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. 2010 Dec 20;5(12):e15651.
doi: 10.1371/journal.pone.0015651.

V体育平台登录 - Tiling histone H3 lysine 4 and 27 methylation in zebrafish using high-density microarrays

Leif C Lindeman  1 , Andrew H Reiner, Sinnakaruppan Mathavan, Peter Aleström, Philippe Collas
Affiliations

V体育2025版 - Tiling histone H3 lysine 4 and 27 methylation in zebrafish using high-density microarrays

Leif C Lindeman et al. PLoS One. .

Abstract

Background: Uncovering epigenetic states by chromatin immunoprecipitation and microarray hybridization (ChIP-chip) has significantly contributed to the understanding of gene regulation at the genome-scale level VSports手机版. Many studies have been carried out in mice and humans; however limited high-resolution information exists to date for non-mammalian vertebrate species. .

Principal findings: We report a 2. 1-million feature high-resolution Nimblegen tiling microarray for ChIP-chip interrogations of epigenetic states in zebrafish (Danio rerio) V体育安卓版. The array covers 251 megabases of the genome at 92 base-pair resolution. It includes ∼15 kb of upstream regulatory sequences encompassing all RefSeq promoters, and over 5 kb in the 5' end of coding regions. We identify with high reproducibility, in a fibroblast cell line, promoters enriched in H3K4me3, H3K27me3 or co-enriched in both modifications. ChIP-qPCR and sequential ChIP experiments validate the ChIP-chip data and support the co-enrichment of trimethylated H3K4 and H3K27 on a subset of genes. H3K4me3- and/or H3K27me3-enriched genes are associated with distinct transcriptional status and are linked to distinct functional categories. .

Conclusions: We have designed and validated for the scientific community a comprehensive high-resolution tiling microarray for investigations of epigenetic states in zebrafish, a widely used developmental and disease model organism. V体育ios版.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Design of the zebrafish ChIP-chip tiling array.
Array probe design (upper chart) and median promoter coverage within the tiled regions, shown for the positive strand. Probes are represented by the dashed lines. Spacing between dashes symbolizes probe spacing. Note: the array was designed from the Zv7 assembly as the Zv8 assembly was unpublished at the time. Zv7 reported a genome size of 1,440,582,308 bp; Zv8 currently reports a genome size of 1,441,241,298 bp.
Figure 2
Figure 2. Reproducibility of zebrafish ChIP-chip experiments.
(A) Two-dimensional scatter plots of MaxSixty values for H3K4me3 and H3K27me3 log2 signal intensities detected in each of two ChIP-chip replicates from ZF4 cells. Correlation coefficient (R) and regression line are shown. (B) H3K4me3 and H3K27me3 profiles detected by ChIP-chip in two independent replicates through 310 kb of zebrafish chromosome 10. Data are expressed as log2 ChIP/input ratios. Position of methylation peaks are shown as blue horizontal bars. Tracks representing primary transcripts and tiled regions are also shown. Primary transcripts included in the region are as follows: 1) sin2; 2) NM_001003421; 3) NM_200663; 4) NM_001008616; 5) ENSDART00000081978; 6) ripply3, 7) ENSDART00000081992; 8) dyrk1aa; 9) ENSDART00000058411; 10) ENSDART00000088605; 11) NM_001037708; 12) hyou1; 13) hist2h2l; 14) znf259. Red bars in the H3K4me3 tracks indicate probes with out-of-scale signal intensity.
Figure 3
Figure 3. Quantitative PCR validation of ChIP-chip data.
(A) ChIP-on-chip profiles of H3K4me3 and H3K27me3 enrichment on indicated genes. Position of primary transcripts and TSS (arrow) are shown. (B) ChIP-qPCR analysis of H3K4me3 and H3K27me3 enrichment on the same genes as in (A) from separate duplicate ChIPs. ChIP DNA was not WGA amplified prior to PCR. Position of amplicons and primer sequences for each gene are shown in Table S3. Note the correlation between ChIP- chip and ChIP-qPCR data.
Figure 4
Figure 4. H3K4me3 and H3K27me3 enrichment profiles in ZF4 cells.
(A) Distinct H3K4me3 and H3K27me3 enrichment profiles on indicated genomic regions. Genomic positioning is indicated by nucleotide number of the first (5′) and last (3′) probe in the tiled region. Gene names or accession numbers as well as their genomic position are shown in blue. (B) 2-D scatter plot of averaged MaxSixty values for H3K4me3 vs. H3K27me3 log2 signal intensities. Data points (all points being shown in gray) were colored to visualize classification according to peak calling highlighting H3K4me3-enriched promoters (purple; N = 6315), H3K27me3-enriched promoters (green; N = 1079) and H3K4me3/K27me3-co-enriched promoters (blue; N = 2120). Red line is the regression line through all data points. (C) Venn diagram analysis of H3K4me3 and H3K27me3 genes.
Figure 5
Figure 5. Distribution of H3K4me3 and H3K27me3 on promoters.
Metagene analysis of the distribution of H3K4me3 and H3K27me3 occupancy on (A) H3K4me3-only, (B) H3K27me3-only and (C) H3K4me3/K27me3 tiled regions, relative to the TSS (red vertical bar). (D) Sequential ChIP analysis of H3K4me3 and H3K27me3 co-enrichment on the sox3, sox2 promoters and on bactin1, downstream of the coding region. Panels on the left show results from the first ChIP using antibodies indicated on the x-axis. The graph on the right shows results of the re-ChIP experiment as indicated on the x-axis.
Figure 6
Figure 6. Genes marked by H3K4me3 and/or H3K27me3 are linked to distinct functional GO terms.
(A) GO term enrichment of genes containing H3K4me3, H3K27me3 or H3K4/K27me3 promoters in ZF4 cells. The twelve most significant GO terms are shown as a function of significance from bottom (highest significance) to top. (B) Representation of all enriched GO terms among H3K4/K27me3 genes. All enriched GO terms are listed in Table S1. (C) H3K4me3 and H3K27me3 enrichment profiles on the developmentally regulated hoxc locus, expressed as log2 ChIP/Input (y axis).
Figure 7
Figure 7. Gene expression status in relation to histone modification enrichment.
(A) Percentage of expressed and non-expressed genes marked by indicated histone modifications, and among all RefSeq genes co-represented on both the Agilent and Nimblegen arrays (n = 11,971). (B) Percentile analysis of expression levels of genes marked by indicated modifications. Low, 25th percentile; Mid, 25thth percentile; High, >75th percentile. Only expressed genes (blue bars in A) are taken into the analysis. Numbers within the pie charts indicate the number of genes in each category; numbers are the bottom indicate the number of expressed genes (Present call) marked by either or both histone modifications.
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