Louis Lefebvre

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 imprintedregulation of both Mest and Copg2 has been well documented, the mechanism of Klf14imprinting is unknown. Klf14 expression is lost in embryos lacking maternal methylation iiigenome-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.

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The Mest locus is regulated by genomic imprinting in mammals and only the paternally inherited allele is expressed. A targeted mutation at this locus revealed that it plays an important role in the regulation of embryonic growth and adult behavior. The Mest locus is located in a conserved imprinted domain on mouse chromosome 6 where it is thought to play a key role in the regulation of neighboring maternally expressed genes Copg2 and Klf14 since it contains the only potential imprinting center (IC) identified thus far in this domain, a differentially methylated region (DMR) methylated in oogenesis. Here we describe new larger isoforms of the Mest mRNA, referred to as MestXL, that are generated via alternative polyadenylation and transcribed more than 10kb into the adjacent antisense gene Copg2 exclusively in the developing central nervous system. The MestXL isoforms appear to regulate the allelic usage at Copg2, but not at Klf14, in embryonic neural tissues, as Copg2 is preferentially maternally expressed only in these tissues presumably due to transcriptional interference from MestXL on the paternal chromosome. Our results therefore establish the Mest DMR as an IC and propose a new mechanism to regulate allelic usage and imprinting at sense-antisense gene pairs in mammalian genomes, via tissue-specific alternative polyadenylation and transcriptional interference. Imprinted transcription at the Mest locus also produces a microRNA, miR-335, that acts to down-regulate target genes via binding to their 3’UTRs and ultimately repressing their translation. Here we show that production of miR-335 is imprinted and that its levels are reduced from the mutant MestKO allele. Additionally, we identify several candidate target genes of miR-335 by RNA-seq analysis on primary mouse fibroblasts that under-express miR-335. Our investigation of MestXL and miR-335, two unique alternative functions of the Mest locus, demonstrates that the Mest locus is involved in two types of RNA-mediated regulation and ultimately contributes to the understanding of genomic imprinting and microRNAs in mammalian biology.

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Imprinted genes are expressed either from the maternal or paternal allele during development and tend to be found in clusters throughout the mammalian genome, suggesting they may be regulated by long-range mechanisms. Many of them have important roles in placental development. The Beckwith-Wiedemann Syndrome (BWS) region on human chromosome 11p15.5 contains two imprinted subdomains each regulated by their own differentially methylated regions, known as imprinting centres (IC1 and IC2). These two imprinted subdomains are separated by an evolutionarily conserved region of about 300 kilobases. Distal mouse chromosome 7 (MMU7) shares syntenic homology with the human BWS region. Since the mechanisms by which imprinting occurs are unclear, we sought to characterize this region further using two mouse lines carrying deletions within the BWS imprinted region. The first mouse line, called DelTel7/IC2KO, allows us to dissect out the role of imprinting centre 2 in the silencing of imprinted genes. We demonstrate that all of the distal MMU7 imprinted genes implicated in placental function are silenced by IC2 and the noncoding RNA Kcnq1ot1. The second mouse line, called Del⁷AI, allows us to determine whether placental imprinting is perturbed when the region between IC1 and IC2 is deleted. We found that maternal inheritance of Del⁷AI leads to partial loss of the gene Ascl2, and we show that this affects all three layers of the mature mouse placenta. We found that paternal inheritance of Del⁷AI leads to partial loss of Ascl2 imprinting. Detailed investigation of the underlying mechanisms of imprinting and phenotypes in these mouse lines provides us with new fundamental insights into placental biology and the regulation of gene expression by imprinting centres on distal mouse chromosome 7.

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Regulation of inserted transcriptional units by epigenetic means has been reported for many years, and has been used to study characteristics of epigenetic regulation. Some of these transgenes have become regulated by genomic imprinting, and thus are expressed from only one of the two parental chromosomes and, occasionally, acquire parent-of-origin-specific epigenetic markings such as DNA methylation. These transgenes in particular have been useful in elucidating mechanisms of imprinted regulation. Here is described the first imprinted fluorescent transgene, a green fluorescent protein (GFP) gene inserted in the distal MMU7 imprinted domain between the imprinting centers 1 and 2 (IC1 and IC2) regulated regions. This transgene, called Tel7KI, exhibits imprinted expression only from the maternal allele, and is silenced and DNA methylated on the paternal allele in post-implantation embryos. In the embryo this allele-specificity is consistent throughout all tissues and developmental stages analyzed except the developing germ line, making Tel7KI a potential reporter of epigenetic reprogramming in that lineage. In the placenta, imprinted expression and DNA methylation of Tel7KI is lost, and both alleles are expressed and methylated at moderate levels. Finally, an analysis of the effect of IC2 on silencing of Tel7KI in an embryonic stem cell differentiation assay revealed a possible extension of the region of influence for that imprinting centre a further 300kb proximal. Thus, Tel7KI has the potential to be an extremely useful tool in the study of genomic imprinting.

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