Evolutionary and functional analyses of cytochrome P4501A promoter polymorphisms in natural populations

The functional importance of variable, transcriptional regulatory sequences within and among natural populations is largely unexplored. We analysed the cytochrome P4501A (CYP1A) promoter in three populations of the minnow, Fundulus heteroclitus, because two SNPs in the promoter and first intron of CYP1A are under selection in populations adapted to pollutants. To define the importance of these SNPs, 1630 bp of the CYP1A promoter and first intron and exon were sequenced in eight individuals from three populations: a population from a polluted environment resistant to some aromatic pollutants and two flanking reference populations. CYP1A is induced by many aromatic pollutants, but in populations adapted to pollutants, CYP1A has been shown to be refractory to induction. We were interested in understanding whether variation in the CYP1A promoter explains mechanism(s) of adaptation to these aromatic pollutants. The CYP1A promoter was extremely variable (an average of 9.3% of the promoter nucleotides varied among all populations) and exhibited no fixed differences between populations. As CYP1A is poorly inducible in adapted fish, we hypothesized that CYP1A promoter regions might vary functionally between populations. Unexpectedly, in vitro analysis showed significantly greater transcription from CYP1A promoters found in the population from the polluted environment relative to promoters found in both reference populations. Thus, despite extensive variation among populations and lack of fixed differences between populations, individuals from a polluted environment have significantly enhanced promoter activity. These data demonstrate that intraspecific variation, which provides the raw material for natural selection to act on, can occur while maintaining promoter function.