Figure 5: Atp2a1 is a direct target gene of Ebf3.
From: Ebf factors and MyoD cooperate to regulate muscle relaxation via Atp2a1
(a) Schematic overview of the promoter structure of the Atp2a1 gene. Red boxes indicate potential binding sites for Ebf proteins, numbers give the position relative to the transcriptional start site of Atp2a1. The sequence of each potential binding site is indicated and mutations used in the experiments in this figure are shown in small letters. (b) EMSA of HEK293T protein extract containing flag-tagged Ebf3 with oligonucleotides corresponding to potential binding sites. For specificity control non-radioactive (cold) or mutated oligonucleotides were used, identity was confirmed by supershift using α-Flag antibody (Ab); n=3. (c) Chromatin immunoprecipitation (ChIP) of flag-tagged Ebf3 protein from C2C12 cells; qPCR analysis of ChIP using primers specific for all four 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. (d) Analysis of the transactivation potential of Ebf3 from the Atp2a1 locus. HEK293T cells were transfected with plasmids comprising all four potential binding sites in wild-type or mutated form as indicated in a. Activity of luciferase was determined, and values were normalized to β-galactosidase. Transfections of the pBL-Luc plasmid not containing promoter elements together with pCMV-Ebf3 were set to 1; 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) Quantitative PCR analysis of Atp2a1 expression in C2C12 cells 2 and 5 days after transfection of mock- or Ebf3-plasmid and initiation of in vitro differentiation by horse serum (HS). n=3; error bars=s.d.; P-values are relative to mock; ***P<0.001.
