Multiple distinct domains of human XIST are required to coordinate gene silencing and subsequent heterochromatin formation

Abstract

Background Mammalian dosage compensation is achieved by the inactivation of one X chromosome in XX individuals. In eutheria this process is initiated early in development by the long non-coding RNA XIST. Studies of the initiation of silencing by XIST have focussed on mouse models, so the domains of XIST required to induce silencing in humans, and their relationship with domains required to establish heterochromatin remain to be determined. Methods We have previously established an inducible XIST cDNA in somatic cells and shown it can induce silencing and recruit heterochromatic features. We now assess a series of deletions across the construct for the ability to induce silencing and integrate these results with time-course and chromatin remodelling inhibitor treatments to follow the steps of XIST-induced silencing and heterochromatinization. Discussion We find that in addition to the previously reported necessity of the 5’ A repeat region for XIST-induced silencing, the F repeat region and a non-repetitive region at the 3’ end of the RNA are also required to silence genes. Silencing of genes up to 17Mb from the XIST integration occurs within two days, while formation of a Cot-1 depleted domain is slower, and more dependent on repeat F. The role of Repeat F in both the silencing of actively transcribed genes, the spread of H3K27me3 and the formation of a transcriptionally inert domain suggests a role in a pathway crucial for the spread of XIST across the chromatin to target distal regions of inactivation. Histone deacetylation requires only the A repeat region, with HDAC3 inhibition showing limited effect on silencing, but an impact on H3K27me3 recruitment, and as a result the recruitment of MacroH2A. Global HDAC inhibition impacted silencing in both a distance and dose-dependent fashion. The E repeat region was required for CIZ1 and H4K20me1 recruitment as well as H3K27me3; however, these appeared to act relatively independently. The H3K27me3 mark established by PRC2 integrated silencing and many of the heterochromatic features, while the PRC1 mark ubH2A appeared to be downstream of silencing in these human somatic cells.

Derivation of a minimal functional XIST by combining human and mouse interaction domains

“…Induction of an XIST cDNA integrated into an FRT site on chromosome 8p results in both silencing of flanking 8p genes and recruitment of heterochromatin marks 43 . Assessment of deletions spanning this full XIST reinforced that the 5’ end of XIST including the conserved A repeats was critical for silencing 44 . While the 5’ A repeats are essential for silencing, they are only sufficient to induce local silencing 45 .…”

“…Thus, based on the current knowledge about the different functions of each XIST domain, we tried to generate a smaller construct that could recapitulate all the XIST functions. Domain A is crucial for silencing by recruiting proteins at the initiation of XCI in both, mice and humans 5,6,15,44 , and domain E is relevant for XIST/Xist localization also through protein interactions 12,28,29,48 . While A and E domains are clearly critical, our recent results suggested that the functional pathways utilized in human somatic cells may differ from those described in mouse differentiation (reviewed in 7 ).…”

Targeting RNA with small molecules: lessons learned from Xist RNA

“…and what protein partners may be bound to them (Wutz et al 2002;Colognori et al 2019;Lee et al 2019b;Colognori et al 2020;Dixon-McDougall and Brown 2022). RepA was an obvious target because multiple studies have confirmed a requirement for initiating XCI silencing, and biochemical and cellular studies have identified a large suite of interacting protein partners, including PRC2 and SPEN (Zhao et al 2008;McHugh et al 2015;Monfort et al 2015).…”