41,213 research outputs found
CpG islands of the pig
No full textWe describe an analysis of the CpG islands (CGIs) of the pig. We have used both database survey and a porcine genomic library that is enriched for CGIs. Approximately half of 41 pig genomic database sequences had CGIs with an average G + C content of 65.3%, an average CpG observed/expected frequency of 0.85, and an average size of 978 bp. Of 27 CGI library clones, 16 were nonrepetitive, nonribosomal DNA and CGI-like. CGI library clones had similar average values for G + C and CpG frequency to CGIs of database genes, and an average size of 670 bp, as MseI cuts within some islands. Library clones were also shown to be low copy number and unmethylated in genomic DNA. The presence in the library of seven previously known CGI sequences was confirmed as was the absence of one nonisland sequence. The CGI library exhibits an R-band pattern for many chromosomes in FISH analysis. The pig chromosome arms that show the most dense CGI population are homologous to segments of human chromosomes that are known to be gene rich
In Vivo Control of CpG and Non-CpG DNA Methylation by DNA Methyltransferases
No full textThe enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs
CpG oligonucleotide activates Toll-like receptor 9 and causes lung inflammation in vivo
No full textBackground Bacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-ODN) initiate an innate immune response mediated by the pattern recognition receptor Toll-like receptor 9 (TLR9). This leads in particular to the expression of proinflammatory mediators such as tumor necrosis factor (TNF-alpha) and interleukin-1beta (IL-1beta). TLR9 is expressed in human and murine pulmonary tissue and induction of proinflammatory mediators has been linked to the development of acute lung injury. Therefore, the hypothesis was tested whether CpG-ODN administration induces an inflammatory response in the lung via TLR9 in vivo. Methods Wild-type (WT) and TLR9-deficient (TLR9-D) mice received CpG-ODN intraperitoneally (1668-Thioat, 1 nmol/g BW) and were observed for up to 6 hrs. Lung tissue and plasma samples were taken and various inflammatory markers were measured. Results In WT mice, CpG-ODN induced a strong activation of pulmonary NFKB as well as a significant increase in pulmonary TNF-alpha and IL-1beta mRNA/protein. In addition, cytokine serum levels were significantly elevated in WT mice. Increased pulmonary content of lung myeloperoxidase (MPO) was documented in WT mice following application of CpG-ODN. Bronchoalveolar lavage (BAL) revealed that CpG-ODN stimulation significantly increased total cell number as well as neutrophil count in WT animals. In contrast, the CpG-ODN-induced inflammatory response was abolished in TLR9-D mice. Conclusion This study suggests that bacterial CpG-ODN causes lung inflammation via TLR9
Computational analysis of CpG site DNA methylation
No full textThis thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Epigenetics is the study of factors that can change DNA and passed to next generation without change to DNA sequence. DNA methylation is one of the categories of epigenetic change. DNA methylation is the attachment of methyl group (CH3) to DNA. Most of the time it occurs in the sequences that G is followed by C known as CpG sites and by addition of methyl to the cytosine residue. As science and technology progress new data are available about individual’s DNA methylation profile in different conditions. Also new features discovered that can have role in DNA methylation. The availability of new data on DNA methylation and other features of DNA provide challenge to bioinformatics and the opportunity to discover new knowledge from existing data. In this research multiple data series were used to identify classes of methylation DNA to CpG sites. These classes are a) Never methylated CpG sites,b) Always methylated CpG sites, c) Methylated CpG sites in cancer/disease samples and non-methylated in normal samples d) Methylated CpG sites in normal samples and non-methylated in cancer/disease samples. After identification of these sites and their classes, an analysis was carried out to find the features which can better classify these sites a matrix of features was generated using four applications in EMBOSS software suite. Features matrix was also generated using the gUse/WS-PGRADE portal workflow system. In order to do this each of the four applications were grid enabled and ported to BOINC platform. The gUse portal was connected to the BOINC project via 3G-bridge. Each node in the workflow created portion of matrix and then these portions were combined together to create final matrix. This final feature matrix used in a hill climbing workflow. Hill climbing node was a JAVA program ported to BOINC platform. A Hill climbing search workflow was used to search for a subset of features that are better at classifying the CpG sites using 5 different measurements and three different classification methods: support vector machine, naïve bayes and J48 decision tree. Using this approach the hill climbing search found the models which contain less than half the number of features and better classification results. It is also been demonstrated that using gUse/WS-PGRADE workflow system can provide a modular way of feature generation so adding new feature generator application can be done without changing other parts. It is also shown that using grid enabled applications can speedup both feature generation and feature subset selection. The approach used in this research for distributed workflow based feature generation is not restricted to this study and can be applied in other studies that involve feature generation. The approach also needs multiple binaries to generate portions of features. The grid enabled hill climbing search application can also be used in different context as it only requires to follow the same format of feature matrix
Studies on CpG distribution in genomes
No full textMS, DBTESDNA methylation is an epigenetic modification that plays very important role in vertebrate
genomes. In vertebrate genomes, DNA methylation occurs at CpG sites and leads to gene
regulation, gene imprinting, X-chromosome inactivation and cancer. DNA methylation has
resulted in depletion in CpG sites in vertebrate genome. CpG islands are the clusters of CpGs in
regions of high GC content and are usually unmethylated. However all the clusters of CpGs are
not CpG islands but seem to affects the DNA methylation levels in vertebrate genomes. The
present study has used CpG gap size (number of nucleotides between two adjacent CpG) to
investigate the distribution of CpGs in genomes. Mean CpG gap sizes have been observed to be
larger in methylated genome while smaller values are associated with poorly and non-methylated
genomes. Similarly, dispersion index of CpG gap values also shows higher value for methylated
genomes and lower value in poorly or unmethylated genomes indicating relationship between
DNA methylation and clustered distribution of CpGs in the genomes. A similar result was
obtained when exon, intron and intergenic region of different organisms were studied separately.
Further CpG gap analysis were compared against five other related dinucleotides gaps (GpC,
TpG, GpT, CpA and ApC) in differently methylated genomes and higher value of mean CG gaps
and dispersion index was found in methylated human genome in a pronounced manner
Colony-stimulating factor-1 suppresses responses to CpG DNA and expression of toll-like receptor 9 but enhances responses to lipopolysaccharide in murine macrophages
No full textDuring bacterial infections, the balance between resolution of infection and development of sepsis is dependent upon the macrophage response to bacterial products. We show that priming of murine bone marrow-derived macrophages (BMMs) with CSF-1 differentially regulates the response to two such stimuli, LPS and immunostimulatory (CpG) DNA. CSF-1 pretreatment enhanced IL-6, IL-12, and TNF-alpha production in response to LPS but suppressed the same response to CpG DNA. CSF-1 also regulated cytokine gene expression in response to CpG DNA and LPS; CpG DNA-induced IL-12 p40, IL-12 p35, and TNF-alpha mRNAs were all suppressed by CSF-1 pretreatment. CSF-1 pretreatment enhanced LPS-induced IL-12 p40 mRNA but not TNF-alpha and IL-12 p35 mRNAs, suggesting that part of the priming effect is posttranscriptional. CSF-1 pretreatment also suppressed CpG DNA-induced nuclear translocation of NF-kappaB and phosphorylation of the mitogen-activated protein kinases p38 and extracellular signal-related kinases-1/2 in BMMs, indicating that early events in CpG DNA signaling were regulated by CSF-1. Expression of Toll-like receptor (TLR)9, which is necessary for responses to CpG DNA, was markedly suppressed by CSF-1 in both BMMs and thioglycolate-elicited peritoneal macrophages. CSF-1 also down-regulated expression of TLR1, TLR2, and TLR6, but not the LPS receptor, TLR4, or TLR5. Hence, CSF-1 may regulate host responses to pathogens through modulation of TLR expression. Furthermore, these results suggest that CSF-1 and CSF-1R antagonists may enhance the efficacy of CpG DNA in vivo
Orphan CpG islands identify numerous conserved promoters in the mammalian genome
No full textThis work was funded by grants from the Wellcome Trust (077224; http://www.wellcome.ac.uk/) and the Medical Research Council (G0800026; http:// www.mrc.ac.uk/index.htm).CpG islands (CGIs) are vertebrate genomic landmarks that encompass the promoters of most genes and often lack DNA methylation. Querying their apparent importance, the number of CGIs is reported to vary widely in different species and many do not co-localise with annotated promoters. We set out to quantify the number of CGIs in mouse and human genomes using CXXC Affinity Purification plus deep sequencing (CAP-seq). We also asked whether CGIs not associated with annotated transcripts share properties with those at known promoters. We found that, contrary to previous estimates, CGI abundance in humans and mice is very similar and many are at conserved locations relative to genes. In each species CpG density correlates positively with the degree of H3K4 trimethylation, supporting the hypothesis that these two properties are mechanistically interdependent. Approximately half of mammalian CGIs (>10,000) are "orphans'' that are not associated with annotated promoters. Many orphan CGIs show evidence of transcriptional initiation and dynamic expression during development. Unlike CGIs at known promoters, orphan CGIs are frequently subject to DNA methylation during development, and this is accompanied by loss of their active promoter features. In colorectal tumors, however, orphan CGIs are not preferentially methylated, suggesting that cancer does not recapitulate a developmental program. Human and mouse genomes have similar numbers of CGIs, over half of which are remote from known promoters. Orphan CGIs nevertheless have the characteristics of functional promoters, though they are much more likely than promoter CGIs to become methylated during development and hence lose these properties. The data indicate that orphan CGIs correspond to previously undetected promoters whose transcriptional activity may play a functional role during development.Peer reviewe
RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies
No full textMutating RNA virus genomes to alter codon pair (CP) frequencies and reduce translation efficiency has been advocated as a method to generate safe, attenuated virus vaccines. However, selection for disfavoured CPs leads to unintended increases in CpG and UpA dinucleotide frequencies that also attenuate replication. We designed and phenotypically characterised mutants of the picornavirus, echovirus 7, in which these parameters were independently varied to determine which most influenced virus replication. CpG and UpA dinucleotide frequencies primarily influenced virus replication ability while no fitness differences were observed between mutants with different CP usage where dinucleotide frequencies were kept constant. Contrastingly, translation efficiency was unaffected by either CP usage or dinucleotide frequencies. This mechanistic insight is critical for future rational design of live virus vaccines and their safety evaluation; attenuation is mediated through enhanced innate immune responses to viruses with elevated CpG/UpA dinucleotide frequencies rather the viruses themselves being intrinsically defective
Adaptive CPG Based Coordinated Control of Healthy and Robotic Lower Limb Movements
No full textThis paper proposes an adaptive CPG based controller for a lower limb prosthesis consisting of online trajectory generation and interlimb coordination. The adaptive CPG can produce multidimensional rhythmic patterns and modulate their frequency by tuning relevant parameters in an autonomously way adapting to a changing periodicity ofexternal signals. Also, to increase the stability of the prosthesis, a spring-damper component is attached between the hip and ankle joints, allowing the absorption of impulsive ground reaction forces at landing. We verify the validity of the proposed controller with a simulated humanoid robot through the investigation of the self-coordination between the healthy and robotic legs
Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains
No full textMBD1 is a vertebrate methyl-CpG binding domain protein (MBD) that can bring about repression of methylated promoter DNA sequences. Like other MBD proteins, MBD1 localizes to nuclear foci that in mice are rich in methyl-CpG. In methyl-CpG-deficient mouse cells, however, Mbd1 remains localized to heterochromatic foci whereas other MBD proteins become dispersed in the nucleus. We find that Mbd1a, a major mouse isoform, contains a CXXC domain (CXXC-3) that binds specifically to nonmethylated CpG, suggesting an explanation for methylation-independent localization. Transfection studies demonstrate that the CXXC-3 domain indeed targets nonmethylated CpG sites in vivo. Repression of nonmethylated reporter genes depends on the CXXC-3 domain, whereas repression of methylated reporters requires the MBD. Our findings indicate that MBD1 can interpret the CpG dinucleotide as a repressive signal in vivo regardless of its methylation status
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