Genomic expression over batch growth of mupirocin producing strain Pseudomonas synxantha NCIMB10586
ABSTRACT: The Pseudomonas synxantha strain NCIMB10586 produces the antibiotic mupirocin / Pseudomonic acid A from a 75 kb gene cluster; expression of this is regulated through quorum-sensing. We wished to examine expression of the strain during batch growth, in particular of the mupirocin cluster and any other operons regulated in a similar manner. We took samples from three independent cultures at 7 time-points - sampling every 2 hours from 6 to 18 hours.
Project description:Cytosine DNA methylation is a heritable epigenetic mark present in many eukaryotic organisms. While 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 of the organisms. Furthermore, genes appear to be the major target of methylation in Ciona and honeybee. 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, we demonstrate that 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. Comparison of methylation across eight eukaryotic organisms
Project description:Differentiation into diverse cell lineages requires orchestration of gene regulatory networks guiding cell fate choices. Genetic factors acting through changes in transcriptional levels can contribute to cardiovascular disease risk by impacting early stages of development and have cell type-specific effects. We set out to characterize lineage trajectory progression of subpopulations and identify potential disease-related genes by examining their expression changes in single cells during early stages of cardiac lineage specification. Using 43,168 single-cell transcriptomes, we developed novel classification and trajectory analysis methods to dissect cellular composition and gene networks across five discrete time points underlying lineage derivation of mesoderm, definitive endoderm, vascular endothelium, cardiac precursors, and definitive cell types that comprise cardiomyocytes and a previously unrecognized cardiac outflow tract population.
Project description:Selective transcriptional activation and repression of genes throughout signaling cascades and development are poorly understood. Transcription factors (TF) orchestrate patterns and magnitude of transcriptional response, but TF action, or inaction, is highly dependent upon TF kinetics, distance from genes, chromatin architecture, and the local occupancy of other TFs. We integrated genomic transcription, chromosome looping, TF binding, and chromatin structure data to analyze the molecular cascade that results from estradiol-induced (E2) signaling in human MCF-7 breast cancer cells and addressed the context-specific nature of gene regulation. We analyzed kinetic ChIP-seq that profiled the master regulator of the E2-mediated response, estrogen receptor (ER), and found that transient ER binding sites are specifically associated with enhancers of repressed genes. We performed replicate ChIP-seq experiments prior to estrogen treatment and 2min, 5min, 10min, 40min, and 160min after E2 treatment.
Project description:This study contributes large-scale genes expression data of molecular function for a plant system under combined of two CO2 concentration and three Mg levels. It provides an additional example of the power of integrated analyses for the comprehensive study of the molecular physiology of complex biological systems. Moreover, taking into consideration the particular interest of the two investigated perturbations in plant biotechnology, enhanced understanding of the molecular physiology of the plants under climate change conditions could lead to the design of novel metabolic engineering strategies to copy with crops to magnesium problems. Examination of two CO2 concentration and three Mg levels of shoot and root Arabidopsis
Project description:The thymus shapes the T cell receptor repertoire, and is one of the first organs to rapidly age, with gross changes in cellularity and architecture. To resolve the nature of these changes we used SMART-Seq2 on FACS purified thymic epithelial cells (TEC) across the first year of mouse life. We sorted 4 TEC populations in each of 5 mice at each age (1 week, 4 weeks, 16 weeks, 32 weeks and 52 weeks old).
Project description:piRNAs are required to maintain germline integrity and fertility but their mechanism of action is poorly understood. Here we demonstrate that C. elegans piRNAs silence transcripts in trans through imperfectly complementary sites. We find that target silencing is independent of Piwi endonuclease activity or “slicing”. Instead, we show that piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous protein-coding gene, pseudogene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are derepressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes but not pseudogenes or transposons. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNAi in C. elegans. 7 small RNA libraries were sequenced as part of 25 flow cell lanes on the Illumina GA II platform. Samples were treated with tobacco acid pyrophosphatase to allow cloning of small RNAs with a 5'-triphosphate. Samples were labelled for multiplexing using 4-bp 5'-barcodes or barcodes included in Illumina TruSeq adapters. In most cases a single flow cell lane included several multiplexed libraries.
Project description:Human epidemiologic and animal model data indicate that early environmental influences can persistently alter an individual’s risk of obesity. Environmental effects on hypothalamic developmental epigenetics provide a strong candidate mechanism to explain such ‘developmental programming’ of obesity. To advance our understanding of these processes, it is essential to determine to what extent the diversity of hypothalamic cell types is regulated by epigenetic differences, and when these are established. By performing genome-scale DNA methylation profiling in hypothalamic neurons and non-neuronal cells at postnatal day 0 (P0) and P21, we found that most of the DNA methylation differences distinguishing these two cell types are established postnatally. We found dramatic neuron-specific increases in DNA methylation from P0 to P21. Gene ontology analyses indicated that cell-type specific P0 to P21 methylation changes are key regulators of hypothalamic development. Quantitative bisulfite pyrosequencing verified our methylation profiling results in 16 of 16 selected regions. Expression differences were associated with DNA methylation in several genes analyzed. Our data indicate that future studies of hypothalamic epigenetics in developmental programming of obesity will gain far greater sensitivity and insight by examining outcomes at the cell-type specific level. Moreover, our results provide new evidence that early postnatal life is a critical period for murine hypothalamic developmental epigenetics. Hypothalami were dissected from inbred male C57 mice at postnatal day 0 (P0) and P21. Non-neuronal and neuronal nuclei were separated via fluorescence-activated sorting based on staining for the neuron-specific nuclear surface marker NeuN; each sample for sorting was comprised of 2 age-matched hypothalami. Genome-scale DNA methylation profiling was performed by methylation specific amplification coupled with next generation sequencing (MSA-seq) as decribed below (5 independent samples per age).
Project description:During transcription the nascent RNA can invade the DNA template, forming extended RNA-DNA duplexes (R-loops). Here we employ ChIP-seq in strains expressing or lacking RNase H to map targets of RNase H activity throughout budding yeast genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region transcribed by Pol I and over the 5S rDNA transcribed by Pol III. In strains lacking RNase H activity, R-loops were elevated over other Pol III genes notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5?-flanking regions of tRNA genes. Unexpectedly, R-loops were also associated with mitochondrial genes in the absence of RNase H1, but not of RNase H2. Finally, R-loops were detected on highly expressed protein-coding genes in the wild-type, notably over the second exon of spliced ribosomal protein genes. ChIP-seq of RNA-DNA hybrids using antibody S9.6
Project description:The mammalian circadian clock involves a transcriptional feedback loop in which CLOCK and BMAL1 activate the Period and Cryptochrome genes, which then feedback and repress their own transcription. We have interrogated the transcriptional architecture of the circadian transcriptional regulatory loop on a genome scale in mouse liver and find a stereotyped, time-dependent pattern of transcription factor binding, RNA polymerase II (RNAPII) recruitment, RNA expression and chromatin states. We find that the circadian transcriptional cycle of the clock consists of three distinct phases - a poised state, a coordinated de novo transcriptional activation state, and a repressed state. Interestingly only 22% of mRNA cycling genes are driven by de novo transcription, suggesting that both transcriptional and post-transcriptional mechanisms underlie the mammalian circadian clock. We also find that circadian modulation of RNAPII recruitment and chromatin remodeling occurs on a genome-wide scale far greater than that seen previously by gene expression profiling. Examination of 9 transcriptional regulators, 2 RNAPII and 6 histone modifications every 4hr during the circadian cycle in mouse liver
Project description: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 E coli, and in the mouse are expressed in tissues in which demethylation occurs. Here we profiled DNA methylation throughout the genome by unbiased bisulfite Next Generation Sequencing (BS-Seq) in wildtype and Aid deficient PGCs at E13.5. Wildtype PGCs revealed dramatic genome-wide erasure of methylation to a level below that of methylation deficient (Np95-/-) ES cells, with female PGCs being less methylated than male ones. By contrast, Aid deficient PGCs were up to three times more methylated than wildtype 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, suggesting that Aid has a critical function in epigenetic reprogramming and potentially in restricting the inheritance of epimutations in mammals. Comparison of methylation in wild-type and Aid deficient mouse tissues