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1.
Synth Biol (Oxf) ; 8(1): ysad005, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37073283

RESUMEN

Computational tools addressing various components of design-build-test-learn (DBTL) loops for the construction of synthetic genetic networks exist but do not generally cover the entire DBTL loop. This manuscript introduces an end-to-end sequence of tools that together form a DBTL loop called Design Assemble Round Trip (DART). DART provides rational selection and refinement of genetic parts to construct and test a circuit. Computational support for experimental process, metadata management, standardized data collection and reproducible data analysis is provided via the previously published Round Trip (RT) test-learn loop. The primary focus of this work is on the Design Assemble (DA) part of the tool chain, which improves on previous techniques by screening up to thousands of network topologies for robust performance using a novel robustness score derived from dynamical behavior based on circuit topology only. In addition, novel experimental support software is introduced for the assembly of genetic circuits. A complete design-through-analysis sequence is presented using several OR and NOR circuit designs, with and without structural redundancy, that are implemented in budding yeast. The execution of DART tested the predictions of the design tools, specifically with regard to robust and reproducible performance under different experimental conditions. The data analysis depended on a novel application of machine learning techniques to segment bimodal flow cytometry distributions. Evidence is presented that, in some cases, a more complex build may impart more robustness and reproducibility across experimental conditions. Graphical Abstract.

2.
Synth Biol (Oxf) ; 7(1): ysac018, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36285185

RESUMEN

We describe an experimental campaign that replicated the performance assessment of logic gates engineered into cells of Saccharomyces cerevisiae by Gander et al. Our experimental campaign used a novel high-throughput experimentation framework developed under Defense Advanced Research Projects Agency's Synergistic Discovery and Design program: a remote robotic lab at Strateos executed a parameterized experimental protocol. Using this protocol and robotic execution, we generated two orders of magnitude more flow cytometry data than the original experiments. We discuss our results, which largely, but not completely, agree with the original report and make some remarks about lessons learned. Graphical Abstract.

3.
ACS Synth Biol ; 11(2): 608-622, 2022 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-35099189

RESUMEN

Synthetic biology is a complex discipline that involves creating detailed, purpose-built designs from genetic parts. This process is often phrased as a Design-Build-Test-Learn loop, where iterative design improvements can be made, implemented, measured, and analyzed. Automation can potentially improve both the end-to-end duration of the process and the utility of data produced by the process. One of the most important considerations for the development of effective automation and quality data is a rigorous description of implicit knowledge encoded as a formal knowledge representation. The development of knowledge representation for the process poses a number of challenges, including developing effective human-machine interfaces, protecting against and repairing user error, providing flexibility for terminological mismatches, and supporting extensibility to new experimental types. We address these challenges with the DARPA SD2 Round Trip software architecture. The Round Trip is an open architecture that automates many of the key steps in the Test and Learn phases of a Design-Build-Test-Learn loop for high-throughput laboratory science. The primary contribution of the Round Trip is to assist with and otherwise automate metadata creation, curation, standardization, and linkage with experimental data. The Round Trip's focus on metadata supports fast, automated, and replicable analysis of experiments as well as experimental situational awareness and experimental interpretability. We highlight the major software components and data representations that enable the Round Trip to speed up the design and analysis of experiments by 2 orders of magnitude over prior ad hoc methods. These contributions support a number of experimental protocols and experimental types, demonstrating the Round Trip's breadth and extensibility. We describe both an illustrative use case using the Round Trip for an on-the-loop experimental campaign and overall contributions to reducing experimental analysis time and increasing data product volume in the SD2 program.


Asunto(s)
Proyectos de Investigación , Programas Informáticos , Automatización/métodos , Humanos , Estándares de Referencia , Biología Sintética/métodos
4.
PLoS Genet ; 16(10): e1008623, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-33052904

RESUMEN

Plant cells undergo two types of cell cycles-the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2'-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Comparison of the sequence-based RT profiles showed that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle and the fact that endocycling is typically associated with cell differentiation. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small with a median size of 135 kb, shift to a later RT in the endocycle, and are enriched for genes expressed in the root tip. We found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere, which ranged between 1 and 2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells but primarily in late S phase of the endocycle. In contrast, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in 8C vs 2C nuclei suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and possible reduction in CENH3 enrichment after endocycle replication is consistent with a hypothesis that centromeres are inactivated when their function is no longer needed.


Asunto(s)
Momento de Replicación del ADN/genética , Replicación del ADN/efectos de los fármacos , Raíces de Plantas/genética , Zea mays/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Centrómero/efectos de los fármacos , Centrómero/genética , Replicación del ADN/genética , Momento de Replicación del ADN/efectos de los fármacos , ADN de Plantas/efectos de los fármacos , ADN de Plantas/genética , Desoxiuridina/análogos & derivados , Desoxiuridina/farmacología , Endocitosis/efectos de los fármacos , Meristema/efectos de los fármacos , Meristema/genética , Mitosis/efectos de los fármacos , Mitosis/genética , Nucleosomas/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Fase S/genética , Zea mays/crecimiento & desarrollo
5.
Plant Physiol ; 183(1): 206-220, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32205451

RESUMEN

The selection and firing of DNA replication origins play key roles in ensuring that eukaryotes accurately replicate their genomes. This process is not well documented in plants due in large measure to difficulties in working with plant systems. We developed a new functional assay to label and map very early replicating loci that must, by definition, include at least a subset of replication origins. Arabidopsis (Arabidopsis thaliana) cells were briefly labeled with 5-ethynyl-2'-deoxy-uridine, and nuclei were subjected to two-parameter flow sorting. We identified more than 5500 loci as initiation regions (IRs), the first regions to replicate in very early S phase. These were classified as strong or weak IRs based on the strength of their replication signals. Strong initiation regions were evenly spaced along chromosomal arms and depleted in centromeres, while weak initiation regions were enriched in centromeric regions. IRs are AT-rich sequences flanked by more GC-rich regions and located predominantly in intergenic regions. Nuclease sensitivity assays indicated that IRs are associated with accessible chromatin. Based on these observations, initiation of plant DNA replication shows some similarity to, but is also distinct from, initiation in other well-studied eukaryotic systems.


Asunto(s)
Arabidopsis/metabolismo , Cromatina/metabolismo , ADN de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Replicación del ADN/genética , Replicación del ADN/fisiología , ADN de Plantas/fisiología , Origen de Réplica/genética , Origen de Réplica/fisiología
6.
Plant J ; 100(3): 641-654, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31350781

RESUMEN

Improvements in next-generation sequencing technologies have resulted in dramatically reduced sequencing costs. This has led to an explosion of '-seq'-based methods, of which RNA sequencing (RNA-seq) for generating transcriptomic data is the most popular. By analysing global patterns of gene expression in organs/tissues/cells of interest or in response to chemical or environmental perturbations, researchers can better understand an organism's biology. Tools designed to work with large RNA-seq data sets enable analyses and visualizations to help generate hypotheses about a gene's function. We present here a user-friendly RNA-seq data exploration tool, called the 'eFP-Seq Browser', that shows the read map coverage of a gene of interest in each of the samples along with 'electronic fluorescent pictographic' (eFP) images that serve as visual representations of expression levels. The tool also summarizes the details of each RNA-seq experiment, providing links to archival databases and publications. It automatically computes the reads per kilobase per million reads mapped expression-level summaries and point biserial correlation scores to sort the samples based on a gene's expression level or by how dissimilar the read map profile is from a gene splice variant, to quickly identify samples with the strongest expression level or where alternative splicing might be occurring. Links to the Integrated Genome Browser desktop visualization tool allow researchers to visualize and explore the details of RNA-seq alignments summarized in eFP-Seq Browser as coverage graphs. We present four cases of use of the eFP-Seq Browser for ABI3, SR34, SR45a and U2AF65B, where we examine expression levels and identify alternative splicing. The URL for the browser is https://bar.utoronto.ca/eFP-Seq_Browser/. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. Tool is at https://bar.utoronto.ca/eFP-Seq_Browser/; RNA-seq data at https://s3.amazonaws.com/iplant-cdn/iplant/home/araport/rnaseq_bam/ and https://s3.amazonaws.com/iplant-cdn/iplant/home/araport/rnaseq_bam/Klepikova/. Code is available at https://github.com/BioAnalyticResource/eFP-Seq-Browser.


Asunto(s)
Arabidopsis/genética , Visualización de Datos , Genoma de Planta/genética , Transcriptoma , Navegador Web , Empalme Alternativo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , ARN de Planta/genética , Alineación de Secuencia , Análisis de Secuencia de ARN , Estrés Fisiológico , Temperatura
7.
G3 (Bethesda) ; 8(6): 1921-1932, 2018 05 31.
Artículo en Inglés | MEDLINE | ID: mdl-29618467

RESUMEN

DNA methylation is a chromatin modification that can provide epigenetic regulation of gene and transposon expression. Plants utilize several pathways to establish and maintain DNA methylation in specific sequence contexts. The chromomethylase (CMT) genes maintain CHG (where H = A, C or T) methylation. The RNA-directed DNA methylation (RdDM) pathway is important for CHH methylation. Transcriptome analysis was performed in a collection of Zea mays lines carrying mutant alleles for CMT or RdDM-associated genes. While the majority of the transcriptome was not affected, we identified sets of genes and transposon families sensitive to context-specific decreases in DNA methylation in mutant lines. Many of the genes that are up-regulated in CMT mutant lines have high levels of CHG methylation, while genes that are differentially expressed in RdDM mutants are enriched for having nearby mCHH islands, implicating context-specific DNA methylation in the regulation of expression for a small number of genes. Many genes regulated by CMTs exhibit natural variation for DNA methylation and transcript abundance in a panel of diverse inbred lines. Transposon families with differential expression in the mutant genotypes show few defining features, though several families up-regulated in RdDM mutants show enriched expression in endosperm tissue, highlighting the potential importance for this pathway during reproduction. Taken together, our findings suggest that while the number of genes and transposon families whose expression is reproducibly affected by mild perturbations in context-specific methylation is small, there are distinct patterns for loci impacted by RdDM and CMT mutants.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/genética , Elementos Transponibles de ADN/genética , Silenciador del Gen , Genes de Plantas , ARN de Planta/genética , Zea mays/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Sitios Genéticos , Mutación/genética , ARN de Planta/metabolismo , Regulación hacia Arriba/genética
8.
IEEE J Biomed Health Inform ; 22(2): 318-324, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29505399

RESUMEN

Biomedical data are quickly growing in volume and in variety, providing clinicians an opportunity for better clinical decision support. Here, we demonstrate a robust platform that uses software automation and high performance computing (HPC) resources to achieve real-time analytics of clinical data, specifically magnetic resonance imaging (MRI) data. We used the Agave application programming interface to facilitate communication, data transfer, and job control between an MRI scanner and an off-site HPC resource. In this use case, Agave executed the graphical pipeline tool GRAphical Pipeline Environment (GRAPE) to perform automated, real-time, quantitative analysis of MRI scans. Same-session image processing will open the door for adaptive scanning and real-time quality control, potentially accelerating the discovery of pathologies and minimizing patient callbacks. We envision this platform can be adapted to other medical instruments, HPC resources, and analytics tools.


Asunto(s)
Metodologías Computacionales , Interpretación de Imagen Asistida por Computador/métodos , Programas Informáticos , Humanos , Imagen por Resonancia Magnética
9.
Plant Physiol ; 176(3): 2166-2185, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29301956

RESUMEN

Eukaryotes use a temporally regulated process, known as the replication timing program, to ensure that their genomes are fully and accurately duplicated during S phase. Replication timing programs are predictive of genomic features and activity and are considered to be functional readouts of chromatin organization. Although replication timing programs have been described for yeast and animal systems, much less is known about the temporal regulation of plant DNA replication or its relationship to genome sequence and chromatin structure. We used the thymidine analog, 5-ethynyl-2'-deoxyuridine, in combination with flow sorting and Repli-Seq to describe, at high-resolution, the genome-wide replication timing program for Arabidopsis (Arabidopsis thaliana) Col-0 suspension cells. We identified genomic regions that replicate predominantly during early, mid, and late S phase, and correlated these regions with genomic features and with data for chromatin state, accessibility, and long-distance interaction. Arabidopsis chromosome arms tend to replicate early while pericentromeric regions replicate late. Early and mid-replicating regions are gene-rich and predominantly euchromatic, while late regions are rich in transposable elements and primarily heterochromatic. However, the distribution of chromatin states across the different times is complex, with each replication time corresponding to a mixture of states. Early and mid-replicating sequences interact with each other and not with late sequences, but early regions are more accessible than mid regions. The replication timing program in Arabidopsis reflects a bipartite genomic organization with early/mid-replicating regions and late regions forming separate, noninteracting compartments. The temporal order of DNA replication within the early/mid compartment may be modulated largely by chromatin accessibility.


Asunto(s)
Arabidopsis/genética , Cromatina/genética , Cromosomas de las Plantas , Momento de Replicación del ADN , Cromatina/metabolismo , Elementos Transponibles de ADN , Citometría de Flujo , Genoma de Planta , Estudio de Asociación del Genoma Completo , Fase S/genética , Análisis de Secuencia de ADN/métodos
10.
Plant Cell ; 29(9): 2126-2149, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28842533

RESUMEN

All plants and animals must replicate their DNA, using a regulated process to ensure that their genomes are completely and accurately replicated. DNA replication timing programs have been extensively studied in yeast and animal systems, but much less is known about the replication programs of plants. We report a novel adaptation of the "Repli-seq" assay for use in intact root tips of maize (Zea mays) that includes several different cell lineages and present whole-genome replication timing profiles from cells in early, mid, and late S phase of the mitotic cell cycle. Maize root tips have a complex replication timing program, including regions of distinct early, mid, and late S replication that each constitute between 20 and 24% of the genome, as well as other loci corresponding to ∼32% of the genome that exhibit replication activity in two different time windows. Analyses of genomic, transcriptional, and chromatin features of the euchromatic portion of the maize genome provide evidence for a gradient of early replicating, open chromatin that transitions gradually to less open and less transcriptionally active chromatin replicating in mid S phase. Our genomic level analysis also demonstrated that the centromere core replicates in mid S, before heavily compacted classical heterochromatin, including pericentromeres and knobs, which replicate during late S phase.


Asunto(s)
Momento de Replicación del ADN/genética , Genómica , Meristema/citología , Meristema/genética , Mitosis/genética , Fase S/genética , Zea mays/citología , Zea mays/genética , Secuencia de Bases , Cromosomas de las Plantas/genética , Elementos Transponibles de ADN/genética , Genes de Plantas , Modelos Genéticos , Secuencias Repetidas en Tándem/genética , Factores de Tiempo , Transcripción Genética
11.
BMC Bioinformatics ; 18(1): 362, 2017 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-28784090

RESUMEN

BACKGROUND: Replication timing experiments that use label incorporation and high throughput sequencing produce peaked data similar to ChIP-Seq experiments. However, the differences in experimental design, coverage density, and possible results make traditional ChIP-Seq analysis methods inappropriate for use with replication timing. RESULTS: To accurately detect and classify regions of replication across the genome, we present Repliscan. Repliscan robustly normalizes, automatically removes outlying and uninformative data points, and classifies Repli-seq signals into discrete combinations of replication signatures. The quality control steps and self-fitting methods make Repliscan generally applicable and more robust than previous methods that classify regions based on thresholds. CONCLUSIONS: Repliscan is simple and effective to use on organisms with different genome sizes. Even with analysis window sizes as small as 1 kilobase, reliable profiles can be generated with as little as 2.4x coverage.


Asunto(s)
Momento de Replicación del ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Genoma , Tamaño del Genoma
12.
Plant Cell Physiol ; 58(1): e4, 2017 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-28013278

RESUMEN

ThaleMine (https://apps.araport.org/thalemine/) is a comprehensive data warehouse that integrates a wide array of genomic information of the model plant Arabidopsis thaliana. The data collection currently includes the latest structural and functional annotation from the Araport11 update, the Col-0 genome sequence, RNA-seq and array expression, co-expression, protein interactions, homologs, pathways, publications, alleles, germplasm and phenotypes. The data are collected from a wide variety of public resources. Users can browse gene-specific data through Gene Report pages, identify and create gene lists based on experiments or indexed keywords, and run GO enrichment analysis to investigate the biological significance of selected gene sets. Developed by the Arabidopsis Information Portal project (Araport, https://www.araport.org/), ThaleMine uses the InterMine software framework, which builds well-structured data, and provides powerful data query and analysis functionality. The warehoused data can be accessed by users via graphical interfaces, as well as programmatically via web-services. Here we describe recent developments in ThaleMine including new features and extensions, and discuss future improvements. InterMine has been broadly adopted by the model organism research community including nematode, rat, mouse, zebrafish, budding yeast, the modENCODE project, as well as being used for human data. ThaleMine is the first InterMine developed for a plant model. As additional new plant InterMines are developed by the legume and other plant research communities, the potential of cross-organism integrative data analysis will be further enabled.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/genética , Proteínas de Arabidopsis/metabolismo , Biología Computacional/métodos , Ontología de Genes , Genómica/métodos , Almacenamiento y Recuperación de la Información/métodos , Internet , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/genética , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN
13.
Curr Protoc Plant Biol ; 1(3): 510-529, 2016 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31725961

RESUMEN

Epigenetic modification of DNA through methylation is known to be involved in multiple biological processes such as gene suppression. However, the exact mechanism of how DNA methylations play their part is yet unclear. In mammals, CpG islands (CGI) have been studied extensively for their involvement in cancer. Whereas in plants, despite the fact that there are not only CpG but also CHG and CHH contexts of methylation, an efficient and easy-to-use pipeline to decipher these phenomena is still to be developed. Both ZED-align and BisuKit are user-friendly apps deployed on CyVerse infrastructure where users can use their bisulfite sequence files to run multiple command line-based packages with minimal intervention. © 2016 by John Wiley & Sons, Inc.

14.
Proc Natl Acad Sci U S A ; 112(47): 14728-33, 2015 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-26553984

RESUMEN

The maize genome is relatively large (∼ 2.3 Gb) and has a complex organization of interspersed genes and transposable elements, which necessitates frequent boundaries between different types of chromatin. The examination of maize genes and conserved noncoding sequences revealed that many of these are flanked by regions of elevated asymmetric CHH (where H is A, C, or T) methylation (termed mCHH islands). These mCHH islands are quite short (∼ 100 bp), are enriched near active genes, and often occur at the edge of the transposon that is located nearest to genes. The analysis of DNA methylation in other sequence contexts and several chromatin modifications revealed that mCHH islands mark the transition from heterochromatin-associated modifications to euchromatin-associated modifications. The presence of an mCHH island is fairly consistent in several distinct tissues that were surveyed but shows some variation among different haplotypes. The presence of insertion/deletions in promoters often influences the presence and position of an mCHH island. The mCHH islands are dependent upon RNA-directed DNA methylation activities and are lost in mop1 and mop3 mutants, but the nearby genes rarely exhibit altered expression levels. Instead, loss of an mCHH island is often accompanied by additional loss of DNA methylation in CG and CHG contexts associated with heterochromatin in nearby transposons. This suggests that mCHH islands and RNA-directed DNA methylation near maize genes may act to preserve the silencing of transposons from activity of nearby genes.


Asunto(s)
Metilación de ADN/genética , Eucromatina/genética , Genoma de Planta , Heterocromatina/genética , ARN de Planta/metabolismo , Zea mays/genética , Secuencia Conservada/genética , Islas de CpG/genética , ADN Intergénico/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Genotipo , Mutación INDEL/genética , Secuencias Invertidas Repetidas/genética , Sitio de Iniciación de la Transcripción
15.
Plant Physiol ; 168(4): 1262-74, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25869653

RESUMEN

DNA methylation is a stable modification of chromatin that can contribute to epigenetic variation through the regulation of genes or transposons. Profiling of DNA methylation in five maize (Zea mays) inbred lines found that while DNA methylation levels for more than 99% of the analyzed genomic regions are similar, there are still 5,000 to 20,000 context-specific differentially methylated regions (DMRs) between any two genotypes. The analysis of identical-by-state genomic regions that have limited genetic variation provided evidence that DMRs can occur without local sequence variation, but they are less common than in regions with genetic variation. Characterization of the sequence specificity of DMRs, location of DMRs relative to genes and transposons, and patterns of DNA methylation in regions flanking DMRs reveals a distinct subset of DMRs. Transcriptome profiling of the same tissue revealed that only approximately 20% of genes with qualitative (on-off) differences in gene expression are associated with DMRs, and there is little evidence for association of DMRs with genes that show quantitative differences in gene expression. We also identify a set of genes that may represent cryptic information that is silenced by DNA methylation in the reference B73 genome. Many of these genes exhibit natural variation in other genotypes, suggesting the potential for selection to act upon existing epigenetic natural variation. This study provides insights into the origin and influences of DMRs in a crop species with a complex genome organization.


Asunto(s)
Metilación de ADN , Epigénesis Genética , Variación Genética , Genoma de Planta/genética , Zea mays/genética , Cruzamiento , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genotipo
16.
Plant Cell ; 26(12): 4602-16, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25527708

RESUMEN

DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana.


Asunto(s)
Metilación de ADN , Regulación de la Expresión Génica de las Plantas , Zea mays/genética , Alelos , Cruzamientos Genéticos , ADN (Citosina-5-)-Metiltransferasas/genética , Epigenómica , Genes de Plantas , Mutación
17.
PLoS One ; 9(8): e105267, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25122127

RESUMEN

DNA methylation and dimethylation of lysine 9 of histone H3 (H3K9me2) are two chromatin modifications that can be associated with gene expression or recombination rate. The maize genome provides a complex landscape of interspersed genes and transposons. The genome-wide distribution of DNA methylation and H3K9me2 were investigated in seedling tissue for the maize inbred B73 and compared to patterns of these modifications observed in Arabidopsis thaliana. Most maize transposons are highly enriched for DNA methylation in CG and CHG contexts and for H3K9me2. In contrast to findings in Arabidopsis, maize CHH levels in transposons are generally low but some sub-families of transposons are enriched for CHH methylation and these families exhibit low levels of H3K9me2. The profile of modifications over genes reveals that DNA methylation and H3K9me2 is quite low near the beginning and end of genes. Although elevated CG and CHG methylation are found within gene bodies, CHH and H3K9me2 remain low. Maize has much higher levels of CHG methylation within gene bodies than observed in Arabidopsis and this is partially attributable to the presence of transposons within introns for some maize genes. These transposons are associated with high levels of CHG methylation and H3K9me2 but do not appear to prevent transcriptional elongation. Although the general trend is for a strong depletion of H3K9me2 and CHG near the transcription start site there are some putative genes that have high levels of these chromatin modifications. This study provides a clear view of the relationship between DNA methylation and H3K9me2 in the maize genome and how the distribution of these modifications is shaped by the interplay of genes and transposons.


Asunto(s)
Metilación de ADN , Genoma de Planta , Histonas/metabolismo , Zea mays/genética , Zea mays/metabolismo , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Elementos Transponibles de ADN , Epigénesis Genética , Evolución Molecular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Intrones , Regiones Promotoras Genéticas , Sitio de Iniciación de la Transcripción
18.
Plant Cell ; 26(1): 102-20, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24488963

RESUMEN

Scaffold or matrix attachment regions (S/MARs) are found in all eukaryotes. The pattern of distribution and genomic context of S/MARs is thought to be important for processes such as chromatin organization and modulation of gene expression. Despite the importance of such processes, much is unknown about the large-scale distribution and sequence content of S/MARs in vivo. Here, we report the use of tiling microarrays to map 1358 S/MARs on Arabidopsis thaliana chromosome 4 (chr4). S/MARs occur throughout chr4, spaced much more closely than in the large plant and animal genomes that have been studied to date. Arabidopsis S/MARs can be divided into five clusters based on their association with other genomic features, suggesting a diversity of functions. While some Arabidopsis S/MARs may define structural domains, most occur near the transcription start sites of genes. Genes associated with these S/MARs have an increased probability of expression, which is particularly pronounced in the case of transcription factor genes. Analysis of sequence motifs and 6-mer enrichment patterns show that S/MARs are preferentially enriched in poly(dA:dT) tracts, sequences that resist nucleosome formation, and the majority of S/MARs contain at least one nucleosome-depleted region. This global view of S/MARs provides a framework to begin evaluating genome-scale models for S/MAR function.


Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , Regiones de Fijación a la Matriz , Nucleosomas/metabolismo , Poli dA-dT/metabolismo , Factores de Transcripción/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Regulación de la Expresión Génica de las Plantas , Motivos de Nucleótidos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
19.
Proc Natl Acad Sci U S A ; 110(48): 19639-44, 2013 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-24218619

RESUMEN

In plants, a subset of genes exhibit imprinting in endosperm tissue such that expression is primarily from the maternal or paternal allele. Imprinting may arise as a consequence of mechanisms for silencing of transposons during reproduction, and in some cases imprinted expression of particular genes may provide a selective advantage such that it is conserved across species. Separate mechanisms for the origin of imprinted expression patterns and maintenance of these patterns may result in substantial variation in the targets of imprinting in different species. Here we present deep sequencing of RNAs isolated from reciprocal crosses of four diverse maize genotypes, providing a comprehensive analysis that allows evaluation of imprinting at more than 95% of endosperm-expressed genes. We find that over 500 genes exhibit statistically significant parent-of-origin effects in maize endosperm tissue, but focused our analyses on a subset of these genes that had >90% expression from the maternal allele (69 genes) or from the paternal allele (108 genes) in at least one reciprocal cross. Over 10% of imprinted genes show evidence of allelic variation for imprinting. A comparison of imprinting in maize and rice reveals that 13% of genes with syntenic orthologs in both species exhibit conserved imprinting. Genes that exhibit conserved imprinting between maize and rice have elevated nonsynonymous to synonymous substitution ratios compared with other imprinted genes, suggesting a history of more rapid evolution. Together, these data suggest that imprinting only has functional relevance at a subset of loci that currently exhibit imprinting in maize.


Asunto(s)
Alelos , Evolución Molecular , Variación Genética , Impresión Genómica/genética , Oryza/genética , Zea mays/genética , Secuencia de Bases , Teorema de Bayes , Cruzamientos Genéticos , Genética de Población , Hibridación Genética , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ARN , Especificidad de la Especie
20.
Plant Cell ; 25(8): 2783-97, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23922207

RESUMEN

DNA methylation is a chromatin modification that is frequently associated with epigenetic regulation in plants and mammals. However, genetic changes such as transposon insertions can also lead to changes in DNA methylation. Genome-wide profiles of DNA methylation for 20 maize (Zea mays) inbred lines were used to discover differentially methylated regions (DMRs). The methylation level for each of these DMRs was also assayed in 31 additional maize or teosinte genotypes, resulting in the discovery of 1966 common DMRs and 1754 rare DMRs. Analysis of recombinant inbred lines provides evidence that the majority of DMRs are heritable. A local association scan found that nearly half of the DMRs with common variation are significantly associated with single nucleotide polymorphisms found within or near the DMR. Many of the DMRs that are significantly associated with local genetic variation are found near transposable elements that may contribute to the variation in DNA methylation. Analysis of gene expression in the same samples used for DNA methylation profiling identified over 300 genes with expression patterns that are significantly associated with DNA methylation variation. Collectively, our results suggest that DNA methylation variation is influenced by genetic and epigenetic changes that are often stably inherited and can influence the expression of nearby genes.


Asunto(s)
Metilación de ADN/genética , Epigénesis Genética , Variación Genética , Zea mays/genética , Análisis por Conglomerados , Genotipo , Endogamia , Patrón de Herencia/genética , Modelos Genéticos , Recombinación Genética/genética , Reproducibilidad de los Resultados
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