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  • 1
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    CDD: NCBI's conserved domain database (2015)
    Oxford University Press
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    Publication Date: 2015-01-16
    Description: NCBI's CDD, the Conserved Domain Database, enters its 15 th year as a public resource for the annotation of proteins with the location of conserved domain footprints. Going forward, we strive to improve the coverage and consistency of domain annotation provided by CDD. We maintain a live search system as well as an archive of pre-computed domain annotation for sequences tracked in NCBI's Entrez protein database, which can be retrieved for single sequences or in bulk. We also maintain import procedures so that CDD contains domain models and domain definitions provided by several collections available in the public domain, as well as those produced by an in-house curation effort. The curation effort aims at increasing coverage and providing finer-grained classifications of common protein domains, for which a wealth of functional and structural data has become available. CDD curation generates alignment models of representative sequence fragments, which are in agreement with domain boundaries as observed in protein 3D structure, and which model the structurally conserved cores of domain families as well as annotate conserved features. CDD can be accessed at http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml .
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 2
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    Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors (2014)
    Oxford University Press
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    Publication Date: 2014-04-15
    Description: The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 ( Pou5f1 ) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around –120 to –80 bp, while highly effective sgRNAs targeted from –147 to –89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4 . Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 3
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    Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing (2015)
    Oxford University Press
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    Publication Date: 2015-12-02
    Description: Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineering of bacterial species. Systematic identification of DNA methylation in R-M systems, including N 6 -methyladenine (6mA), 5-methylcytosine (5mC) and N 4 -methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing technology is capable of detecting 4mC directly for any bacterial species regardless of whether an assembled genome exists or not, it is not as scalable to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method that rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome. In 4mC-TAB-seq, both cytosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We applied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor , in which 4mC-related restriction is a major barrier to DNA transformation from other species. In combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bacteria in the future.
    Keywords: Nucleic acid modification, Phsyical and Biochemical Characterisation of DNA
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    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 4
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    Young intragenic miRNAs are less coexpressed with host genes than old ones: implications of miRNA-host gene coevolution (2012)
    Oxford University Press
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    Publication Date: 2012-05-13
    Description: MicroRNAs (miRNAs) have emerged as key regulators of gene expression. Intragenic miRNAs account for ~50% of mammalian miRNAs. Classic studies reported that they are usually coexpressed with host genes. Here, using genome-wide miRNA and gene expression profiles from five sample sets, we show that evolutionarily conserved (‘old’) intragenic miRNAs tend to be coexpressed with host genes, but non-conserved (‘young’) ones rarely do so. This result is robust: in all sample sets, the coexpression rate of young miRNAs is significantly lower than that of conserved ones even after controlling for abundance. As a result, although young miRNAs dominate in human genome, the majority of intragenic miRNAs that show coexpression with host genes are phylogenetically old ones. For younger miRNAs, extrapolation of their expression profiles from those of their host genes should be treated with caution. We propose a model to explain this phenomenon in which the majority of young miRNAs are unlikely to be coexpressed with host genes; however, for some fraction of young miRNAs coexpression with their host genes, initially imbued by chromatin level effects, is advantageous and these are the ones likely to embed into the system and evolve ever higher levels of coexpression, possibly by evolving piggybacking mechanisms.
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    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 5
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    Molecular mechanisms of two-component system RhpRS regulating type III secretion system in Pseudomonas syringae (2014)
    Oxford University Press
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    Publication Date: 2014-10-10
    Description: Pseudomonas syringae uses the two-component system RhpRS to regulate the expression of type III secretion system (T3SS) genes and bacterial virulence. However, the molecular mechanisms and the regulons of RhpRS have yet to be fully elucidated. Here, we show that RhpS functions as a kinase and a phosphatase on RhpR and as an autokinase upon itself. RhpR is phosphorylated by the small phosphodonor acetyl phosphate. A specific RhpR-binding site containing the inverted repeat (IR) motif GTATC-N 6 -GATAC, was mapped to its own promoter by a DNase I footprint analysis. Electrophoretic mobility shift assay indicated that P-RhpR has a higher binding affinity to the IR motif than RhpR. To identify additional RhpR targets in P. syringae , we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) and detected 167 enriched loci including the hrpR promoter, suggesting the direct regulation of T3SS cascade genes by RhpR. A genome-wide microarray analysis showed that, in addition to the T3SS cascade genes, RhpR differentially regulates a large set of genes with various functions in response to different growth conditions. Together, these results suggested that RhpRS is a global regulator that allows P. syringae to sense and respond to environmental changes by coordinating T3SS expression and many other biological processes.
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    Topics: Biology
    Published by Oxford University Press
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  • 6
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    A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands (2016)
    Oxford University Press
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    Publication Date: 2016-10-14
    Description: Ten Eleven Translocation (TET) protein-catalyzed 5mC oxidation not only creates novel DNA modifications, such as 5hmC, but also initiates active or passive DNA demethylation. TETs’ role in the crosstalk with specific histone modifications, however, is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domains underlying methylated DNA CpG islands (CGIs). Overexpression of wild type (WT), but not catalytic inactive mutant (Mut), TET2 in low-TET-expressing cells results in an increase in the level of 5hmC with accompanying DNA demethylation at a subset of CGIs. Most importantly, this alteration is sufficient in making de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of essential developmental gene promoters, which are located within CGIs and are previously silenced due to DNA methylation. On the other hand, deletion of Tet1 and Tet2 in mouse embryonic stem (ES) cells results in an apparent loss of H3K27me3 at bivalent domains, which are associated with a particular set of key developmental gene promoters. Collectively, this study demonstrates the critical role of TET proteins in regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at CGIs associated with developmental genes.
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    Topics: Biology
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  • 7
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    Nucleosome eviction and multiple co-factor binding predict estrogen-receptor-alpha-associated long-range interactions (2014)
    Oxford University Press
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    Publication Date: 2014-06-21
    Description: Many enhancers regulate their target genes via long-distance interactions. High-throughput experiments like ChIA-PET have been developed to map such largely cell-type-specific interactions between cis -regulatory elements genome-widely. In this study, we integrated multiple types of data in order to reveal the general hidden patterns embedded in the ChIA-PET data. We found characteristic distance features related to promoter–promoter, enhancer–enhancer and insulator–insulator interactions. Although a protein may have many binding sites along the genome, our hypothesis is that those sites that share certain open chromatin structure can accommodate relatively larger protein complex consisting of specific regulatory and ‘bridging’ factors, and may be more likely to form robust long-range deoxyribonucleic acid (DNA) loops. This hypothesis was validated in the estrogen receptor alpha (ERα) ChIA-PET data. An efficient classifier was built to predict ERα-associated long-range interactions solely from the related ChIP-seq data, hence linking distal ERα-dependent enhancers to their target genes. We further applied the classifier to generate additional novel interactions, which were undetected in the original ChIA-PET paper but were validated by other independent experiments. Our work provides a new insight into the long-range chromatin interactions through deeper and integrative ChIA-PET data analysis and demonstrates DNA looping predictability from ordinary ChIP-seq data.
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    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 8
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    Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A (2015)
    Oxford University Press
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    Publication Date: 2015-11-17
    Description: Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC.
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    Topics: Biology
    Published by Oxford University Press
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  • 9
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    Widespread occurrence of N6-methyladenosine in bacterial mRNA (2015)
    Oxford University Press
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    Publication Date: 2015-07-25
    Description: N 6 -methyladenosine (m 6 A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA). Recent discoveries of demethylases and specific binding proteins of m 6 A as well as m 6 A methylomes obtained in mammals, yeast and plants have revealed regulatory functions of this RNA modification. Although m 6 A is present in the ribosomal RNA of bacteria, its occurrence in mRNA still remains elusive. Here, we have employed ultra-high pressure liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) to calculate the m 6 A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m 6 A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m 6 A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved m 6 A pattern that is distinct from those in eukaryotes. Most m 6 A peaks are located inside open reading frames and carry a unique consensus motif of GCCAU. Functional enrichment analysis of bacterial m 6 A peaks indicates that the majority of m 6 A-modified genes are associated with respiration, amino acids metabolism, stress response and small RNAs, suggesting potential functional roles of m 6 A in these pathways.
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    Topics: Biology
    Published by Oxford University Press
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  • 10
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    FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function (2016)
    Oxford University Press
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    Publication Date: 2016-10-08
    Description: Forkhead box A1 (FOXA1) is an FKHD family protein that plays pioneering roles in lineage-specific enhancer activation and gene transcription. Through genome-wide location analyses, here we show that FOXA1 expression and occupancy are, in turn, required for the maintenance of these epigenetic signatures, namely DNA hypomethylation and histone 3 lysine 4 methylation. Mechanistically, this involves TET1, a 5-methylcytosine dioxygenase. We found that FOXA1 induces TET1 expression via direct binding to its cis-regulatory elements. Further, FOXA1 physically interacts with the TET1 protein through its CXXC domain. TET1 thus co-occupies FOXA1-dependent enhancers and mediates local DNA demethylation and concomitant histone 3 lysine 4 methylation, further potentiating FOXA1 recruitment. Consequently, FOXA1 binding events are markedly reduced following TET1 depletion. Together, our results suggest that FOXA1 is not only able to recognize but also remodel the epigenetic signatures at lineage-specific enhancers, which is mediated, at least in part, by a feed-forward regulatory loop between FOXA1 and TET1.
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    Topics: Biology
    Published by Oxford University Press
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