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-vt (C/EBP alpha) has a vital role in cell growth and differentiation. To delineate further a mechanism for C/EBP alpha -mediated differentiation, we screened C/EBP alpha - interacting proteins through far-Western screening. One of the strongest interactions was with RFC140, the large subunit of the replication factor C complex. C/EBPa 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/EBPa interacted with the DNA-binding region of RFC140. Overexpression of RFC140 in mammalian cells increased the transactivation activity of C/EBPa on both minimal and native promoters. Consistent with the enhanced transactivation, a complex of C/EBP alpha and RFC140 proteins with the cognate DNA element was detected in vitro. The specific interaction between C/EBP alpha and RFC140 was detected in the terminal differentiation of 3T3-LI 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/EBPa 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.

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