Evolutionary analysis of TATA-less proximal promoter function

Many molecular studies describe how components of the proximal promoter aff ect transcriptional processes. However, these studies do not account for th e likely effects of distant enhancers or chromatin structure, and thus it i s difficult to conclude that the sequence variation in proximal promoters a cts to modulate transcription in the natural context of the whole genome. T his problem, the biological importance of proximal promoter sequence variat ion, can be addressed using a combination of molecular and evolutionary ana lyses. Provided here are molecular and evolutionary analyses of the variati on in promoter function and sequence within and between populations of Fund ulus heteroclitus for the lactate dehydrogenase-B (Ldh-B) proximal promoter . Approximately one third of the Ldh-B proximal promoter contains intersper sed regions that are functionally important: (1) they bind transcription fa ctors in vivo, (2) they effect a change in transcription as assayed by tran sient transfection into two different fish cell lines, and (3) they bind pu rified transcription factors in vitro. Evolutionary analyses that compare s equence variation in these functional regions versus the nonfunctional regi ons indicate that the changes in the Ldh-B proximal promoter sequences are due to directional selection. Thus, the Ldh-B proximal promoter sequence va riations that affect transcriptional processes constitute a phenotypic chan ge that is subject to natural selection, suggesting that proximal promoter sequence variation affects transcription in the natural context of the whol e genome.