Mapping Transcriptome-Wide and Genome-Wide RNA-DNA Contacts with Chromatin-Associated RNA Sequencing (ChAR-seq).
Methods in molecular biology (Clifton, N.J.)
2020; 2161: 115–42
RNAs play key roles in the cell as molecular intermediates for protein synthesis and as regulators of nuclear processes such as splicing, posttranscriptional regulation, or chromatin remodeling. Various classes of non-coding RNAs, including long non-coding RNAs (lncRNAs), can bind chromatin either directly or via interaction with chromatin binding proteins. It has been proposed that lncRNAs regulate cell-state-specific genes by coordinating the locus-dependent activity of chromatin-modifying complexes. Yet, the vast majority of lncRNAs have unknown functions, and we know little about the specific loci they regulate. A key step toward understanding chromatin regulation by RNAs is to map the genomic loci with which every nuclear RNA interacts and, reciprocally, to identify all RNAs that target a given locus. Our ability to generate such data has been limited, until recently, by the lack of methods to probe the genomic localization of more than a few RNAs at a time. Here, we describe a protocol for ChAR-seq, an RNA-DNA proximity ligation method that maps the binding loci for thousands of RNAs at once and without the need for specific RNA or DNA probe sequences. The ChAR-seq approach generates chimeric RNA-DNA molecules in situ and then converts those chimeras to DNA for next-generation sequencing. Using ChAR-seq we detect many types of chromatin-associated RNA, both coding and non-coding. Understanding the RNA-DNA interactome and its changes during differentiation or disease with ChAR-seq will likely provide key insights into chromatin and RNA biology.
View details for DOI 10.1007/978-1-0716-0680-3_10
View details for PubMedID 32681510
Chromatin-Associated RNA Sequencing (ChAR-seq).
Current protocols in molecular biology
RNA is a fundamental component of chromatin. Noncoding RNAs (ncRNAs) can associate with chromatin to influence gene expression and chromatin state; many also act at long distances from their transcriptional origin. Yet we know almost nothing about the functions or sites of action for most ncRNAs. Current methods to identify sites of RNA interaction with the genome are limited to the study of a single RNA at a time. Here we describe a protocol for ChAR-seq, a strategy to identify all chromatin-associated RNAs and map their DNA contacts genome-wide. In ChAR-seq, proximity ligation of RNA and DNA to a linker molecule is used to construct a chimeric RNA-DNA molecule that is converted to DNA for sequencing. In a single assay, ChAR-seq can discover de novo chromatin interactions of distinct RNAs, including nascent transcripts, splicing RNAs, and long noncoding RNAs (lncRNAs). Resulting "maps" of genome-bound RNAs should provide new insights into RNA biology. © 2019 by John Wiley & Sons, Inc.
View details for PubMedID 30786161
Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts
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.
View details for PubMedID 29648534
Phosphorylated SIRT1 associates with replication origins to prevent excess replication initiation and preserve genomic stability
NUCLEIC ACIDS RESEARCH
2017; 45 (13): 7807–24
Chromatin structure affects DNA replication patterns, but the role of specific chromatin modifiers in regulating the replication process is yet unclear. We report that phosphorylation of the human SIRT1 deacetylase on Threonine 530 (T530-pSIRT1) modulates DNA synthesis. T530-pSIRT1 associates with replication origins and inhibits replication from a group of 'dormant' potential replication origins, which initiate replication only when cells are subject to replication stress. Although both active and dormant origins bind T530-pSIRT1, active origins are distinguished from dormant origins by their unique association with an open chromatin mark, histone H3 methylated on lysine 4. SIRT1 phosphorylation also facilitates replication fork elongation. SIRT1 T530 phosphorylation is essential to prevent DNA breakage upon replication stress and cells harboring SIRT1 that cannot be phosphorylated exhibit a high prevalence of extrachromosomal elements, hallmarks of perturbed replication. These observations suggest that SIRT1 phosphorylation modulates the distribution of replication initiation events to insure genomic stability.
View details for DOI 10.1093/nar/gkx468
View details for Web of Science ID 000406776400033
View details for PubMedID 28549174
View details for PubMedCentralID PMC5570034