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Epigenomic roadmap of X-chromosome reactivation in female primordial germ cells

proposé par [Maud BORENSZTEIN >], Institut de Génétique Moléculaire de Montpellier (IGMM), UMR5535 1919 route de Mende, 34090 Montpellier

Projet de stage :

Our recent results elicit tantalizing questions about epigenetic reprogramming : why do some X-linked genes behave differently (early/late reactivation) ? Does an epigenetic memory of the silent state exist for the inactive X chromosome in female germ cells ? To tackle these fundamental questions, the master student will use in vivo approaches in the mouse, complemented by low-input epigenomic profiling to determine the detailed epigenomic roadmap of X-chromosome reactivation in female primordial germ cells.

Removal of repressive histone marks is a relevant signature of X-chromosome reactivation. Cytological loss of trimethylation of lysine 27 of histone H3 (H3K27me3) is among the earliest, most striking and conserved chromatin events during X-chromosome reactivation in different cellular contexts, including the germline, the inner cell mass of the blastocyst and induced pluripotent stem cells. The student will use an emerging genome-wide approach to monitor chromatin states in low cell numbers, CUT&RUN (an alternative to chromatin immunoprecipitation) to assay for loss, gain or redistribution of H3K27me3 on the inactive X chromosome. It will be crucial to evaluate the dynamic changes in both male and female primordial germ cells separately, with allele-specific information, and at different developmental time points of germline specification. In details, from each single embryo - males and females-, germ cells will be sorted by flow cytometry and pooled according to the embryo’s sex at different developmental time points of germline specification (Embryonic day 8.5, E10.5 and E12.5). This precious material will allow us to monitor H3K27me3 mark on both the active and the inactive X chromosomes by CUT&RUN followed by sequencing. This will also give access to allele-specific autosomal information in both males and females, to study potential H3K27me3 distribution differences between the sexes at genomic imprinting loci or on repeats for instance.

Techniques mises en œuvre par le stagiaire : Mouse embryo dissection, Genotyping (DNA extraction and PCR), FACS, CUT&RUN, quantitative PCR

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