The Experts below are selected from a list of 309 Experts worldwide ranked by ideXlab platform
John T Lis - One of the best experts on this subject based on the ideXlab platform.
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EmPC-seq: Accurate RNA-sequencing and Bioinformatics Platform to Map RNA Polymerases and Remove Background Error.
Bio-protocol, 2021Co-Authors: Yuqing Wang, John T Lis, Tin Hang Chong, Ilona Christy Unarta, Gianmarco Domenico Suarez, Jiguang Wang, Xuhui Huang, Peter P. CheungAbstract:Transcription errors can substantially affect metabolic processes in organisms by altering the epigenome and causing misincorporations in mRNA, which is translated into aberrant mutant proteins. Moreover, within eukaryotic genomes there are specific Transcription Error-Enriched genomic Loci (TEELs) which are transcribed by RNA polymerases with significantly higher error rates and hypothesized to have implications in cancer, aging, and diseases such as Down syndrome and Alzheimer's. Therefore, research into transcription errors is of growing importance within the field of genetics. Nevertheless, methodological barriers limit the progress in accurately identifying transcription errors. Pro-Seq and NET-Seq can purify nascent RNA and map RNA polymerases along the genome but cannot be used to identify transcriptional mutations. Here we present background Error Model-coupled Precision Nuclear Run-On Circular-sequencing (EmPC-seq), a method combining a Nuclear Run-On assay and circular sequencing with a background error model to precisely detect nascent transcription errors and effectively discern TEELs within the genome.
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Identifying Transcription Error-Enriched Genomic Loci Using Nuclear Run-On Circular-Sequencing Coupled with Background Error Modeling
Journal of molecular biology, 2020Co-Authors: Pak Hang Peter Cheung, Gregory T Booth, John T Lis, Biaobin Jiang, Tin Hang Chong, Ilona Christy Unarta, Yuqing Wang, Gianmarco Domenico Suarez, Jiguang Wang, Xuhui HuangAbstract:Abstract RNA polymerase transcribes certain genomic loci with higher errors rates. These transcription error-enriched genomic loci (TEELs) have implications in disease. Current deep-sequencing methods cannot distinguish TEELs from post-transcriptional modifications, stochastic transcription errors, and technical noise, impeding efforts to elucidate the mechanisms linking TEELs to disease. Here, we describe background error model-coupled precision Nuclear Run-On circular-sequencing (EmPC-seq) to discern genomic regions enriched for transcription misincorporations. EmPC-seq innovatively combines a Nuclear Run-On assay for capturing nascent RNA before post-transcriptional modifications, a circular-sequencing step that sequences the same nascent RNA molecules multiple times to improve accuracy, and a statistical model for distinguishing error-enriched regions among stochastic polymerase errors. Applying EmPC-seq to the ribosomal RNA transcriptome, we show that TEELs of RNA polymerase I are not randomly distributed but clustered together, with higher error frequencies at nascent transcript 3′ ends. Our study establishes a reliable method of identifying TEELs with nucleotide precision, which can help elucidate their molecular origins.
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Base-pair-resolution genome-wide mapping of active RNA polymerases using precision Nuclear Run-On (PRO-seq)
Nature Protocols, 2016Co-Authors: Dig Bijay Mahat, Hojoong Kwak, Gregory T Booth, Iris H Jonkers, Charles G Danko, Ravi K Patel, Colin T Waters, Katie Munson, Leighton J Core, John T LisAbstract:Mahat et al . describe how to map the genome-wide positions of active RNA polymerases using a modified Nuclear Run-On approach called PRO-seq. Details for PRO-cap, a modification that identifies transcription start sites, are also included. We provide a protocol for precision Nuclear Run-On sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under Nuclear Run-On conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3′ end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3′ end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4–5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast.
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Base-pair-resolution genome-wide mapping of active RNA polymerases using precision Nuclear Run-On (PRO-seq)
Nature protocols, 2016Co-Authors: Dig Bijay Mahat, Hojoong Kwak, Gregory T Booth, Charles G Danko, Ravi K Patel, Colin T Waters, Katie Munson, Leighton J Core, Iris Jonkers, John T LisAbstract:We provide a protocol for precision Nuclear Run-On sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under Nuclear Run-On conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3' end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3' end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4-5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast.
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Nuclear Run-On assays: assessing transcription by measuring density of engaged RNA polymerases.
Methods in enzymology, 1999Co-Authors: Kazunori Hirayoshi, John T LisAbstract:Publisher Summary This chapter describes the use of Nuclear Run-On assays as a direct measure of the density of elongating RNA polymerases. Assuming a relatively constant rate of elongation, this density provides a measure of transcription at the moment of nuclei isolation. Transcriptionally engaged RNA polymerases in isolated nuclei are allowed to continue elongation in the presence of labeled nucleoside triphosphate. The labeled RNA generated from a specific gene sequence is then quantified by hybridization to specific unlabeled DNAs bound to filters. The levels of hybridization to these DNAs, which are present in vast numbers over specific labeled RNAs, are proportional to polymerase density on these sequences in the isolated nuclei. Probing several DNAs simultaneously by simply spotting the single-stranded DNA sequence on a filter and hybridizing the labeled Run-On RNA allow the relative density of transcriptionally active polymerase on different genes or on fragments within a single gene to be compared directly. The chapter describes the standard protocols used by researchers in a variety of cell and animal systems. It also describes protocols used with mammalian cells and with yeast for different cell or tissue types.
Alan C Sartorelli - One of the best experts on this subject based on the ideXlab platform.
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activation of transiently transfected reporter genes in 3t3 swiss cells by the inducers of differentiation apoptosis dimethylsulfoxide hexamethylene bisacetamide and trichostatin a
FEBS Journal, 2004Co-Authors: Kimiko Ishiguro, Alan C SartorelliAbstract:Despite decades of investigation, the primary site of action of the prototypical inducers of differentiation, dimethylsulfoxide and hexamethylene bisacetamide (HMBA), has not been delineated. During studies designed to analyze cis-acting elements responsible for induction of stage-specific globin genes, we discovered the capacity of HMBA and dimethylsulfoxide to enhance the expression of transiently transfected reporter genes derived from globin and nonglobin gene promoters, prominently in nonerythroid 3T3 Swiss cells. The action of HMBA and dimethylsulfoxide in the transient transfection system resembled that of the inhibitor of histone deacetylases (HDACs), trichostatin A (TSA), in that the three agents enhanced reporter gene expression (a) regardless of the promoter employed, (b) with similar kinetics and (c) with an increase in the steady-state level of reporter mRNA. Transiently transfected DNA was assembled rapidly into a chromatinized structure in 3T3 cells, suggesting that transcription of reporter genes was at least in part repressed by chromatin organization. Nuclear Run-On analyses indicated that dimethylsulfoxide and HMBA enhanced transcriptional initiation of the reporter and p21/WAF1/Cip1 genes. In contrast, TSA produced negligible effects on Nuclear Run-On transcription of these genes. HMBA and dimethylsulfoxide did not change the acetylation, phosphorylation, or methylation status of histones and did not activate stably transfected genes. Despite these differences, the three agents modulated the expression of common sets of cellular genes and induced differentiation or apoptosis in intact cells. The findings imply that HMBA and dimethylsulfoxide modulate transcription by a mechanism independent of histone acetylation.
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Activation of transiently transfected reporter genes in 3T3 Swiss cells by the inducers of differentiation/apoptosis--dimethylsulfoxide, hexamethylene bisacetamide and trichostatin A.
European journal of biochemistry, 2004Co-Authors: Kimiko Ishiguro, Alan C SartorelliAbstract:Despite decades of investigation, the primary site of action of the prototypical inducers of differentiation, dimethylsulfoxide and hexamethylene bisacetamide (HMBA), has not been delineated. During studies designed to analyze cis-acting elements responsible for induction of stage-specific globin genes, we discovered the capacity of HMBA and dimethylsulfoxide to enhance the expression of transiently transfected reporter genes derived from globin and nonglobin gene promoters, prominently in nonerythroid 3T3 Swiss cells. The action of HMBA and dimethylsulfoxide in the transient transfection system resembled that of the inhibitor of histone deacetylases (HDACs), trichostatin A (TSA), in that the three agents enhanced reporter gene expression (a) regardless of the promoter employed, (b) with similar kinetics and (c) with an increase in the steady-state level of reporter mRNA. Transiently transfected DNA was assembled rapidly into a chromatinized structure in 3T3 cells, suggesting that transcription of reporter genes was at least in part repressed by chromatin organization. Nuclear Run-On analyses indicated that dimethylsulfoxide and HMBA enhanced transcriptional initiation of the reporter and p21/WAF1/Cip1 genes. In contrast, TSA produced negligible effects on Nuclear Run-On transcription of these genes. HMBA and dimethylsulfoxide did not change the acetylation, phosphorylation, or methylation status of histones and did not activate stably transfected genes. Despite these differences, the three agents modulated the expression of common sets of cellular genes and induced differentiation or apoptosis in intact cells. The findings imply that HMBA and dimethylsulfoxide modulate transcription by a mechanism independent of histone acetylation.
John J. Reiners - One of the best experts on this subject based on the ideXlab platform.
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Quantitative RT-PCR on CYP1A1 heterogeneous Nuclear RNA: a surrogate for the in vitro transcription Run-On assay.
BioTechniques, 1996Co-Authors: Cornelis J. Elferink, John J. ReinersAbstract:A quantitative reverse transcription polymerase chain reaction (RT-PCR) assay was developed to amplify a region of the CYP1A1 heterogeneous Nuclear RNA (hnRNA) transcript encompassing the first intron-exon boundary. The RT-PCR protocol uses a CYP1A1 recombinant RNA internal standard identical to the target hnRNA except for an engineered unique internal restriction site. Its inclusion enables normalization between reactions and a measurement of the absolute number of target hnRNA transcripts. Specificity for the hnRNA was achieved by using introndirected primers in both the RT and the PCR. Nuclear Run-On assays and the hnRNA RT-PCR assay detected an equivalent increase in transcription of Cypla-1 in cultured murine Hepa 1c1c7 cells following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The RT-PCR assay also revealed TCDD-dependent transcriptional activation of the Cypla-1 gene in murine skin, a tissue unsuited to the Nuclear Run-On assay because of inherent difficulties associated with the isola...
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Quantitative RT-PCR on CYP1A1 heterogeneous Nuclear RNA: a surrogate for the in vitro transcription Run-On assay.
BioTechniques, 1996Co-Authors: Cornelis J. Elferink, John J. ReinersAbstract:A quantitative reverse transcription polymerase chain reaction (RT-PCR) assay was developed to amplify a region of the CYP1A1 heterogeneous Nuclear RNA (hnRNA) transcript encompassing the first intron-exon boundary. The RT-PCR protocol uses a CYP1A1 recombinant RNA internal standard identical to the target hnRNA except for an engineered unique internal restriction site. Its inclusion enables normalization between reactions and a measurement of the absolute number of target hnRNA transcripts. Specificity for the hnRNA was achieved by using intron-directed primers in both the RT and the PCR. Nuclear Run-On assays and the hnRNA RT-PCR assay detected an equivalent increase in transcription of Cyp1a-1 in cultured murine Hepa 1c1c7 cells following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The RT-PCR assay also revealed TCDD-dependent transcriptional activation of the Cyp1a-1 gene in murine skin, a tissue unsuited to the Nuclear Run-On assay because of inherent difficulties associated with the isolation of nuclei. These examples demonstrate that the hnRNA RT-PCR assay is a facile surrogate for the Nuclear Run-On assay. Moreover, the sensitivity and design characteristics of the RT-PCR assay suggest the potential for its broad application in general transcriptional research.
Kevin V. Morris - One of the best experts on this subject based on the ideXlab platform.
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Quantification of nascent transcription by bromouridine immunocapture Nuclear Run-On RT-qPCR
Nature Protocols, 2015Co-Authors: Thomas C. Roberts, Jonathan R. Hart, Minna U. Kaikkonen, Marc S. Weinberg, Peter K. Vogt, Kevin V. MorrisAbstract:Nuclear Run-On (NRO) is a method that measures transcriptional activity via the quantification of biochemically labeled nascent RNA molecules derived from Nuclear isolates. Widespread use of this technique has been limited because of its technical difficulty relative to steady-state total mRNA analyses. Here we describe a detailed protocol for the quantification of transcriptional activity in human cell cultures. Nuclei are first isolated and NRO transcription is performed in the presence of bromouridine. Labeled nascent transcripts are purified by immunoprecipitation, and transcript levels are determined by reverse-transcription quantitative PCR (RT-qPCR). Data are then analyzed using standard techniques described elsewhere. This method is rapid (the protocol can be completed in 2 d) and cost-effective, exhibits negligible detection of background noise from unlabeled transcripts, requires no radioactive materials and can be performed from as few as 500,000 nuclei. It also takes advantage of the high sensitivity, specificity and dynamic range of RT-qPCR. Roberts et al . describe how to measure transcriptional activity using Nuclear Run-On RT-qPCR assays. The approach is ideal for analyzing a single gene (or small number of genes) or for follow-up or validation of genome-wide analyses.
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Quantification of nascent transcription by bromouridine immunocapture Nuclear Run-On RT-qPCR
Nature protocols, 2015Co-Authors: Thomas C. Roberts, Jonathan R. Hart, Minna U. Kaikkonen, Marc S. Weinberg, Peter K. Vogt, Kevin V. MorrisAbstract:Roberts et al. describe how to measure transcriptional activity using Nuclear Run-On RT-qPCR assays. The approach is ideal for analyzing a single gene (or small number of genes) or for follow-up or validation of genome-wide analyses.
Kimiko Ishiguro - One of the best experts on this subject based on the ideXlab platform.
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activation of transiently transfected reporter genes in 3t3 swiss cells by the inducers of differentiation apoptosis dimethylsulfoxide hexamethylene bisacetamide and trichostatin a
FEBS Journal, 2004Co-Authors: Kimiko Ishiguro, Alan C SartorelliAbstract:Despite decades of investigation, the primary site of action of the prototypical inducers of differentiation, dimethylsulfoxide and hexamethylene bisacetamide (HMBA), has not been delineated. During studies designed to analyze cis-acting elements responsible for induction of stage-specific globin genes, we discovered the capacity of HMBA and dimethylsulfoxide to enhance the expression of transiently transfected reporter genes derived from globin and nonglobin gene promoters, prominently in nonerythroid 3T3 Swiss cells. The action of HMBA and dimethylsulfoxide in the transient transfection system resembled that of the inhibitor of histone deacetylases (HDACs), trichostatin A (TSA), in that the three agents enhanced reporter gene expression (a) regardless of the promoter employed, (b) with similar kinetics and (c) with an increase in the steady-state level of reporter mRNA. Transiently transfected DNA was assembled rapidly into a chromatinized structure in 3T3 cells, suggesting that transcription of reporter genes was at least in part repressed by chromatin organization. Nuclear Run-On analyses indicated that dimethylsulfoxide and HMBA enhanced transcriptional initiation of the reporter and p21/WAF1/Cip1 genes. In contrast, TSA produced negligible effects on Nuclear Run-On transcription of these genes. HMBA and dimethylsulfoxide did not change the acetylation, phosphorylation, or methylation status of histones and did not activate stably transfected genes. Despite these differences, the three agents modulated the expression of common sets of cellular genes and induced differentiation or apoptosis in intact cells. The findings imply that HMBA and dimethylsulfoxide modulate transcription by a mechanism independent of histone acetylation.
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Activation of transiently transfected reporter genes in 3T3 Swiss cells by the inducers of differentiation/apoptosis--dimethylsulfoxide, hexamethylene bisacetamide and trichostatin A.
European journal of biochemistry, 2004Co-Authors: Kimiko Ishiguro, Alan C SartorelliAbstract:Despite decades of investigation, the primary site of action of the prototypical inducers of differentiation, dimethylsulfoxide and hexamethylene bisacetamide (HMBA), has not been delineated. During studies designed to analyze cis-acting elements responsible for induction of stage-specific globin genes, we discovered the capacity of HMBA and dimethylsulfoxide to enhance the expression of transiently transfected reporter genes derived from globin and nonglobin gene promoters, prominently in nonerythroid 3T3 Swiss cells. The action of HMBA and dimethylsulfoxide in the transient transfection system resembled that of the inhibitor of histone deacetylases (HDACs), trichostatin A (TSA), in that the three agents enhanced reporter gene expression (a) regardless of the promoter employed, (b) with similar kinetics and (c) with an increase in the steady-state level of reporter mRNA. Transiently transfected DNA was assembled rapidly into a chromatinized structure in 3T3 cells, suggesting that transcription of reporter genes was at least in part repressed by chromatin organization. Nuclear Run-On analyses indicated that dimethylsulfoxide and HMBA enhanced transcriptional initiation of the reporter and p21/WAF1/Cip1 genes. In contrast, TSA produced negligible effects on Nuclear Run-On transcription of these genes. HMBA and dimethylsulfoxide did not change the acetylation, phosphorylation, or methylation status of histones and did not activate stably transfected genes. Despite these differences, the three agents modulated the expression of common sets of cellular genes and induced differentiation or apoptosis in intact cells. The findings imply that HMBA and dimethylsulfoxide modulate transcription by a mechanism independent of histone acetylation.
