Contribution of epigenetic landscapes and transcription factors to X-chromosome reactivation in the inner cell mass.

X-chromosome inactivation is established during early development. In mice, transcriptional repression of the paternal X-chromosome (Xp) and enrichment in epigenetic marks such as H3K27me3 is achieved by the early blastocyst stage. X-chromosome inactivation is then reversed in the inner cell mass. The mechanisms underlying Xp reactivation remain enigmatic. Using in vivo single-cell approaches (allele-specific RNAseq, nascent RNA-fluorescent in situ hybridization and immunofluorescence), we show here that different genes are reactivated at different stages, with more slowly reactivated genes tending to be enriched in H3meK27. We further show that in UTX H3K27 histone demethylase mutant embryos, these genes are even more slowly reactivated, suggesting that these genes carry an epigenetic memory that may be actively lost. On the other hand, expression of rapidly reactivated genes may be driven by transcription factors. Thus, some X-linked genes have minimal epigenetic memory in the inner cell mass, whereas others may require active erasure of chromatin marks

A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary

cis-Regulatory communication is crucial in mammalian development and is thought to be restricted by the spatial partitioning of the genome in topologically associating domains (TADs). Here, we discovered that the Xist locus is regulated by sequences in the neighboring TAD. In particular, the promoter of the noncoding RNA Linx (LinxP) acts as a long-range silencer and influences the choice of X chromosome to be inactivated. This is independent of Linx transcription and independent of any effect on Tsix, the antisense regulator of Xist that shares the same TAD as Linx. Unlike Tsix, LinxP is well conserved across mammals, suggesting an ancestral mechanism for random monoallelic Xist regulation. When introduced in the same TAD as Xist, LinxP switches from a silencer to an enhancer. Our study uncovers an unsuspected regulatory axis for X chromosome inactivation and a class of cis-regulatory effects that may exploit TAD partitioning to modulate developmental decisions.Galupa et al. uncover elements important for Xist regulation in its neighboring TAD and reveal that these elements can influence gene regulation both within and between topological domains. These findings, in a context where dynamic, developmental expression is necessary, challenge current models for TAD-based gene-regulatory landscapes

Xist-dependent imprinted X inactivation and the early developmental consequences of its failure

The long noncoding RNA Xist is expressed from only the paternal X chromosome in mouse preimplantation female embryos and mediates transcriptional silencing of that chromosome. In females, absence of Xist leads to postimplantation lethality. Here, through single-cell RNA sequencing of early preimplantation mouse embryos, we found that the initiation of imprinted X-chromosome inactivation absolutely requires Xist. Lack of paternal Xist leads to genome-wide transcriptional misregulation in the early blastocyst and to failure to activate the extraembryonic pathway that is essential for postimplantation development. We also demonstrate that the expression dynamics of X-linked genes depends on the strain and parent of origin as well as on the location along the X chromosome, particularly at the first 'entry' sites of Xist. This study demonstrates that dosage-compensation failure has an effect as early as the blastocyst stage and reveals genetic and epigenetic contributions to orchestrating transcriptional silencing of the X chromosome during early embryogenesis.This work was funded by a fellowship of Région Ile-de-France (DIM STEMP OLE) to M.B., the Paris Alliance of Cancer Research Institutes (PACRI-ANR) to LS and ERC Advanced Investigator award (ERC-2010-AdG–No.250367), EU FP7 grants SYBOSS (EU 7th Framework G.A. no. 242129) and MODHEP (EU 7th Framework G.A. no. 259743), La Ligue, Fondation de France, Labex DEEP (ANR-11-LBX-0044) part of the IDEX Idex PSL (ANR-10-IDEX-0001-02 PSL) and ABS4NGS (ANR-11-BINF-0001) to E.H and France Genomique National infrastructure (ANR-10-INBS09) to EH, NS, EB

Diversity of X-inactivation initiation mechanisms in mammals

X-chromosome inactivation (XCI) in female mammals enables dosage compensation for X- linked gene products between the sexes. Since its discovery fifty years ago (Lyon, 1961), the developmental regulation of this process has been extensively investigated in mice (see Morey and Avner,2011; Augui et al, 2011; Gendrel and Heard, 2011 for reviews), but hardly at all in non-murine species. In mice, the X chromosome of paternal origin (Xp) is silenced during early embryogenesis due to imprinted expression of the regulatory RNA, Xist (X- inactive-specific-transcript). Paternal XCI initiates early in mice and is then reversed in the inner cell mass (ICM) of the blastocyst. Random inactivation of either paternal or maternal X then ensues in epiblast cells. We investigated the developmental regulation of XCI in rabbit and human embryos (Okamoto et al, 2011) and were able to show that in these mammals, Xist is not subject to imprinting and X inactivation begins much later than in the mouse. Furthermore, Xist is up-regulated on both X chromosomes in a high proportion of rabbit and human embryo cells, even in the ICM. In rabbits, we showed that this triggers XCI on both X chromosomes, implying that the choice of which X chromosome will finally become inactive occurs downstream of Xist up-regulation, a situation that is totally different to the mouse, where choice occurs upstream of Xist RNA accumulation. In humans, on the other hand, XCI is not triggered, even by the blastocyst stage, despite the up-regulation of Xist. Xist is thus expressed, rather than repressed, in Oct4/Nanog positive ICM cells, in both non- murine species. In fact, the status of the two X chromosomes in the ICM is very different in rabbit, human and mouse embryos. In human ICM cells, the two X chromosomes seem to be active, while in the rabbit ICM, the two Xs are initially active (day 4) but then XCI initiates one day later (day 5) in the ICM. This is exactly the opposite situation to that found in the ICM of mouse blastocysts, where the paternal X is initially inactive and then becomes reactivated in the epiblast cells of the ICM. All these results demonstrate the remarkable diversity of X-inactivation initiation mechanisms and highlight major differences between mammals in the requirement for dosage compensation during early embryogenesis

Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development

 X-chromosome inactivation (XCI) in female mammals allows dosage compensation for X-linked gene products between the sexes1. The developmental regulation of this process has been extensively investigated in mice, where the X chromosome of paternal origin (Xp) is silenced during early embryogenesis owing to imprinted expression of the regulatory RNA, Xist (X-inactive specific transcript). Paternal XCI is reversed in the inner cell mass of the blastocyst and random XCI subsequently occurs in epiblast cells. Here we show that other eutherian mammals have very different strategies for initiating XCI. In rabbits and humans, the Xist homologue is not subject to imprinting and XCI begins later than in mice. Furthermore,Xist is upregulated on both X chromosomes in a high proportion of rabbit andhuman embryo cells, even in the inner cell mass. In rabbits, this triggers XCI on both X chromosomes in some cells. In humans, chromosome-wide XCI has not initiated even by the blastocyst stage, despite the upregulation of XIST. The choice of which X chromosome will finally become inactive thus occurs downstream of Xist upregulation in both rabbits and humans, unlike in mice. Our study demonstrates the remarkable diversity in XCI regulation and highlights differences between mammals in their requirement for dosage compensation during early embryogenesis