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1.
Plant Sci ; 292: 110375, 2020 Mar.
Article in English | MEDLINE | ID: mdl-32005381

ABSTRACT

The AP2/ERF (APETALA2/ethylene-responsive factor) family of transcription factors (TF) is involved in regulating biotic and abiotic stress responses in plants. To explore the role of AP2/ERFs in cold tolerance in woody plants, BpERF13 was cloned and characterized in Betula platyphylla (white birch), a species primarily found in Asia in temperate and boreal climates. Based on phylogenetic analysis, BpERF13 is a member of the IXb subfamily of ERFs. Using qRT-PCR, we found that BpERF13 was differentially expressed in different tissues, and its expression could be induced by cold treatment (4 °C). BpERF13 protein, fused with GFP, was exclusively localized to nuclei. To further assess the role of BpERF13 in cold tolerance, BpERF13 overexpression (OE) transgenic lines were generated in B. platyphylla and used for cold stress treatment and biochemical/physiological studies. BpERF13 overexpression lines had significantly increased tolerance to subfreezing treatment and reduced reactive oxygen species. Using a TF-centered yeast one-hybrid (Y1H) experimental system, we showed that BpERF13 could bind to LTRECOREATCOR15 and MYBCORE cis-elements to activate a reporter gene. ChIP-seq and ChIP-PCR experiments further demonstrated that BpERF13 bound to these cis-elements when present in the 5' proximal regions of superoxide dismutase (SOD), peroxidase (POD), and C-repeat-binding factor (CBF) genes. qRT-PCR was employed to examine the expression levels of these genes in response to cold stress; SOD, POD, and CBF genes were significantly upregulated in BpERF13 transgenic lines compared to wild-type plants in response to cold stress. These results indicate that the transcription factor BpERF13 regulates physiological processes underlying cold tolerance in woody plants.


Subject(s)
Betula/physiology , Cold Temperature , Gene Expression Regulation, Plant/physiology , Plant Proteins/genetics , Reactive Oxygen Species/metabolism , Transcription Factors/genetics , Up-Regulation , Betula/genetics , Plant Proteins/metabolism , Sequence Analysis, DNA , Transcription Factors/metabolism
3.
Anal Chem ; 87(1): 821-8, 2015 Jan 06.
Article in English | MEDLINE | ID: mdl-25495696

ABSTRACT

Affinity reagent pairs that recognize distinct epitopes on a target protein can greatly improve the sensitivity and specificity of molecular detection. Importantly, such pairs can be conjugated to generate reagents that achieve two-site "bidentate" target recognition, with affinities greatly exceeding either monovalent component. DNA aptamers are especially well-suited for such constructs, because they can be linked via standard synthesis techniques without requiring chemical conjugation. Unfortunately, aptamer pairs are difficult to generate, primarily because conventional selection methods preferentially yield aptamers that recognize a dominant "hot spot" epitope. Our array-based discovery platform for multivalent aptamers (AD-MAP) overcomes this problem to achieve efficient discovery of aptamer pairs. We use microfluidic selection and high-throughput sequencing to obtain an enriched pool of aptamer sequences. Next, we synthesize a custom array based on these sequences, and perform parallel affinity measurements to identify the highest-affinity aptamer for the target protein. We use this aptamer to form complexes that block the primary binding site on the target, and then screen the same array with these complexes to identify aptamers that bind secondary epitopes. We used AD-MAP to discover DNA aptamer pairs that bind distinct sites on human angiopoietin-2 with high affinities, even in undiluted serum. To the best of our knowledge, this is the first work to discover new aptamer pairs using arrays. We subsequently conjugated these aptamers with a flexible linker to construct ultra-high-affinity bidentate reagents, with equilibrium dissociation constants as low as 97 pM: >200-fold better than either component aptamer. Functional studies confirm that both aptamers critically contribute to this ultrahigh affinity, highlighting the promise of such reagents for research and clinical use.


Subject(s)
Angiopoietin-2/metabolism , Aptamers, Nucleotide/metabolism , Microfluidics/methods , Oligonucleotide Array Sequence Analysis , SELEX Aptamer Technique/methods , Angiopoietin-2/genetics , Aptamers, Nucleotide/chemistry , Binding Sites , Fluorescence , High-Throughput Nucleotide Sequencing , Humans
4.
Proc Natl Acad Sci U S A ; 110(46): 18460-5, 2013 Nov 12.
Article in English | MEDLINE | ID: mdl-24167271

ABSTRACT

Aptamers are promising affinity reagents that are potentially well suited for high-throughput discovery, as they are chemically synthesized and discovered via completely in vitro selection processes. Recent advancements in selection, sequencing, and the use of modified bases have improved aptamer quality, but the overall process of aptamer generation remains laborious and low-throughput. This is because binding characterization remains a critical bottleneck, wherein the affinity and specificity of each candidate aptamer are measured individually in a serial manner. To accelerate aptamer discovery, we devised the Quantitative Parallel Aptamer Selection System (QPASS), which integrates microfluidic selection and next-generation sequencing with in situ-synthesized aptamer arrays, enabling simultaneous measurement of affinity and specificity for thousands of candidate aptamers in parallel. After using QPASS to select aptamers for the human cancer biomarker angiopoietin-2 (Ang2), we in situ synthesized arrays of the selected sequences and obtained equilibrium dissociation constants (Kd) for every aptamer in parallel. We thereby identified over a dozen high-affinity Ang2 aptamers, with Kd as low as 20.5 ± 7.3 nM. The same arrays enabled us to quantify binding specificity for these aptamers in parallel by comparing relative binding of differentially labeled target and nontarget proteins, and by measuring their binding affinity directly in complex samples such as undiluted serum. Finally, we show that QPASS offers a compelling avenue for exploring structure-function relationships for large numbers of aptamers in parallel by coupling array-based affinity measurements with next-generation sequencing data to identify nucleotides and motifs within the aptamer that critically affect Ang2 binding.


Subject(s)
Aptamers, Peptide/metabolism , High-Throughput Screening Assays/methods , Proteins/metabolism , Proteomics/methods , Aptamers, Peptide/genetics , DNA Primers/genetics , Enzyme-Linked Immunosorbent Assay , Fluorescence , Gene Library , High-Throughput Nucleotide Sequencing/methods , Humans , Least-Squares Analysis , Microfluidics/methods , Protein Binding
5.
PLoS One ; 8(8): e71798, 2013.
Article in English | MEDLINE | ID: mdl-23967247

ABSTRACT

BACKGROUND: DNA aptamers generated by cell-SELEX offer an attractive alternative to antibodies, but generating aptamers to specific, known membrane protein targets has proven challenging, and has severely limited the use of aptamers as affinity reagents for cell identification and purification. METHODOLOGY: We modified the BJAB lymphoblastoma cell line to over-express the murine c-kit cell surface receptor. After six rounds of cell-SELEX, high-throughput sequencing and bioinformatics analysis, we identified aptamers that bound BJAB cells expressing c-kit but not wild-type BJAB controls. One of these aptamers also recognizes c-kit endogenously expressed by a mast cell line or hematopoietic progenitor cells, and specifically blocks binding of the c-kit ligand stem cell factor (SCF). This aptamer enables better separation by fluorescence-activated cell sorting (FACS) of c-kit(+) hematopoietic progenitor cells from mixed bone marrow populations than a commercially available antibody, suggesting that this approach may be broadly useful for rapid isolation of affinity reagents suitable for purification of other specific cell types. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel procedure for the efficient generation of DNA aptamers that bind to specific cell membrane proteins and can be used as high affinity reagents. We have named the procedure STACS (Specific TArget Cell-SELEX).


Subject(s)
Aptamers, Nucleotide , SELEX Aptamer Technique , Animals , Aptamers, Nucleotide/genetics , Aptamers, Nucleotide/metabolism , Bone Marrow Cells/metabolism , Cell Line, Tumor , Computational Biology/methods , Flow Cytometry , Gene Expression , High-Throughput Nucleotide Sequencing , Male , Mice , Protein Binding , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/metabolism , Stem Cell Factor/metabolism
6.
PLoS Comput Biol ; 9(3): e1002936, 2013.
Article in English | MEDLINE | ID: mdl-23505351

ABSTRACT

The salamander has the remarkable ability to regenerate its limb after amputation. Cells at the site of amputation form a blastema and then proliferate and differentiate to regrow the limb. To better understand this process, we performed deep RNA sequencing of the blastema over a time course in the axolotl, a species whose genome has not been sequenced. Using a novel comparative approach to analyzing RNA-seq data, we characterized the transcriptional dynamics of the regenerating axolotl limb with respect to the human gene set. This approach involved de novo assembly of axolotl transcripts, RNA-seq transcript quantification without a reference genome, and transformation of abundances from axolotl contigs to human genes. We found a prominent burst in oncogene expression during the first day and blastemal/limb bud genes peaking at 7 to 14 days. In addition, we found that limb patterning genes, SALL genes, and genes involved in angiogenesis, wound healing, defense/immunity, and bone development are enriched during blastema formation and development. Finally, we identified a category of genes with no prior literature support for limb regeneration that are candidates for further evaluation based on their expression pattern during the regenerative process.


Subject(s)
Ambystoma mexicanum/physiology , Gene Expression Profiling/methods , Gene Expression Regulation , Oncogenes , Sequence Analysis, RNA/methods , Ambystoma mexicanum/genetics , Amputation, Surgical , Animals , Cluster Analysis , Extremities/injuries , Extremities/physiology , Regeneration/genetics , Regeneration/physiology , Up-Regulation , Wound Healing/genetics , Wound Healing/physiology
7.
PLoS One ; 7(11): e50411, 2012.
Article in English | MEDLINE | ID: mdl-23226279

ABSTRACT

BACKGROUND: Constructing coexpression networks and performing network analysis using large-scale gene expression data sets is an effective way to uncover new biological knowledge; however, the methods used for gene association in constructing these coexpression networks have not been thoroughly evaluated. Since different methods lead to structurally different coexpression networks and provide different information, selecting the optimal gene association method is critical. METHODS AND RESULTS: In this study, we compared eight gene association methods - Spearman rank correlation, Weighted Rank Correlation, Kendall, Hoeffding's D measure, Theil-Sen, Rank Theil-Sen, Distance Covariance, and Pearson - and focused on their true knowledge discovery rates in associating pathway genes and construction coordination networks of regulatory genes. We also examined the behaviors of different methods to microarray data with different properties, and whether the biological processes affect the efficiency of different methods. CONCLUSIONS: We found that the Spearman, Hoeffding and Kendall methods are effective in identifying coexpressed pathway genes, whereas the Theil-sen, Rank Theil-Sen, Spearman, and Weighted Rank methods perform well in identifying coordinated transcription factors that control the same biological processes and traits. Surprisingly, the widely used Pearson method is generally less efficient, and so is the Distance Covariance method that can find gene pairs of multiple relationships. Some analyses we did clearly show Pearson and Distance Covariance methods have distinct behaviors as compared to all other six methods. The efficiencies of different methods vary with the data properties to some degree and are largely contingent upon the biological processes, which necessitates the pre-analysis to identify the best performing method for gene association and coexpression network construction.


Subject(s)
Gene Expression , Gene Regulatory Networks , Systems Biology/statistics & numerical data , Transcription Factors/genetics , Algorithms , Gene Expression Profiling , Humans , Oligonucleotide Array Sequence Analysis , Statistics, Nonparametric , Systems Biology/methods
8.
Angew Chem Int Ed Engl ; 51(50): 12449-53, 2012 Dec 07.
Article in English | MEDLINE | ID: mdl-23125174

ABSTRACT

In a single round: By combining the high-efficiency enrichment through the continuous-flow magnetic separation (CFMS) technique with the analytical power of next-generation sequencing, the generation of antibody mimetics with a single round of mRNA display is made possible. This approach eliminates iterative selection cycles and provides a path to fully automated ligand generation (see picture).


Subject(s)
Biomimetic Materials/metabolism , RNA, Messenger/chemistry , Amino Acid Sequence , Antibodies/chemistry , Antibodies/metabolism , Biomimetic Materials/chemistry , Enzyme-Linked Immunosorbent Assay , Fibronectins/chemistry , Fibronectins/metabolism , Gene Library , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/metabolism , Immunomagnetic Separation , Ligands , Maltose-Binding Proteins/chemistry , Maltose-Binding Proteins/metabolism , RNA, Messenger/isolation & purification
9.
Anal Chem ; 84(12): 5365-71, 2012 Jun 19.
Article in English | MEDLINE | ID: mdl-22624874

ABSTRACT

Many analytical techniques benefit greatly from the use of affinity reagent pairs, wherein each reagent recognizes a discrete binding site on a target. For example, antibody pairs have been widely used to dramatically increase the specificity of enzyme linked immunosorbent assays (ELISA). Nucleic acid-based aptamers offer many advantageous features relative to protein-based affinity reagents, including well-established chemical synthesis, thermostability, and low production cost. However, the generation of suitable aptamer pairs has posed a significant challenge, and few such pairs have been reported to date. To address this important challenge, we present multivalent aptamer isolation systematic evolution of ligands by exponential enrichment (MAI-SELEX), a technique designed for the efficient selection of aptamer pairs. In contrast to conventional selection methods, our method utilizes two selection modules to generate separate aptamer pools that recognize distinct binding sites on a single target. Using MAI-SELEX, we have isolated two groups of 2'-fluoro-modified RNA aptamers that specifically recognize the αV or ß3 subunits of integrin αVß3. These aptamers exhibit low nanomolar affinities for their targets, with minimal cross-reactivity to other closely related integrin homologues. Moreover, we show that these aptamer pairs do not interfere with each other's binding and effectively detect the target even in complex mixtures such as undiluted serum.


Subject(s)
Aptamers, Nucleotide/metabolism , Integrin alphaVbeta3/metabolism , SELEX Aptamer Technique/methods , Animals , Binding Sites , Cattle , Integrin alphaVbeta3/chemistry , Ligands , Protein Subunits/chemistry , Protein Subunits/metabolism , Sequence Homology, Amino Acid , Substrate Specificity
10.
BMC Syst Biol ; 5: 53, 2011 Apr 15.
Article in English | MEDLINE | ID: mdl-21496241

ABSTRACT

BACKGROUND: Identifying the key transcription factors (TFs) controlling a biological process is the first step toward a better understanding of underpinning regulatory mechanisms. However, due to the involvement of a large number of genes and complex interactions in gene regulatory networks, identifying TFs involved in a biological process remains particularly difficult. The challenges include: (1) Most eukaryotic genomes encode thousands of TFs, which are organized in gene families of various sizes and in many cases with poor sequence conservation, making it difficult to recognize TFs for a biological process; (2) Transcription usually involves several hundred genes that generate a combination of intrinsic noise from upstream signaling networks and lead to fluctuations in transcription; (3) A TF can function in different cell types or developmental stages. Currently, the methods available for identifying TFs involved in biological processes are still very scarce, and the development of novel, more powerful methods is desperately needed. RESULTS: We developed a computational pipeline called TF-Cluster for identifying functionally coordinated TFs in two steps: (1) Construction of a shared coexpression connectivity matrix (SCCM), in which each entry represents the number of shared coexpressed genes between two TFs. This sparse and symmetric matrix embodies a new concept of coexpression networks in which genes are associated in the context of other shared coexpressed genes; (2) Decomposition of the SCCM using a novel heuristic algorithm termed "Triple-Link", which searches the highest connectivity in the SCCM, and then uses two connected TF as a primer for growing a TF cluster with a number of linking criteria. We applied TF-Cluster to microarray data from human stem cells and Arabidopsis roots, and then demonstrated that many of the resulting TF clusters contain functionally coordinated TFs that, based on existing literature, accurately represent a biological process of interest. CONCLUSIONS: TF-Cluster can be used to identify a set of TFs controlling a biological process of interest from gene expression data. Its high accuracy in recognizing true positive TFs involved in a biological process makes it extremely valuable in building core GRNs controlling a biological process. The pipeline implemented in Perl can be installed in various platforms.


Subject(s)
Systems Biology/methods , Algorithms , Animals , Arabidopsis/genetics , Cell Differentiation , Cluster Analysis , Computational Biology/methods , Embryonic Stem Cells/cytology , Gene Expression Profiling , Gene Expression Regulation , Genome , Humans , Models, Genetic , Plant Roots/physiology , Software
11.
Proc Natl Acad Sci U S A ; 108(16): 6537-42, 2011 Apr 19.
Article in English | MEDLINE | ID: mdl-21464322

ABSTRACT

Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a unique approach to gene therapy. Here, we begin to assess whether the mutational load acquired during gene correction of iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected after standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine mutations in protein coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base-pair change, no additional mutations or copy number variation were identified in iPS cells after the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.


Subject(s)
Gyrate Atrophy/enzymology , Gyrate Atrophy/genetics , Ornithine-Oxo-Acid Transaminase/genetics , Ornithine-Oxo-Acid Transaminase/metabolism , Pluripotent Stem Cells/enzymology , Cells, Cultured , Gene Targeting/methods , Genome-Wide Association Study , Genomic Instability/genetics , Gyrate Atrophy/pathology , Gyrate Atrophy/therapy , Humans , Pluripotent Stem Cells/pathology , Recombination, Genetic
12.
Proc Natl Acad Sci U S A ; 107(35): 15373-8, 2010 Aug 31.
Article in English | MEDLINE | ID: mdl-20705898

ABSTRACT

We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with K(d) < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have approximately 3-8-fold higher affinity and approximately 2-4-fold higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions.


Subject(s)
Aptamers, Nucleotide/chemistry , Microfluidics/methods , SELEX Aptamer Technique/methods , Sequence Analysis, DNA/methods , Aptamers, Nucleotide/genetics , Aptamers, Nucleotide/metabolism , Base Sequence , Becaplermin , Binding, Competitive , Cloning, Molecular , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , Gene Library , Kinetics , Platelet-Derived Growth Factor/genetics , Platelet-Derived Growth Factor/metabolism , Protein Binding , Proto-Oncogene Proteins c-sis , Reproducibility of Results
13.
Stem Cells ; 27(7): 1524-8, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19544458

ABSTRACT

Human embryonic stem (ES) cells exhibit a shorter G(1) cell cycle phase than most somatic cells. Here, we examine the role of an abundant, human ES cell-enriched microRNA, miR-92b, in cell cycle distribution. Inhibition of miR-92b in human ES cells results in a greater number of cells in the G(1) phase and a lower number in the S phase. Conversely, overexpression of miR-92b in differentiated cells results in a decreased number of cells in G1 phase and an increased number in S-phase. p57, a gene whose product inhibits G(1) to S-phase progression, is one of the predicted targets of miR-92b. Inhibition of miR-92b in human ES cells increases p57 protein levels, and miR-92b overexpression in differentiated cells decreases p57 protein levels. Furthermore, miR-92b inhibits a luciferase reporter construct that includes part of the 3' untranslated region of the p57 gene containing the predicted target of the miR-92b seed sequence. Thus, we show that the miRNA miR-92b directly downregulates protein levels of the G(1)/S checkpoint gene p57. STEM CELLS 2009;27:1524-1528.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p57/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , G1 Phase/genetics , MicroRNAs/physiology , S Phase/genetics , Blotting, Western , Cell Cycle/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinase Inhibitor p57/genetics , Flow Cytometry , Humans , MicroRNAs/genetics , Models, Genetic
14.
Nucleic Acids Res ; 36(9): 2926-38, 2008 May.
Article in English | MEDLINE | ID: mdl-18385155

ABSTRACT

Well-defined relationships between oligonucleotide properties and hybridization signal intensities (HSI) can aid chip design, data normalization and true biological knowledge discovery. We clarify these relationships using the data from two microarray experiments containing over three million probes from 48 high-density chips. We find that melting temperature (T(m)) has the most significant effect on HSI while length for the long oligonucleotides studied has very little effect. Analysis of positional effect using a linear model provides evidence that the protruding ends of probes contribute more than tethered ends to HSI, which is further validated by specifically designed match fragment sliding and extension experiments. The impact of sequence similarity (SeqS) on HSI is not significant in comparison with other oligonucleotide properties. Using regression and regression tree analysis, we prioritize these oligonucleotide properties based on their effects on HSI. The implications of our discoveries for the design of unbiased oligonucleotides are discussed. We propose that isothermal probes designed by varying the length is a viable strategy to reduce sequence bias, though imposing selection constraints on other oligonucleotide properties is also essential.


Subject(s)
Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Oligonucleotide Probes/chemistry , Humans , Nucleic Acid Conformation , Nucleic Acid Denaturation , Regression Analysis , Sequence Homology, Nucleic Acid , Temperature
15.
Science ; 318(5858): 1917-20, 2007 Dec 21.
Article in English | MEDLINE | ID: mdl-18029452

ABSTRACT

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.


Subject(s)
Cell Line , Cellular Reprogramming , Fibroblasts/cytology , Pluripotent Stem Cells/cytology , Animals , Cell Differentiation , Cell Proliferation , Cell Shape , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Embryonic Stem Cells/cytology , Fetus , HMGB Proteins/genetics , HMGB Proteins/physiology , Homeodomain Proteins/genetics , Homeodomain Proteins/physiology , Humans , Infant, Newborn , Karyotyping , Mice , Mice, SCID , Nanog Homeobox Protein , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/physiology , Oligonucleotide Array Sequence Analysis , Pluripotent Stem Cells/physiology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/physiology , SOXB1 Transcription Factors , Stem Cell Transplantation , Teratoma/pathology , Transcription Factors/genetics , Transcription Factors/physiology , Transduction, Genetic , Transgenes
16.
Cell Stem Cell ; 1(3): 299-312, 2007 Sep 13.
Article in English | MEDLINE | ID: mdl-18371364

ABSTRACT

We mapped Polycomb-associated H3K27 trimethylation (H3K27me3) and Trithorax-associated H3K4 trimethylation (H3K4me3) across the whole genome in human embryonic stem (ES) cells. The vast majority of H3K27me3 colocalized on genes modified with H3K4me3. These commodified genes displayed low expression levels and were enriched in developmental function. Another significant set of genes lacked both modifications and was also expressed at low levels in ES cells but was enriched for gene function in physiological responses rather than development. Commodified genes could change expression levels rapidly during differentiation, but so could a substantial number of genes in other modification categories. SOX2, POU5F1, and NANOG, pluripotency-associated genes, shifted from modification by H3K4me3 alone to colocalization of both modifications as they were repressed during differentiation. Our results demonstrate that H3K27me3 modifications change during early differentiation, both relieving existing repressive domains and imparting new ones, and that colocalization with H3K4me3 is not restricted to pluripotent cells.


Subject(s)
Embryonic Stem Cells/metabolism , Genome, Human/genetics , Histones/metabolism , Lysine/metabolism , Cell Differentiation , Cell Lineage , Embryonic Stem Cells/cytology , Gene Expression Regulation , Humans , Methylation , Promoter Regions, Genetic/genetics , Protein Transport
17.
Physiol Genomics ; 23(2): 246-56, 2005 Oct 17.
Article in English | MEDLINE | ID: mdl-16106031

ABSTRACT

The broad goal of physiological genomics research is to link genes to their functions using appropriate experimental and computational techniques. Modern genomics experiments enable the generation of vast quantities of data, and interpretation of this data requires the integration of information derived from many diverse sources. Computational biology and bioinformatics offer the ability to manage and channel this information torrent. The Rat Genome Database (RGD; http://rgd.mcw.edu) has developed computational tools and strategies specifically supporting the goal of linking genes to their functional roles in rat and, using comparative genomics, to human and mouse. We present an overview of the database with a focus on these unique computational tools and describe strategies for the use of these resources in the area of physiological genomics.


Subject(s)
Databases, Genetic , Genome/genetics , Genomics/methods , Rats/genetics , Rats/physiology , Animals , Cloning, Molecular , Gene Expression Profiling
18.
Nucleic Acids Res ; 33(Database issue): D485-91, 2005 Jan 01.
Article in English | MEDLINE | ID: mdl-15608243

ABSTRACT

The Rat Genome Database (RGD) (http://rgd.mcw.edu) aims to meet the needs of its community by providing genetic and genomic infrastructure while also annotating the strengths of rat research: biochemistry, nutrition, pharmacology and physiology. Here, we report on RGD's development towards creating a phenome database. Recent developments can be categorized into three groups. (i) Improved data collection and integration to match increased volume and biological scope of research. (ii) Knowledge representation augmented by the implementation of a new ontology and annotation system. (iii) The addition of quantitative trait loci data, from rat, mouse and human to our advanced comparative genomics tools, as well as the creation of new, and enhancement of existing, tools to enable users to efficiently browse and survey research data. The emphasis is on helping researchers find genes responsible for disease through the use of rat models. These improvements, combined with the genomic sequence of the rat, have led to a successful year at RGD with over two million page accesses that represent an over 4-fold increase in a year. Future plans call for increased annotation of biological information on the rat elucidated through its use as a model for human pathobiology. The continued development of toolsets will facilitate integration of these data into the context of rat genomic sequence, as well as allow comparisons of biological and genomic data with the human genomic sequence and of an increasing number of organisms.


Subject(s)
Databases, Nucleic Acid , Genomics , Phenotype , Rats/genetics , Animals , Chromosome Mapping , Database Management Systems , Disease Models, Animal , Genetic Markers , Genome , Quantitative Trait Loci , Rats/physiology , Systems Integration
19.
Genome Res ; 14(4): 651-60, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15060006

ABSTRACT

Integration of the large variety of genome maps from several organisms provides the mechanism by which physiological knowledge obtained in model systems such as the rat can be projected onto the human genome to further the research on human disease. The release of the rat genome sequence provides new information for studies using the rat model and is a key reference against which existing and new rat physiological results can be aligned. Previously, we described comparative maps of the rat, mouse, and human based on EST sequence comparisons combined with radiation hybrid maps. Here, we use new data and introduce the Integrated Genomics Environment, an extensive database of curated and integrated maps, markers, and physiological results. These results are integrated by using VCMapview, a java-based map integration and visualization tool. This unique environment allows researchers to relate results from cytogenetic, genetic, and radiation hybrid studies to the genome sequence and compare regions of interest between human, mouse, and rat. Integrating rat physiology with mouse genetics and clinical results from human by using the respective genomes provides a novel route to capitalize on comparative genomics and the strengths of model organism biology.


Subject(s)
Genomics/methods , Multifactorial Inheritance/genetics , Quantitative Trait Loci/genetics , Animals , Chromosome Mapping/methods , Computational Biology , Databases, Genetic , Expressed Sequence Tags , Gene Order/genetics , Genetic Markers/genetics , Genome , Genome, Human , Humans , Mice , Quantitative Trait, Heritable , Radiation Hybrid Mapping/methods , Rats , Software
20.
Genome Res ; 14(4): 750-7, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15060019

ABSTRACT

The laboratory rat is a major model organism for systems biology. To complement the cornucopia of physiological and pharmacological data generated in the rat, a large genomic toolset has been developed, culminating in the release of the rat draft genome sequence. The rat draft sequence used a variety of assembly packages, as well as data from the Radiation Hybrid (RH) map of the rat as part of their validation. As part of the Rat Genome Project, we have been building a high-density RH map to facilitate data integration from multiple maps and now to help validate the genome assembly. By incorporating vectors from our lab and several other labs, we have doubled the number of simple sequence length polymorphisms (SSLPs), genes, expressed sequence tags (ESTs), and sequence-tagged sites (STSs) compared to any other genome-wide rat map, a total of 24,437 elements. During the process, we also identified a novel approach for integrating the RH placement results from multiple maps. This new integrated RH map contains approximately 10 RH-mapped elements per Mb on the genome assembly, enabling the RH maps to serve as a scaffold for a variety of data visualization tools.


Subject(s)
Expressed Sequence Tags , Genes/genetics , Genome , Polymorphism, Genetic/genetics , Radiation Hybrid Mapping/methods , Animals , Chromosome Mapping/methods , Chromosomes/genetics , Crosses, Genetic , Databases, Genetic , Genetic Markers/genetics , Genetic Markers/physiology , Lod Score , Rats , Rats, Inbred ACI , Rats, Inbred BN , Rats, Inbred OLETF , Rats, Inbred SHR , Repetitive Sequences, Nucleic Acid , Sequence Tagged Sites
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