RESUMO
BACKGROUND: Jasmonates (JAs) mediate trade-off between responses to both biotic and abiotic stress and growth in plants. The Arabidopsis thaliana HISTONE DEACETYLASE 6 is part of the CORONATINE INSENSITIVE1 receptor complex, co-repressing the HDA6/COI1-dependent acetic acid-JA pathway that confers plant drought tolerance. The decrease in HDA6 binding to target DNA mirrors histone H4 acetylation (H4Ac) changes during JA-mediated drought response, and mutations in HDA6 also cause depletion in the constitutive repressive marker H3 lysine 27 trimethylation (H3K27me3). However, the genome-wide effect of HDA6 on H4Ac and much of the impact of JAs on histone modifications and chromatin remodelling remain elusive. RESULTS: We performed high-throughput ChIP-Seq on the HDA6 mutant, axe1-5, and wild-type plants with or without methyl jasmonate (MeJA) treatment to assess changes in active H4ac and repressive H3K27me3 histone markers. Transcriptional regulation was investigated in parallel by microarray analysis in the same conditions. MeJA- and HDA6-dependent histone modifications on genes for specialized metabolism; linolenic acid and phenylpropanoid pathways; and abiotic and biotic stress responses were identified. H4ac and H3K27me3 enrichment also differentially affects JAs and HDA6-mediated genome integrity and gene regulatory networks, substantiating the role of HDA6 interacting with specific families of transposable elements in planta and highlighting further specificity of action as well as novel targets of HDA6 in the context of JA signalling for abiotic and biotic stress responses. CONCLUSIONS: The findings demonstrate functional overlap for MeJA and HDA6 in tuning plant developmental plasticity and response to stress at the histone modification level. MeJA and HDA6, nonetheless, maintain distinct activities on histone modifications to modulate genetic variability and to allow adaptation to environmental challenges.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Desacetilase 6 de Histona , Acetilação , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , MetilaçãoRESUMO
Epigenetic variations of phenotypes, especially those associated with DNA methylation, are often inherited over multiple generations in plants. The active and inactive chromatin states are heritable and can be maintained or even be amplified by positive feedback in a transgenerational manner. However, mechanisms controlling the transgenerational DNA methylation dynamics are largely unknown. As an approach to understand the transgenerational dynamics, we examined long-term effect of impaired DNA methylation in Arabidopsis mutants of the chromatin remodeler gene DDM1 (Decrease in DNA Methylation 1) through whole genome DNA methylation sequencing. The ddm1 mutation induces a drastic decrease in DNA methylation of transposable elements (TEs) and repeats in the initial generation, while also inducing ectopic DNA methylation at hundreds of loci. Unexpectedly, this ectopic methylation can only be seen after repeated self-pollination. The ectopic cytosine methylation is found primarily in the non-CG context and starts from 3' regions within transcription units and spreads upstream. Remarkably, when chromosomes with reduced DNA methylation were introduced from a ddm1 mutant into a DDM1 wild-type background, the ddm1-derived chromosomes also induced analogous de novo accumulation of DNA methylation in trans. These results lead us to propose a model to explain the transgenerational DNA methylation redistribution by genome-wide negative feedback. The global negative feedback, together with local positive feedback, would ensure robust and balanced differentiation of chromatin states within the genome.
Assuntos
Proteínas de Arabidopsis/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Retroalimentação Fisiológica , Genoma de Planta , Fatores de Transcrição/genética , Arabidopsis , Proteínas de Arabidopsis/biossíntese , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Citosina , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/biossíntese , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Mutação , Fatores de Transcrição/biossínteseRESUMO
Exposure to short-term cold stress delays flowering by activating the floral repressor FLOWERING LOCUS C (FLC) in Arabidopsis thaliana. The cold signaling attenuator HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 (HOS1) negatively regulates cold responses. Notably, HOS1-deficient mutants exhibit early flowering, and FLC expression is suppressed in the mutants. However, it remains unknown how HOS1 regulates FLC expression. Here, we show that HOS1 induces FLC expression by antagonizing the actions of FVE and its interacting partner histone deacetylase 6 (HDA6) under short-term cold stress. HOS1 binds to FLC chromatin in an FVE-dependent manner, and FVE is essential for the HOS1-mediated activation of FLC transcription. HOS1 also interacts with HDA6 and inhibits the binding of HDA6 to FLC chromatin. Intermittent cold treatments induce FLC expression by activating HOS1, which attenuates the activity of HDA6 in silencing FLC chromatin, and the effects of intermittent cold are diminished in hos1 and fve mutants. These observations indicate that HOS1 acts as a chromatin remodeling factor for FLC regulation under short-term cold stress.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Transporte/metabolismo , Montagem e Desmontagem da Cromatina/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Domínio MADS/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Núcleo Celular/genética , Cromatina/metabolismo , Temperatura Baixa , Flores/genética , Regulação da Expressão Gênica de Plantas , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Domínio MADS/genética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Plantas Geneticamente Modificadas , Estresse Fisiológico , Fatores de Transcrição , Transcrição GênicaRESUMO
Heterochromatin silencing is pivotal for genome stability in eukaryotes. In Arabidopsis, a plant-specific mechanism called RNA-directed DNA methylation (RdDM) is involved in heterochromatin silencing. Histone deacetylase HDA6 has been identified as a component of such machineries; however, its endogenous targets and the silencing mechanisms have not been analyzed globally. In this study, we investigated the silencing mechanism mediated by HDA6. Genome-wide transcript profiling revealed that the loci silenced by HDA6 carried sequences corresponding to the RDR2-dependent 24-nt siRNAs, however their transcript levels were mostly unaffected in the rdr2 mutant. Strikingly, we observed significant overlap of genes silenced by HDA6 to those by the CG DNA methyltransferase MET1. Furthermore, regardless of dependence on RdDM pathway, HDA6 deficiency resulted in loss of heterochromatic epigenetic marks and aberrant enrichment for euchromatic marks at HDA6 direct targets, along with ectopic expression of these loci. Acetylation levels increased significantly in the hda6 mutant at all of the lysine residues in the H3 and H4 N-tails, except H4K16. Interestingly, we observed two different CG methylation statuses in the hda6 mutant. CG methylation was sustained in the hda6 mutant at some HDA6 target loci that were surrounded by flanking DNA-methylated regions. In contrast, complete loss of CG methylation occurred in the hda6 mutant at the HDA6 target loci that were isolated from flanking DNA methylation. Regardless of CG methylation status, CHG and CHH methylation were lost and transcriptional derepression occurred in the hda6 mutant. Furthermore, we show that HDA6 binds only to its target loci, not the flanking methylated DNA, indicating the profound target specificity of HDA6. We propose that HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1, possibly recruiting MET1 to specific loci, thus forming the foundation of silent chromatin structure for subsequent non-CG methylation.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Inativação Gênica , Heterocromatina/metabolismo , Histona Desacetilases/genética , Acetilação , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Loci Gênicos , Histona Desacetilases/metabolismo , Ligação ProteicaRESUMO
Jasmonate (JA) and ethylene (ET) are two major plant hormones that synergistically regulate plant development and tolerance to necrotrophic fungi. Both JA and ET induce the expression of several pathogenesis-related genes, while blocking either signaling pathway abolishes the induction of these genes by JA and ET alone or in combination. However, the molecular basis of JA/ET coaction and signaling interdependency is largely unknown. Here, we report that two Arabidopsis ET-stabilized transcription factors (EIN3 and EIL1) integrate ET and JA signaling in the regulation of gene expression, root development, and necrotrophic pathogen defense. Further studies reveal that JA enhances the transcriptional activity of EIN3/EIL1 by removal of JA-Zim domain (JAZ) proteins, which physically interact with and repress EIN3/EIL1. In addition, we find that JAZ proteins recruit an RPD3-type histone deacetylase (HDA6) as a corepressor that modulates histone acetylation, represses EIN3/EIL1-dependent transcription, and inhibits JA signaling. Our studies identify EIN3/EIL1 as a key integration node whose activation requires both JA and ET signaling, and illustrate transcriptional derepression as a common mechanism to integrate diverse signaling pathways in the regulation of plant development and defense.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Etilenos/metabolismo , Proteínas Nucleares/metabolismo , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Sequência de Bases , Botrytis/patogenicidade , DNA de Plantas/genética , Proteínas de Ligação a DNA , Histona Desacetilases/metabolismo , Modelos Biológicos , Proteínas Nucleares/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Acetic acid is a simple and universal compound found in various organisms. Recently, acetic acid was found to play an essential role in conferring tolerance to water deficit stress in plants. This novel mechanism of drought stress tolerance mediated by acetic acid via networks involving phytohormones, genes, and chromatin regulation has great potential for solving the global food crisis and preventing desertification caused by global warming. We highlight the functions of acetic acid in conferring tolerance to water deficit stress.
RESUMO
Histone acetylation ranks with DNA methylation as one of major epigenetic modifications in eukaryotes. Deacetylation of histone N-terminal tails is intimately correlated with gene silencing and heterochromatin formation. In Arabidopsis, histone deacetylase 6 (HDA6) is a well-studied histone deacetylase that functions in gene silencing. Recently, it has been reported that HDA6 cooperates with DNA methylation on its direct target locus in the gene silencing mechanism. HDA6 has the multifaceted role in regulation of genome maintenance, development and environmental stress responses in plants. Elucidation of HDA6 function provides important information for understanding of epigenetic regulation in plants. In this review, we highlight recent progress in elucidating the HDA6-mediated gene silencing mechanisms and deciphering the biological function of HDA6.
Assuntos
Meio Ambiente , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Histona Desacetilases/metabolismo , Desenvolvimento Vegetal , Estresse Fisiológico/genética , Plantas/genéticaRESUMO
Changes in chromatin status are correlated with gene regulation of biological processes such as development and stress responses in plants. In this study, we focused on the transition of chromatin status toward gene repression during the process of recovery from drought stress of drought-inducible genes (RD20, RD29A and AtGOLS2) and a rehydration-inducible gene (ProDH). In response to drought, RNA polymerase II was recruited on the drought-inducible genes and rapidly disappeared after rehydration, although mRNA levels of these genes were maintained to some degree after rehydration, suggesting that the transcriptional activities of these genes were rapidly inactivated by rehydration treatment. Histone H3K9ac was enriched by drought and rapidly removed from these regions by rehydration. In contrast, histone H3K4me3 was gradually decreased by rehydration but was maintained at low levels after rehydration, suggesting that H3K4me3 functions as an epigenetic mark of stress memory. These results show that the transcriptional activity and chromatin status are rapidly changed from an active to inactive mode during the recovery process. Our results demonstrate that histone modifications are correlated with the inactivation of drought-inducible genes during the recovery process by rehydration.
Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Cromatina/metabolismo , Secas , Estresse Fisiológico/genética , Acetilação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas/genética , Histonas/metabolismo , Lisina/metabolismo , Metilação , Modelos Biológicos , Nucleossomos/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Cytosine is methylated in both CpG and non-CpG contexts (mCG and mCH, respectively) in plant genomes. Although mCG and mCH are almost independent in regard to their "maintenance," recent studies uncovered crosstalk between them during their "establishment," which unexpectedly functions in both RNAi-dependent and -independent pathways. In addition, the importance of linker histone H1 and variants of histone H2A to DNA methylation dynamics is starting to be understood. We summarize these new aspects of mechanisms to generate DNA methylomes and discuss future prospects.
Assuntos
Metilação de DNA , Epigenoma , Metilação de DNA/genéticaRESUMO
Among hymenopteran insects, aculeate species such as bees, ants, and wasps have enlarged and morphologically elaborate mushroom bodies (MBs), a higher-order brain center in the insect, implying their relationship with the advanced behavioral traits of aculeate species. The molecular bases leading to the acquisition of complicated MB functions, however, remains unclear. We previously reported the constitutive and MB-preferential expression of an ecdysone-signaling related transcription factor, Mblk-1/E93, in the honey bee brain. Here, we searched for target genes of Mblk-1 in the worker honey bee MBs using chromatin immunoprecipitation sequence analyses and found that Mblk-1 targets several genes involved in synaptic plasticity, learning, and memory abilities. We also demonstrated that Mblk-1 expression is self-regulated via Mblk-1-binding sites, which are located upstream of Mblk-1. Furthermore, we showed that the number of the Mblk-1-binding motif located upstream of Mblk-1 homologs increased associated with evolution of hymenopteran insects. Our findings suggest that Mblk-1, which has been focused on as a developmental gene transiently induced by ecdysone, has acquired a novel expression pattern to play a role in synaptic plasticity in honey bee MBs, raising a possibility that molecular evolution of Mblk-1 may have partly contributed to the elaboration of MB function in insects.
Assuntos
Ecdisona , Corpos Pedunculados , Animais , Abelhas/genética , Corpos Pedunculados/metabolismo , Ecdisona/metabolismo , Fatores de Transcrição/metabolismo , Plasticidade Neuronal/genética , Regulação da Expressão Gênica , Encéfalo/metabolismoRESUMO
Transposable elements (TEs) are robustly silenced by multiple epigenetic marks, but dynamics of crosstalk among these marks remains enigmatic. In Arabidopsis, TEs are silenced by cytosine methylation in both CpG and non-CpG contexts (mCG and mCH) and histone H3 lysine 9 methylation (H3K9me). While mCH and H3K9me are mutually dependent for their maintenance, mCG and mCH/H3K9me are independently maintained. Here, we show that establishment, rather than maintenance, of mCH depends on mCG, accounting for the synergistic colocalization of these silent marks in TEs. When mCG is lost, establishment of mCH is abolished in TEs. mCG also guides mCH in active genes, though the resulting mCH/H3K9me is removed thereafter. Unexpectedly, targeting efficiency of mCH depends on relative, rather than absolute, levels of mCG within the genome, suggesting underlying global negative controls. We propose that local positive feedback in heterochromatin dynamics, together with global negative feedback, drive robust and balanced DNA methylome patterning.
Assuntos
Arabidopsis/genética , Metilação de DNA/fisiologia , Elementos de DNA Transponíveis/genética , Genoma de Planta/genética , Heterocromatina/metabolismo , Nucleotídeos de Citosina/metabolismo , Epigênese Genética/genética , Regulação da Expressão Gênica de Plantas/genética , Heterocromatina/genética , Histonas/metabolismoRESUMO
Many plants exhibit altered gene expression patterns in response to low nonfreezing temperatures and an increase in freezing tolerance in a phenomenon known as cold acclimation. Here we show, for the first time, that the histone deacetylase gene HDA6 is required for cold acclimation and freezing tolerance in Arabidopsis. HDA6 is transcriptionally upregulated during long-term cold treatment. Cold-treated hda6 mutants showed reduced freezing tolerance compared with the cold-treated wild-type plants. Freezing-caused electrolyte leakage increased in the cold-treated hda6 mutant. In contrast, the non-cold-treated hda6 mutants showed no significant difference in survivability and electrolyte leakage compared to wild-type plants. Transcriptome analysis identified the genes that showed aberrant expression in the hda6 mutant after cold acclimation. We conclude that HDA6 plays a critical role in regulating cold acclimation process that confers freezing resistance on Arabidopsis.
Assuntos
Aclimatação/genética , Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Congelamento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Histona Desacetilases/fisiologia , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Perfilação da Expressão Gênica , Histona Desacetilases/genética , Análise de Sequência com Séries de Oligonucleotídeos , Regulação para CimaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
DNA methylation is important for silencing transposable elements (TEs) in diverse eukaryotes, including plants. In plant genomes, TEs are silenced by methylation of histone H3 lysine 9 (H3K9) and cytosines in both CG and non-CG contexts. The role of RNA interference (RNAi) in establishing TE-specific silent marks has been extensively studied, but the importance of RNAi-independent pathways remains largely unexplored. Here, we directly investigated transgenerational de novo DNA methylation of TEs after the loss of silent marks. Our analyses uncovered potent and precise RNAi-independent pathways for recovering non-CG methylation and H3K9 methylation in most TE genes (that is, coding regions within TEs). Characterization of a subset of TE genes without the recovery revealed the effects of H3K9 demethylation, replacement of histone H2A variants and their interaction with CG methylation, together with feedback from transcription. These chromatin components are conserved among eukaryotes and may contribute to chromatin reprogramming in a conserved manner.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Metilação de DNA/genética , Elementos de DNA Transponíveis/genética , Inativação Gênica , Histonas/genética , Histonas/metabolismo , Interferência de RNA , Regulação da Expressão Gênica de Plantas , Genes de PlantasRESUMO
Nuclear genomes are always faced with the modification of themselves by insertions and integrations of foreign DNAs and intrinsic parasites such as transposable elements. There is also substantial number of integrations from symbiotic organellar genomes to their host nuclear genomes. Such integration might have acted as a beneficial mutation during the evolution of symbiosis, while most of them have more or less deleterious effects on the stability of current genomes. Here we report the pattern of DNA substitution and methylation on organellar DNA fragments integrated from plastid into plant nuclear genomes. The genome analyses of 17 plants show homology-dependent DNA substitution bias. A certain number of these sequences are DNA methylated in the nuclear genome. The intensity of DNA methylation also decays according to the increase of relative evolutionary times after being integrated into nuclear genomes. The methylome data of epigenetic mutants shows that the DNA methylation of organellar DNA fragments in nuclear genomes are mainly dependent on the methylation maintenance machinery, while other mechanisms may also affect on the DNA methylation level. The DNA methylation on organellar DNA fragments may contribute to maintaining the genome stability and evolutionary dynamics of symbiotic organellar and their host's genomes.
RESUMO
Post-translational modification of histone N-tails affects eukaryotic gene activity. In Arabidopsis, the histone modification level correlates with gene activation and repression in vernalization and flowering processes, but there is little information on changes in histone modification status and nucleosome structure under abiotic stresses. We determined the temporal and spatial changes in nucleosome occupancy and levels of H3K4me3, H3K9ac, H3K14ac, H3K23ac and H3K27ac in the histone H3 N-tail on the regions of four Arabidopsis drought stress-inducible genes, RD29A, RD29B, RD20 and At2g20880 [corrected], under drought stress conditions by chromatin immunoprecipitation analysis. We found two types of regulatory mechanisms of nucleosome occupancy function in the drought stress response. For RD29A and RD29B genes, nucleosome occupancy of promoter regions is low compared with that of coding regions, and no notable nucleosome loss occurs under drought stress. In contrast, nucleosome density is gradually decreased in response to drought stress on RD20 and At2g20880 [corrected] genes. Enrichments of H3K4me3 and H3K9ac correlate with gene activation in response to drought stress in all four genes. Interestingly, establishment of H3K4me3 occurs after accumulation of RNAPII on the coding regions of RD29A and At2g20880 [corrected]. Enrichment of H3K23ac and H3K27ac occurs in response to drought stress on the coding regions of RD29B, RD20 and At2g20880 [corrected], but not on the coding region of At2g20880 [corrected]. Our results indicate that histone modifications on the H3 N-tail are altered with gene activation on the coding regions of drought stress-responsive genes under drought stress conditions and that several patterns of nucleosome changes function in the drought stress response.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Histonas/metabolismo , Lisina/metabolismo , Acetilação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Imunoprecipitação da Cromatina , Desastres , Perfilação da Expressão Gênica , Metilação , Nucleossomos/metabolismo , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação TranscricionalRESUMO
Water deficit caused by global climate changes seriously endangers the survival of organisms and crop productivity, and increases environmental deterioration1,2. Plants' resistance to drought involves global reprogramming of transcription, cellular metabolism, hormone signalling and chromatin modification3-8. However, how these regulatory responses are coordinated via the various pathways, and the underlying mechanisms, are largely unknown. Herein, we report an essential drought-responsive network in which plants trigger a dynamic metabolic flux conversion from glycolysis into acetate synthesis to stimulate the jasmonate (JA) signalling pathway to confer drought tolerance. In Arabidopsis, the ON/OFF switching of this whole network is directly dependent on histone deacetylase HDA6. In addition, exogenous acetic acid promotes de novo JA synthesis and enrichment of histone H4 acetylation, which influences the priming of the JA signalling pathway for plant drought tolerance. This novel acetate function is evolutionarily conserved as a survival strategy against environmental changes in plants. Furthermore, the external application of acetic acid successfully enhanced the drought tolerance in Arabidopsis, rapeseed, maize, rice and wheat plants. Our findings highlight a radically new survival strategy that exploits an epigenetic switch of metabolic flux conversion and hormone signalling by which plants adapt to drought.
Assuntos
Acetatos/metabolismo , Arabidopsis/fisiologia , Secas , Aclimatação , Aldeído Oxirredutases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Epigênese Genética , Glicólise , Histona Desacetilases/metabolismo , Oxilipinas/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Piruvato Descarboxilase/metabolismo , Transdução de SinaisRESUMO
This corrects the article DOI: 10.1038/nplants.2017.97.
RESUMO
Gene activity is regulated via chromatin dynamics in eukaryotes. In plants, alterations of histone modifications are correlated with gene regulation for development, vernalization, and abiotic stress responses. Using ChIP, ChIP-on-chip, and ChIP-seq analyses, the direct binding regions of transcription factors and alterations of histone modifications can be identified on a genome-wide level. We have established reliable and reproducible ChIP and ChIP-on-chip methods that have been optimized for the Arabidopsis model system. These methods are not only useful for identifying the direct binding of transcription factors and chromatin status but also for scanning the regulatory network in Arabidopsis.