A platform to directly test the role of histone residues in animals
Post-translational histone modifications are thought to be a central mechanism of epigenetic regulation for many DNA-dependent processes, including gene regulation. However, the role of histone post-translational modifications (PTMs) in animals has largely been inferred from mutations in the enzymes that catalyze a PTM (aka “writers”) and the proteins that bind to and mediate the effect of a PTM (aka “readers”). Because readers and writers often have many substrates and non-catalytic functions, causal roles of histone PTMs in gene regulation cannot be determined solely through reader/writer mutation. We have established a genetic platform in Drosophila for generating and analyzing any desired histone genotype by using BAC-based arrays of histone genes to complement deletion of the endogenous histone locus (Figure 1). Due to the genomic organization of its replication-dependent histone genes and the sophisticated suite of tools available for genetic analysis, Drosophila is the only model organism in which a gene replacement strategy can be used in animals. This work is part of a broader collaborative project with the Duronio, Matera, and Strahl labs at UNC (see Links).

Questions we ask:

Which histone residues contribute to transcriptional regulation?

Do histone PTM writer mutant phenotypes match those of mutant histone residues?  If not, why?

How do histone PTMs contribute to initiation and maintenance of cellular identity?

Do histone PTMs control 3D genome organization?

Figure 1. Multimerization of histone gene arrays for BAC-dependent transgenesis.

Figure 2. H3K27 is required for Polycomb target gene repression. (A) Mitotic clones of Enhancer of Zeste (E(z)), the writer of H3K27me3, show de-repression of the Polycomb target gene Ubx (red) in wing imaginal discs. (B) Histone locus mutant clones covered by H3K27A transgenes also de-repress Ubx. (C) Histone locus mutant clones covered by wild-type histone transgenes do not de-repress Ubx. Mutant clones are indicated by loss of GFP signal.

Figure 3. Heatmap depicting the change in Hi-C contacts made in wild-type and histone H3K9 mutants. In wild-type cells, the euchromatic and heterochromatic portions of the genome are spatially segregated, but the underlying mechanisms are unclear. We tested the hypothesis that H3K9me3 and its reader protein, HP1, two hallmarks of constitutive heterochromatin, contribute to this segregation. Reciprocal mutation of H3K9 and HP1 revealed an increase in contacts between heterochromatic pericentromeres and euchromatic chromosome arms (orange and gray shading on the chromosome ideogram, respectively). However, self-association of pericentromeres is largely preserved despite loss of H3K9me3 and HP1 binding (see Stutzman, Mol Cell 2024).