Independent domains for recruitment of PRC1 and PRC2 by human XIST

Abstract

XIST establishes inactivation across its chromosome of origin, even when expressed from autosomal transgenes. To identify the regions of human XIST essential for recruiting heterochromatic marks we generated a series of overlapping deletions in an autosomal inducible XIST transgene present in 8p of the HT1080 male fibrosarcoma cell line. We examined the ability of each construct to enrich its unified XIST territory with the histone marks established by PRC1 and PRC2 as well as the heterochromatin factors MacroH2A and SMCHD1. Chromatin enrichment of ubH2A by PRC1 required four distinct regions of XIST, and these were completely distinct from the two domains crucial for enrichment of H3K27me3 by PRC2. Both the domains required, as well as the impact of PRC1 and PRC2 inhibitors, suggest that PRC1 is required for SMCHD1 while PRC2 function is necessary for MacroH2A recruitment, although incomplete overlap of regions implicates roles for additional factors. This cooperativity between factors contributes to the requirement for multiple separate domains being required for each feature examined. The independence of the PRC1/PRC2 pathways was observed when XIST was expressed both autosomally or from the X chromosome suggesting that these observations are not purely a result of the context in which XIST operates. Although independent domains were required for the PRC1 and PRC2 pathways overall all regions tested were important for some aspect of XIST functionality, demonstrating both modularity and cooperativity across the XIST lncRNA.

Multiple distinct domains of human XIST are required to coordinate gene silencing and subsequent heterochromatin formation

“…In addition to the well-studied Repeat A region, we also observed that Repeat F and a 3' region were critical for silencing. While those domains were not required for H4K20me1 or CIZ1 recruitment, we previously demonstrated that H3K27me3 enrichment required the Repeat F region, while the 3' region was critical for ubH2A recruitment (13).…”

“…1C and Additional le 1: Table S2 (see ref. (13))). The change in allelic ratio caused by each deletion construct was normalized to the change in allelic ratio for Full length XIST and presented as strength of silencing, where a value of 1 indicated comparable silencing to Full XIST and 0 indicated no change in allelic contribution from the pre-induction levels.…”

“…The isolation of RNA, its puri cation and reverse transcription were all performed using the same methodology described in our previous publication in order to ensure consistency of results (13). In brief, RNA was extracted and puri ed from cells with TRIZOL according to the protocol of the manufacturer, Invitrogen.…”

“…In order to examine if and how these various XIST factors and pathways are associated with each other, we used the previously established HT1080 inducible XIST model system described in previous studies (13,16,17). A full-length XIST cDNA transgene in these male cells is integrated as a single copy on an autosome (8p) and induced with doxycycline (dox).…”

Long Non-Coding RNA (lncRNA) Roles in Cell Biology, Neurodevelopment and Neurological Disorders

“…The well-characterized lncRNAs HOTAIR , ANRIL , XIST and KCNQ1OT1 are able to recruit epigenetic modifiers to specific loci for reprogramming the chromatin state [ 64 , 80 ]. For example, HOTAIR acts as a scaffold for coordinating the targeting of specific repressive, histone-modifying complexes to target loci [ 80 ]; XIST mediates X chromosome inactivation by recruiting repressive histone complexes such as PRC1 and PRC2 [ 81 , 82 , 83 ] and DNA-methylating complex [ 84 ]. Additionally, lncRNAs affect allelic gene expression through imprinting.…”