title: 5'UTR of the neurogenic bHLH Nex1/MATH-2/NeuroD6 gene is regulated by two distinct promoters through CRE and C/EBP binding sites.
abstract: Expression of the bHLH transcription factor Nex1/MATH-2/NeuroD6, a member of the NeuroD subfamily, parallels overt neuronal differentiation and synaptogenesis during brain development. Our previous studies have shown that Nex1 is a critical effector of the NGF pathway and promotes neuronal differentiation and survival of PC12 cells in the absence of growth factors. In this study, we investigated the transcriptional regulation of the Nex1 gene during NGF-induced neuronal differentiation. We found that Nex1 expression is under the control of two conserved promoters, Nex1-P1 and Nex1-P2, located in two distinct non-coding exons. Both promoters are TATA-less with multiple transcription start sites, and are activated on NGF or cAMP exposure. Luciferase-reporter assays showed that the Nex1-P2 promoter activity is stronger than the Nex1-P1 promoter activity, which supports the previously reported differential expression levels of Nex1 transcripts throughout brain development. Using a combination of DNaseI footprinting, EMSA assays, and site-directed mutagenesis, we identified the essential regulatory elements within the first 2 kb of the Nex1 5'UTR. The Nex1-P1 promoter is mainly regulated by a conserved CRE element, whereas the Nex1-P2 promoter is under the control of a conserved C/EBP binding site. Overexpression of wild-type C/EBPbeta resulted in increased Nex1-P2 promoter activity in NGF-differentiated PC12 cells. The fact that Nex1 is a target gene of C/EBPbeta provides new insight into the C/EBP transcriptional cascade known to promote neurogenesis, while repressing gliogenesis.
authors.affiliation: Department of Anatomy and Cell Biology, George Washington University Medical Center, Washington, DC, USA.
authors.forename: Martine
authors.initials: M
authors.lastname: Uittenbogaard
authors.forename: Debra L
authors.initials: DL
authors.lastname: Martinka
authors.forename: Peter F
authors.initials: PF
authors.lastname: Johnson
authors.forename: Charles
authors.initials: C
authors.lastname: Vinson
authors.forename: Anne
authors.initials: A
authors.lastname: Chiaramello
mesh_terms: D020121:5' Untranslated Regions / Q000502:physiology*; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D001665:Binding Sites / Q000502:physiology; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D003001:Cloning, Molecular / Q000379:methods; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D005786:Gene Expression Regulation / Q000502:physiology; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D020932:Nerve Growth Factor / Q000494:pharmacology; D016716:PC12 Cells / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000502:physiology*; D051381:Rats; D016385:TATA Box; D015533:Transcriptional Activation / Q000502:physiology
chemical_list: D020121:5' Untranslated Regions; D051792:Basic Helix-Loop-Helix Proteins; D022782:CCAAT-Enhancer-Binding Protein-beta; D017362:Cyclic AMP Response Element-Binding Protein; D008156:Luciferases; D020932:Nerve Growth Factor