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PubMed2005

A regulator of nutritional copper signaling in Chlamydomonas is an SBP domain protein that recognizes the GTAC core of copper response element.

The CRR1 (Copper Response Regulator) locus, required for both activating and repressing target genes of a copper- and hypoxia-sensing pathway in Chlamydomonas, encodes a 1,232-residue candidate transcription factor with a plant-specific DNA-binding domain named SBP, ankyrin repeats, and a C-terminal Cys-rich region, with similarity to a Drosophila metallothionein. The recombinant SBP domain of Crr1 shows zinc-dependent binding to functionally defined copper-response elements associated with the CYC6 and CPX1 promoters that contain a critical GTAC core sequence. Competition experiments indicate equivalent selectivity for copper-response elements from either promoter and 10-fold greater selectivity for the wild-type sequence vs. a sequence carrying a single mutation in the GTAC core. The SBP domain of Chlamydomonas Crr1 binds also to a related GTAC-containing sequence in the Arabidopsis AP1 promoter that is the binding site of a defining member of the SBP family of DNA-binding proteins. Chlamydomonas Crr1 is most similar to a subset of the Arabidopsis SBP domain proteins, which include SPL1, SPL7, and SPL12. The abundance of the CRR1 mRNA is only marginally copper-responsive, and although two mRNAs that differ with respect to splicing of the first intron are detected, there is no indication that the splicing event is regulated by metal nutrition or hypoxia. It is likely that the dramatic copper-responsive action of Crr1 occurs at the level of the polypeptide.

Algal Proteins↗
PubMed1990

Transcriptional activation by TGF beta 1 mediated by the dyad symmetry element (DSE) and the TPA responsive element (TRE).

Transforming growth factor beta 1 (TGF beta 1) is a multifunctional regulator of growth and differentiation. However, both cytoplasmatic and nuclear signal transduction mechanisms leading to the biological effects of TGF beta 1 are largely unknown. In this report we show, that TGF beta 1 induces the expression of the immediate early genes c-jun and jun B, that encode trans-acting factors regulating transcription of a variety of genes in response to growth factors and phorbol esters. The jun genes are induced by TGF beta 1 in a protein synthesis independent fashion both in quiescent mouse 3T3 fibroblasts, which are growth stimulated by TGF beta 1, as well as in mink lung CCL64 (ML-CCL64) epithelial cells, which are growth inhibited by TGF beta 1. The PDGF inducible JE gene was induced by TGF beta 1 in 3T3, but not in ML-CCL64 cells. Furthermore, we show that chimaeric reporter-CAT constructs containing the TPA responsive element (TRE) or the dyad symmetry element (DSE) are activated by TGF beta 1 in transient transfection assays in both growth inhibited and growth stimulated cells. These results show that the early genomic responses to TGF beta 1 resemble changes in gene expression induced by serum, growth factors and phorbol esters, suggesting common mechanisms of transcriptional activation.

Animals↗
PubMed1999

Identification of a mineralocorticoid/glucocorticoid response element in the human Na/K ATPase alpha1 gene promoter.

Sodium-potassium ATPase (Na/K ATPase) is a major target of mineralocorticoids. Both aldosterone and glucocorticoids activate the human Na/K ATPase alpha1 and beta1 genes transcriptionally. The mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) have been shown to bind the glucocorticoid response element (GRE); however, a specific element responsible for the activation of the MR is not known. Sequence analysis of the putative regulatory region of the Na/K ATPase alpha1 gene revealed the presence of a hormone response element that allows the MR to interact with it, at least as well as if not better than the GR. This response element is designated MRE/GRE. In this investigation, we demonstrated the MR and GR induced gene expression in COS-1 cells by cotransfecting with respective expression plasmids (RshMR and RshGR) along with a luciferase reporter. The synthetic MRE/GRE linked to a neutral promoter was activated by MR (6-fold); however, the GR induced a lower level of expression (3.8-fold), suggesting that the element may be preferably MR responsive. Mutations in the synthetic MRE/GRE could not induce the expression with MR, whereas GR had a small effect. Electrophoretic mobility shift analyses demonstrated a direct interaction of MR and GR with the MRE/GRE that was supershifted by an antiMR antibody and the complex was partially cleared by an antiGR antibody, respectively, whereas nonimmune serum had no effect. Footprinting analyses of the promoter region showed that a portion of the DNA containing this element is protected by recombinant MR and GR. Thus these data confirm that this MRE/GRE interacts with both MR and GR but interaction with receptors may be more MR-responsive than response elements previously described.

Adrenal Cortex Hormones↗
PubMed1992

Molecular cloning of human CREB-2: an ATF/CREB transcription factor that can negatively regulate transcription from the cAMP response element.

The cAMP response element (CRE) is an octanucleotide motif (TGACGTCA) that mediates diverse transcriptional regulatory effects. In this report we describe the isolation and characterization of a full-length cDNA that encodes a CRE binding protein called CREB-2. Like other ATF/CREB transcription factors, the 351-amino acid CREB-2 protein contains a COOH-terminal leucine-zipper motif and an adjacent basic domain. CREB-2 mRNA is expressed ubiquitously in human tumor cell lines and mouse organs suggesting that it is involved in regulating transcription in a wide variety of cell types. Overexpression of CREB-2 resulted in a consistent and significant repression of CRE-dependent transcription in CV-1 cells. Deletional analyses localized the transcriptional repressor activity of CREB-2 to a 102-amino acid COOH-terminal region (amino acids 249-351) that contains the leucine-zipper and basic domains of the molecule. These results demonstrate that CRE-dependent transcription can be both positively and negatively regulated by structurally related members of the ATF/CREB family.

Amino Acid Sequence↗
PubMed1995

Characterization of the constitutive and inducible components of a T cell IL-4 activation responsive element.

An Il-4 regulatory element, activation responsive element (ARE), located between -88 and -60, contributes to activation-dependent transcription of IL-4/CAT reporter gene constructs in T cells. It was previously demonstrated that nuclear proteins present in both unstimulated and stimulated T cells specifically interact with the ARE. In this study, these proteins were further characterized. UV cross-linking experiments established that multiple proteins are associated with the ARE in both the constitutive and activation-dependent complexes and several of these have identical apparent m.w. The formation of both complexes is dependent on the same ARE subsequence. In addition, activator protein 1 family members are uniquely associated with the activation-dependent complex. These results support a model in which activation-dependent proteins, including jun/fos family members, associate with a preexisting transcription complex to influence inducible IL-4 gene transcription. The ARE shares 9 bp of sequence identity with the IL-2 nuclear factor of activated T cell (NF-AT) binding site within the critical protein binding region, and several features of ARE-protein interactions are similar to the NF-AT transcription complex. However, we demonstrate that the constitutive nuclear ARE-associated factors react with Abs, raised to NF-ATp and NF-ATc, preferentially bind to the ARE but not to the NF-AT binding site and are cyclosporin A sensitive. Taken together, these data indicate that there are IL-4 gene-specific factors associated with the ARE and that the formation of the ARE and NF-AT complexes are regulated differently.

Animals↗
PubMed1988

Isolation and properties of cDNA clones encoding SRF, a transcription factor that binds to the c-fos serum response element.

The serum response element (SRE) is a sequence required for transient transcriptional activation of genes in response to growth factors. We have isolated cDNA clones encoding serum response factor (SRF), a ubiquitous nuclear protein that binds to the SRE. The SRF gene is highly conserved through evolution, and in cultured cells its transcription is itself transiently increased following serum stimulation. A cDNA clone of SRF expressed in vitro generates protein that forms complexes indistinguishable from those formed with HeLa cell SRF, as judged by DNA binding specificity and the ability to promote SRE-dependent in vitro transcription. SRF binds DNA as a dimer, and the DNA binding/dimerization domain of the protein exhibits striking homology to two yeast regulatory proteins.

Base Sequence↗
PubMed1999

Transcriptional regulation of the gonadotropin-releasing hormone receptor gene is mediated in part by a putative repressor element and by the cyclic adenosine 3',5'-monophosphate response element.

The levels of the GnRH receptor (GnRHR) and its messenger RNA depend on the pattern of administration of GnRH. In this study, internal deletion mutants in a luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment of mouse GnRHR gene were used to examine the regulation of GnRHR gene transcription in GGH3 cells. Our results indicate that the mouse GnRHR promoter contains one putative repressor element located at position -343/-335. When this sequence was deleted, the GnRHR promoter activity was significantly increased in both basal and GnRH agonist (Buserelin)-, phorbol ester-, and forskolin-stimulated cells. Gel mobility shift assay showed that the sequence -343/-335 is capable of binding GGH3 nuclear proteins. With deletion of the cAMP response element (-107/-100), basal and Buserelin-stimulated transcription was decreased. The same response was observed after stimulation with forskolin. Stimulation with (Bu)2cAMP did not alter transcription above basal levels. The stimulation with phorbol ester resulted in an attenuated increase in transcriptional activity, suggesting that this sequence of the GnRHR promoter is a cAMP response element. These results suggest that the transcriptional activity of the GnRHR gene is mediated in part by a putative repressor element and by the cAMP response element.

Animals↗
PubMed2002

The as-1 promoter element is an oxidative stress-responsive element and salicylic acid activates it via oxidative species.

The activation sequence-1 (as-1)-like element found in the promoter of some glutathione S-transferase (GST) genes, has been previously described as a salicylic acid (SA)- and auxin-responsive element. In this paper, we tested the hypothesis that the activating effect of SA on the as-1 element is mediated by oxidative species. Supporting this hypothesis, our results show that the antioxidants dimethylthiourea (DMTU) and 3-t-butyl-4-hydroxy-anizole (BHA) inhibit the SA-induced transcription of genes controlled by as-1 elements in tobacco (Nicotiana tabacum) plants [i.e. GNT35 gene coding for a GST and (as-1)(4)/beta-glucuronidase (GUS) reporter transgene]. DMTU and BHA also inhibit SA-activated as-1-binding activity in nuclear extracts. Further support for the hypothesis that the as-1 element is activated by oxidative species comes from our result showing that light potentiates the SA-induced activation of the as-1 element. Furthermore, methyl viologen, a known oxidative stress inducer in plants, also activates the as-1 element. Increasing H(2)O(2) levels by incubation with H(2)O(2) or with the catalase inhibitor 3-amino-1,2,5-triazole does not activate the (as-1)(4)/GUS gene. On the contrary, 3-amino-1,2,5-triazole inhibits the activating effect of SA on the (as-1)(4)/GUS gene. These results suggest that oxidative species other than H(2)O(2) mediate the activation of the as-1 element by SA. Our results also suggest that even though the as-1 binding activity is stimulated by oxidative species, this is not sufficient for the transactivation of genes controlled by this element. The complex interplay between SA and reactive oxygen species in the transcriptional activation of defense genes is discussed.

Amitrole↗
PubMed2000

Effect of opioids on Ca2+/cAMP responsive element binding protein.

Ca2+/cAMP response element binding protein (CREB) is an important factor linking the opioid-regulated secondary messenger systems to alterations in gene expression. Opioids regulate CREB level, its phosphorylation and binding to its corresponding response element in the promoters of several genes implicated in drug addiction. CREB mediates the action of opioids on the expression of several genes in brain regions responsible for drug-seeking behavior and manifestation of signs of dependence. Moreover, alterations in CREB level can effect the rewarding properties of morphine and regulate the self-administration of cocaine. At the cellular level CREB acts as convergence point for different cellular pathways. Opioids affect two different intracellular mediator systems: inhibitory--connected with cAMP, and stimulatory--involving calcium and the PKC pathway. Both can affect CREB but in different phases of opiate action. The presence of this biphasic mechanism can explain the phenomenon of the induction of some CRE-controlled genes after both acute and chronic morphine administration. Cellular studies also highlight the relevance of other ATF/CREB family members which can affect Ca2+/cAMP response element (CRE) controlled transcription as well as other transcription factors which make the opioid induction longer lasting.

Animals↗
PubMed2000

The tetracycline-responsive promoter contains functional interferon-inducible response elements.

Tetracycline (tet)-responsive expression vectors allow controlled inducible expression of proteins in mammalian cells. This system is widely used for experimental research both in vivo and in vitro. In our attempts to use this system to study the antiviral effect of IFNalpha on hepatitis B virus, we discovered an unexpected feature of the tet-responsive promoter (tet promoter) of the currently available expression vectors. IFNalphawas found to stimulate tet promoter activity after transient transfection in a dose- and cell type-dependent manner. By sequence inspection, an IFNalpha-stimulated response element (ISRE)-like sequence was identified in the linker regions located between the heptameric tet operator sequences. Gel shift assays revealed binding of IFN-stimulated gene factors to these sequences, indicating that they mediate the IFNalpha-mediated promoter stimulation. These data demonstrate an unexpected feature of the tet-responsive expression system which needs to be taken into account when using this system for analysis of cytokine functions in vitro and in vivo. The data also imply that the tet promoter-based expression system can be rendered non-responsive to IFNalpha by mutagenesis of the ISREs and this may be essential when considering gene therapy in vivo.

Anti-Bacterial Agents↗
PubMed1997

Hyperosmolarity-induced gene stimulation is mediated by the negative calcium responsive element.

The negative calcium responsive elements of the parathyroid hormone gene bind to a specific set of nuclear proteins in an extracellular calcium (Ca2+e)-dependent manner. We have found that one of the negative calcium responsive elements, named oligo B, is found in the 5'-flanking region of such vasoactive genes as the vasopressin and atrial natriuretic polypeptide genes. Furthermore, the oligo B-like sequence in the former gene is conserved throughout evolution. Because expression of some of these vasoactive genes is altered by external stimuli which change cell volume, we examined whether oligo B is involved in gene regulation by hyperosmolarity. Here, we demonstrate that the binding between oligo B and its binding nuclear proteins including a redox factor 1 was reduced by hyperosmolarity generated by sodium chloride but not by urea. Such attenuated binding was reversed by dephosphorylating nuclear proteins by a potato acid phosphatase, suggesting that NaCl treatment elicited phosphorylation of these nuclear proteins to weaken their binding activity to oligo B. Furthermore, these nuclear events led to hyperosmolarity-mediated transcriptional stimulation of the genes bearing this DNA element in the cultured cells.

Calcium↗
PubMed1990

Transcription factors different from the estrogen receptor stimulate in vitro transcription from promoters containing estrogen response elements.

The estrogen response element (ERE) directly linked to a TATA box induces CAT activity in a hormone-dependent manner in Fe 33 cells, the rat hepatoma cell line FTO-2B, stably transfected with the human estrogen receptor (ER). The same promoter construct mediates the stimulation of in vitro transcription. This stimulation is dependent on the presence of the ERE. Induction of transcription in a variety of nuclear extracts derived from mammalian cells is of the same magnitude irrespective of the presence of ER. Similarly, transcription in vitro mediated by B1 vitellogenin 5' flanking sequences in different nuclear extracts is not due to the interaction of the ER with the ERE. Competition analyses with a variety of oligonucleotides reveal that proteins different from the ER, which recognize ERE-like DNA elements, functionally interact with the ERE in vitro. These experiments suggest that ubiquitous proteins related or even identical to the transcription factor USF (MLTF) activate in vitro transcription in an ERE-dependent manner.

Animals↗
PubMed1992

Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common feature which defines the capacity of these elements to confer T3 induction.

Animals↗
PubMed1994

Homodimer and heterodimer DNA binding and transcriptional responsiveness to triiodothyronine (T3) and 9-cis-retinoic acid are determined by the number and order of high affinity half-sites in a T3 response element.

T3 (triiodothyronine) response elements (TREs) consist of pairs of strong and weak (S and W), 10-nucleotide T3 receptor (TR) monomer binding sites (half-sites). We report that the number and order of S and W half-sites in a direct repeat TRE determines whether it mediates ligand-dependent or independent transcriptional activation or inhibition in the presence of TR or TR and 9-cis-retinoic acid receptor (RXR); and whether a TRE is preferentially bound by TR homodimers, TR-RXR heterodimers, or CV1 cell TR accessory protein (TRAP)-TR heterodimers. TR homodimers bound equally to TREs composed of the 5'-S and 3'-W (SW) and the opposite (WS) arrangement of half-sites. TR-RXR gamma heterodimers bound SW better than WS. TR-TRAP heterodimers bound WS better than SW. Transcription of a reporter gene cis-linked to WS responded to unliganded TR and RXR, and either ligand stimulated expression 2-fold more. Reporter expression cis-linked to SW was not altered by unliganded receptors, and T3 stimulated transcription only in the presence of both TR and RXR. SS was strongly activated by liganded, but not by unliganded TR. SS was activated by unliganded TR and RXR gamma together, and T3 further stimulated transcription 2-fold. Under these conditions, transcription was inhibited 60% by 9-cis-retinoic acid.

Animals↗
PubMed1990

Transcriptional regulation of the rat glutathione S-transferase Ya subunit gene. Characterization of a xenobiotic-responsive element controlling inducible expression by phenolic antioxidants.

We have identified previously a xenobiotic-responsive element, which we termed the beta-naphthoflavone-responsive element, between nucleotide -722 and -682 in the 5'-flanking region of the rat glutathione S-transferase Ya subunit gene (Rushmore, T.H., King, R.G., Paulson, K.E., and Pickett, C.B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3826-3830). The beta-naphthoflavone-responsive element is responsible for part of the transcriptional activation of the Ya subunit gene by planar aromatic compounds but has a sequence distinct from the xenobiotic-responsive element found in multiple copies in the cytochrome P-450 IA1 gene and as a single copy in the Ya subunit gene. In the present study, we demonstrate that the beta-naphthoflavone-responsive element is required for the transcriptional activation of the Ya subunit gene by phenolic antioxidants such as t-butylhydroquinone through a mechanism that does not require functional Ah receptors. Furthermore, we present evidence that planar aromatic compounds must be metabolized before they transcriptionally activate the Ya subunit gene through the beta-naphthoflavone-responsive element. The transcriptional activation of the Ya subunit gene by planar aromatic compounds requires a functional Ah receptor. These data provide evidence that transcriptional activation of the glutathione S-transferase Ya subunit gene can be mediated by a novel xenobiotic-responsive element which is directly responsive to phenolic antioxidants such as t-butylhydroquinone. Hence we have named this new xenobiotic-responsive element the antioxidant-responsive element or ARE.

Animals↗
PubMed2000

Vascular endothelial growth factor (VEGF) modulation by targeting hypoxia-inducible factor-1alpha--> hypoxia response element--> VEGF cascade differentially regulates vascular response and growth rate in tumors.

Although tumors can activate vascular endothelial growth factor (VEGF) promoter in host stromal cells, the relative contribution to VEGF production of host versus tumor cells and the resulting vascular response have not been quantitated to date. To this end, we implanted VEGF-/- and wild-type (WT) embryonic stem (ES) cells in transparent dorsal skin windows in severe combined immunodeficient mice. VEGF-/- ES cell-derived tumors produced approximately 50% of VEGF compared with the WT tumors, suggesting significant contribution of host stromal cells. To discern the hypoxia-induced hypoxia inducible factor (HIF)-1alpha --> hypoxia response element (HRE) --> VEGF signaling cascade, we also examined tumors derived from HIF-1alpha-/- and HRE-/- ES cells. As expected, the VEGF protein level in HIF-1alpha-/- ES tumors was intermediate between VEGF-/- and WT ES cell tumors. Surprisingly, HRE-/- ES tumors produced the same level of VEGF as the VEGF-/- ES tumors, suggesting a critical role of HRE in tumor cell VEGF production. Angiogenesis in these tumors was proportional to their VEGF levels (VEGF-/- approximate to HRE-/- < HIF-1alpha-/- < WT). In contrast, vascular permeability, leukocyte-endothelial adhesion, and tumor growth were reduced in VEGF-/- and HRE-/- tumors but were comparable in HIF-1a-/- and WT tumors. This discrepancy suggests that different intracellular signaling pathways may be involved in each of these functions of VEGF. More importantly, these data suggest that host cells are active players in tumor angiogenesis and growth and need to be taken into account in the design of any therapeutic strategy.

Animals↗
PubMed1998

Transactivation of the human apolipoprotein CII promoter by orphan and ligand-dependent nuclear receptors. The regulatory element CIIC is a thyroid hormone response element.

The regulatory elements CIIC (-159/-116) and CIIB (-102/-81) of the apolipoprotein CII (apoCII) promoter have distinct specificities for orphan nuclear receptors (Vorgia, P., Zannis, V. I., and Kardassis, D. (1998) J. Biol. Chem. 273, 4188-4199). In this communication we investigated the contribution of ligand-dependent and orphan nuclear receptors on the transcriptional regulation of the human apoCII gene. It was found that element CIIC in addition to ARP-1 and EAR-2 binds RXRalpha/T3Rbeta heterodimers strongly, whereas element CIIB binds hepatic nuclear factor 4 (HNF-4) exclusively. Binding is abolished by mutations that alter the HRE binding motifs. Transient cotransfection experiments showed that in the presence of T3, RXRalpha/T3Rbeta heterodimers transactivated the -205/+18 apoCII promoter 1.6- and 11-fold in HepG2 and COS-1 respectively. No transactivation was observed in the presence of 9-cis-retinoic acid. Transactivation requires the regulatory element CIIC, suggesting that this element contains a thyroid hormone response element. HNF-4 did not affect the apoCII promoter activity in HepG2 cells. However, mutations in the HNF-4 binding site on element CIIB and inhibition of HNF-4 synthesis in HepG2 cells by antisense HNF-4 constructs decreased the apoCII promoter activity to 25-40% of the control, indicating that HNF-4 is a positive regulator of the apoCII gene. ARP-1 repressed the -205/+18 but not the -104/+18 apoCII promoter activity in HepG2 cells, indicating that the repression depends on the regulatory element CIIC. In contrast, combination of ARP-1 and HNF-4 transactivated different apoCII promoter segments as well as a minimal adenovirus major late promoter driven by the regulatory element CIIB. Mutagenesis or deletion of elements CIIB or CIIC established that the observed transactivation requires DNA binding of one of the two factors and may result from HNF-4-ARP-1 interactions that elicit the transactivation functions of HNF-4. The combined data indicate that RXRalpha/T3Rbeta in the presence of T3 and HNF-4 can upregulate the apoCII promoter activity by binding to the regulatory elements CIIC and CIIB, respectively. In addition, ARP-1 can either have inhibitory or stimulatory effects on the apoCII promoter activity via different mechanisms.

Animals↗
PubMed2005

Role of basic region leucine zipper transcription factors cyclic AMP response element binding protein (CREB), CREB2, activating transcription factor 2 and CAAT/enhancer binding protein alpha in cyclic AMP response element-mediated transcription.

The transcription factor cAMP response element binding protein (CREB), a member of the basic region leucine zipper (bZIP) family of proteins, is the major cAMP response element (CRE) binding. Other bZIP proteins, including CREB2, activating transcription factor 2 (ATF2), or CAAT/enhancer binding protein (C/EBP) have been reported to transactivate CRE-containing genes or to interfere with transactivation by CREB. We have designed a simple transactivation assay using expression of either a constitutively active CREB mutant or a nuclear targeted mutant of the catalytic subunit of cAMP-dependent protein kinase. In both cases, a striking stimulation of transcription of CRE-containing reporter genes was observed in noradrenergic locus coeruleus-like CATH.a cells. In addition, a constitutively active mutant of ATF2 specifically transactivated a secretogranin II promoter/luciferase reporter gene, but had no effect on the tyrosine hydroxylase promoter. In contrast, CREB2 and C/EBPalpha did not transactivate CRE-containing reporter genes, indicating that these bZIP proteins target distinct genetic elements. Experiments involving dominant-negative bZIP mutants revealed that CREB does not heterodimerize with CREB2, ATF2, c-Jun or C/EBP. Rather, CREB and ATF2 compete for binding to the CRE, and are independently able to up-regulate transcription of genes containing CRE motifs in their regulatory regions.

Activating Transcription Factor 2↗