Figure 3.

Time-course data, promoter reporter assays, and MRTF-B-YAP cooperation. MRTF-B was overexpressed in different cell types (200 MOI), and cells were harvested at different times. RNA was subsequently isolated, and CHRM3 was measured by RT-qPCR. (A) Shows time-dependent upregulation of CHRM3 in human coronary artery SMCs. Significant increases were seen at times exceeding 48h, and a 597-fold increase was seen at 120h (p=0.0004, n=3). Because there was no indication that the increase of CHRM3 reached a plateau at longer transduction times, we designed an experiment using even longer incubations in human bladder SMCs (B). Again, there was no tendency of a plateau. Moreover, the maximal increase was somewhat smaller than in coronary artery SMCs. Similar results were obtained in human coronary artery endothelial cells [(C), 200 MOI]. (D) Shows that CHRM3 was reduced by Latrunculin B (100nM, gray bar) in MRTF-B-transduced ECs. Inspection of the CHRM3 gene locus on chromosome 1 (E) revealed SRF binding (green vertical bars) and TEAD binding (red vertical bars) to many sequences 5' of, and over, the longest transcript (blue). Direct examination of a commercial promoter reporter sequence (NM_000740, transcript variant 2, FIGURE 3 | hg38; chr1+: 239627686–239,629,364; TSS=239,629,073) did not reveal any true CArGs, but 11 motifs with 2 deviations from the classical CArG sequence [CC(A/T)₆GG, green highlights, deviations underlined, (F)] were present, along with 9 TEAD motifs [red highlights, (F)]. The transcription start site for the promoter is highlighted in yellow with red lettering. This “CArG-deficient” promoter responded to MRTFs in a luciferase reporter assay (G) run using HEK 293 cells. (H) Shows CHRM3 mRNA expression in MCF10 cells transfected with YAP, MRTF-B, and two MRTF-B mutants; the YBD mutant does not bind YAP, and the SBD mutant does not bind SRF. (I) Shows the effects of YAP and MRTF-B transduction, alone and in combination, on CHRM3 in human coronary artery SMCs. Ct values for CHRM3 were sometimes too high for reliable detection (null and YAP). This is the reason why the sample size is less than n=6 for CHRM3 in the null and YAP groups, even if six experiments were run for the panel. (J) Shows the effect of the YAP-TEAD inhibitor verteporfin in MRTF-B-transduced coronary artery SMCs. Two samples were lost in the verteporfin group again due to lack of amplification. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05.