The genomic binding sites of a noncoding RNA

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 genomewide 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.chromatin-associated RNAs | chromatin-modifying complexes | RNase H mapping G enerating cellular diversity from genetic information requires the regulatory interplay between cis-acting elements encoded at specific loci in chromatin and trans-acting factors that bind them (1). Although the importance of trans-acting proteins (e.g., transcription factors) has long been appreciated, there is growing interest in the role of long noncoding RNAs (lncRNAs) (2) as factors that can regulate specific chromatin loci. This interest is enhanced by the recent discovery that the majority of eukaryotic genomes are transcribed (3) and that many of the resulting transcripts are developmentally regulated (4) but do not encode proteins. Although the functional scope of these RNAs remains unknown (5-7), several lncRNAs play important regulatory roles at the level of chromatin (8). Determining where these ncRNAs bind on the genome is central to determining their function.Examples of lncRNAs that influence chromatin include the roX ncRNAs in flies and Xist in mammals, both having wellestablished roles in dosage compensation (8, 9); Kcnq1ot1 and Air ncRNAs, which are expressed from genomically imprinted loci and affect chromatin silencing (10-13); Evf2, HSR1, and other ncRNAs that positively regulate transcription (14-16); lncRNAs that target the dihydrofolate reductase promoter and the rDNA promoters through triplex formation (17, 18); and the human HOTAIR and HOTTIP lncRNAs, which regulate polycomb-repressed and trithorax-activated chromatin, respectively (19,20). Dysregulation of several of these lncRNAs has been associated with disease (21, 22). Our understanding of the biochemical roles of these RNAs comes largely from their interactions with specific proteins-insights gained from classical biochemical techniques developed for studying translation and RNA-processing complexes and also more recent technological advances using RNA immunoprecipitation (23) and cross-linking and immunoprecipitation (24)(25)(26). These experiments suggest that several lncRNAs specifically interact with chromatin-m odifying machine...

Long Noncoding RNAs: Past, Present, and Future

“…Based on principles of both chromatin immunoprecipitation (ChIP) and RIP, chromatin RNA immunoprecipitation (ChRIP) can be used to identify RNAs associated with a particular chromatin mark (Pandey et al 2008). On the other hand, techniques such as chromatin oligo-affinity precipitation (ChOP) (Mariner et al 2008), chromatin isolation by RNA purification (ChIRP) (Chu et al 2011), and capture hybridization of RNA targets (CHART) (Simon et al 2011) use tagged complementary oligonucleotides to identify DNA loci that interact with an RNA of interest. (C) Structural features: ncRNAs form specific secondary (base pairing) and tertiary (three-dimensional) structures to carry out their functions.…”

Dosage Compensation inDrosophila

“…Further understanding of the targeting process will be facilitated by the molecular analysis of evolving sex chromosomes in different Drosophila species with fully sequenced genomes (see www.FlyBase .org). An understanding of the function of the roX RNAs in spreading of the MSL complex will make use of new RNA, rather than protein-based ChIP mapping techniques (Chu et al 2011;Simon et al 2011). The role of histone modifications, namely H4K16 acetylation and H2B ubiquitination, will be assessed by replacing canonical histones with multiple copies of modified histone transgenes (Gunesdogan et al 2010).…”

The emerging molecular biology toolbox for the study of long noncoding RNA biology

“…Thereafter by, for example, tiling antisense oligonucleotide probes along the lncRNA to capture the transcript, components of the complex can be isolated and determined by numerous approaches. Variations to this RNA-centric purification method exist and include chromatin isolation by RNA purification (ChIRP) [62], capture hybridization analysis of RNA targets (Chart) [63] and RAP [21]. Using these methods in conjunction with high-throughput sequencing and mass spectrometry, interacting DNA, RNA and proteins can be detected.…”

“…Quantitative reads of the entire transcriptome [27] CAGE Quantitative reads of the transcriptome by capturing the 5 -end of the transcript [28] 3C (and its derivatives) Mapping of chromosomal interaction for the identification of spatially proximal genes and genetic elements [29][30][31] ChIP-Seq Genome wide mapping and quantification of epigenetic marks and protein on DNA [32] smFISH (and its derivatives) Abundance and subcellular localization of transcripts in single cells [33][34][35][36][37][38][39] Genome and epigenome editing tools Perturbation of lncRNA transcription and position to determine function [44,45,50,51,[53][54][55]58,60] ChIRP Identification of interacting RNA, DNA and protein partners through the capture of the lncRNA transcript [61] Chart Identification of interacting DNA and protein partners through the capture of the lncRNA transcript [62] RAP Identification of interacting RNA, DNA and protein partners through the capture of the lncRNA transcript [20] PAR-CLIP Identification of lncRNAs that are interacting with a particular protein of interest through the immunoprecipitation of the protein [63,64] SHAPE LncRNA secondary structure at single nucleotide resolution [65] the recruitment of proteins, such as PRC2, the entire X-chromosome is epigenetically silenced [20]. Through the use of these molecular tools, these studies have defined the functional mechanism of Xist, which has now been established as a common modality for a subclass of lncRNAs [5,25].…”