title: Melatonin synthesis in retina: cAMP-dependent transcriptional regulation of chicken arylalkylamine N-acetyltransferase by a CRE-like sequence and a TTATT repeat motif in the proximal promoter.
abstract: Arylalkylamine N-acetyltransferase (AANAT) is the key regulatory enzyme controlling the daily rhythm of melatonin biosynthesis. In chicken retinal photoreceptor cells, Aanat transcription and AANAT activity are regulated in part by cAMP-dependent mechanisms. The purpose of this study was to identify regulatory elements within the chicken Aanat promoter responsible for cAMP-dependent induction. Photoreceptor-enriched retinal cell cultures were transfected with a luciferase reporter construct containing up to 4 kb of 5'-flanking region and the first exon of Aanat. Forskolin treatment stimulated luciferase activity driven by the ∼4 kb promoter construct and by all 5'-deletion constructs except the smallest, Aanat (-217 to +120)luc. Maximal basal and forskolin-stimulated expression levels were generated by the Aanat (-484 to +120)luc construct. This construct lacks a canonical cyclic AMP-response element (CRE), but contains two other potentially important elements in its sequence: an eight times TTATT repeat (TTATT₈) and a CRE-like sequence. Electrophoretic mobility shift assays, luciferase reporter assays, chromatin immunoprecipitation, and siRNA experiments provide evidence that these elements bind c-Fos, JunD, and CREB to enhance basal and forskolin-stimulated Aanat transcription. We propose that the CRE-like sequence and TTATT₈ elements in the 484 bp proximal promoter interact to mediate cAMP-dependent transcriptional regulation of Aanat.
authors.affiliation: Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: Rashidul
authors.initials: R
authors.lastname: Haque
authors.forename: Nelson W
authors.initials: NW
authors.lastname: Chong
authors.forename: Fatima
authors.initials: F
authors.lastname: Ali
authors.forename: Shyam S
authors.initials: SS
authors.lastname: Chaurasia
authors.forename: Trisha
authors.initials: T
authors.lastname: Sengupta
authors.forename: Eugene
authors.initials: E
authors.lastname: Chun
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Howell
authors.forename: David C
authors.initials: DC
authors.lastname: Klein
authors.forename: P Michael
authors.initials: PM
authors.lastname: Iuvone
mesh_terms: D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D047068:Arylalkylamine N-Acetyltransferase / Q000096:biosynthesis* / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D047369:Chromatin Immunoprecipitation; D000242:Cyclic AMP / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D008156:Luciferases / Q000235:genetics; D008550:Melatonin / Q000096:biosynthesis* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis; D034741:RNA, Small Interfering; D012091:Repetitive Sequences, Nucleic Acid; D012160:Retina / Q000378:metabolism*; D014162:Transfection
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D034741:RNA, Small Interfering; D000242:Cyclic AMP; D008156:Luciferases; D047068:Arylalkylamine N-Acetyltransferase; D008550:Melatonin