Unknown,Transcriptomics,Genomics,Proteomics

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Developmental establishment of DNA methylation in hypothalamic neurons and glia.

ABSTRACT: 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).

ORGANISM(S): Mus musculus

SUBMITTER: Ge Li

PROVIDER: E-GEOD-48256 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:Purpose: to characterize epigenetic changes following Twist1 mediated Epithelial-Mesenchymal Transition in human Methods: we characterized the epigenetic and transcriptome landscapes using whole genome transcriptome analysis by RNA-seq, DNA methylation by digital restriction enzyme analysis of methylation (DREAM) and histone modifications by CHIP-seq of H3K4me3 and H3K27me3 in immortalized human mammary epithelial cells relative to cells induced to undergo EMT by Twist1. Results: EMT is accompanied by focal hypermethylation and widespread global DNA hypomethylation, predominantly within transcriptionally repressed gene bodies. At the chromatin level, the number of gene promoters marked by H3K4me3 increases by more than one fifth; H3K27me3 undergoes dynamic genomic redistribution characterized by loss at half of gene promoters and overall reduction of peak size by almost one-half. This is paralleled by increased phosphorylation of EZH2 at serine 21. Among genes with highly altered mRNA expression, 23.1% switch between H3K4me3 and H3K27me3 marks, and those point to the master EMT targets and regulators CDH1, PDGFRA and ESRP1. Strikingly, Twist1 increases the number of bivalent genes by more than two fold. Inhibition of the H3K27 methyltransferases EZH2 and EZH1, which form part of the PRC2 complex, results in blocking EMT and stemness properties. Conclusion: Our findings demonstrate that the EMT program requires epigenetic remodeling by the Polycomb/Trithorax complexes leading to increased cellular plasticity which suggests that its inhibition will prevent EMT, and the associated breast cancer metastasis. DREAM profiles of human mammary epithelial cells before (HMLE_parental) and after Twist1 transfection (HMLE_Twist) were generated in monolayer (HMLE_Twist2D) and sphere culture by deep sequencing using Illumina GAIIx or Illumina hiseq2000. Furthermore, DREAM profile was also obtained in parental human mammary epithelial cells transfected with GFP

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Developmental establishment of DNA methylation in hypothalamic neurons and glia.

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