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

Nature. 2015 May 14;521(7551):232-6. doi: 10.1038/nature14443. Epub 2015 Apr 27.

Abstract

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 silencing of one X chromosome during 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 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Animals
  • Cell Line
  • DNA-Binding Proteins
  • Embryonic Stem Cells / enzymology
  • Embryonic Stem Cells / metabolism
  • Female
  • Gene Silencing*
  • Heterogeneous-Nuclear Ribonucleoprotein U / metabolism
  • Histone Deacetylases / metabolism*
  • Histones / metabolism
  • Lamin B Receptor
  • Male
  • Mass Spectrometry / methods*
  • Mice
  • Nuclear Proteins / metabolism*
  • Nuclear Receptor Co-Repressor 2 / metabolism
  • Polycomb Repressive Complex 2 / metabolism
  • Protein Binding
  • RNA Polymerase II / metabolism
  • RNA, Long Noncoding / genetics
  • RNA, Long Noncoding / metabolism*
  • RNA-Binding Proteins / analysis
  • RNA-Binding Proteins / metabolism
  • Receptors, Cytoplasmic and Nuclear / metabolism
  • Transcription, Genetic / genetics*
  • X Chromosome / genetics*
  • X Chromosome / metabolism
  • X Chromosome Inactivation / genetics

Substances

  • DNA-Binding Proteins
  • Heterogeneous-Nuclear Ribonucleoprotein U
  • Histones
  • Nuclear Proteins
  • Nuclear Receptor Co-Repressor 2
  • RNA, Long Noncoding
  • RNA-Binding Proteins
  • Receptors, Cytoplasmic and Nuclear
  • SAF-A protein, mouse
  • Spen protein, mouse
  • XIST non-coding RNA
  • Polycomb Repressive Complex 2
  • RNA Polymerase II
  • Histone Deacetylases
  • histone deacetylase 3