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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 the homology between the 5′ and intronic regulatory regions of the zebrafish prep1.1 and human Prep1.The middle part of the Figure shows a scheme of the 5′ region of human Prep1 gene, including the first two exons (red boxes) and the first intron. The positions of the transcription start site (TSS) and of the translation start sequence (ATG) are indicated. Moreover, the promoter fragments used for the analysis of the Prep1 promoter, measuring 0.1, 0.7, 1.8 and 5.0 Kb, are reported (blue lines). In the bottom left corner there is a schematic representation of the homology in the first 5 kb between the zebrafish and human region, performed blasting 500 bp segments of the zebrafish prep1.1 regulatory region agaibnst the whole human 5 Kb. The red numbers indicate the percentage of identity, the black numbers show the position of the 500 bp zebrafish promoter segments. The bottom right corner shows the sequence comparison between the first (untranslated) exons (red) and the first 100 bp of the first intron (black) of human and zebrafish Prep1 gene. Prep1/Meis-Pbx and Pbx-Hox binding sites are indicated.
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pone-0015047-g002: Analysis of the homology between the 5′ and intronic regulatory regions of the zebrafish prep1.1 and human Prep1.The middle part of the Figure shows a scheme of the 5′ region of human Prep1 gene, including the first two exons (red boxes) and the first intron. The positions of the transcription start site (TSS) and of the translation start sequence (ATG) are indicated. Moreover, the promoter fragments used for the analysis of the Prep1 promoter, measuring 0.1, 0.7, 1.8 and 5.0 Kb, are reported (blue lines). In the bottom left corner there is a schematic representation of the homology in the first 5 kb between the zebrafish and human region, performed blasting 500 bp segments of the zebrafish prep1.1 regulatory region agaibnst the whole human 5 Kb. The red numbers indicate the percentage of identity, the black numbers show the position of the 500 bp zebrafish promoter segments. The bottom right corner shows the sequence comparison between the first (untranslated) exons (red) and the first 100 bp of the first intron (black) of human and zebrafish Prep1 gene. Prep1/Meis-Pbx and Pbx-Hox binding sites are indicated.

Mentions: The sequence of the region around the transcription start site of human Prep1 is conserved in chimpanzee, mouse, fugu Prep1 and zebrafish prep1.1 genes. Figure S2 shows that this region is conserved both in the 3′ direction to include most or part of intron 1, and in the 5′ direction. The region of homology to the human sequence is much more extended in chimpanzee (where it includes almost the entire intron 1) than in mouse and even less in zebrafish. The details of the human and zebrafish DNA sequence are reported in Figure 2 (bottom right section).


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 the homology between the 5′ and intronic regulatory regions of the zebrafish prep1.1 and human Prep1.The middle part of the Figure shows a scheme of the 5′ region of human Prep1 gene, including the first two exons (red boxes) and the first intron. The positions of the transcription start site (TSS) and of the translation start sequence (ATG) are indicated. Moreover, the promoter fragments used for the analysis of the Prep1 promoter, measuring 0.1, 0.7, 1.8 and 5.0 Kb, are reported (blue lines). In the bottom left corner there is a schematic representation of the homology in the first 5 kb between the zebrafish and human region, performed blasting 500 bp segments of the zebrafish prep1.1 regulatory region agaibnst the whole human 5 Kb. The red numbers indicate the percentage of identity, the black numbers show the position of the 500 bp zebrafish promoter segments. The bottom right corner shows the sequence comparison between the first (untranslated) exons (red) and the first 100 bp of the first intron (black) of human and zebrafish Prep1 gene. Prep1/Meis-Pbx and Pbx-Hox binding sites are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015047-g002: Analysis of the homology between the 5′ and intronic regulatory regions of the zebrafish prep1.1 and human Prep1.The middle part of the Figure shows a scheme of the 5′ region of human Prep1 gene, including the first two exons (red boxes) and the first intron. The positions of the transcription start site (TSS) and of the translation start sequence (ATG) are indicated. Moreover, the promoter fragments used for the analysis of the Prep1 promoter, measuring 0.1, 0.7, 1.8 and 5.0 Kb, are reported (blue lines). In the bottom left corner there is a schematic representation of the homology in the first 5 kb between the zebrafish and human region, performed blasting 500 bp segments of the zebrafish prep1.1 regulatory region agaibnst the whole human 5 Kb. The red numbers indicate the percentage of identity, the black numbers show the position of the 500 bp zebrafish promoter segments. The bottom right corner shows the sequence comparison between the first (untranslated) exons (red) and the first 100 bp of the first intron (black) of human and zebrafish Prep1 gene. Prep1/Meis-Pbx and Pbx-Hox binding sites are indicated.
Mentions: The sequence of the region around the transcription start site of human Prep1 is conserved in chimpanzee, mouse, fugu Prep1 and zebrafish prep1.1 genes. Figure S2 shows that this region is conserved both in the 3′ direction to include most or part of intron 1, and in the 5′ direction. The region of homology to the human sequence is much more extended in chimpanzee (where it includes almost the entire intron 1) than in mouse and even less in zebrafish. The details of the human and zebrafish DNA sequence are reported in Figure 2 (bottom right section).

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