Dissecting how gene regulatory redundancy ensures robustness of mammalian development

PROJECT SUMMMARY/ABSTRACT Transcriptional enhancers are non-coding DNA elements that control the expression of their target genes with spatiotemporal specificity. Transcription factors confer the specificity of enhancer function by recruiting core transcriptional machinery to the enhancer’s target promoter during gene activation. Altered enhancer function may yield a variety of pathologies, including congenital anomalies. However, even under adverse environmental and genetic conditions, most births occur without congenital anomalies, attesting to the resilience of developmental programs. This “developmental robustness” may rely on the fact that most genes expressed during development are controlled by multiple redundant enhancers with overlapping spatiotemporal activities, so-called shadow enhancers. However, despite the ubiquity of these shadow enhancers, we do not completely understand how they confer developmental robustness. Using a mouse model of limb morphogenesis and genetic engineering at the mouse Gli3 genomic locus, I generated preliminary data suggesting that shadow enhancers are activated by distinct transcription factor inputs, despite their apparent redundancy. This “separation of transcription factor inputs” may be significant to shadow enhancers’ roles in supporting developmental robustness. The primary objective of this proposal is to identify the distinct transcription factors driving each shadow enhancer (Aim1) and characterize how two distinct shadow enhancers organize in the three-dimensional space of the nucleus during gene activation (Aim2). To achieve Aim 1, I will perform a genomic footprinting technique to determine which transcription factor binding sites are occupied within each shadow enhancer during gene activation. This approach will be complemented with an in vivo enhancer-reporter assay to functionally test for the requirement of each binding site. To accomplish Aim 2, I will perform three-color DNA fluorescence in situ hybridization (DNA-FISH), which will allow me to measure the co-localization of two shadow enhancers and the Gli3 promoter under different genetic conditions. The proposed research will unveil the molecular and spatial mechanisms of gene regulatory redundancy during mammalian development. Providing his expertise in gene regulation, mouse transgenics, and developmental biology, Dr. Evgeny Kvon will serve as my sponsor during the fellowship period. With consultation from Dr. Kvon, I have developed a robust training plan to facilitate my transition to becoming an independent investigator and biology educator. Under this proposal, I will learn new skills in genomics, DNA-FISH, transgenics, and bioinformatic analysis. My professional development will encompass training in scientific communication, teaching, and mentorship.