Treating messenger RNA transcript abundances as quantitative traits and mapping gene expression quantitative trait loci for these traits has been pursued in gene-specific ways. Transcript abundances often serve as a surrogate for classical quantitative traits in that the levels of expression are significantly correlated with the classical traits across members of a segregating population. The correlation structure between transcript abundances and classical traits has been used to identify susceptibility loci for complex diseases such as diabetes and allergic asthma. One study recently completed the first comprehensive dissection of transcriptional regulation in budding yeast, giving a detailed glimpse of a genome-wide survey of the genetics of gene expression. Unlike classical quantitative traits, which often represent gross clinical measurements that may be far removed from the biological processes giving rise to them, the genetic linkages associated with transcript abundance affords a closer look at cellular biochemical processes. Here we describe comprehensive genetic screens of mouse, plant and human transcriptomes by considering gene expression values as quantitative traits. We identify a gene expression pattern strongly associated with obesity in a murine cross, and observe two distinct obesity subtypes. Furthermore, we find that these obesity subtypes are under the control of different loci.

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Individual risk of type 2 diabetes (T2D) is modified by perturbations of adipose mass, distribution and function. To investigate mechanisms responsible, we explored the molecular, cellular, and whole-body effects of T2D-associated alleles near KLF14. We show that KLF14 diabetes-risk alleles act in adipose tissue to reduce KLF14 expression, and modulate, in trans, expression of 385 genes. We demonstrate that, in human cellular studies, reduced KLF14 expression increases pre-adipocyte proliferation but disrupts lipogenesis, and, in mice, adipose-specific deletion of Klf14 partially recapitulates the human phenotype of insulin resistance, dyslipidemia and T2D. We show that KLF14 T2D risk-allele carriers shift body fat from gynoid to abdominal stores, and display a marked increase in adipocyte cell size: these effects on fat distribution, and the T2D-association, are female-specific. Metabolic risk associated with variation at this imprinted locus depends on both the sex of the subject, and of the parent from whom the risk-allele derives.

SUMMARY Emerging evidence suggests that microbes resident in the human intestine represent a key environmental factor contributing to obesity-associated disorders. Here we demonstrate that the gut microbiota-initiated trimethylamine-N-oxide (TMAO)-generating pathway is linked to obesity and energy metabolism. In multiple clinical cohorts, systemic levels of TMAO were observed to strongly associate with type 2 diabetes. In addition, circulating TMAO levels were associated with obesity traits in the different inbred strains represented in the Hybrid Mouse Diversity Panel. Further, antisense oligonucleotide-mediated knockdown or genetic deletion of the TMAO-producing enzyme, flavin-containing monooxygenase 3 (FMO3), conferred protection against obesity in mice. Complimentary mouse and human studies indicate a negative regulatory role for FMO3 in the beiging of white adipose tissue. Collectively, our studies reveal a link between the TMAO-producing enzyme FMO3 and obesity and the beiging of white adipose tissue.

SUMMARY Many common diseases have an important inflammatory component mediated in part by macrophages. Here we used a systems genetics strategy to examine the role of common genetic variation in macrophage responses to inflammatory stimuli. We examined genome-wide transcript levels in macrophages from 92 strains of the Hybrid Mouse Diversity Panel. We exposed macrophages to control media, bacterial lipopolysaccharide, or oxidized phospholipids. We performed association mapping under each condition and identified several thousand expression quantitative trait loci (eQTL), gene-by-environment interactions and several eQTL “hotspots” that specifically control LPS responses. We validated an eQTL hotspot in chromosome 8 using siRNA knock-down of candidate genes and identified the gene 2310061C15Rik, as a novel regulator of inflammatory responses in macrophages. We have created a public database where the data presented here can be used as a resource for understanding many common inflammatory traits which are modeled in the mouse, and for the dissection of regulatory relationships between genes.

The etiology of non-alcoholic fatty liver disease (NAFLD), the most common form of chronic liver disease, is poorly understood. To understand the causal mechanisms underlying NAFLD, we conducted a multi-omics, multi-tissue integrative study using the Hybrid Mouse Diversity Panel, consisting of ∼100 strains of mice with various degrees of NAFLD. We identified both tissue-specific biological processes and processes that were shared between adipose and liver tissues. We then used gene network modeling to predict candidate regulatory genes of these NAFLD processes, including Fasn, Thrsp, Pklr, and Chchd6. In vivo knockdown experiments of the candidate genes improved both steatosis and insulin resistance. Further in vitro testing demonstrated that downregulation of both Pklr and Chchd6 lowered mitochondrial respiration and led to a shift toward glycolytic metabolism, thus highlighting mitochondria dysfunction as a key mechanistic driver of NAFLD.

The genetic factors contributing to the complex disorder of myocardial calcification are largely unknown. Using a mouse model, we fine-mapped the major locus (Dyscalc1) contributing to the dystrophic cardiac calcification (DCC) to an 840-kb interval containing 38 genes. We then identified the causal gene by using an approach integrating genetic segregation and expression array analyses to identify, on a global scale, cis-acting DNA variations that perturb gene expression. By studying two intercrosses, in which the DCC trait segregates, a single candidate gene (encoding the ATPbinding cassette transporter ABCC6) was identified. Transgenic complementation confirmed Abcc6 as the underlying causal gene for Dyscalc1. We demonstrate that in the cross, the expression of Abcc6 is highly correlated with the local mineralization regulatory system and the BMP2-Wnt signaling pathway known to be involved in the systemic regulation of calcification, suggesting potential pathways for the action of Abcc6 in DCC. Our results demonstrate the power of the integrative genomics in discovering causal genes and pathways underlying complex traits.expression quantitative trait locus ͉ transgenic ͉ positional cloning ͉ osteopontin C haracterized by hydroxyapatite deposition in necrotic myocytes, myocardial calcification is common in specific forms of cardiomyopathy and in myocardial infarction. It has been estimated that Ϸ8% of patients with severe myocardial infarction develop myocardial calcification within 6 years, suggesting a genetic predisposition for postinjury healing and remodeling processes (1). Historically, dystrophic cardiac calcification (DCC) has been considered a spontaneous form of cardiomyopathy in mice, associated with a variety of predisposing factors, but with normal blood levels of calcium and phosphate. Experimentally, it can be reproducibly initiated using a transdiaphragmal freeze-thaw injury or a high-phosphorous (HP) diet (2, 3). Several inbred mouse strains, including C3H/HeJ (C3H) and DBA/2J (DBA), are highly susceptible, whereas many other inbred mouse strains, including C57BL/6J (B6), C57BL/10J (B10), A/J, MRL/MpJ, and BALB/cJ are resistant (refs. 4 and 5; X.W., T.A.D., and A.J.L., unpublished data). In DCC susceptible strains, calcification has also been observed in skeletal muscle, including in the tongue and diaphragm, and kidney, suggesting a systemic defect (2, 5).Using quantitative trait locus (QTL) analysis of an F 2 intercross between B6 and C3H mice (BxH), we previously mapped four DCC loci (6, 7). The locus on chromosome 7 (Dyscalc1), which exhibits recessive inheritance, explains 31% of the total genetic variance and is the major contributor. Dyscalc1 was confirmed by separate intercrosses of B6 and DBA mice (BxD) (5, 8). The C3H strain was originally derived from an outbreeding experiment of the DBA strain, leading us to hypothesize that the susceptible strains C3H and DBA share a common diseasecausing allele (8). To fine map the Dyscalc1 locus, we screened a panel of recombinant congenic (RC) s...

Objective-To test the hypothesis that NF-E2-related factor 2 (Nrf2) expression plays an antiatherogenic role by its vascular antioxidant and anti-inflammatory properties. Methods and Results-Nrf2 is an important transcription factor that regulates the expression of phase 2 detoxifying enzymes and antioxidant genes. Its expression in vascular cells appears to be an important factor in the protection against vascular oxidative stress and inflammation. We developed Nrf2 heterozygous (HET) and homozygous knockout (KO) mice on an apolipoprotein (apo) E-null background by sequential breeding, resulting in Nrf2 Ϫ/Ϫ , apoE Ϫ/Ϫ (KO), Nrf2, apoE Ϫ/Ϫ (HET) and Nrf2, and apoE Ϫ/Ϫ wild-type littermates. KO mice exhibited decreased levels of antioxidant genes with evidence of increased reactive oxygen species generation compared with wild-type controls. Surprisingly, KO males exhibited 47% and 53% reductions in the degree of aortic atherosclerosis compared with HET or wild-type littermates, respectively. Decreased atherosclerosis in KO mice correlated with lower plasma total cholesterol in a sex-dependent manner. KO mice also had a decreased hepatic cholesterol content and a lower expression of lipogenic genes, suggesting that hepatic lipogenesis could be reduced. In addition, KO mice exhibited atherosclerotic plaques characterized by a lesser macrophage component and decreased foam cell formation in an in vitro lipid-loading assay. This was associated with a lower rate of cholesterol influx, mediated in part by decreased expression of the scavenger receptor CD36. A therosclerosis is a chronic inflammatory disease with both genetic and environmental components, 1,2 characterized by the deposition of lipids in the artery wall and the infiltration of inflammatory cells, such as monocytes and lymphocytes. Vascular oxidative stress is thought to be an important element in its pathogenesis. Indeed, oxidative modifications of low-density lipoprotein (LDL) in the subendothelial space with subsequent generation of foam cells is 1 of the earliest events in atherogenesis. 3 In addition, endothelial cells, macrophages, and smooth muscle cells are all sources of reactive oxygen species (ROS). 4 -6 Therefore, the interplay between pro-oxidant and antioxidant factors is likely to be of key importance, 7 although this notion has been questioned on the basis that oxidative modifications could be consequential, rather than causal, to atherosclerotic lesion formation. 8 Vascular cells express an extensive repertoire of antioxidant genes and phase 2 detoxifying enzymes regulated by a transcription factor, NF-E2-related factor 2 (Nrf2). Nrf2 is a member of the Cap and Collar subfamily of basic leucine zipper transcription factors that act through the formation of a heterodimer with 1 of the small Maf proteins 9 and bind to a cis-acting enhancer sequence, known as the antioxidant response element, 10 -13 in the 5Ј-flanking regions of its target genes. 12 Although the regulation of Nrf2 activity is not completely understood, it appears that ...