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RNA-DamID reveals cell-type-specific binding of roX RNAs at chromatin-entry sites.

ABSTRACT: Thousands of long noncoding RNAs (lncRNAs) have been identified in eukaryotic genomes, many of which are expressed in spatially and temporally restricted patterns. Nonetheless, the roles of the majority of these transcripts are still unknown. One of the mechanisms by which lncRNAs function is through the modulation of chromatin states. To assess the functions of lncRNAs, we developed RNA-DamID, a novel approach that detects lncRNA-genome interactions in a cell-type-specific manner in vivo with high sensitivity and accuracy. Identifying the cell-type-specific genome occupancy of lncRNAs is vital to understanding their mechanisms of action in development and disease. We used RNA-DamID to investigate targeting of the lncRNAs in the Drosophila dosage-compensation complex (DCC) and show that initial targeting is cell-type specific.

SUBMITTER: Cheetham SW

PROVIDER: S-EPMC5813796 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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RNA-DamID reveals cell-type-specific binding of roX RNAs at chromatin-entry sites.

Cheetham Seth W SW Brand Andrea H AH

Nature structural & molecular biology 20171218 1

Thousands of long noncoding RNAs (lncRNAs) have been identified in eukaryotic genomes, many of which are expressed in spatially and temporally restricted patterns. Nonetheless, the roles of the majority of these transcripts are still unknown. One of the mechanisms by which lncRNAs function is through the modulation of chromatin states. To assess the functions of lncRNAs, we developed RNA-DamID, a novel approach that detects lncRNA-genome interactions in a cell-type-specific manner in vivo with h ...[more]

PMID: 29323275

Similar Datasets

RNA-DamID reveals cell-type-specific binding of roX RNAs at chromatin entry sites

Project description:Thousands of long noncoding RNAs (lncRNAs) have been identified in eukaryotic genomes, many of which are expressed in spatially and temporally restricted patterns. Nonetheless, the roles of the majority of these transcripts are still unknown. One of the mechanisms by which lncRNAs function is through the modulation of chromatin state. To assess the functions of lncRNAs we developed RNA-DamID, a novel approach that detects lncRNA-genome interactions in a cell-type specific manner in vivo with high sensitivity and accuracy. Identifying the cell-type-specific genome occupancy of lncRNAs is key to understanding their mechanisms of action in development and disease. We used RNA-DamID to investigate targeting of the lncRNAs in the Drosophila dosage compensation complex (DCC) and show that initial targeting is cell-type-specific.

2017-12-04 | GSE97456 | GEO

RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent Pre-mRNAs and chromatin sites.

Project description:Intermolecular RNA-RNA interactions are used by many noncoding RNAs (ncRNAs) to achieve their diverse functions. To identify these contacts, we developed a method based on RNA antisense purification to systematically map RNA-RNA interactions (RAP-RNA) and applied it to investigate two ncRNAs implicated in RNA processing: U1 small nuclear RNA, a component of the spliceosome, and Malat1, a large ncRNA that localizes to nuclear speckles. U1 and Malat1 interact with nascent transcripts through distinct targeting mechanisms. Using differential crosslinking, we confirmed that U1 directly hybridizes to 5' splice sites and 5' splice site motifs throughout introns and found that Malat1 interacts with pre-mRNAs indirectly through protein intermediates. Interactions with nascent pre-mRNAs cause U1 and Malat1 to localize proximally to chromatin at active genes, demonstrating that ncRNAs can use RNA-RNA interactions to target specific pre-mRNAs and genomic sites. RAP-RNA is sensitive to lower abundance RNAs as well, making it generally applicable for investigating ncRNAs.

| S-EPMC4177037 | biostudies-literature

Incorporation of the noncoding roX RNAs alters the chromatin-binding specificity of the Drosophila MSL1/MSL2 complex.

Project description:The male-specific lethal (MSL) protein-RNA complex is required for X chromosome dosage compensation in Drosophila melanogaster. The MSL2 and MSL1 proteins form a complex and are essential for X chromosome binding. In addition, the MSL complex must integrate at least one of the noncoding roX RNAs for normal X chromosome binding. Here we find the amino-terminal RING finger domain of MSL2 binds as a complex with MSL1 to the heterochromatic chromocenter and a few sites on the chromosome arms. This binding required the same amino-terminal basic motif of MSL1 previously shown to be essential for binding to high-affinity sites on the X chromosome. While the RING finger domain of MSL2 is sufficient to increase the expression of roX1 in females, activation of roX2 requires motifs in the carboxyl-terminal domain. Binding to hundreds of sites on the X chromosome and efficient incorporation of the roX RNAs into the MSL complex require proline-rich and basic motifs in the carboxyl-terminal domain of MSL2. We suggest that incorporation of the roX RNAs into the MSL complex alters the binding specificity of the chromatin-binding module formed by the amino-terminal domains of MSL1 and MSL2.

| S-EPMC2258739 | biostudies-literature

Targeted DamID reveals differential binding of mammalian pluripotency factors.

Project description:The precise control of gene expression by transcription factor networks is crucial to organismal development. The predominant approach for mapping transcription factor-chromatin interactions has been chromatin immunoprecipitation (ChIP). However, ChIP requires a large number of homogeneous cells and antisera with high specificity. A second approach, DamID, has the drawback that high levels of Dam methylase are toxic. Here, we modify our targeted DamID approach (TaDa) to enable cell type-specific expression in mammalian systems, generating an inducible system (mammalian TaDa or MaTaDa) to identify genome-wide protein/DNA interactions in 100 to 1000 times fewer cells than ChIP-based approaches. We mapped the binding sites of two key pluripotency factors, OCT4 and PRDM14, in mouse embryonic stem cells, epiblast-like cells and primordial germ cell-like cells (PGCLCs). PGCLCs are an important system for elucidating primordial germ cell development in mice. We monitored PRDM14 binding during the specification of PGCLCs, identifying direct targets of PRDM14 that are key to understanding its crucial role in PGCLC development. We show that MaTaDa is a sensitive and accurate method for assessing cell type-specific transcription factor binding in limited numbers of cells.

| S-EPMC6215400 | biostudies-literature

Identification of Tox chromatin binding properties and downstream targets by DamID-Seq.

Project description:In recent years, DNA adenine methyltransferase identification (DamID) has emerged as a powerful tool to profile protein-DNA interaction on a genome-wide scale. While DamID has been primarily combined with microarray analyses, which limits the spatial resolution and full potential of this technique, our group was the first to combine DamID with sequencing (DamID-Seq) for characterizing the binding loci and properties of a transcription factor (Tox) (sequencing data available at NCBI's Gene Expression Omnibus under the accession number GSE64240). Our approach was based on the combination and optimization of several bioinformatics tools that are here described in detail. Analysis of Tox proximity to transcriptional start sites, profiling on enhancers and binding motif has allowed us to identify this transcription factor as an important new regulator of neural stem cells differentiation and newborn neurons maturation during mouse cortical development. Here we provide a valuable resource to study the role of Tox as a novel key determinant of mammalian somatic stem cells during development of the nervous and lymphatic system, in which this factor is known to be active, and describe a useful pipeline to perform DamID-Seq analyses for any other transcription factor.

| S-EPMC4778673 | biostudies-literature

Detection of RNA-DNA binding sites in long noncoding RNAs.

Project description:Long non-coding RNAs (lncRNAs) can act as scaffolds that promote the interaction of proteins, RNA, and DNA. There is increasing evidence of sequence-specific interactions of lncRNAs with DNA via triple-helix (triplex) formation. This process allows lncRNAs to recruit protein complexes to specific genomic regions and regulate gene expression. Here we propose a computational method called Triplex Domain Finder (TDF) to detect triplexes and characterize DNA-binding domains and DNA targets statistically. Case studies showed that this approach can detect the known domains of lncRNAs Fendrr, HOTAIR and MEG3. Moreover, we validated a novel DNA-binding domain in MEG3 by a genome-wide sequencing method. We used TDF to perform a systematic analysis of the triplex-forming potential of lncRNAs relevant to human cardiac differentiation. We demonstrated that the lncRNA with the highest triplex-forming potential, GATA6-AS, forms triple helices in the promoter of genes relevant to cardiac development. Moreover, down-regulation of GATA6-AS impairs GATA6 expression and cardiac development. These data indicate the unique ability of our computational tool to identify novel triplex-forming lncRNAs and their target genes.

| S-EPMC6451187 | biostudies-literature

Direct nanopore RNA sequencing of umbra-like virus-infected plants reveals long non-coding RNAs, specific cleavage sites, D-RNAs, foldback RNAs, and temporal- and tissue-specific profiles.

Project description:The traditional view of plus (+)-strand RNA virus transcriptomes is that infected cells contain a limited variety of viral RNAs, such as full-length (+)-strand genomic RNA(s), (-)-strand replication intermediate(s), 3' co-terminal subgenomic RNA(s), and viral recombinant defective (D)-RNAs. To ascertain the full complement of viral RNAs associated with the simplest plant viruses, long-read direct RNA nanopore sequencing was used to perform transcriptomic analyses of two related umbra-like viruses: citrus yellow vein-associated virus (CY1) from citrus and CY2 from hemp. Analysis of different timepoints/tissues in CY1- and CY2-infected Nicotiana benthamiana plants and CY2-infected hemp revealed: (i) three 5' co-terminal RNAs of 281 nt, 442 nt and 671 nt, each generated by a different mechanism; (ii) D-RNA populations containing the 671 fragment at their 5'ends; (iii) many full-length genomic RNAs and D-RNAs with identical 3'end 61 nt truncations; (iv) virtually all (-)-strand reads missing 3 nt at their 3' termini; (v) (±) foldback RNAs comprising about one-third of all (-)-strand reads and (vi) a higher proportion of full-length gRNAs in roots than in leaves, suggesting that roots may be functioning as a gRNA reservoir. These findings suggest that viral transcriptomes are much more complex than previously thought.

| S-EPMC11327873 | biostudies-literature

Photoactivatable ribonucleosides mark base-specific RNA-binding sites.

Project description:RNA-protein interaction can be captured by crosslinking and enrichment followed by tandem mass spectrometry, but it remains challenging to pinpoint RNA-binding sites (RBSs) or provide direct evidence for RNA-binding. To overcome these limitations, we here developed pRBS-ID, by incorporating the benefits of UVA-based photoactivatable ribonucleoside (PAR; 4-thiouridine and 6-thioguanosine) crosslinking and chemical RNA cleavage. pRBS-ID robustly detects peptides crosslinked to PAR adducts, offering direct RNA-binding evidence and identifying RBSs at single amino acid-resolution with base-specificity (U or G). Using pRBS-ID, we could profile uridine-contacting RBSs globally and discover guanosine-contacting RBSs, which allowed us to characterize the base-specific interactions. We also applied the search pipeline to analyze the datasets from UVC-based RBS-ID experiments, altogether offering a comprehensive list of human RBSs with high coverage (3,077 RBSs in 532 proteins in total). pRBS-ID is a widely applicable platform to investigate the molecular basis of posttranscriptional regulation.

| S-EPMC8519950 | biostudies-literature

DamID profiling of dynamic Polycomb-binding sites in Drosophila imaginal disc development and tumorigenesis.

Project description:BackgroundTracking dynamic protein-chromatin interactions in vivo is key to unravel transcriptional and epigenetic transitions in development and disease. However, limited availability and heterogeneous tissue composition of in vivo source material impose challenges on many experimental approaches.ResultsHere we adapt cell-type-specific DamID-seq profiling for use in Drosophila imaginal discs and make FLP/FRT-based induction accessible to GAL driver-mediated targeting of specific cell lineages. In a proof-of-principle approach, we utilize ubiquitous DamID expression to describe dynamic transitions of Polycomb-binding sites during wing imaginal disc development and in a scrib tumorigenesis model. We identify Atf3 and Ets21C as novel Polycomb target genes involved in scrib tumorigenesis and suggest that target gene regulation by Atf3 and AP-1 transcription factors, as well as modulation of insulator function, plays crucial roles in dynamic Polycomb-binding at target sites. We establish these findings by DamID-seq analysis of wing imaginal disc samples derived from 10 larvae.ConclusionsOur study opens avenues for robust profiling of small cell population in imaginal discs in vivo and provides insights into epigenetic changes underlying transcriptional responses to tumorigenic transformation.

| S-EPMC5987561 | biostudies-literature

Non-coding roX RNAs prevent the binding of the MSL-complex to heterochromatic regions.

Project description:Long non-coding RNAs contribute to dosage compensation in both mammals and Drosophila by inducing changes in the chromatin structure of the X-chromosome. In Drosophila melanogaster, roX1 and roX2 are long non-coding RNAs that together with proteins form the male-specific lethal (MSL) complex, which coats the entire male X-chromosome and mediates dosage compensation by increasing its transcriptional output. Studies on polytene chromosomes have demonstrated that when both roX1 and roX2 are absent, the MSL-complex becomes less abundant on the male X-chromosome and is relocated to the chromocenter and the 4th chromosome. Here we address the role of roX RNAs in MSL-complex targeting and the evolution of dosage compensation in Drosophila. We performed ChIP-seq experiments which showed that MSL-complex recruitment to high affinity sites (HAS) on the X-chromosome is independent of roX and that the HAS sequence motif is conserved in D. simulans. Additionally, a complete and enzymatically active MSL-complex is recruited to six specific genes on the 4th chromosome. Interestingly, our sequence analysis showed that in the absence of roX RNAs, the MSL-complex has an affinity for regions enriched in Hoppel transposable elements and repeats in general. We hypothesize that roX mutants reveal the ancient targeting of the MSL-complex and propose that the role of roX RNAs is to prevent the binding of the MSL-complex to heterochromatin.

| S-EPMC4263465 | biostudies-literature

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