Supplementary Figure 5: Functional validation of CRMs as transcriptional enhancers in pancreatic MPCs.

(a) Functional validation of CRMs as transcriptional enhancers in human progenitors. Thirty-two CRMs and 8 negative control regions were cloned into the pGL4.23 vector and tested in reporter assays. Reporter activity was compared to empty pGL4.23. ^∗ Two-tailed Student’s t test P<0.05 (P values fully listed in Supplementary Table 22). n = 3-4 independent transfections per enhancer, 8 of 32 constructs were tested in an independent experiment that yielded comparable results. (b) Functional validation of unannotated CRMs identified in the vicinity of MPC-enriched genes in 24 hpf zebrafish embryos. Eight out of 10 TEAD1-bound CRMs yielded activation of a minimal promoter driving GFP (see also Fig. 5c–e and Supplementary Table 21). Pancreatic progenitors were identified by co-staining Nkx6.1 and either Pdx1 or insulin. Note that in zebrafish Nkx6.1 is expressed in early pancreatic progenitors but not in endocrine cells, unlike in mammalian embryos, which show Nkx6.1 expression in both cellular compartments⁶⁸. The percentage of transgenics showing activation of GFP in the pancreatic domain for each CRM and in control injections is presented in the bar plot in Fig. 5e. Dashed lines demarcate the pancreatic progenitor domain (Nkx6.1⁺ cells). y: yolk autofluorescence, s: somites showing crossreactivity with anti-Pdx1 serum.