Biology subjects

Deal, R. B.

Publications and source records attributed to Deal, R. B..

9 recordsLinked to original sources

bioRxiv2024

Temporal profiling of the phosphate starvation response in Arabidopsis root hair cells reveals that induction of polycomb target genes does not depend on removal of H3K27me3 or H2A.Z

Altered nutrient conditions can trigger massive transcriptional reprogramming in plants, leading to the activation and silencing of thousands of genes. To gain a deeper understanding of the phosphate starvation response and the relationships between transcriptional and epigenomic changes that occur during this reprogramming, we conducted a time-resolved analysis of transcriptome and chromatin alterations in root hair cells of Arabidopsis thaliana during phosphate (P) starvation and subsequent resupply. We found that 96 hours of P starvation causes induction or repression of thousands of transcripts, and most of these recover to pre-starvation levels within 4 hours of P resupply. Among the phosphate starvation-induced genes are many polycomb targets with high levels of H3K27me3 and histone variant H2A.Z. When induced, these genes often show increased H3K4me3 consistent with active transcription, but surprisingly minimal loss of H3K27me3 or H2A.Z. These results indicate that the removal of silencing marks is not a prerequisite for activation of these genes. Our data provide a cell type- and time-resolved resource for studying the dynamics of a systemic nutrient stress and recovery and suggest that our current understanding of the mechanisms for switching between silent and active transcriptional states is incomplete.

plant biology↗

bioRxiv2024

SWR1 is recruited to activated ABA response genes to maintain gene body H2A.Z in Arabidopsis thaliana

The histone variant H2A.Z is important for transcriptional regulation across eukaryotes, where it can alternately promote or repress transcription. In plants, actively transcribed genes show H2A.Z enrichment in nucleosomes immediately downstream of the transcription start site (TSS), while silent genes show H2A.Z enrichment across the gene body. Previous work showed that silent genes responsive to temperature and far-red light lose gene body H2A.Z upon activation, but whether H2A.Z loss is generally required for transcription is not clear. We profiled H2A.Z and components of its deposition complex, SWR1, before and after treating Arabidopsis thaliana with the hormone abscisic acid (ABA). Our results show that transcribed genes with TSS-enriched H2A.Z have high SWR1 binding at steady-state, indicating continuous replacement of H2A.Z, while silent genes with gene body H2A.Z show lower SWR1 binding. Surprisingly, upon ABA treatment, thousands of previously silent genes activate, coincident with recruitment of SWR1 and retention of gene body H2A.Z enrichment. We also found that the SWR1-interacting protein MBD9 is not required for SWR1 recruitment to activated genes. These results provide new insights into the relationship between H2A.Z and transcription and the mechanics of H2A.Z targeting to chromatin.

plant biology↗

bioRxiv2023

Differences in transcription initiation directionality underlie distinctions between plants and animalsin 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.

genomics↗

bioRxiv2023

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 each of the three human H2A.Z variants 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 significant divergence in the N-terminal tail sequences of Arabidopsis and human H2A.Zs. In contrast, the brain-specific splice variant H2A.Z.2.2 has a dominant-negative effect in wild-type plants, mimicking an H2A.Z deficiency phenotype. Furthermore, H2A.Z.1 almost completely re-establishes normal H2A.Z chromatin occupancy in h2a.z plants and restores the expression of more than 84% of misexpressed genes. Finally, we used a series of N-terminal tail truncations of Arabidopsis HTA11 to reveal that the N-terminal tail of Arabidopsis H2A.Z is not necessary for normal plant development but does play an important role in mounting proper environmental stress responses. Article SummaryH2A.Z is a histone variant conserved across the eukaryotes that plays important roles in transcription. Despite high overall conservation of H2A.Z protein sequence between organisms, sequence variation is seen in the N-terminal region, a site for many posttranslational modifications implicated in H2A.Z function. We replaced Arabidopsis thaliana H2A.Z with each of the human H2A.Z orthologs and found that both human H2A.Z isoforms rescue the severe phenotypes of plant H2A.Z null mutants, despite divergent N-terminal sequences. We further analyzed N-terminal sequence requirements by progressively deleting the endogenous plant H2A.Z N-terminus. Plants with a tailless version of H2A.Z developed normally but were impaired in stress responsiveness. Our results indicate that human H2A.Zs are broadly functional in plants and that the N-terminus of H2A.Z is not essential for normal development but is implicated in stress responsiveness.

genetics↗

bioRxiv2021

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 present altered H3K27me3 modifications at a subset of PRC2 targets throughout the genome. 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. Author summaryPlants form organs continuously throughout their lives, and the number and shape of their organs is determined in a flexible manner according to the internal and external circumstances. Alongside this flexibility, plants maintain basic developmental programs to ensure proper functioning. Among the ways by which plants achieve flexible development is by tuning the pace of their maturation and differentiation, at both the plant and organ levels. One of the ways plants regulate the rate of maturation and differentiation is by changing gene expression. Here, we identified a gene that promotes plant and organ maturation and differentiation. This gene, CRAWLING ELEPHANT (CREL) acts by bringing a repressing complex to target genes. We show the importance of CREL in multiple developmental processes and in the expression of multiple genes throughout the tomato genome.

plant biology↗

bioRxiv2018

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 characterized in yeast and animals but had not been purified from plants. 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). Since mbd9 mutant plants were phenotypically similar to arp6 mutants, we further 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 regions normally enriched with H2A.Z. Genetic analyses showed that arp6;mbd9 double mutants have far more severe phenotypes than either single mutant. In conjunction with the finding that MBD9 does not appear to be a core subunit of the Arabidopsis SWR1 complex, this suggests that MBD9 also has important roles beyond H2A.Z deposition. Our data establish the SWR1 complex as being conserved across eukaryotes and also provide new insights into the mechanisms that target H2A.Z to chromatin.

plant biology↗

bioRxiv2018

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

Plants adapt to changes in their environment by regulating transcription and chromatin organization. The histone H2A variant H2A.Z and the SWI2/SNF2 ATPase BRAHMA have overlapping roles in positively and negatively regulating environmentally responsive genes in Arabidopsis, but the extent of this overlap was uncharacterized. Both 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 act both cooperatively and antagonistically to contribute directly to transcriptional repression and activation of genes involved in development and response to environmental stimuli. We identified 8 classes of genes that show distinct relationships between H2A.Z and BRM and their roles in transcription. We found that H2A.Z contributes to a range of different nucleosome properties, while BRM stabilizes nucleosomes where it binds and destabilizes and/or repositions flanking nucleosomes. H2A.Z and BRM contribute to +1 nucleosome destabilization, especially where they coordinately regulate transcription. We also found that at genes regulated by both BRM and H2A.Z, both factors overlap with the binding sites of light-regulated transcription factors PIF4, PIF5, and FRS9, and that some of the 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 their interrelated roles in chromatin organization. The variability observed in their individual functions implies that both BRM and H2A.Z have more context-specific roles within diverse chromatin environments than previously assumed.

plant biology↗

bioRxiv2017

Chromatin accessibility changes between Arabidopsis stem cells and mesophyll cells illuminate cell type-specific transcription factor networks

BackgroundCell differentiation is driven by changes in transcription factor (TF) activity and subsequent alterations in transcription. To study this process, differences in TF binding between cell types can be deduced by methods that probe chromatin accessibility. We used cell type-specific nuclei 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 of Arabidopsis thaliana.\n\nResultsChromatin accessibility profiles of SAM stem cells and leaf mesophyll cells were highly similar at a qualitative level, yet thousands of regions of quantitatively different chromatin accessibility were also identified. We found that chromatin regions preferentially accessible in mesophyll cells tended to also be substantially accessible in the stem cells as compared to the genome-wide average, whereas the converse was not true. Analysis of genomic regions preferentially accessible in each cell type identified hundreds of overrepresented TF binding motifs, highlighting a set of TFs that are likely important for each cell type. Among these, we found evidence for extensive co-regulation of target genes by multiple TFs that are preferentially expressed in one cell type or the other. For example, a set of zinc-finger TFs appear to control a suite of growth-and development-related genes specifically in stem cells, while another TF set co-regulates genes involved in light responses and photosynthesis specifically in mesophyll cells. Interestingly, the TFs within both of these sets also show evidence of extensively co-regulating each other.\n\nConclusionsQuantitative analysis of chromatin accessibility differences between stem cells and differentiated mesophyll cells allowed us to identify TF regulatory networks and downstream target genes that are likely to be functionally important in each cell type. Our findings that mesophyll cell-enriched accessible sites tend to already be substantially accessible in stem cells, but not vice versa, suggests that widespread regulatory element accessibility may be important for the developmental plasticity of stem cells. This work also demonstrates the utility of cell type-specific chromatin accessibility profiling in quickly developing testable models of regulatory control differences between cell types.

plant biology↗

bioRxiv2017

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.

plant biology↗