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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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Determination of the Transcription Start Region of human and zebrafish Prep1 genes.A) DNA sequence of the region upstream to the published cDNA sequence (shown in bold). Underlined sequences are the primers employed in the primer extension analysis (Ex1-3, Rev40, Rev10 and Ex-1). The red bold sequence identifies an EGR-1 binding element. The predicted transcription start site (TSS) was identified in this experiment on the basis of the migration of the amplified primers. Below, primer extension analysis using HeLa cells RNA and the primers indicated in A. OX174 lanes show the migration of molecular weight markers (indicated on the side). B) DNA sequence of the zebrafish genomic region which includes a predicted TATA box and TSR of prep1.1 gene. Underlined sequences are the primers used for the RT-PCR assay, F0 to F8 and REV3 (see Table 1). The red bold sequence identifies an EGR-1 binding element. Below, agarose gel electrophoresis of the RT-PCR: F7 identifies the last primer able to amplify, together with REV3, the zebrafish cDNA.
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pone-0015047-g001: Determination of the Transcription Start Region of human and zebrafish Prep1 genes.A) DNA sequence of the region upstream to the published cDNA sequence (shown in bold). Underlined sequences are the primers employed in the primer extension analysis (Ex1-3, Rev40, Rev10 and Ex-1). The red bold sequence identifies an EGR-1 binding element. The predicted transcription start site (TSS) was identified in this experiment on the basis of the migration of the amplified primers. Below, primer extension analysis using HeLa cells RNA and the primers indicated in A. OX174 lanes show the migration of molecular weight markers (indicated on the side). B) DNA sequence of the zebrafish genomic region which includes a predicted TATA box and TSR of prep1.1 gene. Underlined sequences are the primers used for the RT-PCR assay, F0 to F8 and REV3 (see Table 1). The red bold sequence identifies an EGR-1 binding element. Below, agarose gel electrophoresis of the RT-PCR: F7 identifies the last primer able to amplify, together with REV3, the zebrafish cDNA.

Mentions: We first identified the transcription start site (TSS) of the human Prep1 gene using a primer extension assay (see Methods). Figure 1A (top) shows the DNA sequence upstream of the 5′-UTR region of the published cDNA (which is shown in bold). The figure also shows the oligonucleotides (underlined) which were 32P-labeled and used for primer extension (see Methods). HeLa cells total mRNA (50 µg) was primer-extended with reverse transcriptase and the material analyzed on acrylamide-urea gel electrophoresis. Figure 1A (bottom) shows that primer Ex1, localized in the 5′-UTR, amplified a band higher than 300 bp. No amplification was obtained with primer Ex1-3 indicating that the TSS was downstream of this sequence. Rev40 amplified a band of 90 bp, setting the TSS 68 bp upstream of this primer. This site corresponds to a thymidine indicated in green in Figure 1A (top) and is considered position +1. Primer Rev10, indeed, gives a band of about 140 nucleotides in good agreement with the TSS identified with Rev40. As the TSS is determined through the length of the amplified fragment comparing its migration with that of size markers, there can be a minor uncertainty (2-4 nucleotides) on the exact nucleotide from which transcription starts. However, since this uncertainty does not dramatically affect the identification of the upstream regulatory region, we have not investigated this point further. The region does not present an obvious TATA box, but is surrounded by several CpG dinucleotides (Figure 1A, top). The sequence is well conserved in the mouse, thus it is likely to be the TSS also in the mouse gene (Figure S1).


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)

Determination of the Transcription Start Region of human and zebrafish Prep1 genes.A) DNA sequence of the region upstream to the published cDNA sequence (shown in bold). Underlined sequences are the primers employed in the primer extension analysis (Ex1-3, Rev40, Rev10 and Ex-1). The red bold sequence identifies an EGR-1 binding element. The predicted transcription start site (TSS) was identified in this experiment on the basis of the migration of the amplified primers. Below, primer extension analysis using HeLa cells RNA and the primers indicated in A. OX174 lanes show the migration of molecular weight markers (indicated on the side). B) DNA sequence of the zebrafish genomic region which includes a predicted TATA box and TSR of prep1.1 gene. Underlined sequences are the primers used for the RT-PCR assay, F0 to F8 and REV3 (see Table 1). The red bold sequence identifies an EGR-1 binding element. Below, agarose gel electrophoresis of the RT-PCR: F7 identifies the last primer able to amplify, together with REV3, the zebrafish cDNA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008670&req=5

pone-0015047-g001: Determination of the Transcription Start Region of human and zebrafish Prep1 genes.A) DNA sequence of the region upstream to the published cDNA sequence (shown in bold). Underlined sequences are the primers employed in the primer extension analysis (Ex1-3, Rev40, Rev10 and Ex-1). The red bold sequence identifies an EGR-1 binding element. The predicted transcription start site (TSS) was identified in this experiment on the basis of the migration of the amplified primers. Below, primer extension analysis using HeLa cells RNA and the primers indicated in A. OX174 lanes show the migration of molecular weight markers (indicated on the side). B) DNA sequence of the zebrafish genomic region which includes a predicted TATA box and TSR of prep1.1 gene. Underlined sequences are the primers used for the RT-PCR assay, F0 to F8 and REV3 (see Table 1). The red bold sequence identifies an EGR-1 binding element. Below, agarose gel electrophoresis of the RT-PCR: F7 identifies the last primer able to amplify, together with REV3, the zebrafish cDNA.
Mentions: We first identified the transcription start site (TSS) of the human Prep1 gene using a primer extension assay (see Methods). Figure 1A (top) shows the DNA sequence upstream of the 5′-UTR region of the published cDNA (which is shown in bold). The figure also shows the oligonucleotides (underlined) which were 32P-labeled and used for primer extension (see Methods). HeLa cells total mRNA (50 µg) was primer-extended with reverse transcriptase and the material analyzed on acrylamide-urea gel electrophoresis. Figure 1A (bottom) shows that primer Ex1, localized in the 5′-UTR, amplified a band higher than 300 bp. No amplification was obtained with primer Ex1-3 indicating that the TSS was downstream of this sequence. Rev40 amplified a band of 90 bp, setting the TSS 68 bp upstream of this primer. This site corresponds to a thymidine indicated in green in Figure 1A (top) and is considered position +1. Primer Rev10, indeed, gives a band of about 140 nucleotides in good agreement with the TSS identified with Rev40. As the TSS is determined through the length of the amplified fragment comparing its migration with that of size markers, there can be a minor uncertainty (2-4 nucleotides) on the exact nucleotide from which transcription starts. However, since this uncertainty does not dramatically affect the identification of the upstream regulatory region, we have not investigated this point further. The region does not present an obvious TATA box, but is surrounded by several CpG dinucleotides (Figure 1A, top). The sequence is well conserved in the mouse, thus it is likely to be the TSS also in the mouse gene (Figure S1).

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