The role of CCAAT/enhancer-binding protein alpha and a protein that binds to the activator-protein-1 site in the regulation of liver-specific expression of the winter flounder antifreeze protein gene.
Winter flounder, Pleuronectes americanus, produces antifreeze proteins (AFPs) to avoid freezing during winter. The AFP in the blood is synthesized by the liver and its annual level is regulated by environmental factors and somatotropin. The only intron (+106 to +602) of the AFP gene contains putative binding sites for liver-enriched transcription activators. Due to the lack of an appropriate fish liver cell line, a mammalian model system was used to demonstrate the liver-specific enhancer activity of the intron. Deletion analysis of the intron indicated nucleotide sequences between +192 bp and +334 bp were important for the enhancer activity. Further refinement showed that the element B (+303 to +322 bp) resided in this region interacted with proteins from rat nuclear extracts by DNase I footprinting and mobility shift analysis. Gel retardation with competition and supershift experiments defined element B to include binding sites for CCAAT/enhancer binding protein a (C/EBP alpha), a liver-enriched transcription factor, and a novel activator protein-1 (AP-1) site binding protein, designated as antifreeze enhancer binding protein (AEP). Residues important to DNA-protein interaction in element B were mapped by methylation interference. Mutations in element B by site-directed mutagenesis destroyed the enhancer activity of the intron. Ultraviolet crosslinking experiments using element B revealed the specific binding of two proteins of approximately 43 and 80 kDa. The present findings provide a mechanism(s) by which the tissue specificity of AFP gene expression can be achieved. Integration of the present information with the somatotropin-mediated signal transduction pathways has generated a working model for the hormonal regulation and seasonal expression of the AFP gene.
Chan
S L
SL
Research Institute, Hospital for Sick Children and Department of Biochemistry, University of Toronto, Canada.
Miao
M
M
Fletcher
G L
GL
Hew
C L
CL
D000818:Animals; D021301:Antifreeze Proteins; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D005432:Flounder / Q000235:genetics*; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008099:Liver / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000502:physiology*
D021301:Antifreeze Proteins; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D006023:Glycoproteins; D009687:Nuclear Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors