Transcriptional regulation of the gene coding for human protein C.
The promoter for the gene coding for human protein C has been characterized as to nucleotide sequences that regulate the synthesis of mRNA. The major transcription start site was found 65 nucleotides upstream from the first intron/exon boundary along with two minor sites. Functional characterization of 1528 base pairs at the 5'-end of the gene was then carried out by chloramphenicol acetyltransferase reporter assays, protection from DNase I digestion, and electrophoretic mobility shift assays employing HepG2 and HeLa cells. One of the upstream regions (nucleotides -25 to +9) contained binding sites for at least two different transcription factors, including a hepatic nuclear factor 1-binding site (-10 to +9) and two overlapping and oppositely oriented hepatic nuclear factor 3-binding sites (-25 to -11). A second major region (PCE1) (+12 to +30) appeared to be a unique, liver-specific regulatory sequence. An Sp1-binding site in exon I (+58 to +65) was also recognized by cotransfection experiments with an Sp1 expression plasmid. Specific mutations in these promoter elements reduced transcriptional activity and abolished the binding of hepatic nuclear proteins. Finally, a strong silencer element (PCS1) (between -162 and -82) and two possible liver-specific enhancer regions (PCE2 and PCE3), which interact coordinately with the promoter elements, were also found (between -1462 and -162).
Miao
C H
CH
Department of Biochemistry, University of Washington, Seattle, Washington 98195-7350, USA.
Ho
W T
WT
Greenberg
D L
DL
Davie
E W
EW
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D018983:DNA Footprinting; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D005786:Gene Expression Regulation; D015698:Genomic Library; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011486:Protein C / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011486:Protein C; D012333:RNA, Messenger; D011994:Recombinant Proteins; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I