Abstract 4881: Analysis of long-noncoding RNA interaction at chromatin by chromatin isolation by RNA purification (ChIRP)

Abstract

Abstract Gene regulation plays a critical role during development and progression of Cancer. While the regulation of gene expression by transcription factors and epigenetic influences has been well studied over time, pervasive genomic transcription and the role of non-coding RNAs in this process is a rapidly evolving field that remains to be thoroughly explored. Chromatin Isolation by RNA Purification (ChIRP) is one of the methods, which allows discovery of the sites of interaction of chromatin-associated RNAs (e.g. lncRNAs) with genomic DNA sequences by using probe-based hybridization to target RNA molecules in chromatin. To perform ChIRP multiple biotinylated oligonucleotide probes complementary to the RNA of interest are used. To help eliminate non-specific signals two different pools of probes are used (even and odd probe sets). These probe sets are combined with chromatin and hybridized to the chromatin-associated RNA. Complexes containing biotinylated-probes bound to the chromatin-associated RNA are then isolated using streptavidin magnetic beads. DNA can then be recovered and analyzed by quantitative PCR or next generation sequencing (ChIRP-seq). Alternatively, RNA may also be isolated from an aliquot of the recovered chromatin to detect other RNA molecules that may be associated with the RNA of interest. To enable the exploration of these RNA interactions in chromatin regulation, we have optimized the methods and developed ChIRP reagents. Using these reagents ChIRP experiments can be performed with reliable recovery of chromatin using lncRNA or other chromatin associated RNA as targets. Additionally, negative and positive control probe sets, and detection primers were developed to ensure first time success. We have performed ChIRP experiments with a HeLa cell lysate and capture oligos for the NEAT1 lncRNA. Isolated DNA was subjected to NGS library construction and sequenced on an Illumina HiSeq™ instrument. Sequences were aligned to the reference genome (hg19). The peaks were called separately for odd and even reactions, and only common peaks were selected. We successfully identified several NEAT1 binding sites in the genomic DNA sequence. In summary the methods developed allow unbiased discovery of RNA-associated genomic DNA sequences, RNA sequences, and potentially proteins. Citation Format: Kan Saito, Konstantin Taganov, John M. Rosenfeld, Nick Asbrock, Vi Chu. Analysis of long-noncoding RNA interaction at chromatin by chromatin isolation by RNA purification (ChIRP). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4881. doi:10.1158/1538-7445.AM2015-4881