Identification of an imprinted master trans regulator at the KLF14 locus related to multiple metabolic phenotypes.
ABSTRACT: Genome-wide association studies have identified many genetic variants associated with complex traits. However, at only a minority of loci have the molecular mechanisms mediating these associations been characterized. In parallel, whereas cis regulatory patterns of gene expression have been extensively explored, the identification of trans regulatory effects in humans has attracted less attention. Here we show that the type 2 diabetes and high-density lipoprotein cholesterol-associated cis-acting expression quantitative trait locus (eQTL) of the maternally expressed transcription factor KLF14 acts as a master trans regulator of adipose gene expression. Expression levels of genes regulated by this trans-eQTL are highly correlated with concurrently measured metabolic traits, and a subset of the trans-regulated genes harbor variants directly associated with metabolic phenotypes. This trans-eQTL network provides a mechanistic understanding of the effect of the KLF14 locus on metabolic disease risk and offers a potential model for other complex traits.
Project description:Subcutaneous adipose tissue stores excess lipids and maintains energy balance. We performed expression quantitative trait locus (eQTL) analyses by using abdominal subcutaneous adipose tissue of 770 extensively phenotyped participants of the METSIM study. We identified cis-eQTLs for 12,400 genes at a 1% false-discovery rate. Among an approximately 680 known genome-wide association study (GWAS) loci for cardio-metabolic traits, we identified 140 coincident cis-eQTLs at 109 GWAS loci, including 93 eQTLs not previously described. At 49 of these 140 eQTLs, gene expression was nominally associated (p < 0.05) with levels of the GWAS trait. The size of our dataset enabled identification of five loci associated (p < 5 × 10-8) with at least five genes located >5 Mb away. These trans-eQTL signals confirmed and extended the previously reported KLF14-mediated network to 55 target genes, validated the CIITA regulation of class II MHC genes, and identified ZNF800 as a candidate master regulator. Finally, we observed similar expression-clinical trait correlations of genes associated with GWAS loci in both humans and a panel of genetically diverse mice. These results provide candidate genes for further investigation of their potential roles in adipose biology and in regulating cardio-metabolic traits.
Project description:Individual risk of type 2 diabetes (T2D) is modified by perturbations to the mass, distribution and function of adipose tissue. To investigate the mechanisms underlying these associations, 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, the expression of 385 genes. We demonstrate, in human cellular studies, that reduced KLF14 expression increases pre-adipocyte proliferation but disrupts lipogenesis, and in mice, that adipose tissue-specific deletion of Klf14 partially recapitulates the human phenotype of insulin resistance, dyslipidemia and T2D. We show that carriers of the KLF14 T2D risk allele shift body fat from gynoid stores to abdominal stores and display a marked increase in adipocyte cell size, and that these effects on fat distribution, and the T2D association, are female specific. The metabolic risk associated with variation at this imprinted locus depends on the sex both of the subject and of the parent from whom the risk allele derives.
Project description:Metabolic syndrome (MetSyn) is a combination of metabolic abnormalities that lead to the development of cardiovascular disease (CVD) and Type 2 Diabetes (T2D). Although various criteria for defining MetSyn exist, common abnormalities include abdominal obesity, elevated serum triglyceride, insulin resistance, and blood glucose, decreased high-density lipoprotein cholesterol (HDL-C), and hypertension. MetSyn prevalence has been increasing with the rise of obesity worldwide, with significantly higher prevalence in women compared with men and in Hispanics compared with Whites. Affected individuals are at a higher risk of developing T2D (5-fold) and CVD (2-fold). Heritability estimates for individual components of MetSyn vary between 40 and 70%, suggesting a strong contribution of an individual's genetic makeup to disease pathology. The advent of next-generation sequencing technologies has enabled large-scale genome-wide association studies (GWAS) into the genetics underlying MetSyn pathogenesis. Several such studies have implicated the transcription factor KLF14, a member of the Krüpple-like factor family (KLF), in the development of metabolic diseases, including obesity, insulin resistance, and T2D. How KLF14 regulates these metabolic traits and increases the risk of developing T2D, atherosclerosis, and liver dysfunction is still unknown. There have been some debate and controversial results with regards to its expression profile and functionality in various tissues, and a systematic review of current knowledge on KLF14 is lacking. Here, we summarize the research progress made in understanding the function of KLF14 and describe common attributes of its biochemical, physiological, and pathophysiological roles. We also discuss the current challenges in understanding the role of KLF14 in metabolism and provide suggestions for future directions.
Project description:Recent genome-wide association studies have revealed that variations near the gene locus encoding the transcription factor Krüppel-like factor 14 (KLF14) are strongly associated with HDL cholesterol (HDL-C) levels, metabolic syndrome, and coronary heart disease. However, the precise mechanisms by which KLF14 regulates lipid metabolism and affects atherosclerosis remain largely unexplored. Here, we report that KLF14 is dysregulated in the liver of 2 dyslipidemia mouse models. We evaluated the effects of both KLF14 overexpression and genetic inactivation and determined that KLF14 regulates plasma HDL-C levels and cholesterol efflux capacity by modulating hepatic ApoA-I production. Hepatic-specific Klf14 deletion in mice resulted in decreased circulating HDL-C levels. In an attempt to pharmacologically target KLF14 as an experimental therapeutic approach, we identified perhexiline, an approved therapeutic small molecule presently in clinical use to treat angina and heart failure, as a KLF14 activator. Indeed, in WT mice, treatment with perhexiline increased HDL-C levels and cholesterol efflux capacity via KLF14-mediated upregulation of ApoA-I expression. Moreover, perhexiline administration reduced atherosclerotic lesion development in apolipoprotein E-deficient mice. Together, these results provide comprehensive insight into the KLF14-dependent regulation of HDL-C and subsequent atherosclerosis and indicate that interventions that target the KLF14 pathway should be further explored for the treatment of atherosclerosis.
Project description:Krüppel-like factor 14 (KLF14) is a member of the Cys2/His2 zinc-finger DNA-binding proteins. Despite strong evidence showing that a polymorphism in the Klf14 gene is closely linked to the development of type 2 diabetes, the physiological and metabolic functions of KLF14 still remain unclear. In the present study, we investigated the role of KLF14 in the regulation of hepatic gluconeogenesis. Adenoviruses expressing KLF14 (Ad-Klf14) or KLF14-specific shRNAs (Ad-shKlf14) were injected into normal C57BL/6J, db/db diabetic, or high-fat diet-induced obese (DIO) mice. Gene expression, hepatic glucose production, glucose tolerance, and insulin resistance were tested in C57BL/6J, db/db, and DIO mice and primary hepatocytes. Our results demonstrate that KLF14 expression is induced in the livers of normal C57BL/6J mice upon fasting and significantly up-regulated in the livers of db/db mice, suggesting a physiological link between KLF14 and gluconeogenesis. Adenovirus-mediated overexpression of KLF14 in primary hepatocytes increased both the mRNA and protein levels of peroxisome proliferator-activated receptor-? coactivator 1? (Pgc-1?, also known as Ppargc1a), thereby stimulating cellular glucose production. Conversely, knockdown of KLF14 expression led to a reduction in PGC-1?, subsequently inhibiting glucose output in primary hepatocytes. Finally, forced expression of KLF14 in the livers of normal mice increased the plasma glucose levels and impaired glucose tolerance; in contrast, KLF14 knockdown in diabetic mouse livers improved glucose tolerance. Taken together, our data strongly indicate that KLF14 modulates hepatic gluconeogenesis.
Project description:<h4>Introduction</h4>Imprinted genes are preferentially expressed from one parentally inherited allele, and many are crucial to the regulation of placental function and fetal growth. Murine Krüppel-like factor 14 (Klf14) is a maternally expressed imprinted transcription factor that is a component of the Mest imprinted gene cluster on mouse chromosome 6. We sought to determine if loss of Klf14 expression alters the course of normal mouse extraembryonic development. We also used high-throughput RNA sequencing (RNAseq) to identify a set of differentially expressed genes (DEGs) in placentas with loss of Klf14.<h4>Methods</h4>We generated a Klf14 knockout (Klf14<sup>null</sup>) mouse using recombineering and transgenic approaches. To identify DEGs in the mouse placenta we compared mRNA transcriptomes derived from 17.5dpc Klf14<sup>matKO</sup> and wild-type littermate placentas by RNAseq. Candidate DEGs were confirmed with quantitative reverse transcription PCR (qPCR) on an independent cohort of male and female gestational age matched Klf14<sup>matKO</sup> placentas.<h4>Results</h4>We found that 17.5dpc placentas inheriting a maternal null allele (Klf14<sup>matKO</sup>) had a modest overgrowth phenotype and a near complete ablation of Klf14 expression. However, there was no effect on fetal growth. We identified 20 DEGs differentially expressed in Klf14<sup>matKO</sup> placentas by RNAseq, and subsequently validated five that are highly upregulated (Begain, Col26a1, Fbln5, Gdf10, and Nell1) by qPCR. The most enriched functional gene-networks included those classified as regulating cellular development and metabolism.<h4>Conclusion</h4>These results suggest that loss of the maternal Klf14 locus in the mouse placenta acts results in changes in gene expression patterns that modulate placental growth.
Project description:The genetics of gene expression in recombinant inbred lines (RILs) can be mapped as expression quantitative trait loci (eQTLs). So-called "genetical genomics" studies have identified locally acting eQTLs (cis-eQTLs) for genes that show differences in steady-state RNA levels. These studies have also identified distantly acting master-modulatory trans-eQTLs that regulate tens or hundreds of transcripts (hotspots or transbands). We expand on these studies by performing genetical genomics experiments in two environments in order to identify trans-eQTL that might be regulated by developmental exposure to the neurotoxin lead. Flies from each of 75 RIL were raised from eggs to adults on either control food (made with 250 microM sodium acetate), or lead-treated food (made with 250 microM lead acetate, PbAc). RNA expression analyses of whole adult male flies (5-10 days old) were performed with Affymetrix DrosII whole genome arrays (18,952 probesets). Among the 1389 genes with cis-eQTL, there were 405 genes unique to control flies and 544 genes unique to lead-treated ones (440 genes had the same cis-eQTLs in both samples). There are 2396 genes with trans-eQTL which mapped to 12 major transbands with greater than 95 genes. Permutation analyses of the strain labels but not the expression data suggests that the total number of eQTL and the number of transbands are more important criteria for validation than the size of the transband. Two transbands, one located on the 2nd chromosome and one on the 3rd chromosome, co-regulate 33 lead-induced genes, many of which are involved in neurodevelopmental processes. For these 33 genes, rather than allelic variation at one locus exerting differential effects in two environments, we found that variation at two different loci are required for optimal effects on lead-induced expression.
Project description:Centrosome amplification is frequent in cancer, but the underlying mechanisms remain unclear. Here we report that disruption of the Kruppel-like factor 14 (KLF14) gene in mice causes centrosome amplification, aneuploidy and spontaneous tumorigenesis. Molecularly, KLF14 functions as a transcriptional repressor of Plk4, a polo-like kinase whose overexpression induces centrosome overduplication. Transient knockdown of KLF14 is sufficient to induce Plk4-directed centrosome amplification. Clinically, KLF14 transcription is significantly downregulated, whereas Plk4 transcription is upregulated in multiple types of cancers, and there exists an inverse correlation between KLF14 and Plk4 protein expression in human breast and colon cancers. Moreover, KLF14 depletion promotes AOM/DSS-induced colon tumorigenesis. Our findings reveal that KLF14 reduction serves as a mechanism leading to centrosome amplification and tumorigenesis. On the other hand, forced expression of KLF14 leads to mitotic catastrophe. Collectively, our findings identify KLF14 as a tumour suppressor and highlight its potential as biomarker and therapeutic target for cancer.
Project description:While over ten thousand genetic loci have been associated with phenotypic traits and inherited diseases in genome-wide association studies, in most cases only a relatively small proportion of the trait heritability is explained and biological mechanisms underpinning these traits have not been clearly identified. Expression quantitative trait loci (eQTL) are subsets of genomic loci shown experimentally to influence gene expression. Since gene expression is one of the primary determinants of phenotype, the identification of eQTL may reveal biologically relevant loci and provide functional links between genomic variants, gene expression and ultimately phenotype. Skeletal muscle (gluteus medius) gene expression was quantified by RNA-seq for 111 Thoroughbreds (47 male, 64 female) in race training at a single training establishment sampled at two time-points: at rest (n = 92) and four hours after high-intensity exercise (n = 77); n = 60 were sampled at both time points. Genotypes were generated from the Illumina Equine SNP70 BeadChip. Applying a False Discovery Rate (FDR) corrected P-value threshold (P FDR < 0.05), association tests identified 3,583 cis-eQTL associated with expression of 1,456 genes at rest; 4,992 cis-eQTL associated with the expression of 1,922 genes post-exercise; 1,703 trans-eQTL associated with 563 genes at rest; and 1,219 trans-eQTL associated with 425 genes post-exercise. The gene with the highest cis-eQTL association at both time-points was the endosome-associated-trafficking regulator 1 gene (ENTR1; Rest: P FDR = 3.81 × 10-27, Post-exercise: P FDR = 1.66 × 10-24), which has a potential role in the transcriptional regulation of the solute carrier family 2 member 1 glucose transporter protein (SLC2A1). Functional analysis of genes with significant eQTL revealed significant enrichment for cofactor metabolic processes. These results suggest heritable variation in genomic elements such as regulatory sequences (e.g. gene promoters, enhancers, silencers), microRNA and transcription factor genes, which are associated with metabolic function and may have roles in determining end-point muscle and athletic performance phenotypes in Thoroughbred horses. The incorporation of the eQTL identified with genome and transcriptome-wide association may reveal useful biological links between genetic variants and their impact on traits of interest, such as elite racing performance and adaptation to training.
Project description:BACKGROUND: We aimed to assess whether whole blood expression quantitative trait loci (eQTLs) with effects in cis and trans are robust and can be used to identify regulatory pathways affecting disease susceptibility. MATERIALS AND METHODS: We performed whole-genome eQTL analyses in 890 participants of the KORA F4 study and in two independent replication samples (SHIP-TREND, N?=?976 and EGCUT, N?=?842) using linear regression models and Bonferroni correction. RESULTS: In the KORA F4 study, 4,116 cis-eQTLs (defined as SNP-probe pairs where the SNP is located within a 500 kb window around the transcription unit) and 94 trans-eQTLs reached genome-wide significance and overall 91% (92% of cis-, 84% of trans-eQTLs) were confirmed in at least one of the two replication studies. Different study designs including distinct laboratory reagents (PAXgene™ vs. Tempus™ tubes) did not affect reproducibility (separate overall replication overlap: 78% and 82%). Immune response pathways were enriched in cis- and trans-eQTLs and significant cis-eQTLs were partly coexistent in other tissues (cross-tissue similarity 40-70%). Furthermore, four chromosomal regions displayed simultaneous impact on multiple gene expression levels in trans, and 746 eQTL-SNPs have been previously reported to have clinical relevance. We demonstrated cross-associations between eQTL-SNPs, gene expression levels in trans, and clinical phenotypes as well as a link between eQTLs and human metabolic traits via modification of gene regulation in cis. CONCLUSIONS: Our data suggest that whole blood is a robust tissue for eQTL analysis and may be used both for biomarker studies and to enhance our understanding of molecular mechanisms underlying gene-disease associations.