Functional classification of noncoding RNAs associated with distinct histone modifications by PIRCh-seq

Abstract

32 33 Many long noncoding RNAs (lncRNAs) regulate gene transcription through 34 binding to histone modification complexes. Therefore, a comprehensive study of nuclear 35RNAs in a histone modification-specific manner is critical to understand their regulatory 36 mechanisms. Here we develop a method named Profiling Interacting RNAs on Chromatin 37 by deep sequencing (PIRCh-seq), in which we profile chromatin-associated transcriptome 38in 5 different cell types using antibodies recognizing histone H3 and 6 distinct histone 39 modifications associated with active or repressive chromatin states. PIRCh-seq identified 40chromatin-associated RNAs with substantially less contamination by nascent transcripts, 41as compared to existing methods. We classified chromatin-enriched lncRNAs into 6 42 functional groups based on the patterns of their association with specific histone 43 modifications. LncRNAs were enriched with different chromatin modifications in 44 different cell types, suggesting lncRNAs' regulation may also be cell type-specific. By 45 integrating profiles of RNA secondary structure and RNA m 6 A modification, we found 46 that RNA bases which bind to chromatin tend to be more single stranded. We discovered 47 hundreds of allele-specific RNA-chromatin interactions, nominating specific single 48 nucleotide variants that alter RNA association with chromatin. These results provide a 49 unique resource to globally study the functions of chromatin-associated lncRNAs and 50 elucidate the basic mechanisms of chromatin-RNA interaction.

Structural modularity of the XIST ribonucleoprotein complex

“…Together, the XIST RNP complex acts as a bridge to bring the X chromosome to the nuclear periphery for remodeling and silencing. To understand how the multiple XIST-associated proteins coordinate the localization of Xi and silencing, we used PIRCh sequencing 37 to identify regions of RNA that associate with chromatin ( Fig. 5a) and analyzed the binding sites of these proteins on XIST (Fig.…”

“…We found that stochastic folding of the A-repeat domain serves as a multivalent platform to recruit SPEN. To determine the structural of the multi-functional XIST RNP complex, we develop and integrate several methods, including PARIS (psoralen crosslinking) 27 , formaldehyde RNA immunoprecipitation sequencing (fRIP-seq; formaldehyde) 35 , enhanced crosslinking and immunoprecipitation (eCLIP; ultraviolet (UV) crosslinking) 36 , and PIRCh (purification of interacting RNA on chromatin, using glutaraldehyde crosslinking) 37 . Together, we find that the entire XIST RNA-protein complex is folded in a modular manner.…”

Understanding Long Noncoding RNA and Chromatin Interactions: What We Know So Far

“…PIRCh-seq was developed to identify chromatin-associated transcriptome using antibodies recognizing histone H3, and six other distinct histone modifications associated with both active and repressive chromatin states [95]. This study additionally integrated the profiles of RNA secondary structure and RNA m6A modification to identify RNA sequences that are in contact with chromatin.…”

Multimodal Long Noncoding RNA Interaction Networks: Control Panels for Cell Fate Specification

“…Profiling interacting RNAs on chromatin: Profiling interacting RNAs on chromatin (PIRCh-Seq) is an antibody-dependent approach that profiles RNA-chromatin interactions, with less enrichment of nascent RNAs cotranscriptionally tethered by RNA polymerases to chromatin. PIRCh-Seq has been used to identify the chromatin-associated transcriptome in both human and mouse ES cells and fibroblasts, as well as mouse neuronal progenitors, where the authors found cell-and allele-specific RNA-chromatin interactions (Fang et al 2019).…”