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

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.

Figures

FIGURE 1.

Immunoprecipitation of native HNF4α and its interactive proteins. The immunoprecipitated native HNF4α and its complexes from HepG2 nuclear extract were (A) stained with SYPRO Ruby protein staining and (B) immunoblotted with the H1415 horseradish peroxidase (HRP)-conjugated anti-HNF4α F domain antibody. From a single gel band, pointed out by the arrow in A, HNF4α was identified by database search and inspection of individual MS/MS spectra in the raw files.

FIGURE 2.

Identification of steady-state native HNF4α phosphorylation and isoforms. A, schematic illustration of the identified regions of HNF4α phosphorylation. The box represents the domain structure of human HNF4α isoform 2. The numbers above the box represent the amino acid number in each domain. The regions and amino acid number corresponding to phosphorylation are represented by lines and the numbers under the box, respectively. B, the identified sites of native HNF4α phosphorylation. The identified phosphorylated peptides in human HNF4α isoform 2 are represented by single letter notation of the amino acid, and phosphorylated residues are represented by yellow circles. C, schematic illustration of the identified peptides that correspond to the HNF4α isoforms. The upper six boxes represent the domain structure of the human HNF4α isoforms. The lower bars represent the identified isoform-specific peptides. Each colored region indicates the isoform-specific amino acid residues that were identified by proteomic analysis. D, physical conformation of the native HNF4α isoform heterodimer. HepG2 nuclear extract was used for immunoprecipitation. Immunoblotting was carried out using the horseradish peroxidase (HRP)-conjugated anti-HNF4α F domain antibody H1415, anti-HNF4α P1 driven A/B domain antibody K9218, and anti-HNF4α P2 driven A/B-domain antibody H6939. The bands indicated by the open arrowheads are the HNF4α isoforms that did not react with the monoclonal antibodies used for immunoprecipitation.

FIGURE 3.

Physical conformation of the native HNF4α and HNF4γ heterodimerization. A, physical interaction of native HNF4α and HNF4γ. HepG2 nuclear extract was used for immunoprecipitation. B, schematic diagrams of the HNF4α and HNF4γ mutation constructs. The box represents the domain structure of human HNF4α isoform 2 and HNF4γ. The numbers above the box represent the amino acid number in each domain. C, the pulldown assays of the co-expressed HNF4α and HNF4γ in combination with the heterodimerizing mutation. D, localization of the native HNF4α and HNF4γ in HepG2 cells by immunofluorescence staining. HepG2 cells were visualized with the anti-HNF4α F domain monoclonal antibody H1415 (green) and the anti-HNF4γ monoclonal antibody B6502A (red). The left bottom panel shows the merged image of HNF4α and HNF4γ. HepG2 cells were also visualized by 4,6-diamidino-2-phenylindole (DAPI) staining (blue). All immunoprecipitation results were subjected to immunoblotting using HRP-conjugated antibodies H1415 and B6502A.

FIGURE 4.

Genome-wide analysis of HNF4α and HNF4γ binding genes and the regulation of their expression in the steady-state. A, venn diagrams of HNF4α and HNF4γ co-localization (gene body and −/+ 20,000 base pairs surrounding the gene body) by ChIP-seq analyses. The circles designate the genes for which the signal is observed with each antibody. The numbers in each set denote the number of genes. The overlapping region indicates that signal overlap is observable. HNF4α F-domain (blue), anti-HNF4α F domain antibody H1415; HNF4α A/B-domain (red), anti-HNF4α A/B domain antibody K9218; HNF4γ (green), anti-HNF4γ antibody B6502A. B, siRNA-mediated HNF4α and HNF4γ double knockdown. HepG2 cells were transfected with either HNF4α and -γ-specific siRNAs (4α+4γ) or control siRNA (control). After transfection, the cells were harvested for the isolation of total RNA and whole cell lysate. C, identification of enriched motifs in the HNF4 binding sequence. The height of each letter represents the relative frequency of nucleotides at different positions in the consensus region. D, the correlations between each of the HNF4 binding levels and gene expression. Averages of the microarray expression dataset of the control siRNA-transfected HepG2 cells were used for the steady-state HepG2 expression level. Genes were grouped as 100 gene sets (one dot in the figure) according to the steady-state expression level. Each HNF4 binding level was calculated for the same 100 gene sets. The y axis indicates the HNF4 binding level, and the x axis indicates the steady-state expression level.

FIGURE 5.

The transcriptional activation of the HGD and CIDEB genes by HNF4α-HNF4γ heterodimerization through the same DR1 sites. A, transcriptional down-regulation of HGD and CIDEB by siRNA-mediated HNF4α and -γ double knockdown in HepG2 cells. The ID of each probe is indicated under each gene name. B, the ChIP-seq analysis of HNF4α and HNF4γ overlap binding on the CIDEB and HGD promoter regions in HepG2 cells. The arrows denote the regions of the primer sets for ChIP-qPCR. C, ChIP-qPCR (left) and ChIP-reChIP-qPCR (right) analysis of HNF4α and HNF4γ overlap binding of the CIDEB short transcript and HGD promoter region in HepG2 cells. The details of the primer sets are provided under supplemental Table S1. D, left, schematic representation of the human CIDEB and HGD promoters, illustrating the DR1 motifs. Right, alignment of the two DR1 motifs in the human CIDEB and HGD promoters. The sequences of the DR1 motifs identified in the ChIP-seq study (Fig. 4C) (identified heterodimer motif) and the reported HNF4α binding motifs in HepG2 (Reported HNF4α motif) (72) are aligned. E, luciferase analysis of the HNF4α and HNF4γ cooperative transcriptional activity effect of the CIDEB and HGD genes on DR1 sequences. The relative luciferase activities of HNF4α (left box), HNF4γ (center box), and both HNF4α and HNF4γ (right box) are indicated. The numbers above the bars refer to the increases induced by each HNF4. F, EMSAs. The bands of DNA proteins in the HepG2 nuclear extract complex formations are indicated by the arrow. The bands of the complexes supershifted by the addition of each anti-HNF4 antibody are indicated by an asterisk. The oligonucleotide sequences used for probes and competitors are shown under supplemental Table S1. HNF4α F domain, anti-HNF4α F domain antibody H1415; HNF4α A/B domain, anti-HNF4α A/B domain antibody K9218; HNF4γ, anti-HNF4γ antibody B6502A.