The genomic binding sites of a noncoding RNA

Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2011; 108(51):20497-502. DOI: 10.1073/pnas.1113536108
Source: PubMed


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.

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Available from: Jason West, Jul 18, 2014
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    • "We also identify that the predominant means of tethering of cheRNAs (including hundreds of annotated lncRNAs) is through active RNAPII. Although widely speculated (Bonasio et al., 2010; Guttman and Rinn, 2012; Quinodoz and Guttman, 2014; Rinn and Chang, 2012), we are not aware of tests of this proposed chromatin association beyond a few anecdotal cases (Mao et al., 2011; Simon et al., 2011). This result is consistent with the cis-activating correlation observed, leading to a model where ongoing or paused transcription of noncoding RNAs influences the expression of proximal genes (Bonasio et al., 2010; Guttman and Rinn, 2012). "
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    ABSTRACT: A number of long noncoding RNAs (lncRNAs) have been reported to regulate transcription via recruitment of chromatin modifiers or bridging distal enhancer elements to gene promoters. However, the generality of these modes of regulation and the mechanisms of chromatin attachment for thousands of unstudied human lncRNAs remain unclear. To address these questions, we performed stringent nuclear fractionation coupled to RNA sequencing. We provide genome-wide identification of human chromatin-associated lncRNAs and demonstrate tethering of RNA to chromatin by RNAPII is a pervasive mechanism of attachment. We also uncovered thousands of chromatin-enriched RNAs (cheRNAs) that share molecular properties with known lncRNAs. Although distinct from eRNAs derived from active prototypical enhancers, the production of cheRNAs is strongly correlated with the expression of neighboring protein-coding genes. This work provides an updated framework for nuclear RNA organization that includes a large chromatin-associated transcript population correlated with active genes and may prove useful in de novo enhancer annotation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 08/2015; 12(7). DOI:10.1016/j.celrep.2015.07.033 · 8.36 Impact Factor
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    • "The ability of nuclear localised lncRNAs to act in trans at distal genomic locations to regulate gene expression programs has been poorly understood. This is in large part because the direct transcriptional targets of only a small number of such transcripts (for example, Paupar (mouse), HOTAIR, NEAT1, TERC, RMST (all human), and rox2 (Drosophila)) have been identified thus far (Chu et al., 2011; Simon et al., 2011; Ng et al., 2013; Vance et al., 2014). Consequently, it has been unclear how these transcripts are targeted to distal functional elements and whether thereafter they alter chromatin structure in situ. "
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    ABSTRACT: Many intergenic long noncoding RNA (lncRNA) loci regulate the expression of adjacent protein coding genes. Less clear is whether intergenic lncRNAs commonly regulate transcription by modulating chromatin at genomically distant loci. Here, we report both genomically local and distal RNA-dependent roles of Dali, a conserved central nervous system expressed intergenic lncRNA. Dali is transcribed downstream of the Pou3f3 transcription factor gene and its depletion disrupts the differentiation of neuroblastoma cells. Locally, Dali transcript regulates transcription of the Pou3f3 locus. Distally, it preferentially targets active promoters and regulates expression of neural differentiation genes, in part through physical association with the POU3F3 protein. Dali interacts with the DNMT1 DNA methyltransferase in mouse and human and regulates DNA methylation status of CpG island-associated promoters in trans. These results demonstrate, for the first time, that a single intergenic lncRNA controls the activity and methylation of genomically distal regulatory elements to modulate large-scale transcriptional programmes. - See more at:
    eLife Sciences 11/2014; 3. DOI:10.7554/eLife.04530#sthash.9o6XQjd1.dpuf · 9.32 Impact Factor
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    • "By contrast, lncRNA transcripts have also been proposed to regulate gene expression in trans, without influencing transcription of their genomically neighbouring genes (Guttman et al, 2011; Hung et al, 2011). Trans-acting lncRNAs include p53-induced lncRNAs involved in mediating the DNA damage response (Huarte et al, 2010; Hung et al, 2011), lncRNAs transcribed from within the promoters of cell cycle genes (Hung et al, 2011), lncRNAs that function in the control of pluripotency and lineage differentiation (Guttman et al, 2011) and those that are regulators of dosage compensation (Chu et al, 2011; Simon et al, 2011). Other examples include Evf-2 which binds and modulates the activity of the homeodomain containing transcription factor Dlx2 (Feng et al, 2006), and Hotair, a lncRNA transcribed from the HoxC locus, which regulates the activity of HoxD cluster genes in trans and interacts with chromatin at over 800 regions genome-wide (Chu et al, 2011; Rinn et al, 2007). "
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    ABSTRACT: Although some long noncoding RNAs (lncRNAs) have been shown to regulate gene expression in cis, it remains unclear whether lncRNAs can directly regulate transcription in trans by interacting with chromatin genome-wide independently of their sites of synthesis. Here, we describe the genomically local and more distal functions of Paupar, a vertebrate-conserved and central nervous system-expressed lncRNA transcribed from a locus upstream of the gene encoding the PAX6 transcription factor. Knockdown of Paupar disrupts the normal cell cycle profile of neuroblastoma cells and induces neural differentiation. Paupar acts in a transcript-dependent manner both locally, to regulate Pax6, as well as distally by binding and regulating genes on multiple chromosomes, in part through physical association with PAX6 protein. Paupar binding sites are enriched near promoters and can function as transcriptional regulatory elements whose activity is modulated by Paupar transcript levels. Our findings demonstrate that a lncRNA can function in trans at transcriptional regulatory elements distinct from its site of synthesis to control large-scale transcriptional programmes.
    The EMBO Journal 02/2014; 33(4). DOI:10.1002/embj.201386225 · 10.43 Impact Factor
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