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. 2019 May 6;21(5):628-639.
doi: 10.1093/neuonc/noz021.

Identification of genes functionally involved in the detrimental effects of mutant histone H3.3-K27M in Drosophila melanogaster

Johannes Berlandi  1 , Amel Chaouch  2 , Nicolas De Jay  3   4 , Isabel Tegeder  1 , Katharina Thiel  1 , Margret Shirinian  5 , Claudia L Kleinman  3   4 , Astrid Jeibmann  1 , Paul Lasko  2 , Nada Jabado  6 , Martin Hasselblatt  1
Affiliations

Identification of genes functionally involved in the detrimental effects of mutant histone H3.3-K27M in Drosophila melanogaster

Johannes Berlandi et al. Neuro Oncol. .

Abstract

Background: Recurrent specific mutations in evolutionarily conserved histone 3 (H3) variants drive pediatric high-grade gliomas (HGGs), but little is known about their downstream effects. The aim of this study was to identify genes involved in the detrimental effects of mutant H3.3-K27M, the main genetic driver in lethal midline HGG, in a transgenic Drosophila model.

Methods: Mutant and wild-type histone H3.3-expressing flies were generated using a φC31-based integration system. Genetic modifier screens were performed by crossing H3.3-K27M expressing driver strains and 194 fly lines expressing short hairpin RNA targeting genes selected based on their potential role in the detrimental effects of mutant H3. Expression of the human orthologues of genes with functional relevance in the fly model was validated in H3-K27M mutant HGG.

Results: Ubiquitous and midline glia-specific expression of H3.3-K27M but not wild-type H3.3 caused pupal lethality, morphological alterations, and decreased H3K27me3. Knockdown of 17 candidate genes shifted the lethal phenotype to later stages of development. These included histone modifying and chromatin remodeling genes as well as genes regulating cell differentiation and proliferation. Notably, several of these genes were overexpressed in mutant H3-K27M mutated HGG.

Conclusions: Rapid screening, identification, and validation of relevant targets in "oncohistone" mediated pathogenesis have proven a challenge and a barrier to providing novel therapies. Our results provide further evidence on the role of chromatin modifiers in the genesis of H3.3-K27M. Notably, they validate Drosophila as a model system for rapid identification of relevant genes functionally involved in the detrimental effects of H3.3-K27M mutagenesis.

Keywords: PRC2; chromatin remodeling; diffuse midline glioma; histone H3-K27M.

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Figures

Fig. 1
Fig. 1
Phenotype upon ubiquitous expression of wild-type and mutant H3.3 in Drosophila. Phenotype of third instar control larvae (A) compared with ubiquitous expression of H3.3 wild-type (WT, B), H3.3-K27M (C), and H3.3-G34R (D). Note formation of melanotic masses only upon ubiquitous H3.3-K27M expression. H3K27me3 expression in third instar larval wild-type brains (control, E) compared with ubiquitous expression of H3.3 wild-type (WT, F), H3.3-K27M (G), and H3.3-G34R (H). Note complete loss of H3K27me3 only upon ubiquitous H3.3-K27M expression. Nuclear staining by 4′,6′-diamidino-2-phenylindole serves as control. For each condition, at least 5 animals were examined.
Fig. 2
Fig. 2
Phenotype upon midline glia specific expression of wild-type and mutant H3.3 in Drosophila. H3K27me3 expression in third instar larval wild-type brains (control, A) compared with midline glia specific expression of H3.3 wild-type (WT, B), H3.3-K27M (C), and H3.3-G34R (D). Note loss of H3K27me3 in the midline only upon ubiquitous H3.3-K27M expression. Nuclear staining by 4′,6′-diamidino-2-phenylindole (DAPI) serves as control. Expression of slit protein in third instar larval wild-type brains (control, E) compared with midline glia specific expression of H3.3 wild-type (WT, F), H3.3-K27M (G), and H3.3-G34R (H). Note perturbed distribution of slit protein only upon midline glia specific H3.3-K27M expression. Nuclear DAPI staining serves as control. For each condition, at least 5 animals were examined.
Fig. 3
Fig. 3
Genes involved in the detrimental effects of H3.3-K27M expression. The potential of 194 candidate RNAi strains to shift the lethal phenotype encountered upon H3.3-K27M expression to earlier or later stages of development was investigated upon (A) ubiquitous and (B) midline glia specific H3.3-K27M expression. Each bar represents one of the 194 candidate genes, and the effect of shRNA knockdown on the lethal phenotype observed in the F1 generation is depicted. Note that shRNA knockdown of some genes caused a positive shift of the lethal phenotype compared with controls expressing H3.3-K27M and UAS-mCherry-RNAi (arrows), and the phenotype observed upon ubiquitous H3.3-K27M expression (larval lethality: red; pupal lethality with pupation defects: orange; pupal lethality: lime-green; only pupal lethality: olive green; hatched: green) was more severe than that observed upon midline glial H3.3-K27M expression (larval lethality: red; pupal lethality with pupation defects: orange; pupal lethality: lime-green; only pupal lethality: olive green; hatched: green). (C) Genes whose knockdown rescued pupation defects observed upon ubiquitous H3.3-K27M expression or lethality observed upon midline glial H3.3-K27M expression were examined in 3 independent experiments. Note that shRNA knockdown of HmgZ, HmgD, and trol shifted the phenotype to later stages of development upon both ubiquitous and midline glial specific H3.3-K27M expression.
Fig. 4
Fig. 4
Orthologues of Drosophila candidate genes are overexpressed in H3-K27M-HGG. (A) Expression of human orthologues of all fly candidate genes shifting the lethal phenotype to later stages of development are overrepresented in H3-K27M HGG (N = 5) compared with H3 wild-type HGG (N = 4) and normal brain samples (N = 3; **P < 0.01; ***P < 0.001). The number of detected reads (RPKM) for each transcript is shown for each tumor sample. (B) Single-sample GSEA of human orthologues of the genes identified in the fly model, as in (A) (*P < 0.05). (C) Volcano plot of differentially expressed genes in H3-K27M HGG compared with normal brain samples. Highlighted are human orthologues of Drosophila genes inducing a positive shift upon downregulation in H3.3-K27M expressing flies (upregulated: blue; downregulated: red). Note that expression of 5 individual genes (ASCL1, HMGB2, CCND1, EFNB1, HSPG2) is significantly upregulated. (D) Volcano plot of differentially expressed genes in H3-K27M HGG compared with H3 wild-type HGG. Highlighted are human orthologues of Drosophila genes inducing a positive shift upon downregulation in H3.3-K27M expressing flies (upregulated: blue; downregulated: red).
Fig. 5
Fig. 5
Protein expression of CCND1, BMI1, and HMGB2 in histone H3-K27M HGGs compared with histone H3 wild-type HGGs. In H3-K27M mutated HGGs (N = 8), overexpression of CCND1, BMI1, and HMGB2 was confirmed on protein level, CCND1 also being significantly overexpressed compared with histone H3 wild-type HGGs (8/8 vs 3/9 tumors; chi-square = 8.242, P < 0.01). Representative staining results are shown.
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