Systems Biology Approaches Identify G-Quadruplex DNA (Telomere-Like Motifs) in a Genome-Wide Regulatory Role

^1,2Shantanu Chowdhury, ¹Anjali Verma, ¹Kangkan Halder, ¹Praveen Kumar, ¹Ramkrishna Thakur, ¹Anirban Kar, ²Vinod Yadav, ²Rashi Kulshreshta, ³Farhan Mohammed, ⁴Jean-Luc Parent, ³Abhay Sharma
¹Institute of Genomics and Integrative Biology, Proteomics and Structural Biology Unit, Delhi, India, ²Institute of Genomics and Integrative Biology, G.N.R. Knowledge Centre for Genome Informatics, Delhi, India, ³Institute of Genomics and Integrative Biology, Comparative Genomics and Gene Expression Unit, Delhi, India, ⁴University of Sherbrooke, Division of Rheumatology, Faculty of Medicine, Quebec, Canada

Comprehensive characterization of all encoded elements is a fundamental prerequisite for understanding biological function. DNA sequence as regulatory elements is well-established; however, DNA structure is relatively unexplored in this context. Using G-quadruplex or G4 DNA (structural motifs commonly found within eukaryote telomores) as a model we researched the role of DNA structure in transcription. Computational analyses of human, chimpanzee, mouse and rat genomes (>90,000 promoters) indicated significant G4 DNA enrichment within proximal promoters and more than 700 promoters were found to conserve G4 motifs 'orthologously' across mouse, rat and human. Genes harboring conserved motifs within putative regulatory regions show significantly enriched expression in more than 75 human tissues and indicate evolutionary role on comparing expression across brain, heart, liver, kidney and testis in human and chimpanzee. We experimentally investigated the role of the G4 motif in gene regulation by whole genome expression using microarrays in HeLa and A549 cells, after pre-treatment with molecules that are G4 DNA-specific and further validated the results using real-time quantitative PCR. Results support widespread role of G4 DNA in regulation. This was further investigated at the molecular level by establishing the possible mechanism of G4 DNA mediated regulation by showing for the first time that the metastasis suppressor protein NM23-H2 binds to G4 DNA in vitro and in vivo. We demonstrate that expression of the proto-oncogene MYC, thymidine kinase (TK1) and recombination activating gene (RAG1) are regulated by NM23-H2 binding to G4 DNA structural motif in vivo using chromatin immunoprecipation; this is also supported by in vitro binding of recombinant NM23-H2 to G4 DNA. We also used ChIP-on-chip experiments to demonstrate genome-wide NM23-H2 - G4 DNA interaction as direct evidence of in vivo presence of G4 DNA within promoters.