A 5' fragment of Xist can sequester the RNA of adjacent genes on chromatin

Xist requires Repeat-A, a protein binding module in its first two kilobases (2kb), to repress transcription. We report that in mouse embryonic stem cells (ESCs), when expressed as a standalone transcript, the first 2kb of Xist (Xist-2kb) represses the expression of adjacent genes not at the transcriptional level, but by sequestering their mRNA on chromatin. Sequestration does not spread beyond adjacent genes, requires the same sequence elements in Repeat-A that full-length Xist requires to repress transcription, and can be induced by lncRNAs whose sequence composition is related to Xist-2kb. We did not detect local sequestration by full-length Xist, but did detect it by mutant forms of Xist with attenuated transcriptional silencing capability. SPEN and RBM15, Repeat-A binding proteins required for Xist-induced transcriptional silencing, also bound Repeat-A in Xist-2kb, but neither protein was necessary for sequestration. Thus, in mouse ESCs, expression of a 5¢ fragment of Xist that contains Repeat-A can repress gene expression by sequestering nearby mRNAs on chromatin, but it does not induce transcriptional silencing. Rather, Xist-induced transcriptional silencing requires synergy between Repeat-A and additional sequence elements in Xist. These results highlight an important gap in our understanding of the mechanisms through which Repeat-A functions within Xist. We discuss how the mechanisms that underpin sequestration may be relevant to the biology of Xist and of other lncRNAs. Overall design: RNA-seq data compare relative gene expression and transcript subcellular localization upon expression of Xist