Abstract 397: Smooth Muscle Cell-specific Lncrna Carmn Is Regulated By Srf/myocd Complex

Background: Cell identify is established by strict control of cell type-specific gene expression programs through regulatory enhancers binding of transcriptional factors. Vascular smooth muscle cells (VSMCs) are critical in maintaining the vascular homeostasis by expressing an array of contractile genes that are primarily governed by SRF/MYOCD complex via binding to the DNA motif called CArG element. However, most of these previous studies regarding regulatory mechanism underlying SM-specific gene expression were performed in vitro, outside of their in situ genomic contexts. Methods and Results: To define the regulatory atlas of VSMC, we analyzed epigenetic datasets of multiple human tissues and cell types. We found CARMN , that we previously reported as a SMC-specific lncRNA and a critical regulator of SMC contractile phenotype by binding to MYOCD, is enriched with SMCs-specific enhancers. Co-expression analysis using RNA-seq datasets of different human tissues identified CARMN expression is positively correlated with both MYOCD and SRF . We further demonstrated overexpressing MYOCD activates, while silencing MYOCD or SRF inhibits CARMN expression in human coronary VSMCs. Subsequent analysis of SRF ChIP-seq data revealed 4 SRF binding peaks with CARMN locus, including 2 located within promoter (namely CArG 1 and 2) and the other 2 (namely CArG 3 and 4) located within distal enhancers. Further luciferase reporter assays revealed mutation of CArG 1 or 3 dramatically abolish MYOCD-induced reporter activity. To confirm these findings in vivo, we generated single CArG element mutant mice for each of the 4 CArG elements and CArG 3/4 double mutant mice by CRISPR-Cas9 genome editing approach. Analysis of Carmn expression in SMC-enriched tissues from these mutant mice revealed a ~50% decrease of Carmn expression in tissues of CArG 1 mutant mice while mutation of other CArG elements appears no effect on Carmn expression. Conclusions: Our results suggest the SM-specific expression of Carmn is dependent, at least partially, on SRF/MYOCD complex and CArG 1 is critical for this regulation. Together with our previous findings that CARMN potentiates MYOCD function, we propose CARMN and SRF/MYOCD complex form a feedforward loop promoting SMC contractile phenotype.