Factor substitution in a human HSP70 gene promoter: TATA-dependent and TATA-independent interactions.

To investigate interactions between transcription factors on mammalian promoters, we constructed a set of 24 variations of the human HSP70 gene promoter in which six upstream sequence motifs are paired in every possible combination with four TATA motifs. These promoters were analyzed for in vivo expression, and selected constructs were examined by in vitro template commitment studies. Activation transcription factor (ATF) and CP1 showed dramatically different interactions with the factor(s) bound to the TATA region. CP1 functioned in vivo regardless of the TATA motif that it was paired with and was not capable of sequestering the core promoter complex in a template commitment assay. ATF activity was dramatically altered by changing the TATA motif, and ATF was able to sequester the core promoter complex. These data suggest that CP1 and ATF function by distinct mechanisms that differ with respect to interaction with the factor(s) at the TATA box. Factor Sp1 also appeared to function by a TATA-independent mechanism. These data imply that the ability of a factor to function is determined not only by the intrinsic properties of the factor but also by promoter context.