TCF21 regulates basic cellular functions in vascular smooth muscle cells in vitro.

A) HCASMC transduced with TCF21 overexpressing lentivirus (pWPI-TCF21) or empty lentivirus (pWPI empty) were labeled with the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU), which was visualized with a fluorescent azide, allowing identification and quantification of proliferating cells with immunofluorescence microscopy, reported here as a percent with baseline being all DAPI positive cells. HCASMC showed an increase in the percentage of TCF21 overexpressing cells compared to DAPI stained cells (33.1% ± 2.3 control vs. 51% ± 4.1 overexpressing cells, P<0.001). B) HCASMC transduced with knockdown lentiviruses showed a decreased percentage of dividing cells (43.4% ± 4.4 control vs. 30.8% ± 2.4 knockdown cells, P<0.01). C) siTCF21 produced a decrease in apoptosis in HCASMC as measured by a caspase activity assay (41,824±1872 vs. 18,837±1302, P<0.0001). D) TCF21 regulation of HCASMC migration was evaluated with a gap closure assay. TCF21 overexpressing cells transduced with pWPI-TCF21 lentivirus covered a significantly larger surface area after 12 hours of study compared to cells transduced with the empty pWPI vector (27.8 ± 2.7 vs. 15.8 ± 1.16, P<0.001). E) HCASMC treated with siTCF21 compared to siCTRL showed significantly increased expression of ACTA2, TAGLN, and MYH11 SMC marker genes (P<0.05/P<0.01/P<0.05 respectively). F) The ACTA2 locus as visualized on the University of California Santa Cruz genome browser. Data provided here reveals evidence for a likely enhancer region in the first intron, as indicated by DNase hypersensitivity measured in human aortic SMC, and histone modification data showing enrichment of H3K27Ac and H3K4me1 at this same site, as well as clustering of a number of transcription factor binding sites. G) Chromatin immunoprecipitation for TCF21 binding to the enhancer region of the ACTA2 locus by ChIP-qPCR (P<0.05). H) Dual luciferase assays in rat aortic smooth muscle cells with a reporter construct containing the human ACTA2 promoter and first intron (SMA-luc). A TCF21 expression construct was transfected with human TCF3 (E12), TCF3 (E47), TCF12, and Twist1 murine expression vectors, showing specific suppression of transcription of the SMA-luc reporter (1.0 ± 0.01 vs. 0.1 ± 0.009, P<0.01 for TCF21 alone). I) Similar dual luciferase assays using a 3 E-box containing minimal promoter construct (E-luc) based on the nucleotide sequence of the first intron (n = 3, 3 replicates), again showing TCF21 mediated suppression of transcription (1.0 ± 0.29 vs. 0.21 ± 0.02, P<0.01 for TCF21 alone).