Designing synthetic regulatory domains to understand gene expression

ongoing

How the genome controls precise, cell-type specific gene expression is crucial for cell identity and differentiation. Short cis-regulatory elements, such as enhancers, are distributed in extended regulatory domains surrounding their target gene, which in mammals typically span hundreds of kilobases. Although all evidence indicates that gene regulation is controlled by the combined action of large domains, our current understanding mostly focuses on enhancers. If and how intervening DNA sequences, enhancer order, or spacing within regulatory domains contribute to gene regulation is unknown. The reason for this largely unexplored aspect lies in our limited ability to systematically alter large genomic regions. In this study, we will use recent advances in synthetic biology to overcome this limitation. From synthetic DNA, we will create variants of entire gene regulatory domains and integrate these into mouse embryonic stem cells. We will test synthetic regulatory domain function in tailored gene expression analyses using cell culture, organoid, and in vivo models. By investigating regulatory elements with respect to their surrounding regulatory domain, we will uncover hidden rules of gene regulation. In variant regulatory domains we will systematically assay understudied parameters, such as enhancer spacing, order, or inter-enhancer sequences. We will use single-molecule methods to measure gene expression level; use organoid and in vivo systems to profile cell-type specific changes in expression patterns, and we will use the unique potential of synthetic DNA to understand the fundamental link between DNA sequence and chromatin composition of regulatory domains. With this interdisciplinary approach, we aim to uncover previously hidden layers of genomic information. The insights generated from this work will provide a novel and unique view on the organization of the non-coding genome, with the potential to expand our ability to read and write genomic DNA sequences.