Figure 4.

Set7 is required for H3K4me1 and the recruitment of SRF at the promoters of SM-associated genes. (A) SRF gene targets are down-regulated in Set7KD Sca1+ cells. GSEA identifies regulatory targets implicated in SM-associated gene expression in Set7KD cells (Left panel). A negative ES indicates down-regulated transcription factor gene sets by Set7KD. # FDR q-value = 0.01. GSEA identifies SRF gene targets in Set7KD Sca1+ cells (Right panel). SM-associated transcription factors were derived from mSigDB. (B) SM-associated gene expression is subject to SRF and H3K4me1 dependent mechanisms regulated by Set7 methyltransferase. Soluble chromatin was isolated from the Sca1+ cells transduced with shRNA for non-target control (NTC; black) or Set7KD (white) and immunoprecipitated with antibodies against SRF, H3K4me1, H3K4me2, H3K9/14ac and H4ac. Enrichment at CArG sites were assessed at Cnn1, Tagln, Acta2 and Myh11 genes by qPCR. Data are calculated as relative to the IgG-negative control and represented as ChIP DNA normalized against input DNA. Error bars represent the standard error of the mean of at least five independent experiments. *P < 0.05 and **P < 0.01. (C) Set7 interacts with SRF. 293FT cells were co-transfected with Flag-tagged Set7 (Flag-Set7) and HA-tagged SRF (HA-SRF) expression plasmids. Reciprocal immunoprecipitaion assays were performed with anti-Flag or anti-HA beads. Immunoprecipitations were detected by immuno-blotting using anti-FLAG, HA, SET7 and SRF antibodies. The data presented are a representative of four independent experiments. (D) Set7 methylates recombinant SRF protein. Immunopurified Flag-Set7 (black circle) was incubated with (H³)-S-adenosyl-methione and HA-SRF protein for 1 h at 30°C. Tritiated HA-SRF protein was measured by liquid scintillation. Tritiated HA-SRF proteins without Flag-Set7 (white circle) represent background activity. Data are presented as mean ± SD, n = 3.