Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
DSpace
UBCV
Gao, Xiaochen
Master of Science - MSc
University of British Columbia
Chromatin disruption caused by polymerase passage during transcription or DNA replication has the potential to lead to cryptic transcription originating within aberrant initiation sites across the genome. We developed a sfGFP fluorescence reporter to track cryptic transcription initiating from an intragenic cryptic promoter within the FLO8 coding region and confirmed reporter utility in a spt16-197 mutant strain. Using this reporter, our results showed that the proteins, Spt10, Spt21, Cac1, and Asf1 are required for chromatin stability during DNA replication, while Spt2 plays a replication-independent role in maintaining chromatin. We also showed that blocking DNA replication only partially suppresses cryptic transcription in Spt5, Spt6, or Spt16-depleted cells, indicative of these factors functioning in a process independent of replication. Taken together, our results demonstrate that restoration of nucleosome stability following disruption is facilitated by a number of collaborating factors functioning in various pathways.
INVESTIGATION OF CRYPTIC TRANSCRIPTION SUPPRESSING FACTORS USING A FLO8:sfGFP REPORTER by Xiaochen Gao B.Sc., The University of British Columbia, 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) March 2023 © Xiaochen Gao, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Investigation of cryptic transcription suppressing factors using a FLO8:sfGFP reporter submitted by Xiaochen Gao in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry and Molecular Biology Examining Committee: LeAnn Howe, Professor, Biochemistry and Molecular Biology, UBC Supervisor Sheila Teves, Assistant Professor, Biochemistry and Molecular Biology, UBC Supervisory Committee Member Annie Ciernia, Assistant Professor, Biochemistry and Molecular Biology, UBC Supervisory Committee Member Vivien Measday, Associate Professor, Faculty of Land and Food Systems, UBC Additional Examiner iii Abstract Chromatin disruption caused by polymerase passage during transcription or DNA replication has the potential to lead to cryptic transcription originating within aberrant initiation sites across the genome. We developed a sfGFP fluorescence reporter to track cryptic transcription initiating from an intragenic cryptic promoter within the FLO8 coding region and confirmed reporter utility in a spt16-197 mutant strain. Using this reporter, our results showed that the proteins, Spt10, Spt21, Cac1, and Asf1 are required for chromatin stability during DNA replication, while Spt2 plays a replication-independent role in maintaining chromatin. We also showed that blocking DNA replication only partially suppresses cryptic transcription in Spt5, Spt6, or Spt16-depleted cells, indicative of these factors functioning in a process independent of replication. Taken together, our results demonstrate that restoration of nucleosome stability following disruption is facilitated by a number of collaborating factors functioning in various pathways. iv Lay Summary Genetic information is stored in the form of DNA, which is wrapped around histones and compacted into chromatin. The integrity of the chromatin landscape is paramount in regulating the process of manufacturing protein from DNA, as only the correct starting points are exposed to worker proteins so that the assembly line does not start in the middle of genes and create cryptic products. Cellular processes like DNA replication temporarily disrupt chromatin, which is quickly restored by chaperones. Using a fluorescence reporter system, we tracked the spontaneous production of cryptic products resulting from disrupted chromatin. Moreover, by depleting specific proteins separately, we successfully identified whether a protein is responsible for restoring replication or transcription-induced chromatin damage. v Preface The identification and design of the research program were completed in collaboration with Dr. LeAnn Howe. Text and data in Chapter 3 were a collaboration with Dr. LeAnn Howe and Stephanie Jung. Analysis of research data and figure generation were works of myself. vi Table of Contents Abstract .......................................................................................................................................... ii Lay Summary ................................................................................................................................ iv Preface ............................................................................................................................................ v Table of Contents .......................................................................................................................... vi List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................ x List of Abbreviations .................................................................................................................... xi Acknowledgements ..................................................................................................................... xiv Dedication ..................................................................................................................................... xv Chapter 1: Introduction ................................................................................................................ 1 1.1 The nucleosome landscape is crucial for transcription regulation ...................................... 1 1.1.1 Nucleosomes pose barriers for transcription factors and the transcription machinery..1 1.1.2 Histone chaperones both facilitate and prevent histone turnover during transcription..4 1.1.3 Nucleosome assembly during replication ...................................................................... 7 1.2 Cryptic transcription ............................................................................................................ 9 1.2.1 Characteristics of cryptic promoters ............................................................................ 9 1.2.2 Cryptic transcription suppressing factors .................................................................. 10 1.2.2.1 FACT ................................................................................................................. 12 1.2.2.2 Spt6 .................................................................................................................... 13 1.2.2.3 Spt5 .................................................................................................................... 15 1.2.2.4 Spt2 .................................................................................................................... 15 vii 1.2.2.5 Spt10/21 ............................................................................................................. 16 1.2.2.6 Asf1 ................................................................................................................... 17 1.2.2.7 CAF-1 ................................................................................................................ 18 1.2.2.8 Rtt106 ................................................................................................................ 19 1.2.3 Classical and novel methods for detecting cryptic transcription ............................... 20 1.3 Thesis objectives ................................................................................................................ 20 Chapter 2: Materials and Methods ............................................................................................ 22 2.1 Yeast Strains ...................................................................................................................... 22 2.2 Plasmids and nucleotides ................................................................................................... 28 2.3 Immunoblot ....................................................................................................................... 30 2.4 Flow cytometry .................................................................................................................. 31 2.5 Drug treatments ................................................................................................................. 31 2.6 Dilution plating .................................................................................................................. 31 2.7 Statistical analysis .............................................................................................................. 31 Chapter 3: Results ....................................................................................................................... 33 3.1 Generation of a fluorescent reporter for cryptic transcription ........................................... 33 3.2 A fluorescent reporter of cryptic transcription can differentiate chromatin disruption due to transcription or DNA replication........................................................................................36 3.3 Blocking the cell cycle does not suppress cryptic transcription following Spt5, Spt6 or Spt16 depletion .................................................................................................................. 44 Chapter 4: Summary and Discussion ........................................................................................ 47 3.1 Summary ............................................................................................................................ 47 3.3 Discussion and future directions ........................................................................................ 48 viii Bibliography ................................................................................................................................. 51 ix List of Tables Table 1. Yeast strains used in this study ........................................................................................ 22 Table 2. Nucleotide sequences ...................................................................................................... 28 x List of Figures Figure 1.1 Histone chaperones involved in transcription ................................................................ 5 Figure 1.2 Nucleosome assembly during replication ...................................................................... 8 Figure 3.1 Development of FLO8:sfGFP reporter for cryptic transcription detection .................. 34 Figure 3.2 Confirmation of spt16-197 degradation and controlling for differences due to arrest or cell sizes ......................................................................................................................................... 35 Figure 3.3 Cryptic tanscription in cells lacking Spt2 is dependent on active FLO8 transcription.37 Figure 3.4 Cryptic tanscription in cells depleted of Spt10 or Spt21 is dependent on replication.....39 Figure 3.5 Test for cryptic transcription following Cac1 depletion .............................................. 41 Figure 3.6 Test for cryptic transcription following Asf1 depletion ............................................... 42 Figure 3.7 Test for cryptic transcription following Rtt106 depletion ............................................ 43 Figure 3.8 Replication inhibition partially suppress cryptic transcription in cells depleted of Spt5, Spt16 or Spt6 ................................................................................................................................. 45 xi List of Abbreviations ADE5/7 Adenine requiring 5/7 ATP Adenosine triphosphate Abf1 ARS-binding factor 1 Air1/2 Arginine methyltransferase-interacting RING finger protein 1/2 Asf1 Anti-silencing function 1 BrdU 5’-bromo-2’-deoxyuridine CAF-1 Chromatin assembly factor 1 CRISPR Clustered regularly interspaced short palindromic repeats Cryo-EM Cryogenic electron microscope DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DSIF DRB sensitivity inducing factor FACT Facilitates chromatin transaction FLO8 Flocculation 8 GAL1 Galactose metabolism 1 GFP Green fluorescent protein HIR Histone regulation HIS3 Histidine 3 HMG High mobility group LYS2 Lysine requiring 2 Leu3 Leucine biosynthesis 3 MBF MCB-binding factor xii MEC1 Mitosis entry checkpoint 1 Mcm2 Minichromosome maintenance 2 Mcm4 Minichromosome maintenance 4 NDR Nucleosome depleted region Nap1 Nucleosome assembly protein 1 ORF Open reading frame PAF1 RNA polymerase II associated factor 1 PCA1 P-type Cation-transporting ATPase PCNA Proliferating cell nuclear antigen PCR Polymerase chain reaction PHO5 Phosphate metabolism 5 PHO8 Phosphate metabolism 8 PTM Post-translational modification Pho4 Phosphate metabolism 4 RCAF Replication-coupling assembly factor RFC Replication factor C RLF2 Rap1 protein localization factor RNAPII Ribonucleic acid polymerase II RSC Remodel the structure of chromatin Rap1 Repressor/activator site binding protein 1 Rtt106 Regulator of Ty1 transposition 106 Rtt109 Regulator of Ty1 transposition 109 SBF SCB-binding factor xiii SH2 Src homology 2 SPT Suppressor of Ty STE11 Sterile 11 SUC2 Sucrose 2 SWI/SNF Switch/sucrose non-fermentable TBP TATA-binding protein UAS Upstream activating sequence mRNA Messenger RNA xiv Acknowledgements I would like to offer my genuine appreciation to my supervisor Dr. LeAnn Howe, for her consistent support in both academic and mental aspects during my degree. Without her I would not be able to surpass the many difficulties encountered during my study. Secondly, I would also thank my supervisory committee members, Dr. Sheila Teves and Dr. Annie Ciernia, for their valuable feedbacks and suggestions guiding my research. Thirdly, special appreciation to Stephanie Jung, who offered a great lot of help in generating data for the project. Furthermore, to my colleagues and friends Maggie Ling, Josh Brown and Jonah Lu, for their kind help and support in the lab. Lastly, I would like to thank the granting institutes, the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada for all the funding. xv Dedication To my Parents 1 Chapter 1: Introduction 1.1 The Nucleosome landscape is crucial for transcription regulation Genetic information is compacted in a nucleoprotein complex named chromatin, which consists of regularly distributed nucleosomes often illustrated as a “beads-on-a-string” structure (1). Two copies of each core histone proteins H2A, H2B, H3 and H4, along with 147 base pairs of DNA, constitute one nucleosome (Luger et al., 1997). This compaction restricts the accessibility of DNA by various factors, notably RNAPII and transcription factors (Zhu et al., 2018; Teves et al., 2014; Morse, 2003). Sequences such as promoters and enhancers that require frequent interaction with transcription factors are often located in nucleosome-depleted regions (NDRs), as opposed to sequences in gene bodies which are tightly wrapped around nucleosomes (Ichikawa et al., 2016; You et al., 2011; Bal et al., 2011; Müller and Tora, 2014; Hartley and Madhani, 2009). Therefore, maintenance of correct nucleosomal patterns is crucial for transcription activation at correct regions. The perturbation caused by RNAPII progression through nucleosomes is faithfully reversed following transcription, owing to the cohesive contribution made by various histone chaperones (Gurova et al., 2018; Tanae et al., 2012; Viktorovskaya et al., 2021; Avvakumov et al., 2011; Jeronimo et al., 2019). This prevents disturbance of nucleosome integrity and any consequential alteration of transcription initiation sites. (DeGennaro et al., 2013; Han and Grunstein, 1988; Almer et al., 1986). 1.1.1 Nucleosomes pose barriers to transcription factors and the transcription machinery Despite the importance of nucleosomes in maintaining organized DNA compaction, they represent a major pathway for regulating transcription initiation. In vitro studies demonstrate that transcription factors bind to nucleosomal DNA at a much lower rate compared to naked DNA (Owen-2 Hughes and Workman, 1994). But the initial in vivo evidence suggesting nucleosome packaging may regulate transcription factors binding came from the observed alteration of nucleosome positions upon gene activation. PHO5 encodes an acid phosphatase and under gene repression induced by high phosphate conditions, promoter nucleosomes are positioned to overlap the regulatory elements, the TATA box and two upstream activating sequences (UAS) (Almer et al., 1986; Vogel and Hinnen, 1990; Almer and Hörz, 1986; Svaren and Hörz, 1995). During transcription activation, Pho4 binding to UAS1 is required for nucleosome removal from UAS2. Following the deletion of UAS1, nucleosomes are detained at UAS2 even under activating conditions, which precludes the transcription factor Pho4 from binding to UAS2 to activate transcription. Loss of four promoter nucleosomes allows PHO5 activation: Pho4 gains access to DNA and binds the two UASs in the active promoter (Almer et al., 1986; Venter et al., 1994; Zhou and O'Shea, 2011). Accompanying PHO5 gene repression, nucleosomes are quickly redeposited, accompanied by loss of Pho4 and the transcription machinery (Adkins and Tyler, 2006). Similarly, differences in the bound sites of the transcription factor Leu3 in naked and nuclear DNA are mostly accounted for by DNA-nucleosome interactions. Leu3 binding sites in vitro and in vivo are overlapping but distinct sets, in which some identified binding sites in naked DNA lack Leu3 association in a chromatin context due to restrictions provided by nucleosomes (Liu et al., 2006). Leu3 binding is not the cause of nucleosome depletion in bound regions, since nucleosome occupancy over Leu3 binding sites is not affected by Leu3 overexpression or a mutation that disables Leu3 binding. In summary, nucleosomes pose a hindrance to the protein-DNA interactions that are crucial for transcription initiation. Nucleosome positioning is not random but well patterned, often with low nucleosome density over regulatory elements, granting access to transcription factors (Lee et al., 2007; Yuan et al., 2005; Mavrich et al., 2008). It was found that gene expression correlates negatively with nucleosome occupancy at promoter regions: constitutive genes tend to contain prominent 3 nucleosome-depleted regions (NDRs), while the promoters of repressed genes are occupied by nucleosomes (Lee et al., 2007). NDRs are sites for the initial binding of transcription factors, as well as transcription machinery assembly. The formation of the NDR is partially directed by DNA sequences, with long stretches of poly(dA:dT) or poly(dG:dC) elements favoring nucleosome exclusion and capable of stimulating transcription activation (Anderson and Widom, 2001; Iyer and Struhl, 1995; Morohashi et al., 2006). Additionally, the NDRs at the promoters of constitutive genes are due to the activity of many factors, including the constitutively available pioneer factors and the only essential yeast remodeler, RSC (Badis et al., 2008). Pioneer factors are a special class of transcription factors that are capable of binding nucleosome-covered sites and opening chromatin by out-competing histones for DNA binding (Ganapathi et al., 2011; Peterson and Workman, 2000; Yan et al., 2018). For example, Abf1 and Rap1 were reported to prevent nucleosome positioning around their binding sites, and contribute to the activation of genes including HIS4, ADE5/7 and HIS7 (Yarragudi et al., 2004). The RSC (remodel the structure of chromatin) complex can be recruited by pioneer factors or bind on its own, shifting nucleosome positions or even evicting them in an ATP-dependent manner (Chaban et al., 2008; Yague-Sanz et al., 2017). Mutation in one of its components, Rsc3, results in a dramatic increase in nucleosome occupancy at certain promoters (Badis et al., 2008). At regulated genes, transcription factors tend to bind only in activating conditions when histones are evicted by chaperones. A different remodeler, SWI/SNF, is then recruited to the scene. The SWI/SNF family of remodelers are highly conserved in eukaryotic species and are responsible for the initial eviction of nucleosomes over some promoters (Jaskelioff et al., 2000). This is crucial for transcription initiation of inducible or stress-responsive genes, as their promoters are not often in nucleosome-depleted regions (Ioshikhes et al., 2006). For example, Pho4 recruits SWI/SNF that rapidly removes promoter 4 nucleosomes to activate PHO5 (Ferreira et al., 2009). This dynamic nature of DNA-nucleosome interactions with remodelers allows for transcription factors to access DNA with flexibility as a crucial part of transcription regulation. 1.1.2 Histone chaperones both facilitate and prevent histone turnover during transcription Both transcription initiation and elongation can disrupt the intact nucleosome, potentially leading to histone loss and subsequent replacement with newly synthesized histones or histone variants in a process known as histone turnover. Histone chaperones are a class of proteins responsible for binding histones and regulating nucleosome dynamics by either disassembling or reassembling them. During gene activation and repression, chaperones can facilitate the eviction of histones from DNA and deposit new ones for nucleosome assembly respectively. Histone chaperones can promote histone turnover by replacing histones with new ones or prevent turnover by retaining the original histones during nucleosome disruption. A study utilizing differently-tagged histone H3 to measure its turnover rate in G1-arrested cells found that despite a high turnover rate in the coding regions of highly expressed genes, promoters are the primary place for rapid histone turnover. This is possibly to expose promoter sequences to various transcription initiation factors as well as to erase all activation-associated chromatin marks for rapid transcription repression to ensure that reinitiation only occurs in the presence of activating signals (Dion et al., 2007). Histone turnover at promoters is facilitated by various histone chaperones. For example, Asf1, a histone H3/H4 chaperone, was found to facilitate PHO5 and PHO8 activation by inducing histone eviction at their promoters, followed by histone deposition during gene repression (Adkins et al., 2004; Korber et al., 2006) (Fig 1.1). The FACT 5 Figure 1.1 Histone chaperones involved in transcription. Cited from Avvakumov et al., 2011. Over the promoter region, Asf1 facilitates the removal of H3-H4 dimers, while the HIRA complex serves in a repressive pathway. In the coding region, histones displaced by the passage of RNAPII are collected by FACT and Spt6 and redeposited onto DNA, while Asf1 functions in the placement of newly synthesized histones. 6 complex facilitates nucleosome removal upon activation of several inducible promoters including PHO5 and HO, while its role in histone deposition is suggested by FACT depletion leading to increased transcription of many other genes (Ransom et al., 2009; Takahata et al., 2009; McCullough et al., 2019; Malone et al., 1991; Yan et al., 2018) (Fig. 1.1). In contrast, the HIR (histone regulatory) complex is primarily responsible for transcription repression by depositing histones in promoter nucleosomes, and its mutation leads to derepression of histone genes outside of S phase (Prochasson et al., 2005). Similarly, Rtt106 is also recruited to the promoters of the HTA1-HTB1 histone genes in the repressive pathway to regulate histone expression (Fillingham et al., 2009). Histone turnover over gene coding regions is less profound, and this is important to prevent the transcription initiation machinery from accessing the underlying DNA, as well as to maintain the many histone post-translational modifications (PTMs) over these regions. Histone PTMs, including methylation, acetylation, phosphorylation, and ubiquitination on various residues of core histones, serve in many gene regulatory processes such as establishing the border between heterochromatin and euchromatin, as well as regulating transcription through interactions with an immense network of regulatory factors (Millán-Zambrano et al., 2022). As these marks are absent from newly synthesized histones, they can be used to study histone turnover and the chaperones involved in this process. One study by Jeronimo et al. found that histone modifications are scrambled in cells depleted of FACT or Spt6, both of which are essential histone chaperones involved in transcription elongation (Jeronimo et al., 2019). This defect can be alleviated by inhibiting transcription, suggesting that FACT and Spt6 are responsible for the retention of original histones during RNAPII-induced nucleosome disruption, possibly by acting as “sinks” for evicted histones before depositing them back following polymerase passage (Kato et al., 2013; Holla et 7 al., 2020; Svensson et al., 2015). Spt5, another transcription elongation factor, was also suggested to act similarly in binding histones and preventing turnover (Evrin et al., 2022). In the absence of FACT or Spt6, Asf1 and HIR direct histone turnover by binding the evicted histones and re-incorporating them to random places or facilitating nucleosome assembly with newly synthesized histones. 1.1.3 Nucleosome assembly during replication DNA replication also induces nucleosome disruption and requires precise chromatin restoration. The process of strand separation implies that histones must be fully removed from the parent strand and redeposited on the nascent strands, while maintaining histone PTM patterns (Alabert and Groth, 2012). During nucleosome reassembly, the first step is the deposition of the H3/H4 tetramer. Although chromatin assembly on nascent DNA uses a combination of old and newly synthesized histones, studies have shown that H3/H4 tetramers do not contain a mixture of old and new H3/H4 dimers, while such mixing can exist between H3/H4 tetramers and H2A/H2B dimers (Leffak et al., 1977; Russev and Hancock, 1981). Besides its role in transcription, Asf1 also serves as a histone chaperone that binds newly synthesized H3/H4 dimers before transferring them to CAF-1 and Rtt106 for tetramer assembly and deposition onto DNA (English et al., 2005; Schulz and Tyler, 2006; Mattiroli et al., 2017; Fazly et al., 2012) (Fig. 1.2). Unlike Rtt106 that binds one H3/H4 tetramer directly, two copies of CAF-1 are needed to position two H3/H4 dimers in an intermediate form that favours oligomerization before depositing the formed tetramer onto DNA (Mattiroli et al., 2017; Verreault et al., 1996). In contrast to yeast cells where the absence of CAF-1 does not affect cell proliferation, depletion of CAF-1 in human cells leads to nucleosome assembly defects that result in cells accumulating in S-phase and reduced cell viability (Nabatiyan 8 1.2. Nucleosome assembly during replication. Cited from Hammond et al., 2017. Chromatin assembly on nascent DNA randomly uses old and new histones. FACT engages with the MCM helicase to retain parental histones and deposit them upstream of the replication fork. Asf1 escorts new H3-H4 dimers to CAF-1 or Rtt106 (not shown) for tetramer assembly and deposition onto DNA. A mixture of old and new H2A-H2B dimer is assembled last on either side of the H3-H4 tetramer to form complete nucleosomes. 9 and Krude, 2004). The deposition of parental H3/H4 tetramers is less understood. It is proposed that Mcm2, a component of the replicative helicase, can engage in the retention of parental histones before escorting them to other histone chaperones such as Asf1 or FACT for deposition (Foltman et al., 2013; Richet et al., 2015; Tan et al., 2006) (Fig. 1.2). Following H3/H4 tetramer deposition with either new or recycled histones, H2A/H2B dimers are incorporated on either side of the tetramer through the activities of Nap1 or FACT (Formosa and Winston, 2020; Miyaji-Yamaguchi et al., 2003). A recent study also found a role for Spt6, a transcription elongation factor, in assembling nascent chromatin as it interacts with Mcm2/4, and depletion of Spt6 is associated with replication impairment and increased sensitivity to replication stress (Miller and Winston, 2022). All histone chaperones described in this section that are related to this study are discussed in further detail in later sections. 1.2 Cryptic transcription As mentioned previously, transcription initiates with the recognition of promoter sequences by activators and transcription factors. Due to the permutation of nucleotides in the genome, sequences that resemble a promoter, termed cryptic promoters, can exist in intragenic regions and potentially attract transcription factors when chromatin is disrupted. When transcription is initiated from a cryptic promoter, it is referred to as cryptic transcription. 1.2.1 Characteristics of cryptic promoters Besides the nucleosome-depleted regions typically found at promoter regions, loss of pathways required for chromatin integrity can potentially lead to histone displacement in gene bodies. The discovery of cryptic promoters arose from the observation that depletion of Spt6 10 induces global nucleosome loss and aberrant transcription initiation from within coding regions (DeGennaro et al., 2013; Kaplan et al., 2003; Uwimana et al., 2017; Doris et al., 2018). At these initiation sites, there is an observed depletion of DNA binding motifs for Abf1 and Reb1, two factors commonly required for NDR formation at canonical promoters, explaining why cryptic promoters do not occupy defined NDRs in wild-type cells. Nevertheless, they share many common characteristics with canonical promoters, including similarly organized nucleosome arrays downstream and transcription factor binding sites upstream from the transcription start sites (Doris et al., 2018; Wei et al., 2019). Sequence-wise, cryptic promoters are enriched in poly(AT) motifs and TATA elements that perfectly match the consensus sequence for TBP binding. By conservative estimate, there are over 1000 cryptic promoters present in the genome of S. cerevisiae. Some well-characterized ones are located in the gene bodies of FLO8, STE11, and PCA1. Cryptic promoters, which often generate truncated mRNA products with altered open reading frames, can compete with canonical promoters for transcription factors and reduce the level of productive mRNA (Kaplan et al., 2003). Cryptic transcription can also be bi-directional starting from the anti-sense strand, interfering with canonical transcription (Hainer et al., 2011). Besides transcription and replication, yeast aging also leads to a loss of nucleosome occupancy and positioning with sequential activation of cryptic promoters (Hu et al., 2014). Additionally, cell cycle acceleration makes the chromatin of cancer cells especially unstable, and in turn opens up cryptic promoters that interfere with the transcription of tumor-suppressor genes (Terrinoni et al., 2001). 11 1.2.2 Cryptic transcription suppressing factors Proper restoration of chromatin integrity, and thus suppression of cryptic transcription, following transcription and DNA replication is mediated by many factors, and key among these were identified through a Suppressor of Ty (SPT) screen. Insertion of transposable Ty elements or just the long terminal δ repeats at the 5’ non-coding region or within a gene often leads to altered gene expression due to the additional cis-acting sequence elements provided (Winston et al., 1984; Simchen et al., 1984). Insertions of δ elements adjacent to HIS4 or LYS2 that disrupted expression of the normal gene products were used to screen for spt mutants that reversed this effect (Fassler and Winston, 1988). Besides these spt mutants, high copy expression of some factors also suppresses Ty insertion mutations. Among these, SPT16 was identified as both a mutant and high-copy suppressor (Malone et al., 1991). SPT11 and SPT12, which were later identified to encode histones H2A and H2B, suppress the effects of δ insertions when either mutated or present on a high-copy-number plasmid (Clark-Adams et al., 1988). It was further suggested that altering the ratios of H2A/H2B to H3/H4 can suppress the effect of δ insertions and thus proper histone stoichiometry is important for proper chromatin structure. Overall, the SPT screen discovered the following key regulators of transcription and chromatin structure: 1) Spt1 (Hir2), Spt2, Spt4, Spt5, Spt6 and Spt16 as histone chaperones or transcription elongation factors; 2) Spt10 and Spt21 as histone regulatory proteins; 3) Spt11 (histone H2A) and Spt12 (histone H2B); 4) Spt13 (Gal11) as a Mediator component (Yamaguchi et al., 2001). In a subsequent screen led by the Winston lab, additional factors responsible for suppressing cryptic transcription initiating from an intragenic promoter within FLO8 were identified and classified (Cheung et al., 2008). Besides the SPT proteins, they also identified 12 proteins involved in chromatin remodeling, histone deacetylation, as well as components of the Mediator complex. As many factors identified were histone chaperones acting during transcription, activation of the FLO8 cryptic promoter is often dependent on FLO8 transcription, or in other words, nucleosome disruption induced by RNAPII passage. However, in certain mutants, cryptic transcription was also observed in the absence of upstream transcription, suggesting the participation of those proteins in maintaining chromatin structure during other processes, such as replication. 1.2.2.1 FACT FACT (FAcilitates Chromatin Transcription) was first identified and named for its ability to aid RNAPII in overcoming nucleosome-induced arrest during transcription elongation in vitro (Orphanides et al., 1998). In yeast, FACT is a heterodimeric complex composed of Spt16 and Pob3 (Pol one binder), which are both essential for cell viability. FACT regulates nucleosome dynamics during DNA-related cellular processes, as it interacts with RNA polymerase-disrupted nucleosomes during transcription elongation as well as with the MCM helicase and DNA polymerase α during replication (Tan et al., 2006; Martin et al., 2018). Global transcription derepression and activation of cryptic transcription in FACT mutant cells also indicate a role for FACT in regulating transcription initiation through the establishment of proper nucleosome architecture (Feng et al., 2016; McCullough et al., 2019). FACT contains multiple contact sites with core histones and DNA that allows it to retain histones and reestablish histone-DNA contacts following RNAPII-induced nucleosome disruption (Wang et al., 2018; Stuwe et al., 2008; VanDemark et al., 2008; Schwabish and Struhl, 2004). Cryo-EM analysis captured FACT binding partially unwrapped histone-DNA intermediates, suggesting FACT is able to tether components 13 together to prevent histone loss and enhance assembly (Ehara et al., 2022; Farnung et al., 2021; Wang et al., 2018; Jamai et al., 2019). Consistent with this, FACT depletion leads to the scrambling of histone variant and histone PTM localizations, suggesting FACT is responsible for preventing histone dispersion (Jeronimo et al., 2019; Jeronimo et al., 2015). FACT also plays a role in chromatin maintenance and nucleosome assembly following replication, which is as important for cell survival. Physical interactions were found between Spt16 and the replicative helicase subunits Mcm2/4, and this interaction can promote the firing of replication forks as well as chromatin reestablishment of newly replicated DNA (Tan et al., 2006; Tan et al., 2010; Foltman et al., 2013). In vitro analysis showed that despite having no effect on the replication rate on naked DNA, FACT functions in a bilateral manner as it promotes not only replication through a chromatin template, but also rapid nucleosome assembly behind the replisome (Kurat et al., 2017). In addition to Pob3 interacting with polymerase α as suggested by its name, mutations in POB3 were also identified to have genetic interactions with multiple replication factors. For example, a dual mutation of POB3 and the S phase checkpoint gene MEC1 causes rapid cell death (Wittmeyer and Formosa, 1997; Schlesinger and Formosa, 2000). The increased sensitivity if a pob3 mutant to the replication inhibitor hydroxyurea, also suggests that Pob3 plays an important role in S phase progression. Additionally, FACT also cooperates with other histone chaperones such as CAF-1 and Rtt106 in depositing newly synthesized H3/H4 onto DNA (Yang et al., 2016). Taken together, FACT is an important histone chaperone that functions during both transcription and replication. 14 1.2.2.2 Spt6 Identified through the SPT screen, an SPT6 mutant was found to suppress δ insertion mutations in HIS4 and LYS2 (Winston et al., 1984; Clark-Adams and Winston, 1987). Spt6 is an essential transcription elongation factor and histone chaperone that contains an SH2 (Src homology 2) domain that recognizes and interacts with RNAPII through its phosphorylated C-terminal tail (Diebold et al., 2010; Sdano et al., 2017). Additionally, physical interactions were found between Spt6 and H3/H4 that allow it to bind histones and reassemble nucleosomes following RNAPII progression (Bortvin and Winston, 1996; Li et al., 2022; McCullough et al., 2015). Spt6 inactivation leads to failure in promoter nucleosome reassembly following PHO5 repression, resulting in continued transcription activation despite the displacement of activators Pho4 and Pho2 (Adkins and Tyler, 2006). As Spt6 is also essential in maintaining chromatin structure within coding regions, Spt6 depletion leads to reduced histone levels and histone PTM scrambling similar to mutations in FACT subunits (Jeronimo et al., 2019; Kato et al., 2013). Additionally, cryptic transcription in spt6-1004 temperature-sensitive cells was detected from intragenic promoters within multiple genes including FLO8, RAD18 and STE11 (Kaplan et al., 2003; Cheung et al., 2008; Hainer et al., 2011). While previous works demonstrated that SPT6 mutant cells exhibit phenotypes indicative of DNA replication defects including an increase in recombination frequency and higher sensitivity to replication inhibitors, it is only recently that Spt6 was confirmed to have a direct role in replication (Viktorovskaya et al., 2021; Malagón and Aguilera, 1996; Malagón and Aguilera, 2001). Physical interactions exist between Spt6 and Mcm2/4 that are resistant to nuclease, indicative of direct, not DNA-mediated interaction (Miller and Winston, 2022). In Spt6-depleted cells, incorporation of the thymidine analog BrdU into newly synthesized DNA was hindered 15 along with an observed delay in DNA synthesis. Although not fully clear, the role of Spt6 in DNA replication is likely similar as in transcription to reestablish chromatin stability as well as to mediate parental histone reincorporation by binding and redepositing histones in the wake of replication forks. 1.2.2.3 Spt5 Like Spt6, Spt5 is a conserved histone chaperone and eukaryotic elongation factor that was first identified in yeast as a suppressor of certain δ insertion mutations in the 5’ non-coding region of HIS4 (Winston et al., 1984). Often in a complex with Spt4, termed DSIF, Spt5 interacts with elongating RNAPII across the genome through the RNAPII clamp domain in a manner that enhances transcription processivity (Martinez-Rucobo et al., 2011; Hirtreiter et al., 2010; Hartzog et al., 1998). Direct interactions were also identified between Spt5 and the non-coding DNA strand as well as nascent mRNA, with mutations that impair these interactions resulting in aberrant transcription arrest in vitro (Crickard et al., 2016; Meyer et al., 2015). Additionally, Spt5 also signals for recruitment of other factors including mRNA capping enzyme and the PAF1 complex, the latter of which is important for RNAPII elongation (Schneider et al., 2010; Wen and Shatkin, 1999; Wier et al., 2013). As a histone chaperone, Spt5 contains a conserved histone binding motif for H3/H4 that is essential for cell viability (Evrin et al., 2022). In cells depleted of Spt5, a reduction in global nucleosome occupancy levels was observed, indicating a role of Spt5 in capturing and retaining histones during transcription. The positive correlation between histone loss and gene expression level in Spt5-defective cells indicates Spt5 function is coupled to active transcription, possibly by tethering histones to DNA as the transcription machinery progresses along. 16 1.2.2.4 Spt2 Aside from suppressing Ty/δ insertion mutations, SPT2 (SIN1) mutation leads to activation of HO transcription in the absence of Swi1, a subunit of the SWI/SNF remodeler (Kruger and Herskowitz, 1991), suggesting Spt2 functions in nucleosome-mediated gene repression. Spt2 shares the properties of an HMG protein, as in amino acid composition and function in binding DNA without sequence specificity in vitro. Thus, it was first considered a non-histone component of chromatin with linkage to transcription. Later, structural analysis of human Spt2 showed that it is able to bind an H3/H4 tetramer, exhibiting the properties of a histone chaperone (Chen et al., 2015). Mutation in the Spt2 C-terminal histone contact site leads to a higher rate of H3/H4 exchange over coding regions in vivo, suggesting Spt2 plays a role in histone retention during transcription (Osakabe et al., 2013; Chen et al., 2015). Additionally, in a spt2 mutant, FLO8 transcription is required for cryptic transcription to initiate from the FLO8 internal cryptic promoter, emphasizing its role in maintaining chromatin stability and suppressing cryptic transcription over transcribed regions. 1.2.2.5 Spt10/21 Again, identified from the SPT screen, mutations in SPT10 or SPT21 suppress the URA3 silencing induced by Ty1 insertion, resulting in a Ura+ phenotype (Natsoulis et al., 1991). In addition, these mutations were also found to cause inappropriate transcription at regulated genes, manifested as increased transcription in the repressed state or decreased transcription during activation. Specifically, these mutations were reported to cause transcription defects at a subset of histone genes, SUC2 transcription in the absence of proper upstream activating sequences, and altered chromatin structure (Prelich and Winston, 1993). Spt10/21 is known to promote the 17 production of histones encoded by the HTA2/HTB2 and HHF2/HHT2 genes in S phase, thus many gene regulation defects observed in the spt10/21 mutants could be due to global indirect effects caused by insufficient histones (Dollard et al., 1994). Additionally, Spt21 is required for Spt10 recruitment to histone promoters, and their physical interaction is crucial for their function (Hess et al., 2004). Despite the seemingly overlapping functions, spt10Δ mutants do exhibit a greater degree of transcription alteration and growth defects compared to spt21Δ mutants. Since neither Spt10 nor Spt21 contain a DNA-binding motif, it is suggested that they are recruited to histone promoters by interactions with other transcription activation complexes such as MBF (MCB-binding factor) and SBF (SCB-binding factor) (Hess and Winston, 2005). 1.2.2.6 Asf1 Asf1, or anti-silencing factor 1, was identified and named as a high-copy suppressor of telomeric silencing in Saccharomyces cerevisiae, but was later recognized to be a conserved H3/H4 histone chaperone across eukaryotes (Singer et al., 1998). Asf1 has broad functions in most DNA-related cellular processes including transcription, DNA replication, and repair. During transcription initiation, Asf1 aids in histone eviction from the PHO5 and PHO8 promoters, with potential functional interactions with the SWI/SNF remodeling complex, as double mutation in asf1 and snf2 results in a synthetic defect in PHO5 induction kinetics (Korber et al., 2006). Asf1 is also responsible for the replication-independent turnover of the histone H3 flanking the NDRs of promoters of inducible genes, which is possibly linked to their rapid induction and repression as required (Rufiange et al., 2007). Later evidence reaffirmed a role for Asf1 in nucleosome disassembly during transcription elongation, as Asf1 associates with active RNAPII throughout elongation and asf1 mutants exhibit increased H3 occupancy across coding regions. Structural 18 analysis showed that Asf1 binds H3 at the H3/H4 dimerization surface, consequently blocking H3/H4 tetramer formation. This observation suggested a potential “strand-capture” mechanism in which the C-terminal tail of H4, which adopts a dramatic conformational change upon Asf1 binding, serves as a handle for H3/H4 tetramer disassembly and reassembly (English et al., 2006). Furthermore, activation of cryptic promoters in the absence of Asf1 also suggested Asf1 conversely functions in reassembling nucleosomes following RNAPII passage, like FACT (Cheung et al., 2008; Schwabish and Struhl, 2006). Active FLO8 transcription, although not required, increases cryptic transcription from the internal cryptic promoter in an asf1 mutant, suggesting its function is at least partially related to transcription (Cheung et al., 2008). Cooperativity also exists between Asf1 and other factors, including the HIR complex and Rtt109, to enhance nucleosome assembly efficiency in a replication-independent manner (Green et al., 2005; Driscoll et al., 2007). Co-purifying with Drosophila RCAF (replication-coupling assembly factor) complex, Asf1 also takes a part in chromatin assembly during replication (Tyler et al., 1999). Cells lacking Asf1 showed increased sensitivity to replication-inhibiting drugs including hydroxyurea and camptothecin (Ramey et al., 2004). Additionally, Asf1 was reported to preserve several replication factors, including replication factor C (RFC), PCNA, and DNA polymerase ε at stalled replication forks, maintaining fork integrity (Franco et al., 2005). With direct interaction with the replisome, Asf1 was proposed to capture displaced H3/H4 heterodimers and pass them along to CAF-1 or Rtt106 for tetramer formation and deposition onto DNA (Mattiroli et al., 2017; Fazly et al., 2012). 19 1.2.2.7 CAF-1 In Saccharomyces cerevisiae, the chromatin assembly factor 1 (CAF-1) complex is a major replication-coupled H3/H4 chaperone and consists of three subunits known as Cac1 (Rlf2), Cac2 and Cac3 (Msi1). CAF-1 was first isolated from human cells and named based on its function in reassembling histones on SV40 plasmids undergoing replication (Stillman, 1986; Smith and Stillman, 1989). Differing from the transcription-coupled histone chaperones, which are recruited by components of the transcription machinery, CAF-1 activity is triggered by cooperating proteins such as PCNA or the specific state of supercoiled DNA in the wake of DNA polymerase, and was found to colocalize with replication foci (Kaufman, 1996; Shibahara and Stillman, 1999; Krude, 1995). Through structural analysis, it was discovered that orthologs of Cac3 in human and fly cells bind to the first helix of histone H4, which is incompatible with the normal organization of a H3/H4 dimer, indicating CAF-1 binding induces allosteric changes in histone structure, possibly in a way that favours substantial histone deposition (Song et al., 2008; Murzina et al., 2008). As mentioned previously, CAF-1 also interacts with Asf1 to receive H3/H4 dimers during the assembly of newly replicated chromatin. Although depletion of CAF-1 alone is not lethal to yeast cells, dual mutation of Asf1 and CAF-1 leads to severe gene silencing and cell cycle progression defects, suggesting they function redundantly in an essential pathway of chromatin reassembly during replication (Tyler et al., 1999; Enomoto et al., 1997; Kaufman et al., 1997; Sharp et al., 2001). 1.2.2.8 Rtt106 Rtt106 was first identified to regulate Ty1 transposition, as the inactivation of Rtt106 leads to an accumulation of Ty1 cDNA (Scholes et al., 2001). Although Rtt106 receives H3/H4 dimers from Asf1, similar to CAF-1, Rtt106 is able to bind H3/H4 tetramers before depositing them onto 20 DNA, in a mechanism differing from that of CAF-1, which bind to H3/H4 heterodimers before oligomerization (Fazly et al., 2012). Also distinct from CAF-1, Rtt106 was found to localize to transcribed regions of chromatin and function independently of replication to promote new histone deposition over coding regions of active genes. Genetic interactions were found between RTT106 and genes encoding several transcription elongation factors, including Spt6, DSIF and TFIIS. Finally, like other transcription-coupled histone chaperones, depletion of Rtt106 leads to cryptic transcription originating from intragenic cryptic promoters (Imbeault et al., 2008). While all this evidence indicates a role of Rtt106 in transcription elongation and chromatin stability, it was also later characterized as another histone chaperone responsible for chromatin assembly on newly replicated DNA. Rtt106 copurifies with histone H3 and H4 both in vitro and in vivo, while also promoting nucleosome assembly on a DNA template in vitro (Huang et al., 2005). 1.2.3 Classical and novel methods for detecting cryptic transcription Early studies used Northern blots to detect the smaller transcripts indicative of cryptic transcription. Although this approach allowed for a direct comparison of the relative abundance of canonical versus cryptic transcripts, it is time-consuming and laborious. Moreover, as a whole cell population is analyzed, no information on heterogeneity between individual cells could be obtained. A less laborious, high-throughput approach was developed by the Winston lab, which employed a HIS3 reporter inserted downstream of the FLO8 cryptic promoter, allowing the presence of cryptic transcription activation to be assessed by growth on media lacking histidine (Cheung et al., 2008). Despite the ease of this system, it still lacked the capability of measuring cryptic transcription in single cells. Further, as multiple rounds of proliferation are required for 21 plate growth, mutations or experimental approaches that require a cell cycle arrest cannot be implemented. 1.3 Thesis objective In this study, we aimed to develop a new reporter system that allows for rapid and real-time measurements of cryptic transcription in single cells, allowing us to better characterize the roles played by suppressors of cryptic transcription during transcription and DNA replication. To this end, we inserted a fluorescence reporter downstream of the FLO8 intragenic cryptic promoter and confirmed the successful detection of cryptic transcription following the depletion of FACT. Next, we used the assay to test whether passage through S phase is required for cryptic transcription following the depletion of replication-coupled factors. This work revealed novel insight into the roles played by histone chaperones in stabilizing chromatin during transcription and replication-induced nucleosome disruption and allowed us to further dissect the functions of certain factors that are utilized in both processes. 22 Chapter 2: Materials and Methods 2.1 Yeast Strain All strains in this study, listed in Table 1, were isogenic to S288C. Genetic manipulations were performed using standard protocols, except for strains with spt16-197, spt6-1004 and null alleles, which were genetically edited using a standard CRISPR protocol (DiCarlo et al., 2013). Strain verification was performed using standard PCR protocol from either purified genomic DNA or colonies (Kushnirov, 2000; Bonnet et al., 2013). Yeast strains were grown at 30°C in YPD (yeast extract, bacto peptone, 2% dextrose) unless otherwise specified. Table 1. Yeast Strains used in this study Strain Parent Mating type Genotype yLH1086 yLH101 Mat a his3D200 leu2D1 lys2-128d ura3-52 trp1D63 FLO8::sfGFP-HpH yLH1088 yLH1086 Mat a his3D200 leu2D1 lys2-128d ura3-52 trp1D63 FLO8::sfGFP-HpH spt16-197 yLH2009 yLH975 Mat a his3D200 leu2D1 lys2-128d ura3-52 trp1D63 spt16G132D-HA6::HIS3 yLH975 yLH101 Mat a his3D200 leu2D1 lys2-128d ura3-52 trp1D63 SPT16-HA6::HIS3 23 yA002 yA001 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH yA003 yA002 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH SPT2-FRB::HISMX6 yA004 yA002 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH SPT21-FRB::HISMX6 yA005 yA002 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH SPT6-1004::HISMX6 yA006 yA002 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH SPT5-FRB::HISMX6 yA007 yA002 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH SPT10-FRB::HISMX6 24 yA008 yA002 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH ASF1-FRB::HISMX6 yA009 yA002 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH RTT106-FRB::HISMX6 yA010 yA002 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH RLF2-FRB::HISMX6 yA011 yA002 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH CAC2-FRB::HISMX6 yA102 yA002 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH GAL1pr-mCherry:URA3 yA103 yA003 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-25 2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH SPT2-FRB::HISMX6 GAL1pr-mCherry:URA3 yA104 yA004 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH SPT21-FRB::HISMX6 GAL1pr-mCherry:URA3 yA105 yA005 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH SPT6-1004::HISMX6 GAL1pr-mCherry:URA3 yA106 yA006 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH SPT5-FRB::HISMX6 GAL1pr-mCherry:URA3 yA107 yA007 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH SPT10-FRB::HISMX6 GAL1pr-mCherry:URA3 yA108 yA008 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH ASF1-FRB::HISMX6 GAL1pr-mCherry:URA3 26 yA200 yA002 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH asf1Δ yA201 yA005 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH SPT6-1004::HISMX6 asf1Δ yA202 yA006 Mat a tor1-1 fpr1::loxP-LEU2-loxP ade 2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ RPL13A-2xFKB12::loxP NAT:GAL1pr-FLO8::sfGFP-HpH SPT5-FRB::HISMX6 asf1Δ yLH2036 yEG100 Mat a his3D200 leu2D1 lys2-128d ura3-52 trp1D63 NAT:GAL1pr-FLO8::sfGFP-HpH LEU2::PGK1pr-mCherry yLH2037 yEG100 Mat a his3D200 leu2D1 lys2-128d ura3-52 trp1D63 NAT:GAL1pr-FLO8::sfGFP-HpH LEU2::PGK1pr-mCherry asf1Δ yLH2038 yEG100 Mat a his3D200 leu2D1 lys2-128d ura3-52 trp1D63 NAT:GAL1pr-FLO8::sfGFP-HpH LEU2::PGK1pr-mCherry cac2Δ 27 yLH2039 yEG100 Mat a his3D200 leu2D1 lys2-128d ura3-52 trp1D63 NAT:GAL1pr-FLO8::sfGFP-HpH LEU2::PGK1pr-mCherry rlf2Δ yLH2040 yEG100 Mat a his3D200 leu2D1 lys2-128d ura3-52 trp1D63 NAT:GAL1pr-FLO8::sfGFP-HpH LEU2::PGK1pr-mCherry rtt106Δ yLH2041 FY2173 Mat a his3D200 leu2D1 lys2-128D trp1D63 ura3-52 kanMX-GAL1pr-FLO8-HIS3 yLH2042 yLH2041 Mat a his3D200 leu2D1 lys2-128D trp1D63 ura3-52 kanMX-GAL1pr-FLO8-HIS3 asf1Δ yLH2043 yLH2041 Mat a his3D200 leu2D1 lys2-128D trp1D63 ura3-52 kanMX-GAL1pr-FLO8-HIS3 cac2Δ yLH2044 yLH2041 Mat a his3D200 leu2D1 lys2-128D trp1D63 ura3-52 kanMX-GAL1pr-FLO8-HIS3 rlf2Δ yLH2045 yLH2041 Mat a his3D200 leu2D1 lys2-128D trp1D63 ura3-52 kanMX-GAL1pr-FLO8-HIS3 rtt106Δ yLH101 FY602 Mat a his3D200 leu2D1 lys2-128d ura3-52 trp1D63 yEG001 yLH1086 Mat a his3D200 leu2D1 lys2-128d ura3-52 trp1D63 FLO8::sfGFP-HpH URA3::PGK1pr-mCherry yEG100 yEG001 Mat a his3D200 leu2D1 lys2-128d ura3-52 trp1D63 NAT:GAL1pr-FLO8::sfGFP-HpH LEU2::PGK1pr-mCherry 28 yEG110 yEG100 Mat a his3D200 leu2D1 lys2-128d ura3-52 trp1D63 NAT:GAL1pr-FLO8::sfGFP-HpH LEU2::PGK1pr-mCherry spt16G-197 2.2 Allele generation Nucleotide sequences are listed in Table 2. The FLO8::sfGFP-HpH allele was synthesized by amplifying the sfGFP-HpH cassette from pMaM60 (pFA6a-mCherry-sfGFP-hphNT1) (Zhang et al., 2019) with primers FLO8_S3 and FLO8_inTSS_S2, transforming it into yeast, and selecting for integration on YPD with hygromycin. Subsequently, a GAL1 promoter was inserted by transforming PCR products amplified from either pFA6a-NAT-pGAL1 or pFA6a-KANMX6-pGAL1. The FRB tags were integrated by transforming PCR products generated using pFA6a-FRB-HIS3MX6 as a template with F2/R1 primers specific to each gene. The GAL1pr-mCherry allele was synthesized by transforming PCR products using pRS406 GAL1pr-mCherry-PGK1t as template. Table 2. Nucleotide sequences Name Sequence FLO8_S3 CATTATCTTCAGTGACAGAGAGTTGCTTCGATCCAGAAAGTGGCAAGATTGCCGGAAAGAGGAGCAGGTGCTGGTGCTG FLO8_inTSS_S2 TTATTATGTTTCCTGTCATTAAGAGTTTTTATTTTTTATTATAATACTCAACACGTGACTATCGATGAATTCGAGCTCG SPT2_F2 GAGGAAGCTTGGTTAAAAAAGCATGAAGAGGAGAAGAGACGCCGTAAGAAGGGCATACGCCGGATCCCCGGGTTAATTAA 29 SPT2_R1 ACTCTCTACGACTTCATTAGTCATTTCACGTCCATATATCAAAACATATATCAATATTCCGAATTCGAGCTCGTTTAAAC SPT21_F2 GGAATAAGAGTAAGTCAGGTAGTAACAAGTCCCATCGGTGAATTTCAGTCAATAAAACACCGGATCCCCGGGTTAATTAA SPT21_R1 AGTATATATATATATATATATATATACATGCTGTGCTAGGAATAAGTTCATGTAATATTTGAATTCGAGCTCGTTTAAAC SPT10_F2 ATCTAATACATTTACAGAGCACAATTCTAATATTTATTATCGGATCCCCGGGTTAATTAA SPT10_R1 CTAGGGTTGGTGATGTGACCGTCTCTGGCAGAGTCGCAATGAATTCGAGCTCGTTTAAAC ASF1_F2 ATAGAAGGGGGGTCCACGGATATTGAATCCACTCCAAAGGATGCGGCACGTTCAACGAATCGGATCCCCGGGTTAATTAA ASF1_R1 ATACATTTTATAAAGTGTACCTCTCTTGCAGGTACCATTAATCTTATAACCCATAAATTCGAATTCGAGCTCGTTTAAAC RTT106_F2 CGATGAAGATGATGAGGATGGATCCGGAGTAGAATACGATCGGATCCCCGGGTTAATTAA RTT106_R1 CATATGCGTATTCATGCTATATTATAATATCGAATCTAAGGAATTCGAGCTCGTTTAAAC RLF2_F2 AAAGACGCACAAAACTGGGAGAATCTTCGAGCCAATGCAAATATGCCAACCCCGTCTTTGCGGATCCCCGGGTTAATTAA RLF2_R1 TGTATACCAATAAATAATCAGTTTATCTGTATGTTTCTATATACTAAAGATCCGTTCAAGGAATTCGAGCTCGTTTAAAC 30 SPT5_F2 TAAGTCAAACTATGGTGGTAACAGTACATGGGGAGGTCATCGGATCCCCGGGTTAATTAA SPT5_R1 TTGATTTCTTCTTGGGTGATATTGGTTCTCCTTTTGGTGAGAATTCGAGCTCGTTTAAAC 2.3 Immunoblots Protein levels for Spt16 and loading control Pgk1 were measured using a western blot as shown in Fig. S1. Cell pellets obtained from individual samples were washed with water and resuspended in 2M NaOH before incubating at room temperature for 15 minutes. Samples were prepared by adding 3xSDS solvent (180 mM Tris-HCl pH 6.8, 9% SDS, 27% glycerol, 10% 2-mercaptoethanol, 0.03% bromophenol blue), 5x electrophoresis buffer (9.4% glycerol, 1.5% Tris base, 5% SDS) and boiling for 5 minutes at 100°C. Proteins were run and separated via 8% SDS-PAGE before transferring to nitrocellulose blotting membrane (Life Science, lot no. A29466444) in 30V overnight. The membrane was blocked with 1% Bovine Serum Albumin (BSA) (Life Science) for 30 minutes and blotted with α-HA (Roche, ref no. 11583816001) or α-Pgk1 (Novex, part/lot no. 459250/J3282) primary antibodies at 4°C overnight. The membrane was washed 1x15 min and 2x5 min with PBST (4% NaCl, 0.1% KCl, 0.72% Na2HPO4, 0.12% KH2PO4, 1% Tween, pH 7.4) before blotting with anti-mouse secondary antibody (LI-COR, lot no. D10825-11) for 1 hour at room temperature, followed by another round of PBST wash. Visualization was performed using a BIO-RAD Image Lab instrument. 31 2.4 Flow cytometry Yeast strains were grown at 30°C in YPD (yeast extract, bacto peptone, 2% dextrose) unless otherwise specified. Cells in the exponential growth phase were resuspended in cold 1x PBS. Samples were measured using a Guava® easyCyte™ Flow Cytometer. Fluorescence was detected with 525/30 nm and 620/52 nm fluorochromes for sfGFP and mCherry, respectively. The mean fluorescence intensity, forward scatter, and side scatter of each individual yeast cell was recorded. Subsequent data analysis was performed in R Studio. Histograms were generated using 10,000 cells measured in each sample using three independent cultures as replicates. 2.5 Drug treatments Alpha factor (Genescript, catalog no. SC1208, sequence WHWLQLKPGQPMY) was dissolved in 0.1 M NaAc and added in a final concentration of 10 nM for two hours. Rapamycin (Bio Basic Canada Inc., lot no. E62010LR2T) was dissolved in DMSO (Life Science, lot no. 3177C021) and added in a final concentration of 1 μl/ml for two hours. DMSO was added in a final concentration of 1μl/ml for two hours. 2.6 Dilution plating Yeast cultures were grown to saturation and blotted on plates with 1x, 10x, 100x, 1000x and 10,000x dilutions. Images were taken after plates were incubated at 30°C for two days. 2.7 Statistical analysis Mean values of GFP signal from the three technical replicates were calculated for each treatment, and the three mean values were compared between two treatment groups using a non-parametric 32 one-tailed Mann-Whitney U test. The significance level was set to be 0.05. Any comparison that met this threshold (i.e. Ustat ≤ Ucrit) will be described as significant in the text. 33 Chapter 3: Results 3.1 Generation of a fluorescent reporter for cryptic transcription To rapidly track cryptic transcription in single cells, we followed a similar approach as Cheung et al., but inserted a fluorescent sfGFP reporter gene downstream of the cryptic promoter in the FLO8 gene (Fig. 3.1A). The site of insertion places the sfGFP ORF in a +1-frameshift relative to the FLO8 start codon and thus only transcripts originating from the cryptic promoter, and not the FLO8 promoter, are translated into a fluorescent gene product. To test our reporter system, we chose a well-characterized mutation in SPT16, which encodes a component of the FACT complex. Previous work has shown that in this mutant, spt16-197, Spt16 is rapidly degraded at the non-permissive temperature (37°C), resulting in genome-wide loss of nucleosome occupancy and positioning (Malone et al., 1991; Morillo-Huesca et al., 2010; Johnson et al., 2013). We confirmed protein degradation by immunoblot (Fig. 3.2A) and used flow cytometry to track the appearance of sfGFP signal at varying intervals following the temperature shift. While wild-type cells showed a minimal level of fluorescence (Fig. 3.1B), spt16-197 cells demonstrated a time-dependent increase in GFP signal (Fig. 3.1C). The spt16-197 mutant arrests in G1 at the non-permissive temperature. To ensure that the GFP signal was not due to cells spending increased time in G1, we treated wild-type cells carrying the sfGFP reporter with alpha factor to induce a similar arrest. Wildtype arrested cells produced an almost identical GFP signal compared to asynchronous cells (Fig. 3.2B). To rule out the possibility that the difference in cell sizes between WT and spt16-197 cell populations was driving the difference in GFP signal, we also compared results analyzed either from the whole population or a subpopulation containing only the smaller- 34 Figure 3.1. Development of a FLO8-sfGFP fluorescence reporter for cryptic transcription detection. A) Diagram of the FLO8:sfGFP reporter. The sfGFP ORF is inserted downstream of the cryptic promoter shown as the approximate position with a +1-framshift to the FLO8 start codon. The expected full-length and cryptic transcript, as well as the GFP phenotype, are indicated beneath the diagram. The FLO8 promoter was replaced with the GAL1 promoter for subsequent experiments. B) Histogram of GFP signal measured by flow cytometry in both WT and spt16-197 cells grown in non-permissive temperature (37°C) over a three-hour duration in rich media. Relative distribution over 10,000 cells was plotted against the GFP signal measured as the mean fluorescence intensity of each individual cell. 35 Figure 3.2. Confirmation of spt16-197 depletion and controlling for differences due to arrest or cell sizes. A) Immunoblot showing degradation of wildtype Spt16 and Spt16-197 tagged with 6xHA in a three-hour time interval (T0-T3). Pgk1 was used as a loading control to compare the overall protein level in each sample. B) Histograms comparing GFP signal between WT asynchronous and alpha factor arrested cells. C) Histograms of GFP signal measured by flow cytometry in both WT and spt16-197 cells grown in non-permissive temperature (37°C) over a three-hour duration. Out of the 10,000 cells measured, only smaller-sized cells (FSC<200, SSC<150) were used to generate this plot. 36 sized cells from each sample (Fig. 3.2C). As the overall trend was very similar between the two, we concluded that the effect of cell cycle arrest on the GFP signal can be taken out of consideration for further study. Furthermore, the level of sfGFP in wildtype cells was substantially less than that of spt16-197 cells indicating that our reporter can be used to detect chromatin disruption. 3.2 A fluorescent reporter of cryptic transcription can differentiate chromatin disruption due to transcription or DNA replication Transcription and DNA replication are two cellular processes that destabilize chromatin and evidence suggests that cells use dedicated factors to reestablish chromatin structure following each process. Using a regulatable GAL1pr-FLO8-HIS3 reporter Cheung et al. directly tested whether transcription from an upstream promoter was required to disrupt chromatin over the cryptic promoter in specific mutants. One mutant that showed cryptic transcription only in the presence of upstream transcription was a null allele of SPT2. To determine whether we could recapitulate this result using our system, we designed an experiment to compare the GFP signal between arrested and asynchronous cells with and without upstream transcription (Fig. 3.3A). We depleted Spt2 from the nucleus using anchor-away in cells grown in dextrose or galactose and performed flow cytometry for sfGFP. Figure 3.3B shows that sfGFP could be detected following Spt2 depletion (i.e. rapamycin treatment) from nuclei in cells grown in galactose, but not dextrose (Fig. 3.3B bottom panel, one-tailed Mann-Whitney U test, WT rapamycin vs. Spt2-FRB rapamycin). The elevation of GFP signal in Spt2-FRB strain treated with DMSO (Fig. 3.3B bottom panel, one-tailed Mann-Whitney U test, WT DMSO vs. Spt2-FRB DMSO) might be due to FRB tag interfering with Spt2 function, creating a chromatin instability defect. In addition to the observation that cryptic transcription was only activated when FLO8 transcription was present, 37 Figure 3.3. Cryptic transcription in cells lacking Spt2 is dependent on active FLO8 transcription. A) Experimental scheme for comparing cryptic transcription levels in asynchronous and arrested cells. All cells were grown to log-phase in dextrose or galactose before being separated into asynchronous and arrested groups. Both cultures were then treated with rapamycin or vehicle control DMSO for two hours. Note that cells were grown in galactose overnight in the case of C) lower panel. B) Histograms of GFP signal comparing both asynchronous and arrested WT and Spt2 depleted cells treated with either rapamycin or DMSO. Each strain was tested using three independent cultures as illustrated with different colors as shown in the legend. The two experiments were performed with an identical setup in different carbon sources (dextrose/galactose). C) Histograms of mCherry signal measured in strains with a GALpr-mCherry reporter depleted with different factors after growth in galactose. α-factor α-factor α-factor α-factor 38 replication inhibition also did not affect the GFP signal in cells depleted of Spt2 grown in either media. This suggested that Spt2 functions exclusively during transcription to maintain chromatin stability over actively transcribed regions. Contrary to Spt2, Cheung et al demonstrated that cryptic transcription in cells lacking SPT21 did not require transcription from an upstream promoter. Because Spt21 is required for induction of the histone genes in S phase, a likely explanation for this is that in the absence of Spt21, cells are unable to synthesize sufficient histones in S phase to properly package chromatin over the cryptic promoter. To test this, we blocked DNA replication in cells containing a GAL1pr-FLO8-sfGFP reporter by arresting in G1 with alpha factor and then depleting Spt21 from the nucleus using anchor-away. Importantly, these cells were grown on dextrose to prevent chromatin disruption due to transcription from the upstream GAL1 promoter, but it should be noted that depletion of Spt21 also results in a severe GAL1 induction defect (Fig. 3.3C). While depletion of Spt21 induced a significant increase in cryptic transcription in asynchronous cells (Fig. 3.4A, one-tailed Mann-Whitney U test, WT rapamycin vs. Spt21-FRB rapamycin), prior treatment with alpha factor reduced the level of fluorescence to that of cells treated with DMSO (Fig. 3.4A, one-tailed Mann-Whitney U test, Spt21-FRB α-factor rapamycin vs. Spt21-FRB rapamycin), suggesting that passage through the cell cycle is required for cryptic transcription following depletion of this protein. Similar results were observed following the depletion of Spt10, a second factor required for full histone gene induction in S phase (Fig. 3.4B, one-tailed Mann-Whitney U tests, WT rapamycin vs. Spt10-FRB rapamycin; Spt10-FRB α-factor rapamycin vs. Spt10-FRB rapamycin). Thus, Spt10 and Spt21, but not Spt2, are important for maintaining chromatin following DNA replication. The fact that cell cycle arrest can block sfGFP expression following Spt10 and Spt21 39 Figure 3.4. Cryptic transcription in cells depleted of Spt10 or Spt21 is dependent on replication. A) Histograms of GFP signal comparing both asynchronous and arrested WT and Spt21 depleted cells treated with either rapamycin or DMSO. Each strain was tested using three independent cultures as illustrated with different colors as shown in the legend. All cells were grown in rich media with dextrose as a carbon source. B) Histograms of GFP signal comparing both asynchronous and arrested WT and Spt10 depleted cells treated with either rapamycin or DMSO. α-factor α-factor α-factor α-factor 40 depletion suggests that failure to properly establish chromatin structure following DNA replication can make cryptic promoters accessible to the transcription machinery. Indeed, multiple histone chaperones with roles in DNA replication have been shown to suppress transcription from cryptic promoters. Interestingly, although the CAF-1 complex has a well-characterized function in depositing histones H3 and H4 onto newly synthesized DNA, depletion of the subunits of this complex has not been reported to result in cryptic transcription. To test this with our system, we depleted Cac1 with anchor-away and measured the GFP signal. We observed a significant increase in GFP signal with Cac1 depletion compared to wildtype cells (Fig. 3.5A, one-tailed Mann-Whitney U test, WT rapamycin vs. Cac1-FRB rapamycin), and this effect was suppressed in arrested cells, consistent with its primary role in replication (Fig. 3.5A, one-tailed Mann-Whitney U test, Cac1-FRB α-factor rapamycin vs. Cac1-FRB rapamycin). Interestingly, when we delete CAC1 in a strain containing the GALpr-FLO8-HIS3 reporter designed by Cheung et al., there was no growth on the -histidine plate suggesting an absence of cryptic transcription from this reporter (Fig. 3.5B). Although we cannot rule out the possibility that low levels of HIS3 expression were insufficient for growth on -histidine media, it is also possible that yeast cells have a way to compensate for long-term depletion of Cac1. Additional suppressors of cryptic transcription that have been linked to replication are Asf1 and Rtt106. We sought to confirm these roles using our assay with cells grown in dextrose. Depletion of Asf1 with anchor-away showed a significant increase in GFP signal (Fig. 3.6A, one-tailed Mann-Whitney U test, WT rapamycin vs. Asf1-FRB rapamycin), which was suppressed by cell cycle arrest (Fig. 3.6A, one-tailed Mann-Whitney U test, Asf1-FRB α-factor rapamycin vs. Asf1-FRB rapamycin), implying a role of Asf1 in stabilizing chromatin on newly replicated DNA. 41 Figure 3.5. Test for cryptic transcription following Cac1 depletion. A) Histograms of GFP signal comparing both asynchronous and arrested WT and Cac1 depleted cells treated with either rapamycin or DMSO. Each sample was tested using three independent cultures as illustrated with different colors as shown in the legend. All cells were grown in rich media with dextrose as a carbon source. B) Dilution plates of both WT and cac1 knock-out cells grown in either synthetic complete (SC) media or SC -His media. Dilution levels from left to right are: 1x, 10x, 100x, 1000x and 10,000x. α-factor α-factor 42 Figure 3.6. Test for cryptic transcription following Asf1 depletion. A) Histograms of GFP signal comparing both asynchronous and arrested WT and Rtt106 depleted cells treated with either rapamycin or DMSO. Each strain was tested using three independent cultures as illustrated with different colors as shown in the legend. All cells were grown in rich media with dextrose as a carbon source. B) Histograms of GFP signal comparing WT and asf1 knock-out cells grown overnight. C) Dilution plates of both WT and asf1 knock-out cells grown in either synthetic complete (SC) media or SC -His media. Dilution levels from left to right are: 1x, 10x, 100x, 1000x and 10,000x. AGFP Signal BWTasf1KOCWTasf1 KOSCSC -HISWTasf1 KODMSO RapamycinWTWTa-factorAsf1-FRBAsf1-FRBa-factorα-f t r α-f t r 43 Interestingly however, depletion of Rtt106 did not result in a significant change in GFP levels (Fig. 3.7A, one-tailed Mann-Whitney U test, WT rapamycin vs. Rtt106-FRB rapamycin). To rule out the possibility that the lack of a significant effect in the Rtt106 mutant strain was due to the incomplete depletion of this factor, we repeated the analysis using null alleles. To this end, RTT106, as well as ASF1, were deleted using CRISPR, and the presence of cryptic transcripts following the deletion of these genes was confirmed using the FLO8-HIS3 reporter (Fig. 3.6C and 3.7C). Deletion of these genes in cells bearing the FLO8-sfGFP resulted in a significant level of cryptic transcription compared to WT cells (Fig. 3.6B, one-tailed Mann-Whitney U test, WT vs. asf1 KO; Fig. 3.7B, one-tailed Mann-Whitney U test, WT vs. rtt106 KO). While these results suggest that the nuclear depletion of Rtt106 by anchor-away was incomplete, an alternate possibility is that long-term depletion of Rtt106 might be needed for an observable increase in cryptic transcription. Thus, although we were able to provide support for a role for Asf1 in stabilizing chromatin following DNA replication, we were unable to do the same for Rtt106 due to a requirement for complete or long-term depletion of this protein to observe the generation of cryptic transcripts. 3.3 Replication inhibition partially suppresses cryptic transcription following Spt5, Spt6, or Spt16 depletion Multiple essential factors, including Spt5, 6, and 16, are important for maintaining chromatin structure on transcribed genes. Later work however showed that Spt6 and Spt16 also play roles in DNA replication, but whether the bulk of chromatin disruption following the depletion of these proteins is due to transcription or replication is unknown (Tan et al., 2006; Kurat et al., 2017; Miller and Winston, 2022). We sought to test this using our reporter assay. Cells 44 Figure 3.7. Test for cryptic transcription following Rtt106 depletion. A) Histograms of GFP signal comparing both asynchronous and arrested WT and Rtt106 depleted cells treated with either rapamycin or DMSO. Each sample was tested using three independent cultures as illustrated with different colors as shown in the legend. All cells were grown in rich media with dextrose as a carbon source. B) Histograms of GFP signal comparing WT and rtt106 knock-out cells grown overnight. C) Dilution plates of both WT and rtt106 knock-out cells grown in either synthetic complete (SC) media or SC -His media. Dilution levels from left to right are: 1x, 10x, 100x, 1000x and 10,000x. D) Histograms of GFP signal comparing WT and rtt106 knock-out cells grown overnight in either YPD or CSM drop-out media. α-factor α-factor 45 were grown to log phase in dextrose to repress the GAL1 promoter upstream of FLO8 and the proteins were depleted using anchor away in the case of Spt5, or temperature-sensitive alleles in the case of Spt6 and Spt16. As all three factors were previously shown to be responsible for suppressing cryptic transcription, the strong increase in GFP signal following protein depletion in asynchronous cells compared to wild-type again showed the reliability of our reporter (Fig. 3.8A-C, one-tailed Mann-Whitney U tests, WT rapamycin vs. Spt5-FRB rapamycin; WT 37°C vs. spt6-1004/spt16-197 37°C). Interestingly, when replication was inhibited, we saw a partial suppression of cryptic transcription as the GFP signal decreased in arrested cells (Fig. 3.8A-C, one-tailed Mann-Whitney U tests, Spt5-FRB α-factor rapamycin vs. Spt5-FRB rapamycin; spt6-1004/spt16-197 α-factor 37°C vs. spt6-1004/spt16-197 37°C), indicating that these factors counteract replication-induced chromatin disruption. The extent of rescue was less distinct in the case of Spt6 depleted cells compared to Spt16, which has a better-established role in replication. The same partial suppression observed in cells depleted of Spt5 was surprising since there haven’t been any studies supporting a role for this factor in depositing histones during replication. However, as it is only recently reported that Spt6 plays a role in reestablishing intact chromatin on nascent DNA, this is not out of the realm of possibility. Overall, these results are indicative that Spt5, Spt6 and Spt16 function during replication as well as in a replication-independent mechanism. The origin of cryptic transcripts from a non-transcribed, non-replicating locus was surprising. One possible explanation for our observations is that it is an effect of “transcription-coupled histone redistribution”. The passage of RNA polymerase targets many factors to transcribed genes that work cohesively in maintaining nucleosome structure, preventing histone loss. In the absence of Spt5, 6, or 16, other histone chaperones may use non-transcribed regions as a source of histones for transcription-linked histone deposition. Consistent with this, previous 46 Figure 3.7. Replication inhibition partially suppresses cryptic transcription in cells depleted of Spt5, Spt16 or Spt6. A) Histograms of GFP signal comparing both asynchronous and arrested WT and Spt5 depleted cells treated with either rapamycin or DMSO. Each sample was tested using three independent cultures as illustrated with different colors as shown in the legend. All cells were grown in rich media with dextrose as a carbon source. B) Histograms of GFP signal comparing both asynchronous and arrested WT and spt16-197 cells grown in 28°C or 37°C. C) Histograms of GFP signal comparing both asynchronous and arrested WT and spt6-1004 cells grown in 30°C or 37°C. Dotted lines are aligned with the approximate GFP level in asynchronous cells depleted of each specific factor for easier comparison with arrested cells. α-factor α-factor α-factor α-factor α-factor α-factor 47 works have shown poor correlations between Spt6 and Spt16 occupancy and nucleosome loss following the depletion of these factors (Doris et al., 2018; Jeronimo et al., 2019). An alternate explanation is that loss of Spt5, 6, and 16 results in the activation of promoters upstream of the FLO8 cryptic promoter that drives sense transcription or downstream promoters driving anti-sense transcription, and it is this transcription that is disrupting chromatin over the FLO8 cryptic promoter. Regardless of the cause, these results underscore the importance of Spt5, 6, and 16 in directing RNAPII to canonical promoters. 48 Chapter 4: Summary and Discussion 4.1 Summary of study In this study, we generated a fluorescent reporter for detecting cryptic transcription in single cells. Distinct from previous reporters, our FLO8:sfGFP reporter enables us to examine cryptic transcription in single cells. Using a spt16-197 mutant, we successfully showed the utility of the reporter in detecting cryptic transcription induced by chromatin instability. With the aim to differentiate cryptic transcription-suppressing factors that function during transcription or replication, we repressed FLO8 transcription through the incorporation of the GAL1 promoter. We identified Spt10 and Spt21 to be required for chromatin stability on newly replicated DNA, as cryptic transcription induced by their depletion was suppressed by cell cycle arrest. On the contrary, cryptic transcription induced by Spt2 depletion was only present when FLO8 was actively transcribed, suggesting Spt2 functions during transcription. Interestingly, although Cac1 depletion through anchor-away induced an increase in GFP signal, long-term depletion alleviated detectable cryptic transcription, suggesting that cells might have a way to compensate for long term loss of certain factors. However, since these results were generated using different reporters, we cannot rule out the possibility that the FLO8-sfGFP reporter is more sensitive for detecting cryptic transcripts than the FLO8-HIS3 reporter. Similar to Cac1, we were also able to provide support for a role for Asf1 in chromatin assembly following DNA replication, but were unable to do so for Rtt106 using our reporter system. This was likely due to the fact that cryptic transcription in the absence of Rtt106 requires either complete or long-term depletion of this factor. Finally, we showed that the depletion of Spt5, Spt6 and Spt16 induced a significant increase in cryptic transcription, which was partially suppressed by replication inhibition. This suggested that all three 49 factors are responsible for maintaining chromatin stability during replication, while also functioning in other pathways independent of replication. 4.2 Discussion and future directions As our reporter assay is able to examine the level of cryptic transcription within each individual cell, we observed that in mutants with significant levels of cryptic transcription, nearly all cells tested showed some level of GFP increase and there was no second peak in histograms. This suggested that the effects of factor loss through anchor-away had a high penetrant effect. The extent of cryptic transcription in cells depleted of each factor is different, probably because the level of chromatin disruption varied. Depletion of Spt5/6/16 led to the relatively high GFP signals compared to other factors including Spt2, suggesting their function in maintaining chromatin stability is rather paramount and non-redundant. In Spt5/6/16 mutants, cryptic transcription was detected in G1-arrested cells with repressed FLO8 transcription, suggesting that chromatin structure was unstable without an evident source of disruption. One possibility is that during the long arrest in G1 phase, cells lack newly synthesized histones to replenish disrupted nucleosomes, leading to chromatin instability. To fulfill the requirement of chromatin regeneration on newly synthesized DNA in S phase, histone genes are regulated to actively transcribe in early S phase (Osley, 1991; Nelson et al., 2002). Thus, replication is important in maintaining histone stock and refreshing chromatin. Human cells contain histone variant H3.3 which is expressed throughout the cell cycle and can be incorporated in a replication-independent way (Shi et al., 2017). Yeast do not have this luxury and even human cells lack a replication-independent H4 variant. The Van Leeuwen lab suggested that histone synthesis occurs in G1, but is probably not sufficient to maintain chromatin structure in the absence of replication. The lack of strong GFP signal increase 50 following the depletion of replication-linked factors including Spt10, Spt21, Asf1, Rtt106 and Cac1 might be due to redundancy as they are all non-essential. As an alternative, it was demonstrated that in the minutes following replication, nucleosomes are re-aligned in their position with promoters and the inter-space between nucleosomes is decreased (Fennessy and Owen-Hughes, 2016). This “over-chromatinization” leads to reduced NDR and nucleosome phasing on newly replicated DNA. Although it was later discovered that remodelers are responsible for the nucleosome phasing redistribution, it is possible that this mechanism protects promoters from transcription machinery and leaves room for subtle defects during replication. It will be interesting to test whether the depletion of two or more factors can cause a more significant chromatin disruption during replication. Alternatively, the greater effect induced by Spt10/21 removal compared to the replication-linked chaperones might suggest that chaperones are dispensable for histone transfer during replication. Previous work showed that displaced histone octamers can be passively transferred back to DNA via the formation of a DNA loop in the absence of any histone chaperones (Brennan et al., 2016). As Spt10 and Spt21 are required for histone gene activation prior to replication, their depletion leads to reduced histone stock and defects in both “over-chromatinization” and chaperone-independent histone transfer. There were some discrepancies in our study when investigating cryptic transcription following the depletion of the same factor with different approaches. We observed a modest but significant increase in GFP signal when depleting Cac1 with anchor-away, whereas a null allele did not produce a detectable level of cryptic transcription. It is possible that cells have a compensatory mechanism for coping for long-term loss of replication-linked factors. A discrepancy was also observed when Rtt106 was depleted through anchor-away or with a null allele. As both the FLO8-sfGFP and the FLO8-HIS3 reporters showed a substantial increase in 51 cryptic transcription using a rtt106 null allele, Rtt106 depletion through anchor-away may not be sufficient enough to produce a detectable level of chromatin disruption. According to microarray analysis, there are over 1000 cryptic promoters in the S. cerevisiae genome. Although the reason for the abundance of cryptic promoters and their potential function are yet unknown, our study provided further insights into some plausible hypotheses described by Cheung et al. Firstly, cryptic promoters may control the transcription of conditionally expressed proteins under certain environmental changes. Although our artificially inserted GFP gene does not possess an actual cellular function, it provides evidence that transcripts from internal promoters are translated into proteins. Previous studies also support the idea of using internal promoters for alternative protein expression under different growth conditions (Carlson and Botstein, 1982; Gammie et al., 1999). Secondly, the product of a cryptic promoter may provide a growth advantage under selective conditions. Finally, cryptic transcripts could just be by-products induced by aberrant promoters during chromatin disruption. Transcripts from normally repressed intragenic regions are eliminated by a polyadenylation-assisted degradation mechanism employing the exosome as well as factors Trf4 and Air1/2 (Wyers et al., 2005). As transcription from cryptic promoters can interfere with canonical transcription, many factors investigated in our study are required to suppress cryptic promoters and restore normal transcription. As none of these hypotheses are incompatible with the others, further studies are needed to validate whether one or more of them is true. Current studies mostly utilized the few well-studied internal promoters including the ones within FLO8, STE11 and PCA1, and investigating the function of transcripts from other cryptic promoters may yield further insights into the reason behind their existence. 52 References Adkins, M.W., Howar, S.R., and Tyler, J.K. (2004) Chromatin disassembly mediated by the histone chaperone Asf1 is essential for transcriptional activation of the yeast PHO5 and PHO8 genes. Mol Cell 14: 657–666. Adkins, M.W., and Tyler, J.K. (2006) Transcriptional activators are dispensable for transcription in the absence of Spt6-mediated chromatin reassembly of promoter regions. Mol Cell 21: 405–416. Alabert, C., and Groth, A. (2012) Chromatin replication and epigenome maintenance. Nat Rev Mol Cell Biol 13: 153–167. Almer, A., and Hörz, W. (1986) Nuclease hypersensitive regions with adjacent positioned nucleosomes mark the gene boundaries of the PHO5/PHO3 locus in yeast. EMBO J 5: 2681–2687. Almer, A., Rudolph, H., Hinnen, A., and Hörz, W. (1986) Removal of positioned nucleosomes from the yeast PHO5 promoter upon PHO5 induction releases additional upstream activating DNA elements. EMBO J 5: 2689–2696. Anderson, J.D., and Widom, J. (2001) Poly(dA-dT) promoter elements increase the equilibrium accessibility of nucleosomal DNA target sites. Mol Cell Biol 21: 3830–3839. Avvakumov, N., Nourani, A., and Côté, J. (2011) Histone chaperones: modulators of chromatin marks. Mol Cell 41: 502–514. Badis, G., Chan, E.T., Bakel, H. van, Pena-Castillo, L., Tillo, D., Tsui, K., et al. (2008) A library of yeast transcription factor motifs reveals a widespread function for Rsc3 in targeting nucleosome exclusion at promoters. Mol Cell 32: 878–887. Bai, L., Ondracka, A., and Cross, F.R. (2011) Multiple sequence-specific factors generate the nucleosome-depleted region on CLN2 promoter. Mol Cell 42: 465–476. Bonnet, C., Rigaud, C., Chanteclaire, E., Blandais, C., Tassy-Freches, E., Arico, C., and Javaud, C. (2013) PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae. BMC Res Notes 6: 201. Bortvin, A., and Winston, F. (1996) Evidence that Spt6p controls chromatin structure by a direct interaction with histones. Science 272: 1473–1476. Brennan, L.D., Forties, R.A., Patel, S.S., and Wang, M.D. (2016) DNA looping mediates nucleosome transfer. Nat Commun 7: 13337. 53 Carlson, M., and Botstein, D. (1982) Two differentially regulated mRNAs with different 5’ ends encode secreted with intracellular forms of yeast invertase. Cell 28: 145–154. Chaban, Y., Ezeokonkwo, C., Chung, W.-H., Zhang, F., Kornberg, R.D., Maier-Davis, B., et al. (2008) Structure of a RSC-nucleosome complex and insights into chromatin remodeling. Nat Struct Mol Biol 15: 1272–1277. Chen, S., Rufiange, A., Huang, H., Rajashankar, K.R., Nourani, A., and Patel, D.J. (2015) Structure-function studies of histone H3/H4 tetramer maintenance during transcription by chaperone Spt2. Genes Dev 29: 1326–1340. Cheung, V., Chua, G., Batada, N.N., Landry, C.R., Michnick, S.W., Hughes, T.R., and Winston, F. (2008) Chromatin- and transcription-related factors repress transcription from within coding regions throughout the Saccharomyces cerevisiae genome. PLoS Biol 6: e277. Clark-Adams, C.D., Norris, D., Osley, M.A., Fassler, J.S., and Winston, F. (1988) Changes in histone gene dosage alter transcription in yeast. Genes Dev 2: 150–159. Clark-Adams, C.D., and Winston, F. (1987) The SPT6 gene is essential for growth and is required for delta-mediated transcription in Saccharomyces cerevisiae. Mol Cell Biol 7: 679–686. Crickard, J.B., Fu, J., and Reese, J.C. (2016) Biochemical Analysis of Yeast Suppressor of Ty 4/5 (Spt4/5) Reveals the Importance of Nucleic Acid Interactions in the Prevention of RNA Polymerase II Arrest. J Biol Chem 291: 9853–9870. DeGennaro, C.M., Alver, B.H., Marguerat, S., Stepanova, E., Davis, C.P., Bähler, J., et al. (2013) Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast. Mol Cell Biol 33: 4779–4792. DiCarlo, J.E., Norville, J.E., Mali, P., Rios, X., Aach, J., and Church, G.M. (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41: 4336–4343. Diebold, M.-L., Loeliger, E., Koch, M., Winston, F., Cavarelli, J., and Romier, C. (2010) Noncanonical tandem SH2 enables interaction of elongation factor Spt6 with RNA polymerase II. J Biol Chem 285: 38389–38398. Dion, M.F., Kaplan, T., Kim, M., Buratowski, S., Friedman, N., and Rando, O.J. (2007) Dynamics of replication-independent histone turnover in budding yeast. Science 315: 1405–1408. 54 Dollard, C., Ricupero-Hovasse, S.L., Natsoulis, G., Boeke, J.D., and Winston, F. (1994) SPT10 and SPT21 are required for transcription of particular histone genes in Saccharomyces cerevisiae. Mol Cell Biol 14: 5223–5228. Doris, S.M., Chuang, J., Viktorovskaya, O., Murawska, M., Spatt, D., Churchman, L.S., and Winston, F. (2018) Spt6 Is Required for the Fidelity of Promoter Selection. Mol Cell 72: 687-699.e6. Driscoll, R., Hudson, A., and Jackson, S.P. (2007) Yeast Rtt109 promotes genome stability by acetylating histone H3 on lysine 56. Science 315: 649–652. Ehara, H., Kujirai, T., Shirouzu, M., Kurumizaka, H., and Sekine, S.-I. (2022) Structural basis of nucleosome disassembly and reassembly by RNAPII elongation complex with FACT. Science 377: eabp9466. English, C.M., Adkins, M.W., Carson, J.J., Churchill, M.E.A., and Tyler, J.K. (2006) Structural basis for the histone chaperone activity of Asf1. Cell 127: 495–508. English, C.M., Maluf, N.K., Tripet, B., Churchill, M.E.A., and Tyler, J.K. (2005) ASF1 binds to a heterodimer of histones H3 and H4: a two-step mechanism for the assembly of the H3-H4 heterotetramer on DNA. Biochemistry 44: 13673–13682. Enomoto, S., McCune-Zierath, P.D., Gerami-Nejad, M., Sanders, M.A., and Berman, J. (1997) RLF2, a subunit of yeast chromatin assembly factor-I, is required for telomeric chromatin function in vivo. Genes Dev 11: 358–370. Eriksson, P.R., Ganguli, D., Nagarajavel, V., and Clark, D.J. (2012) Regulation of histone gene expression in budding yeast. Genetics 191: 7–20. Evrin, C., Serra-Cardona, A., Duan, S., Mukherjee, P.P., Zhang, Z., and Labib, K.P.M. (2022) Spt5 histone binding activity preserves chromatin during transcription by RNA polymerase II. EMBO J 41: e109783. Farnung, L., Ochmann, M., Engeholm, M., and Cramer, P. (2021) Structural basis of nucleosome transcription mediated by Chd1 and FACT. Nat Struct Mol Biol 28: 382–387. Fassler, J.S., and Winston, F. (1988) Isolation and analysis of a novel class of suppressor of Ty insertion mutations in Saccharomyces cerevisiae. Genetics 118: 203–212. 55 Fazly, A., Li, Q., Hu, Q., Mer, G., Horazdovsky, B., and Zhang, Z. (2012) Histone chaperone Rtt106 promotes nucleosome formation using (H3-H4)2 tetramers. J Biol Chem 287: 10753–10760. Feng, J., Gan, H., Eaton, M.L., Zhou, H., Li, S., Belsky, J.A., et al. (2016) Noncoding Transcription Is a Driving Force for Nucleosome Instability in spt16 Mutant Cells. Mol Cell Biol 36: 1856–1867. Fennessy, R.T., and Owen-Hughes, T. (2016) Establishment of a promoter-based chromatin architecture on recently replicated DNA can accommodate variable inter-nucleosome spacing. Nucleic Acids Res 44: 7189–7203. Ferreira, M.E., Prochasson, P., Berndt, K.D., Workman, J.L., and Wright, A.P.H. (2009) Activator-binding domains of the SWI/SNF chromatin remodeling complex characterized in vitro are required for its recruitment to promoters in vivo. FEBS J 276: 2557–2565. Fillingham, J., Kainth, P., Lambert, J.-P., Bakel, H. van, Tsui, K., Peña-Castillo, L., et al. (2009) Two-color cell array screen reveals interdependent roles for histone chaperones and a chromatin boundary regulator in histone gene repression. Mol Cell 35: 340–351. Foltman, M., Evrin, C., De Piccoli, G., Jones, R.C., Edmondson, R.D., Katou, Y., et al. (2013) Eukaryotic replisome components cooperate to process histones during chromosome replication. Cell Rep 3: 892–904. Formosa, T., and Winston, F. (2020) The role of FACT in managing chromatin: disruption, assembly, or repair? Nucleic Acids Res 48: 11929–11941. Franco, A.A., Lam, W.M., Burgers, P.M., and Kaufman, P.D. (2005) Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C. Genes Dev 19: 1365–1375. Gammie, A.E., Stewart, B.G., Scott, C.F., and Rose, M.D. (1999) The two forms of karyogamy transcription factor Kar4p are regulated by differential initiation of transcription, translation, and protein turnover. Mol Cell Biol 19: 817–825. Ganapathi, M., Palumbo, M.J., Ansari, S.A., He, Q., Tsui, K., Nislow, C., and Morse, R.H. (2011) Extensive role of the general regulatory factors, Abf1 and Rap1, in determining genome-wide chromatin structure in budding yeast. Nucleic Acids Res 39: 2032–2044. 56 Green, E.M., Antczak, A.J., Bailey, A.O., Franco, A.A., Wu, K.J., Yates, J.R., and Kaufman, P.D. (2005) Replication-independent histone deposition by the HIR complex and Asf1. Curr Biol 15: 2044–2049. Gurova, K., Chang, H.-W., Valieva, M.E., Sandlesh, P., and Studitsky, V.M. (2018) Structure and function of the histone chaperone FACT - Resolving FACTual issues. Biochim Biophys Acta Gene Regul Mech S1874-9399(18)30159–7. Hainer, S.J., Pruneski, J.A., Mitchell, R.D., Monteverde, R.M., and Martens, J.A. (2011) Intergenic transcription causes repression by directing nucleosome assembly. Genes Dev 25: 29–40. Hammond, C.M., Strømme, C.B., Huang, H., Patel, D.J., and Groth, A. (2017) Histone chaperone networks shaping chromatin function. Nat Rev Mol Cell Biol 18: 141–158. Han, M., and Grunstein, M. (1988) Nucleosome loss activates yeast downstream promoters in vivo. Cell 55: 1137–1145. Hartley, P.D., and Madhani, H.D. (2009) Mechanisms that specify promoter nucleosome location and identity. Cell 137: 445–458. Hartzog, G.A., Wada, T., Handa, H., and Winston, F. (1998) Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae. Genes Dev 12: 357–369. Hess, D., Liu, B., Roan, N.R., Sternglanz, R., and Winston, F. (2004) Spt10-dependent transcriptional activation in Saccharomyces cerevisiae requires both the Spt10 acetyltransferase domain and Spt21. Mol Cell Biol 24: 135–143. Hess, D., and Winston, F. (2005) Evidence that Spt10 and Spt21 of Saccharomyces cerevisiae play distinct roles in vivo and functionally interact with MCB-binding factor, SCB-binding factor and Snf1. Genetics 170: 87–94. Hirtreiter, A., Damsma, G.E., Cheung, A.C.M., Klose, D., Grohmann, D., Vojnic, E., et al. (2010) Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif. Nucleic Acids Res 38: 4040–4051. Holla, S., Dhakshnamoorthy, J., Folco, H.D., Balachandran, V., Xiao, H., Sun, L.-L., et al. (2020) Positioning Heterochromatin at the Nuclear Periphery Suppresses Histone Turnover to Promote Epigenetic Inheritance. Cell 180: 150-164.e15. 57 Hu, Z., Chen, K., Xia, Z., Chavez, M., Pal, S., Seol, J.-H., et al. (2014) Nucleosome loss leads to global transcriptional up-regulation and genomic instability during yeast aging. Genes Dev 28: 396–408. Huang, S., Zhou, H., Katzmann, D., Hochstrasser, M., Atanasova, E., and Zhang, Z. (2005) Rtt106p is a histone chaperone involved in heterochromatin-mediated silencing. Proc Natl Acad Sci U S A 102: 13410–13415. Ichikawa, Y., Morohashi, N., Tomita, N., Mitchell, A.P., Kurumizaka, H., and Shimizu, M. (2016) Sequence-directed nucleosome-depletion is sufficient to activate transcription from a yeast core promoter in vivo. Biochem Biophys Res Commun 476: 57–62. Imbeault, D., Gamar, L., Rufiange, A., Paquet, E., and Nourani, A. (2008) The Rtt106 histone chaperone is functionally linked to transcription elongation and is involved in the regulation of spurious transcription from cryptic promoters in yeast. J Biol Chem 283: 27350–27354. Ioshikhes, I.P., Albert, I., Zanton, S.J., and Pugh, B.F. (2006) Nucleosome positions predicted through comparative genomics. Nat Genet 38: 1210–1215. Iyer, V., and Struhl, K. (1995) Poly(dA:dT), a ubiquitous promoter element that stimulates transcription via its intrinsic DNA structure. EMBO J 14: 2570–2579. Jamai, A., Puglisi, A., and Strubin, M. (2009) Histone chaperone spt16 promotes redeposition of the original h3-h4 histones evicted by elongating RNA polymerase. Mol Cell 35: 377–383. Jaskelioff, M., Gavin, I.M., Peterson, C.L., and Logie, C. (2000) SWI-SNF-mediated nucleosome remodeling: role of histone octamer mobility in the persistence of the remodeled state. Mol Cell Biol 20: 3058–3068. Jeronimo, C., Poitras, C., and Robert, F. (2019) Histone Recycling by FACT and Spt6 during Transcription Prevents the Scrambling of Histone Modifications. Cell Rep 28: 1206-1218.e8. Jeronimo, C., Watanabe, S., Kaplan, C.D., Peterson, C.L., and Robert, F. (2015) The Histone Chaperones FACT and Spt6 Restrict H2A.Z from Intragenic Locations. Mol Cell 58: 1113–1123. Johnson, J.M., French, S.L., Osheim, Y.N., Li, M., Hall, L., Beyer, A.L., and Smith, J.S. (2013) Rpd3- and spt16-mediated nucleosome assembly and transcriptional regulation on yeast ribosomal DNA genes. Mol Cell Biol 33: 2748–2759. 58 Kaplan, C.D., Laprade, L., and Winston, F. (2003) Transcription elongation factors repress transcription initiation from cryptic sites. Science 301: 1096–1099. Kato, H., Okazaki, K., Iida, T., Nakayama, J.-I., Murakami, Y., and Urano, T. (2013) Spt6 prevents transcription-coupled loss of posttranslationally modified histone H3. Sci Rep 3: 2186. Kaufman, P.D. (1996) Nucleosome assembly: the CAF and the HAT. Curr Opin Cell Biol 8: 369–373. Kaufman, P.D., Kobayashi, R., and Stillman, B. (1997) Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I. Genes Dev 11: 345–357. Korber, P., Barbaric, S., Luckenbach, T., Schmid, A., Schermer, U.J., Blaschke, D., and Hörz, W. (2006a) The histone chaperone Asf1 increases the rate of histone eviction at the yeast PHO5 and PHO8 promoters. J Biol Chem 281: 5539–5545. Korber, P., Barbaric, S., Luckenbach, T., Schmid, A., Schermer, U.J., Blaschke, D., and Hörz, W. (2006b) The histone chaperone Asf1 increases the rate of histone eviction at the yeast PHO5 and PHO8 promoters. J Biol Chem 281: 5539–5545. Krude, T. (1995) Chromatin assembly factor 1 (CAF-1) colocalizes with replication foci in HeLa cell nuclei. Exp Cell Res 220: 304–311. Kruger, W., and Herskowitz, I. (1991) A negative regulator of HO transcription, SIN1 (SPT2), is a nonspecific DNA-binding protein related to HMG1. Mol Cell Biol 11: 4135–4146. Kurat, C.F., Yeeles, J.T.P., Patel, H., Early, A., and Diffley, J.F.X. (2017) Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates. Mol Cell 65: 117–130. Kushnirov, V.V. (2000) Rapid and reliable protein extraction from yeast. Yeast 16: 857–860. Lee, W., Tillo, D., Bray, N., Morse, R.H., Davis, R.W., Hughes, T.R., and Nislow, C. (2007) A high-resolution atlas of nucleosome occupancy in yeast. Nat Genet 39: 1235–1244. Leffak, I.M., Grainger, R., and Weintraub, H. (1977) Conservative assembly and segregation of nucleosomal histones. Cell 12: 837–845. Li, S., Edwards, G., Radebaugh, C.A., Luger, K., and Stargell, L.A. (2022) Spn1 and Its Dynamic Interactions with Spt6, Histones and Nucleosomes. J Mol Biol 434: 167630. 59 Lin, A., Du, Y., and Xiao, W. (2020) Yeast chromatin remodeling complexes and their roles in transcription. Curr Genet 66: 657–670. Liu, X., Lee, C.-K., Granek, J.A., Clarke, N.D., and Lieb, J.D. (2006) Whole-genome comparison of Leu3 binding in vitro and in vivo reveals the importance of nucleosome occupancy in target site selection. Genome Res 16: 1517–1528. Luger, K., Mäder, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J. (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389: 251–260. Malagón, F., and Aguilera, A. (1996) Differential intrachromosomal hyper-recombination phenotype of spt4 and spt6 mutants of S. cerevisiae. Curr Genet 30: 101–106. Malagón, F., and Aguilera, A. (2001) Yeast spt6-140 mutation, affecting chromatin and transcription, preferentially increases recombination in which Rad51p-mediated strand exchange is dispensable. Genetics 158: 597–611. Malone, E.A., Clark, C.D., Chiang, A., and Winston, F. (1991) Mutations in SPT16/CDC68 suppress cis- and trans-acting mutations that affect promoter function in Saccharomyces cerevisiae. Mol Cell Biol 11: 5710–5717. Martin, B.J.E., Chruscicki, A.T., and Howe, L.J. (2018) Transcription Promotes the Interaction of the FAcilitates Chromatin Transactions (FACT) Complex with Nucleosomes in Saccharomyces cerevisiae. Genetics 210: 869–881. Martinez-Rucobo, F.W., Sainsbury, S., Cheung, A.C.M., and Cramer, P. (2011) Architecture of the RNA polymerase-Spt4/5 complex and basis of universal transcription processivity. EMBO J 30: 1302–1310. Mattiroli, F., Gu, Y., Yadav, T., Balsbaugh, J.L., Harris, M.R., Findlay, E.S., et al. (2017) DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication. Elife 6: e22799. Mavrich, T.N., Ioshikhes, I.P., Venters, B.J., Jiang, C., Tomsho, L.P., Qi, J., et al. (2008) A barrier nucleosome model for statistical positioning of nucleosomes throughout the yeast genome. Genome Res 18: 1073–1083. McCullough, L., Connell, Z., Petersen, C., and Formosa, T. (2015) The Abundant Histone Chaperones Spt6 and FACT Collaborate to Assemble, Inspect, and Maintain Chromatin Structure in Saccharomyces cerevisiae. Genetics 201: 1031–1045. 60 McCullough, L.L., Pham, T.H., Parnell, T.J., Connell, Z., Chandrasekharan, M.B., Stillman, D.J., and Formosa, T. (2019) Establishment and Maintenance of Chromatin Architecture Are Promoted Independently of Transcription by the Histone Chaperone FACT and H3-K56 Acetylation in Saccharomyces cerevisiae. Genetics 211: 877–892. Meyer, P.A., Li, S., Zhang, M., Yamada, K., Takagi, Y., Hartzog, G.A., and Fu, J. (2015) Structures and Functions of the Multiple KOW Domains of Transcription Elongation Factor Spt5. Mol Cell Biol 35: 3354–3369. Millán-Zambrano, G., Burton, A., Bannister, A.J., and Schneider, R. (2022) Histone post-translational modifications - cause and consequence of genome function. Nat Rev Genet 23: 563–580. Miller, C.L., and Winston, F. (2022) The conserved histone chaperone Spt6 facilitates DNA replication and mediates genome instability. Molecular Biology, . http://biorxiv.org/lookup/doi/10.1101/2022.04.27.489770. Accessed November 26, 2022. Miyaji-Yamaguchi, M., Kato, K., Nakano, R., Akashi, T., Kikuchi, A., and Nagata, K. (2003) Involvement of nucleocytoplasmic shuttling of yeast Nap1 in mitotic progression. Mol Cell Biol 23: 6672–6684. Morillo-Huesca, M., Maya, D., Muñoz-Centeno, M.C., Singh, R.K., Oreal, V., Reddy, G.U., et al. (2010) FACT prevents the accumulation of free histones evicted from transcribed chromatin and a subsequent cell cycle delay in G1. PLoS Genet 6: e1000964. Morohashi, N., Yamamoto, Y., Kuwana, S., Morita, W., Shindo, H., Mitchell, A.P., and Shimizu, M. (2006) Effect of sequence-directed nucleosome disruption on cell-type-specific repression by alpha2/Mcm1 in the yeast genome. Eukaryot Cell 5: 1925–1933. Morse, R.H. (2003) Getting into chromatin: how do transcription factors get past the histones? Biochem Cell Biol 81: 101–112. Müller, F., and Tora, L. (2014) Chromatin and DNA sequences in defining promoters for transcription initiation. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1839: 118–128. Murzina, N.V., Pei, X.-Y., Zhang, W., Sparkes, M., Vicente-Garcia, J., Pratap, J.V., et al. (2008) Structural basis for the recognition of histone H4 by the histone-chaperone RbAp46. Structure 16: 1077–1085. 61 Nabatiyan, A., and Krude, T. (2004) Silencing of chromatin assembly factor 1 in human cells leads to cell death and loss of chromatin assembly during DNA synthesis. Mol Cell Biol 24: 2853–2862. Natsoulis, G., Dollard, C., Winston, F., and Boeke, J.D. (1991) The products of the SPT10 and SPT21 genes of Saccharomyces cerevisiae increase the amplitude of transcriptional regulation at a large number of unlinked loci. New Biol 3: 1249–1259. Nelson, D.M., Ye, X., Hall, C., Santos, H., Ma, T., Kao, G.D., et al. (2002) Coupling of DNA synthesis and histone synthesis in S phase independent of cyclin/cdk2 activity. Mol Cell Biol 22: 7459–7472. Orphanides, G., LeRoy, G., Chang, C.H., Luse, D.S., and Reinberg, D. (1998) FACT, a factor that facilitates transcript elongation through nucleosomes. Cell 92: 105–116. Osakabe, A., Tachiwana, H., Takaku, M., Hori, T., Obuse, C., Kimura, H., et al. (2013) Vertebrate Spt2 is a novel nucleolar histone chaperone that assists in ribosomal DNA transcription. J Cell Sci 126: 1323–1332. Osley, M.A. (1991) The regulation of histone synthesis in the cell cycle. Annu Rev Biochem 60: 827–861. Owen-Hughes, T., and Workman, J.L. (1994) Experimental analysis of chromatin function in transcription control. Crit Rev Eukaryot Gene Expr 4: 403–441. Peterson, C.L., and Workman, J.L. (2000) Promoter targeting and chromatin remodeling by the SWI/SNF complex. Curr Opin Genet Dev 10: 187–192. Prajapati, H.K., Ocampo, J., and Clark, D.J. (2020) Interplay among ATP-Dependent Chromatin Remodelers Determines Chromatin Organisation in Yeast. Biology (Basel) 9: 190. Prelich, G., and Winston, F. (1993) Mutations that suppress the deletion of an upstream activating sequence in yeast: involvement of a protein kinase and histone H3 in repressing transcription in vivo. Genetics 135: 665–676. Prochasson, P., Florens, L., Swanson, S.K., Washburn, M.P., and Workman, J.L. (2005) The HIR corepressor complex binds to nucleosomes generating a distinct protein/DNA complex resistant to remodeling by SWI/SNF. Genes Dev 19: 2534–2539. 62 Ramey, C.J., Howar, S., Adkins, M., Linger, J., Spicer, J., and Tyler, J.K. (2004) Activation of the DNA damage checkpoint in yeast lacking the histone chaperone anti-silencing function 1. Mol Cell Biol 24: 10313–10327. Ransom, M., Williams, S.K., Dechassa, M.L., Das, C., Linger, J., Adkins, M., et al. (2009) FACT and the proteasome promote promoter chromatin disassembly and transcriptional initiation. J Biol Chem 284: 23461–23471. Richet, N., Liu, D., Legrand, P., Velours, C., Corpet, A., Gaubert, A., et al. (2015) Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork. Nucleic Acids Res 43: 1905–1917. Rufiange, A., Jacques, P.-E., Bhat, W., Robert, F., and Nourani, A. (2007) Genome-wide replication-independent histone H3 exchange occurs predominantly at promoters and implicates H3 K56 acetylation and Asf1. Mol Cell 27: 393–405. Russev, G., and Hancock, R. (1981) Formation of hybrid nucleosomes cantaining new and old histones. Nucleic Acids Res 9: 4129–4137. Schlesinger, M.B., and Formosa, T. (2000) POB3 is required for both transcription and replication in the yeast Saccharomyces cerevisiae. Genetics 155: 1593–1606. Schneider, S., Pei, Y., Shuman, S., and Schwer, B. (2010) Separable functions of the fission yeast Spt5 carboxyl-terminal domain (CTD) in capping enzyme binding and transcription elongation overlap with those of the RNA polymerase II CTD. Mol Cell Biol 30: 2353–2364. Scholes, D.T., Banerjee, M., Bowen, B., and Curcio, M.J. (2001) Multiple regulators of Ty1 transposition in Saccharomyces cerevisiae have conserved roles in genome maintenance. Genetics 159: 1449–1465. Schulz, L.L., and Tyler, J.K. (2006) The histone chaperone ASF1 localizes to active DNA replication forks to mediate efficient DNA replication. FASEB J 20: 488–490. Schwabish, M.A., and Struhl, K. (2004) Evidence for eviction and rapid deposition of histones upon transcriptional elongation by RNA polymerase II. Mol Cell Biol 24: 10111–10117. Schwabish, M.A., and Struhl, K. (2006) Asf1 mediates histone eviction and deposition during elongation by RNA polymerase II. Mol Cell 22: 415–422. 63 Sdano, M.A., Fulcher, J.M., Palani, S., Chandrasekharan, M.B., Parnell, T.J., Whitby, F.G., et al. (2017) A novel SH2 recognition mechanism recruits Spt6 to the doubly phosphorylated RNA polymerase II linker at sites of transcription. Elife 6: e28723. Sharp, J.A., Fouts, E.T., Krawitz, D.C., and Kaufman, P.D. (2001) Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing. Curr Biol 11: 463–473. Shi, L., Wen, H., and Shi, X. (2017) The Histone Variant H3.3 in Transcriptional Regulation and Human Disease. J Mol Biol 429: 1934–1945. Shibahara, K., and Stillman, B. (1999) Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell 96: 575–585. Simchen, G., Winston, F., Styles, C.A., and Fink, G.R. (1984) Ty-mediated gene expression of the LYS2 and HIS4 genes of Saccharomyces cerevisiae is controlled by the same SPT genes. Proc Natl Acad Sci U S A 81: 2431–2434. Singer, M.S., Kahana, A., Wolf, A.J., Meisinger, L.L., Peterson, S.E., Goggin, C., et al. (1998) Identification of high-copy disruptors of telomeric silencing in Saccharomyces cerevisiae. Genetics 150: 613–632. Smith, S., and Stillman, B. (1989) Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro. Cell 58: 15–25. Song, J.-J., Garlick, J.D., and Kingston, R.E. (2008) Structural basis of histone H4 recognition by p55. Genes Dev 22: 1313–1318. Stillman, B. (1986) Chromatin assembly during SV40 DNA replication in vitro. Cell 45: 555–565. Stuwe, T., Hothorn, M., Lejeune, E., Rybin, V., Bortfeld, M., Scheffzek, K., and Ladurner, A.G. (2008) The FACT Spt16 “peptidase” domain is a histone H3-H4 binding module. Proc Natl Acad Sci U S A 105: 8884–8889. Svaren, J., and Hörz, W. (1995) Interplay between nucleosomes and transcription factors at the yeast PHO5 promoter. Semin Cell Biol 6: 177–183. Svensson, J.P., Shukla, M., Menendez-Benito, V., Norman-Axelsson, U., Audergon, P., Sinha, I., et al. (2015) A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin. Genome Res 25: 872–883. 64 Takahata, S., Yu, Y., and Stillman, D.J. (2009) FACT and Asf1 regulate nucleosome dynamics and coactivator binding at the HO promoter. Mol Cell 34: 405–415. Tan, B.C.-M., Chien, C.-T., Hirose, S., and Lee, S.-C. (2006) Functional cooperation between FACT and MCM helicase facilitates initiation of chromatin DNA replication. EMBO J 25: 3975–3985. Tan, B.C.-M., Liu, H., Lin, C.-L., and Lee, S.-C. (2010) Functional cooperation between FACT and MCM is coordinated with cell cycle and differential complex formation. J Biomed Sci 17: 11. Tanae, K., Horiuchi, T., Matsuo, Y., Katayama, S., and Kawamukai, M. (2012) Histone chaperone Asf1 plays an essential role in maintaining genomic stability in fission yeast. PLoS One 7: e30472. Terrinoni, A., Dell’Arciprete, R., Fornaro, M., Stella, M., and Alberti, S. (2001) Cyclin D1 gene contains a cryptic promoter that is functional in human cancer cells. Genes Chromosomes Cancer 31: 209–220. Teves, S.S., Weber, C.M., and Henikoff, S. (2014) Transcribing through the nucleosome. Trends Biochem Sci 39: 577–586. Tyler, J.K., Adams, C.R., Chen, S.R., Kobayashi, R., Kamakaka, R.T., and Kadonaga, J.T. (1999) The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402: 555–560. Uwimana, N., Collin, P., Jeronimo, C., Haibe-Kains, B., and Robert, F. (2017) Bidirectional terminators in Saccharomyces cerevisiae prevent cryptic transcription from invading neighboring genes. Nucleic Acids Res 45: 6417–6426. VanDemark, A.P., Xin, H., McCullough, L., Rawlins, R., Bentley, S., Heroux, A., et al. (2008) Structural and functional analysis of the Spt16p N-terminal domain reveals overlapping roles of yFACT subunits. J Biol Chem 283: 5058–5068. Venter, U., Svaren, J., Schmitz, J., Schmid, A., and Hörz, W. (1994) A nucleosome precludes binding of the transcription factor Pho4 in vivo to a critical target site in the PHO5 promoter. EMBO J 13: 4848–4855. Verreault, A., Kaufman, P.D., Kobayashi, R., and Stillman, B. (1996) Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4. Cell 87: 95–104. 65 Viktorovskaya, O., Chuang, J., Jain, D., Reim, N.I., López-Rivera, F., Murawska, M., et al. (2021) Essential histone chaperones collaborate to regulate transcription and chromatin integrity. Genes Dev 35: 698–712. Vogel, K., and Hinnen, A. (1990) The yeast phosphatase system. Mol Microbiol 4: 2013–2017. Wang, T., Liu, Y., Edwards, G., Krzizike, D., Scherman, H., and Luger, K. (2018) The histone chaperone FACT modulates nucleosome structure by tethering its components. Life Sci Alliance 1: e201800107. Wei, W., Hennig, B.P., Wang, J., Zhang, Y., Piazza, I., Pareja Sanchez, Y., et al. (2019) Chromatin-sensitive cryptic promoters putatively drive expression of alternative protein isoforms in yeast. Genome Res 29: 1974–1984. Wen, Y., and Shatkin, A.J. (1999) Transcription elongation factor hSPT5 stimulates mRNA capping. Genes Dev 13: 1774–1779. Wier, A.D., Mayekar, M.K., Héroux, A., Arndt, K.M., and VanDemark, A.P. (2013) Structural basis for Spt5-mediated recruitment of the Paf1 complex to chromatin. Proc Natl Acad Sci U S A 110: 17290–17295. Williams, S.K., Truong, D., and Tyler, J.K. (2008) Acetylation in the globular core of histone H3 on lysine-56 promotes chromatin disassembly during transcriptional activation. Proc Natl Acad Sci U S A 105: 9000–9005. Winston, F., Chaleff, D.T., Valent, B., and Fink, G.R. (1984) Mutations affecting Ty-mediated expression of the HIS4 gene of Saccharomyces cerevisiae. Genetics 107: 179–197. Wittmeyer, J., and Formosa, T. (1997) The Saccharomyces cerevisiae DNA polymerase alpha catalytic subunit interacts with Cdc68/Spt16 and with Pob3, a protein similar to an HMG1-like protein. Mol Cell Biol 17: 4178–4190. Wyers, F., Rougemaille, M., Badis, G., Rousselle, J.-C., Dufour, M.-E., Boulay, J., et al. (2005) Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell 121: 725–737. Yague-Sanz, C., Vázquez, E., Sánchez, M., Antequera, F., and Hermand, D. (2017) A conserved role of the RSC chromatin remodeler in the establishment of nucleosome-depleted regions. Curr Genet 63: 187–193. 66 Yamaguchi, Y., Narita, T., Inukai, N., Wada, T., and Handa, H. (2001) SPT genes: key players in the regulation of transcription, chromatin structure and other cellular processes. J Biochem 129: 185–191. Yan, C., Chen, H., and Bai, L. (2018a) Systematic Study of Nucleosome-Displacing Factors in Budding Yeast. Mol Cell 71: 294-305.e4. Yan, X., Yang, J., Xu, J., Feng, J., and Li, Q. (2018b) Histone chaperone Spt16p is required for heterochromatin mediated silencing in budding yeast. Protein Cell 9: 652–658. Yang, J., Zhang, X., Feng, J., Leng, H., Li, S., Xiao, J., et al. (2016) The Histone Chaperone FACT Contributes to DNA Replication-Coupled Nucleosome Assembly. Cell Rep 14: 1128–1141. Yarragudi, A., Miyake, T., Li, R., and Morse, R.H. (2004) Comparison of ABF1 and RAP1 in chromatin opening and transactivator potentiation in the budding yeast Saccharomyces cerevisiae. Mol Cell Biol 24: 9152–9164. You, J.S., Kelly, T.K., De Carvalho, D.D., Taberlay, P.C., Liang, G., and Jones, P.A. (2011) OCT4 establishes and maintains nucleosome-depleted regions that provide additional layers of epigenetic regulation of its target genes. Proc Natl Acad Sci U S A 108: 14497–14502. Yuan, G.-C., Liu, Y.-J., Dion, M.F., Slack, M.D., Wu, L.F., Altschuler, S.J., and Rando, O.J. (2005) Genome-scale identification of nucleosome positions in S. cerevisiae. Science 309: 626–630. Zhang, H., Linster, E., Gannon, L., Leemhuis, W., Rundle, C.A., Theodoulou, F.L., and Wirtz, M. (2019) Tandem Fluorescent Protein Timers for Noninvasive Relative Protein Lifetime Measurement in Plants. Plant Physiol 180: 718–731. Zhou, X., and O’Shea, E.K. (2011) Integrated approaches reveal determinants of genome-wide binding and function of the transcription factor Pho4. Mol Cell 42: 826–836. Zhu, F., Farnung, L., Kaasinen, E., Sahu, B., Yin, Y., Wei, B., et al. (2018) The interaction landscape between transcription factors and the nucleosome. Nature 562: 76–81.
Thesis/Dissertation
eng
Biochemistry and Molecular Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Howe, LeAnn
Investigation of cryptic transcription suppressing factors using a FLO8:sfGFP reporter
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