Role of histone methylation and variants in genome function

The eukaryotic genome is compacted in the form of chromatin, which is a complex of DNA and histone proteins. Regulation of chromatin structure influences all aspects of cellular processes, especially the DNA -dependent processes. The basic unit of chromatin is composed of DNA wrapped around a core of octameric histones, forming what is called the nucleosome core particle. The dynamic nature of this structure implies that there will be well-regulated processes and pathways that help in the interchanges between one form of chromatin and another. In chapter 1, I outline the current state of literature for mechanisms that regulate chromatin structure, with special emphasis on histone modifications and histone variants. I also review the literature for genome maintenance and how chromatin regulates genomic integrity. One of the most critical histone modifications that regulate chromatin structure is the methylation of histone H3 at lysine 36 (H3K36me). Set2 catalyzes H3K36me and its function is well established in regulation of chromatin structure during transcription elongation, but its function in maintaining the integrity of yeast genome was not known. In chapter 2, I describe its novel function in regulating chromatin structure after double strand break (DSB). Work from chapter 2 reveals that Set2-dependent H3K36me (2/3) and its interaction with RNA polymerase II (RNAPII) is critical for surviving DSBs after phleomycin. It also shows that H3K36me is critical for full activation of a DSB checkpoint. Furthermore, I show that H3K36me is also important for chromatin remodeling around a DSB, abrogation of which subsequently facilitates inappropriate end-processing. In chapter 3, I describe our ongoing efforts to delineate the dynamic incorporation/eviction of the histone variant Htz1 in yeast. I show that deletion of NAP1 and CHZ1 results in increased retention of Htz1 in yeast chromatin, and show that there are two non-overlapping surfaces on the Htz1-H2B nucleosome. Furthermore we show that specific point mutations of these residues have biochemical and biological effects on cells. In chapter 4, I describe the implications of my research, place it in the wider context of chromatin research and discuss the contribution of H3K36me and Htz1 in tumorigenesis.Doctor of Philosoph

Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork

International audienceMCM2 is a subunit of the replicative helicase machinery shown to interact with histones H3 and H4 during the replication process through its N-terminal domain. During replication, this interaction has been proposed to assist disassembly and assembly of nu-cleosomes on DNA. However, how this interaction participates in crosstalk with histone chaperones at the replication fork remains to be elucidated. Here, we solved the crystal structure of the ternary complex between the histone-binding domain of Mcm2 and the histones H3-H4 at 2.9 ˚ A resolution. Histones H3 and H4 assemble as a tetramer in the crystal structure , but MCM2 interacts only with a single molecule of H3-H4. The latter interaction exploits binding surfaces that contact either DNA or H2B when H3-H4 dimers are incorporated in the nucleosome core particle. Upon binding of the ternary complex with the histone chaperone ASF1, the histone tetramer dissociates and both MCM2 and ASF1 interact simultaneously with the histones forming a 1:1:1:1 het-eromeric complex. Thermodynamic analysis of the quaternary complex together with structural model-ing support that ASF1 and MCM2 could form a chaperoning module for histones H3 and H4 protecting them from promiscuous interactions. This suggests an additional function for MCM2 outside its helicase function as a proper histone chaperone connected to the replication pathway

An integrated view of the essential eukaryotic chaperone FACT in complex with histones H2A-H2B

Summary: Structure of the FACT chaperone domain in complex with histones H2A-H2B, and a model for FACT-mediated nucleosome reorganization Nucleosomes are the smalles unit of chromatin: two coils of DNA are wrapped around a histone octamer core, which neutralizes its charge and `packs' the lengthy molecule. Nucleosomes confer a barrier to processes that require access to the eukaryotic genome such as transcription, DNA replication and repair. A variety of nucleosome remodeling machines and histone chaperones facilitate nucleosome dynamics by depositing or evicting histones and unwrapping the DNA. The eukaryotic FACT complex (composed of the subunits Spt16 and Pob3) is an essential and highly conserved chaperone. It assists the progression of DNA and RNA polymerases, for example by facilitating transcriptional initiation and elongation. Further, it promotes the genome-wide integrity of chromatin structure, including the suppression of cryptic transcription. Genetic and biochemical assays have shown that FACT's chaperone activity is crucially mediated by a direct interaction with histones H2A-H2B. However, the structural basis for how H2A-H2B are recognized and how this integrates with FACT’s other functions, including the recognition of histones H3-H4 and of other nuclear factors, is unknown. In my PhD research project, I was able to reveal the structure of the yeast chaperone domain in complex with the H2A-H2B heterodimer and show that the Spt16M module in FACT’s Spt16 subunit establishes the evolutionarily conserved H2A-H2B binding and chaperoning function. The structure shows how an alpha-helical `U-turn' motif in Spt16M interacts with the alpha-1-helix of H2B. The U-turn motif scaffolds onto a tandem pleckstrin-homology-like (PHL) module, which is structurally and functionally related to the H3-H4 chaperone Rtt106 and the Pob3M domain of FACT. Biochemical and in vivo assays validate the crystal structure and dissect the contribution of histone tails and H3-H4 toward FACT binding. My results show that Spt16M makes multiple interactions with histones, which I suggest allow the module to gradually invade the nucleosome and ultimately block the strongest interaction surface of H2B with nucleosomal DNA by binding the H2B alpha-1-helix. Together, these multiple contact points establish an extended surface that could reorganize the first 30 base-pairs of nucleosomal histone–DNA contacts. Further, I report a brief biochemical analysis of FACT’s heterodimerization domain. Its PHL fold indicates shared evolutionary origin with the H3-H4-binding Spt16M, Pob3M and Rtt106 tandem PHL modules. However, the Spt16D–Pob3N heterodimer does not bind histones, rather it connects FACT to replicative DNA polymerases. The snapshots of FACT’s engagement with H2A-H2B and structure-function analysis of all its domains lay the foundation for the systematic analysis of FACT’s vital chaperoning functions and how the complex promotes the activity of enzymes that require nucleosome reorganization.Zusammenfassung: Struktur der FACT Chaperon-Domäne im Komplex mit Histonen H2A-H2B, und ein Modell für die FACT-vermittelte Restrukturierung des Nukleosoms Nukleosomen sind die kleinsten Bausteine des Chromatin: das DNA Molekül wickelt sich in zwei Windungen um einen Oktamer aus Histon-Proteinen, die seine Ladung neutralisieren und es ordentlich `verpacken'. Deshalb sind Nukleosomen ein Hindernis für alle nukleären Prozesse, die Zugang zur DNA erfordern, wie zum Beispiel Transkription, Replikation oder Reparatur der DNA. Verschiedene Protein-Komplexe (ATP-abhängige `Remodeler' und ATP-unabhängige Histon-Chaperone) halten Nukleosomen in einem dynamischen und zugänglichen Zustand, indem sie Histone aus- oder ein-bauen, oder die DNA vom Oktamer abwickeln. Der eukaryotische FACT Komplex ist ein hochkonserviertes, heterodimeres Histon-Chaperon (aus den Unterheiten Spt16 und Pob3), das DNA und RNA Polymerasen unterstützt, durch Nukleosomen hindurchzuschreiben. Gleichzeitig stellt es sicher, dass die Chromatin-Integrität erhalten bleibt und unterdrückt dadurch z.B. Transkription von sogenannten kryptischen Promotoren. Genetische und biochemische Experimente haben gezeigt, dass die Interaktion mit Histonen, vor allem mit dem H2A-H2B Histon-Dimer, entscheidend für die Funktionalität von FACT als Histon Chaperon ist. Es fehlten jedoch molekulare oder strukturelle Informationen wie die Histone gebunden werden und wie dies mit den anderen biologischen Funktionen von FACT zusammenspielt, wie zum Beispiel der Interaktion mit Histonen H3-H4 oder anderen nukleären Faktoren, und letztendlich wie das reorganisierte Nukleosom aussehen könnte. In dieser Arbeit habe ich die H2A-H2B bindende Domäne von FACT, Spt16M, identifiziert und ihre Struktur im Komplex mit H2A-H2B gelöst. Die H2A-H2B Bindung habe ich biochemisch verifiziert, verfeinert und den Phänotyp von wichtigen Spt16M-Aminosäuren in vivo in Hefe analysiert. Ein strukturell und funktionell konserviertes, neuartiges `U-turn' (Kehrtwende) Motif interagiert mit der alpha-1-Helix des globulären Kerns von Histon H2B; diese hydrophobe Interaktion mit mikromolarer Affnität ist essentiell für die Komplex-Stabilität. Ein konservierter `acidic patch' (`negativ geladene Partie') interagiert zusätzlich mit dem unstrukturierten N-terminalen Ende von H2B und stabilisiert dadurch den Komplex kinetisch. Das Spt16M U-turn Motif ist auf ein Tandem-PHL (pleckstrin-homology like) Modul aufgebaut, das hohe strukturelle Verwandtschaft zu den Histon-Chaperonen Rtt106 und Pob3M aufweist. Wie Rtt106 und Pob3M bindet auch Spt16M Histone H3-H4. Die Interaktion wurde biochemisch auf die alpha-N-Helix von H3 eingegrenzt. Zusammenfassend bindet Spt16M an drei Stellen auf der Histon-Oktamer Oberfläche des Nukleosoms. Diese bilden eine zusammenhängende Fläche, welche die ersten 30 Basenpaare der nukleosomalen DNA koordiniert. Vermutlich erfolgt die Interaktion von Spt16M mit dem Nukleosom schrittweise: Zunächst bindet Spt16M über das frei zugänglichen N-terminale Ende von H2B an das Nukleosom. Dort `verharrt' das Chaperon bis die beiden stärkeren Interaktions-Stellen (die alpha-N Helix von H3 und die alpha-1 Helix von H2B), welche meist von DNA bedeckt sind, durch spontanes Ablösen der DNA freigelegt werden. Letztendlich würde die vollständige Bindung von FACT an das Nukleosom die ersten 30 Basenpaare DNA verdrängen und dadurch das Nukleosom destablisieren, so dass andere nukläere Prozesse (z.B. Polymerasen) auf die DNA Stränge zugreifen können. Des Weiteren habe ich die Heterodimerisierungs-Domäne von FACT biochemisch analysiert. Spt16D-Pob3N besteht ebenfalls aus PHL Domänen, diese können jedoch keine Histone binden. Stattdessen koppeln sie den Chaperon-Komplex an die DNA Replikations-Maschinerie. Die vorgestellten Ergebnisse legen den Grundstein für strukturelle und mechanistische Studien wie der holo-FACT Komplex mit dem Nukleosom interagiert, und wie sich dies in den Replikations- und Transkriptions-Prozess eingliedert

Characterisation of the (H3-H4)2-tetramer and its interaction with histone chaperones

Histones are a major protein component of chromatin, yet mechanisms which control synthesis, post translational modification, deposition and removal of histones are not fully understood. Although atomic resolution structures have been solved for the nucleosome core particle, there is limited information regarding the conformation of the core histones outside of chromatin. Insight into the structure of soluble histones, and the complexes they form, is likely to further our understanding of important nuclear processes such as transcription, chromatin replication and epigenetic inheritance. In this study we employ novel biochemical and biophysical techniques to address two key questions in the field: the structure of the soluble (H3-H4)2-tetramer, and the conformation of H3 and H4 in complex with histone chaperones.Firstly, we determined the conformation of histones H3 and H4 when in complex with two histone chaperones from S. cerevisaie, Nap1 and Vps75. Within the nucleosome H3 and H4 form a heterotetrameric structure sustained by the interface between two histone H3 proteins. Interestingly, when bound to the histone chaperone Asf1 the H3-H3’ interaction is disrupted, thus Asf1 effectively splits the tetramer binding a single H3-H4 dimer. Using targeted protein crosslinking and pulsed EPR we determine that, unlike Asf1, the Nap1 family of histone chaperones can bind H3-H4 in their tetrameric conformation, analogous to that observed within the nucleosome. The ability to bind H3 and H4 as a tetramer has implications in the prevalence of chromatin states during DNA replication and transcription, and may be in part responsible for the alternate in vivo functions of these two classes of chaperones.Secondly, using site direct spin labelling in conjunction with pulsed EPR we probe in detail the structure of the soluble (H3-H4)2-tetramer. Whilst the core crescent shape of the tetramer surrounding the H3-H3’ interface is retained, discrete regions such as the aN helix of H3 are more structurally heterogeneous than in the histone octamer or nucleosome. Such structural heterogeneity in the aN helix of H3 highlights potential roles in the post translational modification of histones and in their binding to histone-chaperones. These new findings reveal possible modes of interaction between a tetramer of H3-H4 and Nap1 proteins, and highlight the need for further investigation into histone – chaperone complexes.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF

Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B. Here, we show that the acidic domain of DNA repair factor APLF (APLF AD) can assemble the histone octamer in a single step and deposit it on DNA to form nucleosomes. The crystal structure of the APLF AD-histone octamer complex shows that APLF AD tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLF AD affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted

UNCOVERING THE BIOPHYSICAL MECHANISMS OF HISTONE COMPLEX ASSEMBLY

At the most basic level, inheritance in living beings occurs by passing the genomic information such as the DNA sequences from the parent generation to the offspring generation. Hence, it is a fundamental goal for every generation to efficiently express the genomic information and safely pass it on to the next generation. In human and other eukaryotic species, this mission is mediated via chromatin, a macromolecule with intricate hierarchical structure. The fundamental unit of chromatin is called a nucleosome, a complex of histone proteins wrapped around with DNA. To carry out diverse biological functions such as transcription and DNA replication, the DNA-protein complex must dynamically transition between more compact, closed states and more accessible, open ones. To fully understand the chromatin structure and dynamics, it is essential to comprehend the basic structural unit of chromatin, nucleosome. In this dissertation, I present my doctoral research in the exploration of the nucleosome dynamics problem, focusing on the assembly process of histone proteins. From histone monomer to dimer, then to tetramer, octamer, and nucleosome, I used different computational modeling theories and techniques, together with different experimental collaborations, to investigate the overall thermodynamics and specific mechanistic details of nucleosome dynamics at different levels. My work has shed light on the fundamental principles governing the histone protein folding and histone complex assembly, in particular, highlighting similarities and differences between the canonical and variant CENP-A histones

Polycation-π Interactions Are a Driving Force for Molecular Recognition by an Intrinsically Disordered Oncoprotein Family

Molecular recognition by intrinsically disordered proteins (IDPs) commonly involves specific localized contacts and target-induced disorder to order transitions. However, some IDPs remain disordered in the bound state, a phenomenon coined "fuzziness", often characterized by IDP polyvalency, sequence-insensitivity and a dynamic ensemble of disordered bound-state conformations. Besides the above general features, specific biophysical models for fuzzy interactions are mostly lacking. The transcriptional activation domain of the Ewing's Sarcoma oncoprotein family (EAD) is an IDP that exhibits many features of fuzziness, with multiple EAD aromatic side chains driving molecular recognition. Considering the prevalent role of cation-π interactions at various protein-protein interfaces, we hypothesized that EAD-target binding involves polycation- π contacts between a disordered EAD and basic residues on the target. Herein we evaluated the polycation-π hypothesis via functional and theoretical interrogation of EAD variants. The experimental effects of a range of EAD sequence variations, including aromatic number, aromatic density and charge perturbations, all support the cation-π model. Moreover, the activity trends observed are well captured by a coarse-grained EAD chain model and a corresponding analytical model based on interaction between EAD aromatics and surface cations of a generic globular target. EAD-target binding, in the context of pathological Ewing's Sarcoma oncoproteins, is thus seen to be driven by a balance between EAD conformational entropy and favorable EAD-target cation-π contacts. Such a highly versatile mode of molecular recognition offers a general conceptual framework for promiscuous target recognition by polyvalent IDPs. © 2013 Song et al

The impact of the chromatin regulators, Abo1 and HIRA, on global nucleosome architecture

PhD ThesisHIRA is an evolutionarily conserved histone H3-H4 chaperone that mediates replication-independent nucleosome deposition and is important in a variety of contexts such as transcription, the response to DNA damage and cellular quiescence. Here the genome-wide contribution of HIRA to nucleosome organization in Schizosaccharomyces pombe was determined using a chromatin sequencing approach. Cells lacking HIRA (hip1Δ) experience a global reduction in nucleosome occupancy over the 3’ end of genes, consistent with the proposed role for HIRA in nucleosome re-assembly in the wake of RNA polymerase II. In addition, at HIRA-regulated promoters, it commonly maintains the proper occupancy of the -1 and +1 nucleosomes. Thus HIRA likely exerts its transcriptional regulatory roles through assembly/disassembly of specific target nucleosomes. In addition to transcription-coupled functions, HIRA has been implicated in the DNA damage response pathway. Indeed HIRA deficient cells present with increased sensitivity to DNA damaging agents and experience delays to the repair of DNA double strand breaks. Furthermore, hip1+ exhibits interactions with components of both the homologous recombination (HR) and non-homologous end joining (NHEJ) repair pathways. HIRA has also been identified as a regulator of nitrogen-starvation induced quiescence in S. pombe. Cells lacking HIRA are defective in both their ability to maintain and exit quiescence. Consistent with this, quiescent hip1Δ cells fail to properly induce MBF-dependent gene transcription in response to the restoration of a nitrogen source. During the course of this study Abo1, a bromodomain containing AAA-ATPase, was identified as a factor whose function potentially overlaps with histone chaperones such as HIRA. Therefore the contribution of Abo1 to global chromatin architecture was also assessed. Consistent with a nucleosome assembly function, abo1Δ cells have widespread changes to nucleosome occupancy and positioning in both euchromatic and heterochromatic regions of the genome. Furthermore, Abo1 physically interacts with the FACT histone chaperone and the distribution of Abo1 on chromatin is perturbed by loss of FACT subunits.Medical Research Council (MRC) and the National Institute for Health Research (NIHR)