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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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A transgenic fish expressing GFP under the 1.8 Kb zebrafish prep1.1 promoter recapitulates the expression of the endogenous gene during embryogenesis.A–F: live Tg(-1.8pknox1.1:eGFP)io009 embryos of three different developmental stages (indicated) visualized by confocal (A) or fluorescence microscopy (D, F). Embryos in B, C and E are the same of A, D and F respectively, visualized in transmission (A) and bright field (C, E). G, H, I: In situ hybridization of embryos at the same stage of development with the pknox1.1 antisense RNA-probe, shown in lateral view.
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pone-0015047-g007: A transgenic fish expressing GFP under the 1.8 Kb zebrafish prep1.1 promoter recapitulates the expression of the endogenous gene during embryogenesis.A–F: live Tg(-1.8pknox1.1:eGFP)io009 embryos of three different developmental stages (indicated) visualized by confocal (A) or fluorescence microscopy (D, F). Embryos in B, C and E are the same of A, D and F respectively, visualized in transmission (A) and bright field (C, E). G, H, I: In situ hybridization of embryos at the same stage of development with the pknox1.1 antisense RNA-probe, shown in lateral view.

Mentions: Embryos injected with the various constructs were cultured and bred in order to obtain germ-line transmission. In two of the embryos the transgene (-1.8prep1.1:GFP) integrated into the germ line but an F2 line could be derived only from one of them. In this line, we compared the expression of GFP in live embryos and that of endogenous Prep1.1 by in situ hybridization (ISH) using an antisense prep1.1 probe. The results are reported in Figure 7, that shows confocal fluorescence of 128-cells embryos (Figure 7A) and transversal transmission microscopy (Figure 7D and 7F) in 24 and 32 hpf embryos. Immunohistochemistry result is shown in Figures 7G, 7H and 7I. The two techniques largely identify the same regions in particular in the anterior areas. We conclude that the 1.8 Kb construct reproduces the expression of prep1.1 fairly faithfully during the first three days of development. In particular, it reproduces the initial ubiquitous expression of the first hours and the subsequent focusing to the anterior region [14].


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)

A transgenic fish expressing GFP under the 1.8 Kb zebrafish prep1.1 promoter recapitulates the expression of the endogenous gene during embryogenesis.A–F: live Tg(-1.8pknox1.1:eGFP)io009 embryos of three different developmental stages (indicated) visualized by confocal (A) or fluorescence microscopy (D, F). Embryos in B, C and E are the same of A, D and F respectively, visualized in transmission (A) and bright field (C, E). G, H, I: In situ hybridization of embryos at the same stage of development with the pknox1.1 antisense RNA-probe, shown in lateral view.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015047-g007: A transgenic fish expressing GFP under the 1.8 Kb zebrafish prep1.1 promoter recapitulates the expression of the endogenous gene during embryogenesis.A–F: live Tg(-1.8pknox1.1:eGFP)io009 embryos of three different developmental stages (indicated) visualized by confocal (A) or fluorescence microscopy (D, F). Embryos in B, C and E are the same of A, D and F respectively, visualized in transmission (A) and bright field (C, E). G, H, I: In situ hybridization of embryos at the same stage of development with the pknox1.1 antisense RNA-probe, shown in lateral view.
Mentions: Embryos injected with the various constructs were cultured and bred in order to obtain germ-line transmission. In two of the embryos the transgene (-1.8prep1.1:GFP) integrated into the germ line but an F2 line could be derived only from one of them. In this line, we compared the expression of GFP in live embryos and that of endogenous Prep1.1 by in situ hybridization (ISH) using an antisense prep1.1 probe. The results are reported in Figure 7, that shows confocal fluorescence of 128-cells embryos (Figure 7A) and transversal transmission microscopy (Figure 7D and 7F) in 24 and 32 hpf embryos. Immunohistochemistry result is shown in Figures 7G, 7H and 7I. The two techniques largely identify the same regions in particular in the anterior areas. We conclude that the 1.8 Kb construct reproduces the expression of prep1.1 fairly faithfully during the first three days of development. In particular, it reproduces the initial ubiquitous expression of the first hours and the subsequent focusing to the anterior region [14].

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