Histone Modification ChIP-seq on Arabidopsis thaliana Plantlets.

Characterizing the molecular mechanisms regulating gene expression is crucial for understanding the regulatory processes underlying physiological responses to environmental and developmental signals in eukaryotes. The covalent modification of histones contributes to the compaction levels of chromatin, as well as the recruitment of the transcriptional machinery to specific loci, facilitating metastable changes in gene activity. ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) has become the gold standard method for determining histone modification profiles among different organisms, tissues, and genotypes. In the current protocol, we describe a highly robust method for performing ChIP-seq of histone modifications in Arabidopsis thaliana plantlets. Besides its robustness, this method uses in-house-prepared buffers for chromatin extraction, immunoprecipitation, washing, and elusion, making it cost-effective in contrast to commercial kits.

Polycomb-dependent differential chromatin compartmentalization determines gene coregulation in Arabidopsis.

In animals, distant H3K27me3-marked Polycomb targets can establish physical interactions forming repressive chromatin hubs. In plants, growing evidence suggests that H3K27me3 acts directly or indirectly to regulate chromatin interactions, although how this histone modification modulates 3D chromatin architecture remains elusive. To decipher the impact of the dynamic deposition of H3K27me3 on the Arabidopsis thaliana nuclear interactome, we combined genetics, transcriptomics, and several 3D epigenomic approaches. By analyzing mutants defective for histone H3K27 methylation or demethylation, we uncovered the crucial role of this chromatin mark in short- and previously unnoticed long-range chromatin loop formation. We found that a reduction in H3K27me3 levels led to a decrease in the interactions within Polycomb-associated repressive domains. Regions with lower H3K27me3 levels in the H3K27 methyltransferase clf mutant established new interactions with regions marked with H3K9ac, a histone modification associated with active transcription, indicating that a reduction in H3K27me3 levels induces a global reconfiguration of chromatin architecture. Altogether, our results reveal that the 3D genome organization is tightly linked to reversible histone modifications that govern chromatin interactions. Consequently, nuclear organization dynamics shapes the transcriptional reprogramming during plant development and places H3K27me3 as a key feature in the coregulation of distant genes.

Wheat chromatin architecture is organized in genome territories and transcription factories.

BACKGROUND: Polyploidy is ubiquitous in eukaryotic plant and fungal lineages, and it leads to the co-existence of several copies of similar or related genomes in one nucleus. In plants, polyploidy is considered a major factor in successful domestication. However, polyploidy challenges chromosome folding architecture in the nucleus to establish functional structures. RESULTS: We examine the hexaploid wheat nuclear architecture by integrating RNA-seq, ChIP-seq, ATAC-seq, Hi-C, and Hi-ChIP data. Our results highlight the presence of three levels of large-scale spatial organization: the arrangement into genome territories, the diametrical separation between facultative and constitutive heterochromatin, and the organization of RNA polymerase II around transcription factories. We demonstrate the micro-compartmentalization of transcriptionally active genes determined by physical interactions between genes with specific euchromatic histone modifications. Both intra- and interchromosomal RNA polymerase-associated contacts involve multiple genes displaying similar expression levels. CONCLUSIONS: Our results provide new insights into the physical chromosome organization of a polyploid genome, as well as on the relationship between epigenetic marks and chromosome conformation to determine a 3D spatial organization of gene expression, a key factor governing gene transcription in polyploids.

GCN5 modulates salicylic acid homeostasis by regulating H3K14ac levels at the 5' and 3' ends of its target genes.

The modification of histones by acetyl groups has a key role in the regulation of chromatin structure and transcription. The Arabidopsis thaliana histone acetyltransferase GCN5 regulates histone modifications as part of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) transcriptional coactivator complex. GCN5 was previously shown to acetylate lysine 14 of histone 3 (H3K14ac) in the promoter regions of its target genes even though GCN5 binding did not systematically correlate with gene activation. Here, we explored the mechanism through which GCN5 controls transcription. First, we fine-mapped its GCN5 binding sites genome-wide and then used several global methodologies (ATAC-seq, ChIP-seq and RNA-seq) to assess the effect of GCN5 loss-of-function on the expression and epigenetic regulation of its target genes. These analyses provided evidence that GCN5 has a dual role in the regulation of H3K14ac levels in their 5' and 3' ends of its target genes. While the gcn5 mutation led to a genome-wide decrease of H3K14ac in the 5' end of the GCN5 down-regulated targets, it also led to an increase of H3K14ac in the 3' ends of GCN5 up-regulated targets. Furthermore, genome-wide changes in H3K14ac levels in the gcn5 mutant correlated with changes in H3K9ac at both 5' and 3' ends, providing evidence for a molecular link between the depositions of these two histone modifications. To understand the biological relevance of these regulations, we showed that GCN5 participates in the responses to biotic stress by repressing salicylic acid (SA) accumulation and SA-mediated immunity, highlighting the role of this protein in the regulation of the crosstalk between diverse developmental and stress-responsive physiological programs. Hence, our results demonstrate that GCN5, through the modulation of H3K14ac levels on its targets, controls the balance between biotic and abiotic stress responses and is a master regulator of plant-environmental interactions.

Plant Immunity: From Signaling to Epigenetic Control of Defense.

Pathogen recognition by plants results in the activation of signaling pathways that induce defense reactions. There is growing evidence indicating that epigenetic mechanisms directly participate in plant immune memory. Here, we discuss current knowledge of diverse epigenomic processes and elements, such as noncoding RNAs, DNA and RNA methylation, histone post-translational modifications, and chromatin remodeling, that have been associated with the regulation of immune responses in plants. Furthermore, we discuss the currently limited evidence of transgenerational inheritance of pathogen-induced defense priming, together with its potentials, challenges, and limitations for crop improvement and biotechnological applications.

Modify the Histone to Win the Battle: Chromatin Dynamics in Plant-Pathogen Interactions.

Relying on an immune system comes with a high energetic cost for plants. Defense responses in these organisms are therefore highly regulated and fine-tuned, permitting them to respond pertinently to the attack of a microbial pathogen. In recent years, the importance of the physical modification of chromatin, a highly organized structure composed of genomic DNA and its interacting proteins, has become evident in the research field of plant-pathogen interactions. Several processes, including DNA methylation, changes in histone density and variants, and various histone modifications, have been described as regulators of various developmental and defense responses. Herein, we review the state of the art in the epigenomic aspects of plant immunity, focusing on chromatin modifications, chromatin modifiers, and their physiological consequences. In addition, we explore the exciting field of understanding how plant pathogens have adapted to manipulate the plant epigenomic regulation in order to weaken their immune system and thrive in their host, as well as how histone modifications in eukaryotic pathogens are involved in the regulation of their virulence.

The Arabidopsis SWI/SNF protein BAF60 mediates seedling growth control by modulating DNA accessibility.

BACKGROUND: Plant adaptive responses to changing environments involve complex molecular interplays between intrinsic and external signals. Whilst much is known on the signaling components mediating diurnal, light, and temperature controls on plant development, their influence on chromatin-based transcriptional controls remains poorly explored. RESULTS: In this study we show that a SWI/SNF chromatin remodeler subunit, BAF60, represses seedling growth by modulating DNA accessibility of hypocotyl cell size regulatory genes. BAF60 binds nucleosome-free regions of multiple G box-containing genes, opposing in cis the promoting effect of the photomorphogenic and thermomorphogenic regulator Phytochrome Interacting Factor 4 (PIF4) on hypocotyl elongation. Furthermore, BAF60 expression level is regulated in response to light and daily rhythms. CONCLUSIONS: These results unveil a short path between a chromatin remodeler and a signaling component to fine-tune plant morphogenesis in response to environmental conditions.

Draft Genome Sequence of Plant Growth-Promoting Micrococcus luteus Strain K39 Isolated from Cyperus conglomeratus in Saudi Arabia.

Micrococcus luteus strain K39 is an endophyte bacterium isolated from roots of the desert plant Cyperus conglomeratus collected from the Red Sea shore, Thuwal, Saudi Arabia. The draft genome sequence of strain K39 revealed a number of enzymes involved in salinity and oxidative stress tolerance or having herbicide-resistance activity.

Draft Genome Sequence of Halomonas elongata Strain K4, an Endophytic Growth-Promoting Bacterium Enhancing Salinity Tolerance In Planta.

Halomonas elongata strain K4 is an endophytic bacterial strain that was isolated from roots of Cyperus conglomeratus collected at the Red Sea coast in Thuwal, Saudi Arabia. Here, we present a draft genome sequence of this strain, highlighting a number of pathways involved in plant growth promotion under salt stress.

Put your 3D glasses on: plant chromatin is on show.

The three-dimensional organization of the eukaryotic nucleus and its chromosomal conformation have emerged as important features in the complex network of mechanisms behind gene activity and genome connectivity dynamics, which can be evidenced in the regionalized chromosomal spatial distribution and the clustering of diverse genomic regions with similar expression patterns. The development of chromatin conformation capture (3C) techniques has permitted the elucidation of commonalities between the eukaryotic phyla, as well as important differences among them. The growing number of studies in the field performed in plants has shed light on the structural and regulatory features of these organisms. For instance, it has been proposed that plant chromatin can be arranged into different conformations such as Rabl, Rosette-like, and Bouquet, and that both short- and long-range chromatin interactions occur in Arabidopsis. In this review, we compile the current knowledge about chromosome architecture characteristics in plants, as well as the molecular events and elements (including long non-coding RNAs, histone and DNA modifications, chromatin remodeling complexes, and transcription factors) shaping the genome three-dimensional conformation. Furthermore, we discuss the developmental outputs of genome topology-mediated gene expression regulation. It is becoming increasingly clear that new tools and techniques with higher resolution need to be developed and implemented in Arabidopsis and other model plants in order to better understand chromosome architecture dynamics, from an integrative perspective with other fields of plant biology such as development, stress biology, and finally agriculture.