4/3/2023 0 Comments Air video server hd ac3![]() ![]() 1c, d and Supplementary Table 1), while auxin had no effect on XCI in wild-type cells ( Extended Data Fig. However, gene silencing was almost completely abolished in the absence of SPEN, along the entire X chromosome ( Fig. 1c, e), confirming that SPEN is dispensable for Xist localization 2– 5. Loss of SPEN had no effect on the formation of Xist RNA clouds ( Extended Data Fig. To evaluate the immediate consequences of SPEN loss on initiation of XCI, we acutely depleted SPEN for 4 hours prior to inducing Xist expression for 24 hours and performed RNA-seq. d, e, h, horizontal lines denote the median, box limits correspond to upper and lower quartiles. h, X-chromosomal transcript allelic ratio distribution (n=256 genes) in WT (N=2), maternal-only Spen ko (N=3), maternal-zygotic Spen ko (N=5), and Xist ko E3.5 embryos (N=30 single-cells, *see Borensztein et al., two-sided Wilcoxon rank-sum test). g, Mouse cross schematic for Spen KO experiment. c, d, e, and f, show averages of 2 independent clones. ![]() f, Pyrosequencing assay of 7 X-linked transcripts in mESCs after 0h, 24h dox or 24h dox+auxin. e, Boxplot representation of gene silencing defect upon SPEN loss in three groups of genes differing by their SPEN-dependence level for Xist-mediated silencing. c, Heatmap and d, violin plots showing X-chromosomal transcript allelic ratios after 0h, 24h dox or 24h dox+auxin treatment in SPEN-degron mESCs (n=434 genes, two-sided Student’s t-test). This experiment was repeated at least twice with similar results. b, Western blot showing auxin-induced degradation of endogenous Halo-tagged SPEN. SPEN mediates gene silencing across the entire X chromosome in vitro and in vivo.Ī, Schematic of SPEN-degron Xist-inducible mESCs. ![]() We propose that SPEN acts as a molecular integrator for initiation of XCI, bridging Xist RNA with the transcription machinery as well as nucleosome remodelers and histone deacetylases, at active enhancers and promoters. We identify SPOC’s protein partners which include NCOR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in regulation of transcription initiation and elongation. ![]() We define SPEN’s SPOC domain as a major effector of SPEN’s gene silencing function, and show that tethering SPOC to Xist RNA is sufficient to mediate gene silencing. SPEN rapidly disengages from chromatin upon gene silencing, implying a need for active transcription to tether it to chromatin. We show that SPEN is immediately recruited to the X-chromosome upon Xist up-regulation, and is targeted to enhancers and promoters of active genes. SPEN is dispensable for maintenance of XCI in neural progenitors, although it significantly dampens expression of genes that escape XCI. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and embryonic stem cells. Here we demonstrate that SPEN is a key orchestrator of XCI in vivo and unravel its mechanism of action. Multiple Xist-RNA binding proteins have recently been identified, including SPEN 1– 3, the loss of which has been associated with deficient XCI at multiple loci 2– 6. Xist represents a paradigm for long non-coding RNA function in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. ![]()
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