Abstract
RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here, we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. Using Drosophila cells, we show that ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.
Highlights
Much of the eukaryotic genome is transcribed into non-coding RNA, and several studies have established that a subset of these ncRNAs form ribonucleoprotein complexes that bind and regulate chromatin (Guttman and Rinn, 2012; Meller et al, 2015; Cech and Steitz, 2014)
We observed a correlation between RNA expression level and chromatin-RNA contacts; a cluster of RNAs clearly generated more chromatin interactions that would be expected from the overall expression levels (Figure 2E). Using both the length and read normalized contacts (CPKM) and the fold-enrichment over RNA expression as measured by RNA-seq, we identified 138 RNAs that had more than 100 contacts per kilobase million reads (CPKM) and were enriched more than ten-fold, though many were enriched by 2–5 orders of magnitude (Figure 2E, red symbols; Figure 2—figure supplement 1)
When we correlated genome-wide binding signal within this class, we found that the distribution patterns of the major spliceosome small nuclear RNA (snRNA) U1, U2, U4, U5, U6 clustered together along with snRNA:7SK (Figure 4B), which is part of the p-TEFb complex that relieves pausing of RNA Polymerase II at promoters (Kwak and Lis, 2013) and may participate in the release of paused polymerase during RNA splicing (Barboric et al, 2009)
Summary
Much of the eukaryotic genome is transcribed into non-coding RNA (ncRNA), and several studies have established that a subset of these ncRNAs form ribonucleoprotein complexes that bind and regulate chromatin (Guttman and Rinn, 2012; Meller et al, 2015; Cech and Steitz, 2014). In Drosophila, roX1 and roX2 are part of the male-specific lethal (MSL) complex that coats the single male X chromosome to acetylate histone H4K16 and increase transcription (Conrad and Akhtar, 2012). Repetitive ncRNA transcripts have roles at chromosomal loci essential in maintaining genomic integrity over many cell divisions, including TERRA at telomeres (Bunting et al, 2010) and alpha-satellites near centromeres (Hall et al, 2012). Despite these well-studied examples, the genomic targets of most chromatin-associated ncRNAs are unknown, and the mechanisms by which these ncRNAs regulate the epigenetic and spatial organization of chromatin remain largely unexplored
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