Long G4 regions (LG4s) in the human genome constitute functional enhancers that coordinate neighboring gene expressions

University Of South Alabama, Mobile AL

Investigators

Abstract

A promoter is a region of DNA upstream of a gene where proteins bind to initiate transcription of (turn on) a gene by producing an RNA copy of the gene sequence. Promoters can be very complex and typically work together with other DNA regions known as enhancers to ensure specific, robust gene transcription. Fortunately, despite their complexity, promoters are easy to identify because they generally correspond to the 100-1,000 base pairs (bp) just before the start of a gene. This has allowed detailed characterization of a massive number of promoters over the past several decades. However, enhancer characterizations have proven much more challenging. Like promoters, enhancers are typically short, 100-1,000 bp DNA sequences that markedly regulate gene transcription but, unlike promoters, an enhancer can be found up to 1 million bp away from the gene it influences. How do enhancers work across such large distances? Genomic DNA can bend, bringing enhancers and promoters close to one another to initiate transcription. Interestingly, numerous studies attempting to link genomic mutations with various traits and/or diseases have found that more than 90% of disease-associated mutations lie within the noncoding portions of the genome, and even more strikingly, most of these mutations lie within regions of the genome thought to function as enhancers rather than promoters. Despite their importance, there is still only a very limited ability to define enhancer sequences, and major questions about their mechanisms of action and targeting determinants remain unanswered. Understanding what drives enhancer:promoter interaction can significantly improve our ability to address a number of societal challenges (e.g. disease, improving crop resistance to adverse environmental conditions via gene modulation, etc.). This project also provides unique interdisciplinary training for students combining molecular biology and computational genetics/bioinformatics. A better understanding of the interplay between gene promoters and gene-distal enhancers will unquestionably lead to fundamental new insights into the basic control of gene transcription and the action of regulatory mutations involved in disease. However, the fundamental mechanisms responsible for how enhancers target and transfer information to their cognate promoters, broadly speaking, remain key gaps in our knowledge. The work outlined in this proposal will directly address the long-standing question of how enhancers, which are often located far (hundreds of kilobase pairs) away, make contact with specific promoters in the context of the nucleus. Notably, enhancers are often significantly enriched with G4-capable sequences and, interestingly, the promoters regulated by G4-enhancers are likewise disproportionately enriched with G4-capable sequences. As hybrid G4 structures can be formed between distinct DNA strands, together, these observations suggest that direct G4:G4 interactions can contribute to enhancer:promoter targeting. The work outlined in this proposal is significant in that it will employ an array of genetic manipulations, biochemical assays, and novel NGS-IP strategies to directly characterize the role of G4 DNA in enhancer::promoter targeting and generate novel methodologies that will be of use in defining specific enhancer::promoter interactions. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.