Loss-of-function mutations in Zn-finger DNA-binding domain of HNF4A cause aberrant transcriptional regulation in liver cancer

Hepatocyte nuclear factors (HNF) are transcription factors that crucially regulate cell-specific gene expression in many tissues, including the liver. Of these factors, HNF4A acts both as a master regulator of liver organogenesis and a tumor suppressor in the liver. In our whole genome sequencing analysis, we found seven somatic mutations (three Zn-finger mutations, three deletion mutants, and one intron mutation) of HNF4A in liver cancers. Interestingly, three out of seven mutations were clustered in its Zn-finger DNA-binding domain; G79 and F83 are positioned in the DNA recognition helix and the sidechain of M125 is sticking into the core of domain. These mutations are likely to affect DNA interaction from a structural point of view. We then generated these mutants and performed in-vitro promoter assays as well as DNA binding assays. These three mutations reduced HNF4 transcriptional activity at promoter sites of HNF4A-target genes. Expectedly, this decrease in transcriptional activity was associated with a change in DNA binding. RNA-Seq analysis observed a strong correlation between HNF4A expression and expression of its target genes, ApoB and HNF1A, in liver cancers. Since knockdown of HNF4A caused a reduction in ApoB and HNF1A expression, possibly loss of HNF4 reduces the expression of these genes and subsequently tumor growth is triggered. Therefore, we propose that HNF4A mutations G79C, F83C, and M125I are functional mutations found in liver cancers and that loss of HNF4A function, through its mutation, leads to a reduction in HNF1A and ApoB gene expression with a concomitant increased risk of liver tumorigenesis.