer time points may reveal additional information but the ability of aphidicolin to maintain S phase arrest > 8 hours would need to be assessed.   6.4! Experimental considerations when affecting basal chromatin architecture and analyzing DSB repair It is clear that chromatin structure plays an influential role in DNA repair processes, both dictating the mechanism of repair and being dynamically altered during its course. Thus, it is critical to ask targeted questions when designing experiments to mitigate confounding, and sometimes opposing, effects that may take place simultaneously to the processes being investigated. For example, heterochromatic DSBs require sustained localized ATM kinase activity to retain pS824 KAP-1 and permit an open structure amenable to repair (Goodarzi et al., 111  2011; Goodarzi et al., 2008; Lee et al., 2012; Noon et al., 2010; Woodbine et al., 2011; Ziv et al., 2006). In euchromatin however, ATM activity on KAP-1 is transient and both factors are quickly released from DSBs in order to compact chromatin structure and promote repair (Ayrapetov et al., 2014; Geuting et al., 2013). Methylation also plays an important role since decreasing basal H3K9me3 levels decreases genomic stability and damage-induced ATM activity (Peng and Karpen, 2009; Peters et al., 2001; Sun et al., 2009), while inhibiting DNA methylation can increase ATM signaling in response to damage and make cell cycle checkpoints hypersensitive to DSBs (Brunton et al., 2011). Additionally, it has been seen that HP1α knockdown or HDAC inhibition alone can induce ATM signaling and checkpoint arrest with no detectable change in DNA damage (Kaidi and Jackson, 2013). The proliferative status of cells can also influence the effects of altering basal chromatin structure. It has been seen that cells replicating in the absence of HC factors have daughter cells with improper HC reconstitution and pan-nuclear chromatin relaxation; whereas the same knockdown in G0 cells still possessed a heterochromatic barrier to overcome during repair (Klement et al., 2014). Therefore, it is critical to have a robust understanding of how experimental design can impact the chromatin context at the time of damage.  There are also spatiotemporal dynamics in DDR that are increasingly becoming appreciated. Repair foci do not form within heterochromatic regions, and are instead found on their periphery (Brunton et al., 2011; Cowell et al., 2007; Goodarzi et al., 2008; Kim et al., 2007). There are numerous studies of HR in yeast and Drosophila revealing that end resection can take place within HC but Rad51 filament formation can only occur once the DSB has been repositioned to the heterochromatic periphery, or associates with nuclear pore complexes, in a process dependent 112  on HP1 orthologs (Chiolo et al., 2011; Dronamraju and Mason, 2011; Horigome et al., 2014; Kalocsay et al., 2009; Nagai et al., 2008; Oza et al., 2009; Ryu et al., 2015). This is an attractive model in which to include MCL-1 given its interaction with HP1α and putative function downstream of RPA foci formation. But an analogous mechanism in mammalian cells has yet to be robustly described, and there are no orthologs of MCL-1 below vertebrates to test this hypothesis in model organisms. There is recent evidence though, in mammalian cells, that pathway choice and repair kinetics are affected by whether the DSB was induced at the nuclear membrane, pore, or interior (Lemaitre et al., 2014). As the understanding of these processes and methodologies to investigate them evolve it will be interesting to study MCL-1 in the context of HC DSB repositioning.  6.5! Future directions In addition to the mechanistic elucidations already discussed, there are at least three main avenues to pursue in future experiments. First, it will prove immensely useful to have high quality and specific immunofluorescent staining of MCL-1. To date, there has never been robust IF images showing punctate staining of endogenous MCL-1, especially within the nucleus. Throughout the course of this work several commercial antibodies and IF staining protocols were utilized but failed to yield conclusive results. The most striking images were obtained using a rabbit monoclonal antibody from Abcam (Cat #ab32087), which had strong staining of sub-nuclear compartments that were verified to be nucleoli and PML nuclear bodies. However, siRNA knockdown of MCL-1 failed to diminish the intensity of the IF staining while immunoblot samples prepared in parallel confirmed efficient knockdown in nuclear fractionated lysates. Testing the siRNA transfection from 24 – 72 hours consistently yielded this result and 113  Figure 3-1 suggests that it should in fact be easier to manipulate nuclear MCL-1 levels. Furthermore, the Dharmacon SMARTpool MCL-1 siRNA used is validated to knock down all known mRNA splice variant transcripts of MCL-1. Collectively, attempts to visualize endogenous MCL-1 by IF yielded either poor quality structural resolution or confirmed non-specificity.   Thus, a concerted effort should be made to visualize endogenous MCL-1 because IF is a powerful technique in the field of DNA repair research. This would make it easier to study the localization of MCL-1 within sub-nuclear compartments and chromatin architecture, kinetics of its focal recruitment and resolution, and co-localization with any number of established repair factors. Such MCL-1 antibodies could be raised using standard approaches in host animals (e.g. rabbit, mouse) or by generating recombinant single chain antibodies. The Infection and Immunity Research Center, of which our group is apart, has the capabilities to manufacture the latter. Additionally, CRISPR-Cas9 methodologies are increasingly being used to introduce fluorescent proteins or epitope tags within endogenous genomic sequences of interest (Harrison et al., 2014; Pinder et al., 2015).  Secondly, we have shown that MCL-1 participates in HR and utilized techniques to quantify repair foci in G2 cells specifically. HR is dynamically regulated during the cell cycle (Branzei and Foiani, 2008; Delacote and Lopez, 2008; Mathiasen and Lisby, 2014) and we could similarly investigate the role of MCL-1 on DSB repair during S phase. Pulse-labeling cells with BrdU in conjunction with aphidicolin just prior to IR treatment allows for identification of S phase cells for time course RPA/Rad51 foci enumeration (Kocher et al., 2012). Using these approaches it 114  has been shown that the Artemis nuclease is required for HR in G2 cells but is dispensable in S phase (Beucher et al., 2009; Kocher et al., 2012). MCL-1 may too be differentially required for HR processing events throughout the cell cycle. As well, while my investigations of protein interactions showed that MCL-1 constitutively binds HP1α and p150CAF-1, and is dispensable for their established interaction, this work was done on asynchronous cell populations. By performing CoIPs at different time points following release from cell synchronization (e.g. double thymidine block) we could interrogate whether cell cycle phase affects MCL-1 interactions or the composition of other established complexes containing, ATM, Tip60, HP1α, p150CAF-1, KAP-1, SUV39H1/2, and SETDB1.  Lastly, and most importantly, a robust set of structure/function studies should be performed to determine if the nuclear functions of MCL-1 depend on its BH domains that govern its canonical BCL-2 family function, or on other parts of the protein. For these experiments, a series of MCL-1 mutants should be generated with truncations of the overall structure, targeted mutations within specific domains, phosphosite substitutions, and constructs representing the products of splice variation. Furthermore, the chromoshadow domain of HP1 isoforms interact with PxVxL motifs in their binding partners (Canzio et al., 2014; Cowieson et al., 2000; Smothers and Henikoff, 2000; Thiru et al., 2004), and amino acids 138 – 144 of MCL-1 (PAVLPLL) closely fit this motif. This region is within extended N-terminus of MCL-1 that bears no similarity with other BCL-2 family members and its mutation could reveal a structural basis for delineating the protein’s cytoplasmic and nuclear functions. These exogenous MCL-1 constructs should be used for CoIP experiments to see how its interactions with HP1α and p150CAF-1, and the composition of other protein complexes listed above, are impacted. The easiest functional 115  experiment to start with is the LacO array size quantification in U2OS 2-6-3 cells. Each construct should be cloned into the mCherry-LacR vector to see which domain(s) are required for HC compaction, and correlate this with the protein interaction results. Additionally, the U2OS 2-6-3 cells could be used to detect the concomitant presence/absence of HP1α, p150CAF-1, KAP-1, SUV39H1/2, and SETDB1 with each MCL-1 mutant construct.   If mutating the molecular determinants of MCL-1 needed for the DDR and chromatin compaction do not affect its canonical apoptosis regulating functions, then synthetic lethality experiments could be performed. A mainstay of DNA repair research is to show that cells deficient in one repair pathway exhibit synergistic toxicity with treatments that impair a separate pathway capable of repairing the same break. The best example of this is that cells with diminished HR capacity from BRCA1/2 deficiency experience synthetic lethality with PARP inhibitors (Aly and Ganesan, 2011; Dedes et al., 2011; Helleday, 2011; O'Connor, 2015; Polyak and Garber, 2011). This rationale led to the development and first-ever FDA approval in December 2014 of a DNA repair inhibiting drug, Lynparza™ (Olaparib), used to treat ovarian cancers expressing germline BRCA1/2 mutations. With MCL-1 however, its canonical function as a pro-survival BCL-2 family member confounds interpretation of any such synthetic lethality experiments, unless its apoptotic and DNA repair functions could be delineated.  Conversely, if the BH domains of MCL-1 are required for its role in DNA repair and HC dynamics then drugs targeted against them would have two simultaneous therapeutic effects for cancer treatment, affecting apoptosis regulation and DNA repair. Navitoclax and Venetoclax are BH3-mimetic compounds currently undergoing numerous ongoing Phase II and III clinical trials. 116  But it is well documented that resistance to BH3-mimetics and several chemotherapeutic compounds is mediated by high expression of MCL-1 (Akagi et al., 2013; Chen et al., 2011; Chen et al., 2007a; Choudhary et al., 2015; Inuzuka et al., 2011; Konopleva et al., 2006; Long et al., 2015; Phillips et al., 2015; Tahir et al., 2007; van Delft et al., 2006; Wei et al., 2008; Wertz et al., 2011). Thus, the recent validation of the first-ever potent and selective BH3-mimetic compound against MCL-1 (Bruncko et al., 2015; Leverson et al., 2015) was met with great enthusiasm and the field is poised to widely embrace such compounds for cancer therapy studies. If they simultaneously inhibit DNA repair processes and prime cells for apoptosis, then they could provide an immensely effective therapeutic tool.  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