Leveraging a budding yeast model to characterize the functional consequences of mutations in histone genes that are implicated in human disease

PROJECT SUMMARY Histone proteins are crucial for regulating gene expression and accessibility to genomic DNA. Mutations in genes encoding histones cause abnormal gene expression, disrupt DNA compaction, and even result in disease, such as neurological disorders or cancer. Many of these mutations alter residues that are sites of extensive post

translational modification, including acetylation and methylation. One of the first histone mutants identified is histone H3K36M, which is implicated in driving various cancers. Elucidating the molecular basis for abnormal growth caused by H3K36M will shed light on key biological functions of histone proteins. Histone proteins are

highly conserved, supporting the use of model systems to explore histone function. For example, budding yeast and human histone H3 proteins share 97% sequence identity. Studies in budding yeast have revealed that H3K36 mutants show sensitivity to a number of drugs that perturb different cellular pathways. A budding yeast

high copy suppressor screen was previously performed to identify proteins that suppress drug-sensitive growth defects in H3K36 mutant cells. One candidate suppressor identified in the screen is Esa1/Tip60, which is the catalytic component of the NuA4 lysine acetyl transferase complex. This complex targets lysines within the N-

terminal tails of histones H4 and H2A, though Esa1/Tip60 also has non-histone targets. The NuA4 complex is recruited to nucleosomes via methylation at H3K36, particularly in the presence of DNA double strand breaks (DSBs), in both yeast and humans. Both H3K36 methylation and H4 tail acetylation play roles in DSB repair.

Thus, I hypothesize that Esa1/Tip60 promotes normal growth in H3K36 mutant cells through compensating for H4 tail acetylation and that Esa1/Tip60 inhibition in H3K36 mutant cells could severely impair growth. I will test this hypothesis through three aims: 1) Elucidate the mechanism of Esa1-mediated

suppression, by examining whether there is a specific requirement for the catalytic subunit of NuA4, Esa1, compared to other lysine acetyl transferases and whether the lysines present in the H4 histone tail are critical for this Esa1-mediated suppression. The functional impact of Esa1 overexpression on the transcriptome in these

mutants will also be analyzed; 2) Examine the consequence of Tip60 inhibition in H3K36M-expressing human cells on DNA repair and growth properties; and 3) Initiate characterization of a different histone variant, which disrupts growth and is implicated in human disease, by performing a new high copy suppressor screen of

H3G34V mutants to identify suppressors related to DSB repair. Characterizing the molecular basis of these disease-implicated histone mutations will enhance the understanding of fundamental chromatin processes. This work will also establish a paradigm of investigating current and future histone variants via rapid discovery in

budding yeast and then translating findings to a human system. As a training fellowship, the proposed work will provide a thorough training in yeast as a model system and novel technical approaches. The environment of co- sponsors with complementing expertise is ideal for the successful completion of this project.