The genomic binding sites of a noncoding RNA

TLDR

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 enriches endogenous RNAs and their chromatin targets from reversibly cross‑linked extracts. CHART was applied to enrich DNA and protein targets of endogenous lncRNAs in flies and humans and was extended to genome‑wide mapping of the dosage‑compensation ncRNA roX2 in Drosophila. CHART identified roX2 binding at genomic sites overlapping male‑specific lethal complex proteins, revealing its dosage‑compensation targets and demonstrating CHART as an RNA‑analog of chromatin immunoprecipitation.

Abstract

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 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 male-specific lethal 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 chromatin immunoprecipitation for proteins.