title: Characterization of the regulatory region of the zebrafish Prep1.1 gene: analogies to the promoter of the human PREP1.
abstract: Prep1 is a developmentally essential TALE class homeodomain transcription factor. In zebrafish and mouse, Prep1 is already ubiquitously expressed at the earliest stages of development, with important tissue-specific peculiarities. The Prep1 gene in mouse is developmentally essential and has haploinsufficient tumor suppressor activity [1]. We have determined the human Prep1 transcription start site (TSS) by primer extension analysis and identified, within 20 bp, the transcription start region (TSR) of the zebrafish Prep1.1 promoter. The functions of the zebrafish 5' upstream sequences were analyzed both by transient transfections in Hela Cells and by injection in zebrafish embryos. This analysis revealed a complex promoter with regulatory sequences extending up to -1.8, possibly -5.0 Kb, responsible for tissue specific expression. Moreover, the first intron contains a conserved tissue-specific enhancer both in zebrafish and in human cells. Finally, a two nucleotides mutation of an EGR-1 site, conserved in all species including human and zebrafish and located at a short distance from the TSS, destroyed the promoter activity of the -5.0 Kb promoter. A transgenic fish expressing GFP under the -1.8 Kb zebrafish promoter/enhancer co-expressed GFP and endogenous Prep1.1 during embryonic development. In the adult fish, GFP was expressed in hematopoietic regions like the kidney, in agreement with the essential function of Prep1 in mouse hematopoiesis. Sequence comparison showed conservation from man to fish of the sequences around the TSS, within the first intron enhancer. Moreover, about 40% of the sequences spread throughout the 5 Kbof the zebrafish promoter are concentrated in the -3 to -5 Kb of the human upstream region.
authors.affiliation: IFOM-FIRC Institute of Molecular Oncology Foundation, Milan, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Bernardi
authors.forename: Gianluca
authors.initials: G
authors.lastname: Deflorian
authors.forename: Federica
authors.initials: F
authors.lastname: Pezzimenti
authors.forename: Federica
authors.initials: F
authors.lastname: Pezzinenti
authors.forename: Victor M
authors.initials: VM
authors.lastname: Diaz
authors.forename: Marina
authors.initials: M
authors.lastname: Mione
authors.forename: Francesco
authors.initials: F
authors.lastname: Blasi
mesh_terms: D000818:Animals; D001665:Binding Sites; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D051379:Mice; D008957:Models, Genetic; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*; D024363:Transcription Initiation Site; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics*
chemical_list: D018398:Homeodomain Proteins; D009711:Nucleotides; C106682:PKNOX1 protein, human; D014157:Transcription Factors; D029961:Zebrafish Proteins; C559064:pknox1.1 protein, zebrafish; D049452:Green Fluorescent Proteins