TRIC: A novel method for genome-wide identification and functional characterization of ncRNA:DNA triplexes

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Many non-coding RNAs (ncRNAs) act as epigenetic regulators at distinct genomic sites by impacting the association of transcription factors and chromatin modifiers. However, little is known about how ncRNAs recognize and interact with their target sites. Sequence-specific DNA-recognition can be mediated by formation of a triple helix (triplex), in which a part of a ncRNA intercalates into the DNA duplex and forms hydrogen bonds. Indeed, a few recent studies provide evidence for ncRNA:DNA triplexes in humans and mice, but so far the direct and unbiased detection of these structures in cells was not possible. To overcome this obstacle, I have developed a method termed "TRIC" (triplex recognition in cells), which uses the triplex-binding protein Stm1TBD to isolate ncRNA:DNA triplexes from cells. In the course of establishing this approach, I have discovered a ncRNA:DNA triplex at the promoter of the mouse Kras gene.One aim of this project is to use TRIC for dissecting the regulatory function of the Kras triplex. In this regard we will also follow up preliminary results showing that formation of the ncRNA:DNA triplex competes with another non-canonical nucleic acid structure, a G-quadruplex. The opposing effects of both structures on Kras expression correlate with differential binding of the transcription factors hnRNP K, Miz1 and Myc. By elucidating the interplay between triplex, G-quadruplex and the three transcription factors we will gain important insights into how the Kras proto-oncogene is regulated. Given that many proto-oncogene promoters contain sequences with the potential to form either triplexes or G-quadruplexes, the Kras promoter is likely to serve as a paradigm for such a "conformational switch". The second part of the project aims at the systematic identification of ncRNA:DNA triplexes by combining TRIC with next-generation sequencing. Pilot experiments in which TRIC was followed by RNA-sequencing confirmed the existence of the Kras triplex and uncovered another novel ncRNA:DNA triplex at the Rab5c promoter. In the proposed project, we will increase the RNA sequencing depth and will conduct additional DNA sequencing after TRIC to comprehensively detect triplex-forming RNA- and DNA-sequences in the human and mouse cells. The genomic distribution of ncRNA:DNA triplexes will then be bioinformatically deduced according to the defined triplex base pairing rules. The results will establish fundamental knowledge about the role of ncRNA:DNA triplexes in gene regulation, thereby initiating new research on the connection between ncRNAs and epigenetics.