Lefebvre, Louis | DOOR - DOuglas Open Repository

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Louis Lefebvre

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The interval between Ins2 and Ascl2 is dispensable for imprinting centre function in the murine Beckwith–Wiedemann region

Digital Document

Imprinted genes are commonly clustered in domains across the mammalian genome, suggesting a degree of coregulation via long-range coordination of their monoallelic transcription. The distal end of mouse chromosome 7 contains two clusters of imprinted genes within a ~1 Mb domain. This region is conserved on human 11q15.5 where it is implicated in the Beckwith-Wiedemann syndrome. In both species, imprinted regulation requires two critical cis-acting imprinting centres, carrying different germline epigenetic marks and mediating imprinted expression in the proximal and distal sub-domains. The clusters are separated by a region containing the gene for tyrosine hydroxylase (Th) as well as a high density of short repeats and retrotransposons in the mouse. We have used the Cre-loxP recombination system in vivo to engineer an interstitial deletion of this ~280-kb intervening region previously proposed to participate in the imprinting mechanism or to act as a boundary between the two sub-domains. The deletion allele, Del7AI, is silent with respect to epigenetic marking at the two flanking imprinting centres. Reciprocal inheritance of Del7AI demonstrates that the deleted region, which represents more than a quarter of the previously defined imprinted domain, is associated with intrauterine growth restriction in maternal heterozygotes. In homozygotes, the deficiency behaves as a Th null allele and can be rescued pharmacologically by bypassing the metabolic requirement for TH in utero. Our results show that the deleted interval is not required for normal imprinting on distal Chr 7 and uncover a new imprinted growth phenotype.

Rescue of placental phenotype in a mechanistic model of Beckwith-Wiedemann syndrome

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Background: Several imprinted genes have been implicated in the process of placentation. The distal region of mouse chromosome 7 (Chr 7) contains at least ten imprinted genes, several of which are expressed from the maternal homologue in the placenta. The corresponding paternal alleles of these genes are silenced in cis by an incompletely understood mechanism involving the formation of a repressive nuclear compartment mediated by the long noncoding RNA Kcnq1ot1 initiated from imprinting centre 2 (IC2). However, it is unknown whether some maternally expressed genes are silenced on the paternal homologue via a Kcnq1ot1-independent mechanism. We have previously reported that maternal inheritance of a large truncation of Chr7 encompassing the entire IC2-regulated domain (DelTel7 allele) leads to embryonic lethality at mid-gestation accompanied by severe placental abnormalities. Kcnq1ot1 expression can be abolished on the paternal chromosome by deleting IC2 (IC2KO allele). When the IC2KO mutation is paternally inherited, epigenetic silencing is lost in the region and the DelTel7 lethality is rescued in compound heterozygotes, leading to viable DelTel7/IC2KO mice. <p>Results: Considering the important functions of several IC2-regulated genes in placentation, we set out to determine whether these DelTel7/IC2KO rescued conceptuses develop normal placentae. We report no abnormalities with respect to the architecture and vasculature of the DelTel7/IC2KO rescued placentae. Imprinted expression of several of the IC2-regulated genes critical to placentation is also faithfully recapitulated in DelTel7/IC2KO placentae. <p>Conclusion: Taken together, our results demonstrate that all the distal chromosome 7 imprinted genes implicated in placental function are silenced by IC2 and Kcnq1ot1 on the paternal allele. Furthermore, our results demonstrate that the methylated maternal IC2 is not required for the regulation of nearby genes. The results show the potential for fully rescuing LQ trans placental abnormalities that are caused by imprinting defects. [ABSTRACT FROM AUTHOR]

Digital Document

The development of gene targeting approaches has had a tremendous impact on the functional analysis of the mouse genome. A specific application of this technique has been the adaptation of the bacteriophage P1 Cre/loxP site-specific recombinase system which allows for the precise recombination between two loxP sites, resulting in deletion or inversion of the intervening sequences. Because of the efficiency of this system, it can be applied to conditional deletions of relatively short coding sequences or regulatory elements but also to more extensive chromosomal rearrangement strategies. Both mechanistic and functional studies of genomic imprinting have benefited from the development of the Cre/loxP technology. Since imprinted genes within large chromosomal regions are regulated by the action of cis-acting sequences known as imprinting centers, chromosomal engineering approaches are particularly well suited to the elucidation of long-range mechanisms controlling the imprinting of autosomal genes. Here we review the applications of the Cre/loxP technology to the study of genomic imprinting, highlight important insights gained from these studies and discuss future directions in the field. [ABSTRACT FROM AUTHOR]

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

Digital Document

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.

Partial loss of Ascl2 function affects all three layers of the mature placenta and causes intrauterine growth restriction

Digital Document

Several imprinted genes have been implicated in the regulation of placental function and embryonic growth. On distal mouse chromosome 7, two clusters of imprinted genes, each regulated by its own imprinting center (IC), are separated by a poorly characterized region of 280 kb (the IC1–IC2 interval). We previously generated a mouse line in which this IC1–IC2 interval has been deleted (Del7AI allele) and found that maternal inheritance of this allele results in low birth weights in newborns. Here we report that Del7AI causes a partial loss of Ascl2, a maternally expressed gene in the IC2 cluster, which when knocked out leads to embryonic lethality at midgestation due to a lack of spongiotrophoblast formation. The hypomorphic Ascl2 allele causes embryonic growth restriction and an associated placental phenotype characterized by a reduction in placental weight, reduced spongiotrophoblast population, absence of glycogen cells, and an expanded trophoblast giant cell layer. We also uncovered severe defects in the labyrinth layer of maternal mutants including increased production of the trilaminar labyrinth trophoblast cell types and a disorganized labyrinthine vasculature. Our results have important implications for our understanding of the role played by the spongiotrophoblast layer during placentation and show that regulation of the dosage of the imprinted gene Ascl2 can affect all three layers of the chorio-allantoic placenta.

Transcription factor ASCL2 is required for development of the glycogen trophoblast cell lineage

Digital Document

The basic helix-loop-helix (bHLH) transcription factor ASCL2 plays essential roles in diploid multipotent trophoblast progenitors, intestinal stem cells, follicular T-helper cells, as well as during epidermal development and myogenesis. During early development, Ascl2 expression is regulated by genomic imprinting and only the maternally inherited allele is transcriptionally active in trophoblast. The paternal allele-specific silencing of Ascl2 requires expression of the long non-coding RNA Kcnq1ot1 in cis and the deposition of repressive histone marks. Here we show that Del7AI, a 280-kb deletion allele neighboring Ascl2, interferes with this process in cis and leads to a partial loss of silencing at Ascl2. Genetic rescue experiments show that the low level of Ascl2 expression from the paternal Del7AI allele can rescue the embryonic lethality associated with maternally inherited Ascl2 mutations, in a level-dependent manner. Despite their ability to support development to term, the rescued placentae have a pronounced phenotype characterized by severe hypoplasia of the junctional zone, expansion of the parietal trophoblast giant cell layer, and complete absence of invasive glycogen trophoblast cells. Transcriptome analysis of ectoplacental cones at E7.5 and differentiation assays of Ascl2 mutant trophoblast stem cells show that ASCL2 is required for the emergence or early maintenance of glycogen trophoblast cells during development. Our work identifies a new cis-acting mutation interfering with Kcnq1ot1 silencing function and establishes a novel critical developmental role for the transcription factor ASCL2. [ABSTRACT FROM AUTHOR]

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