Role of Nuclear Hat1p Complex and Acetylation of Newly Synthesized Histone H4 in
Chromatin Assembly
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of The Ohio State University
By
Zhongqi Ge
Molecular, Cellular and Developmental Biology Graduate Program
The Ohio State University
2013
Dissertation Committee:
Professor Mark Parthun, advisor
Professor Paul Herman
Professor James Hopper
Professor Jiyan Ma
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Copyright by
Zhongqi Ge
2013
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Abstract
Although Hat1p has long been presumed to be involved in chromatin assembly through the acetylation of newly synthesized histone H4 on lysine 5 and 12, there has been lack of straightforward evidence linking Hat1p to this process. We utilized the yeast Gal-HO system to study the function of nuclear Hat1p-containing type B histone acetyltransferase complex (NuB4) in DNA double strand break repair linked chromatin assembly. We found that each of the NuB4 components affects repair-linked chromatin reassembly but that their contributions are not equivalent. In particular, deletion of the catalytic subunit,
Hat1p, caused a significant defect in chromatin reassembly. In addition, loss of the histone chaperone Hif1p, when combined with an allele of H3 that mutates lysines 14 and
23 to arginine, has a pronounced defect in chromatin reassembly that is similar to that observed in an asf1∆. Lastly, the role of Hat1p and Hif1p is at least partially redundant with the role of the histone chaperone Asf1p in the DNA repair linked chromatin reassembly process.
The acetylation on lysine 5 and 12 of newly synthesized histone H4 is highly conserved and is temporally correlated with the process of chromatin assembly. However, this pattern of modification has not been shown to be essential for either cell viability or chromatin assembly in any model organism. Using genetic assays in S. cerevisiae, we
ii demonstrated that mutations in histone H4 lysines 5 and 12 confer hypersensitivity to replication stress and DNA damaging agents when combined with mutations in histone
H4 lysine 91, which has also been found to be a site of acetylation in the core domain of soluble histone H4. We also showed that mutation of the sites of acetylation on newly synthesized histone H4 results in defects in the reassembly of chromatin structure that accompanies the repair of HO-mediated double strand breaks in yeast cells. Intriguingly, mutations that alter the sites of newly synthesized histone H4 acetylation are also defective in DNA damage response signaling as the mutants show a marked decrease in the levels of phosphorylated H2A on chromatin 20 kb from double strand break. This decrease is not the result of an inability to generate phosphorylated histone H2A but to an inability to localize this modified histone to chromatin. Therefore, acetylation on newly synthesized histone H4 is required for proper chromatin assembly and maintenance of chromatin structure.
Using the technology called iPOND (isolation of proteins on nascent DNA), we were able to show direct biochemical evidence that mammalian Hat1p is involved in DNA replication coupled chromatin assembly. We found that mouse Hat1p influences newly synthesized histone H4 lysine 5 and 12 acetylation levels in the cell as well as on nascent chromatin. Also, we showed that in the absence of Hat1p, acetylation pattern of histone
H3 NH2-terminal tail is different from wild type cells. Finally, we provided evidence that mouse Hat1p is associated with replication forks.
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Dedicated to my parents
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Acknowledgements
I would like to express my deepest gratitude to my advisor, Dr. Mark Parthun, who has guided me through these six years of my PhD life with constant support and endless encouragement. As a scientist and mentor, he is creative, insightful and resourceful. His optimistic attitude towards research and scientific discovery has led me through difficulties and profited me.
I would also like to thank the members of my dissertation committee, Dr. Paul Herman,
Dr. Jiyan Ma and Dr. James Hopper for the critical reviews of this thesis, scientific input and valuable comments for my PhD research. Their knowledge and suggestions also provide me with guidance to my future career.
I also thank my colleagues in the Parthun lab both past and present: Amy Knapp, Xi Ai,
Jianxin Ye, Qin Song, Erica Mersfelder, Devi Nair, Raghuvir Tomar, Huanyu Wang,
Prabakaran Nagarajan, Rajbir singh, Neha Rastogi, Pei Zhang and Paula Agudelo. In particular, I would like to thank Amy, Erica, Prabakaran and Huanyu for teaching me all the techniques and discuss with me scientific questions with great patience. I thank
Huanyu for her valuable scientific suggestions for my research as well as friendship in my life in Columbus. I am grateful to Rajbir and Neha for real-time PCR and SILAC techniques.
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In addition, I would like to thank the Pelotonia Fellowship Program from the Ohio State
University Comprehensive Cancer Center for supporting the last two years of my PhD study. And I would like to thank the Bell Lab, Ma Lab, Rafael Lab, Scheonberg lab,
Burghes Lab, Kolb Lab Freitas lab and Cortez Lab for communications and collaborations.
Finally, I would like to thank my parents for always believed in me, supported me and be there for me. I could not have done this without your love.
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Vita March 24, 1983……………………………………………………...Born, Hefei, Anhui, China PR
Sep 2001 – July 2005………………...……………...B.S. Tsinghua University, Beijing, China PR
Jan 2011 – June 2012……………………….Master Applied Statistics, The Ohio State University
Sep 2006 – Present…………….Graduate Research Associate, MCDB, The Ohio State University
Publications
1. Ge, Z., Wang, H., and Parthun, M.R., Nuclear Hat1p complex (NuB4) components participate in DNA repair-linked chromatin reassembly. 2011, Journal of Biological Chemistry, 286, 16790-16799. 2. Nair, D.M., Ge, Z., Mersfelder, E.M. and Parthun, M.R., Genetic Interactions between POB3 and the Acetylation of Newly Synthesized Histones. 2011, Current Genetics, 57, 271-286. 3. Wang, Huanyu; Ge, Zhongqi; Walsh, Scott; Parthun, Mark, The Human Histone Chaperone sNASP. 2011, Nucleic Acid Research, 40, 660-669. 4. Shang-feng Liu, Chao Ai, Zhong-qi Ge, Hai-luo Liu, Bo-wen Liu, Shan He and Zhao Wang, Molecular Cloning and Bioinformatic Analysis of SPATA4 Gene, 2005, Journal of Biochemistry and Molecular Biology, 38, 739-747. 5. Ge, Z. and Parthun, M.R., Role of acetylation of newly synthesized histone H4 in DNA damage response, submitted. 6. Prabakaran Nagarajan, Zhongqi Ge, Bianca M Sirbu, Paula A. Agudelo Garcia, Cheryl Doughty, Antyony Annunziato, David Cortez, Lukas Kenner, Mark Parthun, HAT1 is essential for mammalian development, genome stability and the processing of newly synthesized histone H3 and H4, submitted.
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Fields of study
Major Field: Molecular, Cellular and Developmental Biology
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Table of Contents
Abstract ...... ii Dedicated to my parents...... iv Vita ...... vii List of Tables ...... xiii List of Figures ...... xiv Chapter 1 Introduction ...... 1 1.1 Histone Posttranslational Modifications ...... 2
1.2 Chromatin Assembly ...... 4
1.3 Chromatin assembly related histone acetylation ...... 6
1.4 DNA double strand break repair mechanisms ...... 8
1.4.1 yeast mating type switching system as a model for DNA DSB repair ...... 10
1.4.2 DNA DSB repair related histone acetylation ...... 10
1.4.3 histone H2AX phosphorylation and dephosphorylation in response to DNA
DSBs ...... 13
1.4.4 DNA damage checkpoint response ...... 14
List of abbreviations ...... 18
Chapter 2 Nuclear Hat1p Complex (NuB4) Components Participate in DNA Repair- linked Chromatin Reassembly ...... 20 2.1 Abstract ...... 20
2.2 Introduction ...... 21 ix
2.3 Experimental Procedures ...... 25
2.3.1 Yeast Strains ...... 25
2.3.2 HO Endonuclease Sensitivity Assays ...... 25
2.3.3 DNA Damage and Repair Analysis ...... 25
2.3.4 Chromatin Immunoprecipitation Analyses ...... 26
2.3.5 Quantitative Real Time PCR Analysis ...... 26
2.3.6 Whole Cell Extracts ...... 27
2.3.7 Column Chromatography ...... 27
2.3.8 Western Blotting ...... 28
2.3.9 Histone Acetyltransferase Assays ...... 28
2.3.10 HO Gene Expression Analysis ...... 28
2.4 Result ...... 29
2.4.1 Involvement of NuB4 Complex Components in DNA Repair-linked Chromatin
Reassembly ...... 29
2.4.2 Non-overlapping Action of Asf1p and NuB4 Complex Components in
Chromatin Reassembly ...... 32
2.4.3 Hif1p Is Present in High Molecular Weight Complexes Independent of Hat1p
and Hat2p and Influences a Histone H3-specific HAT ...... 34
2.5 Discussion ...... 36
Chapter 3 Sites of Acetylation on Newly Synthesized Histone H4 are required for Chromatin Assembly and DNA damage Damage Response Signaling...... 51 3.1 Abstract ...... 51
3.2 Introduction ...... 52
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3.3 Experimental Procedures ...... 57
3.3.1 Yeast strains...... 57
3.3.2 Yeast phenotypic assays...... 58
3.3.3 Cell extracts, chromatin fractionation and immunoblotting...... 58
3.3.4 DNA damage and repair analysis...... 59
3.3.5 ChIP and realtime PCR...... 59
3.4 Results ...... 60
3.4.1 Functional redundancy in the sites of acetylation found on newly synthesized
histones H3 and H4...... 60
3.4.2 Histone H4 lysines 5, 12 and 91 are involved in DNA repair-linked chromatin
reassembly...... 63
3.4.3 Sites of newly synthesized histone H4 acetylation are required for normal
DNA damage response signaling...... 66
3.4.4 Histone H4 lysine 91 acetylation and lysine 5, 12 acetylation affect different
aspects in the DNA damage response...... 69
3.5 Discussion ...... 71
Chapter 4 Role of mammalian Hat1p in DNA replication coupled chromatin assembly . 89 4.1 Abstract ...... 89
4.2 Introduction ...... 89
4.3 Materials and Methods ...... 91
4.3.1 Cell culture ...... 91
4.3.2 IPOND Method ...... 91
4.4 Results ...... 93 xi
4.4.1 Hat1p is essential for normal H4 K5, 12Ac level at the replication fork ...... 93
4.4.2 Hat1p influences acetylation on newly synthesized histone H3 ...... 93
4.4.3 Hat1p can be detected at the replication fork ...... 94
4.5 Discussion ...... 95
Bibliography ...... 101
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List of Tables
Table 2.1 Strains used in this study ...... 41
Table 3.1 Strains used in this study ...... 76
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List of Figures
Figure 2.1 Sensitivity of NuB4 complex and histone H3 mutants to an HO- induced DNA double strand break...... 42
Figure 2.2 Impact of NuB4 complex and histone H3 mutations on DNA repair- linked chromatin reassembly...... 43
Figure 2.3 Effect of combining the H3K14R, K23R allele with mutations of the
NuB4 complex components on DNA repair-linked chromatin reassembly...... 45
Fig 2.4 Functional redundancy between NuB4 complex components, histone H3, and Asf1p in DNA repair-linked chromatin reassembly...... 46
Figure 2.5 Hif1p is present in a high molecular weight complex independent of HAT1 and HAT2 and influences an H3-specific HAT activity...... 48
Figure 2.6 Transcriptional induction of the HO gene...... 50
Figure 3.1 Histone H4 lysines 5, 12 and 91 are functionally redundant...... 77
Figure 3.2 Histone H4 lysines 5, 12 and 91 function in chromatin assembly...... 78
Figure 3.3 Newly synthesized H4 acetylation site mutants are defective in the formation of γ-H2AX domains near a double strand break...... 80
Figure 3.4 Sites of acetylation of histone H4 are not necessary for the formation of γ-H2AX...... 82
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Figure 3.5 The redundant function of newly synthesized histone H4 acetylation sites in the NH2-terminal tail and core domains requires Mec1p...... 84
Figure 3.6 Model for the involvement of chromatin assembly or histone exchange in DNA damage response signaling...... 85
Figure 3.7 Functional redundancy with histone H4 lysine 91 requires lysines 5 and 12ac...... 86
Figure 3.8 Repair of an HO-induced DNA double strand break...... 87
Figure 4.1 Mouse Hat1p is important for H4 lysine 5 and 12 acetylations in the cell ...... 98
Figure 4.2 Mouse Hat1p is important for H3 acetylations in the cell...... 99
Figure 4.3 Mammalian Hat1p is a replication fork associated protein...... 100
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Chapter 1 Introduction
The eukaryotic genome is tightly packaged into chromatin, which interacts with nuclear machineries and mediates processes such as DNA replication, transcription, and repair
(Kornberg and Lorch, 1999). The fundamental component of the chromatin is the nucleosome core particle, which is composed of approximately 147 bp of DNA wrapped around an octamer that consists of two copies of each of the core histones H2A, H2B, H3 and H4 (Kornberg, 1974; Luger et al., 1997). Nucleosomes are separated by varying lengths of linker DNA, which can be associated with linker histones to promote higher order and more compact structures (Widom, 1998).
The tightly packaged chromatin represents a formidable obstacle for regulatory proteins to gain access and three mechanisms have been used to adjust the nucleosomal structure and increase the accessibility of chromatin. First, ATP dependent chromatin remodelers use ATP hydrolysis to load, eject or restructure nucleosome structure (reviewed in
(Becker and Horz, 2002; Clapier and Cairns, 2009). Currently there are four different families of chromatin remodeling complexes, SWI/SNF, ISW, CHD and INO80, of which an ATPase domain is shared while the unique subunits target them to specialized biological processes. Second, chromatin structure can be altered by introducing variants of histone H2A as well as H3 into nucleosomes (Henikoff et al., 2004a). In contrast to canonical histones, these histone variants are synthesized and incorporated independent 1 of DNA replication and play important roles in a broad range of nuclear processes. The best characterized histone variants are histone H2AZ and histone H3.3, which are required for active transcription and are dynamically displaced and reloaded at transcription sites (Henikoff et al., 2004b; Santisteban et al., 2000). Third, chromatin can be modulated through covalent histone posttranslational modification (PTM). Most of the histone PTMs such as acetylation, phosphorylation, methylation, ubiquitination and
ADP-ribosylation, take place on the NH2-terminal tails of histones, while core domain and C-terminal tail modifications (for histone H2A and H2B) have been shown to also play important roles. The biological outcome of histone posttranslational modification is conveyed either through direct electrostatic change to the histone DNA interaction, or by providing platforms for downstream effectors (Cosgrove et al., 2004). Examples of translational recognition of histone codes by protein modules are the bromodomain which specifically recognize the acetylated lysines in the NH2-terminal tail and chromodomain, which targets metylation marks (Eissenberg, 2001; Horn and Peterson, 2001).
1.1 Histone Posttranslational Modifications
Histones are subject to an enormous number of posttranslational modifications, including acetylation and methylation on lysines and arginines, phosporylation on serines and threonines, ubiquitylation, sumoylation and ADP-ribosylation on lysines. This complexity is furthered by the fact that the lysine residues can accept mono-, di- or tri- methylation. The majority of these posttranslational marks are seen on the NH2-terminal tail domains, although more and more modifications have been discovered to be within the core domains of the histones, such as H3 lysine 56 acetylation, H4 lysine 91
2 acetylation and H3 lysine 79 methylation. These modifications are thought to regulate transcription, by promoting either active or silent promoter regions, and facilitate progression through DNA repair, barriers to silencing and DNA replication.
Many classical models proposed that histone modifications may directly influence nucleosome structure or chromatin folding arrays. For example, histone hyper-acetylation is thought to weaken DNA-histone contacts by neutralizing the positive charges on histone NH2-terminal tails and decreasing the affinity for negatively charged DNA sugar phosphate backbone, and vice versa. Thus histone hyperacetylation leads to an active chromatin structure and increased gene transcription, while histone hypoacetylation promotes a silent chromatin structure and, hence, decreased transcription (Edmondson and Roth, 1996; Lee et al., 1993).
The histone code hypothesis, on the other hand, suggests that histone posttranslational modifications provide binding platforms for nonhistone proteins to the chromatin fiber
(Cosgrove et al., 2004). In support of this hypothesis, structural studies showed that many chromatin-binding proteins share structural modules that recognize hisone posttranslational modifications. For example, chromodomains bind to methylated lysines, whereas bromodomains bind to acetylated lysines (Eissenberg, 2001; Horn and Peterson,
2001). A variety of proteins can be recruited, such as histone-modifying enzymes, transcription factors, heterochromatin-associated proteins, as well as ATP-dependent nucleosome-remodeling enzymes. Several lines of evidence suggest that recruited proteins either stabilize or remodel specific chromatin states.
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1.2 Chromatin Assembly
Chromatin assembly is the process by which genomic DNA is packaged into nucleosomes and the subsequent formation of higher-order structures (Haushalter and
Kadonaga, 2003). The basic chromatin assembly process requires the cooperation of histone chaperones, which prevent nonspecific interactions between positively charged histones and negatively charged DNA, and ATP-utilizing factors which catalyze the deposition of the histones onto DNA to yield periodic arrays (Haushalter and Kadonaga,
2003; Tyler, 2002). There are two main types of chromatin assembly, namely replication- conjugated chromatin assembly and replication independent chromatin assembly.
DNA replication dependent chromatin assembly takes place in S phase and is tightly coupled to DNA replication and possibly DNA repair. Before the replication fork, pre- existing nucleosomes are disrupted to allow passage of the replication fork. Parental histones are recycled and loaded back onto nascent DNA with no preference for leading or lagging strand, which only provide half of the histone needed for nucleosome assembly. Newly synthesized histones are also deposited onto nascent DNA, which provide the other half of the histones required to complete nucleosome assembly. The replication-dependent de novo nucleosome deposition starts from the synthesis of core histones in the cytoplasm, which are then acetylated at specific lysine residues.
Acetylated histones will then form heterodimeric complexes as H3/H4 and H2A/H2B dimmers, which will be recognized by specific histone chaperones and soluble transport factors called karyopherins or importins that shuttle them into the nucleus (Greiner et al.,
2004). Once in the nucleus, core histones are deposited onto DNA in the order that
4 histones H3 and H4 are deposited first followed by histones H2A and H2B. DNA replication coupled deposition of histone H3 and H4 is mediated by the chromatin assembly factor 1 (CAF-1) complex, which deposits newly synthesized histone H3/H4 at the replication fork by directly interacting with proliferating cell nuclear antigen (PCNA)
(Smith and Stillman, 1989). Another histone chaperone nucleosome assembly protein 1
(NAP-1) is responsible for H2A/H2B deposition (Park and Luger, 2006). The last step is to generate physiologically spaced arrays of nucleosomes, which requires the ATP- dependent chromatin remodeling factors such as ATP-utilizing chromatin assembly and remodeling factor (ACF) and remodeling and spacing factor (RSF).
The second type of chromatin assembly occurs outside of S phase and usually involves incorporation of histone variants, which alters chromatin structure and therefore the switch of epigenetic states. Histone variants, such as H2A.Z and H3.3 are synthesized outside of S phase and can be deposited onto chromatin as needed throughout the cell cycle (Ray-Gallet et al., 2002). For instance, the assembly of histone variant H3.3 is mediated by histone chaperone HIRA protein and is targeted to transcriptionally active loci such as the rDNA arrays (Ahmad and Henikoff, 2002). The exchange of histone variant H2AZ for canonical H2A is catalyzed by the SWR1 chromatin remodeling complex, which also takes place in transcriptionally active domains near yeast telomeres and acts as a buffer to gene silencing imposed by the spread of Sir proteins (Meneghini et al., 2003).
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1.3 Chromatin assembly related histone acetylation
Histone acetylation is a reversible modification, in which histone acetyltransferases
(HATs) transfer acetyl groups from acetyl-CoA to the -NH2 group of the side chain of conserved lysine residues (Brownell and Allis, 1996). When it was first discovered, acetylation of lysine residues was thought to be neutralizing positive charges, thus reducing affinity between histone and DNA and allowing access to proteins such as transcription factors.
Newly synthesized histone H3 and H4 are found to be acetylated at specific lysine residues before they are deposited onto chromatin (Annunziato and Hansen, 2000). The acetylaion of the NH2-terminal tails of newly synthesized histone H3 and H4 is evolutionary conserved among species. Newly synthesized histone H4 is diacetylated at lysine 5 and 12, but not at lysine 8 and 16, which is conserved from yeast to humans.
Unlike histone H4, the acetylation pattern on newly synthesized histone H3 is not the same in different organisms. In yeast cells, out of the five acetylatable lysine residues (K9,
K14, K18, K23 and K27), newly synthesized histone H3 is predominantly acetylated at lysine 9, followed by lysine 27. In addition to the acetylation on the NH2-terminal tail domains, newly synthesized histone H3 and H4 are also acetylated at their globular core domains. For instance, histone H3 is actylated at lysine 56 and histone H4 is acetylated at lysine 91 (Ozdemir et al., 2005; Ye et al., 2005). No particular pattern of acetylation on newly synthesized histone H2A and H2B has been discovered (Annunziato and Hansen,
2000).
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Histone acetylation is catalyzed by histone acetyltransferases (HATs), which can be classified into two groups with respect to their intracellular locations and substrate specificity. Type A HATs, usually subunits of chromatin modifying multiprotein complexes, localize to the nucleus and are capable of acetylating chromosomal histones.
On the other hand, type B HATs acetylate free histones in the cytoplasm, which is thought to be the first step of de novo chromatin assembly. Histone H4 diacetylation at lysine 5 and 12 is catalyzed by the type B HAT Hat1p (Parthun et al., 1996). The yeast
Hat1p forms a complex with Hat2p, which is essential for a high level of Hat1p catalytic activity in the cytoplasm. Horologes of the Hat1p/Hat2p complex have been identified in a variety of organisms, hence, both the acetylation pattern on newly synthesized histone
H4 and the enzyme complex that modify it are highly conserved throughout eukaryotes
(Chang et al., 1997; Imhof and Wolffe, 1999; Lusser et al., 1999; Verreault et al., 1998).
Interestingly, the Hat1p/Hat2p complex is not restricted to the cytoplasm but is also found in the nucleus (Ai and Parthun, 2004; Parthun et al., 1996). In yeast cells, the nuclear Hat1p (NuB4) complex contains Hat1p, Hat2p and Hif1p. Hif1p was shown to be a histone H3/H4 chaperone and has intrinsic chromatin assembly activity (Ai and Parthun,
2004). Newly synthesized histone H3 is acetylated at lysine 56 and lysine 9 by Rtt109, which appears during S phase and disappears at the G2/M phase of the cell cycle
(Driscoll et al., 2007; Han et al., 2007b; Schneider et al., 2006). Lysine 56 on histone H3 is located at the end of the alpha-helix and in close proximity to the entry of exist point of the DNA. Rtt109 co-purifies with Vps75 and the native Rtt109-Vps75 complex purified
7 from yeast cells acetylates only free, but not chromatin associated histone H3 (Han et al.,
2007c).
1.4 DNA double strand break repair mechanisms
DNA double strand break (DSB) formation is essential for cellular processes such as genetic recombination in mitosis and meiosis. On the other hand, DNA double strand breaks generated by environmental stress can be detrimental to the genome and inappropriately repaired DSBs can cause genetic instability, which could lead to cancer.
Thus, efficient repair of DSBs is indispensable for the integrity and functionality of the genome. There are several mechanisms for DSB-repair, such as homologous recombination (HR), single strand annealing (SSA), break induced replication (BIR) and non-homologous end joining (NHEJ).
The two main pathways are NHEJ, which is merely a simple ligation of the broken DNA ends, and HR, which requires homologous sequences and new DNA synthesis. The key components of the NHEJ pathway in Sacchromyces cerevisiae are the yKu70/80 heterodimers, the MRX (Mre11, Rad50 and Xrs2) complex, and the specialized DNA ligase IV (Dnl4) with its associated factors Lif1 and Nej1 (Daley et al., 2005). The broken DNA ends are held together by the yKu70/yKu80 heterodimer, which forms a ring-like structure encircling the duplex DNA, as well as the MRX complex, which bridges two DNA ends via the intermolecular interaction of the Rad50 coiled coil domain
(de Jager et al., 2001; Hopfner et al., 2002; Wiltzius et al., 2005). NHEJ is completed by the subsequent ligation of the two broken DNA ends by the DNA ligase Dnl4 and Lif1, which stabilize Dnl4 and is required for Dnl4’s ligase activity (Zhang et al., 2007). The
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NHEJ pathway is chosen for repair of the DNA double strand break mainly through binding of the Ku complex onto DSB ends, which is stabilized by Dnl4/Lif1, thus preventing exonuclease-mediated resection of the double strand DNA.
Homologous recombination is the major pathway to repair DNA double strand breaks in the presence of a homologous template. The first step involves 5’ to 3’ resection from the
DNA lesion, which reveals 3’ tailed single strand DNA (ssDNA) (Lee et al., 1998). This step requires multiple exonucleases such as Mre11, Exo1 and certain unidentified factors
(Moreau et al., 2001). As the resection proceeds, replication protein A complex (RPA) binds to ssDNA and facilitates the formation of Rad51 nucleofilament (Krogh and
Symington, 2004). Rad52 is required for targeting Rad51 to RPA bound ssDNA and the subsequent Rad51 mediated RPA displacement since in rad52 cells, Rad51 foci formation in response to an HO endonuclease-induced DSB is abolished and Rad52 localization to DNA damage sites is not detected by chromatin immuno-precipitation
(ChIP) (Miyazaki et al., 2004; New et al., 1998; Sugawara et al., 2003). Besides Rad52,
Rad55/Rad57 also promotes and stabilizes Rad51 nucleofilaments (Sung, 1997). Rad51 coated ssDNA then searches for and invades homologous sequences in the genome and forms a heteroduplex structure (Paques and Haber, 1999). Rad54, a SWI2/SNF2-related protein with ATPase/DNA translocase activity and chromatin remodeling activity, mediates dissociation of the D-loop structure and promotes displacement of Rad51 from the synaptic structure through its ATPase activity (Bugreev et al., 2006). After successful homology search and formation of a synaptic structure, cells can use three different HR pathways. Gene conversion (GC), when both ends of broken DNA share homology with
9 template sequence, break induced repair (BIR) if only one end of the broken DNA is homologous to the template DNA, and single strand annealing (SSA) if the DSB occurs between two flanking homologous sequences. The result of HR is the one way transfer of genetic information from the template to the damaged site, which is faithful and accurate repair of the harmful DSB.
1.4.1 yeast mating type switching system as a model for DNA DSB repair
Yeast cells have two mating types, a and , that are dependent on the mating type pheromones expressed from the MAT mating locus. Besides the MAT locus, yeast cells have two silent mating loci HML and HMR, which contain mating type information for mating type and a, respectively. A yeast cell changes its mating type by expressing the endonuclease HO, which then cuts specifically at the YZ junction of the MAT locus, generating a DNA DSB. The cell then uses homologous sequences present at the one of the silent mating loci to repair the break, usually resulting in the mating type change
(Haber, 1995). By putting the HO endonuclease gene under the GAL-10 promoter and integrated into the yeast genome, the induction or repression of HO can be regulated by adding galactose or glucose in to the media, respectively. The yeast mating type switching system is well characterized and a useful tool to study a single defined DNA
DSB by homologous recombination (Tsukuda et al., 2009).
1.4.2 DNA DSB repair related histone acetylation
In yeast cells, mutation of all four lysines on the histone H4 NH2-terminal tail to glutamine results in a marked delay during G2/M phase of the cell cycle, which is checkpoint protein Rad9 dependent (Megee et al., 1995). This mutant also shows 10 sensitivity to DNA damaging agents such as Camptothecin (CPT), which inhibits topoisomerase I and methane methyl sulfonate (MMS), which is a DNA alkylating agent that causes single and double strand breaks (Bird et al., 2002). Similarly, mutations in histone H3 NH2-terminal lysines, especially changing lysines 14 and 23 to arginine, leads to sensitivity to genotoxic stress (Qin and Parthun, 2002). Also, acetyation of multiple sites on the histone H3 NH2-terminal tail increases following a single DNA DSB
(Tamburini and Tyler, 2005). Histone H3 lysine 56 acetylation is also involved in the
DNA DSB response by turning off the DNA damage checkpoint signal (Chen et al.,
2008). These studies showed that histone H3 and H4 lysine acetylation are important in genome integrity.
Histone acetyltransferases (HATs) have also been shown to be involved in the DNA damage response. When Esa1, the catalytic subunit of NuA4, is mutated to lose its catalytic activity, the mutant cell is hypersensitive to DNA damaging agents and shows a
Rad9 dependent delay in G2/M progression, reminiscent of mutating lysine residues on
H4 NH2-terminal tails (Clarke et al., 1999; Downs et al., 2004). Association of NuA4 subunits such as Arp4, Esa1, Epl1 and Eaf1 to the site of a HO endonuclease induced
DNA DSB can be detected by ChIP. It is then demonstrated that the Arp4 subunit of the
NuA4 complex recognizes specifically the - H2A induced in DNA damage response pathway and recruits the rest of the complex to the DNA lesion (Downs et al., 2004).
Notably, another histone H4 acetyltransferase, Hat1p, is also detected by ChIP near the
HO endonuclease induced DNA DSB (Qin and Parthun, 2006). Gcn5p is also recruited to
DNA DSB sites and gcn5 cells are sensitive to DNA damaging reagents. Recently,
11 research has shown that HATs such as Rtt109 and Hat1p promote nucleosome reassembly following DNA repair which is required for DNA damage recovery (Chen et al., 2008; Ge et al., 2011). Also, it has been shown that H3 NH2-terminal tail acetylation by Gcn5 following DNA damage may aid in the recruitment of the chromatin remodeling
SWI/SNF complex, which facilitate DNA damage signal transduction (Lee et al., 2010).
Thus, HATs mediated histone lysine acetylations may impact various aspects of the DNA damage repair process including cell cycle arrest, chromatin reassembly, and checkpoint recovery.
Histone deactylases (HDACs) also have roles in DNA DSB-induced repair pathways as demonstrated by several studies. Disruption of SIN3 leads to hypersensitivity to the DNA
DSB inducing drug Phleomycin, with little or no sensitivity towards UV light induced pyrimidine dimmers, which are repaired by nucleotide excision repair (Jazayeri et al.,
2004). It was also shown that Sin3p and Rpd3p are required for efficient nonhomologous end joining repair of DNA DSBs (Jazayeri et al., 2004). In response to an HO endonuclease induced DNA DSB, histone H4 lysine 16 is first acetylated and then deactylated following homologous recombination. Using ChIP assays, the histone deactylases such as Sir2p, Hst1p and Rpd3p are recruited to the sites of the DNA lesion concurrent with the time recombination occurs (Tamburini and Tyler, 2005). The fact that the recruitment of HATs and HDACs to the DNA lesion in a timely manner explains the paradox that enzymes with opposite effects are both required for DNA DSB repair. It also provides examples for the interplay among histone modifications to fulfill certain cellular functions (Utley et al., 2005).
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1.4.3 histone H2AX phosphorylation and dephosphorylation in response to DNA DSBs
Histone H2AX (H2A in S. cerevisiae) phosphorylation is the earliest and most abundant histone modification in response to DNA damage. H2AX has a carboxy-terminal SQE motif which is recognized by phosphatidylinositol 3-kinase family proteins ATR (Mec1 in yeast), ATM (Tel1 in yeast) and DNA-dependent protein kinase (only in mammals).
DNA DSBs invariably lead to H2AX phosphorylation regardless of the source of the damage. Phosphorylated H2AX, referred to as - H2AX is rapidly detected at the DNA damage site by immunofluorescence and by ChIP (Shroff et al., 2004). The - H2AX signal spans a large region up to 100 kb in yeast cells, while ChIP results showed that the level of - H2AX adjacent to the break site is very low (Kim et al., 2007; Shroff et al.,
2004).
Although H2AX phosphorylation is highly coordinately regulated, the precise role of this modification is still not completely understood. H2AX-/- mouse embryonic cells or cells with H2AX-S139A mutation are viable but show sensitivity to ionizing radiation or camptothecin and genomic instability (Bassing et al., 2002; Celeste et al., 2002). In yeast cells, H2A phosphorylation, mediated by Mec1 and Tel1, is required for the efficient repair of chromosomal DSBs by NHEJ but not by homologous recombination (Downs et al., 2000). It is believed that - H2AX is not essential for DNA repair per se, nor the initial formation of DNA repair foci in vivo, rather it is important for retaining DNA repair factors near the DNA damage site (Celeste et al., 2003). For instance, the DNA end-binding Mre11-Rad50-Xrs2 (MRX) complex is required for recognition, signal transduction and DNA end resection in the early DNA damage responses and contains a
13 subunit, Xrs2 (Nbs1 in mammals), which specifically binds to - H2AX (Kobayashi et al., 2002). 53BP1 and its yeast homolog Rad9p are also shown to interact with - H2AX and depend on the interaction for their functions as adaptors between the sensing kinases
ATM and ATR and the effector kinases Chk1 and Chk2 (Rad53p in yeast) (Toh et al.,
2006; Ward et al., 2003). However, the initial recruitment of 53BP1 is normal in H2AX-/- cells while its accumulation is impaired. Similarly MRX complex foci upon DNA damage is detected but not sustained in the absence of - H2AX. Another important function of H2AX phosphorylation is the recruitment of cohesins to DNA lesions (Strom et al., 2004; Unal et al., 2004). Studies in yeast showed that the HO endonuclease induced
DNA DSB leads to cohesin recruitment within 45 min of the break formation. Cohesin accumulates to the same extent as - H2AX and up to 50 kb around the break, which is dependent on - H2AX. Therefore, - H2AX is used to tether sister chromatids through
DNA DSB induced cohesins and to facilitate homologous recombinational repair of the break.
1.4.4 DNA damage checkpoint response
In response to a DNA double strand break, cells activate cell cycle checkpoint pathway to stall cell cycle in G2/M phase to allow DNA repair. The DNA damage signal is first detected by sensors, and is then amplified through downstream mediators and effectors.
Phosphatidylinositol 3-kinase like kinases (PI3KKs), ATM (Tel1 in S. cerevisiae) and
ATR (Mec1 in S. cerevisiae), and DNA-PK (only in mammals) are key players in the
DNA damage response pathway, which trigger a kinase cascade (Pellicioli et al., 2001).
14
Maintenance of the kinase activities of the PI3KKs is required for the checkpoint signal, which will be deactivated once DNA is repaired (Harrison and Haber, 2006).
The MRX complex (Mre11-Rad50-Xrs2), which recognizes the DNA DSB ends, is recruited to the lesion at an early stage. MRX complex functions in ssDNA formation and deletion of MRX proteins reduces the rate of DSB resection. The ssDNA is then coated with RPA, which provides the platform for PI3KKs to be recruited. In S. cerevisiae,
Mec1 forms a complex with Ddc2 (ATRIP in mammals), which is the binding partner of
Mec1 and is indispensable for the recruitment of Mec1 to DNA lesion (Rouse and
Jackson, 2000). The largest subunit of the RPA complex, Rfa1, is important for loading of the Mec1/Ddc2 complex since a mutant form, rfa-11, reduced association of Ddc2 at the HO-induced DNA DSB detected either by ChIP or by GFP-Ddc2 (Lisby et al., 2004;
Nakada et al., 2004). Interestingly, although the rfa-11 mutant is defective in recombinational repair of DNA DSBs, the mutant is competent to execute a DNA damage checkpoint (Lee et al., 1998). It is shown that Mec1p recruitment to the ssDNA is required for it to phosphorylate downstream targets. Activation of Tel1 is also dependent on the MRX complex. More specifically, Tel1 is recruited to the DSB lesion by binding to the C-terminus of Xrs2 (Nakada et al., 2003). Unlike the Mec1-Ddc2 complex, which binds to RPA coated ssDNA and spreads outward from the DNA lesion, MRX and Tel1 bind to blunt or minimally processed DSB ends.
The 9-1-1 clamp (Rad17-Mec3-Ddc1 complex), which shares structural homology to the
DNA replication clamp, PCNA, is also loaded at the DNA lesion immediately after the induction of DNA DSBs (Lisby et al., 2004; Nakada et al., 2004). In S. cerevisiae,
15 loading of the 9-1-1 clamp is mediated by the checkpoint clamp loader, the RFC complex containing Rad24 instead of Rfc1 (Ellison and Stillman, 2003). RPA coated ssDNA is also required for the recruitment of the 9-1-1 complex while Mec1 is dispensable
(Nakada et al., 2004). However, Mec1-dependent phosphorylation of checkpoint mediator Rad9 is reduced in 9-1-1 mutant cells (Emili, 1998). Therefore, the 9-1-1 complex is considered a DNA DSB sensor which promotes Mec1 activity.
The DNA damage response signal is amplified by a kinase cascade downstream of
Mec1/Tel1. In S. cerevisiae, Rad53 (Chk2 in mammals) is the major downstream effecter for signal amplification. Rad53 is phosphorylated in a Mec1- and Tel1- dependent manner. Rad9, which is also phosphorylated by Mec1 and Tel1, has been shown to play a role as an adaptor for signal transduction as the FHA domain of Rad53 binds to phosphorylated Rad9 (Schwartz et al., 2002). This interaction helps bring Rad53 to close vicinity of Mec1 and facilitates in trans autophosphorylation of Rad53 and thus is required for Rad53 hyperphosphorylation. Activated Rad53 is then released and phosphorylates downstream effectors such as Dun1, which negatively regulates the ribonucleotide reductase inhibitor Sml1 (Zhao and Rothstein, 2002). Another transducer kinase, Chk1, is also phosphorylated by Mec1 and is required for Pds1 phosphorylation.
Phosphorylated Pds1 is resistant to anaphase promoting complex (APC) mediated ubiquitination and degradation and thus is required for normal cell cycle arrest during
DNA damage repair (Agarwal et al., 2003).
When DNA damage is repaired and chromatin structure is restored, the DNA damage checkpoint needs to be turned off so that cells can go back to normal cell cycle
16 progression. In S cerevisiae, the process when cells are released from cell cycle arrest after DNA repair is called “recovery”. Although the exact mechanism of DNA damage recovery is not known, it is shown that at least γ – H2A and Rad53 need to be dephosphorylated (Pellicioli et al., 2001). The protein phosphatase 2C (PP2C) family member Ptc2 and Ptc3 are required for Rad53 dephosphorylation since in ptc2∆ptc3∆ cells, there is prolonged Rad53 hyperphosphorylation and cells fail to resume the cell cycle even after DNA is repaired (Leroy et al., 2003). Dephosphorylation of γ – H2A in yeast cells by the PP4C phosphatase complex (Pph3, Psy2 and Ybl046w) also influences the duration of cell cycle arrest. Cell lacking any of the complex components has H2A hyperphophorylation even in the absence of DNA damage and has prolonged cell cycle arrest despite normal DNA repair (Keogh et al., 2006). On the other hand, the persistence of an unrepairable HO-endouclease induced DNA DSB (induced in the absence of the
HML and HMR sequences) does not prevent the checkpoint from being turned off in yeast cells, a phenomenon called “adaptation”. Mutants that have adaptation defects include yku70∆, which has increased resection rates at the DNA DSB, and ptc2∆ptc3∆, which fails to dephosphorylate checkpoint proteins (Harrison and Haber, 2006; Leroy et al., 2003). Although the significance of adaptation is not clear, especially for cells of multicellular eukaryotes, understanding its mechanism will give insight to how cells inactivate cell cycle arrest.
17
List of abbreviations
PTM posttranslational modification
HAT histone acetyltransferase
HDAC histone deactylase
ChIP Chromatin Immuno-precipitation
DSB DNA double strand break
HR homologous recombination
SSA single strand annealing
BIR break induced replication
NHEJ non-homologous end joining ssDNA single strand DNA
RPA replication protein A
D-loop displacement loop
GC gene converstion
CPT camptothecin
MMS methane methyl sulfonate
MRX Mre11-Rad50-Xrs2
PI3KK phosphatidylinositol 3-kinase like kinase
9-1-1 clamp Rad17-Mec3-Ddc1 complex
APC anaphase promoting complex
18
PP2C protein phosphates 2C
19
Chapter 2 Nuclear Hat1p Complex (NuB4) Components Participate in
DNA Repair-linked Chromatin Reassembly
2.1 Abstract
Chromatin is disassembled and reassembled during DNA repair. To assay chromatin reassembly accompanying DNA double strand break repair, ChIP analysis can be used to monitor the presence of histone H3 near the lesion. The chromatin assembly factor
Asf1p, as well as the acetylation of histone H3 lysine 56, have been shown to promote chromatin reassembly when DNA double strand break repair is complete. Using Gal-HO- mediated double strand break repair, we have tested each of the components of the nuclear Hat1p-containing type B histone acetyltransferase complex (NuB4) and have found that they can affect repair-linked chromatin reassembly but that their contributions are not equivalent. In particular, deletion of the catalytic subunit, Hat1p, caused a significant defect in chromatin reassembly. In addition, loss of the histone chaperone
Hif1p, when combined with an allele of H3 that mutates lysines 14 and 23 to arginine, has a pronounced effect on chromatin reassembly that is similar to that observed in an asf1∆. The role of Hat1p and Hif1p is at least partially redundant with the role of Asf1p.
Consistent with a more prominent role for Hif1p in chromatin reassembly than either
Hat1p or Hat2p, Hif1p exists in complex(es) independent of Hat1p and Hat2p and
20 influences the activity of an H3-specific histone acetyltransferase activity. Our data directly demonstrate the role of the nuclear HAT1 complex (NuB4) components in DNA repair linked chromatin reassembly.
2.2 Introduction
The post-translational acetylation of the core histone NH2-terminal tails has been shown to be an important mechanism by which cells regulate the accessibility of chromatin
(Kouzarides, 2007). Histone acetylation plays an important role in the initial formation of chromatin structure, as well. A role for histone acetylation in chromatin assembly was first suggested by the observation that histones H3 and H4 are rapidly acetylated on their
NH2-terminal tail domains in the cytoplasm following their synthesis (Jackson et al.,
1976; Ruiz-Carrillo et al., 1975). Once incorporated into chromatin, this acetylation is removed during the process of chromatin maturation (Annunziato and Seale, 1983).
The acetylation of the NH2-terminal tails of newly synthesized histones H3 and H4 is an evolutionarily conserved phenomenon. The acetylation state of newly synthesized molecules of histone H4 has been analyzed in a diverse collection of eukaryotic organisms. In each case, new H4 is found to be diacetylated at lysine residues 5 and 12
(Annunziato and Hansen, 2000; Chicoine et al., 1986; Sobel et al., 1995). Whereas the presence of acetylation on the NH2-terminal tail of newly synthesized histone H3 has been conserved, distinct patterns of modification are found in different organisms
(Masumoto et al., 2005; Sobel et al., 1995). In addition to the acetylation of the NH2- terminal tail domains of newly synthesized H3 and H4, it has recently been found that these molecules are also acetylated in their globular core domains. Histone H3 is
21 acetylated on lysine 56 and histone H4 is acetylated on lysine 91 (Masumoto et al., 2005;
Ozdemir et al., 2005; Xu et al., 2005; Ye et al., 2005). These sites of acetylation have been observed in both yeast and mammalian cells and, hence, may also be evolutionarily conserved modifications (Basu et al., 2009; Das et al., 2009; Xie et al., 2009; Zhang et al., 2003).
The acetylation of newly synthesized histones is catalyzed by type B histone acetyltransferases. Type B histone acetyltransferases were originally defined as cytoplasmic enzymes that acetylate free, but not chromatin-associated, histones
(Brownell and Allis, 1996). The first type B histone acetyltransferase complex was isolated from Saccharomyces cerevisiae cytoplasmic extracts and consisted of two proteins, Hat1p and Hat2p (Kleff et al., 1995; Parthun et al., 1996). Hat1p, the catalytic subunit, specifically acetylates lysine residues at positions 5 and 12 of free histone H4.
Hat2p enhances the catalytic activity of Hat1p through facilitating the interaction of
Hat1p with its histone substrate (Parthun et al., 1996).
Although the acetylation of newly synthesized histones by type B histone acetyltransferases is presumed to play a role in replication-coupled histone deposition,
HAT1 is a non-essential gene in yeast (Kleff et al., 1995; Parthun et al., 1996). In vivo investigations into the function of Hat1p in S. cerevisiae demonstrated that loss of Hat1p resulted in defects in the telomeric silent chromatin structure and sensitivity to DNA damaging agents (Kelly et al., 2000b; Qin and Parthun, 2002). A role in DNA damage repair may be evolutionarily conserved as this phenotype is also seen in
22
Schizosaccharomyces pombe and chicken DT40 cells that lack Hat1p (Barman et al.,
2008; Benson et al., 2007).
The involvement of Hat1p in DNA damage repair has been most extensively studied in S. cerevisiae. Cells lacking Hat1p are specifically sensitive to agents that generate DNA double strand breaks, such as high concentrations of MMS or induction of endonucleases
(EcoRI or HO), due to a defect in recombinational repair. Interestingly, these phenotypes are only observed when hat1Δ is combined with mutations that alter specific sets of lysine residues in the histone H3 NH2-terminal tail (such as H3 K14R,K23R) suggesting that acetylation of the newly synthesized H3 and H4 may be functionally redundant (Qin and Parthun, 2002).
Several observations suggest that Hat1p has a more extensive role in modulating chromatin structure than merely acetylating histone H4 in the cytoplasm. First, Hat1p is predominantly localized in the nucleus (Ai and Parthun, 2004; Poveda et al., 2004).
Second, when in the nucleus, Hat1p is found in a distinct complex (NuB4), in which
Hat1p/Hat2p is joined by Hif1p (a H3/H4-specific histone chaperone) and histones H3 and H4 (Ai and Parthun, 2004). Finally, both Hat1p and Hif1p have been shown to be directly recruited to chromatin within a relatively small domain surrounding a DNA double strand break (Qin and Parthun, 2006).
The repair of damaged DNA occurs in a chromatin context and it has been shown that the removal of histones from DNA and their subsequent reassembly onto DNA accompanies
DNA repair (Chen et al., 2008; Osley and Shen, 2006; Tsukuda et al., 2005; Tsukuda et al., 2009). The S. cerevisiae mating-type switching system has proven to be a valuable
23 tool for the study of DNA double strand break repair (Tsukuda et al., 2009). A GAL- inducible copy of the HO endonuclease is integrated into the genome. Switching of the mating type is initiated by the induction of the HO endonuclease, which then cuts at the
MAT locus leaving a single double strand break. Sequences homologous to the HO cut site present at the silent mating loci, HML and HMR, are then used by the homologous recombination machinery to repair the break, resulting in a change of mating type (Haber,
1995).
The inducible HO system has been used to examine the reassembly of chromatin structure that occurs following repair of the double strand break (Chen et al., 2008;
Tsukuda et al., 2005). By monitoring the presence of histone H3 by ChIP, it was demonstrated that histones are lost from near the site of an HO-induced double strand break as single strand DNA resection occurs. H3 levels then return following DNA repair. The involvement of chromatin assembly factors in this reassembly process was indicated by defects in restoration of histone H3 levels near the break site in the absence of the histone chaperone Asf1p or in the presence of mutations that prevent the acetylation of histone H3 lysine 56 (Chen et al., 2008).
To determine whether the nuclear Hat1p-containing type B histone acetyltransferase
(NuB4) complex participates in the DNA double strand break repair-linked chromatin reassembly process, we employed the inducible GAL-HO endonuclease system and ChIP analysis to investigate the requirement of each of its components. We found that subunits of the NuB4 complex can affect repair-linked chromatin reassembly but that their contributions are not equivalent. Although loss of Hat1p causes a moderate defect in
24 chromatin reassembly, deletion of the HIF1 gene, in combination with a mutation in the histone H3 tail, has a pronounced effect on chromatin reassembly that is similar to that seen in the absence of Asf1p. In addition, our results suggest that Hat1p complex components may function in a pathway that is distinct from Asf1p. Finally, biochemical evidence indeed indicated that Hif1p may exist in complexes distinct from the NuB4 complex and the presence of Hif1p is necessary for the activity of a histone H3-specific
HAT.
2.3 Experimental Procedures
2.3.1 Yeast Strains
Yeast culture and genetic manipulation were done by standard methods (32). Gene deletions were generated by PCR-mediated gene disruption with a nutritional marker.
The genotypes of yeast strains used in this study are shown in Table 1.
2.3.2 HO Endonuclease Sensitivity Assays
Yeast strains were grown overnight in rich medium with 2% raffinose. Cells were diluted and grown to an optical density at 600 nm (A600) of ∼0.8 and concentrated to an A600 of 1 to plate in 10-fold serial dilutions onto rich medium or medium with 2% galactose.
2.3.3 DNA Damage and Repair Analysis
Primers flanking the HO site in the MAT locus were used to determine the degree of cutting and repair of mating type by PCR amplification. Cells were grown overnight in rich medium containing 2% raffinose. Galactose and then glucose were added to 2% at the times indicated in the figure legends. The number of PCR cycles to produce 25 amplification in the linear range was determined empirically. PCR products were resolved by agarose gel electrophoresis. Gels were stained with ethidium bromide and
PCR products were quantitated with 1D image analysis software (Kodak).
2.3.4 Chromatin Immunoprecipitation Analyses
Cultures were grown overnight in rich medium containing 2% raffinose, diluted, and grown until the cells reached an A600 of ∼0.5. Galactose and then glucose were added to
2% at the times indicated in the figure legends. Samples were taken for chromatin immunoprecipitation (ChIP) analysis at the time points indicated in the figure legends and processed as described previously. Samples were analyzed using quantitative real time PCR in a multiplex reaction with primers and probes designed as described previously (Chen et al., 2008). All experiments were performed with two or three biological replicates.
2.3.5 Quantitative Real Time PCR Analysis
Real time PCR was used to quantitate fragments in the immunoprecipitated samples from the ChIP analyses, using the ABI 7300 sequence detector and TaqMan PCR Master Mix protocol. Each PCR was performed in sextuplet with cycling conditions as follows: 50 for
2 min, 95 for 10 min, and then 40 cycles, with 1 cycle consisting of 95 for 15 s and 60 for
1 min. The cycle threshold (CT) value was set so that the fluorescence signal was above the base line noise and as low as possible in the exponential amplification phase. The amount of change compared with the SMC2 control was calculated for each immunoprecipitation using the standard comparative CT method.
26
2.3.6 Whole Cell Extracts
Yeast whole cell extracts were prepared from overnight yeast culture in YPD as described previously. Briefly, cells were harvested at midlog phase and washed with cold
H2O and extraction buffer (100 mM HEPES, pH 7.9, 245 mM KCl, 5 mM EGTA, 1 mM
EDTA, 0.5 mM PMSF, and 0.3% β-mercaptoethanol). The cell pellets were passed through a 3-ml syringe into a 50-ml tube containing liquid N2. The frozen pellets were ground to a fine powder in the presence of liquid N2 and incubated with extraction buffer
(150 μl of buffer/g of cells) on ice for 20 min, followed by centrifugation at 30,000 × g for 1 h at 4 °C. Supernatant was collected as whole cell extracts and dialyzed against dialysis buffer DN(50) (20 mM HEPES, pH 7.9, 50 mM NaCl, 5 mM MgCl2, 1 mM
EDTA, 10% glycerol) before use.
2.3.7 Column Chromatography
Dialyzed yeast whole cell extracts were centrifuged at 10,000 × g for 10 min. The resulting clarified extracts were applied to a Mono Q column (GE Healthcare) equilibrated with DN(50). The column was washed with 10 column volumes, and proteins were eluted with a 20 column volume gradient from DN(50) to DN(1000) (25 mM Tris, pH 7.9, 1 M NaCl, 0.1 mM EDTA, and 10% glycerol). The elution profile of protein was determined by Western blot. The peak fractions containing Hif1p that were determined by Western blot were pooled and concentrated to a volume of 200 μl and then resolved by gel filtration chromatography (Superose 6 column, GE Healthcare) run with
DN(300) buffer (25 mM Tris, pH 7.0, 0.1 mM EDTA, 10% glycerol, and 300 mM NaCl).
The elution profiles of proteins were determined by Western blot.
27
2.3.8 Western Blotting
Western blots were performed and visualized using an ECL Plus chemiluminescent detection kit according to the manufacturer's instructions (Amersham Biosciences). The signal was detected by scanning on a Storm PhosphorImager.
2.3.9 Histone Acetyltransferase Assays
Liquid histone acetyltransferase assays were performed using free chicken histones as the substrate. Reactions were performed in a final volume of 50 μl in buffer DN(50) containing 0.1 μM [3H]acetyl-coenzyme A (6.1 Ci/mmol, ICN) and 1 mg/ml of chicken erythrocyte core histones. Chicken histones were purified as previously described.
Reactions were incubated at 37 °C for 60 min. The assay mixture was resolved on an
18% SDS-PAGE and stained with Coomassie Blue. The gel was then incubated in fluorohance, dried, and exposed to x-ray film to identify the labeled histone.
2.3.10 HO Gene Expression Analysis
RT-PCR assays were performed as described previously (Durairaj et al., 2010). Briefly, total mRNA from 10 ml of yeast culture with an A600 of 0.8–1.0 was prepared using the purelink RNA minikit (Invitrogen). Reverse transcription is carried out using the high capacity cDNA reverse transcription kit (Applied Biosystems) following the manufacturer's protocol. PCR and real time PCR were then performed using synthesized first strand cDNA as template and the primer pairs targeted to the HO and ADH1 coding sequences (sequences available upon request).
28
2.4 Result
2.4.1 Involvement of NuB4 Complex Components in DNA Repair-linked Chromatin
Reassembly
The use of an inducible HO endonuclease has proven to be a valuable tool for the study of recombinational repair and the chromatin assembly and disassembly that must accompany it (Chen et al., 2008). To use this model system to look specifically at the role of the nuclear Hat1p (NuB4) complex in chromatin reassembly accompanying DNA double strand break repair, we integrated a copy of a galactose-inducible HO endonuclease gene into the genome that enables us to introduce a single double strand break at the MAT locus. We then individually deleted each of the NuB4 complex components to generate hat1Δ, hat2Δ, and hif1Δ strains. The strain background that was used for these studies has also been deleted for all of the endogenous genes encoding histones H3 and H4, which allows for the introduction of mutant alleles of H3 and H4 as the only copies of these histones (Kelly et al., 2000b). Previous studies have indicated that mutations in specific sets of lysine residues in the histone H3 NH2-terminal tail (such as H3 K14R, K23R) cause sensitivity to DNA damaging agents and that this sensitivity is increased by combining these mutations with mutations in the components of the NuB4 complex (Ai and Parthun, 2004; Qin and Parthun, 2002).
To determine whether the NuB4 complex and histone H3 mutants were sensitive to a single double strand break at the MAT locus, we tested their ability to grow on medium containing galactose, which induced HO expression. As seen in Figure 2.1, none of the single mutants (H3 K14R, K23R, hat1Δ, hat2Δ, or hif1Δ) showed a growth defect on
29 galactose. Surprisingly, when the H3 K14R, K23R allele was combined with each of the
NuB4 complex deletions, only the H3 K14R, K23R hif1Δ combination showed a significant synthetic sensitivity to HO induction suggesting that Hif1p may play a more prominent role in DNA repair or chromatin reassembly than Hat1p and Hat2p.
To assess the impact of these mutations on the reassembly of chromatin structure, we used ChIP analysis to monitor the presence of histone H3 near the HO cleavage site as the repair process proceeded (diagramed in Figure 2.2A). As a positive control for these experiments, we generated an asf1Δ in this strain background as this histone chaperone has been shown to be necessary for chromatin reassembly following the repair of an HO- induced double strand break (Chen et al., 2008). Results from the asf1Δ strain are included in each of the figures for comparison.
Cultures were grown to mid-log phase in raffinose, and galactose was added to induce expression of HO. After 2 h, glucose was added to repress HO and allow for repair of the
HO-induced double strand break to proceed. The introduction of the double strand break at the MAT locus and its subsequent repair were monitored by a PCR reaction that spanned the HO cut site and that generates distinct fragments from MATa and MATα cells (see Figure 2.2A). In a wild type strain (which starts as MATα), the HO site at MAT is efficiently cut in the presence of HO and then repaired following repression of HO
(Figure 2.2B). Similar kinetics of digestion and repair were also seen in hat1Δ, hat2Δ, hif1Δ, and H3 K14R, K23R strains (Figure 2.2, B and C). Interestingly, we reproducibly observed inefficient generation of the HO-induced double strand break in asf1Δ cells
(Figures 2.2B and C, and 2.4B and C). One explanation is that Asf1p might be necessary
30 for the proper transcriptional induction of the GAL-regulated HO gene (Adkins et al.,
2004; Korber et al., 2006). However, we detected only minor differences in the kinetics and level of HO induction in asf1Δ cells (Figure 2.6). Whether Asf1p plays a direct role in facilitating the HO-mediated cleavage of chromatin remains a possibility.
We then used ChIP to monitor the presence of histone H3 at a position 600 bp from the
HO cut site. As reported previously, there is a loss of histone H3 as the MAT locus is cut and single strand resection occurs at the break site (Chen et al., 2008). The levels of histone H3 then returned as the recombinational repair proceeded and chromatin structure was reassembled (Fig 2.2D). In the absence of Asf1p, there was also loss of H3 near the
HO cut site. However, also as previously reported, there was a dramatic decrease in chromatin reassembly as indicated by the observation that there was only a slight increase in the levels of histone H3 near the break site following its repair (Chen et al., 2008). The hat1Δ and H3 K14R, K23R mutations also caused a significant defect in repair-linked chromatin reassembly that was reproducibly observed in multiple trials (Figure 2.2D).
The rate of reassembly in these mutants was intermediate between that in the wild type and asf1Δ cells. Loss of Hat2p and Hif1p resulted in a minor defect on chromatin reassembly (Figure 2.2D). The fact that both the hat1Δ and H3 K14R, K23R mutations cause a clear defect in chromatin reassembly without an effect on cell viability suggests that the cells can tolerate a suboptimal level of chromatin reassembly during the DNA repair process.
We then examined chromatin reassembly when the H3 K14R, K23R allele was combined with the mutations in NuB4 complex components. Again, similar kinetics of repair was
31 observed in each of the strains (Figure 2.3, A and B). When the hat1Δ and H3 K14R,
K23R mutations were combined, the level of chromatin reassembly was similar to that observed in the single mutants (Figure 2.3C). The lack of an additive effect on chromatin reassembly suggests that the loss of Hat1p and the H3 K14R, K23R mutation may impact the same aspect of chromatin reassembly and is consistent with the observation that this mutant combination also does not cause a synthetic sensitivity to HO expression (Figure
2.1). Combining hat2Δ with the H3 K14R, K23R allele did not increase this reassembly defect. However, when hif1Δ was combined with the H3 K14R, K23R allele, there was a dramatic loss of chromatin reassembly (Figure 2.3C). In fact, the hif1Δ/H3 K14R, K23R mutant had a defect in chromatin reassembly similar to that of the asf1Δ. The synthetic defect in chromatin reassembly with this combination of mutations mirrors the synthetic growth defect observed when these cells are grown on galactose (Figure 2.1).
2.4.2 Non-overlapping Action of Asf1p and NuB4 Complex Components in Chromatin
Reassembly
Asf1p is an important histone chaperone that physically interacts with other histone chaperones that are involved in distinct pathways of chromatin assembly. For example,
Asf1p interacts with both the CAF-1 complex and the Hir-Hpc complex that, respectively, are core components of the replication-coupled and replication-independent chromatin assembly pathways (Green et al., 2005; Tyler et al., 2001). These interactions are thought to allow Asf1p to shuttle H3/H4 complexes into each of these assembly pathways (Das et al., 2010). Recent evidence suggests that Asf1p also physically interacts with the NuB4 complex and that the NuB4 complex acts upstream of Asf1p in the
32 process of chromatin assembly (Campos et al., 2010; Das et al., 2010; Fillingham et al.,
2008). Therefore, given that mutations in NuB4 complex components (and histone H3) cause defects in DNA repair-linked chromatin reassembly that are similar to those observed in an asf1Δ, we sought to determine whether the NuB4 complex and Asf1p act in concert or whether they function in distinct pathways to promote chromatin reassembly following DNA repair.
As expected from its importance in repair-linked chromatin reassembly, asf1Δ leads to a significant decrease in viability under HO-inducing conditions (galactose, Figure 2.4A).
When pairwise combinations of the hat1Δ, hat2Δ, hif1Δ, and H3 K14R, K23R alleles were constructed with an asf1Δ, only the H3 K14R, K23R allele showed a synthetic phenotype. The H3 K14R, K23R allele accentuated the slow growth phenotype of the asf1Δ strain on glucose and increased the sensitivity of asf1Δ to HO induction. When triple mutant combinations were examined, both the hat1Δ and hif1Δ increased the severity of the H3 K14R, K23R asf1Δ phenotypes with loss of Hif1p having a somewhat greater effect. Loss of Hat2p had no effect on any of the mutants (Figure 2.4A).
The genetic interactions observed between ASF1 mutants and the HAT1, HIF1, and H3 mutants suggested that they are functioning in at least partially distinct pathways in the context of DNA repair-linked chromatin reassembly. To determine whether the synthetic sensitivity of these mutants to a double strand break resulted from a further decrease in chromatin reassembly activity, ChIP analysis was used to monitor chromatin structure at the MAT locus during repair. For these experiments the time course of HO induction was extended to 3 h to allow for more double strand break formation in the asf1Δ
33 backgrounds (Figure 2.4, B and C). As seen in Figure 2.4D, following repression of HO synthesis, H3 levels gradually return during the course of repair in the asf1Δ mutant.
However, in asf1Δ H3 K14R, K23R, asf1Δ H3 K14R, K23R hat1Δ, and asf1Δ H3 K14R,
K23R hif1Δ mutants, there was no apparent restoration of histone H3 following repression of HO and, in fact, the levels of H3 continued to decrease. Intriguingly, the magnitude of the effect on chromatin reassembly correlated with the level of HO sensitivity of these mutants suggesting that the in vivo phenotype was a result of the chromatin reassembly defect.
2.4.3 Hif1p Is Present in High Molecular Weight Complexes Independent of Hat1p and
Hat2p and Influences a Histone H3-specific HAT
Hif1p was originally isolated and identified as a protein that interacts with the
Hat1p/Hat2p complex in the nucleus (Ai and Parthun, 2004; Poveda et al., 2004). The analysis of the role of these factors in DNA repair-mediated chromatin reassembly indicated that Hif1p plays a more significant role in this process than Hat1p and Hat2p suggesting that Hif1p may act independently of these factors. This led to the prediction that a portion of the native Hif1p in cells should exist in a form that is not physically associated with Hat1p/Hat2p. Therefore, to determine whether Hif1p was a component of complexes distinct from NuB4, we performed a biochemical characterization of the native protein isolated from yeast. Yeast whole cell extracts (made from a strain in which the endogenous Hif1p was fused to a Myc epitope tag) were fractionated over a Mono Q column and Hif1p containing fractions were concentrated and further fractionated by a gel filtration column (Superose 6) to resolve proteins and complexes by size. Hif1p eluted
34 in a broad peak centered at ∼500 kDa (Figure 2.5A). Hat2p eluted in two distinct peaks.
The larger peak overlapped with the Hif1p peak and the molecular weight was consistent with that of the NuB4 complex (Hat1p/Hat2p/Hif1p) previously identified. The apparent molecular mass of the smaller peak, 150 to 200 kDa, closely matched the cytoplasmic
Hat1p/Hat2p complex. In addition, these two peaks co-eluted with a strong histone H4- specific HAT activity (Figure 2.5B).
We then determined how the elution profile of Hat2p depended on the presence of Hif1p.
As expected, deletion of the HIF1 gene caused a significant alteration in the Hat2p elution profile; with the loss of the higher molecular weight Hat2p peak and the corresponding H4-specific HAT activity (Figure 2.5, A and B). This result strongly suggests that a significant fraction of Hat1p/Hat2p complex is associated with Hif1p.
Surprisingly, the converse was not true. Deletion of both HAT1 and HAT2 did not significantly affect the elution profile of Hif1p as the broad high molecular weight peak remained. This elution profile is not an intrinsic property of the protein as recombinant
Hif1p eluted in a single narrow peak with an apparent molecular mass of ∼150 kDa. In addition, we observed a significant decrease in the level of an H3-specific HAT activity that elutes at >1 MDa in the hif1Δ cells that is not affected by the loss of Hat1p and
Hat2p. Taken together, these observations suggest that native Hif1p is present in one, or more, high molecular weight complexes that are independent of Hat1p and Hat2p. In addition, whereas Hif1p does not appear to make a stable interaction with an H3-specific
HAT complex, it is necessary for its full activity. These results confirm that Hif1p
35 participates in Hat1p/Hat2p independent interactions as predicted by its more significant role in DNA repair-linked chromatin reassembly.
2.5 Discussion
Although long hypothesized to be involved in chromatin assembly, directly linking Hat1p to this process has been problematic. One of the difficulties in studying the role of histone acetylation in chromatin assembly is that unlike transcription, which occurs at known places and times, chromatin assembly is a more fluid and transient phenomenon. An important advance in this area was reported in a recent study that exploited the HO- induced double strand break repair model system to monitor the disassembly and reassembly of the chromatin structure that occurs during the recombinational repair process (Chen et al., 2008; Tsukuda et al., 2005). This study demonstrated that Asf1p and the acetylation of histone H3 lysine 56 are required for the efficient reassembly of chromatin following repair of an HO-mediated double strand break at the MAT locus
(Chen et al., 2008).
Several lines of evidence suggested that Hat1p might play a role in the reassembly of chromatin structure during the recombinational repair of a DNA double strand break.
First, DNA damage sensitivity accompanies the loss of Hat1 activity in several eukaryotes (Barman et al., 2008; Benson et al., 2007; Qin and Parthun, 2002). Second, the DNA damage sensitivity of S. cerevisiae hat1Δ cells is a result of defects in recombinational repair (Qin and Parthun, 2002). Finally, Hat1p is recruited to chromatin at the MAT locus following an HO-induced double strand break (Qin and Parthun, 2006).
Therefore, we have used a similar strategy to determine whether Hat1p, as well as the
36 other components of the NuB4 complex (Hat2p and Hif1p), also play a role in this repair- linked chromatin reassembly. We have demonstrated that there is a significant decrease in the rate at which chromatin is reformed near the site of a double strand break in hat1Δ cells providing direct evidence that Hat1p functions in a chromatin assembly process.
To date, yeast Hat1p has been found to be a component of two distinct complexes. In the cytoplasm, Hat1p is associated with Hat2p to form the HAT-B complex that is thought to acetylate newly synthesized histone H4 (Parthun et al., 1996). In the nucleus, Hat1p is a subunit of a larger complex (termed the NuB4 complex) that contains Hat2p and the
H3/H4-specific histone chaperone Hif1p (Ai and Parthun, 2004; Poveda et al., 2004;
Ruiz-Garcia et al., 1998). Both complexes have been found to be stably associated with histones H3 and H4 (Ai and Parthun, 2004; Mosammaparast et al., 2002). Surprisingly, the components of these complexes have diverse effects on the repair-linked chromatin reassembly process.
Based on both genetic and biochemical evidence, Hat2p has little influence on chromatin reassembly. Previous results have shown that the catalytic activity of Hat1p isolated from cells lacking Hat2p is decreased 10-fold (Parthun et al., 1996). This suggests a number of possibilities. First, the role of Hat1p in the context of repair-linked chromatin reassembly may not be strongly dependent on its catalytic activity. Alternatively, Hat1p may have substrates other than histone H4 involved in chromatin reassembly and the ability of
Hat1p to modify these proteins may not require Hat2p. Finally, in the context of the cell,
Hat2p may not be as important for the catalytic activity of Hat1p as when the enzyme is assayed in vitro.
37
Conversely, the genetic and biochemical data indicate that Hif1p has functions in repair- linked chromatin reassembly that are independent of Hat1p. Hif1p may participate in multiple pathways of chromatin assembly of which only a subset involve Hat1p (or
Hat2p). This is consistent with the more pronounced effect of hif1Δ on repair-linked chromatin reassembly. Alternatively, Hif1p may have both direct and indirect roles in chromatin reassembly. For example, in addition to acting directly in reassembly as a histone chaperone, Hif1p may indirectly affect chromatin reassembly through its influence on histone H3 acetylation. In the absence of Hif1p, there was a significant decrease in the activity of a histone H3-specific HAT despite the fact that Hif1p did not appear to form a stable complex with this enzyme activity (based on the lack of overlap in their elution profiles). The effect of Hif1p on this HAT activity is reminiscent of that between Asf1p and Rtt109p where Asf1p is required for Rtt109p activity in the absence of a stable association between them (Adkins et al., 2007; Fillingham et al., 2008; Han et al., 2007b, c; Selth and Svejstrup, 2007; Tsubota et al., 2007). Alternatively, Hif1p may indirectly influence histone H3 acetylation, perhaps through transcriptional regulation of an H3-specific HAT. Although the H3-specific HAT activity that is affected by Hif1p has not been identified, if this activity targets residues other than H3 lysines 14 and 23, the synthetic interactions observed between the hif1Δ and the H3 K14R, K23R allele could be explained by a cumulative effect on H3 acetylation.
The possibility that Hif1p functions in pathways independent of Hat1p and Hat2p is consistent with the biochemical data presented here that indicated that Hif1p may be a component of a high molecular weight complex (or complexes) that does not contain
38
Hat1p and Hat2p. This possibility is also consistent with results from Poveda and colleagues (Poveda et al., 2004) where precipitation of epitope-tagged Hat1p co- precipitated only a small fraction of the Hif1p present in the extract. It will be interesting to determine whether the high molecular weight Hif1p-containing complexes are related to the multichaperone containing complexes from mammalian cells of which NASP, the human homolog of Hif1p, is a component (Campos and Reinberg, 2010; Drane et al.,
2010; Lewis et al., 2010; Tagami et al., 2004).
Current models of chromatin assembly generally suggest that Asf1p plays a central role
(Das et al., 2010; De Koning et al., 2007). This histone chaperone is thought to participate in multiple pathways of chromatin assembly (as well as disassembly) by functioning to shuttle H3/H4 complexes into these pathways. This model is supported by the observation that Asf1p physically interacts with other histone chaperones that are specific for distinct chromatin assembly pathways, such as CAF-1 and the Hir-Hpc complex (Green et al., 2005; Tyler et al., 2001). Hat1p and its associated factors are thought to act upstream of Asf1p as Asf1p has been found to be associated with newly synthesized histones that carry the acetylation pattern characteristic of Hat1p action (Das et al., 2010; Groth et al., 2007; Groth et al., 2005; Jasencakova et al., 2010). In addition, a direct physical association between Asf1 and the NuB4 complex was recently reported suggesting the possibility that the NuB4 complex may directly transfer newly synthesized histones to Asf1p (Fillingham et al., 2008). Despite this biochemical data, the genetic results presented here indicated that mutations in HIF1, HAT1, and histone H3 show genetic interactions with mutations in ASF1 where combinations of these mutations
39 generate increasing sensitivities to the HO-induced DNA double strand breaks and generate greater defects in chromatin reassembly. There are a number of interpretations for these results. First, Asf1p and NuB4 complex components may function in a common pathway but may be partially redundant for the optimal functioning of this pathway. For example, in the absence of Asf1p, the NuB4 complex may be capable of providing
H3/H4 complexes to other chromatin assembly factors. Alternatively, the NuB4 complex may act in a chromatin assembly pathway that is distinct from Asf1p, perhaps exploiting the chromatin assembly activity of Hif1p.
The results presented here demonstrate that Hat1p and Hif1p function in the reassembly of chromatin that accompanies the recombinational repair of a DNA double strand break.
It is not clear how this type of chromatin assembly relates to the replication-coupled and replication-independent pathways of chromatin assembly. It will be of interest to determine whether the NuB4 complex components influence these pathways of chromatin assembly, as well.
40
Reference Strain Genotype or source MATα ade2Δ::hisG his3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63 ura3Δ0 ADE3::GAL10-HO HHF2- SQY501 This study HHT2::LEU2 HHF1-HHT1::ADE3 (TRP1 CEN ARS)- HHF2-HHT2 MATα ade2Δ::hisG his3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63 ursa3Δ0 ADE3::GAL10-HO HHF2- ZGY101 This study HHT2::LEU2 HHF1-HHT1::ADE3 (TRP1 CEN ARS)- HHF2-HHT2 HAT1::URA3 MATα ade2Δ::hisG his3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63 ura3Δ0 ADE3::GAL10-HO HHF2- ZGY102 This study HHT2::LEU2 HHF1-HHT1::ADE3 (TRP1 CEN ARS)- HHF2-HHT2 HAT2::URA3 MATα ade2Δ::hisG his3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63 ura3Δ0 ADE3::GAL10-HO HHF2- ZGY103 This study HHT2::LEU2 HHF1-HHT1::ADE3 (TRP1 CEN ARS)- HHF2-HHT2 HIF1::URA3 MATα ade2Δ::hisG his3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63 ura3Δ0 ADE3::GAL10-HO HHF2- ZGY104 This study HHT2::LEU2 HHF1-HHT1::ADE3 (TRP1 CEN ARS)- HHF2-HHT2 ASF1::URA3 MATα ade2Δ::hisG his3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63 ura3Δ0 ADE3::GAL10-HO HHF2- ZGY105 This study HHT2::LEU2 HHF1-HHT1::ADE3 (TRP1 CEN ARS)- HHF2-HHT2 ASF1::URA3 HIF1::HIS3 MATα ade2Δ::hisG his3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63 ura3Δ0 ADE3::GAL10-HO HHF2- ZGY106 This study HHT2::LEU2 HHF1-HHT1::ADE3 (TRP1 CEN ARS)- HHF2-HHT2 ASF1::URA3 HAT1::HIS3
Table 2.1 Strains used in this study.
41
Figure 2.1 Sensitivity of NuB4 complex and histone H3 mutants to an HO-induced
DNA double strand break.
10-Fold serial dilutions of strains with the indicated genotypes were spotted on plates containing minimal media with either glucose or galactose as carbon source. Plates were incubated at 30 °C for 3 days and then photographed.
42
Figure 2.2 Impact of NuB4 complex and histone H3 mutations on DNA repair- linked chromatin reassembly.
A) schematic diagram of the inducible HO-mediated mating type switch system used to model recombinational DNA repair. Locations of primers used for PCR analysis of the double strand break at the MAT locus and for ChIP analysis of histone H3 disassembly and reassembly are indicated. B) introduction and repair of the double strand break at the
MAT locus was monitored by PCR assay in the indicated strains. Galactose was added at the 0-h time point and glucose was added at the 2-h time point. Reaction products were resolved on a 1.5% agarose gel and visualized by ethidium bromide staining. The migration of the MAT and MATa-specific bands is indicated. Amplification of a region of the RAD27 locus was used as a control. C) quantitation of double strand break formation and repair. Stained agarose gels were photographed and the MAT, MATa, and RAD27 bands were quantitated using one-dimensional Image Analysis software
(Kodak). MAT and MATa fragments were normalized to the RAD27 fragment. The
MAT and MATa fragments were plotted relative to the 0h and 5h time points, respectively. D) ChIP analysis of the abundance of histone H3 at a site 600 bp from the double strand break at the MAT locus. The graph shows a comparison of the indicated
NuB4 complex or histone H3 mutant to a wild type strain and an asf1 strain. Histone H3 levels were normalized to H3 levels at the SMC2 locus. Subsequent time points are normalized to the 0h time point.
43
44
Figure 2.3 Effect of combining the H3K14R, K23R allele with mutations of the
NuB4 complex components on DNA repair-linked chromatin reassembly.
A) cutting and repair of the MAT locus in the indicated strains was monitored as described in the legend to Figure. 2.2B, the levels of MAT and MATa fragments were quantitated as described in the legend to Figure. 2.2C, ChIP analyses of H3 levels near the MAT locus in the indicated strains were performed and analyzed as described in the legend to Figure. 2.2.
45
Figure 2.4 Functional redundancy between NuB4 complex components, histone H3, and Asf1p in DNA repair-linked chromatin reassembly.
A) 10-fold serial dilutions of the indicated strains were spotted on plates containing either glucose or galactose as carbon source. Plates were incubated at 30 °C for 3 days and then photographed. B) cutting and repair of the MAT locus in the indicated strains was monitored as described in the legend to Figure. 2.2B, the levels of MAT and MATa fragments were quantitated as described in the legend to Figure. 2.2 except that the
MATa levels are plotted relative to the 6h time point. D) ChIP analysis of H3 levels near the MAT locus at the indicated times. Galactose was added to cultures at time 0h.
Glucose was added after 3 h. ChIP experiments were performed and analyzed as described in the legend to Figure. 2.2.
46
47
Figure 2.5 Hif1p is present in a high molecular weight complex independent of HAT1 and HAT2 and influences an H3-specific HAT activity.
A) whole cell extracts were resolved by Superose 6 chromatography and the elution of
Hif1p and Hat2p was determined by Western blot analysis as indicated.
The elution position of size standards is indicated above the fraction numbers. The whole cell extracts were derived from strains with the genotypes indicated on the right. The bottom panel shows the elution pattern of rHif1p isolated from Escherichia coli. B)
Superose 6 column fractions from the indicated strains were assayed for histone acetyltransferase activity using free histones [3H]acetyl-CoA as substrates. Reaction products were resolved by 18% SDS-PAGE and processed for fluorography. Position of radiolabeled histones was visualized by exposure of x-ray film. Migration of histones H3 and H4, as determined by Coomassie Blue staining, is indicated.
48
49
Figure 2.6 Transcriptional induction of the HO gene. A) WT and asf1 mutant strains were grown as described in Figure 1. Cells were collected at indicated time points and
RT-PCR assays were carried out. RT-PCR products were separated on a 2.2% agarose gel and stained with ethidium bromide. B) Same samples as in A) except that SYBR
Green real-time PCR was carried out to quantitate the level of HO mRNA relative to
ADH1 mRNA levels.
50
Chapter 3 Sites of Acetylation on Newly Synthesized Histone H4 are required for Chromatin Assembly and DNA damage Damage Response
Signaling
3.1 Abstract
The acetylation of newly synthesized histone H4 is temporally correlated with the process of chromatin assembly. The most well characterized acetylation of newly synthesized histone H4 is the diacetylation of the NH2-terminal tail on lysines 5 and 12. Despite its’ evolutionary conservation, this pattern of modification has not been shown to be essential for either viability or chromatin assembly in any model organism. Using genetic assays in
S. cerevisiae, we demonstrate that mutations in histone H4 lysines 5 and 12 confer hypersensitivity to replication stress and DNA damaging agents when combined with mutations in histone H4 lysine 91, which has also been found to be a site of acetylation on soluble histone H4. In addition, mutations that alter these three sites of histone H4 acetylation are synthetically lethal when combined with mutations in histone H3 lysine
56, a site that is also modified on newly synthesized molecules. Using an inducible HO endonuclease model of DNA double strand break repair, we also show that mutation of the sites of acetylation on newly synthesized histone H4 results in defects in the reassembly of chromatin structure that accompanies the repair of HO-mediated double
51 strand breaks. Intriguingly, mutations that alter the sites of newly synthesized histone H4 acetylation are also defective in DNA damage response signaling as they show a marked decrease in the levels of phosphorylated H2A in chromatin surrounding the double strand break. This decrease is not the result of an inability to generate phosphorylated histone
H2A but to an inability to localize this modified histone to chromatin. These results indicate that the sites of acetylation on newly synthesized histones H3 and H4 can function in non-overlapping ways that are required for chromatin assembly and viability.
In addition, mutations that influence the assembly of H3 and H4 into chromatin can alter either the assembly or stability of phosphorylated histone H2A on chromatin.
3.2 Introduction
Each time a eukaryotic cell divides it must duplicate not only its genomic DNA but also the chromatin structure in which it is packaged. At its most fundamental level, chromatin structure consists of ~147 base pairs of DNA wrapped around the histone octamer (which contains two molecules of each of the core histones, H2A, H2B, H3 and H4) to form a nucleosome. The linear strings of nucleosomes that package eukaryotic chromosomes can then form a number of successively higher order structures to achieve the necessary level of compaction. The rapid and efficient regeneration of chromatin structure during
DNA synthesis is essential for the mechanical packaging of the enormous linear length of eukaryotic chromosomes within the confines of the nucleus as well as for the physical protection of the DNA that ensures genomic integrity. Importantly, as post-translational modifications to histones are an integral component of epigenetic regulation, the process
52 of chromatin assembly also plays a critical role in the inheritance of distinct chromatin states.
The process of chromatin assembly actually begins in the cytoplasm where histone proteins are produced. Analysis of these cytoplasmic histones gave the first indication that acetylation of histone NH2-terminal tails might play a role in chromatin assembly. In mid-1970’s, it was shown that newly synthesized histone H4 was rapidly acetylated in the cytoplasm following its synthesis (Altheim and Schultz, 1999b; Annunziato, 2012;
Annunziato and Hansen, 2000). Subsequently, histone H3 was also found to be acetylated soon after translation (Sobel et al., 1995). In the case of histone H4, acetylation of newly synthesized molecules was found to occur in a precise pattern that is highly conserved across eukaryotic evolution. Of the four lysine residues in the NH2- terminal tail that are subject to acetylation (at positions 5, 8, 12 and 16) there are high levels of modification at lysines 5 and 12 and little or no modification on lysines 8 and 16
(Chicoine et al., 1986; Sobel et al., 1995). For histone H3, while it appears most organisms acetylate newly synthesized molecules, different patterns of acetylation can be found on the five NH2-terminal tail lysine residues that can be acetylated (at positions 9,
14,18, 23 and 27) (Kuo et al., 1996; Sobel et al., 1995).
In addition to the acetylation that occurs on the NH2-teriminal tails, acetylation of lysine residues in the core domain of newly synthesized histones H3 and H4 has recently been observed. The most well-characterized core domain acetylation occurs on lysine 56 of histone H3 (Masumoto et al., 2005; Ozdemir et al., 2005; Xu et al., 2005). Lysine 56 is located at the end of the a-N helix of histone H3 and is a point of contact with DNA at the
53 entry/exit point of the nucleosome. Thus, H3 lysine 56 acetylation may be capable of physically altering the contact between histone H3 and DNA. Histone H3 lysine 56 acetylation occurs on newly synthesized histones, peaks during S-phase and is removed from histones in G2/M (Kaplan et al., 2008; Maas et al., 2006; Masumoto et al., 2005;
Recht et al., 2006; Yuan et al., 2009; Zhou et al., 2006). Mutations in yeast that alter H3 lysine 56 to mimic the constitutively unacetylated state (H3 K56R) result in cells that have increased levels of chromosomal breaks and are sensitive to DNA damaging agents
(Celic et al., 2006; Celic et al., 2008; Driscoll et al., 2007; Han et al., 2007a; Maas et al.,
2006; Masumoto et al., 2005; Recht et al., 2006; Schneider et al., 2006). While originally thought to be limited to yeast, H3 lysine 56 acetylation is also found in higher eukaryotes and may play important roles in stem cell biology and cancer progression (Das et al.,
2009; Xie et al., 2009; Yuan et al., 2009).
A second site of histone core domain acetylation that that has been observed on the soluble pool of histones is H4 lysine 91 (Ye et al., 2005). H4 lysine 91 lies along the interface between the H3/H4 tetramer and the H2A/H2B dimers. In fact, H4 lysine 91 normally forms a salt bridge with an aspartic acid residue in histone H2B (Cosgrove et al., 2004; Mersfelder and Parthun, 2006). Hence, neutralization of the positive charge of
H4 lysine 91 may function to destabilize tetramer-dimer interactions and, thus, regulate the process of histone octamer assembly. Histone H4 lysine 91 is also a highly conserved modification that has been observed in human, bovine and yeast cells (Yan et al., 2009;
Zhang et al., 2003). Mutations in yeast that alter H4 lysine 91 cause severe defects in silent chromatin formation and sensitivity to DNA damaging agents. Consistent with a
54 role for H4 lysine 91 acetylation in chromatin assembly, genetic analysis of these mutations indicate that this modification functions in common pathways with the histone chaperones Asf1 and CAF-1 in the context of DNA repair (Nair et al., 2011; Ye et al.,
2005).
While it has been known for decades that histones H3 and H4 can exist in an acetylated state during the process of chromatin assembly, the acetylation of newly synthesized histones has never been shown to be absolutely essential for histone deposition or viability in any organism. For example, mutating any of the sites of acetylation on newly synthesized H3 or H4 results in viable cells that are capable of chromatin assembly (Ma et al., 1998; Megee et al., 1990). In addition, none of the histone acetyltransferases that are solely involved in the acetylation of newly synthesized histones are essential for viability in yeast (Driscoll et al., 2007; Han et al., 2007a; Han et al., 2007b; Kleff et al.,
1995; Parthun et al., 1996; Schneider et al., 2006). However, a number of recent studies have provided a direct functional link between the acetylation of newly synthesized histones and histone deposition. Mutations that alter histone H3 lysine 56, or deletion of
RTT109 (the histone acetyltransferase responsible of H3 lysine 56 acetylation) and ASF1
(which is required for Rtt109p-mediated acetylation of H3 lysine 56), cause a defect in the reassembly of chromatin following the recombinational repair of a DNA double strand break in yeast (Chen et al., 2008). Histone H3 lysine 56 acetylation has also been directly linked to replication-independent chromatin assembly (or histone exchange)
(Kaplan et al., 2008; Rufiange et al., 2007). In addition, deletion of HAT1, which encodes the histone acetyltransferase that is thought to generate the diacetylation pattern
55 on newly synthesized histone H4, also results in defects in DNA repair-linked chromatin reassembly and histone exchange (Ge et al., 2011; Verzijlbergen et al., 2011).
Studies of histone H3 lysine 56 acetylation have also provided a link between the acetylation of newly synthesized histones and the DNA damage checkpoint response.
The DNA damage checkpoint is activated to facilitate the repair of DNA lesions by providing time for the repair process through arrest of the cell cycle (Harrison and Haber,
2006). In budding yeast, the recruitment of the Mec1 and Tel1 kinases to the site of a
DNA lesion is believed to initiate the checkpoint cascade through phosphorylation downstream kinases such as Rad53p and Chk1p. In addition, Mec1p and Tel1p also phosphorylate histone H2A (H2AX in mammalian cells) in a large domain of chromatin near site of a DNA double strand break. The phosphorylation of H2A is an early event in the DNA damage checkpoint that is important for maintaining the checkpoint and for recruiting factors to chromatin that are involved in modulating chromatin structure during the repair process. The mechanism of checkpoint recovery is not well understood and the minimum requirement seems to be deactivation of Rad53 and dephosphorylation of gamma H2A (Keogh et al., 2006; Leroy et al., 2003; O'Neill et al., 2007). The fact that fully repaired DNA does not turn off the DNA damage checkpoint suggests that a proper chromatin state needs to be restored in order for cells to reenter the cell cycle. In fact, recent results have demonstrated that deactivation of the DNA damage checkpoint requires the reassembly of chromatin containing histone H3 that is acetylatyed on lysine
56 (Chen et al., 2008; Chen and Tyler, 2008; Das et al., 2009; Yuan et al., 2009).
56
In this study, we have explored the function of the acetylation sites on newly synthesized histone H4, namely lysines 5, 12 and 91. We have found that combining a mutation that mimics the constitutively acetylated state of H4 lysine 91 (H4 K91Q) with mutations that alter both H4 lysines 5 and 12 to arginine (H4 K5,12R) results in a pronounced sensitivity to DNA damage and DNA replication stress. Furthering the connection between these sites of newly synthesized histone acetylation on H4, mutations of these sites are synthetically lethal with the mutation of histone H3 lysine 56. We also use the inducible
HO endonuclease system to directly demonstrate the requirement for the sites of newly synthesized histone H4 acetylation in the reassembly of chromatin structure that accompanies the recombinational repair of a DNA double strand break. Strikingly, we also find that, while these sites of modification are not required for the phosphorylation of histone H2A, they are required for the association of this modified histone with chromatin suggesting a role for chromatin assembly in DNA damage signaling.
3.3 Experimental Procedures
3.3.1 Yeast strains.
Yeast culture and genetic manipulation were done by standard methods. Strains used in the phenotypic assays are derivatives of MPY302 (MATa hhf2-hht2::LEU2 hhf1- hht1::MET15 (LYS2 CEN ARS)-HHF2-HHT2), with MEC1, MEC3, RAD9 and MRC1 deleted using PCR-mediated gene disruption with URA3 and shuffled with TRP1-based plasmids carrying the corresponding histone H4 mutations. Strains used in the histone H3 and γ-H2AX ChIP assays are derivatives of ZGY110 (MAT ade3::GAL10-HO hhf2- hht2::LEU2 hhf1-hht1::ADE3 (LYS2 CEN ARS)-HHF2-HHT2) shuffled with TRP1-
57 based plasmids carrying the corresponding histone H4 mutations. Tests of synthetic lethality were performed in a derivative of MPY302 that contained a wild type copy of
HHT2 and HHF2 on a URA3-based plasmid (RMY200U) (Mann and Grunstein, 1992).
3.3.2 Yeast phenotypic assays.
Yeast strains were grown overnight in rich medium with 2% glucose or raffinose. Log phase cells were collected and concentrated to an A600 of 1 to plate in 10-fold serial dilutions onto rich medium or medium with 2% galactose or plates with the indicated concentration of DNA damaging drugs.
3.3.3 Cell extracts, chromatin fractionation and immunoblotting.
Cell extracts were prepared by trichloroacetic acid addition as described in (Kim and
Haber, 2009) and were subjected to Western blot analysis. γ-H2AX was detected by polyclonal anti-yeast γ-H2A antibody (Abcam). Yeast cell fractionation is carried out as described (Keogh et al., 2006). Briefly, cells were collected and spheroplasts were made using Zymolyase 100T. Spheroplasts were then washed and lysed with cell lysis buffer with 0.5% Triton X-100, which is then layered over a 60% sucrose cushion and spun for
10 min in the cold room. The upper layer was taken as the cytosolic fraction that was acid extracted to isolate histones. The white glassy pellet at the bottom of the cushion is further lysed with nuclear lysis buffer with 1% Triton X-100. The nuclear lysate was spun for 10 min in the cold room and the supernatant is taken as the soluble nuclear fraction and acid extracted to isolate histones. The pellet was the chromatin fraction and was boiled in SDS dye prior to electrophoresis.
58
3.3.4 DNA damage and repair analysis.
Primers flanking the HO site in the MAT locus were used to determine the degree of cutting and repair of mating type by PCR amplification. Cells were grown overnight in rich media containing 2% raffinose. Galactose and then glucose were added to 2% at the times indicated in the figure legends. The number of PCR cycles to produce amplification in the linear range was determined empirically. PCR products were resolved by agarose gel electrophoresis. Gels were stained with ethidium bromide and PCR products were quantitated with 1D image analysis software (Kodak).
3.3.5 ChIP and realtime PCR.
Cultures were grown overnight in rich media containing 2% raffinose, diluted and grown until the cells reached an OD600 of approximately 0.5. Galactose and then glucose were added to 2% at the times indicated in the figure legends. Samples were taken for ChIP analysis at the time points indicated in the figure legends and were processed as described previously (Ge et al., 2011). Samples were analyzed using quantitative real-time PCR in a multiplex reaction with primers and probes designed as described previously (Chen et al.,
2008). All experiments were performed with two or three biological replicates. Real-time
PCR was used to quantitate amounts of DNA fragments in the immunoprecipitated (IP) samples from the ChIP analyses, using the ABI 7300 sequence detector and Taqman PCR
Master Mix protocol or Sybrgreen Master Mix protocol. Each PCR was performed in triplet with cycling conditions as follows: 50 for 2 min, 95 for 10 min, and then 40 cycles, with 1 cycle consisting of 95 for 15 s and 60 for 1 min. The cycle threshold (CT) value was set so that the fluorescence signal was above the base line noise and as low as
59 possible in the exponential amplification phase. The amount of change compared to the
SMC2 control was calculated for each IP using the standard comparative CT method.
3.4 Results
3.4.1 Functional redundancy in the sites of acetylation found on newly synthesized histones H3 and H4.
Histone H4 lysine 91 lies in an interface between histones H4 and H2B and appears to form a salt bridge with a glutamic acid residue at position 74 of H2B (yeast). H4 lysine
91 is acetylated in the soluble fraction of H4 molecules that co-purify with the nuclear
Hat1p-containing complex type B histone acetyltransferase complex (NuB4). Based on the potential for modulating this H4-H2B salt bridge, acetylation of lysine 91 was proposed to influence chromatin assembly via modulating the association of H3/H4 tetramers and H2A/H2B dimers (Ye et al., 2005). To further test the model that H4 lysine 91 acetylation functions in the process of chromatin assembly, we tested whether genetic interactions exist between this site and the other sites of modification on newly synthesized histones H3 and H4.
As previously reported, H4 K91Q mutants (which mimic the constitutive acetylation of this residue) were sensitive to DNA damage and replication stress (MMS and HU) while
H4 K91R mutants are not (Figure 3.1A) (Ye et al., 2005). In addition, mutating H4 lysines 5 and 12 to arginine did not increase the sensitivity of cells to HU or MMS.
However, when the H4 K91Q and H4 K5,12R mutations were combined, there was a defect in cell growth as well as a striking increase in HU and MMS sensitivity. H4
K5,12R K91R mutants did not display any of these phenotypes. The genetic interactions
60 between sites on the H4 NH2-terminal tail and the H4 core domain required mutating both H4 lysines 5 and 12 (Figure 3.7). These results indicate that mutations in all three sites of acetylation on newly synthesized histone H4 can cause phenotypes that are consistent with a role in chromatin assembly and that the H4 NH2-terminal tail is functionally redundant with H4 lysine 91.
We next tested whether the sites of newly synthesized histone acetylation on histone H4 showed genetic interactions with histone H3 lysine 56. It has previously been shown that
H4 lysine 5 and 12 mutations enhance the growth defect and DNA damage sensitivity phenotypes of H3 K56R mutants (Fillingham et al., 2008; Li et al., 2008). Therefore, we focused on potential genetic interactions between histone H4 lysine 91 and histone H3 lysine 56. Mutation of H3 lysine 56 showed an opposite pattern of phenotypes relative to
H4 lysine 91 when comparing the effect of altering these residues to glutamine or arginine. As reported previously, mimicking constitutive deacetylation of H3 lysine 56
(H3 K56R) resulted in sensitivity to DNA damaging agents and replication stress while there were no defects seen when mimicking the constitutive acetylation of this site (H3
K56Q, Figure 3.1B) (Celic et al., 2006; Celic et al., 2008; Driscoll et al., 2007; Han et al.,
2007a; Maas et al., 2006; Masumoto et al., 2005; Recht et al., 2006; Schneider et al.,
2006). Interestingly, mutating H4 lysine 91 to arginine (H4 K91R) suppressed the phenotypes of the H3 K56R mutant. However, the converse was not true as the H3
K56Q allele did not suppress the phenotypes of the H4 K91Q mutant. In addition, while both the H3 K56R and H4 K91Q alleles individually display sensitivity to DNA damage and replication stress, combining these alleles did not lead to a synthetic increase in
61 sensitivity. These genetic interactions strongly support the hypothesis that H4 lysine 91 plays a functional role in chromatin assembly and that H4 lysine 91 and H3 lysine 56 are functioning in a common pathway.
We also sought to determine whether additional genetic interactions existed with H4 lysines 5 and 12. A plasmid shuffle assay was used in which cells contained both an
ADE2-based plasmid with a wild type copy of yeast H3 and H4 genes (HHT2/HHF2) and a second TRP1-based plasmid that contained the mutated alleles of H3 and H4 (the test plasmid). As the starting strain is ade2, only cells that can lose ADE2-based plasmid will form red colonies. Using this assay, we were unable to obtain red colonies when the test plasmid contained the H3 K56R, H4 K91Q and H4 K5,12R mutations. This suggested that the yeast could not survive with the mutant forms of H3 and H4 as the sole copies of these histones. To confirm this synthetic lethality, a separate strain of yeast that contained a wild type copy of the HHT2 and HHF2 genes on a plasmid that contained a
URA3 selectable marker was transformed with a second plasmid that contained either the wild type, the H4 K5,12R K91Q or the H4 K5,12R K91Q H3 K56Q alleles (TRP1 marker). These cells were grown extensively in media that selected for the test plasmid
(media lacking tryptophan) but in the absence of selection for the URA3 plasmid. Serial dilutions were then plated on synthetic media with and without 5-fluoroorotic acid (5-
FOA). The 5-FOA is lethal to cells that are expressing the URA3 enzyme (Boeke et al.,
1987). Therefore, cells that are not able to retain viability following the loss of the
URA3-based plasmid that that contains wild type H3 and H4 genes will not be able to grow on media containing 5-FOA. As expected, cells containing a test plasmid with wild
62 type H3 and H4 genes showed full viability on 5-FOA media (Figure 3.1C). The H4
K91Q K5,12R allele supported moderate growth on 5-FOA consistent with the growth defect seen with this combination of mutations. However, combining the H4 K91Q, H4
K5,12R and H3 K56R mutations resulted in a nearly complete loss of viability on 5-FOA media. This indicated that the sites of newly synthesized histone acetylation on both H3 and H4 are functionally redundant and essential for viability in yeast.
3.4.2 Histone H4 lysines 5, 12 and 91 are involved in DNA repair-linked chromatin reassembly.
One important aspect of the DNA damage response is the reassembly of chromatin after the completion of DNA repair (Chen et al., 2008). The repair of an HO-induced DNA double strand break has provided a powerful model system for the study of chromatin dynamics during DNA repair (Haber, 1995). By expressing HO from a regulated promoter, a double strand break can be initiated at a specific place (the MAT locus) and at a specific time allowing for the use of chromatin immunoprecipitation (ChIP) to measure histone occupancy near the break site during the course of repair (Tsukuda et al.,
2005). This system was used to show that the Rtt109p- and Asf1p-mediated acetylation of histone H3 lysine 56 was involved in the reassembly of chromatin structure that accompanies the recombinational repair of a DNA double strand break (Chen et al.,
2008). We have employed the inducible HO system to determine whether the sites of acetylation on newly synthesized histone H4 are also involved in DNA repair-linked chromatin reassembly.
63
We first tested whether the H4 mutants were sensitive to a single double strand break at the MAT locus (Sugawara et al., 2003). Plasmids containing various combinations of histone H4 mutations were introduced into strains containing a galactose inducible HO gene integrated into the genome. Serial dilutions of equal numbers of cells from each strain were spotted on synthetic media containing either glucose or galactose (Figure
3.2A). The H4 K5, 12R, H4 K91R, and H4 K5, 12R, 91R alleles did not show sensitivity to a single double strand break, as reflected in the ability to grow on galactose-containing media. The H4 K91Q allele showed a slight decrease in viability in the presence of galactose. However, combining the H4 K5,12R and K91Q mutations resulted in a pronounced decrease in viability following induction of HO that was similar to that seen with an asf1D. These results again demonstrate the functional redundancy between the
NH2-terminal tail and core domain acetylation sites on newly synthesized histone H4 and are consistent with a role for these sites of modification in chromatin assembly.
To directly test whether these histone H4 mutations cause a defect in DNA-repair-linked chromatin reassembly, we monitored chromatin structure near the site of the HO- mediated double strand break by ChIP. For these experiments, galactose was added at time 0 to induce expression of HO and then glucose was added at 2 hours to repress HO expression and allow for DNA repair. The introduction of the HO-induced double strand break and its subsequent repair was detected by a PCR reaction that spanned the HO cut site and that generates distinct fragments from MATa and MAT cells (Figure 3.8). The kinetics of DNA double strand break formation and repair was similar in all of the strains examined (Figure 3.8).
64
In a wild type strain, following induction of HO, levels of histone H3 found at a point
~600 bp from the break site began to decrease. During the course of repair, the level of histone H3 restored (Figure 3.2B). The involvement of chromatin assembly in this histone restoration is indicated by decrease in H3 restoration seen in the absence of the key histone chaperone Asf1p (Figure 3.2B) (Chen et al., 2008). We found that while H4
K91R did not show any defect in chromatin reassembly, H4 K5, 12R and H4 K5, 12, 91R displayed a level of reassembly that is intermediate between that seen wild type cells and asf1D cells. Interestingly, this level of reassembly is similar to that seen in the absence of
Hat1p, which is thought to be responsible for the acetylation of H4 lysines 5 and 12 (Ge et al., 2011). In the presence of the H4 K91Q allele, chromatin reassembly near the double strand break was similar to that seen in the asf1D cells. Combining the H4
K5,12R and H4 K91Q mutations (H4 K5,12R K91Q) resulted in an further decrease in chromatin reassembly.
Replication-coupled chromatin assembly is not easily visualized by ChIP as the sites at which assembly occurs are continuously changing. Therefore, we used a surrogate assay to determine whether the sites of acetylation on newly synthesized histones are important for generalized chromatin assembly. The levels of soluble histones are typically very low as the synthesis of histones is tightly linked to DNA synthesis. However, a disruption in chromatin assembly caused by a block to DNA replication causes in increase in the level of non-chromatin associated histones (Bonner et al., 1988; Groth et al., 2005). Therefore, mutations that decrease the rates of chromatin assembly would be predicted to increase the soluble pools of histones. Therefore, we analyzed the levels of soluble histones in the
65
H4 K91Q and H4 K5,12R K91Q cells. Soluble cytosolic and nuclear fractions were analyzed by SDS-PAGE and Western blotting for histone H4. As seen in Figure 3.2C, the H4 K91Q allele caused a significant increase in soluble histone H4 and this increase was enhanced by the H4 K5,12R K91Q allele. In addition, treatment of these cells with the DNA damaging agent MMS further accentuated the levels of soluble histones. The presence of excess soluble histones is detrimental to cell viability and a Rad53p- dependent pathway has been identified that is responsible for the degradation of these excess histones (Gunjan and Verreault, 2003). Consistent with this, we were not able to introduce the H4 K5,12R K91Q allele into rad53D cells (data not shown). Taken together, these results are direct evidence that that sites of acetylation on newly synthesized histone H4 are involved in chromatin assembly and are consistent with the genetic evidence that they function in non-overlapping ways.
3.4.3 Sites of newly synthesized histone H4 acetylation are required for normal DNA damage response signaling.
Mutations that block the acetylation of histone H3 lysine 56 have a significant impact on the DNA damage response. In the absence of H3 lysine 56 acetylation, the DNA damage checkpoint is activated normally but, following the completion of repair, the checkpoint is not deactivated (Chen et al., 2008; Chen and Tyler, 2008; Yuan et al., 2009). This suggests that assembly of chromatin that contains H3 acetylated at lysine 56 is an essential step for recovery from the DNA damage checkpoint.
To determine whether the acetylation sites on newly synthesized histone H4 have a similar effect, we monitored the status of the DNA damage checkpoint during DNA
66 repair. Phosphorylated histone H2A, which will be referred to here as γ-H2AX to be consistent with the mammalian homolog, is detected soon after DNA damage (within 1hr in budding yeast around an HO-mediated double strand break at the MAT locus) and is found to span a large region (up to 100 kb in yeast cells)(Kim et al., 2007; Shroff et al.,
2004). ChIP assays were carried out to monitor the γ-H2A levels at 10 kb from the MAT locus. We found that the kinetics and abundance of γ-H2AX in wild type cells was similar to that seen with the H4 K5,12R, H4 K91R, H4 K91Q and H5 K5,12R K91R mutants. However, there was a dramatic loss of γ-H2AX specifically in the H4 K5,12R
K91Q cells (Figure 3.3A). We also observed a similar decrease in γ-H2AX levels at 20 kb from the break in the H4 K5,12R K91Q cells (Figure 3.3B). These results indicated that the sites of acetylation on newly synthesized histone H4 function redundantly in regulating the DNA damage response.
Previous results have shown that the RSC ATP-dependent chromatin remodeler is required to maintain normal γ-H2A levels on chromatin and that the enrichment of
Mec1p and Tel1p at the DNA lesion is reduced in rsc2 cells (Liang et al., 2007). To test the hypothesis that DNA damage checkpoint kinase recruitment was impaired in the H4
K5,12R K91Q mutant, we monitored the chromatin localization of Ddc2p. Ddc2p is the binding partner of Mec1p and its absence impairs Mec1p recruitment to DNA double strand breaks and the formation of γ-H2AX domains (Paciotti et al., 2000; Rouse and
Jackson, 2000; Wakayama et al., 2001). As assayed by ChIP utilizing a Ddc2p-myc fusion, we found that in a wild type strain, Ddc2p accumulation near the break site peak begins following induction of the HO endonuclease and then decreases as repair proceeds
67
(Figure 3.4A). In all of the histone H4 mutants tested, including the H4 K5,12R K91Q mutant, we observed that Ddc2p was recruited with similar kinetics and to similar levels.
This suggests that a defect in Mec1p recruitment was not responsible for the loss of chromatin associated γ-H2AX in the H4 K5,12R K91Q mutant.
If DNA damage checkpoint kinase recruitment is not altered in the H4 K5,12R K91Q mutant, another explanation for the loss of γ-H2AX is that a specific modification state or structure of chromatin may be necessary for the catalytic activity of the checkpoint kinases. To test this model, we isolated total histones from WT, H4 K91Q and H4
K5,12R K91Q cells before and after treatment with MMS. As expected, MMS treatment resulted in an increase in γ-H2AX in WT cells (Figure 3.4B). In addition, we saw identical increases in γ-H2AX in the histone H4 mutant cells. This indicated that the defect in localization of γ-H2AX near DNA double strand breaks was not due to an inability to generate this phosphorylated form of H2A.
As the defect in the formation of a γ-H2AX chromatin domain is not due to a decrease in total γ-H2AX in the cell, another possibility is that the H4 K5,12R K91Q mutation may be influencing either the assembly of γ-H2AX into chromatin or the stability of γ-H2AX in chromatin. Either scenario would predict that there would be an increase in soluble γ-
H2AX in these cells. Therefore, we purified soluble histones from cytosolic extracts from wild type, H4 K91Q and H4 K5,12R K91Q cells. In both the wild type and H4
K91Q mutant there were undetectable levels of γ-H2AX in the cytosol in the absence of
DNA damage (MMS treatment). Following MMS treatment, there was a similar increase in soluble γ-H2AX in both strains. However, in the H4 K5,12R K91Q mutant, there was
68 a significant increase in the level of γ-H2AX in untreated cells that was increased further by MMS treatment. This indicated that the H4 K5,12R K91Q mutation specifically caused an increase in the soluble pool of γ-H2AX consistent with a defect in either γ-
H2AX assembly or stability.
3.4.4 Histone H4 lysine 91 acetylation and lysine 5, 12 acetylation affect different aspects in the DNA damage response.
As seen above, the H4 K5,12R and H4 K91Q mutations display a pronounced synthetic increase in sensitivity to DNA damage and DNA replication stress. In addition, these mutations display defects in chromatin assembly and in the chromatin localization of γ-
H2AX. We used a series of genetic assays to determine whether the DNA damage sensitivity of the H4 K5,12R K91Q mutant is primarily due to the defect in γ-H2AX chromatin localization. First, alleles that contain combinations of mutations in the acetylation sites on newly synthesized histone H4 were combined with a deletion of
MEC1. Similar to what was previously reported for an H4 K91A allele, the H4 K91Q allele increased the DNA damage sensitivity of a mec1D strain (Ye et al., 2005). We did not detect an increase in sensitivity to DNA replication stress at the low levels of HU that are necessary to use with the mec1D cells. In addition, the H4 K5,12R allele also increased the DNA damage sensitivity of the mec1D. However, there was no further increase in sensitivity to MMS when the H4 K5,12R and H4 K91Q mutations were combined in the absence of Mec1p (Figure 3.5A). One interpretation of these results is that the sites of acetylation on newly synthesized histone H4 impact multiple aspects of the cellular response to DNA damage. One role for these acetylation sites is outside of
69 the DNA damage checkpoint and is likely to involve the process of chromatin assembly.
A second function in DNA repair, which H4 lysines 5 and 12 and H4 lysine 91 impact in a functionally redundant way, requires the function of Mec1p.
Mec1p (and Tel1p) phosphorylate a number of proteins in addition of histone H2A in response to DNA damage. To determine whether the interactions observed between the sites of acetylation on newly synthesized histone H4 and Mec1p are restricted to the γ-
H2AX pathway or also include other Mec1p activated pathways, we combined the same set of histone H4 alleles with deletions of key factors involved in other aspects of the
DNA damage checkpoint. When the H4 alleles were introduced into rad9D, mec3D or mrc1D strains, there was a synthetic increase in sensitivity to both MMS and HU when the H4 K5,12R and H4 K91Q alleles were combined (Figure 3.5B-D). This suggests that these sites of acetylation on histone H4 influenced the function of γ-H2AX but not other pathways activated by Mec1p phosphorylation.
Our current study makes a number of advances in our understanding of the function of the acetylation of newly synthesized histones. First, the acetylation sites in the NH2- terminal tail and globular core domain of histone H4 act in a functionally redundant manner. This is evidenced by synthetic increases in sensitivity to DNA damage and
DNA replication stress. Second, the sites of acetylation on newly synthesized histone H4 are essential for viability in the absence of histone H3 lysine 56 acetylation. Third, we provide direct evidence that the sites of acetylation on newly synthesized histone H4 participate in chromatin assembly. Finally, these sites of acetylation are required for the
70 formation of γ-H2AX domains on chromatin surrounding the sites of DNA double strand breaks through facilitating either the assembly or stability of this modified histone.
3.5 Discussion
The diacetylation of newly synthesized histone H4 on lysines 5 and 12 was the first pattern of post-translational modification to be identified on a histone. Despite this, the functional significance of this modification pattern is not known (Annunziato, 2012;
Annunziato and Hansen, 2000). Initial genetic studies in yeast using mutations that altered H4 lysines 5 and 12 to arginine suggested that the absence of this modification was well tolerated (Ma et al., 1998; Megee et al., 1990; Park and Szostak, 1990; Zhang et al., 1998). More recent studies have shown that the H4 K5,12R allele can have a significant effect on cell viability in strain backgrounds that have mutations in the Pob3p subunit of the yeast FACT complex (Nair et al., 2011; VanDemark et al., 2006). These studies suggest that FACT may be involved in nucleosome assembly but, given the multi- functional nature of the FACT complex, do not shed light on the specific function of H4 lysine 5/12 acetylation. However, experiments in P. physarum and HeLa cell systems suggest that the acetylation of H4 lysines 5 and 12 can influence the nuclear import of histone H4 (Altheim and Schultz, 1999a; Ejlassi-Lassallette et al., 2011).
The results presented here suggest that one reason for the minor phenotypes associated with the loss of the H4 lysine 5/12 acetylation pattern in yeast is functional redundancy with other sites of modification on newly synthesized histones H3 and H4. In particular, while combining the H4 K5,12R mutations with a H4 K91Q mutation caused a modest decrease in cell viability/growth, it also produced a pronounced sensitivity to DNA
71 damaging agents and DNA replication stress. Importantly, the observation that mutations of H4 lysine 91 and H3 lysine 56 are synthetically lethal with the H4 K5,12R allele suggests that the evolutionarily conserved diacetylation of the H4 NH2-terminal tail may, in fact, have an essential function in the cell.
While the sites of acetylation on newly synthesized histones appear to play essential and functionally redundant roles in the cell, it is still an open question whether this essential function is chromatin assembly. As with any alterations to the histones, these mutations may impact the regulation of transcription in such a way as to negatively impact viability.
However, the idea that the sites of acetylation on newly synthesized H3 and H4 primarily function through histone deposition is supported by a number of observations. First, these modifications are predominantly S-phase specific and are found on soluble histones that are in the process of chromatin assembly (Benson et al., 2006; Jasencakova et al.,
2010; Loyola et al., 2006; Masumoto et al., 2005; Recht et al., 2006; Ye et al., 2005;
Zhou et al., 2006). Second, histone H3 lysine 56 has been directly shown to participate in chromatin assembly and can influence the binding of histones to histone chaperones that are involved in chromatin assembly (Chen et al., 2008; Kaplan et al., 2008; Li et al.,
2008). Finally, this study provides a direct demonstration of a role for H4 lysines 5, 12 and 91 in DNA repair-linked chromatin reassembly and the accumulation of soluble histones when these sites are mutated strongly suggests that chromatin assembly has been globally altered.
Studies of the DNA damage checkpoint pathway have made the interesting observation that the regions of chromatin with which the checkpoint kinases Mec1p and Tel1p
72 associate do not overlap with the domains of chromatin that contain γ-H2AX (Shroff et al., 2004). Tel1p is recruited to unprocessed double strand breaks by the MRX complex while Mec1p is recruited to resected double strand breaks through interactions with
Ddc2p and the single strand binding complex RPA. The recruitment of these kinases to chromatin is necessary for the subsequent phosphorylation of histone H2A (Harrison and
Haber, 2006). The generation of large domains of chromatin that contain γ-H2AX may be the result of spreading of the kinases from their initial point of recruitment. However, these kinases are only found at significant levels near the break sites. Another possibility is that chromatin looping brings distal nucleosomes into contact with Mec1p/Tel1p located at the break, which allows for long stretches of chromatin to be modified.
Our results show that mutations in the sites of acetylation on histone H4 do not affect the production of γ-H2AX but do have a profound effect on the localization of γ-H2AX to chromatin. This suggests that the localization of γ-H2AX to chromatin may be a distinct step in the DNA damage checkpoint pathway (Figure 3.6). There are a number of models that can explain the involvement of sites of acetylation on newly synthesized histone H4 in this pathway. The first is a model in which chromatin assembly or histone exchange plays a critical role in the chromatin localization of γ-H2AX. In this model, the recruitment of Mec1p and Tel1p to chromatin brings them into contact with soluble pools of histones that are concentrated in close proximity to chromatin. Mec1p/Tel1p can then phosphorylate soluble histone H2A, which is then incorporated into nearby chromatin through chromatin assembly or histone exchange. The incorporation of γ-H2AX may require concomitant assembly of H3/H4, which would be facilitated by the proper
73 modification state of histone H4. Conversely, the mutations in the sites of acetylation on newly synthesized histone H4 might actually increase the rate of histone exchange leading to a loss of γ-H2AX from chromatin.
An alternative model is that combining the H4 K5,12R and H4 K91Q alleles destabilizes nucleosomes such that γ-H2AX is lost from chromatin. Indeed, it has been demonstrated that a mutation that eliminates the ability of H4 lysine 91 to form a salt bridge with glutamic acid 74 of histone H2B leads to a destabilized histone octamer (Ye et al., 2005).
However, the crystal structure of a nucleosome that contains a histone H4 lysine 91 to glutamine mutation shows that, while the salt bridge is lost, there are no significant structural alterations to the nucleosome (Iwasaki et al.). In addition, γ-H2AX localization is not effected by the H4 K91Q allele alone. Therefore, the mutation at H4 lysines 5 and
12 must be contributing to the structural destabilization of the nucleosome. As these residues are not visible in nucleosome crystal structure, it is not clear whether they would alter nucleosome stability through a direct or indirect mechanism (Luger et al., 1997).
The involvement of histone exchange/chromatin assembly in the generation of γ-H2AX domains would be analogous to the mechanisms that have been identified as being responsible for the removal of H2A phosphorylation from chromatin in yeast. Pph3p is the phosphatase that is responsible for the dephosphorylation of γ-H2AX. However, in the absence of Pph3p, γ-H2AX is still lost from chromatin with normal kinetics (Keogh et al., 2006). This indicates that γ-H2AX is removed from chromatin, perhaps by histone exchange, prior to dephosphorylation. Hence, modulation of the phosphorylation state of
74 histone H2A may occur on soluble histones and presence of γ-H2AX may be controlled by chromatin assembly and histone exchange pathways.
75
Strain Genotype Reference SQY501 MAT ade2::hisG his3200 leu20 lys20 met150 (Ge et al., trp163 ura30 ADE3::GAL10-HO HHF2-HHT2::LEU2 2011) HHF1-HHT1::ADE3 (TRP1 CEN ARS)-HHF2-HHT2 ZGY104 MAT ade::hisG his3200 leu20 lys20 met150 (Ge et al., trp163 ura30 ADE3::GAL10-HO HHF2-HHT2::LEU2 2011) HHF1-HHT1::ADE3 (LYS CEN ARS)-HHF2-HHT2 ASF1::URA3 UCC1111 MAT ade2::hisG his3200 leu20 lys20 met150 (Kelly et trp163 ura30 adh3-URA3-TEL(VII-L) HHF2- al., 2000a) HHT2::MET15 HHF1-HHT1::LEU2 (LYS2 CEN ARS)- HHF2-HHT2 MPY302 MATa ade2::hisG his3200 leu20 lys20 met150 This study trp163 ura30 HHF2-HHT2::MET15 HHF1-HHT1::LEU2 (ADE2 CEN ARS)-HHF2-HHT2 ZGY201 MATa ade2::hisG his3200 leu20 lys20 met150 This study trp163 ura30 HHF2-HHT2::MET15 HHF1-HHT1::LEU2 (ADE2 CEN ARS)-HHF2-HHT2 SML1::URA3 ZGY202 MATa ade2::hisG his3200 leu20 lys20 met150 This study trp163 ura30 HHF2-HHT2::MET15 HHF1-HHT1::LEU2 (ADE2 CEN ARS)-HHF2-HHT2 SML1::URA3 ZGY203 MATa ade2::hisG his3200 leu20 lys20 met150 This study trp163 ura30 HHF2-HHT2::MET15 HHF1-HHT1::LEU2 (ADE2 CEN ARS)-HHF2-HHT2 SML1::URA3 MEC1::HIS3 ZGY204 MATa ade2::hisG his3200 leu20 lys20 met150 This study trp163 ura30 HHF2-HHT2::MET15 HHF1-HHT1::LEU2 (ADE2 CEN ARS)-HHF2-HHT2 RAD9::HIS3 ZGY205 MATa ade2::hisG his3200 leu20 lys20 met150 This study trp163 ura30 HHF2-HHT2::MET15 HHF1-HHT1::LEU2 (ADE2 CEN ARS)-HHF2-HHT2 MRC1::HIS3
Table 3.1 Strains used in this study.
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Figure 3.1 Histone H4 lysines 5, 12 and 91 are functionally redundant.
A) and B) Ten-fold serial dilutions of cells with the indicated alleles of histone H3 and
H4 were spotted on plates containing synthetic complete (SC) media with or with out the indicated concentrations of hydroxyurea (HU) or MMS. Plates were incubated at 30 for
3 days. C) Cells with the indicated alleles of histone H3 and H4 on a TRP1-based plasmid were grown for several passages in liquid SC media lacking tryptophan. Ten- fold serial dilutions were then plated on SC media with or without 5-FOA. Plates were incubated at 30 for 3 days.
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Figure 3.2 Histone H4 lysines 5, 12 and 91 function in chromatin assembly.
A) Ten-fold serial dilutions of cells with the indicated genotype were spotted onto plates containing either glucose or galactose as carbon source. Plates were incubated at 30 for
3 days. B) Cultures of strains with the indicated genotypes were grown in raffinose and galactose was added at time 0 hr to induce expression of HO. HO expression was then repressed by the addition of glucose at 2 hr. DNA repair of a single double strand break introduced at the MAT locus. Galactose was added at the 0 hr time point and glucose was added at the 2 hr time point. ChIP analysis was used to determine histone H3 levels 600 bp from the DSB at the MAT locus. The large bar graph shows a comparison of the indicated histone H4 mutant to a wild type strain as well as an asf1 strain. Histone H3 levels were normalized to H3 levels at the SMC2 locus at each time point. Subsequent time points are then normalized to the 0 hr time point. Smaller bar graphs replot the data for the H4 H91Q and H4 K5,12R K91Q alleles for clarity. C) Top. Western blots of cytosolic and soluble nuclear extracts of the indicated strains. Cultures were treated with
0.03% MMS as indicated. Bottom. Cytosolic and nuclear extracts were resolved by SDS-
PAGE, transferred to nitrocellulose and stained with Ponceau Red.
78
79
Figure 3.3 Newly synthesized H4 acetylation site mutants are defective in the formation of γ-H2AX domains near a double strand break.
A) ChIP analysis of H2A levels 10 kb from the DSB. Each bar graph shows a comparison of the indicated histone H4 mutant to a wild type strain. Galactose was added at the 0 hr time point and glucose was added at the 2 hr time point. -H2A levels are normalized to the internal control SMC2 and to 0 hr time point. B) ChIP analysis of
H2A level 20 kb from the DSB was performed as described in A.
80
81
Figure 3.4 Sites of acetylation of histone H4 are not necessary for the formation of γ-
H2AX.
A) ChIP analysis of Ddc2-myc level at 600 kb from the DSB. Galactose was added at the
0 hr time point and glucose was added at the 2 hr time point. Ddc2 levels were normalized to Ddc2 levels at the SMC2 locus at each time point. Subsequent time points are then normalized to the 0 hr time point. B) Indicated strains were grown to log phase and 0.03% MMS was added. Whole cell extracts were resolved by SDS-PAGE and analyzed by Western blots probed with a-H2A and a--H2A antibodies. C) Indicated strains were grown to log phase and 0.03% MMS was added. Soluble cell extracts were resolved by SDS-PAGE and analyzed by Western blots probed with a-H2A and a--H2A antibodies.
82
83
Figure 3.5 The redundant function of newly synthesized histone H4 acetylation sites in the NH2-terminal tail and core domains requires Mec1p.
Strains containing the indicated genotype and alleles of histone H4 were plated in ten- fold serial dilutions on SC media containing the indicated concentrations of HU or MMS.
Plates were incubated at 30 for 3 days.
84
Figure 3.6. Model for the involvement of chromatin assembly or histone exchange in DNA damage response signaling.
The formation of domains of γ-H2AX is proposed to be a two step process. The first step involves the phosphorylation of soluble histone H2A by Mec1p or Tel1p. Chromatin assembly or histone exchange then incorporates the phosphorylated H2A into chromatin near the break site.
85
Figure 3.7. Functional redundancy with histone H4 lysine 91 requires lysines 5 and
12ac.
Ten-fold serial dilutions of cells with the indicated alleles of histone H4 were spotted on plates containing synthetic complete (SC) media with or with out the indicated concentrations of hydroxyurea (HU). Plates were incubated at 30° for 3 days.
86
Figure 3.8. Repair of an HO-induced DNA double strand break.
A) Schematic diagram depicting the MAT locus and the silent mating loci (HMLα and
HMRa). The site of HO cutting is indicated by the red triangle. The repair status of the
MAT locus was assayed by a pcr reaction that spanned the HO digestion site and generates a longer fragment from a MATα cell than from a MATa cell (black lines). For
ChIP experiments in the manuscript, pcr reactions applified fragments that were 0.6 kb,
10 kb and 20 kb from the HO cut site as indicated. B) Galactose was added to cultures containing the indicated alleles of histone H4 at the 0 hr time point and glucose was added at the 2 hr time point. Reaction products were resolved on a 1.5% agarose gel and visualized by ethidium bromide staining. MATα and MATa products are indicated and
RAD27 locus was used as a control. C) Quantitation of DSB formation and repair.
Stained agarose gels were photographed and the MATα, MATa and RAD27 bands were quantitated using 1D Image Analysis software (Kodak). MATα and MATa bands were normalized to the RAD27 bands and plotted relative to the 0 hr or 5 hr time points.
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88
Chapter 4 Role of mammalian Hat1p in DNA replication coupled
chromatin assembly
4.1 Abstract
Although Hat1p has long been presumed to be involved in the acetylation of newly synthesized histone H4 which consists of the very first step of chromatin assembly, there has been lack of straightforward evidence linking Hat1p to this process. Using the technology called iPOND (isolation of proteins on nascent DNA), we were able to show for the first time that the presence of Hat1p influences H4 lysine 5 and 12 acetylation levels in the cell as well as on nascent chromatin. Also, we showed that in the absence of
Hat1p, histone H3 lysine 9, 18 and 27 acetylation levels, but not lysine 14 and 23 acetylation levels decreased at the replication fork. Finally, we showed that Hat1p specifically associates with the sites of chromatin assembly and localizes to nascent DNA.
4.2 Introduction
Hat1p is the first type B histone acetyltransferase discovered that modifies newly synthesized histone H4 at lysine 5 and 12, presumably in the cytoplasm. Biochemical studies showed that the yeast Hat1p exists in at least 2 distinct complexes. One is a cytoplasmic complex that consists of Hat1p and Hat2p (homolog of mammalian protein
Rbap46), the other one is a nuclear complex that also contains a third protein, Hif1p
(homolog of mammalian protein NASP). Not only the Hat1p itself, but also the Hat1p 89 complex is highly conserved throughout eukaryotic evolution and Hat1p complex homologs have been isolated from human, chicken, maize, Xenopus laevis and
Saccharomyces cerevisiae (Parthun et al., 1996; Verreault et al., 1998).
Since the enzymatic specificity of Hat1p corresponds to the replication dependent acetylation pattern of histone H4, Hat1p is thought to be involved in replication-coupled chromatin assembly. There is evidence suggesting that Hat1p has functions other than acetylating newly synthesized histone H4 in the cytoplasm. First, Hat1p is found in the nucleus in a complex together with Hat2p and a histone H3/H4 chaperone, Hif1p, as well as histone H3 and H4. Second, both Hat1p’s nuclear localization as well as its catalytic activity are required for its in vivo function, as a construct that prevents its accumulation in the nucleus caused defects in telomere silencing. Third, Hat1p and Hif1p have been shown to be recruited to HO endonuclease induced DNA double strand break (Qin and
Parthun, 2006).
Despite of this genetic and biochemical evidence from studies in yeast cells, there has been a lack of direct evidence in vivo, especially in mammalian cells. To explore the role of mammalian Hat1p in replication coupled chromatin assembly, we generated a conditional HAT1 knock out mouse model and immortalized mouse embryonic fibroblast
(MEFs) were derived from HAT1+/+ MEFs as well as HAT1-/- MEFs. The new technique called iPOND (isolation of proteins on nascent DNA) was carried out, which can be used to isolate proteins at active, stalled and collapsed replication forks and can also be used to monitor the process of chromatin maturation(Sirbu et al., 2012). The iPOND technique involves pulse-labeling cells with the thymidine analog EdU, which will be incorporated
90 into DNA that is being synthesized. Following cross-linking with formaldehyde, Click chemistry is then used to covalently attach biotin molecules onto the EdU moieties.
Streptavidin beads can then be used to fish out proteins that are cross-linked to EdU labeled DNA.
In this study, we used the technique IPOND to show that mammalian Hat1p indeed contributes to replication coupled chromatin assembly. We showed that mouse Hat1p is responsible for normal H4 lysine 5 and 12 acetylation levels at the replication fork as well as that of histone H3 lysine 9, 18 and 27 acetylation levels, but not lysine 14 and 23 acetylation levels. We also showed that mouse Hat1p physically associates with nascent
DNA.
4.3 Materials and Methods
4.3.1 Cell culture
U2OS cells were cultured in McCoy’s media supplemented with 10% FBS at 37 in 5%
+/+ -/- CO2 supply. Hat1 and Hat1 mouse embryonic fibroblasts (MEFs) were culture in
Dulbecco’s Modified Eagle Media supplemented with 10% FBS at 37 in 5% CO2 supply.
4.3.2 IPOND Method
1.5 108 cells (U2OS or MEFs) were incubated with 10 M EdU (Invitrogen) for various time periods. For thymidine chase experiment, EdU labeled cells were washed once with pre-equilibrated (temperature, pH and thymidine) medium and then incubated with 10 M thymidine for various time. After labeling and/or pulse-chase, cells were cross-linked with 1% formaldehyde/PBS for 20 min, quenched with 1.25 M glycine and
91 scraped off the plates and collected. After washing three times with PBS, the cell pellet was then resuspended in 0.25% Triton-X 100/PBS to permeabilize in room temperature.
Cells were spun down after permeabilization and washed first with 0.5% BSA/PBS and then with PBS. Cells were incubated with either click reaction buffer (10 M biotin azide, 10 mM sodium ascorbate, 2 M CuSO4 in PBS) or control buffer (as reaction buffer but DMSO added instead of biotin azide) at a concentration of about 3 107 cells/ml for 1 hr at room temperature, protected from light. After incubation, cells were again washed with 0.5% BSA/PBS and PBS. Cells were then lysed with lysis buffer (1%
SDS, 50 mM Tris pH 8.0, 1 g/ml Leupeptin, 1 g/ml aportinin) at a concentration of 1.5
107 cells/l. Samples were then sonicated using the Bioruptor (Degenode) for 30 sec on and 60 sec off per cycle for 12 cycles. Samples were then spun down and supernatant is filtered through 90 micro nylon mesh (small parts company) and diluted with PBS containing protease inhibitors. An aliquot of the lysate was kept as input, the rest was incubated with prewashed Streptavidin-agarose beads (Novagen) for 16 hrs at 4 C. The beads were then washed once with lysis buffer, once with 1 M NaCl, and twice with lysis buffer. Beads were boiled with 2 SDS dye for 25 min at 95 C. Proteins can be resolved by SDS-PAGE and detected by western blot. Antibodies used in this study include:
PCNA (Santa Cruz Biotechnology), Hat1p (Abcam), H3 K9Ac (Abcam), H3 K14Ac
(Abcam), H3 K18Ac (Upstate), H3 K23Ac (Upstate), H3 K27Ac (Upstate).
92
4.4 Results
4.4.1 Hat1p is essential for normal H4 K5, 12Ac level at the replication fork
In order to determine whether Hat1p mediated histone H4 lysine acetylation is involved in replication coupled chromatin assembly, we used Hat1+/+ and Hat1-/- MEFs in the
IPOND analysis to monitor histone modification dynamics. Immortalized Hat1+/+ and
Hat1-/- MEFs were pulsed either 10 or 30 minutes with EdU. Following the 30 min EdU pulse, the cells were given a thymidine chase for 10, 60 or 120 minutes. In wild type cells, as expected, PCNA levels increased during the EdU pulse and then decreased as the cells go into thymidine chase (Figure 4.1). Also as previously reported, levels of H4 lysine 5 and lysine 12 acetylation were high with EdU label but then decrease over thymidine chase in wild type cells. As a control, histone H4 levels remained constant through the thymidine chase in wild type cells (Figure 4.1). In Hat1-/- MEFs, there was about a two fold decrease in the H4 K5Ac and H4 K12Ac levels in the input samples compared to wild type MEFs (Figure 4.1 left penal). H4 lysine 5 and 12 aceylation levels on the nascent DNA were further decreased as can be seen in the IPOND samples compared to wild type MEFs (Figure 4.1 right penal). Also note that PCNA and histone
H4 levels are the same in Hat1+/+ and Hat1-/- MEFs. A decrease in H4 lysine 5 and 12 acetylation levels in Hat1-/- cells was also confirmed by native iPond results (data not shown).
4.4.2 Hat1p influences acetylation on newly synthesized histone H3
We also examined dynamics of histone H3 acetylation during the chromatin maturation process using iPOND. Hat1+/+ and Hat1-/- MEFs were processed the same as in 4.4.1 with
93 the same time points monitored. As expected PCNA levels in Hat1+/+ and Hat1-/- MEFs increased during EdU pulse and then decreased during thymidine chase indicative of active replication fork associated proteins (Figure 4.2). On the contrary, histone H3 levels in Hat1+/+ and Hat1-/- MEFs remained constant throughout thymidine chase indicative of a stable component of chromatin (Figure 4.2). In wild type MEFs, we showed that histone H3 lysine 9, 18 and 27 acetylations showed characteristics of replication fork specific histone modifications as they are associated with EdU lableled DNA and can be chased away with thymidine (Figure 4.2). Histone H3 lysine 14 and 23 acetylation, on the other hand, seems to be constant once incorporated into chromatin (Figure 4.2). In the absence of Hat1p, we found that there was decreased histone H3 lysine 9, 18 and 27 acetylations at the replication fork, while no observable difference on histone H3 lysine
14 and 23 acetylation signals were found (Figure 4.2, compare HAT1 and hat1 click rxn samples). Also, there is a decrease in whole cell levels of histone H3 lysine 9, 18 and 27 acetylations but not in H3 lysine 14 and 23 acetylations in Hat1-/- MEFs compared to
Hat1+/+ MEFs (Figure 4.2, compare HAT1 and hat1 input samples). Therefore, although
Hat1p has no activity towards histone H3, it is required for the integrity of histone H3 acetylation on nascent DNA.
4.4.3 Hat1p can be detected at the replication fork
Previous research has shown that yeast Hat1p becomes associated with chromatin near the DNA break induced by the HO endonuclease and is responsible for H4 lysine 12 acetylation level changes that occur during the course of recombinational DNA repair
(Qin and Parthun, 2006). This prompted us to determine whether a similar role is true for
94 mammalian Hat1p during the course of DNA replication. Using U2OS cells for IPOND, we were able to show that the mammalian Hat1p is indeed associated with chromatin
(Figure 4.3A). H4 K5Ac and K12Ac, as expected, are detected in the EdU labeled samples (Figure 4.3A). Next, by adding thymidine into the medium to chase away EdU labeled replication fork, we found gradual loss of the Hat1p signal with similar kinetics as the gradual loss of H4 K5Ac and K12Ac signals (Figure 4.3B). This implies that
Hat1p is specifically recruited to the DNA replication fork when DNA replication is taking place.
4.5 Discussion
Soluble, mostly newly synthezised and some parental histones H3 and H4 are found to be within multiple distinct complexes, which lead to the hypothesis that newly synthesized histones H3 and H4 are processed in a sequential pathway through distinct modifications before they get loaded onto nascent DNA. Several recent studies support this hypothesis.
For example, the very first modification on newly synthesized histones was found to be histone H3 monomethylation on lysine 9 (Loyola et al., 2006), followed by H3 acetylation on lysine 14 and 18 at an early stage (Alvarez et al., 2011). Then, the H3/H4 tetramer is modified by Hat1p on H4 lysine 5 and 12. Our study provided direct evidence for the role of mammalian Hat1p in the acetylation and processing of newly synthesized histone H3 and H4 during replication coupled chromatin assembly. Also, we provided evidence for the first time that Hat1p is recruited to the close vicinity of the replication fork.
95
The fact that histone H4 lysine 5 and 12 acetylation levels are dramatically decreased at the replication fork in the absence of Hat1p could be explained in two ways. Firstly, newly synthesized histone H4 could not get acetylated at lysine 5 and 12 efficiently in the absence of Hat1p, which is partially supported by the fact that the whole cell level of histone H4 lysine 5 and 12 acetylation levels are about 2 fold less in hat1-/- MEFs.
Secondly, Hat1p could be physically interacting with acetylated histone H4 NH2-tail at the replication fork and protect it from histone deacetylases. One possible way to test the second possibility is to generate HAT1-NES MEFs to eliminate Hat1p function in the nucleus. This experiment will distinctly tell the difference between cytoplasmic and nuclear Hat1p complexes in mammalian cells.
Our iPOND analysis indicated that histone H3 lysine 9, 18 and 27 acetylations showed clear characteristics of replication fork specific modification as they are gradually erased as chromatin matures. This suggests that this could be a specific acetylation pattern associated with newly synthesized histone H3 in murine cells. On the contrary, histone
H3 lysine 14 and 23 acetylations seem constant on chromatin, which suggests that they may be histone marks associated with parental histones recycled and then reloaded on to replicating DNA. This pattern of histone H3 acetylation, however, does not match recent studies that observed soluble histones in other mammalian systems. For example, soluble histone H3 from HeLa cells showed acetylation on lysine 14 and 18 in the cytoplasm
(Loyola et al., 2006). The reason for this discrepancy could be two fold. First, the pattern of newly synthesized histone H3 acetylation is not as conserved as that of histone H4 and could be different among different cell types. Second, histone H3 could be further
96 modified (aceylation and/or deactylation) on its N terminal tails and thus present different actylation pattern from those in the cytoplasm. The fact that acetylation of newly synthesized histone H3 in MEFs is dependent on the presence of Hat1p is intriguing since the substrate specificity of Hat1p is highly conserved. One explanation would be that the acetylation state of histone H4 promotes downstream acetylation of histone H3 by promoting the activity of a specific H3 HAT. Another explanation could be that the H4
K5 and K12 acetylation is important for the recognition of a histone chaperone that is necessary for H3 acetylation.
Studies in S cerevisiae and Physarum polycephalum have shown evidence that the Hat1p complex can physically and functionally associate with the origin recognition complex
(ORC) (Suter et al., 2007) and replicating chromatin (Ejlassi-Lassallette et al., 2011;
Suter et al., 2007), which suggest that Hat1p could function directly at the replication fork besides its role as a type B HAT which acetylates newly synthesized histone H4 in the cytoplasm. Here we showed that mammalian Hat1p physically associates with the replication fork using the iPOND method and we propose two possible functions of
Hat1p with the replication fork. One possibility is that Hat1p could be part of the complex for replication coupled chromatin assembly, the other is that Hat1p is acetylating histone H4 in the vicinity of nascent DNA prior of H4 loading. Mammalian
Hat1p is recruited to replication fork and is responsible for H4 diaceylation is also reminiscent of the fact that yeast Hat1p is recruited to sites of DNA lesion and influences the H4 diacetylation pattern around the break (Qin and Parthun, 2006).
97
Figure 4.1 Mouse Hat1p is important for H4 lysine 5 and 12 acetylations in the cell.
WT and HAT1-/- MEFS were labeled with EdU for 10 min or 30 min, or after EdU labeling of 30 min, chased into thymidine containing medium for the indicated times.
IPOND was performed and pull-down samples as well as inputs were run on SDS-PAGE followed by western blot analysis using -PCNA, -H4 K5ac and -H4K12ac antibodies.
98
Figure 4.2 Mouse Hat1p is important for H3 lysine acetylations in the cell. WT and
HAT1-/- MEFS were labeled with EdU for 10 min or 30 min, or after EdU labeling of 30 min, chased into thymidine containing medium for the indicated times. IPOND was performed and pull-down samples as well as inputs were run on SDS-PAGE followed by western blot analysis using -H3 K9Ac, -H3 K14Ac, -H3 K18ac, -H3 K23ac and -
H3 K27ac antibodies.
99
B A Thymidine Chase (0 – 60 min)
input click rxn - 30 60 - 30 60 Thd chase (min) HAT1
H4 K5 ac
H4 K12 ac
Figure 4.3 Mammalian Hat1p is a replication fork associated protein. U2OS cells were labeled with 10 M EdU for 30 min (A) or labeled with EdU for 30 min first and then chased with 10 M thymidine for 30 min or 60 min (B). IPOND was performed and pull-down samples as well as inputs were run on SDS-PAGE followed by western blot analysis using -HAT1, -H4 K5ac and -H4K12ac antibodies.
100
Bibliography
Adkins, M.W., Carson, J.J., English, C.M., Ramey, C.J., and Tyler, J.K. (2007). The histone chaperone anti-silencing function 1 stimulates the acetylation of newly synthesized histone H3 in S-phase. J Biol Chem 282, 1334-1340.
Adkins, M.W., Howar, S.R., and Tyler, J.K. (2004). Chromatin disassembly mediated by the histone chaperone Asf1 is essential for transcriptional activation of the yeast PHO5 and PHO8 genes. Mol Cell 14, 657-666.
Agarwal, R., Tang, Z., Yu, H., and Cohen-Fix, O. (2003). Two distinct pathways for inhibiting pds1 ubiquitination in response to DNA damage. J Biol Chem 278, 45027-
45033.
Ahmad, K., and Henikoff, S. (2002). The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol Cell 9, 1191-1200.
Ai, X., and Parthun, M.R. (2004). The nuclear Hat1p/Hat2p complex: a molecular link between type B histone acetyltransferases and chromatin assembly. Mol Cell 14, 195-205.
Altheim, B.A., and Schultz, M.C. (1999a). Histone modification governs the cell cycle regulation of a replication- independent chromatin assembly pathway in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 96, 1345-1350.
101
Altheim, B.A., and Schultz, M.C. (1999b). Histone modification governs the cell cycle regulation of a replication-independent chromatin assembly pathway in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 96, 1345-1350.
Alvarez, F., Munoz, F., Schilcher, P., Imhof, A., Almouzni, G., and Loyola, A. (2011).
Sequential establishment of marks on soluble histones H3 and H4. J Biol Chem 286,
17714-17721.
Annunziato, A.T. (2012). Assembling chromatin: The long and winding road. Biochim
Biophys Acta 1819, 196-210.
Annunziato, A.T., and Hansen, J.C. (2000). Role of histone acetylation in the assembly and modulation of chromatin structures. Gene Expr 9, 37-61.
Annunziato, A.T., and Seale, R.L. (1983). Histone deacetylation is required for the maturation of newly replicated chromatin. J Biol Chem 258, 12675-12684.
Barman, H.K., Takami, Y., Nishijima, H., Shibahara, K., Sanematsu, F., and Nakayama,
T. (2008). Histone acetyltransferase-1 regulates integrity of cytosolic histone H3-H4 containing complex. Biochem Biophys Res Commun 373, 624-630.
Bassing, C.H., Chua, K.F., Sekiguchi, J., Suh, H., Whitlow, S.R., Fleming, J.C., Monroe,
B.C., Ciccone, D.N., Yan, C., Vlasakova, K., et al. (2002). Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX. Proc Natl Acad Sci U
S A 99, 8173-8178.
Basu, A., Rose, K.L., Zhang, J., Beavis, R.C., Ueberheide, B., Garcia, B.A., Chait, B.,
Zhao, Y., Hunt, D.F., Segal, E., et al. (2009). Proteome-wide prediction of acetylation substrates. Proc Natl Acad Sci U S A 106, 13785-13790.
102
Becker, P.B., and Horz, W. (2002). ATP-dependent nucleosome remodeling. Annu Rev
Biochem 71, 247-273.
Benson, L.J., Gu, Y., Yakovleva, T., Tong, K., Barrows, C., Strack, C.L., Cook, R.G.,
Mizzen, C.A., and Annunziato, A.T. (2006). Modifications of H3 and H4 during chromatin replication, nucleosome assembly, and histone exchange. J Biol Chem 281,
9287-9296.
Benson, L.J., Phillips, J.A., Gu, Y., Parthun, M.R., Hoffman, C.S., and Annunziato, A.T.
(2007). Properties of the type B histone acetyltransferase Hat1: H4 tail interaction, site preference, and involvement in DNA repair. J Biol Chem 282, 836-842.
Bird, A.W., Yu, D.Y., Pray-Grant, M.G., Qiu, Q., Harmon, K.E., Megee, P.C., Grant,
P.A., Smith, M.M., and Christman, M.F. (2002). Acetylation of histone H4 by Esa1 is required for DNA double-strand break repair. Nature 419, 411-415.
Boeke, J.D., Trueheart, J., Natsoulis, G., and Fink, G.R. (1987). 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol 154, 164-175.
Bonner, W.M., Wu, R.S., Panusz, H.T., and Muneses, C. (1988). Kinetics of accumulation and depletion of soluble newly synthesized histone in the reciprocal regulation of histone and DNA synthesis. Biochemistry 27, 6542-6550.
Brownell, J.E., and Allis, C.D. (1996). Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation. Curr Opin Genet Dev 6,
176-184.
Bugreev, D.V., Mazina, O.M., and Mazin, A.V. (2006). Rad54 protein promotes branch migration of Holliday junctions. Nature 442, 590-593.
103
Campos, E.I., Fillingham, J., Li, G., Zheng, H., Voigt, P., Kuo, W.H., Seepany, H., Gao,
Z., Day, L.A., Greenblatt, J.F., et al. (2010). The program for processing newly synthesized histones H3.1 and H4. Nat Struct Mol Biol 17, 1343-1351.
Campos, E.I., and Reinberg, D. (2010). New chaps in the histone chaperone arena. Genes
Dev 24, 1334-1338.
Celeste, A., Fernandez-Capetillo, O., Kruhlak, M.J., Pilch, D.R., Staudt, D.W., Lee, A.,
Bonner, R.F., Bonner, W.M., and Nussenzweig, A. (2003). Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat Cell Biol 5,
675-679.
Celeste, A., Petersen, S., Romanienko, P.J., Fernandez-Capetillo, O., Chen, H.T.,
Sedelnikova, O.A., Reina-San-Martin, B., Coppola, V., Meffre, E., Difilippantonio, M.J., et al. (2002). Genomic instability in mice lacking histone H2AX. Science 296, 922-927.
Celic, I., Masumoto, H., Griffith, W.P., Meluh, P., Cotter, R.J., Boeke, J.D., and
Verreault, A. (2006). The sirtuins hst3 and Hst4p preserve genome integrity by controlling histone h3 lysine 56 deacetylation. Curr Biol 16, 1280-1289.
Celic, I., Verreault, A., and Boeke, J.D. (2008). Histone H3 K56 hyperacetylation perturbs replisomes and causes DNA damage. Genetics 179, 1769-1784.
Chang, L., Loranger, S.S., Mizzen, C., Ernst, S.G., Allis, C.D., and Annunziato, A.T.
(1997). Histones in transit: cytosolic histone complexes and diacetylation of H4 during nucleosome assembly in human cells. Biochemistry 36, 469-480.
104
Chen, C.C., Carson, J.J., Feser, J., Tamburini, B., Zabaronick, S., Linger, J., and Tyler,
J.K. (2008). Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair. Cell 134, 231-243.
Chen, C.C., and Tyler, J. (2008). Chromatin reassembly signals the end of DNA repair.
Cell Cycle 7, 3792-3797.
Chicoine, L.G., Schulman, I.G., Richman, R., Cook, R.G., and Allis, C.D. (1986).
Nonrandom utilization of acetylation sites in histones isolated from Tetrahymena.
Evidence for functionally distinct H4 acetylation sites. J Biol Chem 261, 1071-1076.
Clapier, C.R., and Cairns, B.R. (2009). The biology of chromatin remodeling complexes.
Annu Rev Biochem 78, 273-304.
Clarke, A.S., Lowell, J.E., Jacobson, S.J., and Pillus, L. (1999). Esa1p is an essential histone acetyltransferase required for cell cycle progression. Mol Cell Biol 19, 2515-2526.
Cosgrove, M.S., Boeke, J.D., and Wolberger, C. (2004). Regulated nucleosome mobility and the histone code. Nat Struct Mol Biol 11, 1037-1043.
Daley, J.M., Laan, R.L., Suresh, A., and Wilson, T.E. (2005). DNA joint dependence of pol X family polymerase action in nonhomologous end joining. J Biol Chem 280, 29030-
29037.
Das, C., Lucia, M.S., Hansen, K.C., and Tyler, J.K. (2009). CBP/p300-mediated acetylation of histone H3 on lysine 56. Nature 459, 113-117.
Das, C., Tyler, J.K., and Churchill, M.E. (2010). The histone shuffle: histone chaperones in an energetic dance. Trends Biochem Sci 35, 476-489.
105 de Jager, M., Dronkert, M.L., Modesti, M., Beerens, C.E., Kanaar, R., and van Gent, D.C.
(2001). DNA-binding and strand-annealing activities of human Mre11: implications for its roles in DNA double-strand break repair pathways. Nucleic Acids Res 29, 1317-1325.
De Koning, L., Corpet, A., Haber, J.E., and Almouzni, G. (2007). Histone chaperones: an escort network regulating histone traffic. Nat Struct Mol Biol 14, 997-1007.
Downs, J.A., Allard, S., Jobin-Robitaille, O., Javaheri, A., Auger, A., Bouchard, N., Kron,
S.J., Jackson, S.P., and Cote, J. (2004). Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites. Mol Cell 16, 979-990.
Downs, J.A., Lowndes, N.F., and Jackson, S.P. (2000). A role for Saccharomyces cerevisiae histone H2A in DNA repair. Nature 408, 1001-1004.
Drane, P., Ouararhni, K., Depaux, A., Shuaib, M., and Hamiche, A. (2010). The death- associated protein DAXX is a novel histone chaperone involved in the replication- independent deposition of H3.3. Genes Dev 24, 1253-1265.
Driscoll, R., Hudson, A., and Jackson, S.P. (2007). Yeast Rtt109 promotes genome stability by acetylating histone H3 on lysine 56. Science 315, 649-652.
Durairaj, G., Chaurasia, P., Lahudkar, S., Malik, S., Shukla, A., and Bhaumik, S.R.
(2010). Regulation of chromatin assembly/disassembly by Rtt109p, a histone H3 Lys56- specific acetyltransferase, in vivo. J Biol Chem 285, 30472-30479.
Edmondson, D.G., and Roth, S.Y. (1996). Chromatin and transcription. FASEB J 10,
1173-1182.
Eissenberg, J.C. (2001). Molecular biology of the chromo domain: an ancient chromatin module comes of age. Gene 275, 19-29.
106
Ejlassi-Lassallette, A., Mocquard, E., Arnaud, M.C., and Thiriet, C. (2011). H4 replication-dependent diacetylation and Hat1 promote S-phase chromatin assembly in vivo. Mol Biol Cell 22, 245-255.
Ellison, V., and Stillman, B. (2003). Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5' recessed
DNA. PLoS Biol 1, E33.
Emili, A. (1998). MEC1-dependent phosphorylation of Rad9p in response to DNA damage. Mol Cell 2, 183-189.
Fillingham, J., Recht, J., Silva, A.C., Suter, B., Emili, A., Stagljar, I., Krogan, N.J., Allis,
C.D., Keogh, M.C., and Greenblatt, J.F. (2008). Chaperone control of the activity and specificity of the histone H3 acetyltransferase Rtt109. Mol Cell Biol 28, 4342-4353.
Ge, Z., Wang, H., and Parthun, M.R. (2011). Nuclear Hat1p Complex (NuB4)
Components Participate in DNA Repair-linked Chromatin Reassembly. J Biol Chem 286,
16790-16799.
Green, E.M., Antczak, A.J., Bailey, A.O., Franco, A.A., Wu, K.J., Yates, J.R., 3rd, and
Kaufman, P.D. (2005). Replication-independent histone deposition by the HIR complex and Asf1. Curr Biol 15, 2044-2049.
Greiner, M., Caesar, S., and Schlenstedt, G. (2004). The histones H2A/H2B and H3/H4 are imported into the yeast nucleus by different mechanisms. Eur J Cell Biol 83, 511-520.
Groth, A., Corpet, A., Cook, A.J., Roche, D., Bartek, J., Lukas, J., and Almouzni, G.
(2007). Regulation of replication fork progression through histone supply and demand.
Science 318, 1928-1931.
107
Groth, A., Ray-Gallet, D., Quivy, J.P., Lukas, J., Bartek, J., and Almouzni, G. (2005).
Human Asf1 regulates the flow of S phase histones during replicational stress. Mol Cell
17, 301-311.
Gunjan, A., and Verreault, A. (2003). A Rad53 kinase-dependent surveillance mechanism that regulates histone protein levels in S. cerevisiae. Cell 115, 537-549.
Haber, J.E. (1995). In vivo biochemistry: physical monitoring of recombination induced by site-specific endonucleases. Bioessays 17, 609-620.
Han, J., Zhou, H., Horazdovsky, B., Zhang, K., Xu, R.M., and Zhang, Z. (2007a). Rtt109 acetylates histone H3 lysine 56 and functions in DNA replication. Science 315, 653-655.
Han, J., Zhou, H., Li, Z., Xu, R.M., and Zhang, Z. (2007b). Acetylation of lysine 56 of histone H3 catalyzed by RTT109 and regulated by ASF1 is required for replisome integrity. J Biol Chem 282, 28587-28596.
Han, J., Zhou, H., Li, Z., Xu, R.M., and Zhang, Z. (2007c). The Rtt109-Vps75 histone acetyltransferase complex acetylates non-nucleosomal histone H3. J Biol Chem 282,
14158-14164.
Harrison, J.C., and Haber, J.E. (2006). Surviving the breakup: the DNA damage checkpoint. Annu Rev Genet 40, 209-235.
Haushalter, K.A., and Kadonaga, J.T. (2003). Chromatin assembly by DNA-translocating motors. Nat Rev Mol Cell Biol 4, 613-620.
Henikoff, S., Furuyama, T., and Ahmad, K. (2004a). Histone variants, nucleosome assembly and epigenetic inheritance. Trends Genet 20, 320-326.
108
Henikoff, S., McKittrick, E., and Ahmad, K. (2004b). Epigenetics, histone H3 variants, and the inheritance of chromatin states. Cold Spring Harb Symp Quant Biol 69, 235-243.
Hopfner, K.P., Craig, L., Moncalian, G., Zinkel, R.A., Usui, T., Owen, B.A., Karcher, A.,
Henderson, B., Bodmer, J.L., McMurray, C.T., et al. (2002). The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair. Nature 418, 562-
566.
Horn, P.J., and Peterson, C.L. (2001). The bromodomain: a regulator of ATP-dependent chromatin remodeling? Front Biosci 6, D1019-1023.
Imhof, A., and Wolffe, A.P. (1999). Purification and properties of the Xenopus Hat1 acetyltransferase: association with the 14-3-3 proteins in the oocyte nucleus.
Biochemistry 38, 13085-13093.
Iwasaki, W., Tachiwana, H., Kawaguchi, K., Shibata, T., Kagawa, W., and Kurumizaka,
H. Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains. Biochemistry 50,
7822-7832.
Jackson, V., Shires, A., Tanphaichitr, N., and Chalkley, R. (1976). Modifications to histones immediately after synthesis. J Mol Biol 104, 471-483.
Jasencakova, Z., Scharf, A.N., Ask, K., Corpet, A., Imhof, A., Almouzni, G., and Groth,
A. (2010). Replication stress interferes with histone recycling and predeposition marking of new histones. Mol Cell 37, 736-743.
Jazayeri, A., McAinsh, A.D., and Jackson, S.P. (2004). Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair. Proc Natl Acad Sci U S A 101, 1644-1649.
109
Kaplan, T., Liu, C.L., Erkmann, J.A., Holik, J., Grunstein, M., Kaufman, P.D., Friedman,
N., and Rando, O.J. (2008). Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast. PLoS Genet 4, e1000270.
Kelly, T.J., Qin, S., Gottschling, D.E., and Parthun, M.R. (2000a). Type B histone acetyltransferase Hat1p participates in telomeric silencing. Mol Cell Biol 20, 7051-7058.
Kelly, T.J., Qin, S., Gottschling, D.E., and Parthun, M.R. (2000b). Type B histone acetyltransferase Hat1p participates in telomeric silencing. Mol Cell Biol 20, 7051-7058.
Keogh, M.C., Kim, J.A., Downey, M., Fillingham, J., Chowdhury, D., Harrison, J.C.,
Onishi, M., Datta, N., Galicia, S., Emili, A., et al. (2006). A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery. Nature 439,
497-501.
Kim, J.A., and Haber, J.E. (2009). Chromatin assembly factors Asf1 and CAF-1 have overlapping roles in deactivating the DNA damage checkpoint when DNA repair is complete. Proc Natl Acad Sci U S A 106, 1151-1156.
Kim, J.A., Kruhlak, M., Dotiwala, F., Nussenzweig, A., and Haber, J.E. (2007).
Heterochromatin is refractory to gamma-H2AX modification in yeast and mammals. J
Cell Biol 178, 209-218.
Kleff, S., Andrulis, E.D., Anderson, C.W., and Sternglanz, R. (1995). Identification of a gene encoding a yeast histone H4 acetyltransferase. J Biol Chem 270, 24674-24677.
Kobayashi, J., Tauchi, H., Sakamoto, S., Nakamura, A., Morishima, K., Matsuura, S.,
Kobayashi, T., Tamai, K., Tanimoto, K., and Komatsu, K. (2002). NBS1 localizes to
110 gamma-H2AX foci through interaction with the FHA/BRCT domain. Curr Biol 12, 1846-
1851.
Korber, P., Barbaric, S., Luckenbach, T., Schmid, A., Schermer, U.J., Blaschke, D., and
Horz, W. (2006). The histone chaperone Asf1 increases the rate of histone eviction at the yeast PHO5 and PHO8 promoters. J Biol Chem 281, 5539-5545.
Kornberg, R.D. (1974). Chromatin structure: a repeating unit of histones and DNA.
Science 184, 868-871.
Kornberg, R.D., and Lorch, Y. (1999). Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome. Cell 98, 285-294.
Kouzarides, T. (2007). Chromatin modifications and their function. Cell 128, 693-705.
Krogh, B.O., and Symington, L.S. (2004). Recombination proteins in yeast. Annu Rev
Genet 38, 233-271.
Kuo, M.H., Brownell, J.E., Sobel, R.E., Ranalli, T.A., Cook, R.G., Edmondson, D.G.,
Roth, S.Y., and Allis, C.D. (1996). Transcription-linked acetylation by Gcn5p of histones
H3 and H4 at specific lysines. Nature 383, 269-272.
Lee, D.Y., Hayes, J.J., Pruss, D., and Wolffe, A.P. (1993). A positive role for histone acetylation in transcription factor access to nucleosomal DNA. Cell 72, 73-84.
Lee, H.S., Park, J.H., Kim, S.J., Kwon, S.J., and Kwon, J. (2010). A cooperative activation loop among SWI/SNF, gamma-H2AX and H3 acetylation for DNA double- strand break repair. EMBO J 29, 1434-1445.
111
Lee, S.E., Moore, J.K., Holmes, A., Umezu, K., Kolodner, R.D., and Haber, J.E. (1998).
Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94, 399-409.
Leroy, C., Lee, S.E., Vaze, M.B., Ochsenbein, F., Guerois, R., Haber, J.E., and
Marsolier-Kergoat, M.C. (2003). PP2C phosphatases Ptc2 and Ptc3 are required for DNA checkpoint inactivation after a double-strand break. Mol Cell 11, 827-835.
Lewis, P.W., Elsaesser, S.J., Noh, K.M., Stadler, S.C., and Allis, C.D. (2010). Daxx is an
H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres. Proc Natl Acad Sci U S A 107, 14075-14080.
Li, Q., Zhou, H., Wurtele, H., Davies, B., Horazdovsky, B., Verreault, A., and Zhang, Z.
(2008). Acetylation of histone H3 lysine 56 regulates replication-coupled nucleosome assembly. Cell 134, 244-255.
Liang, B., Qiu, J., Ratnakumar, K., and Laurent, B.C. (2007). RSC functions as an early double-strand-break sensor in the cell's response to DNA damage. Curr Biol 17, 1432-
1437.
Lisby, M., Barlow, J.H., Burgess, R.C., and Rothstein, R. (2004). Choreography of the
DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell 118, 699-713.
Loyola, A., Bonaldi, T., Roche, D., Imhof, A., and Almouzni, G. (2006). PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state. Mol Cell 24,
309-316.
112
Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J. (1997).
Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251-260.
Lusser, A., Eberharter, A., Loidl, A., Goralik-Schramel, M., Horngacher, M., Haas, H., and Loidl, P. (1999). Analysis of the histone acetyltransferase B complex of maize embryos. Nucleic Acids Res 27, 4427-4435.
Ma, X.J., Wu, J., Altheim, B.A., Schultz, M.C., and Grunstein, M. (1998). Deposition- related sites K5/K12 in histone H4 are not required for nucleosome deposition in yeast.
Proc Natl Acad Sci U S A 95, 6693-6698.
Maas, N.L., Miller, K.M., DeFazio, L.G., and Toczyski, D.P. (2006). Cell cycle and checkpoint regulation of histone H3 K56 acetylation by Hst3 and Hst4. Mol Cell 23, 109-
119.
Mann, R.K., and Grunstein, M. (1992). Histone H3 N-terminal mutations allow hyperactivation of the yeast GAL1 gene in vivo. Embo J 11, 3297-3306.
Masumoto, H., Hawke, D., Kobayashi, R., and Verreault, A. (2005). A role for cell- cycle-regulated histone H3 lysine 56 acetylation in the DNA damage response. Nature
436, 294-298.
Megee, P.C., Morgan, B.A., Mittman, B.A., and Smith, M.M. (1990). Genetic analysis of histone H4: essential role of lysines subject to reversible acetylation. Science 247, 841-
845.
Megee, P.C., Morgan, B.A., and Smith, M.M. (1995). Histone H4 and the maintenance of genome integrity. Genes Dev 9, 1716-1727.
113
Meneghini, M.D., Wu, M., and Madhani, H.D. (2003). Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of silent heterochromatin. Cell 112, 725-
736.
Mersfelder, E.L., and Parthun, M.R. (2006). The tale beyond the tail: histone core domain modifications and the regulation of chromatin structure. Nucleic Acids Res 34, 2653-
2662.
Miyazaki, T., Bressan, D.A., Shinohara, M., Haber, J.E., and Shinohara, A. (2004). In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair. EMBO J 23, 939-949.
Moreau, S., Morgan, E.A., and Symington, L.S. (2001). Overlapping functions of the
Saccharomyces cerevisiae Mre11, Exo1 and Rad27 nucleases in DNA metabolism.
Genetics 159, 1423-1433.
Mosammaparast, N., Guo, Y., Shabanowitz, J., Hunt, D.F., and Pemberton, L.F. (2002).
Pathways mediating the nuclear import of histones H3 and H4 in yeast. J Biol Chem 277,
862-868.
Nair, D.M., Ge, Z., Mersfelder, E.L., and Parthun, M.R. (2011). Genetic interactions between POB3 and the acetylation of newly synthesized histones. Curr Genet 57, 271-
286.
Nakada, D., Hirano, Y., and Sugimoto, K. (2004). Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway. Mol Cell Biol 24,
10016-10025.
114
Nakada, D., Matsumoto, K., and Sugimoto, K. (2003). ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism. Genes Dev 17, 1957-1962.
New, J.H., Sugiyama, T., Zaitseva, E., and Kowalczykowski, S.C. (1998). Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A. Nature 391, 407-
410.
O'Neill, B.M., Szyjka, S.J., Lis, E.T., Bailey, A.O., Yates, J.R., 3rd, Aparicio, O.M., and
Romesberg, F.E. (2007). Pph3-Psy2 is a phosphatase complex required for Rad53 dephosphorylation and replication fork restart during recovery from DNA damage. Proc
Natl Acad Sci U S A 104, 9290-9295.
Osley, M.A., and Shen, X. (2006). Altering nucleosomes during DNA double-strand break repair in yeast. Trends Genet 22, 671-677.
Ozdemir, A., Spicuglia, S., Lasonder, E., Vermeulen, M., Campsteijn, C., Stunnenberg,
H.G., and Logie, C. (2005). Characterization of lysine 56 of histone H3 as an acetylation site in Saccharomyces cerevisiae. J Biol Chem 280, 25949-25952.
Paciotti, V., Clerici, M., Lucchini, G., and Longhese, M.P. (2000). The checkpoint protein Ddc2, functionally related to S. pombe Rad26, interacts with Mec1 and is regulated by Mec1-dependent phosphorylation in budding yeast. Genes Dev 14, 2046-
2059.
Paques, F., and Haber, J.E. (1999). Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 63, 349-404.
Park, E.C., and Szostak, J.W. (1990). Point mutations in the yeast histone H4 gene prevent silencing of the silent mating type locus HML. Mol Cell Biol 10, 4932-4934.
115
Park, Y.J., and Luger, K. (2006). The structure of nucleosome assembly protein 1. Proc
Natl Acad Sci U S A 103, 1248-1253.
Parthun, M.R., Widom, J., and Gottschling, D.E. (1996). The major cytoplasmic histone acetyltransferase in yeast: links to chromatin replication and histone metabolism. Cell 87,
85-94.
Pellicioli, A., Lee, S.E., Lucca, C., Foiani, M., and Haber, J.E. (2001). Regulation of
Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced
G2/M arrest. Mol Cell 7, 293-300.
Peterson, C.L., and Laniel, M.A. (2004). Histones and histone modifications. Curr Biol
14, R546-551.
Poveda, A., Pamblanco, M., Tafrov, S., Tordera, V., Sternglanz, R., and Sendra, R.
(2004). Hif1 is a component of yeast histone acetyltransferase B, a complex mainly localized in the nucleus. J Biol Chem 279, 16033-16043.
Qin, S., and Parthun, M.R. (2002). Histone H3 and the histone acetyltransferase Hat1p contribute to DNA double-strand break repair. Mol Cell Biol 22, 8353-8365.
Qin, S., and Parthun, M.R. (2006). Recruitment of the type B histone acetyltransferase
Hat1p to chromatin is linked to DNA double-strand breaks. Mol Cell Biol 26, 3649-3658.
Ray-Gallet, D., Quivy, J.P., Scamps, C., Martini, E.M., Lipinski, M., and Almouzni, G.
(2002). HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis. Mol Cell 9, 1091-1100.
Recht, J., Tsubota, T., Tanny, J.C., Diaz, R.L., Berger, J.M., Zhang, X., Garcia, B.A.,
Shabanowitz, J., Burlingame, A.L., Hunt, D.F., et al. (2006). Histone chaperone Asf1 is
116 required for histone H3 lysine 56 acetylation, a modification associated with S phase in mitosis and meiosis. Proc Natl Acad Sci U S A 103, 6988-6993.
Rouse, J., and Jackson, S.P. (2000). LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae.
EMBO J 19, 5801-5812.
Rufiange, A., Jacques, P.E., Bhat, W., Robert, F., and Nourani, A. (2007). Genome-wide replication-independent histone H3 exchange occurs predominantly at promoters and implicates H3 K56 acetylation and Asf1. Mol Cell 27, 393-405.
Ruiz-Carrillo, A., Wangh, L.J., and Allfrey, V.G. (1975). Processing of newly synthesized histone molecules. Science 190, 117-128.
Ruiz-Garcia, A.B., Sendra, R., Galiana, M., Pamblanco, M., Perez-Ortin, J.E., and
Tordera, V. (1998). HAT1 and HAT2 proteins are components of a yeast nuclear histone acetyltransferase enzyme specific for free histone H4. J Biol Chem 273, 12599-12605.
Santisteban, M.S., Kalashnikova, T., and Smith, M.M. (2000). Histone H2A.Z regulats transcription and is partially redundant with nucleosome remodeling complexes. Cell 103,
411-422.
Schneider, J., Bajwa, P., Johnson, F.C., Bhaumik, S.R., and Shilatifard, A. (2006). Rtt109 is required for proper H3K56 acetylation: a chromatin mark associated with the elongating RNA polymerase II. J Biol Chem 281, 37270-37274.
Schwartz, M.F., Duong, J.K., Sun, Z., Morrow, J.S., Pradhan, D., and Stern, D.F. (2002).
Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint. Mol Cell 9, 1055-1065.
117
Selth, L., and Svejstrup, J.Q. (2007). Vps75, a new yeast member of the NAP histone chaperone family. J Biol Chem 282, 12358-12362.
Shroff, R., Arbel-Eden, A., Pilch, D., Ira, G., Bonner, W.M., Petrini, J.H., Haber, J.E., and Lichten, M. (2004). Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break. Curr Biol 14, 1703-1711.
Sirbu, B.M., Couch, F.B., and Cortez, D. (2012). Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA. Nat Protoc 7, 594-605.
Smith, S., and Stillman, B. (1989). Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro. Cell 58, 15-
25.
Sobel, R.E., Cook, R.G., Perry, C.A., Annunziato, A.T., and Allis, C.D. (1995).
Conservation of deposition-related acetylation sites in newly synthesized histones H3 and
H4. Proc Natl Acad Sci U S A 92, 1237-1241.
Strom, L., Lindroos, H.B., Shirahige, K., and Sjogren, C. (2004). Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair. Mol Cell 16,
1003-1015.
Sugawara, N., Wang, X., and Haber, J.E. (2003). In vivo roles of Rad52, Rad54, and
Rad55 proteins in Rad51-mediated recombination. Mol Cell 12, 209-219.
Sung, P. (1997). Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. Genes
Dev 11, 1111-1121.
118
Suter, B., Pogoutse, O., Guo, X., Krogan, N., Lewis, P., Greenblatt, J.F., Rine, J., and
Emili, A. (2007). Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p. BMC Biol 5, 38.
Tagami, H., Ray-Gallet, D., Almouzni, G., and Nakatani, Y. (2004). Histone H3.1 and
H3.3 complexes mediate nucleosome assembly pathways dependent or independent of
DNA synthesis. Cell 116, 51-61.
Tamburini, B.A., and Tyler, J.K. (2005). Localized histone acetylation and deacetylation triggered by the homologous recombination pathway of double-strand DNA repair. Mol
Cell Biol 25, 4903-4913.
Toh, G.W., O'Shaughnessy, A.M., Jimeno, S., Dobbie, I.M., Grenon, M., Maffini, S.,
O'Rorke, A., and Lowndes, N.F. (2006). Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation. DNA Repair (Amst) 5, 693-703.
Tsubota, T., Berndsen, C.E., Erkmann, J.A., Smith, C.L., Yang, L., Freitas, M.A., Denu,
J.M., and Kaufman, P.D. (2007). Histone H3-K56 acetylation is catalyzed by histone chaperone-dependent complexes. Mol Cell 25, 703-712.
Tsukuda, T., Fleming, A.B., Nickoloff, J.A., and Osley, M.A. (2005). Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae. Nature 438,
379-383.
Tsukuda, T., Trujillo, K.M., Martini, E., and Osley, M.A. (2009). Analysis of chromatin remodeling during formation of a DNA double-strand break at the yeast mating type locus. Methods 48, 40-45.
119
Tyler, J.K. (2002). Chromatin assembly. Cooperation between histone chaperones and
ATP-dependent nucleosome remodeling machines. Eur J Biochem 269, 2268-2274.
Tyler, J.K., Collins, K.A., Prasad-Sinha, J., Amiott, E., Bulger, M., Harte, P.J.,
Kobayashi, R., and Kadonaga, J.T. (2001). Interaction between the Drosophila CAF-1 and ASF1 chromatin assembly factors. Mol Cell Biol 21, 6574-6584.
Unal, E., Arbel-Eden, A., Sattler, U., Shroff, R., Lichten, M., Haber, J.E., and Koshland,
D. (2004). DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain. Mol Cell 16, 991-1002.
Utley, R.T., Lacoste, N., Jobin-Robitaille, O., Allard, S., and Cote, J. (2005). Regulation of NuA4 histone acetyltransferase activity in transcription and DNA repair by phosphorylation of histone H4. Mol Cell Biol 25, 8179-8190.
VanDemark, A.P., Blanksma, M., Ferris, E., Heroux, A., Hill, C.P., and Formosa, T.
(2006). The structure of the yFACT Pob3-M domain, its interaction with the DNA replication factor RPA, and a potential role in nucleosome deposition. Mol Cell 22, 363-
374.
Verreault, A., Kaufman, P.D., Kobayashi, R., and Stillman, B. (1998). Nucleosomal
DNA regulates the core-histone-binding subunit of the human Hat1 acetyltransferase.
Curr Biol 8, 96-108.
Verzijlbergen, K.F., van Welsem, T., Sie, D., Lenstra, T.L., Turner, D.J., Holstege, F.C.,
Kerkhoven, R.M., and van Leeuwen, F. (2011). A barcode screen for epigenetic regulators reveals a role for the NuB4/HAT-B histone acetyltransferase complex in histone turnover. PLoS Genet 7, e1002284.
120
Wakayama, T., Kondo, T., Ando, S., Matsumoto, K., and Sugimoto, K. (2001). Pie1, a protein interacting with Mec1, controls cell growth and checkpoint responses in
Saccharomyces cerevisiae. Mol Cell Biol 21, 755-764.
Ward, I.M., Minn, K., Jorda, K.G., and Chen, J. (2003). Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX. J
Biol Chem 278, 19579-19582.
Widom, J. (1998). Chromatin structure: linking structure to function with histone H1.
Curr Biol 8, R788-791.
Wiltzius, J.J., Hohl, M., Fleming, J.C., and Petrini, J.H. (2005). The Rad50 hook domain is a critical determinant of Mre11 complex functions. Nat Struct Mol Biol 12, 403-407.
Xie, W., Song, C., Young, N.L., Sperling, A.S., Xu, F., Sridharan, R., Conway, A.E.,
Garcia, B.A., Plath, K., Clark, A.T., et al. (2009). Histone h3 lysine 56 acetylation is linked to the core transcriptional network in human embryonic stem cells. Mol Cell 33,
417-427.
Xu, F., Zhang, K., and Grunstein, M. (2005). Acetylation in histone H3 globular domain regulates gene expression in yeast. Cell 121, 375-385.
Yan, Q., Dutt, S., Xu, R., Graves, K., Juszczynski, P., Manis, J.P., and Shipp, M.A.
(2009). BBAP monoubiquitylates histone H4 at lysine 91 and selectively modulates the
DNA damage response. Mol Cell 36, 110-120.
Yang, X., Yu, W., Shi, L., Sun, L., Liang, J., Yi, X., Li, Q., Zhang, Y., Yang, F., Han, X., et al. (2011). HAT4, a Golgi apparatus-anchored B-type histone acetyltransferase, acetylates free histone H4 and facilitates chromatin assembly. Mol Cell 44, 39-50.
121
Ye, J., Ai, X., Eugeni, E.E., Zhang, L., Carpenter, L.R., Jelinek, M.A., Freitas, M.A., and
Parthun, M.R. (2005). Histone H4 lysine 91 acetylation a core domain modification associated with chromatin assembly. Mol Cell 18, 123-130.
Yuan, J., Pu, M., Zhang, Z., and Lou, Z. (2009). Histone H3-K56 acetylation is important for genomic stability in mammals. Cell Cycle 8, 1747-1753.
Zhang, L., Eugeni, E.E., Parthun, M.R., and Freitas, M.A. (2003). Identification of novel histone post-translational modifications by peptide mass fingerprinting. Chromosoma 112,
77-86.
Zhang, W., Bone, J.R., Edmondson, D.G., Turner, B.M., and Roth, S.Y. (1998). Essential and redundant functions of histone acetylation revealed by mutation of target lysines and loss of the Gcn5p acetyltransferase. Embo J 17, 3155-3167.
Zhang, Y., Hefferin, M.L., Chen, L., Shim, E.Y., Tseng, H.M., Kwon, Y., Sung, P., Lee,
S.E., and Tomkinson, A.E. (2007). Role of Dnl4-Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination. Nat Struct Mol
Biol 14, 639-646.
Zhao, X., and Rothstein, R. (2002). The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1. Proc Natl Acad Sci U S A 99,
3746-3751.
Zhou, H., Madden, B.J., Muddiman, D.C., and Zhang, Z. (2006). Chromatin assembly factor 1 interacts with histone h3 methylated at lysine 79 in the processes of epigenetic silencing and DNA repair. Biochemistry 45, 2852-2861.
122