Characterization of transcriptional regulatory elements in the promoter region of the murine blood coagulation factor VII gene

Daniel R. Stauffer, Beatrice N. Chukwumezie, Julie A. Wilberding, Elliot Rosen, Francis J. Castellino

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

To identify the 5' sequences of the murine coagulation factor VII (fVII) gene that resulted in its efficient transcription, a variety of 5'-flanking sequences up to 7 kilobase pairs upstream of the translation ATG initiation codon were fused to the reporter gene, bacterial chloramphenicol acetyltransferase, and relative expression levels of this gene in mouse Helm 1-6 cells were determined. It was found that the 5' region extending approximately 85 base pairs (bp) upstream of the transcriptional initiation site served as the minimal DNA region that provided full relative promotor activity for chloramphenicol acetyltransferase expression. This region of the gene also contains consensus sequences for liver-enriched transcription factors, C/EBPβ and HNF4, as well as for the ubiquitous protein factors, AP1, H4TF1, NF1, and Sp1. In vitro DNase I footprinting of the 200-bp proximal region of the promoter with a murine Hepa 1-6 cell nuclear extract revealed a clear footprint of a region corresponding to -80 to -28 bp of the murine fVII gene, suggesting that liver factors interact with this region of the DNA. Competitive gel shift and supershift assays with different synthetic oligonucleotide probes demonstrate that proteins contained in the nuclear extract, identified as C/EBPβ, H4TF1, and HNF4, bind to a region of the murine fVII DNA from 85 to 32 bp upstream of the transcription start site. Purified Sp1 also interacts with this region of the DNA at a site that substantially overlaps, but is not identical to, the H4TF1 binding locus. Binding of Sp1 to the mouse DNA was not observed with the nuclear extract as the source of the transcription factors, suggesting that Sp1 is likely displaced from its binding site by H4TF1 in the crude extract. In vivo dimethyl sulfate footprint analysis confirmed the existence of these sites and additionally revealed two other binding regions slightly upstream of the CCAAT/enhancer-binding protein (C/EBP) binding locus that are homologous to NF1 binding sequences. The data demonstrate that appropriate transcription factor binding sites exist in the proximal promoter region of the murine fVII gene that are consistent with its strong liver-based expression in a highly regulated manner.

Original languageEnglish (US)
Pages (from-to)2277-2287
Number of pages11
JournalJournal of Biological Chemistry
Volume273
Issue number4
DOIs
StatePublished - Jan 23 1998
Externally publishedYes

Fingerprint

Transcriptional Regulatory Elements
Factor VII
Genetic Promoter Regions
CCAAT-Enhancer-Binding Proteins
Base Pairing
Genes
DNA
Chloramphenicol O-Acetyltransferase
Liver
Transcription Factors
Binding Sites
Sp1 Transcription Factor
Initiator Codon
Oligonucleotide Probes
5' Flanking Region
Transcription Initiation Site
Deoxyribonuclease I
Consensus Sequence
Cell Extracts
Complex Mixtures

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of transcriptional regulatory elements in the promoter region of the murine blood coagulation factor VII gene. / Stauffer, Daniel R.; Chukwumezie, Beatrice N.; Wilberding, Julie A.; Rosen, Elliot; Castellino, Francis J.

In: Journal of Biological Chemistry, Vol. 273, No. 4, 23.01.1998, p. 2277-2287.

Research output: Contribution to journalArticle

Stauffer, Daniel R. ; Chukwumezie, Beatrice N. ; Wilberding, Julie A. ; Rosen, Elliot ; Castellino, Francis J. / Characterization of transcriptional regulatory elements in the promoter region of the murine blood coagulation factor VII gene. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 4. pp. 2277-2287.
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abstract = "To identify the 5' sequences of the murine coagulation factor VII (fVII) gene that resulted in its efficient transcription, a variety of 5'-flanking sequences up to 7 kilobase pairs upstream of the translation ATG initiation codon were fused to the reporter gene, bacterial chloramphenicol acetyltransferase, and relative expression levels of this gene in mouse Helm 1-6 cells were determined. It was found that the 5' region extending approximately 85 base pairs (bp) upstream of the transcriptional initiation site served as the minimal DNA region that provided full relative promotor activity for chloramphenicol acetyltransferase expression. This region of the gene also contains consensus sequences for liver-enriched transcription factors, C/EBPβ and HNF4, as well as for the ubiquitous protein factors, AP1, H4TF1, NF1, and Sp1. In vitro DNase I footprinting of the 200-bp proximal region of the promoter with a murine Hepa 1-6 cell nuclear extract revealed a clear footprint of a region corresponding to -80 to -28 bp of the murine fVII gene, suggesting that liver factors interact with this region of the DNA. Competitive gel shift and supershift assays with different synthetic oligonucleotide probes demonstrate that proteins contained in the nuclear extract, identified as C/EBPβ, H4TF1, and HNF4, bind to a region of the murine fVII DNA from 85 to 32 bp upstream of the transcription start site. Purified Sp1 also interacts with this region of the DNA at a site that substantially overlaps, but is not identical to, the H4TF1 binding locus. Binding of Sp1 to the mouse DNA was not observed with the nuclear extract as the source of the transcription factors, suggesting that Sp1 is likely displaced from its binding site by H4TF1 in the crude extract. In vivo dimethyl sulfate footprint analysis confirmed the existence of these sites and additionally revealed two other binding regions slightly upstream of the CCAAT/enhancer-binding protein (C/EBP) binding locus that are homologous to NF1 binding sequences. The data demonstrate that appropriate transcription factor binding sites exist in the proximal promoter region of the murine fVII gene that are consistent with its strong liver-based expression in a highly regulated manner.",
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AU - Castellino, Francis J.

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N2 - To identify the 5' sequences of the murine coagulation factor VII (fVII) gene that resulted in its efficient transcription, a variety of 5'-flanking sequences up to 7 kilobase pairs upstream of the translation ATG initiation codon were fused to the reporter gene, bacterial chloramphenicol acetyltransferase, and relative expression levels of this gene in mouse Helm 1-6 cells were determined. It was found that the 5' region extending approximately 85 base pairs (bp) upstream of the transcriptional initiation site served as the minimal DNA region that provided full relative promotor activity for chloramphenicol acetyltransferase expression. This region of the gene also contains consensus sequences for liver-enriched transcription factors, C/EBPβ and HNF4, as well as for the ubiquitous protein factors, AP1, H4TF1, NF1, and Sp1. In vitro DNase I footprinting of the 200-bp proximal region of the promoter with a murine Hepa 1-6 cell nuclear extract revealed a clear footprint of a region corresponding to -80 to -28 bp of the murine fVII gene, suggesting that liver factors interact with this region of the DNA. Competitive gel shift and supershift assays with different synthetic oligonucleotide probes demonstrate that proteins contained in the nuclear extract, identified as C/EBPβ, H4TF1, and HNF4, bind to a region of the murine fVII DNA from 85 to 32 bp upstream of the transcription start site. Purified Sp1 also interacts with this region of the DNA at a site that substantially overlaps, but is not identical to, the H4TF1 binding locus. Binding of Sp1 to the mouse DNA was not observed with the nuclear extract as the source of the transcription factors, suggesting that Sp1 is likely displaced from its binding site by H4TF1 in the crude extract. In vivo dimethyl sulfate footprint analysis confirmed the existence of these sites and additionally revealed two other binding regions slightly upstream of the CCAAT/enhancer-binding protein (C/EBP) binding locus that are homologous to NF1 binding sequences. The data demonstrate that appropriate transcription factor binding sites exist in the proximal promoter region of the murine fVII gene that are consistent with its strong liver-based expression in a highly regulated manner.

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