An Animal Model for Genetic Analysis of Multi-Gene Families: Cloning and Transgenesis of Large Tandemly Repeated Histone Gene Clusters

Abstract

Histone post-translational modifications (PTMs) are thought to participate in a range of essential molecular and cellular processes, including gene expression, replication, and nuclear organization. Importantly, histone PTMs are also thought to be prime candidates for carriers of epigenetic information across cell cycles and generations. However, directly testing the necessity of histone PTMs themselves in these processes by mutagenesis has been extremely difficult to carry out because of the highly repetitive nature of histone genes in animal genomes. We developed a transgenic system to generate Drosophila melanogaster genotypes in which the entire complement of replication-dependent histone genes is mutant at a residue of interest. We built a BAC vector containing a visible marker for lineage tracking along with the capacity to clone large (60-100 kb) inserts that subsequently can be site-specifically integrated into the D. melanogaster genome. We demonstrate that artificial tandem arrays of the core 5 kb replication-dependent histone repeat can be generated with relative ease. This genetic platform represents the first histone replacement system to leverage a single tandem transgenic insertion for facile genetics and analysis of molecular and cellular phenotypes. We demonstrate the utility of our system for directly preventing histone residues from being modified, and studying the consequent phenotypes. This system can be generalized to the cloning and transgenic insertion of any tandemly repeated sequence of biological interest.

Drosophilahistone locus body assembly and function involves multiple interactions

“…Engineered 5-kb histone repeats were then arrayed to 12 copies in pMultiBac ( McKay et al. , 2015 ; Meers et al , 2018 ). All histone gene arrays were inserted into the VK33 attP site on chromosome 3L (65B2) ( Venken et al.…”

Distinct roles for canonical and variant histone H3 lysine 36 in Polycomb silencing

“…All experimental animals were raised at 25°C. Details regarding construction of bacterial artificial chromosome (BAC) transgenes containing the 12xH3.2 histone gene arrays can be found here (10). The 12x HWT, K27R, K36R, K9R alleles were generated previously (9,13).…”

“…In animal cells, histone PTM functions have largely been inferred from genetic analyses of histone modifying factors (readers, writers, erasers), rather than from studying the histone residues themselves. To help decipher the metazoan 'histone code' (8), we developed an experimental system in Drosophila that allows for sophisticated phenotypic analysis following loss of a specific site of histone modification (9,10). Interestingly, we found that histone missense mutants often exhibit a subset of the phenotypes caused by mutations in their cognate chromatin-modifying enzymes (9,(11)(12)(13)(14).…”

Distinct developmental phenotypes result from mutation of Set8/KMT5A and histone H4 lysine 20 in Drosophila melanogaster

“…This strategy allows us to engineer histone genotypes encoding mutant histone proteins in which a given residue is changed to one that is not a substrate for its cognate modifying enzyme. 5,41,[49][50][51][52]65,66 Using this strategy, we demonstrated previously that H4 K20A mutant animals can survive to adulthood 49 (Figure 5C, D). By contrast, 100% of Set8 20/20 null animals die as larvae or early pupae 40 (Figure 2C).…”

“…This strategy allows us to engineer histone genotypes encoding mutant histone proteins in which a given residue is changed to one that is not a substrate for its cognate modifying enzyme. 5,41,49–52,65,66…”