Contributions of Specificity Protein-1 and Steroidogenic Factor 1 to Adcy4 Expression in Y1 Mouse Adrenal Cells

Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario, Canada.
Endocrinology (Impact Factor: 4.5). 08/2008; 149(7):3668-78. DOI: 10.1210/en.2008-0203
Source: PubMed


The type 4 adenylyl cyclase, Adcy4, is the least abundant of five different adenylyl cyclase isoforms expressed in the Y1 mouse adrenocortical cell line and is deficient in a Y1 mutant with impaired steroidogenic factor 1 (SF1) activity. This study examines the contributions of SF1 and other DNA promoter/regulatory elements to Adcy4 expression in the Y1 cell line and its derivative Adcy4-deficient mutant. Primer extension and in silico analyses indicate that Adcy4 transcription initiates from multiple sites just downstream of a GC-rich sequence. Luciferase reporter gene assays identify a 124-bp sequence, situated 19 bp upstream of the major transcription start site and highly conserved among several mammalian species, as the major determinant of Adcy4 expression in Y1 cells and as a site of compromised activity in the Adcy4-deficient mutant. EMSAs using competitor nucleotides and specific antibodies indicate that this conserved region contains three specificity protein (Sp)-1/Sp3-binding sites and one SF1-binding site. As determined by site-specific mutagenesis, the 5'-most Sp1/Sp3-site enhances promoter activity, whereas the middle Sp1/Sp3 and SF1 sites each repress Adcy4 promoter activity. In the Adcy4-deficient mutant, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with small interfering RNAs increases Adcy4 expression, confirming the contribution of SF1 to the mutant phenotype. These studies demonstrate roles for Sp1/Sp3 and SF1 in Adcy4 expression in Y1 cells and establish a repressor function for SF1 in certain promoter contexts.

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    • "On the other hand, it is interesting to notice that a correlation also existed between downregulation of a subset of genes and SF-1 binding in their proximity in both basal and overexpression conditions. This suggests that in certain cases, SF-1 may directly repress gene expression, consistently with previous reports (38,39) and findings for other nuclear receptors classically considered as pure transcriptional activators (40). Finally, indirect mechanisms (e.g. through regulation of the expression of other transcription factors) or long-range effects are likely to account for the regulatory function of different SF-1 dosages on genes in whose proximity the factor does not directly bind. "
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    ABSTRACT: Steroidogenic Factor-1 (SF-1) is a nuclear receptor that has a pivotal role in the development of adrenal glands and gonads and in the control of steroid hormone production, being also implicated in the pathogenesis of adrenocortical tumors. We have analyzed the mechanisms how SF-1 controls gene expression in adrenocortical cells and showed that it regulates different categories of genes according to its dosage. Significant correlations exist between the localization of SF-1-binding sites in chromatin under different dosage conditions and dosage-dependent regulation of gene expression. Our study revealed unexpected functional interactions between SF-1 and Neuron-Restrictive Silencer Factor/RE1-Silencing Transcription Factor (NRSF/REST), which was first characterized as a repressor of neuronal gene expression in non-neuronal tissues, in the regulation of gene expression in steroidogenic cells. When overexpressed, SF-1 reshapes the repertoire of NRSF/REST-regulated genes, relieving repression of key steroidogenic genes. These data show that NRSF/REST has a novel function in regulating gene expression in steroidogenic cells and suggest that it may have a broad role in regulating tissue-specific gene expression programs.
    Nucleic Acids Research 08/2013; 41(19). DOI:10.1093/nar/gkt658 · 9.11 Impact Factor
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    • "For example, the transcription of genes that are required for the uptake and transport of cholesterol and for its metabolism into cortisol and aldosterone requires several transcription factors (Miller and Auchus, 2011; Rainey and Nakamura, 2008; Sewer et al., 2007, 2008), including steroidogenic factor 1 (El-Khairi et al., 2011; Hoivik et al., 2010; Schimmer and White, 2010), nerve growth factor 1B (Bassett et al., 2004a, 2004b), members of the specificity protein family (e.g. Sp1, Sp3) (Huang et al., 2005; Lin et al., 2001; Rui et al., 2008), sterol regulatory element binding protein 1 (Ozbay et al., 2006), GATA proteins (Fluck and Miller, 2004; Jimenez et al., 2003; Nakamura et al., 2007), and cAMP response element modulator (Meier and Clark, 2012; Sugawara et al., 2006; Zwermann et al., 2007). Moreover, emerging evidence implicates the need for the assembly of macromolecular protein complexes that facilitate the movement of cholesterol into the inner mitochondrial membrane (Bose et al., 2002, 2008; Liu et al., 2006; Rone et al., 2012) and for regulatory mechanisms that govern the selective transfer of electrons (Miller, 2005). "
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    ABSTRACT: The production of glucocorticoids and aldosterone in the adrenal cortex is regulated at multiple levels. Biosynthesis of these hormones is initiated when cholesterol, the substrate, enters the inner mitochondrial membrane for conversion to pregnenolone. Unlike most metabolic pathways, the biosynthesis of adrenocortical steroid hormones is unique because some of the enzymes are localized in mitochondria and others in the endoplasmic reticulum (ER). Although much is known about the factors that control the transcription and activities of the proteins that are required for steroid hormone production, the parameters that govern the exchange of substrates between the ER and mitochondria are less well understood. This short review summarizes studies that have begun to provide insight into the role of the cytoskeleton, mitochondrial transport, and the physical interaction of the ER and mitochondria in the production of adrenocortical steroid hormones.
    Molecular and Cellular Endocrinology 11/2012; 371(1-2). DOI:10.1016/j.mce.2012.11.014 · 4.41 Impact Factor
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    • "SF-1 is mostly characterized as a transcriptional activator, so indirect effects likely explain the consequences of SF-1 knockdown on the majority of upregulated genes. However, SF-1 is also known to directly negatively regulate the expression of certain genes, similarly to many other nuclear receptors (Bassett et al., 2002; Rui et al., 2008; Ye et al., 2009). Removal of negative transcriptional outputs by SF-1 may then account for a part of the effect of its knockdown in Y1 cells. "
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    ABSTRACT: Steroidogenic Factor-1 (SF-1) is a nuclear receptor transcription factor that has an essential role in the development of adrenal glands and gonads and in the regulation of steroidogenic gene expression. Recent studies using genomic approaches have revealed that SF-1 also has an important role in regulating proliferation of adrenocortical cells and have revealed its role in the control of a variety of biological processes as diverse as angiogenesis, adhesion to the extracellular matrix, cytoskeleton dynamics, transcriptional and post-transcriptional regulation of gene expression and apoptosis in the adrenal cortex. The identification of the complete set of SF-1 target genes will be of great importance to open new avenues for therapeutic intervention in adrenal diseases.
    Molecular and Cellular Endocrinology 11/2012; 371(1-2). DOI:10.1016/j.mce.2012.11.005 · 4.41 Impact Factor
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