Histone chaperones AtChz1A and AtChz1B are required for H2A.Z deposition and interact with the SWR1 chromatin-remodeling complex in Arabidopsis thaliana.

The histone variant H2A.Z plays key functions in transcription and genome stability in all eukaryotes ranging from yeast to human, but the molecular mechanisms by which H2A.Z is incorporated into chromatin remain largely obscure. Here, we characterized the two homologs of yeast Chaperone for H2A.Z-H2B (Chz1) in Arabidopsis thaliana, AtChz1A and AtChz1B. AtChz1A/AtChz1B were verified to bind to H2A.Z-H2B and facilitate nucleosome assembly in vitro. Simultaneous knockdown of AtChz1A and AtChz1B, which exhibit redundant functions, led to a genome-wide reduction in H2A.Z and phenotypes similar to those of the H2A.Z-deficient mutant hta9-1hta11-2, including early flowering and abnormal flower morphologies. Interestingly, AtChz1A was found to physically interact with ACTIN-RELATED PROTEIN 6 (ARP6), an evolutionarily conserved subunit of the SWR1 chromatin-remodeling complex. Genetic interaction analyses showed that atchz1a-1atchz1b-1 was hypostatic to arp6-1. Consistently, genome-wide profiling analyses revealed partially overlapping genes and fewer misregulated genes and H2A.Z-reduced chromatin regions in atchz1a-1atchz1b-1 compared with arp6-1. Together, our results demonstrate that AtChz1A and AtChz1B act as histone chaperones to assist the deposition of H2A.Z into chromatin via interacting with SWR1, thereby playing critical roles in the transcription of genes involved in flowering and many other processes.

The histone methylation readers MRG1/MRG2 and the histone chaperones NRP1/NRP2 associate in fine-tuning Arabidopsis flowering time.

Histones are highly basic proteins involved in packaging DNA into chromatin, and histone modifications are fundamental in epigenetic regulation in eukaryotes. Among the numerous chromatin modifiers identified in Arabidopsis (Arabidopsis thaliana), MORF-RELATED GENE (MRG)1 and MRG2 have redundant functions in reading histone H3 lysine 36 trimethylation (H3K36me3). Here, we show that MRG2 binds histone chaperones belonging to the NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) family, including NAP1-RELATED PROTEIN (NRP)1 and NRP2. Characterization of the loss-of-function mutants mrg1 mrg2, nrp1 nrp2 and mrg1 mrg2 nrp1 nrp2 revealed that MRG1/MRG2 and NRP1/NRP2 regulate flowering time through fine-tuning transcription of floral genes by distinct molecular mechanisms. In particular, the physical interaction between NRP1/NRP2 and MRG1/MRG2 inhibited the binding of MRG1/MRG2 to the transcription factor CONSTANS (CO), leading to a transcriptional repression of FLOWERING LOCUS T (FT) through impeded H4K5 acetylation (H4K5ac) within the FT chromatin. By contrast, NRP1/NRP2 and MRG1/MRG2 act together, likely in a multiprotein complex manner, in promoting the transcription of FLOWERING LOCUS C (FLC) via an increase of both H4K5ac and H3K9ac in the FLC chromatin. Because the expression pattern of FLC represents the major category of differentially expressed genes identified by genome-wide RNA-sequencing analysis in the mrg1 mrg2, nrp1 nrp2 and mrg1 mrg2 nrp1 nrp2 mutants, it is reasonable to speculate that the NRP1/NRP2-MRG1/MRG2 complex may be involved in transcriptional activation of genes beyond FLC and flowering time control.

MRG1/2 histone methylation readers and HD2C histone deacetylase associate in repression of the florigen gene FT to set a proper flowering time in response to day-length changes.

Day-length changes represent an important cue for modulating flowering time. In Arabidopsis, the expression of the florigen gene FLOWERING LOCUS T (FT) exhibits a 24-h circadian rhythm under long-day (LD) conditions. Here we focus on the chromatin-based mechanism regarding the control of FT expression. We conducted co-immunoprecipitation assays along with LC-MS/MS analysis and identified HD2C histone deacetylase as the binding protein of the H3K4/H3K36 methylation reader MRG2. HD2C and MRG1/2 regulate flowering time under LD conditions, but not under short-day conditions. Moreover, HD2C functions as an effective deacetylase in planta, mainly targeting H3K9ac, H3K23ac and H3K27ac. At dusk, HD2C is recruited to FT to deacetylate histones and repress transcription in an MRG1/2-dependent manner. More importantly, HD2C competes with CO for the binding of MRG2, and the accumulation of HD2C at the FT locus occurs at the end of the day. Our findings not only reveal a histone deacetylation mechanism contributing to prevent FT overexpression and precocious flowering, but also support the model in which the histone methylation readers MRG1/2 provide a platform on chromatin for connecting regulatory factors involved in activating FT expression in response to daylight and decreasing FT expression around dusk under long days.

AtINO80 and AtARP5 physically interact and play common as well as distinct roles in regulating plant growth and development.

The proper modulation of chromatin structure is dependent on the activities of chromatin-remodeling factors and their interplays. Here, we show that the Arabidopsis chromatin-remodeler AtINO80 interacts with the actin-related protein AtARP5 and can form a larger protein complex. Genetic analysis demonstrated that AtARP5 acts in concert with AtINO80 during plant cellular proliferation and replication stress response. At the same time, AtARP5 is not required for AtINO80-mediated control of flowering time and related transcriptional regulation, and their chromatin distribution patterns on regions of flowering-repressor genes FLC/MAF4/MAF5 are also different. An in vitro DNase I digestion assay revealed that the AtINO80N-terminus can weakly bind DNA, an interaction that is significantly inhibited by H2A.Z/H2B addition. AtARP6, a specific subunit of SWR1-C that mediates the H2A.Z exchange, was found to have a previously unexpected inhibitory role in the local chromatin enrichment of AtINO80. Further genetic analyses revealed the functional interplay between AtINO80 and AtARP6 and their critical roles in embryogenesis and post-embryonic organ development, as well as the synergy of AtARP5 and AtARP6 in maintaining genomic stability. Our findings provide insights into the common and distinct roles of AtINO80 and AtARP5 in diverse aspects of plant development.

The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development.

INO80 is a conserved chromatin-remodeling factor in eukaryotes. While a previous study reported that the Arabidopsis thaliana INO80 (AtINO80) is required for somatic homologous recombination (HR), the role of AtINO80 in plant growth and development remains obscure. Here, we identified and characterized two independent atino80 mutant alleles, atino80-5 and atino80-6, which display similar and pleiotropic phenotypes, including smaller plant and organ size, and late flowering. Under standard growth conditions, atino80-5 showed decreased HR; however, after genotoxic treatment, HR in the mutant increased, accompanied by more DNA double-strand breaks and stronger cellular responses. Transcription analysis showed that many developmental and environmental responsive genes are overrepresented in the perturbed genes in atino80-5. These genes significantly overlapped with the category of H2A.Z body-enriched genes. AtINO80 also interacts with H2A.Z, and facilitates the enrichment of H2A.Z at the ends of the key flowering repressor genes FLC and MAF4/5. Our characterization of the atino80-5 and atino80-6 mutants confirms and extends the previous AtINO80 study, and provides perspectives for linking studies of epigenetic mechanisms involved in plant chromatin stability with plant response to developmental and environmental cues.