Global Analysis of DNA Methylation Variation in Adipose Tissue from Twins Reveals Links to Disease-Associated Variants in Distal Regulatory Elements

Epigenetic modifications such as DNA methylation play a key role in gene regulation and disease susceptibility. However, little is known about the genome-wide frequency, localization, and function of methylation variation and how it is regulated by genetic and environmental factors. We utilized the Multiple Tissue Human Expression Resource (MuTHER) and generated Illumina 450K adipose methylome data from 648 twins. We found that individual CpGs had low variance and that variability was suppressed in promoters. We noted that DNA methylation variation was highly heritable (h²_median = 0.34) and that shared environmental effects correlated with metabolic phenotype-associated CpGs. Analysis of methylation quantitative-trait loci (metQTL) revealed that 28% of CpGs were associated with nearby SNPs, and when overlapping them with adipose expression quantitative-trait loci (eQTL) from the same individuals, we found that 6% of the loci played a role in regulating both gene expression and DNA methylation. These associations were bidirectional, but there were pronounced negative associations for promoter CpGs. Integration of metQTL with adipose reference epigenomes and disease associations revealed significant enrichment of metQTL overlapping metabolic-trait or disease loci in enhancers (the strongest effects were for high-density lipoprotein cholesterol and body mass index [BMI]). We followed up with the BMI SNP rs713586, a cg01884057 metQTL that overlaps an enhancer upstream of ADCY3, and used bisulphite sequencing to refine this region. Our results showed widespread population invariability yet sequence dependence on adipose DNA methylation but that incorporating maps of regulatory elements aid in linking CpG variation to gene regulation and disease risk in a tissue-dependent manner.     

Common STAT3 variants are not associated with obesity or insulin resistance in female twins

In animal models, STAT3 action in the hypothalamus and liver appears essential for normal body weight and glucose homeostasis in response to insulin. We hypothesized that variation in the STAT3 gene may be associated with body fat and/or insulin resistance in the general population. Five tagging SNPs spanning the STAT3 gene, rs8074524, rs2293152, rs2306580, rs6503695, and rs7211777 were genotyped in 2776 white female twins (mean age, 47.4+/-12.5 yrs) from the St Thomas' United Kingdom Adult Twin Registry (Twins UK). Minor allele frequencies were as follows: rs8074524 (0.19), rs2293152 (0.37), rs2306580 (0.06), rs6503695 (0.35), and rs7211777 (0.34). The minor allele of rs2293152 was associated with higher homeostasis model assessment index of insulin resistance (p=0.013) in the full cohort and confirmed in sib-transmission/disequilibrium test (TDT): (p=0.015; n=60). However, there were no associations with fasting serum insulin or glucose or with obesity variables. Although defective STAT3 action results in obesity and insulin resistance in animal models, we failed to establish any indicative associations with common SNPs in this human study.     

A proteome analysis of the cadmium response in Saccharomyces cerevisiae

Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood. We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins. A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal. Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium.     

A genetic investigation of the essential role of glutathione: mutations in the proline biosynthesis pathway are the only suppressors of glutathione auxotrophy in yeast

In an attempt to elucidate the essential function of glutathione in Saccharomyces cerevisiae, we searched for suppressors of the GSH auxotrophy of Deltagsh1, a strain lacking the rate-limiting enzyme of glutathione biosynthesis. We found that specific mutations of PRO2, the second enzyme in proline biosynthesis, permitted the growth of Deltagsh1 in the absence of exogenous GSH. The suppression mechanism by alleles of PRO2 involved the biosynthesis of a trace amount of glutathione. Deletion of PRO1, the first enzyme of the proline biosynthesis pathway, or PRO2 eliminated the suppression, suggesting that gamma-glutamyl phosphate, the product of Pro1 and the physiological substrate of Pro2, is required as an obligate substrate of suppressor alleles of PRO2 for glutathione synthesis. A mutagenesis of a Deltagsh1 strain also lacking the proline pathway failed to generate any suppressor mutants under either aerobic or anaerobic conditions, confirming that glutathione is essential in yeast. This essential function is not related to DNA synthesis based on the terminal phenotype of glutathione-depleted cells or to toxic accumulation of non-native protein disulfides. Analysis of the suppressor strain demonstrates that normal glutathione levels are required for the tolerance to oxidants under acute, but not chronic stress conditions.     

A mutation in COL9A1 causes multiple epiphyseal dysplasia: further evidence for locus heterogeneity

Multiple epiphyseal dysplasia (MED) is an autosomal dominantly inherited chondrodysplasia. It is clinically highly heterogeneous, partially because of its complex genetic background. Mutations in four genes, COL9A2, COL9A3, COMP, and MATR3, all coding for cartilage extracellular matrix components (i.e., the alpha2 and alpha 3 chains of collagen IX, cartilage oligomeric matrix protein, and matrilin-3), have been identified in this disease so far, but no mutations have yet been reported in the third collagen IX gene, COL9A1, which codes for the alpha1(IX) chain. MED with apparently recessive inheritance has been reported in some families. A homozygous R279W mutation was recently found in the diastrophic dysplasia sulfate transporter gene, DTDST, in a patient with MED who had a club foot and double-layered patella. The series consisted of 41 probands with MED, 16 of whom were familial and on 4 of whom linkage analyses were performed. Recombination was observed between COL9A1, COL9A2, COL9A3, and COMP and the MED phenotype in two of the families, and between COL9A2, COL9A3, and COMP and the phenotype in the other two families. Screening of COL9A1 for mutations in the two probands from the families in which this gene was not involved in the recombinations failed to identify any disease-causing mutations. The remaining 37 probands were screened for mutations in all three collagen IX genes and in the COMP gene. The probands with talipes deformities or multipartite patella were also screened for the R279W mutation in DTDST. The analysis resulted in identification of three mutations in COMP and one in COL9A1, but none in the other two collagen IX genes. Two of the probands with a multipartite patella had the homozygous DTDST mutation. The results show that mutations in COL9A1 can cause MED, but they also suggest that mutations in COL9A1, COL9A2, COL9A3, COMP, and DTDST are not the major causes of MED and that there exists at least one additional locus.     

Nerve regeneration-induced recovery of quinine avoidance after complete gustatory deafferentation of the tongue

The concentration-dependent decrease in quinine licking by rats is substantially attenuated by combined bilateral transection of the chorda tympani (CT) and glossopharyngeal (GL) nerves, but transection of either nerve alone produces marginal impairments at most. Here we tested whether regeneration of one or both of these nerves after combined transection would result in recovery of taste avoidance. Water-restricted rats were presented with a series of brief-access (5 s) taste trials (water and 0.003-3.0 mM quinine-HCl) in a 5-day test block of 40-min sessions both before nerve transection and starting 75-77 days after transection. Licking avoidance returned to presurgical levels when both nerves were allowed to regenerate. When only the GL was allowed to regenerate, performance did not differ from that of sham-transected animals. This suggests that even after considerable gustatory deafferentation, regeneration has the capacity to restore normal taste-guided behavior. Surprisingly, when only the CT was allowed to regenerate, avoidance behavior was severely impaired and was not different from that of rats in which regeneration of both nerves was prevented. Taking into account prior findings, it appears that the absence of the GL in the presence of an intact CT is fundamentally different from the absence of the GL in the presence of a regenerated CT with respect to some taste functions. This represents the first reported instance to our knowledge in which the capacity of a regenerated nerve to maintain taste-guided behavior was distinctly different from that of an intact nerve in a rodent model.     

Effect of soluble epoxide hydrolase inhibition on epoxyeicosatrienoic acid metabolism in human blood vessels

We investigated the effects of soluble epoxide hydrolase (sEH) inhibition on epoxyeicosatrienoic acid (EET) metabolism in intact human blood vessels, including the human saphenous vein (HSV), coronary artery (HCA), and aorta (HA). When HSV segments were perfused with 2 micromol/l 14,15-[3H]EET for 4 h, >60% of radioactivity in the perfusion medium was converted to 14,15-dihydroxyeicosatrienoic acid (DHET). Similar results were obtained with endothelium-denuded vessels. 14,15-DHET was released from both the luminal and adventitial surfaces of the HSV. When HSVs were incubated with 14,15-[3H]EET under static (no flow) conditions, formation of 14,15-DHET was detected within 15 min and was inhibited by the selective sEH inhibitors N,N'-dicyclohexyl urea and N-cyclohexyl-N'-dodecanoic acid urea (CUDA). Similarly, CUDA inhibited the conversion of 11,12-[3H]EET to 11,12-DHET by the HSV. sEH inhibition enhanced the uptake of 14,15-[3H]EET and facilitated the formation of 10,11-epoxy-16:2, a beta-oxidation product. The HCA and HA converted 14,15-[3H]EET to DHET, and this also was inhibited by CUDA. These findings in intact human blood vessels indicate that conversion to DHET is the predominant pathway for 11,12- and 14,15-EET metabolism and that sEH inhibition can modulate EET metabolism in vascular tissue.     

Stimulus processing of glycine is dissociable from that of sucrose and glucose based on behaviorally measured taste signal detection in Sac 'taster' and 'non-taster' mice

Mouse strains have been divided into 'tasters' and 'non-tasters' based on their relatively high and low preference, respectively, for low concentrations of sucrose and saccharin. These phenotypic differences appear to be due to a polymorphism in the gene at the Sac locus encoding for the T1R3 taste receptor selectively affecting the functionality of the T1R2+3 heterodimer. To psychophysically examine whether these phenotypes are due to sensory sensitivity as opposed to hedonic responsiveness, we measured taste signal detection of sucrose, glucose, and glycine by Sac taster (C57BL/6J and SWR/J) and non-taster (129P3/J and DBA/2J) strains in an operant conditioning paradigm using a gustometer. The taster mice had lower detection thresholds for sucrose and glucose compared with the non-taster mice. The detection thresholds corresponded well with reported responsiveness to low concentrations of these sugars in two-bottle intake tests suggesting that the Sac taster phenotype has a sensory basis and is not simply a matter of strain differences in the hedonic evaluation of weak intensities of the stimuli. Taster status did not entirely account for the strain differences in detection thresholds for glycine, a 'sweet' tasting amino acid. Collapsed across strains, detection thresholds for sucrose and glucose were highly correlated with each other (r = 0.81), but only modestly correlated with those for glycine (r < or = 0.43). This suggests that stimulus processing of glycine in the perithreshold intensity domain can be dissociated from that of sucrose and glucose. The mechanism underlying this difference may be related to the ability of glycine to bind with the T1R1+3 heterodimer.