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. 2020 Feb 28;9:e53375. doi: 10.7554/eLife.53375

Figure 1. Effect of H2A.Z.1 and H2A.Z.2 depletion on gene expression.

(A) WI38 cells were transfected with the indicated siRNAs. 72 hr later, total RNA was prepared. The amount of H2A.Z.1 and H2A.Z.2 mRNA was quantified by RT-qPCR, standardised using GAPDH mRNA levels and calculated relative to one for cells transfected with the control siRNA. The mean and SDOM from five independent experiments are shown. (B) Genome edited U2OS cells expressing either tagged H2A.Z.1 (top) or tagged H2A.Z.2 (bottom) were transfected with the indicated siRNAs. 72 hr later, total cell extracts were prepared and subjected to western blot analysis using an anti H2A.Z antibody. The star * indicates a band probably corresponding to a post-translationally modified untagged H2A.Z isoform. (C) Same as in A, except that total cell extracts were prepared and subjected to western blot analysis using the indicated antibody, then protein signals were standardised using GAPDH protein levels and calculated relative to1 for cells transfected with the control siRNA. A representative experiment out of two is shown. (D) Gene ontology analyses (Genecodis) of genes downregulated upon H2A.Z.1or H2A.Z.2 depletion or upregulated upon H2A.Z.1 or H2A.Z.2 depletion (from top to bottom). The top 10 most significant enrichments are shown.

Figure 1—source data 1. Source data of the histogramme representing the depletion of H2A.
Z.1 and H2A.Z.2 in response to siRNAs in Figure 1A.

Figure 1.

Figure 1—figure supplement 1. Characterisation of U2OS cells genome-edited to express 3xFlag-2xStrep H2A.Z.1 or H2A.Z.2.

Figure 1—figure supplement 1.

(A) Genomic DNA samples were subjected to a PCR reaction using primers located outside of the inserted sequence as shown in the scheme (Bottom). The top panels show an analysis on agarose gel of the PCR products obtained on negative clones and on the genome-edited clones used in this study. Note the shift of the band in the genome-edited clones expressing Flag-H2A.Z.1 (Left) or Flag-H2A.Z.2 (Right), indicating the recombination. We chose the B clone from U2OS-Flag H2A.Z.1 and the A clone from U2OS-Flag H2A.Z.2 for further analyses. The bands were excised and sequenced to check the accuracy of the recombination. (B) Nuclear extracts from U2OS cells expressing Flag-H2A.Z.1 (Left) or Flag-H2A.Z.2 (Right) transfected with the indicated siRNA were subjected to a Flag western blot. Note the band migrating close to 30 kDa which likely corresponds to a post-translationally modified tagged histone. Note that the H2A.Z.2 siRNA is the one used in Figure 2—figure supplement 2 (si Z2#), ie? a different siRNA than in Figure 1.