Histone post-translational modifications (PTMs) are key determinants of the local chromatin landscape and critical for regulation of eukaryotic gene expression. These histone marks are deposited by a vast number of chromatin modifying enzymes and preferentially recognized by specific associated histone reader domains. Recognition of histone PTMs by histone reader domains is important for either targeting these complexes to chromatin or regulating their enzymatic activity once there. The Polycomb repressive complex 1 and 2 (PRC1 and PRC2) are two such chromatin modifying complexes that are critical for developmental gene repression. The enzymatic activity of PRC2 is tightly regulated by many histone reader domains whereas the PRC1 complex is targeted to chromatin through these domains. In this thesis, I explore how PRC1 and PRC2 functions are regulated by histone reader domains. I identify a previously unrecognized histone reader domain within the PRC2 complex, the EZH2 SANT1 domain, which has important implications for regulating PRC2 enzymatic activity. In addition, I explore the mechanism through which the CBX8 chromodomain targets the PRC1 complex to chromatin. Together, these studies provide significant insight into the regulation of chromatin modifying complexes by histone reader domains and how this occurs via multiple mechanisms.

The dynamic changes in chromatin play a significant role in lineage commitment and differentiation. These epigenetic modifications control gene expression through recruitment of transcription factors. While the active mark H3K4me3 is present around the transcription start site on the gene, the function of the H3K36me3 mark is unknown. A number of H3K36-specific histone methyltransferases (HMTs) have been identified, however the focus of this study is the HMT Hypb. To elucidate the role of H3K36me3 in mediating expression of developmentally-regulated loci, native chromatin immunoprecipitation (N-ChIP) was performed at a subset of genes. Upon differentiation, we observe that H3K36me3 becomes enriched at the 3’ end of several muscle-specific genes. To further investigate the role of H3K36me3 in myogenesis, a lentiviral-mediated knockdown of the H3K36 HMT Hypb was performed in muscle myoblasts using shRNA. Upon Hypb knockdown, we were surprised to observe enhanced myogenesis. N-ChIP was also performed on differentiated Hypb knockdown cell lines in order to look at H3K36me3 enrichment on genes involved in muscle differentiation. N-ChIP data show a drop in H3K36me3 enrichment levels on myogenin and Ckm genes. The possible occupancy of Hypb on the coding regions of muscle-specific genes was experimentally observed by cross-linked chromatin immunoprecipitation (X-ChIP) on differentiated C2C12 cells and subsequently confirmed by X-ChIP on knockdown lines where the occupancy was lost. A model is proposed that links the observed phenotype with H3K36me3.

The human SRC gene encodes pp60c-Src (or c-Src), a 60 kDa, non-receptor tyrosine kinase frequently activated in colon and other tumors. Many studies have demonstrated c-Src activation can be accounted for by overexpression of c-Src protein, and that this overexpression is important for the fully transformed phenotype of cancer cells. The general goal of this thesis, therefore, was to determine the mechanism of this overexpression in human cancer cells. Examination of c-Src expression and activity in human colon cancer cell lines showed that c-Src activation was due to transcriptional activation of the SRC gene. SRC transcription is directed by the ubiquitous, Sp1 regulated SRC1A promoter, and the HNF-1alpha regulated, tissue restricted SRC1alpha promoter. To study the mechanism of SRC transcriptional activation in human cancer cell lines, a dual SRC promoter reporter construct was generated with both these promoters in their natural, physiologically linked context. Very low activity of the SRC1alpha promoter, relative to SRC1A, was consistently observed from this construct, leading to the conclusion that an enhancer element elevates SRC1alpha promoter activity. Interestingly, the HNF binding site in the SRC1alpha promoter enhanced SRC1A promoter activity in the dual promoter construct, but only in a colon cancer cell line with activated SRC. These results therefore suggest SRC transcriptional activation results from enhancer action and/or SRC promoter cross-talk in subsets of human cancer cells. <p> This study has also determined that histone deacetylase inhibitors (HDIs), compounds with documented anti-neoplastic properties, repress transcription from both SRC promoters in various cancer cell lines. To identify the mechanism of this repression, various deletion and mutant SRC promoter constructs were assayed, but HDI response elements were not identified. However, it was discovered that both promoters shared a common requirement for functional TAF1/TAF(II)250, a component of the general transcription factor TFIID. Compromised TAF1 function impaired SRC transcription, but also blocked SRC repression by HDIs. Experiments with SRC:WAF1 promoter chimeras showed the SRC promoters' TAF1 requirement could be conferred on the heterologous, TAF1-independent promoter for the WAF1 gene, which encodes the cell cycle inhibitor p21. These chimeras were also repressed by HDIs, despite WAF1 normally being strongly induced by these agents. These results therefore provide a potential functional link between promoter architecture, TAF1 dependence, and HDI mediated transcriptional repression.

The human SRC gene encodes pp60c-Src (or c-Src), a 60 kDa, non-receptor tyrosine kinase frequently activated in colon and other tumors. Many studies have demonstrated c-Src activation can be accounted for by overexpression of c-Src protein, and that this overexpression is important for the fully transformed phenotype of cancer cells. The general goal of this thesis, therefore, was to determine the mechanism of this overexpression in human cancer cells. Examination of c-Src expression and activity in human colon cancer cell lines showed that c-Src activation was due to transcriptional activation of the SRC gene. SRC transcription is directed by the ubiquitous, Sp1 regulated SRC1A promoter, and the HNF-1alpha regulated, tissue restricted SRC1alpha promoter. To study the mechanism of SRC transcriptional activation in human cancer cell lines, a dual SRC promoter reporter construct was generated with both these promoters in their natural, physiologically linked context. Very low activity of the SRC1alpha promoter, relative to SRC1A, was consistently observed from this construct, leading to the conclusion that an enhancer element elevates SRC1alpha promoter activity. Interestingly, the HNF binding site in the SRC1alpha promoter enhanced SRC1A promoter activity in the dual promoter construct, but only in a colon cancer cell line with activated SRC. These results therefore suggest SRC transcriptional activation results from enhancer action and/or SRC promoter cross-talk in subsets of human cancer cells. <p> This study has also determined that histone deacetylase inhibitors (HDIs), compounds with documented anti-neoplastic properties, repress transcription from both SRC promoters in various cancer cell lines. To identify the mechanism of this repression, various deletion and mutant SRC promoter constructs were assayed, but HDI response elements were not identified. However, it was discovered that both promoters shared a common requirement for functional TAF1/TAF(II)250, a component of the general transcription factor TFIID. Compromised TAF1 function impaired SRC transcription, but also blocked SRC repression by HDIs. Experiments with SRC:WAF1 promoter chimeras showed the SRC promoters' TAF1 requirement could be conferred on the heterologous, TAF1-independent promoter for the WAF1 gene, which encodes the cell cycle inhibitor p21. These chimeras were also repressed by HDIs, despite WAF1 normally being strongly induced by these agents. These results therefore provide a potential functional link between promoter architecture, TAF1 dependence, and HDI mediated transcriptional repression.

During early mammalian embryogenesis, the embryonic genome undergoes critical reprogramming events that include changes in both DNA methylation and histone modifications necessary to control chromatin structure and thus, gene expression. Improper reprogramming of the epigenome during this window of development can lead to a vast number of imprinting anomalies, which are increased in children and livestock conceived in vitro. In the bovine, which closely resembles human preimplantation development, epigenetic changes occur from fertilization through the blastocyst stages. In particular, and concurrent with embryonic genome activation (EGA), de novo DNA methylation begins at the 8-cell stage. In order to explore the roles of histone-modifying enzymes during this crucial period of development, we characterized the transcript expression of several enzymes key enzymes across in vitro bovine preimplantation development using quantitative real-time PCR. Two of the 7 genes analyzed (Suz12 and Lsh) exhibited notable increases at the 8-16 cell stages, with basal levels observed both before and after this. These increases coincided with both EGA and de novo DNA methylation. We further explored their roles in bovine preimplantation embryos by knocking down expression via the use of gene-specific targeting siRNAs. Independent suppression of either Suz12 or Lsh via cytoplasmic microinjection of targeting siRNAs resulted in lower development rates (p < 0.0001), and poorer embryo quality of the morulas and blastocysts that survived. In addition, Suz12 suppression led to reductions in both H3K27 (p < 0.0001) and H3K9 (p = 0.07) trimethylation, and an increase in DNA methylation levels (p < 0.0001), as compared to the null-injected controls. Lsh suppression did not change H3K27, but led to a reduction in H3K9 trimethylation (p = 0.006) and an increase in DNA methylation (p < 0.0001). Clearly our data demonstrate that these epigenetic modifiers play a critical role in formation of the embryonic epigenome, but further research would be necessary in order to fully characterize gene activities during this developmental window.

In S. cerevisiae, the ribosomal DNA locus is silent for RNA polymerase II (Pol II) transcription and recombination (rDNA silencing). Our goal is to understand how histones and histone-modifying enzymes regulate the silent chromatin at the rDNA locus. Sir2, a NAD+-dependent histone deacetylase, is required for rDNA silencing. To understand how Sir2 regulates rDNA silencing, we performed chromatin immunoprecipitation to measure the association of modified histones across the rDNA repeat in wild-type and sir2Δ cells. We found that in sir2Δ cells, histone H3 at the rDNA became hyperacetylated and hypermethylated. High levels of K4-methylated H3 correlate with Pol II transcription. Consistent with this, we found that the nontranscribed spacer (NTS) region was transcribed by Pol II in sir2Δ cells. To investigate if transcription of the NTS region regulates rDNA silencing, we overexpressed this region both in trans and in cis. Our data showed that overexpression of the NTS region in cis caused Pol II silencing defect and hyperrecombination at the rDNA. These data suggest that Sir2 contributes to maintain the silent chromatin at the rDNA by repressing Pol II transcription in the NTS region. We also found that the NTS transcripts could be translated in vitro and that they copurified with polysomes, suggesting that the transcripts may encode proteins or that the transcripts are somehow involved in the process of translation. Additionally, we examined the role of linker histone H1 in regulating rDNA silencing. We found that, unlike Sir2 that represses both Pol II transcription and recombination, histone H1 only represses recombination at the rDNA. The hyperrecombination defect at the rDNA is more severe in sir2Δ hho1Δ double mutant than in either single mutant, suggesting histone H1 and Sir2 act independently. Consistently, hho1Δ cells did not accumulate extrachromosomal rDNA circles (ERCs) or the Holliday junction intermediates, which accumulate in sir2Δ cells. These data suggest that histone H1 and Sir2 regulate different recombination pathways. In summary, my research has provided insight into the mechanism of how silent chromatin at the rDNA locus is regulated, which will help us understand how fundamental components of chromosomes affect gene expression and genome stability.

Zugl.: Göttingen, Univ., Diss., 2009

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 90-97).

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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