The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.
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.
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Colleen A
CA
McHugh
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Chun-Kan
CK
Chen
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Amy
A
Chow
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Christine F
CF
Surka
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Christina
C
Tran
Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02139, USA.
Patrick
P
McDonel
1] Department of Biological Chemistry, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90095, USA [2] Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90095, USA.
Amy
A
Pandya-Jones
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Mario
M
Blanco
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Christina
C
Burghard
Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, California 91125, USA.
Annie
A
Moradian
Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, California 91125, USA.
Michael J
MJ
Sweredoski
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Alexander A
AA
Shishkin
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Julia
J
Su
Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02139, USA.
Eric S
ES
Lander
Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, California 91125, USA.
Sonja
S
Hess
1] Department of Biological Chemistry, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90095, USA [2] Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90095, USA.
Kathrin
K
Plath
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Mitchell
M
Guttman
D000107:Acetylation; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins; D053595:Embryonic Stem Cells / Q000201:enzymology / Q000378:metabolism; D005260:Female; D020868:Gene Silencing; D034722:Heterogeneous-Nuclear Ribonucleoprotein U / Q000378:metabolism; D006655:Histone Deacetylases / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D056985:Nuclear Receptor Co-Repressor 2 / Q000378:metabolism; D063151:Polycomb Repressive Complex 2 / Q000378:metabolism; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D014960:X Chromosome / Q000235:genetics* / Q000378:metabolism; D049951:X Chromosome Inactivation / Q000235:genetics; D000097682:Lamin B Receptor; D000099186:Histone Deacetylase 3
D004268:DNA-Binding Proteins; D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D006655:Histone Deacetylases; D006657:Histones; D009687:Nuclear Proteins; D056985:Nuclear Receptor Co-Repressor 2; D063151:Polycomb Repressive Complex 2; D012319:RNA Polymerase II; D062085:RNA, Long Noncoding; D016601:RNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D000097682:Lamin B Receptor; D000099186:Histone Deacetylase 3; C558468:SAF-A protein, mouse; C121253:Spen protein, mouse; C077885:XIST non-coding RNA