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The ING1b protein facilitates accessibility to UV-damaged nucleosomal DNA Kuo, Weihong 2006

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THE ING IB PROTEIN FACILITATES ACCESSIBILITY TO UV-DAMAGED NUCLEOSOMAL DNA by Wei Hung Kuo B.Sc, University of British Columbia, 2003 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine) i THE UNIVERSITY OF BRITISH COLUMBIA June 2006 ©Wei Hung Kuo, 2006 Abstract INGlb is the most studied ING-family member protein and perhaps the most ubiquitous and highly expressed. This protein is involved in the regulation of various biological functions ranging from senescence, cell cycle arrest, apoptosis, to D N A repair. INGlb is upregulated by U V irradiation and enhances the removal of bulky nucleic acid photoproducts. We provide evidence that INGlb mediates nucleotide excision repair by facilitating access to damaged nucleosomal DNA. Our data suggest that this enhancement effect precedes the actions of damage recognition factor XPC, implicating a role of INGlb in the global genomic repair pathway. Through immunofluorescent imaging we demonstrate that INGlb does not colocalize with UV-induced D N A lesions and is therefore not part of core repair factors. INGlb also undergoes a nucleoli-nucleoplasm shift and increasingly associates with chromatin upon U V irradiation. Microccocal nuclease digestion of genomic DNA reveals however that INGlb facilitates chromatin relaxation and may allow better access to nucleotide excision repair machinery. INGlb can also alter histone acetylation dynamics upon exposure to U V light. Our preliminary observations suggest that p300 is likely a modulator of this event. More importantly, we found that INGlb facilitates DNA access to nucleotide excision repair factor, XPA, and is therefore important in the early steps of the 'Access, Repair, Restore' model. 11 Table of Contents Abstract i i Table of Contents iii List of Tables vii List of Figures viii List of Abbreviations x Acknowledgements xii 1 Introduction 1 1.1 D N A Repair 1 1.1.1 Major D N A Repair Pathways in Mammalian Cells 1 1.1.2 NER and Human Disorders 2 1.1.3 NER Factors 3 X P A 4 RPA 5 XPC 6 TFIIH 7 X P G 7 XPF-ERCC1 8 1.1.4 NER Subpathway - Global Genomic Repair 9 1.1.5 NER Subpathway - Transcription-coupled Repair 11 1.2 ING Family Tumour Suppressors 15 1.2.1 Discovery of ING Family Members 15 1.2.2 ING Family Protein Structures 17 1.2.3 ING Family Biological Functions 18 iii D N A Repair 18 Chromatin Remodeling 21 Yeast Chromatin Remodeling 21 Mammalian Chromatin Remodeling 25 Transcriptional Regulation 27 Cell Death and Cell Cycle Arrest 29 1.3 NER in the Chromatin 32 1.3.1 Dynamic Nature of Chromatin 32 1.3.2 Chromatin Rearrangement During NER 34 Chromatin is Refractory to Repair of Damaged D N A 34 'Access, Repair, Restore' Model 35 1.3.3 Chromatin Remodeling During NER 36 Histone Acetylation 36 ATP-dependent Chromatin Remodeling 38 1.3.4 Interplay between NER and Transcription 39 1.4 Hypotheses 41 1.5 Thesis Objectives 41 2 Materials and Methods 43 2.1 Cell Culture 43 2.2 siRNA and Transfections 43 2.3 Ultraviolet Irradiation 44 2.4 Host-cell-reactivation Assay 44 2.5 Reverse-transcription Polymerase Chain Reaction 45 iv 2.6 Chromatin Immunoprecipitation (ChIP) Assay 46 2.7 Immunofluorescence 47 2.8 Sulforhodamine-B (SRB) assay 48 2.9 Western Blot Analysis 48 2.10 Biochemical Fractionation 49 2.11 Micrococcal Nuclease Digestion Assay 50 2.12 Triton-X Protein Extraction 50 2.13 Flow Cytometric Analysi s 51 2.14 Immunoprecipitation 51 2.15 Statistical Analysis 52 3 Results 53 3.1 ING Family Members Enhance the Repair of UV-damaged Plasmid DNA 53 3.2 INGlb Does not Colocalize with Core Repair Factors 57 3.3 Histone Hyperacetylation Bypasses the NER Requirement for INGlb 57 3.4 siRNA knockdown of INGlb Reduces Global Histone H4 Acetylation • 62 3.5 Overexpression of INGlb Induces Chromatin Relaxation 65 3.6 Expression of INGlb Is Essential for DNA Lesion Detection 66 3.7 INGlb Depletion Causes Defective S phase D N A Damaging Response to U V Irradiation 75 3.8 Possible Involvement of p300 in ING lb-mediated Chromatin Relaxation 77 v 4 Discussion 81 4.1 ING Family Members NER of U V Lesions 81 4.2 ING lb-mediated Histone H4 Acetylation and Chromatin Relaxation Is Induced by U V 83 4.3 Benefits of Chromatin Relaxation 86 4.4 UV-induced INGlb Intranuclear Relocalization 89 5 Conclusion 92 6 References 96 vi List of Tables Table 1-1ING Protein Association with HAT and HDAC Components Vll List of Figures Figure 1-1 Step-wise Assembly of XP Factors During GGR 12 Figure 1-2 Emerging Model of TCR 16 Figure 1-3 ING Protein Structures 19 Figure 1-4 Hypothesis of ING lb-mediated Chromatin Relaxation During Early NER Response 42 Figure 3-1 Host-cell-reactivation (HCR) Assay of the Five ING Family Members 55 Figure 3-2 INGlb does not Colocalize with UV-induced 6-4 Photoproducts 58 Figure 3-3 Histone Deacetylase Inhibitors and Wild-type INGlb Enhance HCR at Comparable Levels 61 Figure 3-4 Cytotoxicity of U V Irradiation 63 Figure 3-5 INGlb Induced Histone Acetylation after UV-irradiation 64 Figure 3-6 INGlb Promotes Chromatin Accessibility to Micrococcal Nuclease Digestion after U V Irradiation 67 Figure 3-7 Biochemical Fractionation of Cytosolic, Nuclear and Chromatin-bound INGlb Protein 69 Figure 3-8 INGlb Intranuclear Localization Upon U V Irradiation 70 Figure 3-9 INGlb Binds to Chromatin and Confers Retention of Chromatin-bound X P A Protein after UV Irradiation 71 Figure 3-10 Efficient Recruitments of X P A and XPB to Damaged Chromatin were Absent in XPC-deficient Cells upon U V Irradiation 73 viii Figure 3-11 ING 1 b-mediated NER Enhancement Effect is Limited to the Presence of Functional XPC Protein 74 Figure 3-12 Depletion of INGlb Causes Defective S-phase D N A Damaging Response to U V Irradiation 76 Figure 3-13 U V Irradiation of S-phase Chromatin Generates an Accumulation of y-H2A.X 78 Figure 3-14 p300 Associates with INGlb and Modulates Global Histone H4 Acetylation upon U V Irradiation 79 Figure 5-1 A Working Model for the Chromatin Relaxation during GGR ... 95 ix List of Abbreviations Abbreviation Full Name 6-4 PP pyrimidine (6-4) pyrimidone photoproducts Y-H2A.X phospho-H2A.X AcH3 histone H3 acetylation/acetylated histone H3 AcH4 histone H4 acetylation/acetylated histone H4 ARR access, repair, restore CAF-1 chromatin assembly factor CAT chloramphenicol acetyltransferase CDK cyclin-dependent kinase ChIP chromatin immunoprecipitation CPD cyclobutane pyrimidine dimer CS cockayne syndrome DDB DNA damage-binding protein DSB double strand break GADD45 growth arrest & DNA damage 45 GGR global genomic repair HAT histone acetyltransferase HDAC histone deacetylase HCR host-cell-reactivation HR homologous recombination ING inhibitor of growth NER nucleotide excision repair NHEJ non-homologous end-joining NLS nuclear localization signal NTS nucleolar targeting sequence PCNA proliferating cell nuclear antigen PCR polymerase chain reaction PCR domain potential chromatin regulatory domain PHD plant homeodomain PIC preincision complex picNuA4 picclo-NuA4 PIP domain PCNA-interaction protein domain PtdIns(5)P phosphatidylinositol-5-phosphate RPA replication protein A RNAPII RNA polymerase II SB sodium butyrate siRNA small interfering RNA SRB sulforhodamine-B TCR transcription-coupled repair TFTC TBP-free TAF(II) complex Top topoisomerase TSA trichostatin A x trichothiodystrophy ultraviolet xeroderma pigmentosum xi Acknowledgements I would like to show my most sincere gratitude to the Michael Smith Foundation for Health Research, the UBC & Vancouver Hospital Foundation, and the National Science and Engineering Research Council for supporting my research. The recognitions awarded by these funding agencies mean tremendously to me as a scientist-in-training and open up so many opportunities for my future. Special thanks go to my research supervisor, Dr. Gang L i for his guidance. I am also very grateful for the fruitful scientific discussions with my committee members, Dr. Olive and Dr. Rennie. Thank you for your patience and help on my Master's study. I will always remember the friendship from Sarwat, Eric, Maryam, and Joseph. You have provided so much fun and laughter in the lab and made my time in the lab especially enjoyable. Lastly, I would like to thank Dr. Hao Xiao (ImmuneChem Pharmaceuticals Inc.) and Dr. Chi Chin-Wen (Taipei Veteran's General Hospital) for helping me to achieve my dreams and encourage me along the way to do better in my research and personal life. xii 1. Introduction 1.1 DNA Repair 1.1.1 Major DNA Repair Pathways in Mammalian Cells There are various endogenous and exogenous agents in our environment that can damage DNA. Inability to efficiently correct the DNA damage often leads to mutations, cell malfunctioning, or even cell death. In order to meet various genotoxic challenges, different organisms have developed a spectrum of mechanisms to maintain the integrity of DNA and thus the genome. There have been so far -130 human DNA repair genes cloned in human. However, a complete characterization of their functions is still lacking (Christmann et al, 2003). The DNA repair genes can be subcategorized into the damage response genes that regulates D N A repair and the actual genes that encode proteins with distinct functions that separate different DNA repair mechanisms (Scharer, 2003). There are 5 major D N A repair pathways in mammalian cells that have discrete or overlapping specificity to a variety of mutagenic chemically modified D N A lesions. The 'reversion repair' pathway employs principally the 06-alkylguanine transferase to directly revert the O^-alkylguanine adduct to guanine (Christmann et al, 2003). The 'base excision repair' pathway removes DNA damages result from deamination, alkylation, and oxidation through recognition and excision by D N A glycosylases (Scharer, 2003). The 'nucleotide excision repair' (NER) pathway removes primarily the bulky base adducts such as cyclobutane pyrimidine dimmer (CPD) and 6-4 pyrimidine pyrimidone photoproducts (6-4 PPs) caused by U V light. This pathway uses a sequential assembly strategy to recognize, excise, and resynthesize the D N A damage site. This pathway will be discussed in detail in the following sections. The 'mismatch repair' 1 pathway removes D N A damages arise from spontaneous or induced deamination, oxidation, methylation, and replication errors. The D N A strand containing an erroneous base is discriminated against from the normal template strand. This triggers the assembly of the repairosome that excises the DNA lesion and resynthesizes the single-stranded DNA gap (Scharer, 2003). Lastly, the 'double-strand break (DSB) repair' can be subdivided into the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways. The NHEJ system ligates the two ends of a DSB without sequence homology. HR repairosome mediates a physical contact between the damaged and undamaged DNA molecules with sequence homology. In this pathway, the undamaged homologous DNA is used as template for repair (Christmann et al., 2003). 1.1.2 NER and Human Disorders Xeroderma pigmentosum (XP) is an autosomal recessive disorder characterized by the physician Moritz Kaposi in the late nineteenth century. Later work by James Cleaver and Richard B. Setlow found that cells isolated from the XP patients are defective in NER (Friedberg, 2001). Typical X P symptoms are high susceptibility to skin cancer development with high occurrence of squamous cell carcinoma and basal cell carcinoma (de Snoo and Hayward, 2005). A wealth of evidence also suggests that the XP cells are particularly unable to remove base damages, especially those generated by exposure to the U V irradiation, leading to high mutation burden and neoplastic transformation. Seven genetic groups X P A - X P G have been identified in a series of complementation studies in which, nuclei from different XP patients were fused together to detect 2 restoration of defective repair synthesis phenotype (Cleaver, 2005). An additional XP variant, XPV, also produces the XP phenotype but does not confer a defective NER phenotype. X P V cells display different molecular pathology associating with an impairment of the accurate replicational bypass of UV-induced base damage (Masutani et al, 1999). Thus, XP arises primarily from the accumulation of mutations in skin cells that were unable to remove base damages through NER or replication-dependent bypass of DNA lesions. The correlation between XP clinical manifestations and defective NER or replicative bypass of UV-generated DNA damages supports the classic somatic mutation theory of cancer (Friedberg, 2001). There is clinical overlap between XP, Cockayne syndrome (CS), and trichothiodystrophy and (TTD). These latter conditions display neurological disorders and other obvious features (Friedberg, 2001). They do not, however, show predisposition to skin cancer. The two most common X P groups, X P A and XPC, show no CS or TTD syndromes. XPA patients have higher chances to develop anterior eye and tongue cancers and 20% of them have neurological disorders (de Snoo and Hayward, 2005). X P C is commonly mutated in the X P patients who have high susceptibility to developing skin cancer. Closer examination of cancer histology shows that the XP patients predominantly carry the lentigo malignant melanoma that associates with older age. This is in contrast with the general population whose melanoma type is superficial spreading (Cleaver, 2005). 1.1.3 NER Factors In mammalian cells, NER eliminates a broad spectrum of DNA lesions by excising 24-32 nucleotide-long oligomers surrounding the DNA damage. There are three major 3 steps involved in the NER pathway: (1) damage recognition; (2) dual incision and release of the excised oligomer; (3) resynthesis of the excised gap and ligation. NER is the main repair system for bulky D N A adducts such as cyclobutane pyridimine dimmer (CPD), 6-4 photoproduct, benzo(a)pyrene-guanine adduct, acetylaminofluorene-guanine, and cisplatin-d(GpG) diadduct (Friedberg, 2001). In humans, the only known pathway in removing CPD and 6-4 photoproducts that result from sunlight exposure is NER (Friedberg, 2001). The basic strategies employed in NER by prokaryotes and eukaryotes are similar. They involve in the sequential steps coordinated by specific protein-DNA interactions. The DNA damage is first recognized by an ATP-independent mechanism to form an unstable DNA-protein complex. This is followed by formation of the stable preincision complex by actions of the ATPase subunit to unwind DNA duplex at the expense of hydrolyzing ATP, allowing more intimate contact between protein and DNA. The last step involves in the incision of the nucleotide oligomers harboring D N A damage with 3' incision precedes slightly of the 5' incision (Araujo and Wood, 1999). The NER proteins and a number of structural motifs and protein-protein interactions play an essential role in aiding these NER steps. XPA X P A is a metalloprotein that binds to double-stranded DNA with a small preference for damaged DNA substrates (Reardon and Sancar, 2003). The central C-terminus DNA-binding domain has been mapped and structurally resolved by NMR. Two regions can be found within this region: a zinc-binding core that is important for protein-protein 4 interactions and a loop-rich domain involved in DNA binding (Buchko et al, 1998; Ikegami et al, 1998). Although the mechanism of X P A binding to D N A damage is still unclear, the loop-rich domain has recently been found to bind to the deformed helical kinks in the DNA substrate (Camenisch et al, 2006). X P A also binds cooperatively to DNA damage substrate with RPA and ERCC1. Besides these interactions, X P A also interacts avidly with XPF-ERCC1, RPA, and TFIIH (Reardon and Sancar, 2005). RPA Replication protein A (RPA) is a heterotrimeric polypeptides initially discovered to be essential for the initiation and elongation of replication. It was subsequently found to be able to bind damaged DNA. RPA consists of three subunits - p70, p32, and pl4. It has a profound impact on the recognition and the dual incision steps of NER (Reardon and Sancar, 2005). Various attempts to characterize the substrate specificity of RPA suggest that it binds primarily to single-stranded DNA. The bulky DNA adducts produce D N A helical distortion. RPA binds especially well to the single-stranded DNA loop opposite to the damage bases in the disrupted Watson-Crick in the DNA duplex (Patrick and Turchi, 1999). In addition, single-stranded nucleotide oligomers containing UV-induced damage also bind to RPA at higher affinity than the control DNA. This binding interaction is believed to be aided by both the non-specific DNA binding domain, the OB-fold domain, and the damage-specific zinc-finger domain in the p70 subunit (Lao et al, 2000). In addition to the cooperative binding to damaged base by X P A and RPA proteins, RPA engages in interactions with XPF-ERCC1 and XPG. In a pull-down essay of 5 XPF-ERCC1 and X P G nucleases using a nucleotide bubble that mimics preincision complex, it was found that RPA loads the two endonucleases to the bubble structure to allow proper NER (Matsunaga et al, 1996). It has also been suggested that RPA helps to orient the ERCC1 and X P G to the 5' and 3' of the damage base, respectively (de Laat et al, 1998a). XPC XPC is a polypeptide lacking any known structural motif except the transglutaminase-fold domain that localizes in the central region. This domain is believed to enhance protein-protein interactions. XPC binds preferentially to the damaged DNA with a high specificity to the distorted, single-stranded D N A helices (Kusumoto et al., 2001; Sugasawa et al, 2002). The DNA binding domain situates in the C-terminus that also partially overlaps with the HR23B protein binding motif. The extreme C-terminal 125 amino acids are crucial for efficient loading of the TFIIH complex onto the damaged D N A (Yokoi et al, 2000). Deletion of this peptide extremity completely abrogates the XPC activity on DNA damaged in vitro (Uchida et al, 2002). XPC can be copurified with the HR23B protein (Masutani et al, 1994). HR23B harbors the ubiquitin-associated and ubiquitin-like motifs (Masutani et al, 1997). The C-terminal of HR23B has been mapped to interact with the XPC partner (Uchida et al, 2002). Several in vitro studies suggested that addition of HR23B modestly stimulates the in vitro NER on the UV-damaged minichromosome (Sugasawa et al, 2996). The XPC-HR23B interaction has been found to stabilize XPC by protection against the proteosomal degradation (Ng et al, 2003). In addition, one centrosome protein (centrin 2) 6 also copurifies with XPC. It has been found that binding of CEN2 to X P C potentiates the DNA damage recognition function of NER. (Nishi et al, 2005) TFIIH TFIIH is a general RNA polymerase II transcription factor composed of several polypeptides. Analysis has shown that the purified TFIIH can participate in both transcription and D N A excision repair. The nomenclature of TFIIH subunits is defined as the ERCC genes (Reardon and Sancar, 2005). XPB (ERCC3) and XPD (ERCC2) have been found to be stable components of the TFIIH complex that are required for the reconstitution of excision nuclease activity in a purification study (Drapkin et al, 1994). XPB and XPD are both helicases that open up the D N A duplex surrounding the DNA damage site. XPB has a 3'-5' polarity while XPD has a 5'-3' polarity on the DNA substrate (Friedberg, 2001). Other TFIIH subunit such as p44 interacts with XPD, thereby stimulating its activity (Coin et al, 1998). The N-terminus of p44 is responsible for binding with XPD and the C-terminus is cysteine rich proposed to mediate protein-protein interactions (Reardon and Sancar, 2005). The p52 protein, like p44, attaches the XPB protein within TFIIH (Jawhari et al, 2002). XPG belongs to the FEN-1 family of nucleases with a 5'-3' exonuclease domain (Harrington and Lieber, 1994). X P G cleaves bubble, splayed D N A arms, and flap structures in vicinity to the 5' single-stranded DNA (Reardon and Sancar, 2005). The catalytic activity of X P G localizes to the 3' of the D N A lesion in D N A with a high 7 specificity at the single-stranded/double-stranded junction (Matsunaga et al, 1995; Hohl et al, 2003). Functional domains within XPG include X P G N at the N-terminus and the XPGI at the internal peptide region. Together, these two motifs make up the catalytic core of XPG (Constantinou et al, 1999). X P G can be copurified with the TFIIH subunits XPB, XPD, p62, and p42 (Mu et al, 1995; Iyer et al, 1996). X P G also interacts with RPA which stimulates its activity. The region of this interaction, however, has not been determined (Matsunaga et al, 1996). XPF-ERCC1 ERCC1 was copurified with XPF (ERCC4) from the HeLa cells that can complement the impaired excision activity of the XPF mutant cell extract (Park et al, 1995). ERCC1-XPF heterodimer has four helix-turn-helix motifs thought to bind to the DNA helix. The catalytic site of the complex is a metal-binding domain resides in the C-terminus of XPF (Enzlin and Scharer, 2002). XPF-ERCC1 cuts 5' to the D N A lesions. Additionally, it also cuts the loop and bubble structure at the D N A duplex close to the 5' of the single-stranded DNA junction (Matsunaga et al, 1996). Within the XPF-ERCC1 heterodimer, each subunit forms a tight interaction, making it difficult to discern the structure-specific nuclease activities to each subunit. Without any one of the subunits, the nuclease action is dramatically reduced (Gaillard and Wood, 2001). The peptide mapping study showed that the C-termini of XPF and ERCC1 are involved in the heterodimer formation (de Laat et al, 1998b). It has also been found that X P A can interact with XPF-ERCC1 to proper formation of the preincision complex (Nagai et al, 1995). 8 1.1.4 NER Subpathway-Global Genomic Repair NER consists of two distinct subpathways: the global genomic repair (GGR) and transcription-coupled repair (TCR). GGR is carried out by the six NER factors: XPA, RPA, XPC, TFIIH, X P G , and XPF-ERCC1. This is the key feature of NER characterized by sequential assembly of different proteins to generate the stable repairosome (preincision complex) (Friedberg, 2001). The DNA damage is first recognized by XPA, RPA, and XPC. This leads to recruitment of TFIIH and the unwinding of D N A duplex to create NER bubble. The latter step involves in the excision of the oligomer containing the DNA damage by X P G and XPF-ERCC1 (Figure 1-1). DNA damage recognition is considered the most important but the least understood step during NER. It is an unsolved question of whether XPC-HR23B, XPA, or RPA binds to the DNA lesion first and thus acts as the "damage sensor". The XPA-RPA complex binds to 6-4 photoproduct but at a low efficiency. In addition, XPC also appears to mainly recognize specific secondary DNA structure than binding specific D N A damage. Therefore, the current acceptable model lies in the cooperative binding between all the three damage detection factors in which the DNA lesion is randomly recognized by any of the three factors (Reardon and Sancar, 2005). The first bound factor then facilitates the loading of the remaining two factors that lead to a conformational change of the unstable protein complex into a stable, tightly bound complex (Reardon and Sancar, 2005). This event then recruits the remaining NER factors to the damage site. However, it has also recently been found that DDB, a heterodimer consisting of DDB1 and DDB2 (XPE) subunits, can bind to the D N A lesion first, particularly 6-4 photoproduct, and acts as the damage sensor (Hwang et al, 1999). It was proposed that the bound DDB recruits 9 and polyubiquitinates XPC through its interaction with cullin 4 A (ubiquitin ligase E3 complex). The polyubiquitinated XPC then binds at high affinity to 6-4 photoproduct and CPD to trigger the subsequent NER events (Sugasawa et al, 2005). This model is currently under intensive investigations by various laboratories world-wide. After the initial detection of D N A damage, the general transcription factor TFIIH is then recruited to the site of D N A damage with the help of XPC (Friedberg, 2001). The helicase activity of XPB and XPD in TFIIH unwind the DNA duplex to create an NER bubble around the DNA lesion about twenty nucleotides long by hydrolyzing ATP (Evans et al, 1997). This generates discrete double-stranded/single-stranded DNA junctions at the edge of the NER bubble that are important for the incision of DNA during NER. This leads to the formation of a stable protein complex termed preincision complex 1 (PIC1) (Reardon and Sancar, 2005). Next, XPC uses the energy derived from ATP hydrolysis to promote the entry of XPG to the D N A damage site. The complex is then termed PIC2 after X P C departs. XPF-ERCC1 then binds to PIC2 to form PIC3 (Reardon and Sancar, 2005). The 3' incision is then made by XPG. 5' incision is performed by XPF-ERCC1 (Christmann et al, 2003). The excised oligomer and most of the NER factors then leave the DNA duplex with RPA remaining bound to the gap. Lastly, the excised gap is filled by repair synthesis activity. RFC/PCNA and DNA polymerase 5/s mediate this process. DNA ligase 1 accessory factor then seals the gap (Friedberg, 2001; Christmann et al, 2005). This concludes the NER activity on D N A lesion site. 10 1.1.5 Nucleotide Excision Repair Subpathway-Transcription-coupled Repair In the mid 1980's, Hanawalt and his group presented a novel observation that NER in mammalian cells is faster in the transcriptionally active region than the silenced region of the genome (Hanawalt, 1994). Later studies showed that the faster NER kinetics is accounted for by more rapid repair of the transcribed strand of the gene compared with non-transcribed strand. This led to the identification of transcription-coupled repair (TCR) in NER (Christmann et al, 2003). Human and mice defective for XPC protein retain the capacity for TCR. Thus, XPC is not required for this pathway (Friedberg, 2001). The recognition of DNA lesions is thought to be caused by the stalled RNA polymerase II (RNAP II) by the blocking damage in the transcribed strand. So far, there is not an available in vitro model in which transcription by RNAPII can stimulate excision repair of base damage (Reardon and Sancar, 2005). Therefore, it is currently not possible to elucidate the mechanistic details of the TCR pathway. Two proteins are required for TCR. These proteins are associated with the two genetic complementation groups CSA and CSB. CS cells are hypersensitive to U V irradiation and display deficient TCR but proficient GGR (Christmann et al, 2003). CSA is a protein harboring the WD40 family repeats proposed to be a domain in multi-protein complex assembly (Henning et al, 1995). The biological function of CSA is poorly understood. It has been shown that CSA translocates into the nuclear matrix immediately after UV irradiation and colocalizes with hyperphosphorylated form of RNAPII in a 11 Figure 1-1. Step-wise assembly of XP factors during GGR. The global genomic repair (GGR) pathway involves stepwise recruitment of xeroderma pigmentosum (XP) factors onto a damaged D N A template during NER. The damage recognition factors X P A , RPA, XPC-TFIIH bind cooperatively in a random fashion to the photolesion. TFIIH (containing XPB and XPD helicases) then unwinds the DNA duplex to create the preincision complex 1. Departure of XPC and recruitment of 3' endonuclease XPG, forms the preincision complex 2. The recognition of this complex by 5' endonuclease XPF-ERCC1 results in formation of the preincision complex 3. X P G and XPF-ERCC1 then excise out the nucleotide oligomer containing the photolesion and the complex leaves the DNA damage site. RPA remains bound to the gap, allowing subsequent gap resynthesis by DNA polymerase 5. Figure adapted from Reardon and Sancar (2005). 12 X 1 / 111111 r^ Tl 1111111 Pre-incision complex 1 Pre-incision complex 2 5'Incision 3" Incision Pre-incision complex 3 Resynthesis 13 CSB-dependent fashion (Kamiuchi et al, 2002). CSB is an ATPase-containing protein with DNA translocase activity that can allow RNAPII to progress through the transcription blocking site (Selby and Sancar, 1997a; Selby and Sancar, 1997b). There is also a helicase motif within CSB protein but its function has not been assigned (Reardon and Sancar, 2005). The molecular mechanisms of TCR are just starting to emerge. In the current model of TCR (Figure 1-2), it is believed that the CSB and XPG protein are the initiators of the pathway by recognizing and binding to the stalled RNAPII. The bound CSB and XPG then cooperatively recruit TFIIH to the transcription bubble. The presence of XPG also stimulates the ATPase activity of the CSB to remodel histone or other DNA-bound proteins (Sarker et al, 2005). TFIIH then hydrolyzes ATP to remodel RNAPII complex, allowing access of X P G to upstream of the transcription bubble. Subsequent recruitments of XPA, RPA, and XPF-ERCC1 then lead to excision of DNA lesion without removal of RNAPII (Reardon and Sancar, 2005; Sarker et al., 2005). In vitro evidences also show that the excision gap synthesis can occur in the presence of RNAPII (Tremeau-Bravard et al, 2004). How CSA participates in TCR in remains unclear. Other models of TCR have also been proposed but are less likely to be the predominant TCR pathway. RNAPII may skip the DNA lesion to allow TCR (Friedberg, 2001). However, this has been shown to introduce high mutation rate in the DNA strand opposite to the DNA lesion (termed "transcriptional mutagenesis") (Bregeon et al, 2003). Moreover, RNAPII can also back up 1 nucleotide from the D N A lesion with the aid of transcription elongation factor TFIIS (Tornaletti et al, 1999). This remains to be 14 vigorously tested as CSB can antagonize the activity of TFIIS (Selby and Sancar, 1997b; Tremeau-Bravard et al, 2004). Lastly, the classic proteosomal degradation of RNAPII to allow proper NER has been suggested to be the backup mechanism when all attempts to repair DNA lesion via TCR fail (Sarker et al, 2005). 1.2 ING Family Tumour Suppressors 1.2.1 Discovery of ING Family Members In 1996, Dr. Riabowol at University of Calgary identified the first member of the Inhibitor of Growth Family (ING), ING1, through subtractive hybridization in breast cancer cell lines. Once overexpressed in primary cells, ING1 construct promoted inhibitory growth phenotype while chronic anti-sense ING1 oligonucleotide expression led to cellular transformation in primary cell cultures (Garkavtsev et al, 1996). Since discovery of ING 1, four additional ING genes ING2-5, have been identified in humans and classified as ING family proteins through sequence homology search with ING1 (Shimada et al, 1998; Nagashima et al, 2003; Shiseki et al, 2003). Phylogenic analysis shows that ING proteins are present not only in other vertebrates but also other kingdoms including plants, fungi and protista (He et al, 2005). ING family members have been shown to impinge different aspects of cell physiology including D N A repair, chromatin remodeling, transcriptional regulation, cell death and cell cycle control (Campos et al, 2004a) and are currently under intensive investigations. 15 Figure 1-2. Emerging model of TCR. The elongating RNAPII stalled at the photolesion is recognized in a cooperative manner by the cockayne syndrome B (CSB) and XPG proteins. Recognition of this complex by TFIIH and CSA with the help of CSB and X P G leads to remodeling of RNAPII complex by TFIIH. This exposes the photolesion allowing access of X P G to the transcription bubble. Subsequent recruitment of XPA, RPA, and XPF-ERCC1 then enables excision of DNA lesion in the presence of RNAPII. The gap is then resynthesize by polymerase 8. Figure adapted from Sarker et al. (2005). Pol II A Damage of Pol II Incision & Repair 16 1.2.2 ING Fam ily Protein Structures ING 1-5 proteins have several highly conserved domains proposed to be responsible for different functions. As Figure 1-3 shows, the two most obvious prominent features are the N-terminal potential chromatin regulatory (PCR) domain and the C-terminus plant homeodomain (PHD) finger. The PCR domain spans 125 amino acids and is proposed to bind to various HAT/HDAC complexes and serve as a critical region to regulate ING-mediated chromatin remodeling (He et al., 2005). The PHD finger has a prominent Cys4-His-Cys3 feature that binds to positively charged zinc ions. It has been recently found to bind to phosphatidylinositol-5-phosphate during D N A damage response (Gozani et al., 2003). In fact, the PHD finger is found in a variety of chromatin-associated proteins and is essential for their functions (Aasland et al., 1995). For example, the PHD finger of CBP HAT is crucial for its catalytic HAT activity (Kalkhoven et al, 2002). ING proteins are mostly nuclear. A basic region identified as the nuclear localization sequence (NLS) located between the PCR and PHD domain is essential for ING protein nuclear localization (Campos et al., 2004a). Within the NLS domain lays three nucleolar targeting signal (NTS) motifs that are responsible for shuttling the ING proteins to the nucleolus (He et al., 2005). The exact functions of the ING intranuclear locale remains to be functionally identified. One novel conserved domain recently proposed is the leucine-zipper like (LZL) motif consisting of leucine residues spanning every seven amino acids, forming a hydrophobic patch (Campos et al, 2004a). This N-terminal L Z L motif is found on ING2-5 and has been proposed to mediate homodimerization or heterodimerization between ING proteins and other transcription factors (He et al., 2005). 17 The PCNA-interaction protein (PIP) domain is only present in INGlb and is responsible for interaction with PCNA, a protein involved in growth inhibition, replication, and repair (Campos et al, 2004a). 1.2.3 ING Gamily Biological Functions DNA Repair The first study reporting INGlb's involvement in nucleotide excision repair of UV-induced D N A lesions was published in 2001. Cheung et al. (2001) found that overexpression of INGlb construct promoted the repair of UVC-damaged DNA plasmid to a level 2-4 fold higher than that of the vector control. In addition, INGlb overexpression also enhanced the clearance of the CPD lesions generated by U V B irradiation. By comparing to control, INGlb transfected cells displayed accelerated lesion clearance in the human melanoma M M R U cells. Through immunoprecipitation of the endogenous protein, INGlb was also found to associate with a UV-inducible factors growth arrest and D N A damage 45 (GADD45), which enhances cell survival upon genotoxic stresses (Gupta et al, 2006), but not X P A and XPB. In this thesis we propose that the enhancement effect on DNA repair by the ING family members is related to their associated chromatin remodeling activities. Histone acetylation adds moieties to the N-terminal histone tails in the nucleosomes to induce relaxed, chromatin structures. This confers accessibility of damaged D N A to different protein machineries including DNA repair factors. ING family members reside in a spectrum of HAT/HDAC complexes that regulate chromatin structures and it was found 18 Figure 1-3. ING protein structures. Several domains are highly conserved among ING family proteins. The N-terminal potential chromatin regulatory (PCR) domain and the C-terminal plant homeodomain (PHD) motif are two prominent features which define ING proteins. The nucleolar targeting sequence (NTS) locates within the nucleolar localization signal (NLS) of the ING 1-2 proteins. In addition, the hydrophobic leucine zipper domain is common to ING2-5 but not INGlb. Lastly, the PCNA-interaction protein (PIP) domain is unique to INGlb. PCR , ,NLS& NTS PHD INGlb PCR PHD 1 PCR NLS & NTS PHD | 1 I Leucine • 1 * Zipper 1 l * V „ " * * PCR NLS & NTS "* PHD 1 1 I 1 ING1a ING1c ING2 Leucine Zipper PCR NLS PHD 1NG3 Leucine Zipper PCR NLS PHD NLS Leucine' Zipper PCR -'••NL'S S , PHD NLS ING4 INGS 19 that the mammalian ING1 yeast homologue Yng2 mutant or INGlb siRNA-treated cells present significant decreased level of histone H4 acetylation. These cells are also sensitive to either DNA damaging agent, U V and topoisomerase inhibitors (Choy and Kron, 2002). In addition, ING lb-mediated histone H4 acetylation also enhances the relaxation of chromatin and the binding of NER factor, XPA, to UV-damaged chromatin. Results from this study link ING lb-mediated chromatin remodeling to DNA repair. One recent report from our laboratory also indirectly supports this hypothesis. In the attempt to search for naturally occurring INGlb mutations, Campos et al., (2004b) identified two mutations R102L and N260S as common alterations in human melanoma biopsies. Further analysis of the R l 02L and N260S found that the two mutants are detrimental to NER. It should be noted that codon 102 and 260 reside in the putative PCR domain and PHD domain, respectively. Mutations within these regions are thought to perturb the chromatin remodeling functions of ING family proteins and their ability to facilitate DNA repair. Besides histone acetylation, ING family members are also known to acetylate the p53 tumour suppressor protein. ING2, ING4, and ING5 cooperate with the p300 HAT to acetylate Lys-382 residue on the p53 protein, while INGlb is proposed to antagonize HDACs capable of de-acetylating p53 (Nagashima et al, 2001; Kataoka et al, 2003; Shiseki et al, 2003). Acetylation of p53 at this site after various D N A damages, such as U V irradiation, has been shown to increase its protein stability by antagonizing the MDM2-regulated protein ubiquitination and degradation pathway (Nagashima et al, 2001; Campos et al, 2004a). The acetylated p53 avidly binds D N A and is able to activate wafl transcription after generation of DNA damage (Gu and Roeder, 1997; 20 Sakaguchi et al, 1998; Kataoka et al, 2003). It is likely that ING family members influence D N A repair activities by modulation of p53 acetylation. This possibility, however, remains to be thoroughly tested as ING family members can also induce acetylated p53 to upregulate box to induce cell death following D N A damage (Kataoka, 2003). Chromatin Remodeling Yeast Chromatin Remodeling Studies from various groups suggest that the ING family members associate with a spectrum of HAT cofactors from yeast to human (Table 1-1). There are five families of HATs: Gcn5-related acetyltransferases (GNATs), the MYST-related HATs, the p300/CBP HATs, general transcription factor HATs and nuclear hormone-related HATs (Carrozza et al, 2003; Sterner and Berger, 2000). The mammalian and yeast ING proteins have been found to be a stable component of the MYST-related HATs, GNATs complexes and p300/CBP. The first report relating the ING family to histone acetylation and chromatin remodeling was published in year 2000. Through database searching, three Saccharomyces cerevisae (Yngl, Yng2, and Pho23) and two Schizosaccharomyces pombe (Pngl and Png2) proteins were found to share high homology to human INGlb. Deletion of yng2 leads to pleiotropic phenotypes, including slow growth and increased sensitivity to U V 21 Table 1-1 ING Protein Association with HAT & HDAC Components Protein Associated Components Complex Involved Species Metho d Reference INGlb RBP1; Sin3; HDAC1/2: RbAp48; RbAp46; Sap30; Brg1; BAF155; p42; p35 mSin3A Human MS; AP Skowyra et al., J Biol Chem 276(12):8734-9, 2001; Kuzmichev et al., Mol Cel Biol. 22(3):835-48, 2002 INGlb D300/CBP; PCNA p300 Human IP Vieyra et al., J Biol Chem 277(33):29832-9, 2002 INGlb P/CAF P/CAF Human IP Vieyra et al., J Biol Chem 277(33):29832-9, 2002 INGlb hSir2 hSir2 Human IP Kataoka et al., Cancer Res 63(18):5785-92, 2003 ING1a HDAC1 HDAC1 Human IP Vieyra et al., J Biol Chem 277(33):29832-9, 2002 ING2 Sin3: HDAC1/2: RbAp48; RbAp46; Sap30 mSin3A Human MS; AP Kuzmichev et al., Mol Cel Biol. 22(3):835-48, 2002 ING3 Tip60; TRAPP; BAF53a; RUVBL1/2; EPC1; ' MRG15; DMAP1; hDomino; Brd8; Actin hNuA4/Ti p60 Human IP; AP; MS Doyon et al., Mol Cell Biol 24(5): 1884-96, 2004 ING4 p300; p53 p300 Human IP Shiseki et al., Cancer Res 63(10):2373-8, 2003 ING4 hEaf6; HB01; JADE1/2/3 HBOT Human AP; MS Doyon et al., Mol Cell 21(1 ):51 -64, 2006 ING5 p300; p53 p300 Human IP Shiseki et al., Cancer Res 63(10):2373-8, 2003 ING5 hEaf6; HB01; JADE1/2/3 HB01 Human AP; MS Doyon et al., Mol Cell 21(1):51-64, 2006 ING5 hEaf6; Brpf 1/2/3; MOZ/MORF MOZ/MO RF Human AP; MS Doyon et al., Mol Cell 21(1 ):51 -64, 2006 Yng1 Sas3; Anc1/Taf30 NuA3 Yeast IP; MS; AP Howeet al., Mol Cell Biol 22(14):5047-53, 2002; Nourani et al., J Biol Chem 278(21 ):19171-3, 2003 Yng2 Tra1; Esa1; Epl1; Arp4; Act1 ; Eaf 1; Eaf2; Eaf3; Eaf4; Eaf5; Eaf6, p53 NuA4 . Yeast Y2H; IP; GST; AP Loewith etal., Mol Cell Biol 20(11):3807-16, 2000; Choy et al., J Biol Chem 276(47):43653-62, 2001; Nourani et al., Mol Cell Biol. 21(22):7629-40, 2001; Nourani et al., J Biol Chem 278(21 ):19171-5, 2003 Pho23 Rpd3; Sap30; Sin3 Rpd3-Sin3 Yeast AP Nourani et al., J Biol Chem 278(21 ):19171-5, 2003 Y2H = Yeast-two-hybrid IP = Immunoprecipitation MS = Mass Spectrometry GST = Glutathione-S-transferase Pulldown Assay AP = Affinity Purification 22 irradiation. These defects can be, however, rescued by expression of the human INGlb or S. pombe Pngl, indicative the highly conserved functional properties (Loewith et al, 2000). The authors also found Yng2p can interact with Tral, an ATM/phosphatidylinositol 3-kinase-related homolog of the human TRRAP cofactor that acts as an accessory subunit of various HAT complexes (Loewith et al, 2000). Further biochemical purification reveals that Yng2p is a stable component of the NuA4 histone H4 and H2A HAT complexes (Nourani et al., 2001). In the absence of Yng2p, the NuA4 complex is present suggesting the role of Yng2p in activating or maintaining NuA4 HAT activity (Choy et al., 2001). In support of this, the Ayng2 mutant displays a global loss of acetylated histone H4 and H2A with delayed mitotic progression. Treatment of the HDAC inhibitor, trichostatin A (TSA), could restore both the histone H4/H2A acetylation status and the mitotic defects in the yeast mutants. Interestingly, introduction of the N-terminus of Yng2p could rescue the mutant phenotypes. The PHD-fmger remains dispensable for the Yng2p-mediated phenotypic consequences but affects the NuA4 activity on nucleosomal histones (Choy et al, 2001; Nourani et al, 2001; Selleck et al, 2005). Recently, a trimeric form of NuA4, termed Piccolo NuA4 (picNuA4), compromised of Yng2p, Esal (catalytic subunit of NuA4), and Epl l has also been purified and characterized in vitro. Both NuA4 and picNuA4 show a preference on chromatin over free histones. The substrate specificity is preserved in picNuA4 to target histone H4 and H2A, like NuA4 counterpart. However, unlike NuA4, picNuA4 does not interact with well-defined transcription activators VP 16, Gcn4, and Hap4, to acetylate histones in vitro. This leads to the proposal that picNuA4 is responsible for the global, nontargeted 23 histone H4/H2A acetylation whereas NuA4 is mainly recruited to specific genomic loci and perturb the local histone acetylation/deacetylation dynamic (Boudreault et al, 2003). Ynglp has been purified together with the MYST histone H3/H4 HAT, NuA3 complex whose catalytic subunit Sas3 is the mammalian MOZ proto-oncogene product. Deletion of yngl does not affect the NuA3 HAT activity but affects the nucleosomal substrate recognition. This indicates that Ynglp might tether the NuA3 complex to nucleosome for histone acetylation. In addition, Ynglp also genetically interacts with the Gcn5 and Sas3 to produce a lethal phenotype, implicating its role in maintaining genomic integrity similar to Yng2p (Howe et al, 2002). Surprisingly, deletion of sas3 or yngl leads to increased transcriptional activation by p53 (Nourani et al., 2003). This opens a new avenue for the study of undefined HAT-specific transcriptional repression. The last yeast ING family member, Pho23p, is a stable component of the Rpd3/Sin3 HDAC complex, which are the mammalian HDAC1 and HDAC2 homologs (Loewith et al, 2001; Nourani et al, 2003). Deletion of Pho23p not only abrogates Rpd3/Sin3 HDAC activity but also generates cellular hypersensitivity to cyclohexamide and heat shock and, more importantly, enhanced silencing at the rDNA, telomeric, and the yeast mating type HMR loci. These phenotypes are reminiscent to the A.rpd3 mutant and underscore the importance of Pho23/Rpd3/Sin3 complex in antagonizing gene silencing at the centromeric regions (Loewith et al, 2001). However, unlike Yng2p, Pho23p has been found to negatively regulate p53-mediated transcription of p21 w a f l . This fits to the classic histone deacetylation/transcription repression model and suggests Pho23/Rpd3 have distinct functions to modulate chromatin structures at different genomic regions. 24 Mammalian Chromatin Remodeling The majority of ING1 is part of the mSin3/HDAC complexes, similarly to the yeast Pho23p ING homolog (Skowyra et al, 2001; Kuzmichev et al; 2002 Vieyra, et al, 2002). ING2 is also co-purified in the same complexes albeit with a few novel polypeptides (Skowyra et al, 2001; Doyon et al, 2006) (Table 1-1). In addition, in vitro HDAC analysis showed that ING 1-associated complex deacetylates both acetylated histone H3 and H4 and lead to repression at the reporter gene promoters. Although it was not tested, ING2-associated complex profile is believed to share the same substrate specificity. It is interesting to note that a subset of ING1 also associate with the Brgl-based-Swi/Snf complex whose activity also enhances ING 1-mediated gene repression (Kuzmichev et al, 2002). Besides histone deacetylation, overexpression of INGlb induces a global increase in histone H3 and H4 acetylation whereas INGlb causes a decrease of histone acetylation. Precipitation of INGlb could recover a greater amount of HAT activity compare with INGla, suggesting that INGlb possesses a more robust association with HAT complex. This suggestion is corroborated by the interaction of INGlb with p300 (histone H3/H4 HAT complex), TRRAP (accessory subunit to M Y S T HAT complexes), PCAF (subunit of the Gcn5 GNAT HAT complex involved in histone H3 acetylation), and CBP (involved in acetylation of histone H2A, H2B, H3 and H4) while INGla associates predominantly with HDA'Cl complex (Table 1-1). Additionally, depletion of INGlb protein also leads to a significant decrease in histone H4 acetylation (Vieyra et al, 2002). ING3 is identified as a component of the Tip60 complex (Table 1-1), an equivalent complex to yeast NuA4. The Tip60 complex preferentially acetylates histone H2A, and 25 H4 requires the PHD domain of ING3 for nucleosomal HAT activity (Doyon et al, 2004; Doyon et al., 2006). Like Yng2p, ING3-Tip60-p53 pathway is essential for upregulation of the p53-responsive gene wafl. Interestingly, the recombinant trimeric complex formed by Tip60, EPC1, and ING3 can reconstitute nucleosomal histone H2A and H4 acetylation like the yeast picNuA4 (Doyon et al, 2004). ING4 is purified in a novel tetrameric H B O l HAT complex (including H B O l , JADE1/2/3, ING4, and hEaf6) (Table 1-1). The complex targets not only histone H4 but also H3 to a lesser extent and is responsible for the majority of the histone H4 acetylation in cells. Depletion of HB01-ING4 HAT activity leads to impaired cell growth, a phenotype analogous to yeast ING1 homolog mutant. ING5 is purified in either H B O l or the MOZ/MORF complex (Table 1-1), a HAT known to induce oncogenic transformation. The HB01-ING5 shows a preference for histone H4 on chromatin. In contrary, the MOZ/MORF-ING5 targets only histone H3. Functional characterization reveals that the HOB1-ING5 can coprecipitate with stoichiometric amount of the prereplicative initiation complex M C M helicases (MCM4, 6, and 7). Depletion of ING5 also results in defective D N A synthesis, demonstrating that the HB01-ING5 and MOZ/MORF-ING5 HAT activity in S phase. Because of the similarity of the complex architecture and also substrate specificity, Doyon et al. (2006) categorized the ING4/ING5 complex as the picNuA4-related HAT. The role of HOB1-ING4 HAT remains to be identified. It is also worth noting that ING4 and ING5 have both been discovered to interact with p300 HAT by immunoprecipitation but functions of such interaction have never been tested (Shiseki et al, 2003). 26 Transcriptional Regulation INGlb was initially linked to transcriptional regulation by the virtue of its association with p53 and enhancement of transcriptional activity at the p21 w a f l promoter. Microarray analysis identified several INGlb-downregulated onco- or protooncogenes including AFP (marker of hepatocellular carcinoma), CCNB1 (cyclin BI , overexpressed in various cancers), TIS11 (involved in growth and survival pathway), D E K (proto-oncogene in acute myeloid leukemia), TDE (lung tumour marker), osteopontin (cancer metastasis), TPT1 (protein in malignant transformation) as well as upregulation of other genes, including various ribosomal genes (Takahashi et al, 2002). Most cases seem to involve interdependence between the INGlb and p53 tumour suppressors. For example, both INGlb and p53 are thought to be required to efficiently upregulate p21 w a f l (Campos et al., 2004b). In p53-null cell lines ING1 also failed to properly regulate the expression of cyclin B I , suggestive of the high dependency of ING1 function on the functional status of p53 (Takahashi et al., 2002). Like INGlb, the other human ING family members are also implicated in transcriptional regulation. ING2-3 proteins have been found to enhance Bax transcription (Kataoka et ai, 2003; Nagashima et al., 2003). In addition, like INGlb/c, ING2-5 can enhance the p21 w a f l promoter activity (Nagashima et al., 2001; Shiseki et al, 2003). ING4 is capable of binding N F - K B and promote the downregulation of various angiogenic factors, including interleukins IL-6, IL-8, prostaglandin-endoperoxide synthase 2 and colony-stimulating factor 3. ING4-mediated transcriptional activity has also been shown to inhibit angiogenesis of brain tumours in a murine model (Garkavtsev et al, 2004). Furthermore, ING4 has been implicated in the attenuation of the HIF transcriptional 27 activity under hypoxia as to inhibit tumour progression (Ozer et al, 2005). Besides negative gene regulation, Doyon et al. (2006) showed that ING3 and ING5 can enhance the expression of RUNX2-dependent promoters (important for T cell lymphomagenesis). The exact mechanism of ING family regulation of gene transcription is still under scrutiny. Studies done in yeast suggest a direct linkage of HAT/HDAC chromatin remodeling activities to promoter activation/suppression. In S. cerevisiae, p53 has been shown to physically interact with the NuA4 complex. Although it does not interact with the p53 directly, the ING family member Yng2p is shown to be a critical mediator of the p53-responsive genes and other NuA4 target genes. Deletion of the Yng2p gene results in localized deficiency of histone H4 acetylation at the wafl promoter and abrogates p53-dependent transcription. It was hypothesized that recruitment of ING 1-containing HAT complexes by p53 enhances its transcriptional activity at its downstream promoters (Nourani et al, 2001; Nourani et al, 2003). On the contrary, the Rpd3/Pho23 complex is proposed to cause histone deacetylation at the promoter and inhibit transcription. Deletion of either pho23 or rpd3 leads to upregulation of p21 w a f I . This is supported by evidence that the mSin3 subcomplex, Brgl-mSin3 can enhance reporter gene Gal4 promoter (Kuzmichev et al, 2002). NuA3, however, presents a unique scenario whose activities lead to a novel HAT-mediated transcription repression. Expression of NuA3/Yngl complex promotes reduced wafl promoter activity. Nourani et al. (2003) proposed that NuA3-induced histone H3 acetylation is detrimental to the p53 transactivation at the wafl promoter. Nonetheless, this scheme remains to be tested as NuA3 HAT action can also antagonize the silencing effect at HMR mating loci (Howe et al, 2002). 28 Cell Death and Cell Cycle Arrest ING proteins have also been implicated in cell cycle checkpoints. Expression of INGlb, lc, and ING2-5 leads to the upregulation of the cyclin-dependent kinase inhibitor (CDKI) p 2 1 w a n ( G a r k a v t s e v e t a / 5 1 9 9 8 . Kataoka et al, 2003; Shiseki et al, 2003). Wafl is a potent regulator of the G\ checkpoint that binds to cyclin-Cdk complexes to achieve inactivation. The inhibited Cdk complexes display less phosphorylation activities towards the retinoblastoma protein (Rb) and causes cell cycle arrest at the G i / S boundary (Harper et al, 1993). For this reason, overexpression of ING family proteins produces to a Gi cell cycle arrest phenotype with reduced colony formation in the tissue cultures (Nagashima et al, 2001; Nagashima et al, 2003; Shiseki et al, 2003). Interestingly, these functions are highly dependent on the functional presence of p53. Deletion or mutation of p53 proteins in various cell types significantly reduces the ING family-mediated p21 w a f l transcriptional regulation and Gi arrest (Zhu et al, 2005). One recent study also reports reduced cyclin E-associated kinase activity on recombinant Rb protein in cells overexpressing INGlb (Ohgi et al, 2002). Since cyclin E is involved in Gi/S phase progression, ING proteins are likely to regulate Gi arrest through various mechanisms that negatively affect C D K activities. Several reports suggest a regulatory role of ING proteins in G 2 cell cycle control. INGlb can attenuate expression of cyclin B l (Takahashi et al, 2002), a regulatory subunit of the G 2 cyclin-dependent-kinase 1 (CDKI). Since p53 can repress both cyclin B l mRNA and protein levels, this suggests that there exists a cooperative mechanism between the ING family and p53 tumour suppressor. Indeed, co-expression of ING1, 4, and5 and p53 can induce a more prominent G 2 / M arrests than expression of either suppressor gene alone 29 (Shiseki et al, 2003; Tsang et al, 2003). It was also been found that the INGlb protein level is inversely correlated to the cyclin BI level (Campos et al, 2004a). While INGlc protein level increases in late Gi and decreases in G2, cyclin BI level rises during G2/M phase of the cell cycle and is rapidly degraded at the end of mitosis (Porter and Donoghue, 2003). This suggests a role of ING proteins in the cell cycle checkpoint regulation. Recent publications also implicate the ING family members in the S phase cell cycle regulation. In a screen for synthetic lethal interaction with the yeast Ayng2 mutant, genes that affect DNA metabolism have been found to produce toxic phenotype in combination with yng2 deletion mutant. Examples of these genes are CDC7 (origin firing), CDC9 (DNA ligation), GCN5 (histone H3 HAT), TOPI (topoisomerase 1), RAD50 (break resection during and homologous recombination and non-homologous recombination repair), and RAD 52 (homologous repair) (Choy and Kron, 2002). Mutant Ayng2 cells also display impaired meiotic and delayed intra-S-phase checkpoint response (Choy and Kron, 2002). It was proposed that the histone H4 HAT mediated by Yng2p is important to maintain a relaxed chromatin environment for DNA metabolic activity, since treatment of Ayng2 mutant with HDAC inhibitor trichostatin A (TSA), could rescue all the above-mentioned phenotypes (Choy and Kron, 2002). This is further reinforced by the recent discovery that the ING4-HOB1 HAT complex and ING5-MOZ/MORF HAT complexes are essential for the normal S phase progression in mammalian cells (Doyon et al, 2006). 30 Initial discovery of the ING proteins in apoptosis was reported in the Xenopus tadpole metamorphosis model. It was observed that both ING1 and ING2 expressions are induced in tissues, such as tail and brain, which undergo extensive apoptosis during metamorphosis while the ING expressions remained low in limbs where cells proliferate and grow (Wagner et al, 2001). Consistent with this, senescent human fibroblasts were shown to express more ING1 mRNA compared to early passage fibroblasts, while early passage fibroblasts were able to upregulate INGlb expression and enter apoptosis upon growth factor deprivation (Garkavtsev and Riabowol, 1997). In later studies, it was also reported that expression of either ING2 or ING3 also enhances apoptosis to various chemotherapeutic agents (Nagashima et al., 2003). INGs and p53 also cooperate in sensitizing cells to apoptosis. By stable transfection of either ING1 or p53, or both, it was found the two proteins have a synergistic effect in suppressing colony formation by inducing apoptosis in fibroblasts, glioma, esophageal carcinoma, and melanoma cells (Garkavtsev et al, 1996; Garkavtsev et al, 1998; Shimada et al, 2002; Shinoura et al, 1999). It has also been found that INGlb enhances transactivation of proapoptotic Bcl-2 family protein Bax in response to U V irradiation (Cheung et al, 2002). Other than transcriptional regulation of p21 w a f l and Bax, ING proteins also have a distinct mechanism to promote apotosis. INGlb harbors a PCNA-interacting protein (PIP) box that permits physical interaction with PCNA (Figure 1-3). In addition, all ING proteins contain a nucleolar targeting sequence (NTS) that allows protein translocation into the nucleolus, the transcriptionally active area in the nucleus. Upon U V irradiation, 3 1 INGlb quickly mobilizes to the nucleolus and increasingly associates with PCNA to induce apoptosis (Scott et al, 2001; Campos et al, 2004a). Disruption of any of these molecular events protects cells from entering apoptosis. 1.3 NER in the Chromatin 1.3.1 Dynamic Nature of Chromatin The fundamental repeating subunits of chromatin structure is the nucleosome core particle, formed by 147bp of D N A wrapping twice around the histone octomers consisting of 2 copies of each histone (H2A, H2B, H3, and H4). In a typical nucleosome core particle, the D N A contacts the histone octomer 14 times with a 10 base periodicity (Luger, 2003). This generates high steric hindrance to the access of various proteins. The chromatin structure has inherent flexibility contributed by the molecular nucleosomal properties - the intrinsic fluidity of nucleosome structure and mechanisms that regulate the histone-DNA contacts (Becker, 2002; Peterson, 2002). Cells employ molecular players such as ATP-dependent chromatin remodellers, post-translational histone modifiers, histone chaperones, and DNA-processing events such as transcription and replication to affect the nucleosomal states (Gontijo et al, 2003). Most of the D N A repair events happen at the nucleosomal level. It is, thus important to understand the interplay between chromatin and repair factors. With the nucleosome reconstituted in vitro from DNA and purified histone octomers, it has been found that it can exist in the closed, inaccessible state and the open, transiently exposed state (Gontijo et al, 2003). The degree of DNA relaxation gradually decreases towards the center of nucleosome core. It has been observed that the restriction enzyme can digest even the 32 most inner, in accessible part of the core, albeit at a very low efficiency (Polach and Widom, 1995). Subsequent crystal structural analysis of the nucleosome later confirmed the plasticity of the nucleosome particle (Luger, 2003). For D N A repair, this means that the damage recognition factor can eventually bind to the damaged D N A substrate if sufficient time is given. Various factors need however to be taken into consideration, such as a high degree of heterogeneity within genomic nucleosomal structures, differences in D N A sequences, the presence of histone variants as well as non-histone chromatin structural proteins (Green and Almouzni, 2002). Together the higher order structure of D N A can present a physical barrier to any DNA metabolic activity. For this reason, D N A damage surveillance within chromatin is the rate-limiting event in a repair pathway as the higher chromatin structures in the cells also have profound impact on the accessibility of DNA repair factors to DNA. The linker histone H1 that facilitates higher-order folding of chromatin from the linear nucleosome fiber has been found inhibitory to the DNA nuclease digestion, such as micrococcal nuclease (Vincent et al, 2002; Woodcock and Dimitrov, 2002). On the other hand, the chromatin chaperone high-mobility group (HMG) proteins that fluidize chromatin were observed to enhance the enzymatic entry to the chromatin environment (Agresti and Bianchi, 2003). As chromatin constituents vary between different intranuclear domains (euchromatin versus heterochromatin) and genomic loci (silenced genes versus actively transcribed genes), the rate of DNA repair in these regions will also differ (Gontijo et al, 2003). 33 1.3.2 Chromatin Rearrangement during NER Chromatin is Refractory to Repair of Damaged DNA Chromatin is constantly under the assault of DNA-damaging agents. It also serves as the primary structure D N A repair factors operate upon. Multiple evidences have shown that DNA damage in the chromatin structure is initially refractory to repair. In 1974, Wilkins and Hart first discovered that UV-irradiated human permeable cell chromatin is less accessible to the CPD cleavage enzyme than chromatin treated with high salt to strip away histones. This observation is substantiated by increased D N A nuclease sensitivity in mouse mammary tumour cells treated with methanesulfonate and in human fibroblasts irradiated with U V light. Using human cell extracts or purified NER factors, it has also been shown that the SV40 minichromosomes or plasmid DNA loaded with nucleosome are less accessible to NER activities than the naked DNA (Araki et ai, 2000). Dr. Smerdon and his colleagues also reported that the hyperacetylated chromatin induced by the histone deacetylase (HDAC) inhibitor, sodium butyrate, has enhancing effects on excision repair. The impact of chromatin structure on NER is further supported by studies in Saccharomyces cerevisiae. By analyzing NER activities in the four different regions of the minichromosome, it has been found that the repair activity correlates highly with the transcription rate. The NER efficiency is particularly high in the transcribed strand of the reporter marker URA3 gene. When the URA3 gene is inserted into the telomeric region of the genome, the spread of the heterochromatic chromatin structures into the reporter gene block the entry of the photolyase enzyme that can directly reverse the UV-induced DNA 34 lesions. Together, these results suggest the 'open' state of the chromatin is facilitative to the NER reaction. The nucleosomal array can also affect the NER activities. The repair of CPD is significantly inhibited by the mononucleosome regardless of the nucleosomal position (Liu and Smerdon, 2000). By employing the dinucleosome template, the NER on 6-4 photoproduct is strongly reduced even when the lesion is in the linker D N A region. This result suggests that NER machinery requires a space greater than the linker DNA to excise the DNA damage substrate (Ura et al., 2001). In addition, as opposed to naked DNA, nucleosomes also significantly inhibit NER of the site-specific platinum-DNA adduct upon incubation with mammalian cell free extracts. Furthermore, the D N A damage detection factors, XPC-HR23B, XPA, and RPA also show less affinity to the nucleosomal D N A compared with naked DNA (Hara et al, 2000). As a result, it appears that nucleosome is the primary determinant on NER kinetics. 'Access, Repair, Restore'Model In 1970's, initial observations of chromatin alterations during repair were published that led to the development of the 'unfolding-refolding' model during NER. In the UV-irradiated human chromatin, the newly synthesized D N A becomes more accessible to nuclease digestion. However, this nuclease sensitivity gradually decreases over time, suggestive of the incorporation of nucleosomes into the D N A after initial repair. This model is extended by Green and Almouzni (2001) into the 'Access, Repair, Restore' (ARR) model which depicts the initial perturbation of chromatin structure to allow repair machinery to access the D N A damage with the final restoration of canonical 35 nucleosomal organization after the departure of repair factors. The A A R model has been applied to several other major DNA repair pathways including DSB repair and base excision repair. Recent research advances enable us to understand the A A R model in better details. The novel repair-coupled nucleosome assembly was discovered by simultaneous nucleosome assembly during DNA synthesis stage in NER (Gaillard et al, 1996). Kosmoski et al. (2001) also observed similar phenomenon in which nucleosome formation occurs favorably on the UV-damaged naked DNA in the presence of excessive amounts of undamaged DNA (Kosmoski et al, 2001). Biochemical studies determined that the chromatin assembly factor I (CAF-1), which deposits histones on newly replicated DNA, mechanistically links repair synthesis to nucleosome formation (Gaillard et al, 1996). In addition, the histone chaperone anti-silencing function 1 (Asfl) is also required for this pathway by directly interacting with CAF-1 (Mello et al, 2002). The human proliferating cell nuclear antigen (PCNA), an essential component of the DNA replication fork, is also required in this repair-coupled nucleosome assembly pathway way by loading CAF-1 to the repair site (Moggs et al, 2000). 1.3.3 Chromatin Remodeling during NER Histone Acetylation Early studies show that histone hyperacetylation induced by sodium butyrate can maximally enhance NER repair in the nucleosome. Recent findings further support the role of histone acetylation in facilitating NER. The p300 HAT can physically interact with PCNA through out the cell cycle and the formed complex enhances DNA synthesis 36 in vitro. Chromatin immunoprecipitation (ChIP) assay shows that p300 associates with newly synthesized DNA after U V irradiation. It is likely that p300 exerts its chromatin remodeling actions to assist PCNA function during DNA synthesis in N E R (Hasan et al, 2001). One in vitro study using cisplatin-damaged nucleosomes to analyze histone post-translational modifications shows that the anti-cancer drugs caused a hyperacetylation of histone H4 to enhance NER (Wang et al., 2003; Gong et al, 2005). Additionally, one group also observed hGCN5 histone H3 HAT associates with TBP-free TAF(II) complex (TFTC). One subunit, SAP130, of TFTC binds preferentially to UV-damaged DNA. A parallel recruitment of both TFTC complex and X P A to UV-damaged DNA has been observed in HeLa cells. This leads to acetylation of histone H3 both in vivo and in vitro (Brand et al, 2001). Immunoprecipitation experiment also shows that X P A binds to p300 HAT (Hasan et al, 2001). These data suggest that TFTC-or p300-mediated histone acetylation allows better entry of D N A damage recognition factors to the chromatin. Furthermore, another DNA damage binding factor, DDB2 (XPE) associates with the CBP/p300 HAT in vivo and in vitro. According to this finding, it is proposed that DDB2 brings the CPB/p300 HAT activities to damaged-chromatin during GGR to recruit other repair factors (Datta et al, 2001). Additionally, another hGCN5-containing HAT complex, STAGA, also copurifies with the DDB1 protein (Martinez et al, 2001). Together, these reports suggest a dependency of DNA damage recognition on histone acetylation activities during NER. 37 Lately, different groups have observed that UV-irradiation leads to a genomic histone H3 and H4 hyperacetylation that precedes the DNA damage recognition event in both yeast and mammalian cells (Yu et al, 2005). Using the yeast repressed MFA2 promoter, it was found that histone H3 acetylation at Lys-9 and Lys-14 is induced by U V irradiation. This acetylation spike enhances the removal of nucleosome-embedded CPD lesions and the accessibility of restriction to the DNA. This event is, however, independent of the Rad4 (XPC) and Radl4 (XPA) D N A damage recognition factors. In the same study, it was determined that GCN5 is the major HAT that mediates this process (Yu et al, 2005). UV-promoted histone H3 acetylation at Lys-9 by p53 in mammalian cells also share similar biological activity. It allows greater accessibility of micrococcal nuclease to the damaged genome upon U V irradiation. This effect is thought to be mediated by the p53-p300 complex (Rubbi and Milner, 2003a). These studies indicate that the relaxation of chromatin prior to the actual NER events might be crucial for the DNA lesion detection process. ATP-dependent Chromatin Remodeling There is accumulating evidence of the involvement of ATP-dependent chromatin remodeling factors in D N A repair. The SWI/SNF2 family proteins share a conserved ATPase subunit which might allow better access of repair factors to the damaged DNA. The ACF complex contains the SNF2 family member imitation switch (ISWI) and Acfl can catalyze nucleosome movement, allowing timely removal of DNA lesions from the internucleosomal linker regions (Ura et al, 2001). Incubation of the in vitro NER system with SWI/SNF and IWS2 factors can also modulate damage reversion repair by photolyase (Gaillard et al, 2003). The activity of SWI/SNF can also be stimulated by 38 XPA, RPA, and XPC in vitro to accelerate removal of 6-4 photoproduct but not CPD from the internucleosomal core (Hara and Sancar, 2002; Hara and Sancar, 2003). Although the exact mechanisms remain to be identified, it would be of great interest to understand how ATP-dependent chromatin remodeling complexes are recruited to the DNA damaged site and how they influence the dynamics and fluidity of nucleosomes for promoting DNA repair. 1.3.4 Interplay between NER and Transcription TCR removes DNA lesions from the transcribed strand of the gene. DNA is compacted in the condensed chromatin structure. The pathway utilizes the transcription machinery to survey DNA lesions by the stalled RNAPII within the 'open' transcription bubble and avoids the inhibitory effect imposed by chromatin on repair (Gong et al, 2005). Whether the stalled RNAPII blocks the repair of DNA lesion remains controversial. It has been shown that the RNAPII stalled at the pyridimine dimmer does not inhibit excision of lesions by the reconstituted human excision nucleases (Selby et al, 1997). However, by comparing the half-life of nearby lesions on the complimentary strand (same position, opposite strand) in the yeast CSB-deficient cells, it has been found that certain internucleosomal D N A lesions are repaired more slowly in the transcribed strand than in the non-transcribed strand. NER in these more accessible sites is thought to be hindered by RNAPII in the transcribed strand (Tijstermand and Brouwer, 1999). The CSB protein belongs to the SNF2 family. It has been hypothesized to use its SNF2-like ATPase activity to alter the molecular architecture of stalled RNAPII-DNA complex during TCR (Svejstrup, 2002). The rearranged interactions will facilitate 39 smooth repair by allowing the entry of other repair factors. The recombinant CSB has recently shown to alter the digestion profile of nucleosomes in vitro, suggesting this protein does indeed have remodelling activity (Citterio et al, 2000). Another challenge of TCR is the large heterogenous genomic organization (Gong et al, 2005). In eukaryotes, TCR initiation sites vary from gene to gene. For example, in the yeast RPB2 and GAL1 genes, TCR starts approximately 40 and 180 nucleotides upstream of the transcription start sites, respectively (Li and Smerdon, 2002). However, in the yeast URA3 genes, TCR start sites is only limited to the transcribed region (Tijsterman et al, 1997). The human Jun, CDC2, and PGK upstream promoter sites have also been observed to have slow NER activity. It has been proposed that the bound transcription factors in the promoter sequence might inhibit the repair machinery accessibility to the DNA lesions (Gong et al, 2005). On the other hand, one in vitro study using reconstituted chromatin also shows that transcription activators bound to the gene promoters can activate NER (Frit et al, 2002). It was shown that the bound transcription activators can induce a localized ATP-dependent chromatin remodeling and histone acetylation to enhance NER (Frit et al, 2002). Therefore, transcription factors may facilitate NER indirectly by allowing TCR through RNA polymerase recruitment or by GGR through chromatin remodellers recruitment. 40 1.4 Hypotheses We hypothesize that the INGlb protein can be recruited to sites of DNA damage and promote a localized chromatin relaxation. This event will then lead to an increased recruitment and binding of NER lesion detection factors to their damaged D N A substrate in the chromatin. Depletion of the INGlb protein will disrupt or at least reduce the DNA repair efficiency. Finally, since ING family members share several conserved domains, we further hypothesize that like their INGlb counterpart, the remaining family members can also enhance NER of UV-induced DNA lesions (Figure 1-4). 1.5 Thesis Objectives The notion that the compact nature of chromatin is obtrusive to NER is well supported by published literature (Reardon and Sancar, 2005). Factors that facilitate the relief of condensed chromatin during NER have only begun to be identified. It has recently been shown that the p53 tumour suppressor can act as an accessibility factor that facilitates chromatin relaxation through histone H3 hyperacetylation, improving global lesion detection following U V irradiation (Rubbi and Milner, 2003a). Since ING lb-mediated enhancement of NER is dependent on the p53 protein (Cheung et al, 2001), we suspect that INGlb may also share a similar molecular mechanism which enhances the removal of UV-induced D N A lesions. Thus, the objectives of this thesis are to: (1) determine if INGlb is part of the core NER machinery at the site of DNA lesions; (2) determine if INGlb can promote a global chromatin relaxation upon UV irradiation; (3) determine ING lb-induced chromatin relaxation enhances core NER factors recruitment (e.g. lesion detection); and (4) determine if there is a potential cooperative mechanism between p53 and ING family proteins in NER. 41 Figure 1-4. Hypothesis of INGlb-mediated chromatin relaxation during early N E R response. INGlb and its associated HAT complex is hypothesized to mediate early NER response by enhancing histone acetylation leading to local/global chromatin relaxation. The highly conserved PHD domain is responsible for tethering the ING-HAT complexes to the chromatin for efficient histone acetylation. This process grants accessibility to early NER factors to recognize photolesions in the UV-damaged chromatin. Latter assembly of repairosome can also be affected by this early event. Time post-UV irradiation 42 2. Material and Methods 2.1 Cell Culture The M M R U melanoma cell line was a kind gift from Dr. R. Byers, Boston University School of Medicine. The U87 (passage 7) and SF88 glioblastoma cell lines were kindly Dr. W. Jia, University of British Columbia. Human fibroblasts NHFB, XP44RO (XPC deficient) and XP44RO+XPC (stably expressing wild-type XPC) cell lines were gifts from Dr. A. Sarasin, Centre National de la Recherche Scientifique UPR2169. These cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen, Burlington, ON, Canada) in the presence of 100 units/ml penicillin, 100 pg/ml streptomycin and 25 ug/ml amphotericin B in a 5% C02 at 37°C atmosphere. Only cells cultured before passage 13 were used in our experiments. 2.2 siRNA and Transfections The chloramphenicol acetyltransferase (CAT) reporter plasmid p C M V o r was kindly provided by Dr. L. Grossman, Johns Hopkins University. The pCIneo-INGlb-FLAG plasmid has been previously described (Campos et al, 2004). Plasmids were introduced into cells using the Effectene transfection kit (Qiagen, Mississauga, ON, Canada) at a ratio of 1 pg DNA per 25 pi Effectene as recommended by the manufacturer. Three synthesized siRNA oligomers (Qiagen, Mississauga, ON, Canada) specific for the following INGlb target sequences were used: siRNA-1, 5' -C A A G A C C T C C A A G A A G A A G A A - 3 ' ; siRNA-2, 43 5 ' -ACCCACGTACTGTCTGTGCAA-3 ' ; and siRNA-3 (against the ING1 UTR region), 5' -TTGGTACACGTGTAACAAGAA-3 ' . siRNA was introduced into cultured cells using the SilentFect reagent (Bio-Rad, Mississauga, ON, Canada) according to the manufacturer's instruction. siRNA and plasmid co-transfections were also carried out using the SilentFect reagent as described with the addition of 1 u-g plasmid DNA per 10 cm2 of culture surface. 2.3 Ultraviolet Irradiation U V irradiation was performed by removing media and exposing cells to controlled doses of UVB (290-320 nm) light using a bank of four unfiltered FS40 sunlamps (Westinghouse, Bloomfield, NJ) or U V C (220-290 nm) light using a UltraLum crosslinker. For in vivo localized irradiation of nuclear DNA, cells were grown on glass cover-slips and media removed. An isopore disc PC hydrophilic filter with 5-um pores (Millipore, Cambridge, ON, Canada) was gently placed over cells prior to U V irradiation. Cells were immediately fixed upon exposure to UV or incubated for a determined amount of time with culture media prior fixing. 2.4 Host-cell-reactivation Assay The host-cell-reactivation assay was performed as previously described (Campos et al., 2004). In summary, the CAT reporter plasmid p C M V o r was irradiated ex vivo at 40 mJ/cm2 using an UV-cross-linker and used for transfection. The irradiated plasmid was then co-transfected with one of the five major human ING genes (ING1-5) or one of the 44 INGlb mutant N260S and R102L constructs. Forty hours post transfection, cells were harvested and the cell-free extracts were obtained through repeated freeze/thaw cycles using liquid nitrogen. Each assay reaction mixture contained 7.5 pi of 5 mM chloramphenicol, 1 pi of 2.5 mM [3H]-acetyl-CoA, and 16.5 pi of distilled water in addition to collected cell-free extract. The reaction was then incubated at 37°C for 90 min. To terminate the reaction, 200 pi of water-saturated ethyl acetate was added. The organic phase was back-extracted in distilled water, isolated and dried. The amount of radioactivity was determined using a scintillation counter. A l l reactions were performed in triplicates. Controls include co-transfection with an undamaged reporter plasmid and transfection with empty vector. To induce histone hyperacetylation, 200 ug/ml trichostatin A (TSA) or 5 mM sodium butyrate (SB) was added to the cell culture media 24 h after transfection. Fresh media were replaced 9 h after incubation. 2.5 Reverse-transcription Polymerase Chain Reaction Transfected M M R U cells were extracted with TriZol to obtain mRNA. Five micrograms of total RNA were reverse transcribed into cDNA in the presence of lOunits/uL of Superscript II RNase H reverse transcriptase (Canadian Life Technologies), 5 x first strand buffer (250mM/L Tris-HCl pH 8-3, 375 mM/L, KCI and 15mM/L MgCl 2), 100 mM/L dithiothreitol, 10 mM/L dNTP mix (10 mM/L of each of dATP, dCTP, dGTP, and dTTP at pH 70) and 2 pmol of the INGlb forward primer (5'-GATCCTGAAGGAGCTAGACG-3*) in a total volume of 20 uL. The reverse transcription (RT) mix was then incubated at 42 °C for 2 min. Reaction was inactivated by heating at 70 °C for 15 min. The polymerase chain reaction (PCR) reaction contained 45 10% of the first strand reaction, 10 x PCR buffer (200mM/L Tris-HCl pH 8-4 and 500 mM/L KC1), 50 mM/L MgCl 2 , 10 mM/L dNTP mix, 10 pM/L of the INGlb forward primer (5 '-GATCCTGAAGGAGCTAGACG-3') , 10 mM/L of the reverse primer (5 ' -AGAAGTGGAACCACTCGATG-3') and 5 units/uL of Taq D N A polymerase. PCR was performed under the following conditions: 94 °C, 3 min; 60 °C, 1 min; 72 °C, 2 min; 25 cycles. Thermal cycles were terminated by polymerization at 72 °C for 10 min. PCR products were then resolved by electrophoresis in a 1.5% agarose gel and visualized under U V light after ethidium bromide staining. 2.6 Chromatin Immunoprecipitation (ChIP) Assay M M R U cells were seeded at 60% confluency and transfected with the pCMV or pCMVCAT plasmid. Twenty-four hours later, cells were washed with PBS and cross-linked with 1% formaldehyde for 10 min at room temperature. The reaction was then stopped with addition of 125 mM glycine for 5 min at room temperature. Cells were washed once with PBS and lysed by scraping with 0.25% Triton-X solubilization buffer (Triton-X, 50 mM Tris-Cl[pH 7.7], 10% glycerol, 100 mM NaCl, 2.5 mM EDTA) containing 100 mg/L salmon sperm DNA and protease inhibitor cocktail described above. Extracts were sonicated 5 times for 5 sec at the setting 8 using the Microson sonicator. Samples were centrifuged for 5 min at 12,000 xg, resuspended in solubilization buffer, and probed with either rabbit anti-H3 antibody or pre:immune rabbit immunoglobulins (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. Immunoprecipitations were then incubated with protein A agarose beads (Sigma, Missisauga, ON, Canada) at 4°C for 1 h. Immunoprecipitates were elutated with elution 46 buffer (1% SDS, 0.1 M NaHC0 3 ) at room temperature for 15 min. Five M NaCI was subsequently added to reverse cross-linking reaction at 65°C for 2hs. To recover the ChIP DNA, samples were treated with 20 ug Proteinase K (Sigma, Missisauga, ON, Canada) for 1 h at 45°C with I M Tris-Cl (pH 6.5) and 0.5 M EDTA. Sample DNA was then extracted with phenol/chloroform and PCR with the P C M V Q A T primers (forward 5 ' -CCTATAACCAGACCGTTCAG-3' , reverse 5 ' -TCACCGTAACACGCCACATC-3') using the conditions described above. 2.7 Immunofluorescence Cells were cultured at a density of 2 x 104 cells per 10x10 mm glass cover-slips. Prior to fixation, cells were washed three times with PBS, then simultaneously fixed and permeabilized with 2% paraformaldehyde, 0.5% Triton X-100 in PBS at 4°C for 30 min. Cover-slips were then washed three times for 5 min each with PBS and then blocked overnight with normal goat serum diluted to 10% in PBS at 4°C. Cells were then gently inverted into primary antibody (rabbit anti-FLAG (Sigma, Oakville, ON, Canada), mouse anti-6-4PP or mouse anti-CPD antibodies (kind gift from Dr. T. Matsunaga, Kanazawa University), or anti-XPA antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:50 for 1 h at room temperature. Cover-slips were washed three times for 5 min each with PBS and incubated at room temperature for 1 h with Cy3-conjugated goat anti-rabbit IgG or Cy5-conjugated goat anti-mouse at 1:500 dilution (Jackson ImmunoResearch, West Grove, PA). DNA was stained with 2 mg/ml Hoechst 33258 for 1 min. Cover-slip were washed three times for 5 min each and gently inverted into Permount mounting media (Fischer Scientific, Ottawa, ON, Canada) over a 47 microscope glass slide. Pictures were captured using a CCD camera on a Zeiss Axioplan 2 microscope. 2.8 Sulforhodamine-B (SRB) cell survival assay Cells were grown in 24-well plates to reach 50% confluency. The culture was irradiated with UVC at various doses. Twenty-four hours after U V irradiation, cell survival was determined with the sulforhodamine B (SRB) (Sigma-Aldrich, Mississauga, ON) assay as described previously (Casalini et al., 2001). Briefly, after treatment, the medium was removed and the cells were fixed with 500 pi of 10% trichloroacetic acid for 1 h at 4°C. The cells were then washed four times with distilled water and the excess water removed by aspiration. The cells were air-dried and then stained with 500 pi of 0.4% SRB (dissolved in 1% acetic acid) for 30 min at room temperature, washed four times with P/o acetic acid, and air-dried. The cells were then incubated with 500 pi of 10 mM Tris (pH 10.5) on a shaker for 20 min to solubilize the crystallized dye. Spectrometric readings were then taken at 550 nm for 100 pi aliquots. 2.9 Western Blot Analysis Cells were directly solubilized in lysis buffer (50 mM Tris-Cl [pH 8.0], 150 mM NaCI, 0.02% sodium azide, 0.1% SDS, 1% Nonidet P-40) in the presence of protease inhibitors (100 pg/ml phenylmethylsulfonyl fluoride, 1 ug/ml aprotinin, 1 ug/ml leupeptin, 1 ug/ml pepstatin A) (Roche, Mississauga, ON, Canada) and left on ice for 15 min. Cells were then disrupted by sonicating three times for 5 s using a Microson sonicator at setting 8. 48 Cell lysates were then centrifuged at 12,000x g for 30 min at 4°C and supernatants kept for analysis. The concentration of proteins was determined using the DC Protein Assay (Bio-Rad). Thirty pg of proteins were loaded per well and resolved by 15% SDS-PAGE and electrotransferred onto polyvinylidene difluoride (PVDF) filters (Bio-Rad). After drying in methanol, the membranes were subsequently incubated with primary antibodies at 4°C overnight followed by three washes in 0.04% Tween-20 PBS (PBS-T) for 5 min each and subsequent incubation with HRP-conjugated secondary antiserum for 1 h at room temperature. Blots were washed and signals detected using the SuperSignal enhanced chemiluminescence substrate (Pierce, Rockford, IL). The primary antibody dilutions were 1:2000 for rabbit anti-histone H3 and H4, 1:100 for rabbit anti-acetylated histone H3 and histone H4 (Upstate, Charlottesville, VA) , 1:750 for rabbit anti-XPA, 1:250 for mouse anti-INGl, 1:500 for rabbit anti-ILK, 1:250 for mouse anti-p53 (Santa Cruz Biotechnology), 1:1000 for rabbit anti-ERK antibodies, and 1:5000 for mouse anti-/?-actin (Sigma). 2.10 Biochemical Fractionation Chromatin isolation was performed as previously described (Mendez and Stillman, 2000). In brief, chromatin was collected by lysing the cells in membrane lysis buffer (10 mM HEPES [pH. 7.9], 10 mM KC1, 1.5 mM MgCl 2 , 0.34 M sucrose, 10% glycerol, 1 mM DTT, protease inhibitor cocktail as described above with 0.1 % Triton-X. Nuclei were collected by centrifugation at 900x g for 4 min at 4°C and washed once with membrane lysis buffer with 0.1% Triton-X. The supernatant was collected as cytoplasmic fraction for western blot analysis. Nuclei were then lysed with the nuclear 49 lysis buffer (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, protease inhibitor cocktail. Chromatin was pelleted by centrifugation at 2,000x g for 4 min at 4°C and washed once with nuclear lysis buffer. Supernatant was collected as the nucleoplasmic fraction. Chromatin fraction was subsequently resuspended in 2x Laemmli buffer and sonicated three times for 15 s using a Microson sonicator at setting 10. Each fraction was then resolved in 15% SDS-PAGE and analyzed by western blot analysis. 2.11 Microccocal Nuclease Digestion Assay Microccocal nuclease digestion assay was performed as previously described (Smith et al, 1998). In brief, cells were resuspended in lysis buffer (10 m M Tris-HCl [pH 8.0], 10 mM MgC12, 1 m M DTT) with 0.1% NP-40. Nuclei were pelleted by 1,200* g for 10 min and washed twice with the lysis buffer. For digestion, nuclei were resuspended in the micrococcal nuclease digestion buffer (10 mM Tris/HCl [pH 8.0], 50 mM NaCI, 300 mM Sucrose, 3 mM MgC^). Samples were then digested with 0 or 0.1 U of micrococcal nuclease for 5 min at 37°C (Sigma). The reaction was stopped by adding 1% SDS and 20 mM EDTA. Samples were centrifuged at 12,000* g for 15 min. D N A was then extracted twice with phenol/chloroform, dissolved in distilled water, and electrophoresed in 1.5% agarose gel and visualized under U V light after ethidium bromide staining. 2.12 Triton-X Protein Extraction The technique was performed as previously described (Svetlova et al, 1999). Briefly, Normal or transfected cells treated with various U V C doses (from 10-400 mJ/cnr) were 50 harvested by scraping into PBS with 0.1% Triton-X for 5 min on ice. Cells were washed twice with PBS and pelleted by 900x g for 4 min at 4°C. The detergent-resistant proteins were resuspended in lXLaemmli buffer. Proteins were quantitated by DC Protein Assay and analyzed by SDS-PAGE described above. 2.13 Flow Cytometric Analysis Cells were transfected with INGlb siRNA described above. After 24 h, cells were then incubated with 1 mM hydroxyurea for 9 h and irradiated with U V B at doses of 0, 10, 20, and 40 mJ/cm2. After the passage of another 24 h, cells were then trypsinized, pelleted by 2,000x g, and resuspended in the fluorochrome buffer (0.1% Triton X-100, 0.1% sodium citrate, 25 pg/ml RNase A, 50 pg/ml propidium iodide). Samples were incubated at 4°C overnight and run on a Coulter EPICS X L - M C L flow cytometer (Beckman Coulter, Mississauga, ON, Canada) to determine the cell cycle distribution. WinMIDI software was used to analyze the data. 2.14 Immunoprecipitation M M R U cells were grown to 80% confluency in 100-mm tissue culture dishes. Their lysates were harvested and incubated with rabbit anti-p300 (Upstate, Charlottesville, VA) antibody or a nonspecific control pre-immune rabbit polyclonal antibody (Santa Cruz Biotechnology) at 4°C for 1 h and then with protein A-Sepharose at 4°C overnight. The beads were washed three times with 0.25% Triton-X solubilization buffer before boiling 51 for 3 min. The precipitates were then resolved by electrophoresis, followed by Western analysis as described above. 2.15 Statistical Analysis Student's t-test was used for statistical analysis (Microsoft Office Excel 2002). P < 0.05 was considered statistically significant. 52 3. Results 3.1 ING Family Members Enhance the Repair of UV-damaged Plasmid DNA We have previously shown that the ING family founding member INGlb can enhance the repair of UV-induced lesions in the M M R U melanoma line (Cheung et al., 2001). This activity is dependent on the functional status of the tumour suppressor p53, a protein that has been implicated in the repair of DNA damaged by various agents. We set out to determine if the remaining of ING family members also display a similar biological aptitude. The host-cell-reactivation (HCR) assay has previously been validated in the study of nucleotide excision repair of UV-induced D N A lesions (Protic-Sabljic andKraemer, 1985). In our HCR assay, we 'inactivated' a reporter plasmid by U V irradiation and reactivated it using the endogenous cellular NER system. The extent of HCR/DNA repair was then quantified in vitro by measuring the activity of the chloramphenicol acetyltransferase (CAT) enzyme expressed by the UV-damaged reporter plasmid. In the assay we co-expressed a UV-treated (40 mJ/cm2 UVC) or untreated reporter plasmid together with vectors harboring INGlb-5 or an empty vector. Forty hours after transfection, the activity of the damaged reporter is assayed and compared to that of a non-damaged reporter. Interestingly, we found that, like INGlb (Cheung et al, 2001), ING2-5 can also enhance the repair of the UV-damaged reporter plasmid in M M R U cells. By normalizing the NER activity on the UV-treated reporter to undamaged reporter plasmid, overexpression of ING 1-5 was found to enhance the activity of the reporter enzyme to as 53 high as 70% compared to a mere 40% when the reporter is co-expressed with an empty vector (Figure 3-1 A). To our surprise, a similar biological activity on NER by ING family members was also seen in U87 glioblastoma cells (Figure 3-1 B). A l l ING family members also share a high dependency on p53 in DNA repair. ING proteins significantly enhanced NER in the wild-type p53 U87 cells, while this enhancement was abolished in the mutant p53 SF-188 glioblastoma cells (Figure 3-1 B) (Tada et al., 1998). This suggests p53 is central in the regulatory pathway of ING family members in facilitating NER. To investigate the mechanisms of ING enhancement of UV-induced D N A lesions by NER, we focused on the INGlb protein in this study. Through screening of potential ING1 siRNA targets, we achieved a knockdown in INGlb expression using siRNA that targets two ORF (siRNA-1 and siRNA-2) and one 3'UTR region (siRNA-3) of the ING1 gene. When transiently transfected into M M R U cells, the siRNA-2 and siRNA-3 oligomers depleted over 97% and 89% of INGlb mRNA, respectively (Figure 3-1 C). In addition, siRNA treatment markedly reduced INGlb protein levels (Figure 3-1 D). We examined the effect of these two siRNA sequences in our HCR assay in U87 cells and found that the NER activity was reduced by over 90% and 50% for siRNA-2 and siRNA-3, respectively (Figure 3-1 E), impeding cellular NER activity on the reporter plasmid. This further confirms the role of INGlb in the NER of UV-induced DNA lesions. 54 F i g u r e 3-1. H o s t - c e l l - r e a c t i v a t i o n ( H C R ) a s s a y o f t h e f i v e I N G f a m i l y m e m b e r s . (A-B) Overexpression of all five ING proteins enhanced the repair rate of the damaged CAT reporter compared to cells transfected with an empty vector in the M M R U melanoma (A) and U87 glioblastoma cell lines (B). (C) Semi-quantitative RT-PCR of INGlb mRNA levels in M M R U cells 24 h after transfection with a scrambled siRNA (Ctrl) or one of three ING1 -specific siRNA specific. Numbers below each lane represents the reduction in INGlb mRNA levels, corrected for loading, compared to the control siRNA. (D) Western analysis confirming a reduction in INGlb protein levels following transfection with INGlb siRNA. (E) Host-cell-reactivation assay in M M R U cells after transfection with scrambled siRNA, pCIneo-INGlb-FLAG plasmid, or INGlb siRNA. For each one of different ING construct transfections, % CAT activity was calculated as percentage of chloramphenicol acetyltransferase activity from UV-damaged reporter to that of the undamaged reporter. Data represent means + SD from three independent experiments. P < 0.05 for ING1-5 (A and B) and INGlb siRNA transfectants (E) compared to the vector control. 55 3.2 INGlb Does not Colocalize with Core Repair Factors Since INGlb facilitates the removal of UV-induced damage, we thought it important to determine i f INGlb is directly involved in NER activity by localizing to DNA lesions. By applying polycarbonate filter with 5-pM pore size on top of cells, we induced localized DNA damage through UV irradiation (20 mJ/cm2 UVC) (Figure 3-2 A). As previously reported, this technique can generate about 0.4 mJ/cm2 U V C to the cell nucleus and is insufficient to cause cell death (Green and Almouzni, 2003), enabling us to observe the NER response after U V irradiation. We immunolabeled the locally-irradiated cells with antibodies that recognize 6-4PP or CPD D N A lesions and FLAG-tagged INGlb and found that INGlb did not colocalize with UV-induced DNA lesions in the nucleus (Figure 3-2 A). We examined this phenomenon at various time points up to 48 hours after U V irradiation; however, INGlb remained evenly dispersed within the nucleus. In contrast, the XPB helicase, a well known DNA damage-processing factor essential to NER, quickly colocalized with the DNA lesion spots within 15 minutes after localized U V irradiation (Figure 3-2 B). This suggests that INGlb might exert its biological functions at a global level rather than being recruited to sites of DNA damage. 3.3 Histone Hyperacetylation Bypasses the NER Requirement for INGlb We have previously shown that the loss of function INGlb mutants, N260S and R102L, does not enhance the repair of UV-damaged plasmids in M M R U cells (Campos et al, 2004b). INGlb has been identified as a cofactor of various HAT enzymes including the p300 protein. To test if the HAT activity associated with INGlb is an important factor 57 Figure 3-2. I N G l b does not colocalize with UV-induced 6-4 photoproducts. Cells were transfected with FLAG-tagged INGlb and U V irradiated on the entire cellular surface (no filter) or locally over a filter with 5-micron (in diameter) pores. The localization of 6-4PP D N A lesions was compared to that of INGlb (A) and NER factor XPB (B) by immunofluorescence at various time points. Magnification, 400x. 58 59 for the NER of UV-induced D N A lesions, we coexpressed the CAT reporter plasmid with either N260S or R102L mutant and incubated cells with or without SB or TSA, two classic H D A C inhibitors, in the HCR assay with M M R U cells. Treatments with either of these two drugs resulted in overall histone hyperacetylation. Our western analysis confirmed this effect (Figure 3-3 A). While mutations at codons 260 and 102 diminished the NER activity compared to wild-type INGlb positive control as previously reported (Campos et al, 2004b), treatment with SB or TSA can also appreciably bypass the NER requirement for INGlb protein (Figure 3-3 A). The repair activity was elevated to as high as 80% in all cells transfected regardless of the INGlb vector that was introduced in the cells (wt vs. mutant or empty vector) in the presence of one of the two HDAC inhibitors (Figure 3-3 A). This suggests that acetylation is important to NER and that INGlb may facilitate the repair of UV-induced lesions through an unidentified histone acetylation pathway. Recent reports suggest that nucleosomes assemble on UV-damaged or undamaged plasmids (Tagami et al, 2004). Since our plasmid is devoid of nucleosomal structure upon transfection and INGlb may affect NER through histone acetylation, we performed a chromatin immunoprecipitation (ChIP) assay using an antibody that recognizes generic histone H3 to immunoprecipitate the exogenous pCMVCAT reporter plasmid 24 hours after transfection. As shown in Figure 3-3 B, PCR analysis of the CAT reporter gene is positive in pCMVoir plasmid-transfected cells following immunoprecipitation with the anti-histone H3 antibody (lane 4), but not a control pre-immune IgG (lane 3). In fact, the histone H3 ChIP reaction signal (lane 4) is ~2 fold higher than the background (lane 2, 3 60 Figure 3-3. Histone deacetylase inhibitors and wild-type INGlb enhance HCR at comparable levels. (A) Host-cell-reactivation assay of M M R U cells transfected with empty vector (V), wt INGlb, or mutant INGlb (R102L and N260S) and treated with 5 mM SB or 200 pg/ml of TSA. % CAT activity was calculated as percentage of chloramphenicol acetyltransferase activity from UV-damaged reporter to that of the unmodified reporter. (B) Histone deposition onto the reporter plasmid by chromatin immunoprecipitation (ChIP). Lane 1, PCR amplification of the CA T plasmid; lane 2, an empty vector; lane 3, D N A immunoprecipitated with preimmune serum; lane 4, DNA from cells transfected with the CAT reporter plasmid immunoprecipitated with anti-histone H3 antibodies; and lane 5, DNA from cells transfected with an empty vector immunoprecipitated with anti-histone H3 antibodies. Data represent means + SD from three independent experiments. P < 0.001 for various combinations of INGlb construct transfections and drug treatments compared to their respective untreated, vector control. A B + - + Vector + - + + - CAT Plasmid + - - Pre-immune IgG + + Anti-H3 IP 61 and 5). Thus, the association of histones with the reporter plasmid is indicative that there is de novo assembly of canonical nucleosomal structure following transfection and that the INGlb activity on NER may indeed be due to histone acetylation. 3.4 siRNA knockdown of INGlb Reduces Global Histone H4 Acetylation Seminal studies in yeast and mammalian cells have shown that U V irradiation can induce global histone hyperacetylation. This activity, in turn, is implicated in different cell systems to enhance NER. We first studied the toxicity of various doses of genomic UVC irradiation to the U87 and M M R U cells to ensure the biological effects we observe are not due to apoptotic responses but rather bona fide enhancement of NER of UV-induced DNA lesions. In our sulforhodamine-B (SRB) cell survival assay, there is 63% and 67% of viable cells 24 hours after 40 mJ/cm2 of U V C irradiation in U87 and M M R U cell cultures, respectively (Figure 3-4). We decided to use 10 mJ/cm 2 UVC irradiation since this does not generate significant cell death for either cell line. In fact, at this dose, only about 87 % and 79% cells remained viable in U87 and M M R U cultures, respectively (Figure 3-4). Furthermore only viable cells are counted in our host-cell-reactivation NER assay. To study the NER histone acetylation response, we used antibodies that recognize acetylation on lysine 5, 8, 12, and 16 of histone H4 (AcH4) and acetylation of lysine 9 and 14 of histone H3 (AcH3) to examine the histone modification post-UV irradiation. In M M R U cells, there was an increase of acetylation on both histone H3 and H4 as early as 1 minute after U V irradiation (10 mJ/cm2) (Figure 3-5 A). In U87 cells, we also observed 62 Figure 3-4. Cytotoxicity of U V irradiation. Sulforhodamine-B (SRB) assay of U87 and M M R U cells irradiated with of 0, 10, 20, or 40 mJ/cm 2UVC. The bar graph represents relative cell survival, derived as percentage of spectrometric readings from viable cells in each UV treatment to that of the un-treated control (0 mJ/cm2). Spectrometric values were obtained 24 h post-UV irradiation. Data represent means + SD from three independent experiments. P < 0.01 for all U V C doses compared to the untreated control. 63 Figure 3-5. INGlb induced histone acetylation after UV-irradiation. (A-B) Global acetylation response to U V irradiation in M M R U (A) and U87 cells (B). C denotes for samples from undamaged cell lysates. (C) siRNA-mediated knockdown of INGlb levels caused an overall reduction in histone acetylation of U87 cells after 10 mJ/cm2 UVC irradiation. The bar graph indicates the fold increase/decrease comparatively to untreated cells after correction for loading. Data represent means + SD from three independent experiments. B Ac -H3 Ac-H4 P-actin C 1 5 15 30 60 min Ac-H3 Ac-H4 p-actin + + + + + + + + + + + + siRNA-2 Ctrl siRNA min 2.5 -> o INGlb a 2.0 > 0 Ac-H4 8 1.5 c I |1 .0 p-actin 5 -o 0.5 0 u. • Ctrl siRNA Ac-H4 • INGlb siRNA Ac-H4 5 15 30 Time (min) 60 64 a sharp induction of AcH4 1 min after U V irradiation (Figure 3-5 B). It has been previously shown that overexpression of INGlb leads to genomic AcH3 and AcH4 (Vieyra et al, 2002). We believe that INGlb and its associated HAT activity contribute, at least in part, to this early U V irradiation response to enhance NER. By using INGlb siRNAs, we could markedly knockdown the normal AcH4 response in U87 cells, with the most prominent decrease nearing 60% at the 1 minute time point (Figure 3-5 C). We also obtained similar results with M M R U cells at the 1 and 5 minutes time points (not shown). Together, our western analyses illustrate that INGlb plays an important role in UV-induced histone hyperacetylation, particular on histone H4. Since INGlb is reported to associate with HAT and HAT cofactors, such as TRRAP (Vieyra et al, 2002), we believe there a pool of INGlb molecules may exist in HAT complexes whose activity can be stimulated by U V irradiation. The inability to completely knockdown the histone hyperacetylation with INGlb siRNA suggests that other factors might also be involved in this biological response to U V irradiation. 3.5 Overexpression of INGlb Induces Chromatin Relaxation Recently, a new role has been suggested for the p53 tumour suppressor in AcH3 and chromatin relaxation for NER after U V irradiation (Rubbi and Milner, 2003). Analogous to this, one other report also pointed out increased chromatin accessibility to nuclease digestion at silenced yeast MFA2 promoter after U V irradiation (Yu et al, 2005). We have observed that INGlb is crucial for maintaining the early AcH4 response after UV irradiation. To determine i f ING lb-mediated AcH4 is also necessary for inducing a relaxed chromatin environment, we examined the nucleosomal accessibility to 65 micrococcal nuclease digestion at various time points post U V irradiation (10 mJ/cm2) in U87 cells overexpressing INGlb. We found that there was a significantly increased level of chromatin relaxation 30 minutes after U V irradiation with the most profound effect noticeable at 60 minutes post-irradiation. When INGlb-overexpressing cells was compared to the vector control, we found that INGlb overexpression results in altered chromatin structure as a less condensed conformation accompanied by higher nuclease digestion was observed (Figure 3-6 A). As shown in Figure 3-6 B, by normalization to the total loading, ING lb-induced chromatin relaxation increased 2-fold following the 30-min time point and 3-fold 60 minutes following exposure to U V . We also found similar results in M M R U cells (not shown). Taken together, these data suggest that UV-induced ING lb-mediated AcH4 spike is important to relax condensed chromatin to enhance NER of damaged DNA. 3.6 Expression of INGlb Is Essential for DNA Lesion Detection In an attempt to verify if the previous results could translate into enhanced accessibility to DNA, the chromatin bound fraction of cells was examined more closely. M M R U cells were fractionated by differential centrifugation to obtain various cellular compartments. Like p53, INGlb was found to be largely contained within the chromatin fraction (Figure 3-7 A), an observation consistent with previous reports that INGlb is largely nuclear and is also associated with various chromatin remodeling factors (Garkavtsev et al, 1997; Vieyra et al, 2002). By compared with the pre-UV sate, we observed exposure to 10 mJ/cm 2UVC irradiation increased 1.46 and -1.47 folds of the chromatin-bound INGlb 66 Figure 3-6. I N G l b promotes chromatin accessibility to micrococcal nuclease digestion after U V irradiation. (A) Micrococcal nuclease (MNase) digestion of genomic DNA from vector-transfected (top) or FLAG-INGlb-transfected (bottom) M M R U cells. D N A was collected at various time points following irradiation with 10 mJ/cm2 U V C and subjected to 0.1 U MNase digestion. (B) Fold increase in digested DNA of less than 1 kb in ING lb-expressing cells compared to vector-transfected counterparts. Data represent means + SD from three independent experiments. *P < 0.0001 compared to the vector control. 67 and p53 proteins, respectively (Figure 3-7 B). In addition, immunofluorescent examination of the subcellular distribution of FLAG-INGlb protein found most FLAG-INGlb molecules in the nucleoli like previously described by others (Scott et al, 2001) (Figure 3-8). To our surprise, 10 mJ/cm2 genomic U V C irradiation caused a translocation of FLAG-INGlb proteins from nucleolar to diffuse pattern inside the nucleus; it initiates as soon as 1 min after U V irradiation (Figure 3-8 A and B). At 1 hour post-irradiation, around 90% of FLAG-INGlb molecules are diffuse throughout the nucleus (Figure 3-8 B). This further supports the notion that INGlb has a global role to modulate chromatin structure in response to U V irradiation. To verify if INGlb may enhance NER by increasing chromatin accessibility, the retention of X P factors in the chromatin fraction was also examined. We chose X P A as it is an early NER factor which stabilizes open DNA complexes and helps in detection of DNA lesions. Figure 3-9 A shows the levels of chromatin bound X P A in cells overexpressing INGlb or transfected with INGlb siRNA oligomers. As expected, the amount of chromatin-bound X P A nearly doubled following U V irradiation in empty vector transfected cells. Importantly, cells overexpressing INGlb had a 2-fold induction in chromatin bound X P A compared to the vector control, but a 3.7-fold increase in chromatin bound X P A after U V irradiation. In the absence of U V , ING lb-specific siRNA had little effect on the amount of chromatin bound X P A . However, INGlb siRNA transfected cells had 2.2-fold reduction of X P A in the chromatin fraction after UV irradiation compared to UV-treated cells transfected with a scrambled control siRNA, arguing that INGlb facilitates accessibility of DNA repair factor to damaged 68 Figure 3-7. Biochemical fractionation of cytosolic, nuclear and chromatin-bound I N G l b protein. Isolation of various subcellular compartments 1 h after 10 mJ/cm2 UVC irradiation from M M R U cells. (A) Fractions were resolved on a 15% SDS gel and analyzed for the degree of association of INGlb protein with chromatin. Antibodies against INGlb, p53, ILK, Erk l , and total histone H3 were used to identify different cellular compartments. (B) Fold of difference in the levels of chromatin-bound INGlb and p53 protein post-UV irradiation. The bar graph indicates the fold increase comparatively to untreated cells after correction for histone H3 loading. Data represent means + SD from three independent experiments. ^ + + - - - - Cytoplasmic Fraction ^ - - + + - - Chromatin Fraction - - - + + Nuclear Fraction - + - + - + UV 69 Figure 3-8. I N G l b intranuclear localization upon U V irradiat ion. (A) M M R U cells were transfected with FLAG-tagged INGlb and globally irradiated with 10 mJ/cm2 U V C 24 h after transfection. The localization of FLAG-INGlb molecule was examined by immunofluorescence at various time points post-UV irradiation. Magnification, 400x. (B) Percentage of FLAG-INGlb intranuclear localization in nucleoli (nucleolar) or in the nucleus (nucleoplasmic) in response to 10 mJ/cm2 U V C irradiation. In one randomly selected nuclus, relative INGlb level is calculated as percentage of nucleolar or nucleoplasmic INGlb staining compared to that of the total nuclear staining for various post-UV time points. 0 mJ 10 mJ/cm2 15' 10 mJ/cm2 V 10 mJ/cm2 30' 10 mJ/cm2 5' 10 mJ/cm2 60 min B 100 4) > 0) CD • Nucleolar • Nucleoplasmic / y • / / / 70 Figure 3 - 9 . INGlb binds to chromatin and confers retention of chromatin-bound XPA protein after U V irradiation. (A) The level of chromatin bound X P A in untreated M M R U cells (lane 1), 1 h after 10 mJ/cm2 U V C irradiation (lane 2), cells overexpressing INGlb in the absence of U V (lane 3), and 60 min after 10 mJ/cm 2UVC irradiation (lane 4). Data represent means + SD from three independent experiments. (B) Effect of INGlb siRNA on the level of chromatin-bound X P A after U V irradiation. Chromatin-bound proteins were obtained by extraction with 0.1 % Triton-X detergent to remove soluble nuclear proteins. + + - Vector " " + + FLAG-ING1b - + - + 10 mJ/cm 2 UVC 60 - 60 Time (min) B + + + - 60 XPA FLAG H4 XPA H4 3.5 Ctrl siRNA + INGlb siRNA-2 + 10 mJ/cm 2 UVC z 3-0 60 Time (min) SS , _ Vector Vector INGlb INGlb + UV + UV Ctrl Ctrl INGlb INGlb siRNA siRNA siRNA siRNA +UV + UV 71 DNA (Figure 3-9 B, preliminary observation). Similar results were also obtained with XPB, a helicase recruited to DNA lesions downstream of X P A (data now shown). In order to determine the NER pathway through which INGlb enhances the repair of UV-induced DNA lesion, we performed a genetic complementation test using the host cell-reactivation assay. X P C is a damage recognition factor that binds to UV-induced D N A adduct and is required for global genomic repair (GGR) but not transcription-coupled repair (TCR). We performed the HCR assay in NHFB normal fibroblasts, fibroblast deficient for XPC (XP44RO - GGR deficient) or XP44RO cells stably expressing wild-type XPC (XP44RO+XPC - GGR proficient). Since XPC is required for efficient GGR, the XP44RO cells are unable to assemble a competent NER repairosomes (Masson et al., 2003). Our data corroborated these published observations. Figure 3-10 shows that the upon U V irradiation, XP44RO cells did not show a significant recruitment of X P A to the chromatin (lane 2) compared to the XP44RO+XPC cells (NER complemented by stable transfection of wild-type XPC retroviral construct) (lane 4). Once the XPC genetic defect was verified, we performed HCR assay in XP44RO and XP44RO+XPC cells. Our preliminary observations suggest the human fibroblasts have lower tolerance for DNA damage than our tumour cell lines as transfection of U V C - but not UVB-damaged reporter plasmids result in substantial cell death (not shown). For this reason, we UV-damaged the reporter construct with lower-energy U V B rays (250 mJ/cm2). Using the UVB-damaged reporter construct, we showed that INGlb enhances 40% NER activity compared to the vector control in the normal human fibroblasts (NHFB) (Figure 3-11). INGlb expression shows a 32% 72 Figure 3-10. Efficient recruitment of XPA and XPB to damaged chromatin was absent in XPC-deficient cells upon UV irradiation. Lane 1 and 3, the level of chromatin bound X P A and XPB proteins in untreated XPC-deficient XP44RO and XP44RO+XPC cells, respectively. Lane 2 and 4, the level of chromatin-bound X P A and XPB proteins 1 h after 10 mJ/cm2 U V C irradiation in XP44RO and XP44RO+XPC cells, respectively. Chromatin-bound proteins were obtained by extraction with 0.1 % Triton-X detergent to remove soluble nuclear proteins. + + + + 0 1 0 1 XP44RO + X P C XP44RO hrs X P A X P B INGlb Total H4 73 Figure 3-11. INGlb-mediated NER enhancement effect is limited to the presence of functional XPC protein. HCR assay of UVB-damaged (250 mJ/cm2) CAT reporter plasmid in normal human fibroblasts NHFB, XPC-deficient cells XP44RO, and XP44RO+XPC cells. Overexpression of INGlb enhanced the repair rate of the damaged reporter construct compared to empty vector transfection in NHFB and XP44RO+XPC cells, but not in XP44RO cells. % CAT activity was calculated as percentage of chloramphenicol acetyltransferase activity from UV-damaged reporter to that of the unmodified reporter. Data represent means + SD from three independent experiments.*P < 0.01 compared to the vector control. • INGlb • Vector * XP44RO XP44RO +XPC NHFB 74 enhancement on NER in the XP44RO+XPC fibroblasts while no significant enhancement was seen in XP44RO cells (Figure 3-11). Together, our data suggest that INGlb may execute its biological function ahead the DNA lesion detection step. They also implicate INGlb in the GGR subpathway. 3.7 INGlb Depletion Causes Defective S phase DNA Damaging Response to UV Irradiation Murine ingl knockout embryonic stem cells are hypersensitive to radiation and display phenotypes associated with DNA repair defects (Kichina et al., 2006). The yeast ING1, Yng2, was found essential for the DNA damage response during S phase. The yng2 mutant was found to interact with several DNA metabolism mutants, such as type I topoisomerase, to cause synthetic genetic lethality. It has been suggested that Yng2-mediated AcH4 is important for maintaining relaxed chromatin environment for DNA metabolic activities, such as DNA replication (Choy and Kron, 2002). To our knowledge, the role of INGlb has not been defined during S phase damage response. In the previous sections, we showed ING lb-promoted chromatin relaxation is important for clearance of photolesions in the HCR assay (Figure 3-1 A and B). We examined the fitness of ING lb-depleted cells treated with UV-irradiation during S-phase. In comparison to untreated cells, 9 h hydroxy urea incubation results in a significant number of cells arrested at S-phase (Figure 3-12 A and B). Mathematical model proposed by Chadwick and Leenhouts (1983) describes that UV-induced cell killing is proportional to the square of the U V exposure. By calculating the natural logarithm of 75 Figure 3-12. Depletion of INGlb causes defective S-phase DNA damaging response to U V irradiation. S-phase cell death after INGlb siRNA treatment in response U V B irradiation. M M R U cells were arrested at S-phase with 1 mM hydroxy urea for 9 h and treated with 0, 10, and 40 mJ/cm2 U V B irradiation and incubated for 24 h. (A) Normal cell cycle distribution. (B) S-phase arrested cells treated with hydroxy urea. (C) Left: Micrographs of S-phase arrested cell cultures taken 24 h after U V irradiation. Right: Cell survival curve of INGlb siRNA and scramble control transfectants in response to U V irradiation during S-phase. Magnification, lOOx. The percentage of cell survival was determined by manual counting in 5 random fields with a minimal 100 cells in each field. *P < 0.05 compared to the scramble control. ta S3 E 3 z © o % G1 39 % S 23.5 % G2/M 37.5 0J A E 3 a) O DNA Content % G1 28.5 % S 61.4 %G2/M 10.1 DNA Content Control INGlb slRNA-3 0 mJ/cm 2 UVB 10 mJ/cm 2 UVB 40 mJ/cm 2 UVB '" '•"; •} i 0.5 0 u To > -0.5 'E -1 3 V) g_ -1.5 CD 0 -2 —1 -2.5 10 20 40 Doses (mJ/cm 2 ) 60 76 percentage of viable cells INGlb siRNA-transfected cells arrested at the S-phase were found to be more sensitive to U V B irradiation than cells transfected with a scrambled siRNA control. We found a significant difference in the number of viable cells between control and INGlb siRNA transfected cells at all doses tested (Figure 3-12 C). UV irradiation is among various types of DNA damaging agents which can perturb S-phase DNA replication. In M M R U cells, we observed U V B irradiation of the S-phase chromatin leads to a gradual accumulation of Serl39 phosphorylated H2A.X (y-H2A.X), the hall mark of the most lethal D N A lesion—double strand break (Figure 3-13). Together, our observation suggests that INGlb facilitates D N A lesion removal during S-phase by providing an accessible chromatin environment. Depletion of INGlb will reduce S-phase NER efficiency, leading to a buildup of double strand break which ultimately causes cell death. 3.8 Possible Involvement ofp300 in ING lb-mediated Chromatin Relaxation An appreciable amount of literature suggests the involvement of the p300 HAT in NER of UV-induced D N A lesions. Vieyra et al. (2002) found that INGlb can be co-immunoprecipitated with p300. We are currently trying to identify which HAT mediates the prompt AcH4 response after U V irradiation. Figure 3-14 A shows that in comparison with the scrambled control, p300 siRNA can noticeably reduce the UV-induced AcH4, similar to our observation with INGlb siRNA transfection. The AcH3 level, on the other hand, remains unchanged (Figure 3-14 A). To determine if INGlb and p300 interacts in a UV-inducible manner, we performed 77 Figure 3-13. UV irradiation of S-phase chromatin generates an accumulation of Y-H2A .X. M M R U cells were arrested at S-phase with 1 mM hydroxy urea for 9 h and treated with 10 mJ/cm2 U V B irradiation. The appearance of phospho-H2A.X (y-H2A.X) was examined by western blot 1, 3.5, and 24 h post-UV irradiation. P-actin was used as the loading control. 0 1 3 5 24 hrs INGlb Y-H2A.X p-actin 78 Figure 3-14. p300 associates with I N G l b and modulates global histone H4 acetylation upon U V irradiation. (A) siRNA-mediated knockdown of p300 levels caused a reduction in global histone H4 acetylation of M M R U cells 1 min after 10 mJ/cm2 U V C irradiation. (B) Fold of difference of AcH4 and AcH3 levels 1 min post-UVC irradiation in control and p300 siRNA-treated cells. The bar graph indicates the fold increase/decrease comparatively to untreated cells after correction for loading. (C) p300 associates with INGlb in a UV-inducible manner. Immunoprecipitation using anti-p300 antibody detected an increased amount of INGlb protein in the pull-down reaction after 10 mJ/cm U V C irradiation. In the western blot analysis, the rabbit polyclonal antibody IgG was used as the loading control. + + + - - - Ctrl siRNA " + + + p300 siRNA 0 1 5 0 1 5 min 4 M M b 4 H H M ^ t**mm* u -m. • ' : • p300 Ac-H3 Ac-H4 0-actin B + + + + • 5 15 + 30 **** liiMnii liilttti iiillniii mil ii •->"•• Pre-immune Serum (IP) min INGlb IgG 79 INGlb and p300 interacts in a UV-inducible manner, we performed coimmunoprecipitation using antibody against p300. As Figure 3-14 B shows, we could successfully recover a significant amount of INGlb protein in the pull-down reaction (lane 2). Interestingly, this INGlb-p300 interaction steadily increased following UV irradiation (comparing lane 2 and lane 5), suggestive of de novo complex formation stimulated by U V treatment. These results indicate a potential role of INGlb-p300 complex in acetylating histone H4 which contributes to the NER damage detection factors. 80 4. Discussion DNA can be perceived as the physiological script of cellular structure and activity. The packaging of D N A into the nucleosomal and higher order chromatin structures with histone proteins serves as a protective and regulatory mechanism that prevents unspecific DNA metabolic activities in the nucleus. The condensed chromatin needs to be 'opened' to grant access to D N A repair factors for a timely removal of D N A lesions. This has lead to the development of the 'Access, Repair, Restore' (ARR) model. In the recent years, studies have emerged linking chromatin remodeling events to the NER of D N A lesions. Different HATs have been proposed to help neutralize the tight interactions between structural histone proteins on the chromatin fiber by the addition of acetyl moieties. We now describe the ability of INGlb, the most abundant ING1 isoform, to enhance the repair of UV-induced D N A lesions through associated HAT activity. 4.1 ING Family Members Enhance NER of UV Lesions INGlb is known to accelerate the removal of UV-induced DNA lesions in M M R U cells (Cheung et al., 2001). Here we used the host-cell-reactivation (HCR) assay to confirm that this biological function of INGlb is not restricted to our melanoma cell lines but also occurs in the p53-proficient U87 glioblastoma cell line (Figure 3-1 A and B) and normal human fibroblasts (Figure 3-11). We further observed that the ING2-5 proteins can also enhance NER of UV-damaged reporter plasmids in two different cell types (Figure 1 B), and that this property is likely dependent on the functionality of p53 as previously reported for INGlb (Cheung et al., 2001; Campos et al., 2004a). UV irradiation is a potent activator of p53 (Lakin and Jackson, 1999). Since little repair was 81 observed upon overexpression of any of the five ING members in the p53-deficient SF-188 glioblastoma cell line, it is therefore possible that the ING1-5 mediated NER responses are regulated by p53 as a common denominator, although they associate with different HAT enzymes (Doyon et al, 2006). This notion is further supported by the fact that: (1) both INGlb and p53 promote the removal of the same type of U V DNA lesions, namely CPDs (Cheung et al, 2001; Ford and Hanawalt, 1997); (2) both Ingl and p53 knockout mice are hypersensitive to U V irradiation and are prone to various lymphomas (Kichina et al, 2006); and (3) like p53, INGlb, is also involved in the GGR of U V lesions (discussed below). Bioinformatics analyses identified two central features common to ING family members, which have helped up elucidate the molecular mechanisms utilized by ING proteins in NER. At the N-terminus, a potential chromatin regulatory (PCR) domain which aligns almost perfectly between all ING proteins (He et al, 2005), is thought to act as the 'executioner' domain of INGs since it is believed to bind chromatin remodeling factors. In light of our results, we believe that this executioner domain initiates interactions with HAT complexes upon genotoxic injury, an idea that is currently under investigation. At the other extreme, an evolutionarily conserved C-terminal plant homeodomain (PHD) zinc finger motif is also present not only in mammalian but also yeast INGs (He et al, 2005). This domain has been identified as a binding module for nuclear phospohatidylinositol-5-phosphate (PtdIns(5)P). This domain is thought to act as a 'signaling' domain that activates ING-HAT complex activity upon binding to Ptdlns. Since PtdIns(5)P species arise upon genotoxic injury and cause mobilization of ING 82 proteins (Gozani et al, 2003) this signaling domain likely modulates the DNA damage response (discussed below). 4.2 UV Induces ING lb-mediated Histone Acetylation and Chromatin Relaxation The tumour suppressor p53 has also been found to promote a global chromatin relaxation through AcH3. Overexpression of INGlb is reported to increase overall AcH3 and AcH4 (Vieyra et al, 2002). We therefore hypothesized that ING proteins may contribute to NER of UV-induced D N A damage through associated HAT activities. Since the involvement of INGs in D N A damage response is highly conserved from yeast to human, we used INGlb as a representative to study the NER enhancement mechanisms by this gene. In the absence of genotoxic stress, acetylated H4 (AcH4) concentrates in active genomic loci undergoing transcription or replication. In this study, we examined the levels of AcH4 following U V irradiation. UV-induced histone hyperacetylation is a highly dynamic process. We observed a marked increase in AcH4 as early as 1 minute following U V irradiation of asynchronous M M R U and U87 cells (Figure 3-5 A and B). Kinetic studies on the formation of the NER repairosome established a typical maximum incorporation of XPC at damaged D N A sites 10 minutes after U V irradiation (Politi et al, 2005). Since chromatin structure is refractory to NER it is normal to expect a surge of AcH4 prior the formation of NER complex at DNA damage sites since this acetylation 83 spike may contribute to the lesion surveillance process of NER such as that lead by the XPC protein. It is important to note that the antibody used in this report detects a combinatorial effect on histone H4 acetylation (it cannot discriminate between the different acetylation sites on the H4 tail). There may also be additional histone modifications in response to U V that we have not observed. The 'histone code' hypothesis states that individual/combinations of histone post-translational modifications serve as docking sites for chromatin remodeling proteins that regulate accessibility of enzymes and transcription machineries to D N A (Jenuwein and Allis, 2001). Accumulating evidences have pointed to a distinct combination of histone modifications in response to genotoxic stresses. A histone code may therefore be formed to regulate the efficiency of NER acting on the chromatin template. For this reason, the UV-induced AcH4 spike might serve as a chromatin signal to bring forth other chromatin modifications not detected in our study. One such possibility is the recently described UV-induced histone H3 and H4 mono-ubiquitination which also promotes chromatin relaxation for lesion detection (Wang et al, 2006). Another example is the repressed yeast MFA2 locus, in which increase of histone H3 acetylation (AcH3) can be stimulated by U V but is independent of the transcription status of the gene (Yu et al, 2005). Cellular exposure to the HDAC inhibitors, TSA or SB, induced histone hyperacetylation thus mimicking the effect of functional INGlb overexpression, which also leads to histone hyperacetylation (Figure 3-3 A and Figure 3-5 A). Since our hypothesis is 84 dependent on the presence of nucleosomal structure, chromatin immunoprecipitation (ChIP) was used to determine whether our reporter construct associated with histones following transfection. The ChIP assay was performed on the exogenous pCMV CAT 24 hours after transfection after which we found an association between endogenous histone H3 and the exogenous reporter plasmid (Figure 3-3 B). Since histone H3 and H4 are deposited as dimmers during repair-coupled nucleosomal formation following H2A-H2B histones (Tagami et al., 2004), we conclude that nucleosomal structure is likely present on our reporter plasmid after transfection. Our results suggest that INGlb may engage in the chromatin remodeling on the exogenous, UV-damaged plasmid platform. In yeast, mutants lacking the four N-terminal acetyl able lysine residues on histone H4 are hypersensitive to different DNA-damaging agents (Megee et al., 1995; Bird et al., 2002), suggesting that AcH4 is important for DNA repair. In addition, Tip60, an enzyme that associates with ING3, is also reported to facilitate the repair of double strand break through its histone H4 HAT activity (Murr et al, 2006). We speculate INGlb also enhances the AcH4 spike we observed in this study. By using INGlb siRNA, depletion of endogenous INGlb resulted in a significant decrease of AcH4 in all observed time points post-UV irradiation, with the highest reduction at the 1 minute time point (Figure 3-5 C). In contrast, the AcH3 seemed to be affected to a lesser extent. The inability of INGlb siRNA to completely abrogate UV-induced AcH4 spike suggest that other factors might also be involved in this biological phenomenon. Redundancy is quite plausible since the main five human ING proteins associate with different HATs and since there is also redundancy in the number of HATs capable of enhancing double strand break. 85 Namely, Tip60 and Hatl HATs have both been found to enhance chromatin remodeling at the site of double strand break (Murr et al. 2006; Qin and Parthun, 2006). The p300 HAT has been suggested to confer access to NER factors through chromatin remodeling (Rubbi and Milner 2003a). It has been found to specifically enhance p53-mediated histone acetylation upon U V irradiation (Rubbi and Milner, 2003a). Given that p53 is a regulator of INGlb NER functions and that INGlb contributes partly to the UV-induced AcH4 spike, we performed coimmunoprecipitation to detect the interaction between p300 and INGlb when the cellular genome is inflicted with U V light. This experiment suggests that the two proteins associated with each other at an elevated level in a UV-inducible manner (Figure 3-14 B). In addition, when p300 siRNA is employed, the AcH4 spike can be dramatically reduced (Figure 3-14 A). The AcH3 signal remains at the basal, pre-UV level (Figure 3-14 A). Our preliminary data identified p300 as a candidate to regulate ING lb-mediated NER enhancement through AcH4. Most ING proteins are reported to interact with p300 (Campos et ai, 2004a). For future study, it will be interesting to map p300 interaction domain in INGlb and extrapolate the ING-HAT interaction to the rest of family members. We hypothesize the highly conserved PCR domain is a likely region that executes ING functions during NER. 4.3 Benefits of Chromatin Relaxation Hyperacetylation of nucleosomal histones is largely believed to increase chromatin accessibility by reducing overall compaction (Morales and Richard-Foy, 2000; Gorisch 86 et al, 2005). Y u et al. (2005) also found that UV-induced AcH3 leads to increased chromatin accessibility to restriction enzyme Rsal at the yeast MFA2 promoter. We showed that INGlb contributes to the AcH4 spike induced by U V . Our data suggest that overexpression of INGlb promotes digestion of internucleosomal D N A by bacterial micrococcal endonuclease (Figure 3-6 A and B). This is functionally reflected by reduced level of chromatin-bound damage recognition X P A proteins in INGlb siRNA-treated cells irradiated by U V (Figure 3-9 B). Thus INGlb may maintain a favorable chromatin environment for the detection of UV-induced D N A damage. Due to steric hindrance, the bulky 6-4 PP occurs mostly in the linker DNA region while CPD is the most common U V lesion in the tight nucleosomal dyad (center of nucleosome). Since CPD is predominantly removed by the GGR pathway acting mainly on the chromatin template, one dilemma is how accessibility is granted by unknown factors. Like p53, our study shows INGlb is another accessibility factor. Our HCR assay on the XPC human fibroblasts further links the INGlb-pormoted chromatin accessibility to lesion detection. The ING lb-enhanced HCR of UV-damaged report construct is absent in the XPC-deficient XP44RO cells but is present in the XP44RO fibroblasts complemented with wild-type XPC by stable transfection (XP44RO+XPC) (Figure 3-11). This positive genetic interaction suggests that INGlb impacts the GGR by modulating post-UV global chromatin. Three lines of evidence substantiate this notion. First, INGlb enhances removal of CPD in a p53 (a known GGR enhancer) dependent manner (Figure 3-1 B). Second, INGlb only enhances HCR of UV-damaged reporter plasmid when functional XPC protein is present (Figure 3-1-1). Third, the XPC-deficient cells are capable of executing TCR of U V lesions but INGlb is unable to enhance this activity 87 (Figure 3-11). Finally, INGlb-mediated chromatin relaxation enhances the loading of X P A onto the UV-damaged chromatin. Taken together, these novel observations further establish a role of ING proteins in the regulation of chromatin structure through histone acetylation, an event which seems to confer accessibility for D N A damage recognition factors upon cellular genotoxic injury. Chromatin relaxation can also be linked to cellular fitness. Yeast Yng2 has been proposed to promote cell survival by maintaining favorable AcH4 level during S-phase (Choy and Kron, 2002). Deletion mutant Ayng2 is hypersensitive to U V , particularly when arrested at S-phase. We studied the effect of INGlb deletion on S-phase chromatin which constantly undergoes high DNA torsional strength as a result of passage of DNA polymerase on the replication fork. UV lesions impinge the vital D N A metabolic activities, such as replication, to generate cytotoxicity. Our result shows that there is an accumulation of y-H2A.X, a landmark of double strand break, in S-phase M M R U cells U V irradiated at a low dose (Figure 3-13). This is no surprise to us since unrepaired CPD is known to block the replicative polymerase to create replication intermediates (single-stranded or double-stranded) (Garinis et al, 2005). Topoisomerase moves ahead of the replication fork to relieve the DNA superhelical tension. As party of its catalytic cycle, topoisomerase creates a transient protein-linked D N A breaks (Pommier et al, 1998). Blockage of this enzyme by CPD leads to replication fork collapse and double strand break, the most lethal DNA lesions (Garinis et al, 2005). However, it has been reported that apoptotic cells also accumulate y-H2A.X foci (Banath and Olive, 2003). It is, therefore, essential to examine other molecular markers of DSB repair, such as M R N complex formation (Christmann et al, 2003) or phosphorylation of A T M at Serl981 88 (Bakkenist and Kastan, 2003), in order to discern actual strand breaks generated by stalled replication fork at CPD from the apoptotic chromatin. Given that INGlb: (1) enhances chromatin relaxation (this study), (2) accelerates CPD removal (Cheung et al, 2001), (3) causes synthetic lethality with topoisomerase I {topi) mutant in yeast (Choy and Kron, 2002), we U V irradiated INGlb siRNA-treated cells arrested at S-phase. In all U V doses tested, we found that there is higher number of cell death compared with the control (Figure 3-12 C). According to this observation, proper expression of INGlb is essential to facilitate CPD removal and avoid obstructed topoisomerase by maintaining a less condensed global chromatin structure during D N A metabolic activities, such as repair and replication. Our data therefore echoes the S. serevisiae observations. 4.4 UV-induced INGlb Intranuclear Relocalization INGlb is a cofactor of multiple HATs such as p300 (Vieyra et al, 2001). It has also been copurified with the chromatin remodeling complex mSin3A-HDAC complex (Kuzmichev et al, 2002), suggesting INGlb may associate with chromatin. Our results show that INGlb induces AcH4 on the damaged chromatin upon genomic insult by U V irradiation. We tested i f INGlb could associate with chromatin. As expected, like p53, the majority of INGlb molecules are nuclear. In addition, both INGlb and its regulator, p53, associate with chromatin in a UV-inducible manner (Figure 3-7 A and B). It is evident that INGlb mediates UV-induced chromatin rearrangement and that p53 may have a similar role in facilitating its biological function. The obvious interpretation of our data is the ING lb-promoted chromatin relaxation that provides accessibility of the condensed chromatin to NER factors. 89 Rubbi and Milner (2003a) report a new role for p53 as a global chromatin accessibility factor during NER. We speculate that INGlb may also have a parallel role in this process. The intranuclear location of ING lb-mediated chromatin relaxation was examined by micropore U V irradiation which creates genomic lesion spots. First, we observed an increase in the AcH4 staining upon U V irradiation by immunofluorescent staining. Interestingly, the AcH4 signal does not colocalize with 6-4 PP lesion. Thus, ING lb-promoted chromatin relaxation is likely to be global, not localized to U V lesions. This is supported by our inability to observe a significant colocalization between the both types of photolesions and the FLAG-tagged INGlb molecule in asynchronous M M R U cells (Figure 3-2 A). This further suggests that rather than being part of the core NER machinery, INGlb facilitates accessibility to NER factors such as X P A (Figure 3-9). U V irradiation is known to induce the pl4ARF tumour suppressor, a protein that sequesters INGlb in the nucleolus, to remobilize from nucleolus to nucleus (Gonzales et al, 2006). We believe that INGlb may also change its intranuclear locale upon U V treatment. Indeed, genomic U V irradiation causes an instant displacement of INGlb proteins from nucleoli to nucleoplasm (Figure 3-8 A and B). This is in agreement with an observation by Rubbi and Milner (2003b) which describes nucleolar disruption, acting as an upstream DNA damage sensor, is necessary to activate p53-dependent damage response after U V treatment. Theoretically, disruption of such intranuclear structure would lead to leakage of its compartmental proteins, such as p l4ARF and INGlb, allowing them to diffuse to the global genome. Our understanding is that, INGlb, a 90 protein largely nucleolar, can be induced to mobilize and associate with global chromatin to enhance AcH4 and chromatin relaxation upon U V irradiation. 91 5. Conclusion Based on the 'Access, Repair, Restore' model, we examined the ING lb-promoted gross chromatin rearrangement in response to U V irradiation. Our previous study has demonstrated that INGlb is an enhancer of NER, but in the presence of functional p53 protein. We now demonstrate that like p53, INGlb can also promote chromatin relaxation through associated HAT activities. This is likely an early event that is later amplified into a large scale post-genotoxic injury and grants better D N A accessibility to NER factors, such as X P A . By using the INGlb siRNA oligos that we have screened in our laboratory, we successfully knocked-down INGlb expression in both M M R U and U87 cell lines. We discovered that INGlb participates in the AcH4 spike response upon U V irradiation that may either directly affect and relax chromatin structure or indirectly lay down an 'epigenetic' histone code to recruit other chromatin remodeling factor to alleviate the histone-DNA interactions. We prefer the former proposition, since tetra-acetylated (hyperacetylation) involves a maximal number of negative charges on histone H4 that can neutralize the positively charged lysine histone tails. It is likely that a specific set of histone codes exist in the event of NER. At any rate, this ING lb-mediated chromatin relaxation is beneficial for the detection of photolesions. We have shown that in addition to the bacterial micrococcal nuclease, the cellular damage recognition factor X P A gains better access to the UV-damaged chromatin. Genetic complementation between XPC and INGlb in our HCR assay further supports this notion and implicates INGlb in the GGR pathway. The adverse effect of hypoacetylated chromatin on damage removal is exemplified by S-phase chromatin when INGlb expression is reduced. This leads to an 92 elevated blocked topoisomerase by CPD that leads to lethal double strand break, causing cell death. Lastly, we also found p300 HAT as a potential modulator of this ING lb-mediated response, although further experimentation should be done to confirm the role of p300 in ING lb-enhancement of NER. The INGlb intranuclear milieu was also examined in our study. We found that U V can induce an increased association between INGlb protein and chromatin. In addition, we found that INGlb is displaced from the nucleoli upon U V irradiation to disperse into the nucleoplasm. Recent publications by Gozani et al. (2003, 2005) have shown that the PHD finger, the signaling domain of ING family, is a binding module of the nuclear phosphatidylinosotol-5-phosphate (PtdIns(5)P). Interestingly, binding of PtdIns(5)P also causes a relocalization of ING 1 and ING2 proteins from nucleoli to nucleoplasm (Gozani et ah, 2003). How this nuclear phospholipid species is generated remains to be determined. It is thought however, that U V irradiation can promote the generation of PtdIns(5)P which in turn may induce the INGlb nucleoli-nucleoplasm shift, a phenomenom also proposed by Jones and Divecha (2004). Therefore, it is likely that ING-PtdIns(5)P complexes participate in the nucleolar disruption event that serves as a DNA damage sensor to initiate cellular response such as p53 activation. A direct link between Ptdlns signaling and ING-mediated chromatin relaxation also needs to be established. According to results obtained in the study, we would like to propose a working model entailing ING lb-promoted chromatin relaxation upon U V irradiation. Since ING family members can all enhance NER of photolesions, this model may also be extended to all 93 ING proteins. Upon U V irradiation, the production of PtdIns(5)P from Ptdlns increases. The lipid signal then binds to the PHD domain of ING proteins and activates their functions. Activated INGs then mobilize from nucleolus to nucleoplasm where they form de novo interactions with p300 HAT and possibly p53. This newly formed ternary complex is high mobile and engages in dynamic global chromatin relaxation though histone H4 acetylation for inspection of DNA lesions. The PtdIns(5)P-bound PHD finger domain forms a new interface that binds to specific epigenetic histone marks and tethers the ING-p53-HAT complex to the damaged chromatin. Although the trimethylated histone H3 at Lys 4 can bind to the ING2 PHD domain (Shi et al, 2006), it is less likely to be the epigenetic mark for chromatin relaxation since its interaction with PHD domain is independent of PtdIns(5)P (Shi et al, 2006). Lastly, once the lesion is recognized, subsequent recruitments of NER factors result in efficient assembly of repairosome that ultimately removes D N A damage in a timely manner. Prompt histone deacetylation then ensues to restore the condensed chromatin structure and protect the integrity of the genome. 94 Figure 5-1. A working model for the chromatin relaxation during GGR. UV irradiation stimulates production of PtdIns(5)P that binds to the PHD domain of nucleolar INGlb, inducing nucleolar disintegration and INGlb activation. Activated INGlb then diffuses into nucleoplasm where it associates with p53 and p300 to form a ternary complex, capable of promoting AcH4 that leads to chromatin relaxation. GGR is then initiated by lesion detectors (XPA, XPC-HR23B, and UV-DDB complex) binds to photolesions in the damaged chromatin. Nuclear Enveloppe Chromatin Nucleolus Ptd(5)lns DNA Lesion ING1b-p53-p300 HAT complex Chromatin Relaxation Lesion Detection Factors Global Lesion Detection 95 6. References Aasland R, Gibson TJ, Stewart AF. (1995) The PHD finger: implications for chromatin-mediated transcriptional regulation. Trends Biochem Sci. 20(2):56-9. 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