C/EBPα 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/EBP alpha), but not by the related C/EBP beta or C/EBP delta proteins. Formation of the C/EBP alpha-HNF6 protein complex required the HNF6 cut domain and the C/EBP alpha activation domain (AD) 1/AD2 sequences. This C/EBP alpha-HNF6 transcriptional synergy required both the N-terminal HNF6 polyhistidine and serine/threonine/proline box sequences, as well as the C/EBP alpha 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/EBP alpha-HNF6 transcriptional synergy in cotransfection assays. Co-immuno-precipitation assays with liver protein extracts demonstrate an association between the HNF6 and C/EBP alpha transcription factors and the CBP coactivator protein in vivo. Furthermore, chromatin immuno-precipitation assays with hepatoma cells demonstrated that increased levels of both C/EBP alpha 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/EBP alpha-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.