Genome-wide maps of SMAD2-chromatin binding state in TGFbeta-treated vascular smooth muscle cells

To further explore the role of these two factors in SMC reprogramming, we treated human aortic smooth muscle (HASMCs) in vitro with TGFβ and mapped binding of Smad2/3, which reflects TGFβ activity, and Pol2-Ser2p, which reflects transcriptional activity, by ChIP-seq analysis. Smad2/3 bound to numerous regulatory regions in the genome differentially regulating expression of numerous genes as demonstrate by alterations in Pol2-Serp2 binding profile and analysis of bulk RNA sequencing. Examination of the top 20 transcription factors identified by sequencing of Smad2/3 binding regulatory regions identified KLF4 gene as one of the most prominently regulated genes. Further analysis showed Smad2/3 binding to multiple KLF4 gene regulatory elements that increased following TGFβ treatment, indicating that KLF4 is a direct target of Smad2/3. At the same time, the amount of bound Pol2-Ser2p decreased, pointing to reduced transcriptional activity. In contrast, Smad2/3 did not bind to KLF2 or KLF5 regulatory elements, demonstrating that these genes are not directly regulated by TGFβ. Examination of the binding of SMAD in chromatin of vascular smooth muscle cell.