Extrachromosomal DNA (microDNA) in Human, Mouse and Chicken
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ABSTRACT: MicroDNAs are <400-base long extrachromosomal circles found in mammalian cells. Tens of thousands of microDNAs were found in all tissue types, including sperm. MicroDNAs arose preferentially from areas with high gene density, high GC content, high exon density, promoters with activating chromatin modifications and in sperm from the 5'UTR of full-length LINE-1 elements, but were depleted from lamin-associated heterochromatin. Furthermore, analysis of microDNAs from a set of human cancer cell lines revealed lineage-specific patterns of microDNA origins. A survey of microDNAs from chicken cell lines defective in various DNA repair proteins revealed that homologous recombination repair and non-homologous end joining repair pathways are not required for microDNA production. A deletion of the MSH3 protein, involved in DNA mismatch repair, resulted in a significant decrease in microDNA abundance specifically from non-CpG areas of the genome. Thus microDNAs arise as part of normal cellular physiology, either from DNA breaks associated with RNA metabolism or from replication slippage followed by mismatch repair. Circular DNA profiling by high throughput sequencing. Five human, ten mouse and nine chicken samples are analyzed
Project description:We have identified tens of thousands of short extrachromosomal circular DNAs (microDNA) in mouse tissues as well as mouse and human cell lines. These microDNAs are 200-400 bp long, derived from unique non-repetitive sequence and are enriched in the 5' untranslated regions of genes, exons and CpG islands. Chromosomal loci that are enriched sources of microDNA in adult brain are somatically mosaic for micro-deletions that appear to arise from the excision of microDNAs. Germline microdeletions identified by the "Thousand Genomes" project may also arise from the excision of microDNAs in the germline lineage. We have thus identified a new DNA entity in mammalian cells and provide evidence that their generation leaves behind deletions in different genomic loci. Circular DNA profiling by high throughput sequencing
Project description:Formation of the blood from self-renewing hematopoietic stem cells to terminal lineages necessarily involves epigenomic modifications of the genome to control regulator and signature gene expression. By analysing the global expression profiles of hematopoietic stem cells (HSCs), in vivo differentiated CD4+ T cells and CD19+ B cells as well as in vitro differentiated erythrocyte precursor cells, we identified hundreds of transcripts showing type-specific expression in these cell types. To understand the epigenomic changes related to tissue-specific expression during HSC differentiation, we examined the genome-wide distribution of H3K4me1, H3K4me3, H3K27me1, H3K27me3, histone variant H2A.Z, chromatin remodeler BRG1, and RNA Polymerase II in the same four cell types, as well as embryonic stem cells. Analysis of these datasets revealed that numerous key differentiation genes are primed for expression by Brg1 and Pol II binding, as well as bivalent modifications in the HSCs prior to their expression in downstream differentiated cell types. Much of this bivalency in HSC is retained from embryonic stem cells. After differentiation, these modified regions resolve to active chromatin modification configuration in the specific lineage, while in parallel differentiated lineages the bivalent modification remains; Pol II and Brg1 are lost in closer lineages but bivalency resolves to silent monovalency in more distant lineages. Correlation of tissue-specific gene expression with the epigenomic changes predicts tens of thousands of potential common enhancers and tissue-specific enhancers, which may critically contribute to the expression patterns. We provide a valuable dataset for further understanding the regulatory mechanisms of differentiation and function of blood lineages. ChIP-Seq: profiling of DNA-associated proteins in blood cell subsets.
Project description:Although lincRNAs are implicated in regulating gene expression in various tissues, little is known about lincRNA transcriptomes in the T cell lineages. Here we identify 1,524 lincRNAs in 42 T cell samples from early T cell progenitors to terminally differentiated T helper subsets. Our analysis revealed highly dynamic and cell-specific expression patterns of lincRNAs during T cell differentiation. Importantly, these lincRNAs are located in genomic regions enriched for protein-coding genes with immune-regulatory functions. Many of these transcripts are bound and regulated by the key T cell transcription factors, T-bet, GATA3, STAT4 and STAT6. We demonstrate that the lincRNA LincR-Ccr2-5'AS, together with GATA3, is an essential component of a regulatory circuit in Th2-specific gene expression. To obtain comprehensive profiles of lincRNA expression during the development and differentiation of T cell lineages, we purified CD4-CD8 double negative (DN) cells (DN1, DN2, DN3 and DN4), double positive (DP) cells (CD4+CD8+CD3low and CD4+CD8intCD69+), single positive (SP) CD4 and CD8 cells, and thymic-derived regulatory T cells (tTreg) from thymi of C57BL/6 mice. Additionally, we obtained Th1, Th2, Th17 and iTreg cells by in vitro differentiation of naM-CM-/ve CD4 T cells for a various length of time in culture (4 hrs, 8 hrs, 12 hrs, 24 hrs, 48 hrs, 72 hrs, 1 week, 2weeks). Total and/or polyadenylated RNAs from these cells was analyzed using RNA-Seq. To understand the regulation of lincRNAs by T cell master regulator T-bet, we compared the transcriptiomes between T-bet deficient Th1 cells and control Th1 cells. We did similar experiments and data analysis for STAT4 (Th1), GATA3 (Th2) and STAT6 (Th2). Finally, to address the funcation of a Th2-specifically expressed lincRNA, lincR-Ccr2-5'AS, we compared the transcriptomes between lincR-Ccr2-5'AS knockdown Th2 cells and control Th2 cells.
Project description:Idiopathic Pulmonary Fibrosis (IPF) is a chronic progressive lung disease that affects more than 5 million people worldwide with a steady increase in both incidence and mortality. There is currently no effective therapy and the median survival without transplant is 2-5 years. The etiological factor is unknown, but several observational and pathogenesis studies suggest that environmental agents may cause IPF. DNA methylation is a type of chemical modification of DNA such environmental and occupational factors, that can induced a changes in the regulation of biological processes and link to diseases such as a cancer. We hypothesize that the global changes in methylation patterns of IPF lungs caused by environmental factors. In this study we will identify the global methylation signatures of the IPF lung and to compare to methylation signature of lung cancer. The DNA methylation profiles of IPF lung tissue differs from control lung but it shares great similarity with that of lung cancer. Immunoprecipitated methylated DNA from 12 IPF lungs, 10 lung adenocarcinomas and 10 normal histology lungs obtained from the same group of adenocarcinoma patients was hybridized to Agilent human CpG Islands Microarrays. Only probes with a hybridization Tm value between 79 C and 93C were included in the analysis because these show higher quality signal. All probes were divided according to their Tm into 14 groups/bins differing by 1C. Probe signals in each bin were standardized to have an average of 0 and a standard deviation of 1. To work in a CpG island oriented manner, we scored each island for its likelihood to be methylated. For that purpose, each probe was mapped to the genome and the signals of the probes that were mapped to a single CpG island were averaged to obtain the islandM-bM-^@M-^Ys methylation score. Data analysis was performed using BRB-Array Tools and DAVID Bioinformatics Resources software packages.
Project description:Comparison of epigenome and Tcf1 occupancy between control and Tcf1/Lef1-deficient CD8 T cells Control mice or those are deficient for Tcf1 and Lef1 transcription factors (deleted by CD4-Cre) were used to isolate thymocytes. The thymocytes were surface-stained to identify TCRbeta high, CD69â, CD24â CD8+ subsets. These cells were sorted for ChIPseq analysis of various histone marks. Control mice or those are deficient for Tcf1 (deleted by CD4-Cre) were used to isolate thymocytes. The splenocytes were surface-stained to identify TCRbeta high, CD8+ subsets. These cells were sorted for ChIPseq analysis of Tcf1 binding locations.
Project description:Formation of the blood from self-renewing hematopoietic stem cells to terminal lineages necessarily involves epigenomic modifications of the genome to control regulator and signature gene expression. By analysing the global expression profiles of hematopoietic stem cells (HSCs), in vivo differentiated CD4+ T cells and CD19+ B cells as well as in vitro differentiated erythrocyte precursor cells, we identified hundreds of transcripts showing type-specific expression in these cell types. To understand the epigenomic changes related to tissue-specific expression during HSC differentiation, we examined the genome-wide distribution of H3K4me1, H3K4me3, H3K27me1, H3K27me3, histone variant H2A.Z, chromatin remodeler BRG1, and RNA Polymerase II in the same four cell types, as well as embryonic stem cells. Analysis of these datasets revealed that numerous key differentiation genes are primed for expression by Brg1 and Pol II binding, as well as bivalent modifications in the HSCs prior to their expression in downstream differentiated cell types. Much of this bivalency in HSC is retained from embryonic stem cells. After differentiation, these modified regions resolve to active chromatin modification configuration in the specific lineage, while in parallel differentiated lineages the bivalent modification remains; Pol II and Brg1 are lost in closer lineages but bivalency resolves to silent monovalency in more distant lineages. Correlation of tissue-specific gene expression with the epigenomic changes predicts tens of thousands of potential common enhancers and tissue-specific enhancers, which may critically contribute to the expression patterns. We provide a valuable dataset for further understanding the regulatory mechanisms of differentiation and function of blood lineages. This SuperSeries is composed of the SubSeries listed below. Refer to individual Series.
Project description:We investigated the genomic landscape of histone modifications in antigen-experienced CD8+ T cells. Using a ChIP-Seq approach coupled with global gene expression profiling [GSE67825], we generated genome-wide histone H3 lysine 4 (H3K4me3) and H3 lysine 27 (H3K27me3) trimethylation maps in distinct subsets of CD8+ T cells - naïve, stem cell memory, central memory, and effector memory. To gain insight into how histone architecture is remodeled during the differentiation of activated T cells
Project description:Transcriptional profiling was carried out on lung and ileum samples at 1dpi and 3dpi from chickens infected with either low pathogenic (H5N2) or highly pathogenic (H5N1) avian influenza. Infected birds were compared to control birds at each time point.
Project description:CD4+ T cells that selectively produce interleukin (IL)-17, are critical for host defense and autoimmunity. Crucial for T helper17 (Th17) cells in vivo, IL-23 has been thought to be incapable of driving initial differentiation. Rather, IL-6 and transforming growth factor (TGF)-β1 have been argued to be the factors responsible for initiating specification. Herein, we show that Th17 differentiation occurs in the absence of TGF-β signaling. Neither IL-6 nor IL-23 alone efficiently generated Th17 cells; however, these cytokines in combination with IL-1β effectively induced IL-17 production in naïve precursors, independently of TGF-β. Epigenetic modification of the Il17a/Il17f and Rorc promoters proceeded without TGF-β1, allowing the generation of cells that co-expressed Rorγt and T-bet. T-bet+Rorγt+ Th17 cells are generated in vivo during experimental allergic encephalomyelitis (EAE), and adoptively transferred Th17 cells generated with IL-23 in the absence of TGF-β1 were more pathogenic in this experimental disease. These data suggest a new model for Th17 differentiation. Consistent with genetic data linking the IL23R with autoimmunity, our findings re-emphasize the role of IL-23 and therefore have important implications for the development of new therapies. Examination of Stat3 binding and H3K4me and H3Ac in helper T cells.
Project description:iCLIP-seq experiment to asses the binding of mitochondrially targeted MRB8170 and MRB4160 involved in RNA editing on a genomic scale. Furthermore, to investigate what subsets of maxicircles transcripts (pan-edited, minimally-edited and never-edited) are bound to both the above proteins in vivo.