Ubc9 and Protein Inhibitor of Activated STAT 1 Activate Chicken Ovalbumin Upstream Promoter-Transcription Factor I-mediated Human CYP11B2 Gene Transcription

Abstract

Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the ؊129/؊114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast twohybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.Aldosterone is exclusively produced in adrenal zona glomerulosa cells due to its unique expression of aldosterone synthase cytochrome P450 (CYP11B2), the enzyme required for the final steps of aldosterone biosynthesis. In aldosterone-producing adrenal cortical adenomas of patients with primary aldosteronism, overexpression of CYP11B2 is demonstrated at the transcriptional level (1, 2). Although the reason for aberrant expression of CYP11B2 in these adenomas is not known, mutations in the CYP11B2 gene do not appear to be the cause (3, 4). We therefore postulated that transcription factors and/or coregulators may play important roles in CYP11B2 overexpression in the tumors.The trans-acting factors that regulate CYP11B2 expression remain poorly defined. The orphan nuclear receptor, steroidogenic factor-1 (SF-1), 1 is shown to play a crucial regulator of most steroid hydroxylase genes, including CYP17 and CYP11B1 (5, 6). However, SF-1 actually represses rather than activates expression of hCYP11B2 (7-9). In addition, other transcription factors that are expressed in the adrenal cortex include the NGFI-B family of or...

Regulation of the Ets-1 transcription factor by sumoylation and ubiquitinylation

“…Nonetheless, in agreement with a previous report (Hahn et al, 1997), we found that the WT version of Ubc9 both enhanced the transcriptional response to Ets-1 WT and K15R/K227R mutant (data not shown). It remains possible that Ubc9 in addition to its sumoylation activity can function as a co-activator in transcription and that these activities are separable, as previously demonstrated for the COUP-TF1 transcription factor (Kurihara et al, 2005). To further examine the contribution of the enzymes of the SUMO pathway, we analysed the effect of the SENP1 protease, that we demonstrated to be effective in desumoylating Ets-1 (Figure 3d).…”

Coactivation of the N-terminal Transactivation of Mineralocorticoid Receptor by Ubc9

“…Plasmid Constructs-Several Ubc9 constructs, such as pcDNA3.1/His-Ubc9, pcDNA3.1/His-Ubc9(C93S), pGADT7-Ubc9, pGEX4T-1-Ubc9, pGEX4T-1-Ubc9(C93S), and pEGFPUbc9 were described previously (49,50). pCMV-YFP-SRC-1 was a generous gift from Dr. Toshihiko Yanase (Kyushu University).…”

“…Western Blot Analysis and Coimmunoprecipitation-The cells were lysed with lysis buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride), and Western blots were performed before the immunoprecipitation (IP) steps to confirm protein expression by corresponding antibodies as described previously (49,50). The same samples for the Western blots were diluted to 1 ml in IP buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM dithiothreitol, 5 ng/l aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 0.1% Tween 20) and precleared with protein G plusagarose beads (Santa Cruz Biotechnology, Santa Cruz, CA), and antibodies were added for 1 h. Immune complexes were adsorbed to protein G plus-agarose beads and washed four times in IP buffer.…”

“…Yeast Y187 cells were transformed with yeast expression plasmids encoding hMR and Ubc9. ␤-Galactosidase activity in liquid culture was determined with chlorophenol red ␤-D-galactopyranoside as described previously (49,50,52,53).…”

NF-YC Functions as a Corepressor of Agonist-bound Mineralocorticoid Receptor

“…ChIP Assay-A chromatin immunoprecipitation (ChIP) assay was performed as described previously (17,26). The crosslinked, sheared chromatin solution was used for immunoprecipitation with 3 g of anti-NF-YC (C-19) antibody, anti-MR (N-17) antibody, anti-SRC-1 (M-341) antibody (Santa Cruz Biotechnology), anti-NF-YA antibody, anti-NF-YB antibody (Abcam), and normal IgG.…”