Figure 5.

Functional validation of CRMs as transcriptional enhancers. (a) Thirty two CRMs were cloned into the pGL4.23 vector and tested in reporter assays, where 20 (62.5%) yielded significant activation of a minimal promoter driving luciferase in human pancreatic MPCs. Lines represent median with IQR. Two-tailed Mann-Whitney test P value is shown (n=4 replicate wells). (See also Supplementary Fig. 5a). (b) TEAD binding sites are essential for MPC enhancer activity. Mutation of one or more canonical TEAD binding sites in three CRMs abolished their activity in luciferase reporter assays in in vitro MPCs. Locations of the FGFR2 and MAP3K1 CRMs are highlighted in Figure 4a and Supplementary Figure 4c, respectively. Two-tailed t-test P values are listed in Supplementary Table 22 (n=3-4 transfections per construct, in 1-2 independent experiments). Error bars represent SEM. (c,d) A TEAD1-bound CRM near SOX9 (Fig. 7e) was fused to a minimal promoter and GFP, and injected into zebrafish embryos. In (c), a SOX9 CRM drove strong GFP expression in the pancreatic domain of 48 hpf zebrafish embryos (dotted circle, left panel), which was disrupted by a mutation in the TEAD recognition sequence (right). A midbrain-specific enhancer was used as internal control of transgenesis. Note that this experiment assessed activity of a single SOX9 CRM, which does not necessarily fully recapitulate the expression of endogenous sox9b. In the graph, +, +/− and − represent strong, weak and absent GFP expression in the pancreatic domain, respectively (n=110-140 embryos per condition, Chi-squared test P=1.37×10⁻⁸³). (d) Immunofluorescence analysis of pancreatic MPCs in zebrafish embryos injected at one- to two-cell stage with constructs containing SOX9, MAP3K1 and FOXA2 CRMs driving GFP. Images show GFP in Pdx1⁺/Nkx6.1⁺ cells at 24/48 hpf, as indicated. In total, 8/10 CRMs yielded activity in Pdx1⁺/Nkx6.1⁺ progenitors (see also Supplementary Fig. 5b). The pancreatic progenitor domain is revealed by co-expression of Pdx1⁺ and Nkx6.1⁺ (dashed lines). Note that in zebrafish Nkx6.1 is specific to MPCs within embryonic pancreas⁴⁷. g: Pdx1⁺ gut cells, s: somites showing crossreactivity with anti-Pdx1 serum. (e) Percentage of transgenic embryos with CRM-driven GFP expression in MPCs, or in negative controls (neg.) (quantifications shown in Supplementary Table 21).