DECIPHERING THE ROLE OF DNMT3B IN REGULATING DNA METHYLATION AND EPIGENETIC MODULATION OF TRANSCRIPTIONAL SILENCING

Cancer;epigenetics;DNA methylation;Human Genetics and Molecular Biology

Mitchell, Khadijah AishaCasero, Robert A. (Bob) ;

Johns Hopkins University

关键词: Cancer;    epigenetics;    DNA methylation;    Human Genetics and Molecular Biology;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39304/MITCHELL-DISSERTATION-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

DNA methylation is one of several epigenetic mechanisms used by cells tocontrol gene expression. DNA methylation patterns are not randomly distributed, instead they are compartmentalized by region. In normal cells heterochromatin is hypermethylated and transcriptionally silent. The rest of the genome is predominantly CpG poor, except for CpG islands, which tend to be hypomethylated and associated with active genes. In tumors this normal compartmentalization is reversed. Cancers are characterized by global hypomethylationthat affects repetitive DNA found in heterochromatin, leading to genomic instability. Concurrently, site-specific hypermethylation of CpG islands at the promoters of tumor suppressor genes leads to silencing.This work focused on how DNMT3B influences methylation patterns on a global and local scale. We hypothesized DNMT3B regulated DNA methylation outside of promoter regions. Using an isogenic colorectal cancer cell line panel we profiled global DNA methylation patterns across the genome in the presence and absence of DNMT3B. Upon DNMT3B removal there was a statistically significant loss of methylation in Gene Bodies, 3;; UTRs, Shores, and Shelves. Interestingly, loss of DNMT3B had a greater effect on the methylation status of non-CpG island genes when compared to CpG island genes. Re-introduction of wild-type DNMT3B resulted in a gain of methylation in these areas. Unexpectedly, mutant DNMT3B also caused increased methylation, suggesting DNMT3A induction. This data supports the notion DNMT3B regulates DNA methylation in a context-dependent manner at distinct regions of the human genome. Local studies identified endogenous target genes and demonstrated that DNMT3B is capable of modulating gene expression independently of its methyltransferase activity. Both wild-type and catalytically dead DNMT3B repressed target genes to similar degrees, suggestive of protein-protein interactions at the N-terminus. A DNMT3B-specific loss of methylationmay help to explain expression changes seen in non-CpG island genes. However, it was not required for gene repression at CpG island genes. Further studies in genetic knockout cells alluded to changes in chromatin status upon DNMT3B overexpression that explained transcriptional repression at this subset of genes, such as decreased enrichment for a histone modification associated with transcriptional activity (H3K4me3) and the recruitment of a repressive epigenetic protein (LSD1).