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Proteomic analysis of native hepatocyte nuclear factor-4? (HNF4?) isoforms, phosphorylation status, and interactive cofactors.

ABSTRACT: Hepatocyte nuclear factor-4? (HNF4?, NR2A1) is a nuclear receptor that has a critical role in hepatocyte differentiation and the maintenance of homeostasis in the adult liver. However, a detailed understanding of native HNF4? in the steady-state remains to be elucidated. Here we report the native HNF4? isoform, phosphorylation status, and complexes in the steady-state, as shown by shotgun proteomics in HepG2 hepatocarcinoma cells. Shotgun proteomic analysis revealed the complexity of native HNF4?, including multiple phosphorylation sites and inter-isoform heterodimerization. The associating complexes identified by label-free semiquantitative proteomic analysis include the following: the DNA-dependent protein kinase catalytic subunit, histone acetyltransferase complexes, mRNA splicing complex, other nuclear receptor coactivator complexes, the chromatin remodeling complex, and the nucleosome remodeling and histone deacetylation complex. Among the associating proteins, GRB10 interacting GYF protein 2 (GIGYF2, PERQ2) is a new candidate cofactor in metabolic regulation. Moreover, an unexpected heterodimerization of HNF4? and hepatocyte nuclear factor-4? was found. A biochemical and genomewide analysis of transcriptional regulation showed that this heterodimerization activates gene transcription. The genes thus transcribed include the cell death-inducing DEF45-like effector b (CIDEB) gene, which is an important regulator of lipid metabolism in the liver. This suggests that the analysis of the distinctive stoichiometric balance of native HNF4? and its cofactor complexes described here are important for an accurate understanding of transcriptional regulation.

SUBMITTER: Daigo K

PROVIDER: S-EPMC3013027 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Proteomic analysis of native hepatocyte nuclear factor-4α (HNF4α) isoforms, phosphorylation status, and interactive cofactors.

Daigo Kenji K Kawamura Takeshi T Ohta Yoshihiro Y Ohashi Riuko R Katayose Satoshi S Tanaka Toshiya T Aburatani Hiroyuki H Naito Makoto M Kodama Tatsuhiko T Ihara Sigeo S Hamakubo Takao T

The Journal of biological chemistry 20101103 1

Hepatocyte nuclear factor-4α (HNF4α, NR2A1) is a nuclear receptor that has a critical role in hepatocyte differentiation and the maintenance of homeostasis in the adult liver. However, a detailed understanding of native HNF4α in the steady-state remains to be elucidated. Here we report the native HNF4α isoform, phosphorylation status, and complexes in the steady-state, as shown by shotgun proteomics in HepG2 hepatocarcinoma cells. Shotgun proteomic analysis revealed the complexity of native HNF4 ...[more]

PMID: 21047794

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Project description:HNF4α is a nuclear receptor regulating the transcription of genes involved mainly in development, cell differentiation and metabolism. Opposite functions for the two classes of P1 and P2 isoforms of HNF4α have recently been highlighted. These classes include 12 variants of HNF4α that can be expressed by the use of two promoters and by alternative splicing. Until now, the characterization of this transcription factor has ignored this diversity and has remained confined to the study of a fraction of the isoforms. We therefore wanted to clarify the situation by specifically characterizing the transcriptional functions of the 12 isoforms of HNF4α. We have generated for this purpose stable lines expressing each isoform of HNF4α in HCT 116 cells. We analyzed the whole transcriptome associated with each isoform by sequencing RNA, as well as their proteome by a BioID approach coupled to quantitative mass spectrometry. We noted major differences in the transcriptional function of the 12 isoforms. The α4, α5 and α6 isoforms have been characterized for the first time, and show a greatly reduced transcriptional potential. We have shown that these isoforms are unable to recognize the consensus response element of HNF4α. The α1 and α2 isoforms are the most potent regulators of gene expression, while the α3 isoform exhibits significantly reduced activity. Several transcription factors and coregulators have been identified as potential specific partners for certain HFH4α isoforms. The IRF-2BP2 co-repressor interacts specifically with isoforms which include the long form of the F domain of HNF4α. This specific interaction could explain the large number of genes modulated negatively by α1 and α2 compared to α3. The analysis integrating the vast amount of transcriptomic and proteomic data allows the identification of transcriptional regulatory mechanisms specific to certain isoforms, demonstrating the importance of considering all isoforms which can have diverse functions.

Dataset Information

Proteomic analysis of native hepatocyte nuclear factor-4? (HNF4?) isoforms, phosphorylation status, and interactive cofactors.

Publications

Proteomic analysis of native hepatocyte nuclear factor-4α (HNF4α) isoforms, phosphorylation status, and interactive cofactors.

Similar Datasets

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