Fig 1.
In vitro phosphorylation assay of HNF4α by ERK1 kinase.
The phosphorylation assay was carried out with in vitro translated human recombinant HNF4α protein, ERK1 kinase and radioactively labelled [γ-32P] ATP. HNF4α does not contain any post-translational modifications, but it has a GST-tag at N-terminal. The samples were run on SDS-PAGE and subjected to autoradiography. ERK1 kinase is capable of autophosphorylation.
Fig 2.
Phosphorylated sites on HNF4α protein by ERK1 kinase detected by mass spectrometry.
Non-radioactively phosphorylated HNF4α by ERK1/2 was cut out from the polyacrilamid gel. Tryptic peptide fragments were analysed by mass spectrometry. Affected phosphorylation sites (indicated by lollipops) appear in the DNA binding domain (DBD), the hinge, the ligand-binding domain (LBD) and the C-terminus of HNF4α, as well. The phosphorylated amino acid residues can be attributed to either previously described sites (S138/T139, S142/S143, S147/S148, S151, T166/S167, S313) or new positions reported first here (S95, S262/S265, S451, T457/T459).
Table 1.
Phosphorylated amino acid residues identified by mass spectrometry.
In vitro phosphorylated HNF4α was subjected to mass spectrometry analysis. The fragments containing the phosphorylated amino acid residues correspond to phosphorylation sites on the HNF4α protein. Detected phosphorylation sites (serine/threonine residues) in the cryptic fragments are marked bold and underlined. Affected phosphorylation sites appear in the DNA binding domain (DBD), the hinge, the ligand-binding domain (LBD) and the C-terminus of HNF4α, as well.
Fig 3.
Luciferase assay measuring ABCC6 promoter activity of HNF4α phosphomimetic mutants in HeLa cells.
Mutations were designed for serine or threonine phosphorylation sites creating phosphomimetic (glutamate or aspartate) or neutral (alanine) mutants. For luciferase assays, triple co-transfection was performed with the phACCC6(-332/+72)Luc construct composed of the ABCC6 promoter fragment, pcDNA5-FRT/TO plasmid encoding HNF4α variants and pRL-TK Renilla luciferase Control Reporter Vector. Luciferase activity was measured 48 hours after transfection. Relative luciferase activity was calculated by normalizing for background noise and for transfection efficiency by the co-transfected control reporter vector. The error bars represent S.D. Tukey-HSD test was performed. Significance versus WT HNF4 alpha is indicated by asterisks: *p<0.05; **p<0.01.
Fig 4.
(A) Read distribution plot of HNF4α and H3K27ac upon vehicle treatment, relative to the 8,748 HNF4α peaks in 2-kb frames. Peaks are sorted according to HNF4α tag density. (B) Area-proportional Venn diagram illustrating the overlap between H3K27ac regions and HNF4α peaks. (C) Histogram shows the average tag density of vehicle-treated HNF4α peaks and H3K27ac signals at the sites of 8,748 HNF4α binding sites. (D) Motif enrichment of 8,748 HNF4α peaks. The P value and target and background (Bg) percentages are included for each motif. (E) IGV snapshot of HNF4α and H3K27ac ChIP-seq coverage representing eight selected genomic regions upon vehicle treatment. HBB promoter: negative control region. The interval scale is 50 in both cases. Peaks, highlighted with grey lines, represent the sites of the investigated HNF4α-target genomic regions.
Table 2.
Top 15 biological pathways related to the 8,748 HNF4α binding sites.
Data derived from KEGG database.
Fig 5.
ChIP-qPCR results showing HNF4α occupancy on seven genomic regions of hepatic HNF4α target genes.
Immunoprecipitation of chromatin from HepG2 cells was performed with anti-HNF4α or IgG antibody untreated or treated with EGF for 30 mins. Enrichment was compared to % input (y axis). HNF4α binding (black columns) is decreased after short EGF treatment (grey). BLVR A: Biliverdin A, BLVR B: Biliverdin B, HPD: 4-hydroxyphenylpyruvate dioxygenase, PKLR: Pyruvate kinase, liver and RBC, ABCC6: ATP-binding cassette subfamily C, member 6 and APOA1: Apolipoprotein A1. Beta globin: negative control region. Beads represent immunoprecipitation performed without any antibody. S.D. is indicated on the figure. Results of a representative experiment (n = 7) are shown.
Table 3.
List of qPCR primers.