Innovation, conservation, and repurposing of gene function in root cell type development.

Plant species have evolved myriads of solutions, including complex cell type development and regulation, to adapt to dynamic environments. To understand this cellular diversity, we profiled tomato root cell type translatomes. Using xylem differentiation in tomato, examples of functional innovation, repurposing, and conservation of transcription factors are described, relative to the model plant Arabidopsis. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Comparative translatome analyses of rice, tomato, and Arabidopsis cell populations suggest increased expression conservation of root meristems compared with other homologous populations. In addition, the functions of constitutively expressed genes are more conserved than those of cell type/tissue-enriched genes. These observations suggest that higher order properties of cell type and pan-cell type regulation are evolutionarily conserved between plants and animals.

The VIL gene CRAWLING ELEPHANT controls maturation and differentiation in tomato via polycomb silencing.

VERNALIZATION INSENSITIVE 3-LIKE (VIL) proteins are PHD-finger proteins that recruit the repressor complex Polycomb Repressive Complex 2 (PRC2) to the promoters of target genes. Most known VIL targets are flowering repressor genes. Here, we show that the tomato VIL gene CRAWLING ELEPHANT (CREL) promotes differentiation throughout plant development by facilitating the trimethylation of Histone H3 on lysine 27 (H3K27me3). We identified the crel mutant in a screen for suppressors of the simple-leaf phenotype of entire (e), a mutant in the AUX/IAA gene ENTIRE/SlIAA9, involved in compound-leaf development in tomato. crel mutants have increased leaf complexity, and suppress the ectopic blade growth of e mutants. In addition, crel mutants are late flowering, and have delayed and aberrant stem, root and flower development. Consistent with a role for CREL in recruiting PRC2, crel mutants show drastically reduced H3K27me3 enrichment at approximately half of the 14,789 sites enriched in wild-type plants, along with upregulation of many underlying genes. Interestingly, this reduction in H3K27me3 across the genome in crel is also associated with gains in H3K27me3 at a smaller number of sites that normally have modest levels of the mark in wild-type plants, suggesting that PRC2 activity is no longer limiting in the absence of CREL. Our results uncover a wide role for CREL in plant and organ differentiation in tomato and suggest that CREL is required for targeting PRC2 activity to, and thus silencing, a specific subset of polycomb targets.

Global analysis of the RNA-protein interaction and RNA secondary structure landscapes of the Arabidopsis nucleus.

Posttranscriptional regulation in eukaryotes requires cis- and trans-acting features and factors including RNA secondary structure and RNA-binding proteins (RBPs). However, a comprehensive view of the structural and RBP interaction landscape of nuclear RNAs has yet to be compiled for any organism. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approaches to globally profile these features in Arabidopsis seedling nuclei in vivo. We reveal anticorrelated patterns of secondary structure and RBP binding throughout nuclear mRNAs that demarcate sites of alternative splicing and polyadenylation. We also uncover a collection of protein-bound sequence motifs, and identify their structural contexts, co-occurrences in transcripts encoding functionally related proteins, and interactions with putative RBPs. Finally, using these motifs, we find that the chloroplast RBP CP29A also interacts with nuclear mRNAs. In total, we provide a simultaneous view of the RNA secondary structure and RBP interaction landscapes in a eukaryotic nucleus.

Methyl-CpG-binding domain 9 (MBD9) is required for H2A.Z incorporation into chromatin at a subset of H2A.Z-enriched regions in the Arabidopsis genome.

The SWR1 chromatin remodeling complex, which deposits the histone variant H2A.Z into nucleosomes, has been well characterized in yeast and animals, but its composition in plants has remained uncertain. We used the conserved SWR1 subunit ACTIN RELATED PROTEIN 6 (ARP6) as bait in tandem affinity purification experiments to isolate associated proteins from Arabidopsis thaliana. We identified all 11 subunits found in yeast SWR1 and the homologous mammalian SRCAP complexes, demonstrating that this complex is conserved in plants. We also identified several additional proteins not previously associated with SWR1, including Methyl-CpG-BINDING DOMAIN 9 (MBD9) and three members of the Alfin1-like protein family, all of which have been shown to bind modified histone tails. Since mbd9 mutant plants were phenotypically similar to arp6 mutants, we explored a potential role for MBD9 in H2A.Z deposition. We found that MBD9 is required for proper H2A.Z incorporation at thousands of discrete sites, which represent a subset of the genomic regions normally enriched with H2A.Z. We also discovered that MBD9 preferentially interacts with acetylated histone H4 peptides, as well as those carrying mono- or dimethylated H3 lysine 4, or dimethylated H3 arginine 2 or 8. Considering that MBD9-dependent H2A.Z sites show a distinct histone modification profile, we propose that MBD9 recognizes particular nucleosome modifications via its PHD- and Bromo-domains and thereby guides SWR1 to these sites for H2A.Z deposition. Our data establish the SWR1 complex as being conserved across eukaryotes and suggest that MBD9 may be involved in targeting the complex to specific genomic sites through nucleosomal interactions. The finding that MBD9 does not appear to be a core subunit of the Arabidopsis SWR1 complex, along with the synergistic phenotype of arp6;mbd9 double mutants, suggests that MBD9 also has important roles beyond H2A.Z deposition.

The Chromatin Remodelers PKL and PIE1 Act in an Epigenetic Pathway That Determines H3K27me3 Homeostasis in Arabidopsis.

Selective, tissue-specific gene expression is facilitated by the epigenetic modification H3K27me3 (trimethylation of lysine 27 on histone H3) in plants and animals. Much remains to be learned about how H3K27me3-enriched chromatin states are constructed and maintained. Here, we identify a genetic interaction in Arabidopsis thaliana between the chromodomain helicase DNA binding chromatin remodeler PICKLE (PKL), which promotes H3K27me3 enrichment, and the SWR1-family remodeler PHOTOPERIOD INDEPENDENT EARLY FLOWERING1 (PIE1), which incorporates the histone variant H2A.Z. Chromatin immunoprecipitation-sequencing and RNA-sequencing reveal that PKL, PIE1, and the H3K27 methyltransferase CURLY LEAF act in a common gene expression pathway and are required for H3K27me3 levels genome-wide. Additionally, H3K27me3-enriched genes are largely a subset of H2A.Z-enriched genes, further supporting the functional linkage between these marks. We also found that recombinant PKL acts as a prenucleosome maturation factor, indicating that it promotes retention of H3K27me3. These data support the existence of an epigenetic pathway in which PIE1 promotes H2A.Z, which in turn promotes H3K27me3 deposition. After deposition, PKL promotes retention of H3K27me3 after DNA replication and/or transcription. Our analyses thus reveal roles for H2A.Z and ATP-dependent remodelers in construction and maintenance of H3K27me3-enriched chromatin in plants.

Profiling of Accessible Chromatin Regions across Multiple Plant Species and Cell Types Reveals Common Gene Regulatory Principles and New Control Modules.

The transcriptional regulatory structure of plant genomes remains poorly defined relative to animals. It is unclear how many cis-regulatory elements exist, where these elements lie relative to promoters, and how these features are conserved across plant species. We employed the assay for transposase-accessible chromatin (ATAC-seq) in four plant species (Arabidopsis thaliana, Medicago truncatula, Solanum lycopersicum, and Oryza sativa) to delineate open chromatin regions and transcription factor (TF) binding sites across each genome. Despite 10-fold variation in intergenic space among species, the majority of open chromatin regions lie within 3 kb upstream of a transcription start site in all species. We find a common set of four TFs that appear to regulate conserved gene sets in the root tips of all four species, suggesting that TF-gene networks are generally conserved. Comparative ATAC-seq profiling of Arabidopsis root hair and non-hair cell types revealed extensive similarity as well as many cell-type-specific differences. Analyzing TF binding sites in differentially accessible regions identified a MYB-driven regulatory module unique to the hair cell, which appears to control both cell fate regulators and abiotic stress responses. Our analyses revealed common regulatory principles among species and shed light on the mechanisms producing cell-type-specific transcriptomes during development.

The histone variant H2A.Z and chromatin remodeler BRAHMA act coordinately and antagonistically to regulate transcription and nucleosome dynamics in Arabidopsis.

Plants adapt to environmental changes by regulating transcription and chromatin organization. The histone H2A variant H2A.Z and the SWI2/SNF2 ATPase BRAHMA (BRM) have overlapping roles in positively and negatively regulating environmentally responsive genes in Arabidopsis, but the extent of this overlap was uncharacterized. Both factors have been associated with various changes in nucleosome positioning and stability in different contexts, but their specific roles in transcriptional regulation and chromatin organization need further characterization. We show that H2A.Z and BRM co-localize at thousands of sites, where they interact both cooperatively and antagonistically in transcriptional repression and activation of genes involved in development and responses to environmental stimuli. We identified eight classes of genes that show distinct relationships between H2A.Z and BRM with respect to their roles in transcription. These include activating and silencing transcription both redundantly and antagonistically. We found that H2A.Z contributes to a range of different nucleosome properties, while BRM stabilizes nucleosomes where it binds and destabilizes or repositions flanking nucleosomes. We also found that, at many genes regulated by both BRM and H2A.Z, both factors overlap with binding sites of the light-regulated transcription factor FAR1-Related Sequence 9 (FRS9) and that a subset of these FRS9 binding sites are dependent on H2A.Z and BRM for accessibility. Collectively, we comprehensively characterized the antagonistic and cooperative contributions of H2A.Z and BRM to transcriptional regulation, and illuminated several interrelated roles in chromatin organization. The variability observed in their individual functions implies that both BRM and H2A.Z have more context-dependent roles than previously assumed.

Changes in chromatin accessibility between Arabidopsis stem cells and mesophyll cells illuminate cell type-specific transcription factor networks.

Cell differentiation is driven by changes in the activity of transcription factors (TFs) and subsequent alterations in transcription. To study this process, differences in TF binding between cell types can be deduced by probing chromatin accessibility. We used cell type-specific nuclear purification followed by the assay for transposase-accessible chromatin (ATAC-seq) to delineate differences in chromatin accessibility and TF regulatory networks between stem cells of the shoot apical meristem (SAM) and differentiated leaf mesophyll cells in Arabidopsis thaliana. Chromatin accessibility profiles of SAM stem cells and leaf mesophyll cells were very similar at a qualitative level, yet thousands of regions having quantitatively different chromatin accessibility were also identified. Analysis of the genomic regions preferentially accessible in each cell type identified hundreds of overrepresented TF-binding motifs, highlighting sets of TFs that are probably important for each cell type. Within these sets, we found evidence for extensive co-regulation of target genes by multiple TFs that are preferentially expressed in each cell type. Interestingly, the TFs within each of these cell type-enriched sets also showed evidence of extensively co-regulating each other. We further found that preferentially accessible chromatin regions in mesophyll cells tended to also be substantially accessible in the stem cells, whereas the converse was not true. This observation suggests that the generally higher accessibility of regulatory elements in stem cells might contribute to their developmental plasticity. This work demonstrates the utility of cell type-specific chromatin accessibility profiling for the rapid development of testable models of regulatory control differences between cell types.

Nuclear Transcriptomes at High Resolution Using Retooled INTACT.

Isolated nuclei provide access to early steps in gene regulation involving chromatin as well as transcript production and processing. Here, we describe transfer of the isolation of nuclei from tagged specific cell types (INTACT) to the monocot rice (Oryza sativa L.). The purification of biotinylated nuclei was redesigned by replacing the outer nuclear-envelope-targeting domain of the nuclear tagging fusion (NTF) protein with an outer nuclear-envelope-anchored domain. This modified NTF was combined with codon-optimized Escherichia coli BirA in a single T-DNA construct. We also developed inexpensive methods for INTACT, T-DNA insertion mapping, and profiling of the complete nuclear transcriptome, including a ribosomal RNA degradation procedure that minimizes pre-ribosomal RNA (pre-rRNA) transcripts. A high-resolution comparison of nuclear and steady-state poly(A)+ transcript populations of seedling root tips confirmed the capture of pre-messenger RNA (pre-mRNA) and exposed distinctions in diversity and abundance of the nuclear and total transcriptomes. This retooled INTACT can enable high-resolution monitoring of the nuclear transcriptome and chromatin in specific cell types of rice and other species.

On the origin and evolutionary consequences of gene body DNA methylation.

In plants, CG DNA methylation is prevalent in the transcribed regions of many constitutively expressed genes (gene body methylation; gbM), but the origin and function of gbM remain unknown. Here we report the discovery that Eutrema salsugineum has lost gbM from its genome, to our knowledge the first instance for an angiosperm. Of all known DNA methyltransferases, only CHROMOMETHYLASE 3 (CMT3) is missing from E. salsugineum Identification of an additional angiosperm, Conringia planisiliqua, which independently lost CMT3 and gbM, supports that CMT3 is required for the establishment of gbM. Detailed analyses of gene expression, the histone variant H2A.Z, and various histone modifications in E. salsugineum and in Arabidopsis thaliana epigenetic recombinant inbred lines found no evidence in support of any role for gbM in regulating transcription or affecting the composition and modification of chromatin over evolutionary timescales.

Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling.

An understanding of developmental processes requires knowledge of transcriptional and epigenetic landscapes at the level of tissues and ultimately individual cells. However, obtaining tissue- or cell-type-specific expression and chromatin profiles for animals has been challenging. Here we describe a method for purifying nuclei from specific cell types of animal models that allows simultaneous determination of both expression and chromatin profiles. The method is based on in vivo biotin-labeling of the nuclear envelope and subsequent affinity purification of nuclei. We describe the use of the method to isolate nuclei from muscle of adult Caenorhabditis elegans and from mesoderm of Drosophila melanogaster embryos. As a case study, we determined expression and nucleosome occupancy profiles for affinity-purified nuclei from C. elegans muscle. We identified hundreds of genes that are specifically expressed in muscle tissues and found that these genes are depleted of nucleosomes at promoters and gene bodies in muscle relative to other tissues. This method should be universally applicable to all model systems that allow transgenesis and will make it possible to determine epigenetic and expression profiles of different tissues and cell types.

Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model.

Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato.

Differences in transcription initiation directionality underlie distinctions between plants and animals in chromatin modification patterns at genes and cis-regulatory elements.

Transcriptional initiation is among the first regulated steps controlling eukaryotic gene expression. High-throughput profiling of fungal and animal genomes has revealed that RNA Polymerase II often initiates transcription in both directions at the promoter transcription start site, but generally only elongates productively into the gene body. Additionally, Pol II can initiate transcription in both directions at cis-regulatory elements such as enhancers. These bidirectional RNA Polymerase II initiation events can be observed directly with methods that capture nascent transcripts, and they are also revealed indirectly by the presence of transcription-associated histone modifications on both sides of the transcription start site or cis-regulatory elements. Previous studies have shown that nascent RNAs and transcription-associated histone modifications in the model plant Arabidopsis thaliana accumulate mainly in the gene body, suggesting that transcription does not initiate widely in the upstream direction from genes in this plant. We compared transcription-associated histone modifications and nascent transcripts at both transcription start sites and cis-regulatory elements in A. thaliana, Drosophila melanogaster, and Homo sapiens. Our results provide evidence for mostly unidirectional RNA Polymerase II initiation at both promoters and gene-proximal cis-regulatory elements of A. thaliana, whereas bidirectional transcription initiation is observed widely at promoters in both D. melanogaster and H. sapiens, as well as cis-regulatory elements in Drosophila. Furthermore, the distribution of transcription-associated histone modifications around transcription start sites in the Oryza sativa (rice) and Glycine max (soybean) genomes suggests that unidirectional transcription initiation is the norm in these genomes as well. These results suggest that there are fundamental differences in transcriptional initiation directionality between flowering plant and metazoan genomes, which are manifested as distinct patterns of chromatin modifications around RNA polymerase initiation sites.

Replacement of Arabidopsis H2A.Z with human H2A.Z orthologs reveals extensive functional conservation and limited importance of the N-terminal tail sequence for Arabidopsis development.

The incorporation of histone variants, distinct paralogs of core histones, into chromatin affects all DNA-templated processes in the cell, including the regulation of transcription. In recent years, much research has been focused on H2A.Z, an evolutionarily conserved H2A variant found in all eukaryotes. In order to investigate the functional conservation of H2A.Z histones during eukaryotic evolution we transformed h2a.z deficient plants with three human H2A.Z proteins to assess their ability to rescue the mutant defects. We discovered that human H2A.Z.1 and H2A.Z.2.1 fully complement the phenotypic abnormalities of h2a.z plants despite the fact that Arabidopsis and human H2A.Z N-terminal tail sequences are quite divergent. In contrast, the brain-specific splice variant H2A.Z.2.2 has a dominant-negative effect in wild-type plants. Furthermore, H2A.Z.1 almost completely re-establishes normal H2A.Z chromatin occupancy in h2a.z plants and restores the transcript levels of more than 84 % of misexpressed genes. Finally, our hypothesis that the N-terminal tail of Arabidopsis H2A.Z is not crucial for its developmental functions was supported by the ability of N-terminal end truncations of Arabidopsis HTA11 to largely rescue the defects of h2a.z mutants.

Differences in transcription initiation directionality underlie distinctions between plants and animals in chromatin modification patterns at genes and cis-regulatory elements.

Transcriptional initiation is among the first regulated steps controlling eukaryotic gene expression. High-throughput profiling of fungal and animal genomes has revealed that RNA Polymerase II (Pol II) often initiates transcription in both directions at the promoter transcription start site (TSS), but generally only elongates productively into the gene body. Additionally, Pol II can initiate transcription in both directions at cis-regulatory elements (CREs) such as enhancers. These bidirectional Pol II initiation events can be observed directly with methods that capture nascent transcripts, and they are also revealed indirectly by the presence of transcription-associated histone modifications on both sides of the TSS or CRE. Previous studies have shown that nascent RNAs and transcription-associated histone modifications in the model plant Arabidopsis thaliana accumulate mainly in the gene body, suggesting that transcription does not initiate widely in the upstream direction from genes in this plant. We compared transcription-associated histone modifications and nascent transcripts at both TSSs and CREs in Arabidopsis thaliana, Drosophila melanogaster, and Homo sapiens. Our results provide evidence for mostly unidirectional Pol II initiation at both promoters and gene-proximal CREs of Arabidopsis thaliana, whereas bidirectional transcription initiation is observed widely at promoters in both Drosophila melanogaster and Homo sapiens, as well as CREs in Drosophila. Furthermore, the distribution of transcription-associated histone modifications around TSSs in the Oryza sativa (rice) and Glycine max (soybean) genomes suggests that unidirectional transcription initiation is the norm in these genomes as well. These results suggest that there are fundamental differences in transcriptional initiation directionality between flowering plant and metazoan genomes, which are manifested as distinct patterns of chromatin modifications around RNA polymerase initiation sites.

Histone Variants in the Specialization of Plant Chromatin.

The basic unit of chromatin, the nucleosome, is an octamer of four core histone proteins (H2A, H2B, H3, and H4) and serves as a fundamental regulatory unit in all DNA-templated processes. The majority of nucleosome assembly occurs during DNA replication when these core histones are produced en masse to accommodate the nascent genome. In addition, there are a number of nonallelic sequence variants of H2A and H3 in particular, known as histone variants, that can be incorporated into nucleosomes in a targeted and replication-independent manner. By virtue of their sequence divergence from the replication-coupled histones, these histone variants can impart unique properties onto the nucleosomes they occupy and thereby influence transcription and epigenetic states, DNA repair, chromosome segregation, and other nuclear processes in ways that profoundly affect plant biology. In this review, we discuss the evolutionary origins of these variants in plants, their known roles in chromatin, and their impacts on plant development and stress responses. We focus on the individual and combined roles of histone variants in transcriptional regulation within euchromatic and heterochromatic genome regions. Finally, we highlight gaps in our understanding of plant variants at the molecular, cellular, and organismal levels, and we propose new directions for study in the field of plant histone variants.

Gene regulatory networks shape developmental plasticity of root cell types under water extremes in rice.

Understanding how roots modulate development under varied irrigation or rainfall is crucial for development of climate-resilient crops. We established a toolbox of tagged rice lines to profile translating mRNAs and chromatin accessibility within specific cell populations. We used these to study roots in a range of environments: plates in the lab, controlled greenhouse stress and recovery conditions, and outdoors in a paddy. Integration of chromatin and mRNA data resolves regulatory networks of the following: cycle genes in proliferating cells that attenuate DNA synthesis under submergence; genes involved in auxin signaling, the circadian clock, and small RNA regulation in ground tissue; and suberin biosynthesis, iron transporters, and nitrogen assimilation in endodermal/exodermal cells modulated with water availability. By applying a systems approach, we identify known and candidate driver transcription factors of water-deficit responses and xylem development plasticity. Collectively, this resource will facilitate genetic improvements in root systems for optimal climate resilience.

Histone H2A.Z regulates the expression of several classes of phosphate starvation response genes but not as a transcriptional activator.

Phosphate (Pi) availability is a major constraint to plant growth. Consequently, plants have evolved complex adaptations to tolerate low Pi conditions. Numerous genes implicated in these adaptations have been identified, but their chromatin-level regulation has not been investigated. The nuclear actin-related protein ARP6 is conserved among all eukaryotes and is an essential component of the SWR1 chromatin remodeling complex, which regulates transcription via deposition of the H2A.Z histone variant into chromatin. Here, we demonstrate that ARP6 is required for proper H2A.Z deposition at a number of Pi starvation response (PSR) genes in Arabidopsis (Arabidopsis thaliana). The loss of H2A.Z at these target loci results in their derepression in arp6 mutants and correlates with the presence of multiple Pi-starvation-related phenotypes, including shortened primary roots and increases in the number and length of root hairs, as well as increased starch accumulation and phosphatase activity in shoots. Our data suggest a model for chromatin-level control of Pi starvation responses in which ARP6-dependent H2A.Z deposition modulates the transcription of a suite of PSR genes.