The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3

• Published in: Nature (London) Vol. 521; no. 7551; pp. 232 - 236
• Main Authors: McHugh, Colleen A., Chen, Chun-Kan, Chow, Amy, Surka, Christine F., Tran, Christina, McDonel, Patrick, Pandya-Jones, Amy, Blanco, Mario, Burghard, Christina, Moradian, Annie, Sweredoski, Michael J., Shishkin, Alexander A., Su, Julia, Lander, Eric S., Hess, Sonja, Plath, Kathrin, Guttman, Mitchell
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 14-05-2015; Nature Publishing Group
• Subjects: 631/208/176/1433; 631/208/200; 631/337/100/101; 631/337/384/2568; Acetylation; Animals; Cell Line; Chromosomes; DNA-Binding Proteins; Embryonic Stem Cells - enzymology; Embryonic Stem Cells - metabolism; Female; Gene Silencing; Genetic aspects; Genetic research; Heterogeneous-Nuclear Ribonucleoprotein U - metabolism; Histone Deacetylases - metabolism; Histones - metabolism; Humanities and Social Sciences; Lamin B Receptor; letter; Male; Mass spectrometry; Mass Spectrometry - methods; Methods; Mice; multidisciplinary; Nuclear Proteins - metabolism; Nuclear Receptor Co-Repressor 2 - metabolism; Polycomb Repressive Complex 2 - metabolism; Protein Binding; Proteins; Receptors, Cytoplasmic and Nuclear - metabolism; RNA; RNA Polymerase II - metabolism; RNA, Long Noncoding - genetics; RNA, Long Noncoding - metabolism; RNA-Binding Proteins - analysis; RNA-Binding Proteins - metabolism; RNA-protein interactions; Science; Scientific imaging; Transcription, Genetic - genetics; X Chromosome - genetics; X Chromosome - metabolism; X Chromosome Inactivation - genetics
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature14443

The mechanisms by which
Xist
, a long non-coding RNA, silences one X chromosome in female mammals are unknown; here a mass spectrometry-based approach is developed to identify several proteins that interact directly with
Xist
, including the transcriptional repressor SHARP that is required for transcriptional silencing through the histone deacetylase HDAC3.
Gene silencing mechanism of
Xist
Xist
is a long non-coding RNA required for transcriptional silencing of one X chromosome in female mammals. Mitchell Guttman and colleagues have used a mass spectrometry based approach to identify several proteins that directly interact with
Xist
, including the transcriptional repressor SHARP. SHARP and another repressor HDAC3 are required for silencing transcription and the
Xist
-mediated recruitment of polycomb across the X chromosome in embryonic stem cells.
Many long non-coding RNAs (lncRNAs) affect gene expression
1
, but the mechanisms by which they act are still largely unknown
2
. One of the best-studied lncRNAs is
Xist
, which is required for transcriptional silencing of one X chromosome during development in female mammals
3
,
4
. Despite extensive efforts to define the mechanism of
Xist
-mediated transcriptional silencing, we still do not know any proteins required for this role
3
. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell
5
. 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
6
that activates HDAC3
7
, 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 HDAC3 are also required for silencing and Pol II exclusion. In addition to silencing transcription, SHARP and HDAC3 are required for
Xist
-mediated recruitment of the polycomb repressive complex 2 (PRC2) across the X chromosome. Our results suggest that
Xist
silences transcription by directly interacting with SHARP, recruiting SMRT, activating HDAC3, and deacetylating histones to exclude Pol II across the X chromosome.