Fig. 2

Twist1 was an essential, positive transcriptional regulator of Prrx1 expression in both normal fibroblasts and CAFs. Twist1 enhanced the expression of Prrx1, a potential TNC inducer, by direct transcriptional activation. a Analysis of the FANTOM database revealed 11 candidate genes whose expressions were significantly correlated with both Twist1 and TNC expression in fibroblasts and stem cells. b Induction of Twist1 expression increased Prrx1 expression in normal fibroblasts. c The knockdown of Twist1 expression in CAFs led to decreased Prrx1 expression. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: **p<0.001, ***p<0.0001). d To determine whether Twist1 regulates Prrx1 promoter activity, Prrx1 promoter-driven luciferase gene and Twist1 expression vector were cotransfected into HT1080 fibrosarcoma cells. The luciferase reporter assay revealed that the ectopic expression of Twist1 increased the Prrx1 promoter activity. Of the four different Prrx1 promoter constructs, construct #2 containing two E-boxes between −798 and −592 bp from the TSS of Prrx1 showed the most obvious difference in response to transient cotransfection with a Twist1 expression vector. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.001). e Two E-box mutations in E-box 1 (CATCTG (−619) to GGTCTG) and E-box 2 (CAACTG (−787) to TCACTG) were introduced in HT1080 cells. The Twist1-induced Prrx1 promoter activity was negated by the E-box mutation that was located at 619 bp of the TSS of Prrx1. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.05). f The schematic illustration of the ChIP assay PCR primer binding sites in the Prrx1 promoter region. We used the E-box sequence in the SDF1a promoter as a positive control. Compared with the IgG and positive controls, the Prrx1 promoter region that contained E-box 1 and E-box 2 was specifically enriched by each primer set after chromatin immunoprecipitation. That is, the ChIP assay revealed that Twist1 binds to the Prrx1 promoter sites (−819 to −597 bp) that contain two E-boxes. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.05). g Schematic diagram of the regulatory relationship among Twist1, Prrx1, and TNC