Many long non-coding RNAs (lncRNAs) affect gene expression1, but the mechanisms by which they act are still largely unknown2. One of the best-studied lncRNAs is Xist, which is required for transcriptional silencing of one X-chromosome during development in female mammals3,4. Despite extensive efforts to define the mechanism of Xist-mediated transcriptional silencing, we still do not know any proteins required for this role3. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell5. Here we develop a method to purify a lncRNA and identify its direct interacting proteins using quantitative mass spectrometry. We identify 10 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-repressor6 that activates HDAC37, is not only essential for silencing, but is also required for the exclusion of RNA Polymerase II (PolII) from the inactive X. Both SMRT and HDAC3 are also required for silencing and PolII 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 PolII across the X-chromosome.

Many large noncoding RNAs (lncRNAs) regulate chromatin, but the mechanisms by which they localize to genomic targets remain unexplored. Here we investigate the localization mechanisms of the Xist lncRNA during X-chromosome inactivation (XCI), a paradigm of lncRNA-mediated chromatin regulation. During the maintenance of XCI, Xist binds broadly across the X-chromosome. During initiation of XCI, Xist initially transfers to distal regions across the X-chromosome that are not defined by specific sequences. Instead, Xist identifies these regions by exploiting the three-dimensional conformation of the X-chromosome. Xist requires its silencing domain to spread across actively transcribed regions and thereby access the entire chromosome. This suggests a model where Xist coats the X-chromosome by searching in three dimensions, modifying chromosome structure, and spreading to newly accessible locations.

SUMMARY Macrophages respond to inflammatory stimuli by modulating the expression of hundreds of genes in a defined temporal cascade, with diverse transcriptional and post-transcriptional mechanisms contributing to the regulatory network. We examined pro-inflammatory gene regulation in activated macrophages by performing RNA-Seq with fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts. This methodological approach allowed us to separate the synthesis of nascent transcripts from transcript processing and the accumulation of mature mRNAs. In addition to documenting the sub-cellular locations of coding and non-coding transcripts, the results provide a high-resolution view of the relationship between defined promoter and chromatin properties and the temporal regulation of diverse classes of co-expressed genes. The data also reveal a striking accumulation of full-length yet incompletely spliced transcripts in the chromatin fraction, suggesting that splicing often occurs after transcription has been completed, with transcripts retained on the chromatin until fully spliced.