Sunday, November 24, 2024
11/24/2024
PROJECT SUMMARY Hundreds of thousands of distant-acting enhancers control the function of the human genome by orchestrating the transcription of genes during pre- and postnatal development and in normal and disease states of cells and tissues. They display remarkable cell type specificity and dynamic spatial and temporal activity patterns. While there is now abundant indirect evidence that sequence changes in enhancers are likely to impact substantially on human phenotypic variation and many disease processes, our understanding of how biological function is encoded within enhancer sequences remains incomplete. This represents a major challenge for the interpretation of variation in enhancer sequences observed by whole-genome sequencing (WGS) in patients and for linking sequence variants within enhancers to diseases and other phenotypes. Over the past 16 years, the research program supported by this R01 has provided major insights into enhancer biology and groundbreaking tools for enhancer discovery and characterization. This included the first demonstration of ChIP-seq from mammalian tissues for enhancer discovery, and a constantly evolving suite of mouse engineering methods for studying enhancers in vivo. This program has also produced the largest cohesive collection of human and mouse in vivo-characterized enhancers available to date and provides the VISTA Enhancer Browser as a major community resource. In the next phase of this research program, we propose to leverage the unique capabilities previously developed under this program to further advance our understanding of general enhancer biology, to complement the work of the recently established NHGRI Impact of Genomic Variation on Function (IGVF) consortium with critically needed in vivo assessments of enhancer variants, and to provide enhancer resources for the extended community. Our specific aims include: (1) We will explore the inner anatomy of enhancer sequences through large-scale mutagenesis coupled to in vivo mouse assays. We will also delete large non- coding genome intervals (gene deserts) from the mouse genome to determine the broad functional significance of non-coding DNA beyond individual enhancer elements. (2) Working as Affiliates with the IGVF Consortium, we will perform large-scale in vivo exploration of human variants with predicted impact on enhancer function to assess human variation/mutation through our unique mouse engineering capabilities. This will include careful validation and calibration of massively parallel reporter assays (MPRAs) and CRISPR screens through comparison with in vivo mouse reporter assays. (3) We will continue to provide access to in vivo mouse assays for the community and make our results available through the VISTA Enhancer Browser. In combination, in the next phase of this program we expect to deliver impactful insights into the biology of enhancers as a major category of non-coding genome function, apply innovative tools to demonstrate the in vivo impact of variants identified by the IGVF consortium, and continue to make unique enhancer analysis tools and resources available to the broader community.
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