Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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The Genomic Binding Sites of an Endogenous Non-Coding RNA

ABSTRACT: Long non-coding RNAs (lncRNAs) have important regulatory roles and can function at the level of chromatin. To determine where lncRNAs bind to chromatin, we developed CHART, a hybridization-based technique that specifically enriches endogenous RNAs along with their targets from reversibly-crosslinked chromatin extracts. CHART was used to enrich the DNA and protein targets of endogenous lncRNAs from fly and human. This analysis was extended to genome-wide mapping of roX2, a well-studied ncRNA involved in dosage-compensation in Drosophila. CHART revealed that roX2 binds at specific genomic sites that coincide with the binding sites of proteins from the MSL-complex that affects dosage compensation. These results reveal the genomic targets of roX2 and demonstrate how CHART can be used to study RNAs in a manner analogous to ChIP for proteins. Examination of the binding sites of roX2 ncRNA from S2 cells using two different elution strategies compared with a sense control or input control. Processed data file 'roX2.2.peaks.bed' (for roX2 CHART RNase eluted, combined replicates) linked below as supplementary file.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Mark Borowsky

PROVIDER: E-GEOD-28180 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The genomic binding sites of a noncoding RNA.

Simon Matthew D MD Wang Charlotte I CI Kharchenko Peter V PV West Jason A JA Chapman Brad A BA Alekseyenko Artyom A AA Borowsky Mark L ML Kuroda Mitzi I MI Kingston Robert E RE

Proceedings of the National Academy of Sciences of the United States of America 20111205 51

Long noncoding RNAs (lncRNAs) have important regulatory roles and can function at the level of chromatin. To determine where lncRNAs bind to chromatin, we developed capture hybridization analysis of RNA targets (CHART), a hybridization-based technique that specifically enriches endogenous RNAs along with their targets from reversibly cross-linked chromatin extracts. CHART was used to enrich the DNA and protein targets of endogenous lncRNAs from flies and humans. This analysis was extended to gen ...[more]

PMID: 22143764

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Project description:Long noncoding RNAs (lncRNAs) are important regulators of chromatin; however, the mechanistic roles for many lncRNAs are poorly understood in part because their direct interactions with genomic loci and proteins are difficult to assess. We used CHART-seq to map the genomic binding sites for two highly expressed human lncRNAs, NEAT1 and MALAT1, which localize within the nucleus to paraspeckles and nuclear speckles, respectively. We show that NEAT1 and MALAT1 localize to hundreds of genomic sites in human cells, primarily over active genes. NEAT1 and MALAT1 exhibit colocalization to many of these loci, but display distinct gene body binding patterns at these sites, suggesting independent but complementary functions for these RNAs. Protein mass spectrometry analysis of CHART-enriched material (CHART-MS) identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function, in addition to unique associated proteins. Transcriptional inhibition or stimulation affects the localization of NEAT1 to active chromatin sites, implying that DNA sequence itself does not target NEAT1 to chromatin and that localization responds to cues involved in the transcription process. Paired-end CHART-seq was performed for a single replicate of each capture oligonucleotide in untreated MCF-7 cells to establish binding sites of these RNAs, for a total of 6 samples. To investigate the effects of transcriptional inhibition and E2 stimulation on the localization of these RNAs, we performed paired-end CHART-seq with each capture oligonucleotide for two biological replicates of flavopiridol- and vehicle (DMSO)-treated MCF-7 cells and for two biological replicates of E2- and vehicle (ethanol)-treated MCF-7 cells. To establish the overlap of NEAT1 and MALAT1 binding sites with a known component of paraspeckles (NEAT1-containing subnuclear body), we performed paired-end ChIP-seq for the paraspeckle component PSF in MCF-7 cells, as well as a single-end biological replicate.

Dataset Information

The Genomic Binding Sites of an Endogenous Non-Coding RNA

Publications

The genomic binding sites of a noncoding RNA.

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