FIGURE

Fig. 4

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ZDB-FIG-180620-75
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Murphy et al., 2018 - Placeholder Nucleosomes Underlie Germline-to-Embryo DNA Methylation Reprogramming
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Fig. 4

Anp32e Protein Injection Causes Reduced Nuclear H2AFV and Increased DNA Methylation

(A) Anp32e binding inhibits H2A.Z within the nucleosome. Left: crystal structure for the nucleosome octamer containing mouse H2A.Z (yellow) (Suto et al., 2000). Middle: zoomed-in and rotated view of the H2A.Z nucleosome octamer (dashed box from left), where the C terminus of H2A.Z interacts with histone H3. Right: the same view as middle panel, with the superimposed H2A.Z bound to human ANP32E (Obri et al., 2014).

(B) Purification of recombinant zebrafish Anp32e protein. A comparison to BSA and Coomassie staining to demonstrate purity.

(C) Phenotype of early somite-staged zebrafish following Anp32e protein injection.

(D) H2AFV signal is depleted in nuclei following Anp32e injection in embryos. DAPI and H2AFV:GFP localization in cells from sphere-staged (4hpf) embryos. Injection was into 1-cell embryos.

(E) Microscopy-based quantification of H2AFV following Anp32e injection. Fraction H2AFV:GFP signal in control and injected embryos like in (D). t tests: ∗∗ p < 0.01

(F) Quantification of H2AFV in cellular fractions resulting from Anp32e injection. Western blotting for soluble H2AFV indicates that Anp32e injection causes increased cytoplasmic H2AFV. t tests: ∗∗∗ p < 0.001.

(E and F) Error bars represent SD.

(G) Anp32e injection causes increased DNAme. Scatterplot of CpGs for treatment versus control is displayed, with a bar graph quantifying changing CpGs (>20% absolute change).

(H) Changes in DNA methylation are most significant at regions at which DNA is normally demethylated. t tests: ∗∗∗ p < 0.001. Boxplots are displayed.

(I) A scatterplot of gene transcript abundance (fragments per kilobase of million mapped reads [FPKM]) for Anp32e-injected embryos compared to control samples. Red data points indicate fold change >2 and p < 0.01 false discovery rate (FDR). The number of genes changing is indicated in the top-left and bottom-right corners.

(J) A gallery of browser snapshots for total RNA at developmentally regulated genes.

See also Figure S4.
Expression Data

Expression Detail
Antibody Labeling
Phenotype Data

Phenotype Detail
Acknowledgments
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Reprinted from Cell, 172(5), Murphy, P.J., Wu, S.F., James, C.R., Wike, C.L., Cairns, B.R., Placeholder Nucleosomes Underlie Germline-to-Embryo DNA Methylation Reprogramming, 993-1006.e13, Copyright (2018) with permission from Elsevier. Full text @ Cell
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