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

Abstract

Background. Recurrent specific mutations in evolutionarily conserved histone 3 (H3) variants drive pediatric highgrade 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. Key Points 1. To identify pathways involved in the biology of histone 3 (H3) in pediatric high-grade gliomas, a fly model was developed.

Mutant H3 histones drive human pre-leukemic hematopoietic stem cell expansion and promote leukemic aggressiveness

“…A pUASg.attB Gateway vector containing the human cDNA with 1xHA (C-terminus) was injected into the ZH-attP line on the third chromosome zh-86Fb (3R 86F) for GAL4-UAS expression. A hemocyte-specific driver (Cg-GAL4.A) (#7011) was obtained from BDSC 52 . Flies were crossed at 25 °C.…”

The Leukemic Fly: Promises and Challenges

“…Epigenetic derangements, involving improper histone methylation, constitute important targets to consider when investigating potential AML treatments. Histone mutations were shown to take place early throughout the leukemogenic process and to contribute to the main leukemic clone in AML patient samples [ 56 , 122 ]. Drosophila was used in this context as a model organism harboring a simple hematopoietic system to confirm the effects of histone mutations on hematopoiesis.…”

H2B oncohistones cause homologous recombination defect and genomic instability through reducing H2B monoubiquitination in Schizosaccharomyces pombe

“…Genetic studies in model organisms have been used as alternative approaches to uncover phenotypes and effector genes of oncohistones. The functional consequences or genetic suppressors of H3K27M, H3G34R/V/W, and H3K36M mutations have been studied in budding ( 36 ) and fission yeast ( 28 , 29 ), worm ( 37 ), and fly ( 38 ). Moreover, the cellular effects on growth and viability of noncanonical oncohistones have been systematically screened in the budding yeast Saccharomyces cerevisiae ( S. cerevisiae ) using the “humanized” histones library ( 32 ).…”