Characterization of N1/N2 Family Histone Chaperones: Hif1p and NASP

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy

in the Graduate School of The Ohio State University

Huanyu Wang

Graduate Program in Ohio State Biochemistry Program

The Ohio State University

2010

Dissertation Committee:

Professor Mark Parthun, adviser

Professor Kurt Fredrick

Professor Paul Herman

Professor Jiyan Ma

Huanyu Wang

2010

Abstract

Recent studies revealed that a protein known as Hat1p Interacting Factor-1 (1) forms a complex with Hat1p/Hat2p in the nucleus and functions as a histone chaperone during chromatin assembly. This protein is a yeast homolog of the N1/N2 histone chaperone, which functions in both the storage and assembly of histone H3/H4 tetramers during the rapid rounds of DNA replication which occurs early in X. laevis embryogenesis. Hif1p functions as a chromatin assembly factor in vitro and associates with acetylated histone

H4 in vivo in a Hat1p/Hat2p dependent manner. These findings demonstrated a physical connection between type B HATs and factors directly involved in the process of chromatin assembly. We have performed biochemical experiments to further characterize this protein. By using chromatographic techniques, we demonstrated that Hif1p forms complexes in both Hat1p/Hat2/-dependent and Hat1p/Hat2p-independent manners. We have developed a method combining both conventional and affinity chromatography to isolate and identify proteins associate with Hif1p. Our results also suggested a link between Hif1p and a H3-specific type B HAT.

The human homolog of Hif1p, NASP (2), has been reported to be an H1-specific histone chaperone when all the other members of N1/N2 family are H3/H4-specific histone chaperones. To resolve this paradox, we have performed a detailed and quantitative analysis of the binding specificity of human NASP. Our results confirmed that NASP ii can interact with histone H1 and that this interaction occurs with high affinity. In addition, multiple in vitro and in vivo experiments, including native gel electrophoresis, traditional and affinity chromatography assays and surface plasmon resonance, all indicated that NASP also forms distinct, high specificity complexes with histones H3 and

H4. The interaction between NASP and histones H3 and H4 is functional as NASP is active in in vitro chromatin assembly assays using histone substrates depleted of H1.

We have also further characterized this protein in detail by directly mapping domains that are involved in interactions with each histone in vitro and developing a cell culture model to understand how the association with specific histones contributes to the cellular function of NASP in vivo. We identified two distinct domains that separately interact with linker histone or core histone through different mechanisms. We demonstrated that loss of native NASP increased the sensitivity of chromatin to digestion with micrococcal nuclease, therefore, identified NASP as a significant contributor to global chromatin structure.

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Dedicated to my husband Peng and my parents

Acknowledgments

I would like to express my deepest gratitude to my advisor, Dr. Mark R. Parthun for his constant support, endless encouragement and unfailingful guidance. I want to thank Mark for teaching me how to solve problems, leading me through difficult times during these years and providing me a supportive environment to grow as a scientist. His keen insight and thoughtful reflection on science have inspired me and greatly profited me.

I would also like to thank the members of my dissertation committee, Dr. Jiyan Ma, Dr.

Paul Herman, and Dr. Kurt Fredrick for critical reviews of the thesis, mentoring, encouragement and scientific input. They have provided excellent guidance not only with respect to my dissertation work, but with my future career as well.

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, Zhongqi Ge,

Prabakaran Nagarajan, Rajbir singh and Neha Rastogi. In particular, I would like to express my appreciation to Amy, Xi, Jianxin, Song and Erica, who had taught me all the techniques and helped me with my scientific questions with great patience. More importantly, their spirits of hard working and sharing have benefited me and made my life in the lab a wonderful experience. I am very grateful to Amy for making beautiful relaxed DNA and generously sharing it with me. I thank Zhongqi for her intelligent thoughts and valuable suggestions that inspired me a lot with my research. I am grateful v to Prabakaran and Neha for immunofluorescent microscopy techniques and SILAC techniques.

In addition, I thank members of the Bell Lab, Ma Lab, Rafael Lab, Scheonberg lab,

Burghes Lab and Kolb Lab for their advice, communications and suggestions.

I also thank my friends, Min Li, Wenpeng Zhang, Xudong Zhang, Zhongqi Ge, Jun

Wang’s family, Hongtao Zhang’s family, Lei Zhang’s family and Junwen Yu’s family for all the happy time we have shared together.

Finally, I am deeply indebted to my family members for their unconditional support and understanding. I thank to my mother who went all the way to give me a loving and supportive environment, especially after my father passed away 13 years ago. This work can’t be separated from her love and encouragement. I also thank to my father who had loved me and trusted me the most and raised me to become who I am. His encouragement and blessing is the one of the important motivations I pursue in this field. I would give up anything for him to be here with me. Peng Sun, my loving husband, is the most amazing person in my life. He tried his best to give me a comfortable and worries-free life and supported me every step along the way. He has believed in me and been proud of me all the time. Without his love, I never would have seen this through.

Vita

Jan 26, 1980 ...... Born-Beijing, China PR

Sep 1992 to July 1998...... Beijing No. 8 Middle school

Sep 1998 to Jun 2003...... B.S. BMS, Peking University

March 2004 to present ...... Graduate Research Associate, OSBP, The

Ohio State University

Publications

1. Wang, H., Walsh, S. T., and Parthun, M. R. (2008) Expanded binding specificity

of the human histone chaperone NASP. Nucleic Acids Res 36, 5763-5772

2. McGarry, KG., Walker, SE., Wang, H., and Fredrick, K. (2005) Destabilization of

the P site codon-anticodon helix results from movement of tRNA hybrid state

within the ribosome. Molecular cell 20 613-622

3. Ge, Z., Wang, H., and Parthun, M.R. (2010) Role of nuclear Hat1p complex

(NuB4) components in DNA repair-linked chromatin reassembly. In preparation.

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Fields of Study

Major Field: Ohio State Biochemistry Program

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Table of Contents

Abstract...... ii

Dedicated to my husband Peng and my parents...... iv

Acknowledgments ...... v

Vita ...... vii

List of Tables ...... xiii

List of Figures...... xiv

Chapter 1: Introduction ...... 1 1.1 Histones...... 2 1.2 Chromatin assembly ...... 4 1.3 Nucleosome Assembly Related Histone Acetylation...... 5 1.4 Histone chaperones ...... 9 1.4.2 Chromatin Assembly Factor 1 (CAF-1) ...... 9

1.4.3 Histone regulation proteins (Hir/HirA)...... 11

1.4.4 Asf1...... 11

1.5 N1/N2 family of histone chaperones...... 13 1.5.1 N1/N2 protein ...... 13

1.5.2 Hif1p ...... 14

1.5.3 NASP-1...... 15

1.5.4 Sim3 ...... 15

1.5.5 NASP ...... 16

Chapter 2. Isolation and identification of proteins that associate with Hif1p...... 19 ix

2.1 Abstract...... 19 2.2 Introduction...... 19 2.3 Materials and methods ...... 20 2.3.1 Yeast strain...... 20

2.3.2 Whole cell extracts preparation ...... 21

2.3.3 Column chromatography ...... 21

2.3.4 Western blotting...... 22

2.3.5 HAT activity assays ...... 22

2.3.6 Immunoprecipitation of Hif1p ...... 23

2.4 Results ...... 23 2.4.1 Hif1p associates with other proteins in one or more high molecular weight

complexes ...... 23

2.4.2. Gcn1p may physically interact with Hif1p...... 25

2.4.3 The majority of GCN1p in cells is not associated with Hif1p...... 26

2.4.5 Chd1p is not stably interacting with Hif1p in cells...... 27

2.4.6. Hif1p influences a histone H3 specific HAT activity...... 27

2.5 Discussion ...... 28 2.5.1 Hif1p interacts with proteins other than Hat1p/Hat2p...... 28

2.5.2 Histone H3 HATs and Hif1p ...... 30

Chapter 3 Expanded binding specificity of the human histone chaperone NASP.... 38 3.1 Abstract...... 38 3.2 Introduction...... 39 3.3 Materials and methods ...... 41 3.3.1 Expression and purification of recombinant sNASP ...... 41

3.3.2 Histone isolation ...... 42

3.3.3 Native gel electrophoresis...... 42

3.3.4 Chromatography-based histone binding assays...... 42

3.3.5 Biosensor Analysis...... 43

3.3.6 Immunoprecipitaion of NASP ...... 44

3.3.7 In vitro chromatin assembly...... 45

3.4 Results ...... 45 3.4.1 Multiple sNASP/histone complexes identified by native gel electrophoresis. 46

3.4.2 sNASP forms a stable complex with histones H3 and H4...... 48

3.4.3 sNASP specifically interacts with histones H3 and H4...... 49

3.4.4 sNASP interacts primarily with histone H3...... 51

3.4.5 Biosensor analysis of NASP binding to histones H1 and H3/H4 ...... 51

3.4.6 In vivo interaction of sNASP and histones...... 53

3.4.7 sNASP participates in the deposition of core histones ...... 54

3.5 Discussion ...... 55

Chapter 4 Structure and functional characterization of NASP ...... 68 4.1 Abstract...... 68 4.2 Introduction...... 68 4.3 Materials and methods ...... 70 4.3.1 Plasmid DNA construction ...... 70

4.3.2 Protein expression and purification ...... 71

4.3.3 in vitro chromatin assembly...... 72

4.3.4 in vitro histone binding assays...... 72

4.3.5 Cell culture...... 73 xi

4.3.6 Plasmid Transfection ...... 73

4.3.7 Whole cell extracts preparation ...... 73

4.3.8 in vivo protein pull down experiment...... 73

4.3.9 siRNA transfection...... 74

4.3.10 MNase Digestion ...... 74

4.4 Results ...... 75 4.4.1 Domain analysis of sNASP...... 75

4.4.2 Functional dissection of sNASP...... 75

4.4.3 Construction of inducible NASP cell lines ...... 78

4.4.4 in vivo characterization of binding specificity of full-length sNASP and its

mutants...... 79

4.4.5 Silencing of NASP by RNAi and tet-induction of exogenous sNASP...... 80

4.4.6 sNASP is required to form a regular chromatin structure...... 81

4.5 Discussion ...... 82 4.5.1 What we have learned from in vitro data...... 83

4.5.2 Can the in vitro and in vivo data be reconciled?...... 85

4.5.3 NASP is a significant contributor to global chromatin structure...... 87

Bibliography ...... 98

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List of Tables

Table 3. 1 SPR binding kinetics and affinities for the interactions of NASP with H1 and

H3/H4 tetramera ...... 58

Table 4. 1 SPR binding constants of NASP variants to histone H1 and H3/H4 tetramer 89

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List of Figures

Figure 2. 1 Schematic diagram of the purification scheme used for the Hif1p containing complex...... 32 Figure 2. 2 Presence of Hif1p in a high molecular weight complex independent of HAT1 and HAT2...... 33 Figure 2. 3 Purification of native Hif1p...... 34 Figure 2. 4 The majority of GCN1p in cells is not associated with Hif1p ...... 35 Figure 2. 5 Chd1p is not stably interacting with Hif1p in cells...... 36 Figure 2. 6 Deletion of HIF1 results in significant decrease of a H3-specific HAT activity...... 37 Figure 3. 1 Native gel electrophoresis of sNASP:histone complexes...... 59 Figure 3. 2 NASP forms a stable complex with histones H3 and H4...... 60 Figure 3. 3 orrelation between sNASP:H3/H4 complexes detected by gel filtration chromatography and native gel electrophoresis...... 61 Figure 3. 4 Detection of sNASP:H3/H4 complex formation by affinity chromatography...... 62 Figure 3. 5 Recombinant sNASP primarily interacts with histone H3...... 64 Figure 3. 6 Quantitation of the interactions between sNASP and histones H1 and H3/H4 by SPR ...... 65 Figure 3. 7 NASP interacts with both histone H1 and histone H3 in vivo...... 66 Figure 3. 8 Recombinant sNASP can assemble core histones into chromatin...... 67 Figure 4. 1 Recombinant NASP proteins...... 90 Figure 4. 2 in vitro binding of sNASP variants to core histones...... 91 Figure 4. 3 Nucleosome assembly activity of sNASP variants...... 92 Figure 4. 4 Generation of inducible sNASP-expression U2OS cell lines...... 93 Figure 4. 5 Binding spectrums of sNASP and its mutants in vivo...... 94 xiv

Figure 4. 6 siRNA knockdown and inducible re-expression of sNASP...... 95 Figure 4. 7 sNASP influennces global chromatin structure...... 96 Figure 4. 8 Expression of sNASP mutants can not fully reverse the defects caused by depletion of NASP...... 97

Chapter 1: Introduction

Eukaryotic genomic DNA is packaged into a highly condensed and regulated nucleoprotein structure known as chromatin(4). The principal repeating element of chromatin is the nucleosome in which 146 base pairs of DNA wraps ~1.7 superhelical turns around an octamer containing two copies of each of the core histone proteins: H2A,

H2B, H3 and H4(5). The nucleosome is held together through protein-protein and protein-DNA interactions(6). Linker histone H1 interacts with the DNA sequences between individual nucleosomes and establishes a higher order of compaction and regulation.

Chromatin structure plays an important role in all DNA-dependent cellular processes including DNA replication, transcription, DNA repair and recombination. Therefore, the assembly of eukaryotic DNA is essential not only for the proper and faithful propagation of information contained in the chromosome, but also for normal cellular functions.

There are three mechanisms identified to adjust the nucleosome structure and subsequent cellular processes. First, a wide range of core histone post-translational modifications, including acetylation, ubiquitination, phosphorylation, methylation, sumoylation and ribosylation, affect histone interactions with non-histone proteins as well as histone- histone and histone-DNA interactions. Second, ATP-dependent chromatin remolding complexes can disrupt and remodel the nucleosome structure which increases DNA 1 accessibility(7). Lastly, the composition of nucleosomes can be altered by substituting the major histones with specialized variants (8-11).

1.1 Histones

Histones are the main structural proteins associated with DNA in eukaryotic cells. They are divided into two groups: the core or nucleosomal histones and the linker histones.

Core histones, including histone H2A, H2B, H3 and H4, are small highly basic proteins with molecular weights between 11 and 16 kDa. More than 20% of their amino acid composition consists of lysine and arginine residues. Two of each core histone forms the histone octamer of nucleosome core particle. The core histones have 3 distinct domains: the histone fold domain, formed by three α-helices connected by two loops, is involved in histone/histone and histone/DNA interactions, the relatively unstructured and highly charged N-terminal tail domain, which projects out from the nucleosome into the surrounding environment, is the place for extensive covalent post-translational modifications and a short C-terminal domain that differs in length between the different histone classes.

The synthesis of core histones is highly regulated and balanced. In proliferating cells, the majority of histone synthesis happens during S-phase and is coupled to DNA synthesis to ensure the assembly of chromatin on the newly replicated DNA. Besides the S-phase related histones, minor forms of histones are synthesized outside of S phase. These histone variants, called replacement histones such as H3.3, H2A.X and H2A.Z are generally constitutively synthesized at low levels throughout the entire cell cycle and also

2 in non-proliferating cells during differentiation or quiescence. These variants are deposited onto DNA independent of DNA synthesis and are known to contribute to distinct nucleosomal architectures and play critical roles in the regulation of nuclear functions.

The H1 family of linker histones is the most divergent class of histone proteins. In mammals, H1.1 and H1.5 are expressed in somatic cells during the S phase of the cell cycle. Two others H1.0 and H1x have variant expression pattern in somatic cells and four histone H1 genes are expressed in germ cells(12). Linker histone H1 associates with linker DNA that connects the core particles and seals two rounds of DNA at its entry/exit site on the surface of the nucleosome core(13). Histone H1 contains a highly conserved central globular domain which interacts with DNA at the exit or entry end of the nucleosomal core, a short N-terminal domain that is enriched in basic amino acids and an extended C-terminal domain that is enriched in lysines, serines and prolines. This domain has a major impact on the linker DNA conformation and chromatin condensation(14).

Linker histones are involved in chromatin condensation and limiting the access for regulatory proteins to nucleosomal components(15,16) therefore regulates higher order chromatin structures and modulates the cellular processes that require nucleosome accessibility.

1.2 Chromatin assembly

Chromatin assembly refers to the process in which histone H3/H4 tetramer and H2A/H2B dimers are deposited sequentially onto DNA to form periodic arrays of nucleosomes

(17,18). There now appears to be two fundamental types of chromatin assembly.

The first type is known as replication-coupled chromatin assembly that happens immediately after DNA replication or DNA repair during the cell cycle. Two fundamentally distinct processes affect this basic chromatin structure: First, after pre- existing nucleosomes are transiently disrupted during the passage of the replication fork, parental histones are recycled onto nascent DNA, which only provides half of the histones required. Second, the deposition of newly synthesized histone through a pathway known as replication-dependent de novo nucleosome deposition provides the other half of histones to fulfill the requirement for nucleosome assembly on the two daughter strands.

There is no particular preference for leading and lagging DNA strand when assembly of either parental or newly synthesized histones occurs. The replication-dependent de novo nucleosome deposition starts from synthesis of core histones in the cytoplasm where they form in heterodimeric complexes as H3/H4 dimers and H2A/H2B dimers. These complexes are recognized by specific chaperones and karyopherins that shuttle them into the nucleus(19). Once in the nucleus, core histones are deposited onto the DNA in a specific order with histones H3 and H4 deposited first followed by H2A and H2B. The deposition of H3/H4 dimers is mediated by a three-subunit chromatin assembly factor

(CAF-1) complex, which brings newly synthesized H3/H4 complexes to the sites of DNA replication by directly interacting with proliferating cell nuclear antigen (PCNA) (20,21). 4

Additional histone chaperones NAP-1 and FACT are required for the deposition of

H2A/H2B dimers(22-24). Linker H1 is the last histone that is deposited after DNA replication. In mammalian cells, NASP is the only chaperone that has been reported as linker histone chaperone that is capable of assembling H1 onto DNA(25) while in yeast,

NAP-1 has been reported assemble H1 after DNA replication. After newly formed nucleosomes are in place, ATP-dependent chromatin remodeling factors, such as ACF and RSF, are required to generate the regular spacing of nucleosomes that is characteristic of native chromatin.

The second type of chromatin assembly occurs outside of S phase or in non-dividing cells known as replication-independent chromatin assembly(26-28). Histone variants, which are thought to be involved in this event (26,29), appear to be concentrated in regions of active transcription by incorporating in to these regions. For instance, the assembly of histone variant H3.3 is mediated by the HIR family histone chaperones in a manner that is analogous to the role of CAF-1 in replication-dependent assembly. Histone variant

H2A.Z is loaded by the ATP-dependent chromatin remodeling complex SWR1(30-32).

1.3 Nucleosome Assembly Related Histone Acetylation

Core histone proteins are highly conserved throughout eukaryotes. The NH2-terminal tails of histones are flexible and protrude outward from the nucleosome(33). Histones undergo a variety of post-translational modifications, including acetylation of lysine residues, methylation of lysine and arginine residues, phosphorylation of serine and threonine residues, and ubiquitination and sumolation of lysine residues (33-35). Most of

5 the modifications occur at the NH2-teminal tail domains of histones and have been shown to be involved in the regulation of chromatin dynamics including nucleosome assembly in S phase, transcriptional regulation and the maintenance of genome integrity (36). Over the past decade, extensive studies have been conducted to enhance our understanding of the role of histone acetylation in chromatin assembly. The reversible acetylation reaction involves the transfer of an acetyl group from acetyl coenzyme A onto the ε-amino group of specific lysine residues present in the NH2-terminal tails of each of the core histones(6). This modification is thought to affect chromosome function through two distinct mechanisms. First, they influence chromatin structure through electrostatic effects, which could impact histone-DNA interactions and higher-order chromatin structure. Second, acetylation at histone NH2-tails may modulate the binding of chromatin associated factors (37,38).

During chromatin assembly, newly synthesized histone H4 is diacetylated at K5 and K12 of H4 N-terminal tails before deposition onto the DNA. This modification is highly conserved in organisms ranging from protozoans to humans (39,40). These observations suggest an important role for histone acetylation in the process of chromatin assembly although we still do not understand how H4 NH2-terminal tail acetylations contribute to chromatin assembly. Newly synthesized H3 exhibits a more variable acetylation pattern.

There are five lysine residues on the NH2-terminal tail of H3 that can be acetylated. In

Saccharomyces cerevisiae, H3 K9, K14, K23, and K27 are acetylated with K9 and K27 predominating(41). In Drosophila, K14 and K23 are primarily acetylated, while in

Tetrahymena, K9 and K14 are the favorite sites(42). In Hela cells, K14, K18, and/or K23

6 are preferentially acetylated(42). A recent study demonstrated an important role of H3

NH2-terminal tail acetylation in chromatin assembly in which they indicated that yeast

Gcn5p, a histone acetyltransferase, is required for efficient incorporation of new histones after DNA replication by CAF-1(43). Another lysine residue, histone H3K56 was also discovered to be acetylated after synthesis in many species and this modification persists until G2/M. (44). H3K56 resides within the NH2-terminal alpha helix of H3, which interacts with the DNA as it enters and exits the nucleosome. One identified function of this modification is to alter nucleosome mobility so as to facilitate the efficient repair of replication-associated lesions through S phase. This acetylation is catalyzed by histone

H3 HATs Rtt109 in yeast and CBP/P300 in human(45). The reasons behind this variability are still not clear. No special patterns of transient acetylation of newly synthesized H2A and H2B have been detected (33).

Histone acetyltransferases (HATs) are enzymes that catalyze the transfer of an acetyl group from acetyl coenzyme A onto one or more lysine residues contained in the histone proteins. They have been classified with respect to their intracellular location and substrate specificity into two broad categories. HATs acetylate chromosomal histones are primarily located in the nucleus and usually are components of large multiprotein complexes, including NuA4, SAS-C and SAGA. These HATs are physically or functionally connected to transcriptional regulation. Others acetylate free histones after their translation and prior to their deposition into chromatin. The deposition-associated

H4 K5/K12 HAT is the evolutionarily conserved Hat1 protein, which is a member of the

GNAT superfamily. Hat1p(46) represents the prototypical catalytic subunit of HAT-B

7 complexes(47). It has been noticed that native yeast Hat1p only acetylates H4 K12, which might reflect the in vivo situation(47). The yeast Hat1p is associated with Hat2p, a homolog of the Rbap46/48 proteins that binds to the retinoblastoma protein Rb(47,48).

Hat2p is essential for a high level of Hat1p catalytic activity demonstrated by the fact that

Hat2p directly binds to histone H4, therefore mediates the high affinity binding of Hat1p to H4 (47). The Hat1p/Hat2p enzyme is highly conserved, as a number of divergent organisms have been reported to express complexes that containing homologs of these two proteins (49-51). Interestingly, the distribution of Hat1p/Hat2p complexes is not limited to the cytoplasm. Yeast and maize Hat1p/Hat2p complexes have been reported in both cytoplasmic and nuclear extracts (47,52,53). Surprisingly, hat1Δ mutants display no obvious growth defects or phenotypes, suggesting that some other unidentified HATs with overlapping specificity may exist in yeast (46,47) or that acetylation of the H3 tail can complement for the loss of H4 K12 acetylation (54). Recently, another Hat1p- containing complex was isolated from yeast nuclear extracts and was found to co-purity with Hat2p, as well as a novel component, Hat1 Interacting Factor-1 (Hif1p)(1,55). Hif1p was subsequently shown to have intrinsic chromatin assembly activity (discussed in detail below)(1). The HAT that catalyzes newly synthesized histone H3K56 acetylation is

Rtt109 in yeast and p300/CBP in human. Rtt109 promotes genome stability and resistance to a variety of DNA damaging agents through the direct acetylation of K56 of histone H3(56,57). Subsequently, Rtt109 was reported to acetylate H3K9 as well(58).

1.4 Histone chaperones

Histones are highly basic proteins that bind very avidly and non-specifically to nucleic acids. Mixing histones with negatively charged DNA under physiological condition in vitro results in the formation of insoluble aggregates. Therefore, acidic proteins are required to bind free histones to shield their positive charges, so that ordered nucleosome assembly may occur(18,59). Histone chaperones are a collection of acidic proteins that specifically bind to a subset of histones and facilitate their deposition onto DNA. All the histone chaperones bear long acidic tracts enriched in glutamic acid and aspartic acid.

Biochemical analyses have revealed that histone chaperones interact not only with histones, but also with numerous other chromatin factors that are important for their functions. Based on sequence similarity and histone binding preference, histone chaperones can be categorized in to many families.

1.4.2 Chromatin Assembly Factor 1 (CAF-1)

Chromatin assembly factor 1 (CAF-1) complex that mediates DNA replication-dependent nucleosome assembly has been extensively studied in the past three decades(21,60,61).

CAF-1 complex is highly evolutionarily conserved as its homologs were identified in human, Drosophila, Xenopus and yeast. In most species, CAF-1 consists of three subunits: in humans, they are p150, p60 and p48. CAF-1 binds histones H3 and H4 preferentially and forms a complex with H3 and H4 which is designated as CAC complex. The C-terminal region of p150 is involved in dimer formation and interacts with p60(62). The N-terminal region of p150 is where the DNA polymerase clamp,

PCNA, interacts and co-localizes at DNA replication sites(63). Interestingly, the interaction of CAF-1 with PCNA depends upon phosphorylation of p150 by the replicative kinase Cdc7-Dbf4 in human cells(64). This could provide a means to ensure a tight coordination between histone deposition and ongoing DNA replication. The p150 subunit also interacts with heterochromatin protein 1 (HP1) and PCNA, and localizes to heterochromatin(65,66). The p60 subunit contains a WD40 domain and a Bromo-domain- like motif and mediates the binding between CAF-1 and Asf-1 directly(63,67,68). The smallest subunit p48 is identical to Rbap48, a protein that has WD-repeats and binds to the retinoblastoma protein (33,69). It directly interacts with histone H4 and its homologues have been found to be components of histone deacetylase and chromatin remodeling complexes(51,70). In cells, CAF-1 is found to be associated with acetylated histone H3/H4 in the nucleus with an acetylation pattern the same as newly synthesized histones lysines 5 and 12 of histone H4 tail. However, the N-terminal tails of H3 and H4 are not required for CAF-1 binding or for CAF-1 mediated nucleosome assembly(71).

In yeast, deletion of CAF-1 genes CAC1, CAC2 or CAC3, or all three simultaneously has no effect on yeast cell viability(72). However, deletion of any of them leads to an increased sensitivity to UV radiation and defects in silencing at telomeres and the silent mating loci(72-76). Since CAF-1 is the only chromatin assembly factor whose function is tightly associated with DNA replication and DNA repair. UV sensitivity in these deletion strains is explained by the established role of CAF-1 in nucleosome reassembly following

UV damage-induced nucleotide excision repair(77). Current evidence indicates that this process is very similar to that during replication(78,79): CAF-1 localizes to sites of UV

10 damage and is required for the incorporation of newly synthesized, replication specific histone H3 onto repaired DNA(78).

1.4.3 Histone regulation proteins (Hir/HirA)

Hir proteins were initially identified as transcriptional repressors of gene expression(80).

The HIR genes are highly evolutionarily conserved throughout eukaryotic organisms. In higher eukaryotes, there is only one Hir homologue, called Hira(81,82), while in yeast, the Hir complex contains Hir1, Hir2, Hir3 and Hpc1(83). Recombinant Xenopus HIRA was found to bind specifically to histones H3/H4 and deposit them onto DNA in the absence of DNA replication in vitro(27). In addition, immunoprecipitation against epitope tagged histone H3.1 or H3.3 from Hela cell nuclear extract showed that HIRA selectively interacts with histone H3 variant H3.3 and is the only component in the complex that differs from a machinery co-purifying with H3.1 which mediates DNA replication-dependent nucleosome assembly (29). Hir proteins have also been indicated to facilitate nucleosome assembly in yeast(84).

1.4.4 Asf1

Asf1 was first identified as a factor that de-represses transcriptional silencing when over- expressed(85). Asf1 is the major subunit of Replication-coupling-assembly factor

(RCAF) which also comprises acetylated H3/H4 tetramers harboring the specific acetylation pattern observed in nascent histones and is able to promote DNA replication- dependent nucleosome assembly when supplemented to SV40 DNA-replication- chromatin –assembly reactions(86). The ASF1 gene is found in all examined eukaryotes 11 and it consists of highly conserved N-terminal domain and a variable C-terminal tail.

Although Asf1 was found in a stable complex with histone H3/H4 tetramer(86), a recent study showed that tagged H3.1 or H3.3 soluble pre-deposition complexes purified from

HeLa cells contain heterodimeric forms of H3/H4(29) and this observation is further supported by the discovery that Asf1 binds the C-terminal helix of histone H3 where the tetramerization of H3/H4 heterodimers is mediated(69).

The asf1Δ strains in budding yeast are viable but show phenotypes such as slow growth, silencing defects and sensitivity to DNA-damaging agents(85-87). This DNA-damaging agent sensitivity is different from when CAC1 is deleted. In addition, double mutants show greater sensitivity than either single mutant, suggesting that they have overlapping, yet independent roles(86).

Although Asf1 alone is not able to promote nucleosome assembly in vitro, it appears to function synergistically with CAF-1 to assemble nucleosomes in a DNA replication- dependent manner by directly interacting with the p60 subunit of CAF-1, therefore delivers histones H3/H4 to CAF-1(67,86). The role of Asf1 at the replication fork is clearly important because depletion of human, chicken, or Drosophila Asf1 slows down

DNA replication(88-90). Asf1 also interacts with Hir/HirA that facilitates the DNA replication independent nucleosome assembly and regulates histone gene expression(27,84). In budding yeast, Asf1 interacts with the DNA damage checkpoint protein Rad53, thus to mediate nucleosome assembly after DNA damage(91,92). In addition, Asf1 has been reported to interact with some other factors including histone modification enzymes and modification-recognizing proteins. For example, Asf-1

12 directly activates the HAT activity of Rtt109 that acetylates histone H3 at lysine 56 via its role in presenting H3/H4 dimers to Rtt109(57,93,94). Interestingly, a non-acetylable

H3K56R mutant in yeast showed a phenotype in DNA damage similar to asf1Δ mutant(44) and an H3K56Q mutant which mimics the acetylated state of H356K was found to suppress this defect in DNA damage(93). The mechanism that Asf1 participates in keeping cellular resistance to genotoxic stress by mediating histone H3K56 acetylation has yet to be resolved. One possibility is that H3K56 is located closely to the place where

DNA enters and exits nucleosomes. Acetylation of H3K56 neutralizes the positive charge of K56 so as to weaken the interaction between nucleosome and DNA(44), therefore, increasing the accessibility of nucleosome-bound DNA during DNA damage repair.

Taken together, Asf1 plays an important role in multiple chromatin-related functions including the assembly of nucleosomes, transcriptional silencing and cellular responses to

DNA damage.

1.5 N1/N2 family of histone chaperones

1.5.1 N1/N2 protein

N1 and N2 (Mr ~ 105 and ~110 kDa) histone chaperones were originally isolated from the nuclear extracts of X. laevis(95,96). The DNA sequences of N1 and N2 are identical and the differences in biochemical and immunochemical properties have not been explained, therefore, they were designated N1/N2. Xenopus oocytes have large amounts of stored histones for packaging the chromatin in about 10 thousand somatic cells(59,97).

N1/N2 were shown to associate with core histones H3 and H4, providing a mechanism 13 for the storage of these histones and allow the progressive release of histones and nucleosome assembly right after fertilization, which ensures nucleosome assembly during the rapid cell division in early development(98). Moreover, N1/N2 was shown to have an in vitro nucleosome assembly activity which indicates that it participates in mediating nucleosome assembly during the early embryogenesis most likely by transferring histones from the pool to other chaperones(98). Like a number of nuclear proteins, N1/N2 protein contains extended, negatively charged regions(99) and concentrates in the nucleus at exceptionally high level due to the high rate of nuclear uptake in Xenopus oocyte(99-

102). Homologues of N1/N2 have been found in other species with a conserved domain structure that consists of four TPR motifs and a large domain that is highly enriched in acidic amino acids and inserts into the second TPR motif(103).

1.5.2 Hif1p

The N1/N2 family was thought to be restricted to cells of the higher eukaryotes in the animal lineage because homologs of the Xenopus N1/N2 proteins were only identified in animal species such as mice and human. However, a N1/N2 homolog, Hif1p, was recently identified in budding yeast S. cerevisiae by two independent studies. Two-hybrid analysis revealed that Hif1p associates with the type B histone acetyltransferase Hat1p that catalyzes the acetylation of newly synthesized histone H4 at lysine 5 and 12. This binding is bridged by Hat2p(55), the regulatory subunit of the HAT1 complex. Another study explored the function of Hat1p and Hat2p by purifying complexes containing the corresponding histone H4 HAT activity, in which they identified a nuclear

Hat1p/Hat2p/Hif1p complex(1). Histone H3 and acetylated histone H4 were also found to co-purify with the Hif1p complex in a manner that was dependent on both Hat1p and

Hat2p. Hif1p was found to share a limited sequence similarity to the histone chaperone

N1. Recombinant Hif1p also an H3/H4-specific chaperone was shown to facilitate chromatin assembly when combined with yeast cytosolic extraction. This activity was also confirmed with purified endogenous Hat1p/Hat2p/Hif1p complex. Deletion of either

HIF1 or HAT1 resulted in similar telomeric silencing and DNA repair phenotypes. There was no further defect when two deletions were combined. This suggests that Hif1p and

Hat1p function in the same pathway to promote telomeric silencing and DNA repair.

These studies provided the first direct evidence linking histone acetylation with chromatin assembly in vivo.

1.5.3 NASP-1

A c. elegans homolog NASP-1 was indentified in a transcriptional repressor complex, which cooperates with a transcription, factor TRA-1 to repress male-specific genes in hermaphrodites. It plays a key role in development by functioning as a bridging protein between TRA-4 and HAD-1(104).

1.5.4 Sim3

Sim 3 is the fission yeast homolog of the N1 protein. It was identified from a screen looking for mutants that interrupt the transcriptional silencing of a marker gene inserted within an S. pombe centromere(103,105). Two sim3 mutants were identified and both of them display a variety of abnormal mitotic phenotypes, including hypercondensed 15 chromatin, lagging chromosomes in anaphase, and unequal segregation of chromosomes.

Sim3 was then structurally aligned with the N1/N2 family of histone chaperones including Hif1p, N1/N2 and NASP. It was found that they share a similar organization: 4

TPR motifs and a charged C-terminal motif. Two sim3 mutants isolated have altered residues in one of the conserved TPR motifs and disrupt Sim3-Cenp-A complex formation in vitro and in vivo. A reduced level of CENP-A and elevated levels of H3 were observed at central domain of sim3 mutants suggested that Sim3 is required to ensure that central domain chromatin is composed mainly of CENP-A. Sim3 was found to localize throughout the entire nucleus at all cell-cycle stages and physically interact with H3 by immunoprecipitation experiment. This suggests that one role of Sim3 is to escort CENP-A to the central domain of centromeres. The incorporation of newly synthesized CENP-A into centromeres were compromised in Sim3 mutants while gene expression profiling of these mutants indicated no genes that are involved in centromere function were affected, suggesting Sim3 is required to aid the deposition of newly synthesized CENP-A at centromeres. This nuclear localized protein was found to be important in escorting centromere-specific histone H3 variant CENP-A for assembly into centromere. In addition, Sim3 physically interacts with H3 or H3 variant, CENP-A.

1.5.5 NASP

Mammalian cells also contain an N1/N2 homolog termed NASP (Nuclear Autoantigenic

Sperm Protein) which was originally identified from studies of a sperm-specific protein:

RAS (Rabbit-sperm membrane autoantigen) and screening for proteins that

16 immunologically react with autoantibodies of rabbit spermatozoa (106,107). NASP was then described as a mammalian testis and sperm-specific nuclear autoantigenic protein containing a nuclear translocation signal and up to 25% acidic amino acids. Further studies discovered that NASP is present in two differentially spliced isoforms. The longer form of the protein is known as testicular NASP (tNASP) and is expressed in testis, embryonic tissues and some transformed cells. The shorter form is somatic NASP

(sNASP) that is found in all dividing cells. Mouse tNASP differs from sNASP by two deletions in the coding region and 5’ untranslated region. NASP mRNA levels increase during S-phase and decline during G2 that is concomitant with histone mRNA levels(25).

NASP also has histone binding regions demonstrated by the sequence analysis. Taking together, NASP is a perfect candidate as a histone chaperone during DNA replication.

NASP was found to play an essential role in mammals as demonstrated as followed: 1.

Cells that have been depleted of NASP were arrested at the border of G1/S phase and

DNA replication was compromised as indicated by the decreased incorporation of

BrdU(108). Expression of tNASP protein could not rescue the delay in cell cycle progression. 2. Mice NASP-/- null embryo stops development at blastocyst stage when maternal NASP protein reservoir is depleted(108). 3. NASP, Asf1 and CAF-1 were all found as members of a multichaperone protein complex that appears to shuttle histones during nucleosome assembly that is necessary for progression through S phase(29).

Curiously, although NASP is clearly a member of the N1/N2 family of histone chaperones as indicated by the fact that NASP and N1 share greater than 50% sequence identity and a conserved domain structure, it is described in the literature as a linker

17 histone-specific chaperone(25,109-111). To resolve this paradox, we have performed detailed and quantitative studies to explore this important human histone chaperone that will be extensively discussed in Chapter 3 and Chapter 4.

Chapter 2. Isolation and identification of proteins that associate with

Hif1p

2.1 Abstract

Recent studies revealed that a protein known as Hat1p Interacting Factor-1 (Hif1p) forms a complex with Hat1p/Hat2p in the nucleus and functions as a histone chaperone during chromatin assembly. To better understand Hif1p as a histone chaperone, we performed a biochemical characterization of this protein to isolate and identify proteins that associate with Hif1p in a Hat1p/Hat2p-independent manner. Our results suggest that a fraction of

Hif1p in cells is associated with other proteins. Immunoprecipitation against Hif1p identified some candidate proteins that may interact with Hif1p in vivo. In addition, an

H3-HAT activity was revealed as being influenced by Hif1p.

2.2 Introduction

Recent studies revealed that a protein known as Hat1p Interacting Factor-1 (Hif1p) forms a complex with Hat1p/Hat2p in the nucleus and functions as a histone chaperone during chromatin assembly(1,55). This protein displays sequence similarity to the X. laevis histone chaperone, N1, which functions in both the storage and assembly of histone

H3/H4 tetramers during the rapid rounds of DNA replication that occurs early in X. laevis

19 embryogenesis. Hif1p functions as a chromatin assembly factor in vitro and associates with acetylated histone H4 in vivo in a Hat1p/Hat2p dependent manner. These findings demonstrated a physical connection between type B HATs and factors directly involved in the process of chromatin assembly(1). In addition, the nuclear Hat1p/Hat2p/Hif1p complex may not be the only complex in which Hif1p takes part because co- immunoprecipitation with anti-Hat1 antibodies could not completely immunodeplete

Hif1p (55). To better understand the cellular role of Hif1p, we performed a preliminary biochemical characterization of Hif1p. Our results demonstrated that Hif1p is present in

Hat1p/Hat2p-independent complexes. Immunoprecipitation against Hif1p identified some candidate proteins that might interact with Hif1p in vivo. In addition, an H3-HAT activity was revealed as being influenced by Hif1p.

2.3 Materials and methods

2.3.1 Yeast strain

Yeast culture and genetic manipulation were done according to standard methods(112).

HIF1 was tagged with a Myc tag in UCC1111 (wild type) to generate strain XAY10(1).

GCN1 was tagged with a TAP tag in UCC1111 to generate strain HWY7. The presence of the epitope tags was confirmed by both PCR and Western blot. Gene deletions were generated by PCR-mediated gene disruption with nutrition marker(113). HAT1 and HAT2 were then disrupted in XAY10 to generate XAY15(1). HIF1 was disrupted in UCC1111 to generate XAY4(1). GCN1 was deleted in UCC1111 to generate HWY10. CHD1 was

20 disrupted in UCC1111 and XAY15 to generate HWY1 and HWY4, respectively. PCR was used to confirm all genetic deletions.

2.3.2 Whole cell extracts preparation

Yeast whole cell extracts were prepared from overnight yeast culture in YPD as described previously(114). Briefly, cells were harvested at midlog phase and washed with cold H2O and extraction buffer (100 mM HEPES, pH7.9, 245mM KCl, 5 mM EGTA,

1mM EDTA, 0.5 mM PMSF and 0.3% β-mercaptoethanol). 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 buffer/g 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.

2.3.3 Column chromatography

Dialyzed yeast whole cell extracts were centrifuged at 10,000 X g for 10 min. Resulting clarified extracts were applied to a Mono Q column (Amersham) 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], 1M

NaCl, 0.1 mM EDTA, and 10% Glycerol). The elution profile of protein was determined by western blot. The peak fractions containing Hif1p or Gcn1p that were determined by western blot were pooled and concentrated down to a volume of 200 µl and then resolved 21 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 (Figure 2.1).

2.3.4 Western blotting

Western blots were performed and visualized using an ECL Plus chemifluorecent detection kit according to manufacturer’s instructions (Amersham). The signal was detected by scanning on a Storm Phosphoimager.

2.3.5 HAT activity assays

Liquid histone acetyltransferase assays were performed using free chicken histones as the substrate(47). 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 chicken erythrocyte core histones. Chicken histones were purified as previously described(115).

Reactions were incubated at 37°C for 60 min, and then transferred to P-81 filters

(Whatman). The filters were washed three times with 250 ml of 50 mM NaHCO3 (pH

9.0) for 10 min., followed by a quick acetone rinse and then allowed to air dry. The amount of 3H bound to the filters was quantified by liquid scintillation counting. A portion of the assay mixture was also resolved on an 18% SDS-PAGE and exposed to x- ray in order to identify the histone substrate that was acetylation.

2.3.6 Immunoprecipitation of Hif1p

Four fractions containing Hif1p that were determined by western blot from gel filtration chromatography were pooled and incubated with 50 µl settled anti-c-Myc conjugated agarose beads for 2 hours at 4 degree. The agarose beads were then extensively washed with binding buffer (25mM Hepes-Na, pH7.5, 200mM KCl, 13mM MgCl2, 10%

Glycerol, 0.1% NP-40, 0.3% β-ME). The bound proteins were eluted with 2xSDS loading dye followed by boiling for 5 minutes. The whole cell extract of UCC1111 that contain the non-tagged Hif1p was purified exactly like other whole cell extracts. The corresponding fractions were pooled and served as negative control. Proteins that co- immunoprecipitated with Myc tagged Hif1p were resolved on 10% SDS-PAGE and visualized by coomassie blue staining.

2.4 Results

2.4.1 Hif1p associates with other proteins in one or more high molecular weight complexes

Previous studies suggested that nuclear Hat1p/Hat2p/Hif1p complex may not be the only one in which Hif1p takes part because co-immunoprecipitation with anti-Hat1 antibodies could not completely immunodeplete Hif1p (55). To better understand Hif1p as a histone

H3/H4 chaperone, we decided to explore and identify other Hif1p containing complexes independent of Hat1p/Hat2p. Yeast whole cell extracts were generated from a wild type strain (containing a Hif1p-myc fusion), fractionated over a Mono Q column and Hif1p

23 containing fractions were concentrated and further fractionated by a gel filtration column to resolve proteins by size. Elution profiles of Hat2p and Hif1p were determined by western blot (Figure 2.2). In a wild type strain, Hif1p eluted in a broad peak that centered at approximately at 500kD (Figure 2.2 Fractions 36-48). Hat2p eluted in two distinct peaks. The larger peak overlapped with the Hif1p peak (Figure 2.2 Fractions 38-48) and the apparent molecular weight was consistent with that of the nuclear Hat1p/Hat2p/Hif1p complex described previously(1). The apparent molecular weight of the smaller peak

(Fraction 48-56), 150kD to 200kD, closely matched the cytoplasmic Hat1p/Hat2p complex (1). This is completely consistent with the Hat1p/Hat2p-dependent complexes in cytoplasm and nucleus that have been reported(1,55). We then wondered whether deletion of Hif1p or Hat1p/Hat2p would alter the elution profiles of these proteins. Yeast whole cell extracts generated from hif1 deletion strain or hat1hat2 deletion strain was fractionated exactly like the extracts from wild type strain. Elution profiles of Hat2p in hif1 deletion strain and Hif1p in hat1hat2 deletion strain were determined by western blot. Deletion of the HIF1 gene resulted in a great alteration of the Hat2p elution profile, in which the larger Hat2p peak was significantly less abundant (Figure 2.2, Fractions 38-

48, second panel). This result strongly suggested that a significant fraction of the

Hat1p/Hat2p complex is associated with Hif1p. However, deletion of both HAT1 and

HAT2 did not have any significant effect on the elution profile of Hif1p as was apparent by the broad elution peak which overlapped with the corresponding peak in the wild type strain (Figure 2.2, Fractions 36-48, third panel). The bottom panel of Figure 2.2 represented the elution pattern of recombinant Hif1p, which appeared as narrow peak that

24 centered around 150kD. This was distinct from patterns observed with endogenous Hif1p and therefore demonstrated that the elution profile of Hif1p is not a property that is intrinsic to the Hif1p alone. Taken together, these results suggested that the native Hif1p is associated with other proteins in one, or more high molecular weight complexes.

2.4.2. Gcn1p may physically interact with Hif1p.

In an effort to identify proteins associated with Hif1p in Hat1p/Hat2p independent complexes, fractions corresponding to the Hif1p peak from the gel filtration columns shown in figure 2.2 were immunoprecipitated with anti-Myc antibody. Proteins co- purified with Hif1p were resolved by SDS-PAGE analysis followed by commassie blue staining. When immunoprecipitated from a wild type extract, Hif1p associated with three major polypeptides. The molecular weights of two of these were consistent with the sizes of Hat1p and Hat2p. When extracts from a hat1∆hat2∆ strain were used, these polypeptides were no longer observed. The third Hif1p-associated protein had a molecular weight of ~80 kDa. In addition, there were more polypeptides that co-purified with Hif1p that couldn’t be clearly shown by commassie blue staining due to the low protein amount (data not shown).

We have identified Gcn1p and Chd1p as candidate proteins by Mass spectrometry. At the same time, Gavin (116) and colleagues reported the first genome-wide screen for complexes in budding yeast by using affinity purification and mass spectrometry in which Hif1p and Gcn1 were co-purified with histone H4. This result showed that there

25 may be a physical interaction between Gcn1p and Hif1p. The identification of this protein proved that this is an effective method to purify proteins that associate with Hif1p.

2.4.3 The majority of GCN1p in cells is not associated with Hif1p.

In an effort to better understand the link between Hif1p and Gcn1p we observed previously, we fractionated the whole cell extracts derived from gcn1 deletion strain following the same procedures as described before and determined the elution profile of

Hif1p. Unexpected, deletion of Gcn1p did not affect the elution profile of Hif1p. Hif1p eluted in a peak that overlapped with the corresponding one from a wild type stain

(Figure 2.4 Fractions 38-48). To facilitate isolation and visualization of Gcn1p, we incorporated a tandem affinity purification tag (117) at the COOH-terminus of Gcn1p.

Whole cell extracts were generated from cells that express TAP tagged Gcn1p and then fractionated by Mono-Q column. Chromatography fractions that contained Gcn1p-TAP fusion protein were determined by western blot. Gcn1p containing fractions were pooled, concentrated and further resolved over a Superose 6 column. The elution profile of

Gcn1p was determined. As seen in Figure 2.4, Gcn1p eluted in a narrow peak centered about 700kD (Fractions 34-38 bottom panel). The Gcn1p peak slightly overlapped with the high molecular part of the Hif1p peak in wild type stain. We also did the reciprocal immunoprecipitation experiment to pull down Gcn1p in Gcn1p containing fractions from

Superose 6 column (Fractions 34-38). Unfortunately, Hif1p was not detected co-purified with Gcn1p (data not shown). Combined with the observation that deletion of Gcn1p did not obviously alter the elution profile of Hif1p in wild type strain, our results suggested

26 that the vast majority of Gcn1p in cells is not associated with Hif1p and Gcn1p is not a main component in Hat1p/Hat2p-independent complexes.

2.4.5 Chd1p is not stably interacting with Hif1p in cells.

We have also used the same assay to test whether Chd1p formed a stable complex with

Hif1p. The rationale here is that if a significant fraction of Hif1p forms a stable complex with Chd1p in vivo, deletion of Chd1p will affect the elution profile of Hif1p from gel filtration chromatography due to the loss of one component of this complex. As seen in

Figure 2.5, Hif1p eluted in a broad peak when cell extracts from a wild type strain was resolved by superose 6 column. Unexpectedly, deletion of Chd1p from neither a wild type strain nor a hat1Δhat2Δ strain significantly altered the elution profile of Hif1p.

These results suggested that Chd1p is not stably interacting with Hif1p in cells.

2.4.6. Hif1p influences a histone H3 specific HAT activity.

Since Hif1p was originally identified in a nuclear complex with Hat1p and Hat2p with strong HAT activity that was primarily specific for histone H4(1), we decided to test the

HAT activity in fractions from the superose 6 column described earlier to determine whether Hif1p was associated with additional HAT activities.

Fractions from gel filtration chromatography were analyzed by HAT assay. The fractions were incubated with free core histones and H3 labeled acetyl CoA. The reaction mixture was then resolved by SDS-PAGE and stained with commassie blue to visualize the histones. The gels were then subjected to autoradiography to determine which histones were modified by the addition of a 3H-labeled acetyl group. The specificity of HATs 27 contained within these peak fractions can be inferred from the intensity of each histone band. In a wild type strain, a broad H4 HAT activity peak co-eluted with Hat2p (Figure

2.6 Fractions 38-56), which likely corresponded with the high molecular weight nuclear

Hat1p/Hat2p/Hif1p complex as well as low molecular weight cytoplasmic Hat1p/Hat2p complex. Deletion of HIF1 resulted in the loss of the high molecular weight portion of this H4-specific peak, due to the loss of the nuclear Hat1p/Hat2p/Hif1p complex. The low molecular weight portion of this peak that was related with cytoplasmic Hat1p/Hat2p complex was not significantly affected (Figure 2.6 Fractions 48-56). When Hat1p and

Hat2p were both deleted, there was no detectable H4-specific peak in those fractions

(Fractions 44-56, bottom panel) due to the complete loss of Hat1p/Hat2p dependent complex.

Meanwhile, a histone H3-specific HAT activity, though not as abundant as the H4- specific HAT activity, also decreased following the deletion of HIF1 (Figure 2.6

Fractions 26-32). The deletion of HAT1 and HAT2 does not alter this H3-specific activity.

Although this H3-specific HAT activity peak did not overlapped with Hif1p peak, these results suggested that Hif1p may influence a histone H3 specific type B HAT complex.

2.5 Discussion

2.5.1 Hif1p interacts with proteins other than Hat1p/Hat2p.

Our biochemical chromatography experiments successfully demonstrated that Hif1p is present in complexes other than the nuclear Hat1/Hat2/Hif1 complex.

Immunoprecipitation against myc-Hif1p identified Gcn1p and Chd1p as candidate Hif1p- 28 associated proteins. The association of Gcn1p and Hif1p was also suggested by results reported by another group in which Hif1p and Gcn1p co-purified with histone H4 in a genome wide screen for complexes in budding yeast(116). Unfortunately, further analysis did not definitively demonstrate a physical interaction between Hif1p and Gcn1p or

Chd1p. This was evidenced by the fact that the elution profile of Hif1p from gel filtration chromatography was not significantly affected by deletion of Gcn1p or Chd1p. There are many factors may influence this result such as the multiple purification steps and the use of high ionic concentration buffer to prevent non-specific interaction may limit the outcome or the interaction between Hif1p and Gcn1p or Chd1p. In addition, the interaction between these proteins may be transient. Therefore, this temporary but functional association can merely be detected in a small population of cells. Nevertheless, the narrow peak of Gcn1p containing fractions with an apparent high molecular weight suggested that the broad Hif1p containing peak that we observed was not due to protein degradation and this is still an effective technique to identify protein-protein interaction.

We have also identified Kap123p as binding partner of Hif1p (data not shown). Kap123p is an importin protein that mediates the import of ribosomal proteins(118) and histone H3 and H4(119). Surprisingly, Kap123 also imports Sas2p(120), a subunit of SAS-1 complex that acetylates histone H3K14 and interacts with histone deposition proteins Asf1 and

CAF-1(121,122). Combined with the fact that Hif1p is a histone H3/H4 specific chaperone that functions in chromatin assembly, the discovery of a potential role of Hif1p in complex with these candidate proteins will further complete our understanding of this important protein.

2.5.2 Histone H3 HATs and Hif1p

In a wide range of eukaryotes, newly synthesized H3 and H4 are transiently acetylated at lysine residues within their amino-terminal tails before their deposition. The acetylation pattern of H3, not as conserved as H4, varies among species with 5 important lysines whose acetylation is crucial for efficient incorporation of new histones after DNA replication(43). Moreover, H3 lysine 56 residues are also acetylated after H3 synthesis and this modification is believed to greatly increase the affinity of CAF-1 for H3, therefore facilitating efficient chromatin assembly after DNA replication(123). As a histone chaperone, Hif1p directly links histone acetylation of H4 amino-terminal tails with nucleosome assembly. Our results suggest the possibility that Hif1p may also provide a link between the acetylation of newly synthesized histone H3 and chromatin assembly. We will test this idea by directly monitoring the acetylation level of hif1Δ strain and wild type strain followed by mass spectrometry to identify the specific lysine residues of histone H3 whose acetylation is dependent on Hif1p.

The uncoupled elution profiles of Hif1p and the H3 HAT activity influenced by Hif1p suggests a transient or loose interaction. Recently, the histone H3 acetyltransferases

Rtt109 and Gcn5 were reported as responsible for the posttranslational acetylation of H3 lysine 56 and lysines within the N-terminal tails. The acetylation of H3 K56 by Rtt109 requires the presence of the histone chaperone Asf1 to form a complex with H3-H4 dimers as is indicated by the fact that H3 K56 acetylation is essentially abolished in asf1Δ strain(56,57,94). Intriguingly, Asf1 does not necessarily form a stable complex with

Rtt109. Therefore, it is reasonable to propose that Hif1p and H3 HAT complexes may 30 transiently stay together or be functionally linked with each other to promote the deposition of newly synthesized histones after DNA replication. It will be interesting to explore the genetic interaction between Hif1p and these H3 HATs, which, in turn, will help our understanding of how the highly conserved acetylation of the N-terminal tails of core histones contributes to chromatin assembly and its related cellular events.

Figure 2. 1 Schematic diagram of the purification scheme used for the Hif1p containing complex.

Figure 2. 2 Presence of Hif1p in a high molecular weight complex independent of

HAT1 and HAT2.

Whole cell extracts were resolved by size over superpose 6 column and the elution profiles of Hif1p and Hat2p were determined by western blot analysis. 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 E. coli.

Figure 2. 3 Purification of native Hif1p.

SDS-PAGE analysis of immunoprecipitated Hif1p-myc complexes purified from wild type and Hat1∆Hat2∆ yeast. No tag control is an identical fraction from a strain lacking a Hif1-myc construct.

Figure 2. 4 The majority of GCN1p in cells is not associated with Hif1p.

Whole cell extracts were resolved by size over superpose 6 column and the elution profiles of Hif1p and GCN1p were determined by western blot analysis. 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.

Figure 2. 5 Chd1p is not stably interacting with Hif1p in cells.

Whole cell extracts were resolved by size over superpose 6 column and the elution profile of Hif1p was determined by western blot analysis. 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.

Figure 2. 6 Deletion of HIF1 results in significant decrease of a H3-specific HAT activity.

Whole cell extracts were resolved by size over a superpose 6 column and the elution profiles of HAT activity were determined by standard filter binding HAT assay. The whole cell extracts were derived from strains with the genotypes indicated on the right. The specificity of HAT activity is indicated on the left.

Chapter 3 Expanded binding specificity of the human histone

chaperone NASP

3.1 Abstract

NASP (nuclear autoantigenic sperm protein) has been reported to be an H1-specific histone chaperone. However, NASP shares a high degree of sequence similarity with the

N1/N2 family of proteins; whose members are H3/H4-specific histone chaperones. To resolve this paradox, we have performed a detailed and quantitative analysis of the binding specificity of human NASP. Our results confirm that NASP can interact with histone H1 and that this interaction occurs with high affinity. In addition, multiple in vitro and in vivo experiments, including native gel electrophoresis, traditional and affinity chromatography assays and surface plasmon resonance, all indicate that NASP also forms distinct, high specificity complexes with histones H3 and H4. The interaction between

NASP and histones H3 and H4 is functional as NASP is active in in vitro chromatin assembly assays using histone substrates depleted of H1.

3.2 Introduction

In eukaryotes, genomic DNA is packaged into a highly ordered and regulated structure known as chromatin. The principal repeating element of chromatin is the nucleosome which consists of ~147 base pairs of DNA wrapped around the histone proteins: H2A,

H2B, H3 and H4. The transit of histone proteins throughout the cell and their subsequent deposition into chromatin is facilitated by a collection of proteins known as histone chaperones.

Histone chaperones can be grouped into a large number of families based on sequence similarity. These include the nucleoplasmin, N1/N2, CAF-1, HIR, NAP1, Asf1,

Rbap46/48, Rsf-1, FACT, nucleolin and Arp families of proteins. The defining characteristic of histone chaperones is their ability to bind histone proteins and they are often highly specific with regard to these interactions. For example, chaperones such as

CAF-1 and Asf1 specifically interact with complexes of histones H3 and H4 while other chaperones like nucleoplasmin and nucleolin interact with complexes of histones H2A and H2B. Histone chaperones facilitate a number of events relevant to histone biology such as their storage, transport, deposition and eviction(59,98,124,125).

The N1/N2 family of histone chaperones was originally identified in X. laevis(126-128).

In X. laevis, N1/N2 associates with core histones H3 and H4, providing a mechanism for the storage of these histones in Xenopus oocytes. In addition, N1/N2 also participates in mediating nucleosome assembly. The N1/N2 family was thought to be restricted to cells of the animal lineage but a potential homolog, Hif1p, was recently identified in the

39 budding yeast S. cerevisiae as an H3/H4 specific histone chaperone that associates with a nuclear form of the Hat1p/Hat2p type B histone acetyltransferase complex(1).

A N1/N2 family member has also recently been identified in the fission yeast S. pombe(103). Sim3 was isolated in a screen looking for mutants that disrupted the transcriptional silencing of a marker gene placed in the central core of an S. pombe centromere. Intriguingly, Sim3 was found to be important for the localization of the centromere-specific histone H3 variant CENP-A at S. pombe centromeres. In addition,

Sim3 was found to physically associate with both CENP-A and histone H3.

Mammalian cells contain an N1/N2 homolog termed NASP (Nuclear Autoantigenic

Sperm Protein). In mammals, NASP is present in 2 differentially spliced isoforms. The longer form of the protein is known as testicular NASP (tNASP) and is expressed in the testis, embryonic tissues and some transformed cells. The second form is somatic NASP

(sNASP) which is found in all dividing cells. NASP plays an essential role in mammals as demonstrated by the early embryonic lethality of a mouse knock out model(108). In addition, genetic studies in C. elegans indicate that the NASP homolog in this organism plays a key role in development and may act as a transcriptional regulator(104).

Curiously, although NASP is clearly a member of the N1/N2 family, it has been described in the literature as a linker histone-specific chaperone(2,25,108-110). As the binding specificity of histone chaperones is a critical aspect of their function, we have used multiple methods to explore the biochemical properties of this protein. In vitro binding experiments using purified proteins demonstrate that sNASP binds specifically to both histone H1 and histones H3 and H4. The binding of sNASP with histones H1 and

H3/H4 displays altered binding kinetics that results in high affinity as indicated by sub-

µM dissociation constants for both interactions. Co-immunoprecipitation experiments indicated that NASP retained this spectrum of interactions in vivo. In addition, the interaction of sNASP with histones H3 and H4 was functional as sNASP was active in in vitro chromatin assembly assays that use only core histones.

3.3 Materials and methods

3.3.1 Expression and purification of recombinant sNASP

The human sNASP ORF (Invitrogen) was transferred into the E. coli expression vector pDEST-17, which adds an NH2-terminal His6 tag. The resulting construct was transformed into E.coli BL21 to allow for IPTG-inducible expression. 1L cultures were grown to mid-log phase and sNASP expression was induced for 2 hours. The cells were harvested and the pellets were resuspended in 30 ml start buffer (2 0mM sodium phosphate pH7.4, 500 mM NaCl) and lysed by sonication. PMSF (0.5 mM) and protease inhibitor cocktail[1:100], Sigma) were also added to buffers. Cell lysate was aliquoted into 1.5 ml microcentrifuge tubes and centrifuged at 10,000 RPM for 10 minutes. The supernatant was applied to a Ni2+ charged HiTrap Chelating HP column (5 ml). After washing with 20 mM imidazole and 50mM imidazole, respectively, the recombinant sNASP was eluted with 500 mM imidazole and confirmed by Western blot with anti-His6 antibody. Purified sNASP was dialyzed against DN(200) (25 mM Tris [pH 7.0], 0.1 mM

EDTA, 10% glycerol, 200 mM NaCl) and the protein concentration determined.

3.3.2 Histone isolation

Core and linker histones were isolated from chicken erythrocyte nuclei by acid extraction

(0.4 N H2SO4) followed by extensive dialysis against 50 mM Tris, pH 7.0. Core histones depleted of H1 were isolated from chicken erythrocyte nuclei by hydroxyapatite chromatography as described(115). Purified bovine histone H1 was obtained from

Millipore. Recombinant histones H3 and H4 were purified as described (a generous gift from Dr. M. Poirier)(129).

3.3.3 Native gel electrophoresis

1.5 µg of recombinant sNASP was incubated with increasing amounts of purified histone

H3/H4 or histone H1 in DN(300) for at least 4 hrs at 4° C. The 7% native gel was prepared as follow: 1.75ml Acrylamide 40% stock, 3.75 ml 3M Tris-HCl, pH 8.8, 4.35 ml ddH2O, 100 µl 10% APS and 20 µl TEMED. The protein complexes were resolved by electrophoresis at 120 V with native gel running buffer (0.192 M Glycine, 0.025 M Tris, pH8.3) for 4 hours. The proteins in the native gel were then visualized by coomassie blue staining.

3.3.4 Chromatography-based histone binding assays

Purified sNASP was mixed with histones and incubated overnight at 4 °C in DN (150) or

DN (200) buffer. After incubation, the mixtures were applied to a Ni2+ charged HiTrap

Chelating HP column (1 ml). The column was then washed extensively with wash buffers

(20 mM sodium phosphate pH7.4, 150 mM NaCl 100 mM imidazole and 20 mM sodium

42 phosphate pH7.4, 500mM NaCl, 100mM Imidazole, respectively). 0.1% of NP-40 was added to the wash buffer to minimize non-specific binding between histones and the matrix of column. Bound proteins were eluted with buffer containing 500 mM imidazole.

Flow through and bound fractions were resolved by SDS-PAGE and visualized by coomassie blue staining.

Alternatively, the proteins were 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, 0.1% NP-40 and 300 mM NaCl). Fractions were collected and resolved by

18% SDS-PAGE and visualized by Coomassie blue staining.

3.3.5 Biosensor Analysis

Biosensor experiments were performed using a Biacore 2000 surface plasmon resonance

(SPR) instrument. Histones were coupled to a CM5 sensor chip using HBS-EP (10 mM

Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% TWEEN-20) buffer at 25 oC. In preparation for amine-coupling of the histones, the CM5 sensor chip was first washed with HBS-EP buffer at a flow rate of 10 µl/min for 10 minutes. Each flow cell was injected with 25 µL of 1:1 solution of NHS/EDC (N-hydroxysuccinimide/N-ethy-N’-(3- dimethyl-amino-propyl)-carbodiimide, 75 mg/ml/11.5 mg/ml), followed by a 30-100 µl injection of either H1 or the H3/H4 tetramer at 50-100 µg/ml in PBS, pH 7.4 buffer

(typically 30 µl of the histone(s) diluted into 100 µl of 10 mM sodium acetate, pH 4.5) was injected over flow cells 2-4. No histones were injected over the activated flow cell 1

43 serving as a reference cell. Flow cells were blocked with a 35 µl injection of 1M ethanolamine, pH 8.5.

SPR binding experiments measured at 50 µl/min were performed using either HBS-EP or

TBS-EN (25 mM Tris-HCl, pH 7.0, 300 mM NaCl, 0.1 mM EDTA, and 0.05% NP-40) buffers. Similar NASP binding kinetics were observed over the H1 or H3/H4 tetramer coupled surfaces using either buffer system. Non-specific binding was not observed when

NASP was injected over the underivatized flow cell 1. There were no mass transport effects seen during the association phase during NASP injections. Two-fold serial dilutions of 4-5 NASP concentrations determined the binding kinetics to the histones.

Each 250 µl protein/buffer injection was followed by a 500 sec dissociation period. The surface was regenerated for subsequent runs with 15 µl injections of 3 M NaCl and 4 M

MgCl2.

Sensorgrams were pooled, trimmed, and subtracted using BIAevaluation 4.1. A buffer sensorgram was subtracted from each NASP sensorgram before data analysis (double referencing (130)). The NASP:histone binding kinetics fit well to a simple Langmuir biomolecular reaction model defined by a single on- and off-rate constant. Sensorgrams were globally analyzed using ClampXP (131) and the binding kinetic parameters were determined from three separate experiments.

3.3.6 Immunoprecipitaion of NASP sNASP-containing complexes were immunoprecipitated from Hela cell nuclear extract with rabbit anti-NASP antibody with coupling gel from ProFoundTM Mammalian Co-

44 immunoprecipitation Kit (Pierce)(132). After immobilization of antibody with coupling gel, 100 µl of coupling gel was incubated with 800 µl of Hela cell nuclear extract for 5-6 hrs, at 4 °C. The gel was then washed with 1 ml of DN(500) (25 mM Tris [pH 7.0], 0.1 mM EDTA, 10% glycerol, 0.1% NP-40 and 500 mM NaCl) 6 times to remove unbound protein. The bound proteins were eluted by boiling in 100 µl of 1X SDS loading dye

(0.06M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.001% bromphenol blue and 5% β- mercaptoethanol). The coupling gel without conjugation with antibody was incubated with Hela cell nuclear extract as a negative control. The bound proteins were resolved by

SDS-PAGE and detected by western blot.

3.3.7 In vitro chromatin assembly

Assays contained pUC18 plasmid which had been relaxed by DNA topoisomerase I

(Sigma). The assembly reactions were performed in buffer containing: 10 mM Tris-HCl

(pH 8.0), 1.0 mM EDTA, 100 mM NaCl, 100 mg/ml BSA, 2 mM ATP, and 0.6 µg purified chicken core histones. Reactions were supplemented with 2 µl of yeast cytosolic extract as indicated(1). The reactions were incubated at 37 °C for 1 hr and terminated with 0.2% SDS and 100 µg/ml Protease K. After phenyl extraction and ethanol precipitation, DNA was analyzed on a 1.5% agarose gel and visualized with SYBR gold nucleic acid stain (Invitrogen).

3.4 Results

Human sNASP displays a high level of sequence similarity with X. laevis N1/N2 with greater than 50% amino acid identity. In addition, sNASP and N1/N2 have a shared 45 domain structure that is also conserved in the S. cerevisiae histone chaperone Hif1p

(depicted in Figure 3.1A). Each of these proteins contains a large central domain that is highly enriched in acidic amino acids (glutamic acid and aspartic acid). This acidic domain is flanked by TPR repeats with one repeat NH2-terminal to the acidic domain and two repeats COOH-terminal. Given the sequence and domain conservation between these proteins, we re-examined the histone binding specificity of sNASP to determine whether it shares the H3/H4 specificity of N1/N2 and Hif1p or whether it specifically interacts solely histone H1, as reported in the literature.

3.4.1 Multiple sNASP/histone complexes identified by native gel electrophoresis

Native gel electrophoresis has been one of the primary assays used to assess the in vitro histone binding specificity of sNASP(133). In these experiments, specific complexes were detected between sNASP and histone H1 in the form of discreet bands that were only observed following the incubation of sNASP with linker histones. Conversely, when sNASP was incubated with histones H3 and H4, it was retained in the wells of the native gel leading to the interpretation that sNASP forms non-specific aggregates with histones

H3 and H4(133).

We have re-examined the use of native gel electrophoresis to monitor the interaction of sNASP with histones. For these assays, recombinant human sNASP (which contains an

2+ NH2-terminal His6 tag) was expressed in E.coli and purified by Ni chelate chromatography. Recombinant sNASP was incubated with a mixture of histones isolated

46 from chicken erythrocytes that included both the core and linker histones (see Figure

3.1C, lanes 7 and 15). Native polyacrylamide gel electrophoresis indicated that sNASP formed two distinct complexes with the histones, which had reduced mobility relative to free sNASP.

To determine whether these complexes were the result of interactions between sNASP and histones H1 or H3/H4, sNASP was incubated with either histone H1 or H3/H4 complexes separately. For these experiments, chicken erythrocyte histone H1 was separated from histones H3 and H4 by Ni2+ chelating chromatography, taking advantage of the fact that these histones display varying degrees of non-specific interaction with column resins (see Figure 3.4B). The sNASP was mixed with increasing amounts of either histone H3/H4 complexes or histone H1 and the mixtures resolved by native gel electrophoresis. As seen in Figure 3.1B, sNASP formed complexes with both histones

H3/H4 and histone H1 (lanes 1-6 and lanes 9-14, respectively). While both H1 and

H3/H4 were able to form complexes with sNASP, the interaction between sNASP and histone H1 occurred at lower histone concentrations suggesting that the binding of sNASP to H1 occurred with higher affinity. Surprisingly, the mobility of both the sNASP:H1 and sNASP:H3/H4 complexes was similar to the mobility of the complexes observed when sNASP was incubated with the complete histone mixture. This is likely due to the relatively low resolution afforded by native gel electrophoresis which separates macromolecules based on multiple characteristics such as size, shape and charge.

3.4.2 sNASP forms a stable complex with histones H3 and H4

We employed gel filtration chromatography to obtain a more detailed and higher resolution picture of the interaction between sNASP and histones. In the absence of sNASP, the core histones eluted as a single peak with an apparent molecular weight of

~50 kDa (Figure 3.2B). Histone H1 eluted in two peaks with the first peak having an apparent molecular weight of ~150 kDa and the second peak co-eluting with the core histones suggesting that histone H1 may interact with the core histones (Figure 3.2B).

Following incubation with sNASP, if any of the histones form a complex with this chaperone, they should co-elute from the gel filtration column with sNASP with a larger apparent molecular weight. As seen in Figure 3.2A, histones H3 and H4 perfectly co- eluted with sNASP as a complex with an apparent molecular weight of ~350 kDa.

Histone H1 did not co-purify with sNASP and eluted as a single peak of ~150 KDa apparent molecular weight. Interestingly, in the presence of sNASP, histone H1 did not co-elute with the core histones suggesting that the interaction between histone H1 and the core histones may have been mediated by histones H3/H4. Importantly, the observation that sNASP does not interact with histones H2A, H2B or H1 in this assay indicated that the binding of sNASP to histones is not due to non-specific interactions based solely on electrostatic attraction.

The co-elution of histones H3 and H4 with sNASP during gel filtration chromatography is clear evidence that sNASP forms a distinct stable complex with H3 and H4. To determine whether the complex observed during gel filtration chromatography is related to the complexes observed by native gel electrophoresis, protein fractions from across the 48

Superose 6 sNASP:H3/H4 peak were directly resolved on a native polyacrylamide gel.

As seen in Figure 3.3B, these fractions contain complexes that have electrophoretic mobilities identical to those seen when the sNASP/histone mixtures were directly resolved by native gels. A number of important observations can be made from this result. First, the sNASP:H3/H4-specific bands observed by native gel electrophoresis

(Figure 3.1B) reflect the presence of bone fide, stable complexes. Second, the differences in electrophoretic mobility observed for the sNASP:H3/H4-specific bands during native gel electrophoresis are indicative of complexes with different apparent molecular weights as the complexes that give rise to these bands elute from the gel filtration column with distinct, but over-lapping, profiles. The precise nature of these complexes is not clear but may be related to the observation that sNASP can exist as both a monomer and a dimer(133). Finally, the sNASP that elutes from the gel filtration column is predominantly associated with histones H3 and H4 as there is no free sNASP detected by native gel electrophoresis.

3.4.3 sNASP specifically interacts with histones H3 and H4

A third assay was also used to determine the in vitro binding specificity of sNASP.

Recombinant sNASP (which has an NH2-terminal His6 tag), was incubated with a preparation of chicken erythrocyte histones that contained both the core and linker histones. Following incubation, the sNASP/histone mixture was applied to a Ni2+ chelate column. Unbound proteins were removed by extensive washing (with buffer containing

0.1 M imidazole) and the bound proteins eluted using buffer containing 0.5 M imidazole.

As expected, the His6-tagged sNASP bound to the column and was present in the elution fractions (Figure 3.1B). Histones H3 and H4 were not present in the wash fractions and co-eluted with sNASP. Histones H2A, H2B and H1 were all found in the wash fractions and were not detected in the elution fractions. When the histones were applied to the Ni2+ chelate column in the absence of sNASP, all of the histones, including H3 and H4 were found in the wash fractions (Figure 3.1C).

This result supports the previous observations of a specific interaction between sNASP and histones H3 and H4. However, neither this assay nor the gel filtration chromatography provided evidence for an interaction between sNASP and histone H1.

The lack of interaction between sNASP and histone H1 in these assays may be due to a number of factors. First, these assays contained mixtures of the core and linker histones.

It may be that binding of sNASP to H3/H4 is preferred and the formation of the sNASP:H3/H4 complex precludes the interaction between sNASP and histone H1.

Alternatively, the use of histones derived from chicken erythrocytes may have influenced the results as there is greater sequence identity between chicken and mammals for the core histones than for the linker histones. We tested these possibilities by directly assessing the ability of sNASP to interact with purified bovine histone H1. As seen in

Figure 3.4C, when purified bovine histone H1 was combined with 6His-tagged sNASP, in the absence of other histones, and applied to a Ni2+ chelate column, all of the histone H1 was found in the wash fractions. In addition, no detectable histone H1 co-eluted from the

Ni2+ chelate column with sNASP. This result suggests that the lack of interaction between sNASP and histone H1 is not due to competition or the sequence divergence of chicken

50 histone H1. Hence, while an interaction between sNASP and histone H1 is clearly detectable by native gel electrophoresis, it is not observed using chromatography-based assays. One explanation, which is difficult to rule out, is that the nature of the non- specific interactions that are seen between histone H1 and column resins prevent the formation of sNASP/histone H1 complexes.

3.4.4 sNASP interacts primarily with histone H3.

To further characterize sNASP as a histone H3/H4 specific chaperone, we tested the ability of sNASP bind individually to histone H3 and H4. For these experiments, recombinant X. laevis histones H3 and H4 were each expressed and purified from E. coli

2+ and then combined with His6-tagged sNASP. As shown in Fig 3.5A, following Ni chelate chromatography, all of the histone H3 co-eluted with sNASP. However, even though sNASP is present in excess, histone H4 was only partially retained on the column

(Figure 3.5B). These results suggest that sNASP can bind to both H3 and H4 but the association with histone H3 may be the primary interaction.

3.4.5 Biosensor analysis of NASP binding to histones H1 and H3/H4

The native gel electrophoresis and chromatography-based assays provided evidence that sNASP can form specific complexes with both histone H1 and histones H3/H4. To provide a more quantitative analysis of these interactions, biosensor analysis using surface plasmon resonance (SPR) was performed to determine the binding kinetics and affinities of the sNASP:histone interactions. The H1 and H3/H4 histones were randomly

51 coupled to the sensor chip surface using amine chemistry. Triplicate sNASP samples were injected at various concentrations over the H1 and H3/H4 surfaces (Figure 3.6). The binding kinetics for the NASP:H1 and NASP:H3/H4 interactions were globally fit to a single site Langmuir binding reaction model with the on (kon) (85)- and off (koff)-rate constants listed in Table 1.

NASP displays altered binding kinetics and affinities during the interactions with either the H1 histone or the H3/H4 tetramer. The NASP:H1 interaction yields a kon rate of 1.44

4 -1 -1 -4 -1 x 10 M s and a koff rate of 1.98 x 10 s . The calculated equilibrium dissociation constant KD is 13.8 nM for the NASP:H1 interaction. The NASP:H3/H4 tetramer

3 -1 -1 -4 -1 interaction yields a kon rate of 2.92 x 10 M s and a koff rate of 6.91 x 10 s . The KD is 237 nM for the NASP:H3/H4 tetramer interaction. The 17-fold weaker KD for the

NASP:H3/H4 complex in comparison to the NASP:H1 complex results from an ~5-fold slower kon rate and an ~3.5-fold faster koff rate. The sub-micromolar KD values for both the sNASP:H1 and sNASP:H3/H4 indicated that both complexes form with high affinity.

In addition, the lower KD value for the sNASP:H1 interaction mirrors the results obtained with native gel electrophoresis where lower concentrations of H1 were needed to form the sNASP:histone complex. Thus, the altered binding kinetics and affinities for the sNASP:H1 and sNASP:H3/H4 interactions provide a quantitative description to the qualitative gel electrophoresis and size-exclusion chromatography binding experiments.

3.4.6 In vivo interaction of sNASP and histones

The designation of NASP as a linker histone binding protein was originally based on evidence from the purification of endogenous NASP from cell lysates. NASP was purified from mouse myeloma cell extracts using an anti-NASP antibody affinity column(25). Proteins bound to NASP were then eluted from the column and analyzed by reverse phase HPLC. The presence of histone H1, as well as the absence of core histones, was determined by comparison of the retention times of the NASP-associated proteins to the retention times of histone standards(25). Anti-NASP antibody columns were also used to isolate NASP from mouse testis lystes and HeLa cell lysates(109,111). In each case histone H1 was identified as eluting from the column with NASP by Western blot analysis. However, these samples were not probed for the presence of core histones.

While these experiments did not identify an in vivo interaction between NASP and histones H3/H4, other in vivo evidence to supports an interaction between these proteins.

Tagami and colleagues used epitope tags to affinity purify complexes associated with human histones H3.1 and H3.3(29). While some histone chaperones, such as CAF-1 and

HIRA, selectively associated with only one H3 variant, other chaperones, including Asf1 and NASP, were found complexed with both forms of H3. As histone H1 was not reported to be a component of these complexes, these results indicate that a fraction of the soluble histone H3 in mammalian cells is associated with NASP.

We have used co-immunoprecipitation to evaluate whether the interactions between

NASP and histones seen in vitro are also found in vivo. For these experiments, antibodies were raised in rabbits using purified recombinant sNASP as antigen. Rabbit anti-NASP 53 antibody was conjugated with coupling gel and incubated with Hela cell nuclear extract.

After extensive washing, the bound proteins were eluted. Western blots were then used to determine the presence of specific histones. As seen in Figure 3.7, the antibody immunoprecipitated both sNASP and tNASP. In addition, both histones H1 and H3 were co-immunoprecipitated with the anti-NASP antibody suggesting that NASP interacts with both linker and core histones in vivo. Hence, these results are consistent with the in vitro histone binding data described above.

3.4.7 sNASP participates in the deposition of core histones

It has previously been shown that sNASP can function to restore histone H1 onto chromatin that has been depleted of linker histones(133). To determine whether sNASP:H3/H4 complexes were also functional, we used an in vitro chromatin assembly assay that monitors the formation of nucleosomes through the introduction of supercoils into a relaxed circular plasmid DNA. For these assays we used a preparation of chicken erythrocyte histones that had been purified by hydroxyapatite chromatography to remove histone H1 (data not shown). As seen in Figure 3.8, recombinant sNASP was unable to assemble nucleosomes using purified chicken erythrocyte core histones (compare lanes 3 and 5). As demonstrated previously, recombinant yeast Hif1p is also unable to function alone in the assembly of core histones and requires the presence of factor(s) in yeast cytosolic extracts (see lanes 4, 7-9 and reference(1)). Therefore, we tested whether sNASP could function in a similar manner and replace Hif1p in these assembly reactions.

As seen in Figure 3.8 (compare lane 7 to lanes 10-12), sNASP promotes very robust

54 chromatin assembly in the presence of core histones and a yeast cytosolic extract. These results make two important points. First, the interaction between sNASP and histones H3 and H4 is a productive one that can lead to histone deposition. Hence, sNASP can function as a core histone assembly factor. Second, the similarity in chromatin assembly activity demonstrated by sNASP and Hif1p supports the hypothesis that Hif1p is a functional ortholog of the N1/N2 family of histone chaperones in yeast.

3.5 Discussion

NASP has emerged as a protein that is critical for the proper growth and development of complex eukaryotic organisms(103,104,134). As a histone chaperone, the most fundamental activity of NASP is its histone binding specificity. The experiments presented here indicate that, in vitro, sNASP forms specific, high-affinity complexes with both histone H1 and histones H3/H4. In addition, the interaction of sNASP with histone

H3/H4 complexes is functional as sNASP is able to function in the assembly of core histones onto DNA in vitro. These results are entirely consistent with the high degree of primary sequence and domain structure conservation observed between NASP and members of the N1/N2 family of histones chaperones which have been demonstrated to interact specifically with histones H3 and H4(1,126,127,135).

Our quantitative analysis of the binding of sNASP to histone H1 and histone H3/H4 complexes indicated that sNASP interacts with both sets of histone proteins with high affinity. The dissociation constants that we have observed, 13.8 nM and 237 nM for H1 and H3/H4, respectively, are comparable to those seen for the binding of the histone

55 chaperone p55 to histone H4 and significantly lower than those reported for most other histone binding proteins (typically in the 1 - 10 µM range)(117,136-141).

The effect of sNASP on in vitro plasmid supercoiling assays suggests that sNASP is not merely a histone binding protein but that it is also a chromatin assembly factor. The requirement of an extract in these assays indicates that, under the conditions tested, sNASP is not able to generate stable nucleosomes by itself. Whether sNASP hands off histones to other factors that directly assemble nucleosomes or whether sNASP is more directly involved in mediating the histone/DNA interaction remains to be determined.

The activity of sNASP in in vitro chromatin assembly assays also sheds light on the yeast histone chaperone Hif1p. Despite a low level of primary sequence similarity, Hif1p was suggested to be a fungal ortholog of the N1/N2 family of histone chaperones based on a conserved domain structure and common histone binding specificity(1). The ability of sNASP to functionally substitute for the yeast protein Hif1p in chromatin assays strongly supports this hypothesis that these proteins share a common function.

An interesting issue raised by our results involves the functional significance of a histone chaperone that can bind both histone H1 and histone H3/H4 complexes. One intriguing possibility suggests a potential link between the acetylation of newly synthesized histones

H3 and H4 and the deposition of histone H1. Subsequent to their synthesis, histones H3 and H4 are rapidly acetylated on their NH2-terminal tails by type B histone acetyltransferases. Following deposition of these histones, these modifications are removed during chromatin maturation. While the acetylation of newly synthesized H3 and H4 is an evolutionarily conserved event, the function of this modification is not

56 known. One potential role is suggested by the observation that preventing the deacetylation of H3 and H4 following chromatin assembly prevents association of histone H1 with chromatin(142-144). Thus, the assembly of histone H1 may be regulated by the modification state of histones H3 and H4. Combined with the finding that Hif1p is found associated with the type B histone acetyltransferase Hat1p in yeast nuclei, these observations suggest that histone chaperones such as sNASP and Hif1p may coordinate, either spatially or temporally, the assembly of histone H1 onto nucleosomes containing the proper modification state on histones H3 and H4.

Table 3. 1 SPR binding kinetics and affinities for the interactions of NASP with H1 and H3/H4 tetramer.

Figure 3. 1 Native gel electrophoresis of sNASP:histone complexes.

(A) A schematic diagram representing the conservation of sequence and domain structure between sNASP, N1 and Hif1p. (B and C) A total of 1.5 µg recombinant sNASP was incubated alone (lanes 8 and 16) or with increasing amount of histone H3/H4 (0.36, 0.81, 1.26, 1.71, 2.16 and 2.61 µg, lanes 1–6), histone H1 (0.4, 0.9, 1.4, 1.9, 2.5 and 2.9 µg, lanes 9–14) or 1.5 µg of total chicken histone (lanes 7 and 15) in the presence of DN (300) with a total volume of 30 µl. After incubation, 12 µl of each mixture was analyzed by either native gel electrophoresis (B) or SDS–PAGE (C). Proteins were visualized by Coomassie Blue staining. The migration of sNASP:histone complexes is denoted by asterisks in (B).

Figure 3. 2 NASP forms a stable complex with histones H3 and H4.

(A) Purified recombinant sNASP (150 µg) and chicken erythrocyte histones (200 µg) were mixed (input) and resolved by gel filtration chromatography (Superose 6). Fractions (indicated by numbers at top of gels) were resolved by SDS–AGE and visualized by Coomassie Blue staining. A 4.8% of the input and each fraction were electrophoresed. The elution positions of standards are indicated at the top. (B) Purified chicken erythrocyte histones were chromatographed and visualized as above.

Figure 3. 3 Correlation between sNASP:H3/H4 complexes detected by gel filtration chromatography and native gel electrophoresis.

(A) sNASP (100 µg) was incubated with total chicken erythrocyte histones (150 µg) and resolved on a Superose 6 column. Fractions containing sNASP and histones H3 and H4 (labeled at the top of the gel) were resolved by SDS–PAGE and visualized by Coomassie Blue staining. (B) The indicated fractions from the Superose 6 column were directly resolved by native gel electrophoresis and visualized with Coomassie Blue staining. Recombinant sNASP was also analyzed alone (lane 11) or following incubation with total chicken erythrocyte histones (lane 10). The migration of sNASP:histone complexes is indicated by asterisks.

Figure 3. 4 Detection of sNASP:H3/H4 complex formation by affinity chromatography.

(A) Purified recombinant sNASP (150 µg) and chicken erythrocyte histones (200 µg) were incubated in a total volume of 1.0 ml (input) and chromatographed on a Ni2+ chelate

62 column. The first six fractions to wash off the column after loading (wash) and the first six fractions to elute with 500 mM imidazole buffer (elute) were resolved by SDS–PAGE and visualized by Coomassie Blue staining. A 0.24% of the input and 2.4% of each fraction was loaded on the gel. The position of each protein in the gel is indicated on the left. (B) Purified chicken erythrocyte histones (300 µg) were chromatographed and visualized as above except that 0.75% of the input was run on the gel. (C) Purified sNASP (200 µg) was incubated with bovine histone H1 (500 µg) in a total volume of 1.0 ml and chromatographed on a Ni2+ chelate column. The first five fractions to wash off the column after loading (wash) and the first five fractions to elute with 500 mM imidazole buffer (elute) were resolved by SDS–PAGE and visualized by Coomassie Blue staining. A 0.24% of the input and 2.4% of each fraction was loaded on the gel.

Figure 3. 5 Recombinant sNASP primarily interacts with histone H3.

(A) Purified sNASP (200 µg) was incubated with recombinant X. laevis histone H3 (100 µg) in a total volume of 1.0 ml and chromatographed on a Ni2+ chelate column. The first seven fractions to wash off the column after loading (wash) and the first seven fractions to elute with 500 mM imidazole buffer (elute) were resolved by SDS–PAGE and visualized by Coomassie Blue staining. A 0.24% of the input and 2.4% of each fraction was loaded on the gel. (B) Purified sNASP (200 µg) was incubated with recombinant X. laevis histone H4 (76 µg) in a total volume of 1.0 ml and chromatographed on a Ni2+ chelate column. Fractions were analyzed as described above.

Figure 3. 6 Quantitation of the interactions between sNASP and histones H1 and H3/H4 by SPR

SPR binding kinetic sensorgrams for the interactions of NASP with H1 (A) and H3/H4 (B). The black curves are the trimmed sensorgrams collected at a flow rate of 50 µl/min in HBS-EP buffer at 25°C. Global fits of the data according to the model described in the Materials and methods section are in red, and the resulting kinetic rate constants are tabulated in table 3.1.

Figure 3. 7 NASP interacts with both histone H1 and histone H3 in vivo.

HeLa cell nuclear extract (input) was incubated with coupling gel with (lane 4) or without being conjugated to anti-NASP antibody (lane 4). Five microliters of the input (lane 1) and unbound (lane 2) fractions and 12 µl of the bound fractions were resolved by SDS– PAGE and the proteins visualized by western blots probed with antibodies recognizing the proteins indicated on the right.

Figure 3. 8 Recombinant sNASP can assemble core histones into chromatin.

Chromatin assembly activity of recombinant sNASP with core histones was assayed by incubating the indicated factors with a relaxed circular plasmid. After incubation, the plasmids were extracted and resolved by 1.5% agarose gel electrophoresis, and visualized by SYBR Gold nucleic acid stain. The migration of the supercoiled (S) and relaxed (R) forms of the plasmid are indicated by arrows. Lanes 1 and 2 show the template DNA before and after relaxation, respectively.

Chapter 4 Structure and functional characterization of NASP

4.1 Abstract

In eukaryotes, chromatin assembly is essential for the replication of chromosomes.

Chromatin assembly factors are required to deposit histones onto DNA during chromatin assembly. A histone chaperone NASP, which also had been identified as linker histone specific, was recently demonstrated to be a histone H3/H4-specific chaperone and to function in core histone deposition. To better understand the function of NASP in detail, we mapped the domains of sNASP that are involved in interactions with each histone.

Our results identified two distinct domains to be important in binding to linker histone or core histone exclusively. We have also explored the cellular function of NASP using

RNAi techniques. Our results demonstrated that sNASP is a significant contributor to global chromatin structure.

4.2 Introduction

As discussed in Chapter 3, NASP, a member of the N1/N2 family of histone chaperones, shares greater than 50% sequence identity with N1. It was surprising that, it shares similar sequence and domain structure with other N1/N2 family members which were all described as histone H3/H4 chaperones, NASP was reported to be an H1-specific

68 chaperone in numerous published reports(25,108,145). Therefore, we performed a detailed biochemical and biophysical analysis of sNASP to determine the binding specificity of this important human histone chaperone. We conformed the association of sNASP with histone H1 and identified an interaction between sNASP and histones H3 and H4. We also quantitated the affinity of sNASP for its histone substrates which showed sub-micromolar dissociation constants with both H1 and H3/H4. Co- immunoprecipitation experiments indicated that NASP retained this spectrum of interactions in vivo. Importantly, the interaction of sNASP with histone H3 and H4 was functional as sNASP was active in in vitro chromatin assembly assays that used only core histones.

More and more evidence indicates that N1/N2 family histone chaperones participate in more aspects of chromatin biology than just the storage of H3/H4 complexes in oocytes(103,104,108). NASP is an important member of this family as indicated by a variety in vivo experiments showing that it plays a critical role in mammalian cells(25,108,134). In addition, the biochemical characterization of NASP that indicates binding to both linker histone and core histone H3 suggests a possible role of this unique histone chaperone in coordinating the assembly of core and linker histones. Therefore, a detailed characterization of the mechanisms by which NASP functions in histone dynamics is likely to provide important information regarding the cellular role of NASP in mammalian cells and may also provide fundamental insight into the coordination of core and linker histone assembly. To understand the function of NASP in detail, we mapped the domains of sNASP that are involved in interactions with each histone to help

69 to understand how the association with specific histones contributes to the cellular function of NASP.

We constructed a series of sNASP mutants in which specific domains are either deleted or significantly altered. We used surface plasmon resonance (SPR) to quantitate the interactions between these sNASP mutants and histone H1 and histone H3/H4 tetramers.

We identified two domains that are exclusively important for binding to either linker histone or core histones. These results suggest that sNASP use distinct mechanisms to interact with core histones and linker histone. We have also developed a cell model system for the characterization of sNASP. Our in vivo data suggest that the binding spectrum of sNASP in cells is complicated due to some factors such as the dimerization of NASP. In addition, we have identified sNASP as a significant contributor to global chromatin structure by using RNAi technique to deplete endogenous NASP in cells.

4.3 Materials and methods

4.3.1 Plasmid DNA construction

Human sNASP ORF (Invitrogen) was cloned into an E.coli expression vector pDEST-17, which adds a His6 tag at the NH2-terminus. This construct was transformed into E.coli

BL21 AI competent cell to allow for L-arabinose-inducible expression. Six mutant constructs were generated from this plasmid (Figure4.1). TPR1Δ, TPR3Δ and TPR4Δ were generated by partial overlapping PCR method. Primers were designed to be complementary to the sequences flanking the regions that were targeted for deletion: sNASP-TPR1, TPR3 and TPR4 motifs. The upstream primer partially overlaps with 70 downstream primer to prevent primer self-complementarity. PCR products were then subjected to endonuclease Dpn-1 digestion to remove template DNA and propagated in

E.coli. DH10β competent cells for DNA sequencing and further analysis. sNASP-12E/K was generated by site-directed mutagenesis from pDEST17/sNASP (Quik-Change site- directed mutagenesis kit; Stratagene). The sNASP N-terminal fragment (sNASP-N) that includes all four TPR repeats and acidic domain and C-terminal fragment (sNASP-C) that starts after the final TPR motif and includes the nuclear localization sequence were generated by using PCR technique and Invitrogen gateway cloning system. The DNA fragments that encoding N or C and flanking attB sequences were generated by PCR and then introduced into donor vector pDONR 221 by BP recombination reaction. After being propagated and verified, the donor clones that contained desired gene sequences were subjected to LP recombination reactions by which these sequences were cloned into destination vector pDEST-17. All the mutant constructs were confirmed by DNA sequencing.

Alternatively, all the mutant sequences were introduced into another destination vector pT REX DEST 31 (Invitrogen) which allows the expression in mammalian cells. As with the E. coli expressed proteins, these constructs also contain an NH2-terminal His6 tag that allows for virtualization and protein purification.

4.3.2 Protein expression and purification

Plasmids that contain sequences including full-length sNASP and its mutants were each transformed into E.coli BL21 AI competent cells to allow for L-arabinose-inducible

71 expression. Protein expression and subsequent purification was performed following procedures described in Chapter 3.

4.3.3 in vitro chromatin assembly

The assays contained pUC18 plasmid which had been relaxed by DNA topoisomerase I

(Sigma). The assembly reactions were performed in buffer containing: 10 mM Tris-HCl

(pH 8.0), 1.0 mM EDTA, 100 mM NaCl, 100 mg/ml BSA, 2mM ATP, 0.4 µg purified

Hela core histones (Active Motif) and 0.5µg purified sNASP or sNASP mutants. The reactions were incubated at 37° for 1 hour and stopped by Stop buffer (0.2% SDS,

100µg/ml Protease K). After Phenol extraction and ethanol precipitation, DNA was analyzed on a 1.5% agarose gel and visualized with SYBR Gold nucleic acid stain

(Invitrogen).

4.3.4 in vitro histone binding assays

Purified recombinant sNASP and sNASP mutants were each mixed with chicken histones and Ni2+-NTA beads. The mixtures were incubated at 4° C in DN (200) buffer for 3 hours. After incubation, the beads were extensively washed with wash buffers (20mM sodium phosphate pH7.4, 300mM NaCl, 50mM Imidazole and 0.1% NP-40). Bound proteins were eluted with buffer containing 500mM imidazole. Unbound and bound proteins were resolved by SDS-PAGE and visualized by coomassie blue staining.

4.3.5 Cell culture

U2OS cells that were engineered for tetracycline-inducible expression were cultured in

McCoy’s media supplemented with 10% FBS at 37°C in 5% CO2 supply.

4.3.6 Plasmid Transfection

The U2OS cells were transfected with pT-REX-DEST 31 plasmids (Invitrogen) carrying

His6-tagged full-length sNASP or its mutants using Fugene reagent kit (Roche). Stably transfected clones were selected against 200µg/ml G418. Stable clones that can be induced by tetracycline were confirmed by Western blot.

4.3.7 Whole cell extracts preparation

2-4 × 107 Cells were collected 24 hours after tetracycline induction. Cells were washed with PBS once and then frozen with liquid N2. After being thawed on ice, cell pellets were resuspended in an equal volume of whole cell extract buffer (25% glycerol, 0.42M

NaCl, 1.5mM MgCl2, 0.2mM EDTA. 20mM HEPES pH 7.6 and 0.1% Triton X-100) and incubated on ice for 20min. After incubation, they were pelleted again. The supernants were saved as whole cell extracts.

4.3.8 in vivo protein pull down experiment

Whole cells extracts from cells expressing full-length sNASP or its mutants were incubated with 50 µl Ni2+-NTA beads (settled volume) for 2 hours at 4°C. Beads were then washed with wash buffer (50mM NaPO4 pH 7.5, 300mM NaCl, 10% Glycerol,

10mM Imidazole and 0.05% NP-40) 5 times. Bound proteins were eluted in 2×SDS dye followed by boiling. Western blotting was used to determine bound proteins.

4.3.9 siRNA transfection

U2OS cells were maintained as described previously. siRNA

(GCACAGUUCAGCAAAUCUAdTdT) targeting the human NASP open reading frame was synthesized (108)(Dharmacon, Lafayette, CO). siRNA that had no cellular target served as a negative control. U2OS cells were transfected with NASP and negative control siRNA using siPORT Neofx transfection reagent (Ambion): 2×105 cells were seeded per well for a 6-well plate. The final concentration of siRNA in the culture media was 40nM. After 48 hours, cells were transfected with corresponding siRNA again. Cells were collected 48 hours after second treatment for further analysis.

4.3.10 MNase Digestion

U2OS cell nuclei were isolated as described(146). Approximately 105 nuclei were digested with 5 Worthington Units of MNase (Sigma) for increasing periods of time.

Reactions were stopped by EDTA to a final concentration of 10mM. DNA was deproteinated, phenol extracted, ethanol precipitated and electrophoresed in 1% agarose gel and visualized by ethidium bromide staining.

4.4 Results

4.4.1 Domain analysis of sNASP

When sNASP was structurally aligned with other members of the N1/N2 family, a conserved domain structure with several candidate functional domains was discovered

(103). sNASP contains a large central domain that is highly enriched in acidic amino acids (glutamic acid and aspartic acid) and four TPR motifs. Among these TPR motifs, the second TPR motif is interrupted by the large central acidic domain. Three other TPR motifs flank the acidic domain with one NH2-terminal and two COOH-terminal. There is a nuclear localization signal sequence at position 376-399 which is highly homologous to the one that is found at the COOH-terminus of the N1 protein.

4.4.2 Functional dissection of sNASP.

Previous studies suggested that sNASP has several activities including histone H1 binding, histone H3/H4 binding and nucleosome assembly. We have constructed a series of sNASP mutants in which specific domains are either deleted or significantly altered.

These constructs are: An NH2-terminal fragment that includes all four TPR repeats and the acidic domain, a COOH-terminal fragment that starts after the final TPR motifs and includes the NLS sequence, individual deletions of the 3 intact TPR motifs and a mutant in which 12 of the glutamic acid residues in the acidic domain have been changed to lysines. These constructs allowed us to identify the domains of sNASP that are involved

75 in the known activities of this protein. These mutant proteins were expressed in E. coli and purified to a high degree (Figure 4.1).

SPR methods have been developed to measure the binding kinetics of full length sNASP to linker and core histones(147). Here, we used the same technique to quantitate the interactions between sNASP mutants and histone H1 or histone H3/H4 tetramers (Table

1). Full-length sNASP showed high affinity binding with both H1 and H3/H4 tetramers with Kd values of 13.8 nM and 237 nM, respectively. These values were nearly identical to those that we reported in our recent publication demonstrating the robust reproducibility of our analyses(147). The COOH-terminal fragment of sNASP was unable to bind to any histone ligand. The NH2-terminal fragment of sNASP was capable of binding histone H1 with an affinity approaching that of the full-length molecule. The binding of the NH2-terminal fragment to H3/H4 tetramers was reduced approximately 5- fold which suggests that the COOH-terminus of sNASP contributes to core histone, but not linker histone, binding.

We were able to further localize the domains of sNASP that contribute to linker and core histone interactions. In this respect, two sNASP mutants were particularly informative: sNASP 12 E/K and sNASP TPR4Δ. The sNASP 12E/K mutant contains an altered acidic domain in which 12 of the glutamic acid residues have been changed to lysine. As seen in Table 1, the affinity of this mutant for histone H1 decreased >2000-fold while its affinity for H3/H4 tetramers was relatively unchanged (~2-fold decrease). Conversely, deletion of the fourth TPR motif (sNASP TPR4Δ) had a dramatic effect of H3/H4 tetramer affinity (>500-fold decrease) with no effect on histone H1 binding affinity.

These results suggest that sNASP uses distinct mechanisms to interact with core and linker histones.

We have also directly tested the binding between sNASP mutants and core histones with a chromatography-based assay. This experiment was done using Ni2+ conjugated beads that can bind to the His6-tag at the NH2-terminus of each sNASP mutant. After incubation with core histone and each sNASP variant, the beads were extensively washed to remove the unbound protein. The bound proteins were then eluted and resolved on

18% SDS-PAGE. Consistent with the SPR data and previous results, full-length sNASP co-eluted with histone H3 and H4 with a similar stoichiometry. As expected, the COOH- terminal fragment has very limited ability to bind histone H3 and H4. The NH2-terminal fragment retained most of its binding activity with a similar stoichiometry. In addition, the sNASP TPR4Δ bound less core histone compare to 12E/K. In general, the in vitro pull down data is consistent with what we observed from SPR experiments.

Next we explored the domains that are required for the nucleosome assembly activity of sNASP. in vitro chromatin assembly assays were used to determine the ability of each protein variant to form nucleosomes as indicated by introducing supercoils into a relaxed, circular plasmid. This experiment was conducted with similar amount of each protein variant and Hela core histones. As seen in Figure 4.4, recombinant full-length sNASP promoted very robust chromatin assembly in the presence of Hela core histones (Lane 4). sNASP TPR4Δ lost most of its assembly activity which is consistent with the SPR data that deletion of the fourth TPR motif dramatically decreased its affinity for H3/H4 tetramers. The observation that the COOH-terminal fragment had no assembly activity

77 was also in agreement with the SPR data that it was unable to bind any histone ligand.

Surprisingly, sNASP 12E/K had no obvious chromatin assembly activity either although it bound to H3/H4 tetramers with a relatively high affinity. This result suggests that being able to bind H3/H4 tetramers is necessary but not sufficient to promote the formation of nucleosomes. The highly positive charge possesses in acidic domain also contributes to this function. sNASP TPR1Δ, TPR4Δ and NH2-terminal fragment each possessed chromatin assembly activity that was slightly weaker than the full-length sNASP.

4.4.3 Construction of inducible NASP cell lines

To gain insight into the function of sNASP in vivo, we have designed and constructed vectors that will enable us to express full-length sNASP and its previously described mutants in mammalian cells. As with E. coli expressed recombinant proteins, these constructs also contain an NH2-terminal His6-tag that allows for visualization and protein purification. These vectors have been transfected into a U2OS cell line that has been engineered to provide for tet-inducible expression of these constructs. Following selection, we have isolated cell lines that stably express full-length sNASP or other mutants (Figure 4.4). As seen in Figure 4.4A, U2OS cells expressed both sNASP and tNASP and tetracycline induction resulted in an increase in the levels of sNASP (arrows) without an increase in tNASP. Staining of the western blot with α-His6 antibody confirmed that tet-induction led to the expression of the exogenous full-length sNASP. In addition, it was apparent that the level of expression of the exogenous full-length sNASP was comparable to the levels seen for endogenous sNASP and tNASP. Cell lines that

78 stably express sNASP-N, sNASP-C, sNASP-12E/K, sNASP-TPR1Δ and sNASP-TPR4Δ were all established (Figure 4.4 B). Another mutant, sNASP-NLSΔ, which does not contain the COOH-terminal nuclear localization signal, was also constructed to be expressed in U2OS cells under the control of tetracycline induction.

4.4.4 in vivo characterization of binding specificity of full-length sNASP and its mutants

Taking advantage of being able to express epitope-tagged full-length sNASP and its mutants in U2OS cells, we decided to explore the binding spectrums of these mutants in vivo. Full-length sNASP and its mutants were each stably expressed as fusion proteins

7 with NH2-terminal His6-tag in U2OS cells. 24 hours after tetracycline induction, 2-4×10 cells were collected and subjected to whole cell extraction. Whole cell extracts derived from each cell lines were then incubated with Ni2+-NTA beads to purify proteins that were associated with His6-tagged sNASP variants. As a control, we conducted the purification from untransfected U2OS cells which do not express His6-tagged protein.

SDS-PAGE analysis and western blotting were performed to identify proteins that bound to Ni2+ chelating beads (Figure 4.5). U2OS Cells expressed both native sNASP and tNASP that can be found in input fractions (Figure 4.5 In). In addition to native NASP,

His6-tagged version of full-length sNASP and its mutants were each found in the corresponding input fractions. As expected, His6-tagged full-length sNASP bound to

Ni2+-chelating beads and was detected by anti-NASP antibody in the elution fractions

(EL). Histone H3 and H4 all co-eluted with full-length sNASP as indicated by western

79 blot. This is completely consistent with our previous results. In vitro, sNASP-TPR1Δ bound to histone H3/H4 tetramer with a relatively high affinity and promoted nucleosome assembly with core histones (Table 4.1 and Figure 4.3). Consistent with the in vitro data, both histones H3 and H4 co-purified with TPR1Δ, although with a decreased affinity.

Surprisingly, sNASP 12E/K, a full-length protein with an altered acidic domain did not bind to histone H3 or H4 while the in vitro data suggested a high level affinity to histone

H3/H4 tetramers. sNASP TPR4Δ showed the robust binding of histones H3/H4. This was surprising given that the in vitro data indicated 500 fold decreases in affinity to H3/H4 tetramers compared to full-length sNASP.

In addition to histone H3/H4 affinity, we were intrigued by the observations that sNASP-

TPR1Δ and sNASP-TPR4Δ were able to pull down tNASP which was not seen with full- length sNASP or sNASP-12E/K.

4.4.5 Silencing of NASP by RNAi and tet-induction of exogenous sNASP

In addition to in vivo pull down experiments, we investigated the role of NASP in U2OS cells by selectively silencing the expression of both tNASP and sNASP. Richardson and colleagues have identified an siRNA that efficiently knocks down the expression of

NASP in U2OS cells(108). The constructs that we have used to create the NASP expression cell lines do not contain the sequences that are targeted by these siRNA.

Hence, we could use these siRNA to deplete these stable cell lines of endogenous NASP without effecting the expression of the synthetic NASP constructs. 48 hours after the second treatment with siRNA, cells were collected and subjected to the whole cell

80 extraction. The expression of NASP in cells was determined by western blot. As seen in

Figure 4.6, expression of both sNASP and tNASP was specifically and significantly reduced. In contrast, expression of GAPDH, a product of a housekeeping gene, was not affected by the silencing of NASP. When a full-length sNASP was induced, there was an increase in the level of sNASP in cells treated with a control siRNA and there was no change in the level of tNASP. When endogenous NASP was depleted by the siRNA, the expression of the synthetic sNASP restored the sNASP to normal levels without effecting tNASP.

4.4.6 sNASP is required to form a regular chromatin structure

We have established stable U2OS cell lines with N-terminally His6 tagged full-length sNASP and sNASP mutants under the control of a tetracycline-inducible promoter. Since sNASP has been shown to be a histone chaperon and was able to promote nucleosome assembly with core histones in vitro, we decided to use these U2OS cells to identify defects that result from loss of NASP activity by directly testing for general defects in chromatin structure. The use of Micrococonuclease (MNase) to probe chromatin structure has proven to be a valuable technique. MNase digests DNA that is in the linker region between nucleosomes but is blocked by the presence of nucleosomes. Hence, disruption of regular chromatin structure leads to increased sensitivity of the underlying DNA to digestion. Nuclei were isolated from U2OS cells treated with either a non-specific control siRNA or and siRNA targeting NASP and digested with MNase over the course of 15 minutes. Digested chromatin was sampled at specific timepoints, deproteinized and

81 resolved by agarose gel electrophoresis. As seen in Figure 4.7, loss of NASP led to a significant increase in the digestion of chromatin. Regular nucleosomal ladders were apparent in both samples, indicating that the majority of chromatin still formed regularly spaced chromatin when NASP was depleted. This result suggests that NASP is a significant contributor to global chromatin structure. When full-length sNASP was induced in the cells that were depleted of NASP, exogenous sNASP largely reversed the defect caused by the loss of native NASP.

We then tested whether the induction of sNASP mutants was also able to reverse this increased MNase sensitivity. 24 hours after tetracycline induction, nuclei were collected from the cells that were treated with control siRNA or siRNA specific for NASP and then subjected to MNase digestion. Digested chromatin was prepared as described earlier and analyzed on agarose gels electrophoresis. Induction of sNASP-TPR1Δ or sNASP-TPR4Δ did not completely reverse the increased MNase sensitivity of chromatin due to the loss of NASP. Interestingly, induction of these TPR4Δ in the cells that were treated with control siRNA increased the MNase sensitivity of chromatin. This result suggests that sNASP mutants may interfere with native NASP or proteins that are associated with

NASP, therefore, cause defects in global chromatin structure.

4.5 Discussion

NASP is a histone chaperone that is critical for the proper growth and development of mammals(103,104,134). As a member of the N1/N2 family of histone chaperones, it differs from others in that it functions as a chaperone for both core histones H3/H4 and

82 linker histone H1(147). This interaction is functional that capable of directly participating in deposition of core histone(147). Human NASP was found in multichaperone complexes with H3/H4 and H3/H4 specific chaperones(29). On the other hand, NASP was reported to bind to H1 in vitro and in vivo and can incorporate H1 onto nucleosome arrays(110,147).

4.5.1 What we have learned from in vitro data

We have quantitated the binding affinity of sNASP to both core histones and linker histones in the previous chapter. Here, we used the same method to determine the binding affinity of each purified mutant to histone H1 or histone H3/H4 tetramers. We identified two domains that are important for linker histone binding or core histone binding. Since we used purified recombinant proteins in these experiments, we directly identified the intrinsic binding properties of each protein.

Our results suggest that the binding of sNASP to linker histone H1 is mostly dependent on the negative charge possessed by the acidic domain. The ionic nature of this interaction to explain why we could not observe binding between sNASP and linker histone in chromatography experiments. The high NaCl concentration in the wash buffer

(300mM NaCl), which was originally used for preventing non-specific interaction of core histones to the matrix of the columns, shielded the negative charge of sNASP and therefore, decreased the binding between these two proteins. Unlike core histones, linker histones have a tripartite structure with an unstructured NH2-terminal and COOH- terminal domains (CTD) flanking a globular domain. The CTD of linker histone has 40%

83 lysine and arginine residues that provides sufficient screening of the DNA negative charge which is required for chromatin condensation. Hence, H1 plays a key role of chromatin compaction primarily based on electrostatic attraction between H1 and linker

DNA. sNASP carries more than 40% acidic amino acids, so sNASP may bind to linker histones primarily through electrostatic attraction and hold them during DNA replication in S phase until their turn to be deposited. It will be of interest to explore whether the

CTD of linker histone H1 is the place where sNASP interacts.

When sNASP is structurally aligned with other members of N1/N2 family, four TPR related motifs are identified that are shared among all the members. The tetratricopeptide repeat (TPR) motif is a well-studied example of a module facilitating protein-protein interactions ubiquitous in all kingdoms of life. Each TPR motif forms two α-helical domains that are antiparallel to each other. Most TPR domains contain multiple motifs arrayed in tandem thereby forming an extended, right-handed superhelical arrangement.

One of NASP’s binding partners, heat shock protein 90(111) was reported to be associated with other proteins by directly interacting with their TPR motifs(148-150). In our experiment, deletion of sNASP’s fourth TPR motif significantly reduced the binding affinity of sNASP to histone H3/H4 tetramer with no affect on H1 binding in vitro. This suggests that the TPR motif contained by sNASP is indeed a module that facilitates protein-protein interaction.

Under physiological conditions, simply mixing purified histones with DNA results in an amorphous aggregate due to the non-specific interactions between histones and DNA.

The negatively charged histone chaperones prevent non-specific charge-charge

84 interactions by competing with DNA and other charged species in the cell for histones, thereby facilitating the energetically downhill process of nucleosome assembly. From this point of view, our experiments identified two domains that mainly contribute to the two definitive characteristics of sNASP as a histone chaperone: 1) specifically recognizing and binding to histones 2) neutralizing the charges of histones. Importantly, these two characteristics are tightly connected in that sNASP functions as a chromatin assembly factor as indicated by the fact that the chromatin assembly activity of sNASP is significantly decreased by deletion of any of these two domains. The importance of separate domains of sNASP that drive electrostatic or conformational specific interaction between sNASP and histone H1 and H3/H4 as well as subsequent nucleosome assembly and possible disassembly requires further efforts and will shed light on the formation of higher order chromatin structures and epigenetic inheritance.

4.5.2 Can the in vitro and in vivo data be reconciled?

In addition to in vitro experiments, we have also extended our studies to a relatively physiological condition. We purified proteins that were associated with His6-tagged full- length sNASP or its mutants from cell extracts derived from corresponding cell lines. Our results shed light on how NASP behave in the complicated protein-protein interaction network in cells and how NASP indirectly interacts with other proteins. Consistent with previous data, sNASP associated with both histones H3 and H4 in cells. To our surprised, sNASP mutants presented different and complicated binding spectrums compared with those when purified recombinant proteins were studies.

85 sNASP 12E/K has a high affinity for binding H3/H4 tetramers in vitro. However, neither is associated with this mutant in vivo. This suggests that the interaction of sNASP with

H3/H4 tetramers was significantly decreased in cells by the mutation of some of the acidic amino acids in acidic domain. NASP has been reported being a component in many complexes associated with histone H3 or H3 variants(29,151) that are an important for both histone H3.1 or histone H3.3 nucleosome pre-assembly machinery. The mutations in the acidic domain of sNASP may have altered the interaction profile of sNASP with other proteins, in turn, affected the binding between sNASP and histone

H3/H4. Histone H3/H4-specific chaperones, including CAF-1, Asf-1 and HIRA were all found to co-elute with NASP in histone H3 containing complexes. We hypothesize that these chaperones compete with each other for histones substrates in a functionally redundant way and, therefore, facilitate ordered and regulated nucleosome assembly to occur. This is supported by a recent study in which Drane and colleagues showed that when a histone H3.3 assembly factor, DAXX, was depleted, a fraction of histone H3.3 was deposited in a DNA-replication dependent way by CAF-1. More interestingly, an increased amount of tNASP was detected being associated with H3.3 concomitant with the depletion of DAXX.

The TPR4Δ that had a dramatically decreased affinity for histone H3/H4 tetramers were found co-elute with both histone H3 and H4. In addition, it was also associated with tNASP in vivo. This will be discussed later.

It was also surprising that tNASP co-eluted with sNASP-TPR1Δ and sNASP-TPR4Δ. sNASP was reported forming dimers in a head-to-tail way and assembling histone H1 in

86 vitro(110). sNASP dimerization was also suggested by our native gel electrophoresis assay in which sNASP alone resolved on native gel as two distinct bands (data not shown). There had been no evidence that sNASP and tNASP formed heterodimers.

A number of TPR proteins not only bind heterologous ligands but can also self-assemble into higher order structures, either intrinsically or in response to external stimuli (152-

154). The deletion of one of the TPR motifs may dramatically change the protein confirmation under physiological conditions and as a result, alter the binding profile of sNASP in cells. We believe that the observation that a fraction of the histone H3/H4 co- eluted with sNASP-TPR1Δ and sNASP-TPR4Δ was originally associated with tNASP and was not the intrinsic property of these two mutants. Nevertheless, it will be interesting to study the binding profiles of each mutant to further our understanding of how sNASP functions in vivo.

4.5.3 NASP is a significant contributor to global chromatin structure

As a histone chaperone, NASP was proposed to play an important role in cells. This is suggested by the observation that depletion of NASP from cells resulted in a delay of the cell cycle and that NASP mRNA expression parallels that of major histone mRNA during the cell cycle(25,108,134). Our results showed that depletion of NASP significantly increased the sensitivity of chromatin to MNase which digests chromatin at sequence between nucleosomes. This suggests that NASP is important for maintaining a normal chromatin structure. Combined with the fact that loss of NASP resulted in a decrease in

DNA replication(108), we hypothesize that NASP is required for normal progression

87 through S phase. We do not know whether it was depositional defects of core histones or linker histone H1 or both that led to the instability of chromatin structure. It will be interesting to study phenotypes that are caused by decreased incorporation of histone H1 in NASP depletion cells, such as increased nuclear size and low H1 stoichiometry with short linker DNA sequences.

We were intrigued to find that ectopic expression of sNASP mutant TPR4Δ caused an increased sensitivity in MNase digestion. One possible explanation is that these mutant proteins from dysfunctional dimers with native sNASP and with tNASP as shown by our pull down experiments (Figure 4.5). Further experiments are required to identify components in NASP containing complexes, and more importantly, understand the physical interaction and functional links among these proteins.

Table 4. 1 SPR binding constants of NASP variants to histone H1 and H3/H4 tetramer.

Figure 4. 1 Recombinant NASP proteins.

A) Schematic diagrams of the sNASP constructs that have been generated. Each of the constructs also contains an NH2-terminal His6 tag. B) Each of the sNASP constructs shown in (A) was expressed in E. coli, purified by Ni2+-chelate chromatography and resolved by SDS-PAGE. Proteins were visualized by Coomassie Blue staining.

Figure 4. 2 in vitro binding of sNASP variants to core histones.

Full-length sNASP or sNASP mutants were each incubated with chicken histones and Ni2+-chelating beads. After extensive wash, bound proteins were eluted. Input proteins (IN), unbond proteins (FT) and bond proteins (EL) were resolved on SDS-PAGE and visualized by coomassie blue staining.

Figure 4. 3 Nucleosome assembly activity of sNASP variants.

Nucleosome assembly activity of recombinant full-length sNASP and its mutants with core histones was assayed by incubating the indicated factors with a relaxed circular plasmid. After incubation, the plasmids were extracted and resolved by 1.5% agarose gel electrophoresis, and visualized by SYBR Gold nucleic acid stain. The migration of the supercoiled (S) and relaxed (R) forms of the plasmid are indicated. Lane 1 and 2 show the template DNA before and after relaxation, respectively.

Figure 4. 4 Generation of inducible sNASP-expression U2OS cell lines

A) A U2OS cell line containing a stably integrated tet-inducible construct that expresses the full-length sNASP (with a His6 tag) was grown in the presence or absence of tetracycline as indicated. Whole cell extracts were resolved by SDS-PAGE and NASP proteins were visualized on Western blots probed with the indicated antibodies. Bands represented sNASP and tNASP are labeled. B) Cell lines that stablely express the sNASP-N, sNASP-C, sNASP-12E/K, sNASP-TPR4Δ and sNASP-NLSΔ were grown in the presence or absence of tetracyclines as indicated. The migration of specific forms of sNASP is indicated.

Figure 4. 5 Binding spectrums of sNASP and its mutants in vivo

Whole cell extracts were generated from U2OS cells containing stably integrated tet- inducible constructs that express the full-length sNASP or its mutants (with His6 tags). After incubation with Ni2+-chelating beads, unbound proteins were removed and bound proteins were eluted. Input proteins (IN), unbond proteins (FT) and bond proteins (EL) were resolved on SDS-PAGE. Proteins eluted with sNASP or its mutants were determined by western blot with antibodies indicated on the right.

Figure 4. 6 siRNA knockdown and inducible re-expression of sNASP.

U2OS cells were treated with either a non-specific controls siRNA (lane 1 and 3) or an siRNA specific for NASP (lane 2 and 4). Following siRNA treatment, one aliquot of cells was treated with tetracycline to induce expression of an sNASP construct that is not susceptible to the NASP siRNA treatment (lane 3 and 4).

Figure 4. 7 sNASP influences global chromatin structure.

U2OS cells were treated with either non-specific control siRNA or siRNA targeting NASP (as indicated). Expression of an siRNA-insensitive form of sNASP was induced with tetracycline (bottom). Isolated nuclei were digested with MNase for the indicated times and chromatin fragments were isolated and resolved by agarose gel electrophoresis.

Figure 4. 8 Expression of sNASP mutants cannot fully reverse the defects caused by depletion of NASP.

U2OS cells were treated with either non-specific control siRNA or siRNA targeting NASP (as indicated in the bottom). Expression of sNASP mutants was induced and indicated on the right. Isolated nuclei were digested with MNase for the indicated times and chromatin fragments were isolated and resolved by agarose gel electrophoresis.

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