SUV39H1 Regulates KLF4 and Chromatin Remodeling in Smooth Muscle Cell Phenotypic Plasticity

Reversible DNA methylation contributes to vascular smooth muscle cell (VSMC) phenotypic plasticity. This plasticity contributes to vascular growth and remodeling but also underlies pathologies, including intimal hyperplasia. We investigated the role of SUV39H1 (suppressor of variegation 3-9 homolog 1), a histone methyltransferase that generates trimethylation at histone H3 lysine 9 (H3K9me3), a repressive heterochromatin-associated epigenetic mark, in VSMC plasticity.

We applied knockdown, quantitative polymerase chain reaction, Western blotting, chromatin immunoprecipitation, assay for transposase-accessible chromatin using sequencing, and RNA-sequencing in human coronary artery smooth muscle cells and murine carotid ligation to assess SUV39H1 functions in VSMC plasticity.

In normal mouse carotid artery, SUV39H1 and H3K9me3 were markedly increased, while the cognate H3K9me3 demethylase KDM4A (lysine demethylase 4A) rapidly decreased with carotid ligation and neointimal hyperplasia. In human coronary artery smooth muscle cells, SUV39H1 knockdown induced contractile genes, morphology, and contractility but inhibited migration and proliferation. We found that SUV39H1 was required for PDGF (platelet-derived growth factor) induction of KLF4 (Krüppel-like factor 4)-regulating miR143, KLF4 mRNA stability, and promoter accessibility. PDGF-induced SUV39H1 expression and SUV39H1-dependent H3K9me3 modification at contractile gene promoters. SUV39H1 knockdown increased KDM4A expression and binding to contractile promoters, suggesting an opposing regulatory relationship between the H3K9me3 writer and eraser in VSMCs. Chromatin immunoprecipitation assays with SUV39H1 knockdown revealed that SUV39H1 modifies H3K9me3 but also promotes a repressive state (increased 5mC and reduced H3K27Ac [histone K27 acetylation]) at contractile gene promoters. Conversely, SUV39H1 induced an active state at the KLF4 promoter, reducing DNMT1 (DNA methyltransferases 1) recruitment and 5mC (5-methylcytosine) levels. Assay for transposase-accessible chromatin using sequencing revealed that SUV39H1 oppositely modifies chromatin accessibility at phenotype-specific human coronary artery smooth muscle cell promoters genome-wide. Consistently, transcriptomic profiling showed that SUV39H1 and TET2 (Tet methylcytosine dioxygenase 2) oppositely influence SMC gene expression.

We identify SUV39H1 as a potent PDGF-induced epigenetic regulator that promotes KLF4 expression and VSMC dedifferentiation. SUV39H1 regulates dynamic trimethylation at histone H3 lysine 9 in phenotypic switching, regulating mark deposition and the KDM4A demethylase. We report that SUV39H1 coordinately regulates DNA and histone methylation and histone acetylation. This altered chromatin accessibility by a heterochromatin-associated enzyme represents a new mechanism underlying VSMC plasticity.