The Arabidopsis transcription factor NLP2 regulates early nitrate responses and integrates nitrate assimilation with energy and carbon skeleton supply.

Nitrate signaling improves plant growth under limited nitrate availability and, hence, optimal resource use for crop production. Whereas several transcriptional regulators of nitrate signaling have been identified, including the Arabidopsis thaliana transcription factor NIN-LIKE PROTEIN7 (NLP7), additional regulators are expected to fine-tune this pivotal physiological response. Here, we characterized Arabidopsis NLP2 as a top-tier transcriptional regulator of the early nitrate response gene regulatory network. NLP2 interacts with NLP7 in vivo and shares key molecular features such as nitrate-dependent nuclear localization, DNA-binding motif, and some target genes with NLP7. Genetic, genomic, and metabolic approaches revealed a specific role for NLP2 in the nitrate-dependent regulation of carbon and energy-related processes that likely influence plant growth under distinct nitrogen environments. Our findings highlight the complementarity and specificity of NLP2 and NLP7 in orchestrating a multitiered nitrate regulatory network that links nitrate assimilation with carbon and energy metabolism for efficient nitrogen use and biomass production.

Deciphering Plant Chromatin Regulation via CRISPR/dCas9-Based Epigenome Engineering.

CRISPR-based epigenome editing uses dCas9 as a platform to recruit transcription or chromatin regulators at chosen loci. Despite recent and ongoing advances, the full potential of these approaches to studying chromatin functions in vivo remains challenging to exploit. In this review we discuss how recent progress in plants and animals provides new routes to investigate the function of chromatin regulators and address the complexity of associated regulations that are often interconnected. While efficient transcriptional engineering methodologies have been developed and can be used as tools to alter the chromatin state of a locus, examples of direct manipulation of chromatin regulators remain scarce in plants. These reports also reveal pitfalls and limitations of epigenome engineering approaches that are nevertheless informative as they are often associated with locus- and context-dependent features, which include DNA accessibility, initial chromatin and transcriptional state or cellular dynamics. Strategies implemented in different organisms to overcome and even take advantage of these limitations are highlighted, which will further improve our ability to establish the causality and hierarchy of chromatin dynamics on genome regulation.

A network of transcriptional repressors modulates auxin responses.

The regulation of signalling capacity, combined with the spatiotemporal distribution of developmental signals themselves, is pivotal in setting developmental responses in both plants and animals1. The hormone auxin is a key signal for plant growth and development that acts through the AUXIN RESPONSE FACTOR (ARF) transcription factors2-4. A subset of these, the conserved class A ARFs5, are transcriptional activators of auxin-responsive target genes that are essential for regulating auxin signalling throughout the plant lifecycle2,3. Although class A ARFs have tissue-specific expression patterns, how their expression is regulated is unknown. Here we show, by investigating chromatin modifications and accessibility, that loci encoding these proteins are constitutively open for transcription. Through yeast one-hybrid screening, we identify the transcriptional regulators of the genes encoding class A ARFs from Arabidopsis thaliana and demonstrate that each gene is controlled by specific sets of transcriptional regulators. Transient transformation assays and expression analyses in mutants reveal that, in planta, the majority of these regulators repress the transcription of genes encoding class A ARFs. These observations support a scenario in which the default configuration of open chromatin enables a network of transcriptional repressors to regulate expression levels of class A ARF proteins and modulate auxin signalling output throughout development.

Mitotic Inheritance of PRC2-Mediated Silencing: Mechanistic Insights and Developmental Perspectives.

Maintenance of gene repression by Polycomb Repressive Complex 2 (PRC2) that catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3) is integral to the orchestration of developmental programs in most multicellular eukaryotes. Faithful inheritance of H3K27me3 patterns across replication ensures the stability of PRC2-mediated transcriptional silencing over cell generations, thereby safeguarding cellular identities. In this review, we discuss the molecular and mechanistic principles that underlie H3K27me3 restoration after the passage of the replication fork, considering recent advances in different model systems. In particular, we aim at emphasizing parallels and differences between plants and other organisms, focusing on the recycling of parental histones and the replenishment of H3K27me3 patterns post-replication thanks to the remarkable properties of the PRC2 complex. We then discuss the necessity for fine-tuning this genuine epigenetic memory system so as to allow for cell fate and developmental transitions. We highlight recent insights showing that genome-wide destabilization of the H3K27me3 landscape during chromatin replication participates in achieving this flexible stability and provides a window of opportunity for subtle transcriptional reprogramming.

Histone acetylation orchestrates wound-induced transcriptional activation and cellular reprogramming in Arabidopsis.

Plant somatic cells reprogram and regenerate new tissues or organs when they are severely damaged. These physiological processes are associated with dynamic transcriptional responses but how chromatin-based regulation contributes to wound-induced gene expression changes and subsequent cellular reprogramming remains unknown. In this study we investigate the temporal dynamics of the histone modifications H3K9/14ac, H3K27ac, H3K4me3, H3K27me3, and H3K36me3, and analyze their correlation with gene expression at early time points after wounding. We show that a majority of the few thousand genes rapidly induced by wounding are marked with H3K9/14ac and H3K27ac before and/or shortly after wounding, and these include key wound-inducible reprogramming genes such as WIND1, ERF113/RAP2.6 L and LBD16. Our data further demonstrate that inhibition of GNAT-MYST-mediated histone acetylation strongly blocks wound-induced transcriptional activation as well as callus formation at wound sites. This study thus uncovered a key epigenetic mechanism that underlies wound-induced cellular reprogramming in plants.

The m6A pathway protects the transcriptome integrity by restricting RNA chimera formation in plants.

Global, segmental, and gene duplication-related processes are driving genome size and complexity in plants. Despite their evolutionary potentials, those processes can also have adverse effects on genome regulation, thus implying the existence of specialized corrective mechanisms. Here, we report that an N6-methyladenosine (m6A)-assisted polyadenylation (m-ASP) pathway ensures transcriptome integrity in Arabidopsis thaliana Efficient m-ASP pathway activity requires the m6A methyltransferase-associated factor FIP37 and CPSF30L, an m6A reader corresponding to an YT512-B Homology Domain-containing protein (YTHDC)-type domain containing isoform of the 30-kD subunit of cleavage and polyadenylation specificity factor. Targets of the m-ASP pathway are enriched in recently rearranged gene pairs, displayed an atypical chromatin signature, and showed transcriptional readthrough and mRNA chimera formation in FIP37- and CPSF30L-deficient plants. Furthermore, we showed that the m-ASP pathway can also restrict the formation of chimeric gene/transposable-element transcript, suggesting a possible implication of this pathway in the control of transposable elements at specific locus. Taken together, our results point to selective recognition of 3'-UTR m6A as a safeguard mechanism ensuring transcriptome integrity at rearranged genomic loci in plants.

Genome-wide identification of RETINOBLASTOMA RELATED 1 binding sites in Arabidopsis reveals novel DNA damage regulators.

Retinoblastoma (pRb) is a multifunctional regulator, which was likely present in the last common ancestor of all eukaryotes. The Arabidopsis pRb homolog RETINOBLASTOMA RELATED 1 (RBR1), similar to its animal counterparts, controls not only cell proliferation but is also implicated in developmental decisions, stress responses and maintenance of genome integrity. Although most functions of pRb-type proteins involve chromatin association, a genome-wide understanding of RBR1 binding sites in Arabidopsis is still missing. Here, we present a plant chromatin immunoprecipitation protocol optimized for genome-wide studies of indirectly DNA-bound proteins like RBR1. Our analysis revealed binding of Arabidopsis RBR1 to approximately 1000 genes and roughly 500 transposable elements, preferentially MITES. The RBR1-decorated genes broadly overlap with previously identified targets of two major transcription factors controlling the cell cycle, i.e. E2F and MYB3R3 and represent a robust inventory of RBR1-targets in dividing cells. Consistently, enriched motifs in the RBR1-marked domains include sequences related to the E2F consensus site and the MSA-core element bound by MYB3R transcription factors. Following up a key role of RBR1 in DNA damage response, we performed a meta-analysis combining the information about the RBR1-binding sites with genome-wide expression studies under DNA stress. As a result, we present the identification and mutant characterization of three novel genes required for growth upon genotoxic stress.

Polycomb Repressive Complex 2 attenuates the very high expression of the Arabidopsis gene NRT2.1.

PRC2 is a major regulator of gene expression in eukaryotes. It catalyzes the repressive chromatin mark H3K27me3, which leads to very low expression of target genes. NRT2.1, which encodes a key root nitrate transporter in Arabidopsis, is targeted by H3K27me3, but the function of PRC2 on NRT2.1 remains unclear. Here, we demonstrate that PRC2 directly targets and down-regulates NRT2.1, but in a context of very high transcription, in nutritional conditions where this gene is one of the most highly expressed genes in the transcriptome. Indeed, the mutation of CLF, which encodes a PRC2 subunit, leads to a loss of H3K27me3 at NRT2.1 and results, exclusively under permissive conditions for NRT2.1, in a further increase in NRT2.1 expression, and specifically in tissues where NRT2.1 is normally expressed. Therefore, our data indicates that PRC2 tempers the hyperactivity of NRT2.1 in a context of very strong transcription. This reveals an original function of PRC2 in the control of the expression of a highly expressed gene in Arabidopsis.

Chromatin Immunoprecipitation Sequencing (ChIP-Seq) for Transcription Factors and Chromatin Factors in Arabidopsis thaliana Roots: From Material Collection to Data Analysis.

Chromatin immunoprecipitation combined with next-generation sequencing (ChIP-seq) is a powerful technique to investigate in vivo transcription factor (TF) binding to DNA, as well as chromatin marks. Here we provide a detailed protocol for all the key steps to perform ChIP-seq in Arabidopsis thaliana roots, also working on other A. thaliana tissues and in most non-ligneous plants. We detail all steps from material collection, fixation, chromatin preparation, immunoprecipitation, library preparation, and finally computational analysis based on a combination of publicly available tools.

Cell Type-Specific Profiling of Chromatin Modifications and Associated Proteins.

Progression of a cell along a differentiation path is characterized by changes in gene expression profiles. Alterations of these transcriptional programs result from cell type-specific transcription factors that act in a dynamic chromatin environment. Understanding the precise contribution of these molecular factors during the differentiation process requires accessing specific cell types within a developing organ. This chapter describes a streamlined and alternative version of INTACT, a method enabling the isolation of specific cell populations by affinity-purification of tagged nuclei and the subsequent analysis of gene expression, transcription factor binding profiles, as well as chromatin state at a genome-wide scale. In particular, modifications of the nuclei isolation, capture, and purification procedures are proposed that improve time scale, yield, and purity. In addition, the combination of different tags enables the analysis of distinct cell populations from a single transgenic line and the subtractive purification of subpopulations of cells, including those for which no specific promoter is available. Finally, we describe a chromatin immunoprecipitation protocol that has been successfully used to profile histone modifications and other chromatin-associated proteins such as RNA Polymerase II in different cell populations of the Arabidopsis root, including the quiescent center of the stem cell niche.

Comparative epigenomics in the Brassicaceae reveals two evolutionarily conserved modes of PRC2-mediated gene regulation.

BACKGROUND: Polycomb Repressive Complexes 2 (PRC2) are multi-protein chromatin modifiers that are evolutionarily conserved among eukaryotes and play key roles in the regulation of gene expression, notably through the trimethylation of lysine 27 of histone H3 (H3K27me3). Although PRC2-mediated gene regulation has been studied in many organisms, few studies have explored in depth the evolutionary conservation of PRC2 targets. RESULTS: Here, we compare the H3K27me3 epigenomic profiles for the two closely related species Arabidopsis thaliana and Arabidopsis lyrata and the more distant species Arabis alpina, three Brassicaceae that diverged from each other within the past 24 million years. Using a robust set of gene orthologs present in the three species, we identify two classes of evolutionarily conserved PRC2 targets, which are characterized by either developmentally plastic or developmentally constrained H3K27me3 marking across species. Constrained H3K27me3 marking is associated with higher conservation of promoter sequence information content and higher nucleosome occupancy compared to plastic H3K27me3 marking. Moreover, gene orthologs with constrained H3K27me3 marking exhibit a higher degree of tissue specificity and tend to be involved in developmental functions, whereas gene orthologs with plastic H3K27me3 marking preferentially encode proteins associated with metabolism and stress responses. In addition, gene orthologs with constrained H3K27me3 marking are the predominant contributors to higher-order chromosome organization. CONCLUSIONS: Our findings indicate that developmentally plastic and constrained H3K27me3 marking define two evolutionarily conserved modes of PRC2-mediated gene regulation that are associated with distinct selective pressures operating at multiple scales, from DNA sequence to gene function and chromosome architecture.

DNA methylation dynamics during early plant life.

BACKGROUND: Cytosine methylation is crucial for gene regulation and silencing of transposable elements in mammals and plants. While this epigenetic mark is extensively reprogrammed in the germline and early embryos of mammals, the extent to which DNA methylation is reset between generations in plants remains largely unknown. RESULTS: Using Arabidopsis as a model, we uncovered distinct DNA methylation dynamics over transposable element sequences during the early stages of plant development. Specifically, transposable elements and their relics show invariably high methylation at CG sites but increasing methylation at CHG and CHH sites. This non-CG methylation culminates in mature embryos, where it reaches saturation for a large fraction of methylated CHH sites, compared to the typical 10-20% methylation level observed in seedlings or adult plants. Moreover, the increase in CHH methylation during embryogenesis matches the hypomethylated state in the early endosperm. Finally, we show that interfering with the embryo-to-seedling transition results in the persistence of high CHH methylation levels after germination, specifically over sequences that are targeted by the RNA-directed DNA methylation (RdDM) machinery. CONCLUSION: Our findings indicate the absence of extensive resetting of DNA methylation patterns during early plant life and point instead to an important role of RdDM in reinforcing DNA methylation of transposable element sequences in every cell of the mature embryo. Furthermore, we provide evidence that this elevated RdDM activity is a specific property of embryogenesis.

Epigenetic memory and cell fate reprogramming in plants.

Plants have a high intrinsic capacity to regenerate from adult tissues, with the ability to reprogram adult cell fates. In contrast, epigenetic mechanisms have the potential to stabilize cell identity and maintain tissue organization. The question is whether epigenetic memory creates a barrier to reprogramming that needs to be erased or circumvented in plant regeneration. Early evidence suggests that, while chromatin dynamics impact gene expression in the meristem, a lasting constraint on cell fate is not established until late stages of plant cell differentiation. It is not yet clear whether the plasticity of plant cells arises from the ability of cells to erase identity memory or to deploy cells that may exhibit cellular specialization but still lack an epigenetic restriction on cell fate alteration.

Direct conversion of root primordium into shoot meristem relies on timing of stem cell niche development.

To understand how the identity of an organ can be switched, we studied the transformation of lateral root primordia (LRP) into shoot meristems in Arabidopsis root segments. In this system, the cytokinin-induced conversion does not involve the formation of callus-like structures. Detailed analysis showed that the conversion sequence starts with a mitotic pause and is concomitant with the differential expression of regulators of root and shoot development. The conversion requires the presence of apical stem cells, and only LRP at stages VI or VII can be switched. It is engaged as soon as cell divisions resume because their position and orientation differ in the converting organ compared with the undisturbed emerging LRP. By alternating auxin and cytokinin treatments, we showed that the root and shoot organogenetic programs are remarkably plastic, as the status of the same plant stem cell niche can be reversed repeatedly within a set developmental window. Thus, the networks at play in the meristem of a root can morph in the span of a couple of cell division cycles into those of a shoot, and back, through transdifferentiation.

Transcriptional Regulation of Arabidopsis Polycomb Repressive Complex 2 Coordinates Cell-Type Proliferation and Differentiation.

Spatiotemporal regulation of transcription is fine-tuned at multiple levels, including chromatin compaction. Polycomb Repressive Complex 2 (PRC2) catalyzes the trimethylation of Histone 3 at lysine 27 (H3K27me3), which is the hallmark of a repressive chromatin state. Multiple PRC2 complexes have been reported in Arabidopsis thaliana to control the expression of genes involved in developmental transitions and maintenance of organ identity. Here, we show that PRC2 member genes display complex spatiotemporal gene expression patterns and function in root meristem and vascular cell proliferation and specification. Furthermore, PRC2 gene expression patterns correspond with vascular and nonvascular tissue-specific H3K27me3-marked genes. This tissue-specific repression via H3K27me3 regulates the balance between cell proliferation and differentiation. Using enhanced yeast one-hybrid analysis, upstream regulators of the PRC2 member genes are identified, and genetic analysis demonstrates that transcriptional regulation of some PRC2 genes plays an important role in determining PRC2 spatiotemporal activity within a developing organ.

Emerging concepts in chromatin-level regulation of plant cell differentiation: timing, counting, sensing and maintaining.

Plants are characterized by a remarkable phenotypic plasticity that meets the constraints of a sessile lifestyle and the need to adjust constantly to the environment. Recent studies have begun to reveal how chromatin dynamics participate in coordinating cell proliferation and differentiation in response to developmental cues as well as environmental fluctuations. In this review, we discuss the pivotal function of chromatin-based mechanisms in cell fate acquisition and maintenance, within as well as outside meristems. In particular, we highlight the emerging role of specific epigenomic factors and chromatin pathways in timing the activity of stem cells, counting cell divisions and positioning cell fate transitions by sensing phytohormone gradients.

A versatile Multisite Gateway-compatible promoter and transgenic line collection for cell type-specific functional genomics in Arabidopsis.

Multicellular organisms are composed of many cell types that acquire their specific fate through a precisely controlled pattern of gene expression in time and space dictated in part by cell type-specific promoter activity. Understanding the contribution of highly specialized cell types in the development of a whole organism requires the ability to isolate or analyze different cell types separately. We have characterized and validated a large collection of root cell type-specific promoters and have generated cell type-specific marker lines. These benchmarked promoters can be readily used to evaluate cell type-specific complementation of mutant phenotypes, or to knockdown gene expression using targeted expression of artificial miRNA. We also generated vectors and characterized transgenic lines for cell type-specific induction of gene expression and cell type-specific isolation of nuclei for RNA and chromatin profiling. Vectors and seeds from transgenic Arabidopsis plants will be freely available, and will promote rapid progress in cell type-specific functional genomics. We demonstrate the power of this promoter set for analysis of complex biological processes by investigating the contribution of root cell types in the IRT1-dependent root iron uptake. Our findings revealed the complex spatial expression pattern of IRT1 in both root epidermis and phloem companion cells and the requirement for IRT1 to be expressed in both cell types for proper iron homeostasis.

Genome expansion of Arabis alpina linked with retrotransposition and reduced symmetric DNA methylation.

Despite evolutionary conserved mechanisms to silence transposable element activity, there are drastic differences in the abundance of transposable elements even among closely related plant species. We conducted a de novo assembly for the 375 Mb genome of the perennial model plant, Arabis alpina. Analysing this genome revealed long-lasting and recent transposable element activity predominately driven by Gypsy long terminal repeat retrotransposons, which extended the low-recombining pericentromeres and transformed large formerly euchromatic regions into repeat-rich pericentromeric regions. This reduced capacity for long terminal repeat retrotransposon silencing and removal in A. alpina co-occurs with unexpectedly low levels of DNA methylation. Most remarkably, the striking reduction of symmetrical CG and CHG methylation suggests weakened DNA methylation maintenance in A. alpina compared with Arabidopsis thaliana. Phylogenetic analyses indicate a highly dynamic evolution of some components of methylation maintenance machinery that might be related to the unique methylation in A. alpina.