Summary RNA 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.

The pervasive transcription of our genome presents a possibility of revealing new genomic functions by investigating RNA interactions. Current methods for mapping RNA–RNA interactions have to rely on an ‘anchor' protein or RNA and often require molecular perturbations. Here we present the MARIO (Mapping RNA interactome in vivo) technology to massively reveal RNA–RNA interactions from unperturbed cells. We mapped tens of thousands of endogenous RNA–RNA interactions from mouse embryonic stem cells and brain. We validated seven interactions by RNA antisense purification and one interaction using single-molecule RNA–FISH. The experimentally derived RNA interactome is a scale-free network, which is not expected from currently perceived promiscuity in RNA–RNA interactions. Base pairing is observed at the interacting regions between long RNAs, including transposon transcripts, suggesting a class of regulatory sequences acting in trans. In addition, MARIO data reveal thousands of intra-molecule interactions, providing in vivo data on high-order RNA structures.

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...

Chromatin-associated RNA (caRNA) has been proposed as a type of epigenomic modifier. Here, we test whether environmental stress can induce cellular dysfunction through modulating RNA-chromatin interactions. We induce endothelial cell (EC) dysfunction with high glucose and TNFα (H + T), that mimic the common stress in diabetes mellitus. We characterize the H + T-induced changes in gene expression by single cell (sc)RNA-seq, DNA interactions by Hi-C, and RNA-chromatin interactions by iMARGI. H + T induce inter-chromosomal RNA-chromatin interactions, particularly among the super enhancers. To test the causal relationship between H + T-induced RNA-chromatin interactions and the expression of EC dysfunction-related genes, we suppress the LINC00607 RNA. This suppression attenuates the expression of SERPINE1, a critical pro-inflammatory and pro-fibrotic gene. Furthermore, the changes of the co-expression gene network between diabetic and healthy donor-derived ECs corroborate the H + T-induced RNA-chromatin interactions. Taken together, caRNA-mediated dysregulation of gene expression modulates EC dysfunction, a crucial mechanism underlying numerous diseases.