Abstract
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 HNF4α, 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 HNF4α and hepatocyte nuclear factor-4γ 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 HNF4α and its cofactor complexes described here are important for an accurate understanding of transcriptional regulation.
Highlights
Hepatocyte nuclear factor-4␣ (HNF4␣)3 is an orphan nuclear receptor (NR), which plays a critical role in hepatocyte differentiation [1,2,3] as well as the maintenance of homeostasis of the adult liver, intestine, and pancreatic  cells (4 –7)
HNF4␣ consists of six distinct functional domains (A to F) [13], an A/B domain, which is associated with activation function 1 (AF-1), a C domain, which binds certain specific DNA sequences, a 6-base pair repeat segment with a 1-base pair spacer called direct repeat 1 (DR1), an E domain, which is the homodimerization region and the ligand-binding domain associated with activation function 2, and an F domain, which has a negative regulatory function
Strategy for the Proteomic Analysis of the Native HNF4␣ Status—To clarify the transcriptional control of HNF4␣ and its cofactor complex in the steady-state native condition, HNF4␣ was immunopurified from HepG2 nuclear extract (Fig. 1) and analyzed using highly sensitive shotgun proteomics
Summary
HNF4␣, hepatocyte nuclear factor-4␣; ChIPseq, chromatin immunoprecipitation sequencing; CIDEB, cell death-inducing DFFA-like effector b; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; DR1, direct repeat 1; HAT, histone acetyltransferase; HGD, homogentisate 1,2-dioxygenase; HNF4␥, hepatocyte nuclear factor-4␥; IP, immunoprecipitation; NR, nuclear receptor; STAGA, SPT3TAFII31-GCN5L acetylase; HPRD, Human Protein Reference Database. Recent reports showed that the cofactor-mediated function results in histone modification, regulation of chromatin conformation, and immature mRNA metabolism [27] Whereas these key factors might be linked with each other and have a central role in the fine tuning of the multiple transcriptional regulation activities performed by HNF4␣, the details of the steady-state of native HNF4␣ are, as yet, poorly understood. The data indicate the complexity of the native HNF4␣ states and cofactors obtained via stoichiometry In confirmation of this proteomic analysis, we unexpectedly demonstrated HNF4␣ and HNF4␥ heterodimerization and transcriptional activation. The results support the concept that the fine tuning of the multiple transcriptional regulation activities arose from a distinctive stoichiometric balancing of the nuclear receptor and interacting cofactors The application of this method to dynamic proteomics should help provide a means to obtain an adequate understanding of transcriptional regulation in extracellular/intracellular signaling and/or the developmental cascade
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