Chromatin Immunoprecipitation Assay for H3K4me2

Dimethylation of lysine 4 on histone 3 (H3K4me2) of the smooth muscle myosin heavy chain 11 (Myh11) promoter was detected by in situ hybridization (ISH) and Proximity Ligation Assay (PLA) as previously published^{24 (link)}. Briefly, the 2 kb promoter of Myh11 was amplified by PCR, cloned into pCR2.1 vector for amplification (TOPO cloning kit, ThermoFisher Scientific) and biotin labeled probes were generated by Nick Translation (Roche) using biotin-14-ATP (ThermoFisher Scientific). Probes were denatured in hybridization buffer (2X SSC, 50% high grade formamide, 10% dextran sulfate, 1 μg mouse Cot-1 DNA) for 5 min at 80 °C. Abdominal aorta sections were incubated with 0.5% pepsin at 37 °C for 15 min, followed by incubation with hybridization buffer containing biotinylated probe for 5 min at 80 °C then followed by incubation for 16–24 h at 37 °C. Following hybridization, slides were washed in 2X SSC, 0.1% NP-40 buffer. Sections were blocked, incubated with mouse H3K4dime (5 μg/mL, Millipore Sigma, clone CMA303) and rabbit Biotin (5 μg/mL, Abcam #ab53494) antibodies overnight at 4 °C, followed by incubated with secondary antibodies containing PLA probe at 37 °C for 1 h. Ligation and amplification were performed (Duolink detection kit Orange 555 nm) and mounting medium with DAPI was used. Immunofluorescence was quantified with Image J.