Chromatin immunoprecipitation (ChIP) is a powerful tool for the characterization of covalent histone modifications and DNA-histone interactions in vivo. The procedure includes DNA-histone cross-linking in chromatin, shearing DNA into smaller fragments, immunoprecipitation with antibodies against the histone modifications of interest, followed by PCR identification of associated DNA sequences. In this protocol, we describe a simplified and optimized version of ChIP assay by reducing the number of experimental steps and isolation solutions and shortening preparation times. We include a nuclear isolation step before chromatin shearing, which provides a good yield of high-quality DNA resulting in at least 15 mug of DNA from each immunoprecipitated sample (from 0.2 to 0.4 g of starting tissue material) sufficient to test > or =25 genes of interest. This simpler and cost-efficient protocol has been applied for histone-modification studies of various Arabidopsis thaliana tissues and is easy to adapt for other systems as well.

ATX1 functions as an activator of homeotic genes, like Trithorax in animal systems. The histone methylating activity of the ATX1-SET domain argues that the molecular basis of these effects is the ability of ATX1 to modify chromatin structure. Our results suggest a conservation of trxG function between the animal and plant kingdoms despite the different structural nature of their targets.

Gene duplication followed by functional specialization is a potent force in the evolution of biological diversity. A comparative study of two highly conserved duplicated genes, ARABIDOPSIS TRITHORAX-LIKE PROTEIN1 (ATX1) and ATX2, revealed features of both partial redundancy and of functional divergence. Although structurally similar, their regulatory sequences have diverged, resulting in distinct temporal and spatial patterns of expression of the ATX1 and ATX2 genes. We found that ATX2 methylates only a limited fraction of nucleosomes and that ATX1 and ATX2 influence the expression of largely nonoverlapping gene sets. Even when coregulating shared targets, ATX1 and ATX2 may employ different mechanisms. Most remarkable is the divergence of their biochemical activities: both proteins methylate K4 of histone H3, but while ATX1 trimethylates it, ATX2 dimethylates it. ATX2 and ATX1 provide an example of separated K4 di from K4 trimethyltransferase activity.

The Arabidopsis homolog of trithorax, ATX1, regulates numerous functions in Arabidopsis beyond the homeotic genes. Here, we identified genome-wide targets of ATX1 and showed that ATX1 is a receptor for a lipid messenger, phosphatidylinositol 5-phosphate, PI5P. PI5P negatively affects ATX1 activity, suggesting a regulatory pathway connecting lipid-signaling with nuclear functions. We propose a model to illustrate how plants may respond to stimuli (external or internal) that elevate cellular PI5P levels by altering expression of ATX1-controlled genes.epigenetic regulation ͉ lipid signaling P roteins of the trithorax family activate the early homeotic genes that regulate animal development and embryonic pattern formation (1, 2). A major difference in the developmental process in plants is that organ formation is not restricted to the embryonic state, differentiation and organogenesis occurring throughout the lifespan of the organism. In plants, as in animals, homeosis is a consequence of a mutation of a homeotic gene. Usually, homeotic genes encode transcription factors. Unlike the animal counterparts, however, many of the plant homeotic genes belong to the MADSbox family (3). Despite the difference in structure, plant homeotic genes, like animal counterparts, are controlled by factors belonging to the trithorax family (4). Mutation of the Arabidopsis homolog of trithorax, ATX1, causes numerous developmental defects in the formation, placement, and identity of flower organs: Petals (second-whorl organs) were seen to develop stems, a third-whorl feature; stamens (third-whorl organs) developed ovules, a fourthwhorl characteristic (4).The signature feature of all trithorax proteins is the presence of the highly conserved SET [SuVar (3-9)-E(z)-trithorax] domain. The discovery that the SET domain peptides carry histone methyltransferase activity (5) provided critical evidence that chromatinmodifying activities function as epigenetic regulators. Certain lysines at the histone tails can be either acetylated or methylated, creating recognition sites for cellular repressive or activating complexes (6). SET domains of the trithorax family can methylate lysine 4 of histone H3, a modification associated with transcriptional activation (7). The SET domain of ATX1 has histone H3-K4 methyltransferase activity and can activate expression of Arabidopsis genes (4, 8). Thus, biochemical and genetic evidence define ATX1 as a functional homolog of the animal trithorax genes.Regulation of homeotic genes is only one possible role for trithorax (9, 10). In Arabidopsis, atx1 mutants displayed stem-, root-, and leaf-growth defects, indicating that the plant homolog of trithorax has pleiotropic roles (4). By whole genome expression profiling, we determined that Ϸ1,700 genes changed robust expression as a result of ATX1 loss of function. The altered expression of these genes provides a probable molecular basis underlying the pleiotropic functions of ATX1.The most important result of the study reported here is the finding that ATX1 can specifi...

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Covalent modifications of histone-tail amino acid residues communicate information via a specific ‘histone code’. Here, we report histone H3-tail lysine methylation profiles of several Arabidopsis genes in correlation with their transcriptional activity and the input of the epigenetic factor ARABIDOPSIS HOMOLOG OF TRITHORAX (ATX1) at ATX1-regulated loci. By chromatin immunoprecipitation (ChIP) assays, we compared modification patterns of a constitutively expressed housekeeping gene, of a tissue-specific gene, and among genes that differed in degrees of transcriptional activity. Our results suggest that the di-methylated isoform of histone H3-lysine4 (m2K4/H3) provide a general mark for gene-related sequences distinguishing them from non-transcribed regions. Lys-4 (K4/H3), lys-9 (K9/H3) and lys-27 (K27/H3) nucleosome methylation patterns of plant genes may be gene-, tissue- or development-regulated. Absence of nucleosomes from the LTP-promotor was not sufficient to provoke robust transcription in mutant atx1-leaf chromatin, suggesting that the mechanism repositioning nucleosomes at transition to flowering functioned independently of ATX1.

Tightly balanced antagonism between the Polycomb group (PcG) and the Trithorax group (TrxG) complexes maintain Hox expression patterns in Drosophila and murine model systems. Factors belonging to the PcG/TrxG complexes control various processes in plants as well but whether they participate in mechanisms that antagonize, balance or maintain each other's effects at a particular gene locus is unknown. CURLY LEAF (CLF), an Arabidopsis homolog of enhancer of zeste (EZ) and the ARABIDOPSIS HOMOLOG OF TRITHORAX (ATX1) control the expression of the flower homeotic gene AGAMOUS (AG). Disrupted ATX1 or CLF function results in misexpression of AG, recognizable phenotypes and loss of H3K4me3 or H3K27me3 histone H3-tail marks, respectively. A novel idea suggested by our results here, is that PcG and TrxG complexes function as a specific pair generating bivalent chromatin marks at the silent AG locus. Simultaneous loss of ATX1 and CLF restored AG repression and normalized leaf phenotypes. At the molecular level, disrupted ATX1 and CLF functions did not lead to erasure of the CLF- and ATX1-generated epigenetic marks, as expected: instead, in the double mutants, H3K27me3 and H3K4me3 tags were partially restored. We demonstrate that ATX1 and CLF physically interact linking mechanistically the observed effects.

To survive in adverse conditions, plants have evolved complex mechanisms that “prime” their defense system to respond and adapt to stresses. Their competence to respond to such stresses fundamentally depends on its capacity to modulate the transcriptome rapidly and specifically. Thus, chromatin dynamics is a mechanism linked to transcriptional regulation and enhanced defense in plants. For example, in Arabidopsis, priming of the SA-dependent defense pathway is linked to histone lysine methylation. Such modifications could create a memory of the primary infection that is associated with an amplified gene response upon exposure to a second stress-stimulus. In addition, the priming status of a plant for induced resistance can be inherited to its offspring. However, analyses on the molecular mechanisms of generational and transgenerational priming in the common bean (Phaseolus vulagris L.), an economically important crop, are absent. Here, we provide evidence that resistance to P. syringae pv. phaseolicola infection was induced in the common bean with the synthetic priming activators BABA and INA. Resistance was assessed by evaluating symptom appearance, pathogen accumulation, changes in gene expression of defense genes, as well as changes in the H3K4me3 and H3K36me3 marks at the promoter-exon regions of defense-associated genes. We conclude that defense priming in the common bean occurred in response to BABA and INA and that these synthetic activators primed distinct genes for enhanced disease resistance. We hope that an understanding of the molecular changes leading to defense priming and pathogen resistance will provide valuable knowledge for producing disease-resistant crop varieties by exposing parental plants to priming activators, as well as to the development of novel plant protection chemicals that stimulate the plant's inherent disease resistance mechanisms.