An RNAi-based platform for spatiotemporal control of histone gene expression during animal development

Mutational analysis of histones provides an important means of studying the function of histone post-translational modifications (PTMs) in epigenetic gene regulation. However, several technical challenges have impeded direct tests of histone residue function in metazoans, including the massive abundance of histone gene products, multiple copies of histone genes in the genome, and the necessity of histones for cell viability.

Here, we describe a new experimental approach in Drosophila for selective depletion of the replication-dependent histone H3.2. Using short hairpin RNA (shRNA) transgenes, we demonstrate effective depletion of endogenous H3.2 gene expression, which causes defects in cell proliferation and organ development. We further show that a histone replacement transgene, engineered to be insensitive to RNA interference (RNAi) fully rescues shRNA-mediated developmental defects. Last, we demonstrate that this selective depletion platform recapitulates phenotypes caused by histone gene mutation.

We conclude that shRNA-mediated depletion of endogenous histone H3.2, coupled with histone replacement transgenes engineered to be insensitive to RNAi, is an effective experimental approach for studying the role of histone PTMs in animal development.