Two adjacent C/EBP-binding sequences that participate in the cell-specific expression of the mouse serum amyloid A3 gene.
Serum amyloid A (SAA) is a major acute-phase protein synthesized primarily in the liver. Its expression, very low in normal animals, is increased several hundredfold following acute inflammation. To examine DNA sequences involved in liver-specific expression, 5'-flanking regions of the mouse SAA3 gene were analyzed by transient transfection, band shift, and DNase I protection assays. We found that a 56-bp fragment immediately 5' to the TATA box spanning the region -93 to -38 relative to the transcription start site was sufficient to confer liver cell-specific transcriptional activation onto a heterologous promoter in a dose-dependent and orientation-independent manner. This DNA fragment could form DNA-protein complexes with heat-stable nuclear proteins, and the complexes formed could be specifically competed for by excess oligomers corresponding to the C/EBP- or DBP-binding sites but not by binding sites for three other liver-specific factors, HNF1, HNF3, and HNF4. Footprint analysis using Hep3B nuclear extracts revealed two adjacent footprint regions within this 56-bp fragment, the distal region having at least fivefold-greater affinity than the proximal region. Identical footprint patterns were observed when purified recombinant C/EBP protein was used. These results indicated that binding of C/EBP to this 56-bp fragment plays an important role in vivo in enhancing expression of the mouse SAA3 gene in hepatocytes.
Li
X X
XX
Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Huang
J H
JH
Rienhoff
H Y
HY
Liao
W S
WS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D005786:Gene Expression Regulation; D008099:Liver / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009710:Nucleotide Mapping; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D000685:Serum Amyloid A Protein / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D000685:Serum Amyloid A Protein; D014157:Transcription Factors; D004247:DNA; D003850:Deoxyribonuclease I