No abstract

Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, less than 5% of thousands of recently discovered phosphosites have been functionally annotated. Here, we devised a chemical genetic approach to study the functional relevance of phosphosites in S. cerevisiae. We generated 474 yeast strains with mutations in specific phosphosites that were screened for fitness in 102 conditions, along with a gene deletion library. Of these phosphosites, 42% exhibited growth phenotypes, suggesting that these are more likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions, and validated a subset by thermal proteome profiling and lipidomics. A high fraction exhibited phenotypes not seen in the corresponding gene deletion suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This high-throughput approach allows for functionally characterizing individual phosphosites at scale.

The systematic mutation of histone 3 (H3) genes in model organisms has proven to be a valuable tool to distinguish the functional role of histone residues. No system exists in mammalian cells to directly manipulate canonical histone H3 due to a large number of clustered and multi-loci histone genes. Over the years, oncogenic histone mutations in a subset of H3 have been identified in humans, and have advanced our understanding of the function of histone residues in health and disease. The oncogenic mutations are often found in one allele of the histone variant H3.3 genes, but they prompt severe changes in the epigenetic landscape of cells, and contribute to cancer development. Therefore, mutation approaches using H3.3 genes could be relevant to the determination of the functional role of histone residues in mammalian development without the replacement of canonical H3 genes. In this review, we describe the key findings from the H3 mutation studies in model organisms wherein the genetic replacement of canonical H3 is possible. We then turn our attention to H3.3 mutations in human cancers, and discuss H3.3 substitutions in the N-terminus, which were generated in order to explore the specific residue or associated post-translational modification.

Among Histone post-translational modifications (PTMs), lysine acetylation plays a pivotal role in the epigenetic regulation of gene expression, mediated by chromatin modifying enzymes. Due to their activity in physiology and pathology, several chemical compounds have been developed to inhibit the function of these proteins. However, the pleiotropy of these classes of proteins represents a weakness of epigenetic drugs. Ideally, a new generation of epigenetic drugs should target with molecular precision individual acetylated lysines on the target protein. We exploit a PTM-directed interference, based on an intrabody (scFv-58F) that selectively binds acetylated lysine 9 of histone H3 (H3K9ac), to test the hypothesis that targeting H3K9ac yields more specific effects than inhibiting the corresponding HAT enzyme that installs that PTM. In yeast scFv-58F modulates, gene expression in a more specific way, compared to two well-established HAT inhibitors. This PTM-specific interference modulated expression of genes involved in ribosome biogenesis and function. In mammalian cells, the scFv-58F induces exclusive changes in the H3K9ac-dependent expression of specific genes. These results suggest the H3K9ac-specific intrabody as the founder of a new class of molecules to directly target histone PTMs, inverting the paradigm from inhibiting the writer enzyme to acting on the PTM.

Histone variants, such as histone H3.3, replace canonical histones within the nucleosome to alter chromatin accessibility and gene expression. Although the biological roles of selected histone post-translational modifications (PTMs) have been extensively characterized, the potential differences in the function of a given PTM on different histone variants is almost always elusive. By applying proteomics and genomics techniques, we investigate the role of lysine 27 tri-methylation specifically on the histone variant H3.3 (H3.3K27me3) in the context of mouse embryonic stem cell pluripotency and differentiation as a model system for development. We demonstrate that while the steady state overall levels of methylation on both H3K27 and H3.3K27 decrease during differentiation, methylation dynamics studies indicate that methylation on H3.3K27 is maintained more than on H3K27. Using a custom-made antibody, we identify a unique enrichment of H3.3K27me3 at lineage-specific genes, such as olfactory receptor genes, and at binding motifs for the transcription factors FOXJ2/3. REST, a predicted FOXJ2/3 target that acts as a transcriptional repressor of terminal neuronal genes, was identified with H3.3K27me3 at its promoter region. H3.3K27A mutant cells confirmed an upregulation of FOXJ2/3 targets upon the loss of methylation at H3.3K27. Thus, while canonical H3K27me3 has been characterized to regulate the expression of transcription factors that play a general role in differentiation, our work suggests H3.3K27me3 is essential for regulating distinct terminal differentiation genes. This work highlights the importance of understanding the effects of PTMs not only on canonical histones but also on specific histone variants, as they may exhibit distinct roles.

Histone variants, such as histone H3.3, replace canonical histones within the nucleosome to alter chromatin accessibility and gene expression. Although the biological roles of selected histone post-translational modifications (PTMs) have...