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Characterization of the regulatory region of the zebrafish Prep1.1 gene: analogies to the promoter of the human PREP1.

Bernardi E, Deflorian G, Pezzimenti F, Pezzinenti F, Diaz VM, Mione M, Blasi F - PLoS ONE (2010)

Bottom Line: A transgenic fish expressing GFP under the -1.8 Kb zebrafish promoter/enhancer co-expressed GFP and endogenous Prep1.1 during embryonic development.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.

View Article: PubMed Central - PubMed

Affiliation: IFOM-FIRC Institute of Molecular Oncology Foundation, Milan, Italy.

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.

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Analysis of prep1.1 and Prep1 promoter regions activity in HeLa cells.The graphic data are expressed as relative Luciferase activity (normalized againstb the co-transfected beta-galactosidase), defined as % of luciferase activity. The value of 100% is given to the positive control (SV40 promoter + enhancer). A) Activity of the constructs of zebrafish prep1.1 5′ regulatory regions. B) Activity of the constructs of human Prep1 5′ regulatory regions. zE, zebrafish enhancer element (see text).
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pone-0015047-g003: Analysis of prep1.1 and Prep1 promoter regions activity in HeLa cells.The graphic data are expressed as relative Luciferase activity (normalized againstb the co-transfected beta-galactosidase), defined as % of luciferase activity. The value of 100% is given to the positive control (SV40 promoter + enhancer). A) Activity of the constructs of zebrafish prep1.1 5′ regulatory regions. B) Activity of the constructs of human Prep1 5′ regulatory regions. zE, zebrafish enhancer element (see text).

Mentions: In order to characterize the human and zebrafish promoters, we prepared four kinds of constructs containing 5.0, 1.8, 0.7 and 0.1 Kb upstream of the TSR from both the zebrafish prep1.1 and the human PREP1 gene cloned upstream of a luciferase reporter in the pbasic vector. Figure 2, upper left section, shows schematically the cloned fragments. These constructs were transiently transfected in HeLa cells and their luciferase activity measured (see Methods). Moreover, we cloned in the same vectors also the zebrafish intronic sequence (563 bp, zE) that contains the Pbx-Hox and the Prep1(Meis1)-Pbx binding sites (right bottom section of Figure 2 and Table 2). As controls, we used the promoter-less pbasic luciferase vector, the SV40 promoter and the full SV40 promoter-enhancer (the activity of which was set to 100% in all experiments). The luciferase activity of the zebrafish promoter (resulting from the average of three independent experiments, each performed in triplicate) are presented in Figure 3A. The construct containing the 100 bp upstream of the arbitrary zebrafish TSS (z100 bp) has a very low activity, about 15% of the enhancerless SV40 promoter, but is activated about 10-fold when associated to the zebrafish (zE) enhancer. Moreover, it is activated, although weakly, also by the SV40 enhancer. These data indicate that the 100 bp fragment containing the prep1.1 TSR is, in fact, the basal promoter region. The -700 construct (z700 bp) also showed low promoter activity (about 20% of SV40), was slightly activated by the zebrafish enhancer, and highly by the SV40 enhancer. The −1800 bp construct (z1800 bp) has a stronger basal activity that increases 2-fold in the presence of the SV40 and about 60% in the presence of the zebrafish enhancer. Finally, the −5 Kb construct (z5000 bp) has a basal level which is 30% of SV40 and is activated 4-fold by the zebrafish and SV40 enhancer. These data therefore confirm the functionality of the zebrafish TSS region and indicate that the zE element has enhancer activity.


Characterization of the regulatory region of the zebrafish Prep1.1 gene: analogies to the promoter of the human PREP1.

Bernardi E, Deflorian G, Pezzimenti F, Pezzinenti F, Diaz VM, Mione M, Blasi F - PLoS ONE (2010)

Analysis of prep1.1 and Prep1 promoter regions activity in HeLa cells.The graphic data are expressed as relative Luciferase activity (normalized againstb the co-transfected beta-galactosidase), defined as % of luciferase activity. The value of 100% is given to the positive control (SV40 promoter + enhancer). A) Activity of the constructs of zebrafish prep1.1 5′ regulatory regions. B) Activity of the constructs of human Prep1 5′ regulatory regions. zE, zebrafish enhancer element (see text).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008670&req=5

pone-0015047-g003: Analysis of prep1.1 and Prep1 promoter regions activity in HeLa cells.The graphic data are expressed as relative Luciferase activity (normalized againstb the co-transfected beta-galactosidase), defined as % of luciferase activity. The value of 100% is given to the positive control (SV40 promoter + enhancer). A) Activity of the constructs of zebrafish prep1.1 5′ regulatory regions. B) Activity of the constructs of human Prep1 5′ regulatory regions. zE, zebrafish enhancer element (see text).
Mentions: In order to characterize the human and zebrafish promoters, we prepared four kinds of constructs containing 5.0, 1.8, 0.7 and 0.1 Kb upstream of the TSR from both the zebrafish prep1.1 and the human PREP1 gene cloned upstream of a luciferase reporter in the pbasic vector. Figure 2, upper left section, shows schematically the cloned fragments. These constructs were transiently transfected in HeLa cells and their luciferase activity measured (see Methods). Moreover, we cloned in the same vectors also the zebrafish intronic sequence (563 bp, zE) that contains the Pbx-Hox and the Prep1(Meis1)-Pbx binding sites (right bottom section of Figure 2 and Table 2). As controls, we used the promoter-less pbasic luciferase vector, the SV40 promoter and the full SV40 promoter-enhancer (the activity of which was set to 100% in all experiments). The luciferase activity of the zebrafish promoter (resulting from the average of three independent experiments, each performed in triplicate) are presented in Figure 3A. The construct containing the 100 bp upstream of the arbitrary zebrafish TSS (z100 bp) has a very low activity, about 15% of the enhancerless SV40 promoter, but is activated about 10-fold when associated to the zebrafish (zE) enhancer. Moreover, it is activated, although weakly, also by the SV40 enhancer. These data indicate that the 100 bp fragment containing the prep1.1 TSR is, in fact, the basal promoter region. The -700 construct (z700 bp) also showed low promoter activity (about 20% of SV40), was slightly activated by the zebrafish enhancer, and highly by the SV40 enhancer. The −1800 bp construct (z1800 bp) has a stronger basal activity that increases 2-fold in the presence of the SV40 and about 60% in the presence of the zebrafish enhancer. Finally, the −5 Kb construct (z5000 bp) has a basal level which is 30% of SV40 and is activated 4-fold by the zebrafish and SV40 enhancer. These data therefore confirm the functionality of the zebrafish TSS region and indicate that the zE element has enhancer activity.

Bottom Line: A transgenic fish expressing GFP under the -1.8 Kb zebrafish promoter/enhancer co-expressed GFP and endogenous Prep1.1 during embryonic development.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.

View Article: PubMed Central - PubMed

Affiliation: IFOM-FIRC Institute of Molecular Oncology Foundation, Milan, Italy.

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.

Show MeSH
Related in: MedlinePlus