Doctor of Philosophy

dissertationOne central question in development is how totipotency and pluripotency are established. In mature human sperm, genes of importance for embryo development (i.e. transcription factors) lack DNA methylation and bear nucleosomes with distinctive histone modifications, suggesting the specialized packaging of these developmental genes in the germline. Here, we explored the tractable zebrafish model and found conceptual conservation as well as several new features. Biochemical and mass spectrometric approaches reveal the zebrafish sperm genome packaged in nucleosomes and histone variants (and not protamine), and we find linker histones high and H4K16ac absent-key factors which may contribute to genome condensation. We examined several activating (H3K4me2/3, H3K14ac, H2AFV) and repressing (H3K27me3, H3K36me3, H3K9me3, hypoacetylation) modifications/compositions genome-wide, and find developmental genes packaged in large blocks of chromatin with coincident activating and repressing marks and DNA hypomethylation, revealing complex "multivalent" chromatin. Notably, genes that acquire DNA methylation in the soma (muscle) are enriched in transcription factors for alternative cell fates. Remarkably, we find H3K36me3 located in "silent" developmental gene promoters, and not present at the 3' ends of coding regions of genes heavily transcribed during sperm maturation, suggesting different rules for H3K36me3 in iv the germline and soma. We also reveal the chromatin patterns of transposons, rDNA, and tRNAs. Finally, high levels of H3K4me3 and H3K14ac in sperm are correlated with genes activated in embryos prior to the mid-blastula transition (MBT), whereas multivalent genes are correlated with activation at or after MBT. Taken together, gene sets with particular functions in the embryo are packaged by distinctive types of complex and often atypical chromatin in sperm. Bivalent marks, as the chromatin signature of pluripotency, are not persistent and diluted during early synchronous cell division, making them arguable to be heritable epigenetic marks. Studies in early embryos indicate DNA methylation status is the fundamental to confer totipotency and pluripotency. The anticorrelation between DNA methylation profiles and H2A.Z occupancy is conserved from plants to vertebrates. Here, we examined H2afva occupancy in early embryos in zebrafish by ChIP-seq. We found both H2afva level and enrichment remain consistent from sperm to embryos. H2afva is enriched in proximal promoter region in the first nucleosome. Consistent with previous studies, H2afva occupancy is anticorrelated to DNA methylation both in the promoters and outside of promoters. These data suggest H2afva is potentially a heritable epigenetic mark and sets up DNA methylation profiles of totipotency and pluripotency

The role of the histone variant H2A.Z in early Xenopus laevis development

The genome of eukaryotes is packaged into the small volume of the nucleus in an organised manner. This structure of DNA and associated proteins is called chromatin. The basic unit of chromatin is the nucleosome; an octomer of core histone proteins and associated DNA. Other proteins such as linker histones can also associate with the DNA or the core histones. The modular structure of chromatin allows for structural variation with functional consequences including activation or repression of transcription. Alterations can include post-translational modifications to histones, remodelling by multi-protein complexes, DNA methylation, and non-allelic variants of the canonical histones. Changes to chromatin structure have an important impact on all DNA processing events. This thesis investigated the histone variant H2A.Z, a variant of the canonical core histone H2A. H2A.Z is highly conserved and essential in a number of species suggesting it has a critical function. Preliminary work using the Xenopus laevis developmental model system had revealed that disruption of H2A.Z function resulted in defective embryo morphology consistent with disrupted gastrulation and mesoderm development (Ridgway et al., 2004a). This led to the following hypothesis: H2A.Z is important to gastrulation and mesodermal development in X laevis because it plays a developmental role. Temporal and spatial expression patterns of H2A.Z mRNA demonstrated in this study are consistent with a role in mesoderm development. Peak H2A.Z mRNA expression levels occur during gastrulation. H2A.Z mRNA is enriched in the marginal zone of the late blastula, involuting tissue in the gastrula and in notochord (a mesodermal tissue) in tailbud embryos. Significantly, maternal H2A.Z mRNA is enriched asymmetrically in dorsal cells of the early blastula before zygotic transcription, indicating that H2A.Z may play a role in determining polarity of the dorsal ventral axis. Two important processes for development were examined in this thesis: cell fate and cell movement. Determination of mRNA levels and localisations for a selection of mesodermal marker genes indicates that cell fate programs progress normally in embryos where H2A.Z function is disrupted. However, the localisation of mesoderm derived cells is perturbed suggesting cell movement is perturbed. Taken together these studies suggest the H2A.Z histone variant has a specific role in regulating cell mobility during early Xenopus laevis development

Chromatin Remodeling and Transcriptional Memory: A Dissertation

Transcriptional regulation of gene expression is critical for all unicellular and multicellular organisms. The ability to selectively induce or repress expression of only a few genes from the entire genome gives cells the ability to respond to changing environmental conditions, grow and proliferate. Multicellular organisms begin life as a single totipotent cell, which undergoes many cell divisions during embryonic and later postnatal development. During this process, the dividing cells of the embryo progressively lose their pluripotency and adopt restricted cell fates. Cell fate restriction leads different cell types to gain unique transcriptional profiles. This transcriptional profile or gene expression pattern not only defines the cell types and restricts the ways in which they can respond to signals, it also has to be faithfully re-established in the progeny of these fate-restricted cells when they divide. Different mechanisms have evolved in multicellular organisms to propagate transcriptional memory of cell identity. Most of mechanisms involve modifications of chromatin such as epigenetic modification of DNA or alterations of associated histones. In contrast to multicellular organisms which have considerable cellular diversity and a long lifespan for which cell fates and transcriptional memory needs to be maintained, single celled budding yeast, Sachharomyces cerevisiae have a life cycle of about 90 minutes in normal nutrient rich conditions. However, even budding yeast have tremendous potential to respond to changing environmental conditions like nutrient availability by inducing expression of various genes. We observed that members of the GAL gene cluster, which encodes genes induced in response to and for metabolizing the sugar galactose, showed heritable transcriptional memory of previous activation. This dissertation thesis describes the studies I have done for my graduate research to define this phenomenon of transcriptional memory at the yeast GALgenes and to determine the mechanism by which it can be formed and inherited. Chapter I gives an introduction to different mechanisms of establishing transcriptional memory in unicellular and multicellular organisms. Chromatin based mechanisms have been well studied in multicellular organisms but not observed in budding yeast. We compare chromatin based or nuclear inheritance with cytoplasmic inheritance that can be observed in yeast. Chapter II describes work done to define the phenomenon of transcriptional memory at GAL1 gene. We define this as a faster rate of induction of the GAL1 gene, compared to a naïve gene, after a brief period of repression. We show that this cellular memory persists through mitosis and can be passed on to the next generation. We also show that chromatin remodeling enzymes appear to be required for rapid reinduction, raising the question if yeast may also possess chromatin associated, nuclear mechanisms for cellular memory. Chapter III describes experiments that show that cellular memory observed at GAL1 is cytoplasmic in nature and also compares our work with similar examples observed recently by other groups. Finally, Chapter IV offers a perspective of the significance of such cellular memory mechanisms in budding yeast and outlines some potential further experiments to better understand the control of GAL1 induction kinetics

Analysis of the Ies6 subunit of the INO80 chromatin remodelling complex

The INO80 complex is a large ATPase chromatin remodeller which contains 15 accessory subunits in S.cerevisiae. Its subunits include the highly conserved ATPases Ruvb1 and Ruvb2, the actin-related proteins Arp5, Arp8, Act1 and Arp4, Actin, and a number of IES (I̱noE̱ighty S̱pecific) subunits Ies1, Ies2, Ies3, Ies4, Ies5 and Ies6, in addition to subunits Nhp10 and Taf14. All 15 of the accessory subunits are assembled around a catalytic core component known as Ino80. The INO80 complex has roles in transcription, DNA repair, replication, and chromosome segregation. These roles are in addition to its traditional nucleosome remodelling activities and the dispacement of H2A.Z from chromatin. Recent studies in S. cerevisiae have identified the subunit Ies6 as a critical component of the INO80 complex. Deletion of IES6, which encodes the small accessory subunit, clearly mimics the deletion f the gene encoding the catalytic subunit, INO80. Surprisingly, only one domain within Ies6 has been formally identified based on sequence analysis. This domain belongs to the L1_C class of domains. Such domains are commonly associated with DNA binding activity and transcription factors. This stud has further characterised the Ies6 subunit both genetically and biochemically. Genetically, it has demonstrated that single point mutations at regions of proposed subunit-subunit interaction between the Arp5 or Rvb2 subunits, or within the YL1_C are not sufficient to disrupt Ies6 function. However, expression of a double point mutation, ies6(K114E/Y125A), in combination with rad50 deletion, caused a sensitivity to replication inhibition, but not chromosome segregation inhibition, indicating a potential separation of function in this utant due to the loss due of only one of the biological functions of Ies6. Biochemically, we have confirmed that DBA binding capacity of Ies6 resides within the YL_C domain. In addition, although it has been demonstrated that the removal of H2A.Z acetylation exacerbates the increase in cellular ploidy observed in ies6 null cells, we found that overall levels of H2A.Z acetylation were not influenced by the loss of Ies6. This indicates that the role of H2A.Z acetylation in chromosome segregation may only affect ploidy status upon the loss of Ies6. In addition, work on the R2TP complex (which contains the INO80 APases Ruvb1/Ruvb2, and subunits Tah1 and Phi1) has revealed the recruitment mechanism for the molecular chaperone, Hsp90, and the telomere length regulation protein, Tel2. Together, the R2TP complex, Hsp90 and Tel2 promote the stabilisation and maturation of multi-protein complexes. These include Phosphatidylinositol 3-kinase-related kinases (PIKKs, a family of kinases involved i Serine and Threonine phosphorylation), subunits of the INO80 complex and subunits of the SWR1 chromatin remodelling complex (a partner comlex to INO80 that incorporates H2A.Z into chromatin)

Regulation of Histone Covalent Modifications During Yeast Apoptosis

Chromatin compaction is a hallmark property of apoptosis, a highly coordinated suicide mechanism generally believed to be confined to vertebrates. However, invertebrates such as the budding yeast, Saccharomyces cerevisiae, display an apoptotic-like phenotypes including chromatin condensation, although its functions and mechanism are unclear. One mechanism that alters chromatin structure is the covalent modification of histones, which associates with DNA to form the nucleosome, the fundamental unit of chromatin. Phosphorylation of histone H2B at serine 14 (H2BS14ph), catalyzed by Mst1 kinase, has been linked to chromatin compaction during mammalian apoptosis. I extended these results to yeast by demonstrating that Ste20 kinase, a yeast orthologue of Mst1, directly phosphorylates H2B at serine 10 (H2BS10ph) in a hydrogen peroxide-induced cell death pathway. Unlike Mst1, Ste20 translocates into the nucleus in a caspase-independent fashion to mediate phosphorylation of H2B. H2BS10ph is dependent on the removal of acetylation mark on adjacent lysine residue, (H2BK11ac), which exists in growing yeast. During yeast apoptosis, the HDAC Hos3 deacetylates K11, which in turn, mediates H2BS10ph by Ste20 kinase. My studies underscore a concerted series of enzyme reactions governing histone modifications that promote a switch from cell proliferation to cell death. Moreover, the conservation of targeted H2B phosphorylation and the enzyme system point to an ancient, late-stage chromatin remodeling event that likely governs cellular homeostasis in a wide range of organisms. H2B phosphorylation may mediate apoptotic chromatin compaction by 1) directly affecting internucleosomal contacts and histone DNA interaction (“cis mechanismâ€), or 2) recruiting binding partners that then induce and direct downstream functions (“trans†mechanisms). Peptides corresponding to the phosphorylated form of yeast H2B and human H2B have the intrinsic ability to form “aggregates†in SDS polyacrylamide gel electrophoresis. In addition, nucleosome array containing yeast S10E or human S14E H2B fold into compacted conformation as measured by analytical ultracentrifugation. Moreover, an interaction between the forkhead homology-associated domain 1 (FHA1) of Rad53 and H2BS10ph was uncovered. This interaction inactivates the DNA damage checkpoint pathway and promotes apoptotic chromatin condensation. Thus, both mechanisms may contribute to chromatin remodeling event that govern apoptotic chromatin compaction in a pathway conserved from yeast to humans

Chromatin regulation by the histone acetyltransferase MOF in Drosphila

Like in mammals, sex determination in Drosophila melanogaster involves an unequal distribution of sex chromosomes, with male flies carrying an X and a Y chromosome, as compared to two Xs in females. To prevent the deleterious effects of chromosomal aneuploidy, flies have evolved a dosage compensation system, which upregulates transcription from the single male X chromosome to match transcript levels produced from the two female Xs. This transcriptional activation is achieved by the dosage compensation complex (DCC), a ribonucleoprotein complex consisting of five male specific lethal proteins (MSL) and two non coding RNAs on the X (roX). The DCC is physically tethered to hundreds of target loci along the male X chromosome, where it promotes hyper acetylation of X-linked chromatin at Lysine 16 of histone H4 (H4K16ac). This histone mark is associated with an open, permissive chromatin structure, and its enrichment on the male X chromosome is thought to be required for the twofold increase in X-linked transcription during dosage compensation. However, the exact mechanism by which X-linked transcription is activated in males is still unknown. Responsible for hyper-acetylation of the male X chromosome is the histone acetyltransferase males absent on the first (MOF), which is part of the DCC. Recent studies have shown that MOF plays an additional role in autosomal gene regulation, as it has been found at thousands of autosomal gene promoters as part of the non specific lethal (NSL) complex. However, to what extent H4K16ac at autosomal genes is MOF-dependent, and how MOF is differentially distributed between the two complexes is currently unknown. During the course of my PhD, I used genetic, biochemical, and genomewide approaches to address a wide range of questions, concerning MOF functions in autosomal gene regulation and dosage compensation; the DCC recruitment process to X-linked target genes; and the mechanism of transcriptional upregulation of X-linked genes during dosage compensation. Besides other contributions, investigating the role of the H3K36 specific methyltransferase HypB/Set2 during MSL targeting and dosage compensation, as well as the role of MOF for NSL function at autosomal promoters, I was addressing these questions in the context of two main projects. During the first one of these, I have been able to show that MOF is responsible for genomewide H4K16ac in male and female flies, and that MOF is an essential gene in females. I demonstrated that the Drosophila specific unstructured N-terminus of the MOF protein is required for assembly of the DCC on the male X chromosome, and at the same time constrains MOFs HAT activity. The N-terminus therefore controls MOFs function in X chromosome compensation. I was furthermore able to reveal the biological role of the chromobarrel domain, which is conserved from yeast to human. Unexpectedly, disruption of the MOF chromobarrel domain, which has been shown previously to be required for MOF interaction with roX RNAs, led to a dramatic loss of H4K16ac from all chromosomes. Accordingly, I showed that the chromobarrel domain serves to trigger H4K16ac after the recruitment of MOF to its chromatin targets, revealing for the first time a biological role of this domain in vivo. In a parallel project, to work towards unraveling of the dosage compensation mechanism, I wanted to identify the step in the RNA PolII transcription cycle at which dosage compensation operates in flies. To this end, I generated genomewide profiles of RNA PolII in 3rd instar larva salivary glands from male and female flies, and from male flies with disrupted dosage compensation. Strikingly, we find that the density of PolII is approximately twofold elevated on the male X chromosome as compared to autosomes, including X-linked promoters. This data suggests that dosage compensation operates via enhanced transcription initiation, which constitutes a major advance in our understanding of the dosage compensation process