Louis Lefebvre

 
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Associate Professor

Research Classification

Developmental Genetics
Gene Regulation and Expression
Genomics
Molecular Genetics
Transgenic Model
Embryonic Development

Research Interests

mouse developmental genetics
developmental epigenetics
genomic imprinting
placental biology
germ cell biology

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
Developmental consequences of imprinted transcription at the Mest Iocus (2013)

No abstract available.

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 - 2018)
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)

No abstract available.

Publications

  • Evolution of imprinting via lineage-specific insertion of retroviral promoters (2019)
  • SETD2 regulates the maternal epigenome, genomic imprinting and embryonic development. (2019)
    Nature genetics,
  • Fetal growth restriction in a genetic model of sporadic Beckwith-Wiedemann Syndrome (2018)
    Disease Models & Mechanisms, , dmm.035832
  • LTR retrotransposons transcribed in oocytes drive species-specific and heritable changes in DNA methylation (2018)
    Nature Communications, 9 (1)
  • Talin Autoinhibition Regulates Cell-ECM Adhesion Dynamics and Wound Healing In Vivo. (2018)
    Cell reports,
  • Transcription factor ASCL2 is required for development of the glycogen trophoblast cell lineage (2018)
    PLOS Genetics, 14 (8), e1007587
  • Maternal DNA Methylation Regulates Early Trophoblast Development. (2016)
    Developmental Cell, 36 (2), 152--163
  • Impact of flanking chromosomal sequences on localization and silencing by the human non-coding RNA XIST (2015)
    Genome Biol, 16 (1)
  • Setdb1 is required for germline development and silencing of H3K9me3-marked endogenous retroviruses in primordial germ cells (2014)
    Genes & development, 28 (18), 2041--2055
  • Beckwith-Wiedemann and Silver-Russell syndromes: opposite developmental imbalances in imprinted regulators of placental function and embryonic growth (2013)
    Clinical genetics, 84 (4), 326--334
  • Engineering of large deletions and duplications in vivo (2012)
    Genomic Imprinting, 925, 137--146
  • The placental imprintome and imprinted gene function in the trophoblast glycogen cell lineage (2012)
    Reproductive biomedicine online, 25 (1), 44--57
  • Tissue-specific alternative polyadenylation at the imprinted gene Mest regulates allelic usage at Copg2 (2012)
    Nucleic acids research, 40 (4), 1523--1535
  • An extended domain of Kcnq1ot1 silencing revealed by an imprinted fluorescent reporter (2011)
    Molecular and cellular biology, 31 (14), 2827--2837
  • Developmental regulation of somatic imprints (2011)
    Differentiation, 81 (5), 270--280
  • Partial loss of Ascl2 function affects all three layers of the mature placenta and causes intrauterine growth restriction (2011)
    Developmental biology, 351 (2), 277--286
  • Applications of the site-specific recombinase Cre to the study of genomic imprinting (2010)
    Briefings in functional genomics, , elq017
  • Rescue of placental phenotype in a mechanistic model of Beckwith-Wiedemann syndrome (2010)
    BMC developmental biology, 10 (1), 50
  • An imprinted GFP insertion reveals long-range epigenetic regulation in embryonic lineages (2009)
    Developmental biology, 336 (1), 42--52
  • Human placental-specific epipolymorphism and its association with adverse pregnancy outcomes (2009)
    PloS one, 4 (10), e7389
  • The interval between Ins2 and Ascl2 is dispensable for imprinting centre function in the murine Beckwith--Wiedemann region (2009)
    Human molecular genetics, 18 (22), 4255--4267
  • Epigenetic and phenotypic consequences of a truncation disrupting the imprinted domain on distal mouse chromosome 7 (2008)
    Molecular and cellular biology, 28 (3), 1092--1103
  • A Mouse for All Reasons (2007)
    Cell, 128 (1), 9--13
  • Androgenetic/biparental mosaicism causes placental mesenchymal dysplasia (2006)
    Journal of medical genetics, 43 (2), 187--192
  • Loss-of-imprinting of Peg1 in mouse interspecies hybrids is correlated with altered growth (2004)
    Genesis, 39 (1), 65--72
  • Abnormal maternal behaviour and growth retardation associated with loss of the imprinted gene Mest (1998)
    Nature genetics, 20 (2), 163--169
  • Sl Nuclease Analysis of RNA (1998)
    Molecular Biomethods Handbook, , 35--49
  • Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells (1997)
    The EMBO journal, 16 (21), 6510--6520
  • Genomic structure and parent-of-origin-specific methylation of Peg1 (1997)
    Human molecular genetics, 6 (11), 1907--1915
  • Mutational and functional analysis of dominant SPT2 (SIN1) suppressor alleles in Saccharomyces cerevisiae. (1993)
    Molecular and cellular biology, 13 (9), 5393--5407
  • The ANB1 locus of Saccharomyces cerevisiae encodes the protein synthesis initiation factor eIF-4D (1990)
    J Biol Chem,
 
 

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