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While the yeast Chz1 acts as a specific histone-chaperone for H2A.Z, functions of CHZ-domain proteins in multicellular eukaryotes remain obscure. Here, we report on the functional characterization of OsChz1, a sole CHZ-domain protein identified in rice. OsChz1 interacts with both the canonical H2A-H2B dimer and the variant H2A.Z-H2B dimer. Within crystal structure the C-terminal region of OsChz1 binds H2A-H2B via an acidic region, pointing to a previously unknown recognition mechanism. Knockout of OsChz1 leads to multiple plant developmental defects. At genome-wide level, loss of OsChz1 causes mis-regulations of thousands of genes and broad alterations of nucleosome occupancy as well as reductions of H2A.Z-enrichment. While OsChz1 associates with chromatin regions enriched of repressive histone marks (H3K27me3 and H3K4me2), its loss does not affect the genome landscape of DNA methylation. Taken together, it is emerging that OsChz1 functions as an important H2A/H2A.Z-H2B chaperone in dynamic regulation of chromatin for higher eukaryote development. Function of CHZ-domain proteins in multicellular eukaryotes remains unclear. Here, the authors characterize the sole CHZ-domain protein identified in rice and show that it functions as an H2A/H2A.Z-H2B chaperone in dynamic regulation of chromatin organization and genome function.

The INO80 chromatin remodeling complex plays a critical role in shaping the dynamic chromatin environment. The diverse functions of the evolutionarily conserved INO80 complex have been widely reported. However, the role of INO80 in modulating the histone variant H2A.Z is controversial. Moreover, whether INO80 helps regulate heterochromatin remains unknown. Here, we characterize the regulatory effects of OsINO80 on protein-coding genes and transposable elements (TEs) in rice. Upon OsINO80 overexpression in rice, we found three types of OsINO80-occupied regions with different chromatin signatures: type I (enriched with H2A.Z), type II (enriched with H3K9me2), and type III (deficient in H2A.Z/H3K9me2). Loss of OsINO80 results in a decrease in H3K27me3, but not H2A.Z, at type I regions as well as a decrease in H3K9me2 at type II regions, which correlates with TE activation and transposition. Our findings reveal that OsINO80 facilitates H3K27me3 establishment, promotes H3K9me2 deposition, and maintains TE silencing. This study reveals that the chromatin remodeling factor OsINO80 promotes H3K27me3 and H3K9me2 depositions in rice, which is correlated with gene transcription regulation and transposable element maintenance, providing insights into chromatin dynamics and genome stability.

Chromatin-based epigenetic information plays an important role in developmental gene regulation, in response to environment, and in natural variation of gene expression levels. Histone H3 lysine 4 di/trimethylation (H3K4me2/3) is abundant in euchromatin and is generally associated with transcriptional activation. Strikingly, however, enzymes catalyzing H3K4me2/3 remain poorly characterized in crops so far. Here, we investigated the function of the rice SET DOMAIN GROUP 701 (SDG701) gene by molecular and biochemical characterization of the gene product, and by studying effects of its loss or gain of function on plant growth and development. We demonstrated that SDG701 encodes a methytransferase specifically catalyzing H3K4 methylation. Overexpression and knockdown experiments showed that SDG701 is crucial for proper sporophytic plant development as well as for gametophytic transmission that directly impacts rice grain production. In-depth analysis of plant flowering time revealed that SDG701 promotes rice flowering under either long-day or short-day photoperiods. Consistently, the SDG701 protein was found to bind chromatin to promote H3K4me3 and to enhance expression of the rice Hd3a and RFT1 florigens. Collectively, our results establish SDG701 as a major rice H3K4-specific methyltransferase and provide important insights into function of H3K4me3 deposition in transcription activation of florigens in promoting plant flowering.

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.

The different genome-wide distributions of tri-methylation at H3K36 (H3K36me3) in various species suggest diverse mechanisms for H3K36me3 establishment during evolution. Here, we show that the transcription factor OsSUF4 recognizes a specific 7-bp DNA element, broadly distributes throughout the rice genome, and recruits the H3K36 methyltransferase SDG725 to target a set of genes including the key florigen genes RFT1 and Hd3a to promote flowering in rice. Biochemical and structural analyses indicate that several positive residues within the zinc finger domain are vital for OsSUF4 function in planta. Our results reveal a regulatory mechanism contributing to H3K36me3 distribution in plants. The distribution of H3K36me3 varies between species. Here Liu et al. show that the OsSUF4 transcription factor binds its target motif via a zinc finger domain to promote H3K36 methyltransferase targeting close to the transcription start site of genes including the flowering regulators RFT1 and Hd3a .

Background In animals, H3K4me2 and H3K4me3 are enriched at the transcription start site (TSS) and function as epigenetic marks that regulate gene transcription, but their functions in plants have not been fully characterized. Results We used chromatin immunoprecipitation sequencing to analyze the rice genome-wide changes to H3K4me1/H3K4me2/H3K4me3 following the loss of an H3K4-specific methyltransferase, SDG701. The knockdown of SDG701 resulted in a global decrease in H3K4me2/H3K4me3 levels throughout the rice genome. An RNA-sequencing analysis revealed that many genes related to diverse developmental processes were misregulated in the SDG701 knockdown mutant. In rice, H3K4me3 and H3K36me3 are positively correlated with gene transcription; however, surprisingly, the H3K4me2 level was negatively associated with gene transcription levels. Furthermore, the H3K4me3 level at the TSS region decreased significantly in the genes that exhibited down-regulated expression in the SDG701 knockdown mutant. In contrast, the genes with up-regulated expression in the mutant were associated with a considerable decrease in H3K4me2 levels over the gene body region. Conclusion A comparison of the genome-wide distributions of H3K4me2 in eukaryotes indicated that the H3K4me2 level is not correlated with the gene transcription level in yeast, but is positively and negatively correlated with gene expression in animals and plants, respectively. Our results uncovered H3K4me2 as a novel repressive mark in plants.

Nucleosome Assembly Protein 1 (NAP1) family proteins are evolutionarily conserved histone chaperones that play important roles in diverse biological processes. In this study, we determined the crystal structure of Arabidopsis NAP1-Related Protein 1 (NRP1) complexed with H2A-H2B and uncovered a previously unknown interaction mechanism in histone chaperoning. Both in vitro binding and in vivo plant rescue assays proved that interaction mediated by the N-terminal α-helix (αN) domain is essential for NRP1 function. In addition, the C-terminal acidic domain (CTAD) of NRP1 binds to H2A-H2B through a conserved mode similar to other histone chaperones. We further extended previous knowledge of the NAP1-conserved earmuff domain by mapping the amino acids of NRP1 involved in association with H2A-H2B. Finally, we showed that H2A-H2B interactions mediated by αN, earmuff, and CTAD domains are all required for the effective chaperone activity of NRP1. Collectively, our results reveal multiple interaction modes of a NAP1 family histone chaperone and shed light on how histone chaperones shield H2A-H2B from nonspecific interaction with DNA.

As a key epigenetic modification, the methylation of histone H3 lysine 36 (H3K36) modulates chromatin structure and is involved in diverse biological processes. To better understand the language of H3K36 methylation in rice (Oryza sativa), we chose potential histone methylation enzymes for functional exploration. In particular, we characterized rice SET DOMAIN GROUP 708 (SDG708) as an H3K36‐specific methyltransferase possessing the ability to deposit up to three methyl groups on H3K36. Compared with the wild‐type, SDG708‐knockdown rice mutants displayed a late‐flowering phenotype under both long‐day and short‐day conditions because of the down‐regulation of the key flowering regulatory genes Heading date 3a (Hd3a), RICE FLOWERING LOCUS T1 (RFT1), and Early heading date 1 (Ehd1). Chromatin immunoprecipitation experiments indicated that H3K36me1, H3K36me2, and H3K36me3 levels were reduced at these loci in SDG708‐deficient plants. More importantly, SDG708 was able to directly target and effect H3K36 methylation on specific flowering genes. In fact, knockdown of SDG708 led to misexpression of a set of functional genes and a genome‐wide decrease in H3K36me1/2/3 levels during the early growth stages of rice. SDG708 is a methyltransferase that catalyses genome‐wide deposition of all three methyl groups on H3K36 and is involved in many biological processes in addition to flowering promotion.

The methylation of histone H3 lysine 36 (H3K36) plays critical roles in brassinosteroid (BR)-related processes and is involved in controlling flowering time in rice (Oryza sativa). Although enzymes that catalyze this methylation reaction have been described, little is known about the recognition mechanisms to decipher H3K36 methylation information in rice. In this study, biochemical characterizations showed that MORF-RELATED GENE702 (MRG702) binds to trimethylated H3K4 and H3K36 (H3K4me3 and H3K36me3) in vitro. Similar to the loss-of-function mutants of the rice H3K36 methyltransferase geneSET DOMAIN GROUP725(SDG725), theMRG702knockdown mutants displayed typical BR-deficient mutant and late-flowering phenotypes. Gene transcription analyses showed thatMRG702knockdown resulted in the down-regulation of BR-related genes, includingDWARF11, BRASSINOSTEROD INSENSITIVE1,andBRASSINOSTEROID UPREGULATED1,and several flowering genes, includingEarly heading date1(Ehd1),Ehd2, Ehd3, OsMADS50, Heading date 3a,andRICE FLOWERING LOCUS T1. A binding analysis showed that MRG702 directly binds to the chromatin at target gene loci. This binding is dependent on the level of trimethylated H3K36, which is mediated by SDG725. Together, our results demonstrate that MRG702 acts as a reader protein of H3K4me3 and H3K36me3 and deciphers the H3K36 methylation information set by SDG725. Therefore, the role of MRG702 in the BR pathway and in controlling flowering time in rice is to function as a reader protein to decipher methylation information.

Trithorax group (TrxG) proteins are evolutionarily conserved in eukaryotes and play critical roles in transcriptional activation via deposition of histone H3 lysine 4 trimethylation (H3K4me3) in chromatin. Several Arabidopsis TrxG members have been characterized, and among them SET DOMAIN GROUP 2 (SDG2) has been shown to be necessary for global genome-wide H3K4me3 deposition. Although pleiotropic phenotypes have been uncovered in the sdg2 mutants, SDG2 function in the regulation of stem cell activity has remained largely unclear. Here, we investigate the sdg2 mutant root phenotype and demonstrate that SDG2 is required for primary root stem cell niche (SCN) maintenance as well as for lateral root SCN establishment. Loss of SDG2 results in drastically reduced H3K4me3 levels in root SCN and differentiated cells and causes the loss of auxin gradient maximum in the root quiescent centre. Elevated DNA damage is detected in the sdg2 mutant, suggesting that impaired genome integrity may also have challenged the stem cell activity. Genetic interaction analysis reveals that SDG2 and CHROMATIN ASSEMBLY FACTOR-1 act synergistically in root SCN and genome integrity maintenance but not in telomere length maintenance. We conclude that SDG2-mediated H3K4me3 plays a distinctive role in the regulation of chromatin structure and genome integrity, which are key features in pluripotency of stem cells and crucial for root growth and development.