ABSTRACT: Phosphoenolpyruvate carboxykinase (PEPCK) transcription is induced by cAMP/protein kinase A (PKA) and glucocorticoids [dexamethasone (Dex)] and is inhibited by insulin to regulate blood glucose. Recent reports suggested that CCAAT enhancer binding protein (C/EBP) binding to the PEPCK cAMP response element (CRE) plays a role in Dex induction and that insulin-induces inhibitory forms of C/EBPbeta to inhibit transcription. Here, we assessed the roles of CRE-binding protein (CREB) and C/EBP factors in mediating hormone-regulated transcription. Neither cAMP nor insulin regulated the phosphorylation of C/EBP. Cycloheximide did not block insulin inhibition, indicating that alternate translation of C/EBPbeta is not required. Dominant-negative CREB or C/EBP blocked induction by PKA, but neither affected regulation by Dex or insulin. Tethering the activation domains of CREB or C/EBP to a CRE-->Gal4 (G4) site mediated varying extents of basal and PKA-inducible activity, but neither activation domain affected induction by Dex or inhibition by insulin. Surprisingly, synergistic induction by PKA and Dex did not require the CRE and was unaffected by dominant-negative CREB or C/EBP. PKA and Dex also synergistically induced a minimal 3 x glucocorticoid response element promoter, but inhibited Dex induction of the mouse mammary tumor virus and IGF-binding protein 1 promoters, even though PKA alone did not regulate these promoters. These results suggest that PKA modifies the activity of other factors involved in Dex induction to mediate synergistic induction or inhibition in a promoter-specific manner. Our data indicate that the roles of CREB and C/EBP are restricted to mediating PEPCK induction by PKA, and that other factors mediate PEPCK induction by Dex, synergism between PKA and Dex, and inhibition by insulin.
Molecular Endocrinology 05/2005; 19(4):913-24. · 4.54 Impact Factor
ABSTRACT: Cytochrome P450 17alpha-hydroxylase (CYP17) gene expression and androgen biosynthesis are persistently elevated in theca cells isolated from ovaries of women with polycystic ovary syndrome (PCOS). We previously reported that -235 to -109 bp of the CYP17 promoter confers increased CYP17 promoter function in PCOS theca cells. In this report, additional deletion and mutational analyses of the CYP17 promoter were performed to identify the sequences that contribute to increased CYP17 promoter function in PCOS theca cells. Results of these analyses established that augmented promoter function in PCOS theca cells results from preferentially increased basal regulation conferred by sequences between -188 and -147 bp of the CYP17 promoter. Scanning mutant analysis demonstrated that mutations within a 16-bp sequence, spanning -174 to -158 bp of the promoter, ablated increased basal CYP17 promoter function in PCOS theca cells. EMSA analysis demonstrated that the NF-1 family member, NF-1C, bound this sequence. Cotransfection of several NF-1C isoforms expressed in normal and PCOS cells repressed CYP17 promoter function. NF-1C protein and DNA binding were reduced in PCOS theca cell nuclear extracts, as compared with normal. Another NF-1C site between -102 and -90 bp of the promoter was also identified. However, mutation of this site had no effect on differential promoter function in PCOS theca cells. These studies demonstrate that 1) augmented CYP17 promoter function in PCOS theca cells results from increased basal regulation, and 2) diminished NF-1C-dependent repression may be one mechanism underlying increased basal CYP17 promoter activity and altered gene expression in PCOS theca cells.
Molecular Endocrinology 04/2004; 18(3):588-605. · 4.54 Impact Factor
ABSTRACT: The cAMP response element (CRE)-binding protein (CREB) stimulates basal transcription of CRE-containing genes and mediates induction of transcription upon phosphorylation by protein kinases. The basal activity of CREB maps to a carboxy-terminal constitutive activation domain (CAD), whereas phosphorylation and inducibility map to a central, kinase-inducible domain (KID). The CAD interacts with and recruits the promoter recognition factor TFIID through an interaction with a specific TATA-binding-protein-associated factor (TAF), dTAFII110/ hTAFII135. Interaction between the TAF and the CAD is mediated by a central cluster of hydrophobic amino acids, mutation of which disrupts TAF binding, polymerase recruitment, and transcription activation. Assessment of the contributions of the CAD and KID to recruitment of the polymerase complex versus enhancement of subsequent reaction steps (isomerization, promoter clearance, and reinitiation) showed that the CAD and P-KID act in a concerted mechanism to stimulate transcription. The CAD, but not the KID, mediated recruitment of a complex containing components of a transcription initiation complex, including pol II, IIB, and IID. However, the CAD was relatively ineffective in stimulating subsequent steps in the reaction mechanism. In contrast, phosphorylation of the KID in CREB effectively stimulated isomerization of the recruited polymerase complex and multiple-round transcription. A model for the activation of transcription by phosphorylated CREB is proposed, in which the polymerase is recruited by interaction of the CAD with TFIID and the recruited polymerase is activated further by phosphorylation of the KID in CREB.
Progress in Nucleic Acid Research and Molecular Biology 02/2002; 72:269-305. · 0.31 Impact Factor
ABSTRACT: A specific TATA binding protein-associated factor (TAF), dTAFII110/hTAFII135, interacts with cAMP response element binding protein (CREB) through its constitutive activation domain (CAD), which recruits
a polymerase complex and activates transcription. The simplest explanation is that the TAF is a coactivator, but several studies
have questioned this role of TAFs. Using a reverse two-hybrid analysis in yeast, we previously mapped the interaction between
dTAFII110 (amino acid 1–308) and CREB to conserved hydrophobic amino acid residues in the CAD. That mapping was possible only because
CREB fails to activate transcription in yeast, where all TAFs are conserved, except for the TAF recognizing CREB. To test
whether CREB fails to activate transcription in yeast because it lacks a coactivator, we fused dTAFII110 (amino acid 1–308) to the TATA binding protein domain of the yeast scaffolding TAF, yTAFII130. Transformation of yeast with this hybrid TAF conferred activation by the CAD, indicating that interaction with yTFIID
is sufficient to recruit a polymerase complex and activate transcription. The hybrid TAF did not mediate activation by VP16
or vitamin D receptor, each of which interacts with TFIIB, but not with dTAFII110 (amino acid 1–308). Enhancement of transcription activation by dTAFII110 in mammalian cells required interaction with both the CAD and TFIID and was inhibited by mutation of core hydrophobic
residues in the CAD. These data demonstrate that dTAFII110/hTAFII135 acts as a coactivator to recruit TFIID and polymerase and that this mechanism of activation is conserved in eukaryotes.
Proceedings of the National Academy of Sciences 11/2001; 98(23):13078-13083. · 9.68 Impact Factor
ABSTRACT: The in vivopattern of induction of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription by cAMP and its inhibition by insulin
is reproduced in H4IIe cells and is mediated by a bipartite cAMP/insulin response unit (C/IRU) consisting of a cAMP response
element (−95/−87) and an upstream enhancer, AC (−271/−225). Studies in HepG2 cells showed that binding of AP-1 and CAAT/enhancer-binding
protein (C/EBP) to AC is required for induction by cAMP, but insulin did not inhibit cAMP-induced PEPCK expression in HepG2
cells. Binding of H4IIe nuclear proteins to an AC element probe was inhibited by antibodies or a consensus site for C/EBP,
but not AP-1. Transfection with dominant negative bZIP factors, which prevent endogenous factors from binding to DNA, showed
that elimination of cAMP regulatory element-binding protein CREB or C/EBP activity blocked induction by protein kinase A (PKA),
whereas elimination of AP-1 activity had no effect. In addition, promoters with multiple CREB sites, or a single CREB site
and multiple C/EBP sites, mediated PKA induction, but this was inhibited to no greater extent than basal activity was by insulin.
These results indicate that an AC factor other than C/EBP must mediate insulin inhibition. An A-site probe (−265/−247) or
a probe across the middle of the AC element (−256/−237) competed for complexes formed by factors other than AP-1 or C/EBP.
However, analysis of competitor oligonucleotides and antibodies for candidate factors failed to identify other factors. Scanning
mutations throughout the AC element interfered with induction but allowed us to define five overlapping sites for regulatory
factors in AC and to design probes binding just one or two factors. Comparison of the protein-DNA complexes formed on these
smaller probes revealed that a specific complex present in rat liver and H4IIe cell nuclear extracts differed from those formed
by HepG2 cell nuclear extracts. Our results suggest that multiple factors binding the AC element of the C/IRU interact with
each other and CREB to regulate PEPCK induction by cAMP and inhibition by insulin and that the unique factor expressed in
H4IIe cells is a candidate for involvement in insulin regulation of PKA-induced PEPCK gene transcription.
Journal of Biological Chemistry 06/2000; 275(23):17814-17820. · 4.77 Impact Factor