An Antidiabetic Thiazolidinedione Is a High Affinity Ligand for Peroxisome Proliferator-activated Receptor γ (PPARγ)

Gene regulation by steroid hormones.

The transcription factor CREB -- for 'cyclic AMP response element-binding protein' -- functions in glucose homeostasis, growth-factor-dependent cell survival, and has been implicated in learning and memory. CREB is phosphorylated in response to various signals, but how is specificity achieved in these signalling pathways?

Transcriptional regulation by the phosphorylation-dependent factor CREB

Tumor necrosis factor α (TNFα) is a potent immunomodulator and proinflammatory cytokine that has been implicated in the pathogenesis of autoimmune and infectious diseases. For example, plasma levels of TNFα are positively correlated with severity and mortality in malaria and leishmaniasis. We have previously described a polymorphism at −308 in the TNFα promoter and shown that the rare allele, TNF2, lies on the extended haplotype HLA-A1-B8-DR3-DQ2, which is associated with autoimmunity and high TNFα production. Homozygosity for TNF2 carries a sevenfold increased risk of death from cerebral malaria. Here we demonstrate, with reporter genes under the control of the two allelic TNF promoters, that TNF2 is a much stronger transcriptional activator than the common allele (TNF1) in a human B cell line. Footprint analysis using DNase I and B cell nuclear extract showed the generation of a hypersensitive site at −308 and an adjacent area of protection. There was no difference in affinity of the DNA-binding protein(s) between the two alleles. These results show that this polymorphism has direct effects on TNFα gene regulation and may be responsible for the association of TNF2 with high TNFα phenotype and more severe disease in infections such as malaria and leishmaniasis.

https://www.pnas.org/content/pnas/94/7/3195.full.pdf

Effects of a polymorphism in the human tumor necrosis factor α promoter on transcriptional activation

Extracellular stimuli elicit changes in gene expression in target cells by activating intracellular protein kinase cascades that phosphorylate transcription factors within the nucleus. One of the best characterized stimulus-induced transcription factors, cyclic AMP response element (CRE)-binding protein (CREB), activates transcription of target genes in response to a diverse array of stimuli, including peptide hormones, growth factors, and neuronal activity, that activate a variety of protein kinases including protein kinase A (PKA), pp90 ribosomal S6 kinase (pp90RSK), and Ca2+/calmodulin-dependent protein kinases (CaMKs)[corrected]. These kinases all phosphorylate CREB at a particular residue, serine 133 (Ser133), and phosphorylation of Ser133 is required for CREB-mediated transcription. Despite this common feature, the mechanism by which CREB activates transcription varies depending on the stimulus. In some cases, signaling pathways target additional sites on CREB or proteins associated with CREB, permitting CREB to regulate distinct programs of gene expression under different conditions of stimulation. This review discusses the molecular mechanisms by which Ser133-phosphorylated CREB activates transcription, intracellular signaling pathways that lead to phosphorylation of CREB at Ser133, and features of each signaling pathway that impart specificity at the level of CREB activation.

CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals.

The coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene is wide1y used as an indicator gene in gene transfer experiments dealing with regulation of transcription in eukaryotes. Chimaeric CAT fusion genes are especially useful because no endogenous CAT activity is present in eukaryotic ce11s and because CAT enzyme activity can be monitored by a rapid and sensitive assay (1). In order to simplify the construction of hybrid CAT genes, we have constructed the plasmids pBLCA T2 and pBLCAT3. The coding region of the CA T gene as well as the small t intron and polyadenylation signals from SV40 were inserted into the polylinker region of the high copy number plasmid pUC18 (2). Unique BglII and XhoI restrietion sites were introduced upstream of the CAT coding region by insertion of synthetic linkers. A BamHI site at the 3' end of the transcription unit was converted into adam methylation sensitive ClaI site by partial digestion with BamHI, filling in and re -ligation. In the promoterless construction pBLCAT3 eight unique restriction sites are suitable for insertion of different eukaryotic promoters at the 5' end of the CAT gene. Four additional unique restriction sites rnake the insertion of regulatory signals 3' of the CAT gene possible and enable the excision of the intact fusion gene from the prokaryotic vector. The presence of the Herpes simplex virus tk promoter in pBLCAT2 permits the analysis of the effects of putative regulatory elements on a heterologous eukaryotic promoter. A BamHIIBgllI fragment from the HSV tk linker scanning mutant LS 115/ 105 (3) spanning the promoter from 105 to + 51 was inserted into the corresponding restriction sites of pBLCAT3 thereby generating pBLCAT2. The modified polylinker regions at the 5' and the 3' ends have been sequenced and compiled sequences for both plasmids are available on request.

CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements

Cyclic AMP treatment of hepatoma cells leads to increased protein binding at the cyclic AMP response element (CRE) of the tyrosine aminotransferase (TAT) gene in vivo, as revealed by genomic footprinting, whereas no increase is observed at the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Several criteria establish that the 43 kDa CREB protein is interacting with both of these sites. Two classes of CRE with different affinity for CREB are described. One class, including the TATCRE, is characterized by asymmetric and weak binding sites (CGTCA), whereas the second class containing symmetrical TGACGTCA sites shows a much higher binding affinity for CREB. Both classes show an increase in binding after phosphorylation of CREB by protein kinase A (PKA). An in vivo phosphorylation-dependent change in binding of CREB increases the occupancy of weak binding sites used for transactivation, such as the TATCRE, while high affinity sites may have constitutive binding of transcriptionally active and inactive CREB dimers, as demonstrated by in vivo footprinting at the PEPCK CRE. Thus, lower basal level and higher relative stimulation of transcription by cyclic AMP through low affinity CREs should result, allowing finely tuned control of gene activation.

https://epub.ub.uni-muenchen.de/3200/1/014.pdf

Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription.

Transcriptional activation of gene expression by glucocorticoid hormones is mediated by the interaction of hormone-receptor complexes with specific DNA sequences called glucocorticoid responsive elements (GREs) (refs 1-3, see ref. 4 for review). Deletion of this sequence abolishes glucocorticoid induction of transcription. According to a current model, activation of the cytoplasmic receptor protein by hormone binding leads to its increased affinity for and translocation to the nucleus. However, recent reports that the oestradiol and progesterone receptors are localized in the nucleus in the absence of steroid led us to examine whether the free receptor interacts in vivo with its DNA binding site in the absence of hormone binding. We used the genomic footprinting technique to show that changes in in vivo protein-DNA interactions within the GREs of the tyrosine aminotransferase gene (TAT) can be detected only after hormone treatment in hepatoma cells. Such changes are not detected in fibroblast cells, in which the TAT gene is not expressed. Many of the changes in dimethylsulphate reactivity observed in the living cell are also found in vitro using cloned DNA and a partially purified glucocorticoid receptor.

/pdf/in-vivo-protein-dna-interactions-in-a-glucocorticoid-2qak67qpi2.pdf

In vivo protein-DNA interactions in a glucocorticoid response element require the presence of the hormone

Reporter constructs with low background activity utilizing the cat gene.

We have characterized cDNA clones representing mouse CREB (cyclic AMP responsive element binding protein) mRNA isoforms. These include CREB delta and CREB alpha, of which the rat and human homologues have been previously identified. Both encode proteins with CRE-binding activity and identical transactivation potential. The additional CREB mRNA isoforms potentially encode CREB related proteins. From the structural organization of the mouse CREB gene we conclude that the multiple transcripts are generated by alternative splicing. Furthermore we show that specific CREB mRNA isoforms are expressed at a high level in the adult testis. Expression of these isoforms is induced after commencement of spermatogenesis. In situ hybridization suggests that this expression occurs predominantly in the primary spermatocytes. Comparison of the CREB gene with the recently isolated CREM (cAMP responsive element modulator) cDNAs illustrates that the two genes have arisen by gene duplication and have diverged to encode transcriptional activators and repressors of the cAMP signal transduction pathway.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1992.tb05195.x

Multiple mRNA isoforms of the transcription activator protein CREB: generation by alternative splicing and specific expression in primary spermatocytes.

Günther Schütz

Papers

CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements

Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription.

In vivo protein-DNA interactions in a glucocorticoid response element require the presence of the hormone

Reporter constructs with low background activity utilizing the cat gene.

Multiple mRNA isoforms of the transcription activator protein CREB: generation by alternative splicing and specific expression in primary spermatocytes.