Liver-enriched transcription factor HNF-4 and ubiquitous factor NF-Y are critical for expression of blood coagulation factor X.
Blood coagulation Factor X and its activated form Factor Xa play an essential role in the midphase of the clotting cascade. To delineate the mechanisms governing the liver-specific expression of Factor X, we have previously characterized the complete 2.8 kilobase pairs of the 5'-flanking region of Factor X and demonstrated that the first 209 base pairs is sufficient to confer maximal promoter activity in HepG2 cells, a hepatoma cell line that expresses Factor X. We have also shown that mutations at ACTTTG and CCAAT elements located at -56 to -51 and -120 to -116, respectively, significantly reduce the promoter activity. In this report, we demonstrate that Factor X mRNA is primarily but not exclusively expressed in the liver. Using DNase I footprinting analysis, we determine four protein binding sites within the 209-base pair fragment, designated site 1 (-73) to -44), site 2 (-128 to -94), site 3 (-165 to -132), and site 4(-195 to -169). Using gel mobility shift assays in combination with competition and supershift experiments, we demonstrate that hepatocyte nuclear factor 4 and Sp1 bind at site 1, the site which contains the ACTTTG element. Methylation interference assays reveal that HNF-4 and Sp1 contact adjacent sites with minor overlap. HNF-4 and Sp1 appear to bind site 1 in a mutually exclusive fashion. We also demonstrate that HNF-4 can transactivate the Factor X promoter in HeLa cells; mutation at the adjacent Sp1 site further increases the transactivation. Heteromeric transcription factor NF-Y was identified as the protein that binds the CCAAT box at site 2. We conclude that HNF-4 and NF-Y play crucial roles in modulating the activity of the proximal promoter of Factor X.
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Department of Pediatrics, University of Pennsylvania, Philadelphia, USA.
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D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D001667:Binding, Competitive; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D005170:Factor X / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008099:Liver / Q000502:physiology; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D005170:Factor X