Fig 1: H3.3 mRNA and protein levels do not change when H3.2 gene copy number is reduced.(A) MA plot showing fold change of normalized RNA-seq signal in wild-type (200xWT) vs. 12xHWT for all transcripts (y-axis). Average coverage on the x-axis represents the mean expression level of a transcript. Differentially expressed genes are indicated in dark gray. Values for H3.3A and H3.3B transcripts are indicated (green). (B) Western blot of total H3, H3.3 and tubulin from 12xHWT and 200xWT wandering third instar larval wing imaginal discs. Dilution series with indicated number of wing discs for each genotype. H3.3? third instar larvae were used as a negative control. Bar plot depicting the average fold change in H3.3 signal relative to 200xWT signal normalized to tubulin. Error bars represent standard deviation of three biological replicates. (C) Confocal images of third instar imaginal eye discs stained for DAPI (grey), EdU (magenta), and H3.2 mRNA (cyan) for the four indicated genotypes, denoted as in Figure 1. The maximum projection of four adjacent slices is shown. Bars, 50 µM.
Fig 2: H3.3 is required for viability when H3.2 gene copy number is reduced.(A) Diagram of the genomic locations of the three H3 genes and the 12xHWT transgene. 100x and 12x indicate gene copy number. (B) Schematic model of H3.2 and H3.3 expression during the cell cycle. (C) Amino acid sequence differences of H3.2 and H3.3. (D) Bar plot of viability for the indicated genotypes. Circles represent the full complement of H3.3 or H3.2 gene copies present in the wild-type genome, and the color filling each circle (H3.3 green, H3.2 blue) indicates the number of gene copies present in each experimental genotype, e.g. a solid green circle indicates the 4 H3.3 gene copies of the diploid H3.3A and H3.3B loci. The number of H3.2 genes is shown below the blue circles, as well as whether surviving adults are fertile. Percent of expected genotypic frequencies based on Mendelian ratios. Asterisks indicate fewer than expected survive (chi-square test, ** p value < 0.01, see Figure 1 in File S1 for p values).
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