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Analyzing Top2 Distribution on Yeast Chromosomes by Chromatin ImmunoprecipitationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In vertebrate cells, DNA topoisomerase II (Topo II), named Top2 in yeast, localizes along chromosome axes early in mitosis and concentrates within centromeric chromatin during metaphase. The factors controlling these changes in enzyme distribution are largely unknown. Insight into Topo II dynamics could potentially be derived through genetic approaches in yeast. In practice, however, the small size and limited compaction of yeast chromosomes has precluded a detailed analysis of Top2 localization along mitotic chromosomes. As an alternative approach, we describe a method for examining Top2 distribution using chromatin immun...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Binding of DNA Topoisomerases I and II to Replication OriginsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The interaction of DNA topology modifying enzymes with eukaryotic DNA replication origins can be detected with nucleotide precision exploiting the action of enzyme poisons specific for type I or type II DNA topoisomerases. Using the topoisomerase I poison camptothecin and the topoisomerase II poison VP16, the precise sites of interaction of these enzymes around the lamin B2 origin have been identified at different points in the cell cycle. The procedure can be applied to any origin for which the sequence has been identified within approximately 1 kb.
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Cytometric Assessment of DNA Damage Induced by DNA Topoisomerase InhibitorsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Exposure of cells to inhibitors of DNA topoisomerase I (topo I) or topoisomerase II (topo II) leads to DNA damage that often involves formation of DNA double-strand breaks (DSBs). DNA damage, particularly induction of DSBs, manifests by phosphorylation of histone H2AX on Ser-139 which is mediated by one of the protein kinases of the phosphoinositide kinase family, namely ATM, ATR, and/or DNA-PK. The presence of Ser-139 phosphorylated H2AX (γH2AX) is thus a reporter of DNA damage. This protocol describes quantitative assessment of γH2AX detected immunocytochemically in individual cells combined with quantificati...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Analysis of the Topoisomerase II-Dependent Decatenation G2 Checkpoint and Checkpoint Kinases in Human CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Inhibition of Topo II function using poisons and catalytic inhibitors triggers checkpoints that act to delay progression of G2 cells into mitosis. Topo II poisons induce Topo II-associated DNA double-strand breaks that activate ATM and the DNA damage G2 checkpoint. Topo II catalytic inhibitors do not induce DNA double-strand breaks but block decatenation of intertwined daughter chromatids. Complete decatenation before anaphase of mitosis is required for chromatid segregation. G2 cells appear to sense the degree of chromatid arm catenations and actively delay the onset of mitosis by sustaining the inhibition of mitosis-prom...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Assaying Topoisomerase II Checkpoints in YeastEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Topoisomerase II activity is crucial to maintain genome stability through the removal of catenanes in the DNA formed during DNA replication and scaffolding the mitotic chromosome. Perturbed Topo II activity causes defects in chromosome segregation due to persistent catenations and aberrant DNA condensation during mitosis. Recently, novel top2 alleles in the yeast Saccharomyces cerevisiae revealed a checkpoint control that responds to perturbed Topo II activity. Described in this chapter are protocols for assaying the phenotypes seen in top2 mutants on a cell biological basis in live cells: activation of the Topo II checkpo...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Cytological Analysis of Chromosome Structural Defects that Result from Topoisomerase II DysfunctionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
For analyzing chromosome structural defects that result from topoisomerase II (topo II) dysfunction, we have adapted classical cell cycle experiments, classical cytological techniques, and the use of a potent topo II inhibitor (ICRF-193). In this chapter, we describe in detail the protocols used and we discuss the rationale for our choice and for the adaptations applied. We clarify in which cell cycle stages each of the different chromosomal aberrations induced by inhibiting topo II take place: lack of chromosome segregation, undercondensation, lack of sister chromatid resolution, and lack of chromosome individualization. ...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Top2 SUMO Conjugation in Yeast Cell LysatesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
DNA topoisomerase II (Topo II), named Top2 in budding and fission yeast, is a conserved target of the SUMO modification pathway, with SUMO-conjugated forms of Topo II accumulating specifically during mitosis in both yeast and vertebrate cells (Bachant et al., Mol Cell 9, 1169–82, 2002; Azuma et al., J Cell Biol 163, 477–87, 2003; Dawlaty et al., Cell 133, 103–15, 2008). As with many SUMO substrates, the functional significance of this modification is still incompletely understood and, perhaps surprisingly, better characterized in vertebrates than yeasts. It seems likely, however, that continued analysis o...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Analysis of SUMOylation of Topoisomerase IIα with Xenopus Egg ExtractsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Posttranslational protein modification by the Small Ubiquitin-like MOdifiers (SUMO) is involved in many cellular functions including organization of nuclear structures and chromatin, transcriptional regulation, and nucleo-cytoplasmic transport. Both genetic and biochemical studies indicate that the SUMO modification pathway plays an important role in proper cell cycle control, especially in the normal progression of mitosis. DNA topoisomerase II has been shown to be modified by SUMO in budding yeast as well as in vertebrates. We have shown by biochemical analysis using the Xenopus egg extract (XEE) cell-free assay system t...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

The Dynamics of DNA Topoisomerase IIα in Living CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Here, we describe methods to prepare a mammalian expression plasmid encoding EGFP fused to the amino-terminus of human DNA topoisomerase IIα (Topo IIα) for use in studying the dynamics of Topo IIα in living cells. In previous studies, this plasmid was transfected into LLC-Pk cells, a porcine epithelial cell line that remains relatively flat during mitosis. After selection for stable integration, cells were cloned by serial dilution in microwells and used to grow a stable cell line expressing EGFP-Topo IIα; this cell line was termed LPk-GT2. Using photobleaching methods with conventional and patterne...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Depletion and Mutation of Topoisomerase II in Animal CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Eukaryotic type II topoisomerases (Topo II) are implicated in a wide range of cellular processes. Cells in which Topo II protein has been specifically depleted or mutated provide powerful systems for analysing the normal in vivo functions of Topo II proteins and for assessing their roles in various chemotherapy regimens. Summarised here are the ways in which Topo II has been depleted or mutated in animal cells and the type of information gleaned. The cell lines generated are tabulated and represent a useful resource for further in vivo studies of Topo II function, one that we expect to grow in size and utility in the coming years.
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Introduction: Emerging Themes in DNA Topoisomerase ResearchEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
DNA topoisomerases are enzymes that alter the topology of DNA. They have important functions in DNA replication, transcription, Holliday junction dissolution, chromosome condensation, and sister chromatid separation. Deficiencies in these enzymes are associated with diseases that result from genome instability. The last 10–15 years has seen a great deal of exciting research in the field of topoisomerase. Here we discuss a selection of the new themes that have been recently introduced into the already large body of topoisomerase research.
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Analysis of DNA Topoisomers, Knots, and Catenanes by Agarose Gel ElectrophoresisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Agarose gel electrophoresis is by far the most widely used method for characterizing the topological state of DNA molecules. Although this technique has been used for more than 30 years, the physical mechanism underlying the resolution of topological states remains poorly understood. However, electrophoretic methods remain the most robust and precise techniques for determining the local unwinding of DNA induced by the binding of proteins and small-molecule ligands, analyzing conformational transitions in duplex DNA, measuring changes in helical repeat that accompany shifts in environmental conditions, and characterizing kn...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Two-Dimensional Agarose Gel Electrophoresis of DNA TopoisomersEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The electrophoretic velocity of a duplex DNA ring is mainly determined by its overall shape. Consequently, DNA topoisomers of opposite supercoiling handedness can have identical gel velocity, and topoisomers highly supercoiled cannot be separated beyond some point. These problems are overcome by two-dimensional agarose gel electrophoresis, which involves two successive electrophoresis steps in one gel slab. The first and second electrophoresis steps are conducted in orthogonal directions with different concentrations of DNA intercalating agents. These compounds alter the overall shape of the DNA and, thereby, change the re...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Cleavage of Plasmid DNA by Eukaryotic Topoisomerase IIEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Topoisomerase II is an essential enzyme that is required for a number of critical nuclear processes. All of the catalytic functions of topoisomerase II require the enzyme to generate a transient double-stranded break in the backbone of the double helix. To maintain genomic integrity during the cleavage event, topoisomerase II forms covalent bonds between active site tyrosyl residues and the newly generated 5′-DNA termini. In addition to the critical cellular functions of the type II enzyme, several important anticancer drugs kill cells by increasing levels of covalent topoisomerase II–DNA cleavage complexes. Du...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Assays for the Preferential Binding of Human Topoisomerase I to Supercoiled DNAEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
To assay the preferential binding of eukaryotic type IB topoisomerases to supercoiled DNA, two methods are described that make use of a catalytically inactive mutant form of the enzyme. In the gel shift assay, the preference for binding to supercoiled plasmid DNA is detected in the presence of linear and nicked forms of the same DNA by a reduction in the mobility of the supercoiled plasmid during electrophoresis in agarose. The more quantitative filter binding assay compares the ability of nicked and supercoiled forms of the circular DNA to compete for the binding of a 3H-labeled nicked DNA to the topoisomerase where the e...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Monitoring the Topoisomerase II DNA Gate Conformational Change with Fluorescence Resonance Energy TransferEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Type II DNA topoisomerases are essential, ubiquitous enzymes responsible for performing vital functions in chromosome condensation and segregation and in regulating intracellular DNA supercoiling. Topoisomerase II (topo II) performs these DNA transactions by passing one segment of DNA through the other using a reversible, enzyme-bridged double-stranded DNA break. This cleavage/religation of the DNA backbone is coupled to the opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle. To monitor the opening and closing of the DNA gate, we designed an oligonucleotide substrate with a p...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Single-Molecule Magnetic Tweezers Studies of Type IB TopoisomerasesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The past few years have seen the application of single-molecule force spectroscopy techniques to the study of topoisomerases. Magnetic tweezers are particularly suited to the study of topoisomerases due to their unique ability to exert precise and straightforward control of the supercoiled state of DNA. Here, we illustrate in a stepwise fashion how the dynamic properties of type IB topoisomerases can be monitored using this technique.
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Dissolution of Double Holliday Junctions by the Concerted Action of BLM and Topoisomerase IIIαEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In eukaryotic cells, topoisomerase III forms an evolutionarily conserved complex with a RecQ family helicase and two OB-fold containing proteins, replication protein A (RPA) and RMI1. One role for this complex is to catalyze the completion of homologous recombination reactions in which the recombining DNA molecules are covalently interlinked by a double Holliday junction structure. This process, which requires the single-stranded DNA decatenation activity of topoisomerase III, is termed Holliday junction “dissolution” to distinguish it from Holliday junction “resolution” catalyzed by endonucleases (...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

ChIP-on-Chip Analysis of DNA TopoisomerasesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Here we describe an adapted ChIP-on-chip protocol for the analysis of DNA topoisomerase chromosomal binding in Saccharomyces cerevisiae cells. The ChIP-on-chip technique is based on the immunoprecipitation of crosslinked chromatin (ChIP, chromatin immunoprecipitation), followed by DNA amplification and hybridization to high-density oligonucleotide arrays (Chip). Comparison of the signal intensities of immunoprecipitated and control fractions provides a measurement of the protein–DNA association along entire genomes. ChIP-on-chip analysis of DNA topoisomerase binding to chromosomal DNA opens a window to the understand...
Source: Springer protocols feed by Genetics/Genomics - September 22, 2009 Category: Genetics & Stem Cells Source Type: info

Quantitative Trait Locus Analysis Using J/qtlEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Quantitative trait locus (QTL) analysis is a statistical method to link phenotypes with regions of the genome that affect the phenotypes in a mapping population. R/qtl is a powerful statistical program commonly used for analyzing rodent QTL crosses, but R/qtl is a command line program that can be difficult for novice users to run. J/qtl was developed as an R/qtl graphical user interface that enables even novice users to utilize R/qtl for QTL analyses. In this chapter, we describe the process for analyzing rodent cross data with J/qtl, including data formatting, data quality control, main scan QTL analysis, pair scan QTL an...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2009 Category: Genetics & Stem Cells Source Type: info

Computing Genetic Imprinting Expressed by HaplotypesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Different expression of maternally and paternally inherited alleles at certain genes is called genetic imprinting. Despite its great importance in trait formation, development, and evolution, it remains unclear how genetic imprinting operates in a complex network of interactive genes located throughout the genome. Genetic mapping has proven to be a powerful tool that can estimate the distribution and effects of imprinted genes. While traditional mapping models attempt to detect imprinted quantitative trait loci based on a linkage map constructed from molecular markers, we have developed a statistical model for estimating t...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2009 Category: Genetics & Stem Cells Source Type: info

Haplotype Association Mapping in MiceEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Haplotype Association Mapping (HAM) is a novel phenotype-driven approach to identify genetic loci and was originally developed for mice. This method, which is similar to Genome-Wide Association (GWA) studies in humans, looks for associations between the phenotype and the haplotypes of mouse inbred strains, treating inbred strains as individuals. Although this approach is still in development, we review the current literature, present the different methods and applications that are in use, and provide a glimpse of what is to come in the near future.
Source: Springer protocols feed by Genetics/Genomics - September 15, 2009 Category: Genetics & Stem Cells Source Type: info

Candidate Gene Association AnalysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Candidate gene association study is the most common method for associating human genetic variations with the phenotypes they produce, due to the relative simplicity of acquiring patient samples and genotype data. The study design begins with identifying appropriate DNA samples and an appropriate phenotype for analysis. The candidate genes and polymorphisms must then be chosen. After genotyping the candidate genes in the DNA samples, the results are checked to ensure appropriate quality and association analysis is performed. The raw results are interpreted and placed into context and follow-up analysis is carried out to val...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2009 Category: Genetics & Stem Cells Source Type: info

Genome-Wide Association Study in HumansEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Genome-wide association studies have opened a new era in the study of the genetic basis of common, multifactorial diseases and traits. Before the introduction of this approach only a handful of common genetic variants showed consistent association for any phenotype. Using genome-wide association, scores of novel and unsuspected loci have been discovered and later replicated for many complex traits. The principle is to genotype a dense set of common genetic variants across the genomes of individuals with phenotypic differences and examine whether genotype is associated with phenotype. Because the last common human ancestor ...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2009 Category: Genetics & Stem Cells Source Type: info

Bioinformatics Analysis of Microarray DataEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Gene expression profiling provides unprecedented opportunities to study patterns of gene expression regulation, for example, in diseases or developmental processes. Bioinformatics analysis plays an important part of processing the information embedded in large-scale expression profiling studies and for laying the foundation for biological interpretation.
Source: Springer protocols feed by Genetics/Genomics - September 15, 2009 Category: Genetics & Stem Cells Source Type: info

The Fast Chromatin Immunoprecipitation MethodEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The chromatin immunoprecipitation assay (ChIP assay) has greatly facilitated the recent, dramatic expansion of our knowledge of the protein–DNA interactions involved in regulating gene expression, DNA repair, and cell division. The power of the assay is that it gives a researcher the ability to not only detect a specific protein–DNA interaction in vivo but also determine the relative density of factors along genes or the entire genome. Though powerful, the traditional assay is time consuming (involving 2 days or more) and laborious. With Fast ChIP, we simplified the assay to greatly reduce the time and labor in...
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

μChIP: Chromatin Immunoprecipitation for Small Cell NumbersEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe here a rapid micro (μ)ChIP assay suited for multiple parallel ChIPs from a single chromatin batch from 1,000 cells. The assay is also applicable to a single immunoprecipitation from 100 cells.
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

Fish’n ChIPs: Chromatin Immunoprecipitation in the Zebrafish EmbryoEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We report here a protocol for immunoprecipitation of modified histones from mid-term blastula zebrafish embryos.
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

Epitope Tagging of Endogenous Proteins for Genome-Wide Chromatin Immunoprecipitation AnalysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The development of chromatin imsmunoprecipitation methods coupled with DNA microarray (ChIP-chip) technology has enabled genome-wide identification of cis-DNA regulatory elements to which transcription factors bind. Nonetheless, the ChIP-chip technology requires antibodies with extremely high affinity and specificity for the target transcription factors. Unfortunately, such antibodies are not available for most human transcription factors. In principle, this problem can be circumvented by utilizing ectopically expressed epitope-tagged proteins recognizable by well-characterized antibodies. However, such expression is no lo...
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

Flow Cytometric and Laser Scanning Microscopic Approaches in Epigenetics ResearchEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Our understanding of epigenetics has been transformed in recent years by the advance of technological possibilities based primarily on a powerful tool, chromatin immunoprecipitation (ChIP). However, in many cases, the detection of epigenetic changes requires methods providing a high-throughput (HTP) platform. Cytometry has opened a novel approach for the quantitative measurement of molecules, including PCR products, anchored to appropriately addressed microbeads (Pataki et al. 2005. Cytometry 68, 45–52). Here we show selected examples for the utility of two different cytometry-based platforms of epigenetic ana...
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

Serial Analysis of Binding Elements for Transcription FactorsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The ability to determine genome-wide location of transcription factor binding sites (TFBS) is crucial for elucidating gene regulatory networks in human cells during normal development and disease such as tumorigenesis. To achieve this goal, we developed a method called serial analysis of binding elements for transcription factors (SABE) for globally identifying TFBS in human or other mammalian genomes. In this method, a specific antibody targeting a DNA-binding transcription factor of interest is used to pull down the transcription factor and its bound DNA elements through chromatin immunoprecipitation (ChIP). ChIP DNA fra...
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

Modeling and Analysis of ChIP-Chip ExperimentsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Chromatin immunoprecipitation on microarrays, also known as ChIP-chip, is a popular technique for genome-wide localization of DNA-binding proteins. However, the high density (several million genomic sequences for small eukaryote genomes) and the high noise-to-signal ratio of microarrays make the analysis of ChIP-chip data very challenging. In this chapter, we review some of the issues involved in the analysis of ChIP-chip data and present a few statistical methods that can be used to overcome these issues and improve the detection of DNA–protein binding sites.
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

Use of SNP-Arrays for ChIP Assays: Computational AspectsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The simultaneous genotyping of thousands of single nucleotide polymorphisms (SNPs) in a genome using SNP-Arrays is a very important tool that is revolutionizing genetics and molecular biology. We expanded the utility of this technique by using it following chromatin immunoprecipitation (ChIP) to assess the multiple genomic locations protected by a protein complex recognized by an antibody. The power of this technique is illustrated through an analysis of the changes in histone H4 acetylation, a marker of open chromatin and transcriptionally active genomic regions, which occur during differentiation of human myoblasts into ...
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

DamID: A Methylation-Based Chromatin Profiling ApproachEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Gene expression is a dynamic process and is tightly connected to changes in chromatin structure and nuclear organization (Schneider, R. and Grosschedl, R., 2007, Genes Dev. 21, 3027–3043; Kosak, S. T. and Groudine, M., 2004, Genes Dev. 18, 1371–1384). Our ability to understand the intimate interactions between proteins and the rapidly changing chromatin environment requires methods that will be able to provide accurate, sensitive, and unbiased mapping of these interactions in vivo (van Steensel, B., 2005, Nat. Genet. 37 Suppl, S18–24). One such tool is DamID chromatin profiling, a met...
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

Chromosome Conformation Capture (from 3C to 5C) and Its ChIP-Based ModificationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Chromosome conformation capture (3C) methodology was developed to study spatial organization of long genomic regions in living cells. Briefly, chromatin is fixed with formaldehyde in vivo to cross-link interacting sites, digested with a restriction enzyme and ligated at a low DNA concentration so that ligation between cross-linked fragments is favored over ligation between random fragments. Ligation products are then analyzed and quantified by PCR. So far, semi-quantitative PCR methods were widely used to estimate the ligation frequencies. However, it is often important to estimate the ligation frequencies more precisely w...
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

Determining Spatial Chromatin Organization of Large Genomic Regions Using 5C TechnologyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Spatial organization of chromatin plays an important role at multiple levels of genome regulation. On a global scale, its function is evident in processes like metaphase and chromosome segregation. On a detailed level, long-range interactions between regulatory elements and promoters are essential for proper gene regulation. Microscopic techniques like FISH can detect chromatin contacts, although the resolution is generally low making detection of enhancer–promoter interaction difficult. The 3C methodology allows for high-resolution analysis of chromatin interactions. 3C is now widely used and has revealed that long-...
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

Analysis of Nascent RNA Transcripts by Chromatin RNA ImmunoprecipitationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Biochemical methods to analyze co-transcriptional recruitment of co-activators to nascent RNA molecules have lagged behind for many years. Most of the information on co-transcriptional regulation of nascent RNA came from invaluable in situ studies using single-cell model systems. More recently, the chromatin RNA immunoprecipitation technique has been developed to evaluate at the molecular level the association of proteins with nascent RNA which is still coupled to chromatin. Similar to chromatin immunoprecipitation, the chromatin RNA immunoprecipitation method is suitable to study events along specific genes, and it has be...
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

Methyl DNA ImmunoprecipitationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Epigenetics is the study of heritable changes in gene expression. Chromatin immunoprecipitation (ChIP) and methylation status analysis of genes have been applied to the study of epigenetic modifications, often perturbed in human cancer. ChIP is a technique allowing the analysis of the protein association with specific genomic regions in the context of intact cells. ChIP and immunoprecipitation (IP) of methylated DNA, both rely on the use of well-characterized specific antibodies. The first is described in Chapter 2 and the second is shown here. At Diagenode, a novel METHYL kit has been designed to immunoprecipitate methyla...
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

Immunoprecipitation of Methylated DNAEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
DNA methylation contributes to the regulation of long-term gene repression by enabling the recruitment of transcriptional repressor complexes to methylated cytosines. Several methods for detecting DNA methylation at the gene-specific and genome-wide levels have been developed. Methylated DNA immunoprecipitation, or MeDIP, consists of the selective immunoprecipitation of methylated DNA fragments using antibodies to 5-methylcytosine. The genomic site of interest can be detected by PCR, hybridization to DNA arrays, or by direct sequencing. This chapter describes the MeDIP protocol and quality control tests that should be perf...
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

The State-of-the-Art of Chromatin ImmunoprecipitationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The biological significance of interactions of nuclear proteins with DNA in the context of gene expression, cell differentiation, or disease has immensely been enhanced by the advent of chromatin immunoprecipitation (ChIP). ChIP is a technique whereby a protein of interest is selectively immunoprecipitated from a chromatin preparation to determine the DNA sequences associated with it. ChIP has been widely used to map the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes on the genome or on a given locus. In spite of its power, ChIP has for a long...
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

Characterization and Quality Control of Antibodies Used in ChIP AssaysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present here the very robust characterization and quality control (QC) process that we have established for our polyclonal antibodies, which are mainly directed against targets relevant to the epigenetics field such as modified histones, modifying enzymes, and chromatin-interacting proteins. The final purpose of the characterization and QC is to label antibodies as chromatin immunoprecipitation (ChIP) grade. Indeed, the ChIP method is extensively used in epigenetics to study gene regulation and relies on the use of antibodies to select the protein of interest and then precipitate and identify the DNA associated to it. W...
Source: Springer protocols feed by Genetics/Genomics - July 15, 2009 Category: Genetics & Stem Cells Source Type: info

Methylation Analysis by DNA Immunoprecipitation (MeDIP)Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This article describes the detailed protocol for MeDIP and hybridization of MeDIP DNA to a whole-genome tiling path BAC array.
Source: Springer protocols feed by Genetics/Genomics - February 1, 2009 Category: Genetics & Stem Cells Source Type: info

Combining Chromatin Immunoprecipitation and Oligonucleotide Tiling Arrays (ChIP-Chip) for Functional Genomic StudiesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Central to systems biology are genome-wide technologies and high-throughput experimental approaches. Completion of the sequencing of the human genome as well as those of a number of other higher eukaryotes now allows for the first time the mapping of all of the cis-regulatory regions of genes as well as the details of nucleosome position and modification. One approach to achieving this goal involves chromatin immunoprecipitation combined with DNA oligonucleotide tiling arrays (ChIP-chip). This allows for the identification of genomic regions bound by a given factor, its cistrome, or harboring a given epigenomic modificatio...
Source: Springer protocols feed by Genetics/Genomics - February 1, 2009 Category: Genetics & Stem Cells Source Type: info

ChIP-Chip: Algorithms for Calling Binding SitesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe the use of the respective software packages MAT and MA2C for the analysis of ChIP-chip data from one-color Affymetrix and two-color NimbleGen or Agilent tiling microarrays.
Source: Springer protocols feed by Genetics/Genomics - February 1, 2009 Category: Genetics & Stem Cells Source Type: info

Mapping Regulatory Elements by DNaseI Hypersensitivity Chip (DNase-Chip)Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Historically, the simplest method to robustly identify active gene regulatory elements has been enzymatic digestion of nuclear DNA by nucleases such as DNaseI. Regions of extreme chromatin accessibility to DNaseI, commonly known as DNaseI hypersensitive sites, have been repeatedly shown to be markers for all types of active cis-acting regulatory elements, including promoters, enhancers, silencers, insulators, and locus control regions. However, the original classical method, which for over 25 years relied on Southern blot, was limited to studying only small regions of the genome. Here we describe the detailed protocol for ...
Source: Springer protocols feed by Genetics/Genomics - February 1, 2009 Category: Genetics & Stem Cells Source Type: info

Microarray Analysis of DNA Replication TimingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Although all of the DNA in an eukaryotic cell replicates during the S-phase of cell cycle, there is a significant difference in the actual time in S-phase when a given chromosomal segment replicates. Methods are described here for generation of high-resolution temporal maps of DNA replication in synchronized human cells. This method does not require amplification of DNA before microarray hybridization and so avoids errors introduced during PCR. A major advantage of using this procedure is that it facilitates finer dissection of replication time in S-phase. Also, it helps delineate chromosomal regions that undergo biallelic...
Source: Springer protocols feed by Genetics/Genomics - February 1, 2009 Category: Genetics & Stem Cells Source Type: info

Integration of Diverse Microarray Data TypesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Over the past decade, DNA microarrays have proven to be a powerful tool in biological research for the molecular surveillance of cells and tissues. The expansive utility of DNA microarrays owes its nascence to the development of a multitude of microarray platforms that enable the systematic and comprehensive exploration of diverse genomic properties and processes. Concomitant with the explosive generation of microarray data over the last several years has been an increasing interest in the integration of such diverse data types, thus spurring the development of novel statistical techniques and integrative bioinformatics to...
Source: Springer protocols feed by Genetics/Genomics - February 1, 2009 Category: Genetics & Stem Cells Source Type: info

IntroductionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
DNA microarray technology has revolutionized biological research by enabling genome-scale explorations. This chapter provides an overview of DNA microarray technology and its application to characterizing the physical genome, with a focus on cancer genomes. Specific areas discussed include investigations of DNA copy number alteration (and loss of heterozygosity), DNA methylation, DNA–protein (i.e., chromatin and transcription factor) interactions, DNA replication, and the integration of diverse genome-scale data types. Also provided is a perspective on recent advances and future directions in characterizing the physical genome.
Source: Springer protocols feed by Genetics/Genomics - February 1, 2009 Category: Genetics & Stem Cells Source Type: info

Comparative Genomic Hybridization on BAC ArraysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Alterations in genomic DNA are a key feature of many constitutional disorders and cancer. The discovery of the underlying regions of gene dosage has thus been essential in dissecting complex disease phenotypes and identifying targets for therapeutic intervention and diagnostic testing. The development of array comparative genomic hybridization (aCGH) using bacterial artificial chromosomes (BACs) as hybridization targets has facilitated the discovery and fine mapping of novel genomic alterations allowing rapid identification of target genes.
Source: Springer protocols feed by Genetics/Genomics - February 1, 2009 Category: Genetics & Stem Cells Source Type: info

Comparative Genomic Hybridization on Spotted Oligonucleotide MicroarraysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Recent advances in DNA microarray technology have enabled researchers to comprehensively characterize the complex genomes of higher eukaryotic organisms at an unprecedented level of detail. Array-based comparative genomic hybridization (Array-CGH) has been widely used for detecting DNA copy number alterations on a genomic scale, where the mapping resolution is limited only by the number of probes on the DNA microarray. In this chapter, we present a validated protocol utilizing print-tip spotted HEEBO (Human Exonic Evidence Based Oligonucleotide) microarrays for conducting array-CGH using as little as 25 ng of genomic ...
Source: Springer protocols feed by Genetics/Genomics - February 1, 2009 Category: Genetics & Stem Cells Source Type: info

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