Immunochemical characterization and transacting properties of upstream stimulatory factor isoforms.
The ubiquitous upstream stimulatory factor (USF) transcription factors encoded by two distinct genes (USF1 and USF2) exist under the form of various dimers able to bind E-boxes. We report the molecular cloning and functional characterization of USF2 isoforms, corresponding to a 44-kDa subunit, USF2a, and a new 38-kDa subunit, USF2b, generated by differential splicing. Using specific anti-USF antibodies, we define the different binding complexes in various nuclear extracts. In vivo, the USF1/USF2a heterodimer represents over 66% of the USF binding activity whereas the USF1 and USF2a homodimers represent less than 10%, which strongly suggests an in vivo preferential association in heterodimers. In particular, an USF1/USF2b heterodimer accounted for almost 15% of the USF species in some cells. The preferential heterodimerization of USF subunits was reproduced ex vivo, while the in vitro association of cotranslated subunits, or recombinant USF proteins, appeared to be random. In transiently transfected HeLa or hepatoma cells, USF2a and USF1 homodimers transactivated a minimal promoter with similar efficiency, whereas USF2b, which lacks an internal 67-amino acid domain, was a poor transactivator. Additionally, USF2b was an efficient as USF1 and USF2a homodimers in transactivating the liver-specific pyruvate kinase gene promoter.
Viollet
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B
Institut Cochin de Génétique Moléculaire, U129 INSERM, Université René Descartes, Paris, France.
Lefrançois-Martinez
A M
AM
Henrion
A
A
Kahn
A
A
Raymondjean
M
M
Martinez
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D015723:Gene Library; D014644:Genetic Variation; D006367:HeLa Cells; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D007150:Immunohistochemistry; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D051720:Upstream Stimulatory Factors
D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C497181:USF1 protein, human; C497184:USF2 protein, human; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; C497187:Usf2 protein, rat; D015500:Chloramphenicol O-Acetyltransferase