Investigating the Role of Histone H3.3 in Drosophila Melanogaster Immune Response

Histone proteins are the building blocks of nucleosomes, which control genome organization and regulate gene expression. Histone proteins can be organized into two main categories:  canonical histones whose synthesis is coupled with DNA replication during S phase of the cell cycle and variant histones that are expressed throughout the cell cycle and deposited into chromatin using different chaperone complexes than the canonical histones. Both categories of histones possess N-terminal tails which can be post-translationally modified, and these modifications, such as acetylation, phosphorylation, and methylation, contribute to the regulation of gene expression. Recent studies in mice reported that the phosphorylation of histone H3.3 at position 31 (H3.3S31ph) is critical for proper immune response. This study found that crosstalk between H3.3S31ph and H3K36me3 promotes the expression of rapidly inducible immune genes. However, it is not known whether this mechanism is conserved across eukaryotes. Here, we aimed to identify if H3.3S31 is phosphorylated in Drosophila and whether it is involved in the Drosophila immune response.  To this end, we designed an assay to challenge the immune system of flies lacking H3.3S31 (H3.3S31A) via systemic infection,  but found inconclusive results. Additionally, we conducted western blot assays to determine if H3.3S31ph is present in Drosophila, and whether loss of S31 impacts levels of other H3 PTMs.  We report that two commercially available H3.3S31ph antibodies exhibit nonspecific binding. Interestingly, we observe reduced acetylation of H3K27 (H3K27ac) levels in H3.3S31A flies compared to wild-type, which suggests crosstalk between H3.3S31 and H3K27. As previous groups have reported that H3.3S31ph recruits H3K27 acetyltransferase P300, these data are the first evidence that H3.3S31ph may be conserved in Drosophila melanogaster. These findings have strong implications for future investigations into H3.3’s cellular roles and significance, including the immune response.