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A PEA3 site flanked by SP1, SP4, and GATA sites positively regulates the differentiation-dependent expression of Brachyury in embryonal carcinoma P19 cells.

The promoter sequence of Brachyury was analyzed in mouse embryonal carcinoma P19 cells. The sequence up to -267 bp relative to the transcription start site was sufficient to enhance reporter gene expression depending on the mesodermal differentiation of P19 cells. Footprint analysis by nuclear extract showed binding of a GATA protein and SP4 and mutation of their sites reduced reporter gene expression. Gel-retardation assay in the presence of a series of double-stranded DNA fragments as the competitors showed SP1 and Est sites additionally. Deletion of either sites reduced the reporter gene expression, showing that they are cooperative. Depletion of PEA3 (a transcription factor of the Est family) with a specific antibody diminished the retarded bands only for the nuclear extract from differentiated P19 cells. Thus, the PEA3 site supported by SP1, SP4, and GATA sites positively regulates the differentiation-dependent expression of Brachyury in P19 cells.