RESUMEN
Genome-wide phenotypic screens in the budding yeast Saccharomyces cerevisiae, enabled by its knockout collection, have produced the largest, richest, and most systematic phenotypic description of any organism. However, integrative analyses of this rich data source have been virtually impossible because of the lack of a central data repository and consistent metadata annotations. Here, we describe the aggregation, harmonization, and analysis of ~14,500 yeast knockout screens, which we call Yeast Phenome. Using this unique dataset, we characterized two unknown genes (YHR045W and YGL117W) and showed that tryptophan starvation is a by-product of many chemical treatments. Furthermore, we uncovered an exponential relationship between phenotypic similarity and intergenic distance, which suggests that gene positions in both yeast and human genomes are optimized for function.
Asunto(s)
Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genéticaRESUMEN
The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which > 5,600 yeast genes are engineered for transcriptional inducibility with single-gene precision at their native loci and without plasmids. Each strain contains SGA screening markers and a unique barcode, enabling high-throughput genetics. We characterized YETI using growth phenotyping and BAR-seq screens, and we used a YETI allele to identify the regulon of Rof1, showing that it acts to repress transcription. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from eukaryotic and prokaryotic systems shows that native expression is a variable that can bias promoter-perturbing screens, including CRISPRi. We engineered a second expression system, Z3 EB42, that gives lower expression than Z3 EV, a feature enabling conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.
Asunto(s)
Genes Esenciales , Saccharomyces cerevisiae , Biblioteca de Genes , Plásmidos , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genéticaRESUMEN
Plant xylem cells conduct water and mineral nutrients. Although most plant cells are totipotent, xylem cells are unusual and undergo terminal differentiation. Many genes regulating this process are well characterized, including the Vascular-related NAC Domain 7 (VND7), MYB46, and MYB83 transcription factors, which are proposed to act in interconnected feedforward loops (FFLs). Less is known regarding the molecular mechanisms underlying the terminal transition to xylem cell differentiation. Here, we generate whole-root and single-cell data, which demonstrate that VND7 initiates sharp switching of root cells to xylem cell identity. Based on these data, we identified 4 candidate VND7 downstream target genes capable of generating this switch. Although MYB46 responds to VND7 induction, it is not among these targets. This system provides an important model to study the emergent properties that may give rise to totipotency relative to terminal differentiation and reveals xylem cell subtypes.
Asunto(s)
Activación Transcripcional/fisiología , Xilema/metabolismo , Diferenciación Celular , PlantasRESUMEN
Single-cell transcriptome profiling of heterogeneous tissues can provide high-resolution windows into developmental dynamics and environmental responses, but its application to plants has been limited. Here, we used the high-throughput Drop-seq approach to profile >12,000 cells from Arabidopsis roots. This identified numerous distinct cell types, covering all major root tissues and developmental stages, and illuminated specific marker genes for these populations. In addition, we demonstrate the utility of this approach to study the impact of environmental conditions on developmental processes. Analysis of roots grown with or without sucrose supplementation uncovers changes in the relative frequencies of cell types in response to sucrose. Finally, we characterize the transcriptome changes that occur across endodermis development and identify nearly 800 genes with dynamic expression as this tissue differentiates. Collectively, we demonstrate that single-cell RNA-seq can be used to profile developmental processes in plants and show how they can be altered by external stimuli.
Asunto(s)
Arabidopsis/metabolismo , Perfilación de la Expresión Génica/métodos , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Células Vegetales/metabolismo , Raíces de Plantas/genética , Sacarosa/metabolismoRESUMEN
Plant secondary cell walls constitute the majority of plant biomass. They are predominantly found in xylem cells, which are derived from vascular initials during vascularization. Little is known about these processes in grass species despite their emerging importance as biomass feedstocks. The targeted biofuel crop Sorghum bicolor has a sequenced and well-annotated genome, making it an ideal monocot model for addressing vascularization and biomass deposition. Here we generated tissue-specific transcriptome and DNA methylome data from sorghum shoots, roots and developing root vascular and nonvascular tissues. Many genes associated with vascular development in other species show enriched expression in developing vasculature. However, several transcription factor families varied in vascular expression in sorghum compared with Arabidopsis and maize. Furthermore, differential expression of genes associated with DNA methylation were identified between vascular and nonvascular tissues, implying that changes in DNA methylation are a feature of sorghum root vascularization, which we confirmed using tissue-specific DNA methylome data. Roots treated with a DNA methylation inhibitor also showed a significant decrease in root length. Tissues and organs can be discriminated based on their genomic methylation patterns and methylation context. Consequently, tissue-specific changes in DNA methylation are part of the normal developmental process.
Asunto(s)
Metilación de ADN/genética , Regulación de la Expresión Génica de las Plantas , Haz Vascular de Plantas/genética , Sorghum/genética , Pared Celular/genética , Secuencia Conservada , Genes de Plantas , Raíces de Plantas/genética , Transcriptoma/genéticaRESUMEN
Tissue-specific gene expression is often thought to arise from spatially restricted transcriptional cascades. However, it is unclear how expression is established at the top of these cascades in the absence of pre-existing specificity. We generated a transcriptional network to explore how transcription factor expression is established in the Arabidopsis thaliana root ground tissue. Regulators of the SHORTROOT-SCARECROW transcriptional cascade were validated in planta. At the top of this cascade, we identified both activators and repressors of SHORTROOT. The aggregate spatial expression of these regulators is not sufficient to predict transcriptional specificity. Instead, modeling, transcriptional reporters, and synthetic promoters support a mechanism whereby expression at the top of the SHORTROOT-SCARECROW cascade is established through opposing activities of activators and repressors.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Redes Reguladoras de Genes , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Arabidopsis/crecimiento & desarrollo , Simulación por Computador , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Genes Reporteros , Genes Sintéticos , Modelos Genéticos , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
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.
Asunto(s)
Arabidopsis/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Complejo Represivo Polycomb 2/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
Conserved non-coding sequences (CNS) are islands of non-coding sequence that, like protein coding exons, show less divergence in sequence between related species than functionless DNA. Several CNSs have been demonstrated experimentally to function as cis-regulatory regions. However, the specific functions of most CNSs remain unknown. Previous searches for CNS in plants have either anchored on exons and only identified nearby sequences or required years of painstaking manual annotation. Here we present an open source tool that can accurately identify CNSs between any two related species with sequenced genomes, including both those immediately adjacent to exons and distal sequences separated by >12 kb of non-coding sequence. We have used this tool to characterize new motifs, associate CNSs with additional functions, and identify previously undetected genes encoding RNA and protein in the genomes of five grass species. We provide a list of 15,363 orthologous CNSs conserved across all grasses tested. We were also able to identify regulatory sequences present in the common ancestor of grasses that have been lost in one or more extant grass lineages. Lists of orthologous gene pairs and associated CNSs are provided for reference inbred lines of arabidopsis, Japonica rice, foxtail millet, sorghum, brachypodium, and maize.
RESUMEN
Unlike in mammals, plants rapidly delete functionless, nonrepetitive DNA from their genomes. Following paleopolyploidies, duplicate genes are deleted by intrachromosomal recombination. This may explain how flowering plants have survived multiple whole genome duplications. Genes are disproportionately lost from one parental subgenome, the subgenome that is less expressed in the polyploid. The origin of this unbalanced expression between genomes remains unknown. The consequences of the tradeoffs between transposon repression and gene expression represent one potential explanation of genome dominance. If so, the same mechanisms may act in heterosis: genome dominance is like inbreeding depression. Regulatory DNA deletion following polyploidy combined with abundant RNA-seq expression datasets are being used to generate testable hypothesizes regarding the function of specific cis-regulatory sequences.
Asunto(s)
ADN de Plantas/genética , Plantas/genética , Epigénesis Genética/genética , Regulación de la Expresión Génica de las Plantas/genética , Mutagénesis , PoliploidíaRESUMEN
The steroid and xenobiotic receptor (SXR) is a broad-specificity nuclear hormone receptor that is highly expressed in the liver and intestine, where its primary function is to regulate drug and xenobiotic metabolism. SXR is expressed at lower levels in other tissues, where little is known about its physiological functions. We previously linked SXR with immunity and inflammation by showing that SXR antagonizes the activity of nuclear factor (NF)-κB in vitro and in vivo. SXR(-/-) mice demonstrate aberrantly high NF-κB activity and overexpression of NF-κB target genes. Here we show that SXR(-/-) mice develop B cell lymphoma in an age-dependent manner. SXR(-/-) mice develop multiple hyperplastic lymphoid foci composed of B-1a cells in the intestine, spleen, lymph nodes, peritoneal cavity, and blood. In all circumstances, these lymphocytes possess cell surface and molecular characteristics of either chronic lymphocytic leukemia or non-Hodgkin's lymphoma originating from B-1 lymphocytes. These results demonstrate a novel and unsuspected role for SXR signaling in the B-1 cell compartment, establish SXR as a tumor suppressor in B-1 cells, and may provide a link between metabolism of xenobiotic compounds and lymphomagenesis.