Fig. 8

MLL1 knockdown attenuated TGFβ1-induced increases in H3K4me3 on the promoters of TGFBIp and ECM-associated genes. a Wild-type and GCD2-homozygous corneal fibroblasts were infected with MLL1 or control shRNA lentivirus. After puromycin selection, infected cells were stimulated with TGF-β1 (5 ng/ml) for 8 h, and H3K4me3 levels at the indicated gene promoters were analyzed. ChIP assays were performed with H3K4me3 antibody. Immunoprecipitated DNA and input DNA were subjected to qPCR with primers specific for the indicated gene promoters to measure enrichment levels. qPCR data were analyzed using the 2^-ΔΔCt method, and data are presented as mean fold change of the input ± standard error (SE) of enrichment. (mean ± SE; **P < 0.01 vs. control, n = 3). b Wild-type and GCD2-homozygous corneal fibroblasts were double infected with MLL1 and SET7/9 or control shRNA lentivirus. After puromycin selection, infected cells were stimulated with TGFβ1 (5 ng/ml) for 8 h, and mRNA levels of TGFBIp were analyzed by RT-qPCR. Gene expression was normalized to that of GAPDH (internal control), and results are expressed as fold stimulation over control shRNA-infected wild-type control (mean ± standard error (SE); **P < 0.01 vs. control, n = 3) (c) A model showing that in corneal fibroblasts reaching the quiescent state, transcriptional repression of TGFBIp and ECM-associated genes occurs through heterochromatin formation under no stimulation. Lysine 27 from histone H3 (H3K27) near Smad binding elements (SBEs) within promoters was tri-methylated. During TGFβ1 stimulation, MLL1 and SET7/9 were activated, and their recruitment to SBEs was increased and recruitment of Smad to SBEs was increased. H3K4me1/3 became more methylated, and H3K27me3 was de-methylated on the gene promoters, creating a favorable environment for Smad3 binding