Type of Document	Thesis
Author	Poduch, Kristina Nicole
Author's Email Address	poduch@bio.fsu.edu
URN	etd-08202010-125233
Title	INVESTIGATION OF THE EFFECTS OF G9A KNOCKOUT ON SUBNUCLEAR LOCALIZATION AND REPLICATION TIMING OF G9A REPRESSED GENES WHEN INTEGRATED INTO AN EARLY REPLICATING REGION
Degree	Master of Science
Department	Biological Science, Department of
Advisory Committee	Advisor Name Title David Gilbert, Ph.D Committee Chair Hank Bass, Ph.D Committee Member Karen McGinnis, Ph.D Committee Member Lloyd Epstein, Ph.D Committee Member Akash Gunjan, Ph.D University Representative
Keywords	replication timing replication gene expression nuclear localization cell cycle S-phase G9a
Date of Defense	2010-08-12
Availability	unrestricted
Abstract G9a is a histone methyltransferase that is responsible for dimethylation of histone 3 lysine 9 (H3K9me2) this chromatin marker is typically associated with transcriptional silencing. Following conditional knockout of G9a in mouse embryonic stem cells, genes that are regulated by G9a are strongly upregulated. Typically genes that replicate late in S-phase are transcriptionally repressed while genes that replicate early in S-phase are transcriptionally expressed. Strikingly, although 73% of total genes are early replicating, more than 71% of G9a-repressed genes are late replicating, and a strong correlation was found between H3K9me2 and late replication. However, there were no significant genome-wide replication timing changes when G9a was knocked out. The subnuclear location of genes is generally related to expression levels and replication timing, with expressed genes being early-replicating and located in the interior of the nucleus while repressed, late-replicating genes are located at the periphery. DNA fluorescence in situ hybridization (FISH) can be used to visualize the location of a gene within the nucleus. Thus, I have investigated the subnuclear location of these genes using 2-D DNA FISH, before and after the loss of G9a to determine whether G9a repressed genes are indeed localized to the nuclear periphery and whether they change subnuclear localization when they become transcriptionally activated. This class of genes was found to be located at the periphery of the nucleus before and after the loss of G9a, even though the genes are upregulated when G9a is knocked out. I have demonstrated that these genes are likely direct targets of G9a, since six genes examined by chromatin immunoprecipitation (ChIP) with an anti-H3K9me2 antibody contained modified H3K9me2 nucleosomes at their promoters and lost this modification after G9a knockout. Therefore, I have concluded that late-replication and peripheral localization is not sufficient to silence these genes and that G9a is a gatekeeper for a specific set of genes localized within the late replicating nuclear periphery. Taken together, these studies provide unique insight into the complex relationships between nuclear architecture, DNA replication timing and gene expression.
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