Prdm6 drives ductus arteriosus closure by promoting ductus arteriosus smooth muscle cell identity and contractility
Meng Zou, Kevin D. Mangum, Justin C. Magin, Heidi H. Cao, Michael T. Yarboro, Elaine L. Shelton, Joan M. Taylor, Jeff Reese, Terrence S. Furey, Christopher P. Mack
Meng Zou, Kevin D. Mangum, Justin C. Magin, Heidi H. Cao, Michael T. Yarboro, Elaine L. Shelton, Joan M. Taylor, Jeff Reese, Terrence S. Furey, Christopher P. Mack
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Research Article Vascular biology

Prdm6 drives ductus arteriosus closure by promoting ductus arteriosus smooth muscle cell identity and contractility

Abstract

Based upon our demonstration that the smooth muscle cell–selective (SMC-selective) putative methyltransferase, Prdm6, interacts with myocardin-related transcription factor-A, we examined Prdm6’s role in SMCs in vivo using cell type–specific knockout mouse models. Although SMC-specific depletion of Prdm6 in adult mice was well tolerated, Prdm6 depletion in Wnt1-expressing cells during development resulted in perinatal lethality and a completely penetrant patent ductus arteriosus (DA) phenotype. Lineage tracing experiments in Wnt1Cre2 Prdm6fl/fl ROSA26LacZ mice revealed normal neural crest–derived SMC investment of the outflow tract. In contrast, myography measurements on DA segments isolated from E18.5 embryos indicated that Prdm6 depletion significantly reduced DA tone and contractility. RNA-Seq analyses on DA and ascending aorta samples at E18.5 identified a DA-enriched gene program that included many SMC-selective contractile associated proteins that was downregulated by Prdm6 depletion. Chromatin immunoprecipitation–sequencing experiments in outflow tract SMCs demonstrated that 50% of the genes Prdm6 depletion altered contained Prdm6 binding sites. Finally, using several genome-wide data sets, we identified an SMC-selective enhancer within the Prdm6 third intron that exhibited allele-specific activity, providing evidence that rs17149944 may be the causal SNP for a cardiovascular disease GWAS locus identified within the human PRDM6 gene.

Authors

Meng Zou, Kevin D. Mangum, Justin C. Magin, Heidi H. Cao, Michael T. Yarboro, Elaine L. Shelton, Joan M. Taylor, Jeff Reese, Terrence S. Furey, Christopher P. Mack

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Figure 9

Identification of regulatory elements and genetic variations that control Prdm6 expression in SMCs.

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Identification of regulatory elements and genetic variations that contro...
(A) Schematic illustrating the genome-wide data sets used to prioritize our search for regulatory elements that control the SMC-selective expression of Prdm6. (B) The indicated Prdm6 region was cloned into the appropriate luciferase reporter plasmid and then transfected into HuBrSMCs and mouse ECs. Luciferase activity was measured at 48 hours and is expressed relative to the appropriate empty vector. n ≥ 3 for all experimental groups. *P < 0.05 versus promoter less (t test); #P < 0.05 versus ECs (t test). (C) DNA sequence of the 325 bp conserved region within the Int3.1 enhancer. (D) PCR mutagenesis was used to generate the indicated mutations in the context of the highly active In3.1 conserved regulatory region. Luciferase activity was measured at 48 hours and is expressed relative to the activity of the WT Int3.1 conserved construct. n = 5 for all groups. *P < 0.05 versus WT (t test). (E) Targeted ChIP assays measuring SRF, RBPJ, and TEAD1 binding to the endogenous Int3.1 region. PCR primers used for these experiments are shown in red in C. n = 3 for all groups. *P < 0.05 versus IgG (t test). (F) Int3.1 conserved-luciferase was transfected into ECs and SMCs with/without myocardin. Luciferase activity was measured at 48 hours and is expressed relative to Int3.1 conserved luciferase activity in the presence of empty expression vector. n = 5 for all groups. *P < 0.05 versus plus empty vector (t test). (G) PCR mutagenesis was used to generate an allelic series for the rs17149944 variation within the context of Int3.1-luciferase construct. Note that the presence of the minor A allele significantly reduced the activity of the Int3.1 enhancer. n = 5 per group. *P < 0.05 versus WT (t test).