A competitive mechanism of CArG element regulation by YY1 and SRF: Implications for assessment of Phox1/MHox transcription factor interactions at CArG elements

In the promoters of many immediate early genes, including c-fos, CArG DNA regulatory elements mediate basal constituitive expression and rapid and transient serum induction. CArG boxes also occur in the promoters of muscle-specific genes, including skeletal alpha-actin, where it confers muscle-specific expression, These elements are regulated, at least in part, by the ubiquitous transcription factors serum response factor (SRF) and YY1, The homeobox transcription factor Phox1/MHox has also been implicated in regulation of the c-fos CArG element. and is thought to function by facilitating SRF binding to DNA, Here, we provide in vitro and in vivo evidence that the mechanism of YY1 repression of CArG elements results from competition with SRF for overlapping binding sites, We describe in detail the binding sites of YY1 and SRF through serial point mutations of the skeletal alpha-actin proximal CArG element and identify a mutation that dramatically reduces YY1 binding but retains normal SRF binding, YY1 competes with SRF for binding to wild-type CArG elements, but not to this point mutant in vitro, This mutant is sufficient for muscle-specific expression in vivo but is much less sensitive to repression by YY1 overexpression, We utilized the YY1/SRF competition to address the role of Phox1 at these elements, Phox1 overexpression did not diminish YY1-mediated repression, suggesting that transcriptional activation by Phox1 does not result from enhanced SRF binding to these elements, These methods may prove to be useful for assessing interactions between other CArG element regulatory factors.