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Ha, Amanda

University of British Columbia

Genomic imprinting is an epigenetic phenomenon by which the expression of certain genes follows a parent-of-origin-specific pattern. The sub-proximal end of mouse Chr 6 contains an imprinted domain comprised of the paternally expressed gene Mest, and the maternally expressed genes Copg2 and Klf14. The Mest locus harbors the only gametic differentially methylated region (gDMR) in the region, which may act as an Imprinting Control Region for the coordinated regulation of these three imprinted genes. The Mest-Klf14 imprinted domain shares syntenic homology with human Chr 7q32. Therefore, loss of Mest function, or Klf14 overexpression may contribute to the maternal uniparental disomy 7 (mUPD7) growth retardation phenotype associated with Sliver- Russell syndrome (SRS). Here, I investigate the function of Mest, and its gDMR. In Chapter 3, I leveraged a previously generated Mest KO mouse line to characterize the function of Mest in developmental pathways. Embryonic growth retardation observed in Mest KO mice was independent of gross placental abnormalities in vivo, but associated with altered Wnt signalling in cell assays. Based on additional proteomic and metabolomic characterization, I hypothesize that the putative epoxide hydrolase, known as MEST, acts upon metabolites within the endoplasmic reticulum lumen. In Chapter 4, I explored the function of the Mest gDMR. While the imprinted regulation of both Mest and Copg2 has been well documented, the mechanism of Klf14 imprinting is unknown. Klf14 expression is lost in embryos lacking maternal methylation iii genome-wide, indicating that maternally-inherited DNA methylation (DNAme) is paradoxically required for its expression. Through CRISPR-Cas9 mutagenesis and allele-specific chromosome conformation experiments in F1 embryonic stem cells, I demonstrate that the Mest gDMR regulates imprinting of Klf14 through long-range chromatin looping, permitting differential promoter-enhancer interactions between parental alleles. I propose a model whereby CTCF binding to the unmethylated paternal Mest gDMR structures paternal allele-specific sub-TADs required for Klf14 silencing. Altogether, my results illustrate both the function and mechanisms regulating imprinting at the Mest-Klf14 imprinted domain which may impact the management of patients with Silver-Russell or metabolic syndromes.

Text

2022-12-12

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/83395

Medical Genetics

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/