Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Mellissa Mann

Abstract

Genomic imprinting is an epigenetic phenomenon that restricts gene expression to one parental allele while the other copy is silent. How this duality is regulated is not fully understood. Using the Kcnq1ot1 imprinted domain as a model, previous work in the laboratory identified nucleoporin 107 as a candidate regulator of imprinted domain regulation. Within the Kcnq1ot1 domain resides the imprinting control region, the paternally expressed Kcnq1ot1 (Kcnq1 opposite transcript 1) noncoding RNA, nine maternal-expressed protein-coding genes, as well as genes that escape imprint regulation. On the maternal allele, the Kcnq1ot1 imprinting control region is methylated, silencing the embedded Kcnq1ot1 promoter and its transcription, and thereby permitting expression of neighbouring genes. On the paternal allele, the Kcnq1ot1 imprinting control region is unmethylated, allowing Kcnq1ot1 noncoding RNA transcription, which results in neighbouring protein-coding gene silencing. In the present study, I showed that depletion of nucleoporin 107, nucleoporin 62 and nucleoporin 153 in mouse extraembryonic endoderm stem cells reduced Kcnq1ot1 noncoding RNA expression and volume, decreased Kcnq1ot1 imprinted domain positioning at the nuclear rim and reactivated normally silent paternal alleles of a core group of protein-coding genes in the domain. Paternal alleles of additional protein-coding genes were also reactivated but in a nucleoporin-specific manner. While DNA methylation at the Kcnq1ot1 imprinting control region was not changed, nucleoporin 107, nucleoporin 62 and nucleoporin 153 depletion led to alterations in active and repressive histone modifications and a reduction in cohesin complex protein interactions at the Kcnq1ot1 imprinting control region. Therefore, I identified a novel mechanism of imprinted domain regulation, namely nucleoporin-mediated imprinted domain regulation at the Kcnq1ot1 domain in extraembryonic endoderm stem cells. Next, I demonstrated that this novel nucleoporin-mediated mechanism also regulated the Kcnq1ot1 imprinted domain in embryonic stem and trophoblast stem cells, albeit for a different subset of genes. While CTCF and the cohesin complex interacted at the same sites within the paternal Kcnq1ot1 imprinting control region in a NUP107, NUP62 and NUP153 dependent manner in embryonic stem cells, trophoblast stem cells lacked CTCF and cohesin binding at the Kcnq1ot1 imprinting control region. My results establish an important role for nucleoporins NUP107, NUP62 and NUP153 in mediating imprinted domain regulation in all three cell lineages.

Available for download on Saturday, September 01, 2018


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