Functional interaction of bZIP proteins and the large subunit of replication factor C in liver and adipose cells.
The transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) has a vital role in cell growth and differentiation. To delineate further a mechanism for C/EBPalpha-mediated differentiation, we screened C/EBPalpha-interacting proteins through far-Western screening. One of the strongest interactions was with RFC140, the large subunit of the replication factor C complex. C/EBPalpha specifically interacted with RFC140 from rat liver nuclear extract as determined by a combination of affinity chromatography and co-immunoprecipitation. Subsequent far-Western blotting showed that the bZIP domain of C/EBPalpha interacted with the DNA-binding region of RFC140. Overexpression of RFC140 in mammalian cells increased the transactivation activity of C/EBPalpha on both minimal and native promoters. Consistent with the enhanced transactivation, a complex of C/EBPalpha and RFC140 proteins with the cognate DNA element was detected in vitro. The specific interaction between C/EBPalpha and RFC140 was detected in the terminal differentiation of 3T3-L1 preadipocytes to adipocytes. The synergistic transcription effect of these two proteins increased the promoter activity and protein expression of peroxisome proliferator-activated receptor-gamma, which is a main regulator of adipocyte differentiation. Our results demonstrate that the specific transcription factor C/EBPalpha and the general DNA replication factor RFC140 interact functionally and physically. This observation highlights a unique mechanism by which the levels of the general replication factor can strongly modulate the functional activity of the specific transcription factor as a coactivator.
Hong
S
S
Hormone Research Center, Chonnam National University, Kwangju 500-757, Korea.
Park
S J
SJ
Kong
H J
HJ
Shuman
J D
JD
Cheong
J
J
D017667:Adipocytes / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005347:Fibroblasts / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D051379:Mice; D009687:Nuclear Proteins; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051818:Replication Protein C; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; C486334:Refbp1 protein, mouse; D014157:Transcription Factors; D004247:DNA; D005982:Glutathione Transferase; D051818:Replication Protein C