cis-regulation of Grem1 expression during limb development [ATAC mouse forelimb buds]
Spatiotemporal gene expression programs are orchestrated by transcriptional enhancers which interact with target-gene promoters to regulate gene expression. The BMP antagonist Gremlin1 (Grem1) is an important node in the gene regulatory network that controls vertebrate limb development. In this study, we used a combination of open chromatin profiling (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) in mouse and chick to identify putative cis-regulatory modules (CRMs) that regulate Grem1 in limb buds. Using CRISPR/Cas genome editing we generated different Grem1 regulatory alleles lacking either individual CRMs, combinations or entire enhancer clusters. To study potential interactions of these CRMs with the Grem1 promoter, we generated 4C-seq profiles of specific regulatory alleles. Taken together our results revealed that the spatio-temporal changes in Grem1 expression are caused by cis-regulatory alterations due to the deletions of enhancer clusters rather than global changes in chromatin architecture. Overall design: ATAC-seq analysis on dissected E9.75 mouse forelimb buds