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Find video protocols related to scientific articles indexed in Pubmed.
IL-32 is a molecular marker of a host defense network in human tuberculosis.
Sci Transl Med
PUBLISHED: 08-22-2014
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Tuberculosis is a leading cause of infectious disease-related death worldwide; however, only 10% of people infected with Mycobacterium tuberculosis develop disease. Factors that contribute to protection could prove to be promising targets for M. tuberculosis therapies. Analysis of peripheral blood gene expression profiles of active tuberculosis patients has identified correlates of risk for disease or pathogenesis. We sought to identify potential human candidate markers of host defense by studying gene expression profiles of macrophages, cells that, upon infection by M. tuberculosis, can mount an antimicrobial response. Weighted gene coexpression network analysis revealed an association between the cytokine interleukin-32 (IL-32) and the vitamin D antimicrobial pathway in a network of interferon-?- and IL-15-induced "defense response" genes. IL-32 induced the vitamin D-dependent antimicrobial peptides cathelicidin and DEFB4 and to generate antimicrobial activity in vitro, dependent on the presence of adequate 25-hydroxyvitamin D. In addition, the IL-15-induced defense response macrophage gene network was integrated with ranked pairwise comparisons of gene expression from five different clinical data sets of latent compared with active tuberculosis or healthy controls and a coexpression network derived from gene expression in patients with tuberculosis undergoing chemotherapy. Together, these analyses identified eight common genes, including IL-32, as molecular markers of latent tuberculosis and the IL-15-induced gene network. As maintaining M. tuberculosis in a latent state and preventing transition to active disease may represent a form of host resistance, these results identify IL-32 as one functional marker and potential correlate of protection against active tuberculosis.
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Genome-wide Hi-C analyses in wild-type and mutants reveal high-resolution chromatin interactions in Arabidopsis.
Mol. Cell
PUBLISHED: 08-14-2014
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Chromosomes form 3D structures that are critical to the regulation of cellular and genetic processes. Here, we present a study of global chromatin interaction patterns in Arabidopsis thaliana. Our genome-wide approach confirmed interactions that were previously observed by other methods as well as uncovered long-range interactions such as those among small heterochromatic regions embedded in euchromatic arms. We also found that interactions are correlated with various epigenetic marks that are localized in active or silenced chromatin. Arabidopsis chromosomes do not contain large local interactive domains that resemble the topological domains described in animals but, instead, contain relatively small interactive regions scattered around the genome that contain H3K27me3 or H3K9me2. We generated interaction maps in mutants that are defective in specific epigenetic pathways and found altered interaction patterns that correlate with changes in the epigenome. These analyses provide further insights into molecular mechanisms of epigenetic regulation of the genome.
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Comparison of Molecular Signatures from Multiple Skin Diseases Identifies Mechanisms of Immunopathogenesis.
J. Invest. Dermatol.
PUBLISHED: 08-11-2014
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The ability to obtain gene expression profiles from human disease specimens provides an opportunity to identify relevant gene pathways, but is limited by the absence of data sets spanning a broad range of conditions. Here, we analyzed publicly available microarray data from 16 diverse skin conditions in order to gain insight into disease pathogenesis. Unsupervised hierarchical clustering separated samples by disease as well as common cellular and molecular pathways. Disease-specific signatures were leveraged to build a multi-disease classifier, which predicted the diagnosis of publicly and prospectively collected expression profiles with 93% accuracy. In one sample, the molecular classifier differed from the initial clinical diagnosis and correctly predicted the eventual diagnosis as the clinical presentation evolved. Finally, integration of IFN-regulated gene programs with the skin database revealed a significant inverse correlation between IFN-? and IFN-? programs across all conditions. Our study provides an integrative approach to the study of gene signatures from multiple skin conditions, elucidating mechanisms of disease pathogenesis. In addition, these studies provide a framework for developing tools for personalized medicine toward the precise prediction, prevention, and treatment of disease on an individual level.Journal of Investigative Dermatology advance online publication, 18 September 2014; (2014) 0, 000-000. doi:10.1038/jid.2014.352.
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Selective demethylation and altered gene expression are associated with ICF syndrome in human-induced pluripotent stem cells and mesenchymal stem cells.
Hum. Mol. Genet.
PUBLISHED: 07-15-2014
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Immunodeficiency, centromeric instability and facial anomalies type I (ICF1) syndrome is a rare genetic disease caused by mutations in DNA methyltransferase (DNMT) 3B, a de novo DNA methyltransferase. However, the molecular basis of how DNMT3B deficiency leads to ICF1 pathogenesis is unclear. Induced pluripotent stem cell (iPSC) technology facilitates the study of early human developmental diseases via facile in vitro paradigms. Here, we generate iPSCs from ICF Type 1 syndrome patient fibroblasts followed by directed differentiation of ICF1-iPSCs to mesenchymal stem cells (MSCs). By performing genome-scale bisulfite sequencing, we find that DNMT3B-deficient iPSCs exhibit global loss of non-CG methylation and select CG hypomethylation at gene promoters and enhancers. Further unbiased scanning of ICF1-iPSC methylomes also identifies large megabase regions of CG hypomethylation typically localized in centromeric and subtelomeric regions. RNA sequencing of ICF1 and control iPSCs reveals abnormal gene expression in ICF1-iPSCs relevant to ICF syndrome phenotypes, some directly associated with promoter or enhancer hypomethylation. Upon differentiation of ICF1 iPSCs to MSCs, we find virtually all CG hypomethylated regions remained hypomethylated when compared with either wild-type iPSC-derived MSCs or primary bone-marrow MSCs. Collectively, our results show specific methylome and transcriptome defects in both ICF1-iPSCs and differentiated somatic cell lineages, providing a valuable stem cell system for further in vitro study of the molecular pathogenesis of ICF1 syndrome. GEO accession number: GSE46030.
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Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control.
Plant Cell
PUBLISHED: 05-30-2014
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Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria.
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Phosphoprotein SAK1 is a regulator of acclimation to singlet oxygen in Chlamydomonas reinhardtii.
Elife
PUBLISHED: 05-27-2014
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Singlet oxygen is a highly toxic and inevitable byproduct of oxygenic photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is capable of acclimating specifically to singlet oxygen stress, but the retrograde signaling pathway from the chloroplast to the nucleus mediating this response is unknown. Here we describe a mutant, singlet oxygen acclimation knocked-out 1 (sak1), that lacks the acclimation response to singlet oxygen. Analysis of genome-wide changes in RNA abundance during acclimation to singlet oxygen revealed that SAK1 is a key regulator of the gene expression response during acclimation. The SAK1 gene encodes an uncharacterized protein with a domain conserved among chlorophytes and present in some bZIP transcription factors. The SAK1 protein is located in the cytosol, and it is induced and phosphorylated upon exposure to singlet oxygen, suggesting that it is a critical intermediate component of the retrograde signal transduction pathway leading to singlet oxygen acclimation.DOI: http://dx.doi.org/10.7554/eLife.02286.001.
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Non-exhaustive DNA methylation-mediated transposon silencing in the black truffle genome, a complex fungal genome with massive repeat element content.
Genome Biol.
PUBLISHED: 05-21-2014
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We investigated how an extremely transposon element (TE)-rich organism such as the plant-symbiotic ascomycete truffle Tuber melanosporum exploits DNA methylation to cope with the more than 45,000 repeated elements that populate its genome.
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Intergenerational genomic DNA methylation patterns in mouse hybrid strains.
Genome Biol.
PUBLISHED: 04-30-2014
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DNA methylation is a contributing factor to both rare and common human diseases, and plays a major role in development and gene silencing. While the variation of DNA methylation among individuals has been partially characterized, the degree to which methylation patterns are preserved across generations is still poorly understood. To determine the extent of methylation differences between two generations of mice we examined DNA methylation patterns in the livers of eight parental and F1 mice from C57BL/6J and DBA/2J mouse strains using bisulfite sequencing.
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Nitrogen-Sparing Mechanisms in Chlamydomonas Affect the Transcriptome, the Proteome, and Photosynthetic Metabolism.
Plant Cell
PUBLISHED: 04-18-2014
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Nitrogen (N) is a key nutrient that limits global primary productivity; hence, N-use efficiency is of compelling interest in agriculture and aquaculture. We used Chlamydomonas reinhardtii as a reference organism for a multicomponent analysis of the N starvation response. In the presence of acetate, respiratory metabolism is prioritized over photosynthesis; consequently, the N-sparing response targets proteins, pigments, and RNAs involved in photosynthesis and chloroplast function over those involved in respiration. Transcripts and proteins of the Calvin-Benson cycle are reduced in N-deficient cells, resulting in the accumulation of cycle metabolic intermediates. Both cytosolic and chloroplast ribosomes are reduced, but via different mechanisms, reflected by rapid changes in abundance of RNAs encoding chloroplast ribosomal proteins but not cytosolic ones. RNAs encoding transporters and enzymes for metabolizing alternative N sources increase in abundance, as is appropriate for the soil environmental niche of C. reinhardtii. Comparison of the N-replete versus N-deplete proteome indicated that abundant proteins with a high N content are reduced in N-starved cells, while the proteins that are increased have lower than average N contents. This sparing mechanism contributes to a lower cellular N/C ratio and suggests an approach for engineering increased N-use efficiency.
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Widespread use of non-productive alternative splice sites in Saccharomyces cerevisiae.
PLoS Genet.
PUBLISHED: 04-01-2014
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Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3' splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3'-splice site in GCR1. The use of non-productive alternative splice sites can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing proteome diversity.
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Detecting communities based on network topology.
Sci Rep
PUBLISHED: 03-20-2014
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Network methods have had profound influence in many domains and disciplines in the past decade. Community structure is a very important property of complex networks, but the accurate definition of a community remains an open problem. Here we defined community based on three properties, and then propose a simple and novel framework to detect communities based on network topology. We analyzed 16 different types of networks, and compared our partitions with Infomap, LPA, Fastgreedy and Walktrap, which are popular algorithms for community detection. Most of the partitions generated using our approach compare favorably to those generated by these other algorithms. Furthermore, we define overlapping nodes that combine community structure with shortest paths. We also analyzed the E. Coli. transcriptional regulatory network in detail, and identified modules with strong functional coherence.
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Molecular profiling of premalignant lesions in lung squamous cell carcinomas identifies mechanisms involved in stepwise carcinogenesis.
Cancer Prev Res (Phila)
PUBLISHED: 03-11-2014
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Lung squamous cell carcinoma (SCC) is thought to arise from premalignant lesions in the airway epithelium; therefore, studying these lesions is critical for understanding lung carcinogenesis. Previous microarray and sequencing studies designed to discover early biomarkers and therapeutic targets for lung SCC had limited success identifying key driver events in lung carcinogenesis, mostly due to the cellular heterogeneity of patient samples examined and the interindividual variability associated with difficult to obtain airway premalignant lesions and appropriate normal control samples within the same patient. We performed RNA sequencing on laser-microdissected representative cell populations along the SCC pathologic continuum of patient-matched normal basal cells, premalignant lesions, and tumor cells. We discovered transcriptomic changes and identified genomic pathways altered with initiation and progression of SCC within individual patients. We used immunofluorescent staining to confirm gene expression changes in premalignant lesions and tumor cells, including increased expression of SLC2A1, CEACAM5, and PTBP3 at the protein level and increased activation of MYC via nuclear translocation. Cytoband enrichment analysis revealed coordinated loss and gain of expression in chromosome 3p and 3q regions, respectively, during carcinogenesis. This is the first gene expression profiling study of airway premalignant lesions with patient-matched SCC tumor samples. Our results provide much needed information about the biology of premalignant lesions and the molecular changes that occur during stepwise carcinogenesis of SCC, and it highlights a novel approach for identifying some of the earliest molecular changes associated with initiation and progression of lung carcinogenesis within individual patients.
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An epigenetic signature in peripheral blood associated with the haplotype on 17q21.31, a risk factor for neurodegenerative tauopathy.
PLoS Genet.
PUBLISHED: 03-01-2014
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Little is known about how changes in DNA methylation mediate risk for human diseases including dementia. Analysis of genome-wide methylation patterns in patients with two forms of tau-related dementia--progressive supranuclear palsy (PSP) and frontotemporal dementia (FTD)--revealed significant differentially methylated probes (DMPs) in patients versus unaffected controls. Remarkably, DMPs in PSP were clustered within the 17q21.31 region, previously known to harbor the major genetic risk factor for PSP. We identified and replicated a dose-dependent effect of the risk-associated H1 haplotype on methylation levels within the region in blood and brain. These data reveal that the H1 haplotype increases risk for tauopathy via differential methylation at that locus, indicating a mediating role for methylation in dementia pathophysiology.
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The path to triacylglyceride obesity in the sta6 strain of Chlamydomonas reinhardtii.
Eukaryotic Cell
PUBLISHED: 02-28-2014
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When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen starved in acetate and then "boosted" after 2 days with additional acetate, the cells become "obese" after 8 days, with triacylglyceride (TAG)-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, the sta6 strain and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the 2 days prior and 2 days after the boost, and the data were compared with published reports using other strains and growth conditions. During the 2 h after the boost, ?425 genes are upregulated ?2-fold and ?875 genes are downregulated ?2-fold in each strain. Expression of a small subset of "sensitive" genes, encoding enzymes involved in the glyoxylate and Calvin-Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boosting. Four genes-encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)-are selectively upregulated in the sta6 strain. Although the bulk rate of acetate depletion from the medium is not boost enhanced, three candidate acetate permease-encoding genes in the GPR1/FUN34/YaaH superfamily are boost upregulated, and 13 of the "sensitive" genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is downregulated by the boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in the sta6 strain.
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Nkx2-5 suppresses the proliferation of atrial myocytes and conduction system.
Circ. Res.
PUBLISHED: 02-21-2014
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Tight control of cardiomyocyte proliferation is essential for the formation of four-chambered heart. Although human mutation of NKX2-5 is linked to septal defects and atrioventricular conduction abnormalities, early lethality and hemodynamic alteration in the mutant models have caused controversy as to whether Nkx2-5 regulates cardiomyocyte proliferation.
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All-trans retinoic acid-triggered antimicrobial activity against Mycobacterium tuberculosis is dependent on NPC2.
J. Immunol.
PUBLISHED: 02-05-2014
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A role for vitamin A in host defense against Mycobacterium tuberculosis has been suggested through epidemiological and in vitro studies; however, the mechanism is unclear. In this study, we demonstrate that vitamin A-triggered antimicrobial activity against M. tuberculosis requires expression of NPC2. Comparison of monocytes stimulated with all-trans retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3 (1,25D3), the biologically active forms of vitamin A and vitamin D, respectively, indicates that ATRA and 1,25D3 induce mechanistically distinct antimicrobial activities. Stimulation of primary human monocytes with ATRA did not result in expression of the antimicrobial peptide cathelicidin, which is required for 1,25D3 antimicrobial activity. In contrast, ATRA triggered a reduction in the total cellular cholesterol concentration, whereas 1,25D3 did not. Blocking ATRA-induced cellular cholesterol reduction inhibits antimicrobial activity as well. Bioinformatic analysis of ATRA- and 1,25D3-induced gene profiles suggests that NPC2 is a key gene in ATRA-induced cholesterol regulation. Knockdown experiments demonstrate that ATRA-mediated decrease in total cellular cholesterol content and increase in lysosomal acidification are both dependent upon expression of NPC2. Expression of NPC2 was lower in caseous tuberculosis granulomas and M. tuberculosis-infected monocytes compared with normal lung and uninfected cells, respectively. Loss of NPC2 expression ablated ATRA-induced antimicrobial activity. Taken together, these results suggest that the vitamin A-mediated antimicrobial mechanism against M. tuberculosis requires NPC2-dependent expression and function, indicating a key role for cellular cholesterol regulation in the innate immune response.
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SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation.
Nature
PUBLISHED: 01-22-2014
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RNA-directed DNA methylation in Arabidopsis thaliana depends on the upstream synthesis of 24-nucleotide small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) and downstream synthesis of non-coding transcripts by Pol V. Pol V transcripts are thought to interact with siRNAs which then recruit DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to methylate DNA. The SU(VAR)3-9 homologues SUVH2 and SUVH9 act in this downstream step but the mechanism of their action is unknown. Here we show that genome-wide Pol V association with chromatin redundantly requires SUVH2 and SUVH9. Although SUVH2 and SUVH9 resemble histone methyltransferases, a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-adenosyl methionine (SAM)-binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins. SUVH2 and SUVH9 both contain SRA (SET- and RING-ASSOCIATED) domains capable of binding methylated DNA, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of DNA METHYLTRANSFERASE 1 (MET1) causes loss of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. Furthermore, tethering SUVH2 with a zinc finger to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. These results indicate that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of plant genomes for epigenetic silencing.
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Gene expression and nucleotide composition are associated with genic methylation level in Oryza sativa.
BMC Bioinformatics
PUBLISHED: 01-21-2014
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The methylation of cytosines at CpG dinucleotides, which plays an important role in gene expression regulation, is one of the most studied epigenetic modifications. Thus far, the detection of DNA methylation has been determined mostly by experimental methods, which are not only prone to bench effects and artifacts but are also time-consuming, expensive, and cannot be easily scaled up to many samples. It is therefore useful to develop computational prediction methods for DNA methylation. Our previous studies highlighted the existence of correlations between the GC content of the third codon position (GC?), methylation, and gene expression. We thus designed a model to predict methylation in Oryza sativa based on genomic sequence features and gene expression data.
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A panel of CpG methylation sites distinguishes human embryonic stem cells and induced pluripotent stem cells.
Stem Cell Reports
PUBLISHED: 01-14-2014
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Whether human induced pluripotent stem cells (hiPSCs) are epigenetically identical to human embryonic stem cells (hESCs) has been debated in the stem cell field. In this study, we analyzed DNA methylation patterns in a large number of hiPSCs (n = 114) and hESCs (n = 155), and identified a panel of 82 CpG methylation sites that can distinguish hiPSCs from hESCs with high accuracy. We show that 12 out of the 82 CpG sites were subject to hypermethylation in part by DNMT3B. Notably, DNMT3B contributes directly to aberrant hypermethylation and silencing of the signature gene, TCERG1L. Overall, we conclude that DNMT3B is involved in a wave of de novo methylation during reprogramming, a portion of which contributes to the unique hiPSC methylation signature. These 82 CpG methylation sites may be useful as biomarkers to distinguish between hiPSCs and hESCs.
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FamAnn: an automated variant annotation pipeline to facilitate target discovery for family-based sequencing studies.
Bioinformatics
PUBLISHED: 01-05-2014
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FamAnn is an automated variant annotation pipeline designed for facilitating target discovery for family-based sequencing studies. It can apply a different inheritance pattern or a de novo mutations discovery model to each family and select single nucleotide variants and small insertions and deletions segregating in each family or shared by multiple families. It also provides a variety of variant annotations and retains and annotates all transcripts hit by a single variant. Excel-compatible outputs including all annotated variants segregating in each family or shared by multiple families will be provided for users to prioritize variants based on their customized thresholds. A list of genes that harbor the segregating variants will be provided as well for possible pathway/network analyses. FamAnn uses the de facto community standard Variant Call Format as the input format and can be applied to whole exome, genome or targeted resequencing data.
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A comprehensive resource of genomic, epigenomic and transcriptomic sequencing data for the black truffle Tuber melanosporum.
Gigascience
PUBLISHED: 01-01-2014
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Tuber melanosporum, also known in the gastronomic community as "truffle", features one of the largest fungal genomes (125 Mb) with an exceptionally high transposable element (TE) and repetitive DNA content (>58%). The main purpose of DNA methylation in fungi is TE silencing. As obligate outcrossing organisms, truffles are bound to a sexual mode of propagation, which together with TEs is thought to represent a major force driving the evolution of DNA methylation. Thus, it was of interest to examine if and how T. melanosporum exploits DNA methylation to maintain genome integrity.
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The effects of perinatal testosterone exposure on the DNA methylome of the mouse brain are late-emerging.
Biol Sex Differ
PUBLISHED: 01-01-2014
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The biological basis for sex differences in brain function and disease susceptibility is poorly understood. Examining the role of gonadal hormones in brain sexual differentiation may provide important information about sex differences in neural health and development. Permanent masculinization of brain structure, function, and disease is induced by testosterone prenatally in males, but the possible mediation of these effects by long-term changes in the epigenome is poorly understood.
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Intestinal subepithelial myofibroblasts support the growth of intestinal epithelial stem cells.
PLoS ONE
PUBLISHED: 01-01-2014
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Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.
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Characterizing the strand-specific distribution of non-CpG methylation in human pluripotent cells.
Nucleic Acids Res.
PUBLISHED: 12-16-2013
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DNA methylation is an important defense and regulatory mechanism. In mammals, most DNA methylation occurs at CpG sites, and asymmetric non-CpG methylation has only been detected at appreciable levels in a few cell types. We are the first to systematically study the strand-specific distribution of non-CpG methylation. With the divide-and-compare strategy, we show that CHG and CHH methylation are not intrinsically different in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We also find that non-CpG methylation is skewed between the two strands in introns, especially at intron boundaries and in highly expressed genes. Controlling for the proximal sequences of non-CpG sites, we show that the skew of non-CpG methylation in introns is mainly guided by sequence skew. By studying subgroups of transposable elements, we also found that non-CpG methylation is distributed in a strand-specific manner in both short interspersed nuclear elements (SINE) and long interspersed nuclear elements (LINE), but not in long terminal repeats (LTR). Finally, we show that on the antisense strand of Alus, a non-CpG site just downstream of the A-box is highly methylated. Together, the divide-and-compare strategy leads us to identify regions with strand-specific distributions of non-CpG methylation in humans.
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Systems-Level Analysis of Nitrogen Starvation-Induced Modifications of Carbon Metabolism in a Chlamydomonas reinhardtii Starchless Mutant.
Plant Cell
PUBLISHED: 11-26-2013
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To understand the molecular basis underlying increased triacylglycerol (TAG) accumulation in starchless (sta) Chlamydomonas reinhardtii mutants, we undertook comparative time-course transcriptomics of strains CC-4348 (sta6 mutant), CC-4349, a cell wall-deficient (cw) strain purported to represent the parental STA6 strain, and three independent STA6 strains generated by complementation of sta6 (CC-4565/STA6-C2, CC-4566/STA6-C4, and CC-4567/STA6-C6) in the context of N deprivation. Despite N starvation-induced dramatic remodeling of the transcriptome, there were relatively few differences (5 × 10(2)) observed between sta6 and STA6, the most dramatic of which were increased abundance of transcripts encoding key regulated or rate-limiting steps in central carbon metabolism, specifically isocitrate lyase, malate synthase, transaldolase, fructose bisphosphatase and phosphoenolpyruvate carboxykinase (encoded by ICL1, MAS1, TAL1, FBP1, and PCK1 respectively), suggestive of increased carbon movement toward hexose-phosphate in sta6 by upregulation of the glyoxylate pathway and gluconeogenesis. Enzyme assays validated the increase in isocitrate lyase and malate synthase activities. Targeted metabolite analysis indicated increased succinate, malate, and Glc-6-P and decreased Fru-1,6-bisphosphate, illustrating the effect of these changes. Comparisons of independent data sets in multiple strains allowed the delineation of a sequence of events in the global N starvation response in C. reinhardtii, starting within minutes with the upregulation of alternative N assimilation routes and carbohydrate synthesis and subsequently a more gradual upregulation of genes encoding enzymes of TAG synthesis. Finally, genome resequencing analysis indicated that (1) the deletion in sta6 extends into the neighboring gene encoding respiratory burst oxidase, and (2) a commonly used STA6 strain (CC-4349) as well as the sequenced reference (CC-503) are not congenic with respect to sta6 (CC-4348), underscoring the importance of using complemented strains for more rigorous assignment of phenotype to genotype.
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Epigenetic differences between shoots and roots in Arabidopsis reveals tissue-specific regulation.
Epigenetics
PUBLISHED: 10-29-2013
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DNA methylation and nucleosome densities play a critical role in the regulation of gene expression. While much is known about the mechanisms of transcriptional control that are mediated by these, less is known about the degree to which they are tissue-specific. By comparing DNA methylation, nucleosome densities and transcriptional levels in different tissue types we can gain a clearer understanding of the extent to which these mechanisms influence gene expression in a tissue specific manner. We compared DNA methylation in Arabidopsis shoots and roots and found extensive differences across the genome. We computed DNA methylation differences between roots and shoots at single cytosines and found that one in every 173 cytosines was differentially methylated. In addition we compared DNA methylation with tissue specific gene expression and nucleosome density measurements to identify associations between these. We also identified a group of genes that are strongly correlated with these epigenetic marks and are significantly differentially methylated between roots and shoots. These root-specific genes are part of the extensin family, and are preferentially methylated and have at least 10-fold higher expression and lower nucleosome density in roots relative to shoots.
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Copper response regulator1-dependent and -independent responses of the Chlamydomonas reinhardtii transcriptome to dark anoxia.
Plant Cell
PUBLISHED: 09-06-2013
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Anaerobiosis is a stress condition for aerobic organisms and requires extensive acclimation responses. We used RNA-Seq for a whole-genome view of the acclimation of Chlamydomonas reinhardtii to anoxic conditions imposed simultaneously with transfer to the dark. Nearly 1.4 × 10(3) genes were affected by hypoxia. Comparing transcript profiles from early (hypoxic) with those from late (anoxic) time points indicated that cells activate oxidative energy generation pathways before employing fermentation. Probable substrates include amino acids and fatty acids (FAs). Lipid profiling of the C. reinhardtii cells revealed that they degraded FAs but also accumulated triacylglycerols (TAGs). In contrast with N-deprived cells, the TAGs in hypoxic cells were enriched in desaturated FAs, suggesting a distinct pathway for TAG accumulation. To distinguish transcriptional responses dependent on copper response regulator1 (CRR1), which is also involved in hypoxic gene regulation, we compared the transcriptomes of crr1 mutants and complemented strains. In crr1 mutants, ~40 genes were aberrantly regulated, reaffirming the importance of CRR1 for the hypoxic response, but indicating also the contribution of additional signaling strategies to account for the remaining differentially regulated transcripts. Based on transcript patterns and previous results, we conclude that nitric oxide-dependent signaling cascades operate in anoxic C. reinhardtii cells.
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Remodeling of membrane lipids in iron-starved Chlamydomonas.
J. Biol. Chem.
PUBLISHED: 08-27-2013
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Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24-48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii.
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Long-range chromatin contacts in embryonic stem cells reveal a role for pluripotency factors and polycomb proteins in genome organization.
Cell Stem Cell
PUBLISHED: 08-01-2013
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The relationship between 3D organization of the genome and gene-regulatory networks is poorly understood. Here, we examined long-range chromatin interactions genome-wide in mouse embryonic stem cells (ESCs), iPSCs, and fibroblasts and uncovered a pluripotency-specific genome organization that is gradually reestablished during reprogramming. Our data confirm that long-range chromatin interactions are primarily associated with the spatial segregation of open and closed chromatin, defining overall chromosome conformation. Additionally, we identified two further levels of genome organization in ESCs characterized by colocalization of regions with high pluripotency factor occupancy and strong enrichment for Polycomb proteins/H3K27me3, respectively. Underlining the independence of these networks and their functional relevance for genome organization, loss of the Polycomb protein Eed diminishes interactions between Polycomb-regulated regions without altering overarching chromosome conformation. Together, our data highlight a pluripotency-specific genome organization in which pluripotency factors such as Nanog and H3K27me3 occupy distinct nuclear spaces and reveal a role for cell-type-specific gene-regulatory networks in genome organization.
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BS-Seeker2: a versatile aligning pipeline for bisulfite sequencing data.
BMC Genomics
PUBLISHED: 07-30-2013
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DNA methylation is an important epigenetic modification involved in many biological processes. Bisulfite treatment coupled with high-throughput sequencing provides an effective approach for studying genome-wide DNA methylation at base resolution. Libraries such as whole genome bisulfite sequencing (WGBS) and reduced represented bisulfite sequencing (RRBS) are widely used for generating DNA methylomes, demanding efficient and versatile tools for aligning bisulfite sequencing data.
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Cross-species analysis of genic GC3 content and DNA methylation patterns.
Genome Biol Evol
PUBLISHED: 07-09-2013
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The GC content in the third codon position (GC(3)) exhibits a unimodal distribution in many plant and animal genomes. Interestingly, grasses and homeotherm vertebrates exhibit a unique bimodal distribution. High GC(3) was previously found to be associated with variable expression, higher frequency of upstream TATA boxes, and an increase of GC(3) from 5 to 3. Moreover, GC(3)-rich genes are predominant in certain gene classes and are enriched in CpG dinucleotides that are potential targets for methylation. Based on the GC(3) bimodal distribution we hypothesize that GC(3) has a regulatory role involving methylation and gene expression. To test that hypothesis, we selected diverse taxa (rice, thale cress, bee, and human) that varied in the modality of their GC(3) distribution and tested the association between GC(3), DNA methylation, and gene expression. We examine the relationship between cytosine methylation levels and GC(3), gene expression, genome signature, gene length, and other gene compositional features. We find a strong negative correlation (Pearsons correlation coefficient r = -0.67, P value < 0.0001) between GC(3) and genic CpG methylation. The comparison between 5-3 gradients of CG(3)-skew and genic methylation for the taxa in the study suggests interplay between gene-body methylation and transcription-coupled cytosine deamination effect. Compositional features are correlated with methylation levels of genes in rice, thale cress, human, bee, and fruit fly (which acts as an unmethylated control). These patterns allow us to generate evolutionary hypotheses about the relationships between GC(3) and methylation and how these affect expression patterns. Specifically, we propose that the opposite effects of methylation and compositional gradients along coding regions of GC(3)-poor and GC(3)-rich genes are the products of several competing processes.
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Intrauterine calorie restriction affects placental DNA methylation and gene expression.
Physiol. Genomics
PUBLISHED: 05-21-2013
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Maternal nutrient restriction causes the development of adult onset chronic diseases in the intrauterine growth restricted (IUGR) fetus. Investigations in mice have shown that either protein or calorie restriction during pregnancy leads to glucose intolerance, increased fat mass, and hypercholesterolemia in adult male offspring. Some of these phenotypes are shown to persist in successive generations. The molecular mechanisms underlying IUGR remain unclear. The placenta is a critical organ for mediating changes in the environment and the development of embryos. To shed light on molecular mechanisms that might affect placental responses to differing environments we examined placentas from mice that had been exposed to different diets. We measured gene expression and whole genome DNA methylation in both male and female placentas of mice exposed to either caloric restriction or ad libitum diets. We observed several differentially expressed pathways associated with IUGR phenotypes and, most importantly, a significant decrease in the overall methylation between these groups as well as sex-specific effects that are more pronounced in males. In addition, a set of significantly differentially methylated genes that are enriched for known imprinted genes were identified, suggesting that imprinted loci may be particularly susceptible to diet effects. Lastly, we identified several differentially methylated microRNAs that target genes associated with immunological, metabolic, gastrointestinal, cardiovascular, and neurological chronic diseases, as well as genes responsible for transplacental nutrient transfer and fetal development.
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Evolutionary dynamics and information hierarchies in biological systems.
Ann. N. Y. Acad. Sci.
PUBLISHED: 05-20-2013
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The study of evolution has entered a revolutionary new era, where quantitative and predictive methods are transforming the traditionally qualitative and retrospective approaches of the past. Genomic sequencing and modern computational techniques are permitting quantitative comparisons between variation in the natural world and predictions rooted in neo-Darwinian theory, revealing the shortcomings of current evolutionary theory, particularly with regard to large-scale phenomena like macroevolution. Current research spanning and uniting diverse fields and exploring the physical and chemical nature of organisms across temporal, spatial, and organizational scales is replacing the model of evolution as a passive filter selecting for random changes at the nucleotide level with a paradigm in which evolution is a dynamic process both constrained and driven by the informational architecture of organisms across scales, from DNA and chromatin regulation to interactions within and between species and the environment.
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Proteomic and genomic approaches reveal critical functions of H3K9 methylation and heterochromatin protein-1? in reprogramming to pluripotency.
Nat. Cell Biol.
PUBLISHED: 04-24-2013
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Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) involves a marked reorganization of chromatin. To identify post-translational histone modifications that change in global abundance during this process, we have applied a quantitative mass-spectrometry-based approach. We found that iPSCs, compared with both the starting fibroblasts and a late reprogramming intermediate (pre-iPSCs), are enriched for histone modifications associated with active chromatin, and depleted for marks of transcriptional elongation and a subset of repressive modifications including H3K9me2/me3. Dissecting the contribution of H3K9 methylation to reprogramming, we show that the H3K9 methyltransferases Ehmt1, Ehmt2 and Setdb1 regulate global H3K9me2/me3 levels and that their depletion increases iPSC formation from both fibroblasts and pre-iPSCs. Similarly, we find that inhibition of heterochromatin protein-1? (Cbx3), a protein known to recognize H3K9 methylation, enhances reprogramming. Genome-wide location analysis revealed that Cbx3 predominantly binds active genes in both pre-iPSCs and pluripotent cells but with a strikingly different distribution: in pre-iPSCs, but not in embryonic stem cells, Cbx3 associates with active transcriptional start sites, suggesting a developmentally regulated role for Cbx3 in transcriptional activation. Despite largely non-overlapping functions and the predominant association of Cbx3 with active transcription, the H3K9 methyltransferases and Cbx3 both inhibit reprogramming by repressing the pluripotency factor Nanog. Together, our findings demonstrate that Cbx3 and H3K9 methylation restrict late reprogramming events, and suggest that a marked change in global chromatin character constitutes an epigenetic roadblock for reprogramming.
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Zinc deficiency impacts CO2 assimilation and disrupts copper homeostasis in Chlamydomonas reinhardtii.
J. Biol. Chem.
PUBLISHED: 02-25-2013
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Zinc is an essential nutrient because of its role in catalysis and in protein stabilization, but excess zinc is deleterious. We distinguished four nutritional zinc states in the alga Chlamydomonas reinhardtii: toxic, replete, deficient, and limited. Growth is inhibited in zinc-limited and zinc-toxic cells relative to zinc-replete cells, whereas zinc deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition-responsive changes in gene expression. We identified genes encoding zinc-handling components, including ZIP family transporters and candidate chaperones. Additionally, we noted an impact on two other regulatory pathways, the carbon-concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc deficiency, probably due to reduced carbonic anhydrase activity, validated by quantitative proteomics and immunoblot analysis of Cah1, Cah3, and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in zinc-limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. The Crr1 regulon responds to copper limitation and is turned on in zinc deficiency, and Crr1 is required for growth in zinc-limiting conditions. Zinc-deficient cells are functionally copper-deficient, although they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester copper in a biounavailable form, perhaps to prevent mismetallation of critical zinc sites.
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Plants regenerated from tissue culture contain stable epigenome changes in rice.
Elife
PUBLISHED: 02-11-2013
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Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several regenerated rice lines. We found that all tested regenerated plants had significant losses of methylation compared to non-regenerated plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability. DOI:http://dx.doi.org/10.7554/eLife.00354.001.
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A large-scale zebrafish gene knockout resource for the genome-wide study of gene function.
Genome Res.
PUBLISHED: 02-04-2013
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With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1s predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ~0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome.
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Estrogen and progesterone together expand murine endometrial epithelial progenitor cells.
Stem Cells
PUBLISHED: 01-24-2013
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Synchronous with massive shifts in reproductive hormones, the uterus and its lining the endometrium expand to accommodate a growing fetus during pregnancy. In the absence of an embryo the endometrium, composed of epithelium and stroma, undergoes numerous hormonally regulated cycles of breakdown and regeneration. The hormonally mediated regenerative capacity of the endometrium suggests that signals that govern the growth of endometrial progenitors must be regulated by estrogen and progesterone. Here, we report an antigenic profile for isolation of mouse endometrial epithelial progenitors. These cells are EpCAM(+) CD44(+) ITGA6(hi) Thy1(-) PECAM1(-) PTPRC(-) Ter119(-), comprise a minor subpopulation of total endometrial epithelia and possess a gene expression profile that is unique and different from other cells of the endometrium. The epithelial progenitors of the endometrium could regenerate in vivo, undergo multilineage differentiation and proliferate. We show that the number of endometrial epithelial progenitors is regulated by reproductive hormones. Coadministration of estrogen and progesterone dramatically expanded the endometrial epithelial progenitor cell pool. This effect was not observed when estrogen or progesterone was administered alone. Despite the remarkable sensitivity to hormonal signals, endometrial epithelial progenitors do not express estrogen or progesterone receptors. Therefore, their hormonal regulation must be mediated through paracrine signals resulting from binding of steroid hormones to the progenitor cell niche. Discovery of signaling defects in endometrial epithelial progenitors or their niche can lead to development of better therapies in diseases of the endometrium.
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Retrograde bilin signaling enables Chlamydomonas greening and phototrophic survival.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 01-23-2013
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The maintenance of functional chloroplasts in photosynthetic eukaryotes requires real-time coordination of the nuclear and plastid genomes. Tetrapyrroles play a significant role in plastid-to-nucleus retrograde signaling in plants to ensure that nuclear gene expression is attuned to the needs of the chloroplast. Well-known sites of synthesis of chlorophyll for photosynthesis, plant chloroplasts also export heme and heme-derived linear tetrapyrroles (bilins), two critical metabolites respectively required for essential cellular activities and for light sensing by phytochromes. Here we establish that Chlamydomonas reinhardtii, one of many chlorophyte species that lack phytochromes, can synthesize bilins in both plastid and cytosol compartments. Genetic analyses show that both pathways contribute to iron acquisition from extracellular heme, whereas the plastid-localized pathway is essential for light-dependent greening and phototrophic growth. Our discovery of a bilin-dependent nuclear gene network implicates a widespread use of bilins as retrograde signals in oxygenic photosynthetic species. Our studies also suggest that bilins trigger critical metabolic pathways to detoxify molecular oxygen produced by photosynthesis, thereby permitting survival and phototrophic growth during the light period.
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Stage-specific roles for tet1 and tet2 in DNA demethylation in primordial germ cells.
Cell Stem Cell
PUBLISHED: 01-21-2013
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Primordial germ cells (PGCs) undergo dramatic rearrangements to their methylome during embryogenesis, including initial genome-wide DNA demethylation that establishes the germline epigenetic ground state. The role of the 5-methylcytosine (5mC) dioxygenases Tet1 and Tet2 in the initial genome-wide DNA demethylation process has not been examined directly. Using PGCs differentiated from either control or Tet2(-/-); Tet1 knockdown embryonic stem cells (ESCs), we show that in vitro PGC (iPGC) formation and genome-wide DNA demethylation are unaffected by the absence of Tet1 and Tet2, and thus 5-hydroxymethylcytosine (5hmC). However, numerous promoters and gene bodies were hypermethylated in mutant iPGCs, which is consistent with a role for 5hmC as an intermediate in locus-specific demethylation. Altogether, our results support a revised model of PGC DNA demethylation in which the first phase of comprehensive 5mC loss does not involve 5hmC. Instead, Tet1 and Tet2 have a locus-specific role in shaping the PGC epigenome during subsequent development.
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Methylome reorganization during in vitro dedifferentiation and regeneration of Populus trichocarpa.
BMC Plant Biol.
PUBLISHED: 01-18-2013
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Cytosine DNA methylation (5mC) is an epigenetic modification that is important to genome stability and regulation of gene expression. Perturbations of 5mC have been implicated as a cause of phenotypic variation among plants regenerated through in vitro culture systems. However, the pattern of change in 5mC and its functional role with respect to gene expression, are poorly understood at the genome scale. A fuller understanding of how 5mC changes during in vitro manipulation may aid the development of methods for reducing or amplifying the mutagenic and epigenetic effects of in vitro culture and plant transformation.
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Obsessive-compulsive disorder comorbidity: clinical assessment and therapeutic implications.
Front Psychiatry
PUBLISHED: 10-13-2011
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Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder affecting approximately 1-3% of the population. OCD is probably an etiologically heterogeneous condition. Individuals with OCD frequently have additional psychiatric disorders concomitantly or at some time during their lifetime. Recently, some authors proposed an OCD sub-classification based on comorbidity. An important issue in assessing comorbidity is the fact that the non-response to treatment often involves the presence of comorbid conditions. Non-responsive patients are more likely to meet criteria for comorbid axis I or axis II disorders and the presence of a specific comorbid condition could be a distinguishing feature in OCD, with influence on the treatment adequacy and outcome.
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Lethal iron deprivation induced by non-neutralizing antibodies targeting transferrin receptor 1 in malignant B cells.
Leuk. Lymphoma
PUBLISHED: 08-28-2011
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A number of antibodies have been developed that induce lethal iron deprivation (LID) by targeting the transferrin receptor 1 (TfR1/CD71) and either neutralizing transferrin (Tf) binding, blocking internalization of the receptor and/or inducing its degradation. We have developed recombinant antibodies targeting human TfR1 (ch128.1 and ch128.1Av), which induce receptor degradation and are cytotoxic to certain malignant B-cells. We now show that internalization of TfR1 bound to these antibodies can lead to its sequestration and degradation, as well as reduced Tf uptake, and the induction of a transcriptional response consistent with iron deprivation, which is mediated in part by downstream targets of p53. Cells resistant to these antibodies do not sequester and degrade TfR1 after internalization of the antibody/receptor complex, and accordingly maintain their ability to internalize Tf. These findings are expected to facilitate the rational design and clinical use of therapeutic agents targeting iron import via TfR1 in hematopoietic malignancies.
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Genome-wide binding map of the HIV-1 Tat protein to the human genome.
PLoS ONE
PUBLISHED: 07-12-2011
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The HIV-1 Trans-Activator of Transcription (Tat) protein binds to multiple host cellular factors and greatly enhances the level of transcription of the HIV genome. While Tats control of viral transcription is well-studied, much less is known about the interaction of Tat with the human genome. Here, we report the genome-wide binding map of Tat to the human genome in Jurkat T cells using chromatin immunoprecipitation combined with next-generation sequencing. Surprisingly, we found that ~53% of the Tat target regions are within DNA repeat elements, greater than half of which are Alu sequences. The remaining target regions are located in introns and distal intergenic regions; only ~7% of Tat-bound regions are near transcription start sites (TSS) at gene promoters. Interestingly, Tat binds to promoters of genes that, in Jurkat cells, are bound by the ETS1 transcription factor, the CBP histone acetyltransferase and/or are enriched for histone H3 lysine 4 tri-methylation (H3K4me3) and H3K27me3. Tat binding is associated with genes enriched with functions in T cell biology and immune response. Our data reveal that Tats interaction with the host genome is more extensive than previously thought, with potentially important implications for the viral life cycle.
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Novel stem/progenitor cell population from murine tracheal submucosal gland ducts with multipotent regenerative potential.
Stem Cells
PUBLISHED: 06-29-2011
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The airway epithelium is in direct contact with the environment and therefore constantly at risk for injury. Basal cells (BCs) have been found to repair the surface epithelium (SE), but the contribution of other stem cell populations to airway epithelial repair has not been identified. We demonstrated that airway submucosal gland (SMG) duct cells, in addition to BCs, survived severe hypoxic-ischemic injury. We developed a method to isolate duct cells from the airway. In vitro and in vivo models were used to compare the self-renewal and differentiation potential of duct cells and BCs. We found that only duct cells were capable of regenerating SMG tubules and ducts, as well as the SE overlying the SMGs. SMG duct cells are therefore a multipotent stem cell for airway epithelial repair This is of importance to the field of lung regeneration as determining the repairing cell populations could lead to the identification of novel therapeutic targets and cell-based therapies for patients with airway diseases.
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Regulation of the yeast metabolic cycle by transcription factors with periodic activities.
BMC Syst Biol
PUBLISHED: 04-28-2011
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When growing budding yeast under continuous, nutrient-limited conditions, over half of yeast genes exhibit periodic expression patterns. Periodicity can also be observed in respiration, in the timing of cell division, as well as in various metabolite levels. Knowing the transcription factors involved in the yeast metabolic cycle is helpful for determining the cascade of regulatory events that cause these patterns.
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Systems biology approach in Chlamydomonas reveals connections between copper nutrition and multiple metabolic steps.
Plant Cell
PUBLISHED: 04-15-2011
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In this work, we query the Chlamydomonas reinhardtii copper regulon at a whole-genome level. Our RNA-Seq data simulation and analysis pipeline validated a 2-fold cutoff and 10 RPKM (reads per kilobase of mappable length per million mapped reads) (~1 mRNA per cell) to reveal 63 CRR1 targets plus another 86 copper-responsive genes. Proteomic and immunoblot analyses captured 25% of the corresponding proteins, whose abundance was also dependent on copper nutrition, validating transcriptional regulation as a major control mechanism for copper signaling in Chlamydomonas. The impact of copper deficiency on the expression of several O?-dependent enzymes included steps in lipid modification pathways. Quantitative lipid profiles indicated increased polyunsaturation of fatty acids on thylakoid membrane digalactosyldiglycerides, indicating a global impact of copper deficiency on the photosynthetic apparatus. Discovery of a putative plastid copper chaperone and a membrane protease in the thylakoid suggest a mechanism for blocking copper utilization in the chloroplast. We also found an example of copper sparing in the N assimilation pathway: the replacement of copper amine oxidase by a flavin-dependent backup enzyme. Forty percent of the targets are previously uncharacterized proteins, indicating considerable potential for new discovery in the biology of copper.
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Global phosphoproteomics reveals crosstalk between Bcr-Abl and negative feedback mechanisms controlling Src signaling.
Sci Signal
PUBLISHED: 03-31-2011
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In subtypes and late stages of leukemias driven by the tyrosine kinase fusion protein Bcr-Abl, signaling by the Src family kinases (SFKs) critically contributes to the leukemic phenotype. We performed global tyrosine phosphoprofiling by quantitative mass spectrometry of Bcr-Abl-transformed cells in which the activities of the SFKs were perturbed to build a detailed context-dependent network of cancer signaling. Perturbation of the SFKs Lyn and Hck with genetics or inhibitors revealed Bcr-Abl downstream phosphorylation events either mediated by or independent of SFKs. We identified multiple negative feedback mechanisms within the network of signaling events affected by Bcr-Abl and SFKs and found that Bcr-Abl attenuated these inhibitory mechanisms. The C-terminal Src kinase (Csk)-binding protein Pag1 (also known as Cbp) and the tyrosine phosphatase Ptpn18 both mediated negative feedback to SFKs. We observed Bcr-Abl-mediated phosphorylation of the phosphatase Shp2 (Ptpn11), and this may contribute to the suppression of these negative feedback mechanisms to promote Bcr-Abl-activated SFK signaling. Csk and a kinase-deficient Csk mutant both produced similar globally repressive signaling consequences, suggesting a critical role for the adaptor protein function of Csk in its inhibition of Bcr-Abl and SFK signaling. The identified Bcr-Abl-activated SFK regulatory mechanisms are candidates for dysregulation during leukemia progression and acquisition of SFK-mediated drug resistance.
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Determining DNA methylation profiles using sequencing.
Methods Mol. Biol.
PUBLISHED: 03-25-2011
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Cytosine methylation is an epigenetic mark that has a significant impact on the regulation of transcription and replication of DNA. DNA methylation patterns are highly conserved across cell divisions and are therefore highly heritable. Furthermore, in multicellular organisms, DNA methylation patterning is a key determinant of cellular differentiation and tissue-specific expression patterns. Lastly, DNA demethylases can affect global levels of DNA methylation during specific stages of development. Bisulfite sequencing is considered the gold standard for measuring the methylation state of cytosines. Sodium bisulfite -converts unmethylated cytosines to uracils (which after PCR are converted to thymines), while leaving methylated cytosines unconverted. By mapping bisulfite treated DNA back to the original reference genome, it is then possible to determine the methylation state of individual cytosines. With the advent of next-generation sequencers during the past few years, it is now possible to determine the methylation state of an entire genome. Here, we describe in detail two protocols for preparing bisulfite treated libraries, which may be sequenced using Illumina GAII sequencers. The first of these uses premethylated adapters, which are not affected by bisulfite treatments, while the second uses a two-stage adapter strategy and does not require premethylation of the adapters. We also describe the specialized protocol for mapping bisulfite converted reads. These approaches allow one to determine the methylation state of each cytosine in the genome.
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Identification of novel targets of CSL-dependent Notch signaling in hematopoiesis.
PLoS ONE
PUBLISHED: 03-23-2011
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Somatic activating mutations in the Notch1 receptor result in the overexpression of activated Notch1, which can be tumorigenic. The goal of this study is to understand the molecular mechanisms underlying the phenotypic changes caused by the overexpression of ligand independent Notch 1 by using a tetracycline inducible promoter in an in vitro embryonic stem (ES) cells/OP9 stromal cells coculture system, recapitulating normal hematopoiesis. First, an in silico analysis of the promoters of Notch regulated genes (previously determined by microarray analysis) revealed that the motifs recognized by regulatory proteins known to mediate hematopoiesis were overrepresented. Notch 1 does not bind DNA but instead binds the CSL transcription factor to regulate gene expression. The in silico analysis also showed that there were putative CSL binding sites observed in the promoters of 28 out of 148 genes. A custom ChIP-chip array was used to assess the occupancy of CSL in the promoter regions of the Notch1 regulated genes in vivo and showed that 61 genes were bound by activated Notch responsive CSL. Then, comprehensive mapping of the CSL binding sites genome-wide using ChIP-seq analysis revealed that over 10,000 genes were bound within 10 kb of the TSS (transcription start site). The majority of the targets discovered by ChIP-seq belong to pathways that have been shown by others to crosstalk with Notch signaling. Finally, 83 miRNAs were significantly differentially expressed by greater than 1.5-fold during the course of in vitro hematopoiesis. Thirty one miRNA were up-regulated and fifty two were down-regulated. Overexpression of Notch1 altered this pattern of expression of microRNA: six miRNAs were up-regulated and four were down regulated as a result of activated Notch1 overexpression during the course of hematopoiesis. Time course analysis of hematopoietic development revealed that cells with Notch 1 overexpression mimic miRNA expression of cells in a less mature stage, which is consistent with our previous biological characterization.
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A revised mineral nutrient supplement increases biomass and growth rate in Chlamydomonas reinhardtii.
Plant J.
PUBLISHED: 03-21-2011
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Interest in exploiting algae as a biofuel source and the role of inorganic nutrient deficiency in inducing triacylglyceride (TAG) accumulation in cells necessitates a strategy to efficiently formulate species-specific culture media that can easily be manipulated. Using the reference organism Chlamydomonas reinhardtii, we tested the hypothesis that modeling trace element supplements after the cellular ionome would result in optimized cell growth. We determined the trace metal content of several commonly used Chlamydomonas strains in various culture conditions and developed a revised trace element solution to parallel these measurements. Comparison of cells growing in the revised supplement versus a traditional trace element solution revealed faster growth rates and higher maximum cell densities with the revised recipe. RNA-seq analysis of cultures growing in the traditional versus revised medium suggest that the variation in transcriptomes was smaller than that found between different wild-type strains grown in traditional Hutners supplement. Visual observation did not reveal defects in cell motility or mating efficiency in the new supplement. Ni²?-inducible expression from the CYC6 promoter remained a useful tool, albeit with an increased requirement for Ni²? because of the introduction of an EDTA buffer system in the revised medium. Other advantages include more facile preparation of trace element stock solutions, a reduction in total chemical use, a more consistent batch-to-batch formulation and long-term stability (tested up to 5 years). Under the new growth regime, we analyzed cells growing under different macro- and micronutrient deficiencies. TAG accumulation in N deficiency is comparable in the new medium. Fe and Zn deficiency also induced TAG accumulation, as suggested by Nile Red staining. This approach can be used to efficiently optimize culture conditions for other algal species to improve growth and to assay cell physiology.
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A comparative analysis of DNA methylation across human embryonic stem cell lines.
Genome Biol.
PUBLISHED: 03-18-2011
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We performed a comparative analysis of the genome-wide DNA methylation profiles from three human embryonic stem cell (HESC) lines. It had previously been shown that HESC lines had significantly higher non-CG methylation than differentiated cells, and we therefore asked whether these sites were conserved across cell lines.
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Identification of genes required for de novo DNA methylation in Arabidopsis.
Epigenetics
PUBLISHED: 03-01-2011
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De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1, and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation.
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Algal Functional Annotation Tool: a web-based analysis suite to functionally interpret large gene lists using integrated annotation and expression data.
BMC Bioinformatics
PUBLISHED: 02-08-2011
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Progress in genome sequencing is proceeding at an exponential pace, and several new algal genomes are becoming available every year. One of the challenges facing the community is the association of protein sequences encoded in the genomes with biological function. While most genome assembly projects generate annotations for predicted protein sequences, they are usually limited and integrate functional terms from a limited number of databases. Another challenge is the use of annotations to interpret large lists of interesting genes generated by genome-scale datasets. Previously, these gene lists had to be analyzed across several independent biological databases, often on a gene-by-gene basis. In contrast, several annotation databases, such as DAVID, integrate data from multiple functional databases and reveal underlying biological themes of large gene lists. While several such databases have been constructed for animals, none is currently available for the study of algae. Due to renewed interest in algae as potential sources of biofuels and the emergence of multiple algal genome sequences, a significant need has arisen for such a database to process the growing compendiums of algal genomic data.
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Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli.
Mol. Syst. Biol.
PUBLISHED: 07-13-2010
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Escherichia coli has been engineered to produce isobutanol, with titers reaching greater than the toxicity level. However, the specific effects of isobutanol on the cell have never been fully understood. Here, we aim to identify genotype-phenotype relationships in isobutanol response. An isobutanol-tolerant mutant was isolated with serial transfers. Using whole-genome sequencing followed by gene repair and knockout, we identified five mutations (acrA, gatY, tnaA, yhbJ, and marCRAB) that were primarily responsible for the increased isobutanol tolerance. We successfully reconstructed the tolerance phenotype by combining deletions of these five loci, and identified glucosamine-6-phosphate as an important metabolite for isobutanol tolerance, which presumably enhanced membrane synthesis. The isobutanol-tolerant mutants also show increased tolerance to n-butanol and 2-methyl-1-butanol, but showed no improvement in ethanol tolerance and higher sensitivity to hexane and chloramphenicol than the parental strain. These results suggest that C4, C5 alcohol stress impacts the cell differently compared with the general solvent or antibiotic stresses. Interestingly, improved isobutanol tolerance did not increase the final titer of isobutanol production.
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RNA-seq analysis of sulfur-deprived Chlamydomonas cells reveals aspects of acclimation critical for cell survival.
Plant Cell
PUBLISHED: 06-29-2010
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The Chlamydomonas reinhardtii transcriptome was characterized from nutrient-replete and sulfur-depleted wild-type and snrk2.1 mutant cells. This mutant is null for the regulatory Ser-Thr kinase SNRK2.1, which is required for acclimation of the alga to sulfur deprivation. The transcriptome analyses used microarray hybridization and RNA-seq technology. Quantitative RT-PCR evaluation of the results obtained by these techniques showed that RNA-seq reports a larger dynamic range of expression levels than do microarray hybridizations. Transcripts responsive to sulfur deprivation included those encoding proteins involved in sulfur acquisition and assimilation, synthesis of sulfur-containing metabolites, Cys degradation, and sulfur recycling. Furthermore, we noted potential modifications of cellular structures during sulfur deprivation, including the cell wall and complexes associated with the photosynthetic apparatus. Moreover, the data suggest that sulfur-deprived cells accumulate proteins with fewer sulfur-containing amino acids. Most of the sulfur deprivation responses are controlled by the SNRK2.1 protein kinase. The snrk2.1 mutant exhibits a set of unique responses during both sulfur-replete and sulfur-depleted conditions that are not observed in wild-type cells; the inability of this mutant to acclimate to S deprivation probably leads to elevated levels of singlet oxygen and severe oxidative stress, which ultimately causes cell death. The transcriptome results for wild-type and mutant cells strongly suggest the occurrence of massive changes in cellular physiology and metabolism as cells become depleted for sulfur and reveal aspects of acclimation that are likely critical for cell survival.
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Molecular analyses of human induced pluripotent stem cells and embryonic stem cells.
Cell Stem Cell
PUBLISHED: 05-05-2010
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Recent work from our group and others has argued that human induced pluripotent stem cells (hiPSCs) generated by the introduction of four viruses bearing reprogramming factors differ from human embryonic stem cells (hESCs) at the level of gene expression (Chin et al., 2009). Many of the differences seen were common across independent labs and, at least to some extent, are thought to be a result of residual expression of donor cell-specific genes (Chin et al., 2009; Ghosh et al., 2010; Marchetto et al., 2009). Two new reports reanalyze similar expression data sets as those used in Chin et al. (2009) and come to different conclusions (Newman and Cooper, 2010; Guenther et al., 2010). We compare various approaches to perform gene expression meta-analysis that all support our original conclusions and present new data to demonstrate that polycistronic delivery of the reprogramming factors and extended culture brings hiPSCs transcriptionally closer to hESCs.
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BS Seeker: precise mapping for bisulfite sequencing.
BMC Bioinformatics
PUBLISHED: 04-23-2010
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Bisulfite sequencing using next generation sequencers yields genome-wide measurements of DNA methylation at single nucleotide resolution. Traditional aligners are not designed for mapping bisulfite-treated reads, where the unmethylated Cs are converted to Ts. We have developed BS Seeker, an approach that converts the genome to a three-letter alphabet and uses Bowtie to align bisulfite-treated reads to a reference genome. It uses sequence tags to reduce mapping ambiguity. Post-processing of the alignments removes non-unique and low-quality mappings.
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AID-induced genotoxic stress promotes B cell differentiation in the germinal center via ATM and LKB1 signaling.
Mol. Cell
PUBLISHED: 04-19-2010
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During an immune response, B cells undergo rapid proliferation and activation-induced cytidine deaminase (AID)-dependent remodeling of immunoglobulin (IG) genes within germinal centers (GCs) to generate memory B and plasma cells. Unfortunately, the genotoxic stress associated with the GC reaction also promotes most B cell malignancies. Here, we report that exogenous and intrinsic AID-induced DNA strand breaks activate ATM, which signals through an LKB1 intermediate to inactivate CRTC2, a transcriptional coactivator of CREB. Using genome-wide location analysis, we determined that CRTC2 inactivation unexpectedly represses a genetic program that controls GC B cell proliferation, self-renewal, and differentiation while opposing lymphomagenesis. Inhibition of this pathway results in increased GC B cell proliferation, reduced antibody secretion, and impaired terminal differentiation. Multiple distinct pathway disruptions were also identified in human GC B cell lymphoma patient samples. Combined, our data show that CRTC2 inactivation, via physiologic DNA damage response signaling, promotes B cell differentiation in response to genotoxic stress.
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Evolution of an expanded sex-determining locus in Volvox.
Science
PUBLISHED: 04-17-2010
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Although dimorphic sexes have evolved repeatedly in multicellular eukaryotes, their origins are unknown. The mating locus (MT) of the sexually dimorphic multicellular green alga Volvox carteri specifies the production of eggs and sperm and has undergone a remarkable expansion and divergence relative to MT from Chlamydomonas reinhardtii, which is a closely related unicellular species that has equal-sized gametes. Transcriptome analysis revealed a rewired gametic expression program for Volvox MT genes relative to Chlamydomonas and identified multiple gender-specific and sex-regulated transcripts. The retinoblastoma tumor suppressor homolog MAT3 is a Volvox MT gene that displays sexually regulated alternative splicing and evidence of gender-specific selection, both of which are indicative of cooption into the sexual cycle. Thus, sex-determining loci affect the evolution of both sex-related and non-sex-related genes.
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Conservation and divergence of methylation patterning in plants and animals.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-15-2010
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Cytosine DNA methylation is a heritable epigenetic mark present in many eukaryotic organisms. Although DNA methylation likely has a conserved role in gene silencing, the levels and patterns of DNA methylation appear to vary drastically among different organisms. Here we used shotgun genomic bisulfite sequencing (BS-Seq) to compare DNA methylation in eight diverse plant and animal genomes. We found that patterns of methylation are very similar in flowering plants with methylated cytosines detected in all sequence contexts, whereas CG methylation predominates in animals. Vertebrates have methylation throughout the genome except for CpG islands. Gene body methylation is conserved with clear preference for exons in most organisms. Furthermore, genes appear to be the major target of methylation in Ciona and honey bee. Among the eight organisms, the green alga Chlamydomonas has the most unusual pattern of methylation, having non-CG methylation enriched in exons of genes rather than in repeats and transposons. In addition, the Dnmt1 cofactor Uhrf1 has a conserved function in maintaining CG methylation in both transposons and gene bodies in the mouse, Arabidopsis, and zebrafish genomes.
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Relationship between nucleosome positioning and DNA methylation.
Nature
PUBLISHED: 03-05-2010
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Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana using massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified 10-base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results indicate that nucleosome positioning influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA, indicating that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron-exon and exon-intron boundaries. RNA polymerase II (Pol II) was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is also enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition.
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Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency.
Nature
PUBLISHED: 01-15-2010
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Epigenetic reprogramming including demethylation of DNA occurs in mammalian primordial germ cells (PGCs) and in early embryos, and is important for the erasure of imprints and epimutations, and the return to pluripotency. The extent of this reprogramming and its molecular mechanisms are poorly understood. We previously showed that the cytidine deaminases AID and APOBEC1 can deaminate 5-methylcytosine in vitro and in Escherichia coli, and in the mouse are expressed in tissues in which demethylation occurs. Here we profiled DNA methylation throughout the genome by unbiased bisulphite next generation sequencing in wild-type and AID-deficient mouse PGCs at embryonic day (E)13.5. Wild-type PGCs revealed marked genome-wide erasure of methylation to a level below that of methylation deficient (Np95(-/-), also called Uhrf1(-/-)) embryonic stem cells, with female PGCs being less methylated than male ones. By contrast, AID-deficient PGCs were up to three times more methylated than wild-type ones; this substantial difference occurred throughout the genome, with introns, intergenic regions and transposons being relatively more methylated than exons. Relative hypermethylation in AID-deficient PGCs was confirmed by analysis of individual loci in the genome. Our results reveal that erasure of DNA methylation in the germ line is a global process, hence limiting the potential for transgenerational epigenetic inheritance. AID deficiency interferes with genome-wide erasure of DNA methylation patterns, indicating that AID has a critical function in epigenetic reprogramming and potentially in restricting the inheritance of epimutations in mammals.
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Nonsense-mediated mRNA decay mutes the splicing defects of spliceosome component mutations.
RNA
PUBLISHED: 10-22-2009
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The role of many splicing factors in pre-mRNA splicing and the involvement of these factors in the processing of specific transcripts have often been defined through the analysis of loss-of-function mutants in vivo. Here we show that inactivating the nonsense-mediated mRNA decay (NMD) results in an enhancement of splicing phenotypes associated with several S. cerevisiae splicing factor mutations. Tiling microarrays showed that inactivation of the NMD factor Upf1p in the prp17Delta and prp18Delta mutant strains results in a larger spectrum of splicing defects than what is observed in the single mutants, including new transcripts previously shown unaffected by Prp17p or Prp18p inactivation. Inactivation of Upf1p in the second step/recycling factor prp22-1 mutant and in the nam8Delta and mud1Delta U1 snRNP component mutants also increase unspliced precursor accumulation of several specific transcripts. In addition, deletion of UPF1 partially suppresses the growth defects associated with the prp17Delta or prp22-1 mutations, demonstrating a positive genetic interaction between NMD and splicing factor mutants. These results show that RNA surveillance by NMD can mask some of the effects of splicing factor mutations, and that the roles of splicing factors cannot be fully understood in vivo unless RNA degradation systems that degrade unspliced precursors are also inactivated.
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Copy number variation influences gene expression and metabolic traits in mice.
Hum. Mol. Genet.
PUBLISHED: 07-31-2009
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Copy number variants (CNVs) are genomic segments which are duplicated or deleted among different individuals. CNVs have been implicated in both Mendelian and complex traits, including immune and behavioral disorders, but the study of the mechanisms by which CNVs influence gene expression and clinical phenotypes in humans is complicated by the limited access to tissues and by population heterogeneity. We now report studies of the effect of 19 CNVs on gene expression and metabolic traits in a mouse intercross between strains C57BL/6J and C3H/HeJ. We found that 83% of genes predicted to occur within CNVs were differentially expressed. The expression of most CNV genes was correlated with copy number, but we also observed evidence that gene expression was altered in genes flanking CNVs, suggesting that CNVs may contain regulatory elements for these genes. Several CNVs mapped to hotspots, genomic regions influencing expression of tens or hundreds of genes. Several metabolic traits including cholesterol, triglycerides, glucose and body weight mapped to three CNVs in the genome, in mouse chromosomes 1, 4 and 17. Predicted CNV genes, such as Itlna, Defcr-1, Trim12 and Trim34 were highly correlated with these traits. Our results suggest that CNVs have a significant impact on gene expression and that CNVs may be playing a role in the mechanisms underlying metabolic traits in mice.
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Detecting coordinated regulation of multi-protein complexes using logic analysis of gene expression.
BMC Syst Biol
PUBLISHED: 06-15-2009
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Many of the functional units in cells are multi-protein complexes such as RNA polymerase, the ribosome, and the proteasome. For such units to work together, one might expect a high level of regulation to enable co-appearance or repression of sets of complexes at the required time. However, this type of coordinated regulation between whole complexes is difficult to detect by existing methods for analyzing mRNA co-expression. We propose a new methodology that is able to detect such higher order relationships.
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ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing.
Nat. Struct. Mol. Biol.
PUBLISHED: 04-24-2009
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Constitutive heterochromatin in Arabidopsis thaliana is marked by repressive chromatin modifications, including DNA methylation, histone H3 dimethylation at Lys9 (H3K9me2) and monomethylation at Lys27 (H3K27me1). The enzymes catalyzing DNA methylation and H3K9me2 have been identified; alterations in these proteins lead to reactivation of silenced heterochromatic elements. The enzymes responsible for heterochromatic H3K27me1, in contrast, remain unknown. Here we show that the divergent SET-domain proteins ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6 have H3K27 monomethyltransferase activity, and atxr5 atxr6 double mutants have reduced H3K27me1 in vivo and show partial heterochromatin decondensation. Mutations in atxr5 and atxr6 also lead to transcriptional activation of repressed heterochromatic elements. Notably, H3K9me2 and DNA methylation are unaffected in double mutants. These results indicate that ATXR5 and ATXR6 form a new class of H3K27 methyltransferases and that H3K27me1 represents a previously uncharacterized pathway required for transcriptional repression in Arabidopsis.
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Determining the conservation of DNA methylation in Arabidopsis.
Epigenetics
PUBLISHED: 02-16-2009
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A high-resolution map of DNA methylation in Arabidopsis has recently been generated using high-throughput sequencing of bisulfite-converted DNA. This detailed profile measures the methylation state of most of the cytosines in the Arabidopsis genome, and allows us for the first time to address questions regarding the conservation of methylation across duplicated regions of the genome. To address these questions we measured the degree to which methylation is conserved in both duplicated genes and duplicated non-coding regions of the genome. Methylation is controlled by different mechanisms and methyltransferases depending on the genomic location. Methylation in genes occurs primarily at CG sites and is controlled by the maintenance methyltransferase MET1. In contrast, an RNAi mediated methylation pathway that leads to de novo methylation of asymmetric CHH sites along with CG and CHG sites by the methyltransferase DRM2, drives methylation at tandem and inverted repeats. We find that the cytosine methylation profile is strongly preserved between duplicated genes and repeat regions. The highest level of conservation can be found at CG sites in genes and CHH sites in repeat regions. By constructing substitution matrices between aligned genes we see that methylated cytosines often pair with thymines, which may be explained by the spontaneous deamination of methyl-cytosine to thymine. Despite this observation, we find that methylated cytosines are less often paired with other nucleotides than non-methylated cytosines within gene bodies indicating that they may play an important functional role.
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Mef2C is a lineage-restricted target of Scl/Tal1 and regulates megakaryopoiesis and B-cell homeostasis.
Blood
PUBLISHED: 02-11-2009
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The basic helix-loop-helix transcription factor stem cell leukemia gene (Scl) is a master regulator for hematopoiesis essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However, the critical downstream targets of Scl remain undefined. Here, we identified a novel Scl target gene, transcription factor myocyte enhancer factor 2 C (Mef2C) from Scl(fl/fl) fetal liver progenitor cell lines. Analysis of Mef2C(-/-) embryos showed that Mef2C, in contrast to Scl, is not essential for specification into primitive or definitive hematopoietic lineages. However, adult VavCre(+)Mef2C(fl/fl) mice exhibited platelet defects similar to those observed in Scl-deficient mice. The platelet counts were reduced, whereas platelet size was increased and the platelet shape and granularity were altered. Furthermore, megakaryopoiesis was severely impaired in vitro. Chromatin immunoprecipitation microarray hybridization analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells, but not in erythroid cells. In addition, an Scl-independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice, characterized as severe age-dependent reduction of specific B-cell progenitor populations reminiscent of premature aging. In summary, this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.