C/EBPα and HNF6 protein complex formation stimulates HNF6-dependent transcription by CBP coactivator recruitment in HepG2 cells
C/EBPα and HNF6 protein complex formation stimulates HNF6-dependent transcription by CBP coactivator recruitment in HepG2 cells
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DOI：
10.1002/hep.21044
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发表时间：
2006-02-01
期刊：
影响因子：
13.5
通讯作者：
Costa, RH
中科院分区：
文献类型：
--
作者：
Yoshida, Y;Hughes, DE;Costa, RH
我们先前已经证明，肝细胞核因子6(HNF6)的DNA结合域与叉头盒a2(Foxa2)蛋白形成的复合体可以刺激foxa2的转录活性。在这里，我们使用HepG2细胞共转染实验证明，CCAAT/增强子结合蛋白α(C/EBPα)可刺激HNF6的转录活性，但相关的C/EBPβ或C/EBP Delta蛋白不能刺激HNF6的转录活性。C/EBPα-HNF6蛋白复合体的形成需要HNF6切域和C/EBPα激活结构域(AD)1/AD2序列。这种C/EBPα-HNF6转录协同作用既需要N端的HNF6多组氨酸和丝氨酸/苏氨酸/Pro盒序列，也需要C/EBPαad1/ad2序列，后者已知招募CREB结合蛋白(CBP)转录共激活因子。与这些发现一致的是，腺病毒E1a介导的对p300/CBP组蛋白乙酰转移酶活性的抑制在共转染实验中取消了C/EBPα-HNF6转录协同作用。与肝脏蛋白提取物的免疫共沉淀分析表明，体内HNF6和C/EBPα转录因子与CBP共激活蛋白之间存在关联。此外，对肝癌细胞的染色质免疫沉淀分析表明，需要增加C/EBPα和HNF6蛋白的水平才能刺激这些转录因子和CBP共激活蛋白与内源性小鼠Foxa2启动子区域的联系。总之，C/EBPα-HNF6蛋白复合体的形成刺激了CBP共激活蛋白的募集，以表达Foxa2，Foxa2是一种关键的转录因子，在禁食期间调节肝脏糖异生基因的表达。
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.