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. 1998 Jul;18(7):4245-51.
doi: 10.1128/MCB.18.7.4245.

Targeted disruption of the gene encoding hepatocyte nuclear factor 3gamma results in reduced transcription of hepatocyte-specific genes

K H Kaestner  1 , H Hiemisch, G Schütz
Affiliations

Targeted disruption of the gene encoding hepatocyte nuclear factor 3gamma results in reduced transcription of hepatocyte-specific genes

K H Kaestner et al. Mol Cell Biol. 1998 Jul.

Abstract

The winged helix transcription factor hepatocyte nuclear factor 3gamma (HNF3gamma) is expressed in embryonic endoderm and its derivatives liver, pancreas, stomach, and intestine, as well as in testis and ovary. We have generated mice carrying an Hnf3g-lacZ fusion which deletes most of the HNF3gamma coding sequence as well as 5.5 kb of 3' flanking region. Mice homozygous for the mutation are fertile, develop normally, and show no morphological defects. The mild phenotype change of the Hnf3g-/- mice can be explained in part by an upregulation of HNF3alpha and HNF3beta in the liver of the mutant animals. Analysis of steady-state mRNA levels as well as transcription rates showed that levels of expression of several HNF3 target genes (phosphoenolpyruvate carboxykinase, transferrin, tyrosine aminotransferase) were reduced by 50 to 70%, indicating that HNF3gamma is an important activator of these genes in vivo.

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Figures

FIG. 1
FIG. 1
Targeting strategy for Hnf3g inactivation. (A) (Top line) Gene structure of the Hnf3g locus. (Middle line) Targeting vector used for homologous recombination in embryonic stem cells. (Bottom line) Gene structure of the targeted allele. Probes A, B, and C are referred to in the text. B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; N, NotI; S, SalI; Xh, XhoI; lacZ-neo, fusion gene encoding β-galactosidase and neomycin phosphotransferase. (B) Southern blot analysis of the correctly targeted ES cell clones (heterozygous for the Hnf3g mutation). DNA of wild-type and heterozygous ES cell clones was digested with HindIII (H) or EcoRI (E), size fractionated by agarose gel electrophoresis, blotted onto nylon membrane, and hybridized with probe C. (C) RNase protection analysis of 40 μg of total liver RNA isolated from wild-type (+/+) or homozygous mutant (−/−) mice. M, marker lane; P, undigested probes; tRNA, control. Protected fragments for HNF3γ, HNF3β, HNF3α, and TATA-box binding protein (TBP) are labeled with arrows.
FIG. 2
FIG. 2
The 3′-flanking region of the HNF3γ gene is required for β-galactosidase expression in small intestine, pancreas, and liver. Embryos (E14.5) were obtained from matings between heterozygous parents, genotyped by PCR, and stained for β-galactosidase activity as described in Materials and Methods. (A) Staining in the nasal epithelium. (B) No staining is detectable in the small intestine (i), pancreas (p), and liver (l). (C) A section through the colon of the same embryo shows strong staining in the colonic epithelium, but not in mesenchyme. The scale bar represents 100 μm.
FIG. 3
FIG. 3
Hematoxylin and eosin staining of Hnf3g−/− embryos. Embryos (E16.5) were obtained from matings between heterozygous parents, genotyped by PCR, paraffin embedded, sectioned, and stained. A wild-type embryo (A) and homozygous mutant embryo (B) of the same litter are shown. li, liver; pi, pancreatic islet; mg, midgut.
FIG. 4
FIG. 4
mRNAs for HNF3α and HNF3β, but not HNF1α, HNF1β, and HNF4, are upregulated in liver from Hnf3g−/− mice. (A) RNase protection analysis of HNF3α and HNF3β mRNA levels in liver RNA (40 μg) from wild-type (striped bars) and Hnf3g−/− (open bars) mice were normalized to those of TATA-box binding protein (TBP). Radioactive bands were quantified by phosphorimager analysis. Values are means ± standard errors (n = 5), and differences between wild-type and −/− mRNA levels were statistically significant (P < 0.05 for HNF3β; P < 0.01 for HNF3α) by Student’s t test. (B) RNase protection analysis of HNF3α and HNF3β mRNA levels in stomach RNA (40 μg) from wild-type (striped bars) and Hnf3g−/− (open bars) mice. Values are means ± standard errors (n = 3). (C) RNase protection analysis of HNF3α and HNF3β mRNA levels in colon RNA (40 μg) from wild-type (striped bars) and Hnf3g−/− (open bars) mice. Values are means ± standard errors (n = 3). (D) RNase protection analysis of 40 μg of total liver RNA isolated from wild-type (striped bars) and Hnf3g−/− (open bars) mice was performed with probes specific to HNF1α, HNF1β, and HNF4 and with TBP as a control. Signals were analyzed as by phosphorimager and normalized to TBP (×0.1 for HNF4α). Values are means ± standard errors (n = 5).
FIG. 5
FIG. 5
Analysis of steady-state mRNA levels and transcription rates of potential HNF3γ targets in liver. (A) Total RNA (10 μg) from livers of wild-type (+/+) or homozygous mutant (−/−) mice was separated on denaturing agarose gels, blotted onto nylon membrane, and hybridized to the probes indicated. Cytochrome c oxidase (COX) served as a loading control. (B) The filters obtained in panel A were quantified by phosphorimager analysis, and the mRNA values (arbitrary units [arb.]) were expressed relative to those for COX. Values are means ± standard errors (n = 4); differences between wild-type and −/− mRNA levels were statistically significant (P < 0.05) by Student’s t test. (C) Nuclear run-on transcription assays were performed with liver nuclei isolated from wild-type (hatched bars) and homozygous mutant (open bars) mice as described in Materials and Methods. The signals obtained were quantified by phosphorimager analysis, normalized to those of glyceraldehyde phosphate dehydrogenase, and expressed as percentages of wild-type levels. Values are means ± standard errors (n = 3); differences between wild-type and −/− mRNA levels were statistically significant (P < 0.05) for TAT, PEPCK, and Tf by Student’s t test.
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