HNF1A POU Domain Mutations Found in Japanese Liver Cancer Patients Cause Downregulation of HNF4A Promoter Activity with Possible Disruption in Transcription Networks

Abstract

Hepatocyte nuclear factor 1A (HNF1A) is the master regulator of liver homeostasis and organogenesis and regulates many aspects of hepatocyte functions. It acts as a tumor suppressor in the liver, evidenced by the increased proliferation in HNF1A knockout (KO) hepatocytes. Hence, we postulated that any loss-of-function variation in the gene structure or composition (mutation) could trigger dysfunction, including disrupted transcriptional networks in liver cells. From the International Cancer Genome Consortium (ICGC) database of cancer genomes, we identified several HNF1A mutations located in the functional Pit-Oct-Unc (POU) domain. In our biochemical analysis, we found that the HNF1A POU-domain mutations Y122C, R229Q and V259F suppressed HNF4A promoter activity and disrupted the binding of HNF1A to its target HNF4A promoter without any effect on the nuclear localization. Our results suggest that the decreased transcriptional activity of HNF1A mutants is due to impaired DNA binding. Through structural simulation analysis, we found that a V259F mutation was likely to affect DNA interaction by inducing large conformational changes in the N-terminal region of HNF1A. The results suggest that POU-domain mutations of HNF1A downregulate HNF4A gene expression. Therefore, to mimic the HNF1A mutation phenotype in transcription networks, we performed siRNA-mediated knockdown (KD) of HNF4A. Through RNA-Seq data analysis for the HNF4A KD, we found 748 differentially expressed genes (DEGs), of which 311 genes were downregulated (e.g., HNF1A, ApoB and SOAT2) and 437 genes were upregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed that the DEGs were involved in several signaling pathways (e.g., lipid and cholesterol metabolic pathways). Protein-protein network analysis suggested that the downregulated genes were related to lipid and cholesterol metabolism pathways, which are implicated in hepatocellular carcinoma (HCC) development. Our study demonstrates that mutations of HNF1A in the POU domain result in the downregulation of HNF1A target genes, including HNF4A, and this may trigger HCC development through the disruption of HNF4A-HNF1A transcriptional networks.

Keywords: HNF1A; HNF4A; POU domain; hepatocellular carcinoma; mutation.

Figure 1

(A) Positions of novel mutations are indicated in the human HNF1A protein structure (Pit1, Oct1 and Unc1 (POU) domain-Green; POU homeodomain (POUh) and POU-specific (POUs)). (B) Alignment of the human, mouse, bovine and zebrafish HNF1A amino acid sequences and mutations found in the POU domain. Red color denotes highly conserved (100%) elements among the species. The mutations (indicated by red asterisks) in the POU domain of HNF1A are highly conserved in species.

Figure 2

The ability of the (A) human WT and mutant HNF1A and (B) mouse WT and mutant HNF1A to transactivate HNF4A P1 when overexpressed in HEK293 cells. (C) The ability of the human WT and mutant HNF1A to transactivate the target promoter (HNF4A P1) when overexpressed in Huh7 cells. The cells were co-transfected with the indicated luciferase reporters and either an empty expression vector (serving as a control) or expression vectors (100 ng) for the indicated HNF1A vectors in 24-well culture plates. The bars indicate the fold activation of HNF1A WT and mutants (vs. control) on target promoters. The corresponding promoter activity is reported as fold activation over control (±SEM, n = 3–4). The data reported represent the averages of three experiments, each conducted in duplicate. (*, p < 0.05; ***, p < 0.001). (D) Cellular localization of WT and mutant HNF1A was visualized in HEK293 cells using IFC staining. The nuclei were stained with DAPI, and the images were taken at 20× magnification. (E) Electrophoretic mobility shift assay (EMSA) was used to assess the binding of WT or mutated HNF1A nuclear proteins to a double-stranded oligonucleotide corresponding to the consensus HNF1A-binding elements of the HNF4A promoter region. The HNF1A V259F mutant displayed markedly reduced binding to the HNF4A promoter region for all the experiments. (F) HEK293 cells were transfected with expression vectors encoding HNF1A WT or the indicated mutants. WB analysis showed that all proteins were similarly expressed.

Figure 3

(A) X-ray crystallographic structure of the POUh domain (blue) binding with DNA (orange; PDB ID: 1IC8). The backbone of the N-terminal region is highlighted in red. The residues V259, R203 and R205 are depicted by the stick model. (B) Fluctuations of the WT (blue) and V259F mutant (red) proteins. The RMSF of the backbone was plotted. The fluctuations were calculated using the last 100 ns of 10 runs (1 μs in total). (C) Residue-wise contact-map difference between the WT and V259F mutant proteins. The contacts were calculated using the last 100 ns of 10 runs (1 μs in total). The colors denote the ratios of contact in the simulation time. The five most contacted residue pairs in WT proteins are denoted with numbers. We defined contact as any heavy atom of the residue-pair within 4 Å. (D) (a) The WT structure closest to the averaged structures over 1 μs trajectories. The protein backbone is drawn in a ribbon representation (cyan). The heavy atoms of the five most contacted residue pairs are depicted with a space-filling model; see also (c). (b) The V259Fmutant structure closest to the averaged structures over 1 μs trajectories. The heavy atoms of the five most contacted residue pairs in the WT are depicted in the space-filling model. The residue pairs in contact in the WT were completely lost. (c) Contact ratio of residue pairs in the 1 μs trajectories. The residues in the list are shown in (a,b) with distinct colors. Underlined residues appeared twice in the list. (E) (a) Backbone-RMSD of the whole POUh domain except for the N-terminal region (residues 201 to 206) against the X-ray crystallographic structure. (b) Fluctuation of the N-terminal region. The fluctuation was calculated using the RMSD-fitted structures of (a). (c) Solvent-accessible surface area (SASA) of the sidechain atoms of residue at position 259: Val of the WT is depicted in blue, and mutated residue Phe, in red. Dotted lines show the boundaries of runs. (F) A typical snapshot of the WT (a) and V259F mutant proteins (b) in the trajectory. The 259th residue and the N-terminal region are depicted by red and green/yellow, respectively. (c) The conformations in the N-terminal region of the WT. The images depict 10 structures taken from the last snapshots of 10 runs in green, and V259 is denoted by red in the space-filling model. (d) The conformations in the N-terminal region of the V259F mutant. The images depict 10 structures taken from the last snapshots of 10 runs in yellow, and F259 is denoted by red in the space-filling model.

Figure 4

HuH7 cells were transfected with 20 nM concentrations of either control siRNA (SIC; MISSION® siRNA Universal Negative Control #1) or HNF4A-specific siRNAs. (A) The KD efficiency was verified by RT-PCR. (B) Western blot analysis shows that HNF4A expression was decreased in HNF4A-siRNA-treated HuH7 cells. (C) The number of differentially expressed genes (DEGs) in HNF4A-knockdown (KD) cells. (D) Volcano plot map representing DEGs in KD cells. Red dots represent upregulated genes, green dots show downregulated genes, and gray represents non-DEGs in KD cells. (E) Top 20 KEGG pathways in KD cells. (F) GO analyses of the top 10 biological processes. (G) Protein–protein interaction of downregulated genes. Green asterisk indicates HNF4A, and blue asterisk indicates HNF1A in the network.