SMAD3 and TCF21 have opposing binding behavior and transcriptional regulatory functions in HCASMC.

A) SMAD3 and TCF21 joint binding regions were analyzed for degree occupancy by each factor. DNA occupancy was assessed by comparing peak reads normalized to background reads and this variable graphed. More than half of the colocalized binding regions were biased for either SMAD3 or TCF21 binding, with a 2-fold greater number of normalized reads for one or the other factor. B) To serve as a control for SMAD3 and TCF21 binding patterns, we characterized binding of AP1 heterodimer transcription factors JUN and JUND. Compared to the SMAD3 and TCF21 pattern, there is equivalent binding at the majority of AP1 target sites in the genome. C) SMAD3 binding at the SERPINE1 and COL1A1 loci where it colocalizes with TCF21 was assessed by ChIP-PCR in HCASMC. Knockdown of TCF21 mRNA levels by specific siRNA was associated with increased binding of SMAD3 compared to cells transfected with scrambled siRNA (SCR). D) Local chromatin accessibility was evaluated at the SERPINE1 and COL1A1 loci with the assay for transposase-accessible chromatin coupled with quantitative PCR (ATAC-PCR), in HCASMC with knockdown by siTCF21. E) Reporter gene transfection studies evaluated relative transcriptional activity of SMAD3 and TCF21 at a SERPINE1 enhancer region, in an established human umbilical vein smooth muscle cell line (HUVSMC). F) Identical experiments were conducted in primary cultured HCASMC.