Noncoding RNAs (ncRNAs) are now believed to be transcribed pervasively in the genome, and large numbers of ncRNAs have been identified. However, disproportionally, we still know very little about their functional roles. Many of the known ncRNA functions were inferred by perturbation experiments, which lack the details of what specific target an ncRNA interact with. Technologies like CLIP/RIP-Seq have provided tremendous insights of what kind of ncRNA the protein factors associated, and ChIRP-Seq have generated the chromatin loci for some ncRNAs to interact with, which have suggested that in particular long non-coding RNAs (lncRNAs) are involved in epigenomic regulation of gene expression and chromatin modeling. However, current methods are limited to examine ncRNA or interacting target one at a time. It is desirable to have an unbiased genome-wide strategy to identify the functional targets for all ncRNAs. We hypothesize that if an ncRNA had an epigenetic regulatory role in the nuclear space, it would have to either directly or indirectly interact with chromatin at certain locations in chromosomes, in which functions take place for modulating chromatin states and target gene activity. Hence, we propose to develop a new technology to globally map ncRNA-chromatin interactions through RNA-DNA ligation followed by paired-end-tag sequencing (R&D-PET). In brief, this method includes three main parts: 1) chromatin crosslinking to capture all molecular interaction events between RNA, DNA and proteins in vivo;2) ligation of the tethered interactive RNA and the chromatin DNA fragment through specifically designed RNA linker and DNA linker oligos;3) sequencing and mapping analysis of the RNA-DNA ligation products to localize ncRNAs'transcription sites and their chromatin target sites in the genome. We also realize that this RNA-DNA ligation approach can be applied to study RNA-protein interaction at specific chromatin locations. Thus a ChIP-based R&D-PET method could provide additional specificity of RNA-protein- chromatin interaction information. We have developed a prototype protocol for R&D-PET analysis, and have generated some promising preliminary data from human cells. In this proposal, we plan to further refine the R&D-PET method through systematic optimizations of key experimental conditions and improvement of bioinformatic analysis pipeline. We also plan to apply this method to comprehensively characterize the ncRNA- chromatin interactomes for a number of established human cell lines and stem cells derived from individual cancer patients. The successful development of this method will significantly increase our capability of investigating the immense complex world of RNA functions in regulating the output of the genome, and the successful completion of the proposed characterization of RNA-chromatin interactomes would provide a comprehensive chromatin address book for most of ncRNA species, which would add another dimension of genomic information to help understand how the genome functions in healthy and disease conditions.

Public Health Relevance

The proposed study will identify novel non-coding RNAs (ncRNAs) and the interactions between ncRNAs and their target DNAs. Since ncRNAs are associated with diseases such as cancers, such novel ncRNAs and ncRNA target DNAs have the potential to be diagnostic biomarkers and novel genomic therapeutic targets for disease, which would improve public health in general.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Genomics, Computational Biology and Technology Study Section (GCAT)
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Mietz, Judy
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Jackson Laboratory
Bar Harbor
United States
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