Louis Lefebvre

 
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Associate Professor

Research Classification

Research Interests

Developmental Genetics
developmental epigenetics
Embryonic Development
Gene Regulation and Expression
genomic imprinting
genomics
germ cell biology
Molecular Genetics
mouse developmental genetics
placental biology
Transgenic Model

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Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2021)
Developmental consequences of imprinted transcription at the Mest Iocus (2013)

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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Placental phenotypes associated with abnormal genomic imprinting on distal mouse chromosome 7 (2011)

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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Characterization of a novel fluorescent reporter of genomic imprinting in the mouse (2010)

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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Master's Student Supervision (2010 - 2020)
Telomere position effect in embryonic stem cells: heterogeneity, imprinting, and modifier screen (2018)

Telomeres are repetitive sequences found at the end of linear chromosomes. Their main function is to protect chromosome ends from degradation and to ensure proper DNA replication. Telomere position effect (TPE) refers to the epigenetic phenomenon of a gene being stochastically active or silenced as a consequence of its proximity to telomeric heterochromatin. TPE is a subset of position effect variegation (PEV), which also involves variable transcriptional silencing due to the proximity to centromeric heterochromatin or transposable elements. We have observed a TPE-like effect on GFP expression from the DelTel7 allele in embryonic stem cell (ESC) lines. The DelTel7 allele is an engineered chromosome truncation carrying a GFP reporter next to an array of telomere repeats. The truncation breakpoint is in the middle of a large cluster of imprinted genes found on distal mouse chromosome 7. Our results suggest that the GFP reporter is regulated by TPE in undifferentiated DelTel7/+ ESCs. The studies described in this thesis first addressed the relationship between GFP heterogeneity seen in DelTel7/+ ESCs and the previously described phenotypic heterogeneity existing in undifferentiated ESCs grown in serum. TPE was found to be active in all ESCs grown in serum, irrespective of their state. Growth in KSR+2i serum-free medium, which forces ESCs into a naїve state, up regulates the promoter driving GFP expression, at both the DelTel7 allele and at an interstitial control transgene. My results show that, as previously described in a yeast model of TPE, the silencing imposed by proximity to the telomeres can be at least partially overcome by increased transcription. A parent-of-origin effect at the GFP reporter of the DelTel7 allele was revealed in vivo, but TPE does not spread to nearby imprinted genes in ESCs. Finally, while working on the development of an episomal system to screen for modifiers of TPE, I noted that prolonged zeocin exposure has drastic effects on TPE. My results suggest a previously unappreciated relationship between DSB repair and TPE.

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The function of the imprinted transcription factor ASCL2 in mouse trophoblast development (2013)

The epigenetic phenomenon known as genomic imprinting which leads to the monoallelic expression of genes in a parent-of-origin dependent manner has been linked to the development and function of the placenta in mammals. The imprinted gene, Ascl2, codes for a transcription factor which is expressed from the maternal allele in the placenta and is required for its development. Mice that lack Ascl2 expression from their maternal allele die around mid-gestation of placental failure. The effects of Ascl2 can be studied in vivo, in mice that are Ascl2-deficient and, in vitro in trophoblast stem (TS) cells which provide an excellent model of early placental development. Here we compare the transcriptomes of Ascl2-deficient and wild-type E9.5 placentae and find that a total of 838 coding genes are significantly downregulated in the mutant placentae. These genes were deemed to be potential candidate targets of ASCL2. The downregulation of several genes from this list is verified by qRT-PCR and their location in the placenta investigated by in situ hybridization, verifying their overlap with Ascl2 in the trophoblast. We also investigate the knock-out (KO) placental phenotype of one of these candidate target genes, Hmga2, and recognized a labyrinth phenotype in the Hmga2-KO. We also describe, for the first time, the establishment of Ascl2-deficient TS cells confirming that Ascl2 is dispensable for TS cell establishment and maintenance. We find that Ascl2-deficient TS cells lack expression of several trophoblast cell lineage markers through differentiation suggesting they are unable differentiate into cells of the trophoblast lineage. We also find that Ascl2 candidate gene expression in differentiating Ascl2-deficient TS cells is altered when compared to wild-type. These results provide important insight into the functional role of Ascl2 in the development and differentiation of the cells of the trophoblast lineage.

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Role of Imprinted Genes in Mouse Models of IUGR (2011)

Each year in Canada, 5% of ongoing pregnancies are affected by intrauterine growth restriction (IUGR), a condition diagnosed when a baby's birth weight is less than the bottom 5th percentile. Placental dysfunction is thought to be the main contributor to IUGR and many genetic aberrations can lead to problems in the placenta. The epigenetic phenomenon, genomic imprinting, has evolved with placentation and gene knockout studies of several imprinted genes in the mouse result in IUGR. The main goal of this thesis is to examine how gene expression of all imprinted genes is affected in mouse models of IUGR (Mmp2-/-, Mest+/- and Surgical). The first step is to find suitable IUGR mouse models by comparing the embryonic weights of potential models to normal mouse embryos. Next, I assessed gene expression using genome-wide assays and looked at how expression of imprinted genes is altered in the IUGR mouse model.Amongst the three models, only the surgical model was identified as having IUGR and RNA samples from this model were used in genome-wide expression assays. We found 68 candidate IUGR genes, 42 genes had a 2-fold difference in IUGR embryos or placentae, with 26 genes altered in both tissue. Genes that function in the transport of substances are the most altered in both tissue. The genes that are involved in the development of anatomical structures were affected more in the IUGR embryos whilst stress response genes were more affected in the IUGR placentae. For imprinted genes, only 4 genes in the embryo (H19, Igf2, Slc38a4, and Dlk1) and 6 genes in the placenta (Slc38a4, Sfmbt2, Slc22a3, Phlda2, Cdkn1c, and Dlk1) exhibited significant difference in gene expression between wild-type and IUGR. The majority of these imprinted candidates have been linked to IUGR in either mouse and/or human studies. Overall, imprinted genes as a whole are not more affected in IUGR samples than would be expected by chance based on the chi-square test. These results illustrate that though individual imprinted genes may be important regulator of IUGR, genes regulated by genomic imprinting as a whole are not more affected in this pregnancy complication.

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