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.
Subject(s)
Gene Silencing , Histone Deacetylases/metabolism , Mass Spectrometry/methods , Nuclear Proteins/metabolism , RNA, Long Noncoding/metabolism , Transcription, Genetic/genetics , X Chromosome/genetics , Acetylation , Animals , Cell Line , DNA-Binding Proteins , Embryonic Stem Cells/enzymology , Embryonic Stem Cells/metabolism , Female , Heterogeneous-Nuclear Ribonucleoprotein U/metabolism , Histones/metabolism , Male , Mice , Nuclear Receptor Co-Repressor 2/metabolism , Polycomb Repressive Complex 2/metabolism , Protein Binding , RNA Polymerase II/metabolism , RNA, Long Noncoding/genetics , RNA-Binding Proteins/analysis , RNA-Binding Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , X Chromosome/metabolism , X Chromosome Inactivation/genetics , Lamin B ReceptorABSTRACT
Expanded CGG repeats cause chromosomal fragility and hereditary neurological disorders in humans. Replication forks stall at CGG repeats in a length-dependent manner in primate cells and in yeast. Saccharomyces cerevisiae proteins Tof1 and Mrc1 facilitate replication fork progression through CGG repeats. Remarkably, the fork-stabilizing role of Mrc1 does not involve its checkpoint function. Thus, chromosomal fragility might occur when forks stalled at expanded CGG repeats escape the S-phase checkpoint.
