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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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GFP is expressed in some gata1-positive cells.Combined in situ hybridization for gata1 (purple) and immunohistochemistry for GFP (dark brown) in 24 hpf embryos injected with the 0.1 Kb promoter constructs (-0.1prep1.1:GFP and -0.1prep1.1-Et:GFP) reveals the co-localization of the two signals in the most of embryo.
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pone-0015047-g005: GFP is expressed in some gata1-positive cells.Combined in situ hybridization for gata1 (purple) and immunohistochemistry for GFP (dark brown) in 24 hpf embryos injected with the 0.1 Kb promoter constructs (-0.1prep1.1:GFP and -0.1prep1.1-Et:GFP) reveals the co-localization of the two signals in the most of embryo.

Mentions: We have confirmed the “presumptive” hematopoietic localization of the 0.1 Kb promoter-enhancer construct (-0.1prep1.1-Et:GFP) by performing a combined in situ hybridization for gata1 and immunohistochemistry with anti-GFP antibody. Figure 5 shows two examples in which GFP is expressed in the gata1 positive region.


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)

GFP is expressed in some gata1-positive cells.Combined in situ hybridization for gata1 (purple) and immunohistochemistry for GFP (dark brown) in 24 hpf embryos injected with the 0.1 Kb promoter constructs (-0.1prep1.1:GFP and -0.1prep1.1-Et:GFP) reveals the co-localization of the two signals in the most of embryo.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015047-g005: GFP is expressed in some gata1-positive cells.Combined in situ hybridization for gata1 (purple) and immunohistochemistry for GFP (dark brown) in 24 hpf embryos injected with the 0.1 Kb promoter constructs (-0.1prep1.1:GFP and -0.1prep1.1-Et:GFP) reveals the co-localization of the two signals in the most of embryo.
Mentions: We have confirmed the “presumptive” hematopoietic localization of the 0.1 Kb promoter-enhancer construct (-0.1prep1.1-Et:GFP) by performing a combined in situ hybridization for gata1 and immunohistochemistry with anti-GFP antibody. Figure 5 shows two examples in which GFP is expressed in the gata1 positive region.

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