Date of Award
Doctor of Philosophy (PhD)
Molecular, Cellular, and Developmental Biology
Histones regulate diverse processes in eukaryotes and consequently, can have widespread effects on organismal fitness and development. Histones are a dynamic target for a variety of post-translational modifications (PTMs) and the assessment of histone function has typically been accomplished by mutating enzymes that catalyze and/or recognize these PTMs (i.e., writers and readers, respectively). Although considerable information has been gained in the past several decades by using this strategy, multiple issues such as writer/reader redundancy, unidentified writers/readers of histone PTMs, and writers/readers with additional non-histone targets can preclude the identification of new roles for histones and complicate the assessment of mutant phenotypes. To bypass these issues and provide a complementary strategy to study histones, large-scale histone replacement systems have been developed and optimized in yeast and fly model systems. However, such systems have never been implemented in plants in part due to the difficulty in eliminating endogenous histone genes that are typically present in many copies and different locations in plant genomes. Here, we present the development of a genetic strategy for the plant model organism Arabidopsis thaliana in which the expression of endogenous histone H4 can be completely replaced with modified H4 transgenes. We use histone H4, which is a single variant histone in plants that is encoded by the largest number of genes (8) among all functionally-distinct histone proteins, as a proof-of-concept for an experimental system allowing the direct assessment of histone function in plants. Our CRISPR/Cas9-based strategy allows for the simultaneous targeting of many histone genes for the generation of a background depleted of endogenous histone expression. We validated our platform by showing that a single transformation with our modified H4 transgenes can restore a wild-type phenotype, demonstrating that our system can be used for the rapid establishment of histone replacement in plants. Using this strategy, we established a collection of plants expressing different H4 point mutants targeting residues that may be post-translationally modified in vivo. To demonstrate the utility of this new H4 mutant collection, we screened it to uncover substitutions in H4 that alter flowering time, rosette morphology, DNA replication, chromatin structure, and gene silencing. We identified different mutations in the tail (H4R17A) and the globular domain (H4R36A, H4R39K, H4R39A, and H4K44A) of H4 that strongly accelerate the floral transition. Additionally, we used machine learning to identify H4 mutations that alter different morphometric traits in vegetative tissue. Finally, we identified several novel roles for H4 tail and globular domain residues in the regulation of endoreduplication, chromatin condensation, and transposon silencing. After these broad screens for histone function, we then performed targeted analyses of H4R17A mutants to determine a molecular mechanism responsible for the early flowering displayed by these mutants. We found that a conserved regulatory relationship between H4R17 and the ISWI chromatin remodeling complex in plants is responsible for the phenotypes observed in H4R17A mutants. Similar to other biological systems, H4R17 regulates nucleosome spacing via ISWI, and mutation of H4R17 results in large-scale changes to global nucleosome positioning and gene expression, leading to altered development. Overall, this work provides a large set of H4 mutants to the plant epigenetics community that can be used to systematically assess histone H4 function in A. thaliana and a blueprint to replicate this strategy for studying other histone proteins in plants. As this resource represents the largest collection of H4 point mutants in a multicellular organism, our work will enable new insights into the regulation of chromatin by histone H4 in multicellular eukaryotes.
Corcoran, Emma Tung, "Systematic Screen of Histone H4 in Arabidopsis thaliana" (2022). Yale Graduate School of Arts and Sciences Dissertations. 577.