SETDB1 and HUSH modulate Xist RNA levels during establishment of X chromosome inactivation

The non-coding RNA Xist mediates X chromosome inactivation (XCI) in mammals, functioning in cis to direct heterochromatin formation over a single X chromosome in cells of early female embryos. Prior studies have determined that Xist transcription and turnover are modulated to maintain the correct level of Xist RNA to inactivate a single X chromosome. The underlying molecular mechanisms are poorly understood. In this study we demonstrate that RNA-dependent recruitment of the H3K9me3 methyltransferase SETDB1 and the HUSH complex across the transcribed Xist locus functions to suppress Xist transcription during establishment of XCI. Thus, degron-mediated acute depletion of SETDB1 or core HUSH subunits abrogates allelic H3K9me3 deposition, increasing Xist transcription, accumulation of Xist RNA, and the rate of X-linked gene silencing. Our findings support a model in which the SETDB1/HUSH pathway coordinates Xist transcription rates to maintain optimal Xist RNA levels, ensuring the silencing of a single X chromosome.