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Direct Identification of Insulator Components by Insertional Chromatin Immunoprecipitation  [PDF]
Toshitsugu Fujita, Hodaka Fujii
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0026109
Abstract: Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. However, non-biased methods to identify molecules bound to specific genomic loci in vivo are limited. Here, we applied insertional chromatin immunoprecipitation (iChIP) to direct identification of components of insulator complexes, which function as boundaries of chromatin domain. We found that the chicken β-globin HS4 (cHS4) insulator complex contains an RNA helicase protein, p68/DDX5; an RNA species, steroid receptor RNA activator 1; and a nuclear matrix protein, Matrin-3, in vivo. Binding of p68 and Matrin-3 to the cHS4 insulator core sequence was mediated by CCCTC-binding factor (CTCF). Thus, our results showed that it is feasible to directly identify proteins and RNA bound to a specific genomic region in vivo by using iChIP.
Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain  [PDF]
Toshitsugu Fujita, Hodaka Fujii
Advances in Bioscience and Biotechnology (ABB) , 2012, DOI: 10.4236/abb.2012.35081
Abstract: Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.
Epigenetics & chromatin: interactions and processes  [cached]
Henikoff Steven,Grosveld Frank
Epigenetics & Chromatin , 2013, DOI: 10.1186/1756-8935-6-2
Abstract: On 11 to 13 March 2013, BioMed Central will be hosting its inaugural conference, Epigenetics & Chromatin: Interactions and Processes, at Harvard Medical School, Cambridge, MA, USA. Epigenetics & Chromatin has now launched a special article series based on the general themes of the conference.
Welcome to Epigenetics & Chromatin
Steven Henikoff, Frank Grosveld
Epigenetics & Chromatin , 2008, DOI: 10.1186/1756-8935-1-1
Abstract: We often hear the terms 'epigenetic' and 'chromatin' used interchangeably in the context of inheritance that is not encoded in DNA sequence; however, these two terms have very different histories. The concept of epigenesis was introduced by Aristotle as the unfolding of a developmental program to form an animal or plant from an amorphous egg or spore through differentiation as opposed to the view that the new complex organism is already present in the egg or spore. Much later, Conrad Waddington introduced the more modern term epigenetics by hypothesizing the existence of an epigenetic landscape that channels gene activity to maintain stable inheritance during development [2]. The term chromatin was used by Walther Flemming in the 1880s for what later were called mitotic chromosomes [3], but eventually its meaning shifted, referring instead to the complex of DNA and protein that chromosomes are made of. Although it has long been understood that chromatin is involved in maintaining the epigenetic landscape, just how this happens had long remained mysterious, despite spectacular progress in understanding basic genetic mechanisms. Only in recent years have connections between epigenetics and chromatin been explored at the molecular level, thanks in large part to rapidly improving technologies.As epigenetics and chromatin have converged into a single field of research, it has become increasingly important that researchers at all levels learn about advances made in areas that they might be otherwise unfamiliar with. For example, beginning with the first Epigenetics Gordon Conference [4], which was held in 1995, a concerted effort has been made to invite speakers and attract participants who study epigenetic processes in plant, animal and fungal systems. The wisdom of this strategy is clear in hindsight, insofar as some of the most important advances in the epigenetics and chromatin field have come from unexpected biological systems, such as the role of small RNAs in gene
Molecular epigenetics, chromatin, and NeuroAIDS/HIV: Translational implications  [cached]
Paul Shapshak,Francesco Chiappelli,Deborah Commins,Elyse Singer
Bioinformation , 2008,
Abstract: We describe current research that applies epigenetics to a novel understanding of the immuno-neuropathogenesis of HIV-1 viral infection and NeuroAIDS. We propose the hypothesis that HIV-1 alters the structure-function relationship of chromatin, coding DNA and non-coding DNA, including RNA transcribed from these regions resulting in pathogenesis in AIDS, drug abuse, and NeuroAIDS. We discuss the general implications of molecular epigenetics with special emphasis on drug abuse, bar-codes, pyknons, and miRNAs for translational and clinical research. We discuss the application of the recent recursive algorithm of biology to this field and propose to synthesize the Genomic and Epigenomic views into a holistic approach of HoloGenomics.
Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization
Max Haring, Sascha Offermann, Tanja Danker, Ina Horst, Christoph Peterhansel, Maike Stam
Plant Methods , 2007, DOI: 10.1186/1746-4811-3-11
Abstract: We developed a robust ChIP protocol, using maize (Zea mays) as a model system, and present a general strategy to systematically optimize this protocol for any type of tissue. We propose endogenous controls for active and for repressed chromatin, and discuss various other controls that are essential for successful ChIP experiments. We experienced that the use of quantitative PCR (QPCR) is crucial for obtaining high quality ChIP data and we explain why. The method of data normalization has a major impact on the quality of ChIP analyses. Therefore, we analyzed different normalization strategies, resulting in a thorough discussion of the advantages and drawbacks of the various approaches.Here we provide a robust ChIP protocol and strategy to optimize the protocol for any type of tissue; we argue that quantitative real-time PCR (QPCR) is the best method to analyze the precipitates, and present comprehensive insights into data normalization.Epigenetic regulation of gene expression is crucial for cell differentiation, and thus essential for normal growth and development of higher eukaryotes. Epigenetic control is an intricate interplay between various molecular mechanisms, e.g. DNA methylation and histone modifications (reviewed in [1-4]). Whereas DNA methylation has been studied in great detail for several decades, the role of histone modifications has only been fully appreciated for about 10 years [5]. Since then the number of papers on new histone modifications and their possible functions has exploded.The most widely used procedure to examine histone modifications is Chromatin Immunoprecipitation (ChIP), a technique first established for cultured Drosophila cells [6]. In short, ChIP relies on antibodies to identify the presence of specific histone modifications at DNA regions of interest. Chromatin is extracted from cells or tissue, fragmented and incubated with antibodies against specific histone modifications (Figure 1). The chromatin fragments bound to the antibodie
Molecular epigenetics, chromatin, and NeuroAIDS/HIV: Immunopathological implications  [cached]
Francesco Chiappelli,Paul Shapshak,Deborah Commins,Elyse Singer
Bioinformation , 2008,
Abstract: Epigenetics studies factors related to the organism and environment that modulate inheritance from generation to generation. Molecular epigenetics examines non-coding DNA (ncdDNA) vs. coding DNA (cdDNA), and pertains to every domain of physiology, including immune and brain function. Molecular cartography, including genomics, proteomics, and interactomics, seeks to recognize and to identify the multi-faceted and intricate array of interacting genes and gene products that characterize the function and specialization of each individual cell in the context of cell-cell interaction, tissue, and organ function. Molecular cartography, epigenetics, and chromatin assembly, repair and remodeling (CARR), which, together with the RNA interfering signaling complex (RISC), is responsible for much of the control and regulation of gene expression, intersect. We describe current and ongoing studies aimed to apply these overlapping areas of research, CARR and RISC, to a novel understanding of the immuno-neuropathology of HIV-1 infection, as an example. Taken together, the arguments presented here lead to a novel working hypothesis of molecular immune epigenetics as it pertains to HIV/AIDS, and the immunopathology of HIV-1-infected CD4+ cells. Specifically, we discuss these views in the context of the structure-function relationship of chromatin, the cdDNA/ncdDNA ratio, and possible nucleotide divergence in the untranslated regions (UTRs) of mature mRNA intronic and intergenic DNA sequences, and putative catastrophic consequences for immune surveillance and the preservation of health in HIV/AIDS. Here, we discuss the immunopathology of HIV Infection, with emphasis on CARR in cellular, humoral and molecular immune epigenetics.
A Computational Model of Quantitative Chromatin Immunoprecipitation (ChIP) Analysis
Jingping Xie,Philip S. Crooke,Brett A. McKinney,Joel Soltman
Cancer Informatics , 2008,
Abstract: Chromatin immunoprecipitation (ChIP) analysis is widely used to identify the locations in genomes occupied by transcription factors (TFs). The approach involves chemical cross-linking of DNA with associated proteins, fragmentation of chromatin by sonication or enzymatic digestion, immunoprecipitation of the fragments containing the protein of interest, and then PCR or hybridization analysis to characterize and quantify the genomic sequences enriched. We developed a computational model of quantitative ChIP analysis to elucidate the factors contributing to the method’s resolution. The most important variables identified by the model were, in order of importance, the spacing of the PCR primers, the mean length of the chromatin fragments, and, unexpectedly, the type of fragment width distribution, with very small DNA fragments and smaller amplicons providing the best resolution of TF binding. One of the major predictions of the model was also validated experimentally.
Epigenetics & Chromatin celebrates its first anniversary
Steven Henikoff, Frank Grosveld
Epigenetics & Chromatin , 2009, DOI: 10.1186/1756-8935-2-13
Abstract: Among the papers in our inaugural issue was one from Elizabeth Blackburn and co-workers on the use of novel 4D imaging describing how human telomeres behave in vivo [1]. This landmark study demonstrating the extraordinary potential of modern imaging technology for following chromosome movements remains our most highly accessed paper. A year later, we are delighted to congratulate Dr. Blackburn for sharing the 2009 Nobel Prize in Physiology or Medicine, honoring her pioneering work on telomeres and telomerase. Telomeres have also been of great interest in the field of epigenetics and chromatin, with telomere position-effect and subtelomeric repeats providing important insights into the relationship between chromatin structure and gene silencing. Understanding the role of telomeres in disease continues to be an important area of research represented in E&C [2].Here we review a collection of recent articles that we believe illustrate the scope and quality of articles published in E&C, which have also been collated to form a special anniversary print issue. The most popular model systems for epigenetic research are represented among them, including mice, yeast and flies. The articles range from studies of classical epigenetic phenomena, such as X-chromosome inactivation [3] and position-effect variegation (PEV) [4], to biochemical and biophysical approaches, such as chromatin complex purification [5] and atomic force microscopy [6]. Below, we discuss a few of these findings.The classical phenomenon of PEV, the heritable "spreading" of the silent state into a gene by juxtaposition to heterochromatin, has intrigued geneticists for 80 years. But despite substantial progress in understanding the components of spreading, the mechanism has remained in doubt. In their E&C paper published in January, 2009 [4], Bas van Steensel and colleagues address the question of spreading by determining the precise pattern of binding by Heterochromatin-associated Protein 1 (HP1) in classical
Protocol: methodology for chromatin immunoprecipitation (ChIP) in Chlamydomonas reinhardtii
Daniela Strenkert, Stefan Schmollinger, Michael Schroda
Plant Methods , 2011, DOI: 10.1186/1746-4811-7-35
Abstract: Since several decades the unicellular green alga Chlamydomonas reinhardtii serves as a model organism for studying various aspects of cell biology [1]. However, although all three genetic compartments have been sequenced and are amenable for genetic manipulation [2], transgenic approaches frequently suffer from low transgene expression levels and from transgene silencing [3]. Recent work has shown that this is largely due to epigenetic mechanisms that frequently involve histone modifications. Several factors mediating histone modifications have already been identified in Chlamydomonas, mainly by Cerutti and coworkers: one of them is MUT11, a WD40-repeat protein homologous to human WDR5. Deletion of MUT11 resulted in the activation of single-copy transgenes and of dispersed transposons [4]. MUT11 was shown to interact with SET domain histone methyltransferases and suppression of SET1 by RNAi came along with a reduction in levels of monomethylated H3K4, an epigenetic mark associated with transcriptionally repressed loci [5]. Another factor is the SU(VAR)3-9-related protein SET3p. RNAi-mediated suppression of SET3 released the transcriptional silencing of tandemly repeated transgenes and correlated with a partial loss of monomethyl H3K9 at such loci [6]. Again another factor is the MUT9p kinase which phosphorylates H3T3 and histone H2A and is required for long-term, heritable gene silencing [7]. Furthermore, the Chlamydomonas enhancer of zeste homolog (EZH) catalyzes H3K27 methylation. RNAi-mediated suppression of EZH in Chlamydomonas resulted in a global increase in levels of histone H3K4 trimethylation and H4 acetylation, two characteristic marks for active chromatin, thereby leading to the release of retrotransposons and of silenced, tandemly repeated transgenes [8]. Finally, Yamasaki and coworkers found that silencing of a transgenic RBCS2 promoter, driving the expression of an inverted repeat construct, was associated with low levels of histone H3 acetylation and
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