Interindividual comparison of methylation profiling by MSAM
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ABSTRACT: Interindividual variation in methylation profiling of human DNA samples were detected using two-tissue screening by MSAM. 0.5ug of DNA was serially digested with SmaI and XmaI followed by an adaptor ligation and adaptor mediated PCR amplification HF (hair follicle) and PBL (Peripheral Blood Leukocyte) DNA samples for 8 different individuals, two-color experiment, interindividual paired comparison (same sex and age)
Project description:Investigate the persistent effects of early postnatal overnutrition on the developmental establishment of the DNA methylation in the mouse hypothalamus. Early postnatal overnutrition was induced in mice by reducing the litter size from normally 9 (C) to 4 (SL) pups per litter. Hypothalami were collected from both C and SL mice at the age of postnatal day 180 (P180). Genome-wide DNA methylation difference between SL and C were detected by MSAM. Equal amount of genomic DNA from 5 hypothalami of the same group were pooled as one MSAM sample. Two pooled DNA samples for each group were used for comparison that meant total 10 hypothalami for each group. 500ng pooled DNA was serially digested with SmaI and XmaI followed by adaptor ligation and PCR amplification. Two cohybridizations were performed to compare DNA methylation between SL and C hypothalami, with day swap.
Project description:Investigate the genome-wide DNA methylation changes in the mouse hypothalamus during the suckling period. Hypothalami were collected from new born (P0) mice, or mice of 21 day-old (P21). Two-color experiment was performed as paired comparison of P21 vs. P0 with two biological replicates. Genome-wide DNA methylation changes from P0 to P21 were detected by MSAM. As a brief description of the MSAM: 500ng of genomic DNA was serially digested with SmaI and XmaI followed by an adaptor ligation and adaptor mediated PCR amplification and then cohybridization.
Project description:Investigate the genome-wide DNA methylation and gene expression changes during human embryonic stem cell differentiation. hESCs (H1 and H13) DNA samples for 3 different differentian stage (D0, undifferentiated;D21, 21 days after undirected differentiation; D90, 90 days after undirected differentiation); two-color experiment, paired comparison (differentiated vs undifferentiated; A & B: two biological replicates). Differentian-stage variation in methylation profiling of human DNA samples were detected by MSAM. 2µg of DNA was serially digested with SmaI and XmaI followed by an adaptor ligation and adaptor mediated PCR amplification. Analysis of differentian-stage variation in gene expression of human mRNA samples were implementd following standard Agilent protocol.
Project description:The goal is to compare the DNA methylation patterns between ARH and PVH to examine to what extent DNA methylation is region specific in genome scale. ARH and PVH were micro-dissected from mice brains. Each 7 pieces of ARH or PVH were pooled and used for DNA methylation comparison by methylation specific amplification microarray (MSAM). As a brief description of the MSAM: 500ng of genomic DNA was serially digested with SmaI and XmaI followed by an adaptor ligation and adaptor mediated PCR amplification and then cohybridization. Two-color cohybridizations were performed as paired comparison of PVH vs. ARH with two biological replicates including dye swap.
Project description:DNA methylation profiling of colonic mucosal DNA between P90 and P30 mice. 0.5ug of DNA was serially digested with SmaI and XmaI followed by an adaptor ligation and adaptor mediated PCR amplification Two independent P90 to P30 comparisons were performed as follows. Samples were labelled with Cy3 (P30) and Cy5 (P90) and two independent P90 to P30 comparisons were done on a 2x105k methylation specific amplification microarray (MSAM) containing 90,535 probes, covering 77% of the 31,019 SmaI intervals between 200 bp and 2 kb in the mouse genome (average 3.8 probes per interval)
Project description:We describe a simple method, Digital Restriction Enzyme Analysis of Methylation (DREAM), based on sequential DNA digestion with a pair of methylation-blocked and methylation-tolerant neoschizomeric restriction enzymes SmaI/XmaI followed by end repair and ultra-deep sequencing. DREAM provides information on 160,000 unique CpG sites of which 39,000 are in CpG islands, and 33,000 are at transcription start sites (-1 kb to +1 kb) of 13,139 RefSeq genes. We compared DNA methylation values in white blood cells from 4 healthy individuals and found them to be remarkably uniform. Interindividual differences >30% were observed only at 227 of 28,331 (0.8%) of autosomal CCCGGG sites covered by 100+ sequencing reads. Similarly, differences at only 59 sites were observed between the cord and adult blood. Conserved methylation patterns in healthy blood cells contrasted with extensive changes affecting 18-40% of CpG sites in leukemia. The method is cost effective, quantitative (r2=0.93 when compared to bisulfite pyrosequencing), reproducible (r2=0.997), and can detect differences >25% with false positive rate <0.001. Accurate analysis of changes in DNA methylation will be useful in quantifying epigenetic effects of environment and nutrition, correlating developmental epigenetic variation with phenotypes, understanding epigenetics of cancer and chronic diseases, measuring the effects of drugs on DNA methylation or deriving new biological insights into mammalian genomes. Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the DNA methylation profiles of healthy white blood cells from cord blood and adult blood, acute myeloid leukemia bone marrow, and two leukemia cell lines (HEL and K562). In this approach, genomic DNA is sequentially cut at CCCGGG sites with the methylation-sensitive enzyme SmaI (blunt ends) and its methylation-tolerant neoschizomer XmaI (5'CCGG overhangs), creating different end sequences that represent methylation status of the CCCGGG sites. These end sequences are analyzed by next-generation sequencing, and thereafter the methylation status at individual CCCGGG sites across the genome can be determined.
Project description:Human epidemiologic and animal model data indicate that early environmental influences can persistently alter an individual’s risk of obesity. Environmental effects on hypothalamic developmental epigenetics provide a strong candidate mechanism to explain such ‘developmental programming’ of obesity. To advance our understanding of these processes, it is essential to determine to what extent the diversity of hypothalamic cell types is regulated by epigenetic differences, and when these are established. By performing genome-scale DNA methylation profiling in hypothalamic neurons and non-neuronal cells at postnatal day 0 (P0) and P21, we found that most of the DNA methylation differences distinguishing these two cell types are established postnatally. We found dramatic neuron-specific increases in DNA methylation from P0 to P21. Gene ontology analyses indicated that cell-type specific P0 to P21 methylation changes are key regulators of hypothalamic development. Quantitative bisulfite pyrosequencing verified our methylation profiling results in 16 of 16 selected regions. Expression differences were associated with DNA methylation in several genes analyzed. Our data indicate that future studies of hypothalamic epigenetics in developmental programming of obesity will gain far greater sensitivity and insight by examining outcomes at the cell-type specific level. Moreover, our results provide new evidence that early postnatal life is a critical period for murine hypothalamic developmental epigenetics. Hypothalami were dissected from inbred male C57 mice at postnatal day 0 (P0) and P21. Non-neuronal and neuronal nuclei were separated via fluorescence-activated sorting based on staining for the neuron-specific nuclear surface marker NeuN; each sample for sorting was comprised of 2 age-matched hypothalami. Genome-scale DNA methylation profiling was performed by methylation specific amplification coupled with next generation sequencing (MSA-seq) as decribed below (5 independent samples per age).
Project description:BACKGROUND & AIMS: Toll-like receptor 2 (Tlr2) is important in bacterial pattern recognition and has been recognized as a modifier of intestinal inflammation. In this study we sought to determine the epigenomic, transcriptomic and microbiomic consequences of Tlr2 deficiency in the colonic mucosa of mice to gain insights into biological pathways that shape the interface between the gut microflora and the mammalian host. METHODS: Colonic mucosa from C57BL/6 and Tlr2-/- mice was interrogated by methylation specific amplification microarray (MSAM) to screen for changes in DNA methylation, with bisulfite pyrosequencing validation. Transcriptomic changes in the same tissue were analyzed by microarray expression profiling and real time RT-PCR. The mucosal microbiome was studied by high throughput, detailed pyrosequencing of 16S RNA. RESULTS: Tlr2 deficiency influenced the methylation of about 1% of the interrogated genome and resulted in a significant expression change in a similar percentage of all transcripts studied. Importantly, gene ontology analysis revealed that the expression of genes involved in immune processes is significantly modified by the absence of Tlr2, some of which have been already linked to inflammatory bowel diseases (Stat1, Anpep), for example. Overlaps between DNA methylation and gene expression changes were confirmed at Anpep and Ifit2. The epigenomic and transcriptomic modifications associated with alteration in mucosal microbial composition, affecting 11% of the detected bacterial species in the Tlr2-/- animals. CONCLUSIONS: Tlr2 deficiency induces colonic mucosal epigenomic, transcriptomic and microbiomic changes underscoring the intricate network of biological processes that provide the link between genotype and phenotype in mammals. Our findings bare implications for common gastrointestinal disorders such as IBD and colon cancer. [MSAM]: Methylation profiling. Two independent Tlr2-/- to WT comparisons were performed as follows. Samples were labelled with Cy3 (WT) and Cy5 (Tlr2-/-) and two independent Tlr2-/- to WT comparisons were done on a 2x105k microarray containing probes, covering 33,404 (81% of all) SmaI intervals between 100 bp and 2.2 kb in the mouse genome. [mRNA]: mRNA profiling. 4 independent Tlr2-/- to WT comparisons were performed as follows. RNA (0.4ug) samples were processed and labelled with Cy3 and Cy5 (Quick Amp labelling kit, two color, Agilent technologies) and 4 independent Tlr2-/- to WT comparisons were done on Agilent technologies 4x44k whole genomic expression microarray G2519F, Amadid:014868). The Supplementary files below contain processed data from the MSAM and expression microarrays. For the MSAM: an interval was considered a 'hit' if the averaged probe ratios within an interval showed a >1.6 fold change in both Tlr2-/- vs. WT comparisons. For the expression microarrays: transcripts were considered a 'hit' if probe average intensities showed a >1.6 fold change in at least two microarray comparisons out of four, with the remaining arrays not contradicting the increase or decrease of expression (i.e.: 1 or above 1 in case at least two other >1.6; 1 or less than 1 in case at least two other <0.625).
Project description:Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population worldwide. Recent studies have demonstrated strong genetic associations between AMD and single nucleotide polymorphisms (SNPs) within genes such as CFH and HTRA1. However, we found monozygotic twins had discordant AMD phenotypes (one with disease, the other without disease), suggesting that an epigenetic mechanism may control the pathogenesis of AMD. We obtained genomic DNA from the twins' peripheral blood mononuclear cells (PBMCs) and subjected it to DNA methylation-chip analysis (MeDIP-chip) that profiled genome-wide DNA methylation patterns on promoters of all genes and microRNAs. Our MeDIP-chip analysis identified 256 genes with hypo-methylated promoters only in the twins with AMD and 744 genes with hyper-methylated promoters only in the twins with AMD. Importantly, the promoter region of IL17RC was associated with hypo-methylated CpG sites only in the twins with AMD but not in the twins without AMD. Two pairs of twins with discordant AMD phenotypes. MeDIP-chip analysis of DNA methylation patterns in PBMCs.