C/EBPα and HNF6 protein complex formation stimulates HNFG-dependent transcription by CBP coactivator recruitment in HepG2 cells

Yuichi Yoshida, Douglas E. Hughes, Francisco M. Rausa, Il-man Kim, Yongjun Tan, Gretchen J. Darlington, Robert H. Costa

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Abstract

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

Original languageEnglish (US)
Pages (from-to)276-286
Number of pages11
JournalHepatology
Volume43
Issue number2
DOIs
StatePublished - Feb 1 2006

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Hepatocyte Nuclear Factor 6
CREB-Binding Protein
CCAAT-Enhancer-Binding Proteins
Hep G2 Cells
Proteins
Hepatocyte Nuclear Factor 3-beta
Transcription Factors
Histone Acetyltransferases
Liver Extracts
Chromatin Immunoprecipitation
Threonine

ASJC Scopus subject areas

  • Hepatology

Cite this

Yoshida, Y., Hughes, D. E., Rausa, F. M., Kim, I., Tan, Y., Darlington, G. J., & Costa, R. H. (2006). C/EBPα and HNF6 protein complex formation stimulates HNFG-dependent transcription by CBP coactivator recruitment in HepG2 cells. Hepatology, 43(2), 276-286. https://doi.org/10.1002/hep.21044

C/EBPα and HNF6 protein complex formation stimulates HNFG-dependent transcription by CBP coactivator recruitment in HepG2 cells. / Yoshida, Yuichi; Hughes, Douglas E.; Rausa, Francisco M.; Kim, Il-man; Tan, Yongjun; Darlington, Gretchen J.; Costa, Robert H.

In: Hepatology, Vol. 43, No. 2, 01.02.2006, p. 276-286.

Research output: Contribution to journalArticle

Yoshida, Y, Hughes, DE, Rausa, FM, Kim, I, Tan, Y, Darlington, GJ & Costa, RH 2006, 'C/EBPα and HNF6 protein complex formation stimulates HNFG-dependent transcription by CBP coactivator recruitment in HepG2 cells', Hepatology, vol. 43, no. 2, pp. 276-286. https://doi.org/10.1002/hep.21044
Yoshida, Yuichi ; Hughes, Douglas E. ; Rausa, Francisco M. ; Kim, Il-man ; Tan, Yongjun ; Darlington, Gretchen J. ; Costa, Robert H. / C/EBPα and HNF6 protein complex formation stimulates HNFG-dependent transcription by CBP coactivator recruitment in HepG2 cells. In: Hepatology. 2006 ; Vol. 43, No. 2. pp. 276-286.
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AU - Kim, Il-man

AU - Tan, Yongjun

AU - Darlington, Gretchen J.

AU - Costa, Robert H.

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

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