The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3
2015
Many
long non-coding RNAs(lncRNAs) affect gene expression, but the mechanisms by which they act are still largely unknown. One of the best-studied lncRNAs is
Xist, which is required for transcriptional
silencingof one
X chromosomeduring development in female mammals. Despite extensive efforts to define the mechanism of
Xist-mediated transcriptional
silencing, we still do not know any proteins required for this role. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell. Here we develop a method to purify a lncRNA from cells and identify proteins interacting with it directly using quantitative mass spectrometry. We identify ten proteins that specifically associate with
Xist, three of these proteins—SHARP, SAF-A and LBR—are required for
Xist-mediated transcriptional
silencing. We show that SHARP, which interacts with the SMRT co-repressor that activates
HDAC3, is not only essential for
silencing, but is also required for the exclusion of
RNA polymerase II(Pol II) from the inactive X. Both SMRT and
HDAC3are also required for
silencingand Pol II exclusion. In addition to
silencingtranscription, SHARP and
HDAC3are required for
Xist-mediated recruitment of the polycomb repressive complex 2 (
PRC2) across the
X chromosome. Our results suggest that
Xist
silencestranscription by directly interacting with SHARP, recruiting SMRT, activating
HDAC3, and deacetylating histones to exclude Pol II across the
X chromosome.
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