Different promoter usage for CYP19 gene expression in buffalo ovary and placenta

Abstract

Aromatase is the key enzyme for estrogen biosynthesis and is coded by CYP19 gene. The expression of CYP19 gene is regulated in tissue-specific manner by alternate use of different promoters. In this study, we have analyzed tissue-specific expression and their regulation of CYP19 gene in preovulatory and postovulatory stages of buffalo (Bubalus bubalis) ovary and placenta. RT-PCR analysis showed that the CYP19 gene expression was significantly (p<0.05) higher in granulosa cells of large follicles as compared to the other tissues. The transcript analysis and transcriptional start site identified by 5'-RLM RACE for CYP19 expression indicated different transcriptional start sites within the different sized follicles during folliculogenesis. Sequence analysis showed that the transcription start site of transcript isolated from buffalo granulosa cells of small follicles was 37 bases upstream of the transcript isolated from granulosa cells of large follicles. However, both the transcripts were found to be derived from proximal promoter II. Difference in the transcriptional start site indicates the different promoter sequence usage in granulosa cells of different sized follicles. Further, in silico analysis of the difference in promoter sequence based on the 5'-UTRs isolated from granulosa cells of small follicles (151 bases) and large follicles (114 bases) showed that consensus sequence for certain important trans-elements (viz., TATA binding protein, E2F and CAAT binding protein) would lie in the promoter sequence isolated from the granulosa cells of large follicles. These transcription factors may be involved in regulation of CYP19 gene expression in ovary, either directly or indirectly. The difference in the size of 5'-UTR among the granulosa cells of ovary reflects the possible mechanism for the differential regulation of CYP19 gene during development. The transcripts isolated from buffalo corpus luteum and placental cotyledons were having same 5'-UTR comprises of 168 bases and found to be derived from PI.1. Estimates of CYP19 gene transcript concentration in the different tissues revealed that the CYP19 gene expression in granulosa cells is predominantly regulated by PII and to a minor extent by PI.1. However, PI.1 is almost exclusively responsible for CYP19 gene expression in placenta and residual expression in corpus luteum. In order to understand the complex CYP19 gene regulation in these tissues, further studies are needed to elucidate the activity of different promoters and define regulatory elements for binding of transcription factors.