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Plants contain a great number of genes encoding a distinctive class of SET domain proteins which harbor a plant-specific N-terminal part together with a C-terminal part showing highest sequence similarity to the catalytic domain of the yeast CLR4, the human SUV39H1 and G9a histone-methyltransferases (HMTases). Here we show that NtSET1, a representative member of this class from tobacco, methylated both K9 and K27 of histone H3in vitro. Ectopic expression ofNtSET1, by an inducible promoter, increased the amount of dimethylated H3K9 and induced chromosome-segregation defects in tobacco BY2 cells. Deletion analyses show that the HMTase activity, the association with specific chromatin regions and with condensed chromosomes, and the cellular effects largely depended on the C-terminal region including the SET domain of the protein. Nevertheless, the N-terminal part of NtSET1 was capable of targeting the green fluorescent protein to interphase chromatin. Finally, we show that NtSET1 bound LHP1, theArabidopsishomolog of animal heterochromatin protein 1 (HP1), and that LHP1 co-localized with heterochromatin containing high amounts of dimethylated H3K9, suggesting a role for NtSET1 in heterochromatic function. Taken together, our results provide new insights into the molecular and global chromatin-binding activities of this particular class member of plant SET domain proteins. [ABSTRACT FROM AUTHOR]

Histone chaperones and histone variants play crucial roles in DNA replication, gene transcription, and DNA repair in eukaryotes. Histone chaperones reversibly promote nucleosome assembly and disassembly by incorporating or evicting histones and histone variants to modulate chromatin accessibility, thereby altering the chromatin states and modulating DNA-related biological processes. Cofactors assist histone chaperones to target specific chromatin regions to regulate the exchange of histones and histone variants. In this review, we summarize recent progress in the interplay between histone variants and chaperones in plants. We discuss the structural basis of chaperone–histone complexes and the mechanisms of their cooperation in regulating gene transcription and plant development. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nucleosomal core histones (H2A, H2B, H3 and H4) must be assembled, replaced or exchanged to preserve or modify chromatin organization and function according to cellular needs. Histone chaperones escort histones, and play key functions during nucleosome assembly/disassembly and in nucleosome structure configuration. Because of their location at the periphery of nucleosome, histone H2A-H2B dimers are remarkably dynamic. Here we focus on plant histone H2A/H2B chaperones, particularly members of the NUCLEOSOME ASSEMBLY PROTEIN-1 ( NAP1) and FACILITATES CHROMATIN TRANSCRIPTION ( FACT) families, discussing their molecular features, properties, regulation and function. Covalent histone modifications (e.g. ubiquitination, phosphorylation, methylation, acetylation) and H2A variants (H2A.Z, H2A.X and H2A.W) are also discussed in view of their crucial importance in modulating nucleosome organization and function. We further discuss roles of NAP1 and FACT in chromatin-based processes, such as transcription, DNA replication and repair. Specific functions of NAP1 and FACT are evident when their roles are considered with respect to regulation of plant growth and development and in plant responses to environmental stresses. Future major challenges remain in order to define in more detail the overlapping and specific roles of various members of the NAP1 family as well as differences and similarities between NAP1 and FACT family members, and to identify and characterize their partners as well as new families of chaperones to understand histone variant incorporation and chromatin target specificity. [ABSTRACT FROM AUTHOR]

Summary: Chromatin modifications play important roles in plant adaptation to abiotic stresses, but the precise function of histone H3 lysine 36 (H3K36) methylation in drought tolerance remains poorly evaluated.Here, we report that SDG708, a specific H3K36 methyltransferase, functions as a positive regulator of drought tolerance in rice. SDG708 promoted abscisic acid (ABA) biosynthesis by directly targeting and activating the crucial ABA biosynthesis genes NINE‐CIS‐EPOXYCAROTENOID DIOXYGENASE 3 (OsNCED3) and NINE‐CIS‐EPOXYCAROTENOID DIOXYGENASE 5 (OsNCED5).Additionally, SDG708 induced hydrogen peroxide accumulation in the guard cells and promoted stomatal closure to reduce water loss. Overexpression of SDG708 concomitantly enhanced rice drought tolerance and increased grain yield under normal and drought stress conditions.Thus, SDG708 is potentially useful as an epigenetic regulator in breeding for grain yield improvement. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

Summary: 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. Significance Statement: The identification of a physical interaction between the H3K36me3‐reader MORF‐RELATED GENE (MRG)1/MRG2 and the histone‐chaperone NAP1‐RELATED PROTEIN (NRP)1/NRP2 is significant. This interaction inhibits MRG1/MRG2 to bind CONSTANS in FLOWERING LOCUS T transcription, whereas MRG1/MRG2 and NRP1/NRP2 cooperate in a multiprotein complex manner in promoting FLOWERING LOCUS C (FLC) transcription via elevation of both H4K5ac and H3K9ac in the FLC chromatin. [ABSTRACT FROM AUTHOR]

The Arabidopsis thaliana gain-of-function T-DNA insertion mutant jaw-1D produces miR319A, a microRNA that represses genes encoding CIN-like TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORs (TCPs), a family of transcription factors that play key roles in leaf morphogenesis. In this study, we show that jaw-1D is responsive to paramutation-like epigenetic silencing. A genetic cross of jaw-1D with the polycomb gene mutant curly leaf-29 (clf-29) leads to attenuation of the jaw-1D mutant plant phenotype. This induced mutation, jaw-1D*, was associated with down-regulation of miR319A, was heritable independently from clf-29, and displayed paramutation-like non-Mendelian inheritance. Down-regulation of miR319A in jaw-1D* was linked to elevated levels of histone H3 lysine 9 dimethylation and DNA methylation at the CaMV35S enhancer located within the activation-tagging T-DNA of the jaw-1D locus. Examination of 21 independent T-DNA insertion mutant lines revealed that 11 could attenuate the jaw-1D mutant phenotype in a similar way to the paramutation induced by clf-29. These paramutagenic mutant lines shared the common feature that their T-DNA insertion was present as multi-copy tandem repeats and contained high levels of CG and CHG methylation. Our results provide important insights into paramutation-like epigenetic silencing, and caution against the use of jaw-1D in genetic interaction studies. [ABSTRACT FROM AUTHOR]

Abstract Polycomb group proteins play essential roles in transcriptional gene repression during both animal and plant development. Polycomb repression complex 1 (PRC1) is one of the key functional modules in polycomb group silencing. It acts as both a reader of H3K27me3 (histone H3 lysine 27 trimethylation) and a writer of H2Aub1 (histone H2A monoubiquitination) in establishing stable repression chromatin state. Intriguingly, a recent study showed that Arabidopsis PRC1 contains the H3K4me3-binding proteins of the ALFIN-like (AL) family, pointing to a chromatin state switch from active to repressive transcription of embryonic genes required for vegetative plant development. However, molecular and structural basis of AL–PRC1 complexes are lacking, which harmed insightful mechanistic understanding of AL–PRC1 complex function. In the present study, we report the crystal structures of the PAL domain (DUF3594 domain) of AL2 and AL7 proteins as well as their mechanistic binding to the PRC1 ring-finger proteins (RING1 and BMI1). We found that the PAL domain exists as a homodimer and represents a novel protein fold. We further determined the crystal structures of the PAL domain of AL2 (AL2-PAL) in complex with AtRING1a and AtBMI1b, the two core components of Arabidopsis PRC1. Interestingly, two PAL-binding sites were found on AtRING1a. Each of them can bind AL but with different affinities and distinct structural bases. Based on our results, we propose a mechanistic model to understand how AL proteins target PRC1 to active chromatin to undergo the transition from H3K4me3 to H2Aub1/H3K27me3 in establishing gene silencing. Graphical Abstract Unlabelled Image Highlights • The crystal structure of AL2-PAL reveals a novel protein fold. • Multiple AL2-PAL interaction sites were found on AtRING1a or AtBMI1b. • AL2-PAL binds the proximal site and the distal site of AtRing1a in different modes. • The structures highlight how AL2 recruits PRC1 to facilitate chromatin state switch. [ABSTRACT FROM AUTHOR]

Summary: Stem cells in both plant and animal kingdoms reside in a specialized cellular context called the stem cell niche (SCN). SCN integrity is crucial for organism development. Here we show that the H3/H4 histone chaperone CHROMATIN ASSEMBLY FACTOR‐1 (CAF‐1) and the H2A/H2B histone chaperone NAP1‐RELATED PROTEIN1/2 (NRP1/2) play synergistic roles in Arabidopsis root SCN maintenance. Compared with either the m56‐1 double mutant deprived of NRP1 and NRP2 or the fas2‐4 mutant deprived of CAF‐1, the combined m56‐1fas2‐4 triple mutant displayed a much more severe short‐root phenotype. The m56‐1fas2‐4 mutant root lost the normal organizing center Quiescent Center (QC), and some initial stem cells differentiated precociously. Microarray analysis unraveled the deregulation of 2735 genes within the Arabidopsis genome (representing >8% of all genes) in the m56‐1fas2‐4 mutant roots. Expression of some SCN key regulatory genes (e.g. WOX5, PLT1, SHR) was not limiting, rather the plant hormone auxin gradient maximum at QC was impaired. The mutant roots showed programmed cell death and high levels of the DNA damage marked histone H2A.X phosphorylation (γ‐H2A.X). Knockout of either ATAXIA‐TELANGIECTASIA MUTATED (ATM) or ATR, encoding a DNA damage response kinase, rescued in part the cell death and the short‐root phenotype of the m56‐1fas2‐4 mutant. Collectively, our study indicated that NRP1/2 and CAF‐1 act cooperatively in regulating proper genome transcription, in sustaining chromatin replication and in maintaining genome integrity, which are crucial for proper SCN function during continuous post‐embryonic root development. [ABSTRACT FROM AUTHOR]

Summary: WUSCHEL (WUS) is critical for plant meristem maintenance and determinacy in Arabidopsis, and the regulation of its spatiotemporal expression patterns is complex. We previously found that AGAMOUS (AG), a key MADS‐domain transcription factor in floral organ identity and floral meristem determinacy, can directly suppress WUS expression through the recruitment of the Polycomb group (PcG) protein TERMINAL FLOWER 2 (TFL2, also known as LIKE HETEROCHROMATIN PROTEIN 1, LHP1) at the WUS locus; however, the mechanism by which WUS is repressed remains unclear. Here, using chromosome conformation capture (3C) and chromatin immunoprecipitation 3C, we found that two specific regions flanking the WUS gene body bound by AG and TFL2 form a chromatin loop that is directly promoted by AG during flower development in a manner independent of the physical distance and sequence content of the intervening region. Moreover, AG physically interacts with TFL2, and TFL2 binding to the chromatin loop is dependent on AG. Transgenic and CRISPR/Cas9‐edited lines showed that the WUS chromatin loop represses gene expression by blocking the recruitment of RNA polymerase II at the locus. The findings uncover the WUS chromatin loop as another regulatory mechanism controlling WUS expression, and also shed light on the factors required for chromatin conformation change and their recruitment. [ABSTRACT FROM AUTHOR]

Background: Intron retention (IR), the most prevalent alternative splicing form in plants, plays a critical role in gene expression during plant development and stress response. However, the molecular mechanisms underlying IR regulation remain largely unknown. Results: Knockdown of SDG725, a histone H3 lysine 36 (H3K36)-specific methyltransferase in rice, leads to alterations of IR in more than 4700 genes. Surprisingly, IR events are globally increased at the 5′ region but decreased at the 3′ region of the gene body in the SDG725-knockdown mutant. Chromatin immunoprecipitation sequencing analyses reveal that SDG725 depletion results in a genome-wide increase of the H3K36 mono-methylation (H3K36me1) but, unexpectedly, promoter-proximal shifts of H3K36 di- and tri-methylation (H3K36me2 and H3K36me3). Consistent with the results in animals, the levels of H3K36me1/me2/me3 in rice positively correlate with gene expression levels, whereas shifts of H3K36me2/me3 coincide with position-specific alterations of IR. We find that either H3K36me2 or H3K36me3 alone contributes to the positional change of IR caused by SDG725 knockdown, although IR shift is more significant when both H3K36me2 and H3K36me3 modifications are simultaneously shifted. Conclusions: Our results revealed that SDG725 modulates IR in a position-specific manner, indicating that H3K36 methylation plays a role in RNA splicing, probably by marking the retained introns in plants. [ABSTRACT FROM AUTHOR]

Abstract: The phytohormone gibberellin (GA) plays essential roles in plant growth and development. Here, we report that OsINO80, a conserved ATP‐dependent chromatin‐remodeling factor in rice (Oryza sativa), functions in both GA biosynthesis and diverse biological processes. OsINO80‐knockdown mutants, derived from either T‐DNA insertion or RNA interference, display typical GA‐deficient phenotypes, including dwarfism, reduced cell length, late flowering, retarded seed germination and impaired reproductive development. Consistently, transcriptome analyses reveal that OsINO80 knockdown results in downregulation by more than two‐fold of over 1,000 genes, including the GA biosynthesis genes CPS1 and GA3ox2, and the dwarf phenotype of OsINO80‐knockdown mutants can be rescued by the application of exogenous GA3. Chromatin immunoprecipitation (ChIP) experiments show that OsINO80 directly binds to the chromatin of CPS1 and GA3ox2 loci. Biochemical assays establish that OsINO80 specially interacts with histone variant H2A.Z and the H2A.Z enrichments at CPS1 and GA3ox2 are decreased in OsINO80‐knockdown mutants. Thus, our study identified a rice chromatin‐remodeling factor, OsINO80, and demonstrated that OsINO80 is involved in regulation of the GA biosynthesis pathway and plays critical functions for many aspects of rice plant growth and development. [ABSTRACT FROM AUTHOR]

NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) defines an evolutionarily conserved family of histone chaperones and loss of function of the Arabidopsis thaliana NAP1 family genes NAP1-RELATED PROTEIN1 (NRP1) and NRP2 causes abnormal root hair formation. Yet, the underlying molecular mechanisms remain unclear. Here, we show that NRP1 interacts with the transcription factor WEREWOLF (WER) in vitro and in vivo and enriches at the GLABRA2 (GL2) promoter in a WER-dependent manner. Crystallographic analysis indicates that NRP1 forms a dimer via its N-terminal α-helix. Mutants of NRP1 that either disrupt the α-helix dimerization or remove the C-terminal acidic tail, impair its binding to histones and WER and concomitantly lead to failure to activate GL2 transcription and to rescue the nrp1-1 nrp2-1 mutant phenotype. Our results further demonstrate that WER-dependent enrichment of NRP1 at the GL2 promoter is involved in local histone eviction and nucleosome loss in vivo. Biochemical competition assays imply that the association between NRP1 and histones may counteract the inhibitory effect of histones on the WER-DNA interaction. Collectively, our study provides important insight into the molecular mechanisms by which histone chaperones are recruited to target chromatin via interaction with a gene-specific transcription factor to moderate chromatin structure for proper root hair development. [ABSTRACT FROM AUTHOR]

Polycomb group proteins are important repressors of numerous genes in higher eukaryotes. However, the mechanism by which Polycomb group proteins are recruited to specific genes is poorly understood. In Arabidopsis, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), also known as TERMINAL FLOWER 2, was originally proposed as a subunit of polycomb repressive complex 1 (PRC1) that could bind the tri-methylated lysine 27 of histone H3 (H3K27me3) established by the PRC2. In this work, we show that LHP1 mainly functions with PRC2 to establish H3K27me3, but not with PRC1 to catalyze monoubiquitination at lysine 119 of histone H2A. Our results show that complexes of the transcription factors ASYMMETRIC LEAVES 1 (AS1) and AS2 could help to establish the H3K27me3 modification at the chromatin regions of Class-I KNOTTED1-like homeobox ( KNOX) genes BREVIPEDICELLUS and KNAT2 via direct interactions with LHP1. Additionally, our transcriptome analysis indicated that there are probably more common target genes of AS1 and LHP1 besides Class-I KNOX genes during leaf development in Arabidopsis. [ABSTRACT FROM AUTHOR]

Homologous recombination ( HR) of nuclear DNA occurs within the context of a highly complex chromatin structure. Despite extensive studies of HR in diverse organisms, mechanisms regulating HR within the chromatin context remain poorly elucidated. Here we investigate the role and interplay of the histone chaperones NUCLEOSOME ASSEMBLY PROTEIN1 ( NAP1) and NAP1- RELATED PROTEIN ( NRP) and the ATP-dependent chromatin-remodeling factor INOSITOL AUXOTROPHY80 ( INO80) in regulating somatic HR in Arabidopsis thaliana. We show that simultaneous knockout of the four At NAP1 genes and the two NRP genes in the sextuple mutant m123456-1 barely affects normal plant growth and development. Interestingly, compared with the respective At NAP1 ( m123-1 and m1234-1) or NRP ( m56-1) loss-of-function mutants, the sextuple mutant m123456-1 displays an enhanced plant hypersensitivity to UV or bleomycin treatments. Using HR reporter constructs, we show that At NAP1 and NRP act in parallel to synergistically promote somatic HR. Distinctively, the At INO80 loss-of-function mutation ( atino80-5) is epistatic to m56-1 in plant phenotype and telomere length but hypostatic to m56-1 in HR determinacy. Further analyses show that expression of HR machinery genes and phosphorylation of H2A.X (γ-H2A.X) are not impaired in the mutants. Collectively, our study indicates that NRP and At NAP1 synergistically promote HR upstream of At INO80-mediated chromatin remodeling after the formation of γ-H2A.X foci during DNA damage repair. [ABSTRACT FROM AUTHOR]

Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

INO80 is a conserved chromatin-remodeling factor in eukaryotes. While a previous study reported that the Arabidopsis thaliana INO80 (At INO80) is required for somatic homologous recombination ( HR), the role of At INO80 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. At INO80 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 At INO80 study, and provides perspectives for linking studies of epigenetic mechanisms involved in plant chromatin stability with plant response to developmental and environmental cues. [ABSTRACT FROM AUTHOR]

Histone methylation is a major component in numerous processes such as determination of flowering time, which is fine-tuned by multiple genetic pathways that integrate both endogenous and environmental signals. Previous studies identified SET DOMAIN GROUP 26 ( SDG26) as a histone methyltransferase involved in the activation of flowering, as loss of function of SDG26 caused a late-flowering phenotype in Arabidopsis thaliana. However, the SDG26 function and the underlying molecular mechanism remain largely unknown. In this study, we undertook a genetic analysis by combining the sdg26 mutant with mutants of other histone methylation enzymes, including the methyltransferase mutants Arabidopsis trithorax1 ( atx1), sdg25 and curly leaf ( clf), as well as the demethylase double mutant lsd1-like1 lsd1-like2 ( ldl1 ldl2). We found that the early-flowering mutants sdg25, atx1 and clf interact antagonistically with the late-flowering mutant sdg26, whereas the late-flowering mutant ldl1 ldl2 interacts synergistically with sdg26. Based on microarray analysis, we observed weak overlaps in the genes that were differentially expressed between sdg26 and the other mutants. Our analyses of the chromatin of flowering genes revealed that the SDG26 protein binds at the key flowering integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/ AGAMOUS- LIKE 20 ( SOC1/ AGL20), and is required for histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 36 trimethylation (H3K36me3) at this locus. Together, our results indicate that SDG26 promotes flowering time through a distinctive genetic pathway, and that loss of function of SDG26 causes a decrease in H3K4me3 and H3K36me3 at its target gene SOC1, leading to repression of this gene and the late-flowering phenotype. [ABSTRACT FROM AUTHOR]

ANTI-SILENCING FUNCTION 1 ( ASF1) is an evolutionarily conserved histone chaperone involved in diverse chromatin-based processes in eukaryotes. Yet, its role in transcription and the underlying molecular mechanisms remain largely elusive, particularly in plants. Here, we show that the A rabidopsis thaliana ASF1 homologous genes, AtASF1A and AtASF1B, are involved in gene transcription activation in response to heat stress. The A tasf1ab mutant displays defective basal as well as acquired thermotolerance phenotypes. Heat-induced expression of several key genes, including the HEAT SHOCK PROTEIN ( HSP) genes H sp101, H sp70, H sa32, H sp17.6A and H sp17. 6B - CI, and the HEAT SHOCK FACTOR ( HSF) gene HsfA2 but not HsfB1 is drastically impaired in A tasf1ab as compared with that in wild type. We found that AtASF1A/ B proteins are recruited onto chromatin, and their enrichment is correlated with nucleosome removal and RNA polymerase II accumulation at the promoter and coding regions of HsfA2 and H sa32 but not HsfB1. Moreover, AtASF1A/ B facilitate H3K56 acetylation ( H3K56ac), which is associated with HsfA2 and H sa32 activation. Taken together, our study unravels an important function of AtASF1A/ B in plant heat stress response and suggests that AtASF1A/ B participate in transcription activation of some but not all HSF and HSP genes via nucleosome removal and H3K56ac stimulation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Leaf organogenesis occurs within the peripheral zone of the shoot apical meristem (SAM). The initiation and subsequent development of a leaf requires the stable repression of a highly conserved class of plant genes, namely class-I KNOTTED 1-like homeobox ( KNOX) genes. In Arabidopsis, this class comprises four members: SHOOT MERISTEMLESS ( STM); BREVIPEDICELLUS ( BP); KNAT2 and KNAT6. Two transcription factors, ASYMMETRIC LEAVES 1 (AS1) and AS2, are known to form a protein complex to repress BP, KNAT2 and KNAT6. Here, we show that AS2 physically interacts with the microRNA319 (miR319)-regulated CINCINNATA-like TEOSINTE BRANCHED 1-CYCLOIDEA-PCF (TCP) transcription factors in vitro and in vivo. By chromatin immunoprecipitation, we demonstrated that AS2 and TCPs bind to similar regions of the BP and KNAT2 promoters. In addition, DNA-binding activities of the TCP proteins rely on the presence of AS2, as the activities were dramatically reduced in the as2 mutant. The jaw-D mutant, which overexpresses MIR319a to downregulate several target TCP genes, strongly enhanced the as2 phenotypes and caused more severe ectopic expression of BP, KNAT2 and KNAT6. Our results reveal that KNOX repression requires different types of transcription factors that function together to ensure normal leaf development. [ABSTRACT FROM AUTHOR]

Summary Methylation of histone lysine residues plays an essential role in epigenetic regulation of gene expression in eukaryotes. Enzymes involved in establishment of the repressive H3K9 and H3K27 methylation marks have been previously characterized, but the deposition and function of H3K4 and H3K36 methylation remain uncharacterized in rice. Here, we report that rice SDG725 encodes a H3K36 methyltransferase, and its down-regulation causes wide-ranging defects, including dwarfism, shortened internodes, erect leaves and small seeds. These defects resemble the phenotypes previously described for some brassinosteroid-knockdown mutants. Consistently, transcriptome analyses revealed that SDG725 depletion results in down-regulation by more than two-fold of over 1000 genes, including D11, BRI1 and BU1, which are known to be involved in brassinosteroid biosynthesis or signaling pathways. Chromatin immunoprecipitation analyses showed that levels of H3K36me2/3 are reduced in chromatin at some regions of these brassinosteroid-related genes in SDG725 knockdown plants, and that SDG725 protein is able to directly bind to these target genes. Taken together, our data indicate that SDG725-mediated H3K36 methylation modulates brassinosteroid-related gene expression, playing an important role in rice plant growth and development. [ABSTRACT FROM AUTHOR]

Homologous recombination (HR) is essential for maintaining genome integrity and variability. To orchestrate HR in the context of chromatin is a challenge, both in terms of DNA accessibility and restoration of chromatin organization after DNA repair. Histone chaperones function in nucleosome assembly/disassembly and could play a role in HR. Here, we show that the NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) family histone chaperones are required for somatic HR in Arabidopsis thaliana. Depletion of either the NAP1 group or NAP1-RELATED PROTEIN (NRP) group proteins caused a reduction in HR in plants under normal growth conditions as well as under a wide range of genotoxic or abiotic stresses. This contrasts with the hyperrecombinogenic phenotype caused by the depletion of the CHROMATIN ASSEMBLY FACTOR-1 (CAF-1) histone chaperone. Furthermore, we show that the hyperrecombinogenic phenotype caused by CAF-1 depletion relies on NRP1 and NRP2, but the telomere shortening phenotype does not. Our analysis of DNA lesions, H3K56 acetylation, and expression of DNA repair genes argues for a role of NAP1 family histone chaperones in nucleosome disassembly/reassembly during HR. Our study highlights distinct functions for different families of histone chaperones in the maintenance of genome stability and establishes a crucial function for NAP1 family histone chaperones in somatic HR. [ABSTRACT FROM AUTHOR]

Anti-silencing function1 (ASF1) is an evolutionarily conserved histone chaperone. Studies in yeast and animals indicate that ASF1 proteins play important roles in various chromatin-based processes, including gene transcription, DNA replication and repair. While two genes encoding ASF1 homologues, AtASF1A and AtASF1B, are found in the Arabidopsis genome, their function has not been studied. Here we report that both AtASF1A and AtASF1B proteins bind histone H3, and are localized in the cytoplasm and the nucleus. Loss-of-function of either AtASF1A or AtASF1B did not show obvious defects, whereas simultaneous knockdown of both genes in the double mutant Atasf1ab drastically inhibited plant growth and caused abnormal vegetative and reproductive organ development. The Atasf1ab mutant plants exhibit cell number reduction, S-phase delay/arrest, and reduced polyploidy levels. Selective up-regulation of expression of a subset of genes, including those involved in S-phase checkpoints and the CYCB1;1 gene at the G-to-M transition, was observed in Atasf1ab. Furthermore, the Atasf1ab-triggered replication fork stalling constitutively activates the DNA damage checkpoint and repair genes, including ATM, ATR, PARP1 and PARP2 as well as several genes of the homologous recombination (HR) pathway but not genes of the non-homologous end joining (NHEJ) pathway. In spite of the activation of repair genes, an increased level of DNA damage was detected in Atasf1ab, suggesting that defects in the mutant largely exceed the available capacity of the repair machinery. Taken together, our study establishes crucial roles for the AtASF1A and AtASF1B genes in chromatin replication, maintenance of genome integrity and cell proliferation during plant development. [ABSTRACT FROM AUTHOR]

Histone H3 lysine 4 trimethylation (H3K4me3) is abundant in euchromatin and is in general associated with transcriptional activation in eukaryotes. Although some Arabidopsis thaliana SET DOMAIN GROUP (SDG) genes have been previously shown to be involved in H3K4 methylation, they are unlikely to be responsible for global genome-wide deposition of H3K4me3. Most strikingly, sparse knowledge is currently available about the role of histone methylation in gametophyte development. In this study, we show that the previously uncharacterized SDG2 is required for global H3K4me3 deposition and its loss of function causes wide-ranging defects in both sporophyte and gametophyte development. Transcriptome analyses of young flower buds have identified 452 genes downregulated by more than twofold in the sdg2-1 mutant; among them, 11 genes, including SPOROCYTELESS/NOZZLE (SPL/NZZ) and MALE STERILITY1 (MS1), have been previously shown to be essential for male and/or female gametophyte development. We show that both SPL/NZZ and MS1 contain bivalent chromatin domains enriched simultaneously with the transcriptionally active mark H3K4me3 and the transcriptionally repressive mark H3K27me3 and that SDG2 is specifically required for the H3K4me3 deposition. Our data suggest that SDG2 -mediated H3K4me3 deposition poises SPL/NZZ and MS1 for transcriptional activation, forming a key regulatory mechanism in the gene networks responsible for gametophyte development. [ABSTRACT FROM AUTHOR]

Covalent modifications of histone lysine residues by methylation play key roles in the regulation of chromatin structure and function. In contrast to H3K9 and H3K27 methylations that mark repressive states of transcription and are absent in some lower eukaryotes, H3K4 and H3K36 methylations are considered as active marks of transcription and are highly conserved in all eukaryotes from yeast (Saccharomyces cerevisiae) to Homo sapiens. Paradoxically, protein complexes catalyzing H3K4 and H3K36 methylations are less-extensively characterized in higher eukaryotes, particularly in plants. Arabidopsis (Arabidopsis thaliana) contains 12 SET DOMAIN GROUP (SDG) proteins phylogenetic classified to Trithorax Group (TrxG) and thus potentially involved in H3K4 and H3K36 methylations. So far only some genes of this family had been functionally characterized. Here we report on the genetic and molecular characterization of 5DG25, a previously uncharacterized member of the Arabidopsis TrxG family. We show that the loss-of-function mutant sdg25-1 has an early flowering phenotype associated with suppression of FLOWERING LOCUS C (FLC) expression. Recombinant SDG25 proteins could methylate histone H3 from oligonucleosomes and mutant sdg25-1 plants showed weakly reduced levels of H3K36 dimethylation at FLC chromatin. Interestingly, sdg25-1 transcriptome shared a highly significant number of differentially expressed genes with that of sdg26-1, a previously characterized mutant exhibiting late-flowering phenotype and elevated FLC expression. Taken together, our results provide, to our knowledge, the first demonstration for a biological function of SDG25 and reveal additional layers of complexity of overlap and nonoverlap functions of the TrxG family genes in Arabidopsis. [ABSTRACT FROM AUTHOR]

Abstract: Histone methylation plays crucial roles in epigenetic regulation. The SET domain proteins are now recognized as generally having methyltransferase activity targeted to specific lysine residues of histones. The enzymes and their specific histone lysine methylation have enormous impacts on the regulation of chromatin structure and function. In this review, we discuss recent advances made on histone lysine methylations and their diverse functions in plant growth and development. [Copyright &y& Elsevier]

During leaf organogenesis, a critical step for normal leaf primordium initiation is the repression of the class 1 KNOTTED1-like homeobox ( KNOX) genes. After leaf primordia are formed, they must establish polarity for normal leaf morphogenesis. Recent studies have led to the identification of a number of genes that participate in the class 1 KNOX gene repression and/or the leaf polarity establishment. ASTMMETRIC LEAVES1 and 2 ( AS1 and AS2) are two of these genes, which are critical for both of these two processes. As a first step towards understanding the molecular genetic basis of the AS1–AS2 action, we determined the subcellular localizations of the two proteins in both tobacco BY2 cells and Arabidopsis plants, by fusing them to yellow/cyan fluorescent protein (YFP/CFP). Our data showed that AS1 and AS2 alone were predominantly localized in the nucleolus and the nucleoplasm, respectively. The presence of both AS1 and AS2 proteins in the same interphase cell demonstrated their co-localization in both nucleolus and nucleoplasm. In addition, AS1 alone was able to associate with the condensed chromosome in the metaphase cell. Our data suggest that AS1, AS2 and the AS1-AS2 protein complex may have distinct functions, which are all required for normal plant development. [ABSTRACT FROM AUTHOR]

Abstract: Methylation of histone H3 at lysine 9 (H3K9) is a critical step for heterochromatin formation, which plays essential roles in genome stability and gene regulation. SU(VAR)3-9 was the first H3K9 methyltransferase identified from mammals. In rice and Arabidopsis, each haploid genome contains 15 genes encoding proteins showing high sequence homology to SU(VAR)3-9, referred here as to SU(VAR)3-9 subfamily genes. Here, we report the characterization of three rice SU(VAR)3-9 subfamily genes, SDG704, SDG706 and SDG714. We show that SDG714 specifically methylates H3K9, whereas SDG704 and SDG706 do not show such activity in our assay conditions. All these three proteins are localized in the nucleus, with SDG704 and SDG714 being stably associated with chromatin during mitosis. Deletion analysis revealed that the C-terminus of SDG714 contains enzyme activity, and that both C- and N-termini of SDG704 and SDG714 are required for chromatin binding. Together, our results uncover a functional diversity for different rice SU(VAR)3-9 subfamily genes. [Copyright &y& Elsevier]

The Arabidopsis gene SERRATE ( SE) controls leaf development, meristem activity, inflorescence architecture and developmental phase transition. It has been suggested that SE, which encodes a C2H2 zinc finger protein, may change gene expression via chromatin modification. Recently, SE has also been shown to regulate specific microRNAs (miRNAs), miR165/166, and thus control shoot meristem function and leaf polarity. However, it remains unclear whether and how SE modulates specific miRNA processing. Here we show that the se mutant exhibits some similar developmental abnormalities as the hyponastic leaves1 ( hyl1) mutant. Since HYL1 is a nuclear double-stranded RNA-binding protein acting in the DICER-LIKE1 (DCL1) complex to regulate the first step of primary miRNA transcript (pri-miRNA) processing, we hypothesized that SE could play a previously unrecognized and general role in miRNA processing. Genetic analysis supports that SE and HYL1 act in the same pathway to regulate plant development. Consistently, SE is critical for the accumulation of multiple miRNAs and the trans-acting small interfering RNA (ta-siRNA), but is not required for sense post-transcriptional gene silencing. We further demonstrate that SE is localized in the nucleus and interacts physically with HYL1. Finally, we provide evidence that SE and HYL1 probably act with DCL1 in processing pri-miRNAs before HEN1 in miRNA biogenesis. In plants and animals, miRNAs are known to be processed in a stepwise manner from pri-miRNA. Our data strongly suggest that SE plays an important and general role in pri-miRNA processing, and it would be interesting to determine whether animal SE homologues may play similar roles in vivo. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented about the role of histone H3 lysine 36 (H3K36) methylation in promoting rice flowering.

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. [ABSTRACT FROM AUTHOR]

Abstract The original article [1] contains an error in spelling of author, Yanling Wang's name. The correct version can instead be viewed in this Correction article. [ABSTRACT FROM AUTHOR]

Background: An understanding of the mechanism underlying adipogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) will provide new therapeutic approaches for many diseases, including osteoporosis. This study aimed to investigate the role of miR-431 in adipogenic differentiation of hMSCs. Methods: hMSCs were induced for adipogenic differentiation and miR-431 was detected by polymerase chain reaction (PCR). hMSCs were transfected by miR-431 or small interfering RNA (siRNA) for insulin receptor substance 2 (IRS2). The expression of IRS2 was detected by PCR and Western blot analysis. The targeting of the 3′-untranslated region (UTR) of IRS2 by miR-431 was examined by luciferase assay. Results: miR-431 expression was decreased during adipogenesis of hMSCs. Overexpression of miR-431 inhibited adipogenic differentiation, accompanied by the downregulation of CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ), two key regulators of adipogenesis. Moreover, miR-431 decreased both protein and mRNA levels of IRS2. The expression of IRS2 was increased during adipogenic differentiation of hMSCs in conjunction with decreased levels of miR-431, and knockdown of IRS2 in hMSCs inhibited adipogenic differentiation. Luciferase assay confirmed that miR-431 targeted the 3′-UTR of IRS2 in hMSCs. Conclusions: This is the first study to show that miR-431 inhibits adipogenic differentiation of hMSCs via targeting IRS2. [ABSTRACT FROM AUTHOR]