SUN-013 Estrogen Receptor Alpha Transactivation Function 1 Dependent Uterotropic Estrogenic Genes

Estrogen receptor alpha (ERα) is a ligand dependent transcription regulator, which contains two transactivation domains, AF-1 and AF-2. The activities of these domains are differentially regulated by various estrogenic signals. The chemicals which control the activities of ERα AF-1 and AF-2 in a discriminatory manner in target tissues are named Selective Estrogen Receptor Modulators (SERMs). However, there is very little information on AF-1 or AF-2 predominantly regulated genes within tissues. Therefore, we sought out to determine which estrogenic genes are regulated by AF-1 using the AF-2 mutated knock-in (KI) mouse model, AF2ERKI. AF2ER is an estrogen-insensitive mutant ERα but unique to this mutant, AF-1 can be activated by estrogen-antagonists, such as fulvestrant (ICI) and 4-hydroxytamoxifen, in vitro and in vivo. Uterine microarray analysis revealed that the several genes showed higher fold activation level in the ICI-treated AF2ERKI compared to the estradiol (E2)-treated WT. The genes belong to such category were considered as potential AF-1 regulated genes. We analyzed the promotor activity of estrogen responsive uterine genes using an in vitro cell based luciferase reporter assay using the HepG2 cells. In order to define the promoter functionality, we selected Klk1b21, which fold activation level was higher in the ICI-treated AF2ERKI than E2-treated WT uterus, and Mad2l1, which fold activation level was higher in the E2-treated WT than ICI-treated AF2ERKI uterus. We generated the luciferase reporter plasmids which contain 1k bp 5’-flanking sequence of Klk1b21 or the 1k bp 5’-flanking sequence of Mad2l1. The promoter activity of Klk1b21 was enhanced by AF2ER with ICI and WT-ERα with E2 at the same level. On the other hand, Mad2l1 promoter was activated by WT-ERα with E2 but not AF2ER with ICI. These results suggest that Klk1b21 gene is likely to be regulated by AF-1 predominantly. Further analyses revealed that an 80 bp element of Klk1b21 gene, containing a transcription initiator element (Inr) and a TATA-like sequence contributes to the AF-1 preferential activity of the gene, rather than the sequence of the ERα binding element. These results suggest that the involvement of ERα AF-1 and AF-2 varies in the regulation of each estrogen responsive gene. The AF2ERKI mouse is a useful model to define the AF-1 predominantly regulated genes in the estrogen target tissues. The information would enhance the understanding of the mechanism of selective functionality of SERMs in target organs.