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

• Applicant: Dr. Holger Bierhoff
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 411482956

• Final Report Year: 2024

Final Report Abstract

The role of non-coding RNAs (ncRNAs) as epigenetic regulators is well established, however, the mechanisms that enable ncRNAs to target distinct loci in the chromatin landscape are still under intense research. Sequence-specific DNA-recognition can be mediated by formation of a triple helix (triplex), comprising a ncRNA section that binds to the DNA duplex by Hoogsteen base pairing. Indeed, several studies in human and mouse cells that have focused on specific ncRNAs demonstrated the ncRNAs’ engagement in triplexes. To provide a direct and unbiased detection of ncRNA:DNA triplexes in cells, my lab has developed a triplex capture method based on the triplex-binding protein Stm1TBD and termed it TRIC for ‘triplex recognition in cells’. Applying TRIC in mouse NIH3T3 fibroblasts, we discovered a ncRNA:DNA triplex at the Kras gene promoter, and showed that the ncRNA is a promoter-spanning antisense transcript. Notably, the same triplex-engaging promoter region has also been reported to form an alternative non-canonical nucleic acid structure, a G-quadruplex (G4). By engineering the G4 sensor protein RG4S analogous to Stm1TBD, this project has dissected the Kras regulation conferred by the triplex/G4 interplay. We found that upon druginduced G4 stabilization a repressive complex comprising the transcription factors Miz1 and Myc triggers histone deacetylation and chromatin compaction. This mechanism is antagonized by triplex formation, which hence confers the transcription on-state. Importantly, downregulation of Kras upon cellular senescence entails triplex-to-G4 conversion, demonstrating physiological relevance of this structural switch, which may serve as a new paradigm for epigenetic regulation of promoter activity. In order to assess the prevalence of genome-wide ncRNA:DNA triplex formation, we combined TRIC with RNA sequencing. These experiments confirmed the triplex-engagement of the Kras antisense RNA as well as Oxct1as, another ncRNA previously reported to form triplexes. Moreover, many sequence motifs with high triplex-forming potential were prevalent among the captured RNAs. These data corroborated that TRIC is well suited for de novo detection of cellular triplexes. However, despite several attempts we failed to optimize the signalto-noise ratio for TRIC-mediated DNA isolation and thus refrained from conducting DNA sequencing. Accordingly, further adaptations of the TRIC method are required in the future to enable the simultaneous identification of matching RNA and DNA triplex-moieties.

Publications

• Epigenetic Erosion in Adult Stem Cells: Drivers and Passengers of Aging. Cells, 7(12), 237.
  Kosan, Christian; Heidel, Florian H.; Godmann, Maren & Bierhoff, Holger
  
• RNA:DNA triple helices: from peculiar structures to pervasive chromatin regulators. Essays in Biochemistry, 65(4), 731-740.
  Greifenstein, Andreas Adam; Jo, SoYoung & Bierhoff, Holger
  
• G-quadruplex and RNA:DNA triplex structures: Epigenetic Yin and Yang at the Kras promoter. [Conference presentation]. RNA 2023 - The 28th Annual Meeting of the RNA Society, Suntec Convention Centre, Singapore
  Jo S., Greifenstein A.A., Lange L., Preckwinkel P. & Bierhoff H.