cis-regulation of Grem1 during limb development [4C-seq]
Spatiotemporal gene expression programs are orchestrated by transcriptional enhancers which interact with target-gene promoters to regulate gene expression. In this study, we identified cis regulatory modules (CRMs) within the Fmn1/Grem1 cis-regulatory landscape that participate in controlling spatiotemporal expression of Grem1. We generated different Grem1 alleles lacking either individual CRMs, combinations or entire enhancer clusters (EC1 and EC2) using CRISPR/Cas genome editing. To study potential interactions of these CRMs with the Grem1 promoter, we generated 4C-seq profiles of different alleles. Our analysis of 4C-seq shows that the overall structure of the Grem1 TAD is maintained in CRM2-/- and EC1-/- forelimb buds. Similar to EC1, the chromatin architecture of the Grem1 TAD is not disrupted outside the EC2 and EC1EC2 deletions. In all the genotypes, the contacts of the promoter with the remainder of the Grem1 TAD  are rather increased. Thus, our results revealed that the spatio-temporal changes in Grem1 expression are caused by cis-regulatory alterations due to the deletions of EC1 and EC2 and not global changes in chromatin architecture. Overall design: Circular Chromosome Conformation Capture (4C seq) at Grem1 locus in developing limbs of mutants and WT at E11 Deletion coordinates (mm10) of mutants used in this study: del CRM2: chr2:113689537-113699485 del EC1: chr2:113636776-113707974 del EC2: chr2:113572310-113606580 del EC1 and EC2: chr2:113636776-113707974 and chr2:113572310-113606580 del Cis: chr2:113528475-113709592