Non-Coding RNA: X-Chromosome Inactivation Unravelled

RESEARCH HIGHLIGHTS

Nature Reviews Molecular Cell Biology | AOP, published online 8 May 2015; doi:10.1038/nrm3998

NON-CODING RNA X-chromosome inactivation unravelled The long non-coding RNA (lncRNA) specific and reproducible set of ten suggesting that SAFA acts to localize Xist (X inactive-specific transcript) is proteins that directly interact with Xist to genomic DNA. Depleting required for the transcriptional silenc- Xist. Of these proteins, knocking Xist binds to SHARP led to the retention of Pol II ing of one X chromosome in each cell, down the genes encoding scaffold SHARP to at Xist-coated X chromosomes, in a process known as X-chromosome attachment factor A (SAFA; also recruit SMRT indicating that SHARP might be inactivation (XCI) that occurs during known as HNRNPU), SMRT- and required for initiating transcriptional mammalian female development. HDAC1-associated repressor protein and activate silencing following Xist localization, Owing to technical limitations, little (SHARP; also known as SPEN or HDAC3 … possibly by recruiting the transcrip- is known about the mechanism of MSX2-interacting protein) and resulting in tional co-repressors SMRT and transcriptional silencing during XCI. lamin-B receptor (LBR) largely abol- HDAC3. Indeed, depleting SMRT McHugh et al. now describe using ished the silencing of XCI‑affected gene silencing or HDAC3 (but not other HDACs) RNA antisense purification followed genes in the male ES cells as well as in abrogated Xist-dependent gene by quantitative mass spectrometry differentiating female ES cells. These silencing. Another feature of XCI (RAP–MS) — a novel approach for three proteins are therefore required is the recruitment of the Polycomb identifying proteins that directly for Xist-mediated gene silencing. repressive complex 2 (PRC2), which interact with lncRNAs in vivo — to SAFA was previously shown to tether initiates maintenance of chromatin identify Xist-interacting proteins that Xist to the inactive X chromosome, compaction and gene silencing have a role in XCI. SHARP is known to interact with by catalysing histone H3 Lys27 RAP–MS involves crosslinking the histone deacetylase 3 (HDAC3)- trimethylation. The authors found lncRNAs and proteins in vivo, activator SMRT (silencing mediator that although PRC2 is not required followed by stringent, antisense- of retinoic acid and thyroid hormone for initiating XCI, depleting SHARP mediated purification of the directly receptor; also known as NCOR2) and or HDAC3 resulted in the loss of interacting proteins. SILAC (stable LBR anchors heterochromatin to the PRC2 at inactivated X chromosomes, isotope labelling by amino acids inner nuclear membrane. suggesting that SHARP and HDAC3 in culture) is then used for the The initiation of XCI includes may also have a role in establishing a quantitative comparison of puri- the localization of Xist along the repressive epigenetic state following fied proteins by mass spectrometry X chromosome and the exclusion of the initiation of XCI. between experimental and control RNA polymerase II (Pol II) from the In summary, the data support RNA purifications. The authors chromosome. The depletion of SAFA a model in which SAFA tethers performed RAP–MS on male mouse resulted in diffused localization of Xist to X chromosomes. Xist binds embryonic stem (ES) cells induced to Xist in the nucleus, in agreement to SHARP to recruit SMRT and express Xist and identified a highly with previously published data, activate HDAC3, which directs the removal of histone acetylation marks, resulting in gene silencing and chromatin condensation. Following the initiation of XCI, Xist recruits PRC2 to the X chromosome in a HDAC3- dependent manner to maintain the epigenetic inactive state. The data also show that RAP–MS is highly specific and efficient and thus could be used for identifying directly interacting proteins of any lncRNA. Eytan Zlotorynski

ORIGINAL RESEARCH PAPER McHugh, C. A. et al. The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature GETTY http://dx.doi.org/10.1038/nature14443 (2015)

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 16 | JUNE 2015