Characterization of promoter elements typifying estrogen-responsive genes and downstream pathways in mouse vasculature
Estrogen receptors (ERs) are cell-specific mediators of physiological changes. They act primarily as transcriptional regulators through binding DNA at specific target sequences, such as estrogen response elements (EREs), within gene regulatory regions. Few primary target genes of ERs have been identified, especially in nonreproductive tissues such as the vasculature, where estrogen responses impact both sexes. I initially utilized a bioinformatics approach to characterize known ER-regulated promoters, both with regards to primary estrogen response elements and to secondary transcription factor binding sites that may coregulate estrogen responses. Secondary promoter elements were identified as being overrepresented in promoters containing palindromic EREs The involvement of these predicted promoter elements, distinct from EREs, in estrogen responsiveness was investigated by transient transfection assays. Mutations were constructed in putative transcription factor binding sites neighboring the EREs within promoters of two human estrogen responsive genes. Eight of ten mutated sites within the TFF1 (trefoil factor 1) promoter decreased both basal and ERa-mediated transcription, to similar extents. Only two of eleven lactoferrin promoter mutants altered gene activation, but these increased ERa-mediated transcription, indicating an unexpected inhibitory function of both sites on estrogen regulation. While this screening method presents a potentially significant tool in characterizing promoters of estrogen-responsive genes, these data suggest that investigation of a greater number or a more physiological set of ER-regulated promoters would facilitate greater success in predicting secondary promoter elements involved in estrogen regulation. To elucidate estrogen-responsive gene pathways in a physiologically important primary target tissue, estrogen-responsive gene expression profiling was performed in aortas from wild type, ERa knockout, and ER13 knockout mice. Unexpectedly, mitochondrial respiratory chain and antioxidant encoding genes were identified as a significant target group downstream of estrogen. Computational analysis of the promoters of these estrogen-responsive genes identified overrepresented binding sites for multiple transcription factor families; such factors are predicted to be downstream mediators of liganded ER in regulating gene expression. Furthermore, expression of some of these transcription factors (e.g. Etsl and SRF) is also estrogen-responsive, supporting their importance in regulation of downstream gene sets. Overall, this study charts previously uncharacterized genes and pathways downstream of estrogen action in the vasculature.