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

Abstract

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.

Transcription factor activity rhythms and tissue-specific chromatin interactions explain circadian gene expression across organs

“…4C). Enrichment of both CUX2 and ONECUT (also named HNF6) is consistent with ONECUT1 binding to both ONECUT and CUX2 motifs (Conforto et al 2015). mRNAs of genes with co-occurrence of RORE and liver-specific TF motifs peaked near ZT1, consistent with peak RORE activity (near ZT21) preceding peak mRNA abundance of REV-ERB targets (Supplemental Fig.…”

Hepatic Long Intergenic Noncoding RNAs: High Promoter Conservation and Dynamic, Sex-Dependent Transcriptional Regulation by Growth Hormone

“…Male-biased binding of four GH-regulated transcriptional activators, STAT5, FOXA1, FOXA2, and HNF6 (53,54,56), was significantly enriched at sites near (within 10 kb) the set of 85 stringent (i.e., GH-regulated) male-specific lincRNAs and was significantly depleted near the set of 86 stringent female-specific lincRNAs lincRNAs that had an ortholog in at least 1 of the 10 species investigated. The orthologs were required to span Ն10% of the full-length, mature mouse lincRNA transcript and to be Ն600 bp in length (see Materials and Methods).…”

“…We investigated whether sex-specific lincRNA genes are enriched for nearby sites of sex-dependent chromatin accessibility, which bind TFs in a sex-dependent manner (54)(55)(56) and can be detected as sex-biased DHS (52). We found that the stringent (i.e., GH-regulated) male-specific lincRNAs are significantly enriched for nearby (within 10 kb) male-biased DHS (P ϭ 5.77eϪ11) and depleted of female-biased DHS.…”

“…Next, we investigated whether sex-specific lincRNA genes are enriched for binding TFs previously implicated in GH regulation of sex-specific protein-coding genes in mouse liver (54)(55)(56)83). Male-biased binding of four GH-regulated transcriptional activators, STAT5, FOXA1, FOXA2, and HNF6 (53,54,56), was significantly enriched at sites near (within 10 kb) the set of 85 stringent (i.e., GH-regulated) male-specific lincRNAs and was significantly depleted near the set of 86 stringent female-specific lincRNAs lincRNAs that had an ortholog in at least 1 of the 10 species investigated.…”

“…The resulting sex differences in plasma GH profiles activate the TF STAT5 (48) by JAK2-catalyzed tyrosine phosphorylation (49) in a dynamic, sex-differential manner (50,51), which is linked to downstream sex differences in liver chromatin accessibility and epigenetic (histone) marks (52,53). These sex-differential chromatin states are associated with sex differences in the binding of STAT5 and other GH-regulated TFs (54)(55)(56), which together regulate the sex-biased transcription of ϳ1,000 liver-expressed genes (57)(58)(59). Little is known, however, about the mechanisms whereby GH establishes and maintains the unique, sex-specific chromatin states that are closely linked to sex-biased liver gene transcription (53).…”