DIVERSE MECHANISMS OF CONTROL OF AROMATASE GENE-EXPRESSION

The synthesis of estrogens from androgens is catalyzed by a microsomal cytochrome P450 termed aromatase (P450arom). The expression of this e nzyme is highly regulated in both a developmental and cell-type specif ic fashion. We have chosen to examine the molecular basis of aromatase gene regulation by studying two models of aromatase expression: the S ebright bantam chicken and the R2C rat Leydig tumor cell line. In the first model, affected (Sebright) chickens express aromatase in many ex tragonadal tissues, while normal Leghorn chickens express aromatase on ly in the ovary and hypothalamus. Our studies have demonstrated that i n normal chickens the site of transcription initiation is located appr ox. 147 nucleotides upstream of the initiator methionine. While Sebrig ht animals also express aromatase mRNA initiated at an analogous initi ation site in the ovary, a distinctive species of aromatase mRNA is al so detected and is present in ovary and extragonadal tissues. This mRN A contains an identical coding sequence, but contains an alternatively spliced 5' noncoding exon that is derived from a distinctive promoter . The second model, the R2C Leydig tumor cell line, provides ample con trast. This cell line expresses high basal levels of aromatase (150-20 0 pmol/h/mg protein) that is suppressed with administration of 8 bromo cAMP or forskolin but the activity is not altered by glucocorticoids or epidermal growth factor treatment. Despite this distinctive pattern of regulation, at least three species of aromatase mRNA are detected in Northern blots, each of which is also detected in rat ovary. Primer extension and S1 nuclease assays indicate that both granulosa cells a nd R2C cells utilize a promoter that is located approx. 97 nucleotides upstream of the initiator methionine. These studies suggest that the ''ovarian'' promoter is evolutionarily conserved in both rats and chic kens. These results further imply that the genetic mechanisms controll ing the diversity of aromatase expression among tissues and among diff erent species are likely to fall into two groups: those that employ di stinctive promoters and alternative splicing and those that effect dif ferent patterns of regulation through a common (''ovarian'') promoter.