Figure 7. SoxC proteins directly activate Tead2.
(a) Schematic of the mouse Tead2 locus from upstream of the transcription start site (angled arrow) to downstream of the translation start site (ATG) in the second exon, and analysis of mammalian conservation. Highly conserved, non-coding sequences were found in the promoter and first exon (PE1 box) and in the first intron (InS box). No other highly conserved region was found in other Tead2 introns or in the agenic region around Tead2 (data not shown). Arrows, position of primers used in chromatin immunoprecipitation assay. Numbers 1–4 indicate the position of Sox-binding sites. (b) Tead2 reporters were made by cloning the indicated Tead2 sequences upstream of the firefly luciferase gene. (c) Transient transfection of Cos-1 cells with Tead2 reporters and expression vectors for Sox4, Sox11 or no protein (−). Reporter activities (RLU, relative luciferase units) were normalized for transfection efficiency and are presented as the average with standard deviation of data obtained from three independent cultures. Constructs bearing PE1+InS are significantly activated by SoxC proteins (P<0.001, Student's t-test), unless the SoxC-binding sites are mutated (PE1+InS-Mut). (d) ClustalW alignment of sequences encompassing putative SoxC-binding sites in several mammalian genomes. These sites are boxed. These sequences and mutant versions, as indicated, were used to generate electrophoretic mobility shift assay probes. (e) Electrophoretic mobility shift assay with Tead2 probes and protein extracts from Cos-1 cells transfected with a Sox4 expression plasmid. Sox4 binds to all wild-type probes (arrowhead), but not to mutant probes, except m1b. (f) Chromatin immunoprecipitation assay of SoxC proteins binding to endogenous Tead2. Chromatin extracts from C3H-10T1/2 cells transiently expressing FLAG-Sox proteins were precipitated in the absence (no IgG) or presence of non-immune (n.i.) rabbit IgG, RNA polymerase II antibody or FLAG antibody. PCR products from immunoprecipitated chromatin and input are shown on resolution by agarose gel electrophoresis. DNA markers and the size of PCR products are shown.