Genome-Wide Localization And Novel Deposition Pathways Of Histone Variant H3.3 In Embryonic Stem And Neuronal Precursor Cells

The eukaryotic genome is composed of chromatin, a complex polymer of genomic DNA, RNA, and closely associated proteins. Histone proteins form the core of the nucleosome, the fundamental repeating unit of chromatin. Variant histone proteins play critical roles in the epigenetic regulation of gene expression and in the development of multicellular organisms. In this thesis, I describe the first genome-wide profiles of histone H3 variants in pluripotent mammalian embryonic stem (ES) cells, and I establish the dependence and independence of these patterns on the histone chaperone Hira. To distinguish H3 variants, I use designed zinc finger nucleases (ZFNs) to rapidly knock epitope tags into a single allele of the endogenous histone H3.3B gene in mouse ES cells. Genome-wide analysis reveals that H3.3 is enriched in specific patterns at active and repressed genes with high CpG content promoters, including developmentally repressed bivalent H3K4me3 / H3K27me3 genes in ES cells, in addition to transcribed non-coding regions, telomeres, ribosomal DNA (rDNA), and genic and intergenic transcription factor binding sites (TFBS). Transcriptional termination sites of highly transcribed genes are marked by peaks of H3.3 and phosphorylated RNA polymerase II. Differentiation of ES cells into neural precursor cells (NPCs) leads to specific changes in H3.3 localization, demonstrating that the localization of H3.3 is dependent on cellular state. Targeted gene editing of H3.3B to H3.2 or H3.1 using ZFNs demonstrates that these patterns are dependent on the amino acid sequence of endogenous H3.3. Using wild-type and Hira -/- ES cells, I show that the H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for deposition of H3.3 at rDNA, telomeres, and specific TFBS. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Using Atrxflox and Atrxnull mouse ES cells, I find that Atrx is specifically required for Hira-independent enrichment of H3.3 at telomeres and rDNA, and for repression of telomeric and ribosomal RNA. Overall, the data in this thesis demonstrate that multiple and distinct pathways are responsible for H3.3 deposition at specific genomic locations in mammalian cells

The oncogenic role of histone chaperone ASF1 proteins in solid tumors

Chromatin is the essential medium connecting regulatory signals such as transcription factors and signaling pathways to the alteration of gene activity and cellular phenotypes. Aberrant chromatin (epigenetic) environment plays an important role in carcinogenesis. The fundamental unit of chromatin is the nucleosome which is composed of a histone core wrapped with 145-147 base pairs of DNA around. In the last decades, great efforts have been made to delineate the role of aberrant DNA methylation and chromatin/histone-remodeling factors in oncogenesis. However, recent evidence has merged that the dysregulation of histone chaperones also acts as a cancer-driver. Anti-silencing function 1 (ASF1) is the most conserved histone H3-H4 chaperone, regulating histone metabolism. ASF1 proteins include two paralogs ASF1A and ASF1B in mammals. ASF1A and ASF1B have been reported as oncogenes in human cancers. Data from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases show that ASF1A and ASF1B are overexpressed in 20 and 24 different types of cancers, respectively. Thus, in this thesis, I explored the oncogenic role of histone chaperone ASF1 and underlying molecular mechanisms in several solid tumors. In Paper I, the role for ASF1A in gastrointestinal cancer (GIC) was investigated. We discovered that ASF1A interacted with the oncogenic transcription factor β-catenin and promoted the transcription of β-catenin target genes (c-MYC, cyclin D1, ZEB1 and LGR5). The increased expression of these genes stimulated proliferation, stemness and migration/invasion of GIC cells. Over-expression and knockdown of ASF1A boosts and inhibits in vivo tumor growth and/or metastasis in mouse models, respectively. Higher levels of ASF1A expression predict significantly shorter patient survival in colorectal cancer (CRC). Further analyses of the Gene Expression Omnibus dataset validate higher ASF1A expression predicting a poor prognosis in CRC patients. Taken together, this study reveals the novel function of ASF1A as a transcription co-factor independent of its canonical role and the potential value of ASF1A for outcome prediction and targeted treatment in GIC. In Paper II, we show that ASF1A overexpression is widespread in human malignancies and is required for the infinite proliferation of cancer cells. When ASF1A was knocked-down in wild-type (wt) p53 carrying cells derived from hepatocellular carcinoma (HCC) and prostate cancer (PCa), DNA damage response was activated and up-regulation of p53-p21cip1 expression consequently occurred. These cells eventually underwent cellular senescence. Higher ASF1A expression and/or lower p21cip1 expression predicts a poor outcome in HCC patients. Thus, ASF1A may be a therapeutic target and a prognostic factor in HCC and other cancers. In Paper III, we evaluated whether ASF1B has diagnostic and prognostic values in adrenocortical carcinoma (ACC) and regulates invasion and metastasis. We first analyzed TCGA and GTEx data and found that the ASF1B gene was amplified in two thirds of ACC tumors and associated with its overexpression. ASF1B expression correlates with the ACC diagnostic criteria of the Weiss scoring system. Higher ASF1B expression and ASF1B copy number predict a poor outcome in the TCGA cohort of ACC patients. Knockdown of ASF1B in ACC cells impairs migration and invasion ability by inhibiting expression of the transcription factor FOXM1; whereas ASF1B over-expression exhibits opposing effects. These findings suggest that ASF1B may be a useful factor for ACC diagnostics and prognostication, and potentially a novel target for ACC therapy as well. Collectively, the results presented in this thesis gain profound insights into the oncogenic role of ASF1 in several solid tumors and demonstrated novel activities of ASF1 proteins beyond their conserved histone chaperone function. These findings will inspire further exploration of both the clinical and biological roles of ASF1 in precision oncology

Proteomic analysis of histone mark crosstalk at bivalent domains

The combination and interaction of histone marks and DNA-associated proteins are critical in the regulation of gene transcription. Individual histone marks have been associated with different gene expression states - histone H3 lysine 4 trimethylation (H3K4me3) is associated with “open” chromatin and active transcription while H3K27me3 is associated with “closed” chromatin and a repressive transcriptional state. In certain cases, these marks have been shown to co-localise at genomic loci, and on the same nucleosome. The co-localisation of active H3K4me3 and repressive H3K27me3 marks at CpG promoters is a hallmark of bivalent domains. Bivalent domains have been implicated in priming developmental genes for timely activation. However, the complex network of proteins that bind to these domains to regulate and mediate their influence on transcription is unknown. This study has developed tools to enable the characterisation of the protein networks bound to bivalent domains and other specifically modified nucleosomes. In vitro synthesised specifically modified nucleosomes were utilised in pulldown assays with embryonic stem cell (ESC) nuclear extract to isolate the specific protein binders for different combinations of histone marks. This assay was validated by comparison of proteins bound to symmetrically modified nucleosomes with previously identified protein binders. A comparison of symmetrically and asymmetrically modified nucleosomes has elucidated new binding preferences for known proteins. Analysis of proteins bound to asymmetrically modified nucleosomes showed previously unknown binding affinities and conformational preferences. TAF3, a known H3K4me3 mark binder, prefers to bind symmetrically rather than asymmetrically modified nucleosomes, even when the same amount of the mark is present. Therefore, this preference is not solely dependent on the amount of modification, but also due to the conformation of the marks on the nucleosomes. We have identified multiple key proteins that prefer binding to this specific mark (H3K4me3/K27me3) conformation, including the acetyltransferase KAT6B. Work in mouse ESCs confirmed KAT6B binding to bivalent domains and showed a pronounced differentiation defect in KAT6B-/- cells due to mis-regulation of genes important in development. Further characterisation of these proteins and their interactions will help to clarify bivalent domain function

Characterization of Nucleosomes Containing Specific Forms of the Histone Variant H2A.Z

H2A.Z is a highly conserved histone variant that replaces canonical histone H2A at specific loci to regulate diverse nuclear processes. Amongst these, the role of H2A.Z in transcriptional regulation is of particular interest due to its enrichment at promoters of most genes in yeast and higher eukaryotes. However, its precise role in regulation is complex, as it has been linked to both repression and activation. One possibility is that H2A.Z activity is regulated by post-translational modifications since H2A.Z can be acetylated or monoubiquitylated in mammals. For example, H2A.Z can be multiply acetylated at several lysine residues at its N-terminus, and such modified form is associated with active promoters. In contrast, our lab has previously shown that a fraction of H2A.Z is monoubiquitylated at its C-terminus, and this form is associated with silent chromatin. One aim of this thesis is to characterize monoubiquitylated H2A.Z-nucleosomes in the context of transcriptional regulation. To this end, we devised a biotinylation-based method to enrich for H2A.ZUb1-mononucleosomes, and further characterized their composition and genomic distribution. In the second chapter, I demonstrate that H2A.ZUb1-enriched mononucleosomes are enriched with the histone post-translational modification H3K27me3, but depleted of H3K4 methylation and other modifications associated with transcriptional activity. H2A.ZUb1-eniched mononucleosomes also preferentially co-purify with proteins typically involved in repression, and with CTCF and cohesin. Consistent with these, ChIP-Seq analysis of H2A.ZUb1-nucleosomes identifies non-expressed genes as sites of H2A.ZUb1 enrichment. In addition to post-translational modification, vertebrate H2A.Z is differentiated into non-allelic isoforms H2A.Z-1 and H2A.Z-2. Previously, we used mass-spectrometry to identify proteins that preferentially associate with H2A.Z-mononucleosomes over H2A-mononucleosomes. In the third chapter, I show that one of these proteins, USP39, is enriched on mononucleosomes containing H2A.Z-1 over those containing H2A.Z-2, and that this selectivity can be mapped to an isoform-specific residue in its C-terminal tail. USP39 is a component of the U4/U6.U5 tri-snRNP, and consistent with a functional link between H2A.Z-1 and USP39, we identify a subset of shared alternative splicing events. Altogether, these data support functional diversification of H2A.Z through monoubiquitylation and isoform-specific amino acid substitution, and collectively, contribute to our understanding of biological pathways converging on H2A.Z

Control of telomeric homology-directed repair by poly(ADP-ribose) metabolism

Immediately after single-stranded break (SSB) and double-stranded break (DSB) formation, the synthesis of poly(ADP-ribose) (PAR) reconfigures the local chromatin environment and initiates recruitment of DNA repair proteins. The degradation of PAR chains by poly(ADP-ribose) glycohydrolase (PARG) is essential for DNA repair progression. Here, we show that pharmacological interference of PAR metabolism disrupts the homology-directed repair (HDR) mechanisms that mediate alternative lengthening of telomeres (ALT). Using a proteomics strategy, we uncovered PAR-regulated telomere-associated proteins that coordinate the early stages of the ALT mechanism. These distinct factors exhibit PAR dependency for localization to ALT telomeres in order to orchestrate diverse functions, such as RNA stabilization, actin nucleation, and chromatin remodeling. Most significantly, we identified a key function for PARylation in recruiting the HIRA histone chaperone complex to ALT telomeres, where it is required for deposition of histone H3.3 specifically during G2 Break-Induced Replication (G2-BIR). We propose that HIRA acts to compensate for the loss of a functional ATRX-DAXX complex in ALT cancers and therefore adopts elevated importance in sustaining ALT+ cell viability

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

On the epigenetic ageing clock in humans

Epigenetic clocks are mathematical models that predict the biological age of an organism using DNA methylation data, and which have emerged in the last few years as the most accurate biomarkers of the ageing process. However, little is known about the molecular mechanisms that control the rate of such clocks. In this thesis I focus on the study of the epigenetic ageing clock in humans. First, I review and benchmark statistical and computational tools required for the analysis of DNA methylation data in the context of human ageing. Next, I validate the performance of the Horvath epigenetic clock, the most widely used multi-tissue epigenetic clock in humans, in a control blood dataset and test its behaviour in patients with a variety of developmental disorders, which harbour mutations in proteins of the epigenetic machinery. I demonstrate that loss-of-function mutations in the H3K36 methyltransferase NSD1, which cause Sotos syndrome, substantially accelerate epigenetic ageing. Furthermore, I show that the normal ageing process and Sotos syndrome share methylation changes and the genomic context in which they happen. These results suggest that the H3K36 methylation machinery is a key component of the epigenetic maintenance system in humans, which controls the rate of epigenetic ageing, and this role seems to be conserved in model organisms. Finally, I provide a technological strategy to make epigenetic clocks (or any DNA methylation-based mathematical models) more cost-effective by exploiting the ability of restriction enzymes to perform genomic enrichment. This thesis provides novel insights (statistical, biological, technological) into the epigenetic ageing clock in humans, which will help to shed light on the different processes that erode the human epigenetic landscape during ageing.EMBL PhD programm

Xenopus

This book focuses on the amphibian, Xenopus, one of the most commonly used model animals in the biological sciences. Over the past 50 years, the use of Xenopus has made possible many fundamental contributions to our knowledge in cell biology, developmental biology, molecular biology, and neurobiology. In recent years, with the completion of the genome sequence of the main two species and the application of genome editing techniques, Xenopus has emerged as a powerful system to study fundamental disease mechanisms and test treatment possibilities. Xenopus has proven an essential vertebrate model system for understanding fundamental cell and developmental biological mechanisms, for applying fundamental knowledge to pathological processes, for deciphering the function of human disease genes, and for understanding genome evolution. Key Features Provides historical context of the contributions of the model system Includes contributions from an international team of leading scholars Presents topics spanning cell biology, developmental biology, genomics, and disease model Describes recent experimental advances Incorporates richly illustrated diagrams and color images Related Titles Green, S. L. The Laboratory Xenopus sp. (ISBN 978-1-4200-9109-0) Faber, J. & P. D. Nieuwkoop. Normal Table of Xenopus laevis (Daudin): A Systematical & Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis (ISBN 978-0-8153-1896-5) Jarret, R. L. & K. McCluskey. The Biological Resources of Model Organisms (ISBN 978-1-0320-9095-5

Xenopus

This book focuses on the amphibian, Xenopus, one of the most commonly used model animals in the biological sciences. Over the past 50 years, the use of Xenopus has made possible many fundamental contributions to our knowledge in cell biology, developmental biology, molecular biology, and neurobiology. In recent years, with the completion of the genome sequence of the main two species and the application of genome editing techniques, Xenopus has emerged as a powerful system to study fundamental disease mechanisms and test treatment possibilities. Xenopus has proven an essential vertebrate model system for understanding fundamental cell and developmental biological mechanisms, for applying fundamental knowledge to pathological processes, for deciphering the function of human disease genes, and for understanding genome evolution. Key Features Provides historical context of the contributions of the model system Includes contributions from an international team of leading scholars Presents topics spanning cell biology, developmental biology, genomics, and disease model Describes recent experimental advances Incorporates richly illustrated diagrams and color images Related Titles Green, S. L. The Laboratory Xenopus sp. (ISBN 978-1-4200-9109-0) Faber, J. & P. D. Nieuwkoop. Normal Table of Xenopus laevis (Daudin): A Systematical & Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis (ISBN 978-0-8153-1896-5) Jarret, R. L. & K. McCluskey. The Biological Resources of Model Organisms (ISBN 978-1-0320-9095-5