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: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: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 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: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: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:The transcription factor Zic3 is required for maintenance of embryonic stem (ES) cell pluripotency (Lim LS et al, Mol Biol Cell. 2007;18:1348-1358). By genome-wide chromatin immunoprecipitation (ChIP-chip) in ES cells, we have identified 379 direct Zic3 targets, many of which are functionally associated with pluripotency, cell cycle, proliferation, oncogenesis and early embryogenesis. E14 cells were cultured to a density of 1 x 108 cells for each IP. Two biological replicates were performed per experiment. Cells were cross-linked for 10 minutes at room temperature with 1% (w/v) formaldehyde and the reaction subsequently quenched with 125mM glycine. Nuclear fractions were isolated and the DNA sheared to average lengths of 200-500 bp. For analysis on mouse promoter arrays, purified ChIP material was processed according to the Agilent ChIP-on-chip protocol, and labeled DNA was hybridized to Agilent mouse promoter ChIP-on-chip arrays for 40 hours at 65oC (G4490A; Agilent Technologies, Santa Clara, CA). Chips were washed and scanned as per manufacturer’s protocol and the results were processed with Agilent's ChIP Analytics software v1.3. A p-value cutoff <0.001 was specified in our analysis. To further minimize false positives, we applied a “neighborhood voting” algorithm [2] to filter for high confidence Zic3-enriched sites, wherein binding was considered genuine only in the presence of a second, significantly enriched, neighboring probe (p < 0.005).
Project description:Background: Previously we reported extensive gene expression reprogramming during epithelial to mesenchymal transition (EMT) of primary prostate cells. Here we investigated the hypothesis that specific histone and DNA methylations are involved in coordination of gene expression during EMT and early stages of transformation. Results: Genome-wide profiling of histone methylations (H3K4me3 and H3K27me3) and DNA methylation (DNAMe) was applied on a prostate cell model during EMT and malignant transformation. Integrated analyses of promoter epigenetic modifications and gene expression changes revealed strong correlations between the dynamic changes of histone methylations and gene expression. DNA methylation was weakly associated with global gene repression, but strongly correlated to gene silencing when genes co-modified by H3K4me3 were excluded. In genes labeled with multiple epigenetic marks in their promoters, the level of transcription was associated with the net signal intensity of the activating mark H3K4me3 minus the repressive mark H3K27me3 or DNAMe, indicating that the effect on gene expression of bivalent marks (H3K4/K27me3 or H3K4me3/DNAMe) depends on relative modification intensities. Sets of genes, including epithelial cell junction and EMT associated fibroblast growth factor receptor genes, showed corresponding changes concerning epigenetic modifications and gene expression during EMT. Conclusions: This work presents the first blueprint of epigenetic modifications during EMT in prostate cells and shows that specific histone methylations are extensively involved in gene expression reprogramming during EMT and carcinogenesis. The observation that transcription activity of bivalently marked genes depends on the relative labeling intensity of individual marks provides a new view of quantitative regulation of epigenetic modification.
Project description:Ash1 is a Trithorax Group (TrxG) protein with histone methyl transferase activity that is associated with gene activation. Here we use ChIP-chip to determine the occupancy of Ash1 at promoters in murine embryonic stem cells. This dataset includes singlet ChIP-chip data targeting Ash1 in murine Embryonic stem cells.