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X-Chromosome Inactivation and Plasticity in pre-implantation mouse embryo

Final Report Summary - XCIP (X-Chromosome Inactivation and Plasticity in pre-implantation mouse embryo)

Project context and objectives

In mammals, one of the two X chromosomes is inactivated in females to enable dosage compensation for X-linked gene products. In mice, the first changes associated with X-inactivation occur during early pre-implantation embryogenesis. At the morula-blastocyst transition, paternal and maternal X chromosomes are differentially reprogrammed in the three cell lineages constitutive of the late blastocyst. An imprinted X-inactivation of the paternal X (XP) is observed until the morula stage and subsequently in the trophectoderm layer of the blastocyst, which will give rise to many of the extra-embryonic tissues of the placenta. In contrast, in the cells of the inner cell mass, the XP is reactivated, setting the stage for random X-inactivation involving either the maternal X (XM) or the XP which occurs in the epiblast of the proper embryo. Imprinted X-inactivation is also observed in the extra-embryonic primitive endoderm.

X-inactivation is often presented as a paradigm for large-scale epigenetic regulation of gene expression. The extreme stability of random X-inactivation in somatic tissues, which is associated with the clonal transmission of the inactive state in female mammals through the multiple cell divisions occurring during embryonic development and adult homeostasis, represents an important aspect of this paradigm. This highly stable silencing is triggered by the up-regulation of a key X-linked gene, Xist, which lies within the X-inactivation centre (Xic) and produces large non-coding RNAs, which accumulate on the future inactive X (Xi). This initial event is followed by a series of modifications of the Xi chromatin structure involving the loss of euchromatic marks and the sequential recruitment of the polycomb group complexes PRC2, PRC1 and of PR-Set7, which appose the heterochromatic marks H3K27me3, H2AK119 ubiquitination and H4K20me1 respectively. Additional changes to the Xi, including delayed replication timing, nuclear reorganisation, late-occurring chromatin modifications such as accumulation of the macroH2A histone variants and gene promoter methylation all participate in the final lockdown of the inactive state in female somatic tissues. Attempts to experimentally force chromosome-wide Xi reactivation in somatic cells through release of one or several of these epigenetic locks have proven unsuccessful, further indication of the extreme stability of the random X-inactivation. Genome-wide reprogramming of somatic tissues back to the pluripotent state is the only experimental approach that has achieved high rates of Xi reactivation. Interestingly, several pluripotency factors have been recently shown to directly and indirectly control Xist upregulation in cellular models.

Unlike random X-inactivation, the plasticity of imprinted X-inactivation, which is crucial to allowing the reactivation of the XP at the time of the first embryonic lineage commitments, has been much less extensively characterised. We set out to study in greater details the stability of imprinted X-inactivation in the trophectoderm using trophoblast stem (TS) cells as a model system. The TS cells were derived from female embryos carrying a green fluorescent protein (GFP) transgene on the XP, thus providing a convenient readout of XP activity.

Project results

Using chromatin immune-precipitation (ChIP) and fluorescent in situ hybridisation (FISH) approaches, we identify an unprecedented level of spontaneous reactivation of several X-linked genes distributed along the length of the X-chromosome. We show that such gene reactivation occurs in long pulses that last for several cell divisions before the genes become silent again. Mechanistically, reactivation events are associated with broad losses of histone H3 lysine 27 trimethylation (H3K27me3) and with a three-dimensional relocation of reactivated loci outside of the nuclear compartment of the inactive X. TS cells invalidated for the polycomb PRC2 complex, which is responsible for the deposition of H3K27me3 on the inactive X, display broader X-linked gene reactivations suggesting that PRC2 function cannot fully account for the spontaneous reactivations observed in wild type cells. Strikingly, spontaneous reactivation does not affect randomly X-linked genes but appears preferentially targeted to the most highly conserved genes on the X-chromosome. Given that unstable imprinted X-inactivation is a well-known property of ancient mammals such as marsupials, we suggest that the instability of imprinted X-inactivation in the mouse trophectoderm reflects a mechanism that has been conserved through 150-180 million years of evolution. We believe the type of novel ‘metastable epigenetic’ states that we describe may well turn out to be a recurrent feature of stem cell genome plasticity, as such, it will be of particular interest to define what triggers such local silencing reversal as it may provide us with critical insights into the mechanisms governing the maintenance of genome plasticity in early embryonic lineages.