Quantitative trait locus mapping and identification of Zhx2 as a novel regulator of plasma lipid metabolism

Abstract

Background: We previously mapped a quantitative trait locus on chromosome 15 in mice contributing to high-density lipoprotein cholesterol and triglyceride levels and now report the identification of the underlying gene.

Methods and results: We first fine-mapped the locus by studying a series of congenic strains derived from the parental strains BALB/cJ and MRL/MpJ. Analysis of gene expression and sequencing followed by transgenic complementation led to the identification of zinc fingers and homeoboxes 2 (Zhx2), a transcription factor previously implicated in the developmental regulation of alpha-fetoprotein. Reduced expression of the protein in BALB/cJ mice resulted in altered hepatic transcript levels for several genes involved in lipoprotein metabolism. Most notably, the Zhx2 mutation resulted in a failure to suppress expression of lipoprotein lipase, a gene normally silenced in the adult liver, and this was normalized in BALB/cJ mice carrying the Zhx2 transgene.

Conclusions: We identified the gene underlying the chromosome 15 quantitative trait locus, and our results show that Zhx2 functions as a novel developmental regulator of key genes influencing lipoprotein metabolism.

Figure 1

Genotype of selected subcongenic strains. Genotypes of derived subcongenic strains and parental CON15 strain at the polymorphic microsatellite markers used for genotyping (M indicates MRL/MpJ allele; B, BALB/cJ allele). Segregation of genotypes with male plasma cholesterol levels (depicted as mean±SEM in mg/dL) was used to narrow the Hyplip2 locus within the 5-Mbp critical region bordered by markers D15Mit184 and D15Mit46.

Figure 2

Expression analysis of genes within the critical region and identification of Zhx2 as a Hyplip2 candidate. A, Liver expression of genes located within the critical region (between markers D15Mit184 and D15Mit46) was analyzed by quantitative PCR in BALB/cJ and Sub6 males as described in Methods section. Results are expressed as the mean differences in expression levels of each gene in the Sub6 strain relative to BALB/cJ strain (set to 1) ± 1 SD. *Significantly different mean expression value from control (P<0.05). ND indicates genes below detectable levels. Liver mRNA levels of the BC030396 gene in both strains were very low and bordering detection limits of quantitative PCR. P values are shown only for genes with mean expression values differing by >2-fold. B, Retroviral insertion in Zhx2 gene segregates with plasma cholesterol levels in CON15 and subcongenic strains. DNA isolated from individual strains was subjected to genotyping for the presence of retrovirus insertion in Zhx2. As indicated in Methods section, the presence of a 342-bp product indicates the wild-type allele, whereas absence of the 342-bp product indicates presence of a homozygous mutant allele. As a PCR quality control, the region within the third exon of the Zhx2 gene was also amplified (776-bp product). Plasma cholesterol levels in BALB/cJ, Con15, and subcongenic strains (male mice) are shown below the Zhx2 genotypes. For subcongenic strains, heterozygous and homozygous male cholesterol values were combined and expressed as mean±SEM.

Figure 3

Liver-specific transgenic expression of Zhx2 in BALB/cJ mice increases plasma cholesterol and triglycerides. Liver-specific Zhx2 transgenic mice were repeatedly backcrossed to BALB/cJ, and mice homozygous for endogenous BALB/cJ Zhx2 allele were selected. These mice containing the Zhx2 transgene were then mated with BALB/cJ mice to generate littermates with and without the transgene. Plasma lipid levels were measured in 8-week-old fasted mice. Values are expressed as mean±SEM.

Figure 4

Quantitative PCR analysis of selected genes in livers of Sub13, BALB/cJ, and Zhx2 transgenic strains. BALB/cJ, Sub13, and littermate mice±Zhx2 transgene were fasted overnight and euthanized, and expression of selected genes was analyzed as described in Methods section. In addition to selected genes modulating plasma lipid metabolism, expression of Zhx2 and Ear11 (the most highly differentially expressed gene on the expression array; see supplemental Table VII) was also measured. Results are expressed as the mean differences in expression levels for the indicated strains relative to BALB/cJ littermates (set to 1) ±1 SD. *Significantly different mean expression value from control (P<0.05).

Figure 5

Analysis of mRNA expression levels during murine liver development in Sub13 and BALB/cJ strains. BALB/cJ and Sub13 male mice (n=5 per group) were euthanized at indicated time points after birth, and mRNA levels of Zhx2, Lpl, and Ear11 were analyzed by quantitative PCR as described in Methods. Results are expressed as the mean differences in expression levels for the indicated time points relative to expression in BALB/cJ at birth (set to 1) ±1 SD.