Systematic histone H4 replacement in Arabidopsis thaliana reveals a role for H4R17 in regulating flowering time

Abstract

Despite the broad array of roles for epigenetic mechanisms on regulating diverse processes in eukaryotes, no experimental system is currently available in plants for the direct assessment of histone function. In this work, we present the development of a genetic strategy in Arabidopsis (Arabidopsis thaliana) whereby modified histone H4 transgenes can completely replace the expression of endogenous histone H4 genes. Accordingly, we established a collection of plants expressing different H4 point mutants targeting residues that may be post-translationally modified in vivo. To demonstrate its utility, we screened this new H4 mutant collection to uncover substitutions in H4 that alter flowering time. We identified different mutations in the H4 tail (H4R17A) and the H4 globular domain (H4R36A, H4R39K, H4R39A, and H4K44A) that strongly accelerate the floral transition. Furthermore, we identified a conserved regulatory relationship between H4R17 and the ISWI chromatin remodeling complex in plants: As with other biological systems, H4R17 regulates nucleosome spacing via ISWI. 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 Arabidopsis and a roadmap to replicate this strategy for studying other histone proteins in plants.

Simultaneous Profiling of Chromatin Accessibility and DNA Methylation in Complete Plant Genomes Using Long-Read Sequencing

“…Nucleosome positioning was inferred using MNase libraries previously published [24,36–38]. MNaseq datasets were aligned to the Col-CEN reference genome assembly [39] and libraries with high mappability efficiency and median fragment lengths distribution of ∼147 bp were retained.…”

Maintenance and dynamic reprogramming of chromatin organization during development

“…Instead, in euchromatin that comprises most of the genes, transcriptional activity correlates with enrichment in H3K4me3, H3K9ac and/or H2Bubiquitination (Leng et al, 2020), while gene repression is achieved by H3K27me3 deposition by the Polycomb Repressive complex 2 (PRC2; Mozgova & Hennig, 2015; Figure 1). The importance of these histone modifications, especially the Polycomb mark H3K27me3, for plant development has been revealed by genetic approaches either removing histone modifiers (Goodrich et al, 1997), or more recently expressing histone proteins that cannot be post-translationally modified at specific sites (Corcoran et al, 2022;Fal et al, 2022;Xu et al, 2022). Corcoran et al (2022) probed systematically the importance of modifiable histone H4 residues by replacing endogenous H4 genes by transgenes carrying different point mutations, revealing many residues critical for plant survival or development, including correct flowering time.…”

“…The importance of these histone modifications, especially the Polycomb mark H3K27me3, for plant development has been revealed by genetic approaches either removing histone modifiers (Goodrich et al, 1997), or more recently expressing histone proteins that cannot be post-translationally modified at specific sites (Corcoran et al, 2022;Fal et al, 2022;Xu et al, 2022). Corcoran et al (2022) probed systematically the importance of modifiable histone H4 residues by replacing endogenous H4 genes by transgenes carrying different point mutations, revealing many residues critical for plant survival or development, including correct flowering time. Interfering specifically with modifications set on H3K27 by replacing lysine 27 by alanine in a transgeneencoded H3 resulted in a substantially altered transcriptome when assessed in calli and strong developmental abnormalities partly reminiscent of those found in mutants of histone methyltransferases of the PRC2 complex (Fal et al, 2022).…”

“…Corcoran et al. (2022) probed systematically the importance of modifiable histone H4 residues by replacing endogenous H4 genes by transgenes carrying different point mutations, revealing many residues critical for plant survival or development, including correct flowering time. Interfering specifically with modifications set on H3K27 by replacing lysine 27 by alanine in a transgene‐encoded H3 resulted in a substantially altered transcriptome when assessed in calli and strong developmental abnormalities partly reminiscent of those found in mutants of histone methyltransferases of the PRC2 complex (Fal et al., 2022).…”

Combining multiplex gene editing and doubled haploid technology in maize

“…Many of these plant traits are controlled by a complex interaction between many different genes and require the identification of the right combinations of alleles to have pronounced and desired effects ( Vanhaeren et al, 2014 ; Vanhaeren et al, 2016 ; Sun et al, 2017 ). Having the right combinations of alleles is also valuable in overcoming genetic redundancy, which is common in plants ( Corcoran et al , 2022 ). Hence, the ability to stack multiple gene edits in the same plant is crucial for the engineering of complex traits.…”