The Transcriptional Regulating Protein of 132 kDa (TReP-132) Enhances P450scc Gene Transcription through Interaction with Steroidogenic Factor-1 in Human Adrenal Cells

Oncology and Molecular Endocrinology Research Center, Laval University, Québec GIK 7P4, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2002; 277(42):39144-55. DOI: 10.1074/jbc.M205786200
Source: PubMed


The human P450scc gene is regulated by the tissue-specific orphan nuclear receptor, steroidogenic factor-1 (SF-1), which plays a key role in several physiologic processes including steroid synthesis, adrenal and gonadal development, and sexual differentiation. Several studies have demonstrated the interaction of SF-1 with different proteins. However, it is clear that additional factors not yet identified are involved with SF-1 to regulate different target genes. Recently, it was demonstrated that a novel transcriptional regulating protein of 132 kDa (TReP-132) regulates expression of the human P450scc gene. The overexpression of TReP-132 in adrenal cells increases the production of pregnenolone, which is associated with the activation of P450scc gene expression. Considering the colocalization of TReP-132 and SF-1 in steroidogenic tissues such as the adrenal and testis, and the presence of two putative LXXLL motifs in TReP-132 that can potentially interact with SF-1, the relationship between these two factors on the P450scc gene promoter was determined. The coexpression of SF-1 and TReP-132 in adrenal NCI-H295 cells cooperates to increase promoter activity. Pull-down experiments demonstrated the interaction between TReP-132 and SF-1, and this was further confirmed in intact cells by coimmunoprecipitation/Western blot and two-hybrid analyses. Deletions and mutations of the TReP-132 cDNA sequence demonstrate that SF-1 interaction requires the LXXLL motif found at the amino-terminal region of the protein. Also, the "proximal activation domain" and the "AF-2 hexamer" motif of SF-1 are involved in interaction with TReP-132. Consistent with previous studies showing interaction between CBP/p300 and SF-1 or TReP-132, the coexpression of these three proteins results in a synergistic effect on P450scc gene promoter activity. Taken together the results in this study identify a novel function of TReP-132 as a partner in a complex with SF-1 and CBP/p300 to regulate gene transcription involved in steroidogenesis.

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    • "Since TdIF1 binds to a co-transcriptional factor TReP-132 [4], we suspect that TdIF1 regulates gene transcription together with TReP-132. TReP-132 is involved in the transcription of the p450scc, p21, and p27 genes [9], [29]–[33]. However, the TdIF1-binding DNA sequence does not exist in these promoter regions, implying that TdIF1 is not involved in transcriptional regulation of these genes. "
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    ABSTRACT: TdIF1 was originally identified as a protein that directly binds to DNA polymerase TdT. TdIF1 is also thought to function in transcription regulation, because it binds directly to the transcriptional factor TReP-132, and to histone deacetylases HDAC1 and HDAC2. Here we show that TdIF1 recognizes a specific DNA sequence and regulates gene transcription. By constructing TdIF1 mutants, we identify amino acid residues essential for its interaction with DNA. An in vitro DNA selection assay, SELEX, reveals that TdIF1 preferentially binds to the sequence 5'-GNTGCATG-3' following an AT-tract, through its Helix-Turn-Helix and AT-hook motifs. We show that four repeats of this recognition sequence allow TdIF1 to regulate gene transcription in a plasmid-based luciferase reporter assay. We demonstrate that TdIF1 associates with the RAB20 promoter, and RAB20 gene transcription is reduced in TdIF1-knocked-down cells, suggesting that TdIF1 stimulates RAB20 gene transcription.
    PLoS ONE 07/2013; 8(7):e66710. DOI:10.1371/journal.pone.0066710 · 3.23 Impact Factor
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    • "s shown in Fig. 3B, TdIF1 unexpectedly bound to truncated TReP-132 residues ranging from 387 to 444 without the LXXLL motif and did not bind to del7, suggesting that TdIF1 binds to a confined region from 387 to 407 of TReP-132. Recently, TReP-132 has been reported to bind to SF-1 only through its N-terminal LXXLL, not through its C-terminal LXXLL (Gizard et al . 2002b). Thus, we then examined the possibility of binding through the N-terminal LXXLL. As shown in Fig. 3B (del8), no binding through the N-terminal LXXLL was observed. We therefore conclude that TdIF1 mainly binds to the novel confined region between residues 387 and 407 in TReP-132."
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    ABSTRACT: N regions at the junction of V, D and J DNA segments are synthesized with large protein complexes including terminal deoxynucleotidyltransferase (TdT) during V(D)J recombination in B- or T-cells. TdT directly binds to TdIF1, TdIF2, PCNA and the Ku70/86 heterodimer. Using a yeast two-hybrid system, we isolated a cDNA clone encoding the gene for TReP-132, which is involved in P450scc gene expression in steroid-hormone-producing cells or lymphoid cells. Interaction between TReP-132 and TdIF1 was confirmed by pull-down assay and immunoprecipitation assay using specific antibodies against TReP-132 both in vitro and in vivo. TdT also directly bound to TReP-132 through its confined N-terminal region. Furthermore, the co-expression of TdIF1 and TReP-132 or TdT and TReP-132 in COS7 cells showed that these proteins are co-localized within the nucleus. TReP-132 reduces TdT activity to 2.5% of its maximum value in the in vitro assay system using double-stranded DNA with a 3' protrusion as a primer. These findings suggest that TdT synthesizes N region under a negative control of TReP-132 during V(D)J recombination.
    Genes to Cells 02/2006; 11(1):47-57. DOI:10.1111/j.1365-2443.2005.00916.x · 2.81 Impact Factor
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    • "Plasmid Gal4-tk80-luc was a luciferase reporter gene under the control of the Gal4-responsive element and plasmid pGal4-Smad1 was a human Smad1 sequence fused with the DNA-binding domain of Gal4 (Pearson et al. 1999). P450 scc luciferase reporter constructs containing fragments of the human P450 scc gene spanning from nucleotides –110 to +49, with or without the mutated SF-1-binding site, subcloned in pGL3 vector, were kindly provided by Dr B Staels (Gizard et al. 2002). StAR luciferase reporter construct containing fragments of human StAR gene spanning nucleotides –235 to +39 was a gift from Dr J Strauss (Sugawara et al. 1996). "
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    ABSTRACT: We have recently reported that bone morphogenetic protein-4 (BMP-4) can inhibit progesterone production by ovine granulosa cells (GCs). Here, we have investigated the underlying mechanisms of this effect in basal as well as in FSH-induced conditions. We have confirmed that treatment with BMP-4 decreased basal GC progesterone secretion and totally abolished FSH-stimulating action. This inhibitory action was associated with a decrease in the expression of cAMP-regulated genes, steroidogenic acute regulatory protein (StAR) and P450 side-chain cleavage (P450 scc) at mRNA and protein levels. However, BMP-4 did not alter basal cAMP production by GCs. In contrast, BMP-4 decreased by half the FSH-induced cAMP production and strongly inhibited cAMP-induced progesterone production. Thus, the inhibitory effect of BMP-4 was exerted both upstream and downstream of cAMP signalling. We next examined the downstream effect, focusing on cAMP-dependent transcription factors, steroidogenic factor-1 (SF-1) and CREB, through the BMP factor signalling intermediary, Smad1. As expected, BMP-4 induced phosphorylation and transcriptional activity of Smad1 in ovine GCs. BMP-4-activated Smad1 did not affect CREB activity but inhibited the transcriptional activity of SF-1 on the canonical SF-1 responsive element. Interestingly, this transcriptional inhibitory mechanism occurred on transfected StAR and P450 scc promoter. Based on these results, we propose that SF-1 is a key target in the inhibitory mechanism exerted by BMP-4 on progesterone synthesis by ovine GCs in culture. Because SF-1 plays an essential role in the differentiation of GCs, our findings could have new implications in understanding the role of BMP family members in the control of ovarian folliculogenesis.
    Journal of Molecular Endocrinology 01/2005; 33(3):805-17. DOI:10.1677/jme.1.01545 · 3.08 Impact Factor
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