Role of basic region leucine zipper transcription factors in controlling gene transcription in mammalian cells

The basic region leucine zipper (bZIP) transcription factors are proteins that bind as dimers to specifc sequences within promoter genes to activate or repress the transcription of these genes. The best known member of the bZIP family is cAMP response element (CRE) binding protein CREB that binds to the CRE 5`-TGACGTCA-3`. To investigate gene regulation by bZIP transcription factors constutively active and dominant-negative bZIP mutants were used. The results show that genes encoding GTP cyclohydrolase I, secretogranin II, tumor necrosis factor alpha and Egr-1 are regulated by CREB and ATF2 via CRE motifs or related sequences. CREB and ATF2 do not heterodimerize but they rather compte for DNA-binding. In contrast, the bZIP proteins CREB2/ATF4, ATF5 and C/EBP heterodimerize with each other but not with CREB or ATF2. CREB2/ATF4, ATF5 and C/EBP transactivate the asparagine synthetase gene but has no or marginal effect on CRE-controlled genes. Furthermore, CREB2/ATF4, ATF5 and C/EBP mediate the activation of the asparagine synthetase gene transcription induced by amino acid deprivation. In a second series of experiments the regulation of the inducible nitric oxide synthase (iNOS) has been investigated as a result of Toll-like receptor stimulation. The results show that stress-activated protein kinases and the bZIP proteins ATF2 and/or c-Jun are involved in the upregulation of iNOS gene following stimulation. Finally the regulation of the c-Jun and c-Fos encoding genes has been investiagted in pituitary cells. Stimulation of the gonadotropin-releasing hormone receptor and the muscarinic acetylcholine receptor type III stimulated c-Jun and c-Fos expression indicating that bZIP transcription factor synthesis is controlled by extracellular signals.Basic Region Leucin Zipper (bZIP) Transkriptionfaktoren sind Proteine, die an spezifische Sequenzen im Promotor binden und dadurch die Transkription dieser Gene entweder aktivieren oder hemmen. Das bekannteste Mitglied der bZIP-Familie is das "cAMP responsive element" (CRE)-bindende Protein CREB, welches an die CRE-Consensussequenz bindet. Um die Gen-Regulation durch bZIP-Transkriptionfaktoren zu untersuchen, wurden konstutiv-aktive und dominant-negative-mutanten verwendet. Die Ergebnisse zeigen, dass die GTP cyclohydrolase I, Sekretogranin II, Tumornekrosefaktor alpha und Egr-1 kodierende Gene durch CREB und ATF2 via CRE-Bindungsmotive oder ähnliche Sequenzen reguliert werden. CREB und ATF2 bilden keine Heterodimere, sonderen konkurrieren um dieselbe DNA-Bindungsstelle. Im Gengensatz dazu bilden die bZIP-Proteine CREB2/ATF4, ATF5 und C/EBP zwar untereinander Heteodimere, jedoch dimersieren sie nicht mit CREB oder ATF2. CREB2/ATF4, ATF5 und C/EBP haben keinen order nur geringen Einfluss auf CRE-regulierte Gene. CREB2/ATF4, ATF5 und C/EBP transaktivieren das Asparaginsynthtasegen und regulieren dessen Transkription nach Entzug von Aminosäuren im Kulturmedium. In einer zweiten Studie wurde die Regulation der induzierbaren Stickstoffmonoxidsynthase (iNOS) untersucht. Die Transkription des iNOS-Gens wird nach Stimulation des "toll-like" Rezeptors 4 aktiviert. Die Ergebnisse zeigen, dass stressaktivierte Proteinkinasen und die bZIP Proteine ATF2 und/oder c-Jun die Aktivierung der iNOS-Gentranskription vermitteln. Schließlich wurde die Regulation von c-Jun und c-Fos kodierenden Genen in pituitary Zellen untersucht. Die Stimulation des "Gonadotropin-Releasing-Hormone Rezeptors" oder des muskarinishen-Acetylcholin-Rezeptors Typ III führte zur Aktivierung des c-Jun und des c-Fos-Gens. Dies zeigt, dass die Biosynthese der bZIP-Proteine c-Jun und c-Fos durch extrazelluläre Stimuli kontrolliert wird

cAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

BACKGROUND: The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE). RESULTS: The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP) family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2), known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. CONCLUSIONS: Using a constitutively active CREB2/CREB fusion protein and a mutant of the PKA catalytic subunit that is targeted to the nucleus, we have shown that the glucose-6-phosphatase gene has two distinct genetic elements that function as bona fide CRE. This study further shows that the expression vectors encoding C2/CREB and catalytic subunit of PKA are valuable tools for the study of CREB-mediated gene transcription and the biological functions of CREB

Die Rolle der "Basic Region Leucin Zipper Transkriptionfaktoren" in der Kontrolle der Genexpression in Säugertierzellen

The basic region leucine zipper (bZIP) transcription factors are proteins that bind as dimers to specifc sequences within promoter genes to activate or repress the transcription of these genes. The best known member of the bZIP family is cAMP response element (CRE) binding protein CREB that binds to the CRE 5`-TGACGTCA-3`. To investigate gene regulation by bZIP transcription factors constutively active and dominant-negative bZIP mutants were used. The results show that genes encoding GTP cyclohydrolase I, secretogranin II, tumor necrosis factor alpha and Egr-1 are regulated by CREB and ATF2 via CRE motifs or related sequences. CREB and ATF2 do not heterodimerize but they rather compte for DNA-binding. In contrast, the bZIP proteins CREB2/ATF4, ATF5 and C/EBP heterodimerize with each other but not with CREB or ATF2. CREB2/ATF4, ATF5 and C/EBP transactivate the asparagine synthetase gene but has no or marginal effect on CRE-controlled genes. Furthermore, CREB2/ATF4, ATF5 and C/EBP mediate the activation of the asparagine synthetase gene transcription induced by amino acid deprivation. In a second series of experiments the regulation of the inducible nitric oxide synthase (iNOS) has been investigated as a result of Toll-like receptor stimulation. The results show that stress-activated protein kinases and the bZIP proteins ATF2 and/or c-Jun are involved in the upregulation of iNOS gene following stimulation. Finally the regulation of the c-Jun and c-Fos encoding genes has been investiagted in pituitary cells. Stimulation of the gonadotropin-releasing hormone receptor and the muscarinic acetylcholine receptor type III stimulated c-Jun and c-Fos expression indicating that bZIP transcription factor synthesis is controlled by extracellular signals.Basic Region Leucin Zipper (bZIP) Transkriptionfaktoren sind Proteine, die an spezifische Sequenzen im Promotor binden und dadurch die Transkription dieser Gene entweder aktivieren oder hemmen. Das bekannteste Mitglied der bZIP-Familie is das "cAMP responsive element" (CRE)-bindende Protein CREB, welches an die CRE-Consensussequenz bindet. Um die Gen-Regulation durch bZIP-Transkriptionfaktoren zu untersuchen, wurden konstutiv-aktive und dominant-negative-mutanten verwendet. Die Ergebnisse zeigen, dass die GTP cyclohydrolase I, Sekretogranin II, Tumornekrosefaktor alpha und Egr-1 kodierende Gene durch CREB und ATF2 via CRE-Bindungsmotive oder ähnliche Sequenzen reguliert werden. CREB und ATF2 bilden keine Heterodimere, sonderen konkurrieren um dieselbe DNA-Bindungsstelle. Im Gengensatz dazu bilden die bZIP-Proteine CREB2/ATF4, ATF5 und C/EBP zwar untereinander Heteodimere, jedoch dimersieren sie nicht mit CREB oder ATF2. CREB2/ATF4, ATF5 und C/EBP haben keinen order nur geringen Einfluss auf CRE-regulierte Gene. CREB2/ATF4, ATF5 und C/EBP transaktivieren das Asparaginsynthtasegen und regulieren dessen Transkription nach Entzug von Aminosäuren im Kulturmedium. In einer zweiten Studie wurde die Regulation der induzierbaren Stickstoffmonoxidsynthase (iNOS) untersucht. Die Transkription des iNOS-Gens wird nach Stimulation des "toll-like" Rezeptors 4 aktiviert. Die Ergebnisse zeigen, dass stressaktivierte Proteinkinasen und die bZIP Proteine ATF2 und/oder c-Jun die Aktivierung der iNOS-Gentranskription vermitteln. Schließlich wurde die Regulation von c-Jun und c-Fos kodierenden Genen in pituitary Zellen untersucht. Die Stimulation des "Gonadotropin-Releasing-Hormone Rezeptors" oder des muskarinishen-Acetylcholin-Rezeptors Typ III führte zur Aktivierung des c-Jun und des c-Fos-Gens. Dies zeigt, dass die Biosynthese der bZIP-Proteine c-Jun und c-Fos durch extrazelluläre Stimuli kontrolliert wird

CAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene-5

<p><b>Copyright information:</b></p><p>Taken from "cAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene"</p><p>BMC Molecular Biology 2005;6():2-2.</p><p>Published online 19 Jan 2005</p><p>PMCID:PMC548273.</p><p></p>nal activation domain of CREB2 and the bZIP domain of ATF2, responsible for dimerization and DNA-binding. (B, C, D) HepG2 cells were transfected with one of the reporter plasmids pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc pG6PCRE1/CRE2mutluc (B), pG6PCRE1luc, pG6PCRE2luc (C), or pTNFα(CRE/AP1)luc (D), the pRSVβ internal reference plasmid, and either the "empty" expression vector pCMV5 or an expression vector encoding C2/ATF2 (100 ng expression plasmid/plate). Lysates were prepared forty-eight hours post-transfection and β-galactosidase and luciferase activities were measured. The mean +/- SD is depicted

CAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene-4

<p><b>Copyright information:</b></p><p>Taken from "cAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene"</p><p>BMC Molecular Biology 2005;6():2-2.</p><p>Published online 19 Jan 2005</p><p>PMCID:PMC548273.</p><p></p> (C) (1 μg/plate) was transfected into HepG2 cells together with the pRSVβ internal standard plasmid (2 μg/plate) and the "empty" expression vector pCMV5 or an expression vector encoding either CREB, CREBS133A, or K-CREB (20 ng plasmid/plate). In addition, an expression vector encoding NLSCα (100 ng/plate) was transfected. Forty-eight hours post-transfection cell extracts were prepared and the β-galactosidase and luciferase activities of these extracts were determined. The data are presented as the ratio of luciferase activity (light units) to β-galactosidase units (OD units) measured in the cell extracts. The mean +/- SD is depicted

CAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene-1

<p><b>Copyright information:</b></p><p>Taken from "cAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene"</p><p>BMC Molecular Biology 2005;6():2-2.</p><p>Published online 19 Jan 2005</p><p>PMCID:PMC548273.</p><p></p>The chimeric bZIP protein C2/CREB consists of the constitutively active transcriptional activation domain of CREB2 and the bZIP domain of CREB, responsible for dimerization and DNA-binding. (B) Western Blot analysis of HepG2 cells transfected with an expression vector encoding C2/CREB. As a control, extracts from mock transfected HepG2 cells were analyzed. Western blots were probed with an antibody against the FLAG-tag

CAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene-3

<p><b>Copyright information:</b></p><p>Taken from "cAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene"</p><p>BMC Molecular Biology 2005;6():2-2.</p><p>Published online 19 Jan 2005</p><p>PMCID:PMC548273.</p><p></p>e wild-type enzyme is myristylated as indicated (Myr). The sequence of the nuclear localization signal derived from the SV40 large T antigen and the triple FLAG epitope in NLSCα are shown. (B) Western Blot analysis of HepG2 cells transfected with an expression vector encoding NLSCα. As a control, extracts from mock transfected HepG2 cells were analyzed. Western blots were probed with an antibody against the FLAG-tag. (C) Modular structure of the GAL4-CREB fusion protein. The protein consists of the DNA-binding domain of GAL4 (amino acids 1–147) and the activation domain of CREB (amino acids 1–281). The reporter plasmid pUASluc contains a transcription unit encompassing the luciferase open reading frame and a minimal promoter that consists of five copies of the upstream activating sequence (UAS), a TATA box derived from the HIV long terminal repeat and the initiator element from the adenovirus major late promoter. The reporter plasmid pUASluc (1 μg/plate) was transfected into HepG2 cells together with the pRSVβ internal standard plasmid (2 μg/plate) and either an expression vector encoding the DNA binding domain of GAL4 (plasmid pM1) or the GAL4-CREB fusion protein (1 μg plasmid/plate). In addition, cells were transfected with an expression vector encoding either the wild-type (Cα) or mutated (NLSCα) form of cAMP-dependent protein kinase (100 ng/plate). Forty-eight hours post-transfection cell extracts were prepared and the β-galactosidase and luciferase activities of these extracts were determined

Mapping the human genetic architecture of COVID-19

The genetic make-up of an individual contributes to the susceptibility and response to viral infection. Although environmental, clinical and social factors have a role in the chance of exposure to SARS-CoV-2 and the severity of COVID-191,2, host genetics may also be important. Identifying host-specific genetic factors may reveal biological mechanisms of therapeutic relevance and clarify causal relationships of modifiable environmental risk factors for SARS-CoV-2 infection and outcomes. We formed a global network of researchers to investigate the role of human genetics in SARS-CoV-2 infection and COVID-19 severity. Here we describe the results of three genome-wide association meta-analyses that consist of up to 49,562 patients with COVID-19 from 46 studies across 19 countries. We report 13 genome-wide significant loci that are associated with SARS-CoV-2 infection or severe manifestations of COVID-19. Several of these loci correspond to previously documented associations to lung or autoimmune and inflammatory diseases3–7. They also represent potentially actionable mechanisms in response to infection. Mendelian randomization analyses support a causal role for smoking and body-mass index for severe COVID-19 although not for type II diabetes. The identification of novel host genetic factors associated with COVID-19 was made possible by the community of human genetics researchers coming together to prioritize the sharing of data, results, resources and analytical frameworks. This working model of international collaboration underscores what is possible for future genetic discoveries in emerging pandemics, or indeed for any complex human disease