Distinct roles of the histone chaperones NAP1 and NRP and the chromatin‐remodeling factor INO80 in somatic homologous recombination in Arabidopsis thaliana

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 AtNAP1 genes and the two NRP genes in the sextuple mutant m123456‐1 barely affects normal plant growth and development. Interestingly, compared with the respective AtNAP1 (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 AtNAP1 and NRP act in parallel to synergistically promote somatic HR. Distinctively, the AtINO80 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 AtNAP1 synergistically promote HR upstream of AtINO80‐mediated chromatin remodeling after the formation of γ‐H2A.X foci during DNA damage repair.     

Growth habit determination by the balance of histone methylation activities in Arabidopsis

In Arabidopsis, the rapid‐flowering summer‐annual versus the vernalization‐requiring winter‐annual growth habit is determined by natural variation in FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). However, the biochemical basis of how FRI confers a winter‐annual habit remains elusive. Here, we show that FRI elevates FLC expression by enhancement of histone methyltransferase (HMT) activity. EARLY FLOWERING IN SHORT DAYS (EFS), which is essential for FRI function, is demonstrated to be a novel dual substrate (histone H3 lysine 4 (H3K4) and H3K36)‐specific HMT. FRI is recruited into FLC chromatin through EFS and in turn enhances EFS activity and engages additional HMTs. At FLC, the HMT activity of EFS is balanced by the H3K4/H3K36‐ and H3K4‐specific histone demethylase (HDM) activities of autonomous‐pathway components, RELATIVE OF EARLY FLOWERING 6 and FLOWERING LOCUS D, respectively. Loss of HDM activity in summer annuals results in dominant HMT activity, leading to conversion to a winter‐annual habit in the absence of FRI. Thus, our study provides a model of how growth habit is determined through the balance of the H3K4/H3K36‐specific HMT and HDM activities.     

A plant‐specific histone H3 lysine 4 demethylase represses the floral transition in Arabidopsis

Histone demethylation regulates chromatin structure and gene expression, and is catalyzed by various histone demethylases. Trimethylation of histone H3 at lysine 4 (H3K4) is coupled to active gene expression; trimethyl H3K4 is demethylated by Jumonj C (JmjC) domain‐containing demethylases in mammals. Here we report that a plant‐specific JmjC domain‐containing protein known as PKDM7B (At4g20400) demethylates trimethyl H3K4. PKDM7B mediates H3K4 demethylation in a key floral promoter, FLOWERING LOCUS T (FT), and an FT homolog, TWIN SISTER OF FT (TSF), and represses their expression to inhibit the floral transition in Arabidopsis. Our findings suggest that there are at least two distinct sub‐families of JmjC domain‐containing demethylases that demethylate the active trimethyl H3K4 mark in eukaryotic genes, and reveal a plant‐specific JmjC domain enzyme capable of H3K4 demethylation.     

Arabidopsis O‐GlcNAc transferase SEC activates histone methyltransferase ATX1 to regulate flowering

Post‐translational modification of proteins by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is catalyzed by O‐GlcNAc transferases (OGTs). O‐GlcNAc modification of proteins regulates multiple important biological processes in metazoans. However, whether protein O‐GlcNAcylation is involved in epigenetic processes during plant development is largely unknown. Here, we show that loss of function of SECRET AGENT (SEC), an OGT in Arabidopsis, leads to an early flowering phenotype. This results from reduced histone H3 lysine 4 trimethylation (H3K4me3) of FLOWERING LOCUS C (FLC) locus, which encodes a key negative regulator of flowering. SEC activates ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1), a histone lysine methyltransferase (HKMT), through O‐GlcNAc modification to augment ATX1‐mediated H3K4me3 histone modification at FLC locus. SEC transfers an O‐GlcNAc group on Ser947 of ATX1, which resides in the SET domain, thereby activating ATX1. Taken together, these results uncover a novel post‐translational O‐GlcNAc modification‐mediated mechanism for regulation of HKMT activity and establish the function of O‐GlcNAc signaling in epigenetic processes in plants.     

Brahma is required for proper expression of the floral repressor FLC in Arabidopsis

Background

BRAHMA (BRM) is a member of a family of ATPases of the SWI/SNF chromatin remodeling complexes from Arabidopsis. BRM has been previously shown to be crucial for vegetative and reproductive development.

Methodology/Principal Findings

Here we carry out a detailed analysis of the flowering phenotype of brm mutant plants which reveals that, in addition to repressing the flowering promoting genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), BRM also represses expression of the general flowering repressor FLOWERING LOCUS C (FLC). Thus, in brm mutant plants FLC expression is elevated, and FLC chromatin exhibits increased levels of histone H3 lysine 4 tri-methylation and decreased levels of H3 lysine 27 tri-methylation, indicating that BRM imposes a repressive chromatin configuration at the FLC locus. However, brm mutants display a normal vernalization response, indicating that BRM is not involved in vernalization-mediated FLC repression. Analysis of double mutants suggests that BRM is partially redundant with the autonomous pathway. Analysis of genetic interactions between BRM and the histone H2A.Z deposition machinery demonstrates that brm mutations overcome a requirement of H2A.Z for FLC activation suggesting that in the absence of BRM, a constitutively open chromatin conformation renders H2A.Z dispensable.

Conclusions/Significance

BRM is critical for phase transition in Arabidopsis. Thus, BRM represses expression of the flowering promoting genes CO, FT and SOC1 and of the flowering repressor FLC. Our results indicate that BRM controls expression of FLC by creating a repressive chromatin configuration of the locus.     

Regulation of flowering time by the histone deacetylase HDA5 in Arabidopsis

The acetylation level of histones on lysine residues regulated by histone acetyltransferases and histone deacetylases plays an important but under‐studied role in the control of gene expression in plants. With the aim of characterizing the Arabidopsis RPD3/HDA1 family histone deacetylase HDA5, we present evidence showing that HDA5 displays deacetylase activity. Mutants defective in the expression of HDA5 displayed a late‐flowering phenotype. Expression of the flowering repressor genes FLC and MAF1 was up‐regulated in hda5 mutants. Furthermore, the gene activation markers, histone H3 acetylation and H3K4 trimethylation on FLC and MAF1 chromatin were increased in hda5‐1 mutants. Chromatin immunoprecipitation analysis showed that HDA5 binds to the chromatin of FLC and MAF1. Bimolecular fluorescence complementation assays and co‐immunoprecipitation assays showed that HDA5 interacts with FVE, FLD and HDA6, indicating that these proteins are present in a protein complex involved in the regulation of flowering time. Comparing gene expression profiles of hda5 and hda6 mutants by RNA‐seq revealed that HDA5 and HDA6 co‐regulate gene expression in multiple development processes and pathways.     

Aldehyde dehydrogenase ALDH3F1 involvement in flowering time regulation through histone acetylation modulation on FLOWERING LOCUS C

Flowering time regulation is one of the most important processes in the whole life of flowering plants and FLOWERING LOCUS C (FLC) is a central repressor of flowering time. However, whether metabolic acetate level affects flowering time is unknown. Here we report that ALDEHYDE DEHYDROGENASE ALDH3F1 plays essential roles in floral transition via FLC‐dependent pathway. In the aldh3f1‐1 mutant, the flowering time was significant earlier than Col‐0 and the FLC expression level was reduced. ALDH3F1 had aldehyde dehydrogenase activity to affect the acetate level in plants, and the amino acids of E214 and C252 are essential for its catalytic activity. Moreover, aldh3f1 mutation reduced acetate level and the total acetylation on histone H3. The H3K9Ac level on FLC locus was decreased in aldh3f1‐1, which reduced FLC expression. Expression of ALDH3F1 could rescue the decreased H3K9Ac level on FLC, FLC expression and also the early‐flowering phenotype of aldh3f1‐1, however ALDH3F1^E214A or ALDH3F1^C252A could not. Our findings demonstrate that ALDH3F1 participates in flowering time regulation through modulating the supply of acetate for acetyl‐CoA, which functions as histone acetylation donor to modulate H3K9Ac on FLC locus.     

Requirement of histone acetyltransferases HAM1 and HAM2 for epigenetic modification of FLC in regulating flowering in Arabidopsis

Histone acetylation is an important posttranslational modification associated with gene activation. In Arabidopsis, two MYST histone acetyltransferases HAM1 and HAM2 work redundantly to acetylate histone H4 lysine 5 (H4K5ace) in vitro. The double mutant ham1/ham2 is lethal, which suggests the critical role of HAM1 and HAM2 in development. Here, we used an artificial microRNA (amiRNA) strategy in Arabidopsis to uncover a novel function of HAM1 and HAM2. The amiRNA-HAM1/2 transgenic plants showed early flowering and reduced fertility. In addition, they responded normally to photoperiod, gibberellic acid treatment, and vernalization. The expression of flowering-repressor FLOWERING LOCUS C (FLC) and its homologues, MADS-box Affecting Flowering genes 3/4 (MAF3/4), were decreased in amiRNA-HAM1/2 lines. HAM1 overexpression caused late flowering and elevated expression of FLC and MAF3/4. Mutation of FLC almost rescued the late flowering with HAM1 overexpression, which suggests that HAM1 regulation of flowering time depended on FLC. Global H4 acetylation was decreased in amiRNA-HAM1/2 lines, but increased in HAM1-OE lines, which further confirmed the acetyltransferase activity of HAM1 in vivo. Chromatin immunoprecipitation revealed that H4 hyperacetylation and H4K5ace at FLC and MAF3/4 were less abundant in amiRNA-HAM1/2 lines than the wild type, but were enriched in HAM1-OE lines. Thus, HAM1 and HAM2 may affect flowering time by epigenetic modification of FLC and MAF3/4 chromatins at H4K5 acetylation.