Functional analyses of natural variation in Sp1 binding sites of a TATA-less promoter

Within the lactate dehydrogenase-B (LdhB) proximal promoter is a region with multiple in vivo foot-printed sites that resembles the binding site for the transcription factor SP1. Like many sequences that regulate transcription rate, these Sp1 binding sites are well conserved among species of the teleost fish Fundulus. The only exception is in the northern population of F. het eroclitus, where there are many changes in the Sp1 binding sites. These changes affect footprinting patterns, measures of promoter strength, and are associated with the adaptive increase in Ldh-B transcription rates. Reported here is data that demonstrates that Fundulus hepatocyctes have an SP1-like protein; in comparison to human SP1 protein, it has similar specificity and size and a greater affinity for the consensus Sp1 site. This Fundulus hepatocyte SP1-like protein as well as the human SP1 protein binds the Ldh-B Sp1 sites. Sequence variation in the northern Spl region eliminates the "preferred" Spl binding site, yet these northern Sp1 sites have significantly greater affinity for the SP1 protein than either the Spl sites from southern F. heteroclitus (similar to 1.6-fold) or the consensus Sp1 site (GGGCGG; - 1.8-fold). Furthermore, the Ldh-B Sp1 sites also bind non-SP1 proteins, and the extent of binding is affected by the sequence variation in the proximal promoter. These data suggest that natural variation in Sp1 sites affect binding of transcription factors and may effect a modest change in transcription rates.