Recent studies have suggested that n-3 fatty acids, abundant in fish oil, protect against high-fa... more Recent studies have suggested that n-3 fatty acids, abundant in fish oil, protect against high-fat diet-induced insulin resistance through PPARα activation and a subsequent decrease in intracellular lipid abundance. To directly test this hypothesis we fed PPARα null and wild type (WT) mice for two weeks with isocaloric high-fat diets containing 27% fat from safflower oil (SAFF) or safflower oil with an 8% fish oil replacement (FISH). In both genotypes SAFF diet blunted the insulin-mediated suppression of hepatic glucose production (P<0.02 vs. genotype CONT) and PEPCK gene expression. In WT mice FISH feeding restored hepatic insulin sensitivity (HGP, P<0.002 vs. WT SAFF) whereas in contrast, in PPARα null mice FISH feeding failed to counteract hepatic insulin resistance (HGP n.s. vs. PPARα null SAFF). Hepatic insulin resistance in FISH fed PPARα null mice was dissociated from increases in hepatic triacylglycerol and acyl-CoA but accompanied by a more than threefold increase in hepatic diacylglycerol concentrations (P<0.0001 vs. genotype CONT). These data support the hypothesis that n-3 fatty acids protect from high-fat diet-induced hepatic insulin resistance in a PPARα and diacylglycerol dependent manner.
Eukaryotic genomes are packaged into nucleosome particles that occlude the DNA from interacting w... more Eukaryotic genomes are packaged into nucleosome particles that occlude the DNA from interacting with most DNA binding proteins. Nucleosomes have higher affinity for particular DNA sequences, reflecting the ability of the sequence to bend sharply, as required by the nucleosome structure. However, it is not known whether these sequence preferences have a significant influence on nucleosome position in vivo, and thus regulate the access of other proteins to DNA. Here we isolated nucleosome-bound sequences at high resolution from yeast and used these sequences in a new computational approach to construct and validate experimentally a nucleosome-DNA interaction model, and to predict the genome-wide organization of nucleosomes. Our results demonstrate that genomes encode an intrinsic nucleosome organization and that this intrinsic organization can explain ,50% of the in vivo nucleosome positions. This nucleosome positioning code may facilitate specific chromosome functions including transcription factor binding, transcription initiation, and even remodelling of the nucleosomes themselves.
American journal of physiology. Endocrinology and metabolism, 2001
The underlying mechanism by which skeletal muscle adapts to exercise training or chronic energy d... more The underlying mechanism by which skeletal muscle adapts to exercise training or chronic energy deprivation is largely unknown. To examine this question, rats were fed for 9 wk either with or without beta-guanadinopropionic acid (beta-GPA; 1% enriched diet), a creatine analog that is known to induce muscle adaptations similar to those induced by exercise training. Muscle phosphocreatine, ATP, and ATP/AMP ratios were all markedly decreased and led to the activation of AMP-activated protein kinase (AMPK) in the beta-GPA-fed rats compared with control rats. Under these conditions, nuclear respiratory factor-1 (NRF-1) binding activity, measured using a cDNA probe containing a sequence encoding for the promoter of delta-aminolevulinate (ALA) synthase, was increased by about eightfold in the muscle of beta-GPA-fed rats compared with the control group. Concomitantly, muscle ALA synthase mRNA and cytochrome c content were also increased. Mitochondrial density in both extensor digitorum long...
Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and is strongly assoc... more Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and is strongly associated with obesity. Increased concentrations of intracellular fatty acid metabolites have been postulated to interfere with insulin signaling by activation of a serine kinase cascade involving PKCθ in skeletal muscle. Uncoupling protein 3 (UCP3) has been postulated to dissipate the mitochondrial proton gradient and cause metabolic inefficiency. We therefore hypothesized that overexpression of UCP3 in skeletal muscle might protect against fat-induced insulin resistance in muscle by conversion of intramyocellular fat into thermal energy. Wild-type mice fed a high-fat diet were markedly insulin resistant, a result of defects in insulin-stimulated glucose uptake in skeletal muscle and hepatic insulin resistance. Insulin resistance in these tissues was associated with reduced insulin-stimulated insulin receptor substrate 1-(IRS-1-) and IRS-2-associated PI3K activity in muscle and liver, respectively. In contrast, UCP3-overexpressing mice were completely protected against fat-induced defects in insulin signaling and action in these tissues. Furthermore, these changes were associated with a lower membrane-to-cytosolic ratio of diacylglycerol and reduced PKCθ activity in whole-body fat-matched UCP3 transgenic mice. These results suggest that increasing mitochondrial uncoupling in skeletal muscle may be an excellent therapeutic target for type 2 diabetes mellitus.
American journal of physiology. Endocrinology and metabolism, 2002
To examine the mechanism by which fish oil protects against fat-induced insulin resistance, we st... more To examine the mechanism by which fish oil protects against fat-induced insulin resistance, we studied the effects of control, fish oil, and safflower oil diets on peroxisomal content, fatty acyl-CoA, diacylglycerol, and ceramide content in rat liver and muscle. We found that, in contrast to control and safflower oil-fed rats, fish oil feeding induced a 150% increase in the abundance of peroxisomal acyl-CoA oxidase and 3-ketoacyl-CoA thiolase in liver but lacked similar effects in muscle. This was paralleled by an almost twofold increase in hepatic peroxisome content (both P < 0.002 vs. control and safflower). These changes in the fish oil-fed rats were associated with a more than twofold lower hepatic triglyceride/diacylglycerol, as well as intramuscular triglyceride/fatty acyl-CoA, content. In conclusion, these data strongly support the hypothesis that n-3 fatty acids protect against fat-induced insulin resistance by serving as peroxisome proliferator-activated receptor-alpha l...
The detailed positions of nucleosomes profoundly impact gene regulation and are partly encoded by... more The detailed positions of nucleosomes profoundly impact gene regulation and are partly encoded by the genomic DNA sequence. However, less is known about the functional consequences of this encoding. Here, we address this question using a genome-wide map of ,380,000 yeast nucleosomes that we sequenced in their entirety. Utilizing the high resolution of our map, we refine our understanding of how nucleosome organizations are encoded by the DNA sequence and demonstrate that the genomic sequence is highly predictive of the in vivo nucleosome organization, even across new nucleosome-bound sequences that we isolated from fly and human. We find that Poly(dA:dT) tracts are an important component of these nucleosome positioning signals and that their nucleosome-disfavoring action results in large nucleosome depletion over them and over their flanking regions and enhances the accessibility of transcription factors to their cognate sites. Our results suggest that the yeast genome may utilize these nucleosome positioning signals to regulate gene expression with different transcriptional noise and activation kinetics and DNA replication with different origin efficiency. These distinct functions may be achieved by encoding both relatively closed (nucleosome-covered) chromatin organizations over some factor binding sites, where factors must compete with nucleosomes for DNA access, and relatively open (nucleosome-depleted) organizations over other factor sites, where factors bind without competition.
Nucleosomes occlude their wrapped DNA, strongly influencing the accessibility of functional DNA b... more Nucleosomes occlude their wrapped DNA, strongly influencing the accessibility of functional DNA binding sites. This has led to interest in genome-wide mapping of nucleosome positions and in understanding the principles that govern these positions. We recently compared the positions of nucleosomes reconstituted in vitro to a map of in vivo nucleosome locations 1. We found high similarity between the maps, implying that intrinsic DNA sequence preferences of nucleosomes have a major role in determining the organization of nucleosomes in vivo. A subsequent paper by Struhl and colleagues 2 (henceforth Zhang et al.) used a similar approach but stated an opposite conclusion. We believe that the stated conclusion of Zhang et al. 2 is inconsistent with data in both of these papers and also with previously published results and conclusions, including earlier publications by Struhl and colleagues. Both our study 1 and that of Zhang et al. 2 reconstituted nucleosomes in vitro using purified histone octamers and yeast genomic DNA, then mapped the resulting nucleosomes genomewide using micrococcal nuclease and parallel DNA sequencing. Evidence presented in these and earlier publications that proves that nucleosome sequence preferences contribute substantially to nucleosome organization in vivo includes the following. First, nucleosomebound sequences from yeast, worm, fly, chicken and human have distinctive patterns of
Eukaryotic transcription occurs within a chromatin environment, whose organization plays an impor... more Eukaryotic transcription occurs within a chromatin environment, whose organization plays an important regulatory role and is partly encoded in cis by the DNA sequence itself 1-6. Here, we examine whether evolutionary changes in gene expression are linked to changes in the DNA-encoded nucleosome organization of promoters. We find that in aerobic yeast species, where cellular respiration genes are active under typical growth conditions, the promoter sequences of these genes encode a relatively open (nucleosome-depleted) chromatin organization. This nucleosome-depleted organization requires only DNA sequence information, is independent of any co-factors and of transcription, and is a general property of growth-related genes. In contrast, in anaerobic yeast species, where cellular respiration genes are inactive under typical growth conditions, respiration gene promoters encode relatively closed (nucleosome-occupied) chromatin organizations. Thus, our results suggest a previously unidentified genetic mechanism underlying phenotypic diversity, consisting of DNA sequence changes that directly alter the DNA-encoded nucleosome organization of promoters.
Pu rification of yeast genomic DNA for in vitro nucleosome reconstitution. S. cer evisiae genomic... more Pu rification of yeast genomic DNA for in vitro nucleosome reconstitution. S. cer evisiae genomic DNA was purified from strain YLC8 (MAT(a) ura3(�) leu2(�)his3(�)met15(�)) by standard methods followed by extra steps to remove contaminating RNA. After recovery by ethanol precipitation, the sample was resuspended in TE buffer (TE is 10 mM Tris pH 8.0, 1 mM EDTA), and the DNA concentration was determined by agarose gel electrophoresis using ethidium stain, followed by comparison to mass standards using quantitative fluorometry. The sample was then subjected to RNase A digestion at 50 ⁰C overnight, using 100 µg of RNase A for every 10 µg of DNA, followed by ethanol precipitation of the DNA. After resuspension of the pellet in TE, the genomic DNA was sheared twice each through a 25 gauge needle and then a 27½ gauge needle. The entire mixture was then electrophoresed on a 20 x 20 cm, 1% agarose, 1X TAE gel at 100 V for 6-8 hours. The genomic DNA band was cut out, and the agarose slab cont...
The substrate for the proteins that express genetic information in the cell is not naked DNA but ... more The substrate for the proteins that express genetic information in the cell is not naked DNA but an assembly of nucleosomes, where the DNA is wrapped around histone proteins. The organization of these nucleosomes on genomic DNA is influenced by the DNA sequence. Here, we present a structure-based computational approach that translates sequence information into the energy required to bend DNA into a nucleosome-bound conformation. The calculations establish the relationship between DNA sequence and histone octamer binding affinity. In silico selection using this model identified several new DNA sequences, which were experimentally found to have histone octamer affinities comparable to the highest-affinity sequences known. The results provide insights into the molecular mechanism through which DNA sequence information encodes its organization. A quantitative appreciation of the thermodynamics of nucleosome positioning and rearrangement will be one of the key factors in understanding the regulation of transcription and in the design of new promoter architectures for the purposes of tuning gene expression dynamics.
Primary gene amplification, the mutation from one gene copy per genome to two or more copies per ... more Primary gene amplification, the mutation from one gene copy per genome to two or more copies per genome, is a major mechanism of oncogene overexpression in human cancers. Analysis of the structures of amplifications can provide important evidence about the mechanism of amplification formation. We report here the analysis of the structures of four independent spontaneous circular amplifications of ADH4:CUP1 in the yeast Saccharomyces cerevisiae. The structures of all four amplifications are consistent with their formation by a breakage-fusion-bridge (BFB) mechanism. All four of these amplifications include a centromere as predicted by the BFB model. All four of the amplifications have a novel joint located between the amplified DNA and the telomere, which results in a dicentric chromosome, and is adjacent to all the copies of the amplified DNA as predicted by the BFB model. In addition we demonstrated that two of the amplifications contain most of chromosome VII in an unrearranged form in a 1:1 ratio with the normal copy of chromosome VII, again consistent with the predictions of the BFB model. Finally, all four amplifications are circular, one stable endpoint for molecules after breakage- fusion-bridge.
Primary gene amplification, the mutation from one copy of a gene per genome to two or more genes ... more Primary gene amplification, the mutation from one copy of a gene per genome to two or more genes per genome is a major mechanism of oncogene overexpression. We previously developed a system in the yeast Saccharomyces cerevisiae to phenotypically detect primary amplifications of a reporter cassette, ADH4:CUP1. We present here the sequence analysis of novel joints from four independent, spontaneous circular amplifications identified by the ADH4:CUP1 system. All four novel joints consist of C(1-3) A telomeric repeats joined to short (14- to 16-bp) CA-rich tracts between ADH4 and the telomere of chromosome VII. In three of the four amplifications, the telomeric sequence and the CA-rich tract that are joined in the amplification are normally located in inverted orientation to each other on chromosome VII. In the fourth amplification, the CA-rich tract on chromosome VII is joined to telomere sequences from another chromosome. We suggest that formation of these amplifications was initiated by recombination between these CA-rich tracts and a telomere. The resulting dicentric chromosome could start a breakage-fusion-bridge cycle that could be resolved by the formation of a circular amplification structure.
Recent studies have suggested that n-3 fatty acids, abundant in fish oil, protect against high-fa... more Recent studies have suggested that n-3 fatty acids, abundant in fish oil, protect against high-fat diet-induced insulin resistance through PPARα activation and a subsequent decrease in intracellular lipid abundance. To directly test this hypothesis we fed PPARα null and wild type (WT) mice for two weeks with isocaloric high-fat diets containing 27% fat from safflower oil (SAFF) or safflower oil with an 8% fish oil replacement (FISH). In both genotypes SAFF diet blunted the insulin-mediated suppression of hepatic glucose production (P<0.02 vs. genotype CONT) and PEPCK gene expression. In WT mice FISH feeding restored hepatic insulin sensitivity (HGP, P<0.002 vs. WT SAFF) whereas in contrast, in PPARα null mice FISH feeding failed to counteract hepatic insulin resistance (HGP n.s. vs. PPARα null SAFF). Hepatic insulin resistance in FISH fed PPARα null mice was dissociated from increases in hepatic triacylglycerol and acyl-CoA but accompanied by a more than threefold increase in hepatic diacylglycerol concentrations (P<0.0001 vs. genotype CONT). These data support the hypothesis that n-3 fatty acids protect from high-fat diet-induced hepatic insulin resistance in a PPARα and diacylglycerol dependent manner.
OBJECTIVE-Insulin resistance in skeletal muscle plays a critical role in the pathogenesis of type... more OBJECTIVE-Insulin resistance in skeletal muscle plays a critical role in the pathogenesis of type 2 diabetes, yet the cellular mechanisms responsible for insulin resistance are poorly understood. In this study, we examine the role of serine phosphorylation of insulin receptor substrate (IRS)-1 in mediating fat-induced insulin resistance in skeletal muscle in vivo. RESEARCH DESIGN AND METHODS-To directly assess the role of serine phosphorylation in mediating fat-induced insulin resistance in skeletal muscle, we generated muscle-specific IRS-1 Ser 302 , Ser 307 , and Ser 612 mutated to alanine (Tg IRS-1 Ser3 Ala) and IRS-1 wild-type (Tg IRS-1 WT) transgenic mice and examined insulin signaling and insulin action in skeletal muscle in vivo. RESULTS-Tg IRS-1 Ser3 Ala mice were protected from fatinduced insulin resistance, as reflected by lower plasma glucose concentrations during a glucose tolerance test and increased insulin-stimulated muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. In contrast, Tg IRS-1 WT mice exhibited no improvement in glucose tolerance after high-fat feeding. Furthermore, Tg IRS-1 Ser3 Ala mice displayed a significant increase in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity and Akt phosphorylation in skeletal muscle in vivo compared with WT control littermates. CONCLUSIONS-These data demonstrate that serine phosphorylation of IRS-1 plays an important role in mediating fatinduced insulin resistance in skeletal muscle in vivo.
Recent studies have demonstrated a strong relationship between aging-associated reductions in mit... more Recent studies have demonstrated a strong relationship between aging-associated reductions in mitochondrial function, dysregulated intracellular lipid metabolism, and insulin resistance. Given the important role of the AMP-activated protein kinase (AMPK) in the regulation of fat oxidation and mitochondrial biogenesis, we examined AMPK activity in young and old rats and found that acute stimulation of AMPK-α 2 activity by 5′-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and exercise was blunted in skeletal muscle of old rats. Furthermore, mitochondrial biogenesis in response to chronic activation of AMPK with β-guanidinopropionic acid (β-GPA) feeding was also diminished in old rats. These results suggest that aging-associated reductions in AMPK activity may be an important contributing factor in the reduced mitochondrial function and dysregulated intracellular lipid metabolism associated with aging.
Recent studies have suggested that n-3 fatty acids, abundant in fish oil, protect against high-fa... more Recent studies have suggested that n-3 fatty acids, abundant in fish oil, protect against high-fat diet-induced insulin resistance through PPARα activation and a subsequent decrease in intracellular lipid abundance. To directly test this hypothesis we fed PPARα null and wild type (WT) mice for two weeks with isocaloric high-fat diets containing 27% fat from safflower oil (SAFF) or safflower oil with an 8% fish oil replacement (FISH). In both genotypes SAFF diet blunted the insulin-mediated suppression of hepatic glucose production (P<0.02 vs. genotype CONT) and PEPCK gene expression. In WT mice FISH feeding restored hepatic insulin sensitivity (HGP, P<0.002 vs. WT SAFF) whereas in contrast, in PPARα null mice FISH feeding failed to counteract hepatic insulin resistance (HGP n.s. vs. PPARα null SAFF). Hepatic insulin resistance in FISH fed PPARα null mice was dissociated from increases in hepatic triacylglycerol and acyl-CoA but accompanied by a more than threefold increase in hepatic diacylglycerol concentrations (P<0.0001 vs. genotype CONT). These data support the hypothesis that n-3 fatty acids protect from high-fat diet-induced hepatic insulin resistance in a PPARα and diacylglycerol dependent manner.
Eukaryotic genomes are packaged into nucleosome particles that occlude the DNA from interacting w... more Eukaryotic genomes are packaged into nucleosome particles that occlude the DNA from interacting with most DNA binding proteins. Nucleosomes have higher affinity for particular DNA sequences, reflecting the ability of the sequence to bend sharply, as required by the nucleosome structure. However, it is not known whether these sequence preferences have a significant influence on nucleosome position in vivo, and thus regulate the access of other proteins to DNA. Here we isolated nucleosome-bound sequences at high resolution from yeast and used these sequences in a new computational approach to construct and validate experimentally a nucleosome-DNA interaction model, and to predict the genome-wide organization of nucleosomes. Our results demonstrate that genomes encode an intrinsic nucleosome organization and that this intrinsic organization can explain ,50% of the in vivo nucleosome positions. This nucleosome positioning code may facilitate specific chromosome functions including transcription factor binding, transcription initiation, and even remodelling of the nucleosomes themselves.
American journal of physiology. Endocrinology and metabolism, 2001
The underlying mechanism by which skeletal muscle adapts to exercise training or chronic energy d... more The underlying mechanism by which skeletal muscle adapts to exercise training or chronic energy deprivation is largely unknown. To examine this question, rats were fed for 9 wk either with or without beta-guanadinopropionic acid (beta-GPA; 1% enriched diet), a creatine analog that is known to induce muscle adaptations similar to those induced by exercise training. Muscle phosphocreatine, ATP, and ATP/AMP ratios were all markedly decreased and led to the activation of AMP-activated protein kinase (AMPK) in the beta-GPA-fed rats compared with control rats. Under these conditions, nuclear respiratory factor-1 (NRF-1) binding activity, measured using a cDNA probe containing a sequence encoding for the promoter of delta-aminolevulinate (ALA) synthase, was increased by about eightfold in the muscle of beta-GPA-fed rats compared with the control group. Concomitantly, muscle ALA synthase mRNA and cytochrome c content were also increased. Mitochondrial density in both extensor digitorum long...
Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and is strongly assoc... more Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and is strongly associated with obesity. Increased concentrations of intracellular fatty acid metabolites have been postulated to interfere with insulin signaling by activation of a serine kinase cascade involving PKCθ in skeletal muscle. Uncoupling protein 3 (UCP3) has been postulated to dissipate the mitochondrial proton gradient and cause metabolic inefficiency. We therefore hypothesized that overexpression of UCP3 in skeletal muscle might protect against fat-induced insulin resistance in muscle by conversion of intramyocellular fat into thermal energy. Wild-type mice fed a high-fat diet were markedly insulin resistant, a result of defects in insulin-stimulated glucose uptake in skeletal muscle and hepatic insulin resistance. Insulin resistance in these tissues was associated with reduced insulin-stimulated insulin receptor substrate 1-(IRS-1-) and IRS-2-associated PI3K activity in muscle and liver, respectively. In contrast, UCP3-overexpressing mice were completely protected against fat-induced defects in insulin signaling and action in these tissues. Furthermore, these changes were associated with a lower membrane-to-cytosolic ratio of diacylglycerol and reduced PKCθ activity in whole-body fat-matched UCP3 transgenic mice. These results suggest that increasing mitochondrial uncoupling in skeletal muscle may be an excellent therapeutic target for type 2 diabetes mellitus.
American journal of physiology. Endocrinology and metabolism, 2002
To examine the mechanism by which fish oil protects against fat-induced insulin resistance, we st... more To examine the mechanism by which fish oil protects against fat-induced insulin resistance, we studied the effects of control, fish oil, and safflower oil diets on peroxisomal content, fatty acyl-CoA, diacylglycerol, and ceramide content in rat liver and muscle. We found that, in contrast to control and safflower oil-fed rats, fish oil feeding induced a 150% increase in the abundance of peroxisomal acyl-CoA oxidase and 3-ketoacyl-CoA thiolase in liver but lacked similar effects in muscle. This was paralleled by an almost twofold increase in hepatic peroxisome content (both P < 0.002 vs. control and safflower). These changes in the fish oil-fed rats were associated with a more than twofold lower hepatic triglyceride/diacylglycerol, as well as intramuscular triglyceride/fatty acyl-CoA, content. In conclusion, these data strongly support the hypothesis that n-3 fatty acids protect against fat-induced insulin resistance by serving as peroxisome proliferator-activated receptor-alpha l...
The detailed positions of nucleosomes profoundly impact gene regulation and are partly encoded by... more The detailed positions of nucleosomes profoundly impact gene regulation and are partly encoded by the genomic DNA sequence. However, less is known about the functional consequences of this encoding. Here, we address this question using a genome-wide map of ,380,000 yeast nucleosomes that we sequenced in their entirety. Utilizing the high resolution of our map, we refine our understanding of how nucleosome organizations are encoded by the DNA sequence and demonstrate that the genomic sequence is highly predictive of the in vivo nucleosome organization, even across new nucleosome-bound sequences that we isolated from fly and human. We find that Poly(dA:dT) tracts are an important component of these nucleosome positioning signals and that their nucleosome-disfavoring action results in large nucleosome depletion over them and over their flanking regions and enhances the accessibility of transcription factors to their cognate sites. Our results suggest that the yeast genome may utilize these nucleosome positioning signals to regulate gene expression with different transcriptional noise and activation kinetics and DNA replication with different origin efficiency. These distinct functions may be achieved by encoding both relatively closed (nucleosome-covered) chromatin organizations over some factor binding sites, where factors must compete with nucleosomes for DNA access, and relatively open (nucleosome-depleted) organizations over other factor sites, where factors bind without competition.
Nucleosomes occlude their wrapped DNA, strongly influencing the accessibility of functional DNA b... more Nucleosomes occlude their wrapped DNA, strongly influencing the accessibility of functional DNA binding sites. This has led to interest in genome-wide mapping of nucleosome positions and in understanding the principles that govern these positions. We recently compared the positions of nucleosomes reconstituted in vitro to a map of in vivo nucleosome locations 1. We found high similarity between the maps, implying that intrinsic DNA sequence preferences of nucleosomes have a major role in determining the organization of nucleosomes in vivo. A subsequent paper by Struhl and colleagues 2 (henceforth Zhang et al.) used a similar approach but stated an opposite conclusion. We believe that the stated conclusion of Zhang et al. 2 is inconsistent with data in both of these papers and also with previously published results and conclusions, including earlier publications by Struhl and colleagues. Both our study 1 and that of Zhang et al. 2 reconstituted nucleosomes in vitro using purified histone octamers and yeast genomic DNA, then mapped the resulting nucleosomes genomewide using micrococcal nuclease and parallel DNA sequencing. Evidence presented in these and earlier publications that proves that nucleosome sequence preferences contribute substantially to nucleosome organization in vivo includes the following. First, nucleosomebound sequences from yeast, worm, fly, chicken and human have distinctive patterns of
Eukaryotic transcription occurs within a chromatin environment, whose organization plays an impor... more Eukaryotic transcription occurs within a chromatin environment, whose organization plays an important regulatory role and is partly encoded in cis by the DNA sequence itself 1-6. Here, we examine whether evolutionary changes in gene expression are linked to changes in the DNA-encoded nucleosome organization of promoters. We find that in aerobic yeast species, where cellular respiration genes are active under typical growth conditions, the promoter sequences of these genes encode a relatively open (nucleosome-depleted) chromatin organization. This nucleosome-depleted organization requires only DNA sequence information, is independent of any co-factors and of transcription, and is a general property of growth-related genes. In contrast, in anaerobic yeast species, where cellular respiration genes are inactive under typical growth conditions, respiration gene promoters encode relatively closed (nucleosome-occupied) chromatin organizations. Thus, our results suggest a previously unidentified genetic mechanism underlying phenotypic diversity, consisting of DNA sequence changes that directly alter the DNA-encoded nucleosome organization of promoters.
Pu rification of yeast genomic DNA for in vitro nucleosome reconstitution. S. cer evisiae genomic... more Pu rification of yeast genomic DNA for in vitro nucleosome reconstitution. S. cer evisiae genomic DNA was purified from strain YLC8 (MAT(a) ura3(�) leu2(�)his3(�)met15(�)) by standard methods followed by extra steps to remove contaminating RNA. After recovery by ethanol precipitation, the sample was resuspended in TE buffer (TE is 10 mM Tris pH 8.0, 1 mM EDTA), and the DNA concentration was determined by agarose gel electrophoresis using ethidium stain, followed by comparison to mass standards using quantitative fluorometry. The sample was then subjected to RNase A digestion at 50 ⁰C overnight, using 100 µg of RNase A for every 10 µg of DNA, followed by ethanol precipitation of the DNA. After resuspension of the pellet in TE, the genomic DNA was sheared twice each through a 25 gauge needle and then a 27½ gauge needle. The entire mixture was then electrophoresed on a 20 x 20 cm, 1% agarose, 1X TAE gel at 100 V for 6-8 hours. The genomic DNA band was cut out, and the agarose slab cont...
The substrate for the proteins that express genetic information in the cell is not naked DNA but ... more The substrate for the proteins that express genetic information in the cell is not naked DNA but an assembly of nucleosomes, where the DNA is wrapped around histone proteins. The organization of these nucleosomes on genomic DNA is influenced by the DNA sequence. Here, we present a structure-based computational approach that translates sequence information into the energy required to bend DNA into a nucleosome-bound conformation. The calculations establish the relationship between DNA sequence and histone octamer binding affinity. In silico selection using this model identified several new DNA sequences, which were experimentally found to have histone octamer affinities comparable to the highest-affinity sequences known. The results provide insights into the molecular mechanism through which DNA sequence information encodes its organization. A quantitative appreciation of the thermodynamics of nucleosome positioning and rearrangement will be one of the key factors in understanding the regulation of transcription and in the design of new promoter architectures for the purposes of tuning gene expression dynamics.
Primary gene amplification, the mutation from one gene copy per genome to two or more copies per ... more Primary gene amplification, the mutation from one gene copy per genome to two or more copies per genome, is a major mechanism of oncogene overexpression in human cancers. Analysis of the structures of amplifications can provide important evidence about the mechanism of amplification formation. We report here the analysis of the structures of four independent spontaneous circular amplifications of ADH4:CUP1 in the yeast Saccharomyces cerevisiae. The structures of all four amplifications are consistent with their formation by a breakage-fusion-bridge (BFB) mechanism. All four of these amplifications include a centromere as predicted by the BFB model. All four of the amplifications have a novel joint located between the amplified DNA and the telomere, which results in a dicentric chromosome, and is adjacent to all the copies of the amplified DNA as predicted by the BFB model. In addition we demonstrated that two of the amplifications contain most of chromosome VII in an unrearranged form in a 1:1 ratio with the normal copy of chromosome VII, again consistent with the predictions of the BFB model. Finally, all four amplifications are circular, one stable endpoint for molecules after breakage- fusion-bridge.
Primary gene amplification, the mutation from one copy of a gene per genome to two or more genes ... more Primary gene amplification, the mutation from one copy of a gene per genome to two or more genes per genome is a major mechanism of oncogene overexpression. We previously developed a system in the yeast Saccharomyces cerevisiae to phenotypically detect primary amplifications of a reporter cassette, ADH4:CUP1. We present here the sequence analysis of novel joints from four independent, spontaneous circular amplifications identified by the ADH4:CUP1 system. All four novel joints consist of C(1-3) A telomeric repeats joined to short (14- to 16-bp) CA-rich tracts between ADH4 and the telomere of chromosome VII. In three of the four amplifications, the telomeric sequence and the CA-rich tract that are joined in the amplification are normally located in inverted orientation to each other on chromosome VII. In the fourth amplification, the CA-rich tract on chromosome VII is joined to telomere sequences from another chromosome. We suggest that formation of these amplifications was initiated by recombination between these CA-rich tracts and a telomere. The resulting dicentric chromosome could start a breakage-fusion-bridge cycle that could be resolved by the formation of a circular amplification structure.
Recent studies have suggested that n-3 fatty acids, abundant in fish oil, protect against high-fa... more Recent studies have suggested that n-3 fatty acids, abundant in fish oil, protect against high-fat diet-induced insulin resistance through PPARα activation and a subsequent decrease in intracellular lipid abundance. To directly test this hypothesis we fed PPARα null and wild type (WT) mice for two weeks with isocaloric high-fat diets containing 27% fat from safflower oil (SAFF) or safflower oil with an 8% fish oil replacement (FISH). In both genotypes SAFF diet blunted the insulin-mediated suppression of hepatic glucose production (P<0.02 vs. genotype CONT) and PEPCK gene expression. In WT mice FISH feeding restored hepatic insulin sensitivity (HGP, P<0.002 vs. WT SAFF) whereas in contrast, in PPARα null mice FISH feeding failed to counteract hepatic insulin resistance (HGP n.s. vs. PPARα null SAFF). Hepatic insulin resistance in FISH fed PPARα null mice was dissociated from increases in hepatic triacylglycerol and acyl-CoA but accompanied by a more than threefold increase in hepatic diacylglycerol concentrations (P<0.0001 vs. genotype CONT). These data support the hypothesis that n-3 fatty acids protect from high-fat diet-induced hepatic insulin resistance in a PPARα and diacylglycerol dependent manner.
OBJECTIVE-Insulin resistance in skeletal muscle plays a critical role in the pathogenesis of type... more OBJECTIVE-Insulin resistance in skeletal muscle plays a critical role in the pathogenesis of type 2 diabetes, yet the cellular mechanisms responsible for insulin resistance are poorly understood. In this study, we examine the role of serine phosphorylation of insulin receptor substrate (IRS)-1 in mediating fat-induced insulin resistance in skeletal muscle in vivo. RESEARCH DESIGN AND METHODS-To directly assess the role of serine phosphorylation in mediating fat-induced insulin resistance in skeletal muscle, we generated muscle-specific IRS-1 Ser 302 , Ser 307 , and Ser 612 mutated to alanine (Tg IRS-1 Ser3 Ala) and IRS-1 wild-type (Tg IRS-1 WT) transgenic mice and examined insulin signaling and insulin action in skeletal muscle in vivo. RESULTS-Tg IRS-1 Ser3 Ala mice were protected from fatinduced insulin resistance, as reflected by lower plasma glucose concentrations during a glucose tolerance test and increased insulin-stimulated muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. In contrast, Tg IRS-1 WT mice exhibited no improvement in glucose tolerance after high-fat feeding. Furthermore, Tg IRS-1 Ser3 Ala mice displayed a significant increase in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity and Akt phosphorylation in skeletal muscle in vivo compared with WT control littermates. CONCLUSIONS-These data demonstrate that serine phosphorylation of IRS-1 plays an important role in mediating fatinduced insulin resistance in skeletal muscle in vivo.
Recent studies have demonstrated a strong relationship between aging-associated reductions in mit... more Recent studies have demonstrated a strong relationship between aging-associated reductions in mitochondrial function, dysregulated intracellular lipid metabolism, and insulin resistance. Given the important role of the AMP-activated protein kinase (AMPK) in the regulation of fat oxidation and mitochondrial biogenesis, we examined AMPK activity in young and old rats and found that acute stimulation of AMPK-α 2 activity by 5′-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and exercise was blunted in skeletal muscle of old rats. Furthermore, mitochondrial biogenesis in response to chronic activation of AMPK with β-guanidinopropionic acid (β-GPA) feeding was also diminished in old rats. These results suggest that aging-associated reductions in AMPK activity may be an important contributing factor in the reduced mitochondrial function and dysregulated intracellular lipid metabolism associated with aging.
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Papers by Irene K Moore