published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Vernalization is defined as the response to prolonged cold exposure required for acquiring the molecular competence necessary to undergo floral transition. FLOWERING LOCUS C (FLC), a potent floral repressor in Arabidopsis, is highly expressed before vernalizing cold treatment but is repressed during prolonged vernalization. VERNALIZATION INSENSITIVE 3 (VIN3) is a Plant HomeoDomein (PHD)- containing protein that is required for establishing vernalization-mediated repression of FLC. The induction of VIN3 is one of the earliest molecular events in vernalization response and its expression is intimately linked to prolonged cold exposure. However, mechanisms underlying VIN3 induction remain poorly understood. The constitutive repression of VIN3 in the absence of cold is due to multiple repressive components, including a transposable element-derived sequence, LIKE-HETEROCHROMA TIN PROTEIN 1 (LHP1), and POLYCOMB REPRESSION COMPLEX 2 (PRC2). Furthermore, the full extent of VIN3 induction by vernalization requires activating complex components, including EARLY FLOWERING 7 (ELF7) and EARLY FLOWERING IN SHORT DAYS (EFS). Dynamic changes in the histone modifications present at VIN3 chromatin during vernalization were also observed, indicating that chromatin changes play a critical role in regulating VIN3 induction. However, VIN3 induction by vernalization still occurs in the absence of activation complexes and de- repression of VIN3 in the absence of the repressive complexes is not sufficient for achieving complete induction. Thus, unknown cold-influenced regulators responsible for achieving maximum VIN3 induction during vernalization must exist. Therefore, forward genetic screening was undertaken to elucidate upstream regulators of VIN3. Molecular characterization of T-DNA mutant populations elucidated two interesting mutants: a mutant that ectopically expressed VIN3 before cold (ectopic VIN3 induction, evi1) and mutants that failed to induce VIN3 during vernalization (defects in VIN3 induction, dvi1). FLC is over-expressed in dvi1 despite its failure to induce VIN3 expression during vernalization, suggesting that this mutant may regulate both VIN3 and FLC. In evi1, FLC is hyper-repressed after 40 days of vernalization, leading to an acceleration of flowering time. These results indicate that regulators of VIN3 in the vernalization pathway exist and that these regulators may use different mechanisms in order to influence VIN3 expression. / text

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It is well established that epigenetics and chromatin modifications are important factors that can govern gene activity and nuclear architecture. They are also proven to be essential for normal embryonic development and cell differentiation. One important event during mouse development is the establishment of epigenetic reprogramming which is believed to be essential for normal growth and development, however; the mechanism is still poorly understood. The general objective of this PhD study was to investigate the profiles and mechanisms of epigenetic and chromatin modifications during normal mouse development and in embryonic stem cells. Mouse pre- and postimplantation embryos and ES cells were used in experiments employing a range of different methodologies. The dynamics of epigenetic DNA and histone methylation were captured using laser confocal immunofluorescent microscopy and western blotting. The activity of epigenetic modifiers was monitored by real-time PCR and candidate genes were validated using siRNA technology. The present studies demonstrate that heterochromatin markers H3K9me3, H3K9me2, H4K20me2, H4K20me3, HP1α and HP1β are reprogrammed during early development. Demethylation of H3K9me2, H3K9me3 and H4K20me3 took place at two-cell stage and remethylation occurred at four-cell stage except for H4K20me3. The reestablishment of H4K20me3 was initially observed in early postimplantation embryos in extraembryonic tissue, specifically in the mural trophectoderm. In embryonic tissue, H4K20me3 was not clearly detected until in mid to late postimplantation development. The mechanism of H3K9me2 and H3K9me3 demethylation might be due to either an imbalance of epigenetic modifiers or the presence of Jmjd2a and Jmjd1a histone demethylase postfertilisation. We have also report evidence that HP1α and Suv4-20h are required in heterochromatin before the recruitment of H4K20me3 during mouse development and in ES cells. Therefore H4K20me3 removal was believed to involve the lack of prerequisite heterochromatin complexes such as HP1α and Suv4-20h enzymes. Furthermore, the presence and levels of H4K20me3 and HP1α might be strongly associated with cell differentiation and tissue maturation in mouse in vivo development but not in vitro early differentiated ES cells. Surprisingly, the results showed that chromatin modifications and their modifiers in ES cells are different from ICM and epiblast. Chromatin modifications H4K20me3 and HP1α were absent from ICM and epiblast, but were detected in ES cells. Notably, H4K20me3 and HP1α were established after early incubation of ICM into ES cell medium, but this change was not dependent on the presence of serum and leukaemia inhibiting factor. Epigenetic modifier Jmjd2a but not Jmjd1a was found in ICM. Conversely, Jmjd1a is highly expressed in ES cells while Jmjd2a was inactivated. In addition, the present studies revealed the substantial role of histone demethylases in development, as it may be important for epigenetic reprogramming. The results demonstrated that inhibition of demethylase Jmjd2a and Jmjd1a caused preimplantation embryos to arrest at the twocell stage while Jmjd2c deficient embryos failed to reach blastocyst. Thus it is possible that Jmjd2a and Jmjd1a were essential for epigenetic reprogramming while Jmjd2c is critical for cell fate establishment during blastocyst formation. In conclusion, the global chromatin signature in ES cells differs from ICM and epiblast; heterochromatin reprogramming occurs at two-cell stage; maturation of heterochromatin occurs at postimplantation; and histone demethylases Jmjd1a, Jmjd2a and Jmjd2c are important in preimplantation development. Results from the present studies could provide crucial information for developmental biology and stem cell research, and provide as a model for improvement of reproductive biotechnologies such as somatic cell reprogramming, and diagnosis of epigenetic abnormalities in early development.

Thesis advisor: Anthony T. Annunziato / Thesis advisor: Charles Hoffman / The Hat1 complex was characterized in <italic>S. pombe</italic>. Through tandem affinity purification and mass spectrometry, it was determined that Hat1 is associated with Mis16 (an orthologue of HAT2). Unlike HAT2 in <italic>S. cerevisiae</italic>, we confirm <italic>mis16</italic> to be an essential gene in <italic>S. pombe</italic>. As expected, the <italic>S. pombe</italic> Hat1 complex was found to acetylate lysines 5 and 12 of histone H4. In contrast to budding yeast, deletion of <italic>hat1</italic> alone resulted in the loss of telomeric silencing without concomitant mutations of the H3 N-terminal domain. Deletion of <italic>hat1</italic> caused an increase of H4 acetylation at telomeres. Additionally, the hyperacetylation of histones also results in the loss of telomeric silencing. Loss of Hat1 did not affect silencing at the inner most repeat (imr) or outer repeat (otr) regions of the centromere, but did appear to increase silencing at the central core region (cnt) of the centromere. The experiments described herein demonstrate Hat1 to be essential for the establishment of proper telomeric silencing in fission yeast, and suggest that the timely acetylation of H4 during chromatin assembly is a unique factor in generating the correct epigenetic state at telomeres in <italic>S. pombe</italic>. Additionally, Hat1 and its acetylation of new H4 may have entirely different roles during telomeric silencing than during silencing at the centromeric central core. Our studies in HeLa cells demonstrated that transcription is involved in the exchange of H2A/H2B in acetylated chromatin regions. The finding that cytosolic H2A can be acetylated at lysine 5 is the first demonstration that cytosolic H2A can be specifically modified <italic>in vivo</italic>. Our results support a model in which H2A/H2B exchange during transcription is mediated by the NAP1 chaperone. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Pax6 is a highly conserved developmental regulator with a complex temporal, spatial and quantitative expression pattern, that is crucial for correct development of the central nervous system, the eye, and pancreas. Accordingly, the Pax6 gene resides in a complex genomic locus containing a large array of long-range tissue-specific cis-regulatory elements primarily identified through multispecies sequence conservation and reporter studies. I have set out to understand how the chromatin architecture of the locus contributes to the mechanism and specificity of cis-regulatory interactions. As well as addressing whether the DNA looping model for regulatory interactions applies to the mouse Pax6 locus, I will identify which elements facilitate such interactions and if they vary between cell types. Utilising ChIP-array technology the distribution and variability of key regulatory histone modifications and factors were assessed in a set of Pax6 expressing and non-expressing mouse cell lines, acting as models for different regulatory states of the locus. Work in other loci suggests a key role for CTCF and cohesin (subunit Rad21) in chromatin organisation and long distance regulatory interactions. ChIP-chip for CTCF/Rad21 across the Pax6 locus identified numerous sites within the gene and at distal regulatory locations. The majority of these sites are cell type invariable. The active enhancer modification H3K27ac identified both known and several novel putative enhancer elements distributed through the locus that are highly cell type specific. A subset of CTCF/Rad21 sites also acquire the active enhancer modification H3K27ac in a cell type dependent manor, suggesting that CTCF/Rad21 may facilitate looping to the target gene from these sites. Using reporter based assays, putative regulatory elements marked by the looping factors and active histone modifications showed a diverse range of functional activities. Unexpectedly only 3 of the 7 CTCF sites tested showed classical insulator activity in an enhancer blocking reporter assay. Surprisingly the strongest insulator tested resided within intron 7 the Pax6 gene. Other CTCF/Rad21 sites were neutral or enhancers in the insulator assay. This reveals the disparity between predicting regulatory properties using ChIP binding profiles alone and the actual outcome of functional reporter experiments. A novel element, CTCF6 showed a ChIP signature of CTCF/Rad21/H3K27ac in all Pax6 expressing tissues, and functioned as a strong enhancer in transient transfection and stable LacZ reporter assays. CTCF6 recapitulated a broad range of Pax6 expression patterns, at multiple embryonic stages, including the brain, neural tube and pancreas. A second novel element, E-120 identified in the pancreatic derived cell line, drove stable embryonic reporter expression in the embryonic pancreas and sub set of brain regions. Together this has expanded the repertoire and size of Pax6’s regulatory landscape particular in the upstream region. Chromatin conformation capture (3C) was used to characterise the dynamic chromatin architecture of the locus and identify the interaction profiles from three CTCF/Rad21 binding regulatory locations within the Pax6 locus. This revealed a core set of regulatory interactions with the Pax6 gene, while individual elements showed a more variable set of cell type specific interactions. The CTCF6 enhancer showed highly cell type specific promoter interactions throughout the Pax6 gene, indicative of enhancer-promoter looping not detected in the non-expressing cells. While the downstream site CTCF5 at the edge of a cluster of regulatory elements known as the DRR (differentially regulated region), interacted with both the gene and an upstream element CTCF7 300 kb away only in the Pax6 expressing locus. Together these results reveal Pax6 has a chromatin hub structure with regulatory loops from upstream and downstream bringing distant yet variable active elements in to the vicinity of the Pax6 promoters where they can act. This work has revealed new roles for CTCF/cohesin sites in transcriptional regulation of Pax6 and how the cis-regulatory activity and structure of the locus varies across different cell types.

Transcriptional repression is maintained by many molecular processes, including DNA methylation and polycomb repression. These two systems are both associated with chromatin modification at the promoters of silent genes, and are both essential for mammalian development. Previous work has shown that DNMT proteins are required for correct targeting of polycomb repressive complexes (PRCs). In this thesis, I investigate whether targeting of DNA modification has a reciprocal dependence on the polycomb machinery by mapping DNA modification in wild-type and PRC-mutant ES cells (Ring1B null, EED null, and Ring1B/EED duble null). I find that the loss of PRCs results in increased DNA modification at sites normally targeted by de novo DNA methyltransferase which lose H3K4 methylation upon PRC removal. This increased DNA modificaiton is associated with increased gene expression when found at CpG island shores of genes marked by the PRC-mediated histone modifications H3K27me3 and H2AK119ub, but not genes lacking these marks. Gene misregulation may be further linked to DNA modification changes by increased DNA modification at enhancers. While loss of either Ring1B or EED led primarily to increases in DNA modification at regions dependant on DNMT3A/DNMT3B, the combined loss of Ring1B and EED results in widespread loss of DNA modification at sites more dependent on DNMT1 activity. This thesis suggests an interplay between PRCs and DNA modification placement which is relevant to the cntrol of gene expression.

As a centromere-specific protein complex in direct contact with the DNA, CenH3-containing nucleosomes are generally thought to act as the distinguishing epigenetic mark of active centromere location. Confusingly, seemingly disparate models have been proposed for the structure of CenH3 nucleosomes. The most widely supported model is an octameric structure that, like histone H3 nucleosomes, contains two subunits of each histone. Another more contentious, yet persistent model is the hemisome model proposed for fly and human CenH3 nucleosomes. In this case it is suggested that CenH3 nucleosomes contain only single subunit of each histone. One reason for this lack of consensus is that seemingly contradicting models are often proposed, even with material from the same organism, with little overlap in experimental approaches. For example, the proposed hemisome model for fly and human CenH3 nucleosomes is predominantly based on atomic force microscopy (AFM) imaging where the height of nucleosomes on a surface is measured. These AFM measurements are the main data used by protagonists for the hemisome model. However, data supporting an octameric model for human, and other, CenH3 nucleosomes is largely based on biochemical analysis of nucleosomes prepared in vitro, with little cross-over in the methodology used to generate data to support either model. In order to reach a consensus the same analyses needs to be applied to CenH3 nucleosomes assembled in vitro or extracted from cells. Here, recombinant Schizosaccharomyces pombe CENP-ACnp1 and H3 histones expressed and purified from E. coli have been assembled into nucleosomes. To our knowledge this is the first time that recombinant S. pombe nucleosomes have been produced, allowing the stoichiometry and composition of these nucleosomes to be examined in detail by a variety of biochemical and biophysical assays. The application of AFM has enabled the height of these recombinant nucleosomes to be measured and tests the ability of AFM to infer stoichiometry using defined material. The intriguing conclusion is that octameric CenH3 nucleosomes uniquely behave as tetrameric “hemisomes” as defined by AFM. In recent years the contribution of DNA sequence to directing H3 nucleosome location has received a great deal of interest. Since CENP-ACnp1 nucleosomes wrap DNA differently to H3 nucleosomes their preference for sequences that produce a stable nucleosome is expected to be altered. The development of protocols to assemble recombinant CENP-ACnp1 nucleosomes in vitro has also been used here to assess the contribution of primary DNA sequence to CENP-ACnp1 nucleosome positioning. CENP-ACnp1 and H3 nucleosomes were reconstituted on genomic DNA at low density and the resulting nucleosomal DNA from CENP-ACnp1 and H3 particles compared by Illumina sequencing. The stability of CENP-ACnp1 and H3 nucleosomes on specific ‘H3’ and ‘CENP-ACnp1’ sequences was cross-checked. Comparing these data with in vivo CENP-ACnp1 nucleosome positions has allowed the contribution of primary DNA sequence to CENP-ACnp1 nucleosome positioning to be explored.

In response to low oxygen (hypoxia), cells have evolved sophisticated gene expression programmes for survival and adaption. How the chromatin state coordinates these changes remains largely unknown. Global histone methylation changes occur in response to hypoxia, however, temporal dynamics of histone methylation changes and how they correlate with hypoxia induced gene transcription changes is ill defined. The Jumonji C (Jmjc) histone demethylases are oxygen dependent enzymes and represent a potential link between chromatin structure and oxygen sensing. Many of these enzymes are differentially expressed in hypoxia and some have been found to influence the hypoxic response. Here, the JmjC histone demethylase, KDM2B, is found to be induced at the mRNA level but not at the protein level in response to hypoxia. KDM2B was also found to regulate the transcriptional response hypoxia, in a cell type dependent manner, through control of Hypoxia Inducible Factor (HIF) subunits, HIF 1 and 2α. These findings highlight complex HIF-KDM2B crosstalk involved in the cells response to low oxygen. Additionally, it was found that various histone methylation marks are induced in the early response to hypoxia prior to hypoxia induced gene transcription changes. This demonstrates that chromatin structural marks responds rapidly to changes in oxygen availability. Furthermore the methylation landscape of 2 two active transcription histone methylation marks, H3K4me3 and H3K36me3, were mapped by ChIP sequencing in the acute response to hypoxia. This analyses found specific changes in histone methylation, which correlate with the core gene transcription changes in hypoxia, pointing towards a mechanism by which rapid chromatin changes programs the cell for hypoxic transcription. Finally, KDM5A was identified to, at least in part, regulate early hypoxia H3K4me3 changes and changes in gene expression of a subset of hypoxia responsive genes. Findings described herein provide evidence for the role of chromatin structure dynamics, mediated by chromatin modifying enzymes, in regulating the hypoxic response. Specifically, early histone methylation changes elicited in acute hypoxia may help establish a chromatin landscape for the cell to transcriptionally respond, which is essential for survival and adaptation to hypoxia. Insights into chromatin dynamics in the response to hypoxia and the role played by JmjC histone demethylases in regulating the hypoxic response has the potential for new drug discovery in diseases such as cancer, were hypoxia, epigenetics and JmjC enzymes are often implicated in disease progression.