Elements at the 5′ end of Xist harbor SPEN-independent transcriptional antiterminator activity

Abstract

The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5′ end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of downstream polyadenylation sequences. Readthrough required Repeat A and the ∼750 nucleotides downstream, did not require SPEN, and was attenuated by splicing. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a 5.5-kb Xist transgene robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5′ end of Xist harbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.

The control of polycomb repressive complexes by long noncoding RNAs

“…In the first to identify functional domains within Xist, it was found that a transgenic RNA comprised of the first 3 kb of Xist was incapable of inducing long-distance gene silencing-this fragment contained Repeat A, the domain within Xist essential for long-distance gene silencing, but it lacked all other portions of the transcript, including Repeats B, C, and E (Wutz et al, 2002). Extending these findings, we similarly found that a transgenic RNA comprised of the first 2 kb of Xist was retained on chromatin and bound the critical Xist silencing factor SPEN, but was essentially incapable of inducing gene silencing even of nearby genes (Trotman et al, 2020). In another study, in the context of a 3.9-kb Xist transgene that contained Repeat A, Repeat B, and a portion of Repeat C, deletion of either Repeat A or the Repeat B/C portion of the transgene caused a loss of its ability to silence gene expression and recruit the PRCs (Pintacuda, Wei, et al, 2017).…”

“…In support of this idea, we recently found that a transgene comprising the first 2 kb of Xist, which contains Repeat A but lacks all other known functional domains in Xist, still binds the critical silencing factor SPEN and associates robustly with chromatin, yet it is essentially incapable of inducing gene silencing, even of adjacent genes (Trotman et al, 2020). The reasons for this lack of silencing are currently unclear, but at a minimum our data indicate that Repeat A cooperates with other regions in Xist to induce gene silencing at the onset of XCI (Trotman et al, 2020). The findings from Almeida et al would suggest that the PRCs play a part in filling this cooperative role (Almeida et al, 2017).…”

“…Various forms of mass spectrometry, CLIP, and other methods to screen RNA-protein interactions have identified distinct sets of proteins that associate with each of the tandem repeats in Xist. Repeat A associates with the critical silencing factor SPEN along with a number of RBPs whose roles in XCI remain unknown, including many SR proteins, HNRNPC, and RALY (Chu et al, 2015;Cirillo et al, 2016;Graindorge et al, 2019;Pintacuda, Wei, et al, 2017;Trotman et al, 2020); Repeat B associates predominantly with HNRNPK (Cirillo et al, 2016;Colognori et al, 2019;Nakamoto et al, 2020;Pintacuda, Wei, et al, 2017); Repeat C also associates with HNRNPK and other RBPs, including HNRNPU (Bousard et al, 2019;Cirillo et al, 2016;Graindorge et al, 2019); Repeat D, while not studied as extensively as the other Xist repeats, also binds HNRNPK (Van Nostrand et al, 2016); and Repeat E associates with PTBP1, MATR3, TDP-43, CELF1, and CIZ1, among other proteins (Cirillo et al, 2016;Pandya-Jones et al, 2020;Ridings-Figueroa et al, 2017;Smola et al, 2016;Sunwoo et al, 2017;Van Nostrand et al, 2016). The underlying sequence and structural motifs that are unique to each of these repeats may underlie their ability to recruit distinct subsets of RBPs (Figures 4 and 5; Duszczyk et al, 2011;Fang et al, 2015;F.…”

Independent domains for recruitment of PRC1 and PRC2 by human XIST

“…Previous reports suggested that repeats D, F and A are involved in expression of XIST [ 50 – 54 ], which would not impact our promoter, although a recent report implicates feedback between transcription of mouse Xist and turnover of the RNA [ 55 ]. The effects of repeat A deletion seem to be both size and model system dependent in mouse, with variable effects on expression in particular [ 56 – 58 ]. We previously reported a larger deletion of the A region and surrounding region, Delta XB, in the HT1080 model system.…”

Chromatin Regulator SPEN/SHARP in X Inactivation and Disease

“…As discussed above, SHARP was recently identified in a screen for Xist RNA binding proteins (see Table 1 ) and it was shown to be essential for X-inactivation in embryonic stem cells (ESCs) [ 51 , 52 , 53 , 54 , 55 , 129 ]. However, SHARP does not bind exclusively SRA and Xist but also retroviral RNAs that are characterized by regions with structural similarity to the A-repeat of Xist [ 128 ], which is required for the Xist /SHARP interaction [ 51 , 53 , 130 ]. SHARP was shown to bind to the SRA lncRNA by its RRMs.…”