Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts
- Stanford University, United States
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.
Data availability
All sequence data has been deposited in GEO under accession number GSE97131The software analysis pipeline is available at https://gitlab.com/charseq/flypipe
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Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contactsPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE97131).
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Domain ChIRP reveals the modularity of long noncoding RNA architecture, function, and target genesPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE53020).
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High-Affinity Sites Form an Interaction Network to Facilitate Spreading of the MSL Complex across the X Chromosome in DrosophilaPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE58821).
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GRID-seq reveals the global RNA-chromatin interactomePublicly available at the NCBI Gene Expression Omnibus (accession no: GSE82312).
Article and author information
Author details
Funding
National Institutes of Health (Stanford Center for Systems Biology (NIH P50 GM107615) Seed Grant)
- Jason C Bell
- David Jukam
- Viviana I Risca
- Whitney L Johnson
Howard Hughes Medical Institute (HHMI-Simons Faculty Scholar Award)
- Jan M Skotheim
National Institutes of Health (P50HG00773501)
- William James Greenleaf
National Institutes of Health (R01GM106005)
- Aaron F Straight
Stanford University School of Medicine (Dean's Fellowship)
- Jason C Bell
National Institutes of Health (R01HG009909)
- William James Greenleaf
- Aaron F Straight
National Institutes of Health (R21HG007726)
- William James Greenleaf
National Institutes of Health (NIH Ruth Kirchstein National Research Service Award (F32GM116338))
- Jason C Bell
National Institutes of Health (NIH Ruth Kirchstein National Research Service Award (F32GM108295 ))
- David Jukam
Stanford University (Walter V. and Idun Berry Fellowship)
- Viviana I Risca
National Institutes of Health (Stanford Genetics Training Program (5T32HG000044-19))
- Nicole A Teran
National Institutes of Health (Molecular Pharmacology Training Grant (NIH T32-GM113854-02))
- Owen K Smith
National Institutes of Health (NIH T32 Training Fellowship (GM007276))
- Whitney L Johnson
National Science Foundation (DGE-114747)
- Whitney L Johnson
National Institutes of Health (RO1 HD085135)
- Jan M Skotheim
- Aaron F Straight
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Bell et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts
eLife 7:e27024.
https://doi.org/10.7554/eLife.27024
Further reading
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- Chromosomes and Gene Expression
- Evolutionary Biology
Gene regulation is essential for life and controlled by regulatory DNA. Mutations can modify the activity of regulatory DNA, and also create new regulatory DNA, a process called regulatory emergence. Non-regulatory and regulatory DNA contain motifs to which transcription factors may bind. In prokaryotes, gene expression requires a stretch of DNA called a promoter, which contains two motifs called –10 and –35 boxes. However, these motifs may occur in both promoters and non-promoter DNA in multiple copies. They have been implicated in some studies to improve promoter activity, and in others to repress it. Here, we ask whether the presence of such motifs in different genetic sequences influences promoter evolution and emergence. To understand whether and how promoter motifs influence promoter emergence and evolution, we start from 50 ‘promoter islands’, DNA sequences enriched with –10 and –35 boxes. We mutagenize these starting ‘parent’ sequences, and measure gene expression driven by 240,000 of the resulting mutants. We find that the probability that mutations create an active promoter varies more than 200-fold, and is not correlated with the number of promoter motifs. For parent sequences without promoter activity, mutations created over 1500 new –10 and –35 boxes at unique positions in the library, but only ~0.3% of these resulted in de-novo promoter activity. Only ~13% of all –10 and –35 boxes contribute to de-novo promoter activity. For parent sequences with promoter activity, mutations created new –10 and –35 boxes in 11 specific positions that partially overlap with preexisting ones to modulate expression. We also find that –10 and –35 boxes do not repress promoter activity. Overall, our work demonstrates how promoter motifs influence promoter emergence and evolution. It has implications for predicting and understanding regulatory evolution, de novo genes, and phenotypic evolution.
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- Chromosomes and Gene Expression
- Developmental Biology
The male-specific lethal complex (MSL), which consists of five proteins and two non-coding roX RNAs, is involved in the transcriptional enhancement of X-linked genes to compensate for the sex chromosome monosomy in Drosophila XY males compared with XX females. The MSL1 and MSL2 proteins form the heterotetrameric core of the MSL complex and are critical for the specific recruitment of the complex to the high-affinity ‘entry’ sites (HAS) on the X chromosome. In this study, we demonstrated that the N-terminal region of MSL1 is critical for stability and functions of MSL1. Amino acid deletions and substitutions in the N-terminal region of MSL1 strongly affect both the interaction with roX2 RNA and the MSL complex binding to HAS on the X chromosome. In particular, substitution of the conserved N-terminal amino-acids 3–7 in MSL1 (MSL1GS) affects male viability similar to the inactivation of genes encoding roX RNAs. In addition, MSL1GS binds to promoters such as MSL1WT but does not co-bind with MSL2 and MSL3 to X chromosomal HAS. However, overexpression of MSL2 partially restores the dosage compensation. Thus, the interaction of MSL1 with roX RNA is critical for the efficient assembly of the MSL complex on HAS of the male X chromosome.
