Figure 6: Ebf3 synergises with MyoD in the induction of the Atp2a1 gene.
From: Ebf factors and MyoD cooperate to regulate muscle relaxation via Atp2a1
(a) Schematic overview of the promoter structure of the Atp2a1 gene. Ebf consensus sites are indicated in red, E-boxes X1 to X5 in green, and their position relative to the transcriptional start site is shown. The sequence of each potential binding site is indicated, and mutations used in the experiments in this figure are shown in small letters. (b) Chromatin immunoprecipitation (ChIP) of flag-tagged MyoD protein from C2C12 cells; qPCR analysis of ChIP using primers specific for all five individual binding sites. Fold enrichment indicates the ratio of DNA amplification comparing ChIP with beads-only control; n=3; error bars=s.d.; *P<0.05, **P<0.01. (c) ChIP assay of MyoD protein from diaphragm of E18.5 Ebf3+/+ and Ebf3−/− embryos as indicated; qPCR analysis of ChIP using primers specific for the two binding sites determined in b. Fold enrichment indicates the ratio of DNA amplification comparing ChIP with beads-only control; n=3; error bars=s.d.; *P<0.05, **P<0.01. (d) HEK293 cells were transfected with an expression plasmid for MyoD and a reporter construct containing all indicated consensus sites as wild-type or mutated sequence as shown in a, followed by the luciferase gene. Transfections of the pBL-Luc plasmid not containing promoter elements together with pCMV-Ebf3 were set to 1 for each reporter plasmid; n=3; error bars=s.d.; P-values were calculated in comparison with activation of pBL-Luc-Atp2a1 if not otherwise indicated. *P<0.05, **P<0.01. (e) HEK293 cells were transfected as in d, but expression plasmids for Ebf3 and MyoD were used in parallel, and reporter plasmids with mutations in binding sites were used as indicated. Transfections of the pBL-Luc plasmid not containing promoter elements together with pCMV-Ebf3 were set to 1 for each reporter plasmid; n=3; error bars=s.d.; P-values were calculated in comparison with pBL-Luc-Atp2a1 if not otherwise indicated. *P<0.05, **P<0.01, ***P<0.001. (f) Quantitative PCR analysis of Atp2a1 expression in C2C12 cells 2 and 5 days after transfection of Ebf3 and/or MyoD encoding expression plasmids. Mock (transfection of parental plasmid) is set to 1 for 2 and 5 days. n=3; error bars=s.d.; P-values are relative to mock; **P<0.01, ***P<0.001.
