Project description:RNAseq on Spen-degron TX1072 clones of neural progenitor cells (NPCs). RNAseq in 2 biological replicates at 3 different times of auxin treatment (0h aux, 24h aux, 48h aux).
Project description:CUT&RUN on Spen-degron TX1072 mESCs was performed in 2 biological replicates. Cells were treated with doxycycline (1ug/mL) for 0h, 4h, 8h, 24h and 8h doxycycline and auxin (500uM).
Project description:Xist represents a paradigm for long non-coding RNA function in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Multiple Xist-RNA binding proteins have recently been identified, including SPEN/SHARP, whose knockdown has been associated with deficient XCI at multiple loci. Here we demonstrate that SPEN is a key orchestrator of XCI in vivo and unravel its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and embryonic stem cells. On the other hand, SPEN is dispensable for maintenance of XCI in neural progenitor cells, although it significantly dampens expression of genes that escape from XCI. During initiation of XCI, we show by live-cell imaging and CUT&RUN approaches that SPEN is immediately recruited to the X chromosome upon Xist up-regulation, where it is targeted to enhancers and promoters of actively transcribed genes. SPEN rapidly disengages from chromatin once silencing is accomplished, implying a need for active transcription to tether it to chromatin. We define SPEN’s SPOC (SPEN paralog and ortholog C-terminal) domain as a major effector of SPEN’s gene silencing function, and show that artificial tethering of SPOC to Xist RNA is sufficient to mediate X-linked gene silencing. 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 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.
Project description:HiC on Spen-degron TX1072 clones of neural progenitor cells (NPCs). HiC in 2 biological replicates before and after. 24h of auxin treatment.
