Fig. 1: Multiple CRMs in the Grem1 TAD are enhancers interacting with key transcription complexes.

a Hi-C metadata from mouse embryonic fibroblasts¹⁹ show the chromatin interactions in the Grem1-Fmn1 TAD on mouse chromosome 2. The colour intensity scale shows the contact frequencies. The Grem1 TAD: ~190 kb, indicated by blue dashed lines, Fmn1 TAD: ~240 kb, indicated by green dashed lines. Arhgap11a and Scg5 are part of the genomic region but not located within the Grem1 TAD. b Enlargement of the Grem1 TAD (vertical blue dashed lines) and the delCis region required for Grem1 expression in limb buds (indicated by a horizontal black dashed line). The directions of transcription are indicated by arrows. The ATAC-seq peaks (open chromatin) and histone H3K27 acetylation ChIP-seq peaks (H3K27ac; active enhancers) detected in forelimb buds at E10.5 identify all candidate CRMs located distal to the Grem1 coding region. n = 2 independent biological replicates were analysed for the ATAC-seq and the H3K27ac ChIP-seq. The peak calling function of MACS2 identified the significantly enriched peaks present in both replicates of the ATAC-seq and the H3K27ac ChIP-seq. The genomic regions enriched in both ATAC-seq and H3K27ac ChIP-seq peaks correspond to candidate CRMs that are numbered in 3′ direction starting with CRM1 (Table 1) and CTCF sites that are indicated by black arrowheads. c LacZ reporter assays in transgenic founder embryos, representing independent transgene insertion events, establish robust enhancer activities for CRM2 (n = 7/11 expressors), CRM3 (n = 5/8), CRM4 (n = 6/8) CRM5 (n = 7/13) and CRM7 (n = 3/6) in forelimb buds (E11.0–E11.5). CRM6 displays mostly no or rarely variable activity (n = 2/14 expressors), while CRM8 (n = 0/5) and CRM9 (n = 0/4) are not active in limb buds. The transgenic founder embryos that express LacZ in forelimb buds are indicated as the fraction of all embryos with LacZ expression in limb and non-limb tissues. Scale bar: 250 µm. Ant: anterior, Dist: distal, Post: posterior, Prox: proximal. d ChIP-seq analysis identifies the interaction of SMAD4 chromatin complexes with CRM2 in the Cis region during the onset of forelimb development (E9.5–9.75) and the GLI3, HOXD13 and HOXA13 chromatin complexes during outgrowth (E11.5). n = 2 independent biological replicates were analysed for all ChIP-seq experiments and the peak calling function of MACS2 identified the significantly enriched peaks in both replicates. These peaks overlap the CRMs identified with exception of one HOXA13 ChIP-seq peak that is located in a conserved region of non-accessible and non-H3K27ac marked chromatin (indicated by an arrowhead)²². The only called SMAD4 ChIP-seq peak within the Grem1 TAD is indicated by an arrow. CRM enhancers are indicated in blue, CRMs without LacZ activity in grey. EC1 and EC2: enhancer cluster 1/2. The inputs for the H3K27ac, SMAD4 and GLI3 ChIP-seq analyses (panels b, d) are shown in Supplementary Fig. 3.