Functional analysis of the mouse α-fetoprotein enhancers and their subfragments in primary mouse hepatocyte cultures

Dong Er Zhang, Jeffrey P. Rabek, Ching Chyuan Hsieh, Carlos Torres-Ramos, John Papaconstantinou

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Abstract

We have compared the activities of mouse α-fetoprotein (AFP) enhancers I, II, and III with their minimal enhancer fragments (Mers) I, II, and III and with the entire 7-kilobase pair enhancer domain by transient expression assay in primary fetal mouse liver cells. The level of expression directed by the AFP promoter [p(-1009)AFPcat] alone is stimulated at least 10-fold by the entire AFP enhancer domain (-1009 to -6983). Enhancer I can drive the level of chloramphenicol acetyltransferase activity equivalent to that of the entire enhancer domain, whereas the increase in activity by enhancers II and III is significantly lower (1.5-fold). Mersl, II, and III all mediate a greater increase in activity than their corresponding enhancer regions. The increase with MerI is 16-fold. Using DNase I protection analyses we identified 3 protein-binding regions in MerI; site Ia binds liver and brain nuclear proteins; site Ib binds liver, kidney, and brain nuclear proteins as well as purified C/EBP; site Ic binds liver and kidney nuclear proteins. Site-specific mutation of Ia, Ib, or Ic showed a 10-25% reduction in chloramphenicol acetyltransferase expression; deletion of the C/EBP-binding site in Ib showed a 45% reduction in activity and mutation of all 3 sites (Ia, Ib, and Ic) resulted in a 75% reduction in activity. Our studies indicate no single irons-acting factor is absolutely essential for enhancer activity, and that the enhancer activity of MerI is mediated via a combinatorial and additive mechanism.

Original languageEnglish (US)
Pages (from-to)10676-10682
Number of pages7
JournalJournal of Biological Chemistry
Volume267
Issue number15
StatePublished - May 25 1992

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Fetal Proteins
Functional analysis
Liver
Hepatocytes
Nuclear Proteins
Chloramphenicol O-Acetyltransferase
Brain
Kidney
Mutation
Deoxyribonuclease I
Protein Binding
Assays
Binding Sites
Iron

ASJC Scopus subject areas

  • Biochemistry

Cite this

Functional analysis of the mouse α-fetoprotein enhancers and their subfragments in primary mouse hepatocyte cultures. / Zhang, Dong Er; Rabek, Jeffrey P.; Hsieh, Ching Chyuan; Torres-Ramos, Carlos; Papaconstantinou, John.

In: Journal of Biological Chemistry, Vol. 267, No. 15, 25.05.1992, p. 10676-10682.

Research output: Contribution to journalArticle

Zhang, Dong Er ; Rabek, Jeffrey P. ; Hsieh, Ching Chyuan ; Torres-Ramos, Carlos ; Papaconstantinou, John. / Functional analysis of the mouse α-fetoprotein enhancers and their subfragments in primary mouse hepatocyte cultures. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 15. pp. 10676-10682.
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