Generation of a conditional null allele of the HNF4α gene.
A triple-lox targeting vector was constructed; in this vector a phosphoribosyltransferase (neo) selection cassette flanked by loxP sites was introduced into intron 5 of the mouse HNF4α gene along with a third loxP site in intron 3 (Fig. A). Following standard electroporation and culture of ES cells, homologous recombinants were identified by Southern blotting of genomic DNA. All three loxP sites were incorporated into the HNF4α locus in 8 of 320 ES cells screened (Fig. B). A single clone (clone 300) was injected into C57BL/6 blastocysts to generate chimeric mice. The triple-lox, targeted allele (t) was transmitted through the germ line. However, extensive intercrosses between heterozygous (t/+) mice failed to yield homozygous targeted mice. Thus, the targeted allele was nonfunctional, most likely due to interference with HNF4α gene expression by the neo cassette.
In order to delete the neo
cassette and thus rescue the mouse line, the targeted allele was crossed into a transgenic line expressing Cre under the control of the adenovirus EIIa promoter (EIIaCre) (27
). EIIaCre-mediated recombination occurs early in development (two to eight cells) and at low levels, resulting in chimerism. Thus, it was hoped to selectively delete the neo
cassette and thereby generate a heritable, active conditional null allele (Fig. C). Recombination events were analyzed by Southern blotting of tail DNA (isolated from pups from the cross HNF4αt/+
). This indicated the presence of all possible deletion patterns (Fig. D). Chimeric mice were selected according to the observed pattern of deletion and crossed to wild-type mice in order to generate lines carrying floxed (male 44) and null (male 45) alleles of the HNF4α gene. As predicted, the floxed allele, containing just small loxP sites flanking exons 4 and 5 of the HNF4α gene (Fig. C), was active and viable at homozygosity.
Deletion of exons 4 and 5 is predicted to result in the splicing of exon 3 to exon 6. This results in a frameshift, leading to a premature stop codon. Translation of the truncated mRNA results in an N-terminal peptide of just 128 amino acid residues (118 residues of HNF4α followed by 10 missense residues). While this protein retains both zinc finger motifs, it lacks both the A and T boxes necessary for high-affinity binding to DNA (14
), along with the ligand-binding and activation function 2 domains. Significantly, a mutant HNF4α protein containing 125 N-terminal residues fails to bind DNA at room temperature, although some low-affinity binding is observed at 4°C (18
). The putative truncated protein also lacks sequences required for the recruitment of coactivator proteins CREB binding protein (CBP), SRC-1, and GRIP1 (10
). Given that CBP-mediated acetylation of HNF4α is required for its retention in the nucleus (50
), it is likely that any truncated protein produced would be quickly exported to the cytoplasm and therefore unable to interfere with transcription. While we cannot rule out subtle dominant-negative effects due to retention of some DNA binding, heterozygous animals were normal for all parameters measured (not shown), suggesting that this is not the case. Moreover, consistent with the embryonic lethality of germ line inactivation of HNF4α, crosses between the heterozygous null mice produced here (HNF4α+/−
) failed to yield homozygous knockouts. Thus, the available evidence strongly suggests that deletion of exons 4 and 5 results in a null allele.
High-efficiency, liver-specific disruption of HNF4α.
To examine the role of HNF4α in the maintenance of hepatic gene expression and liver function, the active floxed allele was crossed into mice hemizygous for an albumin-Cre transgene (AlbCre). that express Cre exclusively in the postpartum liver (66
). The (HNF4αfl/+
mice so generated were interbred with HNF4αfl/+
littermates lacking the AlbCre transgene. This breeding scheme yielded (HNF4αfl/fl
mice (designated H4LivKO) and three groups of littermate control mice, HNF4αfl/fl
(AlbCre), and HNF4α+/+
(wild type). Genotypes of all mice were assessed by PCR analysis of tail DNA (Fig. E). All alleles were inherited in a Mendelian fashion (Fig. E), suggesting that neither the floxed allele, the AlbCre transgene, nor the combination thereof resulted in any prenatal lethality.
To assess the time course, extent, and specificity of Cre-mediated recombination at the HNF4α locus, Northern blots of total liver RNA from 2-, 4-, and 6-week-old H4LivKO and control animals were probed with a fragment of the HNF4α cDNA homologous to exons 4 and 5 (Fig. A). Deletion of the floxed exons of HNF4α was about 50% by 4 weeks of age, and, by 6 weeks of age, mRNA signal intensity in H4LivKO livers was below the limit of detection (Fig. A and B). Thus, intact HNF4α mRNA containing exons 4 and 5 is reduced to <10% that seen in control livers at 6 weeks of age. This time course is similar to that reported for a retinoid-X receptor-α (RXRα) floxed allele using the albumin promoter driving Cre expression (60
). All further experiments were conducted using 45-day-old mice.
FIG. 2 Exons 4 and 5 are deleted efficiently and specifically in the livers of H4LivKO mice. (A) Time course of Cre-mediated recombination at the HNF4α locus. Liver RNA from H4LivKO and control animals was subjected to Northern blotting and probed with (more ...)
Northern blots of liver RNA probed with a 3′ fragment of the HNF4α cDNA revealed that H4LivKO mice expressed an mRNA that migrated with greater mobility than that from wild-type, AlbCre, or H4Flox mice (Fig. B). Reverse transcription-PCR of HNF4α cDNA from H4LivKO liver RNA failed to detect the full-length transcript. Sequencing the cloned, truncated cDNA revealed that the expected exon 3-to-exon 6 splice event had occurred (not shown). The steady-state level of the truncated HNF4α mRNA in H4LivKO hepatocytes is about 30% that seen for the undisrupted message in controls. This is consistent with the suggestion that HNF4α regulates its own expression (51
) and/or that the abnormal transcript is less stable than the wild-type message.
Disruption of HNF4α RNA resulted in a corresponding loss of full-length protein as evidenced by Western blots of nuclear protein probed with an antibody raised to a C-terminal peptide of HNF4α (Fig. C). Equal loading of nuclear protein was demonstrated by reprobing the membrane with an antibody to histone H1. Thus, H4LivKO mouse liver nuclei contain less than 10% of the immunoreactive HNF4α protein present in controls.
To confirm that deletion of exons 4 and 5 was restricted to the liver, RNA from kidney and intestine, which also express HNF4α, was subjected to Northern blotting and probed with a probe consisting of exons 4 and 5. As expected, H4LivKO mice expressed full-length HNF4α mRNA in both tissues at levels similar to those found in wild-type, AlbCre, and H4Flox mice (Fig. D). Thus, HNF4α mRNA and protein were efficiently and specifically disrupted in the livers of adult H4LivKO mice by 45 days of age.
Hepatomegaly, hepatocyte hypertrophy, and abnormal glycogen and lipid deposition in livers of H4LivKO mice.
To begin to assess the influence of disruption of HNF4α on hepatic function, liver weights and pathology were examined. Livers from 45-day-old H4LivKO mice were significantly enlarged relative to those of controls (Table ) and had a visibly gray, mottled appearance. Marked pathological lesions were evident in H4LivKO mouse livers but were absent in controls (Fig. ). These lesions were more severe in male mice than in females. Centrilobular hepatocytes in H4LivKO mice were invariably hypertrophic, with pale eosinophilic intracytoplasmic inclusions (Fig. B and C). Hepatocytes throughout the liver lobule were markedly vacuolated (Fig. B). Although the material accumulating in the vacuoles had the typical morphology of glycogen, it did not stain PAS-positive in alcoholic formalin-fixed sections (Fig. D and E). Rather, the majority of this material stained positive for fat with oil red O stain (Fig. F and G).
Liver weights and serum chemistries of conditionally HNF4α-null mice and controlsa
FIG. 3 Histological analysis of the livers of H4LivKO mice. (A) Normal liver of a 45-day-old wild-type (+/+) mouse. Hepatocytes around the central vein have few vacuoles present. Shown is an H&E stain. Magnification, ×300. (B) (more ...)
To further characterize these lesions, ultrastructure was analyzed. This revealed that most hepatocytes had abundant lipid droplets and large mitochondria (Fig. H, l and m, respectively), while others had both lipid droplets and abundant glycogen-like material (Fig. H, l and g, respectively). These abnormal cytoplasmic changes were not seen in control livers.
Liver-specific deletion of HNF4α alters serum lipid levels.
Consistent with the unusual fatty-liver phenotype observed in histological sections of H4LivKO livers, total cholesterol, HDL cholesterol, and triglyceride levels in sera from H4LivKO mice were dramatically reduced relative to those in sera from controls (Table ). Conversely, serum bile acid concentrations were markedly elevated (Table ). These alterations in serum lipid profiles could be due to either a generalized liver failure or to a more specific defect in lipid transport and metabolism. Mild liver dysfunction was indicated by a small elevation in the level of alanine aminotransferase in serum from H4LivKO mice (Table ). However, the levels of albumin, nonesterified fatty acids, and glucose were indistinguishable from those in controls, suggesting the retention of many liver functions (Table ). Thus, at this time point, disruption of HNF4α in the liver appears to induce a specific failure of normal hepatic lipid metabolism and/or transport rather than a generalized liver failure.
FPLC analysis of serum lipids.
To further characterize the observed alterations in blood lipid composition, plasma from H4LivKO and H4Flox mice was subjected to FPLC analysis. Compared to controls, H4LivKO mice had significantly reduced plasma cholesterol (−61%) and phospholipids (−53%) (Fig. ). Moreover, the elution profile of the lipids was altered (Fig. ). Thus, both plasma low-density lipoprotein (LDL) and HDL cholesterol were dramatically decreased in H4LivKO mice compared to controls. Moreover, HDL cholesterol from H4LivKO plasma eluted later than that from controls and contained a significant amount of very-late-eluting, smaller HDL. This elution profile is indicative of production of unusually small, lipid-poor HDL particles. Western blot analysis of lipoprotein content in whole plasma from H4LivKO mice indicated that these mice exhibit reduced ApoB100, ApoA-II and ApoC content, while apolipoproteins A-I, E, and B48 are unaffected (Fig. , inset). Strikingly, HDL cholesterol from H4LivKO mice is virtually devoid of ApoA-II, with ApoA-I as the sole apolipoprotein component (Fig. , inset).
FIG. 4 Serum lipoprotein profiles of control and H4LivKO mice. Lipoproteins were separated from 60 μl of pooled mouse plasma samples by FPLC (n = 4 for each group). Profiles from H4Flox (control; open squares) and H4LivKO (solid squares) mice are shown. (more ...) HNF4α regulates hepatic genes involved in lipid metabolism and transport.
The HNF4α-mediated transactivation of numerous genes involved in lipid metabolism and transport has been demonstrated using cell culture systems. These include genes for ApoA-I (16
), ApoA-II (44
), ApoA-IV (21
), ApoB (37
), ApoC-II (19
), ApoC-III (38
), and ApoE (9
); MCAD (3
); microsomal triglyceride transfer protein (MTP) (15
); and cholesterol 7α-hydroxylase (CYP7A) (8
). To examine whether the expression of these putative target genes was affected by disruption of the hepatic HNF4α gene, liver RNA from 45-day-old H4LivKO mice and controls was analyzed (Fig. ). Strikingly, steady-state mRNA levels for apolipoproteins A-II, A-IV, C-II, and C-III and MTP (Fig. A) and CYP7A1 (Fig. E) were drastically reduced in H4LivKO livers compared to those in controls. In contrast, steady-state mRNA levels for ApoA-I and ApoE were relatively unaffected by disruption of the HNF4α gene (Fig. A), even though both of the genes are HNF4α responsive in transient transfection assays (9
). Paradoxically, while MCAD is positively regulated by HNF4α in cultured cells (4
), it is induced by disruption of the HNF4α gene (Fig. D). To further examine the influence of the deletion of hepatic HNF4α on lipid metabolism, the expression of numerous other key genes involved in lipid transport and metabolism was examined. Expression of the LDL receptor and ATP-binding cassette 1, both of which are essential lipid and cholesterol transporters, was unaffected, while expression of the major HDL receptor, SR-BI, was induced (Fig. B). The expression levels of several nuclear receptors implicated in the control of lipid homeostasis, RXRα, pregnane-X receptor, farsenoid-X receptor (FXR), oxysterol receptor-α, and liver receptor homologue 1, were unchanged by deletion of HNF4α (Fig. C). Expression of the nuclear receptor small heterodimer partner was variable (Fig. C). In contrast, expression of peroxisome proliferator-activated receptor α (PPARα) was lower in H4LivKO livers than in controls (Fig. D). Given this decrease in PPARα, it is interesting to note that expression of some PPARα target genes (carnitoyl-palmitoyl transferase-II, MCAD, and 3-hydroxy-3-methylglutaryl CoA synthase genes) is enhanced in H4LivKO livers relative to controls (Fig. D). The increased levels of serum bile acids suggested that these mice might be deficient in one or more pathways of bile acid trafficking. Consistent with this hypothesis, levels of sodium taurocholate cotransporter protein (Ntcp), organic anion transporter protein 1, liver fatty acid binding protein (L-FABP), and multidrug resistance protein 2 mRNA were markedly decreased in H4LivKO mice relative to controls (Fig. E). In contrast, the steady-state level of bile salt export pump (BSEP) mRNA was mildly elevated (Fig. E). Finally, expression of the key lipogenic genes encoding fatty acid synthase, sterol receptor element binding protein 1c, and spot-14 genes was unaffected in H4LivKO livers (Fig. F). Thus, consistent with the histological and serological alterations observed in these mice, disruption of hepatic HNF4α results in altered expression of genes involved in several pathways of lipid metabolism and transport. Conversely, the unchanged expression of other hepatic genes again argues that the observed dyslipidemia is due to disruption in a specific pathway(s) and not due to a generalized liver failure.
FIG. 5 Northern blot analysis of liver RNA from H4LivKO and control animals. Total liver RNA (10 μg) was separated on 0.22 M formaldehyde–1% agarose gels and blotted to GeneScreen Plus membranes. Blots were hybridized in UltraHyb at 42°C (more ...) Lethality associated with disruption of hepatic HNF4α.
To assess the influence of HNF4α deletion in liver on mouse development, mice were weighed at 10 days and 2 weeks of age and each week thereafter. The H4LivKO mice had no observable phenotype until they reached 5 weeks of age when they lost weight compared with the wild-type, AlbCre transgenic, and H4Flox mice (not shown). This weight loss coincided with the complete loss of HNF4α in the livers (between 4 and 6 weeks). To assess whether this weight loss was terminal, a cohort of mice was allowed to develop without further interference. In this group mortality reached >70% by 8 weeks of age. Thus, while we are as yet unable to explain the observed mortality at the functional level, this indicates that HNF4α activity is essential for the proper functioning of the liver.