INVESTIGATING THE FUNCTION OF PRDM6 IN SMOOTH MUSCLE

The ductus arteriosus is a unique embryonic vessel that shunts blood from the pulmonary circulation to the aorta during embryonic development. Although patent ductus arteriosus (PDA) is a relatively common congenital abnormality that prevents the establishment of normal pulmonary circulation upon birth, the mechanisms that drive ductus closure remain unclear. Based upon our demonstration that the SMC-selective putative methyltransferase, PRDM6, interacted with the serum response co-factor, myocardin-related transcription factor A, we examined the role of PRDM6 function in SMCs in vivo using conditional cell-type specific mouse knockout models. Wnt1Cre2 PRDM6flox/flox mice exhibited a completely penetrant lethal PDA phenotype providing strong evidence that PRDM6 expression in neural crest-derived SMC is required for DA closure. Lineage tracing experiments in Wnt1Cre2 PRDM6flox/flox ROSA26LacZ mice were consistent withnormal neural crest investment of the outflow tract. In contrast, myography measurements of isolated DA segments indicated that PRDM6 depletion significantly reduced DA tone and contractility. Extensive bulk RNAseq analyses on DA and ascending aorta samples isolated from Wnt1Cre2 PRDM6flox/flox and littermate control mice at E18.5 identified a gene program that was selectively enriched in the DA and that was down regulated by PRDM6 depletion. Included in this data set were many genes previously implicated in ductus function and closure including the SMC markers, the prostaglandin receptor EP4, and the ductus selective transcription factor, TFAP2b. ChIPseq was performed in outflow tract SMC cultures and PRDM6 binding sites were identified in about 50% of the genes shown to be altered by PRDM6 depletion by RNAseq. Although PRDM6 binding was frequently associated with promoters, it was more prevalent in early introns suggesting that it may have roles in transcription initiation and maintenance. Using several genome-wide data sets to prioritize our search, we identified a SMC-selective enhancer within the PRDM6 third intron and showed that a genetic variation within this enhancer (rs17149944) regulated its activity. Since this SNP is within an LD block shown to associate with a variety of cardiovascular disease outcomes, is effects on PRDM6 expression may be causal for these associations. Taken together our studies provide novel mechanisms for the role of PRDM6 on DA closure and provide a strong foundation for future studies on the mechanisms that control ductus contractility and the role of PRDM6 in human cardiovascular disease.Doctor of Philosoph