Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2010 Mar 29.
Published in final edited form as: Circ Cardiovasc Genet. 2009 Dec 30;3(1):60–67. doi: 10.1161/CIRCGENETICS.109.902320

Quantitative Trait Locus Mapping and Identification of Zhx2 as a Novel Regulator of Plasma Lipid Metabolism

Peter S Gargalovic 1, Ayça Erbilgin 1, Omid Kohannim 1, Joanne Pagnon 1, Xuping Wang 1, Lawrence Castellani 1, Renee LeBoeuf 1, Martha L Peterson 1, Brett T Spear 1, Aldons J Lusis 1
PMCID: PMC2846770  NIHMSID: NIHMS183227  PMID: 20160197

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 α-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.

Keywords: genetics, lipoproteins, atherosclerosis, genes, mapping

Despite several large-scale genome-wide association studies, the genetic factors contributing to lipoprotein metabolism remain poorly understood. Thus, the loci identified thus far explain <20% of the hereditary component of lipoprotein levels, except for lipoprotein(a).1–5 This is explained in part by complex gene-by-environment interactions, rare variations, and population heterogeneity.6 Moreover, some of the novel loci contain multiple genes or genes with no known connections to lipoprotein metabolism, and establishing their functions based on human studies will be difficult.7

A complementary approach to understand the biology and genetics of lipoprotein metabolism is to study natural variations in experimental organisms such as mice and rats.8 Although the common variations contributing to interindividual differences for complex traits are unlikely to be conserved between species, one would expect a level of conservation of the pathways involved. Unfortunately, efforts to identify genes for complex traits in mice and rats have rarely been successful.9 A major problem has been the lack of mapping resolution in quantitative trait locus (QTL) analyses.10 We report here the dissection of a QTL for lipoprotein levels in mice with a strategy based on the analysis of congenic and subcongenic strains, followed by screening for structural and regulatory gene variations.

We previously mapped a locus on chromosome 15 (Chr.15, named Hyplip 2) contributing to complex variations in total cholesterol, high-density lipoprotein cholesterol, and triglyceride levels in a genetic cross between strains BALB/cJ and MRL/MpJ (MRL).11 To validate the QTL mapping results, the Chr.15 region from MRL was introgressed onto the background of BALB/cJ strain. Analysis of the congenic strain, named CON15, confirmed the QTL findings and revealed a striking influence of the locus on susceptibility to diet-induced atherosclerosis.12 Further biomedical studies of the congenic strain showed that the variation influenced clearance of triglyceride-rich lipoproteins rather than their production.13

In this study, the congenic strain, encompassing >100 Mbp, was narrowed to ≈5 Mbp by analysis of subcongenic strains. Examination of the genes residing in the region by sequencing and quantification of transcript levels identified a strong candidate, the zinc fingers and homeoboxes 2 (Zhx2) transcription factor, previously shown to contribute to the developmental regulation of α-fetoprotein.14 We confirmed that Zhx2 variation was indeed responsible for the lipoprotein alterations in mice by using transgenic complementation and showed that a major impact is altered expression of lipoprotein lipase (Lpl), an enzyme central to the metabolism of triglyceride-rich lipoproteins.

Methods

Animals and Diets

Strain BALB/cJ mice were obtained from the Jackson Laboratory (Bar Harbor, Me). Strain BALB/cH was obtained from Harlan (Indianapolis, Ind). CON15 was constructed as described previously.12 Mice were fed a standard rodent chow diet containing 4% fat (Ralston-Purina Co., St. Louis, Mo.).

Developmental Curves

BALB/cJ and Sub13 mice were euthanized at birth and 5, 14, and 60 days after birth. The livers were removed and immediately frozen in liquid nitrogen. Five livers were taken for each time point from each strain. RNA was extracted with use of the Qiagen RNeasy kit, and cDNA was synthesized with use of the Applied Biosystems cDNA kit. Expression levels were assessed by quantitative real-time polymerase chain reaction (PCR).

Liver-Specific Zhx2 Transgenic Mice

A liver-specific transthyretin promoter/enhancer cassette identical to that used in a previously published study14 was used to generate Zhx2 transgenic mice. Three independent founders were crossed to BALBc/J. All 3 transgenic founders expressed Zhx2 at a very similar level and corrected the elevated α-fetoprotein phenotype. Offspring of 1 representative founder were transferred to the University of California, Los Angeles Animal Facility, and used for further breeding and lipid analysis. To generate mice for data collection, these mice were first bred among themselves, and progeny containing the transgene were backcrossed to BALB/cJ mice. Mice were bled under isoflurane anesthesia after overnight fasting at 8 weeks of age after having been fed a chow diet. Blood was collected through the retro-orbital vein into EDTA anticoagulant as previously described.12 Plasma total cholesterol, high-density lipoprotein cholesterol, and triglyceride levels were measured with enzymatic assays. Transgenic livers for quantitative PCR analysis were also collected at 8 weeks of age from a separate cohort of mice and immediately flash-frozen in liquid nitrogen. RNA was extracted by using the RNeasy kit (Qiagen).

Subcongenic Mapping and Genotyping

In brief, the subcongenic strains were isolated by identifying recombinations within the Chr.15 locus. Parental CON15 mice heterozygous at the Chr.15 locus were intercrossed, and progeny were genotyped at the selected polymorphic microsatellite markers (Research Genetics) by PCR. Near the area of the critical region, a dense series of polymorphic markers (≈1 Mbp) apart were typed (between D15Mit26 and D15Mit101) to pinpoint the recombination break point. Recombinant strains were expanded, and progeny were analyzed for plasma lipid levels. For each subcongenic strain, a minimum of 20 mice were examined. The BALB/cJ mice used for analyses were offspring of a CON15 intercross lacking the MRL allele, to minimize the genetic background variation. The Zhx2 mutation was genotyped by using primers that mapped within the first intron of Zhx2 and that flanked the retrovirus insertion site.14 The resulting 342-bp DNA was amplified in an undisrupted wild-type allele, while the presence of insertion lacked the PCR product. Each DNA sample was also amplified with control PCR primers that mapped within the third exon of Zhx2 (776-bp product; supplemental Table I).

Plasma Lipid Analysis

After overnight fasting, mice under isoflurane anesthesia were subjected to blood collection through the retro-orbital vein into EDTA anticoagulant as described.15 Mice were bled at 8 to10 weeks of age. Plasma total cholesterol, high-density lipoprotein cholesterol, and triglyceride levels were measured by enzymatic assays.15

Real-Time Quantitative PCR Analysis

Two-month-old males from BALB/cJ, Sub6, or Sub13 strains maintained on a chow diet were fasted overnight and euthanized, and livers were removed. Total RNA was isolated from mouse tissues with RNAlater (Ambion) and RNeasy isolation kit (Qiagen) incorporating on-column DNase treatment according to the manufacturer’s instructions. Three micrograms of total RNA was reverse-transcribed with the use of random hexamers and Superscript-III reverse transcriptase (Invitrogen). Quantitative real-time PCR was performed as described previously.16 cDNA sequences for the analyzed genes were obtained from a gene bank and primers were designed with the use of PrimerQuest software (IDT Technologies). For each gene, all available mRNA sequences in the GenBank were analyzed to identify common regions, which were then used for primer selection to ensure targeting of all gene splice variants. Sequences of primers can be found in supplemental Table II. Each individual mouse cDNA was analyzed separately by the relative standard curve method. A standard curve was first constructed for each transcript, including actin (used as endogenous control) by pooling aliquots of sample cDNAs and preparing serial dilutions. Relative expression values of each gene in individual samples were then obtained from the constructed standard curve and corrected for actin expression. The final data are expressed as the mean difference in gene expression level for the indicated strain relative to BALB/cJ litter-mates (set to 1) ±1 SD.

Expression Arrays

BALB/cJ males (n=4) and Sub13 males (n=4) maintained on a chow diet at 2 months of age were fasted overnight and euthanized, and livers were removed. Tissues were stored at −80°C in RNAlater (Ambion), and RNA was isolated with the RNeasy isolation kit (Qiagen) incorporating on-column DNase treatment according to the manufacturer’s instructions. RNA from 8 samples was analyzed on an Agilent 2100 Bioanalyzer (Agilent) to assess the RNA integrity and hybridized to Illumina MouseRef-8 Expression BeadChip according to manufacturer instructions. Data were processed with BeadStudio software (Illumina), and probes were selected on the basis of the threshold of ≥95% probability of positive signal detection. To select differentially expressed genes between the 2 strains, we used expression array analysis software GeneSifter.Net (VizXlabs, Seattle, Wash) with cutoff criteria P<0.05 (Student’s t test with Benjamini and Hochberg correction for the false-discovery rate) and absolute mean value fold change ≥1.5. Gene expression differences passing these criteria were expressed as the mean fold change between the compared strains (supplemental Table III).

Sequencing

PrimerQuest software (IDT Technologies) was used to design PCR primers spanning coding exons (genomic DNA) or in some cases, cDNA of genes within the critical region (supplemental Table I). Sub6 and BALB/cJ liver-derived cDNA was isolated as described previously. Genomic DNA was isolated with the Qiagen DNeasy kit. Amplified PCR products were sequenced on an Applied Biosystems 3730 instrument (Laragen Inc) and sequences were aligned with Vector NTI software (Invitrogen).

Statistical Analysis

Data are presented as mean±SEM. The ANOVA t test was performed with Statview (Abacus Concepts, Inc) to compare differences between groups in lipid and gene expression levels. Differences were considered statistically significant at P<0.05. Because the traits tested were not independent of 1 another, a multiple-comparison adjustment was not performed; but with the exceptions of triglyceride levels in males and total cholesterol levels in females, the results remained significant after Bonferroni correction (supplemental Table IV). Previous studies demonstrated that the lipoprotein traits tested are normally distributed, with the exception of triglycerides. Both parametric and nonparameteric values can be found in supplemental Table IV.

Results

Subcongenic Strain Generation and Fine-Mapping of the Chr. 15 Locus

We have previously demonstrated the feasibility of subdividing the CON15 region (100 Mbp) into 2 smaller segments by using the subcongenic breeding strategy.12 Given this early success, we further subdivided the CON15 region by generating a total of 13 recombinant subcongenic strains, differing in the length and position of the MRL segment. Male and female progeny of each subcongenic strain were analyzed for plasma cholesterol and triglyceride levels and compared with CON15 and BALB/cJ counterparts. Five strains (Sub6, Sub10, Sub11, Sub12, and Sub13) proved to be most informative (Figure 1; supplemental Table V). Because of large nongenetic variations in plasma triglyceride levels, fasted plasma cholesterol levels in males were used as the main phenotype to narrow the locus. The Sub6, Sub11, and Sub13 strains had significantly elevated plasma cholesterol (30% to 40%) compared with BALB/cJ, similar to the CON15 strain. In contrast, strains Sub10 and Sub12 exhibited plasma cholesterol levels similar to those in BALB/cJ and significantly lower than those in CON15. This was true for heterozygous and homozygous animals carrying the MRL alleles at the subcongenic locus, consistent with a dominant pattern of inheritance (supplemental Table V). Cholesterol levels in females showed somewhat smaller differences, intermediate between CON15 and BALB/cJ strains. Based on the segregation of plasma cholesterol levels with individual strain genotypes, the original locus could be reduced to ≈5 Mbp region spanning markers D15Mit184 (24 cM) and D15Mit46 (26 cM) (Figure 1).

Figure 1.

Figure 1

Open in a new tab

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.

Expression Analysis and Sequencing of Genes Within the Critical 5-Mbp Region and Identification of Zhx2 as a Hyplip2 Candidate

At this stage, further subdivision of the locus was not practically feasible. To identify the candidates, we chose a systematic 2-prong approach, combining gene expression profiling and sequence analysis. We reasoned that the underlying genetic variation would either have a pronounced effect on mRNA expression or result in modification of the protein coding sequence. Based on external databases (RefSeq, Uni-Prot, and GenBank), the 5-Mbp critical region harbors 30 genes (supplemental Table VI). We used quantitative PCR to analyze expression of all 30 genes and associated transcript variants in livers of BALB/cJ and Sub6 strains (Figure 2A). Liver was selected because of its key role in modulation of plasma lipid metabolism. These experiments were initiated before isolation of the Sub13 strain, therefore leading to the choice of strain Sub6. Of 30 genes, 28 were detectable in the liver, of which the majority showed remarkably similar expression in the 2 strains. Two genes exhibited a >2-fold difference between the 2 strains: Zhx2 expression was strikingly reduced in BALB/cJ mice (38-fold), and BC030396 expression (coding for hypothetical protein MGC14128) was also reduced in BALB/cJ mice (3-fold), but its expression in the liver was very low, approaching the limits of detection. Even at relatively high RNA (cDNA) input=30 ng, the average fluorescence threshold cycle (Ct) value for BC030396 in the quantitative PCR reaction was >35, nearing single-copy quantities and difficult to precisely quantify. On comparison, the average Ct values for Zhx2 were 26 and 32 in Sub6 and BALB/cJ strains, respectively. Sequencing of coding exons and exon/intron boundaries of genes present in the critical region led to the identification of several single-nucleotide polymorphisms. However, none of the identified single-nucleotide polymorphisms resulted in amino acid alterations and were absent in the exon/intron splice junctions (supplemental Table VI). Thus, Zhx2 became our primary candidate because of the very large difference in mRNA levels between the 2 strains.

Figure 2.

Figure 2

Open in a new tab

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.

During the course of this work, Perincheri et al14 showed that the BALB/cJ strain contains an endogenous retroviral insertion in the first intron of the Zhx2 gene, greatly reducing levels of correctly spliced mRNA and functional protein. This recessive mutation results in a failure to fully repress post-natal expression of the α-fetoprotein and H19 genes in the livers of the BALB/cJ strain. We designed genotyping primers that flank the retrovirus insertion site of the Zhx2 gene. As predicted from the individual strain genotypes (Figure 1), the Zhx2 mutant allele (lack of the 342-bp product) was present in the BALB/cJ, Sub10, and Sub12 strains and absent in the CON15, Sub6, Sub11, and Sub13 strains. Thus, the Zhx2 mutation segregated with reduced plasma cholesterol levels in subcongenic strains (Figure 2B).

Validation of Zhx2 as the Causal Gene

The Zhx2 mutation found in the BALB/cJ strain does not occur in other closely related strains, such as BALB/cH or BALB/cBy.14,17 Consistent with a role for Zhx2 in plasma lipid levels, we observed that cholesterol levels in BALB/cH mice (130±3 mg/dL) were significantly higher than in BALB/cJ mice (103±2 mg/dL) but very similar to those in Sub13 mice (133±4 mg/dL; supplemental Figure I).

To validate the hypothesis that Zhx2 was the gene responsible for the phenotypic differences between BALB/cJ and MRL strains, we used a transgenic complementation approach. The Zhx2 coding region was sub-cloned into an expression vector driven by liver-selective transthyretin promoter to generate transgenic mice. Transgenic founder mice were backcrossed to the BALB/cJ background until homozygous for the Chr.15 BALB/cJ allele and further crossed to BALB/cJ mice to generate offspring littermates, with and without the transgene, which were then used for phenotyping (Figure 3). Both male and female Zhx2 transgenic mice exhibited significantly elevated plasma cholesterol and triglyceride levels, in essence identical with those in the CON15 strain and very similar to those in the subcongenic strains carrying the MRL allele. These data were consistent with the level of Zhx2 expression in transgenic mice, which was very similar to endogenously expressed gene (Figure 4). When compared with BALB/cJ mice, Zhx2 expression was 34-fold higher in transgenic mice and 49- and 38-fold higher in Sub13 and Sub6 strains, respectively. These data thus confirmed that the Zhx2 mutation underlies the Chr. 15 QTL.

Figure 3.

Figure 3

Open in a new tab

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.

Figure 4

Open in a new tab

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).

Gene Expression Profiling and Identification of Lpl as a Gene Regulated by Zhx2

The Zhx2 gene was cloned recently as a member of the ZHX gene family, functioning as a transcriptional repressor and binding partner of nuclear transcription factor Y alpha (NF-YA) in vitro.18,19 In vivo and in vitro studies indicate that Zhx2 functions as a postnatal repressor of the fetal liver genes α-fetoprotein, H19, and Glypican 3.14,20,21 It is therefore highly plausible that Zhx2 also influences lipid metabolism by regulation of downstream gene targets. To help understand the underlying mechanism, we compared global gene expression in livers of the BALB/cJ and Sub13 strains. Because the Sub13 strain is on a BALB/cJ background and Zhx2 is the only gene exhibiting detected variation at the locus, strain differences should originate almost exclusively from the Zhx2 allele variant. We found 1084 differentially expressed genes, several of which are known to be involved in liver and plasma lipid metabolism (supplemental Table VII). Earlier metabolic studies in CON15 and BALB/cJ strains revealed that reduced triglyceride levels in BALB/cJ are caused by enhanced lipoprotein lipase (LPl)-mediated lipolysis and plasma clearance of triglyceride-rich plasma lipoproteins.13 The Lpl gene was among the genes exhibiting elevated expression in the BALB/cJ livers. To confirm the expression profiling data, the Lpl expression was further evaluated by quantitative PCR. Lpl was significantly elevated in BALB/cJ strain (3 to 5 fold) when compared with Sub13 strain (Figure 4A), and its expression was suppressed in BALB/cJ strain expressing the Zhx2 liver transgene (Figure 4B). We also measured expression of other genes known to be involved in modulation of lipase activity, including Apoc1, Apoc2, Apoc3, Apoa5, Angptl3, and Angptl4, and these were not significantly affected (Figure 4). These findings indicate that Zhx2 acts as a suppressor of Lpl expression in the liver. Although low levels of Zhx2 resulted in increased expression of Lpl and most other differentially regulated genes, some genes such as Ear11 exhibited the opposite pattern (Figure 4).

To further examine the effects of Zhx2 on developmental regulation, we monitored expression levels of Lpl and Ear11 in liver during development, beginning at birth. Low levels of functional Zhx2 in BALB/cJ mice led to an increase in Lpl levels compared with Sub13 mice throughout early development, indicating that Zhx2 acts to repress Lpl (Figure 5A and 5B). These results are similar to the effects of Zhx2 on α-fetoprotein and H19. Interestingly, a different developmental expression pattern was observed in the case of Ear11, a member of the eosinophil-associated ribonuclease family. Although this gene may be repressed by Zhx2 immediately after birth, low levels of Zhx2 result in dramatically down-regulated expression of Earl1 at 14 and 60 days (Figure 5C). Thus, the function of Zhx2 seems to be highly context dependent.

Figure 5.

Figure 5

Open in a new tab

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.

Discussion

Using a mouse QTL mapping strategy, we identified the Zhx2 transcription factor as a novel modulator of cholesterol and triglyceride metabolism. Our results indicate that Zhx2 plays a role in the developmental regulation of genes involved in lipid metabolism, although the mechanism by which lipids are affected is unclear.

One striking example of the molecular impact of Zhx2 deficiency that we identified is the repression of Lpl expression during postnatal development. Interestingly, Lpl is normally not expressed in adult liver.22 Studies in rats have shown that Lpl expression is high during the fetal period but rapidly extinguished shortly after birth.23 In fact, the expression pattern of the Lpl gene in hepatocarcinoma cell lines exhibits certain similarities to α-fetoprotein, and Lpl expression can be elevated in mice bearing liver tumors.24,25 In addition, the proximal promoter of the Lpl gene contains NF-YA binding sites known to play important roles in regulation of Lpl expression.26,27 Therefore, the altered plasma lipid phenotype could be a consequence, in part, of changes in Zhx2-mediated regulation of Lpl expression. Indeed, many apolipoproteins (eg, ApoA-I, ApoA-IV, ApoE, ApoB, and ApoC) and lipoprotein enzymes (eg, LPL, lecithin: cholesterol acyltransferase, and hepatic lipase) exhibit substantial temporal and tissue-specific alterations in gene expression during the fetal and neonatal periods.22,28–30 Zhx2 has not been shown to bind DNA directly, and the mechanism by which it modulates Lpl expression is also not clear. One possible mechanism could involve indirect interaction by binding to 1 of its DNA binding partners, such as Zhx3.31 We did not observe transcriptional differences among strains at Zhx3 (quantitative PCR P value=0.59) or at Zhx1 (Figure 2A). Although neither Zhx1 nor Zhx3 variations contributed to lipid metabolism in the MRL×BALB/cJ cross, differences in Zhx3 were associated with high-density lipoprotein cholesterol levels in a CAST/Ei×C57BL/6J intercross (M. Mehrabian, Ph.D., and A.J. Lusis, Ph.D., unpublished data, 2009).

As yet, there have been few examples of the successful identification of genes contributing to QTL for complex traits in mice. Our strategy of narrowing the QTL by using subcongenic strains followed by expression screening and sequencing was effective but laborious. It is noteworthy that Chr.15 QTL in the original cross between MRL and BALB/cJ was large (log of the odds score=11.6), as QTL with very modest effects would clearly be much more difficult to identify with our strategy.11 We previously showed that the Chr.15 congenic interval had a very large impact on atherosclerosis, much larger than would be expected from the relatively modest changes in lipoprotein levels.12 Preliminary studies on a low-density lipoprotein receptor-null background support this conclusion (A. Erbilgin, B.S., P.S. Gargalovic, Ph.D., R.C. LeBoeuf, Ph.D. and A.J. Lusis, Ph.D., unpublished data, 2009). It therefore seems likely that Zhx2 also mediates other pathways related to atherosclerosis. In particular, the effect on the eosinophil-associated ribonuclease A family, member 11 (Ear11), that plays a role in inflammation, is intriguing. Recent evidence suggests that members of the family are expressed in macrophages, an important cell type in atherogenesis.32

Clearly, future studies of Zhx2 and its role in lipoprotein metabolism and atherosclerosis are warranted. There are no reports of associations of Zhx2 polymorphisms with lipoprotein levels in human populations, but polymorphisms of the gene for Zhx3, which forms heterodimers with Zhx2,19 have recently been significantly associated with triglyceride levels (S. Kathiresan, M.D., personal communication, 2009).

CLINICAL PERSPECTIVE

One approach to better understand the biology and genetics of cardiovascular traits is to study natural variations in experimental organisms such as the mouse. In this study, a novel gene contributing to variations in plasma high-density lipoprotein and triglyceride levels in mice was identified by genetic mapping and transgenic complementation. The gene encodes a transcription factor, Zhx2, previously shown to be involved in the suppression of α-fetoprotein expression during postnatal development. Preliminary studies of the mechanism of action of Zhx2 indicate a role in the regulation of lipoprotein lipase, a key enzyme in the catabolism of triglyceride-rich lipoproteins.

Supplementary Material

Acknowledgments

We thank Sarada Charugundla for assistance with lipoprotein measurements.

Sources of Funding

This work was supported by National Institutes of Health grants HL28481, HL094322 (AJL), HL079382 (RL), DK59866 (BTS, MLP) and Ruth L. Kirschstein National Research Service Award GM07185 (AE).

Footnotes

Disclosures

Peter Gargalovic is an employee of Bristol-Myers Squibb Co.

References

  • 1.Aulchenko YS, Ripatti S, Lindqvist I, Boomsma D, Heid IM, Pramstaller PP, Penninx BW, Janssens AC, Wilson JF, Spector T, Martin NG, Pedersen NL, Kyvik KO, Kaprio J, Hofman A, Freimer NB, Jarvelin MR, Gyllensten U, Campbell H, Rudan I, Johansson A, Marroni F, Hayward C, Vitart V, Jonasson I, Pattaro C, Wright A, Hastie N, Pichler I, Hicks AA, Falchi M, Willemsen G, Hottenga JJ, de Geus EJ, Montgomery GW, Whitfield J, Magnusson P, Saharinen J, Perola M, Silander K, Isaacs A, Sijbrands EJ, Uitterlinden AG, Witteman JC, Oostra BA, Elliott P, Ruokonen A, Sabatti C, Gieger C, Meitinger T, Kronenberg F, Doring A, Wichmann HE, Smit JH, McCarthy MI, van Duijn CM, Peltonen L. Loci influencing lipid levels and coronary heart disease risk in 16 European population cohorts. Nat Genet. 2009;41:47–55. doi: 10.1038/ng.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Hegele RA. Plasma lipoproteins: genetic influences and clinical implications. Nat Rev Genet. 2009;10:109–121. doi: 10.1038/nrg2481. [DOI] [PubMed] [Google Scholar]
  • 3.Kathiresan S, Willer CJ, Peloso GM, Demissie S, Musunuru K, Schadt EE, Kaplan L, Bennett D, Li Y, Tanaka T, Voight BF, Bonnycastle LL, Jackson AU, Crawford G, Surti A, Guiducci C, Burtt NP, Parish S, Clarke R, Zelenika D, Kubalanza KA, Morken MA, Scott LJ, Stringham HM, Galan P, Swift AJ, Kuusisto J, Bergman RN, Sundvall J, Laakso M, Ferrucci L, Scheet P, Sanna S, Uda M, Yang Q, Lunetta KL, Dupuis J, de Bakker PI, O’Donnell CJ, Chambers JC, Kooner JS, Hercberg S, Meneton P, Lakatta EG, Scuteri A, Schlessinger D, Tuomilehto J, Collins FS, Groop L, Altshuler D, Collins R, Lathrop GM, Melander O, Salomaa V, Peltonen L, Orho-Melander M, Ordovas JM, Boehnke M, Abecasis GR, Mohlke KL, Cupples LA. Common variants at 30 loci contribute to polygenic dyslipidemia. Nat Genet. 2009;41:56–65. doi: 10.1038/ng.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Ordovas JM, Corella D, Demissie S, Cupples LA, Couture P, Coltell O, Wilson PW, Schaefer EJ, Tucker KL. Dietary fat intake determines the effect of a common polymorphism in the hepatic lipase gene promoter on high-density lipoprotein metabolism: evidence of a strong dose effect in this gene-nutrient interaction in the Framingham Study. Circulation. 2002;106:2315–2321. doi: 10.1161/01.cir.0000036597.52291.c9. [DOI] [PubMed] [Google Scholar]
  • 5.Sabatti C, Service SK, Hartikainen AL, Pouta A, Ripatti S, Brodsky J, Jones CG, Zaitlen NA, Varilo T, Kaakinen M, Sovio U, Ruokonen A, Laitinen J, Jakkula E, Coin L, Hoggart C, Collins A, Turunen H, Gabriel S, Elliot P, McCarthy MI, Daly MJ, Jarvelin MR, Freimer NB, Peltonen L. Genome-wide association analysis of metabolic traits in a birth cohort from a founder population. Nat Genet. 2009;41:35–46. doi: 10.1038/ng.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Manolio TA. Cohort studies and the genetics of complex disease. Nat Genet. 2009;41:5–6. doi: 10.1038/ng0109-5. [DOI] [PubMed] [Google Scholar]
  • 7.Ioannidis JP, Thomas G, Daly MJ. Validating, augmenting and refining genome-wide association signals. Nat Rev Genet. 2009;10:318–329. doi: 10.1038/nrg2544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Lusis AJ, Attie AD, Reue K. Metabolic syndrome: from epidemiology to systems biology. Nat Rev Genet. 2008;9:819–830. doi: 10.1038/nrg2468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Flint J, Valdar W, Shifman S, Mott R. Strategies for mapping and cloning quantitative trait genes in rodents. Nat Rev Genet. 2005;6:271–286. doi: 10.1038/nrg1576. [DOI] [PubMed] [Google Scholar]
  • 10.Flint J, Mott R. Applying mouse complex-trait resources to behavioural genetics. Nature. 2008;456:724–727. doi: 10.1038/nature07630. [DOI] [PubMed] [Google Scholar]
  • 11.Gu L, Johnson MW, Lusis AJ. Quantitative trait locus analysis of plasma lipoprotein levels in an autoimmune mouse model: interactions between lipoprotein metabolism, autoimmune disease, and atherogenesis. Arterioscler Thromb Vasc Biol. 1999;19:442–453. doi: 10.1161/01.atv.19.2.442. [DOI] [PubMed] [Google Scholar]
  • 12.Wang X, Gargalovic P, Wong J, Gu JL, Wu X, Qi H, Wen P, Xi L, Tan B, Gogliotti R, Castellani LW, Chatterjee A, Lusis AJ. Hyplip2, a new gene for combined hyperlipidemia and increased atherosclerosis. Arterioscler Thromb Vasc Biol. 2004;24:1928–1934. doi: 10.1161/01.ATV.0000143385.30354.bb. [DOI] [PubMed] [Google Scholar]
  • 13.Moen CJ, Tholens AP, Voshol PJ, de Haan W, Havekes LM, Gargalovic P, Lusis AJ, van Dyk KW, Frants RR, Hofker MH, Rensen PC. The Hyplip2 locus causes hypertriglyceridemia by decreased clearance of triglycerides. J Lipid Res. 2007;48:2182–2192. doi: 10.1194/jlr.M700009-JLR200. [DOI] [PubMed] [Google Scholar]
  • 14.Perincheri S, Dingle RW, Peterson ML, Spear BT. Hereditary persistence of alpha-fetoprotein and H19 expression in liver of BALB/cJ mice is due to a retrovirus insertion in the Zhx2 gene. Proc Natl Acad Sci U S A. 2005;102:396–401. doi: 10.1073/pnas.0408555102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Castellani LW, Weinreb A, Bodnar J, Goto AM, Doolittle M, Mehrabian M, Demant P, Lusis AJ. Mapping a gene for combined hyperlipidaemia in a mutant mouse strain. Nat Genet. 1998;18:374–377. doi: 10.1038/ng0498-374. [DOI] [PubMed] [Google Scholar]
  • 16.Gargalovic PS, Gharavi NM, Clark MJ, Pagnon J, Yang WP, He A, Truong A, Baruch-Oren T, Berliner JA, Kirchgessner TG, Lusis AJ. The unfolded protein response is an important regulator of inflammatory genes in endothelial cells. Arterioscler Thromb Vasc Biol. 2006;26:2490–2496. doi: 10.1161/01.ATV.0000242903.41158.a1. [DOI] [PubMed] [Google Scholar]
  • 17.Perincheri S, Peyton DK, Glenn M, Peterson ML, Spear BT. Characterization of the ETnII-alpha endogenous retroviral element in the BALB/cJ Zhx2 (Afr1) allele. Mamm Genome. 2008;19:26–31. doi: 10.1007/s00335-007-9077-6. [DOI] [PubMed] [Google Scholar]
  • 18.Kawata H, Yamada K, Shou Z, Mizutani T, Yazawa T, Yoshino M, Sekiguchi T, Kajitani T, Miyamoto K. Zinc-fingers and homeoboxes (ZHX) 2, a novel member of the ZHX family, functions as a transcriptional repressor. Biochem J. 2003;373:747–757. doi: 10.1042/BJ20030171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Kawata H, Yamada K, Shou Z, Mizutani T, Miyamoto K. The mouse zinc-fingers and homeoboxes (ZHX) family; ZHX2 forms a heterodimer with ZHX3. Gene. 2003;323:133–140. doi: 10.1016/j.gene.2003.09.013. [DOI] [PubMed] [Google Scholar]
  • 20.Morford LA, Davis C, Jin L, Dobierzewska A, Peterson ML, Spear BT. The oncofetal gene glypican 3 is regulated in the postnatal liver by zinc fingers and homeoboxes 2 and in the regenerating liver by alpha-fetoprotein regulator 2. Hepatology. 2007;46:1541–1547. doi: 10.1002/hep.21825. [DOI] [PubMed] [Google Scholar]
  • 21.Yamada K, Ogata-Kawata H, Matsuura K, Kagawa N, Takagi K, Asano K, Haneishi A, Miyamoto K. ZHX2 and ZHX3 repress cancer markers in normal hepatocytes. Front Biosci. 2009;14:3724–3732. doi: 10.2741/3483. [DOI] [PubMed] [Google Scholar]
  • 22.Kirchgessner TG, LeBoeuf RC, Langner CA, Zollman S, Chang CH, Taylor BA, Schotz MC, Gordon JI, Lusis AJ. Genetic and developmental regulation of the lipoprotein lipase gene: loci both distal and proximal to the lipoprotein lipase structural gene control enzyme expression. J Biol Chem. 1989;264:1473–1482. [PubMed] [Google Scholar]
  • 23.Peinado-Onsurbe J, Staels B, Deeb S, Ramirez I, Llobera M, Auwerx J. Neonatal extinction of liver lipoprotein lipase expression. Biochim Biophys Acta. 1992;1131:281–286. doi: 10.1016/0167-4781(92)90026-v. [DOI] [PubMed] [Google Scholar]
  • 24.Schoonjans K, Staels B, Devos P, Szpirer J, Szpirer C, Deeb S, Verhoeven G, Auwerx J. Developmental extinction of liver lipoprotein lipase mRNA expression might be regulated by an NF-1-like site. FEBS Lett. 1993;329:89–95. doi: 10.1016/0014-5793(93)80200-e. [DOI] [PubMed] [Google Scholar]
  • 25.Masuno H, Tsujita T, Nakanishi H, Yoshida A, Fukunishi R, Okuda H. Lipoprotein lipase-like activity in the liver of mice with Sarcoma 180. J Lipid Res. 1984;25:419–427. [PubMed] [Google Scholar]
  • 26.Morin CL, Schlaepfer IR, Eckel RH. Tumor necrosis factor-alpha eliminates binding of NF-Y and an octamer-binding protein to the lipoprotein lipase promoter in 3T3-L1 adipocytes. J Clin Invest. 1995;95:1684–1689. doi: 10.1172/JCI117844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Currie RA, Eckel RH. Characterization of a high affinity octamer transcription factor binding site in the human lipoprotein lipase promoter. Arch Biochem Biophys. 1992;298:630–639. doi: 10.1016/0003-9861(92)90459-a. [DOI] [PubMed] [Google Scholar]
  • 28.Warden CH, Langner CA, Gordon JI, Taylor BA, McLean JW, Lusis AJ. Tissue-specific expression, developmental regulation, and chromosomal mapping of the lecithin: cholesterol acyltransferase gene: evidence for expression in brain and testes as well as liver. J Biol Chem. 1989;264:21573–21581. [PubMed] [Google Scholar]
  • 29.Demmer LA, Levin MS, Elovson J, Reuben MA, Lusis AJ, Gordon JI. Tissue-specific expression and developmental regulation of the rat apolipoprotein B gene. Proc Natl Acad Sci U S A. 1986;83:8102–8106. doi: 10.1073/pnas.83.21.8102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Semenkovich CF, Chen SH, Wims M, Luo CC, Li WH, Chan L. Lipoprotein lipase and hepatic lipase mRNA tissue specific expression, developmental regulation, and evolution. J Lipid Res. 1989;30:423–431. [PubMed] [Google Scholar]
  • 31.Liu G, Clement LC, Kanwar YS, Avila-Casado C, Chugh SS. ZHX proteins regulate podocyte gene expression during the development of nephrotic syndrome. J Biol Chem. 2006;281:39681–39692. doi: 10.1074/jbc.M606664200. [DOI] [PubMed] [Google Scholar]
  • 32.Cormier SA, Yuan S, Crosby JR, Protheroe CA, Dimina DM, Hines EM, Lee NA, Lee JJ. T(H)2-mediated pulmonary inflammation leads to the differential expression of ribonuclease genes by alveolar macrophages. Am J Respir Cell Mol Biol. 2002;27:678–687. doi: 10.1165/rcmb.4882. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS183227-supplement-supplement_1.pdf (239.9KB, pdf)

RESOURCES