Chromatin of the early zebrafish embryo lacks H3K9me3. a Developmental stages used for heterochromatin analysis. Zebrafish embryonic stage schematics are reproduced from Kimmel et al.22 with permission from John Wiley & Sons Inc. b Representative images showing H3K9me3 immunofluorescent antibody labeling of nuclei from fixed embryos at 2.7, 3.7, 4.5, and 6 h post fertilization (hpf). Images are representative of three independent experiments with at lease 15 embryos observed at each time point in each experiment. Top panels show H3K9me3 labeling (red). Bottom panels show H3K9me3 (red) overlaid with 4′,6-diamidino-2-phenylindole (DAPI) (blue). All images were taken under identical conditions, the scale bar represents 1 μm. c Western blots for H3K9me3, histone H3, and α-tubulin. For each time point, total protein extracts were isolated from 20 embryos, and one-third of the protein extract for each sample was loaded for western blot. d, e H3K9me3 enrichment measured by chromatin immunoprecipitation with sequencing (ChIP-seq) at 2.5, 4.5, and 6 hpf. d Screen shot of H3K9me3 enrichment at an LTR transposon in a representative genomic region. e Heatmap depicting H3K9me3 enrichment across all 17,621 H3K9me3 peaks identified in 6 hpf embryos. Signals are centered on peak centers and include ±5000 bp. f H3K9me3 enrichment at Satellite-1 (Sat1) pericentromeric repeats at 2.7, 3.7, 4.5, and 6 hpf, measured by ChIP and presented as fold enrichment over an immunoglobulin G (IgG)-only control. P values were calculated by ordinary one-way analysis of variance (ANOVA), with corrections for multiple testing. Error bars indicate the standard error of the mean (SEM). Source data for panel c are provided as a source data file
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