Dimerization of the ETO1 family proteins plays a crucial role in regulating ethylene biosynthesis in Arabidopsis thaliana.

ETHYLENE OVERPRODUCER1 (ETO1), ETO1-LIKE1 (EOL1), and EOL2 are members of the Broad complex, Tramtrack, Bric-a-brac (BTB) protein family that collectively regulate type-2 1-aminocyclopropane-1-carboxylic acid synthase (ACS) activity in Arabidopsis thaliana. Although ETO1 and EOL1/EOL2 encode structurally related proteins, genetic studies suggest that they do not play an equivalent role in regulating ethylene biosynthesis. The mechanistic details underlying the genetic analysis remain elusive. In this study, we reveal that ETO1 collaborates with EOL1/2 to play a key role in the regulation of type-2 ACS activity via protein-protein interactions. ETO1, EOL1, and EOL2 exhibit overlapping but distinct tissue-specific expression patterns. Nevertheless, neither EOL1 nor EOL2 can fully complement the eto1 phenotype under control of the ETO1 promoter, which suggests differential functions of ETO1 and EOL1/EOL2. ETO1 forms homodimers with itself and heterodimers with EOLs. Furthermore, CULLIN3 (CUL3) interacts preferentially with ETO1. The BTB domain of ETO1 is sufficient for interaction with CUL3 and is required for homodimerization. However, domain-swapping analysis in transgenic Arabidopsis suggests that the BTB domain of ETO1 is essential but not sufficient for a full spectrum of ETO1 function. The missense mutation in eto1-5 generates a substitution of phenylalanine with an isoleucine in ETO1F466I that impairs its dimerization and interaction with EOLs but does not affect binding to CUL3 or ACS5. Overexpression of ETO1F466I in Arabidopsis results in a constitutive triple response phenotype in dark-grown seedlings. Our findings reveal the mechanistic role of protein-protein interactions of ETO1 and EOL1/EOL2 that is crucial for their biological function in ethylene biosynthesis.

Arabidopsis JMJ17 promotes cotyledon greening during de-etiolation by repressing genes involved in tetrapyrrole biosynthesis in etiolated seedlings.

Arabidopsis histone H3 lysine 4 (H3K4) demethylases play crucial roles in several developmental processes, but their involvement in seedling establishment remain unexplored. Here, we show that Arabidopsis JUMONJI DOMAIN-CONTAINING PROTEIN17 (JMJ17), an H3K4me3 demethylase, is involved in cotyledon greening during seedling establishment. Dark-grown seedlings of jmj17 accumulated a high concentration of protochlorophyllide, an intermediate metabolite in the tetrapyrrole biosynthesis (TPB) pathway that generates chlorophyll (Chl) during photomorphogenesis. Upon light irradiation, jmj17 mutants displayed decreased cotyledon greening and reduced Chl level compared with the wild-type; overexpression of JMJ17 completely rescued the jmj17-5 phenotype. Transcriptomics analysis uncovered that several genes encoding key enzymes involved in TPB were upregulated in etiolated jmj17 seedlings. Consistently, chromatin immunoprecipitation-quantitative PCR revealed elevated H3K4me3 level at the promoters of target genes. Chromatin association of JMJ17 was diminished upon light exposure. Furthermore, JMJ17 interacted with PHYTOCHROME INTERACTING FACTOR1 in the yeast two-hybrid assay. JMJ17 binds directly to gene promoters to demethylate H3K4me3 to suppress PROTOCHLOROPHYLLIDE OXIDOREDUCTASE C expression and TPB in the dark. Light results in de-repression of gene expression to modulate seedling greening during de-etiolation. Our study reveals a new role for histone demethylase JMJ17 in controlling cotyledon greening in etiolated seedlings during the dark-to-light transition.

ETHYLENE RESPONSE FACTOR 96 positively regulates Arabidopsis resistance to necrotrophic pathogens by direct binding to GCC elements of jasmonate - and ethylene-responsive defence genes.

The ERF (ethylene responsive factor) family is composed of transcription factors (TFs) that are critical for appropriate Arabidopsis thaliana responses to biotic and abiotic stresses. Here we identified and characterized a member of the ERF TF group IX, namely ERF96, that when overexpressed enhances Arabidopsis resistance to necrotrophic pathogens such as the fungus Botrytis cinerea and the bacterium Pectobacterium carotovorum. ERF96 is jasmonate (JA) and ethylene (ET) responsive and ERF96 transcripts accumulation was abolished in JA-insensitive coi1-16 and in ET-insensitive ein2-1 mutants. Protoplast transactivation and electrophoresis mobility shift analyses revealed that ERF96 is an activator of transcription that binds to GCC elements. In addition, ERF96 mainly localized to the nucleus. Microarray analysis coupled to chromatin immunoprecipitation-PCR of Arabidopsis overexpressing ERF96 revealed that ERF96 enhances the expression of the JA/ET defence genes PDF1.2a, PR-3 and PR-4 as well as the TF ORA59 by direct binding to GCC elements present in their promoters. While ERF96-RNAi plants demonstrated wild-type resistance to necrotrophic pathogens, basal PDF1.2 expression levels were reduced in ERF96-silenced plants. This work revealed ERF96 as a key player of the ERF network that positively regulates the Arabidopsis resistance response to necrotrophic pathogens.

The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1.

The dynamics of histone methylation have emerged as an important issue since the identification of histone demethylases. We studied the regulatory function of Rph1/KDM4 (lysine demethylase), a histone H3K36 demethylase, on transcription in Saccharomyces cerevisiae. Overexpression of Rph1 reduced the expression of PHR1 and increased UV sensitivity. The catalytically deficient mutant (H235A) of Rph1 diminished the repressive transcriptional effect on PHR1 expression, which indicates that histone demethylase activity contributes to transcriptional repression. Chromatin immunoprecipitation analysis demonstrated that Rph1 was associated at the upstream repression sequence of PHR1 through zinc-finger domains and was dissociated after UV irradiation. Notably, overexpression of Rph1 and H3K36A mutant reduced histone acetylation at the URS, which implies a crosstalk between histone demethylation and acetylation at the PHR1 promoter. In addition, the crucial checkpoint protein Rad53 acted as an upstream regulator of Rph1 and dominated the phosphorylation of Rph1 that was required for efficient PHR1 expression and the dissociation of Rph1. The release of Rph1 from chromatin also required the phosphorylation at S652. Our study demonstrates that the histone demethylase Rph1 is associated with a specific chromatin locus and modulates histone modifications to repress a DNA damage responsive gene under control of damage checkpoint signaling.

CHITINASE LIKE1 Regulates Root Development of Dark-Grown Seedlings by Modulating Ethylene Biosynthesis in Arabidopsis thaliana.

The plant hormone ethylene plays a regulatory role in development in light- and dark-grown seedlings. We previously isolated a group of small-molecule compounds with a quinazolinone backbone, which were named acsinones (for ACC synthase inhibitor quinazolinones), that act as uncompetitive inhibitors of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). Thus, the triple response phenotype, which consists of shortened hypocotyls and roots, radial swelling of hypocotyls and exaggerated curvature of apical hooks, was suppressed by acsinones in dark-grown (etiolated) ethylene overproducer (eto) seedlings. Here, we describe our isolation and characterization of an Arabidopsis revert to eto1 9 (ret9) mutant, which showed reduced sensitivity to acsinones in etiolated eto1 seedlings. Map-based cloning of RET9 revealed an amino acid substitution in CHITINASE LIKE1 (CTL1), which is required for cell wall biogenesis and stress resistance in Arabidopsis. Etiolated seedlings of ctl1ret9 showed short hypocotyls and roots, which were augmented in combination with eto1-4. Consistently, ctl1ret9 seedlings showed enhanced sensitivity to exogenous ACC to suppress primary root elongation as compared with the wild type. After introducing ctl1ret9 to mutants completely insensitive to ethylene, genetic analysis indicated that an intact ethylene response pathway is essential for the alterations in root and apical hook but not hypocotyl in etiolated ctl1ret9 seedlings. Furthermore, a mild yet significantly increased ethylene level in ctl1 mutants was related to elevated mRNA level and activity of ACC oxidase (ACO). Moreover, genes associated with ethylene biosynthesis (ACO1 and ACO2) and response (ERF1 and EDF1) were upregulated in etiolated ctl1ret9 seedlings. By characterizing a new recessive allele of CTL1, we reveal that CTL1 negatively regulates ACO activity and the ethylene response, which thus contributes to understanding a role for ethylene in root elongation in response to perturbed cell wall integrity.

Structure and dimerization of the kinase domain from yeast Snf1, a member of the Snf1/AMPK protein family.

The Snf1/AMPK kinases are intracellular energy sensors, and the AMPK pathway has been implicated in a variety of metabolic human disorders. Here we report the crystal structure of the kinase domain from yeast Snf1, revealing a bilobe kinase fold with greatest homology to cyclin-dependant kinase-2. Unexpectedly, the crystal structure also reveals a novel homodimer that we show also forms in solution, as demonstrated by equilibrium sedimentation, and in yeast cells, as shown by coimmunoprecipitation of differentially tagged intact Snf1. A mapping of sequence conservation suggests that dimer formation is a conserved feature of the Snf1/AMPK kinases. The conformation of the conserved alphaC helix, and the burial of the activation segment and substrate binding site within the dimer, suggests that it represents an inactive form of the kinase. Taken together, these studies suggest another layer of kinase regulation within the Snf1/AMPK family, and an avenue for development of AMPK-specific activating compounds.

The Histone Acetyltransferase Gcn5 Regulates ncRNA-ICR1 and FLO11 Expression during Pseudohyphal Development in Saccharomyces cerevisiae.

Filamentous growth is one of the key features of pathogenic fungi during the early infectious phase. The pseudohyphal development of yeast Saccharomyces cerevisiae shares similar characteristics with hyphae elongation in pathogenic fungi. The expression of FLO11 is essential for adhesive growth and filament formation in yeast and is governed by a multilayered transcriptional network. Here we discovered a role for the histone acetyltransferase general control nonderepressible 5 (Gcn5) in regulating FLO11-mediated pseudohyphal growth. The expression patterns of FLO11 were distinct in haploid and diploid yeast under amino acid starvation induced by 3-amino-1,2,4-triazole (3AT). In diploids, FLO11 expression was substantially induced at a very early stage of pseudohyphal development and decreased quickly, but in haploids, it was gradually induced. Furthermore, the transcription factor Gcn4 was recruited to the Sfl1-Flo8 toggle sites at the FLO11 promoter under 3AT treatment. Moreover, the histone acetylase activity of Gcn5 was required for FLO11 induction. Finally, Gcn5 functioned as a negative regulator of the noncoding RNA ICR1, which is known to suppress FLO11 expression. Gcn5 plays an important role in the regulatory network of FLO11 expression via Gcn4 by downregulating ICR1 expression, which derepresses FLO11 for promoting pseudohyphal development.

Transcriptional consequence and impaired gametogenesis with high-grade aneuploidy in Arabidopsis thaliana.

Aneuploidy features a numerical chromosome variant that the number of chromosomes in the nucleus of a cell is not an exact multiple of the haploid number, which may have an impact on morphology and gene expression. Here we report a tertiary trisomy uncovered by characterizing a T-DNA insertion mutant (aur2-1/+) in the Arabidopsis (Arabidopsis thaliana) AURORA2 locus. Whole-genome analysis with DNA tiling arrays revealed a chromosomal translocation linked to the aur2-1 allele, which collectively accounted for a tertiary trisomy 2. Morphologic, cytogenetic and genetic analyses of aur2-1 progeny showed impaired male and female gametogenesis to various degrees and a tight association of the aur2-1 allele with the tertiary trisomy that was preferentially inherited. Transcriptome analysis showed overlapping and distinct gene expression profiles between primary and tertiary trisomy 2 plants, particularly genes involved in response to stress and various types of external and internal stimuli. Additionally, transcriptome and gene ontology analyses revealed an overrepresentation of nuclear-encoded organelle-related genes functionally involved in plastids, mitochondria and peroxisomes that were differentially expressed in at least three if not all Arabidopsis trisomics. These observations support a previous hypothesis that aneuploid cells have higher energy requirement to overcome the detrimental effects of an unbalanced genome. Moreover, our findings extend the knowledge of the complex nature of the T-DNA insertion event influencing plant genomic integrity by creating high-grade trisomy. Finally, gene expression profiling results provide useful information for future research to compare primary and tertiary trisomics for the effects of aneuploidy on plant cell physiology.

Dissociation of the H3K36 demethylase Rph1 from chromatin mediates derepression of environmental stress-response genes under genotoxic stress in Saccharomyces cerevisiae.

Cells respond to environmental signals by altering gene expression through transcription factors. Rph1 is a histone demethylase containing a Jumonji C (JmjC) domain and belongs to the C(2)H(2) zinc-finger protein family. Here we investigate the regulatory network of Rph1 in yeast by expression microarray analysis. More than 75% of Rph1-regulated genes showed increased expression in the rph1-deletion mutant, suggesting that Rph1 is mainly a transcriptional repressor. The binding motif 5'-CCCCTWA-3', which resembles the stress response element, is overrepresented in the promoters of Rph1-repressed genes. A significant proportion of Rph1-regulated genes respond to DNA damage and environmental stress. Rph1 is a labile protein, and Rad53 negatively modulates Rph1 protein level. We find that the JmjN domain is important in maintaining protein stability and the repressive effect of Rph1. Rph1 is directly associated with the promoter region of targeted genes and dissociated from chromatin before transcriptional derepression on DNA damage and oxidative stress. Of interest, the master stress-activated regulator Msn2 also regulates a subset of Rph1-repressed genes under oxidative stress. Our findings confirm the regulatory role of Rph1 as a transcriptional repressor and reveal that Rph1 might be a regulatory node connecting different signaling pathways responding to environmental stresses.

A chemical genetics approach reveals a role of brassinolide and cellulose synthase in hypocotyl elongation of etiolated Arabidopsis seedlings.

The development of juvenile seedlings after germination is critical for the initial establishment of reproductive plants. Ethylene plays a pivotal role in the growth of seedlings under light or dark during early development. Previously, we identified small molecules sharing a quinazolinone backbone that suppressed the constitutive triple response phenotype in dark-grown eto1-4 seedlings. We designated these small molecules as ACSinhibitor quinazolinones (acsinones), which were uncompetitive inhibitors of 1-aminocyclopropane-1-carboxylic acid synthase. To explore the additional roles of acsinones in plants, we screened and identified 19 Arabidopsis mutants with reduced sensitivity to acsinone7303, which were collectively named revert to eto1 (ret) because of their recovery of the eto1 phenotype. A map-based cloning approach revealed that CELLULOSE SYNTHASE6 (CESA6) and DE-ETIOLATED2 (DET2) were mutated in ret8 (cesa6(ret8);eto1-4) and ret41 (det2(ret41);eto1-5), respectively. Etiolated seedlings of both ret8 and ret41 exhibit short hypocotyls and roots. Ethylene levels were similar in etiolated cesa6(ret8) and det2-1 and in eto1 mutants treated with acsinone7303. Chemical inhibitors of ethylene biosynthesis and perception did not significantly suppress the etiolated phenotype of cesa6(ret8) and det2(ret41). However, together with eto1, cesa6(ret8) and det2(ret41) exhibited an enhanced phenotype in the hypocotyls and apical hooks of etiolated seedlings. These results confirm that, in addition to ethylene, cellulose synthesis and brassinolides can independently contribute to modulate hypocotyl development in young seedlings.

Identification of histone demethylases in Saccharomyces cerevisiae.

Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Delta, gis1Delta, rph1Delta, jhd1Delta, and jhd2Delta (yjr119cDelta)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Delta. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Delta and jhd2Delta strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Delta and jhd1Delta strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.

Histone H3 phosphorylation can promote TBP recruitment through distinct promoter-specific mechanisms.

Histone phosphorylation influences transcription, chromosome condensation, DNA repair and apoptosis. Previously, we showed that histone H3 Ser10 phosphorylation (pSer10) by the yeast Snf1 kinase regulates INO1 gene activation in part via Gcn5/SAGA complex-mediated Lys14 acetylation (acLys14). How such chromatin modification patterns develop is largely unexplored. Here we examine the mechanisms surrounding pSer10 at INO1, and at GAL1, which herein is identified as a new regulatory target of Snf1/pSer10. Snf1 behaves as a classic coactivator in its recruitment by DNA-bound activators, and in its role in modifying histones and recruiting TATA-binding protein (TBP). However, one important difference in Snf1 function in vivo at these promoters is that SAGA recruitment at INO1 requires histone phosphorylation via Snf1, whereas at GAL1, SAGA recruitment is independent of histone phosphorylation. In addition, the GAL1 activator physically interacts with both Snf1 and SAGA, whereas the INO1 activator interacts only with Snf1. Thus, at INO1, pSer10's role in recruiting SAGA may substitute for recruitment by DNA-bound activator. Our results emphasize that histone modifications share general functions between promoters, but also acquire distinct roles tailored for promoter-specific requirements.

Structural basis for histone and phosphohistone binding by the GCN5 histone acetyltransferase.

Distinct posttranslational modifications on histones occur in specific patterns to mediate certain chromosomal events. For example, on histone H3, phosphorylation at Ser10 can enhance GCN5-mediated Lys14 acetylation to promote transcription. To gain insight into the mechanism underlying this synergism, we determined the structure of Tetrahymena GCN5 (tGCN5) and coenzyme A (CoA) bound to unmodified and Ser10-phosphorylated 19 residue histone H3 peptides (H3p19 and H3p19Pi, respectively). The tGCN5/CoA/H3p19 structure reveals that a 12 amino acid core sequence mediates extensive contacts with the protein, providing the structural basis for substrate specificity by the GCN5/PCAF family of histone acetyltransferases. Comparison with the tGCN5/CoA/H3p19Pi structure reveals that phospho-Ser10 and Thr11 mediate significant histone-protein interactions, and nucleate additional interactions distal to the phosphorylation site. Functional studies show that histone H3 Thr11 is necessary for optimal transcription at yGcn5-dependent promoters requiring Ser10 phosphorylation. Together, these studies reveal how one histone modification can modulate another to affect distinct transcriptional signals.

Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8.

Gene activation and repression regulated by acetylation and deacetylation represent a paradigm for the function of histone modifications. We provide evidence that, in contrast, histone H2B monoubiquitylation and its deubiquitylation are both involved in gene activation. Substitution of the H2B ubiquitylation site at Lys 123 (K123) lowered transcription of certain genes regulated by the acetylation complex SAGA. Gene-associated H2B ubiquitylation was transient, increasing early during activation, and then decreasing coincident with significant RNA accumulation. We show that Ubp8, a component of the SAGA acetylation complex, is required for SAGA-mediated deubiquitylation of histone H2B in vitro. Loss of Ubp8 in vivo increased both gene-associated and overall cellular levels of ubiquitylated H2B. Deletion of Ubp8 lowered transcription of SAGA-regulated genes, and the severity of this defect was exacerbated by codeletion of the Gcn5 acetyltransferase within SAGA. In addition, disruption of either ubiquitylation or Ubp8-mediated deubiquitylation of H2B resulted in altered levels of gene-associated H3 Lys 4 methylation and Lys 36 methylation, which have both been linked to transcription. These results suggest that the histone H2B ubiquitylation state is dynamic during transcription, and that the sequence of histone modifications helps to control transcription.