Identification of RNAs associated with telomeres by enChIP-RNA-Seq
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ABSTRACT: Accumulating evidence suggests important roles of RNAs interacting with genomic regions in the regulation of genome functions including X chromosome inactivation and gene expression. However, no method to identify RNAs interacting with a given genomic region in a non-biased manner has been reported. Here, we used engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) combined with the RNA-Seq analysis (enChIP-RNA-Seq) to perform non-biased search of RNAs interacting with telomeres. In enChIP-RNA-Seq, the target genomic regions are captured with an engineered DNA-binding molecule such as a TAL protein. Subsequently, RNAs are purified and subjected to the RNA-Seq analysis. The detected RNAs contained known telomere-binding RNAs including telomerase RNA and Cajal body-specific RNAs. In addition, we detected many novel telomere-binding RNAs. We confirmed binding of candidate RNAs to telomeres by the enChIP-RT-PCR analysis. Identified novel telomere-binding RNAs may play important roles in telomere functions. In addition, our results suggest that enChIP-RNA-Seq analysis would be useful for identification of RNAs interacting with specific genomic regions. RNAs associated with telomeres were identified by using the enChIP technology combined with deep sequencing using Illumina Miseq. Briefly, to isolate telomeres, a TAL protein, Telomere-TAL (Tel-TAL), recognizing a 19-bp sequence containing an array of TTAGGG (telomere repeats) was fused with 3xFLAG tag and NLS (3xFN-Tel-TAL) and LexA protein (3xFNLDD)11 were expressed in a mouse hematopoietic cell line, Ba/F3, respectively. The cells were crosslinked with formaldehyde, and crosslinked chromatin was fragmented by sonication. Subsequently, chromatin complexes containing 3xFN-Tel-TAL or 3xFNLDD were immunoprepicitated with anti-FLAG M2 Ab. Supplementary URL: http://www.nature.com/srep/2013/131108/srep03171/full/srep03171.html
ORGANISM(S): Mus musculus
SUBMITTER: Daisuke Okuzaki
PROVIDER: E-GEOD-60425 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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