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Fig. 4 The dominant D123N mutation prevents chromatin incorporation and promotes the formation of aberrant H3 homodimers.
a, Western blots show α-FLAG immunostaining of nuclear extracts or purified mononucleosome fractions from HEK cells transfected with vector alone or FLAG-tagged H3.3 (f:H3.3) vectors. Wild-type and D123N f:H3.3 proteins are expressed at equal levels in total extract, but wild-type f:H3.3 is present at much higher levels in the nucleosome fraction (consistent over three replicate experiments). α-FLAG immunoprecipitation from purified nucleosomes shows that wild-type but not D123N f:H3.3 is incorporated into nucleosomes containing H2A, H2B, H3, and H4 (consistent over three replicate experiments). b, Confocal images from H2A.F/Z:GFP embryos expressing wild-type and D123N versions of mCherry(m)H3.3 and mCherry(m)H3.2 fusion proteins. Merged images show that whereas all H3 proteins are nuclear localized in surrounding non-mitotic cells, wild-type mH3.3 and mH3.2, but not D123N mH3.3 and mH3.2, co-localize with H2A.F/Z:GFP in the chromosomes of metaphase/anaphase cells (arrowheads) after nuclear envelope breakdown (wild-type mH3.3, 11/11 cells in 2 embryos; D123N mH3.3, 0/25 cells in 3 embryos; wild-type mH3.2, 21/21 cells in 3 embryos; D123N mH3.2, 0/16 cells in 2 embryos). c, α-FLAG, α-H3 and α-H4 western blots for samples immunoprecipitated by α-FLAG from nuclear extracts of f:H3.3-transfected HEK cells. Recombinant octamer is used as a reference. Whereas both endogenous H3 and H4 co-immunoprecipitate with the wild-type f:H3.3 protein (*), H3 but not H4 co-immunoprecipitates with D123N f:H3.3 (asterisk marks the larger recombinant f:H3.3 protein). Results were consistent over three replicate experiments. d, mRNA injection of D123N mH3.3 (8/17), but not wild-type mH3.3 (0/26), wild-type mH3.2 (0/19), or D123N mH3.2 (0/18), results in loss of the CNC-derived head skeleton at 4 dpf. Scale bars: b, 10 μm; d, 250 μm.