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.

<p>Changes in gene transcription driven by the activation of intracellular calcium signaling pathways play an important role in neural development and plasticity. A growing body of evidence suggests that stimulus-driven modulation of histone modifications play an important role in the regulation of neuronal activity-regulated gene transcription. However, the histone modifying enzymes that are targets of activity-regulated signaling cascades in neurons remain to be identified. The histone demethylases (HDMs) are a large family of enzymes that have selective catalytic activity against specific sites of histone methylation. To identify HDMs that may be important for activity-regulated gene transcription in neurons, we induced seizures in mice and screened for HDMs whose expression is induced in the hippocampus. Among the few HDMs that changed expression, Kdm6b showed the highest induction. Kdm6B is a histone H3K27-specific HDM whose enzymatic activity leads to transcriptionally permissive chromatin environments. In situ hybridization analysis revealed that Kdm6b is highly induced in post-mitotic neurons of the dentate gyrus region of the hippocampus. We can recapitulate the activity-dependent induction of Kdm6b expression in cultured hippocampal neurons by application of Bicuculline, a GABAA receptor antagonist that leads to synaptic NMDA receptor activation and calcium influx. Kdm6b expression is also induced following application of BDNF, a neurotrophic factor that is upregulated in the seized hippocampus. To investigate possible functions of Kdm6b in neuronal development, we performed in situ hybridization analysis that allows for the identification of regions with high Kdm6b expression that could be sites of potential function in the developing mouse brain. We found high levels of Kdm6b expression in the inner layer of the external granule layer of the cerebellum, a region where pre-migratory immature neurons reside and a site of significant apoptosis. On the basis of this data and the fact that intracellular calcium signaling arising from synaptic firing supports neuronal survival, we explored the necessity for Kdm6b in the survival of cultured cerebellar granule cells. Knock down of Kdm6b by RNAi increases cell death, demonstrating that Kdm6b contributes to neuronal survival. Ongoing experiments are addressing the role of Kdm6b in neuronal differentiation. Overall these data raise the possibility that stimulus-dependent regulation of Kdm6b, and perhaps regulation of H3K27 methylation mediated by Kdm6b, may contribute to the regulation of gene expression in neurons and thus to their proper development and plasticity.</p> / Dissertation

The family of Nucleoplasmin (NPM) proteins play an important role in a number of chromatin remodelling processes. The first NPM protein discovered in the eggs and oocytes of Xenopus laevis was NPM2, a tissue specific histone chaperone. In Xenopus, NPM2 has been linked to paternal chromatin decondensation following fertilization through the removal of sperm proteins, nucleosome assembly through the storage and addition of H2A-H2B dimers and apoptosis. In mammals, NPM2 correlates strongly with nucleolus-like bodies, and has been suggested by various groups to differ in its roles when compared to the X. laevis homologue. However, the exact roles of NPM2 in mammals remain to be fully elucidated. In this dissertation, attempts are made to determine the physical interaction sites between mouse NPM2 and core histone proteins, H2A, H2B, H3 and H4, as well as physical interactions between mouse NPM2 and protamines (sperm proteins) P1 and P2. Interaction sites between mouse NPM2 and various chromosomal proteins were investigated using a number of different techniques. First, NPM2: chromosomal protein binding assays were attempted to determine the ratio of NPM2 to both core histones and protamines. When visualized through 12% Native gels, NPM2 was determined to interact with histone octamers at a molar ratio of 1-1.5 mol NPM2/mol histone octamer. Mouse sperm protamines were determined to form complexes with mouse NPM2 at a molar ratio of 2.5 mol protamine/mol NPM2 (or mol protamine/0.4 mol NPM2). Analytical Ultracentrifuge (AUC) analysis was conducted on NPM2 and chromosomal proteins separately and in complex formation. Although determining that isolated, full length mouse NPM2 exists in a pentamer form, attempts with AUC were unsuccessful in determining specific NPM2:chromosomal protein binding affinity and complex formation. Specific physical interaction sites between NPM2 and chromosomal proteins were investigated using Cross Linking Mass Spectrometry. Here, a number of new interaction sites as well as sites previously identified by other groups were determined. In combination, our results present likely interaction sites between NPM2 and chromosomal proteins and represent an interesting point of reference for future work. / Graduate

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 105-114) Also available in print.

Thesis (Ph. D. in Biochemistry)--Vanderbilt University, Aug. 2008. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 104-115).

Thesis (Ph. D.)--Ohio State University, 2008.

Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains viii, 140 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 119-140).

Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains x, 101 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 81-101).