The Experts below are selected from a list of 882 Experts worldwide ranked by ideXlab platform
Bas Van Steensel - One of the best experts on this subject based on the ideXlab platform.
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MOESM1 of The large fraction of heterochromatin in Drosophila neurons is bound by both B-type lamin and HP1a
2018Co-Authors: Alexey Pindyurin, Bas Van Steensel, Artem Ilyin, Anton Ivankin, Mikhail Tselebrovsky, Valentina Nenasheva, Elena Mikhaleva, Ludo Pagie, Yuri ShevelyovAbstract:Additional file 1. Scheme of fly crossing for DamID in neurons
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DamID mapping of in vivo protein genome interactions using tethered dna adenine methyltransferase
Methods in Enzymology, 2006Co-Authors: Frauke Greil, Celine Moorman, Bas Van SteenselAbstract:A large variety of proteins bind to specific parts of the genome to regulate gene expression, DNA replication, and chromatin structure. DamID is a powerful method used to map the genomic interaction sites of these proteins in vivo. It is based on fusing a protein of interest to Escherichia coli DNA adenine methyltransferase (dam). Expression of this fusion protein in vivo leads to preferential methylation of adenines in DNA surrounding the native binding sites of the dam fusion partner. Because adenine methylation does not occur endogenously in most eukaryotes, it provides a unique tag to mark protein interaction sites. The adenine‐methylated DNA fragments are isolated by selective polymerase chain reaction amplification and can be identified by microarray hybridization. We and others have successfully applied DamID to the genome‐wide identification of interaction sites of several transcription factors and other chromatin‐associated proteins. This chapter discusses DamID technology in detail, and a step‐by‐step experimental protocol is provided for use in Drosophila cell lines.
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DamID mapping of in vivo protein genome interactions using tethered dna adenine methyltransferase
Methods in Enzymology, 2006Co-Authors: Frauke Greil, Celine Moorman, Bas Van SteenselAbstract:A large variety of proteins bind to specific parts of the genome to regulate gene expression, DNA replication, and chromatin structure. DamID is a powerful method used to map the genomic interaction sites of these proteins in vivo. It is based on fusing a protein of interest to Escherichia coli DNA adenine methyltransferase (dam). Expression of this fusion protein in vivo leads to preferential methylation of adenines in DNA surrounding the native binding sites of the dam fusion partner. Because adenine methylation does not occur endogenously in most eukaryotes, it provides a unique tag to mark protein interaction sites. The adenine‐methylated DNA fragments are isolated by selective polymerase chain reaction amplification and can be identified by microarray hybridization. We and others have successfully applied DamID to the genome‐wide identification of interaction sites of several transcription factors and other chromatin‐associated proteins. This chapter discusses DamID technology in detail, and a step‐by‐step experimental protocol is provided for use in Drosophila cell lines.
Frauke Greil - One of the best experts on this subject based on the ideXlab platform.
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DamID mapping of in vivo protein genome interactions using tethered dna adenine methyltransferase
Methods in Enzymology, 2006Co-Authors: Frauke Greil, Celine Moorman, Bas Van SteenselAbstract:A large variety of proteins bind to specific parts of the genome to regulate gene expression, DNA replication, and chromatin structure. DamID is a powerful method used to map the genomic interaction sites of these proteins in vivo. It is based on fusing a protein of interest to Escherichia coli DNA adenine methyltransferase (dam). Expression of this fusion protein in vivo leads to preferential methylation of adenines in DNA surrounding the native binding sites of the dam fusion partner. Because adenine methylation does not occur endogenously in most eukaryotes, it provides a unique tag to mark protein interaction sites. The adenine‐methylated DNA fragments are isolated by selective polymerase chain reaction amplification and can be identified by microarray hybridization. We and others have successfully applied DamID to the genome‐wide identification of interaction sites of several transcription factors and other chromatin‐associated proteins. This chapter discusses DamID technology in detail, and a step‐by‐step experimental protocol is provided for use in Drosophila cell lines.
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DamID mapping of in vivo protein genome interactions using tethered dna adenine methyltransferase
Methods in Enzymology, 2006Co-Authors: Frauke Greil, Celine Moorman, Bas Van SteenselAbstract:A large variety of proteins bind to specific parts of the genome to regulate gene expression, DNA replication, and chromatin structure. DamID is a powerful method used to map the genomic interaction sites of these proteins in vivo. It is based on fusing a protein of interest to Escherichia coli DNA adenine methyltransferase (dam). Expression of this fusion protein in vivo leads to preferential methylation of adenines in DNA surrounding the native binding sites of the dam fusion partner. Because adenine methylation does not occur endogenously in most eukaryotes, it provides a unique tag to mark protein interaction sites. The adenine‐methylated DNA fragments are isolated by selective polymerase chain reaction amplification and can be identified by microarray hybridization. We and others have successfully applied DamID to the genome‐wide identification of interaction sites of several transcription factors and other chromatin‐associated proteins. This chapter discusses DamID technology in detail, and a step‐by‐step experimental protocol is provided for use in Drosophila cell lines.
Bas Van Steensel - One of the best experts on this subject based on the ideXlab platform.
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DamID profiling of dynamic Polycomb-binding sites in Drosophila imaginal disc development and tumorigenesis.
Epigenetics & Chromatin, 2018Co-Authors: Marco La Fortezza, Giovanna Grigolon, Andrea Cosolo, Alexey V. Pindyurin, Laura Breimann, Helmut Blum, Bas Van Steensel, Anne-kathrin ClassenAbstract:Tracking dynamic protein–chromatin interactions in vivo is key to unravel transcriptional and epigenetic transitions in development and disease. However, limited availability and heterogeneous tissue composition of in vivo source material impose challenges on many experimental approaches. Here we adapt cell-type-specific DamID-seq profiling for use in Drosophila imaginal discs and make FLP/FRT-based induction accessible to GAL driver-mediated targeting of specific cell lineages. In a proof-of-principle approach, we utilize ubiquitous DamID expression to describe dynamic transitions of Polycomb-binding sites during wing imaginal disc development and in a scrib tumorigenesis model. We identify Atf3 and Ets21C as novel Polycomb target genes involved in scrib tumorigenesis and suggest that target gene regulation by Atf3 and AP-1 transcription factors, as well as modulation of insulator function, plays crucial roles in dynamic Polycomb-binding at target sites. We establish these findings by DamID-seq analysis of wing imaginal disc samples derived from 10 larvae. Our study opens avenues for robust profiling of small cell population in imaginal discs in vivo and provides insights into epigenetic changes underlying transcriptional responses to tumorigenic transformation.
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identification of in vivo dna targets of chromatin proteins using tethered dam methyltransferase
Nature Biotechnology, 2000Co-Authors: Bas Van Steensel, Steven HenikoffAbstract:We have developed a novel technique, named DamID, for the identification of DNA loci that interact in vivo with specific nuclear proteins in eukaryotes. By tethering Escherichia coli DNA adenine methyltransferase (Dam) to a chromatin protein, Dam can be targeted in vivo to native binding sites of this protein, resulting in local DNA methylation. Sites of methylation can subsequently be mapped using methylation-specific restriction enzymes or antibodies. We demonstrate the successful application of DamID both in Drosophila cell cultures and in whole flies. When Dam is tethered to the DNA-binding domain of GAL4, targeted methylation is limited to a region of a few kilobases surrounding a GAL4 binding sequence. Using DamID, we identified a number of expected and unexpected target loci for Drosophila heterochromatin protein 1. DamID has potential for genome-wide mapping of in vivo targets of chromatin proteins in various eukaryotes.
Andrea H Brand - One of the best experts on this subject based on the ideXlab platform.
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mapping rna chromatin interactions in vivo with rna DamID
Methods of Molecular Biology, 2020Co-Authors: Seth W. Cheetham, Andrea H BrandAbstract:Long-noncoding RNAs (lncRNAs) are emerging as regulators of development and disease. lncRNAs are expressed in exquisitely precise expression patterns in vivo and many interact with chromatin to regulate gene expression. However, the limited sensitivity of RNA-purification techniques has precluded the identification of genomic targets of cell-type specific lncRNAs. RNA-DamID is a powerful new approach to understand the mechanisms by which lncRNAs act in vivo. RNA-DamID is highly sensitive and accurate, and can resolve cell-type-specific chromatin binding patterns without cell isolation. The determinants of RNA-chromatin interactions can be identified with RNA-DamID by analyzing RNA and protein cofactor mutants. Here we describe how to implement RNA-DamID and the design considerations to take into account to accurately identify lncRNA-chromatin interactions in vivo.
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Targeted DamID reveals differential binding of mammalian pluripotency factors.
Development, 2018Co-Authors: Seth W. Cheetham, Tony D. Southall, Wolfram H. Gruhn, Jelle Van Den Ameele, Robert Krautz, M. Azim Surani, Toshihiro Kobayashi, Andrea H BrandAbstract:The precise control of gene expression by transcription factor networks is critical to organismal development. The predominant approach for mapping transcription factor-chromatin interactions has been chromatin immunoprecipitation (ChIP). However, ChIP requires a large number of homogeneous cells and antisera with high specificity. A second approach, DamID, has the drawback that high levels of Dam methylase are toxic. Here we modify our Targeted DamID approach (TaDa) to enable cell type-specific expression in mammalian systems, generating an inducible system (mammalian TaDa or MaTaDa) to identify protein/DNA interactions in 100 to 1000 times fewer cells than ChIP. We mapped the binding sites of key pluripotency factors, OCT4 and PRDM14, in mouse embryonic stem cells, epiblast-like cells and primordial germ cell-like cells (PGCLCs). PGCLCs are an important system to elucidate primordial germ cell development in mice. We monitored PRDM14 binding during the specification of PGCLCs, identifying direct targets of PRDM14 that are key to understanding its critical role in PGCLC development. We show that MaTaDa is a sensitive and accurate method to assess cell type specific transcription factor binding in limited numbers of cells.
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DamIDseq_pipeline an automated pipeline for processing DamID sequencing datasets
Bioinformatics, 2015Co-Authors: Owen J Marshall, Andrea H BrandAbstract:Summary: DamID is a powerful technique for identifying regions of the genome bound by a DNA-binding (or DNA-associated) protein. Currently, no method exists for automatically processing next-generation sequencing DamID (DamID-seq) data, and the use of DamID-seq datasets with normalization based on read-counts alone can lead to high background and the loss of bound signal. DamID-seq thus presents novel challenges in terms of normalization and background minimization. We describe here DamIDseq_pipeline, a software pipeline that performs automatic normalization and background reduction on multiple DamID-seq FASTQ datasets. Availability and implementation: Open-source and freely available from http://owenjm.github.io/DamIDseq_pipeline. The DamIDseq_pipeline is implemented in Perl and is compatible with any Unix-based operating system (e.g. Linux, Mac OSX). Contact: ku.ca.mac.nodrug@llahsram.o Supplementary information: Supplementary data are available at Bioinformatics online.
Seth W. Cheetham - One of the best experts on this subject based on the ideXlab platform.
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mapping rna chromatin interactions in vivo with rna DamID
Methods of Molecular Biology, 2020Co-Authors: Seth W. Cheetham, Andrea H BrandAbstract:Long-noncoding RNAs (lncRNAs) are emerging as regulators of development and disease. lncRNAs are expressed in exquisitely precise expression patterns in vivo and many interact with chromatin to regulate gene expression. However, the limited sensitivity of RNA-purification techniques has precluded the identification of genomic targets of cell-type specific lncRNAs. RNA-DamID is a powerful new approach to understand the mechanisms by which lncRNAs act in vivo. RNA-DamID is highly sensitive and accurate, and can resolve cell-type-specific chromatin binding patterns without cell isolation. The determinants of RNA-chromatin interactions can be identified with RNA-DamID by analyzing RNA and protein cofactor mutants. Here we describe how to implement RNA-DamID and the design considerations to take into account to accurately identify lncRNA-chromatin interactions in vivo.
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DamID as a versatile tool for understanding gene regulation
Development, 2019Co-Authors: Gabriel N. Aughey, Seth W. Cheetham, Tony D. SouthallAbstract:The interaction of proteins and RNA with chromatin underlies the regulation of gene expression. The ability to profile easily these interactions is fundamental for understanding chromatin biology DNA adenine methyltransferase identification (DamID) profiles genome-wide protein-DNA interactions without antibodies, fixation or protein pull-downs. Recently, DamID has been adapted for applications beyond simple assaying of protein-DNA interactions, such as for studying RNA-chromatin interactions, chromatin accessibility and long-range chromosome interactions. Here, we provide an overview of DamID and introduce improvements to the technology, discuss their applications and compare alternative methodologies.
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Targeted DamID reveals differential binding of mammalian pluripotency factors.
Development, 2018Co-Authors: Seth W. Cheetham, Tony D. Southall, Wolfram H. Gruhn, Jelle Van Den Ameele, Robert Krautz, M. Azim Surani, Toshihiro Kobayashi, Andrea H BrandAbstract:The precise control of gene expression by transcription factor networks is critical to organismal development. The predominant approach for mapping transcription factor-chromatin interactions has been chromatin immunoprecipitation (ChIP). However, ChIP requires a large number of homogeneous cells and antisera with high specificity. A second approach, DamID, has the drawback that high levels of Dam methylase are toxic. Here we modify our Targeted DamID approach (TaDa) to enable cell type-specific expression in mammalian systems, generating an inducible system (mammalian TaDa or MaTaDa) to identify protein/DNA interactions in 100 to 1000 times fewer cells than ChIP. We mapped the binding sites of key pluripotency factors, OCT4 and PRDM14, in mouse embryonic stem cells, epiblast-like cells and primordial germ cell-like cells (PGCLCs). PGCLCs are an important system to elucidate primordial germ cell development in mice. We monitored PRDM14 binding during the specification of PGCLCs, identifying direct targets of PRDM14 that are key to understanding its critical role in PGCLC development. We show that MaTaDa is a sensitive and accurate method to assess cell type specific transcription factor binding in limited numbers of cells.
