The initiation of X-chromosome inactivation is thought to be tightly correlated with early differentiation events during mouse development. Here, we show that although initially active, the paternal X chromosome undergoes imprinted inactivation from the cleavage stages, well before cellular differentiation. A reversal of the inactive state, with a loss of epigenetic marks such as histone modifications and polycomb proteins, subsequently occurs in cells of the inner cell mass (ICM), which give rise to the embryo-proper in which random X inactivation is known to occur. This reveals the remarkable plasticity of the X-inactivation process during preimplantation development and underlines the importance of the ICM in global reprogramming of epigenetic marks in the early embryo.

https://science.sciencemag.org/content/sci/303/5658/644.full.pdf

Epigenetic dynamics of imprinted X inactivation during early mouse development.

INTRODUCTION The mammal has evolved an epigenetic mechanism to silence one of two X chromosomes in the XX female to equalize gene dosages with the XY male. Once established, the inactivated X chromosome (Xi) is extremely stable and is maintained through the lifetime of the female mammal. The principal regulator, Xist, is a long noncoding RNA that orchestrates the silencing process along the Xi. Xist is believed to operate as a scaffold to recruit and spread repressive complexes, such as Polycomb Repressive Complex 2, along the X chromosome. The identities of crucial interacting factors, however, have remained largely unknown. RATIONALE Although the Xi’s epigenetic stability is a necessary homeostatic property, an ability to unlock this epigenetic state is of great current interest. The X chromosome is home to nearly 1000 genes, at least 50 of which have been implicated in X-linked diseases, such as Rett syndrome and fragile X syndrome. The Xi is therefore a reservoir of functional genes that could be tapped to replace expression of a disease allele on the active X (Xa). A major gap in current understanding is the lack of a comprehensive Xist interactome. Progress toward a full interactome would advance knowledge of epigenetic regulation by long noncoding RNA and potentially inform treatment of X-linked diseases. RESULTS We have developed an RNA-centric proteomic method called iDRiP (identification of direct RNA-interacting proteins). Using iDRiP, we identified 80 to 200 proteins in the Xist interactome. The interactors fall into several functional categories, including cohesins, condensins, topoisomerases, RNA helicases, chromatin remodelers, histone modifiers, DNA methyltransferases, nucleoskeletal factors, and nuclear matrix proteins. Targeted inhibition demonstrates that Xi silencing can be destabilized by disrupting multiple components of the interactome, consistent with the idea that these factors synergistically repress Xi transcription. Triple-drug treatments lead to a net increase of Xi expression and up-regulation of ~100 to 200 Xi genes. We then carry out a focused study of X-linked cohesin sites. Chromatin immunoprecipitation sequencing analysis demonstrates three types of cohesin sites on the X chromosome: Xi-specific sites, Xa-specific sites, and biallelic sites. We find that the Xa-specific binding sites represent a default state. Ablating Xist results in restoration of Xa-specific sites on the Xi. These findings demonstrate that, while Xist attracts repressive complexes to the Xi, it actively repels chromosomal architectural factors such as the cohesins from the Xi. Finally, we examine how Xist and the repulsion of cohesins affect Xi chromosome structure. In wild-type cells, the Xa is characterized by ~112 topologically associated domains (TADs) and the Xi by two megadomains. Intriguingly, loss of Xist and restoration of cohesin binding result in a reversion of the Xi to an Xa-like chromosome conformation. Hi-C analysis shows that TADs return to the Xi in a manner correlated with the reappearance of cohesins and with a transcriptionally permissive state. CONCLUSION Our study unveils many layers of Xi repression and demonstrates a central role for RNA in the topological organization of mammalian chromosomes. Our study also supports a model in which Xist RNA simultaneously acts as (i) a scaffold for the recruitment of repressive complexes to establish and maintain the inactive state and (ii) a repulsion mechanism to extrude architectural factors such as cohesins to avoid acquisition of a transcription-favorable chromatin conformation. Finally, our findings indicate that the stability of the Xi can be perturbed by targeted inhibition of multiple components of the Xist interactome.

http://science.sciencemag.org/content/sci/349/6245/aab2276.full.pdf

A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation

The endoderm of the mouse embryo arises by dynamic widespread intercalation of embryonic and extraembryonic lineages.

In female mammals, one of the two X chromosomes is silenced for dosage compensation between the sexes. X-chromosome inactivation is initiated in early embryogenesis by the Xist RNA that localizes to the inactive X chromosome. During development, the inactive X chromosome is further modified, a specialized form of facultative heterochromatin is formed and gene repression becomes stable and independent of Xist in somatic cells. The recent identification of several factors involved in this process has provided insights into the mechanism of Xist localization and gene silencing. The emerging picture is complex and suggests that chromosome-wide silencing can be partitioned into several steps, the molecular components of which are starting to be defined.

Gene silencing in X-chromosome inactivation: advances in understanding facultative heterochromatin formation

Stereotyped Pruning of Long Hippocampal Axon Branches Triggered by Retraction Inducers of the Semaphorin Family

Summary: A GFP transgene has been integrated on the proximal part of the mouse X chromosome just distal of Timp and Syn1. During development, this X-linked GFP transgene exhibits widespread green fluorescence throughout the embryonic and adult life of male mice but displays mosaic expression in tissues as a result of X-inactivation in females. In living female embryos, inactivation of the transgene is imprinted in extraembryonic regions and random in the embryo proper, demonstrating that this reporter is behaving in a similar fashion to the majority of X-linked loci, and so provides a vital readout of X chromosome activity. This is observation is further supported in T16H/X female mice harboring the GFP transgene on the normal X chromosome where reporter inactivation is observed in somatic cells. The differential expression of GFP activity facilitates fluorescence activated cell sorting for the purification of GFP+ vs. GFP- cells from female embryonic tissues, thereby allowing access to populations of cells that have kept active a particular X chromosome. By tracking the activity of this X-linked GFP transgene, we discovered that the primary and secondary giant cells of the X/X placenta maintain an active paternal copy of this transgene on the presumed silenced paternal X-chromosome. This finding implies that the imprint on the paternal X chromosome may be relaxed in these trophectodermal derivatives. genesis 29:133–140, 2001. © 2001 Wiley-Liss, Inc.

Liza L. Cox

Papers

An X-linked GFP transgene reveals unexpected paternal X-chromosome activity in trophoblastic giant cells of the mouse placenta.