C/EBPalpha and HNF6 protein complex formation stimulates HNF6-dependent transcription by CBP coactivator recruitment in HepG2 cells.
We previously demonstrated that formation of complexes between the DNA-binding domains of hepatocyte nuclear factor 6 (HNF6) and forkhead box a2 (Foxa2) proteins stimulated Foxa2 transcriptional activity. Here, we used HepG2 cell cotransfection assays to demonstrate that HNF6 transcriptional activity was stimulated by CCAAT/enhancer-binding protein alpha (C/EBPalpha), but not by the related C/EBPbeta or C/EBPdelta proteins. Formation of the C/EBPalpha-HNF6 protein complex required the HNF6 cut domain and the C/EBPalpha activation domain (AD) 1/AD2 sequences. This C/EBPalpha-HNF6 transcriptional synergy required both the N-terminal HNF6 polyhistidine and serine/threonine/proline box sequences, as well as the C/EBPalpha AD1/AD2 sequences, the latter of which are known to recruit the CREB binding protein (CBP) transcriptional coactivator. Consistent with these findings, adenovirus E1A-mediated inhibition of p300/CBP histone acetyltransferase activity abrogated C/EBPalpha-HNF6 transcriptional synergy in cotransfection assays. Co-immunoprecipitation assays with liver protein extracts demonstrate an association between the HNF6 and C/EBPalpha transcription factors and the CBP coactivator protein in vivo. Furthermore, chromatin immunoprecipitation assays with hepatoma cells demonstrated that increased levels of both C/EBPalpha and HNF6 proteins were required to stimulate association of these transcription factors and the CBP coactivator protein with the endogenous mouse Foxa2 promoter region. In conclusion, formation of the C/EBPalpha-HNF6 protein complex stimulates recruitment of the CBP coactivator protein for expression of Foxa2, a transcription factor critical for regulating expression of hepatic gluconeogenic genes during fasting.
Yoshida
Yuichi
Y
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, College of Medicine, Chicago, IL 60607-7170, USA.
Hughes
Douglas E
DE
Rausa
Francisco M
FM
Kim
Il-Man
IM
Tan
Yongjun
Y
Darlington
Gretchen J
GJ
Costa
Robert H
RH
D000595:Amino Acid Sequence; D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism / Q000502:physiology*; D050882:CREB-Binding Protein / Q000378:metabolism*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D051542:Hepatocyte Nuclear Factor 3-beta / Q000235:genetics; D051559:Hepatocyte Nuclear Factor 6 / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D051379:Mice; D046912:Multiprotein Complexes / Q000502:physiology; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection
D022763:CCAAT-Enhancer-Binding Protein-alpha; C496502:FOXA2 protein, human; D051559:Hepatocyte Nuclear Factor 6; D046912:Multiprotein Complexes; D051542:Hepatocyte Nuclear Factor 3-beta; D050882:CREB-Binding Protein