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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.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE28180 | GEO | 2011/12/07

SECONDARY ACCESSION(S): PRJNA139533

REPOSITORIES: GEO

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Project description:roX RNAs are involved in the chromosome-wide gene regulation that occurs during dosage compensation in Drosophila. Dosage compensation equalizes expression of X-linked and autosomal genes. Drosophila males increase transcription two-fold from their single X chromosome. This is mediated by the MSL complex, which is composed of the male-specific lethal (MSL) proteins and two noncoding roX RNAs, roX1 and roX2. Upon elimination of both roX transcripts, a global decrease of X-linked gene expression is observed in males. Expression of the genes on the entire 4th chromosome also decreased in the absence of both roX transcripts. roX1 RNA also presents in females in the early stages. To investigate the effect of loss of roX transcripts on gene expression in females, gene expression was analyzed by microarrays in roX1-roX2- female flies. To eliminate inconsistency caused by differences in genetic background, expression of roX1-roX2- females with females of virtually identical genetic background but carrying the [hsp83-roX1+] transgene were compared. Expression of any chromosome did not change in roX1-roX2- females. It was concluded that roX RNAs only effect in males . Experiment Overall Design: Total RNA was prepared from groups of 50 third instar larvae by TRIzol (Invitrogen) extraction and purified using the RNeasy kit (Qiagen). Three independent RNA preparations for each genotype served as templates for probe synthesis. These probes were hybridized to Affymetrix Drosophila Genome 2.0 chips (Santa Clara, CA). Genes were filtered for present/absent calls by a PM-MM (Perfect match-Mismatch) comparison. Affymetrix Gene expression data was background corrected, normalized and summarized into a one expression value per replicate sample and probeset using the RMA (robust multi-array average) algorithm. Changes in gene expression were determined by comparing the mean signal intensities of genes on arrays hybridized with roX1- roX2- (mutant) probes to those hybridized with roX1- roX2-; [hsp83-roX1+] (control) probes.

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The Genomic Binding Sites of an Endogenous Non-Coding RNA

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