Systematic Mapping of RNA-Chromatin Interactions In Vivo

Abstract

SummaryRNA molecules can attach to chromatin. It remains difficult to know what RNAs are associated with chromatin and where are the genomic target loci of these RNAs. Here, we present MARGI (Mapping RNA-genome interactions), a technology to massively reveal native RNA-chromatin interactions from unperturbed cells. The gist of this technology is to ligate chromatin associated RNAs (caRNAs) with their target genomic sequences by proximity ligation, forming RNA-DNA chimeric sequences, which are converted to sequencing library for paired-end sequencing. Using MARGI, we produced RNA-genome interaction maps for human embryonic stem (ES) cells and HEK cells. MARGI revealed hundreds of caRNAs including previously known XIST, SNHG1, NEAT1, MALAT1, as well as each caRNA's genomic interaction loci. Using a cross-species experiment, we estimated that approximately 2.2% of MARGI identified interactions were false positives. In ES and HEK cells, the RNA ends of more than 5% of MARGI read pairs were mapped to distal or inter-chromosomal locations as compared to the locations of their corresponding DNA ends. The majority of transcription start sites are associated with distal or inter-chromosomal caRNAs. ChIP-seq reported H3K27ac and H3K4me3 levels are positively while H3K9me3 is negatively correlated with MARGI reported RNA attachment levels. The MARGI technology should facilitate revealing novel RNA functions and their genomic target regions. Graphical abstractSridhar et al. develop a technology to map global RNA-chromatin interactions in unperturbed cells. They discover hundreds of chromatin associated RNAs. They find that the majority of Correspondence to: Sheng Zhong. 3 Co-first author 4 Lead Contact Supplemental Information: Supplemental Information includes Supplemental Experimental Procedures and four figures and can be found with this article online. All sequencing data are available at Gene Expression Omnibus with access number GSE92345.Author Contributions: B.S., T.C.N., and S.Z. designed the experiments. B.S., T.C.N., and L.H performed the experiments. All authors analyzed and interpreted the data.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. HHS Public Access Results and Discussion Development of the MARGI technologyWe developed MARGI (Mapping RNA-genome interactions), a technology to massively reveal RNA-chromatin interactions from unperturbed cells. MARGI simultaneously identifies all caRNAs and the respective genomic target loci of each caRNA. This changes the paradigm of analyzing one-RNA-at-a-time, and enables the mapping of the native RNAchromatin interaction netwo...

RADICL-seq identifies general and cell type–specific principles of genome-wide RNA-chromatin interactions

“…Comparison of RADICL-seq with existing technologies. RADICL-seq introduces substantial improvements over similar RNA-chromatin proximity ligation approaches [7][8][9] . Compared with MARGI, RADICL-seq minimizes the frequency of spurious interactions in the dataset by performing the in situ ligation in intact nuclei.…”

“…A few technologies have emerged to assess genome-wide RNA-chromatin interactions [7][8][9] , but each has limitations. Mapping RNA-genome interactions (MARGI) is a proximity ligationbased technology that requires a high number of input cells (i.e., hundreds of millions) and the disruption of the nuclear structure 7 , which can result in detection of a large number of spurious interactions 10 ; because of this, it has limited applicability to investigations of RNA-chromatin interactions in multiple cell types. Chromatin-associated RNA-sequencing (CHAR-seq) and global RNA interaction with DNA by deep-sequencing (GRID-seq) utilize in situ approaches to detect genome-wide RNA-chromatin contacts 8,9 .…”

RNAs as Proximity-Labeling Media for Identifying Nuclear Speckle Positions Relative to the Genome

“…These relative sizes suggest that genomic regions in proximity to nuclear speckles may be significantly larger than the typical sizes of ChIP-seq or ATAC-seq peaks. Nevertheless, the enrichment of Xist RNA on X chromosome revealed by imaging ( Jonkers et al., 2008 ) was successfully corroborated by genomics technologies including RAP-seq ( Engreitz et al., 2014 ) and MARGI ( Sridhar et al., 2017 ), offering an example of convergent findings from imaging and genomics approaches. In this work, we tested our “nsaRNA proximity” hypothesis by combining microscopic information and genomics data and aimed for establishing an RNA-based approach for identifying the relative positions of the folded genome and subnuclear structures.…”

“…The recent technology on global mapping of RNA- g enome interactions (MARGI) enabled the identification of interacting genomic sequences of chromatin-interacting RNAs ( Sridhar et al., 2017 ). After cross-linking and genome fragmentation, MARGI ligates RNA, a linker sequence, and proximal DNA to form an RNA-linker-DNA chimeric sequence, which is subsequently converted to double-stranded DNA and subjected to paired-end sequencing (see Figure 1 of Sridhar et al. (2017) ).…”

Central Dogma, Selfish DNA and Noncoding RNAs: A Historical Perspective

“…This indeed has happened. The diverse regulatory and other functions of heterochromatin can now be examined in terms of the large variety of small and long noncoding RNAs generated by and associated with this enigmatic component of eukaryotic genomes (Bierhoff et al, 2014;Chuong et al, 2017;Felden and Paillard, 2017;Lakhotia, 2017aLakhotia, , 2017bSawyer and Dundr, 2017).The presence of chromosomal RNAs, reported in 1960s but quickly debunked in 1970s (see above), may find parallels in the roles of RNAs like the Xist and roX transcripts and in the many other small and long noncoding RNAs that associate with chromatin and affect its structural and functional organization (Bell et al, 2017;Johnson et al, 2017;Lakhotia, 2016Lakhotia, , 2017aLakhotia, , 2017bSridhar et al, 2017;Velazquez Camacho et al, 2017).…”