Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Developmental features of DNA methylation during activation of the embryonic zebrafish genome

ABSTRACT: Zygotic genome activation (ZGA) occurs at the mid-blastula transition (MBT) in zebrafish and is a period of chromatin remodeling. Genome-scale gametic demethylation and remethylation occurs after fertilization, during blastula stages, but how ZGA relates to promoter DNA methylation states is unknown. Using methylated DNA immunoprecipitation coupled to high-density microarray hybridization (MeDIP-ChIP), we characterize genome-wide promoter DNA methylation dynamics before, during and after ZGA onset, in relation changes in post-translational histone modification and gene expression (Series GSE22830). A Kolmogorov-Smirnov (KS) test was applied with P <= 0.01 to identify methylation peaks. MeDIP-chip experiments were performed on24 hpf zebrafish embryos and sperm. Samples were lysed and proteins digested by proteinase k treatment. DNA was extracted with phenol-chloroform-isoamylalcohol and ethanol precipitation. The DNA was RNAse treated and sonicated to fragment lengths between 300-1000 bp. From each stage, duplicate immuneoprecipitations were performed using anti-5-methylcytosine antibody (10 ng/M-BM-5l; Mab-006-100; Diagenode) coupled to Dynabeads M-280 sheep anti-mouse IgG (Invitrogen). MeDIP and input DNA (150 ng each) were amplified (WGA-2; Sigma-Aldrich), cleaned up, eluted and processed for array hybridization. MeDIP and input DNA were labeled and co-hybridized onto the Nimbegen promoter arrays. The array covers 15 kb of upstream regulatory sequence and 5 kb downstream of the TSS of all zebrafish genes. A Kolmogorov-Smirnov (KS) test was applied with P <= 0.01 to identify methylation peaks.

ORGANISM(S): Danio rerio

SUBMITTER: Philippe Collas

PROVIDER: E-GEOD-33236 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Samples from 20 children with B-cell precursor ALL, comprising ten with high hyperdiploidy and ten with the ETV6/RUNX1 fusion, were included in the study. Four Âµg of methylated and input fraction DNA were analyzed with a classic MeDIP assay and subsequent microarray hybridization using the NimbleGen CpG Island-Plus-Promoter Array (Roche NimbleGen, Madison, WI).<br><br>This array platform covers all 28,226 UCSC-annotated CpG islands and promoter regions for all RefSeq genes, including 385,000 probes of 50-75mer length per array, with an upstream and downstream promoter tiling of 800 bp and 200 bp, respectively. Signal intensity data were extracted from the scanned images. Each feature on the array has a corresponding log2 ratio, which is the ratio of the input signals for the methylated DNA and the input fraction DNA. The log2 ratio was scaled in order to center the ratio data around zero by subtracting the bi-weight mean for the log2 ratio values for all features on the array from each log2 ratio value. A modified ACME algorithm, where a fixed-length window (750 bp) is placed around each consecutive probe, and the one-sided Kolmogorov-Smirnov test were then applied on the scaled log2 ratio data to determine whether the probes were drawn from a significantly more positive distribution (peak) of intensity log2 ratios than the other probes in the array. The resulting score for each probe is the -log10 p-value from the windowed Kolmogorov-Smirnov test around that probe. Peak data were generated by searching for probes above a p-value minimum cut-off (-log10) of 2.<br><br>Peaks within 500 bp of each other were merged. Each annotated gene was subsequently searched for peaks appearing in a specified promoter region around the transcription start site. The region searched was design-specific, spanning from 5 kb upstream to 1 kb downstream of the transcription start site. Genes that had at least two probes with peaks above the p-value minimum cut-off were considered to have significant CpG island methylation scores for hypermethylation.

Project description:In mature human sperm, genes of importance for embryo development (i.e. transcription factors) lack DNA methylation and bear nucleosomes with distinctive histone modifications, suggesting the specialized packaging of these developmental genes in the germline. Here, we explored the tractable zebrafish model and found conceptual conservation as well as several new features. Biochemical and mass spectrometric approaches reveal the zebrafish sperm genome packaged in nucleosomes and histone variants (and not protamine), and we find linker histones high and H4K16ac absent - key factors which may contribute to genome condensation. We examined several activating (H3K4me2/3, H3K14ac, H2AFV) and repressing (H3K27me3, H3K36me3, H3K9me3, hypoacetylation) modifications/compositions genome-wide, and find developmental genes packaged in large blocks of chromatin with coincident activating and repressing marks and DNA hypomethylation, revealing complex ‘multivalent’ chromatin. Notably, genes that acquire DNA methylation in the soma (muscle) are enriched in transcription factors for alternative cell fates. Remarkably, we find H3K36me3 located in ‘silent’ developmental gene promoters, and not present at the 3’ ends of coding regions of genes heavily transcribed during sperm maturation, suggesting different rules for H3K36me3 in the germline and soma. We also reveal the chromatin patterns of transposons, rDNA, and tRNAs. Finally, high levels of H3K4me3 and H3K14ac in sperm are correlated with genes activated in embryos prior to the mid-blastula transition (MBT), whereas multivalent genes are correlated with activation at or after MBT. Taken together, gene sets with particular functions in the embryo are packaged by distinctive types of complex and often atypical chromatin in sperm. [DNA profiling]: H3K4me3, H3K4me2, H3K14ac, H3K36me3, H3K27me3, H3K9me3, and H2AFV ChIP-chip in zebrafish sperm; DNA methylation in zebrafish sperm and muscle by MeDIP-chip. (two replicates and dye-swaps for all experiments). Antibodies: H3K4me3 (Abcam ab8580 and Active Motif 39159), H3K4me2 (Abcam ab7766), H3K14Ac (Upstate 07-353), H2AZ (Abcam ab4174), H3K27me3 (Upstate 07-449), H3K9me3 (Active Motif 39161), H3K36me3 (Abcam ab9050), 5-methylcytidine (Eurogentec BI-MECY-1000). Supplementary files: Processed data files reporting calculated enrichment log2 ratio (mean ChIP/mean Input in the log2 ratio). Note: For analysis, "mean Input" from H3K4me3-rep1 ch1, H3K4me2-rep1 ch2, and H3K36me3-rep2 ch1 for all ChIP eluates. [mRNA profiling]: Transcripts in zebrafish mature sperm using zebrafish expression microarray from Agilent. (two replicates)

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Developmental features of DNA methylation during activation of the embryonic zebrafish genome

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