Cross Talk Between GH-Regulated Transcription Factors HNF6 and CUX2 in Adult Mouse Liver.

Hepatocyte-enriched nuclear factor (HNF)6 and CUX2 are GH and STAT5-regulated homeobox transcription factors. CUX2 shows female-specific expression and contributes to liver sex differences by repressing many male-biased genes and inducing many female-biased genes, whereas HNF6 is expressed at similar levels in male and female liver. In cell-based transfection studies, CUX2 inhibited HNF6 transcriptional regulation of the sex-specific gene promoters CYP2C11 and CYP2C12, blocking HNF6 repression of CYP2C11 and HNF6 activation of CYP2C12. These inhibitory actions of CUX2 can be explained by competition for HNF6 DNA binding, as demonstrated by in vitro EMSA analysis and validated in vivo by global analysis of the HNF6 cistrome. Approximately 40 000 HNF6-binding sites were identified in mouse liver chromatin, including several thousand sites showing significant sex differences in HNF6 binding. These sex-biased HNF6-binding sites showed strong enrichment for correspondingly sex-biased DNase hypersensitive sites and for proximity to genes showing local sex-biased chromatin marks and a corresponding sex-biased expression. Further, approximately 90% of the genome-wide binding sites for CUX2 were also bound by HNF6. These HNF6/CUX2 common binding sites were enriched for genomic regions more accessible in male than in female mouse liver chromatin and showed strongest enrichment for male-biased genes, suggesting CUX2 displacement of HNF6 as a mechanism to explain the observed CUX2 repression of male-biased genes in female liver. HNF6 binding was sex independent at a majority of its binding sites, and HNF6 peaks were frequently associated with cobinding by multiple other liver transcription factors, consistent with HNF6 playing a global regulatory role in both male and female liver.

Tumour suppressor p53 down-regulates the expression of the human hepatocyte nuclear factor 4alpha (HNF4alpha) gene.

The liver is exposed to a wide variety of toxic agents, many of which damage DNA and result in increased levels of the tumour suppressor protein p53. We have previously shown that p53 inhibits the transactivation function of HNF (hepatocyte nuclear factor) 4alpha1, a nuclear receptor known to be critical for early development and liver differentiation. In the present study we demonstrate that p53 also down-regulates expression of the human HNF4alpha gene via the proximal P1 promoter. Overexpression of wild-type p53 down-regulated endogenous levels of both HNF4alpha protein and mRNA in Hep3B cells. This decrease was also observed when HepG2 cells were exposed to UV irradiation or doxorubicin, both of which increased endogenous p53 protein levels. Ectopically expressed p53, but not a mutant p53 defective in DNA binding (R249S), down-regulated HNF4alpha P1 promoter activity. Chromatin immunoprecipitation also showed that endogenous p53 bound the HNF4alpha P1 promoter in vivo after doxorubicin treatment. The mechanism by which p53 down-regulates the P1 promoter appears to be multifaceted. The down-regulation was partially recovered by inhibition of HDAC activity and appears to involve the positive regulator HNF6alpha. p53 bound HNF6alpha in vivo and in vitro and prevented HNF6alpha from binding DNA in vitro. p53 also repressed stimulation of the P1 promoter by HNF6alpha in vivo. However, since the R249S p53 mutant also bound HNF6alpha, binding HNF6alpha is apparently not sufficient for the repression. Implications of the p53-mediated repression of HNF4alpha expression in response to cellular stress are discussed.