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.
Highlights
XIST was one of the first long noncoding RNAs to be identified [1], and its role in establishing the complex heterochromatin of the inactive X chromosome (Xi) continues to yield new insights into the process of X-chromosome inactivation (XCI) and the functionality of lncRNAs
CRISPRdirected mutations of an inducible human XIST construct in somatic cells allowed us to discover which regions of the RNA are required for chromatin modification and protein recruitment
Treatment of the cells with 1μg/ml doxycycline for five days (5ddox) resulted in the XIST RNA forming a discrete unified domain that was frequently visibly enriched for heterochromatin marks such as H3K27me3 using immunofluorescence and fluorescent in situ hybridization (IF-FISH) (Fig 1B)
Summary
XIST was one of the first long noncoding RNAs (lncRNA) to be identified [1], and its role in establishing the complex heterochromatin of the inactive X chromosome (Xi) continues to yield new insights into the process of X-chromosome inactivation (XCI) and the functionality of lncRNAs. Human and mouse XIST/Xist show a similar exon/intron structure, including two major splicing isoforms, and ~67% sequence conservation across the 15–19 kb lncRNAs [3]. Both contain a series of tandem repeats (labelled A-F), of which only the A repeat is highly conserved in sequence and size across eutheria [4,5]. In humans there are more copies of the D repeats than in mouse [3,6,7]. The study of human XIST adds an important comparator to our growing understanding of mouse Xist functionality. Roles for XIST variation in human disease will require an understanding of the functional domains of XIST, as will reducing the size of XIST to enable its potential as a ‘chromosome therapeutic’ to silence trisomic chromosomes [8,9]
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