Molecular basis of enhancer versus promoter function of Epromoters – ENHPROM

A major question in molecular biology is to understand how precise regulation of gene expression during normal development and cell differentiation is achieved by the combined action of proximal (promoters) and distal (enhancers) cis-regulatory elements. A breakthrough discovery of our team reported that a subset of promoters, termed Epromoters, works also as enhancers to regulate distal genes. This new paradigm opens novel questions regarding the complexity of our genome and raises the intriguing possibility that genetic variants lying within Epromoters might have pleiotropic effects on various physiological and pathological traits by differentially impacting multiple proximal and distal genes. The general goal of the present project is to understand the molecular determinants that drive enhancer and promoter activity at Epromoters. Building on our interdisciplinary expertise, we will leverage high-throughput reporter assays, synthetic biology and deep learning approaches in a back-and-forth strategy to disentangle the cis- and trans-factors determinants of enhancer and promoter activity of Epromoters. First, we will develop a dual-reporter assay allowing us to quantify enhancer and promoter activity in a quantitative and high-throughput way. Wild-type and mutated Epromoters will be systematically tested along with typical promoters and enhancers. These data will be implemented in deep learning models to infer the DNA sequence determinants that drive enhancer and promoter functions. Subsequently, dual-reporter assays will be performed with loss and gain of function mutations and synthetic DNA sequences to validate and refine the deep learning models. Second, we will develop an integrative reporter strategy to study Epromoter function in a more physiological and chromatinized context. Third, we will perform CRISPR screening to identify transcription factors that are specifically required for either the enhancer or promoter function of Epromoters. Fourth, we will assess the impact of genetic variants on the enhancer and promoter activity of Epromoters and investigate the relevance of enhancer/promoter switch in disease and physiological traits. In particular, we will assess whether Epromoter variants have a pleiotropic or synergistic effect on human diseases by impacting the expression of proximal and distal genes at the same time. In fine, by dissecting the regulatory code of Epromoters, we aim for providing a unifying model of how cis-regulatory elements function to better predict how their dysregulation by genetic variants or other genomic alterations might impact human health.