RESUMO
Isogenic individuals can display seemingly stochastic phenotypic differences, limiting the accuracy of genotype-to-phenotype predictions. The extent of this phenotypic variation depends in part on genetic background, raising questions about the genes involved in controlling stochastic phenotypic variation. Focusing on early seedling traits in Arabidopsis thaliana, we found that hypomorphs of the cuticle-related gene LIPID TRANSFER PROTEIN 2 (LTP2) greatly increased variation in seedling phenotypes, including hypocotyl length, gravitropism and cuticle permeability. Many ltp2 hypocotyls were significantly shorter than wild-type hypocotyls while others resembled the wild-type. Differences in epidermal properties and gene expression between ltp2 seedlings with long and short hypocotyls suggest a loss of cuticle integrity as the primary determinant of the observed phenotypic variation. We identified environmental conditions that reveal or mask the increased variation in ltp2 hypomorphs and found that increased expression of its closest paralog LTP1 is necessary for ltp2 phenotypes. Our results illustrate how decreased expression of a single gene can generate starkly increased phenotypic variation in isogenic individuals in response to an environmental challenge.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Humanos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Interação Gene-Ambiente , Genótipo , Hipocótilo/metabolismo , Fenótipo , Plântula/genética , Plântula/metabolismoRESUMO
Single cell RNA sequencing can yield high-resolution cell-type-specific expression signatures that reveal new cell types and the developmental trajectories of cell lineages. Here, we apply this approach to Arabidopsis (Arabidopsis thaliana) root cells to capture gene expression in 3,121 root cells. We analyze these data with Monocle 3, which orders single cell transcriptomes in an unsupervised manner and uses machine learning to reconstruct single cell developmental trajectories along pseudotime. We identify hundreds of genes with cell-type-specific expression, with pseudotime analysis of several cell lineages revealing both known and novel genes that are expressed along a developmental trajectory. We identify transcription factor motifs that are enriched in early and late cells, together with the corresponding candidate transcription factors that likely drive the observed expression patterns. We assess and interpret changes in total RNA expression along developmental trajectories and show that trajectory branch points mark developmental decisions. Finally, by applying heat stress to whole seedlings, we address the longstanding question of possible heterogeneity among cell types in the response to an abiotic stress. Although the response of canonical heat-shock genes dominates expression across cell types, subtle but significant differences in other genes can be detected among cell types. Taken together, our results demonstrate that single cell transcriptomics holds promise for studying plant development and plant physiology with unprecedented resolution.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Transcriptoma , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Perfilação da Expressão Gênica , Resposta ao Choque Térmico , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Análise de Sequência de RNA , Análise de Célula Única , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Variation in regulatory DNA is thought to drive phenotypic variation, evolution, and disease. Prior studies of regulatory DNA and transcription factors across animal species highlighted a fundamental conundrum: Transcription factor binding domains and cognate binding sites are conserved, while regulatory DNA sequences are not. It remains unclear how conserved transcription factors and dynamic regulatory sites produce conserved expression patterns across species. Here, we explore regulatory DNA variation and its functional consequences within Arabidopsis thaliana, using chromatin accessibility to delineate regulatory DNA genome-wide. Unlike in previous cross-species comparisons, the positional homology of regulatory DNA is maintained among A. thaliana ecotypes and less nucleotide divergence has occurred. Of the â¼50,000 regulatory sites in A. thaliana, we found that 15% varied in accessibility among ecotypes. Some of these accessibility differences were associated with extensive, previously unannotated sequence variation, encompassing many deletions and ancient hypervariable alleles. Unexpectedly, for the majority of such regulatory sites, nearby gene expression was unaffected. Nevertheless, regulatory sites with high levels of sequence variation and differential chromatin accessibility were the most likely to be associated with differential gene expression. Finally, and most surprising, we found that the vast majority of differentially accessible sites show no underlying sequence variation. We argue that these surprising results highlight the necessity to consider higher-order regulatory context in evaluating regulatory variation and predicting its phenotypic consequences.
Assuntos
Arabidopsis/genética , Ecótipo , Elementos Reguladores de Transcrição , Arabidopsis/metabolismo , Sequência de Bases , Desoxirribonuclease I , Variação Estrutural do Genoma , Análise de Sequência de DNARESUMO
The well-studied DNA replication origins of the model budding and fission yeasts are A/T-rich elements. However, unlike their yeast counterparts, both plant and metazoan origins are G/C-rich and are associated with transcription start sites. Here we show that an industrially important methylotrophic budding yeast, Pichia pastoris, simultaneously employs at least two types of replication origins--a G/C-rich type associated with transcription start sites and an A/T-rich type more reminiscent of typical budding and fission yeast origins. We used a suite of massively parallel sequencing tools to map and dissect P. pastoris origins comprehensively, to measure their replication dynamics, and to assay the global positioning of nucleosomes across the genome. Our results suggest that some functional overlap exists between promoter sequences and G/C-rich replication origins in P. pastoris and imply an evolutionary bifurcation of the modes of replication initiation.
Assuntos
Replicação do DNA/genética , DNA/genética , Pichia/genética , Origem de Replicação/genética , Cromatina/genética , Sequência Rica em GC/genética , Sequenciamento de Nucleotídeos em Larga Escala , Nucleossomos/genética , Sítio de Iniciação de TranscriçãoRESUMO
Since their initial discovery in maize, transposable elements (TEs) have emerged as being integral to the evolution of maize, accounting for 80% of its genome. However, the repetitive nature of TEs has hindered our understanding of their regulatory potential. Here, we demonstrate that long-read chromatin fiber sequencing (Fiber-seq) permits the comprehensive annotation of the regulatory potential of maize TEs. We uncover that only 94 LTR retrotransposons contain the functional epigenetic architecture required for mobilization within maize leaves. This epigenetic architecture degenerates with evolutionary age, resulting in solo TE enhancers being preferentially marked by simultaneous hyper-CpG methylation and chromatin accessibility, an architecture markedly divergent from canonical enhancers. We find that TEs shape maize gene regulation by creating novel promoters within the TE itself as well as through TE-mediated gene amplification. Lastly, we uncover a pervasive epigenetic code directing TEs to specific loci, including that locus that sparked McClintock's discovery of TEs.
RESUMO
Argonaute 1 (AGO1), the principal protein component of microRNA-mediated regulation, plays a key role in plant growth and development. AGO1 physically interacts with the chaperone HSP90, which buffers cryptic genetic variation in plants and animals. We sought to determine whether genetic perturbation of AGO1 in Arabidopsis thaliana would also reveal cryptic genetic variation, and if so, whether AGO1-dependent loci overlap with those dependent on HSP90. To address these questions, we introgressed a hypomorphic mutant allele of AGO1 into a set of mapping lines derived from the commonly used Arabidopsis strains Col-0 and Ler. Although we identified several cases in which AGO1 buffered genetic variation, none of the AGO1-dependent loci overlapped with those buffered by HSP90 for the traits assayed. We focused on 1 buffered locus where AGO1 perturbation uncoupled the traits days to flowering and rosette leaf number, which are otherwise closely correlated. Using a bulk segregant approach, we identified a nonfunctional Ler hua2 mutant allele as the causal AGO1-buffered polymorphism. Introduction of a nonfunctional hua2 allele into a Col-0 ago1 mutant background recapitulated the Ler-dependent ago1 phenotype, implying that coupling of these traits involves different molecular players in these closely related strains. Taken together, our findings demonstrate that even though AGO1 and HSP90 buffer genetic variation in the same traits, these robustness regulators interact epistatically with different genetic loci, suggesting that higher-order epistasis is uncommon. Plain Language Summary Argonaute 1 (AGO1), a key player in plant development, interacts with the chaperone HSP90, which buffers environmental and genetic variation. We found that AGO1 buffers environmental and genetic variation in the same traits; however, AGO1-dependent and HSP90-dependent loci do not overlap. Detailed analysis of a buffered locus found that a nonfunctional HUA2 allele decouples days to flowering and rosette leaf number in an AGO1-dependent manner, suggesting that the AGO1-dependent buffering acts at the network level.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Animais , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fenótipo , Alelos , Folhas de Planta/metabolismo , Variação Genética , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismoRESUMO
Isogenic individuals can display seemingly stochastic phenotypic differences, limiting the accuracy of genotype-to-phenotype predictions. The extent of this phenotypic variation depends in part on genetic background, raising questions about the genes involved in controlling stochastic phenotypic variation. Focusing on early seedling traits in Arabidopsis thaliana, we found that hypomorphs of the cuticle-related gene LTP2 greatly increased variation in seedling phenotypes, including hypocotyl length, gravitropism and cuticle permeability. Many ltp2 hypocotyls were significantly shorter than wild-type hypocotyls while others resembled the wild type. Differences in epidermal properties and gene expression between ltp2 seedlings with long and short hypocotyls suggest a loss of cuticle integrity as the primary determinant of the observed phenotypic variation. We identified environmental conditions that reveal or mask the increased variation in ltp2 hypomorphs, and found that increased expression of its closest paralog LTP1 is necessary for ltp2 phenotypes. Our results illustrate how decreased expression of a single gene can generate starkly increased phenotypic variation in isogenic individuals in response to an environmental challenge.
RESUMO
Transcriptional control is exerted primarily through the binding of transcription factor proteins to regulatory elements in DNA. By virtue of eukaryotic DNA being complexed with histones, transcription factor binding to DNA alters or eliminates histone-DNA contacts, leading to increased accessibility of the DNA region to nuclease enzymes. This hypersensitivity to nuclease digestion has been used to define DNA binding events and regulatory elements across genomes, and to compare these attributes between cell types or conditions. These approaches make it possible to define the regulatory elements in a genome as well as to predict the regulatory networks of transcription factors and their target genes in a given cell state. As these chromatin accessibility assays are increasingly used, it is important to consider how to analyze the resulting data to avoid artifactual results or misinterpretation. In this review, we focus on some of the key technical and computational caveats associated with plant chromatin accessibility data, including strategies for sample preparation, sequencing, read mapping, and downstream analyses.
Assuntos
Cromatina , Histonas , DNA , Ligação Proteica , Fatores de TranscriçãoRESUMO
Amino acid substitutions are commonly found in human transcription factors, yet the functional consequences of much of this variation remain unknown, even in well-characterized DNA-binding domains. Here, we examine how six single-amino acid variants in the DNA-binding domain of Ste12-a yeast transcription factor regulating mating and invasion-alter Ste12 genome binding, motif recognition, and gene expression to yield markedly different phenotypes. Using a combination of the "calling-card" method, RNA sequencing, and HT-SELEX (high throughput systematic evolution of ligands by exponential enrichment), we find that variants with dissimilar binding and expression profiles can converge onto similar cellular behaviors. Mating-defective variants led to decreased expression of distinct subsets of genes necessary for mating. Hyper-invasive variants also decreased expression of subsets of genes involved in mating, but increased the expression of other subsets of genes associated with the cellular response to osmotic stress. While single-amino acid changes in the coding region of this transcription factor result in complex regulatory reconfiguration, the major phenotypic consequences for the cell appear to depend on changes in the expression of a small number of genes with related functions.
Assuntos
Regulação Fúngica da Expressão Gênica/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Substituição de Aminoácidos/genética , Sequência de Bases/genética , Proteínas de Ligação a DNA/genética , Fenótipo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
The genome is reprogrammed during development to produce diverse cell types, largely through altered expression and activity of key transcription factors. The accessibility and critical functions of epidermal cells have made them a model for connecting transcriptional events to development in a range of model systems. In Arabidopsis thaliana and many other plants, fertilization triggers differentiation of specialized epidermal seed coat cells that have a unique morphology caused by large extracellular deposits of polysaccharides. Here, we used DNase I-seq to generate regulatory landscapes of A. thaliana seeds at two critical time points in seed coat maturation (4 and 7 DPA), enriching for seed coat cells with the INTACT method. We found over 3,000 developmentally dynamic regulatory DNA elements and explored their relationship with nearby gene expression. The dynamic regulatory elements were enriched for motifs for several transcription factors families; most notably the TCP family at the earlier time point and the MYB family at the later one. To assess the extent to which the observed regulatory sites in seeds added to previously known regulatory sites in A. thaliana, we compared our data to 11 other data sets generated with 7-day-old seedlings for diverse tissues and conditions. Surprisingly, over a quarter of the regulatory, i.e. accessible, bases observed in seeds were novel. Notably, plant regulatory landscapes from different tissues, cell types, or developmental stages were more dynamic than those generated from bulk tissue in response to environmental perturbations, highlighting the importance of extending studies of regulatory DNA to single tissues and cell types during development.
RESUMO
Our understanding of gene regulation in plants is constrained by our limited knowledge of plant cis-regulatory DNA and its dynamics. We mapped DNase I hypersensitive sites (DHSs) in A. thaliana seedlings and used genomic footprinting to delineate â¼ 700,000 sites of in vivo transcription factor (TF) occupancy at nucleotide resolution. We show that variation associated with 72 diverse quantitative phenotypes localizes within DHSs. TF footprints encode an extensive cis-regulatory lexicon subject to recent evolutionary pressures, and widespread TF binding within exons may have shaped codon usage patterns. The architecture of A. thaliana TF regulatory networks is strikingly similar to that of animals in spite of diverged regulatory repertoires. We analyzed regulatory landscape dynamics during heat shock and photomorphogenesis, disclosing thousands of environmentally sensitive elements and enabling mapping of key TF regulatory circuits underlying these fundamental responses. Our results provide an extensive resource for the study of A. thaliana gene regulation and functional biology.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Mapeamento Cromossômico , Códon , Desoxirribonuclease I/metabolismo , Éxons , Redes Reguladoras de Genes , Genoma de Planta , Estudo de Associação Genômica Ampla , Luz , Desenvolvimento Vegetal/genética , Ligação Proteica , Elementos Reguladores de Transcrição/genética , Plântula/genética , Fatores de Transcrição/metabolismoRESUMO
DNA methylation is an epigenetic mark associated with transposable element silencing and gene imprinting in flowering plants and mammals. In plants, imprinting occurs in the endosperm, which nourishes the embryo during seed development. We have profiled Arabidopsis DNA methylation genome-wide in the embryo and endosperm and found that large-scale methylation changes accompany endosperm development and endosperm-specific gene expression. Transposable element fragments are extensively demethylated in the endosperm. We discovered new imprinted genes by the identification of candidates associated with regions of reduced endosperm methylation and preferential expression in endosperm relative to other parts of the plant. These data suggest that imprinting in plants evolved from targeted methylation of transposable element insertions near genic regulatory elements followed by positive selection when the resulting expression change was advantageous.
Assuntos
Arabidopsis/embriologia , Arabidopsis/genética , Metilação de DNA , Elementos de DNA Transponíveis , Impressão Genômica , Sequências Repetitivas de Ácido Nucleico , Sementes/genética , Alelos , Arabidopsis/crescimento & desenvolvimento , Cruzamentos Genéticos , DNA de Plantas/genética , DNA de Plantas/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genoma de Planta , Sementes/crescimento & desenvolvimento , Seleção GenéticaRESUMO
Comparisons between haplotypes from affected patients and the human reference genome are frequently used to identify candidates for disease-causing mutations, even though these alignments are expected to reveal a high level of background neutral polymorphism. This limits the scope of genetic studies to relatively small genomic intervals, because current methods for distinguishing potential causal mutations from neutral variation are inefficient. Here we describe a new strategy for detecting mutations that is based on comparing affected haplotypes with closely matched control sequences from healthy individuals, rather than with the human reference genome. We use theory, simulation, and a real data set to show that this approach is expected to reduce the number of sequence variants that must be subjected to follow-up analysis by at least a factor of 20 when closely matched control sequences are selected from a reference panel with as few as 100 control genomes. We also define a reference data resource that would allow efficient application of this strategy to large critical intervals across the genome.