Saleh Mahmood

Genome-wide survey reveals dynamic widespread tissue-specific changes in DNA methylation during development

BackgroundChanges in DNA methylation in the mammalian genome during development are frequent events and play major roles regulating gene expression and other developmental processes. It is necessary to identify these events so that we may understand how these changes affect normal development and how aberrant changes may impact disease.ResultsIn this study Me thylated D NA I mmunoP recipitation (MeDIP) was used in conjunction with a NimbleGen promoter plus CpG island (CpGi) array to identify T issue and D evelopmental S tage specific D ifferentially M ethylated DNA R egions (T-DMRs and DS-DMRs) on a genome-wide basis. Four tissues (brain, heart, liver, and testis) from C57BL/6J mice were analyzed at three developmental stages (15 day embryo, E15; new born, NB; 12 week adult, AD). Almost 5,000 adult T-DMRs and 10,000 DS-DMRs were identified. Surprisingly, almost all DS-DMRs were tissue specific (i.e. methylated in at least one tissue and unmethylated in one or more tissues). In addition our results indicate that many DS-DMRs are methylated at early development stages (E15 and NB) but are unmethylated in adult. There is a very strong bias for testis specific methylation in non-CpGi promoter regions (94%). Although the majority of T-DMRs and DS-DMRs tended to be in non-CpGi promoter regions, a relatively large number were also located in CpGi in promoter, intragenic and intergenic regions (>15% of the 15,979 CpGi on the array).ConclusionsOur data suggests the vast majority of unique sequence DNA methylation has tissue specificity, that demethylation has a prominent role in tissue differentiation, and that DNA methylation has regulatory roles in alternative promoter selection and in non-promoter regions. Overall, our studies indicate changes in DNA methylation during development are a dynamic, widespread, and tissue-specific process involving both DNA methylation and demethylation.

Identification of DNA methylation in 3′ genomic regions that are associated with upregulation of gene expression in colorectal cancer

Restriction Landmark Genomic Scanning (RLGS), a method for the two-dimensional display of end-labeled DNA restriction fragments, was utilized to identify genomic regions of CpG island methylation associated with Human Colon Cancer. An Average of 1.5% of the RLGS loci/spots are lost or significantly reduced in sporadic primary colon tumors relative to normal colon mucosa from the same patient. This may represent tumor specific methylation of about 400 CpG islands in sporadic colon cancer. A number of RLGS loci exhibiting frequent loss associated with colon cancer were cloned. DNA sequence analysis indicated that the RLGS loci identified genomic regions characteristic of CpG islands. A number of methods including bisulfite genomic sequencing as well as quantitative MassARRAY methylation analysis (www.sequenom.com) confirmed tumor specific methylation at several of these loci. DNA database searches indicated that candidate genes associated with these loci include transcription factors and genes involved in signal transduction (52%), and genes of unknown function (37%). Expression analysis using quantitative real time RT-PCR indicates that methylation of some CpG islands located in non-promoter regions were associated with up-regulation of gene expression in colorectal cancer. These results indicate that alterations in methylation status within CpG islands in colon tumors may have complex consequences on gene expression and tumorigenesis, sometimes resulting in up regulation or ectopic gene expression that may involve novel regulatory mechanisms.

Epigenetic changes in hypothalamic appetite regulatory genes may underlie the developmental programming for obesity in rat neonates subjected to a high-carbohydrate dietary modification.

Earlier, we showed that rearing of newborn rats on a high-carbohydrate (HC) milk formula resulted in the onset of hyperinsulinemia, its persistence in the post-weaning period and adult-onset obesity. DNA methylation of CpG dinucleotides in the proximal promoter region and modifications in the N-terminal tail of histone 3 associated with the neuropeptide Y (Npy) and pro-opiomelanocortin (Pomc) genes were investigated to decipher the molecular mechanisms supporting the development of obesity in HC females. Although there were no differences in the methylation status of CpG dinucleotides in the proximal promoter region of the Pomc gene, altered methylation of specific CpG dinucleotides proximal to the transcription start site was observed for the Npy gene in the hypothalami of 16- and 100-day-old HC rats compared with their methylation status in mother-fed (MF) rats. Investigation of histone tail modifications on hypothalamic chromatin extracts from 16-day-old rats indicated decreased acetylation of lysine 9 in histone 3 (H3K9) for the Pomc gene and increased acetylation for the same residue for the Npy gene, without changes in histone methylation (H3K9) in both genes in HC rats. These findings are consistent with the changes in the levels of Npy and Pomc mRNAs in the hypothalami of HC rats compared with MF animals. Our results suggest that epigenetic modifications could contribute to the altered gene expression of the Npy and Pomc genes in the hypothalami of HC rats and could be a mechanism leading to hyperphagia and the development of obesity in adult female HC rats.

Metabolic programming effects initiated in the suckling period predisposing for adult-onset obesity cannot be reversed by calorie restriction

Neonatal rats reared on high-carbohydrate (HC) milk formula developed chronic hyperinsulinemia and adult-onset obesity due to programming of islets and the hypothalamic energy circuitry. In this study, calorie restriction by pair-feeding was imposed on HC male rats (HC/PF) to normalize food intake similar to that of mother-fed (MF) rats from weaning until postnatal day 140. A group of HC/PF rats was switched over to ad libitum feeding (HC/PF/AL) from days 90 to 140. Pair-feeding reduced body weight gains and serum insulin and leptin levels in HC/PF rats compared with HC rats, but these parameters were restored to HC levels in the HC/PF/AL rats after ad libitum feeding. Interestingly, the heightened insulin secretory response of isolated islets from adult HC/PF and HC/PF/ AL rats to glucose, acetylcholine, and oxymetazoline were not significantly different from the responses of islets from HC rats. Similarly, the expression of neuropeptide Y and proopiomelanocortin in the hypothalamus was not significantly different among HC, HC/PF, and HC/PF/AL rats. Expression of the leptin receptor in the hypothalami from the HC, HC/PF, and HC/PF/AL rats mirrored that of serum leptin, whereas suppressor of cytokine signaling 3 (Socs3) expression remained high in these three groups. The results indicate that, although calorie restriction resulted in reduction in body weight gain and normalized the serum hormonal pattern, the programed predisposition for the hypersecretory capacity of islets and the hypothalamic hyperphagic response in the HC rats could not be permanently overcome by the pair-feeding imposed on HC rats.

Postnatal exposure to a high-carbohydrate diet interferes epigenetically with thyroid hormone receptor induction of the adult male rat skeletal muscle glucose transporter isoform 4 expression.

Early life nutritional intervention causes adult-onset insulin resistance and obesity in rats. Thyroid hormone receptor (TR), in turn, transcriptionally enhances skeletal muscle Glut4 expression. We tested the hypothesis that reduced circulating thyroid-stimulating hormone and T4 concentrations encountered in postnatal (PN4-PN24) high-carbohydrate (HC) milk formula-fed versus the mother-fed controls (MF) would epigenetically interfere with TR induction of adult (100 days) male rat skeletal muscle Glut4 expression, thereby providing a molecular mechanism mediating insulin resistance. We observed increased DNA methylation of the CpG island with enhanced recruitment of Dnmt3a, Dnmt3b and MeCP2 in the glut4 promoter region along with reduced acetylation of histone (H)2A.Z and H4 particularly at the H4.lysine (K)16 residue, which was predominantly mediated by histone deacetylase 4 (HDAC4). This was followed by enhanced recruitment of heterochromatin protein 1β to the glut4 promoter with increased Suv39H1 methylase concentrations. These changes reduced TR binding of the T3 response element of the glut4 gene (TREs; -473 to -450 bp) detected qualitatively in vivo (electromobility shift assay) and quantified ex vivo (chromatin immunoprecipitation). In addition, the recruitment of steroid receptor coactivator and CREB-binding protein to the glut4 promoter-protein complex was reduced. Co-immunoprecipitation experiments confirmed the interaction between TR and CBP to be reduced and HDAC4 to be enhanced in HC versus MF groups. These molecular changes were associated with diminished skeletal muscle Glut4 mRNA and protein concentrations. We conclude that early postnatal exposure to HC diet epigenetically reduced TR induction of adult male skeletal muscle Glut4 expression, uncovering novel molecular mechanisms contributing to adult insulin resistance and obesity.

Adult-onset obesity induced by early life overnutrition could be reversed by moderate caloric restriction

Overnutrition during the suckling period (small litter, SL) results in the development of adult-onset obesity. Our aim was to investigate whether two levels of caloric restriction (CR) in the early postweaning period can reverse obese phenotype in SL rats. The normal litter (NL) had 12 pups/dam and SL had 3 male pups/dam from the postnatal day 3 until day 21. After weaning, rats consumed lab chow as indicated: 1) NL and SL groups were on ad libitum regimen up to day 140, 2) another SL group was pair-fed (SL/PF) to NL(∼14% reduction), 3) SL/PF/AL group was pair-fed up to day 94 and then switched to ad libitum feeding, 4) SL/CR group received 24% reduction (moderate CR) in food intake compared with SL, and 5) SL/CR/AL group was on 24% CR up to day 94 and then switched to ad libitum feeding. Pair-feeding reduced body weight gains and serum insulin and leptin levels compared with SL rats, but these parameters were restored to SL levels in the SL/PF/AL rats after switching to ad libitum feeding. Interestingly, the moderate CR normalized these parameters in SL/CR and SL/CR/AL rats compared with NL. The expression of neuropeptide Y, proopiomelanocortin, and leptin receptor returned to control levels in hypothalami from SL/CR and SL/CR/AL rats. These results indicate that appropriate manipulation of energy intake during the early postweaning period could lead to longer-lasting effects on the regulation of body weight homeostasis via reversal of the early preweaning programming effects on the hypothalamic appetite regulation mechanism.

Lack of mitochondria-generated acetyl-CoA by pyruvate dehydrogenase complex downregulates gene expression in the hepatic de novo lipogenic pathway.

During the absorptive state, the liver stores excess glucose as glycogen and synthesizes fatty acids for triglyceride synthesis for export as very low density lipoproteins. For de novo synthesis of fatty acids from glucose, the mitochondrial pyruvate dehydrogenase complex (PDC) is the gatekeeper for the generation of acetyl-CoA from glucose-derived pyruvate. Here, we tested the hypothesis that limiting the supply of PDC-generated acetyl-CoA from glucose would have an impact on expression of key genes in the lipogenic pathway. In the present study, although the postnatal growth of liver-specific PDC-deficient (L-PDCKO) male mice was largely unaltered, the mice developed hyperinsulinemia with lower blood glucose levels in the fed state. Serum and liver lipid triglyceride and cholesterol levels remained unaltered in L-PDCKO mice. Expression of several key genes (ACL, ACC1) in the lipogenic pathway and their upstream regulators (LXR, SREBP1, ChREBP) as well as several genes in glucose metabolism (Pklr, G6pd2, Pck1) and fatty acid oxidation (FAT, Cpt1a) was downregulated in livers from L-PDCKO mice. Interestingly, there was concomitant upregulation of lipogenic genes in adipose tissue from L-PDCKO mice. Although, the total hepatic acetyl-CoA content remained unaltered in L-PDCKO mice, modified acetylation profiles of proteins in the nuclear compartment suggested an important role for PDC-generated acetyl-CoA in gene expression in de novo fatty acid synthesis in the liver. This finding has important implications for the regulation of hepatic lipid synthesis in pathological states.

Featured Article: Beta cell specific pyruvate dehydrogenase alpha gene deletion results in a reduced islet number and β-cell mass postnatally

The ability of pancreatic β-cells to undertake glucose-stimulated insulin secretion (GSIS) depends on the generation of adenosine triphosphate (ATP) within the mitochondria from pyruvate, a major rate-limiting enzyme being pyruvate dehydrogenase (PDH) complex (PDC). However, glucose metabolism also controls β-cell mass. To examine the role of PDC in the regulation of pancreatic β-cell development and maturation, we generated β-cell-targeted PDHα subunit knock-out male mice (β-PDHKO) and compared these with control males (β-PDHCT) from birth until 6–8 weeks age. Pancreas morphology, transcription factor expression, pancreatic insulin content, and circulating glucose and insulin values were compared. Compared to β-PDHCT male mice, β-PDHKO animals had significantly reduced pancreatic insulin content from birth, a lower serum insulin content from day 15, and relative hyperglycemia from day 30. Isolated islets from β-PDHKO mice demonstrated a reduced GSIS. The number of islets per pancreatic area, mean islet area, and the proportion of islet cells that were β-cells were all reduced in β-PDHKO animals. Similarly the number of insulin-immunopositive, extra-islet small endocrine cell clusters, a possible source of β-cell progenitors, was lower in β-PDHKO mice. Analysis of pancreatic expression of transcription factors responsible for β-cell lineage commitment, proliferation, and maturation, Pdx1, Neurogenin3, and NeuroD1 showed that mRNA abundance was reduced in the β-PDHKO. This demonstrates that PDC is not only required for insulin expression and glucose-stimulated secretion, but also directly influences β-cell growth and maturity, and positions glucose metabolism as a direct regulator of β-cell mass and plasticity.