Proteomic screen defines the hepatocyte nuclear factor 1alpha-binding partners and identifies HMGB1 as a new cofactor of HNF1alpha.
Hepatocyte nuclear factor (HNF)-1alpha is one of the liver-enriched transcription factors involved in many tissue-specific expressions of hepatic genes. The molecular mechanisms for determining HNF1alpha-mediated transactivation have not been explained fully. To identify unknown proteins that interact with HNF1alpha, we developed a co-IP-MS strategy to search HNF1alpha interactions, and high mobility group protein-B1 (HMGB1), a chromosomal protein, was identified as a novel HNF1alpha-interacting protein. In vitro glutathione S-transferase pull-down and in vivo co-immunoprecipitation studies confirmed an interaction between HMGB1 and HNF1alpha. The protein-protein interaction was mediated through the HMG box domains of HMGB1 and the homeodomain of HNF1alpha. Furthermore, electrophoretic mobility shift assay and chromatin-immunoprecipitation assay demonstrated that HMGB1 was recruited to endogenous HNF1alpha-responsive promoters and enhanced HNF1alpha binding to its cognate DNA sequences. Moreover, luciferase reporter analyses showed that HMGB1 potentiated the transcriptional activities of HNF1alpha in cultured cells, and downregulation of HMGB1 by RNA interference specifically affected the HNF1alpha-dependent gene expression in HepG2 cell. Taken together, these findings raise the intriguing possibility that HMGB1 is a new cofactor of HNF1alpha and participates in HNF1alpha-mediated transcription regulation through protein-protein interaction.
Yu
Miao
M
Beijing Institute of Radiation Medicine, Beijing, 100850, Beijing Proteomics Research Center, Beijing, 102206, PR China.
Wang
Jian
J
Li
Wei
W
Yuan
Yan Zhi
YZ
Li
Chang Yan
CY
Qian
Xiao Hong
XH
Xu
Wang Xiang
WX
Zhan
Yi Qun
YQ
Yang
Xiao Ming
XM
D001665:Binding Sites; D002460:Cell Line; D024243:HMGB1 Protein / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D051538:Hepatocyte Nuclear Factor 1-alpha / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D040901:Proteomics; D034622:RNA Interference; D015533:Transcriptional Activation; D000509:alpha-Fetoproteins / Q000235:genetics
D024243:HMGB1 Protein; C496487:HNF1A protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D000509:alpha-Fetoproteins