Abstract
We have recently identified the hADA3 protein, the human homologue of yeast transcriptional coactivator yADA3, as a novel HPV16 E6 target. Using ectopic expression approaches, we further demonstrated that hADA3 directly binds to the 9-cis retinoic acid receptors alpha and beta, and functions as a coactivator for retinoid receptor-mediated transcriptional activation. Here, we examined the role of endogenous hADA3 as a coactivator for estrogen receptor (ER), an important member of the nuclear hormone receptor superfamily. We show that ADA3 directly interacts with ER alpha and ER beta. Using the chromatin immunoprecipitation assay, we also show that hADA3 is a component of the activator complexes bound to the native ER response element within the promoter of the estrogen-responsive gene pS2. Furthermore, using an ER response element-luciferase reporter, we show that overexpression of ADA3 enhances the ER alpha- and ER beta-mediated sequence-specific transactivation. Reverse transcription-PCR analysis showed an ADA3-mediated increase in estrogen-induced expression of the endogenous pS2 gene. More importantly, using RNA interference against hADA3, we demonstrate that inhibition of endogenous hADA3 inhibited ER-mediated transactivation and the estrogen-induced increase in the expression of pS2, cathepsin D, and progesterone receptor, three widely known ER-responsive genes. The HPV E6 protein, by targeting hADA3 for degradation, inhibited the ER alpha-mediated transactivation and the protein expression of ER target genes. Thus, our results demonstrate that ADA3 directly binds to human estrogen receptor and enhances the transcription of ER-responsive genes, suggesting a broader role of mammalian hADA3 as a coactivator of nuclear hormone receptors and the potential role of these pathways in HPV oncogenesis.
Highlights
The nuclear hormone receptors (NRs)1 are a family of structurally related, ligand-induced transcriptional activators that function as critical regulators of diverse physiological processes, including growth, differentiation, development, and reproduction [1, 2]
Whole-cell lysates were prepared in buffer containing 50 mM Tris, pH 8.0, 100 mM NaCl, and 0.5% Nonidet P-40, and 50 ā®g of protein was subjected to Western blotting with anti-PR, anti-c-Myc and anti-tubulin antibodies (Santa Cruz Biotechnology). Both hADA3 and mADA3 Interact Directly with ERā£ and ERā¤āGenetic analyses have shown a requirement for yeast ADA3 for transcriptional activation by human RXR and other NRs when these were expressed in yeast [17, 18]
The overall binding was lower in this assay, both hADA3 and mADA3 could clearly bind to ERā£ in a ligand-independent manner (Fig. 1C, lanes 3, 4, 7, and 8)
Summary
The nuclear hormone receptors (NRs)1 are a family of structurally related, ligand-induced transcriptional activators that function as critical regulators of diverse physiological processes, including growth, differentiation, development, and reproduction [1, 2]. Our results demonstrate that ADA3 directly binds to human estrogen receptor and enhances the transcription of ER-responsive genes, suggesting a broader role of mammalian hADA3 as a coactivator of nuclear hormone receptors and the potential role of these pathways in HPV oncogenesis.
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