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Sample records for addition chromatin immunoprecipitation

  1. Chromatin Immunoprecipitation.

    PubMed

    Wiehle, Laura; Breiling, Achim

    2016-01-01

    Chromatin immunoprecipitation (ChIP) is a valuable method to investigate protein-DNA interactions in vivo. Since its discovery it has been indispensable to identify binding sites and patterns of a variety of DNA-interacting proteins, such as transcription factors and regulators, modified histones, and epigenetic modifiers. The Polycomb repressors were the first proteins that have been mapped using this technique, which provided the mechanistic basis for the understanding of their biological function. Cross-linked (XChIP) or native (NChIP) chromatin from tissues or cultured cells is fragmented and the protein of interest is immunoprecipitated using a specific antibody. The co-precipitated DNA is then purified and subjected to analysis by region-specific PCR, DNA microarray (ChIP-on-chip), or next-generation sequencing (ChIP-seq). The assay can therefore produce information about the localization of the analyzed protein at specific candidate loci or throughout the entire genome. In this chapter, we provide a detailed protocol of the basic standard ChIP assay and some remarks about variations. PMID:27659971

  2. Protein tagging for chromatin immunoprecipitation from Arabidopsis.

    PubMed

    de Folter, Stefan

    2011-01-01

    A powerful method to identify binding sites in target genes is chromatin immunoprecipitation (ChIP), which allows the purification of in vivo formed complexes of a DNA-binding protein and associated DNA. Briefly, the method involves the fixation of plant tissue and the isolation of the total protein-DNA mixture, followed by an immunoprecipitation step with an antibody directed against the protein of interest and, subsequently, the DNA can be purified. Finally, the DNA can be analyzed by PCR for the enrichment of specific regions. A drawback of ChIP is that for each protein another antibody is needed. To overcome this, a generic strategy is possible using tags fused to the protein of interest. In this case, only antibody is needed against the tag. This protocol describes the tagging of proteins and how to perform ChIP. PMID:20931382

  3. Mapping Recombination Initiation Sites Using Chromatin Immunoprecipitation.

    PubMed

    He, Yan; Wang, Minghui; Sun, Qi; Pawlowski, Wojciech P

    2016-01-01

    Genome-wide maps of recombination sites provide valuable information not only on the recombination pathway itself but also facilitate the understanding of genome dynamics and evolution. Here, we describe a chromatin immunoprecipitation (ChIP) protocol to map the sites of recombination initiation in plants with maize used as an example. ChIP is a method that allows identification of chromosomal sites occupied by specific proteins. Our protocol utilizes RAD51, a protein involved in repair of double-strand breaks (DSBs) that initiate meiotic recombination, to identify DSB formation hotspots. Chromatin is extracted from meiotic flowers, sheared and enriched in fragments bound to RAD51. Genomic location of the protein is then identified by next-generation sequencing. This protocol can also be used in other species of plants, animals, and fungi. PMID:27511175

  4. Chromatin Preparation and Chromatin Immuno-precipitation from Drosophila Embryos.

    PubMed

    Löser, Eva; Latreille, Daniel; Iovino, Nicola

    2016-01-01

    This protocol provides specific details on how to perform Chromatin immunoprecipitation (ChIP) from Drosophila embryos. ChIP allows the matching of proteins or histone modifications to specific genomic regions. Formaldehyde-cross-linked chromatin is isolated and antibodies against the target of interest are used to determine whether the target is associated with a specific DNA sequence. This can be performed in spatial and temporal manner and it can provide information about the genome-wide localization of a given protein or histone modification if coupled with deep sequencing technology (ChIP-Seq). PMID:27659972

  5. Chromatin immunoprecipitation analysis of Xenopus embryos.

    PubMed

    Akkers, Robert C; Jacobi, Ulrike G; Veenstra, Gert Jan C

    2012-01-01

    Chromatin immunoprecipitation (ChIP) is a powerful technique to study epigenetic regulation and transcription factor binding events in the nucleus. It is based on immune-affinity capture of epitopes that have been cross-linked to genomic DNA in vivo. A readout of the extent to which the epitope is associated with particular genomic regions can be obtained by quantitative PCR (ChIP-qPCR), microarray hybridization (ChIP-chip), or deep sequencing (ChIP-seq). ChIP can be used for molecular and quantitative analyses of histone modifications, transcription factors, and elongating RNA polymerase II at specific loci. It can also be applied to assess the cellular state of transcriptional activation or repression as a predictor of the cells' capabilities and potential. Another possibility is to employ ChIP to characterize genomes, as histone modifications and binding events occur at specific and highly characteristic genomic elements and locations. This chapter provides a step-by-step protocol of ChIP using early Xenopus embryos and discusses potential pitfalls and other issues relevant for successful probing of protein-genome interactions by ChIP-qPCR and ChIP-seq. PMID:22956095

  6. Sequence analysis of chromatin immunoprecipitation data for transcription factors

    PubMed Central

    Fraenkel, Ernest

    2013-01-01

    Chromatin immunoprecipitation (ChIP) experiments allow the location of transcription factors to be determined across the genome. Subsequent analysis of the sequences of the identified regions allows binding to be localized at a higher resolution than can be achieved by current high-throughput experiments without sequence analysis, and may provide important insight into the regulatory programs enacted by the protein of interest. In this chapter we review the tools, workflow, and common pitfalls of such analyses, and recommend strategies for effective motif discovery from these data. PMID:20827592

  7. Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging.

    PubMed

    Tatavosian, Roubina; Zhen, Chao Yu; Duc, Huy Nguyen; Balas, Maggie M; Johnson, Aaron M; Ren, Xiaojun

    2015-11-20

    Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.

  8. Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging.

    PubMed

    Tatavosian, Roubina; Zhen, Chao Yu; Duc, Huy Nguyen; Balas, Maggie M; Johnson, Aaron M; Ren, Xiaojun

    2015-11-20

    Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes. PMID:26381410

  9. Chromatin immunoprecipitation and microarray-based analysis of protein location

    PubMed Central

    Lee, Tong Ihn; Johnstone, Sarah E; Young, Richard A

    2010-01-01

    Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. The enriched DNA population is then labeled, combined with a differentially labeled reference sample and applied to DNA microarrays to detect enriched signals. Various computational and bioinformatic approaches are then applied to normalize the enriched and reference channels, to connect signals to the portions of the genome that are represented on the DNA microarrays, to provide confidence metrics and to generate maps of protein-genome occupancy. Here, we describe the experimental protocols that we use from crosslinking of cells to hybridization of labeled material, together with insights into the aspects of these protocols that influence the results. These protocols require approximately 1 week to complete once sufficient numbers of cells have been obtained, and have been used to produce robust, high-quality ChIP-chip results in many different cell and tissue types. PMID:17406303

  10. Tagging of MADS domain proteins for chromatin immunoprecipitation

    PubMed Central

    de Folter, Stefan; Urbanus, Susan L; van Zuijlen, Lisette GC; Kaufmann, Kerstin; Angenent, Gerco C

    2007-01-01

    Background Most transcription factors fulfill their role in complexes and regulate their target genes upon binding to DNA motifs located in upstream regions or introns. To date, knowledge about transcription factor target genes and their corresponding transcription factor binding sites are still very limited. Two related methods that allow in vivo identification of transcription factor binding sites are chromatin immunoprecipitation (ChIP) and chromatin affinity purification (ChAP). For ChAP, the protein of interest is tagged with a peptide or protein, which can be used for affinity purification of the protein-DNA complex and hence, the identification of the target gene. Results Here, we present the results of experiments aiming at the development of a generic tagging approach for the Arabidopsis MADS domain proteins AGAMOUS, SEPALLATA3, and FRUITFULL. For this, Arabidopsis wild type plants were transformed with constructs containing a MADS-box gene fused to either a double Strep-tag® II-FLAG-tag, a triple HA-tag, or an eGFP-tag, all under the control of the constitutive double 35S Cauliflower Mosaic Virus (CaMV) promoter. Strikingly, in all cases, the number of transformants with loss-of-function phenotypes was much larger than those with an overexpression phenotype. Using endogenous promoters in stead of the 35S CaMV resulted in a dramatic reduction in the frequency of loss-of-function phenotypes. Furthermore, pleiotropic defects occasionally caused by an overexpression strategy can be overcome by using the native promoter of the gene. Finally, a ChAP result is presented using GFP antibody on plants carrying a genomic fragment of a MADS-box gene fused to GFP. Conclusion This study revealed that MADS-box proteins are very sensitive to fusions with small peptide tags and GFP tags. Furthermore, for the expression of chimeric versions of MADS-box genes it is favorable to use the entire genomic region in frame to the tag of choice. Interestingly, though unexpected

  11. Genome-Wide Chromatin Immunoprecipitation in Candida albicans and Other Yeasts

    PubMed Central

    Lohse, Matthew B.; Kongsomboonvech, Pisiwat; Madrigal, Maria; Hernday, Aaron D.; Nobile, Clarissa J.

    2016-01-01

    Chromatin immunoprecipitation experiments are critical to investigating the interactions between DNA and a wide range of nuclear proteins within a cell or biological sample. In this chapter we outline an optimized protocol for genome-wide chromatin immunoprecipitation that has been used successfully for several distinct morphological forms of numerous yeast species, and include an optimized method for amplification of chromatin immunoprecipitated DNA samples and hybridization to a high-density oligonucleotide tiling microarray. We also provide detailed suggestions on how to analyze the complex data obtained from these experiments. PMID:26483022

  12. Identification of an archaeal mercury regulon by chromatin immunoprecipitation.

    PubMed

    Rudrappa, Deepak; Yao, Andrew I; White, Derrick; Pavlik, Benjamin J; Singh, Raghuveer; Facciotti, Marc T; Blum, Paul

    2015-12-01

    Mercury is a heavy metal and toxic to all forms of life. Metal exposure can invoke a response to improve survival. In archaea, several components of a mercury response system have been identified, but it is not known whether metal transport is a member of this system. To identify such missing components, a peptide-tagged MerR transcription factor was used to localize enriched chromosome regions by chromosome immunoprecipitation combined with DNA sequence analysis. Such regions could serve as secondary regulatory binding sites to control the expression of additional genes associated with mercury detoxification. Among the 31 highly enriched loci, a subset of five was pursued as potential candidates based on their current annotations. Quantitative reverse transcription-PCR analysis of these regions with and without mercury treatment in WT and mutant strains lacking merR indicated significant regulatory responses under these conditions. Of these, a Family 5 extracellular solute-binding protein and the MarR transcription factor shown previously to control responses to oxidation were most strongly affected. Inactivation of the solute-binding protein by gene disruption increased the resistance of mutant cells to mercury challenge. Inductively coupled plasma-MS analysis of the mutant cell line following metal challenge indicated there was less intracellular mercury compared with the isogenic WT strain. Together, these regulated genes comprise new members of the archaeal MerR regulon and reveal a cascade of transcriptional control not previously demonstrated in this model organism.

  13. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles.

    PubMed

    Cejas, Paloma; Li, Lewyn; O'Neill, Nicholas K; Duarte, Melissa; Rao, Prakash; Bowden, Michaela; Zhou, Chensheng W; Mendiola, Marta; Burgos, Emilio; Feliu, Jaime; Moreno-Rubio, Juan; Guadalajara, Héctor; Moreno, Víctor; García-Olmo, Damián; Bellmunt, Joaquim; Mullane, Stephanie; Hirsch, Michelle; Sweeney, Christopher J; Richardson, Andrea; Liu, X Shirley; Brown, Myles; Shivdasani, Ramesh A; Long, Henry W

    2016-06-01

    Extensive cross-linking introduced during routine tissue fixation of clinical pathology specimens severely hampers chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis from archived tissue samples. This limits the ability to study the epigenomes of valuable, clinically annotated tissue resources. Here we describe fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq), a method that enables reliable extraction of soluble chromatin from formalin-fixed paraffin-embedded (FFPE) tissue samples for accurate detection of histone marks. We demonstrate that FiT-seq data from FFPE specimens are concordant with ChIP-seq data from fresh-frozen samples of the same tumors. By using multiple histone marks, we generate chromatin-state maps and identify cis-regulatory elements in clinical samples from various tumor types that can readily allow us to distinguish between cancers by the tissue of origin. Tumor-specific enhancers and superenhancers that are elucidated by FiT-seq analysis correlate with known oncogenic drivers in different tissues and can assist in the understanding of how chromatin states affect gene regulation. PMID:27111282

  14. Chromatin immunoprecipitation and deep sequencing in Xenopus tropicalis and Xenopus laevis

    PubMed Central

    Wills, Andrea E.; Gupta, Rakhi; Chuong, Edward; Baker, Julie C.

    2014-01-01

    Chromatin immunoprecipitation and deep sequencing (ChIP-SEQ) represents a powerful tool for identifying the genomic targets of transcription factors, chromatin remodeling factors, and histone modifications. The frogs Xenopus laevis and Xenopus tropicalis have historically been outstanding model systems for embryology and cell biology, with emerging utility as highly accessible embryos for genome-wide studies. Here we focus on the particular strengths and limitations of Xenopus cell biology and genomics as they apply to ChIP-SEQ, and outline a methodology for ChIP-SEQ in both species, providing detailed strategies for sample preparation, antibody selection, quality control, sequencing library preparation, and basic analysis. PMID:24064036

  15. Protocol: methodology for chromatin immunoprecipitation (ChIP) in Chlamydomonas reinhardtii

    PubMed Central

    2011-01-01

    We report on a detailed chromatin immunoprecipitation (ChIP) protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR. PMID:22050920

  16. A chromatin immunoprecipitation (ChIP) protocol for use in whole human adipose tissue.

    PubMed

    Haim, Yulia; Tarnovscki, Tanya; Bashari, Dana; Rudich, Assaf

    2013-11-01

    Chromatin immunoprecipitation (ChIP) has become a central method when studying in vivo protein-DNA interactions, with the major challenge being the hope to capture "authentic" interactions. While ChIP protocols have been optimized for use with specific cell types and tissues including adipose tissue-derived cells, a working ChIP protocol addressing the challenges imposed by fresh whole human adipose tissue has not been described. Utilizing human paired omental and subcutaneous adipose tissue obtained during elective abdominal surgeries, we have carefully identified and optimized individual steps in the ChIP protocol employed directly on fresh tissue fragments. We describe a complete working protocol for using ChIP on whole adipose tissue fragments. Specific steps required adaptation of the ChIP protocol to human whole adipose tissue. In particular, a cross-linking step was performed directly on fresh small tissue fragments. Nuclei were isolated before releasing chromatin, allowing better management of fat content; a sonication protocol to obtain fragmented chromatin was optimized. We also demonstrate the high sensitivity of immunoprecipitated chromatin from adipose tissue to freezing. In conclusion, we describe the development of a ChIP protocol optimized for use in studying whole human adipose tissue, providing solutions for the unique challenges imposed by this tissue. Unraveling protein-DNA interaction in whole human adipose tissue will likely contribute to elucidating molecular pathways contributing to common human diseases such as obesity and type 2 diabetes.

  17. Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo.

    PubMed

    Komar, Dorota N; Mouriz, Alfonso; Jarillo, José A; Piñeiro, Manuel

    2016-01-14

    Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

  18. Probing flagellar promoter occupancy in wild-type and mutant Caulobacter crescentus by chromatin immunoprecipitation.

    PubMed

    Davis, Nicole J; Viollier, Patrick H

    2011-06-01

    In the asymmetric predivisional cell of Caulobacter crescentus, TipF and TipN mark the cellular pole for future flagellar development. TipF is essential for motility and contains a cyclic-di-GMP phosphodiesterase-like (EAL) domain that is necessary for proper function. TipN is localized to the flagellar pole before TipF and is essential for the proper placement of the flagellum in C. crescentus. Using β-galactosidase promoter-probe assays and quantitative chromatin immunoprecipitation, we investigated the influence of the C. crescentus flagellar assembly regulator TipF on flagellar gene transcription. We compared the transcriptional activity of class II-fliF-lacZ, class III-flgE-lacZ, and class IV-fljL-lacZ fusions in a ΔtipF mutant with that of other flagellar mutants and the wild-type strain. We subsequently verified the in vivo occupancy of the fliF, flgE, and fljL flagellar promoters by the flagellar regulators CtrA, FlbD, and FliX in addition to RNA polymerase. We deduce that TipF contributes to proper expression of flagellar genes in C. crescentus by acting both within and outside of the canonical flagellar gene expression hierarchy.

  19. Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)

    PubMed Central

    Fujita, Toshitsugu; Asano, Yoshinori; Ohtsuka, Junko; Takada, Yoko; Saito, Kazunobu; Ohki, Rieko; Fujii, Hodaka

    2013-01-01

    Biochemical analysis of molecular interactions in specific genomic regions requires their isolation while retaining molecular interactions in vivo. Here, we report isolation of telomeres by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using a transcription activator-like (TAL) protein recognizing telomere repeats. Telomeres recognized by the tagged TAL protein were immunoprecipitated with an antibody against the tag and subjected to identification of telomere-binding molecules. enChIP-mass spectrometry (enChIP-MS) targeting telomeres identified known and novel telomere-binding proteins. The data have been deposited to the ProteomeXchange with identifier PXD000461. In addition, we showed that RNA associated with telomeres could be isolated by enChIP. Identified telomere-binding molecules may play important roles in telomere biology. enChIP using TAL proteins would be a useful tool for biochemical analysis of specific genomic regions of interest. PMID:24201379

  20. Biochemical Analysis of Genome Functions Using Locus-Specific Chromatin Immunoprecipitation Technologies

    PubMed Central

    Fujita, Toshitsugu; Fujii, Hodaka

    2016-01-01

    To isolate specific genomic regions that retain their molecular interactions, allowing direct identification of chromatin-bound molecules, we developed two locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies, insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP) using the clustered regularly interspaced short palindromic repeats (CRISPR) system or transcription activator-like (TAL) proteins. Essentially, a locus-specific ChIP consists of locus-tagging and affinity purification and can be combined with downstream analyses to identify molecules associated with the target genomic regions. In this review, we discuss the applications of locus-specific ChIP to analyze the genome functions, including transcription and epigenetic regulation. PMID:26819551

  1. Chromatin analyses of Zymoseptoria tritici: Methods for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq).

    PubMed

    Soyer, Jessica L; Möller, Mareike; Schotanus, Klaas; Connolly, Lanelle R; Galazka, Jonathan M; Freitag, Michael; Stukenbrock, Eva H

    2015-06-01

    The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein-DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin or H3K9me3 and H3K27me3, which are considered marks for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen.

  2. Identification of Transcribed Enhancers by Genome-Wide Chromatin Immunoprecipitation Sequencing.

    PubMed

    Blinka, Steven; Reimer, Michael H; Pulakanti, Kirthi; Pinello, Luca; Yuan, Guo-Cheng; Rao, Sridhar

    2017-01-01

    Recent work has shown that RNA polymerase II-mediated transcription at distal cis-regulatory elements serves as a mark of highly active enhancers. Production of noncoding RNAs at enhancers, termed eRNAs, correlates with higher expression of genes that the enhancer interacts with; hence, eRNAs provide a new tool to model gene activity in normal and disease tissues. Moreover, this unique class of noncoding RNA has diverse roles in transcriptional regulation. Transcribed enhancers can be identified by a common signature of epigenetic marks by overlaying a series of genome-wide chromatin immunoprecipitation and RNA sequencing datasets. A computational approach to filter non-enhancer elements and other classes of noncoding RNAs is essential to not cloud downstream analysis. Here we present a protocol that combines wet and dry bench methods to accurately identify transcribed enhancers genome-wide as well as an experimental procedure to validate these datasets. PMID:27662872

  3. Optimization of a method for chromatin immunoprecipitation assays in the marine invertebrate chordate Ciona

    PubMed Central

    Aihara, Hitoshi; Katikala, Lavanya; Zeller, Robert W.; Di Gregorio, Anna; Nibu, Yutaka

    2013-01-01

    Chromatin immunoprecipitation (ChIP) assays allow the efficient characterization of the in vivo occupancy of genomic regions by DNA-binding proteins, and thus facilitate the prediction of cis-regulatory sequences in silico and guide their validation in vivo. For these reasons, these assays and their permutations (e.g., ChIP-on-chip, ChIP-Sequencing) are currently being extended to several non-mainstream model organisms, as the availability of specific antibodies increases. Here we describe the development of a polyclonal antibody against the Brachyury protein of the marine invertebrate chordate Ciona intestinalis and provide a detailed ChIP protocol that should be easily adaptable to other marine organisms. PMID:23592257

  4. Weighted enrichment method for prediction of transcription regulators from transcriptome and global chromatin immunoprecipitation data

    PubMed Central

    Kawakami, Eiryo; Nakaoka, Shinji; Ohta, Tazro; Kitano, Hiroaki

    2016-01-01

    Predicting responsible transcription regulators on the basis of transcriptome data is one of the most promising computational approaches to understanding cellular processes and characteristics. Here, we present a novel method employing vast amounts of chromatin immunoprecipitation (ChIP) experimental data to address this issue. Global high-throughput ChIP data was collected to construct a comprehensive database, containing 8 578 738 binding interactions of 454 transcription regulators. To incorporate information about heterogeneous frequencies of transcription factor (TF)-binding events, we developed a flexible framework for gene set analysis employing the weighted t-test procedure, namely weighted parametric gene set analysis (wPGSA). Using transcriptome data as an input, wPGSA predicts the activities of transcription regulators responsible for observed gene expression. Validation of wPGSA with published transcriptome data, including that from over-expressed TFs, showed that the method can predict activities of various TFs, regardless of cell type and conditions, with results totally consistent with biological observations. We also applied wPGSA to other published transcriptome data and identified potential key regulators of cell reprogramming and influenza virus pathogenesis, generating compelling hypotheses regarding underlying regulatory mechanisms. This flexible framework will contribute to uncovering the dynamic and robust architectures of biological regulation, by incorporating high-throughput experimental data in the form of weights. PMID:27131787

  5. Analysis of TGFβ1 and IL-10 transcriptional regulation in CTCL cells by chromatin immunoprecipitation.

    PubMed

    Chang, Tzu-Pei; Kim, Myra; Vancurova, Ivana

    2014-01-01

    The immunosuppressive cytokines transforming growth factor β1 (TGFβ1) and interleukin-10 (IL-10) regulate a variety of biological processes including differentiation, proliferation, tissue repair, tumorigenesis, inflammation, and host defense. Aberrant expression of TGFβ1 and IL-10 has been associated with many types of autoimmune and inflammatory disorders, as well as with many types of cancer and leukemia. Patients with cutaneous T cell lymphoma (CTCL) have high levels of malignant CD4+ T cells expressing IL-10 and TGFβ1 that suppress the immune system and diminish the antitumor responses. The transcriptional regulation of TGFβ1 and IL-10 expression is orchestrated by several transcription factors, including NFκB. However, while the transcriptional regulation of pro-inflammatory and anti-apoptotic genes by NFκB has been studied extensively, much less is known about the NFκB regulation of immunosuppressive genes. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the transcriptional regulation of TGFβ1 and IL-10 by measuring recruitment of NFκB p65, p50, c-Rel, Rel-B, and p52 subunits to TGFβ1 and IL-10 promoters in human CTCL Hut-78 cells. PMID:24908319

  6. Analysis of TGFβ1 and IL-10 transcriptional regulation in CTCL cells by chromatin immunoprecipitation.

    PubMed

    Chang, Tzu-Pei; Kim, Myra; Vancurova, Ivana

    2014-01-01

    The immunosuppressive cytokines transforming growth factor β1 (TGFβ1) and interleukin-10 (IL-10) regulate a variety of biological processes including differentiation, proliferation, tissue repair, tumorigenesis, inflammation, and host defense. Aberrant expression of TGFβ1 and IL-10 has been associated with many types of autoimmune and inflammatory disorders, as well as with many types of cancer and leukemia. Patients with cutaneous T cell lymphoma (CTCL) have high levels of malignant CD4+ T cells expressing IL-10 and TGFβ1 that suppress the immune system and diminish the antitumor responses. The transcriptional regulation of TGFβ1 and IL-10 expression is orchestrated by several transcription factors, including NFκB. However, while the transcriptional regulation of pro-inflammatory and anti-apoptotic genes by NFκB has been studied extensively, much less is known about the NFκB regulation of immunosuppressive genes. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the transcriptional regulation of TGFβ1 and IL-10 by measuring recruitment of NFκB p65, p50, c-Rel, Rel-B, and p52 subunits to TGFβ1 and IL-10 promoters in human CTCL Hut-78 cells.

  7. Evaluation of myc E-box phylogenetic footprints in glycolytic genes by chromatin immunoprecipitation assays.

    PubMed

    Kim, Jung-whan; Zeller, Karen I; Wang, Yunyue; Jegga, Anil G; Aronow, Bruce J; O'Donnell, Kathryn A; Dang, Chi V

    2004-07-01

    Prediction of gene regulatory sequences using phylogenetic footprinting has advanced considerably but lacks experimental validation. Here, we report whether transcription factor binding sites predicted by dot plotting or web-based Trafac analysis could be validated by chromatin immunoprecipitation assays. MYC overexpression enhances glycolysis without hypoxia and hence may contribute to altered tumor metabolism. Because the full spectrum of glycolytic genes directly regulated by Myc is not known, we chose Myc as a model transcription factor to determine whether it binds target glycolytic genes that have conserved canonical Myc binding sites or E boxes (5'-CACGTG-3'). Conserved canonical E boxes in ENO1, HK2, and LDHA occur in 31- to 111-bp islands with high interspecies sequence identity (>65%). Trafac analysis revealed another region in ENO1 that corresponds to a murine region with a noncanonical E box. Myc bound all these conserved regions well in the human P493-6 B lymphocytes. We also determined whether Myc could bind nonconserved canonical E boxes found in the remaining human glycolytic genes. Myc bound PFKM, but it did not significantly bind GPI, PGK1, and PKM2. Binding to BPGM, PGAM2, and PKLR was not detected. Both GAPD and TPI1 do not have conserved E boxes but are induced and bound by Myc through regions with noncanonical E boxes. Our results indicate that Myc binds well to conserved canonical E boxes, but not nonconserved E boxes. However, the binding of Myc to unpredicted genomic regions with noncanonical E boxes reveals a limitation of phylogenetic footprinting. In aggregate, these observations indicate that Myc is an important regulator of glycolytic genes, suggesting that MYC plays a key role in a switch to glycolytic metabolism during cell proliferation or tumorigenesis.

  8. Application of the chromatin immunoprecipitation method to identify in vivo protein-DNA associations in fission yeast.

    PubMed

    Takahashi, K; Saitoh, S; Yanagida, M

    2000-10-31

    The chromatin immunoprecipitation (ChIP) method provides an ideal tool for detecting direct or indirect interactions between proteins of interest and DNAs with known sequences. Here, we introduce the ChIP protocol used in our laboratory to identify in vivo protein-DNA association in the fission yeast Schizosaccharomyces pombe. The cytological and genetic merits of the fission yeast for studying control of the eukaryotic cell cycle and chromosome dynamics are reinforced by application of this ChIP method.

  9. Novel in vivo targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach

    PubMed Central

    Birkaya, Barbara; Ortt, Kori; Sinha, Satrajit

    2007-01-01

    Background p63 is a transcription factor that plays an important role in skin epidermal development and differentiation. The p63 gene encodes for two major protein isoforms, those containing an amino-terminal trans-activation domain (TAp63) and those lacking this domain (ΔNp63). Both the TA and ΔN transcripts are also alternatively spliced at the 3' end producing proteins with unique C-termini that are designated as α, β and γ isoforms. Recent research has suggested that ΔNp63 is the predominant isoform expressed and active in keratinocytes. Results To better elucidate the biological role of p63 in regulating gene expression in keratinocytes we performed chromatin immunoprecipitation (ChIP) experiments with ΔNp63-specific antibodies. We included an additional step in the ChIP procedure to enrich for ΔNp63 targets by screening the library of immunoprecipitated DNA for its ability to bind recombinant GST-ΔNp63. Cloning of ΔNp63-ChIP-derived DNA fragments identified more than 60 potential ΔNp63 target loci that were located close to or embedded within known or predicted genes. Identity of these target genes suggests that they may participate in a myriad of cellular processes including transcriptional regulation, signaling and metabolism. Here we confirm the binding of ΔNp63 to several of these genomic loci both by EMSA and replicate ChIP assays. Finally we show that the expression of many of these target genes is altered when ΔNp63 levels in keratinocytes are reduced by siRNA, further confirming that these are bona fide targets. Conclusion This unbiased genomic approach has allowed us to uncover functional targets of ΔNp63 and serves as the initial step in further analysis of the transcriptional regulatory mechanisms that are governed by p63 in keratinocytes. PMID:17521434

  10. Dynamic chromatin remodelling of ciliate macronuclear DNA as determined by an optimized chromatin immunoprecipitation (ChIP) method for Paramecium tetraurelia.

    PubMed

    Cheaib, Miriam; Simon, Martin

    2013-03-01

    We report the detailed evaluation of crucial parameters for chromatin immunoprecipitation (ChIP) of macronuclear DNA in the unicellular eukaryote Paramecium tetraurelia. Optimized parameters include crosslinking conditions, chromatin sonication and antibody titration thus providing a detailed protocol for successful ChIP in P. tetraurelia. As this ciliate is bacterivorous and RNAi by feeding represents a powerful tool for analysis of gene function, we moreover determined the effects of ingested nucleic acids by food bacteria. Feasibility of our protocol is demonstrated by characterisation of chromatin remodelling at promoters of cytosolic HSP70 isoforms during transcriptional activation under heat shock conditions by analyzing RNA abundance, nucleosome occupancy and levels of H3 lysine 9 acetylation.

  11. Investigation of histone H4 hyperacetylation dynamics in the 5S rRNA genes family by chromatin immunoprecipitation assay.

    PubMed

    Burlibașa, Liliana; Suciu, Ilinca

    2015-12-01

    Oogenesis is a critical event in the formation of female gamete, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the presence of H4 acetylation of the oocyte and somatic 5S rRNA genes in Triturus cristatus, using chromatin immunoprecipitation assay (ChIP). Our findings suggest that some epigenetic mechanisms such as histone acetylation could be involved in the transcriptional regulation of 5S rRNA gene families.

  12. Fully automated high-throughput chromatin immunoprecipitation for ChIP-seq: Identifying ChIP-quality p300 monoclonal antibodies

    PubMed Central

    Gasper, William C.; Marinov, Georgi K.; Pauli-Behn, Florencia; Scott, Max T.; Newberry, Kimberly; DeSalvo, Gilberto; Ou, Susan; Myers, Richard M.; Vielmetter, Jost; Wold, Barbara J.

    2014-01-01

    Chromatin immunoprecipitation coupled with DNA sequencing (ChIP-seq) is the major contemporary method for mapping in vivo protein-DNA interactions in the genome. It identifies sites of transcription factor, cofactor and RNA polymerase occupancy, as well as the distribution of histone marks. Consortia such as the ENCyclopedia Of DNA Elements (ENCODE) have produced large datasets using manual protocols. However, future measurements of hundreds of additional factors in many cell types and physiological states call for higher throughput and consistency afforded by automation. Such automation advances, when provided by multiuser facilities, could also improve the quality and efficiency of individual small-scale projects. The immunoprecipitation process has become rate-limiting, and is a source of substantial variability when performed manually. Here we report a fully automated robotic ChIP (R-ChIP) pipeline that allows up to 96 reactions. A second bottleneck is the dearth of renewable ChIP-validated immune reagents, which do not yet exist for most mammalian transcription factors. We used R-ChIP to screen new mouse monoclonal antibodies raised against p300, a histone acetylase, well-known as a marker of active enhancers, for which ChIP-competent monoclonal reagents have been lacking. We identified, validated for ChIP-seq, and made publicly available a monoclonal reagent called ENCITp300-1. PMID:24919486

  13. AHT-ChIP-seq: a completely automated robotic protocol for high-throughput chromatin immunoprecipitation

    PubMed Central

    2013-01-01

    ChIP-seq is an established manually-performed method for identifying DNA-protein interactions genome-wide. Here, we describe a protocol for automated high-throughput (AHT) ChIP-seq. To demonstrate the quality of data obtained using AHT-ChIP-seq, we applied it to five proteins in mouse livers using a single 96-well plate, demonstrating an extremely high degree of qualitative and quantitative reproducibility among biological and technical replicates. We estimated the optimum and minimum recommended cell numbers required to perform AHT-ChIP-seq by running an additional plate using HepG2 and MCF7 cells. With this protocol, commercially available robotics can perform four hundred experiments in five days. PMID:24200198

  14. Characterization of the Escherichia coli σS core regulon by Chromatin Immunoprecipitation-sequencing (ChIP-seq) analysis

    PubMed Central

    Peano, Clelia; Wolf, Johannes; Demol, Julien; Rossi, Elio; Petiti, Luca; De Bellis, Gianluca; Geiselmann, Johannes; Egli, Thomas; Lacour, Stephan; Landini, Paolo

    2015-01-01

    In bacteria, selective promoter recognition by RNA polymerase is achieved by its association with σ factors, accessory subunits able to direct RNA polymerase “core enzyme” (E) to different promoter sequences. Using Chromatin Immunoprecipitation-sequencing (ChIP-seq), we searched for promoters bound by the σS-associated RNA polymerase form (EσS) during transition from exponential to stationary phase. We identified 63 binding sites for EσS overlapping known or putative promoters, often located upstream of genes (encoding either ORFs or non-coding RNAs) showing at least some degree of dependence on the σS-encoding rpoS gene. EσS binding did not always correlate with an increase in transcription level, suggesting that, at some σS-dependent promoters, EσS might remain poised in a pre-initiation state upon binding. A large fraction of EσS-binding sites corresponded to promoters recognized by RNA polymerase associated with σ70 or other σ factors, suggesting a considerable overlap in promoter recognition between different forms of RNA polymerase. In particular, EσS appears to contribute significantly to transcription of genes encoding proteins involved in LPS biosynthesis and in cell surface composition. Finally, our results highlight a direct role of EσS in the regulation of non coding RNAs, such as OmrA/B, RyeA/B and SibC. PMID:26020590

  15. MOBE-ChIP: a large-scale chromatin immunoprecipitation assay for cell type-specific studies.

    PubMed

    Lau, On Sun; Bergmann, Dominique C

    2015-10-01

    Cell type-specific transcriptional regulators play critical roles in the generation and maintenance of multicellularity. As they are often expressed at low levels, in vivo DNA-binding studies of these regulators by standard chromatin immunoprecipitation (ChIP) assays are technically challenging. We describe here an optimized ChIP protocol termed Maximized Objects for Better Enrichment (MOBE)-ChIP, which enhances the sensitivity of ChIP assays for detecting cell type-specific signals. The protocol, which is based on the disproportional increase of target signals over background at higher scales, uses substantially greater volume of starting materials than conventional ChIPs to achieve high signal enrichment. This technique can capture weak binding events that are ambiguous in standard ChIP assays, and is useful both in gene-specific and whole-genome analysis. This protocol has been optimized for Arabidopsis, but should be applicable to other model systems with minor modifications. The full procedure can be completed within 3 days.

  16. Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans

    PubMed Central

    Mukhopadhyay, Arnab; Deplancke, Bart; Walhout, Albertha J M; Tissenbaum, Heidi A

    2009-01-01

    In order to determine how signaling pathways differentially regulate gene expression, it is necessary to identify the interactions between transcription factors (TFs) and their cognate cis-regulatory DNA elements. Here, we have outlined a chromatin immunoprecipitation (ChIP) protocol for use in whole Caenorhabditis elegans extracts. We discuss optimization of the procedure, including growth and harvesting of the worms, formaldehyde fixation, TF immunoprecipitation and analysis of bound sequences through real-time PCR. It takes ∼10–12 d to obtain the worm culture for ChIP; the ChIP procedure is spaced out over a period of 2.5 d with two overnight incubations. PMID:18388953

  17. Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter

    PubMed Central

    Rogge, George A; Shen, Li-Ling; Kuhar, Michael J.

    2010-01-01

    Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action. PMID:20451507

  18. Genomic response to Wnt signalling is highly context-dependent - Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets

    SciTech Connect

    Railo, Antti; Pajunen, Antti; Itaeranta, Petri; Naillat, Florence; Vuoristo, Jussi; Kilpelaeinen, Pekka; Vainio, Seppo

    2009-10-01

    Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-{beta}-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of {beta}-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays of gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.

  19. PARP1 genomics: chromatin immunoprecipitation approach using anti-PARP1 antibody (ChIP and ChIP-seq)

    PubMed Central

    Lodhi, Niraj; Tulin, Alexei V.

    2011-01-01

    Poly(ADP-ribose) polymerase1 (PARP1) is a global regulator of different cellular mechanisms, ranging from DNA damage repair to control of gene expression. Since PARP1 protein and pADPr have been shown to persist in chromatin through cell cycle, they may both act as epigenetic markers. However, it is not known how many loci are occupied by PARP1 protein during mitosis genome-wide. To reveal the genome-wide PARP1 binding sites, we used the ChIP-seq approach, an emerging technique to study genome-wide PARP1 protein interaction with chromatin. Here, we describe how to perform ChIP-seq in the context of PARP1 binding sites identification in chromatin, using human embryonic kidney cell lines. PMID:21870262

  20. Identification of C/EBPβ Target Genes in ALK+ Anaplastic Large Cell Lymphoma (ALCL) by Gene Expression Profiling and Chromatin Immunoprecipitation

    PubMed Central

    Bonzheim, Irina; Irmler, Martin; Klier-Richter, Margit; Steinhilber, Julia; Anastasov, Nataša; Schäfer, Sabine; Adam, Patrick; Beckers, Johannes; Raffeld, Mark; Fend, Falko; Quintanilla-Martinez, Leticia

    2013-01-01

    C/EBPβ (CCAAT enhancer binding protein) is a transcription factor that plays a crucial role in survival and transformation of ALK+ anaplastic large cell lymphoma (ALCL). The aim of this study was to identify the downstream targets of C/EBPβ responsible for ALK-mediated oncogenesis. C/EBPβ was knocked down in ALK+ ALCL cell lines with a C/EBPβ-shRNA, followed by gene expression profiling (GEP). GEP analysis revealed a reproducible signature of genes that were significantly regulated by C/EBPβ. Classification into biological categories revealed overrepresentation of genes involved in the immune response, apoptosis and cell proliferation. Transcriptional regulation by C/EBPβ was found in 6 of 11 (BCL2A1, G0S2, TRIB1, S100A9, DDX21 and DDIT4) genes investigated by chromatin immunoprecipitation. We demonstrated that BCL2A1, G0S2 and DDX21 play a crucial role in survival and proliferation of ALK+ ALCL cells. DDX21, a gene involved in rRNA biogenesis, was found differentially overexpressed in primary ALK+ ALCL cases. All three candidate genes were validated in primary ALCL cases by either immunohistochemistry or RT-qPCR. In conclusion, we identified and validated several key C/EBPβ-regulated genes with major impact on survival and cell growth in ALK+ ALCL, supporting the central role of C/EBPβ in ALK-mediated oncogenesis. PMID:23741337

  1. Chromatin immunoprecipitation analysis of bortezomib-mediated inhibition of NFκB recruitment to IL-1β and TNFα gene promoters in human macrophages.

    PubMed

    Sanacora, Shannon; Chang, Tzu-Pei; Vancurova, Ivana

    2014-01-01

    Interleukin-1β (IL-1) and tumor necrosis factor-α (TNF) are important pro-inflammatory cytokines involved in the mediation of the immune response, inflammation, tissue repair, and tumor progression. Regulation of IL-1 and TNF expression is mediated at the level of transcription by the transcription factor NFκB. Inhibition of NFκB activity by the proteasome inhibitor bortezomib (BZ) has been used as a frontline therapy in multiple myeloma and other hematological malignancies. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the NFκB recruitment to endogenous IL-1 and TNF promoters in BZ-treated human macrophages. Corresponding to the BZ-suppressed mRNA levels of IL-1 and TNF, we show that BZ inhibits p65 NFκB recruitment to IL-1 and TNF promoters. This study specifically uses U937 macrophages, but the protocol could be easily modified to analyze the regulation of NFκB recruitment in other cell types. PMID:24908318

  2. Chromatin immunoprecipitation analysis of bortezomib-mediated inhibition of NFκB recruitment to IL-1β and TNFα gene promoters in human macrophages.

    PubMed

    Sanacora, Shannon; Chang, Tzu-Pei; Vancurova, Ivana

    2014-01-01

    Interleukin-1β (IL-1) and tumor necrosis factor-α (TNF) are important pro-inflammatory cytokines involved in the mediation of the immune response, inflammation, tissue repair, and tumor progression. Regulation of IL-1 and TNF expression is mediated at the level of transcription by the transcription factor NFκB. Inhibition of NFκB activity by the proteasome inhibitor bortezomib (BZ) has been used as a frontline therapy in multiple myeloma and other hematological malignancies. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the NFκB recruitment to endogenous IL-1 and TNF promoters in BZ-treated human macrophages. Corresponding to the BZ-suppressed mRNA levels of IL-1 and TNF, we show that BZ inhibits p65 NFκB recruitment to IL-1 and TNF promoters. This study specifically uses U937 macrophages, but the protocol could be easily modified to analyze the regulation of NFκB recruitment in other cell types.

  3. Genome-Wide Mapping of the Distribution of CarD, RNAP σ(A), and RNAP β on the Mycobacterium smegmatis Chromosome using Chromatin Immunoprecipitation Sequencing.

    PubMed

    Landick, Robert; Krek, Azra; Glickman, Michael S; Socci, Nicholas D; Stallings, Christina L

    2014-12-01

    CarD is an essential mycobacterial protein that binds the RNA polymerase (RNAP) and affects the transcriptional profile of Mycobacterium smegmatis and Mycobacterium tuberculosis (6). We predicted that CarD was directly regulating RNAP function but our prior experiments had not determined at what stage of transcription CarD was functioning and at which genes CarD interacted with the RNAP. To begin to address these open questions, we performed Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. The distribution of RNAP subunits β and σ(A) were also profiled. We expected that RNAP β would be present throughout transcribed regions and RNAP σ(A) would be predominantly enriched at promoters based on work in Escherichia coli (3), however this had yet to be determined in mycobacteria. The ChIP-seq analyses revealed that CarD was never present on the genome in the absence of RNAP, was primarily associated with promoter regions, and was highly correlated with the distribution of RNAP σ(A). The colocalization of σ(A) and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. Here we describe in detail the data from the ChIP-seq experiments associated with the study published by Srivastava and colleagues in the Proceedings of the National Academy of Science in 2013 (5) as well as discuss the findings from this dataset in relation to both CarD and mycobacterial transcription as a whole. The ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE48164).

  4. Genome-wide mapping of the distribution of CarD, RNAP σA, and RNAP β on the Mycobacterium smegmatis chromosome using chromatin immunoprecipitation sequencing

    PubMed Central

    Landick, Robert; Krek, Azra; Glickman, Michael S.; Socci, Nicholas D.; Stallings, Christina L.

    2014-01-01

    CarD is an essential mycobacterial protein that binds the RNA polymerase (RNAP) and affects the transcriptional profile of Mycobacterium smegmatis and Mycobacterium tuberculosis [6]. We predicted that CarD was directly regulating RNAP function but our prior experiments had not determined at what stage of transcription CarD was functioning and at which genes CarD interacted with the RNAP. To begin to address these open questions, we performed chromatin immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. The distribution of RNAP subunits β and σA were also profiled. We expected that RNAP β would be present throughout transcribed regions and RNAP σA would be predominantly enriched at promoters based on work in Escherichia coli [3], however this had yet to be determined in mycobacteria. The ChIP-seq analyses revealed that CarD was never present on the genome in the absence of RNAP, was primarily associated with promoter regions, and was highly correlated with the distribution of RNAP σA. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. Here we describe in detail the data from the ChIP-seq experiments associated with the study published by Srivastava and colleagues in the Proceedings of the National Academy of Science in 2013 [5] as well as discuss the findings from this dataset in relation to both CarD and mycobacterial transcription as a whole. The ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE48164). PMID:25089258

  5. Chromatin immunoprecipitation and multiplex sequencing (ChIP-Seq) to identify global transcription factor binding sites in the nematode Caenorhabditis elegans.

    PubMed

    Brdlik, Cathleen M; Niu, Wei; Snyder, Michael

    2014-01-01

    The global identification of transcription factor (TF) binding sites is a critical step in the elucidation of the functional elements of the genome. Several methods have been developed that map TF binding in human cells, yeast, and other model organisms. These methods make use of chromatin immunoprecipitation, or ChIP, and take advantage of the fact that formaldehyde fixation of living cells can be used to cross-link DNA sequences to the TFs that bind them in vivo. In ChIP, the cross-linked TF-DNA complexes are sheared by sonication, size fractionated, and incubated with antibody specific to the TF of interest to generate a library of TF-bound DNA sequences. ChIP-chip was the first technology developed to globally identify TF-bound DNA sequences and involves subsequent hybridization of the ChIP DNA to oligonucleotide microarrays. However, ChIP-chip proved to be costly, labor-intensive, and limited by the fixed number of probes available on the microarray chip. ChIP-Seq combines ChIP with massively parallel high-throughput sequencing (see Explanatory Chapter: Next Generation Sequencing) and has demonstrated vast improvement over ChIP-chip with respect to time and cost, signal-to-noise ratio, and resolution. In particular, multiplex sequencing can be used to achieve a higher throughput in ChIP-Seq analyses involving organisms with genomes of lower complexity than that of human (Lefrançois et al., 2009) and thereby reduce the cost and amount of time needed for each result. The multiplex ChIP-Seq method described in this section has been developed for Caenorhabditis elegans, but is easily adaptable for other organisms.

  6. Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis of the Penicillium chrysogenum Velvet Protein PcVelA Identifies Methyltransferase PcLlmA as a Novel Downstream Regulator of Fungal Development

    PubMed Central

    Becker, Kordula; Ziemons, Sandra; Lentz, Katharina; Freitag, Michael

    2016-01-01

    ABSTRACT Penicillium chrysogenum is the sole industrial producer of the β-lactam antibiotic penicillin, which is the most commonly used drug for treating bacterial infections. In P. chrysogenum and other filamentous fungi, secondary metabolism and morphogenesis are controlled by the highly conserved multisubunit velvet complex. Here we present the first chromatin immunoprecipitation next-generation sequencing (ChIP-seq) analysis of a fungal velvet protein, providing experimental evidence that a velvet homologue in P. chrysogenum (PcVelA) acts as a direct transcriptional regulator at the DNA level in addition to functioning as a regulator at the protein level in P. chrysogenum, which was previously described. We identified many target genes that are related to processes known to be dependent on PcVelA, e.g., secondary metabolism as well as asexual and sexual development. We also identified seven PcVelA target genes that encode putative methyltransferases. Yeast two-hybrid and bimolecular fluorescence complementation analyses showed that one of the putative methyltransferases, PcLlmA, directly interacts with PcVelA. Furthermore, functional characterization of PcLlmA demonstrated that this protein is involved in the regulation of conidiosporogenesis, pellet formation, and hyphal morphology, all traits with major biotechnological relevance. IMPORTANCE Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P. chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at

  7. Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis of the Penicillium chrysogenum Velvet Protein PcVelA Identifies Methyltransferase PcLlmA as a Novel Downstream Regulator of Fungal Development.

    PubMed

    Becker, Kordula; Ziemons, Sandra; Lentz, Katharina; Freitag, Michael; Kück, Ulrich

    2016-01-01

    Penicillium chrysogenum is the sole industrial producer of the β-lactam antibiotic penicillin, which is the most commonly used drug for treating bacterial infections. In P. chrysogenum and other filamentous fungi, secondary metabolism and morphogenesis are controlled by the highly conserved multisubunit velvet complex. Here we present the first chromatin immunoprecipitation next-generation sequencing (ChIP-seq) analysis of a fungal velvet protein, providing experimental evidence that a velvet homologue in P. chrysogenum (PcVelA) acts as a direct transcriptional regulator at the DNA level in addition to functioning as a regulator at the protein level in P. chrysogenum, which was previously described. We identified many target genes that are related to processes known to be dependent on PcVelA, e.g., secondary metabolism as well as asexual and sexual development. We also identified seven PcVelA target genes that encode putative methyltransferases. Yeast two-hybrid and bimolecular fluorescence complementation analyses showed that one of the putative methyltransferases, PcLlmA, directly interacts with PcVelA. Furthermore, functional characterization of PcLlmA demonstrated that this protein is involved in the regulation of conidiosporogenesis, pellet formation, and hyphal morphology, all traits with major biotechnological relevance. IMPORTANCE Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P. chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at the

  8. Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis of the Penicillium chrysogenum Velvet Protein PcVelA Identifies Methyltransferase PcLlmA as a Novel Downstream Regulator of Fungal Development.

    PubMed

    Becker, Kordula; Ziemons, Sandra; Lentz, Katharina; Freitag, Michael; Kück, Ulrich

    2016-01-01

    Penicillium chrysogenum is the sole industrial producer of the β-lactam antibiotic penicillin, which is the most commonly used drug for treating bacterial infections. In P. chrysogenum and other filamentous fungi, secondary metabolism and morphogenesis are controlled by the highly conserved multisubunit velvet complex. Here we present the first chromatin immunoprecipitation next-generation sequencing (ChIP-seq) analysis of a fungal velvet protein, providing experimental evidence that a velvet homologue in P. chrysogenum (PcVelA) acts as a direct transcriptional regulator at the DNA level in addition to functioning as a regulator at the protein level in P. chrysogenum, which was previously described. We identified many target genes that are related to processes known to be dependent on PcVelA, e.g., secondary metabolism as well as asexual and sexual development. We also identified seven PcVelA target genes that encode putative methyltransferases. Yeast two-hybrid and bimolecular fluorescence complementation analyses showed that one of the putative methyltransferases, PcLlmA, directly interacts with PcVelA. Furthermore, functional characterization of PcLlmA demonstrated that this protein is involved in the regulation of conidiosporogenesis, pellet formation, and hyphal morphology, all traits with major biotechnological relevance. IMPORTANCE Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P. chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at the

  9. RIP: RNA Immunoprecipitation.

    PubMed

    Gagliardi, Miriam; Matarazzo, Maria R

    2016-01-01

    The relevance of RNA-protein interactions in modulating mRNA and noncoding RNA function is increasingly appreciated and several methods have been recently developed to map them. The RNA immunoprecipitation (RIP) is a powerful method to study the physical association between individual proteins and RNA molecules in vivo. The basic principles of RIP are very similar to those of chromatin immunoprecipitation (ChIP), a largely used tool in the epigenetic field, but with some important caveats. The approach is based on the use of a specific antibody raised against the protein of interest to pull down the RNA-binding protein (RBP) and target-RNA complexes. Any RNA that is associated with this protein complex will also be isolated and can be further analyzed by polymerase chain reaction-based methods, hybridization, or sequencing.Several variants of this technique exist and can be divided into two main classes: native and cross-linked RNA immunoprecipitation. The native RIP allows to reveal the identity of RNAs directly bound by the protein and their abundance in the immunoprecipitated sample, while cross-linked RIP leads to precisely map the direct and indirect binding site of the RBP of interest to the RNA molecule.In this chapter both the protocols applied to mammalian cells are described taking into account the caveats and considerations required for designing, performing, and interpreting the results of these experiments. PMID:27659976

  10. Chromatin immunoprecipitation analysis of the tobacco PR-1a- and the truncated CaMV 35S promoter reveals differences in salicylic acid-dependent TGA factor binding and histone acetylation.

    PubMed

    Butterbrodt, Thomas; Thurow, Corinna; Gatz, Christiane

    2006-07-01

    Salicylic acid (SA) is a plant signalling molecule needed for the induction of defence responses upon attack by a variety of pathogens. Truncation of the Cauliflower Mosaic Virus (CaMV) 35S promoter down to 90 bp has identified activation sequence-1 (as-1) as an autonomous SA-responsive cis element. The as-1-like elements are found in a number of SA-inducible promoters like e.g. the tobacco PR-1a promoter. They are recognized by basic/leucine zipper (bZIP) transcription factors of the TGA family. In tobacco leaves, TGA2.2 is the most abundant TGA factor. TGA2.2 is required for the expression of as-1-containing promoters. Here we unravel clear differences between the "truncated" CaMV 35S and the PR-1a promoter with respect to in vivo TGA binding and histone acetylation. Chromatin immunoprecipitation (ChIP) analysis revealed SA-inducible recruitment of tobacco TGA2.2 as well as SA-inducible histone acetylation at the PR-1a promoter. In contrast, no influence of SA on TGA2.2 binding and histone acetylation was detectable at the "truncated" CaMV 35S promoter. The finding of SA-independent TGA factor binding in the absence of additional flanking regulatory sequences suggests that transcriptional activation is not necessarily mediated by inducible DNA binding of TGA factors. Plants with severely reduced TGA2.2 protein levels also showed SA-induced histone acetylation at the PR-1a promoter indicating that regulatory events independent from TGA2.2 function are initiated at the PR-1a promoter.

  11. Isolation of Specific Genomic Regions and Identification of Their Associated Molecules by Engineered DNA-Binding Molecule-Mediated Chromatin Immunoprecipitation (enChIP) Using the CRISPR System and TAL Proteins

    PubMed Central

    Fujii, Hodaka; Fujita, Toshitsugu

    2015-01-01

    Comprehensive understanding of genome functions requires identification of molecules (proteins, RNAs, genomic regions, etc.) bound to specific genomic regions of interest in vivo. To perform biochemical and molecular biological analysis of specific genomic regions, we developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) to purify genomic regions of interest. In enChIP, specific genomic regions are tagged for biochemical purification using engineered DNA-binding molecules, such as transcription activator-like (TAL) proteins and a catalytically inactive form of the clustered regularly interspaced short palindromic repeats (CRISPR) system. enChIP is a comprehensive approach that emphasizes non-biased search using next-generation sequencing (NGS), microarrays, mass spectrometry (MS), and other methods. Moreover, this approach is not restricted to cultured cell lines and can be easily extended to organisms. In this review, we discuss applications of enChIP to elucidating the molecular mechanisms underlying genome functions. PMID:26370991

  12. Isolation of Specific Genomic Regions and Identification of Their Associated Molecules by Engineered DNA-Binding Molecule-Mediated Chromatin Immunoprecipitation (enChIP) Using the CRISPR System and TAL Proteins.

    PubMed

    Fujii, Hodaka; Fujita, Toshitsugu

    2015-09-09

    Comprehensive understanding of genome functions requires identification of molecules (proteins, RNAs, genomic regions, etc.) bound to specific genomic regions of interest in vivo. To perform biochemical and molecular biological analysis of specific genomic regions, we developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) to purify genomic regions of interest. In enChIP, specific genomic regions are tagged for biochemical purification using engineered DNA-binding molecules, such as transcription activator-like (TAL) proteins and a catalytically inactive form of the clustered regularly interspaced short palindromic repeats (CRISPR) system. enChIP is a comprehensive approach that emphasizes non-biased search using next-generation sequencing (NGS), microarrays, mass spectrometry (MS), and other methods. Moreover, this approach is not restricted to cultured cell lines and can be easily extended to organisms. In this review, we discuss applications of enChIP to elucidating the molecular mechanisms underlying genome functions.

  13. Identification of Pou5f1, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algorithm to time course microarray and genome-wide chromatin immunoprecipitation data

    PubMed Central

    Sharov, Alexei A; Masui, Shinji; Sharova, Lioudmila V; Piao, Yulan; Aiba, Kazuhiro; Matoba, Ryo; Xin, Li; Niwa, Hitoshi; Ko, Minoru SH

    2008-01-01

    Background Target genes of a transcription factor (TF) Pou5f1 (Oct3/4 or Oct4), which is essential for pluripotency maintenance and self-renewal of embryonic stem (ES) cells, have previously been identified based on their response to Pou5f1 manipulation and occurrence of Chromatin-immunoprecipitation (ChIP)-binding sites in promoters. However, many responding genes with binding sites may not be direct targets because response may be mediated by other genes and ChIP-binding site may not be functional in terms of transcription regulation. Results To reduce the number of false positives, we propose to separate responding genes into groups according to direction, magnitude, and time of response, and to apply the false discovery rate (FDR) criterion to each group individually. Using this novel algorithm with stringent statistical criteria (FDR < 0.2) to a compendium of published and new microarray data (3, 6, 12, and 24 hr after Pou5f1 suppression) and published ChIP data, we identified 420 tentative target genes (TTGs) for Pou5f1. The majority of TTGs (372) were down-regulated after Pou5f1 suppression, indicating that the Pou5f1 functions as an activator of gene expression when it binds to promoters. Interestingly, many activated genes are potent suppressors of transcription, which include polycomb genes, zinc finger TFs, chromatin remodeling factors, and suppressors of signaling. Similar analysis showed that Sox2 and Nanog also function mostly as transcription activators in cooperation with Pou5f1. Conclusion We have identified the most reliable sets of direct target genes for key pluripotency genes – Pou5f1, Sox2, and Nanog, and found that they predominantly function as activators of downstream gene expression. Thus, most genes related to cell differentiation are suppressed indirectly. PMID:18522731

  14. Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division in Streptomyces

    PubMed Central

    Chandra, Govind; Bibb, Maureen J.; Findlay, Kim C.; Buttner, Mark J.

    2016-01-01

    ABSTRACT WhiB is the founding member of a family of proteins (the WhiB-like [Wbl] family) that carry a [4Fe-4S] iron-sulfur cluster and play key roles in diverse aspects of the biology of actinomycetes, including pathogenesis, antibiotic resistance, and the control of development. In Streptomyces, WhiB is essential for the process of developmentally controlled cell division that leads to sporulation. The biochemical function of Wbl proteins has been controversial; here, we set out to determine unambiguously if WhiB functions as a transcription factor using chromatin immunoprecipitation sequencing (ChIP-seq) in Streptomyces venezuelae. In the first demonstration of in vivo genome-wide Wbl binding, we showed that WhiB regulates the expression of key genes required for sporulation by binding upstream of ~240 transcription units. Strikingly, the WhiB regulon is identical to the previously characterized WhiA regulon, providing an explanation for the identical phenotypes of whiA and whiB mutants. Using ChIP-seq, we demonstrated that in vivo DNA binding by WhiA depends on WhiB and vice versa, showing that WhiA and WhiB function cooperatively to control expression of a common set of WhiAB target genes. Finally, we show that mutation of the cysteine residues that coordinate the [4Fe-4S] cluster in WhiB prevents DNA binding by both WhiB and WhiA in vivo. PMID:27094333

  15. Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1: application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes.

    PubMed

    Iype, Tessy; Francis, Joshua; Garmey, James C; Schisler, Jonathan C; Nesher, Rafael; Weir, Gordon C; Becker, Thomas C; Newgard, Christopher B; Griffen, Steven C; Mirmira, Raghavendra G

    2005-04-29

    The homeodomain factor Pdx-1 regulates an array of genes in the developing and mature pancreas, but whether regulation of each specific gene occurs by a direct mechanism (binding to promoter elements and activating basal transcriptional machinery) or an indirect mechanism (via regulation of other genes) is unknown. To determine the mechanism underlying regulation of the insulin gene by Pdx-1, we performed a kinetic analysis of insulin transcription following adenovirus-mediated delivery of a small interfering RNA specific for pdx-1 into insulinoma cells and pancreatic islets to diminish endogenous Pdx-1 protein. insulin transcription was assessed by measuring both a long half-life insulin mRNA (mature mRNA) and a short half-life insulin pre-mRNA species by real-time reverse transcriptase-PCR. Following progressive knock-down of Pdx-1 levels, we observed coordinate decreases in pre-mRNA levels (to about 40% of normal levels at 72 h). In contrast, mature mRNA levels showed strikingly smaller and delayed declines, suggesting that the longer half-life of this species underestimates the contribution of Pdx-1 to insulin transcription. Chromatin immunoprecipitation assays revealed that the decrease in insulin transcription was associated with decreases in the occupancies of Pdx-1 and p300 at the proximal insulin promoter. Although there was no corresponding change in the recruitment of RNA polymerase II to the proximal promoter, its recruitment to the insulin coding region was significantly reduced. Our results suggest that Pdx-1 directly regulates insulin transcription through formation of a complex with transcriptional coactivators on the proximal insulin promoter. This complex leads to enhancement of elongation by the basal transcriptional machinery.

  16. Isolation of In Vivo SUMOylated Chromatin-Bound Proteins.

    PubMed

    Bawa-Khalfe, Tasneem

    2016-01-01

    SUMO posttranslational modification directs gene transcription and epigenetic programming to support normal cell function. The dynamic nature of SUMO-modification makes it difficult to identify endogenous protein substrates. Isolation of chromatin-bound SUMO targets is exceptionally challenging, as conventional immunoprecipitation assays are inefficient at concentrating this protein population. This chapter describes a protocol that effectively precipitates chromatin-associated fractions of SUMOylated heterochromatin protein 1α in cultured cells. Techniques to enrich endogenous SUMO substrates at the chromatin are also demonstrated and discussed. This approach could be adapted to evaluate chromatin-bound SUMO targets in additional in vivo systems. PMID:27631808

  17. Machupo virus polypeptides: identification by immunoprecipitation.

    PubMed

    Lukashevich, I S; Lemeshko, N N

    1985-01-01

    The most abundant protein in purified Machupo virions (Corvallo strain) labelled with 14C-Protein hydrolysate is a 64 K polypeptide which is associated with virion RNAs. Another structural polypeptide, 37 K, solubilized by nonionic detergent seems to be a major surface glycoprotein. In addition to this, a 78 K polypeptide and a minor 50 K polypeptide have been detected. In Machupo virus infected cells three virus-specific polypeptides similar in size to those described for structural polypeptides were immunoprecipitated with anti-Machupo virus serum. The most abundant virus-specific polypeptide was nonglycosylated (64 K, NP), and the others were glycosylated polypeptides (78 K and 37 K). The synthesis of NP and 78 K polypeptides was recognized at the beginning of a log phase of virus replication. Pulse-chase experiments as well as experiments with an arginine analogue, canavanine (to block proteolytic processing) suggest that 78 K is a precursor for structural glycoproteins of Machupo virions.

  18. Chromatin Computation

    PubMed Central

    Bryant, Barbara

    2012-01-01

    In living cells, DNA is packaged along with protein and RNA into chromatin. Chemical modifications to nucleotides and histone proteins are added, removed and recognized by multi-functional molecular complexes. Here I define a new computational model, in which chromatin modifications are information units that can be written onto a one-dimensional string of nucleosomes, analogous to the symbols written onto cells of a Turing machine tape, and chromatin-modifying complexes are modeled as read-write rules that operate on a finite set of adjacent nucleosomes. I illustrate the use of this “chromatin computer” to solve an instance of the Hamiltonian path problem. I prove that chromatin computers are computationally universal – and therefore more powerful than the logic circuits often used to model transcription factor control of gene expression. Features of biological chromatin provide a rich instruction set for efficient computation of nontrivial algorithms in biological time scales. Modeling chromatin as a computer shifts how we think about chromatin function, suggests new approaches to medical intervention, and lays the groundwork for the engineering of a new class of biological computing machines. PMID:22567109

  19. Isolation of a thyroid hormone-responsive gene by immunoprecipitation of thyroid hormone receptor-DNA complexes.

    PubMed Central

    Bigler, J; Eisenman, R N

    1994-01-01

    Thyroid hormone (T3) receptor (TR) is a ligand-dependent transcription factor that acts through specific binding sites in the promoter region of target genes. In order to identify new genes that are regulated by T3, we used anti-TR antiserum to immunoprecipitate TR-DNA complexes from GH4 cell nuclei that had previously been treated with a restriction enzyme. Screening of the immunopurified, cloned DNA for TR binding sites by electrophoretic mobility shift assay yielded 53 positive clones. A subset of these clones was specifically immunoprecipitated with anti-TR antiserum and may therefore represent biologically significant binding sites. One of these clones, clone 122, was characterized in detail. It includes sequences highly related to the NICER long terminal repeat-like element and contains three TR binding sites as determined by DNase I footprinting. Two of the clone 122 TR binding sites are located upstream of the TATA box, and one is located downstream. The TR binding site downstream from the promoter was necessary and sufficient to confer T3-dependent regulation in transient transfection experiments. Expression of a reporter construct under the control of the clone 122 promoter region was activated by TR in the absence of ligand and returned to basal levels after T3 addition. Clone 122 sequences hybridize to at least two different mRNAs of approximately 6 and 10 kb from GH4 cells. The levels of both of these mRNAs increased upon removal of T3. Our studies suggest that specific immunoprecipitation of chromatin allows identification of binding sites and target genes for transcription factors. Images PMID:7935476

  20. A Microfluidic Device with Integrated Sonication and Immunoprecipitation for Sensitive Epigenetic Assays.

    PubMed

    Cao, Zhenning; Lu, Chang

    2016-02-01

    Epigenetic studies increasingly require analysis of a small number of cells that are of one specific type and derived from patients or animals. In this report, we demonstrate a simple microfluidic device that integrates sonication and immunoprecipitation (IP) for epigenetic assays, such as chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP). By incorporating an ultrasonic transducer with a microfluidic chamber, we implemented microscale sonication for both shearing chromatin/DNA and mixing/washing of IP beads. Such integration allowed highly sensitive tests starting with 100 cross-linked cells for ChIP or 500 pg of genomic DNA for MeDIP (compared to 10(6)-10(7) cells for ChIP and 1-10 μg of DNA for MeDIP in conventional assays). The entire on-chip process of sonication and IP took only 1 h. Our tool will be useful for highly sensitive epigenetic studies based on a small quantity of sample.

  1. Targeting of cohesin by transcriptionally silent chromatin.

    PubMed

    Chang, Chuang-Rung; Wu, Ching-Shyi; Hom, Yolanda; Gartenberg, Marc R

    2005-12-15

    Eukaryotic DNA replication produces sister chromatids that are linked together until anaphase by cohesin, a ring-shaped protein complex that is thought to act by embracing both chromatids. Cohesin is enriched at centromeres, as well as discrete sites along chromosome arms where transcription positions the complex between convergent gene pairs. A relationship between cohesin and Sir-mediated transcriptional silencing has also begun to emerge. Here we used fluorescence microscopy and site-specific recombination to characterize interactions between newly replicated copies of the silent HMR mating-type locus. HMR was tagged with lac-GFP and flanked by binding sites for an inducible site-specific recombinase. Excision of the locus in cells with sister chromatids produced two chromatin circles that remained associated with one another. Pairing of the circles required silent chromatin, cohesin, and the RSC chromatin-remodeling complex. Chromatin immunoprecipitation showed that targeting of cohesin to the locus is Sir-dependent, and functional tests showed that silent chromatin acts in a continuous fashion to maintain cohesion. Remarkably, loss of silencing led to loss of cohesin from linear chromosomal templates but not from excised chromatin circles. The results are consistent with a model in which cohesin binds silent chromatin via topological linkage to individual chromatids. PMID:16319193

  2. Protein composition of immunoprecipitated synaptic ribbons

    PubMed Central

    Kantardzhieva, A.; Peppi, M.; Lane, W.S.; Sewell, W.F.

    2012-01-01

    The synaptic ribbon is an electron-dense structure found in hair cells and photoreceptors. The ribbon is surrounded by neurotransmitter-filled vesicles and considered to play a role in vesicle release. We generated an objective, quantitative analysis of the protein composition of the ribbon complex using a mass spectrometry-based proteomics analysis. Our use of affinity-purified ribbons and control IgG immunoprecipitations ensure that the identified proteins are indeed associated with the ribbon complex. The use of mouse tissue, where the proteome is complete, generated a comprehensive analysis of the candidates. We identified 30 proteins (comprising 56 isoforms and subunits) associated with the ribbon complex. The ribbon complex primarily comprises proteins found in conventional synapses, which we categorized into 6 functional groups: vesicle handling (38.5%), scaffold (7.3%), cytoskeletal molecules (20.6%), phosphorylation enzymes (10.6%), molecular chaperones (8.2%), and transmembrane proteins from the presynaptic membrane firmly attached to the ribbon (11.3%). The 3 CtBP isoforms represent the major protein in the ribbon whether calculated by molar amount (30%) or by mass (20%). The relatively high quantity of phosphorylation enzymes suggests a very active and regulated structure. The ribbon appears to comprise a concentrated cluster of proteins dealing with vesicle creation, retention and distribution, and consequent exocytosis. PMID:22103298

  3. Rapid induction of chromatin-associated DNA mismatch repair proteins after MNNG treatment

    PubMed Central

    Schroering, Allen G.; Williams, Kandace J.

    2008-01-01

    Treatment with low concentrations of monofunctional alkylating agents induces a G2 arrest only after the second round of DNA synthesis in mammalian cells and requires a proficient mismatch repair (MMR) pathway. Here we have investigated rapid alkylation-induced recruitment of DNA repair proteins to chromosomal DNA within synchronized populations of MMR proficient cells (HeLa MR) after MNNG treatment. Within the first hour, the concentrations of MutSα and PCNA increase well beyond their constitutive chromosomally bound levels and MutLα is newly recruited to the chromatin-bound MutSα. Remarkably, immunoprecipitation experiments demonstrate rapid association of these proteins on the alkylation-damaged chromatin, even when DNA replication is completely blocked. The extent of association of PCNA and MMR proteins on the chromatin is dependent upon the concentration of MNNG and on the specific type of replication block. A subpopulation of the MutSα-associated PCNA also becomes monoubiquitinated, a known requirement for PCNA to interact with translesion synthesis (TLS) polymerases. In addition, chromatin-bound SMC1 and NBS1 proteins, associated with DNA double-strand-breaks (DSBs), become phosphorylated within one to two hours of exposure to MNNG. However, these activated proteins are not colocalized on the chromatin with MutSα in response to MNNG exposure. PCNA, MutSα/MutLα and activated SMC1/NBS1 remain chromatin-bound for at least 6–8 hours after alkylation damage. Thus, cells that are exposed to low levels of alkylation treatment undergo rapid recruitment to and/or activation of key proteins already on the chromatin without the requirement for DNA replication, apparently via different DNA-damage signaling pathways. PMID:18468964

  4. Immunoprecipitation of the parathyroid hormone receptor

    SciTech Connect

    Wright, B.S.; Tyler, G.A.; O'Brien, R.; Caporale, L.H.; Rosenblatt, M.

    1987-01-01

    An /sup 125/I-labeled synthetic analog of bovine parathyroid hormone, (8-norleucine,18-norleucine,34-tyrosine)PTH-(1-34) amide ((Nle)PTH-(1-34)-NH/sub 2/), purified by high-pressure liquid chromatography (HPLC), was employed to label the parathyroid hormone (PTH) receptor in cell lines derived from PTH target tissues: the ROS 17/2.8 rat osteosarcoma of bone and the CV1 and COS monkey kidney lines. After incubation of the radioligand with intact cultured cells, the hormone was covalently attached to receptors by using either a photoaffinity technique or chemical (affinity) crosslinking. In each case, covalent labeling was specific, as evidenced by a reduction of labeling when excess competing nonradioactive ligand was present. After covalent attachment of radioligand, membranes were prepared form the cells and solubilized in the nonionic detergent Nonidet P-40 or octyl glucoside. Analysis of the immunoprecipitate on NaDod-SO/sub 4//polyacrylamide gel electrophoresis followed by autoradiography revealed the presence of a doublet of apparent molecular mass 69-70 kDa. Specifically labeled bands of approximate molecular mass 95 and 28 kDa were also observed. The anti-PTH IgG was affinity purified by passage over a PTH-Sepharose column and used to made an immunoaffinity column. These studies suggest that the use of an anti-PTH antiserum that binds receptor-bound hormone is likely to be a useful step in the further physicochemical characterization and purification of the PTH receptor.

  5. Chromatin Dynamics during Cellular Reprogramming

    PubMed Central

    Apostolou, Effie; Hochedlinger, Konrad

    2014-01-01

    Preface Induced pluripotency is a powerful tool to derive patient-specific stem cells. In addition, it provides a unique assay to study the interplay between transcription factors and chromatin structure. Here, we review the latest insights into chromatin dynamics inherent to induced pluripotency. Moreover, we compare and contrast these events with other physiological and pathological processes involving changes in chromatin and cell state, including germ cell maturation and tumorigenesis. We propose that an integrated view of these seemingly diverse processes could provide mechanistic insights into cell fate transitions in general and might lead to novel approaches in regenerative medicine and cancer treatment. PMID:24153299

  6. Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

    ERIC Educational Resources Information Center

    Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

    2005-01-01

    In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…

  7. Histone Acetylation and Chromatin Remodeling Are Required for UV-B–Dependent Transcriptional Activation of Regulated Genes in Maize[W

    PubMed Central

    Casati, Paula; Campi, Mabel; Chu, Feixia; Suzuki, Nagi; Maltby, David; Guan, Shenheng; Burlingame, Alma L.; Walbot, Virginia

    2008-01-01

    The nuclear proteomes of maize (Zea mays) lines that differ in UV-B tolerance were compared by two-dimensional gel electrophoresis after UV light treatment. Differential accumulation of chromatin proteins, particularly histones, constituted the largest class identified by mass spectrometry. UV-B–tolerant landraces and the B73 inbred line show twice as many protein changes as the UV-B–sensitive b, pl W23 inbred line and transgenic maize expressing RNA interference constructs directed against chromatin factors. Mass spectrometic analysis of posttranslational modifications on histone proteins demonstrates that UV-B–tolerant lines exhibit greater acetylation on N-terminal tails of histones H3 and H4 after irradiation. These acetylated histones are enriched in the promoter and transcribed regions of the two UV-B–upregulated genes examined; radiation-sensitive lines lack this enrichment. DNase I and micrococcal nuclease hypersensitivity assays indicate that chromatin adopts looser structures around the selected genes in the UV-B–tolerant samples. Chromatin immunoprecipitation experiments identified additional chromatin factor changes associated with the nfc102 test gene after UV-B treatment in radiation-tolerant lines. Chromatin remodeling is thus shown to be a key process in acclimation to UV-B, and lines deficient in this process are more sensitive to UV-B. PMID:18398050

  8. Immunoprecipitation of Plasma Membrane Receptor-Like Kinases for Identification of Phosphorylation Sites and Associated Proteins.

    PubMed

    Kadota, Yasuhiro; Macho, Alberto P; Zipfel, Cyril

    2016-01-01

    Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilization, purification, identification of associated proteins, and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins, and to identify post-translational modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases. PMID:26577786

  9. The Chromatin and Transcriptional Landscape of Native Saccharomyces cerevisiae Telomeres and Subtelomeric Domains

    PubMed Central

    Ellahi, Aisha; Thurtle, Deborah M.; Rine, Jasper

    2015-01-01

    Saccharomyces cerevisiae telomeres have been a paradigm for studying telomere position effects on gene expression. Telomere position effect was first described in yeast by its effect on the expression of reporter genes inserted adjacent to truncated telomeres. The reporter genes showed variable silencing that depended on the Sir2/3/4 complex. Later studies examining subtelomeric reporter genes inserted at natural telomeres hinted that telomere position effects were less pervasive than previously thought. Additionally, more recent data using the sensitive technology of chromatin immunoprecipitation and massively parallel sequencing (ChIP-Seq) revealed a discrete and noncontinuous pattern of coenrichment for all three Sir proteins at a few telomeres, calling the generality of these conclusions into question. Here we combined the ChIP-Seq of the Sir proteins with RNA sequencing (RNA-Seq) of messenger RNAs (mRNAs) in wild-type and in SIR2, SIR3, and SIR4 deletion mutants to characterize the chromatin and transcriptional landscape of all native S. cerevisiae telomeres at the highest achievable resolution. Most S. cerevisiae chromosomes had subtelomeric genes that were expressed, with only ∼6% of subtelomeric genes silenced in a SIR-dependent manner. In addition, we uncovered 29 genes with previously unknown cell-type-specific patterns of expression. These detailed data provided a comprehensive assessment of the chromatin and transcriptional landscape of the subtelomeric domains of a eukaryotic genome. PMID:25823445

  10. The Chromatin and Transcriptional Landscape of Native Saccharomyces cerevisiae Telomeres and Subtelomeric Domains.

    PubMed

    Ellahi, Aisha; Thurtle, Deborah M; Rine, Jasper

    2015-06-01

    Saccharomyces cerevisiae telomeres have been a paradigm for studying telomere position effects on gene expression. Telomere position effect was first described in yeast by its effect on the expression of reporter genes inserted adjacent to truncated telomeres. The reporter genes showed variable silencing that depended on the Sir2/3/4 complex. Later studies examining subtelomeric reporter genes inserted at natural telomeres hinted that telomere position effects were less pervasive than previously thought. Additionally, more recent data using the sensitive technology of chromatin immunoprecipitation and massively parallel sequencing (ChIP-Seq) revealed a discrete and noncontinuous pattern of coenrichment for all three Sir proteins at a few telomeres, calling the generality of these conclusions into question. Here we combined the ChIP-Seq of the Sir proteins with RNA sequencing (RNA-Seq) of messenger RNAs (mRNAs) in wild-type and in SIR2, SIR3, and SIR4 deletion mutants to characterize the chromatin and transcriptional landscape of all native S. cerevisiae telomeres at the highest achievable resolution. Most S. cerevisiae chromosomes had subtelomeric genes that were expressed, with only ∼6% of subtelomeric genes silenced in a SIR-dependent manner. In addition, we uncovered 29 genes with previously unknown cell-type-specific patterns of expression. These detailed data provided a comprehensive assessment of the chromatin and transcriptional landscape of the subtelomeric domains of a eukaryotic genome.

  11. An Optimized Protocol for Isolating Primary Epithelial Cell Chromatin for ChIP

    PubMed Central

    Browne, James A.; Harris, Ann; Leir, Shih-Hsing

    2014-01-01

    A critical part of generating robust chromatin immunoprecipitation (ChIP) data is the optimization of chromatin purification and size selection. This is particularly important when ChIP is combined with next-generation sequencing (ChIP-seq) to identify targets of DNA-binding proteins, genome-wide. Current protocols refined by the ENCODE consortium generally use a two-step cell lysis procedure that is applicable to a wide variety of cell types. However, the isolation and size selection of chromatin from primary human epithelial cells may often be particularly challenging. These cells tend to form sheets of formaldehyde cross-linked material in which cells are resistant to membrane lysis, nuclei are not released and subsequent sonication produces extensive high molecular weight contamination. Here we describe an optimized protocol to prepare high quality ChIP-grade chromatin from primary human bronchial epithelial cells. The ENCODE protocol was used as a starting point to which we added the following key steps to separate the sheets of formaldehyde-fixed cells prior to lysis. (1) Incubation of the formaldehyde-fixed adherent cells in Trypsin-EDTA (0.25% room temperature) for no longer than 5 min. (2) Equilibration of the fixed cells in detergent-free lysis buffers prior to each lysis step. (3) The addition of 0.5% Triton X-100 to the complete cell membrane lysis buffer. (4) Passing the cell suspension (in complete cell membrane lysis buffer) through a 25-gauge needle followed by continuous agitation on ice for 35 min. Each step of the modified protocol was documented by light microscopy using the Methyl Green-Pyronin dual dye, which stains cytoplasm red (Pyronin) and the nuclei grey-blue (Methyl green). This modified method is reproducibly effective at producing high quality sheared chromatin for ChIP and is equally applicable to other epithelial cell types. PMID:24971909

  12. AIRE activated tissue specific genes have histone modifications associated with inactive chromatin.

    PubMed

    Org, Tõnis; Rebane, Ana; Kisand, Kai; Laan, Martti; Haljasorg, Uku; Andreson, Reidar; Peterson, Pärt

    2009-12-15

    The Autoimmune Regulator (AIRE) protein is expressed in thymic medullary epithelial cells, where it promotes the ectopic expression of tissue-restricted antigens needed for efficient negative selection of developing thymocytes. Mutations in AIRE cause APECED syndrome, which is characterized by a breakdown of self-tolerance. The molecular mechanism by which AIRE increases the expression of a variety of different genes remains unknown. Here, we studied AIRE-regulated genes using whole genome expression analysis and chromatin immunoprecipitation. We show that AIRE preferentially activates genes that are tissue-specific and characterized by low levels of initial expression in stably transfected HEK293 cell model and mouse thymic medullary epithelial cells. In addition, the AIRE-regulated genes lack active chromatin marks, such as histone H3 trimethylation (H3K4me3) and acetylation (AcH3), on their promoters. We also show that during activation by AIRE, the target genes acquire histone H3 modifications associated with transcription and RNA polymerase II. In conclusion, our data show that AIRE is able to promote ectopic gene expression from chromatin associated with histone modifications characteristic to inactive genes.

  13. Identification of Associated Proteins by Immunoprecipitation and Mass Spectrometry Analysis.

    PubMed

    Cao, Xiumei; Yan, Jianshe

    2016-01-01

    Protein-protein interactions play central roles in intercellular and intracellular signal transduction. Impairment of protein-protein interactions causes many diseases such as cancer, cardiomyopathies, diabetes, microbial infections, and genetic and neurodegenerative disorders. Immunoprecipitation is a technique in which a target protein of interest bound by an antibody is used to pull down the protein complex out of cell lysates, which can be identified by mass spectrometry. Here, we describe the protocol to immunoprecipitate and identify the components of the protein complexes of ElmoE in Dictyostelium discoideum cells. PMID:27271899

  14. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  15. Interpreting results of coulometry and immunoprecipitation in diagnosing iron disorders.

    PubMed

    Rettmer, R L; Labbé, R F; Huebers, H A; Brown, W P; Josephson, B M; Finch, C A

    1987-07-01

    Concentration of iron in plasma, total iron-binding capacity (TIBC), and transferrin saturation are often determined by standard spectrophotometric methods, but iron concentration may be quantified by immunoprecipitation or, electrochemically, by controlled-potential coulometry. Because these iron assays do not all measure the same form(s) of iron, we studied subjects in various states of iron nutriture: normal adults, iron-deficient patients, thalassemia patients with unsaturated transferrin or oversaturated transferrin, and patients with idiopathic hemochromatosis. The spectrophotometric and coulometric methods detected essentially all non-heme iron in plasma; results correlated well but showed a negative bias toward the coulometric method. Results by an immunoprecipitation procedure, which measures only transferrin-bound iron, correlated well with those obtained coulometrically but were slightly higher than the latter. The characteristics of the various methods for iron must be understood by the clinical laboratory if diagnosis of iron disorders is to be accurate.

  16. Proteomics to study DNA-bound and chromatin-associated gene regulatory complexes

    PubMed Central

    Wierer, Michael; Mann, Matthias

    2016-01-01

    High-resolution mass spectrometry (MS)-based proteomics is a powerful method for the identification of soluble protein complexes and large-scale affinity purification screens can decode entire protein interaction networks. In contrast, protein complexes residing on chromatin have been much more challenging, because they are difficult to purify and often of very low abundance. However, this is changing due to recent methodological and technological advances in proteomics. Proteins interacting with chromatin marks can directly be identified by pulldowns with synthesized histone tails containing posttranslational modifications (PTMs). Similarly, pulldowns with DNA baits harbouring single nucleotide polymorphisms or DNA modifications reveal the impact of those DNA alterations on the recruitment of transcription factors. Accurate quantitation – either isotope-based or label free – unambiguously pinpoints proteins that are significantly enriched over control pulldowns. In addition, protocols that combine classical chromatin immunoprecipitation (ChIP) methods with mass spectrometry (ChIP-MS) target gene regulatory complexes in their in-vivo context. Similar to classical ChIP, cells are crosslinked with formaldehyde and chromatin sheared by sonication or nuclease digested. ChIP-MS baits can be proteins in tagged or endogenous form, histone PTMs, or lncRNAs. Locus-specific ChIP-MS methods would allow direct purification of a single genomic locus and the proteins associated with it. There, loci can be targeted either by artificial DNA-binding sites and corresponding binding proteins or via proteins with sequence specificity such as TAL or nuclease deficient Cas9 in combination with a specific guide RNA. We predict that advances in MS technology will soon make such approaches generally applicable tools in epigenetics. PMID:27402878

  17. Gearing up chromatin

    PubMed Central

    Mandemaker, Imke K; Vermeulen, Wim; Marteijn, Jurgen A

    2014-01-01

    During transcription, RNA polymerase may encounter DNA lesions, which causes stalling of transcription. To overcome the RNA polymerase blocking lesions, the transcribed strand is repaired by a dedicated repair mechanism, called transcription coupled nucleotide excision repair (TC-NER). After repair is completed, it is essential that transcription restarts. So far, the regulation and exact molecular mechanism of this transcriptional restart upon genotoxic damage has remained elusive. Recently, three different chromatin remodeling factors, HIRA, FACT, and Dot1L, were identified to stimulate transcription restart after DNA damage. These factors either incorporate new histones or establish specific chromatin marks that will gear up the chromatin to subsequently promote transcription recovery. This adds a new layer to the current model of chromatin remodeling necessary for repair and indicates that this specific form of transcription, i.e., the transcriptional restart upon DNA damage, needs specific chromatin remodeling events. PMID:24809693

  18. Genomic in situ hybridization analysis of Thinopyrum chromatin in a wheat-Th. intermedium partial amphiploid and six derived chromosome addition lines

    PubMed

    Chen; Conner; Laroche; Ji; Armstrong; Fedak

    1999-12-01

    The genomic origin of alien chromosomes present in a wheat-Thinopyrum intermedium partial amphiploid TAF46 (2n = 8x = 56) and six derived chromosome addition lines were analyzed by genomic in situ hybridization (GISH) using S genomic DNA from Pseudoroegneria strigosa (2n = 2x = 14, SS) as a probe. The GISH analysis clearly showed that the chromosome complement of the partial amphiploid TAF46 consists of an entire wheat genome plus one synthetic genome consisting of a mixture of six S genome chromosomes and eight J (=E) genome chromosomes derived from Th. intermedium (2n = 6x = 42, JJJ(s)J(s)SS). There were no Js genome chromosomes present in TAF46. The J genome chromosomes present in TAF46 displayed a unique GISH hybridization pattern with the S genomic DNA probe, in which S genome DNA strongly hybridized at the terminal regions and weakly hybridized over the remaining parts of the chromosomes. This provides a diagnostic marker for distinguishing J genome chromosomes from Js or S genome or wheat ABD genome chromosomes. The genomic origin of the alien chromosomes present in the six derived chromosome addition lines were identified by their characteristic GISH hybridization patterns with S genomic DNA probe. GISH analysis showed that addition lines L1, L2, L3, and L5 carried one pair of J genome chromosomes, while addition lines L4 and L7 each carried one pair of S genome chromosomes. GISH patterns detected by the S genome probe on addition line of L1 were identical to those of the J genome chromosomes present in the partial amphiploid TAF46, suggesting that these chromosomes were not structurally altered when they were transferred from TAF46 to addition lines.

  19. MNase titration reveals differences between nucleosome occupancy and chromatin accessibility

    PubMed Central

    Mieczkowski, Jakub; Cook, April; Bowman, Sarah K.; Mueller, Britta; Alver, Burak H.; Kundu, Sharmistha; Deaton, Aimee M.; Urban, Jennifer A.; Larschan, Erica; Park, Peter J.; Kingston, Robert E.; Tolstorukov, Michael Y.

    2016-01-01

    Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation. PMID:27151365

  20. Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries

    PubMed Central

    Nguyen, Uyen T. T.; Bittova, Lenka; Müller, Manuel M.; Fierz, Beat; David, Yael; Houck-Loomis, Brian; Feng, Vanessa; Dann, Geoffrey P.; Muir, Tom W.

    2014-01-01

    Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone posttranslational modification (‘PTM’) signatures remains a daunting task in the epigenetics field. Here, we introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semi-synthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how pre-existing PTMs, alone or synergistically, affect further PTM deposition via crosstalk mechanisms. We anticipate that the high-throughput and -sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin. PMID:24997861

  1. Effect of post-thaw addition of seminal plasma on motility, viability and chromatin integrity of cryopreserved donkey jack (Equus asinus) spermatozoa.

    PubMed

    Sabatini, C; Mari, G; Mislei, B; Love, Cc; Panzani, D; Camillo, F; Rota, A

    2014-12-01

    Pregnancy rates in donkeys after artificial insemination with cryopreserved semen are still low, compared to the horse species. Addition of autologous seminal plasma to frozen-thawed semen appeared to improve pregnancy rates. The aims of this study were to evaluate (1) sperm motility and plasma membrane integrity after thawing (T0) and after one and 2 h (T1 and T2) of post-thaw incubation in either 0% (SP0) or 70% (SP70) autologous seminal plasma and (2) sperm motility, plasma membrane integrity and DNA quality (%COMP-αt) after thawing (T0) and after 2 and 4 h (T2 and T4) of post-thaw incubation in either 0% (SP0), 5% (SP5) or 20% (SP20) homologous seminal plasma. In experiment 1, seminal plasma decreased total and progressive sperm motility and plasma membrane intact spermatozoa immediately after dilution and at all following time points (p < 0.05). In experiment 2, total and progressive motility did not differ between treatments immediately after dilution and between SP0 and SP5 at T2, while they were lower in both SP5 and SP20 than in SP0 at T4. Plasma membrane intact sperm cells did not differ between SP0 and SP5 and were lower in SP20 at all time points. DNA quality was not affected by treatment immediately after dilution and was significantly worse for SP20 after 4 h of incubation (p < 0.05). The post-thaw addition of seminal plasma at the tested concentrations did not improve donkey frozen semen characteristics in vitro over time. PMID:25256158

  2. A noncanonical bromodomain in the AAA ATPase protein Yta7 directs chromosomal positioning and barrier chromatin activity.

    PubMed

    Gradolatto, Angeline; Smart, Sherri K; Byrum, Stephanie; Blair, Lauren P; Rogers, Richard S; Kolar, Elizabeth A; Lavender, Heather; Larson, Signe K; Aitchison, John D; Taverna, Sean D; Tackett, Alan J

    2009-09-01

    Saccharomyces cerevisiae Yta7 is a barrier active protein that modulates transcriptional states at the silent mating locus, HMR. Additionally, Yta7 regulates histone gene transcription and has overlapping functions with known histone chaperones. This study focused on deciphering the functional role of the noncanonical Yta7 bromodomain. By use of genetic and epistasis analyses, the Yta7 bromodomain was shown to be necessary for barrier activity at HMR and to have overlapping functions with histone regulators (Asf1 and Spt16). Canonical bromodomains can bind to acetylated lysines on histones; however, the Yta7 bromodomain showed an association with histones that was independent of posttranslational modification. Further investigation showed that regions of Yta7 other than the bromodomain conferred histone association. Chromatin immunoprecipitation-chip analyses revealed that the Yta7 bromodomain was not solely responsible for histone association but was also necessary for proper chromosomal positioning of Yta7. This work demonstrates that the Yta7 bromodomain engages histones for certain cellular functions like barrier chromatin maintenance and particular Spt16/Asf1 cellular pathways of chromatin regulation.

  3. Prenucleosomes and Active Chromatin

    PubMed Central

    Khuong, Mai T.; Fei, Jia; Ishii, Haruhiko; Kadonaga, James T.

    2016-01-01

    Chromatin consists of nucleosomes as well as nonnucleosomal histone-containing particles. Here we describe the prenucleosome, which is a stable conformational isomer of the nucleosome that associates with ~80 bp DNA. Prenucleosomes are formed rapidly upon the deposition of histones onto DNA and can be converted into canonical nucleosomes by an ATP-driven chromatin assembly factor such as ACF. Different lines of evidence reveal that there are prenucleosome-sized DNA-containing particles with histones in the upstream region of active promoters. Moreover, p300 acetylates histone H3K56 in prenucleosomes but not in nucleosomes, and H3K56 acetylation is found at active promoters and enhancers. These findings therefore suggest that there may be prenucleosomes or prenucleosome-like particles in the upstream region of active promoters. More generally, we postulate that prenucleosomes or prenucleosome-like particles are present at dynamic chromatin, whereas canonical nucleosomes are at static chromatin. PMID:26767995

  4. Predictive Computational Modeling of Chromatin Folding

    NASA Astrophysics Data System (ADS)

    di Pierro, Miichele; Zhang, Bin; Wolynes, Peter J.; Onuchic, Jose N.

    In vivo, the human genome folds into well-determined and conserved three-dimensional structures. The mechanism driving the folding process remains unknown. We report a theoretical model (MiChroM) for chromatin derived by using the maximum entropy principle. The proposed model allows Molecular Dynamics simulations of the genome using as input the classification of loci into chromatin types and the presence of binding sites of loop forming protein CTCF. The model was trained to reproduce the Hi-C map of chromosome 10 of human lymphoblastoid cells. With no additional tuning the model was able to predict accurately the Hi-C maps of chromosomes 1-22 for the same cell line. Simulations show unknotted chromosomes, phase separation of chromatin types and a preference of chromatin of type A to sit at the periphery of the chromosomes.

  5. Isolation of proteins and protein complexes by immunoprecipitation.

    PubMed

    Kaboord, Barbara; Perr, Maria

    2008-01-01

    Immunoprecipitation (IP) uses the specificity of antibodies to isolate target proteins (antigens) out of complex sample mixtures. Three different approaches for performing IP will be discussed; traditional (classical) method, oriented affinity method and direct affinity method. The traditional method of incubating the IP antibody with the sample and sequentially binding to Protein A or G agarose beads (resin) facilitates the most efficient target antigen recovery. However, this approach results in the target protein becoming contaminated with the IP antibody that can interfere with downstream analyses. The orientated affinity method uses Protein A or G beads to serve as an anchor to which the IP antibody is crosslinked thereby preventing the antibody from co-eluting with the target protein. Similarly, the direct affinity method also immobilizes the IP antibody except in this case it is directly attached to a chemically activated support. Both methods prevent co-elution of the IP antibody enabling reuse of the immunomatrix. All three approaches have unique advantages and can also be used for co-immunoprecipitation to study protein:protein interactions and investigate the functional proteome.

  6. Immunoprecipitation of membrane proteins of cultured human sarcoma cells.

    PubMed

    Grófová, M; Forchhammer, J; Lizonová, A; Popovic, M

    1981-01-01

    Human sarcoma associated antigens (HSAA) have previously been identified by indirect immune fluorescence in human sarcoma cells in culture using sera from patients bearing different types of sarcoma. To further characterize these HSAA, surface proteins of cultured cells were labeled with 125Iodine, [3H]-glucosamine and [35S]-methionine and solubilized. After immunoprecipitation labeled proteins were detected in immune complexes by SDS polyacrylamide gel electrophoresis and autoradiography, which allowed comparison with antigens described by other groups. A surface protein (Mr 96 000) was precipitated with sera from sarcoma bearing patients, and two glycoproteins (Mr 115 000 and 85 000) were preferentially precipitated with antisera from rabbits immunized with membranes from two human sarcoma cell lines. At least two of these proteins were found in each of five human sarcoma cell lines studied (U-4SS, U-3930S, U-20S, B-5GT and B-6FS). None of the proteins were precipitated with three human control sera, and only occasionally a faint band was observed in immunoprecipitates from control cells (B-25F, B-41B, B-42FC, U-2S, and U-393S with the immune sera. These proteins are probably some of the antigens responsible for the immune fluorescence observed in determination of HSAA. However, purification of the proteins and competition experiments are needed before this can be finally established.

  7. Localization of TFIIB binding regions using serial analysis of chromatin occupancy

    PubMed Central

    Yochum, Gregory S; Rajaraman, Veena; Cleland, Ryan; McWeeney, Shannon

    2007-01-01

    Background: RNA Polymerase II (RNAP II) is recruited to core promoters by the pre-initiation complex (PIC) of general transcription factors. Within the PIC, transcription factor for RNA polymerase IIB (TFIIB) determines the start site of transcription. TFIIB binding has not been localized, genome-wide, in metazoans. Serial analysis of chromatin occupancy (SACO) is an unbiased methodology used to empirically identify transcription factor binding regions. In this report, we use TFIIB and SACO to localize TFIIB binding regions across the rat genome. Results: A sample of the TFIIB SACO library was sequenced and 12,968 TFIIB genomic signature tags (GSTs) were assigned to the rat genome. GSTs are 20–22 base pair fragments that are derived from TFIIB bound chromatin. TFIIB localized to both non-protein coding and protein-coding loci. For 21% of the 1783 protein-coding genes in this sample of the SACO library, TFIIB binding mapped near the characterized 5' promoter that is upstream of the transcription start site (TSS). However, internal TFIIB binding positions were identified in 57% of the 1783 protein-coding genes. Internal positions are defined as those within an inclusive region greater than 2.5 kb downstream from the 5' TSS and 2.5 kb upstream from the transcription stop. We demonstrate that both TFIIB and TFIID (an additional component of PICs) bound to internal regions using chromatin immunoprecipitation (ChIP). The 5' cap of transcripts associated with internal TFIIB binding positions were identified using a cap-trapping assay. The 5' TSSs for internal transcripts were confirmed by primer extension. Additionally, an analysis of the functional annotation of mouse 3 (FANTOM3) databases indicates that internally initiated transcripts identified by TFIIB SACO in rat are conserved in mouse. Conclusion: Our findings that TFIIB binding is not restricted to the 5' upstream region indicates that the propensity for PIC to contribute to transcript diversity is far greater

  8. Genome-wide chromatin occupancy analysis reveals a role for ASH2 in transcriptional pausing.

    PubMed

    Pérez-Lluch, Sílvia; Blanco, Enrique; Carbonell, Albert; Raha, Debasish; Snyder, Michael; Serras, Florenci; Corominas, Montserrat

    2011-06-01

    An important mechanism for gene regulation involves chromatin changes via histone modification. One such modification is histone H3 lysine 4 trimethylation (H3K4me3), which requires histone methyltranferase complexes (HMT) containing the trithorax-group (trxG) protein ASH2. Mutations in ash2 cause a variety of pattern formation defects in the Drosophila wing. We have identified genome-wide binding of ASH2 in wing imaginal discs using chromatin immunoprecipitation combined with sequencing (ChIP-Seq). Our results show that genes with functions in development and transcriptional regulation are activated by ASH2 via H3K4 trimethylation in nearby nucleosomes. We have characterized the occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription. ASH2 occupancy correlates with phosphorylated forms of RNA Polymerase II and histone activating marks in expressed genes. Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies. Finally, we have identified specific motifs associated with ASH2 binding in genes that are differentially expressed in ash2 mutants. Our data suggest that recruitment of the ASH2-containing HMT complexes is context specific and points to a function of ASH2 and H3K4me3 in transcriptional pausing control.

  9. DNA immunoprecipitation semiconductor sequencing (DIP-SC-seq) as a rapid method to generate genome wide epigenetic signatures.

    PubMed

    Thomson, John P; Fawkes, Angie; Ottaviano, Raffaele; Hunter, Jennifer M; Shukla, Ruchi; Mjoseng, Heidi K; Clark, Richard; Coutts, Audrey; Murphy, Lee; Meehan, Richard R

    2015-05-14

    Modification of DNA resulting in 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5hmC) has been shown to influence the local chromatin environment and affect transcription. Although recent advances in next generation sequencing technology allow researchers to map epigenetic modifications across the genome, such experiments are often time-consuming and cost prohibitive. Here we present a rapid and cost effective method of generating genome wide DNA modification maps utilising commercially available semiconductor based technology (DNA immunoprecipitation semiconductor sequencing; "DIP-SC-seq") on the Ion Proton sequencer. Focussing on the 5hmC mark we demonstrate, by directly comparing with alternative sequencing strategies, that this platform can successfully generate genome wide 5hmC patterns from as little as 500 ng of genomic DNA in less than 4 days. Such a method can therefore facilitate the rapid generation of multiple genome wide epigenetic datasets.

  10. A DEK Domain-Containing Protein Modulates Chromatin Structure and Function in Arabidopsis[W][OPEN

    PubMed Central

    Waidmann, Sascha; Kusenda, Branislav; Mayerhofer, Juliane; Mechtler, Karl; Jonak, Claudia

    2014-01-01

    Chromatin is a major determinant in the regulation of virtually all DNA-dependent processes. Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. The evolutionarily conserved DEK domain-containing protein is implicated in important chromatin-related processes in animals, but little is known about its DNA targets and protein interaction partners. In plants, the role of DEK has remained elusive. In this work, we identified DEK3 as a chromatin-associated protein in Arabidopsis thaliana. DEK3 specifically binds histones H3 and H4. Purification of other proteins associated with nuclear DEK3 also established DNA topoisomerase 1α and proteins of the cohesion complex as in vivo interaction partners. Genome-wide mapping of DEK3 binding sites by chromatin immunoprecipitation followed by deep sequencing revealed enrichment of DEK3 at protein-coding genes throughout the genome. Using DEK3 knockout and overexpressor lines, we show that DEK3 affects nucleosome occupancy and chromatin accessibility and modulates the expression of DEK3 target genes. Furthermore, functional levels of DEK3 are crucial for stress tolerance. Overall, data indicate that DEK3 contributes to modulation of Arabidopsis chromatin structure and function. PMID:25387881

  11. Chromatin signatures of cancer

    PubMed Central

    Morgan, Marc A.; Shilatifard, Ali

    2015-01-01

    Changes in the pattern of gene expression play an important role in allowing cancer cells to acquire their hallmark characteristics, while genomic instability enables cells to acquire genetic alterations that promote oncogenesis. Chromatin plays central roles in both transcriptional regulation and the maintenance of genomic stability. Studies by cancer genome consortiums have identified frequent mutations in genes encoding chromatin regulatory factors and histone proteins in human cancer, implicating them as major mediators in the pathogenesis of both hematological malignancies and solid tumors. Here, we review recent advances in our understanding of the role of chromatin in cancer, focusing on transcriptional regulatory complexes, enhancer-associated factors, histone point mutations, and alterations in heterochromatin-interacting factors. PMID:25644600

  12. Nucleoporins and chromatin metabolism.

    PubMed

    Ptak, Christopher; Wozniak, Richard W

    2016-06-01

    Mounting evidence has implicated a group of proteins termed nucleoporins, or Nups, in various processes that regulate chromatin structure and function. Nups were first recognized as building blocks for nuclear pore complexes, but several members of this group of proteins also reside in the cytoplasm and within the nucleus. Moreover, many are dynamic and move between these various locations. Both at the nuclear envelope, as part of nuclear pore complexes, and within the nucleoplasm, Nups interact with protein complexes that function in gene transcription, chromatin remodeling, DNA repair, and DNA replication. Here, we review recent studies that provide further insight into the molecular details of these interactions and their role in regulating the activity of chromatin modifying factors. PMID:27085162

  13. Analysis of Chromatin Organisation

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

    Terms to be familiar with before you start to solve the test: chromatin, nucleases, sucrose density gradient centrifugation, melting point, gel electrophoresis, ethidium bromide, autoradiography, Southern blotting, Northern blotting, Sanger sequencing, restriction endonucleases, exonucleases, linker DNA, chloroform extraction, nucleosomes,…

  14. Chromatin assembly using Drosophila systems.

    PubMed

    Fyodorov, Dmitry V; Levenstein, Mark E

    2002-05-01

    To successfully study chromatin structure and activity in vitro, it is essential to have a chromatin assembly system that will prepare extended nucleosome arrays with highly defined protein content that resemble bulk chromatin isolated from living cell nuclei in terms of periodicity and nucleosome positioning. The Drosophila ATP-dependent chromatin assembly system described in this unit meets these requirements. The end product of the reaction described here has highly periodic extended arrays with physiologic spacing and positioning of the nucleosomes.

  15. UV-RNA Immunoprecipitation (UV-RIP) Protocol in Neurons.

    PubMed

    Schaukowitch, Katie; Joo, Jae-Yeol; Kim, Tae-Kyung

    2017-01-01

    With the many advances in genome-wide sequencing, it has been discovered that much more of the genome is transcribed into RNA than previously appreciated. These nonprotein-coding RNAs (ncRNAs) come in many different forms, and they have been shown to have a variety of functions within the cell, influencing processes such as gene expression, mRNA splicing, and transport, just as a few examples. As we delve deeper into studying their mechanisms of action, it becomes important to understand how they play these roles, in particular by understanding what proteins these ncRNAs interact with. This protocol describes one technique that can be used to study this, ultra-violet light cross-linking RNA immunoprecipitation (UV-RIP), which uses an antibody to pull down a specific protein of interest and then detects RNA that is bound to it. This technique utilizes UV light to cross-link the cells, which takes advantage of the fact that UV light will only cross-link proteins and nucleic acids that are directly interacting. This approach can provide key mechanistic insight into the function of these newly identified ncRNAs. PMID:27662868

  16. Searching for biomarkers: humoral response profiling with luciferase immunoprecipitation systems.

    PubMed

    Burbelo, Peter D; Ching, Kathryn H; Bren, Kathleen E; Iadarola, Michael J

    2011-06-01

    B-cell-mediated humoral responses are triggered in many human diseases, including autoimmune diseases, cancer, and neurologic and infectious diseases. However, the full exploitation of the information contained within a patient's antibody repertoire for diagnosis, monitoring and even disease prediction has been limited due to the poor diagnostic performance of many immunoassay formats. We have developed luciferase immunoprecipitation systems (LIPS) that harnesses light-emitting proteins to generate high-definition antibody profiles that are optimal for both diagnostics and biomarker discovery. Here, we describe the results and implications from a range of LIPS-antibody profiling studies performed in our laboratory. These include highly sensitive diagnostics for domestic and global pathogens, insights into infection-related diseases, discovery of new biomarkers for human diseases, subcategorization of symptoms and identification of pathogenic autoantibodies against self-proteins. These investigations highlight the types of humoral response profiles associated with different diseases, provide new information related to disease pathogenesis and offer a framework for incorporating LIPS antibody profiling into global health initiatives and disease monitoring. PMID:21679112

  17. N-DSK gamma-chain binds to immunoprecipitated GP IIb-IIIa

    SciTech Connect

    Thorsen, L.I.; Hessel, B.; Brosstad, F.; Gogstad, G.; Solum, N.O.

    1987-08-01

    The CNBr-split N-terminal disulphide knot of the fibrinogen molecule (N-DSK) binds to ADP-stimulated gel-filtered platelets and immunoprecipitated fibrinogen receptor. To investigate which part of the N-DSK molecule that is involved in this binding, the glycoprotein IIb-IIIa complex (the fibrinogen receptor) was immunoprecipitated in crossed immunoelectrophoresis of Triton X-100 extracts of platelets against rabbit antibodies to whole platelet proteins. The immunoelectrophoresis plates were incubated with solubilized, carboxymethylated /sup 125/I-labelled A alpha -, B beta - or gamma-chains of N-DSK, and investigated for binding by autoradiography. The N-DSK gamma-chain, but not the A alpha - or B beta -chains demonstrated binding to the GP IIb-IIIa complex. These results show that the fibrinogen molecule contains a third sequence of amino acids, in addition to the two previously reported ones that can be involved in binding of fibrinogen to the fibrinogen receptor on the platelets.

  18. Enhancer blocking activity of the insulator at H19-ICR is independent of chromatin barrier establishment.

    PubMed

    Singh, Vikrant; Srivastava, Madhulika

    2008-06-01

    Transcriptional insulators are cis regulatory elements that organize chromatin into independently regulated domains. At the imprinted murine Igf2/H19 locus, the H19-ICR insulator prevents the activation of the Igf2 promoter on the maternal allele by enhancers that activate H19 on the same chromosome. Given the well-demonstrated role of H19-ICR as an enhancer blocker, we investigated its ability to define a chromatin barrier, as the two activities are coincident on several insulators and may act in concert to define a functional chromatin boundary between adjacent genes with distinct transcriptional profiles. Allele-specific association of posttranslationally modified histones, reflecting the presence of active or inactive chromatin, was analyzed in the region encompassing H19-ICR using chromatin immunoprecipitation. The existence of differential histone modifications upstream and downstream of H19-ICR specifically on the maternal chromosome was observed, which is suggestive of a chromatin barrier formation. However, H19-ICR deletion analysis indicated that distinct chromatin states exist despite the absence of an intervening "barrier." Also, the enhancers can activate the Igf2 promoter despite some parts of the intervening chromatin being in the silent state. Hence, H19-ICR insulator activity is not dependent on preventing the enhancer-mediated alteration of the histone modifications in the region between the Igf2 promoter and the cognate enhancers. PMID:18378700

  19. Chromatin Ring Formation at Plant Centromeres.

    PubMed

    Schubert, Veit; Ruban, Alevtina; Houben, Andreas

    2016-01-01

    We observed the formation of chromatin ring structures at centromeres of somatic rye and Arabidopsis chromosomes. To test whether this behavior is present also in other plant species and tissues we analyzed Arabidopsis, rye, wheat, Aegilops and barley centromeres during cell divisions and in interphase nuclei by immunostaining and FISH. Furthermore, structured illumination microscopy (super-resolution) was applied to investigate the ultrastructure of centromere chromatin beyond the classical refraction limit of light. It became obvious, that a ring formation at centromeres may appear during mitosis, meiosis and in interphase nuclei in all species analyzed. However, varying centromere structures, as ring formations or globular organized chromatin fibers, were identified in different tissues of one and the same species. In addition, we found that a chromatin ring formation may also be caused by subtelomeric repeats in barley. Thus, we conclude that the formation of chromatin rings may appear in different plant species and tissues, but that it is not specific for centromere function. Based on our findings we established a model describing the ultrastructure of plant centromeres and discuss it in comparison to previous models proposed for animals and plants. PMID:26913037

  20. Chromatin Ring Formation at Plant Centromeres

    PubMed Central

    Schubert, Veit; Ruban, Alevtina; Houben, Andreas

    2016-01-01

    We observed the formation of chromatin ring structures at centromeres of somatic rye and Arabidopsis chromosomes. To test whether this behavior is present also in other plant species and tissues we analyzed Arabidopsis, rye, wheat, Aegilops and barley centromeres during cell divisions and in interphase nuclei by immunostaining and FISH. Furthermore, structured illumination microscopy (super-resolution) was applied to investigate the ultrastructure of centromere chromatin beyond the classical refraction limit of light. It became obvious, that a ring formation at centromeres may appear during mitosis, meiosis and in interphase nuclei in all species analyzed. However, varying centromere structures, as ring formations or globular organized chromatin fibers, were identified in different tissues of one and the same species. In addition, we found that a chromatin ring formation may also be caused by subtelomeric repeats in barley. Thus, we conclude that the formation of chromatin rings may appear in different plant species and tissues, but that it is not specific for centromere function. Based on our findings we established a model describing the ultrastructure of plant centromeres and discuss it in comparison to previous models proposed for animals and plants. PMID:26913037

  1. Human sperm chromatin epigenetic potential: genomics, proteomics, and male infertility

    PubMed Central

    Castillo, Judit; Estanyol, Josep Maria; Ballescà, Josep Lluis; Oliva, Rafael

    2015-01-01

    The classical idea about the function of the mammalian sperm chromatin is that it serves to transmit a highly protected and transcriptionally inactive paternal genome, largely condensed by protamines, to the next generation. In addition, recent sperm chromatin genome-wide dissection studies indicate the presence of a differential distribution of the genes and repetitive sequences in the protamine-condensed and histone-condensed sperm chromatin domains, which could be potentially involved in regulatory roles after fertilization. Interestingly, recent proteomic studies have shown that sperm chromatin contains many additional proteins, in addition to the abundant histones and protamines, with specific modifications and chromatin affinity features which are also delivered to the oocyte. Both gene and protein signatures seem to be altered in infertile patients and, as such, are consistent with the potential involvement of the sperm chromatin landscape in early embryo development. This present work reviews the available information on the composition of the human sperm chromatin and its epigenetic potential, with a particular focus on recent results derived from high-throughput genomic and proteomic studies. As a complement, we provide experimental evidence for the detection of phosphorylations and acetylations in human protamine 1 using a mass spectrometry approach. The available data indicate that the sperm chromatin is much more complex than what it was previously thought, raising the possibility that it could also serve to transmit crucial paternal epigenetic information to the embryo. PMID:25926607

  2. Chromatin Higher-order Structure and Dynamics

    PubMed Central

    Woodcock, Christopher L.; Ghosh, Rajarshi P.

    2010-01-01

    The primary role of the nucleus as an information storage, retrieval, and replication site requires the physical organization and compaction of meters of DNA. Although it has been clear for many years that nucleosomes constitute the first level of chromatin compaction, this contributes a relatively small fraction of the condensation needed to fit the typical genome into an interphase nucleus or set of metaphase chromosomes, indicating that there are additional “higher order” levels of chromatin condensation. Identifying these levels, their interrelationships, and the principles that govern their occurrence has been a challenging and much discussed problem. In this article, we focus on recent experimental advances and the emerging evidence indicating that structural plasticity and chromatin dynamics play dominant roles in genome organization. We also discuss novel approaches likely to yield important insights in the near future, and suggest research areas that merit further study. PMID:20452954

  3. Drosophila Paf1 modulates chromatin structure at actively transcribed genes.

    PubMed

    Adelman, Karen; Wei, Wenxiang; Ardehali, M Behfar; Werner, Janis; Zhu, Bing; Reinberg, Danny; Lis, John T

    2006-01-01

    The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. We have identified and characterized the Drosophila homologs of three subunits of the yeast Paf1 complex and found striking differences between the yeast and Drosophila Paf1 complexes. We demonstrate that although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure. PMID:16354696

  4. The Arabidopsis SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA Targets Directly to PINs and Is Required for Root Stem Cell Niche Maintenance

    PubMed Central

    Yang, Songguang; Li, Chenlong; Zhao, Linmao; Gao, Sujuan; Lu, Jingxia; Zhao, Minglei; Chen, Chia-Yang; Liu, Xuncheng; Luo, Ming; Cui, Yuhai; Yang, Chengwei; Wu, Keqiang

    2015-01-01

    BRAHMA (BRM), a SWI/SNF chromatin remodeling ATPase, is essential for the transcriptional reprogramming associated with development and cell differentiation in Arabidopsis thaliana. In this study, we show that loss-of-function mutations in BRM led to defective maintenance of the root stem cell niche, decreased meristematic activity, and stunted root growth. Mutations of BRM affected auxin distribution by reducing local expression of several PIN-FORMED (PIN) genes in the stem cells and impaired the expression of the stem cell transcription factor genes PLETHORA (PLT1) and PLT2. Chromatin immunoprecipitation assays showed that BRM could directly target to the chromatin of PIN1, PIN2, PIN3, PIN4, and PIN7. In addition, genetic interaction assays indicate that PLTs acted downstream of BRM, and overexpression of PLT2 partially rescued the stem cell niche defect of brm mutants. Taken together, these results support the idea that BRM acts in the PLT pathway to maintain the root stem cell niche by altering the expression of PINs. PMID:25991732

  5. Direct chromatin PCR (DC-PCR): hypotonic conditions allow differentiation of chromatin states during thermal cycling.

    PubMed

    Vatolin, Sergei; Khan, Shahper N; Reu, Frederic J

    2012-01-01

    Current methods to study chromatin configuration are not well suited for high throughput drug screening since they require large cell numbers and multiple experimental steps that include centrifugation for isolation of nuclei or DNA. Here we show that site specific chromatin analysis can be achieved in one step by simply performing direct chromatin PCR (DC-PCR) on cells. The basic underlying observation was that standard hypotonic PCR buffers prevent global cellular chromatin solubilization during thermal cycling while more loosely organized chromatin can be amplified. Despite repeated heating to >90 °C, 41 of 61 tested 5' sequences of silenced genes (CDKN2A, PU.1, IRF4, FOSB, CD34) were not amplifiable while 47 could be amplified from expressing cells. Two gene regions (IRF4, FOSB) even required pre-heating of cells in isotonic media to allow this differentiation; otherwise none of 19 assayed sequences yielded PCR products. Cells with baseline expression or epigenetic reactivation gave similar DC-PCR results. Silencing during differentiation of CD34 positive cord blood cells closed respective chromatin while treatment of myeloma cells with an IRF4 transcriptional inhibitor opened a site to DC-PCR that was occupied by RNA polymerase II and NFκB as determined by ChIP. Translation into real-time PCR can not be achieved with commercial real-time PCR buffers which potently open chromatin, but even with simple ethidium bromide addition to standard PCR mastermix we were able to identify hits in small molecules screens that suppressed IRF4 expression or reactivated CDKN2A in myeloma cells using densitometry or visual inspection of PCR plates under UV light. While need in drug development inspired this work, application to genome-wide analysis appears feasible using phi29 for selective amplification of open cellular chromatin followed by library construction from supernatants since such supernatants yielded similar results as gene specific DC-PCR.

  6. Chromatin and alternative splicing.

    PubMed

    Alló, M; Schor, I E; Muñoz, M J; de la Mata, M; Agirre, E; Valcárcel, J; Eyras, E; Kornblihtt, A R

    2010-01-01

    Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNA-mediated transcriptional gene silencing.

  7. Inheritance of epigenetic chromatin silencing

    PubMed Central

    David-Rus, Diana; Mukhopadhyay, Swagatam; Lebowitz, Joel L.; Sengupta, Anirvan M.

    2010-01-01

    Maintenance of alternative chromatin states through cell divisions pose some fundamental constraints on the dynamics of histone modifications. In this paper, we study the systems biology of epigenetic inheritance by defining and analyzing general classes of mathematical models. We discuss how the number of modification states involved plays an essential role in the stability of epigenetic states. In addition, DNA duplication and the consequent dilution of marked histones act as a large perturbation for a stable state of histone modifications. The requirement that this large perturbation falls into the basin of attraction of the original state sometimes leads to additional constraints on effective models. Two such models, inspired by two different biological systems, are compared in their fulfilling the requirements of multistability and of recovery after DNA duplication. We conclude that in the presence of multiple histone modifications that characterize alternative epigenetic stable states, these requirements are more easily fulfilled. PMID:19174167

  8. Interaction of the Arabidopsis UV-B-specific signaling component UVR8 with chromatin.

    PubMed

    Cloix, Catherine; Jenkins, Gareth I

    2008-01-01

    Arabidopsis UV RESISTANCE LOCUS8 (UVR8) is a UV-B-specific signaling component that regulates expression of a range of genes concerned with UV protection. Here, we investigate the interaction of UVR8 with chromatin. Using antibodies specific to UVR8 in chromatin immunoprecipitation (ChIP) assays with wild-type plants, we show that native UVR8 binds to chromatin in vivo. Similar experiments using an anti-GFP antibody with plants expressing a GFP-UVR8 fusion show that UVR8 associates with a relatively small region of chromatin containing the HY5 gene. UVR8 interacts with chromatin containing the promoter regions of other genes, but not with all the genes it regulates. UV-B is not required for the interaction of UVR8 with chromatin because association with several gene loci is observed in the absence of UV-B. Pull-down assays demonstrate that UVR8 associates with histones in vivo and competition experiments indicate that the interaction is preferentially with histone H2B. ChIP experiments using antibodies that recognize specific histone modifications indicate that the UV-B-stimulated transcription of some genes may be correlated with histone modification. In particular, the ELIP1 promoter showed a significant enrichment of diacetyl histone H3 (K9/K14) following UV-B exposure. These findings increase understanding of the interaction of the key UV-B-specific regulator UVR8 with chromatin.

  9. ATP Dependent Chromatin Remodeling Enzymes in Embryonic Stem Cells

    PubMed Central

    Saladi, Srinivas Vinod

    2010-01-01

    Embryonic stem (ES) cells are pluripotent cells that can self renew or be induced to differentiate into multiple cell lineages, and thus have the potential to be utilized in regenerative medicine. Key pluripotency specific factors (Oct 4/Sox2/Nanog/Klf4) maintain the pluripotent state by activating expression of pluripotency specific genes and by inhibiting the expression of developmental regulators. Pluripotent ES cells are distinguished from differentiated cells by a specialized chromatin state that is required to epigenetically regulate the ES cell phenotype. Recent studies show that in addition to pluripotency specific factors, chromatin remodeling enzymes play an important role in regulating ES cell chromatin and the capacity to self-renew and to differentiate. Here we review recent studies that delineate the role of ATP dependent chromatin remodeling enzymes in regulating ES cell chromatin structure. PMID:20148317

  10. Cas9 Functionally Opens Chromatin.

    PubMed

    Barkal, Amira A; Srinivasan, Sharanya; Hashimoto, Tatsunori; Gifford, David K; Sherwood, Richard I

    2016-01-01

    Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding. PMID:27031353

  11. High-efficiency solid-phase capture using glass beads bonded to microcentrifuge tubes: immunoprecipitation of proteins from cell extracts and assessment of ras activation.

    PubMed

    Chen, Jeffrey C; von Lintig, Friederike C; Jones, Stephen B; Huvar, Ivana; Boss, Gerry R

    2002-03-15

    We have bonded glass microbeads (425-600 microm diameter) to the inner walls of polypropylene microcentrifuge tubes. In addition to increasing the surface area of the tubes manyfold, the beads provide surface Si groups which can be reacted with a silane compound such as aminopropyltriethoxysilane, yielding a free amino group. The amino group is reacted with another cross-linking reagent, for example, the homobifunctional compound dimethyl suberimidate, which can form a covalent bond with amine groups of proteins. After binding protein A or G to the dimethyl suberimidate, the beads were used to immunoprecipitate proteins from cell extracts; we show that the protein A/G-coated glass beads yield similar amounts of immunoprecipitated proteins as a standard method using protein A- or G-agarose beads, but with fewer contaminating proteins. In addition, we show that when immunoprecipitating Ras from cell extracts and measuring the amounts of Ras-bound GTP and GDP, the new method yielded higher guanine nucleotide levels than protein G-agarose beads, suggesting that it caused less denaturation of Ras. Because the glass beads are bonded to the walls of the tubes, the immunoprecipitates can be washed rapidly and efficiently, and we show that 20-30 tubes can be washed in 1/10 the time required to wash immunoprecipitates on protein A- or G-agarose beads.

  12. Histone variants: key players of chromatin.

    PubMed

    Biterge, Burcu; Schneider, Robert

    2014-06-01

    Histones are fundamental structural components of chromatin. Eukaryotic DNA is wound around an octamer of the core histones H2A, H2B, H3, and H4. Binding of linker histone H1 promotes higher order chromatin organization. In addition to their structural role, histones impact chromatin function and dynamics by, e.g., post-translational histone modifications or the presence of specific histone variants. Histone variants exhibit differential expression timings (DNA replication-independent) and mRNA characteristics compared to canonical histones. Replacement of canonical histones with histone variants can affect nucleosome stability and help to create functionally distinct chromatin domains. In line with this, several histone variants have been implicated in the regulation of cellular processes such as DNA repair and transcriptional activity. In this review, we focus on recent progress in the study of core histone variants H2A.X, H2A.Z, macroH2A, H3.3, and CENP-A, as well as linker histone H1 variants, their functions and their links to development and disease.

  13. Unraveling the mechanisms of chromatin fibril packaging.

    PubMed

    Gavrilov, Alexey A; Shevelyov, Yuri Y; Ulianov, Sergey V; Khrameeva, Ekaterina E; Kos, Pavel; Chertovich, Alexander; Razin, Sergey V

    2016-05-01

    Recent data indicate that eukaryotic chromosomes are organized into Topologically Associating Domains (TADs); however, the mechanisms underlying TAD formation remain obscure. Based on the results of Hi-C analysis performed on 4 Drosophila melanogaster cell lines, we have proposed that specific properties of nucleosomes in active and repressed chromatin play a key role in the formation of TADs. Our computer simulations showed that the ability of "inactive" nucleosomes to stick to each other and the lack of such ability in "active" nucleosomes is sufficient for spatial segregation of these types of chromatin, which is revealed in the Hi-C analysis as TAD/inter-TAD partitioning. However, some Drosophila and mammalian TADs contain both active and inactive chromatin, a fact that does not fit this model. Herein, we present additional arguments for the model by postulating that transcriptionally active chromatin is extruded on the surface of a TAD, and discuss the possible impact of this organization on the enhancer-promoter communication and on the segregation of TADs. PMID:27249516

  14. Strand pairing by Rad54 and Rad51 is enhanced by chromatin.

    PubMed

    Alexiadis, Vassilios; Kadonaga, James T

    2002-11-01

    We investigated the role of chromatin in the catalysis of homologous strand pairing by Rad54 and Rad51. Rad54 is related to the ATPase subunits of chromatin-remodeling factors, whereas Rad51 is related to bacterial RecA. In the absence of superhelical tension, we found that the efficiency of strand pairing with chromatin is >100-fold higher than that with naked DNA. In addition, we observed that Rad54 and Rad51 function cooperatively in the ATP-dependent remodeling of chromatin. These findings indicate that Rad54 and Rad51 have evolved to function with chromatin, the natural substrate, rather than with naked DNA. PMID:12414729

  15. CCSI: a database providing chromatin-chromatin spatial interaction information.

    PubMed

    Xie, Xiaowei; Ma, Wenbin; Songyang, Zhou; Luo, Zhenhua; Huang, Junfeng; Dai, Zhiming; Xiong, Yuanyan

    2016-01-01

    Distal regulatory elements have been shown to regulate gene transcription through spatial interactions, and single nucleotide polymorphisms (SNPs) are linked with distal gene expression by spatial proximity, which helps to explain the causal role of disease-associated SNPs in non-coding region. Therefore, studies on spatial interactions between chromatin have created a new avenue for elucidating the mechanism of transcriptional regulation in disease pathogenesis. Recently, a growing number of chromatin interactions have been revealed by means of 3C, 4C, 5C, ChIA-PET and Hi-C technologies. To interpret and utilize these interactions, we constructed chromatin-chromatin spatial interaction (CCSI) database by integrating and annotating 91 sets of chromatin interaction data derived from published literature, UCSC database and NCBI GEO database, resulting in a total of 3,017,962 pairwise interactions (false discovery rate < 0.05), covering human, mouse and yeast. A web interface has been designed to provide access to the chromatin interactions. The main features of CCSI are (i) showing chromatin interactions and corresponding genes, enhancers and SNPs within the regions in the search page; (ii) offering complete interaction datasets, enhancer and SNP information in the download page; and (iii) providing analysis pipeline for the annotation of interaction data. In conclusion, CCSI will facilitate exploring transcriptional regulatory mechanism in disease pathogenesis associated with spatial interactions among genes, regulatory regions and SNPs. Database URL: http://songyanglab.sysu.edu.cn/ccsi. PMID:26868054

  16. HP1BP3, a Chromatin Retention Factor for Co-transcriptional MicroRNA Processing.

    PubMed

    Liu, Haoming; Liang, Chunyang; Kollipara, Rahul K; Matsui, Masayuki; Ke, Xiong; Jeong, Byung-Cheon; Wang, Zhiqiang; Yoo, Kyoung Shin; Yadav, Gaya P; Kinch, Lisa N; Grishin, Nicholas V; Nam, Yunsun; Corey, David R; Kittler, Ralf; Liu, Qinghua

    2016-08-01

    Recent studies suggest that the microprocessor (Drosha-DGCR8) complex can be recruited to chromatin to catalyze co-transcriptional processing of primary microRNAs (pri-miRNAs) in mammalian cells. However, the molecular mechanism of co-transcriptional miRNA processing is poorly understood. Here we find that HP1BP3, a histone H1-like chromatin protein, specifically associates with the microprocessor and promotes global miRNA biogenesis in human cells. Chromatin immunoprecipitation (ChIP) studies reveal genome-wide co-localization of HP1BP3 and Drosha and HP1BP3-dependent Drosha binding to actively transcribed miRNA loci. Moreover, HP1BP3 specifically binds endogenous pri-miRNAs and facilitates the Drosha/pri-miRNA association in vivo. Knockdown of HP1BP3 compromises pri-miRNA processing by causing premature release of pri-miRNAs from the chromatin. Taken together, these studies suggest that HP1BP3 promotes co-transcriptional miRNA processing via chromatin retention of nascent pri-miRNA transcripts. This work significantly expands the functional repertoire of the H1 family of proteins and suggests the existence of chromatin retention factors for widespread co-transcriptional miRNA processing.

  17. A DNA immunoprecipitation assay used in quantitative detection of in vitro DNA-protein complex binding.

    PubMed

    Kim, Min Young; Chae, Ji Hyung; Oh, Chang-Ho; Kim, Chul Geun

    2013-10-15

    To begin gene transcription, several transcription factors must bind to specific DNA sequences to form a complex via DNA-protein interactions. We established an in vitro method for specific and sensitive analyses of DNA-protein interactions based on a DNA immunoprecipitation (DIP) method. We verified the accuracy and efficiency of the DIP assay in quantitatively measuring DNA-protein binding using transcription factor CP2c as a model. With our DIP assay, we could detect specific interactions within a DNA-CP2c complex, with reproducible and quantitative binding values. In addition, we were able to effectively measure the changes in DNA-CP2c binding by the addition of a small molecule, FQI1 (factor quinolinone inhibitor 1), previously identified as a specific inhibitor of this binding. To identify a new regulator of DNA-CP2c binding, we analyzed several CP2c binding peptides and found that only one class of peptide severely inhibits DNA-CP2c binding. These data show that our DIP assay is very useful in quantitatively detecting the binding dynamics of DNA-protein complex. Because DNA-protein interaction is very dynamic in different cellular environments, our assay can be applied to the detection of active transcription factors, including promoter occupancy in normal and disease conditions. Moreover, it may be used to develop a targeted regulator of specific DNA-protein interaction.

  18. Ultrastructure of bovine sperm chromatin.

    PubMed

    Filho, Romualdo Morandi; Beletti, Marcelo Emilio; de Oliveira, Fabio

    2015-12-01

    Mammalian semen chromatin comprises DNA, protamine, and, at lower levels, other proteins. This constitution confers intense compaction to the chromatin, helping to protect the DNA and causing the head of the sperm to be very small, facilitating the safe transport of its genetic contents. It is known that changes in the sperm chromatin compaction lead to fertility problems in bulls, justifying studies of this structure. Although there are theoretical models of sperm chromatin because of its high compaction, there is no morphological evidence of such models. The aim of this study was to demonstrate the ultrastructure of bovine sperm chromatin in an attempt to corroborate the theoretical chromatin models existing today. The isolated bull sperm heads had their chromatin partially unpacked by chemical treatment using sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) and were then embedded in Epon resin. Using an ultramicrotome, ultrathin sections were obtained, which were contrasted with uranyl acetate and lead citrate, and then viewed under transmission electron microscopy. The methodology used allowed the visualization of toroidal structures interconnected by a filamentous nuclear matrix, which is entirely consistent with the most current theoretical models. PMID:26515508

  19. Single Molecule Studies of Chromatin

    SciTech Connect

    Jeans, C; Colvin, M E; Thelen, M P; Noy, A

    2004-01-06

    The DNA in eukaryotic cells is tightly packaged as chromatin through interactions with histone proteins to form nucleosomes. These nucleosomes are themselves packed together through interactions with linker histone and non-histone proteins. In order for processes such as DNA replication, DNA repair, and transcription to occur, the chromatin fiber must be remodeled such that the necessary enzymes can access the DNA. The structure of the chromatin fiber beyond the level of the single nucleosome and the structural changes which accompany the remodeling process are poorly understood. We are studying the structures and forces behind the remodeling process through the use of atomic force microscopy (AFM). This allows both high-resolution imaging of the chromatin, and manipulation of individual fibers. Pulling a single chromatin fiber apart using the AFM tip yields information on the forces which hold the structure together. We have isolated chromatin fibers from chicken erythrocytes and Chinese hamster ovary cell lines. AFM images of these fibers will be presented, along with preliminary data from the manipulation of these fibers using the AFM tip. The implications of these data for the structure of chromatin undergoing the remodeling process are discussed.

  20. Chromatin remodeling in plant development.

    PubMed

    Jarillo, José A; Piñeiro, Manuel; Cubas, Pilar; Martínez-Zapater, José M

    2009-01-01

    Plant development results from specific patterns of gene expression that are tightly regulated in a spatio-temporal manner. Chromatin remodeling plays a central role in establishing these expression patterns and maintaining epigenetic transcriptional states through successive rounds of mitosis that take place within a cell lineage. Plant epigenetic switches occur not only at the embryo stage, but also during postembryonic developmental transitions, suggesting that chromatin remodeling activities in plants can provide a higher degree of regulatory flexibility which probably underlies their developmental plasticity. Here, we highlight recent progress in the understanding of plant chromatin dynamic organization, facilitating the activation or repression of specific sets of genes involved in different developmental programs and integrating them with the response to environmental signals. Chromatin conformation controls gene expression both in actively dividing undifferentiated cells and in those already fate-determined. In this context, we first describe chromatin reorganization activities required to maintain meristem function stable through DNA replication and cell division. Organ initiation at the apex, with emphasis on reproductive development, is next discussed to uncover the chromatin events involved in the establishment and maintenance of expression patterns associated with differentiating cells; this is illustrated with the complex epigenetic regulation of the Arabidopsis floral repressor FLOWERING LOCUS C (FLC). Finally, we discuss the involvement of chromatin remodeling in plant responses to environmental cues and to different types of stress conditions.

  1. Chromatin organization: form to function.

    PubMed

    de Graaf, Carolyn A; van Steensel, Bas

    2013-04-01

    Recent developments in technology have made it possible to create high resolution genome-wide maps of histone marks, DNA binding proteins and physical interactions along genomic regions. Chromatin features are found together in different combinations, dividing the genome up into domains with distinct functional properties. Microscopy and chromatin conformation capture techniques have shown that the 3D structure of chromosomes is constrained by nuclear features and functional links between different parts of chromatin. These results provide insights about the 3D and domain organization of the genome and their connection to gene regulation and other nuclear functions. PMID:23274160

  2. Chromatin modifications and their function.

    PubMed

    Kouzarides, Tony

    2007-02-23

    The surface of nucleosomes is studded with a multiplicity of modifications. At least eight different classes have been characterized to date and many different sites have been identified for each class. Operationally, modifications function either by disrupting chromatin contacts or by affecting the recruitment of nonhistone proteins to chromatin. Their presence on histones can dictate the higher-order chromatin structure in which DNA is packaged and can orchestrate the ordered recruitment of enzyme complexes to manipulate DNA. In this way, histone modifications have the potential to influence many fundamental biological processes, some of which may be epigenetically inherited. PMID:17320507

  3. Nucleosome positioning and composition modulate in silico chromatin flexibility.

    PubMed

    Clauvelin, N; Lo, P; Kulaeva, O I; Nizovtseva, E V; Diaz-Montes, J; Zola, J; Parashar, M; Studitsky, V M; Olson, W K

    2015-02-18

    The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes-the familiar assemblies of ∼150 DNA base pairs and eight histone proteins-found on chromatin fibers. Here we introduce a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs. We explore the effects of nucleosome positioning and the presence or absence of cationic N-terminal histone tails on the 'local' inter-nucleosomal interactions and the global deformations of the simulated chains. The correspondence between the predicted and observed effects of nucleosome composition and numbers on the long-range communication between the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We also extract effective nucleosome-nucleosome potentials from the simulations and implement the potentials in a larger-scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable effect of nucleosome spacing on chromatin flexibility, with small changes in DNA linker length significantly altering the interactions of nucleosomes and the dimensions of the fiber as a whole. In addition, we find that these changes in nucleosome positioning influence the statistical properties of long chromatin constructs. That is, simulated chromatin fibers with the same number of nucleosomes exhibit polymeric behaviors ranging from Gaussian to worm-like, depending upon nucleosome spacing. These findings suggest that the physical and mechanical properties of chromatin can span a wide range of behaviors, depending on nucleosome positioning, and

  4. Nucleosome positioning and composition modulate in silico chromatin flexibility

    NASA Astrophysics Data System (ADS)

    Clauvelin, N.; Lo, P.; Kulaeva, O. I.; Nizovtseva, E. V.; Diaz-Montes, J.; Zola, J.; Parashar, M.; Studitsky, V. M.; Olson, W. K.

    2015-02-01

    The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes—the familiar assemblies of ˜150 DNA base pairs and eight histone proteins—found on chromatin fibers. Here we introduce a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs. We explore the effects of nucleosome positioning and the presence or absence of cationic N-terminal histone tails on the ‘local’ inter-nucleosomal interactions and the global deformations of the simulated chains. The correspondence between the predicted and observed effects of nucleosome composition and numbers on the long-range communication between the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We also extract effective nucleosome-nucleosome potentials from the simulations and implement the potentials in a larger-scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable effect of nucleosome spacing on chromatin flexibility, with small changes in DNA linker length significantly altering the interactions of nucleosomes and the dimensions of the fiber as a whole. In addition, we find that these changes in nucleosome positioning influence the statistical properties of long chromatin constructs. That is, simulated chromatin fibers with the same number of nucleosomes exhibit polymeric behaviors ranging from Gaussian to worm-like, depending upon nucleosome spacing. These findings suggest that the physical and mechanical properties of chromatin can span a wide range of behaviors, depending on nucleosome

  5. Nucleosome positioning and composition modulate in silico chromatin flexibility

    PubMed Central

    Clauvelin, N.; Lo, P.; Kulaeva, O. I.; Nizovtseva, E. V.; Diaz-Montes, J.; Zola, J.; Parashar, M.; Studitsky, V. M.; Olson, W. K.

    2015-01-01

    The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes—the familiar assemblies of ~ 150 DNA base pairs and eight histone proteins—found on chromatin fibers. Here we introduce a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs. We explore the effects of nucleosome positioning and the presence or absence of cationic N-terminal histone tails on the ‘local’ inter-nucleosomal interactions and the global deformations of the simulated chains. The correspondence between the predicted and observed effects of nucleosome composition and numbers on the long-range communication between the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We also extract effective nucleosome-nucleosome potentials from the simulations and implement the potentials in a larger-scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable effect of nucleosome spacing on chromatin flexibility, with small changes in DNA linker length significantly altering the interactions of nucleosomes and the dimensions of the fiber as a whole. In addition, we find that these changes in nucleosome positioning influence the statistical properties of long chromatin constructs. That is, simulated chromatin fibers with the same number of nucleosomes exhibit polymeric behaviors ranging from Gaussian to worm-like, depending upon nucleosome spacing. These findings suggest that the physical and mechanical properties of chromatin can span a wide range of behaviors, depending on nucleosome

  6. Autoantibodies to IA-2 in insulin-dependent diabetes mellitus. Measurements with a new immunoprecipitation assay.

    PubMed

    Masuda, M; Powell, M; Chen, S; Beer, C; Fichna, P; Rees Smith, B; Furmaniak, J

    2000-01-20

    An immunoprecipitation assay for autoantibodies (Abs) to the human islet cell antigen IA-2 has been developed using 125I-labelled recombinant IA-2 expressed in E. coli. With this assay IA-2 Abs were detected in 103/217 (47%) of IDDM patients of different ages and with different disease duration. IA-2 Ab prevalence was higher in younger patients (at the age of 15 years or below) with the recent onset IDDM (64/113; 57%) compared to patients above the age of 15 years (11/25; 44%). One of 40 (2.5%) Graves' disease patients and five of 204 (2.5%) of NIDDM patients were also positive. IA-2 Abs were not detected in sera from patients with Hashimoto's thyroiditis (n=32), myasthenia gravis (n=20) or systemic lupus erythematosus (n=10). IA-2 Ab measurements based on 125I-labelled IA-2 showed a good correlation with the results of an immunoprecipitation assay based on 35S-labelled IA-2 produced in the in vitro transcription/translation system (r=0.78; n=113; p<0.001). Out of 217 IDDM sera which were tested for IA-2 Abs, 140 (65%) were positive for Abs to glutamic acid decarboxylase (GAD) and 166 (76%) were positive for Abs to IA-2 and/or Abs to GAD. In addition, Abs to IA-2, to GAD and to insulin were analysed in sera from recent onset IDDM patients who had not been treated with insulin (n=117). In all, 76/117 (65%) of these sera were positive for GAD Abs, 66/117 (56%) for IA-2 Abs, 45/117 (38%) for insulin Abs. However, 98/117 (84%) were positive for at least one of the three Abs confirming earlier observations on the complementarity of Ab testing in IDDM. Overall, the IA-2 Ab assay based on 125I-labelled recombinant IA-2 showed good sensitivity, precision and specificity which, combined with an easy and convenient protocol, makes it attractive for routine use.

  7. HAB1–SWI3B Interaction Reveals a Link between Abscisic Acid Signaling and Putative SWI/SNF Chromatin-Remodeling Complexes in Arabidopsis[C][W

    PubMed Central

    Saez, Angela; Rodrigues, Americo; Santiago, Julia; Rubio, Silvia; Rodriguez, Pedro L.

    2008-01-01

    Abscisic acid (ABA) has an important role for plant growth, development, and stress adaptation. HYPERSENSITIVE TO ABA1 (HAB1) is a protein phosphatase type 2C that plays a key role as a negative regulator of ABA signaling; however, the molecular details of HAB1 action in this process are not known. A two-hybrid screen revealed that SWI3B, an Arabidopsis thaliana homolog of the yeast SWI3 subunit of SWI/SNF chromatin-remodeling complexes, is a prevalent interacting partner of HAB1. The interaction mapped to the N-terminal half of SWI3B and required an intact protein phosphatase catalytic domain. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirmed the interaction of HAB1 and SWI3B in the nucleus of plant cells. swi3b mutants showed a reduced sensitivity to ABA-mediated inhibition of seed germination and growth and reduced expression of the ABA-responsive genes RAB18 and RD29B. Chromatin immunoprecipitation experiments showed that the presence of HAB1 in the vicinity of RD29B and RAB18 promoters was abolished by ABA, which suggests a direct involvement of HAB1 in the regulation of ABA-induced transcription. Additionally, our results uncover SWI3B as a novel positive regulator of ABA signaling and suggest that HAB1 modulates ABA response through the regulation of a putative SWI/SNF chromatin-remodeling complex. PMID:19033529

  8. Vernalization-mediated chromatin changes.

    PubMed

    Zografos, Brett R; Sung, Sibum

    2012-07-01

    Proper flowering time is vital for reproductive fitness in flowering plants. In Arabidopsis, vernalization is mediated primarily through the repression of a MADS box transcription factor, FLOWERING LOCUS C (FLC). The induction of a plant homeodomain-containing protein, VERNALIZATION INSENSITIVE 3 (VIN3), by vernalizing cold is required for proper repression of FLC. One of a myriad of changes that occurs after VIN3 is induced is the establishment of FLC chromatin at a mitotically repressed state due to the enrichment of repressive histone modifications. VIN3 induction by cold is the earliest known event during the vernalization response and includes changes in histone modifications at its chromatin. Here, the current understanding of the vernalization-mediated chromatin changes in Arabidopsis is discussed, with a focus on the roles of shared chromatin-modifying machineries in regulating VIN3 and FLC gene family expression during the course of vernalization.

  9. Painting a Clearer Picture of Chromatin.

    PubMed

    Finn, Elizabeth H; Misteli, Tom; Shachar, Sigal

    2016-02-22

    Elucidating chromatin's 3D shape is critical to understanding its function, but the fine structure of chromatin domains remains poorly resolved. In a recent report in Nature, Boettiger et al. (2016) visualize chromatin in super-resolution, gaining unprecedented insight into chromatin architecture. PMID:26906730

  10. Developmental regulation of chromatin conformation by Hox proteins in Drosophila

    PubMed Central

    Agelopoulos, Marios; McKay, Daniel J.; Mann, Richard S.

    2012-01-01

    Summary We present a strategy to examine the chromatin conformation of individual loci in specific cell types during Drosophila embryogenesis. Regulatory DNA is tagged with binding sites (lacO) for LacI, which is used to immunopreciptiate the tagged chromatin from specific cell types. We applied this approach to Distalless (Dll), a gene required for limb development in Drosophila. We show that the local chromatin conformation at Dll depends on the cell type: in cells that express Dll, the 5’ regulatory region is in close proximity to the Dll promoter. In Dll nonexpressing cells this DNA is in a more extended configuration. In addition, transcriptional activators and repressors are bound to Dll regulatory DNA in a cell type specific manner. The pattern of binding by GAGA factor and the variant histone H2Av suggest that they play a role in the regulation of Dll chromatin conformation in expressing and non-expressing cell types, respectively. PMID:22523743

  11. Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils

    PubMed Central

    Greiner, Erin R.; Kelly, Jeffery W.; Palhano, Fernando L.

    2014-01-01

    Amyloid fibrils are associated with many maladies, including Alzheimer’s disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ1–40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ1–40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ1–40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this

  12. Chromatin beacons: global sampling of chromatin physical properties using chromatin charting lines.

    PubMed

    Amini, Aniça; Luo, Chongyuan; Lam, Eric

    2011-01-01

    The extent to which physical properties and intranuclear locations of chromatin can influence transcription output remains unclear and poorly quantified. Because the scale and resolution at which structural parameters can be queried are usually so different from the scale that transcription outputs are measured, the integration of these data is often indirect. To overcome this limitation in quantifying chromatin structural parameters at different locations in the genome, a Chromatin Charting collection with 277 transposon-tagged Arabidopsis lines has been established in order to discover correlations between gene expression and the physical properties of chromatin loci within the nuclei. In these lines, dispersed loci in the Arabidopsis genome are tagged with an identical transgene cassette containing a luciferase gene reporter, which permits the quantification of gene expressions in real time, and an ∼2 kb LacO repeat that acts as a "chromatin beacon" to facilitate the visual tracking of a tagged locus in living plants via the expression of LacI-GFP fusion proteins in trans. In this chapter, we describe the methods for visualizing and tracking these insertion loci in vivo and illustrate the potential of using this approach to correlate chromatin mobility with gene expression in living plants.

  13. Identification of Novel Proteins Co-Purifying with Cockayne Syndrome Group B (CSB) Reveals Potential Roles for CSB in RNA Metabolism and Chromatin Dynamics

    PubMed Central

    Caputo, Manuela; Cipak, Lubos; Gregan, Juraj; Ammerer, Gustav; Frontini, Mattia; Willems, Daniela; Prantera, Giorgio; Balajee, Adayabalam S.; Proietti-De-Santis, Luca

    2015-01-01

    The CSB protein, a member of the SWI/SNF ATP dependent chromatin remodeling family of proteins, plays a role in a sub-pathway of nucleotide excision repair (NER) known as transcription coupled repair (TCR). CSB is frequently mutated in Cockayne syndrome group B, a segmental progeroid human autosomal recessive disease characterized by growth failure and degeneration of multiple organs. Though initially classified as a DNA repair protein, recent studies have demonstrated that the loss of CSB results in pleiotropic effects. Identification of novel proteins belonging to the CSB interactome may be useful not only for predicting the molecular basis for diverse pathological symptoms of CS-B patients but also for unraveling the functions of CSB in addition to its authentic role in DNA repair. In this study, we performed tandem affinity purification (TAP) technology coupled with mass spectrometry and co-immunoprecipitation studies to identify and characterize the proteins that potentially interact with CSB-TAP. Our approach revealed 33 proteins that were not previously known to interact with CSB. These newly identified proteins indicate potential roles for CSB in RNA metabolism involving repression and activation of transcription process and in the maintenance of chromatin dynamics and integrity. PMID:26030138

  14. Chromatin pattern by variogram analysis.

    PubMed

    Diaz, G; Zucca, A; Setzu, M D; Cappai, C

    1997-11-01

    Many cytological processes such as cell proliferation, differentiation, transformation, apoptosis, etc., are accompanied by specific chromatin changes, usually identified on the basis of the relative content of euchromatin and heterochromatin. In order to achieve a quantitative, non-subjective evaluation of the chromatin pattern, two different approaches may be undertaken, one consisting in the analysis of the several morphological features of chromatin grains (size, shape, density, arrangement, and distribution), and the second consisting in the analysis of the chromatin globally considered as a coherent texture. Although the second approach appears to be simpler and more suitable, methods of texture analysis--including those specifically designed for the analysis of the chromatin pattern--are rarely applied due mainly to the unsuitability of sampling procedures and the excessive crypticism of results. As an alternative to traditional texture analysis, we suggest a method supported by a sound mathematical theory and approximately 30 years of applications in the field of geostatistics. The method, called variogram, analyzes the intrinsic structure of data sampled at different distance intervals and directions, and outputs easily understandable results. Recently, variogram analysis has successfully been exported from geostatistics to other fields (for example, ecology and epidemiology) that make use of spatially referenced variables. Based on the fact that pixels represent a perfect array of data ordered at regular distance intervals and directions, the variogram can be adopted to explore nuclear images and recognize chromatin patterns. Variograms of different nuclei can be summarized by multivariate methods without the need of previous standardization of data. This allows comparison and discrimination of chromatin patterns from mixed cell populations. Preliminary data obtained from young neurons undergoing massive apoptosis reveal a self-consistent map of nuclear

  15. Modeling co-occupancy of transcription factors using chromatin features

    PubMed Central

    Liu, Liang; Zhao, Weiling; Zhou, Xiaobo

    2016-01-01

    Regulation of gene expression requires both transcription factor (TFs) and epigenetic modifications, and interplays between the two types of factors have been discovered. However study of relationships between chromatin features and TF–TF co-occupancy remains limited. Here, we revealed the relationship by first illustrating distinct profile patterns of chromatin features related to different binding events, including single TF binding and TF–TF co-occupancy of 71 TFs from five human cell lines. We further implemented statistical analyses to demonstrate the relationship by accurately predicting co-occupancy genome-widely using chromatin features including DNase I hypersensitivity, 11 histone modifications (HMs) and GC content. Remarkably, our results showed that the combination of chromatin features enables accurate predictions across the five cells. For individual chromatin features, DNase I enables high and consistent predictions. H3K27ac, H3K4me 2, H3K4me3 and H3K9ac are more reliable predictors than other HMs. Although the combination of 11 HMs achieves accurate predictions, their predictive ability varies considerably when a model obtained from one cell is applied to others, indicating relationship between HMs and TF–TF co-occupancy is cell type dependent. GC content is not a reliable predictor, but the addition of GC content to any other features enhances their predictive ability. Together, our results elucidate a strong relationship between TF–TF co-occupancy and chromatin features. PMID:26590261

  16. High-resolution mapping of transcription factor binding sites on native chromatin

    PubMed Central

    Kasinathan, Sivakanthan; Orsi, Guillermo A.; Zentner, Gabriel E.; Ahmad, Kami; Henikoff, Steven

    2014-01-01

    Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) is widely used for profiling of TF binding, but is limited by low resolution and poor specificity and sensitivity. We present a simple protocol that starts with micrococcal nuclease-digested uncross-linked chromatin and is followed by affinity purification of TFs and paired-end sequencing. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide highly accurate base-pair resolution maps that are not biased toward accessible chromatin, and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila melanogaster GAGA Factor and Pipsqueak. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions. PMID:24336359

  17. R loops are linked to histone H3 S10 phosphorylation and chromatin condensation.

    PubMed

    Castellano-Pozo, Maikel; Santos-Pereira, José M; Rondón, Ana G; Barroso, Sonia; Andújar, Eloisa; Pérez-Alegre, Mónica; García-Muse, Tatiana; Aguilera, Andrés

    2013-11-21

    R loops are transcription byproducts that constitute a threat to genome integrity. Here we show that R loops are tightly linked to histone H3 S10 phosphorylation (H3S10P), a mark of chromatin condensation. Chromatin immunoprecipitation (ChIP)-on-chip (ChIP-chip) analyses reveal H3S10P accumulation at centromeres, pericentromeric chromatin, and a large number of active open reading frames (ORFs) in R-loop-accumulating yeast cells, better observed in G1. Histone H3S10 plays a key role in maintaining genome stability, as scored by ectopic recombination and plasmid loss, Rad52 foci, and Rad53 checkpoint activation. H3S10P coincides with the presence of DNA-RNA hybrids, is suppressed by ribonuclease H overexpression, and causes reduced accessibility of restriction endonucleases, implying a tight connection between R loops, H3S10P, and chromatin compaction. Such histone modifications were also observed in R-loop-accumulating Caenorhabditis elegans and HeLa cells. We therefore provide a role of RNA in chromatin structure essential to understand how R loops modulate genome dynamics. PMID:24211264

  18. Standardizing chromatin research: a simple and universal method for ChIP-seq

    PubMed Central

    Arrigoni, Laura; Richter, Andreas S.; Betancourt, Emily; Bruder, Kerstin; Diehl, Sarah; Manke, Thomas; Bönisch, Ulrike

    2016-01-01

    Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a key technique in chromatin research. Although heavily applied, existing ChIP-seq protocols are often highly fine-tuned workflows, optimized for specific experimental requirements. Especially the initial steps of ChIP-seq, particularly chromatin shearing, are deemed to be exceedingly cell-type-specific, thus impeding any protocol standardization efforts. Here we demonstrate that harmonization of ChIP-seq workflows across cell types and conditions is possible when obtaining chromatin from properly isolated nuclei. We established an ultrasound-based nuclei extraction method (NEXSON: Nuclei EXtraction by SONication) that is highly effective across various organisms, cell types and cell numbers. The described method has the potential to replace complex cell-type-specific, but largely ineffective, nuclei isolation protocols. By including NEXSON in ChIP-seq workflows, we completely eliminate the need for extensive optimization and sample-dependent adjustments. Apart from this significant simplification, our approach also provides the basis for a fully standardized ChIP-seq and yields highly reproducible transcription factor and histone modifications maps for a wide range of different cell types. Even small cell numbers (∼10 000 cells per ChIP) can be easily processed without application of modified chromatin or library preparation protocols. PMID:26704968

  19. Standardizing chromatin research: a simple and universal method for ChIP-seq.

    PubMed

    Arrigoni, Laura; Richter, Andreas S; Betancourt, Emily; Bruder, Kerstin; Diehl, Sarah; Manke, Thomas; Bönisch, Ulrike

    2016-04-20

    Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a key technique in chromatin research. Although heavily applied, existing ChIP-seq protocols are often highly fine-tuned workflows, optimized for specific experimental requirements. Especially the initial steps of ChIP-seq, particularly chromatin shearing, are deemed to be exceedingly cell-type-specific, thus impeding any protocol standardization efforts. Here we demonstrate that harmonization of ChIP-seq workflows across cell types and conditions is possible when obtaining chromatin from properly isolated nuclei. We established an ultrasound-based nuclei extraction method (NEXSON: Nuclei EXtraction by SONication) that is highly effective across various organisms, cell types and cell numbers. The described method has the potential to replace complex cell-type-specific, but largely ineffective, nuclei isolation protocols. By including NEXSON in ChIP-seq workflows, we completely eliminate the need for extensive optimization and sample-dependent adjustments. Apart from this significant simplification, our approach also provides the basis for a fully standardized ChIP-seq and yields highly reproducible transcription factor and histone modifications maps for a wide range of different cell types. Even small cell numbers (∼10,000 cells per ChIP) can be easily processed without application of modified chromatin or library preparation protocols.

  20. R loops are linked to histone H3 S10 phosphorylation and chromatin condensation.

    PubMed

    Castellano-Pozo, Maikel; Santos-Pereira, José M; Rondón, Ana G; Barroso, Sonia; Andújar, Eloisa; Pérez-Alegre, Mónica; García-Muse, Tatiana; Aguilera, Andrés

    2013-11-21

    R loops are transcription byproducts that constitute a threat to genome integrity. Here we show that R loops are tightly linked to histone H3 S10 phosphorylation (H3S10P), a mark of chromatin condensation. Chromatin immunoprecipitation (ChIP)-on-chip (ChIP-chip) analyses reveal H3S10P accumulation at centromeres, pericentromeric chromatin, and a large number of active open reading frames (ORFs) in R-loop-accumulating yeast cells, better observed in G1. Histone H3S10 plays a key role in maintaining genome stability, as scored by ectopic recombination and plasmid loss, Rad52 foci, and Rad53 checkpoint activation. H3S10P coincides with the presence of DNA-RNA hybrids, is suppressed by ribonuclease H overexpression, and causes reduced accessibility of restriction endonucleases, implying a tight connection between R loops, H3S10P, and chromatin compaction. Such histone modifications were also observed in R-loop-accumulating Caenorhabditis elegans and HeLa cells. We therefore provide a role of RNA in chromatin structure essential to understand how R loops modulate genome dynamics.

  1. The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

    PubMed Central

    Soldi, Monica; Bonaldi, Tiziana

    2014-01-01

    Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations. PMID:24747196

  2. Analysis of Chromatin ADP-Ribosylation at the Genome-wide Level and at Specific Loci by ADPr-ChAP.

    PubMed

    Bartolomei, Giody; Leutert, Mario; Manzo, Massimiliano; Baubec, Tuncay; Hottiger, Michael O

    2016-02-01

    Chromatin ADP-ribosylation regulates important cellular processes. However, the exact location and magnitude of chromatin ADP-ribosylation are largely unknown. A robust and versatile method for assessing chromatin ADP-ribosylation is therefore crucial for further understanding its function. Here, we present a chromatin affinity precipitation method based on the high specificity and avidity of two well-characterized ADP-ribose binding domains to map chromatin ADP-ribosylation at the genome-wide scale and at specific loci. Our ADPr-ChAP method revealed that in cells exposed to oxidative stress, ADP-ribosylation of chromatin scales with histone density, with highest levels at heterochromatic sites and depletion at active promoters. Furthermore, in growth factor-induced adipocyte differentiation, increased chromatin ADP-ribosylation was observed at PPARγ target genes, whose expression is ADP-ribosylation dependent. In combination with deep-sequencing and conventional chromatin immunoprecipitation, the established ADPr-ChAP provides a valuable resource for the bioinformatic comparison of ADP-ribosylation with other chromatin modifications and for addressing its role in other biologically important processes. PMID:26833088

  3. Single Molecule Studies of Chromatin

    SciTech Connect

    Jeans, C; Thelen, M P; Noy, A

    2006-02-06

    In eukaryotic cells, DNA is packaged as chromatin, a highly ordered structure formed through the wrapping of the DNA around histone proteins, and further packed through interactions with a number of other proteins. In order for processes such as DNA replication, DNA repair, and transcription to occur, the structure of chromatin must be remodeled such that the necessary enzymes can access the DNA. A number of remodeling enzymes have been described, but our understanding of the remodeling process is hindered by a lack of knowledge of the fine structure of chromatin, and how this structure is modulated in the living cell. We have carried out single molecule experiments using atomic force microscopy (AFM) to study the packaging arrangements in chromatin from a variety of cell types. Comparison of the structures observed reveals differences which can be explained in terms of the cell type and its transcriptional activity. During the course of this project, sample preparation and AFM techniques were developed and optimized. Several opportunities for follow-up work are outlined which could provide further insight into the dynamic structural rearrangements of chromatin.

  4. Chromatin remodeling in nuclear cloning.

    PubMed

    Wade, Paul A; Kikyo, Nobuaki

    2002-05-01

    Nuclear cloning is a procedure to create new animals by injecting somatic nuclei into unfertilized oocytes. Recent successes in mammalian cloning with differentiated adult nuclei strongly indicate that oocyte cytoplasm contains unidentified remarkable reprogramming activities with the capacity to erase the previous memory of cell differentiation. At the heart of this nuclear reprogramming lies chromatin remodeling as chromatin structure and function define cell differentiation through regulation of the transcriptional activities of the cells. Studies involving the modification of chromatin elements such as selective uptake or release of binding proteins, covalent histone modifications including acetylation and methylation, and DNA methylation should provide significant insight into the molecular mechanisms of nuclear dedifferentiation and redifferentiation in oocyte cytoplasm.

  5. Methods to study transcription-coupled repair in chromatin.

    PubMed

    Gaillard, Hélène; Wellinger, Ralf Erik; Aguilera, Andrés

    2015-01-01

    The effect of endogenous and exogenous DNA damage on the cellular metabolism can be studied at the genetic and molecular level. A paradigmatic case is the repair of UV-induced pyrimidine dimers (PDs) by nucleotide excision repair (NER) in Saccharomyces cerevisiae. To follow the formation and repair of PDs at specific chromosome loci, cells are irradiated with UV-light and incubated in the dark to allow repair by NER. Upon DNA isolation, cyclobutane pyrimidine dimers, which account for about 90 % of PDs, can be cleaved in vitro by the DNA nicking activity of the T4 endonuclease V repair enzyme. Subsequently, strand-specific repair in a suitable restriction fragment is determined by denaturing gel electrophoresis followed by Southern blot and indirect end-labeling using a single-stranded DNA probe. Noteworthy, this protocol could potentially be adapted to other kind of DNA lesions, as long as a DNA nick is formed or a lesion-specific endonuclease is available.Transcription-coupled repair (TC-NER) is a sub-pathway of NER that catalyzes the repair of the transcribed strand of active genes. RNA polymerase II is essential for TC-NER, and its occupancy on a damaged template can be analyzed by chromatin immunoprecipitation (ChIP). In this chapter, we provide an up-dated protocol for both the DNA repair analysis and ChIP approaches to study TC-NER in yeast chromatin. PMID:25827885

  6. Integration of prolactin and glucocorticoid signaling at the beta-casein promoter and enhancer by ordered recruitment of specific transcription factors and chromatin modifiers.

    PubMed

    Kabotyanski, Elena B; Huetter, Markus; Xian, Wa; Rijnkels, Monique; Rosen, Jeffrey M

    2006-10-01

    Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides an excellent system in which to perform kinetic studies of chromatin remodeling and transcriptional activation. Using HC11 cells as a model, we have investigated the effects of prolactin (Prl) and glucocorticoids both singly and in combination at different time points after hormone treatment. Using chromatin immunoprecipitation analysis, we have determined the dynamics of assembly and disassembly of signal transducer and activator of transcription 5, glucocorticoid receptor, CCAAT enhancer binding protein beta, and Ying Yang-1 at the hormonally activated beta-casein proximal promoter as well as the distal mouse beta-casein enhancer located approximately -6 kb upstream of the transcription start site. Prl alone resulted in a rapid recruitment of both signal transducer and activator of transcription 5 and histone deacetylase 1 to the beta-casein promoter and enhancer, and reciprocally the dissociation of Ying Yang-1 from the proximal promoter. In addition, we have examined the recruitment of coactivator p300 and determined chromatin acetylation status as a function of hormonal treatment. Finally, we have established the time course of RNA polymerase II and phospho-RNA polymerase II accumulation at the beta-casein promoter and enhancer after stimulation with hydrocortisone and Prl. Although glucocorticoids alone led to a rapid increase in histone H3 acetylation, treatment with both hormones was required for stable association of p300 and phospho-RNA polymerase II at both the promoter and enhancer. Collectively, these data suggest a model for the assembly of a multiprotein complex that helps to define how the signaling pathways controlled by these lactogenic hormones are integrated to regulate beta-casein gene expression.

  7. Detection of xenoestrogens in serum after immunoprecipitation of endogenous steroidal estrogens.

    PubMed Central

    Natarajan, Kala; Overstreet, James W; Rogers, Jane M; Denison, Michael S; Chen, Jiangang; Lohstroh, Peter N; McConnell, Daniel S; Lasley, Bill L

    2002-01-01

    In this article we report a simple and efficient method for detecting nonsteroidal estrogens in a biologic sample. This method uses polyclonal antibodies to estradiol (E2) to immunoprecipitate these major biologically active steroidal estrogens, leaving behind the nonsteroidal estrogens, which are then detected in a cell-based transcriptional activation bioassay for estrogen receptor agonist. The immunoprecipitation method efficiently removed 99% of radiolabeled E2 and estrone (E1) from human serum. In experiments in which supraphysiologic concentrations of E2 and E1 to human serum, all of the immunoreactive estrogens were still removed by the immunoprecipitation protocol. We carried out an in vivo validation study of this method in which we treated female macaques with the xenoestrogen nonylphenol (NP), during the late follicular phase of the menstrual cycle. We used blood samples collected before and after treatment to evaluate and characterize endogenous and exogenous serum estrogens. An immunoassay for E2 did not detect the NP in treated monkeys. The cell-based bioassay also did not detect the estrogenic activity of NP because of its saturation by the endogenous serum steroidal estrogens. However, when steroidal estrogens were removed by immunoprecipitation, we detected the estrogenic activity of NP in the bioassay. Thus, this approach is appropriate for detecting exogenous, nonsteroidal estrogens in serum samples. PMID:12153760

  8. Role of chromatin in water stress responses in plants.

    PubMed

    Han, Soon-Ki; Wagner, Doris

    2014-06-01

    As sessile organisms, plants are exposed to environmental stresses throughout their life. They have developed survival strategies such as developmental and morphological adaptations, as well as physiological responses, to protect themselves from adverse environments. In addition, stress sensing triggers large-scale transcriptional reprogramming directed at minimizing the deleterious effect of water stress on plant cells. Here, we review recent findings that reveal a role of chromatin in water stress responses. In addition, we discuss data in support of the idea that chromatin remodelling and modifying enzymes may be direct targets of stress signalling pathways. Modulation of chromatin regulator activity by these signaling pathways may be critical in minimizing potential trade-offs between growth and stress responses. Alterations in the chromatin organization and/or in the activity of chromatin remodelling and modifying enzymes may furthermore contribute to stress memory. Mechanistic insight into these phenomena derived from studies in model plant systems should allow future engineering of broadly drought-tolerant crop plants that do not incur unnecessary losses in yield or growth.

  9. PSL, an S phase-related p55 nuclear antigen, associates transiently with chromatin.

    PubMed

    Barque, J P; Lagaye, S; Bendayan, M; Puvion-Dutilleul, F; Danon, F; Larsen, C J

    1985-03-01

    An S phase-related nuclear 55K antigen, also designated PSL, has been characterized in various mammalian cells, using a human serum from a patient with autoimmune disorders (Barque et al., EMBO j 2 (1983) 743). In this report, we show by immunoelectron microscopy that the p55 protein associates in situ with the chromatin of rat hepatocytes. This association is a transient one, as indirect immunofluorescence studies show that PSL does not bind to individualized metaphase chromosomes. Furthermore, immunoprecipitation tests indicate that the majority of PSL is in the non-chromosomal cell fraction. These results suggest that this nuclear antigen is directly involved in the DNA replication process. PMID:3882438

  10. Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

    PubMed Central

    Lin, Guangyun; Tepp, William H.; Bradshaw, Marite; Fredrick, Chase M.

    2014-01-01

    Botulinum neurotoxins (BoNTs) naturally exist as components of protein complexes containing nontoxic proteins. The nontoxic proteins impart stability of BoNTs in the gastrointestinal tract and during purification and handling. The two primary neurotoxin complexes (TCs) are (i) TC1, consisting of BoNT, nontoxin-nonhemagglutinin (NTNH), and hemagglutinins (HAs), and (ii) TC2, consisting of BoNT and NTNH (and possibly OrfX proteins). In this study, BoNT/A subtypes A1, A2, A3, and A5 were examined for the compositions of their TCs in culture extracts using immunoprecipitation (IP). IP analyses showed that BoNT/A1 and BoNT/A5 form TC1s, while BoNT/A2 and BoNT/A3 form TC2s. A Clostridium botulinum host strain expressing recombinant BoNT/A4 (normally present as a TC2) from an extrachromosomal plasmid formed a TC1 with complexing proteins from the host strain, indicating that the HAs and NTNH encoded on the chromosome associated with the plasmid-encoded BoNT/A4. Strain NCTC 2916 (A1/silent B1), which carries both an ha silent bont/b cluster and an orfX bont/a1 cluster, was also examined. IP analysis revealed that NCTC 2916 formed only a TC2 containing BoNT/A1 and its associated NTNH. No association between BoNT/A1 and the nontoxic proteins from the silent bont/b cluster was detected, although the HAs were expressed as determined by Western blotting analysis. Additionally, NTNH and HAs from the silent bont/b cluster did not form a complex in NCTC 2916. The stabilities of the two types of TC differed at various pHs and with addition of KCl and NaCl. TC1 complexes were more stable than TC2 complexes. Mouse serum stabilized TC2, while TC1 was unaffected. PMID:25362065

  11. CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor I with chromatin.

    PubMed

    Jeffery, Daniel C B; Kakusho, Naoko; You, Zhiying; Gharib, Marlene; Wyse, Brandon; Drury, Erin; Weinreich, Michael; Thibault, Pierre; Verreault, Alain; Masai, Hisao; Yankulov, Krassimir

    2015-01-01

    Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28-1 mutant and to a lesser extent in a cdc7-1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.

  12. CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

    PubMed Central

    Jeffery, Daniel CB; Kakusho, Naoko; You, Zhiying; Gharib, Marlene; Wyse, Brandon; Drury, Erin; Weinreich, Michael; Thibault, Pierre; Verreault, Alain; Masai, Hisao; Yankulov, Krassimir

    2015-01-01

    Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28–1 mutant and to a lesser extent in a cdc7–1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly. PMID:25602519

  13. Function of sperm chromatin structural elements in fertilization and development

    PubMed Central

    Ward, W. Steven

    2010-01-01

    Understanding how DNA is packaged in the mammalian sperm cell has important implications for human infertility as well as for the cell biology. Recent advances in the study of mammalian sperm chromatin structure and function have altered our perception of this highly condensed, inert chromatin. Sperm DNA is packaged very tightly to protect the DNA during the transit that occurs before fertilization. However, this condensation cannot sacrifice chromosomal elements that are essential for the embryo to access the correct sequences of the paternal genome for proper initiation of the embryonic developmental program. The primary levels of the sperm chromatin structure can be divided into three main categories: the large majority of DNA is packaged by protamines, a smaller amount (2–15%) retains histone-bound chromatin and the DNA is attached to the nuclear matrix at roughly 50 kb intervals. Current data suggest that the latter two structural elements are transferred to the paternal pronucleus after fertilization where they have important functional roles. The nuclear matrix organization is essential for DNA replication, and the histone-bound chromatin identifies genes that are important for embryonic development. These data support the emerging view of the sperm genome as providing, in addition to the paternal DNA sequence, a structural framework that includes molecular regulatory factors that are required for proper embryonic development. PMID:19748904

  14. The human chromosome. Electron microscopic observations on chromatin fiber organization.

    PubMed

    Abuelo, J G; Moore, D E

    1969-04-01

    Human lymphocytes were grown in short-term tissue culture and were arrested in metaphase with Colcemid. Their chromosomes were prepared by the Langmuir trough-critical point drying technique and were examined under the electron microscope. In addition, some chromosomes were digested with trypsin, Pronase, or DNase. The chromosomes consist entirely of tightly packed, 240 +/- 50-A chromatin fibers. Trypsin and Pronase treatments induce relaxation of fiber packing and reveal certain underlying fiber arrangements. Furthermore, trypsin treatment demonstrates that the chromatin fiber has a 25-50 A trypsin-resistant core surrounded by a trypsin-sensitive sheath. DNase digestion suggests that this core contains DNA.

  15. Chromatin and DNA sequences in defining promoters for transcription initiation.

    PubMed

    Müller, Ferenc; Tora, Làszlò

    2014-03-01

    One of the key events in eukaryotic gene regulation and consequent transcription is the assembly of general transcription factors and RNA polymerase II into a functional pre-initiation complex at core promoters. An emerging view of complexity arising from a variety of promoter associated DNA motifs, their binding factors and recent discoveries in characterising promoter associated chromatin properties brings an old question back into the limelight: how is a promoter defined? In addition to position-dependent DNA sequence motifs, accumulating evidence suggests that several parallel acting mechanisms are involved in orchestrating a pattern marked by the state of chromatin and general transcription factor binding in preparation for defining transcription start sites. In this review we attempt to summarise these promoter features and discuss the available evidence pointing at their interactions in defining transcription initiation in developmental contexts. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.

  16. The Fanconi Anemia Proteins FANCD2 and FANCJ Interact and Regulate Each Other's Chromatin Localization*

    PubMed Central

    Chen, Xiaoyong; Wilson, James B.; McChesney, Patricia; Williams, Stacy A.; Kwon, Youngho; Longerich, Simonne; Marriott, Andrew S.; Sung, Patrick; Jones, Nigel J.; Kupfer, Gary M.

    2014-01-01

    Fanconi anemia is a genetic disease resulting in bone marrow failure, birth defects, and cancer that is thought to encompass a defect in maintenance of genomic stability. Mutations in 16 genes (FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, and Q) have been identified in patients, with the Fanconi anemia subtype J (FA-J) resulting from homozygous mutations in the FANCJ gene. Here, we describe the direct interaction of FANCD2 with FANCJ. We demonstrate the interaction of FANCD2 and FANCJ in vivo and in vitro by immunoprecipitation in crude cell lysates and from fractions after gel filtration and with baculovirally expressed proteins. Mutation of the monoubiquitination site of FANCD2 (K561R) preserves interaction with FANCJ constitutively in a manner that impedes proper chromatin localization of FANCJ. FANCJ is necessary for FANCD2 chromatin loading and focus formation in response to mitomycin C treatment. Our results suggest not only that FANCD2 regulates FANCJ chromatin localization but also that FANCJ is necessary for efficient loading of FANCD2 onto chromatin following DNA damage caused by mitomycin C treatment. PMID:25070891

  17. Clinical and histological findings associated with autoantibodies detected by RNA immunoprecipitation in inflammatory myopathies.

    PubMed

    Suzuki, Shigeaki; Yonekawa, Takahiro; Kuwana, Masataka; Hayashi, Yukiko K; Okazaki, Yuka; Kawaguchi, Yasushi; Suzuki, Norihiro; Nishino, Ichizo

    2014-09-15

    Of 207 adult patients with idiopathic inflammatory myopathies, detection of autoantibodies by RNA immunoprecipitation showed that 99 patients (48%) were antibody-positive. We divided these 99 into five subgroups: anti-signal recognition particle (SRP), anti-aminoacyl transfer RNA synthetase, anti-Ku, anti-U1RNP, and anti-SSA/B. Younger age at onset, severe weakness, muscle atrophy, elevated creatine kinase, and necrosis in muscle fibers without inflammatory cell infiltration were found significantly more frequently among the patients with anti-SRP antibodies (n=41) compared to the antibody-negative patients (n=108). Autoantibody detection by RNA immunoprecipitation can provide useful information associated with clinical and histological findings.

  18. Immunoprecipitation of Cullin-RING Ligases (CRLs) in Arabidopsis thaliana Seedlings.

    PubMed

    Franciosini, Anna; Serino, Giovanna

    2016-01-01

    CRL (Cullin-RING ubiquitin ligase) is the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-RING Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure. PMID:27424742

  19. Chapter 15. Co-immunoprecipitation techniques for assessing RNA-protein interactions in vivo.

    PubMed

    Conrad, Nicholas K

    2008-01-01

    From the moment a nascent transcript emerges from an RNA polymerase until its ultimate destruction, an RNA is bound by proteins that govern its fate. Thus, in order to understand posttranscriptional regulation of gene expression, it is essential to ascertain which proteins bind a given RNA in vivo. This chapter describes three immunoprecipitation-based assays designed to query the in vivo makeup of RNA-protein complexes. Two of these, UV cross-linking and RNA immunoprecipitation (RIP), include cross-linking steps that trap complexes formed in vivo. A third, a cell mixing experiment, verifies that an interaction occurs in vivo by controlling for RNA-protein association subsequent to cell lysis. Using these protocols, this chapter presents evidence that the abundant nuclear RNA-binding protein hnRNP C interacts with the Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA in vivo. PMID:19215765

  20. Organisation of subunits in chromatin.

    PubMed

    Carpenter, B G; Baldwin, J P; Bradbury, E M; Ibel, K

    1976-07-01

    There is considerable current interest in the organisation of nucleosomes in chromatin. A strong X-ray and neutron semi-meridional diffraction peak at approximately 10 nm had previously been attributed to the interparticle specing of a linear array of nucleosomes. This diffraction peak could also result from a close packed helical array of nucleosomes. A direct test of these proposals is whether the 10 nm peak is truly meridional as would be expected for a linear array of nucleosomes or is slightly off the meridian as expected for a helical array. Neutron diffraction studies of H1-depleted chromatin support the latter alternative. The 10 nm peak has maxima which form a cross-pattern with semi-meridional angle of 8 to 9 degrees. This is consistent with a coil of nucleosomes of pitch 10 nm and outer diameter of approximately 30 nm. These dimensions correspond to about six nucleosomes per turn of the coli.

  1. Chromatin Structure in Telomere Dynamics

    PubMed Central

    Galati, Alessandra; Micheli, Emanuela; Cacchione, Stefano

    2013-01-01

    The establishment of a specific nucleoprotein structure, the telomere, is required to ensure the protection of chromosome ends from being recognized as DNA damage sites. Telomere shortening below a critical length triggers a DNA damage response that leads to replicative senescence. In normal human somatic cells, characterized by telomere shortening with each cell division, telomere uncapping is a regulated process associated with cell turnover. Nevertheless, telomere dysfunction has also been associated with genomic instability, cell transformation, and cancer. Despite the essential role telomeres play in chromosome protection and in tumorigenesis, our knowledge of the chromatin structure involved in telomere maintenance is still limited. Here we review the recent findings on chromatin modifications associated with the dynamic changes of telomeres from protected to deprotected state and their role in telomere functions. PMID:23471416

  2. Identification of the immunoproteome of the meningococcus by cell surface immunoprecipitation and MS.

    PubMed

    Newcombe, Jane; Mendum, Tom A; Ren, Chuan-peng; McFadden, Johnjoe

    2014-02-01

    Most healthy adults are protected from meningococcal disease by the presence of naturally acquired anti-meningococcal antibodies; however, the identity of the target antigens of this protective immunity remains unclear, particularly for protection against serogroup B disease. To identify the protein targets of natural protective immunity we developed an immunoprecipitation and proteomics approach to define the immunoproteome of the meningococcus. Sera from 10 healthy individuals showing serum bactericidal activity against both a meningococcal C strain (L91543) and the B strain MC58, together with commercially available pooled human sera, were used as probe antisera. Immunoprecipitation was performed with each serum sample and live cells from both meningococcal strains. Immunoprecipitated proteins were identified by MS. Analysis of the immunoproteome from each serum demonstrated both pan-reactive antigens that were recognized by most sera as well as subject-specific antigens. Most antigens were found in both meningococcal strains, but a few were strain-specific. Many of the immunoprecipitated proteins have been characterized previously as surface antigens, including adhesins and proteases, several of which have been recognized as vaccine candidate antigens, e.g. factor H-binding protein, NadA and neisserial heparin-binding antigen. The data demonstrate clearly the presence of meningococcal antibodies in healthy individuals with no history of meningococcal infection and a wide diversity of immune responses. The identification of the immunoreactive proteins of the meningococcus provides a basis for understanding the role of each antigen in the natural immunity associated with carriage and may help to design vaccination strategies. PMID:24275101

  3. Pr-specific phytochrome phosphorylation in vitro by a protein kinase present in anti-phytochrome maize immunoprecipitates

    NASA Technical Reports Server (NTRS)

    Biermann, B. J.; Pao, L. I.; Feldman, L. J.

    1994-01-01

    Protein kinase activity has repeatedly been found to co-purify with the plant photoreceptor phytochrome, suggesting that light signals received by phytochrome may be transduced or modulated through protein phosphorylation. In this study immunoprecipitation techniques were used to characterize protein kinase activity associated with phytochrome from maize (Zea mays L.). A protein kinase that specifically phosphorylated phytochrome was present in washed anti-phytochrome immunoprecipitates of etiolated coleoptile proteins. No other substrate tested was phosphorylated by this kinase. Adding salts or detergents to disrupt low-affinity protein interactions reduced background phosphorylation in immunoprecipitates without affecting phytochrome phosphorylation, indicating that the protein kinase catalytic activity is either intrinsic to the phytochrome molecule or associated with it by high-affinity interactions. Red irradiation (of coleoptiles or extracts) sufficient to approach photoconversion saturation reduced phosphorylation of immunoprecipitated phytochrome. Subsequent far-red irradiation reversed the red-light effect. Phytochrome phosphorylation was stimulated about 10-fold by a co-immunoprecipitated factor. The stimulatory factor was highest in immunoprecipitates when Mg2+ was present in immunoprecipitation reactions but remained in the supernatant in the absence of Mg2+. These observations provide strong support for the hypothesis that phytochrome-associated protein kinase modulates light responses in vivo. Since only phytochrome was found to be phosphorylated, the co-immunoprecipitated protein kinase may function to regulate receptor activity.

  4. CTCF and CohesinSA-1 Mark Active Promoters and Boundaries of Repressive Chromatin Domains in Primary Human Erythroid Cells

    PubMed Central

    Steiner, Laurie A.; Schulz, Vincent; Makismova, Yelena; Lezon-Geyda, Kimberly; Gallagher, Patrick G.

    2016-01-01

    Background CTCF and cohesinSA-1 are regulatory proteins involved in a number of critical cellular processes including transcription, maintenance of chromatin domain architecture, and insulator function. To assess changes in the CTCF and cohesinSA-1 interactomes during erythropoiesis, chromatin immunoprecipitation coupled with high throughput sequencing and mRNA transcriptome analyses via RNA-seq were performed in primary human hematopoietic stem and progenitor cells (HSPC) and primary human erythroid cells from single donors. Results Sites of CTCF and cohesinSA-1 co-occupancy were enriched in gene promoters in HSPC and erythroid cells compared to single CTCF or cohesin sites. Cell type-specific CTCF sites in erythroid cells were linked to highly expressed genes, with the opposite pattern observed in HSPCs. Chromatin domains were identified by ChIP-seq with antibodies against trimethylated lysine 27 histone H3, a modification associated with repressive chromatin. Repressive chromatin domains increased in both number and size during hematopoiesis, with many more repressive domains in erythroid cells than HSPCs. CTCF and cohesinSA-1 marked the boundaries of these repressive chromatin domains in a cell-type specific manner. Conclusion These genome wide data, changes in sites of protein occupancy, chromatin architecture, and related gene expression, support the hypothesis that CTCF and cohesinSA-1 have multiple roles in the regulation of gene expression during erythropoiesis including transcriptional regulation at gene promoters and maintenance of chromatin architecture. These data from primary human erythroid cells provide a resource for studies of normal and perturbed erythropoiesis. PMID:27219007

  5. Cdt1-binding protein GRWD1 is a novel histone-binding protein that facilitates MCM loading through its influence on chromatin architecture

    PubMed Central

    Sugimoto, Nozomi; Maehara, Kazumitsu; Yoshida, Kazumasa; Yasukouchi, Shuhei; Osano, Satoko; Watanabe, Shinya; Aizawa, Masahiro; Yugawa, Takashi; Kiyono, Tohru; Kurumizaka, Hitoshi; Ohkawa, Yasuyuki; Fujita, Masatoshi

    2015-01-01

    Efficient pre-replication complex (pre-RC) formation on chromatin templates is crucial for the maintenance of genome integrity. However, the regulation of chromatin dynamics during this process has remained elusive. We found that a conserved protein, GRWD1 (glutamate-rich WD40 repeat containing 1), binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner, and that depletion of GRWD1 reduces loading of MCM but not CDC6 and Cdt1. Furthermore, chromatin immunoprecipitation coupled with high-throughput sequencing (Seq) revealed significant genome-wide co-localization of GRWD1 with CDC6. We found that GRWD1 has histone-binding activity. To investigate the effect of GRWD1 on chromatin architecture, we used formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq or FAIRE-quantitative PCR analyses, and the results suggest that GRWD1 regulates chromatin openness at specific chromatin locations. Taken together, these findings suggest that GRWD1 may be a novel histone-binding protein that regulates chromatin dynamics and MCM loading at replication origins. PMID:25990725

  6. Trichomonas vaginalis: chromatin and mitotic spindle during mitosis.

    PubMed

    Gómez-Conde, E; Mena-López, R; Hernández-Jaúregui, P; González-Camacho, M; Arroyo, R

    2000-11-01

    The mitotic phases and the changes that the chromatin and mitotic microtubules undergo during mitosis in the sexually transmitted parasite Trichomonas vaginalis are described. Parasites arrested in the gap 2 phase of the cell cycle by nutrient starvation were induced to mitosis by addition of fresh whole medium. [(3)H] Thymidine labeling of trichomonad parasites for 24 h showed that parasites have at least four synchronic duplications after mitosis induction. Fixed or live and acridine orange (AO)-stained trichomonads analyzed at different times during mitosis by epifluorescence microscopy showed that mitosis took about 45 min and is divided into five stages: prophase, metaphase, early and late anaphase, early and late telophase, and cytokinesis. The AO-stained nucleus of live trichomonads showed green (DNA) and orange (RNA) fluorescence, and the nucleic acid nature was confirmed by DNase and RNase treatment, respectively. The chromatin appeared partially condensed during interphase. At metaphase, it appeared as six condensed chromosomes, as recently reported, which decondensed at anaphase and migrated to the nuclear poles at telophase. In addition, small bundles of microtubules (as hemispindles) were detected only in metaphase with the polyclonal antibody anti-Entamoeba histolytica alpha-tubulin. This antibody showed that the hemispindle and an atractophore-like structure seem to duplicate and polarize during metaphase. In conclusion, T. vaginalis mitosis involves five mitotic phases in which the chromatin undergoes different degrees of condensation, from chromosomes to decondensed chromatin, and two hemispindles that are observed only in the metaphase stage. PMID:11162363

  7. Trichomonas vaginalis: chromatin and mitotic spindle during mitosis.

    PubMed

    Gómez-Conde, E; Mena-López, R; Hernández-Jaúregui, P; González-Camacho, M; Arroyo, R

    2000-11-01

    The mitotic phases and the changes that the chromatin and mitotic microtubules undergo during mitosis in the sexually transmitted parasite Trichomonas vaginalis are described. Parasites arrested in the gap 2 phase of the cell cycle by nutrient starvation were induced to mitosis by addition of fresh whole medium. [(3)H] Thymidine labeling of trichomonad parasites for 24 h showed that parasites have at least four synchronic duplications after mitosis induction. Fixed or live and acridine orange (AO)-stained trichomonads analyzed at different times during mitosis by epifluorescence microscopy showed that mitosis took about 45 min and is divided into five stages: prophase, metaphase, early and late anaphase, early and late telophase, and cytokinesis. The AO-stained nucleus of live trichomonads showed green (DNA) and orange (RNA) fluorescence, and the nucleic acid nature was confirmed by DNase and RNase treatment, respectively. The chromatin appeared partially condensed during interphase. At metaphase, it appeared as six condensed chromosomes, as recently reported, which decondensed at anaphase and migrated to the nuclear poles at telophase. In addition, small bundles of microtubules (as hemispindles) were detected only in metaphase with the polyclonal antibody anti-Entamoeba histolytica alpha-tubulin. This antibody showed that the hemispindle and an atractophore-like structure seem to duplicate and polarize during metaphase. In conclusion, T. vaginalis mitosis involves five mitotic phases in which the chromatin undergoes different degrees of condensation, from chromosomes to decondensed chromatin, and two hemispindles that are observed only in the metaphase stage.

  8. Diet-mediated alteration of chromatin structure.

    PubMed

    Castro, C E; Armstrong-Major, J; Ramirez, M E

    1986-08-01

    Higher-order chromatin structure and the process of transcription are related. The significance of a nutritional state's altering chromatin structure lies in the potential role of that nutritional state in the regulation of gene expression. In rats short-term feeding of semisynthetic diets varying in the proportion of carbohydrate, protein, or fat alters the configuration of liver chromatin as measured by sensitivity to micrococcal nuclease (EC 3.1.31.1). A carbohydrate-rich, fat-free diet increases the sensitivity of rat liver chromatin to micrococcal nuclease and decreases the nucleosome repeat length. In contrast, a protein-free diet or a diet deficient in magnesium or zinc decreases the sensitivity of liver nuclear chromatin to micrococcal nuclease. Diet-mediated mechanisms that alter chromatin structure are now unknown, but the continued study of nutritional interaction with the genome should identify the responsible features as well as their significance to gene function.

  9. Proteomics of a fuzzy organelle: interphase chromatin

    PubMed Central

    Kustatscher, Georg; Hégarat, Nadia; Wills, Karen L H; Furlan, Cristina; Bukowski-Wills, Jimi-Carlo; Hochegger, Helfrid; Rappsilber, Juri

    2014-01-01

    Chromatin proteins mediate replication, regulate expression, and ensure integrity of the genome. So far, a comprehensive inventory of interphase chromatin has not been determined. This is largely due to its heterogeneous and dynamic composition, which makes conclusive biochemical purification difficult, if not impossible. As a fuzzy organelle, it defies classical organellar proteomics and cannot be described by a single and ultimate list of protein components. Instead, we propose a new approach that provides a quantitative assessment of a protein's probability to function in chromatin. We integrate chromatin composition over a range of different biochemical and biological conditions. This resulted in interphase chromatin probabilities for 7635 human proteins, including 1840 previously uncharacterized proteins. We demonstrate the power of our large-scale data-driven annotation during the analysis of cyclin-dependent kinase (CDK) regulation in chromatin. Quantitative protein ontologies may provide a general alternative to list-based investigations of organelles and complement Gene Ontology. PMID:24534090

  10. Pericentric chromatin loops function as a nonlinear spring in mitotic force balance

    PubMed Central

    Stephens, Andrew D.; Haggerty, Rachel A.; Vasquez, Paula A.; Vicci, Leandra; Snider, Chloe E.; Shi, Fu; Quammen, Cory; Mullins, Christopher; Haase, Julian; Taylor, Russell M.; Verdaasdonk, Jolien S.; Falvo, Michael R.; Jin, Yuan; Forest, M. Gregory

    2013-01-01

    The mechanisms by which sister chromatids maintain biorientation on the metaphase spindle are critical to the fidelity of chromosome segregation. Active force interplay exists between predominantly extensional microtubule-based spindle forces and restoring forces from chromatin. These forces regulate tension at the kinetochore that silences the spindle assembly checkpoint to ensure faithful chromosome segregation. Depletion of pericentric cohesin or condensin has been shown to increase the mean and variance of spindle length, which have been attributed to a softening of the linear chromatin spring. Models of the spindle apparatus with linear chromatin springs that match spindle dynamics fail to predict the behavior of pericentromeric chromatin in wild-type and mutant spindles. We demonstrate that a nonlinear spring with a threshold extension to switch between spring states predicts asymmetric chromatin stretching observed in vivo. The addition of cross-links between adjacent springs recapitulates coordination between pericentromeres of neighboring chromosomes. PMID:23509068

  11. Pericentric chromatin loops function as a nonlinear spring in mitotic force balance.

    PubMed

    Stephens, Andrew D; Haggerty, Rachel A; Vasquez, Paula A; Vicci, Leandra; Snider, Chloe E; Shi, Fu; Quammen, Cory; Mullins, Christopher; Haase, Julian; Taylor, Russell M; Verdaasdonk, Jolien S; Falvo, Michael R; Jin, Yuan; Forest, M Gregory; Bloom, Kerry

    2013-03-18

    The mechanisms by which sister chromatids maintain biorientation on the metaphase spindle are critical to the fidelity of chromosome segregation. Active force interplay exists between predominantly extensional microtubule-based spindle forces and restoring forces from chromatin. These forces regulate tension at the kinetochore that silences the spindle assembly checkpoint to ensure faithful chromosome segregation. Depletion of pericentric cohesin or condensin has been shown to increase the mean and variance of spindle length, which have been attributed to a softening of the linear chromatin spring. Models of the spindle apparatus with linear chromatin springs that match spindle dynamics fail to predict the behavior of pericentromeric chromatin in wild-type and mutant spindles. We demonstrate that a nonlinear spring with a threshold extension to switch between spring states predicts asymmetric chromatin stretching observed in vivo. The addition of cross-links between adjacent springs recapitulates coordination between pericentromeres of neighboring chromosomes.

  12. Targeting chromatin binding regulation of constitutively active AR variants to overcome prostate cancer resistance to endocrine-based therapies

    PubMed Central

    Chan, Siu Chiu; Selth, Luke A.; Li, Yingming; Nyquist, Michael D.; Miao, Lu; Bradner, James E.; Raj, Ganesh V.; Tilley, Wayne D.; Dehm, Scott M.

    2015-01-01

    Androgen receptor (AR) variants (AR-Vs) expressed in prostate cancer (PCa) lack the AR ligand binding domain (LBD) and function as constitutively active transcription factors. AR-V expression in patient tissues or circulating tumor cells is associated with resistance to AR-targeting endocrine therapies and poor outcomes. Here, we investigated the mechanisms governing chromatin binding of AR-Vs with the goal of identifying therapeutic vulnerabilities. By chromatin immunoprecipitation and sequencing (ChIP-seq) and complementary biochemical experiments, we show that AR-Vs display a binding preference for the same canonical high-affinity androgen response elements (AREs) that are preferentially engaged by AR, albeit with lower affinity. Dimerization was an absolute requirement for constitutive AR-V DNA binding and transcriptional activation. Treatment with the bromodomain and extraterminal (BET) inhibitor JQ1 resulted in inhibition of AR-V chromatin binding and impaired AR-V driven PCa cell growth in vitro and in vivo. Importantly, this was associated with a novel JQ1 action of down-regulating AR-V transcript and protein expression. Overall, this study demonstrates that AR-Vs broadly restore AR chromatin binding events that are otherwise suppressed during endocrine therapy, and provides pre-clinical rationale for BET inhibition as a strategy for inhibiting expression and chromatin binding of AR-Vs in PCa. PMID:25908785

  13. Investigation of Viral and Host Chromatin by ChIP-PCR or ChIP-Seq Analysis.

    PubMed

    Günther, Thomas; Theiss, Juliane M; Fischer, Nicole; Grundhoff, Adam

    2016-01-01

    Complex regulation of viral transcription patterns and DNA replication levels is a feature of many DNA viruses. This is especially true for those viruses which establish latent or persistent infections (e.g., herpesviruses, papillomaviruses, polyomaviruses, or adenovirus), as long-term persistence often requires adaptation of gene expression programs and/or replication levels to the cellular milieu. A key factor in the control of such processes is the establishment of a specific chromatin state on promoters or replication origins, which in turn will determine whether or not the underlying DNA is accessible for other factors that mediate downstream processes. Chromatin immunoprecipitation (ChIP) is a powerful technique to investigate viral chromatin, in particular to study binding patterns of modified histones, transcription factors or other DNA-/chromatin-binding proteins that regulate the viral lifecycle. Here, we provide protocols that are suitable for performing ChIP-PCR and ChIP-Seq studies on chromatin of large and small viral genomes. PMID:26855283

  14. Investigation of Viral and Host Chromatin by ChIP-PCR or ChIP-Seq Analysis.

    PubMed

    Günther, Thomas; Theiss, Juliane M; Fischer, Nicole; Grundhoff, Adam

    2016-02-08

    Complex regulation of viral transcription patterns and DNA replication levels is a feature of many DNA viruses. This is especially true for those viruses which establish latent or persistent infections (e.g., herpesviruses, papillomaviruses, polyomaviruses, or adenovirus), as long-term persistence often requires adaptation of gene expression programs and/or replication levels to the cellular milieu. A key factor in the control of such processes is the establishment of a specific chromatin state on promoters or replication origins, which in turn will determine whether or not the underlying DNA is accessible for other factors that mediate downstream processes. Chromatin immunoprecipitation (ChIP) is a powerful technique to investigate viral chromatin, in particular to study binding patterns of modified histones, transcription factors or other DNA-/chromatin-binding proteins that regulate the viral lifecycle. Here, we provide protocols that are suitable for performing ChIP-PCR and ChIP-Seq studies on chromatin of large and small viral genomes.

  15. Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

    PubMed Central

    Gentsch, George E.; Patrushev, Ilya; Smith, James C.

    2015-01-01

    The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available. PMID:25742027

  16. Targeting chromatin binding regulation of constitutively active AR variants to overcome prostate cancer resistance to endocrine-based therapies.

    PubMed

    Chan, Siu Chiu; Selth, Luke A; Li, Yingming; Nyquist, Michael D; Miao, Lu; Bradner, James E; Raj, Ganesh V; Tilley, Wayne D; Dehm, Scott M

    2015-07-13

    Androgen receptor (AR) variants (AR-Vs) expressed in prostate cancer (PCa) lack the AR ligand binding domain (LBD) and function as constitutively active transcription factors. AR-V expression in patient tissues or circulating tumor cells is associated with resistance to AR-targeting endocrine therapies and poor outcomes. Here, we investigated the mechanisms governing chromatin binding of AR-Vs with the goal of identifying therapeutic vulnerabilities. By chromatin immunoprecipitation and sequencing (ChIP-seq) and complementary biochemical experiments, we show that AR-Vs display a binding preference for the same canonical high-affinity androgen response elements (AREs) that are preferentially engaged by AR, albeit with lower affinity. Dimerization was an absolute requirement for constitutive AR-V DNA binding and transcriptional activation. Treatment with the bromodomain and extraterminal (BET) inhibitor JQ1 resulted in inhibition of AR-V chromatin binding and impaired AR-V driven PCa cell growth in vitro and in vivo. Importantly, this was associated with a novel JQ1 action of down-regulating AR-V transcript and protein expression. Overall, this study demonstrates that AR-Vs broadly restore AR chromatin binding events that are otherwise suppressed during endocrine therapy, and provides pre-clinical rationale for BET inhibition as a strategy for inhibiting expression and chromatin binding of AR-Vs in PCa.

  17. Mechanism of the Interaction of Plant Alkaloid Vincristine with DNA and Chromatin: Spectroscopic Study

    PubMed Central

    Mohammadgholi, Azadeh; Fallah, Sodabeh

    2013-01-01

    Chromatin has been successfully used as a tool for the study of genome function in cancers. Vincristine as a vinca alkaloid anticancer drug exerts its action by binding to tubulins. In this study the effect of vincristine on DNA and chromatin was investigated employing various spectroscopy techniques as well as thermal denaturation, equilibrium dialysis and DNA–cellulose affinity. The results showed that the binding of vincristine to DNA and chromatin reduced absorbance at both 260 and 210 nm with different extent. Chromopheres of chromatin quenched with the drug and fluorescence emission intensity decreased in a dose-dependent manner. Chromatin exhibited higher emission intensity changes compared to DNA. Upon addition of vincristine, Tm of DNA and chromatin exhibited hypochromicity without any shift in Tm. The binding of the drug induced structural changes in both positive and negative extremes of circular dichroism spectra and exhibited a cooperative binding pattern as illustrated by a positive slope observed in low r values of the binding isotherm. Vincristine showed higher binding affinity to double stranded DNA compared to single stranded one. The results suggest that vincristine binds with higher affinity to chromatin compared to DNA. The interaction is through intercalation along with binding to phosphate sugar backbone and histone proteins play fundamental role in this process. The binding of the drug to chromatin opens a new insight into vincristine action in the cell nucleus. PMID:23590199

  18. Chd1 remodelers maintain open chromatin and regulate the epigenetics of differentiation

    SciTech Connect

    Persson, Jenna; Ekwall, Karl

    2010-05-01

    Eukaryotic DNA is packaged around octamers of histone proteins into nucleosomes, the basic unit of chromatin. In addition to enabling meters of DNA to fit within the confines of a nucleus, the structure of chromatin has functional implications for cell identity. Covalent chemical modifications to the DNA and to histones, histone variants, ATP-dependent chromatin remodelers, small noncoding RNAs and the level of chromatin compaction all contribute to chromosomal structure and to the activity or silencing of genes. These chromatin-level alterations are defined as epigenetic when they are heritable from mother to daughter cell. The great diversity of epigenomes that can arise from a single genome permits a single, totipotent cell to generate the hundreds of distinct cell types found in humans. Two recent studies in mouse and in fly have highlighted the importance of Chd1 chromatin remodelers for maintaining an open, active chromatin state. Based on evidence from fission yeast as a model system, we speculate that Chd1 remodelers are involved in the disassembly of nucleosomes at promoter regions, thus promoting active transcription and open chromatin. It is likely that these nucleosomes are specifically marked for disassembly by the histone variant H2A.Z.

  19. Genome-wide analysis of methylation in bovine clones by methylated DNA immunoprecipitation (MeDIP).

    PubMed

    Kiefer, Hélène

    2015-01-01

    Methylated DNA immunoprecipitation (MeDIP), when coupled to high-throughput sequencing or microarray hybridization, allows for the identification of methylated loci at a genome-wide scale. Genomic regions affected by incomplete reprogramming after nuclear transfer can potentially be delineated by comparing the MeDIP profiles of bovine clones and non-clones. This chapter presents a MeDIP protocol largely inspired from Mohn and colleagues (Mohn et al., Methods Mol Biol 507:55-64, 2009), with PCR primers specific for cattle, and when possible, overviews of experimental designs adapted to the comparison between clones and non-clones.

  20. Quantitation of haptens by homogeneous immunoprecipitation. 2. Centrifugal analysis for tobramycin, phenobarbital, and theophylline in serum.

    PubMed

    Wu, J W; Bunyagidj, C; Hoskin, S; Riebe, S M; Aucker, J; White, K; O'Neill, S P

    1983-08-01

    Our homogeneous immunoprecipitation inhibition assay (Clin. Chem. 28:659-661, 1982) is applied here to tobramycin, phenobarbital, and theophylline. Only 10-50 microL of test sample is needed. No sample treatment, dilution, or extraction is required. A test serum sample is simultaneously mixed with a drug conjugate and its specific antiserum in a centrifugal analyzer. The subsequent reaction and the measurement are completed in 3 min. Within-run and between-run CVs for clinically relevant concentrations were well below 10%. Results for patients' samples correlated well with those by enzyme immunoassay.

  1. A model for chromatin structure.

    PubMed Central

    Li, H J

    1975-01-01

    A model for chromatin structure is presented. (a) Each of four histone species, H2A (IIbl or f2a2), H2B (IIb2 or f2b), H3 (III or f3) and H4 (IV or f2al) can form a parallel dimer. (b) These dimers can form two tetramers, (H2A)2(H2b)2 and (H3)2(H4)2. (C) These two tetramers bind a segment of DNA and condense it into a "C" segments. (d) The adjacent segments, termed extended or "E" segments, are bound by histone H1 (I or fl) for the major fraction of chromatin; the other "E" regions can be either bound by non-histone proteins or free of protein binding. (e) The binding of histones causes a structural distortion of the DNA which, depending upon the external conditions, may generate the formation of either an open structure with a heterogeneous and non-uniform supercoil or a compact structure with a string of beads. The model is supported by experimental data on histone-histone interaction, histone-DNA interaction and histone subunit-DNA interaction. PMID:1101222

  2. Promoter-Targeted Histone Acetylation of Chromatinized Parvoviral Genome Is Essential for the Progress of Infection

    PubMed Central

    Mäntylä, Elina; Salokas, Kari; Oittinen, Mikko; Aho, Vesa; Mäntysaari, Pekka; Palmujoki, Lassi; Kalliolinna, Olli; Ihalainen, Teemu O.; Niskanen, Einari A.; Timonen, Jussi

    2016-01-01

    ABSTRACT The association of host histones with parvoviral DNA is poorly understood. We analyzed the chromatinization and histone acetylation of canine parvovirus DNA during infection by confocal imaging and in situ proximity ligation assay combined with chromatin immunoprecipitation and high-throughput sequencing. We found that during late infection, parvovirus replication bodies were rich in histones bearing modifications characteristic of transcriptionally active chromatin, i.e., histone H3 lysine 27 acetylation (H3K27ac). H3K27ac, in particular, was located in close proximity to the viral DNA-binding protein NS1. Importantly, our results show for the first time that in the chromatinized parvoviral genome, the two viral promoters in particular were rich in H3K27ac. Histone acetyltransferase (HAT) inhibitors efficiently interfered with the expression of viral proteins and infection progress. Altogether, our data suggest that the acetylation of histones on parvoviral DNA is essential for viral gene expression and the completion of the viral life cycle. IMPORTANCE Viral DNA introduced into cell nuclei is exposed to cellular responses to foreign DNA, including chromatinization and epigenetic silencing, both of which determine the outcome of infection. How the incoming parvovirus resists cellular epigenetic downregulation of its genes is not understood. Here, the critical role of epigenetic modifications in the regulation of parvovirus infection was demonstrated. We showed for the first time that a successful parvovirus infection is characterized by the deposition of nucleosomes with active histone acetylation on the viral promoter areas. The results provide new insights into the regulation of parvoviral gene expression, which is an important aspect of the development of parvovirus-based virotherapy. PMID:26842481

  3. Effect of saffron on rat sperm chromatin integrity

    PubMed Central

    Mardani, Mohammad; Vaez, Ahmad; Razavi, Shahnaz

    2014-01-01

    Background: Currently, relation between reactive oxygen species (ROS) ROS concentration and semen quality was indicated. Saffron has traditionally been not only considered as a food additive but also as a medicinal herb, which has a good antioxidant properties. Objective: The aim of this study was to evaluate the protection potency of saffron and vitamin E on sperm chromatin integrity. Materials and Methods: Thirty adult male Wistar rats divided equally into saffron (100 mg/kg), vitamin E (100 mg/kg) and control (0.5cc distilled water /day) groups. After 60 days, cauda epididymis dissected and sperm cells were used for analysis of sperm chromatin packaging by chromomycin A3 (CMA3) staining, and sperm chromatin susceptibility to acid denaturation by acridine orange (AO) staining. Results: The mean percentage of CMA3 positive sperm was significantly decreased in saffron and vitamin E groups relative to control group (p<0.001). Moreover, the AO staining results showed that the mean percentage of sperm with DNA damage was significantly decreased in saffron and vitamin E groups as compared with control group (p<0.001). Conclusion: Our results purposed that saffron can protect sperm against DNA damage and chromatin anomalies. PMID:25031579

  4. Control of trichome branching by Chromatin Assembly Factor-1

    PubMed Central

    Exner, Vivien; Gruissem, Wilhelm; Hennig, Lars

    2008-01-01

    Background Chromatin dynamics and stability are both required to control normal development of multicellular organisms. Chromatin assembly factor CAF-1 is a histone chaperone that facilitates chromatin formation and the maintenance of specific chromatin states. In plants and animals CAF-1 is essential for normal development, but it is poorly understood which developmental pathways require CAF-1 function. Results Mutations in all three CAF-1 subunits affect Arabidopsis trichome morphology and lack of CAF-1 function results in formation of trichomes with supernumerary branches. This phenotype can be partially alleviated by external sucrose. In contrast, other aspects of the CAF-1 mutant phenotype, such as defective meristem function and organ formation, are aggravated by external sucrose. Double mutant analyses revealed epistatic interactions between CAF-1 mutants and stichel, but non-epistatic interactions between CAF-1 mutants and glabra3 and kaktus. In addition, mutations in CAF-1 could partly suppress the strong overbranching and polyploidization phenotype of kaktus mutants. Conclusion CAF-1 is required for cell differentiation and regulates trichome development together with STICHEL in an endoreduplication-independent pathway. This function of CAF-1 can be partially substituted by application of exogenous sucrose. Finally, CAF-1 is also needed for the high degree of endoreduplication in kaktus mutants and thus for the realization of kaktus' extreme overbranching phenotype. PMID:18477400

  5. Characterization of human platelet glycoprotein antigens giving rise to individual immunoprecipitates in crossed-immunoelectrophoresis

    SciTech Connect

    Kunicki, T.J.; Nurden, A.T.; Pidard, D.; Russell, N.R.; Caen, J.P.

    1981-12-01

    Washed human platelets were labeled with 125I by the lactoperoxidase-catalyzed method and solubilized in 1% Triton X-100. The soluble proteins were analyzed by crossed-immunoelectrophoresis in 1% agarose, employing a rabbit antibody raised against whole human platelets. Analysis of autoradiograms developed from dried agarose gels led to the establishment of a normal reference pattern that was consistent for platelets obtained from more than 50 normal individuals. Six platelet membrane glycoprotein antigens contained in four distinguishable precipitates were identified. Each identification was based on direct sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 125I-antigens contained in individually excised precipitates. These platelet antigens include major membrane glycoproteins previously designated la, lb, lla, llb, llla, and lllb. Glycoproteins llb and llla were shown to be contained in a single immunoprecipitate, while glycoproteins la and lla were routinely detected in a single different immunoprecipitate. Analysis of soluble proteins from platelets of five patients with Glanzmann's thrombasthenia demonstrated either a complete absence or a marked reduction of only one radiolabeled precipitate, that containing membrane glycoproteins llb and llla. Platelet samples from two patients with Bernard-Soulier syndrome were devoid of a different precipitate, that containing membrane glycoprotein lb.

  6. A Yeast Display Immunoprecipitation Method for Efficient Isolation and Characterization of Antigens

    PubMed Central

    Cho, Yong Ku; Chen, Irene; Wei, Xin; Li, Lingjun; Shusta, Eric V.

    2009-01-01

    Yeast antibody display has found a wide variety of applications including antibody affinity maturation, epitope mapping, and library screening. Here we report a yeast display immunoprecipitation (YDIP) technique that employs yeast cells displaying single-chain antibody fragments (scFv) on their surface as affinity capture reagents to isolate and characterize antigens. We show that displayed single-chain antibody fragments are active in a variety of detergent solutions commonly used for immunoprecipitation and that the antigen-antibody interaction can be accurately quantified by YDIP coupled with flow cytometry. The YDIP method has also been optimized so that it is compatible with commonly used protein characterization tools such as Western blotting, silver staining, and mass spectrometry. From complex protein mixtures, we have used YDIP to isolate, analyze and sequence both soluble and plasma membrane antigens using tandem mass spectrometry. In the case of the membrane antigen, YDIP coupled with tandem mass spectrometry was successful in identifying neural cell adhesion molecule (NCAM) as the antigen for an antibody previously selected as binding to the plasma membranes of endothelial cells. The presented method therefore has potential to facilitate antibody-antigen characterization. PMID:19041873

  7. Characterization of chick liver chromatin and analysis of its in vitro transcription products

    PubMed Central

    Dierks-Ventling, Christa; Stalder, Jürg; Gautschi, Johannes

    1978-01-01

    Carefully controlled preparation of chromatin from purified chick liver nuclei yielded over 50% native chromatin as shown by the analysis of the nucleosome pattern after micrococcal nuclease digestion. The size of DNA in this chromatin as analyzed on alkaline sucrose gradients varied from 10S to 19S, the majority being 14S. All endogenous RNA polymerases were represented in the chromatin preparation although to different extents: RNA polymerase I was the most and RNA polymerase II the least abundant. Initiation studies showed that endogenous RNA polymerase II was capable of initiating RNA chains during 5 min. Saturation of chromatin with purified homologous RNA polymerase II increased initiation to 10 min. The addition of heparin caused the RNA transcribed to be larger in size and of increased yield. Chromatin transcription with added purified RNA polymerase II in the presence of heparin produced RNA as large as 32S. A chromatin preparation of this kind would therefore be suitable to transcribe any eukaryotic gene invitro provided additional homologous RNA polymerase II is used. Images PMID:566911

  8. Chromatin Remodelers: From Function to Dysfunction.

    PubMed

    Längst, Gernot; Manelyte, Laura

    2015-01-01

    Chromatin remodelers are key players in the regulation of chromatin accessibility and nucleosome positioning on the eukaryotic DNA, thereby essential for all DNA dependent biological processes. Thus, it is not surprising that upon of deregulation of those molecular machines healthy cells can turn into cancerous cells. Even though the remodeling enzymes are very abundant and a multitude of different enzymes and chromatin remodeling complexes exist in the cell, the particular remodeling complex with its specific nucleosome positioning features must be at the right place at the right time in order to ensure the proper regulation of the DNA dependent processes. To achieve this, chromatin remodeling complexes harbor protein domains that specifically read chromatin targeting signals, such as histone modifications, DNA sequence/structure, non-coding RNAs, histone variants or DNA bound interacting proteins. Recent studies reveal the interaction between non-coding RNAs and chromatin remodeling complexes showing importance of RNA in remodeling enzyme targeting, scaffolding and regulation. In this review, we summarize current understanding of chromatin remodeling enzyme targeting to chromatin and their role in cancer development. PMID:26075616

  9. Open chromatin reveals the functional maize genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Every cellular process mediated through nuclear DNA must contend with chromatin. As results from ENCODE show, open chromatin assays can efficiently integrate across diverse regulatory elements, revealing functional non-coding genome. In this study, we use a MNase hypersensitivity assay to discover o...

  10. Expression-Dependent Folding of Interphase Chromatin

    PubMed Central

    Jerabek, Hansjoerg; Heermann, Dieter W.

    2012-01-01

    Multiple studies suggest that chromatin looping might play a crucial role in organizing eukaryotic genomes. To investigate the interplay between the conformation of interphase chromatin and its transcriptional activity, we include information from gene expression profiles into a polymer model for chromatin that incorporates genomic loops. By relating loop formation to transcriptional activity, we are able to generate chromosome conformations whose structural and topological properties are consistent with experimental data. The model particularly allows to reproduce the conformational variations that are known to occur between highly and lowly expressed chromatin regions. As previously observed in experiments, lowly expressed regions of the simulated polymers are much more compact. Due to the changes in loop formation, the distributions of chromatin loops are also expression-dependent and exhibit a steeper decay in highly active regions. As a results of entropic interaction between differently looped parts of the chromosome, we observe topological alterations leading to a preferential positioning of highly transcribed loci closer to the surface of the chromosome territory. Considering the diffusional behavior of the chromatin fibre, the simulations furthermore show that the higher the expression level of specific parts of the chromatin fibre is, the more dynamic they are. The results exhibit that variations of loop formation along the chromatin fibre, and the entropic changes that come along with it, do not only influence the structural parameters on the local scale, but also effect the global chromosome conformation and topology. PMID:22649534

  11. Chromatin conformation in living cells: support for a zig-zag model of the 30 nm chromatin fiber

    NASA Technical Reports Server (NTRS)

    Rydberg, B.; Holley, W. R.; Mian, I. S.; Chatterjee, A.

    1998-01-01

    A new method was used to probe the conformation of chromatin in living mammalian cells. The method employs ionizing radiation and is based on the concept that such radiation induces correlated breaks in DNA strands that are in spatial proximity. Human dermal fibroblasts in G0 phase of the cell cycle and Chinese hamster ovary cells in mitosis were irradiated by X-rays or accelerated ions. Following lysis of the cells, DNA fragments induced by correlated breaks were end-labeled and separated according to size on denaturing polyacrylamide gels. A characteristic peak was obtained for a fragment size of 78 bases, which is the size that corresponds to one turn of DNA around the nucleosome. Additional peaks between 175 and 450 bases reflect the relative position of nearest-neighbor nucleosomes. Theoretical calculations that simulate the indirect and direct effect of radiation on DNA demonstrate that the fragment size distributions are closely related to the chromatin structure model used. Comparison of the experimental data with theoretical results support a zig-zag model of the chromatin fiber rather than a simple helical model. Thus, radiation-induced damage analysis can provide information on chromatin structure in the living cell. Copyright 1998 Academic Press.

  12. Control of RNA synthesis by chromatin proteins.

    PubMed Central

    Cedar, H; Solage, A; Zurucki, F

    1976-01-01

    The effect of chromatin proteins on template activity has been studied. Using both E. coli RNA polymerase and calf thymmus polymerase B we have measured the number of initiation sites on chromatin and various histone-DNA complexes. Chromatin can be reconstituted with histone proteins alone and this complex is still a restricted template for RNA synthesis. The removal of histone f1 causes a large increase in the template activity. Chromatin is then treated with Micrococcal nuclease and the DNA fragments protected from nuclease attack ("covered DNA") are isolated. Alternatively, the chromatin is titrated with poly-D-lysine, and by successive treatment with Pronase and nuclease, the DNA regions accessible to polylysine are isolated ("open DNA"). Both fractions were tested for template activity. It was found that RNA polymerase initiation sites are distributed equally in open and covered region DNA. PMID:787926

  13. Progesterone receptor directly inhibits β-casein gene transcription in mammary epithelial cells through promoting promoter and enhancer repressive chromatin modifications.

    PubMed

    Buser, Adam C; Obr, Alison E; Kabotyanski, Elena B; Grimm, Sandra L; Rosen, Jeffrey M; Edwards, Dean P

    2011-06-01

    Differentiated HC-11 cells ectopically expressing progesterone receptor (PR) were used to explore the molecular mechanisms by which progesterone suppresses β-casein gene transcription induced by prolactin (PRL) and glucocorticoids in the mammary gland. As detected by chromatin immunoprecipitation assays, treatment of cells with the progestin agonist R5020 induced a rapid recruitment (5 min) of PR to the proximal promoter (-235 bp) and distal enhancer (-6 kb upstream of transcription start site) of β-casein. PR remained bound for 4 h and was dissociated by 24 h after treatment. Despite efficient binding, the hormone agonist-occupied PR did not stimulate transcription of the β-casein gene. Recruitment of signal transducer and activator of transcription 5a, glucocorticoid receptor, and the CCAAT enhancer binding protein β to the enhancer and proximal promoter of β-casein induced by PRL and glucocorticoids was blocked by progestin cotreatment, whereas PR binding was induced under these conditions. PRL/glucocorticoid-induced histone acetylation and the recruitment of the coactivator p300 and RNA polymerase II required for gene activation were also inhibited by progestin. In addition, progestin prevented dissociation of the corepressors Yin and Yang 1 and histone deacetylase 3 from the promoter, and demethylation of lysine 9 of histone 3 induced by PRL and glucocorticoids. These studies are consistent with the conclusion that progesterone interferes with PRL/glucocorticoid induction of β-casein transcription by a physical interaction of PR with the promoter and enhancer that blocks assembly of a transcriptional activation complex and dissociation of corepressors and promotes repressive chromatin modifications. These studies define a novel mechanism of steroid receptor-mediated transcriptional repression of a physiologically important gene in mammary gland development and differentiation.

  14. Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin.

    PubMed

    Kaneko, Syuzo; Bonasio, Roberto; Saldaña-Meyer, Ricardo; Yoshida, Takahaki; Son, Jinsook; Nishino, Koichiro; Umezawa, Akihiro; Reinberg, Danny

    2014-01-23

    JARID2 is an accessory component of Polycomb repressive complex-2 (PRC2) required for the differentiation of embryonic stem cells (ESCs). A role for JARID2 in the recruitment of PRC2 to target genes silenced during differentiation has been put forward, but the molecular details remain unclear. We identified a 30-amino-acid region of JARID2 that mediates interactions with long noncoding RNAs (lncRNAs) and found that the presence of lncRNAs stimulated JARID2-EZH2 interactions in vitro and JARID2-mediated recruitment of PRC2 to chromatin in vivo. Native and crosslinked RNA immunoprecipitations of JARID2 revealed that Meg3 and other lncRNAs from the imprinted Dlk1-Dio3 locus, an important regulator of development, interacted with PRC2 via JARID2. Lack of MEG3 expression in human induced pluripotent cells altered the chromatin distribution of JARID2, PRC2, and H3K27me3. Our findings show that lncRNAs facilitate JARID2-PRC2 interactions on chromatin and suggest a mechanism by which lncRNAs contribute to PRC2 recruitment.

  15. Chromatin modifications associated with diabetes.

    PubMed

    Keating, Samuel T; El-Osta, Assam

    2012-08-01

    Accelerated rates of vascular complications are associated with diabetes mellitus. Environmental factors including hyperglycaemia contribute to the progression of diabetic complications. Epidemiological and experimental animal studies identified poor glycaemic control as a major contributor to the development of complications. These studies suggest that early exposure to hyperglycaemia can instigate the development of complications that present later in the progression of the disease, despite improved glycaemic control. Recent experiments reveal a striking commonality associated with gene-activating hyperglycaemic events and chromatin modification. The best characterised to date are associated with the chemical changes of amino-terminal tails of histone H3. Enzymes that write specified histone tail modifications are not well understood in models of hyperglycaemia and metabolic memory as well as human diabetes. The best-characterised enzyme is the lysine specific Set7 methyltransferase. The contribution of Set7 to the aetiology of diabetic complications may extend to other transcriptional events through methylation of non-histone substrates. PMID:22639343

  16. Distinct features of lamin A-interacting chromatin domains mapped by ChIP-sequencing from sonicated or micrococcal nuclease-digested chromatin.

    PubMed

    Lund, Eivind G; Duband-Goulet, Isabelle; Oldenburg, Anja; Buendia, Brigitte; Collas, Philippe

    2015-01-01

    The nuclear lamina has been shown to interact with the genome through lamina-associated domains (LADs). LADs have been identified by DamID, a proximity labeling assay, and more recently by chromatin immunoprecipitation-sequencing (ChIP-seq) of A- and B-type lamins. LADs form megabase-size domains at the nuclear periphery, they are gene-poor and mostly heterochromatic. Here, we show that the mode of chromatin fragmentation for ChIP, namely bath sonication or digestion with micrococcal nuclease (MNase), leads to the discovery of common but also distinct sets of lamin-interacting domains, or LiDs. Using ChIP-seq, we show the existence of lamin A/C (LMNA) LiDs with distinct gene contents, histone composition enrichment and relationships to lamin B1-interacting domains. The extent of genome coverage of lamin A/C (LMNA) LiDs in sonicated or MNase-digested chromatin is similar (∼730 megabases); however over half of these domains are uniquely detected in sonicated or MNase-digested chromatin. Sonication-specific LMNA LiDs are gene-poor and devoid of a broad panel of histone modifications, while MNase-specific LMNA LiDs are of higher gene density and are enriched in H3K9me3, H3K27me3 and in histone variant H2A.Z. LMNB1 LiDs are gene-poor and show no or little enrichment in these marks. Comparison of published LMNB1 DamID LADs with LMNB1 and LMNA LiDs identified here by ChIP-seq further shows that LMNA can associate with 'open' chromatin domains displaying euchromatin characteristics, and which are not associated with LMNB1. The differential genomic and epigenetic properties of lamin-interacting domains reflect the existence of distinct LiD populations identifiable in different chromatin contexts, including nuclease-accessible regions presumably localized in the nuclear interior.

  17. Computational strategies to address chromatin structure problems

    NASA Astrophysics Data System (ADS)

    Perišić, Ognjen; Schlick, Tamar

    2016-06-01

    While the genetic information is contained in double helical DNA, gene expression is a complex multilevel process that involves various functional units, from nucleosomes to fully formed chromatin fibers accompanied by a host of various chromatin binding enzymes. The chromatin fiber is a polymer composed of histone protein complexes upon which DNA wraps, like yarn upon many spools. The nature of chromatin structure has been an open question since the beginning of modern molecular biology. Many experiments have shown that the chromatin fiber is a highly dynamic entity with pronounced structural diversity that includes properties of idealized zig-zag and solenoid models, as well as other motifs. This diversity can produce a high packing ratio and thus inhibit access to a majority of the wound DNA. Despite much research, chromatin’s dynamic structure has not yet been fully described. Long stretches of chromatin fibers exhibit puzzling dynamic behavior that requires interpretation in the light of gene expression patterns in various tissue and organisms. The properties of chromatin fiber can be investigated with experimental techniques, like in vitro biochemistry, in vivo imagining, and high-throughput chromosome capture technology. Those techniques provide useful insights into the fiber’s structure and dynamics, but they are limited in resolution and scope, especially regarding compact fibers and chromosomes in the cellular milieu. Complementary but specialized modeling techniques are needed to handle large floppy polymers such as the chromatin fiber. In this review, we discuss current approaches in the chromatin structure field with an emphasis on modeling, such as molecular dynamics and coarse-grained computational approaches. Combinations of these computational techniques complement experiments and address many relevant biological problems, as we will illustrate with special focus on epigenetic modulation of chromatin structure.

  18. Dynamic chromatin: the regulatory domain organization of eukaryotic gene loci.

    PubMed

    Bonifer, C; Hecht, A; Saueressig, H; Winter, D M; Sippel, A E

    1991-10-01

    It is hypothesized that nuclear DNA is organized in topologically constrained loop domains defining basic units of higher order chromatin structure. Our studies are performed in order to investigate the functional relevance of this structural subdivision of eukaryotic chromatin for the control of gene expression. We used the chicken lysozyme gene locus as a model to examine the relation between chromatin structure and gene function. Several structural features of the lysozyme locus are known: the extension of the region of general DNAasel sensitivity of the active gene, the location of DNA-sequences with high affinity for the nuclear matrix in vitro, and the position of DNAasel hypersensitive chromatin sites (DHSs). The pattern of DHSs changes depending on the transcriptional status of the gene. Functional studies demonstrated that DHSs mark the position of cis-acting regulatory elements. Additionally, we discovered a novel cis-activity of the border regions of the DNAasel sensitive domain (A-elements). By eliminating the position effect on gene expression usually observed when genes are randomly integrated into the genome after transfection, A-elements possibly serve as punctuation marks for a regulatory chromatin domain. Experiments using transgenic mice confirmed that the complete structurally defined lysozyme gene domain behaves as an independent regulatory unit, expressing the gene in a tissue specific and position independent manner. These expression features were lost in transgenic mice carrying a construct, in which the A-elements as well as an upstream enhancer region were deleted, indicating the lack of a locus activation function on this construct. Experiments are designed in order to uncover possible hierarchical relationships between the different cis-acting regulatory elements for stepwise gene activation during cell differentiation. We are aiming at the definition of the basic structural and functional requirements for position independent and high

  19. Dynamic chromatin: the regulatory domain organization of eukaryotic gene loci.

    PubMed

    Bonifer, C; Hecht, A; Saueressig, H; Winter, D M; Sippel, A E

    1991-10-01

    It is hypothesized that nuclear DNA is organized in topologically constrained loop domains defining basic units of higher order chromatin structure. Our studies are performed in order to investigate the functional relevance of this structural subdivision of eukaryotic chromatin for the control of gene expression. We used the chicken lysozyme gene locus as a model to examine the relation between chromatin structure and gene function. Several structural features of the lysozyme locus are known: the extension of the region of general DNAasel sensitivity of the active gene, the location of DNA-sequences with high affinity for the nuclear matrix in vitro, and the position of DNAasel hypersensitive chromatin sites (DHSs). The pattern of DHSs changes depending on the transcriptional status of the gene. Functional studies demonstrated that DHSs mark the position of cis-acting regulatory elements. Additionally, we discovered a novel cis-activity of the border regions of the DNAasel sensitive domain (A-elements). By eliminating the position effect on gene expression usually observed when genes are randomly integrated into the genome after transfection, A-elements possibly serve as punctuation marks for a regulatory chromatin domain. Experiments using transgenic mice confirmed that the complete structurally defined lysozyme gene domain behaves as an independent regulatory unit, expressing the gene in a tissue specific and position independent manner. These expression features were lost in transgenic mice carrying a construct, in which the A-elements as well as an upstream enhancer region were deleted, indicating the lack of a locus activation function on this construct. Experiments are designed in order to uncover possible hierarchical relationships between the different cis-acting regulatory elements for stepwise gene activation during cell differentiation. We are aiming at the definition of the basic structural and functional requirements for position independent and high

  20. SWI/SNF protein component BAF250a regulates cardiac progenitor cell differentiation by modulating chromatin accessibility during second heart field development.

    PubMed

    Lei, Ienglam; Gao, Xiaolin; Sham, Mai Har; Wang, Zhong

    2012-07-13

    ATP-dependent SWI/SNF chromatin remodeling complexes alter the structure of chromatin at specific loci and facilitate tissue-specific gene regulation during development. Several SWI/SNF subunits are required for cardiogenesis. However, the function and mechanisms of SWI/SNF in mediating cardiac progenitor cell (CPC) differentiation during cardiogenesis are not well understood. Our studies of the SWI/SNF chromatin remodeling complex identified that BAF250a, a regulatory subunit of the SWI/SNF, plays a key role in CPC differentiation. BAF250a ablation in mouse second heart field (SHF) led to trabeculation defects in the right ventricle, ventricular septal defect, persistent truncus arteriosus, reduced myocardial proliferation, and embryonic lethality around E13. Using an embryonic stem cell culture system that models the formation and differentiation of SHF CPCs in vivo, we have shown that BAF250a ablation in CPCs specifically inhibits cardiomyocyte formation. Moreover, BAF250a selectively regulates the expression of key cardiac factors Mef2c, Nkx2.5, and Bmp10 in SHF CPCs. Chromatin immunoprecipitation and DNase I digestion assays indicate that BAF250a regulates gene expression by binding selectively to its target gene promoters and recruiting Brg1, the catalytic subunit of SWI/SNF, to modulate chromatin accessibility. Our results thus identify BAF250a-mediated chromatin remodeling as an essential epigenetic mechanism mediating CPC differentiation.

  1. Chromatin maintenance by a molecular motor protein

    PubMed Central

    Sung, Myong-Hee; Misteli, Tom

    2011-01-01

    The kinesin motor protein KIF4 performs essential functions in mitosis. Like other mitotic kinesins, loss of KIF4 causes spindle defects, aneuploidy, genomic instability and ultimately tumor formation. However, KIF4 is unique among molecular motors in that it resides in the cell nucleus throughout interphase, suggesting a non-mitotic function as well. Here we identify a novel cellular function for a molecular motor protein by demonstrating that KIF4 acts as a modulator of large-scale chromatin architecture during interphase. KIF4 binds globally to chromatin and its absence leads to chromatin decondensation and loss of heterochromatin domains. KIF4-dependent chromatin decondensation has functional consequences by causing replication defects and global mis-regulation of gene expression programs. KIF4 exerts its function in chromatin architecture via regulation of ADP-ribosylation of core and linker histones and by physical interaction and recruitment of chromatin assembly proteins during S-phase. These observations document a novel function for a molecular motor protein in establishment and maintenance of higher order chromatin structure. PMID:22130187

  2. Chromatin remodelling initiation during human spermiogenesis

    PubMed Central

    De Vries, Marieke; Ramos, Liliana; Housein, Zjwan; De Boer, Peter

    2012-01-01

    Summary During the last phase of spermatogenesis, spermiogenesis, haploid round spermatids metamorphose towards spermatozoa. Extensive cytoplasmic reduction and chromatin remodelling together allow a dramatic decrease of cellular, notably nuclear volume. DNA packing by a nucleosome based chromatin structure is largely replaced by a protamine based one. At the cytoplasmic level among others the acrosome and perinuclear theca (PNT) are formed. In this study we describe the onset of chromatin remodelling to occur concomitantly with acrosome and PNT development. In spread human round spermatid nuclei, we show development of a DAPI-intense doughnut-like structure co-localizing with the acrosomal sac and sub acrosomal PNT. At this structure we observe the first gradual decrease of nucleosomes and several histones. Histone post-translational modifications linked to chromatin remodelling such as H4K8ac and H4K16ac also delineate the doughnut, that is furthermore marked by H3K9me2. During the capping phase of acrosome development, the size of the doughnut-like chromatin domain increases, and this area often is marked by uniform nucleosome loss and the first appearance of transition protein 2 and protamine 1. In the acrosome phase at nuclear elongation, chromatin remodelling follows the downward movement of the marginal ring of the acrosome. Our results indicate that acrosome development and chromatin remodelling are interacting processes. In the discussion we relate chromatin remodelling to the available data on the nuclear envelope and the linker of nucleoskeleton and cytoskeleton (LINC) complex of spermatids, suggesting a signalling route for triggering chromatin remodelling. PMID:23213436

  3. Chromatin and the genome integrity network

    PubMed Central

    Papamichos-Chronakis, Manolis; Peterson, Craig L.

    2013-01-01

    The maintenance of genome integrity is essential for organism survival and for the inheritance of traits to offspring. Genomic instability is caused by DNA damage, aberrant DNA replication or uncoordinated cell division, which can lead to chromosomal aberrations and gene mutations. Recently, chromatin regulators that shape the epigenetic landscape have emerged as potential gatekeepers and signalling coordinators for the maintenance of genome integrity. Here, we review chromatin functions during the two major pathways that control genome integrity: namely, repair of DNA damage and DNA replication. We also discuss recent evidence that suggests a novel role for chromatin-remodelling factors in chromosome segregation and in the prevention of aneuploidy. PMID:23247436

  4. Chromatin Fiber Dynamics under Tension and Torsion

    PubMed Central

    Lavelle, Christophe; Victor, Jean-Marc; Zlatanova, Jordanka

    2010-01-01

    Genetic and epigenetic information in eukaryotic cells is carried on chromosomes, basically consisting of large compact supercoiled chromatin fibers. Micromanipulations have recently led to great advances in the knowledge of the complex mechanisms underlying the regulation of DNA transaction events by nucleosome and chromatin structural changes. Indeed, magnetic and optical tweezers have allowed opportunities to handle single nucleosomal particles or nucleosomal arrays and measure their response to forces and torques, mimicking the molecular constraints imposed in vivo by various molecular motors acting on the DNA. These challenging technical approaches provide us with deeper understanding of the way chromatin dynamically packages our genome and participates in the regulation of cellular metabolism. PMID:20480035

  5. Unraveling chromatin structure using magnetic tweezers

    NASA Astrophysics Data System (ADS)

    van Noort, John

    2010-03-01

    The compact, yet dynamic organization of chromatin plays an essential role in regulating gene expression. Although the static structure of chromatin fibers has been studied extensively, the controversy about the higher order folding remains. The compaction of eukaryotic DNA into chromatin has been implicated in the regulation of all DNA processes. To understand the relation between gene regulation and chromatin structure it is essential to uncover the mechanisms by which chromatin fibers fold and unfold. We used magnetic tweezers to probe the mechanical properties of individual nucleosomes and chromatin fibers consisting of a single, well-defined array of 25 nucleosomes. From these studies five major features appeared upon forced extension of chromatin fibers: the elastic stretching of chromatin's higher order structure, the breaking of internucleosomal contacts, unwrapping of the first turn of DNA, unwrapping of the second turn of DNA, and the dissociation of histone octamers. These events occur sequentially at the increasing force. Neighboring nucleosomes stabilize DNA folding into a nucleosome relative to isolated nucleosomes. When an array of nucleosomes is folded into a 30 nm fiber, representing the first level of chromatin condensation, the fiber stretched like a Hookian spring at forces up to 4 pN. Together with a nucleosome-nucleosome stacking energy of 14 kT this points to a solenoid as the underlying topology of the 30 nm fiber. Surprisingly, linker histones do not affect the length or stiffness of the fibers, but stabilize fiber folding up to forces of 7 pN. The stiffness of the folded chromatin fiber points at histone tails that mediate nucleosome stacking. Fibers with a nucleosome repeat length of 167 bp instead of 197 bp are significantly stiffer, consistent with a two-start helical arrangement. The extensive thermal breathing of the chromatin fiber that is a consequence of the observed high compliance provides a structural basis for understanding the

  6. The genomic landscape of mantle cell lymphoma is related to the epigenetically determined chromatin state of normal B cells.

    PubMed

    Zhang, Jenny; Jima, Dereje; Moffitt, Andrea B; Liu, Qingquan; Czader, Magdalena; Hsi, Eric D; Fedoriw, Yuri; Dunphy, Cherie H; Richards, Kristy L; Gill, Javed I; Sun, Zhen; Love, Cassandra; Scotland, Paula; Lock, Eric; Levy, Shawn; Hsu, David S; Dunson, David; Dave, Sandeep S

    2014-05-01

    In this study, we define the genetic landscape of mantle cell lymphoma (MCL) through exome sequencing of 56 cases of MCL. We identified recurrent mutations in ATM, CCND1, MLL2, and TP53. We further identified a number of novel genes recurrently mutated in patients with MCL including RB1, WHSC1, POT1, and SMARCA4. We noted that MCLs have a distinct mutational profile compared with lymphomas from other B-cell stages. The ENCODE project has defined the chromatin structure of many cell types. However, a similar characterization of primary human mature B cells has been lacking. We defined, for the first time, the chromatin structure of primary human naïve, germinal center, and memory B cells through chromatin immunoprecipitation and sequencing for H3K4me1, H3K4me3, H3Ac, H3K36me3, H3K27me3, and PolII. We found that somatic mutations that occur more frequently in either MCLs or Burkitt lymphomas were associated with open chromatin in their respective B cells of origin, naïve B cells, and germinal center B cells. Our work thus elucidates the landscape of gene-coding mutations in MCL and the critical interplay between epigenetic alterations associated with B-cell differentiation and the acquisition of somatic mutations in cancer. PMID:24682267

  7. Expanding the roles of chromatin insulators in nuclear architecture, chromatin organization and genome function.

    PubMed

    Schoborg, Todd; Labrador, Mariano

    2014-11-01

    Of the numerous classes of elements involved in modulating eukaryotic chromosome structure and function, chromatin insulators arguably remain the most poorly understood in their contribution to these processes in vivo. Indeed, our view of chromatin insulators has evolved dramatically since their chromatin boundary and enhancer blocking properties were elucidated roughly a quarter of a century ago as a result of recent genome-wide, high-throughput methods better suited to probing the role of these elements in their native genomic contexts. The overall theme that has emerged from these studies is that chromatin insulators function as general facilitators of higher-order chromatin loop structures that exert both physical and functional constraints on the genome. In this review, we summarize the result of recent work that supports this idea as well as a number of other studies linking these elements to a diverse array of nuclear processes, suggesting that chromatin insulators exert master control over genome organization and behavior.

  8. Androgen receptor-driven chromatin looping in prostate cancer.

    PubMed

    Wu, Dayong; Zhang, Chunpeng; Shen, Yanping; Nephew, Kenneth P; Wang, Qianben

    2011-12-01

    The androgen receptor (AR) is important for prostate cancer development and progression. Genome-wide mapping of AR binding sites in prostate cancer has found that the majority of AR binding sites are located within non-promoter regions. These distal AR binding regions regulate AR target genes (e.g. UBE2C) involved in prostate cancer growth through chromatin looping. In addition to long-distance gene regulation, looping has been shown to induce spatial proximity of two genes otherwise located far away along the genomic sequence and the formation of double-strand DNA breaks, resulting in aberrant gene fusions (e.g. TMPRSS2-ERG) that also contribute to prostate tumorigenesis. Elucidating the mechanisms of AR-driven chromatin looping will increase our understanding of prostate carcinogenesis and may lead to the identification of new therapeutic targets.

  9. CpG islands influence chromatin structure via the CpG-binding protein Cfp1.

    PubMed

    Thomson, John P; Skene, Peter J; Selfridge, Jim; Clouaire, Thomas; Guy, Jacky; Webb, Shaun; Kerr, Alastair R W; Deaton, Aimée; Andrews, Rob; James, Keith D; Turner, Daniel J; Illingworth, Robert; Bird, Adrian

    2010-04-15

    CpG islands (CGIs) are prominent in the mammalian genome owing to their GC-rich base composition and high density of CpG dinucleotides. Most human gene promoters are embedded within CGIs that lack DNA methylation and coincide with sites of histone H3 lysine 4 trimethylation (H3K4me3), irrespective of transcriptional activity. In spite of these intriguing correlations, the functional significance of non-methylated CGI sequences with respect to chromatin structure and transcription is unknown. By performing a search for proteins that are common to all CGIs, here we show high enrichment for Cfp1, which selectively binds to non-methylated CpGs in vitro. Chromatin immunoprecipitation of a mono-allelically methylated CGI confirmed that Cfp1 specifically associates with non-methylated CpG sites in vivo. High throughput sequencing of Cfp1-bound chromatin identified a notable concordance with non-methylated CGIs and sites of H3K4me3 in the mouse brain. Levels of H3K4me3 at CGIs were markedly reduced in Cfp1-depleted cells, consistent with the finding that Cfp1 associates with the H3K4 methyltransferase Setd1 (refs 7, 8). To test whether non-methylated CpG-dense sequences are sufficient to establish domains of H3K4me3, we analysed artificial CpG clusters that were integrated into the mouse genome. Despite the absence of promoters, the insertions recruited Cfp1 and created new peaks of H3K4me3. The data indicate that a primary function of non-methylated CGIs is to genetically influence the local chromatin modification state by interaction with Cfp1 and perhaps other CpG-binding proteins. PMID:20393567

  10. Chromatin Domains: The Unit of Chromosome Organization.

    PubMed

    Dixon, Jesse R; Gorkin, David U; Ren, Bing

    2016-06-01

    How eukaryotic chromosomes fold inside the nucleus is an age-old question that remains unanswered today. Early biochemical and microscopic studies revealed the existence of chromatin domains and loops as a pervasive feature of interphase chromosomes, but the biological implications of such organizational features were obscure. Genome-wide analysis of pair-wise chromatin interactions using chromatin conformation capture (3C)-based techniques has shed new light on the organization of chromosomes in interphase nuclei. Particularly, the finding of cell-type invariant, evolutionarily conserved topologically associating domains (TADs) in a broad spectrum of cell types has provided a new molecular framework for the study of animal development and human diseases. Here, we review recent progress in characterization of such chromatin domains and delineation of mechanisms of their formation in animal cells. PMID:27259200

  11. Probabilistic modelling of chromatin code landscape reveals functional diversity of enhancer-like chromatin states

    PubMed Central

    Zhou, Jian; Troyanskaya, Olga G.

    2016-01-01

    Interpreting the functional state of chromatin from the combinatorial binding patterns of chromatin factors, that is, the chromatin codes, is crucial for decoding the epigenetic state of the cell. Here we present a systematic map of Drosophila chromatin states derived from data-driven probabilistic modelling of dependencies between chromatin factors. Our model not only recapitulates enhancer-like chromatin states as indicated by widely used enhancer marks but also divides these states into three functionally distinct groups, of which only one specific group possesses active enhancer activity. Moreover, we discover a strong association between one specific enhancer state and RNA Polymerase II pausing, linking transcription regulatory potential and chromatin organization. We also observe that with the exception of long-intron genes, chromatin state transition positions in transcriptionally active genes align with an absolute distance to their corresponding transcription start site, regardless of gene length. Using our method, we provide a resource that helps elucidate the functional and spatial organization of the chromatin code landscape. PMID:26841971

  12. Chromatin Dynamics During DNA Replication and Uncharacterized Replication Factors determined by Nascent Chromatin Capture (NCC) Proteomics

    PubMed Central

    Alabert, Constance; Bukowski-Wills, Jimi-Carlo; Lee, Sung-Bau; Kustatscher, Georg; Nakamura, Kyosuke; de Lima Alves, Flavia; Menard, Patrice; Mejlvang, Jakob; Rappsilber, Juri; Groth, Anja

    2014-01-01

    SUMMARY To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use Nascent Chromatin Capture (NCC) to profile chromatin proteome dynamics during replication in human cells. NCC relies on biotin-dUTP labelling of replicating DNA, affinity-purification and quantitative proteomics. Comparing nascent chromatin with mature post-replicative chromatin, we provide association dynamics for 3995 proteins. The replication machinery and 485 chromatin factors like CAF-1, DNMT1, SUV39h1 are enriched in nascent chromatin, whereas 170 factors including histone H1, DNMT3, MBD1-3 and PRC1 show delayed association. This correlates with H4K5K12diAc removal and H3K9me1 accumulation, while H3K27me3 and H3K9me3 remain unchanged. Finally, we combine NCC enrichment with experimentally derived chromatin probabilities to predict a function in nascent chromatin for 93 uncharacterized proteins and identify FAM111A as a replication factor required for PCNA loading. Together, this provides an extensive resource to understand genome and epigenome maintenance. PMID:24561620

  13. Sperm chromatin integrity of bucks transgenic for the WAP bGH gene.

    PubMed

    Gogol, P; Bochenek, M; Smorag, Z

    2000-12-01

    The aim of the study was to compare sperm chromatin structure of transgenic and non-transgenic rabbits. In addition, the effect of chromatin structure on semen fertility was determined. Twenty male rabbits transgenic (TG) for WAP bGH gene (Edison Biotechnology Institute Ohio University, USA) and nine non-transgenic (NTG) males were used. Both TG and NTG rabbits were 13-18 months old. Semen was collected at 1-week intervals and 3-7 ejaculates from each rabbit were examined in total. Sperm chromatin abnormalities were measured flow cytometrically according to the Sperm Chromatin Structure Assay method: after chromatin denaturation by low pH, sperm cells were stained with metachromatic fluorochrome acridine orange. Spermatozoa with abnormal chromatin structure and, subsequently, higher degree of denaturation, showed a shift in red fluorescence. Two different methods of semen fertility estimation were used: (1) for TG rabbits, AI of superovulated does and calculation of percentages of fertilised eggs and embryos developing in vitro to the blastocyst stage; (2) for NTG rabbits, AI of non-stimulated does and calculation of percentages of pregnant does and mean litter sizes. The mean value of COMPalpha(t) was 3.71 for TG rabbits and 2.89 for NTG rabbits (no significant difference, t-test). The mean values of S.D.alpha(t) for the TG and NTG rabbits were 10.94 and 10.40 (no significant difference, t-test), respectively. There were no significant correlations between sperm chromatin structure of TG males and the percentages of fertilised eggs or embryos developing to the blastocyst stage. A statistically significant correlation (-0.68, P<0.05) was found between S.D.alpha(t) of NTG males and percentages of pregnant does. The results showed chromatin stability was not different for sperm obtained from TG versus NTG bucks. The presence of WAP bGH gene construct in the genome of transgenic rabbits did not cause any spermatogenesis process disturbances leading to the production

  14. Chromatin remodelers: We are the drivers!!

    PubMed

    Tyagi, Monica; Imam, Nasir; Verma, Kirtika; Patel, Ashok K

    2016-07-01

    Chromatin is a highly dynamic structure that imparts structural organization to the genome and regulates the gene expression underneath. The decade long research in deciphering the significance of epigenetics in maintaining cellular integrity has embarked the focus on chromatin remodeling enzymes. These drivers have been categorized as readers, writers and erasers with each having significance of their own. Largely, on the basis of structure, ATP dependent chromatin remodelers have been grouped into 4 families; SWI/SNF, ISWI, IN080 and CHD. It is still unclear to what degree these enzymes are swayed by local DNA sequences when shifting a nucleosome to different positions. The ability of regulating active and repressive transcriptional state via open and close chromatin architecture has been well studied however, the significance of chromatin remodelers in regulating transcription at each step i.e. initiation, elongation and termination require further attention. The authors have highlighted the significance and role of different chromatin remodelers in transcription, DNA repair and histone variant deposition. PMID:27429206

  15. Links between genome replication and chromatin landscapes.

    PubMed

    Sequeira-Mendes, Joana; Gutierrez, Crisanto

    2015-07-01

    Post-embryonic organogenesis in plants requires the continuous production of cells in the organ primordia, their expansion and a coordinated exit to differentiation. Genome replication is one of the most important processes that occur during the cell cycle, as the maintenance of genomic integrity is of primary relevance for development. As it is chromatin that must be duplicated, a strict coordination occurs between DNA replication, the deposition of new histones, and the introduction of histone modifications and variants. In turn, the chromatin landscape affects several stages during genome replication. Thus, chromatin accessibility is crucial for the initial stages and to specify the location of DNA replication origins with different chromatin signatures. The chromatin landscape also determines the timing of activation during the S phase. Genome replication must occur fully, but only once during each cell cycle. The re-replication avoidance mechanisms rely primarily on restricting the availability of certain replication factors; however, the presence of specific histone modifications are also revealed as contributing to the mechanisms that avoid re-replication, in particular for heterochromatin replication. We provide here an update of genome replication mostly focused on data from Arabidopsis, and the advances that genomic approaches are likely to provide in the coming years. The data available, both in plants and animals, point to the relevance of the chromatin landscape in genome replication, and require a critical evaluation of the existing views about the nature of replication origins, the mechanisms of origin specification and the relevance of epigenetic modifications for genome replication.

  16. Chromatin structure in scrapie and Alzheimer's disease.

    PubMed

    McLachlan, D R; Lukiw, W J; Cho, H J; Carp, R I; Wisniewski, H

    1986-11-01

    Scrapie affected brains exhibit a number of pathological features in common with the human neurodegenerative condition, Alzheimer's disease. The present report describes studies on chromatin structure seen in these two disease processes. Chromatin associated proteins influence transcriptional activity of DNA through an effect upon chromatin structure. We examined chromatin structure by: measuring the capacity of the enzyme micrococcal nuclease to release mono- and dinucleosomes from isolated nuclei and measuring DNA-histone interactions by examining the effect of ambient tonicity upon the release of chromatin proteins. In two strains of mice infected with two strains of scrapie agent there was reduced accessibility to micrococcal nuclease and an increased content on dinucleosomes of the histone H1 and H1(0) types. These changes precede clinical signs of scrapie and resemble those found in the human conditions of Alzheimer's and Pick's disease. Scrapie mouse brain differs from Alzheimer brain in that scrapie does not alter histone-DNA interactions as monitored by ionically induced histone release from chromatin. Despite similarities, the scrapie agent appears to operate upon different molecular mechanisms than those found in Alzheimer's disease.

  17. Chromatin insulation by a transcriptional activator

    PubMed Central

    Sutter, Nathan B.; Scalzo, David; Fiering, Steven; Groudine, Mark; Martin, David I. K.

    2003-01-01

    In eukaryotic genomes, transcriptionally active regions are interspersed with silent chromatin that may repress genes in its vicinity. Chromatin insulators are elements that can shield a locus from repressive effects of flanking chromatin. Few such elements have been characterized in higher eukaryotes, but transcriptional activating elements are an invariant feature of active loci and have been shown to suppress transgene silencing. Hence, we have assessed the ability of a transcriptional activator to cause chromatin insulation, i.e., to relieve position effects at transgene integration sites in cultured cells. The transgene contained a series of binding sites for the metal-inducible transcriptional activator MTF, linked to a GFP reporter. Clones carrying single integrated transgenes were derived without selection for expression, and in most clones the transgene was silent. Induction of MTF resulted in transition of the transgene from the silent to the active state, prolongation of the active state, and a marked narrowing of the range of expression levels at different genomic sites. At one genomic site, prolonged induction of MTF resulted in suppression of transgene silencing that persisted after withdrawal of the induction stimulus. These results are consistent with MTF acting as a chromatin insulator and imply that transcriptional activating elements can insulate active loci against chromatin repression. PMID:12547916

  18. Epigenomic regulation of oncogenesis by chromatin remodeling.

    PubMed

    Kumar, R; Li, D-Q; Müller, S; Knapp, S

    2016-08-25

    Disruption of the intricate gene expression program represents one of major driving factors for the development, progression and maintenance of human cancer, and is often associated with acquired therapeutic resistance. At the molecular level, cancerous phenotypes are the outcome of cellular functions of critical genes, regulatory interactions of histones and chromatin remodeling complexes in response to dynamic and persistent upstream signals. A large body of genetic and biochemical evidence suggests that the chromatin remodelers integrate the extracellular and cytoplasmic signals to control gene activity. Consequently, widespread dysregulation of chromatin remodelers and the resulting inappropriate expression of regulatory genes, together, lead to oncogenesis. We summarize the recent developments and current state of the dysregulation of the chromatin remodeling components as the driving mechanism underlying the growth and progression of human tumors. Because chromatin remodelers, modifying enzymes and protein-protein interactions participate in interpreting the epigenetic code, selective chromatin remodelers and bromodomains have emerged as new frontiers for pharmacological intervention to develop future anti-cancer strategies to be used either as single-agent or in combination therapies with chemotherapeutics or radiotherapy. PMID:26804164

  19. Centromeric chromatin and its dynamics in plants.

    PubMed

    Lermontova, Inna; Sandmann, Michael; Mascher, Martin; Schmit, Anne-Catherine; Chabouté, Marie-Edith

    2015-07-01

    Centromeres are chromatin structures that are required for proper separation of chromosomes during mitosis and meiosis. The centromere is composed of centromeric DNA, often enriched in satellite repeats, and kinetochore complex proteins. To date, over 100 kinetochore components have been identified in various eukaryotes. Kinetochore assembly begins with incorporation of centromeric histone H3 variant CENH3 into centromeric nucleosomes. Protein components of the kinetochore are either present at centromeres throughout the cell cycle or localize to centromeres transiently, prior to attachment of microtubules to each kinetochore in prometaphase of mitotic cells. This is the case for the spindle assembly checkpoint (SAC) proteins in animal cells. The SAC complex ensures equal separation of chromosomes between daughter nuclei by preventing anaphase onset before metaphase is complete, i.e. the sister kinetochores of all chromosomes are attached to spindle fibers from opposite poles. In this review, we focus on the organization of centromeric DNA and the kinetochore assembly in plants. We summarize recent advances regarding loading of CENH3 into the centromere, and the subcellular localization and protein-protein interactions of Arabidopsis thaliana proteins involved in kinetochore assembly and function. We describe the transcriptional activity of corresponding genes based on in silico analysis of their promoters and cell cycle-dependent expression. Additionally, barley homologs of all selected A. thaliana proteins have been identified in silico, and their sequences and domain structures are presented.

  20. Effect of SWI/SNF chromatin remodeling complex on HIV-1 Tat activated transcription

    PubMed Central

    Agbottah, Emmanuel; Deng, Longwen; Dannenberg, Luke O; Pumfery, Anne; Kashanchi, Fatah

    2006-01-01

    Background Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of acquired immunodeficiency virus (AIDS). Following entry into the host cell, the viral RNA is reverse transcribed into DNA and subsequently integrated into the host genome as a chromatin template. The integrated proviral DNA, along with the specific chromatinized environment in which integration takes place allows for the coordinated regulation of viral transcription and replication. While the specific roles of and interplay between viral and host proteins have not been fully elucidated, numerous reports indicate that HIV-1 retains the ability for self-regulation via the pleiotropic effects of its viral proteins. Though viral transcription is fully dependent upon host cellular factors and the state of host activation, recent findings indicate a complex interplay between viral proteins and host transcription regulatory machineries including histone deacetylases (HDACs), histone acetyltransferases (HATs), cyclin dependent kinases (CDKs), and histone methyltransferases (HMTs). Results Here, we describe the effect of Tat activated transcription at the G1/S border of the cell cycle and analyze the interaction of modified Tat with the chromatin remodeling complex, SWI/SNF. HIV-1 LTR DNA reconstituted into nucleosomes can be activated in vitro using various Tat expressing extracts. Optimally activated transcription was observed at the G1/S border of the cell cycle both in vitro and in vivo, where chromatin remodeling complex, SWI/SNF, was present on the immobilized LTR DNA. Using a number of in vitro binding as well as in vivo chromatin immunoprecipitation (ChIP) assays, we detected the presence of both BRG1 and acetylated Tat in the same complex. Finally, we demonstrate that activated transcription resulted in partial or complete removal of the nucleosome from the start site of the LTR as evidenced by a restriction enzyme accessibility assay. Conclusion We propose a model where unmodified Tat

  1. Interrogation of allelic chromatin states in human cells by high-density ChIP-genotyping.

    PubMed

    Light, Nicholas; Adoue, Véronique; Ge, Bing; Chen, Shu-Huang; Kwan, Tony; Pastinen, Tomi

    2014-09-01

    Allele-specific (AS) assessment of chromatin has the potential to elucidate specific cis-regulatory mechanisms, which are predicted to underlie the majority of the known genetic associations to complex disease. However, development of chromatin landscapes at allelic resolution has been challenging since sites of variable signal strength require substantial read depths not commonly applied in sequencing based approaches. In this study, we addressed this by performing parallel analyses of input DNA and chromatin immunoprecipitates (ChIP) on high-density Illumina genotyping arrays. Allele-specificity for the histone modifications H3K4me1, H3K4me3, H3K27ac, H3K27me3, and H3K36me3 was assessed using ChIP samples generated from 14 lymphoblast and 6 fibroblast cell lines. AS-ChIP SNPs were combined into domains and validated using high-confidence ChIP-seq sites. We observed characteristic patterns of allelic-imbalance for each histone-modification around allele-specifically expressed transcripts. Notably, we found H3K4me1 to be significantly anti-correlated with allelic expression (AE) at transcription start sites, indicating H3K4me1 allelic imbalance as a marker of AE. We also found that allelic chromatin domains exhibit population and cell-type specificity as well as heritability within trios. Finally, we observed that a subset of allelic chromatin domains is regulated by DNase I-sensitive quantitative trait loci and that these domains are significantly enriched for genome-wide association studies hits, with autoimmune disease associated SNPs specifically enriched in lymphoblasts. This study provides the first genome-wide maps of allelic-imbalance for five histone marks. Our results provide new insights into the role of chromatin in cis-regulation and highlight the need for high-depth sequencing in ChIP-seq studies along with the need to improve allele-specificity of ChIP-enrichment.

  2. Combining Ultracentrifugation and Peptide Termini Group-specific Immunoprecipitation for Multiplex Plasma Protein Analysis

    PubMed Central

    Volk, Sonja; Schreiber, Thomas D.; Eisen, David; Wiese, Calvin; Planatscher, Hannes; Pynn, Christopher J.; Stoll, Dieter; Templin, Markus F.; Joos, Thomas O.; Pötz, Oliver

    2012-01-01

    Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques. PMID:22527512

  3. Immunoprecipitation and characterization of a binding protein specific for the peptide, intestinal trefoil factor.

    PubMed

    Chinery, R; Cox, H M

    1995-01-01

    Recombinant rat intestinal trefoil factor (rITF) and human spasmolytic polypeptide (hSP) were irreversibly cross-linked to specific binding sites in solubilized rat intestinal epithelial membranes and human adenocarcinoma cells. Analysis of the immunoprecipitates by immunoblotting identified a cross-linked protein complex of approximately 45 kDa, which under reducing conditions appeared as a approximately 28-kDa band and the latter displayed ligand-stimulated phosphorylation of a tyrosine, but not a threonine or serine, residue in the binding complex. [125I]rITF was used to localize binding sites by autoradiography of frozen sections from rat gastrointestinal tissues. A high density of specific [125I]rITF binding sites was present within gastric, colonic, and jejunal mucosal glands. Unlabeled hSP partially inhibited [125I]rITF binding at a concentration of 1 microM when compared with the same concentration of unlabeled rITF. These studies support earlier observations for the existence of trefoil binding sites in the gastrointestinal tract and further suggest that hSP has affinity for the mucosal rITF binding site.

  4. Histone chaperones link histone nuclear import and chromatin assembly.

    PubMed

    Keck, Kristin M; Pemberton, Lucy F

    2013-01-01

    Histone chaperones are proteins that shield histones from nonspecific interactions until they are assembled into chromatin. After their synthesis in the cytoplasm, histones are bound by different histone chaperones, subjected to a series of posttranslational modifications and imported into the nucleus. These evolutionarily conserved modifications, including acetylation and methylation, can occur in the cytoplasm, but their role in regulating import is not well understood. As part of histone import complexes, histone chaperones may serve to protect the histones during transport, or they may be using histones to promote their own nuclear localization. In addition, there is evidence that histone chaperones can play an active role in the import of histones. Histone chaperones have also been shown to regulate the localization of important chromatin modifying enzymes. This review is focused on the role histone chaperones play in the early biogenesis of histones, the distinct cytoplasmic subcomplexes in which histone chaperones have been found in both yeast and mammalian cells and the importins/karyopherins and nuclear localization signals that mediate the nuclear import of histones. We also address the role that histone chaperone localization plays in human disease. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.

  5. Diverse Roles and Interactions of the SWI/SNF Chromatin Remodeling Complex Revealed Using Global Approaches

    PubMed Central

    Davidov, Eugene; Gianoulis, Tara A.; Zhong, Guoneng; Rozowsky, Joel; Bhardwaj, Nitin; Gerstein, Mark B.; Snyder, Michael

    2011-01-01

    A systems understanding of nuclear organization and events is critical for determining how cells divide, differentiate, and respond to stimuli and for identifying the causes of diseases. Chromatin remodeling complexes such as SWI/SNF have been implicated in a wide variety of cellular processes including gene expression, nuclear organization, centromere function, and chromosomal stability, and mutations in SWI/SNF components have been linked to several types of cancer. To better understand the biological processes in which chromatin remodeling proteins participate, we globally mapped binding regions for several components of the SWI/SNF complex throughout the human genome using ChIP-Seq. SWI/SNF components were found to lie near regulatory elements integral to transcription (e.g. 5′ ends, RNA Polymerases II and III, and enhancers) as well as regions critical for chromosome organization (e.g. CTCF, lamins, and DNA replication origins). Interestingly we also find that certain configurations of SWI/SNF subunits are associated with transcripts that have higher levels of expression, whereas other configurations of SWI/SNF factors are associated with transcripts that have lower levels of expression. To further elucidate the association of SWI/SNF subunits with each other as well as with other nuclear proteins, we also analyzed SWI/SNF immunoprecipitated complexes by mass spectrometry. Individual SWI/SNF factors are associated with their own family members, as well as with cellular constituents such as nuclear matrix proteins, key transcription factors, and centromere components, implying a ubiquitous role in gene regulation and nuclear function. We find an overrepresentation of both SWI/SNF-associated regions and proteins in cell cycle and chromosome organization. Taken together the results from our ChIP and immunoprecipitation experiments suggest that SWI/SNF facilitates gene regulation and genome function more broadly and through a greater diversity of interactions

  6. Diverse roles and interactions of the SWI/SNF chromatin remodeling complex revealed using global approaches.

    PubMed

    Euskirchen, Ghia M; Auerbach, Raymond K; Davidov, Eugene; Gianoulis, Tara A; Zhong, Guoneng; Rozowsky, Joel; Bhardwaj, Nitin; Gerstein, Mark B; Snyder, Michael

    2011-03-01

    A systems understanding of nuclear organization and events is critical for determining how cells divide, differentiate, and respond to stimuli and for identifying the causes of diseases. Chromatin remodeling complexes such as SWI/SNF have been implicated in a wide variety of cellular processes including gene expression, nuclear organization, centromere function, and chromosomal stability, and mutations in SWI/SNF components have been linked to several types of cancer. To better understand the biological processes in which chromatin remodeling proteins participate, we globally mapped binding regions for several components of the SWI/SNF complex throughout the human genome using ChIP-Seq. SWI/SNF components were found to lie near regulatory elements integral to transcription (e.g. 5' ends, RNA Polymerases II and III, and enhancers) as well as regions critical for chromosome organization (e.g. CTCF, lamins, and DNA replication origins). Interestingly we also find that certain configurations of SWI/SNF subunits are associated with transcripts that have higher levels of expression, whereas other configurations of SWI/SNF factors are associated with transcripts that have lower levels of expression. To further elucidate the association of SWI/SNF subunits with each other as well as with other nuclear proteins, we also analyzed SWI/SNF immunoprecipitated complexes by mass spectrometry. Individual SWI/SNF factors are associated with their own family members, as well as with cellular constituents such as nuclear matrix proteins, key transcription factors, and centromere components, implying a ubiquitous role in gene regulation and nuclear function. We find an overrepresentation of both SWI/SNF-associated regions and proteins in cell cycle and chromosome organization. Taken together the results from our ChIP and immunoprecipitation experiments suggest that SWI/SNF facilitates gene regulation and genome function more broadly and through a greater diversity of interactions than

  7. A Computer Lab Exploring Evolutionary Aspects of Chromatin Structure and Dynamics for an Undergraduate Chromatin Course

    ERIC Educational Resources Information Center

    Eirin-Lopez, Jose M.

    2013-01-01

    The study of chromatin constitutes one of the most active research fields in life sciences, being subject to constant revisions that continuously redefine the state of the art in its knowledge. As every other rapidly changing field, chromatin biology requires clear and straightforward educational strategies able to efficiently translate such a…

  8. Chk1 protects against chromatin bridges by constitutively phosphorylating BLM serine 502 to inhibit BLM degradation.

    PubMed

    Petsalaki, Eleni; Dandoulaki, Maria; Morrice, Nick; Zachos, George

    2014-09-15

    Chromatin bridges represent incompletely segregated chromosomal DNA connecting the anaphase poles and can result in chromosome breakage. The Bloom's syndrome protein helicase (BLM, also known as BLMH) suppresses formation of chromatin bridges. Here, we show that cells deficient in checkpoint kinase 1 (Chk1, also known as CHEK1) exhibit higher frequency of chromatin bridges and reduced BLM protein levels compared to controls. Chk1 inhibition leads to BLM ubiquitylation and proteasomal degradation during interphase. Furthermore, Chk1 constitutively phosphorylates human BLM at serine 502 (S502) and phosphorylated BLM localises to chromatin bridges. Mutation of S502 to a non-phosphorylatable alanine residue (BLM-S502A) reduces the stability of BLM, whereas expression of a phospho-mimicking BLM-S502D, in which S502 is mutated to aspartic acid, stabilises BLM and prevents chromatin bridges in Chk1-deficient cells. In addition, wild-type but not BLM-S502D associates with cullin 3, and cullin 3 depletion rescues BLM accumulation and localisation to chromatin bridges after Chk1 inhibition. We propose that Chk1 phosphorylates BLM-S502 to inhibit cullin-3-mediated BLM degradation during interphase. These results suggest that Chk1 prevents deleterious anaphase bridges by stabilising BLM.

  9. Differential chromatin proteomics of the MMS-induced DNA damage response in yeast

    PubMed Central

    2011-01-01

    Background Protein enrichment by sub-cellular fractionation was combined with differential-in-gel-electrophoresis (DIGE) to address the detection of the low abundance chromatin proteins in the budding yeast proteome. Comparisons of whole-cell extracts and chromatin fractions were used to provide a measure of the degree of chromatin association for individual proteins, which could be compared across sample treatments. The method was applied to analyze the effect of the DNA damaging agent methyl methanesulfonate (MMS) on levels of chromatin-associated proteins. Results Up-regulation of several previously characterized DNA damage checkpoint-regulated proteins, such as Rnr4, Rpa1 and Rpa2, was observed. In addition, several novel DNA damage responsive proteins were identified and assessed for genotoxic sensitivity using either DAmP (decreased abundance by mRNA perturbation) or knockout strains, including Acf2, Arp3, Bmh1, Hsp31, Lsp1, Pst2, Rnr4, Rpa1, Rpa2, Ste4, Ycp4 and Yrb1. A strain in which the expression of the Ran-GTPase binding protein Yrb1 was reduced was found to be hypersensitive to genotoxic stress. Conclusion The described method was effective at unveiling chromatin-associated proteins that are less likely to be detected in the absence of fractionation. Several novel proteins with altered chromatin abundance were identified including Yrb1, pointing to a role for this nuclear import associated protein in DNA damage response. PMID:21967861

  10. Advance chromatin extraction improves capture performance of protein A affinity chromatography.

    PubMed

    Nian, Rui; Zhang, Wei; Tan, Lihan; Lee, Jeremy; Bi, Xeuzhi; Yang, Yuansheng; Gan, Hui Theng; Gagnon, Pete

    2016-01-29

    Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%. PMID:26774119

  11. UV light-induced DNA lesions cause dissociation of yeast RNA polymerases-I and establishment of a specialized chromatin structure at rRNA genes

    PubMed Central

    Tremblay, Maxime; Charton, Romain; Wittner, Manuel; Levasseur, Geneviève; Griesenbeck, Joachim; Conconi, Antonio

    2014-01-01

    The cytotoxicity of UV light-induced DNA lesions results from their interference with transcription and replication. DNA lesions arrest elongating RNA polymerases, an event that triggers transcription-coupled nucleotide excision repair. Since arrested RNA polymerases reduce the accessibility of repair factors to DNA lesions, they might be displaced. The fate of arrested RNA polymerases-II at DNA lesions has been extensively studied, yielding partially contradictory results. Considerably less is known about RNA polymerases-I that transcribe nucleosomes-depleted rRNA genes at very high rate. To investigate the fate of arrested RNA polymerases-I at DNA lesions, chromatin-immunoprecipitation, electron microscopy, transcription run-on, psoralen-cross-linking and chromatin-endogenous cleavage were employed. We found that RNA polymerases-I density increased at the 5′-end of the gene, likely due to continued transcription initiation followed by elongation and pausing/release at the first DNA lesion. Most RNA polymerases-I dissociated downstream of the first DNA lesion, concomitant with chromatin closing that resulted from deposition of nucleosomes. Although nucleosomes were deposited, the high mobility group-box Hmo1 (component of actively transcribed rRNA genes) remained associated. After repair of DNA lesions, Hmo1 containing chromatin might help to restore transcription elongation and reopening of rRNA genes chromatin. PMID:24097442

  12. Nuclear envelope precursor vesicle targeting to chromatin is stimulated by protein phosphatase 1 in Xenopus egg extracts

    SciTech Connect

    Ito, Hiromi; Koyama, Yuhei; Takano, Makoto; Ishii, Kohei; Maeno, Mitsugu; Furukawa, Kazuhiro; Horigome, Tsuneyoshi . E-mail: thori@chem.sc.niigata-u.ac.jp

    2007-05-15

    The mechanism underlying targeting of the nuclear membrane to chromatin at the end of mitosis was studied using an in vitro cell-free system comprising Xenopus egg membrane and cytosol fractions, and sperm chromatin. The mitotic phase membrane, which was separated from a mitotic phase extract of Xenopus eggs and could not bind to chromatin, became able to bind to chromatin on pretreatment with a synthetic phase cytosol fraction of Xenopus eggs. When the cytosol fraction was depleted of protein phosphatase 1 (PP1) with anti-Xenopus PP1{gamma}1 antibodies, this ability was lost. The addition of recombinant xPP1{gamma}1 to the PP1-depleted cytosol fraction restored the ability. These and other results suggested that dephosphorylation of mitotic phosphorylation sites on membranes by PP1 in the synthetic phase cytosol fraction promoted targeting of the membranes to chromatin. On the other hand, a fragment containing the chromatin-binding domain of lamin B receptor (LBR) but not emerin inhibited targeting of membrane vesicles. It was also shown that PP1 dephosphorylates a phosphate group(s) responsible for regulation of the binding of LBR to chromatin. A possible mechanism involving PP1 and LBR for the regulation of nuclear membrane targeting to chromatin was discussed.

  13. Characterizing RNA-protein interaction using cross-linking and metabolite supplemented nuclear RNA-immunoprecipitation.

    PubMed

    Au, Phil Chi Khang; Helliwell, Chris; Wang, Ming-Bo

    2014-05-01

    RNA-immunoprecipitation (RNA-IP) is a method used to isolate and identify RNA molecules specifically associated with an RNA-binding protein. Non-coding RNAs are emerging as key regulators of many biological and developmental pathways and RNA-IP has become an important tool in studying their function(s). While RNA-IP is successfully used to determine protein-RNA interaction, specific details regarding the level of this association and the metabolic requirement of this interaction which can influence the success of RNA-IP remain unclear. Here, we investigate the conditions required for efficient nuclear RNA-IP using Arabidopsis AGO4 (Argonaute 4) and siRNA binding as the study model. We showed that formaldehyde cross-linking, but not UV cross-linking, allowed for efficient pull-down of 24-nt siRNAs, suggesting that AGO4-siRNA interaction involves other protein(s). We also showed that, while formaldehyde cross-linking could also be performed on purified nuclei, ATP supplementation to the nuclei isolation buffer was needed to efficiently pull down 24-nt siRNAs. This result indicates that ATP is required for efficient siRNA loading onto AGO4. As most of the known RNA-mediated regulatory processes occur in the nucleus, our findings on cross-linking conditions and metabolite requirement for successful AGO4 nuclear RNA-IP provide a valuable insight and future consideration when studying the function of protein-RNA interactions in plants.

  14. Physicochemical characterization of C3b receptors isolated from human erythrocytes by immunoprecipitation.

    PubMed Central

    Gerdes, J; Stein, H

    1980-01-01

    A high yield of active C3b receptors was obtained by solubilizing human erythrocyte membranes with 2 M KBr, whereas other solubilization agents yielded no, or significantly less activity. Gel filtration of the KBr lysates revealed that the apparent molecular wieght of biologically active C3b receptor molecules was greater than 1 x 10(6). Immunoprecipitates prepared with radio-iodinated KBr lysates and anti-C3 receptor sera (AC3RS) were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) or sodium dodecyl gel filtration. Unreduced SDS-PAGE and gel filtration profiles showed three predominant peaks with apparent mol. wts of 1--1.3 x 10(6), 80,000 and 60,000. Whereas the high mol. wt component decreased only slightly after reduction, the 80,000 and 60,000 mol. wt components disappeared and two new peaks with apparent mol. wts of 38,000 and 18,000 appeared in SDS-PAGE profiles. Although the high mol. wt component present in reduced SDS-PAGE profiles was detectable in some of the control experiments, none of the other peaks could be precipitated with control sera, and these components could be demonstrated only when KBr lysates of C3b receptor-positive erythrocytes and AC3RS that were able to inhibit ligand binding of the C3b receptors were used for precipitation. These findings suggest that (a) the C3b receptor of human erythrocytes in its biologically active state is a macromolecule with an apparent mol. wt higher than 1 x 10(6) and (b) the protein moiety consists predominantly of non-covalently linked protein molecules with apparent mol wts of 80,000 and 60,000. These protein molecules are composed of disulphide-bridged polypeptide chains with apparent mol. wts of 38,000 and 18,000. PMID:7461716

  15. Identification of Lysine Acetylation in Mycobacterium abscessus Using LC-MS/MS after Immunoprecipitation.

    PubMed

    Guo, Jintao; Wang, Changwei; Han, Yi; Liu, Zhiyong; Wu, Tian; Liu, Yan; Liu, Yang; Tan, Yaoju; Cai, Xinshan; Cao, Yuanyuan; Wang, Bangxing; Zhang, Buchang; Liu, Chunping; Tan, Shouyong; Zhang, Tianyu

    2016-08-01

    Mycobacterium abscessus (MAB), which manifests in the pulmonary system, is one of the neglected causes of nontuberculous mycobacteria (NTM) infection. Treatment against MAB is difficult, characterized by its intrinsic antibiotic drug resistance. Lysine acetylation can alter the physiochemical property of proteins in living organisms. This study aimed to determine if this protein post-translational modification (PTM) exists in a clinical isolate M. abscessus GZ002. We used the antiacetyl-lysine immunoprecipitation to enrich the low-abundant PTM proteins, followed by the LC-MS/MS analysis. The lysine acetylome of M. abscessus GZ002 was determined. There were 459 lysine acetylation sites found in 289 acetylated proteins. Lysine acetylation occurred in 5.87% of the M. abscessus GZ002 proteome, and at least 25% of them were growth essential. Aerobic respiration and carbohydrate metabolic pathways of M. abscessus GZ002 were enriched with lysine acetylation. Through bioinformatics analysis, we identified four major acetyl motif logos (K(ac)Y, K(ac)F, K(ac)H, and DK(ac)). Further comparison of the reported M. tuberculosis (MTB) acetylomes and that of MAB GZ002 revealed several common features between these two species. The lysine residues of several antibiotic-resistance, virulence, and persistence-related proteins were acetylated in both MAB GZ002 and MTB. There were 51 identical acetylation sites in 37 proteins found in common between MAB GZ002 and MTB. Overall, we demonstrate a profile of lysine acetylation in MAB GZ002 proteome that shares similarities with MTB. Interventions that target at these conserved sections may be valuable as anti-NTM or anti-TB therapies. PMID:27323652

  16. Chromatin States Accurately Classify Cell Differentiation Stages

    PubMed Central

    Larson, Jessica L.; Yuan, Guo-Cheng

    2012-01-01

    Gene expression is controlled by the concerted interactions between transcription factors and chromatin regulators. While recent studies have identified global chromatin state changes across cell-types, it remains unclear to what extent these changes are co-regulated during cell-differentiation. Here we present a comprehensive computational analysis by assembling a large dataset containing genome-wide occupancy information of 5 histone modifications in 27 human cell lines (including 24 normal and 3 cancer cell lines) obtained from the public domain, followed by independent analysis at three different representations. We classified the differentiation stage of a cell-type based on its genome-wide pattern of chromatin states, and found that our method was able to identify normal cell lines with nearly 100% accuracy. We then applied our model to classify the cancer cell lines and found that each can be unequivocally classified as differentiated cells. The differences can be in part explained by the differential activities of three regulatory modules associated with embryonic stem cells. We also found that the “hotspot” genes, whose chromatin states change dynamically in accordance to the differentiation stage, are not randomly distributed across the genome but tend to be embedded in multi-gene chromatin domains, and that specialized gene clusters tend to be embedded in stably occupied domains. PMID:22363642

  17. Chromatin associations in Arabidopsis interphase nuclei

    PubMed Central

    Schubert, Veit; Rudnik, Radoslaw; Schubert, Ingo

    2014-01-01

    The arrangement of chromatin within interphase nuclei seems to be caused by topological constraints and related to gene expression depending on tissue and developmental stage. In yeast and animals it was found that homologous and heterologous chromatin association are required to realize faithful expression and DNA repair. To test whether such associations are present in plants we analyzed Arabidopsis thaliana interphase nuclei by FISH using probes from different chromosomes. We found that chromatin fiber movement and variable associations, although in general relatively seldom, may occur between euchromatin segments along chromosomes, sometimes even over large distances. The combination of euchromatin segments bearing high or low co-expressing genes did not reveal different association frequencies probably due to adjacent genes of deviating expression patterns. Based on previous data and on FISH analyses presented here, we conclude that the global interphase chromatin organization in A. thaliana is relatively stable, due to the location of its 10 centromeres at the nuclear periphery and of the telomeres mainly at the centrally localized nucleolus. Nevertheless, chromatin movement enables a flexible spatial genome arrangement in plant nuclei. PMID:25431580

  18. Active and Repressive Chromatin-Associated Proteome after MPA Treatment and the Role of Midkine in Epithelial Monolayer Permeability

    PubMed Central

    Khan, Niamat; Lenz, Christof; Binder, Lutz; Pantakani, Dasaradha Venkata Krishna; Asif, Abdul R.

    2016-01-01

    Mycophenolic acid (MPA) is prescribed to maintain allografts in organ-transplanted patients. However, gastrointestinal (GI) complications, particularly diarrhea, are frequently observed as a side effect following MPA therapy. We recently reported that MPA altered the tight junction (TJ)-mediated barrier function in a Caco-2 cell monolayer model system. This study investigates whether MPA induces epigenetic changes which lead to GI complications, especially diarrhea. Methods: We employed a Chromatin Immunoprecipitation-O-Proteomics (ChIP-O-Proteomics) approach to identify proteins associated with active (H3K4me3) as well as repressive (H3K27me3) chromatin histone modifications in MPA-treated cells, and further characterized the role of midkine, a H3K4me3-associated protein, in the context of epithelial monolayer permeability. Results: We identified a total of 333 and 306 proteins associated with active and repressive histone modification marks, respectively. Among them, 241 proteins were common both in active and repressive chromatin, 92 proteins were associated exclusively with the active histone modification mark, while 65 proteins remained specific to repressive chromatin. Our results show that 45 proteins which bind to the active and seven proteins which bind to the repressive chromatin region exhibited significantly altered abundance in MPA-treated cells as compared to DMSO control cells. A number of novel proteins whose function is not known in bowel barrier regulation were among the identified proteins, including midkine. Our functional integrity assays on the Caco-2 cell monolayer showed that the inhibition of midkine expression prior to MPA treatment could completely block the MPA-mediated increase in barrier permeability. Conclusions: The ChIP-O-Proteomics approach delivered a number of novel proteins with potential implications in MPA toxicity. Consequently, it can be proposed that midkine inhibition could be a potent therapeutic approach to prevent the

  19. Mechanical model of the nucleosome and chromatin.

    PubMed

    Bishop, Thomas C; Zhmudsky, Oleksandr O

    2002-04-01

    A theoretical framework for evaluating the approximate energy and dynamic properties associated with the folding of DNA into nucleosomes and chromatin is presented. Experimentally determined elastic constants of linear DNA and a simple fold geometry are assumed in order to derive elastic constants for extended and condensed chromatin. The model predicts the Young s modulus of extended and condensed chromatin to within an order of magnitude of experimentally determined values. Thus we demonstrate that the elastic properties of DNA are a primary determinant of the elastic properties of the higher order folded states. The derived elastic constants are used to predict the speed of propagation of small amplitude waves that excite an extension(sound), twist, bend or shear motion in each folded state. Taken together the results demonstrate that folding creates a hierarchy of time, length and energy scales.

  20. [Comparative characteristics of chromatin endonuclease fragments].

    PubMed

    Miul'berg, A A; Tishchenko, L I; Domkina, L K

    1977-05-01

    Soluble fragments of chromatin obtained by Ca, Mg-dependent endonuclease digest of rat liver nuclei, have been separated by gel chromatography on Sepharose 4B into three zones, containing oligomers, tetramers--dimers and monomers, respectively. The content of nonhistone proteins and particularly lysine-rich histones is decreased with a transition from theoligomers to monomers. The average protein/DNA ratio of the monomers is equal to 1.36 and that of histone/DNA ratio--to 0.82. The dependence of the degree of chromatin digest by endonuclease on its protein content and conditions of isolation and incubation of nuclei is discussed. The chromatin monomer formed appears to be made up of a nucleosome and short portions of spacer DNA bound to some part of histone HI and nonhistone proteins. PMID:889964

  1. Chromatin Remodeling, DNA Damage Repair and Aging

    PubMed Central

    Liu, Baohua; Yip, Raymond KH; Zhou, Zhongjun

    2012-01-01

    Cells are constantly exposed to a variety of environmental and endogenous conditions causing DNA damage, which is detected and repaired by conserved DNA repair pathways to maintain genomic integrity. Chromatin remodeling is critical in this process, as the organization of eukaryotic DNA into compact chromatin presents a natural barrier to all DNA-related events. Studies on human premature aging syndromes together with normal aging have suggested that accumulated damages might lead to exhaustion of resources that are required for physiological functions and thus accelerate aging. In this manuscript, combining the present understandings and latest findings, we focus mainly on discussing the role of chromatin remodeling in the repair of DNA double-strand breaks (DSBs) and regulation of aging. PMID:23633913

  2. The Chromatin Fiber: Multiscale Problems and Approaches

    PubMed Central

    Ozer, Gungor; Luque, Antoni; Schlick, Tamar

    2015-01-01

    The structure of chromatin, affected by many factors from DNA linker lengths to posttranslational modifications, is crucial to the regulation of eukaryotic cells. Combined experimental and computational methods have led to new insights into its structural and dynamical features, from interactions due to the flexible core histone tails of the nucleosomes to the physical mechanism driving the formation of chromosomal domains. Here we present a perspective of recent advances in chromatin modeling techniques at the atomic, mesoscopic, and chromosomal scales with a view toward developing multiscale computational strategies to integrate such findings. Innovative modeling methods that connect molecular to chromosomal scales are crucial for interpreting experiments and eventually deciphering the complex dynamic organization and function of chromatin in the cell. PMID:26057099

  3. Nucleosome dynamics during chromatin remodeling in vivo

    PubMed Central

    Ramachandran, Srinivas; Henikoff, Steven

    2016-01-01

    ABSTRACT Precise positioning of nucleosomes around regulatory sites is achieved by the action of chromatin remodelers, which use the energy of ATP to slide, evict or change the composition of nucleosomes. Chromatin remodelers act to bind nucleosomes, disrupt histone-DNA interactions and translocate the DNA around the histone core to reposition nucleosomes. Hence, remodeling is expected to involve nucleosomal intermediates with a structural organization that is distinct from intact nucleosomes. We describe the identification of a partially unwrapped nucleosome structure using methods that map histone-DNA contacts genome-wide. This alternative nucleosome structure is likely formed as an intermediate or by-product during nucleosome remodeling by the RSC complex. Identification of the loss of histone-DNA contacts during chromatin remodeling by RSC in vivo has implications for the regulation of transcriptional initiation. PMID:26933790

  4. Functions of the Proteasome on Chromatin

    PubMed Central

    McCann, Tyler S.; Tansey, William P.

    2014-01-01

    The proteasome is a large self-compartmentalized protease complex that recognizes, unfolds, and destroys ubiquitylated substrates. Proteasome activities are required for a host of cellular functions, and it has become clear in recent years that one set of critical actions of the proteasome occur on chromatin. In this review, we discuss some of the ways in which proteasomes directly regulate the structure and function of chromatin and chromatin regulatory proteins, and how this influences gene transcription. We discuss lingering controversies in the field, the relative importance of proteolytic versus non-proteolytic proteasome activities in this process, and highlight areas that require further investigation. Our intention is to show that proteasomes are involved in major steps controlling the expression of the genetic information, that proteasomes use both proteolytic mechanisms and ATP-dependent protein remodeling to accomplish this task, and that much is yet to be learned about the full spectrum of ways that proteasomes influence the genome. PMID:25422899

  5. Nuclease digestion studies of chromatin structure

    SciTech Connect

    Deutsch, S.M.

    1987-01-01

    Micrococcal nuclease, which preferentially cleaves linker DNA in chromatin, was immobilized by covalent attachment to CNBr-activated agarose beads and used to study the accessibility of linker DNA in chromatin fibers prepared from chicken erythrocyte nuclei. This immobilized nuclease was able to cleave chromatin fibers into the typical pattern of fragments corresponding to multiples of mononucleosomes. Cleavage from only the ends of the fibers was ruled out by examining the products of cleavage of fibers end-labelled with /sup 35/P. Comparison of the rate of digestion by immobilized and soluble micrococcal nuclease indicated that the fiber structure does not significantly affect access to linker DNA. The absence of an effect of reducing temperatures on the rate of digestion of fibers, as compared to short oligonucleosomes, indicated that breathing motions to allow access to the fiber interior were not required for cleavage of linker DNA.

  6. Open chromatin reveals the functional maize genome

    PubMed Central

    Rodgers-Melnick, Eli; Vera, Daniel L.; Bass, Hank W.

    2016-01-01

    Cellular processes mediated through nuclear DNA must contend with chromatin. Chromatin structural assays can efficiently integrate information across diverse regulatory elements, revealing the functional noncoding genome. In this study, we use a differential nuclease sensitivity assay based on micrococcal nuclease (MNase) digestion to discover open chromatin regions in the maize genome. We find that maize MNase-hypersensitive (MNase HS) regions localize around active genes and within recombination hotspots, focusing biased gene conversion at their flanks. Although MNase HS regions map to less than 1% of the genome, they consistently explain a remarkably large amount (∼40%) of heritable phenotypic variance in diverse complex traits. MNase HS regions are therefore on par with coding sequences as annotations that demarcate the functional parts of the maize genome. These results imply that less than 3% of the maize genome (coding and MNase HS regions) may give rise to the overwhelming majority of phenotypic variation, greatly narrowing the scope of the functional genome. PMID:27185945

  7. Hypoxia induces a novel signature of chromatin modifications and global repression of transcription.

    PubMed

    Johnson, Amber Buescher; Denko, Nicholas; Barton, Michelle Craig

    2008-04-01

    Tumor cells respond to the harsh hypoxic microenvironment, in part, by transcriptional regulation of specific target genes. We found that hypoxia-mediated activation of selected genes occurs amidst widespread repression of transcription that is neither cell type-specific nor HIF-1-dependent. Despite overall repression, hypoxia induces a pool of histone modifications typically associated with transcriptional activation or repression. Chromatin immunoprecipitation analyses showed that this global mixture of hypoxia-modified histones is sorted in a gene-specific manner to correlate with transcriptional response to hypoxia. Exceptions to this were unexpected increases in H3K4me3 levels, typically associated with transcriptional activation, and decreased H3K27me3 levels, generally a marker of transcriptional silencing, at core promoters of both hypoxia-activated and -repressed genes. These data suggest that a novel signature of chromatin modifications is induced under hypoxic stress, which may play a role in gene regulatory switches active in proliferating tumor cells undergoing cycles of hypoxia and reoxygenation.

  8. Hypoxia induces a novel signature of chromatin modifications and global repression of transcription

    PubMed Central

    Johnson, Amber Buescher; Denko, Nicholas; Barton, Michelle Craig

    2008-01-01

    Tumor cells respond to the harsh hypoxic microenvironment, in part, by transcriptional regulation of specific target genes. We found that hypoxia-mediated activation of selected genes occurs amidst widespread repression of transcription that is neither cell type-specific nor HIF-1-dependent. Despite overall repression, hypoxia induces a pool of histone modifications typically associated with transcriptional activation or repression. Chromatin immunoprecipitation analyses showed that this global mixture of hypoxia-modified histones is sorted in a gene-specific manner to correlate with transcriptional response to hypoxia. Exceptions to this were unexpected increases in H3K4me3 levels, typically associated with transcriptional activation, and decreased H3K27me3 levels, generally a marker of transcriptional silencing, at core promoters of both hypoxia-activated and -repressed genes. These data suggest that a novel signature of chromatin modifications is induced under hypoxic stress, which may play a role in gene regulatory switches active in proliferating tumor cells undergoing cycles of hypoxia and reoxygenation. PMID:18294659

  9. Targeted chromatin binding and histone acetylation in vivo by thyroid hormone receptor during amphibian development.

    PubMed

    Sachs, L M; Shi, Y B

    2000-11-21

    Amphibian metamorphosis is marked by dramatic, thyroid hormone (TH)-induced changes involving gene regulation by TH receptor (TR). It has been postulated that TR-mediated gene regulation involves chromatin remodeling. In the absence of ligand, TR can repress gene expression by recruiting a histone deacetylase complex, whereas liganded TR recruits a histone acetylase complex for gene activation. Earlier studies have led us to propose a dual function model for TR during development. In premetamorphic tadpoles, unliganded TR represses transcription involving histone deacetylation. During metamorphosis, endogenous TH allows TR to activate gene expression through histone acetylation. Here using chromatin immunoprecipitation assay, we directly demonstrate TR binding to TH response genes constitutively in vivo in premetamorphic tadpoles. We further show that TH treatment leads to histone deacetylase release from TH response gene promoters. Interestingly, in whole animals, changes in histone acetylation show little correlation with the expression of TH response genes. On the other hand, in the intestine and tail, where TH response genes are known to be up-regulated more dramatically by TH than in most other organs, we demonstrate that TH treatment induces gene activation and histone H4 acetylation. These data argue for a role of histone acetylation in transcriptional regulation by TRs during amphibian development in some tissues, whereas in others changes in histone acetylation levels may play no or only a minor role, supporting the existence of important alternative mechanisms in gene regulation by TR.

  10. ChIP-Seq to Analyze the Binding of Replication Proteins to Chromatin.

    PubMed

    Ostrow, A Zachary; Viggiani, Christopher J; Aparicio, Jennifer G; Aparicio, Oscar M

    2015-01-01

    Chromatin immunoprecipitation (ChIP) is a widely used method to study interactions between proteins and discrete chromosomal loci in vivo. ChIP was originally developed for in vivo analysis of protein associations with candidate DNA sequences known or suspected to bind the protein of interest. The advent of DNA microarrays enabled the unbiased, genome-scale identification of all DNA sequences enriched by ChIP, providing a genomic map of a protein's chromatin binding. This method, termed ChIP-chip, is broadly applicable and has been particularly valuable in DNA replication studies to map potential replication origins in Saccharomyces cerevisiae and other organisms based on the specific association of certain replication proteins with these chromosomal elements, which are distributed throughout the genome. More recently, high-throughput sequencing (HTS) technologies have replaced microarrays as the preferred method for genomic analysis of ChIP experiments, and this combination is termed ChIP-Seq. We present a detailed ChIP-Seq protocol for S. cerevisiae that can be adapted for different HTS platforms and for different organisms. We also outline general schemes for data analysis; however, HTS data analyses usually must be tailored specifically for individual studies, depending on the experimental design, data characteristics, and the genome being analyzed.

  11. Androgen receptor DNA binding and chromatin accessibility profiling in prostate cancer

    PubMed Central

    Nevedomskaya, Ekaterina; Stelloo, Suzan; van der Poel, Henk G.; de Jong, Jeroen; Wessels, Lodewyk F.A.; Bergman, Andries M.; Zwart, Wilbert

    2015-01-01

    Prostate cancer (PCa) is the second most common cancer in men. The Androgen Receptor (AR) is the major driver of PCa and the main target of therapy in the advanced setting. AR is a nuclear receptor that binds the chromatin and regulates transcription of genes involved in cancer cell proliferation and survival. In a study by Stelloo et al. (1) we explored prostate cancer on the level of transcriptional regulation by means of Formaldehyde-Assisted Isolation of Regulatory Elements and Chromatin Immunoprecipitation coupled with massive parallel sequencing (FAIRE-seq and ChIP-seq, respectively). We employed these data for the assessment of differences in transcriptional regulation at distinct stages of PCa progression and to construct a prognostic gene expression classifier. Genomics data includes FAIRE-seq data from normal prostate tissue as well as primary, hormone therapy resistant and metastatic PCa. Furthermore, ChIP-seq data from primary and resistant PCa were generated, along with multiple input controls. The data are publicly available through NCBI GEO database with accession number GSE65478. Here we describe the genomics and clinical data in detail and provide comparative analysis of FAIRE-seq and ChIP-seq data. PMID:26981385

  12. A unique chromatin complex occupies young α-satellite arrays of human centromeres

    PubMed Central

    Henikoff, Jorja G.; Thakur, Jitendra; Kasinathan, Sivakanthan; Henikoff, Steven

    2015-01-01

    The intractability of homogeneous α-satellite arrays has impeded understanding of human centromeres. Artificial centromeres are produced from higher-order repeats (HORs) present at centromere edges, although the exact sequences and chromatin conformations of centromere cores remain unknown. We use high-resolution chromatin immunoprecipitation (ChIP) of centromere components followed by clustering of sequence data as an unbiased approach to identify functional centromere sequences. We find that specific dimeric α-satellite units shared by multiple individuals dominate functional human centromeres. We identify two recently homogenized α-satellite dimers that are occupied by precisely positioned CENP-A (cenH3) nucleosomes with two ~100–base pair (bp) DNA wraps in tandem separated by a CENP-B/CENP-C–containing linker, whereas pericentromeric HORs show diffuse positioning. Precise positioning is largely maintained, whereas abundance decreases exponentially with divergence, which suggests that young α-satellite dimers with paired ~100-bp particles mediate evolution of functional human centromeres. Our unbiased strategy for identifying functional centromeric sequences should be generally applicable to tandem repeat arrays that dominate the centromeres of most eukaryotes. PMID:25927077

  13. CHD chromatin remodelers and the transcription cycle.

    PubMed

    Murawska, Magdalena; Brehm, Alexander

    2011-01-01

    It is well established that ATP-dependent chromatin remodelers modulate DNA access of transcription factors and RNA polymerases by "opening" or "closing" chromatin structure. However, this view is far too simplistic. Recent findings have demonstrated that these enzymes not only set the stage for the transcription machinery to act but are actively involved at every step of the transcription process. As a consequence, they affect initiation, elongation, termination and RNA processing. In this review we will use the CHD family as a paradigm to illustrate the progress that has been made in revealing these new concepts.

  14. Chromatin Control of Developmental Dynamics and Plasticity.

    PubMed

    Perino, Matteo; Veenstra, Gert Jan C

    2016-09-26

    Chromatin structure is intimately connected with gene expression and cell identity. Here we review recent advances in the field and discuss how establishment of cell identity during development is accompanied by large-scale remodeling of the epigenetic landscape and how this remodeling drives and supports lineage specification and maintenance. We discuss maternal control of the early embryonic epigenetic landscape, selective usage of enhancer clusters via 3D chromatin contacts leading to activation of transcription factor networks, and conserved regulation of developmental pathways by specific DNA demethylation of key regulatory regions. Together, these processes establish an epigenetic framework regulating different phases of embryonic development. PMID:27676434

  15. Rapid and unbiased extraction of chromatin associated RNAs from purified native chromatin.

    PubMed

    Zhou, Zhongwu; Yang, Yi; Konieczny, Stephen F; Irudayaraj, Joseph M K

    2015-12-24

    An ultra fast and unbiased method that uses salicylic acid coated magnetic nanoparticles (SAMNPs) and magnetophoretic chromatography is developed to extract chromatin associated RNAs (CARs). The SAMNPs were first used for enriching cells from the cell culture media and further used for capturing chromatin after cells were lysed. The formed SAMNPs-chromatin complexes were transferred to a viscous polyethylene glycol (PEG) solution stored in a 200-μl pipette tip. Due to the difference in viscosities, a bi-layer liquid was formed inside the pipette tip. The SAMNPs-chromatin complexes were separated from the free SAMNPs and free RNA-SAMNPs complexes by applying an external magnetic field. The CARs were further extracted from the SAMNP-chromatin complexes directly. The extracted CARs were reverse transcribed as cDNA and further characterized by real-time qPCR. The total assay time taken for cell separation, chromatin purification and chromatin associated RNAs extraction can be accomplished in less than 2h. PMID:26643718

  16. Altered chromatin structure associated with methylation-induced gene silencing in cancer cells: correlation of accessibility, methylation, MeCP2 binding and acetylation

    PubMed Central

    Nguyen, Carvell T.; Gonzales, Felicidad A.; Jones, Peter A.

    2001-01-01

    Silencing of tumor-suppressor genes by hypermethylation of promoter CpG islands is well documented in human cancer and may be mediated by methyl-CpG-binding proteins, like MeCP2, that are associated in vivo with chromatin modifiers and transcriptional repressors. However, the exact dynamic between methylation and chromatin structure in the regulation of gene expression is not well understood. In this study, we have analyzed the methylation status and chromatin structure of three CpG islands in the p14(ARF)/p16(INK4A) locus in a series of normal and cancer cell lines using methylation-sensitive digestion, MspI accessibility in intact nuclei and chromatin immunoprecipitation (ChIP) assays. We demonstrate the existence of an altered chromatin structure associated with the silencing of tumor-suppressor genes in human cancer cell lines involving CpG island methylation, chromatin condensation, histone deacetylation and MeCP2 binding. The data showed that MeCP2 could bind to methylated CpG islands in both promoters and exons; MeCP2 does not interfere with transcription when bound at an exon, suggesting a more generalized role for the protein beyond transcriptional repression. In the absence of methylation, it is demonstrated that CpG islands located in promoters versus exons display marked differences in the levels of acetylation of associated histone H3, suggesting that chromatin remodeling can be achieved by methylation-independent processes and perhaps explaining why non-promoter CpG islands are more susceptible to de novo methylation than promoter islands. PMID:11713309

  17. Centromeric Chromatin and the Pathway that Drives Its Propagation

    PubMed Central

    Falk, Samantha J.; Black, Ben E.

    2011-01-01

    The centromere is the locus that directs chromosomal inheritance at cell division. While centromeres in diverse eukaryotes are commonly found at sites of repetitive DNA, their location is epigenetically specified. The histone H3 variant CENP-A is the prime candidate for epigenetically marking the centromere, and recent work has uncovered several additional proteins that play key roles in centromere assembly and maintenance. We describe advances in the identification and characterization of proteins that form the centromere, and focus on recent findings that have advanced our understanding of the assembly of functional centromeric chromatin. PMID:22154124

  18. In Vivo Chromatin Targets of the Transcription Factor Yin Yang 2 in Trophoblast Stem Cells

    PubMed Central

    Pérez-Palacios, Raquel; Macías-Redondo, Sofía; Climent, María; Contreras-Moreira, Bruno; Muniesa, Pedro; Schoorlemmer, Jon

    2016-01-01

    Background Yin Yang 2 (YY2) is a zinc finger protein closely related to the well-characterized Yin Yang 1 (YY1). YY1 is a DNA-binding transcription factor, with defined functions in multiple developmental processes, such as implantation, cell differentiation, X inactivation, imprinting and organogenesis. Yy2 has been treated as a largely immaterial duplication of Yy1, as they share high homology in the Zinc Finger-region and similar if not identical in vitro binding sites. In contrast to these similarities, gene expression alterations in HeLa cells with attenuated levels of either Yy1 or Yy2 were to some extent gene-specific. Moreover, the chromatin binding sites for YY2, except for its association with transposable retroviral elements (RE) and Endogenous Retroviral Elements (ERVs), remain to be identified. As a first step towards defining potential Yy2 functions matching or complementary to Yy1, we considered in vivo DNA binding sites of YY2 in trophoblast stem (TS) cells. Results We report the presence of YY2 protein in mouse-derived embryonic stem (ES) and TS cell lines. Following up on our previous report on ERV binding by YY2 in TS cells, we investigated the tissue-specificity of REX1 and YY2 binding and confirm binding to RE/ERV targets in both ES cells and TS cells. Because of the higher levels of expression, we chose TS cells to understand the role of Yy2 in gene and chromatin regulation. We used in vivo YY2 association as a measure to identify potential target genes. Sequencing of chromatin obtained in chromatin-immunoprecipitation (ChIP) assays carried out with αYY2 serum allowed us to identify a limited number of chromatin targets for YY2. Some putative binding sites were validated in regular ChIP assays and gene expression of genes nearby was altered in the absence of Yy2. Conclusions YY2 binding to ERVs is not confined to TS cells. In vivo binding sites share the presence of a consensus binding motif. Selected sites were uniquely bound by YY2 as

  19. Chromatin remodelling factor Mll1 is essential for neurogenesis from postnatal neural stem cells

    PubMed Central

    Lim, Daniel A.; Huang, Yin-Cheng; Swigut, Tomek; Mirick, Anika L.; Garcia-Verdugo, Jose Manuel; Wysocka, Joanna; Ernst, Patricia; Alvarez-Buylla, Arturo

    2013-01-01

    Epigenetic mechanisms that maintain neurogenesis throughout adult life remain poorly understood1. Trithorax group (trxG) and Polycomb group (PcG) gene products are part of an evolutionarily conserved chromatin remodelling system that activate or silence gene expression, respectively2. Although PcG member Bmi1 has been shown to be required for postnatal neural stem cell self-renewal3,4, the role of trxG genes remains unknown. Here we show that the trxG member Mll1 (mixed-lineage leukaemia 1) is required for neurogenesis in the mouse postnatal brain. Mll1-deficient subventricular zone neural stem cells survive, proliferate and efficiently differentiate into glial lineages; however, neuronal differentiation is severely impaired. In Mll1-deficient cells, early proneural Mash1 (also known as Ascl1) and gliogenic Olig2 expression are preserved, but Dlx2, a key downstream regulator of subventricular zone neurogenesis, is not expressed. Over-expression of Dlx2 can rescue neurogenesis in Mll1-deficient cells. Chromatin immunoprecipitation demonstrates that Dlx2 is a direct target of MLL in subventricular zone cells. In differentiating wild-type subventricular zone cells, Mash1, Olig2 and Dlx2 loci have high levels of histone 3 trimethylated at lysine 4 (H3K4me3), consistent with their transcription. In contrast, in Mll1-deficient subventricular zone cells, chromatin at Dlx2 is bivalently marked by both H3K4me3 and histone 3 trimethylated at lysine 27 (H3K27me3), and the Dlx2 gene fails to properly activate. These data support a model in which Mll1 is required to resolve key silenced bivalent loci in postnatal neural precursors to the actively transcribed state for the induction of neurogenesis, but not for gliogenesis. PMID:19212323

  20. Imipramine Treatment and Resiliency Exhibit Similar Chromatin Regulation in the Mouse Nucleus Accumbens in Depression Models

    PubMed Central

    Wilkinson, Matthew B.; Xiao, Guanghua; Kumar, Arvind; LaPlant, Quincey; Renthal, William; Sikder, Devanjan; Kodadek, Thomas J.; Nestler, Eric J.

    2009-01-01

    Though it is a widely studied psychiatric syndrome, major depressive disorder remains a poorly understood illness, especially with regard to the disconnect between treatment initiation and the delayed onset of clinical improvement. We have recently validated chronic social defeat stress in mice as a model in which a depression-like phenotype is reversed by chronic, but not acute, antidepressant administration. Here, we use ChIP-chip assays—chromatin immunoprecipitation (ChIP) followed by genome wide promoter array analyses—to study the effects of chronic defeat stress on chromatin regulation in the mouse nucleus accumbens (NAc), a key brain reward region implicated in depression. Our results demonstrate that chronic defeat stress causes widespread and long-lasting changes in gene regulation, including alterations in repressive histone methylation and in phospho-CREB binding, in the NAc. We then show similarities and differences in this regulation to that observed in another mouse model of depression, prolonged adult social isolation. In the social defeat model, we observed further that most of the stress-induced changes in gene expression are reversed by chronic imipramine treatment, and that resilient mice—those resistant to the deleterious effects of defeat stress—show patterns of chromatin regulation in the NAc that overlap dramatically with those seen with imipramine treatment. These findings provide new insight into the molecular basis of depression-like symptoms and the mechanisms by which antidepressants exert their delayed clinical efficacy. They also raise the novel idea that certain individuals resistant to stress may naturally mount antidepressant-like adaptations in response to chronic stress. PMID:19535594

  1. Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin).

    PubMed

    Erwin, Graham S; Grieshop, Matthew P; Bhimsaria, Devesh; Eguchi, Asuka; Rodríguez-Martínez, José A; Ansari, Aseem Z

    2016-01-01

    The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide-DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

  2. Chromatin modifications induced by PML-RARα repress critical targets in leukemogenesis as analyzed by ChIP-Chip

    PubMed Central

    Hoemme, Claudia; Peerzada, Abdul; Behre, Gerhard; Wang, Yipeng; McClelland, Michael; Nieselt, Kay; Zschunke, Matthias; Disselhoff, Christine; Agrawal, Shuchi; Isken, Fabienne; Tidow, Nicola; Berdel, Wolfgang E.; Serve, Hubert

    2008-01-01

    The translocation t(15;17) generates the chimeric PML-RARα transcription factor that is the initiating event of acute promyelocytic leukemia. A global view of PML-RARα transcriptional functions was obtained by genome-wide binding and chromatin modification analyses combined with genome-wide expression data. Chromatin immunoprecipitation (ChIP)–chip experiments identified 372 direct genomic PML-RARα targets. A subset of these was confirmed in primary acute promyelocytic leukemia. Direct PML-RARα targets include regulators of global transcriptional programs as well as critical regulatory genes for basic cellular functions such as cell-cycle control and apoptosis. PML-RARα binding universally led to HDAC1 recruitment, loss of histone H3 acetylation, increased tri-methylation of histone H3 lysine 9, and unexpectedly increased trimethylation of histone H3 lysine 4. The binding of PML-RARα to target promoters and the resulting histone modifications resulted in mRNA repression of functionally relevant genes. Taken together, our results reveal that the transcription factor PML-RARα regulates key cancer-related genes and pathways by inducing a repressed chromatin formation on its direct genomic target genes. PMID:18024792

  3. Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate.

    PubMed

    Wu, Miin-Feng; Yamaguchi, Nobutoshi; Xiao, Jun; Bargmann, Bastiaan; Estelle, Mark; Sang, Yi; Wagner, Doris

    2015-01-01

    Reprogramming of cell identities during development frequently requires changes in the chromatin state that need to be restricted to the correct cell populations. Here we identify an auxin hormone-regulated chromatin state switch that directs reprogramming from transit amplifying to primordium founder cell fate in Arabidopsis inflorescences. Upon auxin sensing, the MONOPTEROS transcription factor recruits SWI/SNF chromatin remodeling ATPases to increase accessibility of the DNA for induction of key regulators of flower primordium initiation. In the absence of the hormonal cue, auxin sensitive Aux/IAA proteins bound to MONOPTEROS block recruitment of the SWI/SNF chromatin remodeling ATPases in addition to recruiting a co-repressor/histone deacetylase complex. This simple and elegant hormone-mediated chromatin state switch is ideally suited for iterative flower primordium initiation and orchestrates additional auxin-regulated cell fate transitions. Our findings establish a new paradigm for nuclear response to auxin. They also provide an explanation for how this small molecule can direct diverse plant responses. PMID:26460543

  4. ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor.

    PubMed

    Citterio, E; Van Den Boom, V; Schnitzler, G; Kanaar, R; Bonte, E; Kingston, R E; Hoeijmakers, J H; Vermeulen, W

    2000-10-01

    The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed. PMID:11003660

  5. Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate.

    PubMed

    Wu, Miin-Feng; Yamaguchi, Nobutoshi; Xiao, Jun; Bargmann, Bastiaan; Estelle, Mark; Sang, Yi; Wagner, Doris

    2015-10-13

    Reprogramming of cell identities during development frequently requires changes in the chromatin state that need to be restricted to the correct cell populations. Here we identify an auxin hormone-regulated chromatin state switch that directs reprogramming from transit amplifying to primordium founder cell fate in Arabidopsis inflorescences. Upon auxin sensing, the MONOPTEROS transcription factor recruits SWI/SNF chromatin remodeling ATPases to increase accessibility of the DNA for induction of key regulators of flower primordium initiation. In the absence of the hormonal cue, auxin sensitive Aux/IAA proteins bound to MONOPTEROS block recruitment of the SWI/SNF chromatin remodeling ATPases in addition to recruiting a co-repressor/histone deacetylase complex. This simple and elegant hormone-mediated chromatin state switch is ideally suited for iterative flower primordium initiation and orchestrates additional auxin-regulated cell fate transitions. Our findings establish a new paradigm for nuclear response to auxin. They also provide an explanation for how this small molecule can direct diverse plant responses.

  6. Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate

    PubMed Central

    Wu, Miin-Feng; Yamaguchi, Nobutoshi; Xiao, Jun; Bargmann, Bastiaan; Estelle, Mark; Sang, Yi; Wagner, Doris

    2015-01-01

    Reprogramming of cell identities during development frequently requires changes in the chromatin state that need to be restricted to the correct cell populations. Here we identify an auxin hormone-regulated chromatin state switch that directs reprogramming from transit amplifying to primordium founder cell fate in Arabidopsis inflorescences. Upon auxin sensing, the MONOPTEROS transcription factor recruits SWI/SNF chromatin remodeling ATPases to increase accessibility of the DNA for induction of key regulators of flower primordium initiation. In the absence of the hormonal cue, auxin sensitive Aux/IAA proteins bound to MONOPTEROS block recruitment of the SWI/SNF chromatin remodeling ATPases in addition to recruiting a co-repressor/histone deacetylase complex. This simple and elegant hormone-mediated chromatin state switch is ideally suited for iterative flower primordium initiation and orchestrates additional auxin-regulated cell fate transitions. Our findings establish a new paradigm for nuclear response to auxin. They also provide an explanation for how this small molecule can direct diverse plant responses. DOI: http://dx.doi.org/10.7554/eLife.09269.001 PMID:26460543

  7. Effete, an E2 ubiquitin-conjugating enzyme with multiple roles in Drosophila development and chromatin organization

    PubMed Central

    Cipressa, Francesca; Cenci, Giovanni

    2013-01-01

    The Drosophila effete gene encodes an extremely conserved class I E2 ubiquitin-conjugating enzyme. Growing evidence indicates that Eff is involved in many cellular processes including eye development, maintenance of female germline stem cells, and regulation of apoptosis. Eff is also a major component of Drosophila chromatin and it is particularly enriched in chromatin with repressive properties. In addition, Eff is required for telomere protection and to prevent telomere fusion. Consistent with its multiple roles in chromatin maintenance, Eff is also one of the rare factors that modulate both telomere-induced and heterochromatin-induced position effect variegation. PMID:24088712

  8. Chromatin modifiers and histone modifications in bone formation, regeneration, and therapeutic intervention for bone-related disease.

    PubMed

    Gordon, Jonathan A R; Stein, Janet L; Westendorf, Jennifer J; van Wijnen, Andre J

    2015-12-01

    Post-translational modifications of chromatin such as DNA methylation and different types of histone acetylation, methylation and phosphorylation are well-appreciated epigenetic mechanisms that confer information to progeny cells during lineage commitment. These distinct epigenetic modifications have defined roles in bone, development, tissue regeneration, cell commitment and differentiation, as well as disease etiologies. In this review, we discuss the role of these chromatin modifications and the enzymes regulating these marks (methyltransferases, demethylases, acetyltransferases, and deacetylases) in progenitor cells, osteoblasts and bone-related cells. In addition, the clinical relevance of deregulated histone modifications and enzymes as well as current and potential therapeutic interventions targeting chromatin modifiers are addressed.

  9. Proteomics Analysis of Cellular Proteins Co-Immunoprecipitated with Nucleoprotein of Influenza A Virus (H7N9).

    PubMed

    Sun, Ningning; Sun, Wanchun; Li, Shuiming; Yang, Jingbo; Yang, Longfei; Quan, Guihua; Gao, Xiang; Wang, Zijian; Cheng, Xin; Li, Zehui; Peng, Qisheng; Liu, Ning

    2015-01-01

    Avian influenza A viruses are serious veterinary pathogens that normally circulate among avian populations, causing substantial economic impacts. Some strains of avian influenza A viruses, such as H5N1, H9N2, and recently reported H7N9, have been occasionally found to adapt to humans from other species. In order to replicate efficiently in the new host, influenza viruses have to interact with a variety of host factors. In the present study, H7N9 nucleoprotein was transfected into human HEK293T cells, followed by immunoprecipitated and analyzed by proteomics approaches. A series of host proteins co-immunoprecipitated were identified with high confidence, some of which were found to be acetylated at their lysine residues. Bioinformatics analysis revealed that spliceosome might be the most relevant pathway involved in host response to nucleoprotein expression, increasing our emerging knowledge of host proteins that might be involved in influenza virus replication activities. PMID:26528969

  10. Chromatin regulation: how complex does it get?

    PubMed

    Meier, Karin; Brehm, Alexander

    2014-11-01

    Gene transcription is tightly regulated at different levels to ensure that the transcriptome of the cell is appropriate for developmental stage and cell type. The chromatin state in which a gene is embedded determines its expression level to a large extent. Activation or repression of transcription is typically accomplished by the recruitment of chromatin-associated multisubunit protein complexes that combine several molecular tools, such as histone-binding and chromatin-modifying activities. Recent biochemical purifications of such complexes have revealed a substantial diversity. On the one hand, complexes that were thought to be unique have been revealed to be part of large complex families. On the other hand, protein subunits that were thought to only exist in separate complexes have been shown to coexist in novel assemblies. In this review we discuss our current knowledge of repressor complexes that contain MBT domain proteins and/or the CoREST co-repressor and use them as a paradigm to illustrate the unexpected heterogeneity and tool sharing of chromatin regulating protein complexes. These recent insights also challenge the ways we define and think about protein complexes in general. PMID:25482055

  11. The great repression: chromatin and cryptic transcription.

    PubMed

    Hennig, Bianca P; Fischer, Tamás

    2013-01-01

    The eukaryotic chromatin structure is essential in correctly defining transcription units. Impairing this structure can activate cryptic promoters, and lead to the accumulation of aberrant RNA transcripts. Here we discuss critical pathways that are responsible for the repression of cryptic transcription and the maintenance of genome integrity.

  12. Trivalent chromatin marks the way in.

    PubMed

    Hysolli, Eriona; Park, In-Hyun

    2013-11-01

    Recently in Cell, Wapinski et al. (2013) investigated the epigenetic mechanisms underlying the direct conversion of fibroblasts to induced neurons (iNs). They found that Ascl1 acts as a pioneer factor at neurogenic loci marked by a closed "trivalent" chromatin state in cells permissive to direct conversion, but not in restrictive cell types. PMID:24209756

  13. Chemical biology: Chromatin chemistry goes cellular

    NASA Astrophysics Data System (ADS)

    Fischle, Wolfgang; Schwarzer, Dirk; Mootz, Henning D.

    2015-05-01

    Analysing post-translational modifications of histone proteins as they occur within chromatin is challenging due to their large number and chemical diversity. A major step forward has now been achieved by using split intein chemistry to engineer functionalized histones within cells.

  14. Chromatin regulation: How complex does it get?

    PubMed Central

    Meier, Karin; Brehm, Alexander

    2014-01-01

    Gene transcription is tightly regulated at different levels to ensure that the transcriptome of the cell is appropriate for developmental stage and cell type. The chromatin state in which a gene is embedded determines its expression level to a large extent. Activation or repression of transcription is typically accomplished by the recruitment of chromatin-associated multisubunit protein complexes that combine several molecular tools, such as histone-binding and chromatin-modifying activities. Recent biochemical purifications of such complexes have revealed a substantial diversity. On the one hand, complexes that were thought to be unique have been revealed to be part of large complex families. On the other hand, protein subunits that were thought to only exist in separate complexes have been shown to coexist in novel assemblies. In this review we discuss our current knowledge of repressor complexes that contain MBT domain proteins and/or the CoREST co-repressor and use them as a paradigm to illustrate the unexpected heterogeneity and tool sharing of chromatin regulating protein complexes. These recent insights also challenge the ways we define and think about protein complexes in general. PMID:25482055

  15. Interplay between mismatch repair and chromatin assembly

    PubMed Central

    Schöpf, Barbara; Bregenhorn, Stephanie; Quivy, Jean-Pierre; Kadyrov, Farid A.; Almouzni, Genevieve; Jiricny, Josef

    2012-01-01

    Single strand nicks and gaps in DNA have been reported to increase the efficiency of nucleosome loading mediated by chromatin assembly factor 1 (CAF-1). However, on mismatch-containing substrates, these strand discontinuities are utilized by the mismatch repair (MMR) system as loading sites for exonuclease 1, at which degradation of the error-containing strand commences. Because packaging of DNA into chromatin might inhibit MMR, we were interested to learn whether chromatin assembly is differentially regulated on heteroduplex and homoduplex substrates. We now show that the presence of a mismatch in a nicked plasmid substrate delays nucleosome loading in human cell extracts. Our data also suggest that, once the mismatch is removed, repair of the single-stranded gap is accompanied by efficient nucleosome loading. We postulated that the balance between MMR and chromatin assembly might be governed by proliferating cell nuclear antigen (PCNA), the processivity factor of replicative DNA polymerases, which is loaded at DNA termini and which interacts with the MSH6 subunit of the mismatch recognition factor MutSα, as well as with CAF-1. We now show that this regulation might be more complex; MutSα and CAF-1 interact not only with PCNA, but also with each other. In vivo this interaction increases during S-phase and may be controlled by the phosphorylation status of the p150 subunit of CAF-1. PMID:22232658

  16. Epigenetic chromatin silencing: bistability and front propagation

    NASA Astrophysics Data System (ADS)

    Sedighi, Mohammad; Sengupta, Anirvan M.

    2007-12-01

    The role of post-translational modification of histones in eukaryotic gene regulation is well recognized. Epigenetic silencing of genes via heritable chromatin modifications plays a major role in cell fate specification in higher organisms. We formulate a coarse-grained model of chromatin silencing in yeast and study the conditions under which the system becomes bistable, allowing for different epigenetic states. We also study the dynamics of the boundary between the two locally stable states of chromatin: silenced and unsilenced. The model could be of use in guiding the discussion on chromatin silencing in general. In the context of silencing in budding yeast, it helps us understand the phenotype of various mutants, some of which may be non-trivial to see without the help of a mathematical model. One such example is a mutation that reduces the rate of background acetylation of particular histone side chains that competes with the deacetylation by Sir2p. The resulting negative feedback due to a Sir protein depletion effect gives rise to interesting counter-intuitive consequences. Our mathematical analysis brings forth the different dynamical behaviors possible within the same molecular model and guides the formulation of more refined hypotheses that could be addressed experimentally.

  17. Epigenetic chromatin silencing: bistability and front propagation

    PubMed Central

    Sedighi, Mohammad; Sengupta, Anirvan M

    2008-01-01

    The role of post-translational modification of histones in eukaryotic gene regulation is well recognized. Epigenetic silencing of genes via heritable chromatin modifications plays a major role in cell fate specification in higher organisms. We formulate a coarse-grained model of chromatin silencing in yeast and study the conditions under which the system becomes bistable, allowing for different epigenetic states. We also study the dynamics of the boundary between the two locally stable states of chromatin: silenced and unsilenced. The model could be of use in guiding the discussion on chromatin silencing in general. In the context of silencing in budding yeast, it helps us understand the phenotype of various mutants, some of which may be non-trivial to see without the help of a mathematical model. One such example is a mutation that reduces the rate of background acetylation of particular histone side chains that competes with the deacetylation by Sir2p. The resulting negative feedback due to a Sir protein depletion effect gives rise to interesting counter-intuitive consequences. Our mathematical analysis brings forth the different dynamical behaviors possible within the same molecular model and guides the formulation of more refined hypotheses that could be addressed experimentally. PMID:17991991

  18. Influenza Virus and Chromatin: Role of the CHD1 Chromatin Remodeler in the Virus Life Cycle

    PubMed Central

    Marcos-Villar, Laura; Pazo, Alejandra

    2016-01-01

    ABSTRACT Influenza A virus requires ongoing cellular transcription to carry out the cap-snatching process. Chromatin remodelers modify chromatin structure to produce an active or inactive conformation, which enables or prevents the recruitment of transcriptional complexes to specific genes; viral transcription thus depends on chromatin dynamics. Influenza virus polymerase associates with chromatin components of the infected cell, such as RNA polymerase II (RNAP II) or the CHD6 chromatin remodeler. Here we show that another CHD family member, CHD1 protein, also interacts with the influenza virus polymerase complex. CHD1 recognizes the H3K4me3 (histone 3 with a trimethyl group in lysine 4) histone modification, a hallmark of active chromatin. Downregulation of CHD1 causes a reduction in viral polymerase activity, viral RNA transcription, and the production of infectious particles. Despite the dependence of influenza virus on cellular transcription, RNAP II is degraded when viral transcription is complete, and recombinant viruses unable to degrade RNAP II show decreased pathogenicity in the murine model. We describe the CHD1–RNAP II association, as well as the parallel degradation of both proteins during infection with viruses showing full or reduced induction of degradation. The H3K4me3 histone mark also decreased during influenza virus infection, whereas a histone mark of inactive chromatin, H3K27me3, remained unchanged. Our results indicate that CHD1 is a positive regulator of influenza virus multiplication and suggest a role for chromatin remodeling in the control of the influenza virus life cycle. IMPORTANCE Although influenza virus is not integrated into the genome of the infected cell, it needs continuous cellular transcription to synthesize viral mRNA. This mechanism implies functional association with host genome expression and thus depends on chromatin dynamics. Influenza virus polymerase associates with transcription-related factors, such as RNA

  19. Understanding RNA-Chromatin Interactions Using Chromatin Isolation by RNA Purification (ChIRP).

    PubMed

    Chu, Ci; Chang, Howard Y

    2016-01-01

    ChIRP is a novel and easy-to-use technique for studying long noncoding RNA (lncRNA)-chromatin interactions. RNA and chromatin are cross-linked in vivo using formaldehyde or glutaraldehyde, and purified using biotinylated antisense oligonucleotides that hybridize to the target RNA. Co-precipitated DNA is then purified and analyzed by quantitative PCR (qPCR) or high-throughput sequencing. PMID:27659979

  20. Chromatin boundary elements organize genomic architecture and developmental gene regulation in Drosophila Hox clusters.

    PubMed

    Ma, Zhibo; Li, Mo; Roy, Sharmila; Liu, Kevin J; Romine, Matthew L; Lane, Derrick C; Patel, Sapna K; Cai, Haini N

    2016-08-26

    The three-dimensional (3D) organization of the eukaryotic genome is critical for its proper function. Evidence suggests that extensive chromatin loops form the building blocks of the genomic architecture, separating genes and gene clusters into distinct functional domains. These loops are anchored in part by a special type of DNA elements called chromatin boundary elements (CBEs). CBEs were originally found to insulate neighboring genes by blocking influences of transcriptional enhancers or the spread of silent chromatin. However, recent results show that chromatin loops can also play a positive role in gene regulation by looping out intervening DNA and "delivering" remote enhancers to gene promoters. In addition, studies from human and model organisms indicate that the configuration of chromatin loops, many of which are tethered by CBEs, is dynamically regulated during cell differentiation. In particular, a recent work by Li et al has shown that the SF1 boundary, located in the Drosophila Hox cluster, regulates local genes by tethering different subsets of chromatin loops: One subset enclose a neighboring gene ftz, limiting its access by the surrounding Scr enhancers and restrict the spread of repressive histones during early embryogenesis; and the other loops subdivide the Scr regulatory region into independent domains of enhancer accessibility. The enhancer-blocking activity of these CBE elements varies greatly in strength and tissue distribution. Further, tandem pairing of SF1 and SF2 facilitate the bypass of distal enhancers in transgenic flies, providing a mechanism for endogenous enhancers to circumvent genomic interruptions resulting from chromosomal rearrangement. This study demonstrates how a network of chromatin boundaries, centrally organized by SF1, can remodel the 3D genome to facilitate gene regulation during development. PMID:27621770

  1. Chromatin boundary elements organize genomic architecture and developmental gene regulation in Drosophila Hox clusters

    PubMed Central

    Ma, Zhibo; Li, Mo; Roy, Sharmila; Liu, Kevin J; Romine, Matthew L; Lane, Derrick C; Patel, Sapna K; Cai, Haini N

    2016-01-01

    The three-dimensional (3D) organization of the eukaryotic genome is critical for its proper function. Evidence suggests that extensive chromatin loops form the building blocks of the genomic architecture, separating genes and gene clusters into distinct functional domains. These loops are anchored in part by a special type of DNA elements called chromatin boundary elements (CBEs). CBEs were originally found to insulate neighboring genes by blocking influences of transcriptional enhancers or the spread of silent chromatin. However, recent results show that chromatin loops can also play a positive role in gene regulation by looping out intervening DNA and “delivering” remote enhancers to gene promoters. In addition, studies from human and model organisms indicate that the configuration of chromatin loops, many of which are tethered by CBEs, is dynamically regulated during cell differentiation. In particular, a recent work by Li et al has shown that the SF1 boundary, located in the Drosophila Hox cluster, regulates local genes by tethering different subsets of chromatin loops: One subset enclose a neighboring gene ftz, limiting its access by the surrounding Scr enhancers and restrict the spread of repressive histones during early embryogenesis; and the other loops subdivide the Scr regulatory region into independent domains of enhancer accessibility. The enhancer-blocking activity of these CBE elements varies greatly in strength and tissue distribution. Further, tandem pairing of SF1 and SF2 facilitate the bypass of distal enhancers in transgenic flies, providing a mechanism for endogenous enhancers to circumvent genomic interruptions resulting from chromosomal rearrangement. This study demonstrates how a network of chromatin boundaries, centrally organized by SF1, can remodel the 3D genome to facilitate gene regulation during development.

  2. Chromatin boundary elements organize genomic architecture and developmental gene regulation in Drosophila Hox clusters

    PubMed Central

    Ma, Zhibo; Li, Mo; Roy, Sharmila; Liu, Kevin J; Romine, Matthew L; Lane, Derrick C; Patel, Sapna K; Cai, Haini N

    2016-01-01

    The three-dimensional (3D) organization of the eukaryotic genome is critical for its proper function. Evidence suggests that extensive chromatin loops form the building blocks of the genomic architecture, separating genes and gene clusters into distinct functional domains. These loops are anchored in part by a special type of DNA elements called chromatin boundary elements (CBEs). CBEs were originally found to insulate neighboring genes by blocking influences of transcriptional enhancers or the spread of silent chromatin. However, recent results show that chromatin loops can also play a positive role in gene regulation by looping out intervening DNA and “delivering” remote enhancers to gene promoters. In addition, studies from human and model organisms indicate that the configuration of chromatin loops, many of which are tethered by CBEs, is dynamically regulated during cell differentiation. In particular, a recent work by Li et al has shown that the SF1 boundary, located in the Drosophila Hox cluster, regulates local genes by tethering different subsets of chromatin loops: One subset enclose a neighboring gene ftz, limiting its access by the surrounding Scr enhancers and restrict the spread of repressive histones during early embryogenesis; and the other loops subdivide the Scr regulatory region into independent domains of enhancer accessibility. The enhancer-blocking activity of these CBE elements varies greatly in strength and tissue distribution. Further, tandem pairing of SF1 and SF2 facilitate the bypass of distal enhancers in transgenic flies, providing a mechanism for endogenous enhancers to circumvent genomic interruptions resulting from chromosomal rearrangement. This study demonstrates how a network of chromatin boundaries, centrally organized by SF1, can remodel the 3D genome to facilitate gene regulation during development. PMID:27621770

  3. Influence of M-phase chromatin on the anisotropy of microtubule asters

    PubMed Central

    1996-01-01

    In many eukaryotic cells going through M-phase, a bipolar spindle is formed by microtubules nucleated from centrosomes. These microtubules, in addition to being "captured" by kinetochores, may be stabilized by chromatin in two different ways: short-range stabilization effects may affect microtubules in close contact with the chromatin, while long- range stabilization effects may "guide" microtubule growth towards the chromatin (e.g., by introducing a diffusive gradient of an enzymatic activity that affects microtubule assembly). Here, we use both meiotic and mitotic extracts from Xenopus laevis eggs to study microtubule aster formation and microtubule dynamics in the presence of chromatin. In "low-speed" meiotic extracts, in the presence of salmon sperm chromatin, we find that short-range stabilization effects lead to a strong anisotropy of the microtubule asters. Analysis of the dynamic parameters of microtubule growth show that this anisotropy arises from a decrease in the catastrophe frequency, an increase in the rescue frequency and a decrease in the growth velocity. In this system we also find evidence for long-range "guidance" effects, which lead to a weak anisotropy of the asters. Statistically relevant results on these long- range effects are obtained in "high-speed" mitotic extracts in the presence of artificially constructed chromatin stripes. We find that aster anisotropy is biased in the direction of the chromatin and that the catastrophe frequency is reduced in its vicinity. In this system we also find a surprising dependence of the catastrophe and the rescue frequencies on the length of microtubules nucleated from centrosomes: the catastrophe frequency increase and the rescue frequency decreases with microtubule length. PMID:8601601

  4. The many faces of plant chromatin: Meeting summary of the 4th European workshop on plant chromatin 2015, Uppsala, Sweden.

    PubMed

    Mozgová, Iva; Köhler, Claudia; Gaudin, Valérie; Hennig, Lars

    2015-01-01

    In June 2015, the fourth European Workshop on Plant Chromatin took place in Uppsala, Sweden, bringing together 80 researchers studying various aspects of plant chromatin and epigenetics. The intricate relationships between plant chromatin dynamics and gene expression change, chromatin organization within the plant cell nucleus, and the impact of chromatin structure on plant development were discussed. Among the main highlights of the meeting were an ever-growing list of newly identified players in chromatin structure establishment and the development of novel tools and approaches to foster our understanding of chromatin-mediated gene regulation, taking into account the context of the plant cell nucleus and its architecture. In this report, we summarize some of the main advances and prospects of plant chromatin research presented at this meeting. PMID:26646904

  5. Chromatin remodelling complex RSC promotes base excision repair in chromatin of Saccharomyces cerevisiae.

    PubMed

    Czaja, Wioletta; Mao, Peng; Smerdon, Michael J

    2014-04-01

    The base excision repair (BER) pathway is a conserved DNA repair system required to maintain genomic integrity and prevent mutagenesis in all eukaryotic cells. Nevertheless, how BER operates in vivo (i.e. in the context of chromatin) is poorly understood. We have investigated the role of an essential ATP-dependent chromatin remodelling (ACR) complex RSC (Remodels the Structure of Chromatin) in BER of intact yeast cells. We show that depletion of STH1, the ATPase subunit of RSC, causes enhanced sensitivity to the DNA alkylating agent methyl methanesulfonate (MMS) and results in a substantial inhibition of BER, at the GAL1 locus and in the genome overall. Consistent with this observation, the DNA in chromatin is less accessible to micrococcal nuclease digestion in the absence of RSC. Quantitative PCR results indicate that repair deficiency in STH1 depleted cells is not due to changes in the expression of BER genes. Collectively, our data indicates the RSC complex promotes efficient BER in chromatin. These results provide, for the first time, a link between ATP-dependent chromatin remodelling and BER in living cells.

  6. A unique chromatin signature uncovers early developmental enhancers in humans.

    PubMed

    Rada-Iglesias, Alvaro; Bajpai, Ruchi; Swigut, Tomek; Brugmann, Samantha A; Flynn, Ryan A; Wysocka, Joanna

    2011-02-10

    Cell-fate transitions involve the integration of genomic information encoded by regulatory elements, such as enhancers, with the cellular environment. However, identification of genomic sequences that control human embryonic development represents a formidable challenge. Here we show that in human embryonic stem cells (hESCs), unique chromatin signatures identify two distinct classes of genomic elements, both of which are marked by the presence of chromatin regulators p300 and BRG1, monomethylation of histone H3 at lysine 4 (H3K4me1), and low nucleosomal density. In addition, elements of the first class are distinguished by the acetylation of histone H3 at lysine 27 (H3K27ac), overlap with previously characterized hESC enhancers, and are located proximally to genes expressed in hESCs and the epiblast. In contrast, elements of the second class, which we term 'poised enhancers', are distinguished by the absence of H3K27ac, enrichment of histone H3 lysine 27 trimethylation (H3K27me3), and are linked to genes inactive in hESCs and instead are involved in orchestrating early steps in embryogenesis, such as gastrulation, mesoderm formation and neurulation. Consistent with the poised identity, during differentiation of hESCs to neuroepithelium, a neuroectoderm-specific subset of poised enhancers acquires a chromatin signature associated with active enhancers. When assayed in zebrafish embryos, poised enhancers are able to direct cell-type and stage-specific expression characteristic of their proximal developmental gene, even in the absence of sequence conservation in the fish genome. Our data demonstrate that early developmental enhancers are epigenetically pre-marked in hESCs and indicate an unappreciated role of H3K27me3 at distal regulatory elements. Moreover, the wealth of new regulatory sequences identified here provides an invaluable resource for studies and isolation of transient, rare cell populations representing early stages of human embryogenesis.

  7. Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining.

    PubMed

    Golan, R; Shochat, L; Weissenberg, R; Soffer, Y; Marcus, Z; Oschry, Y; Lewin, L M

    1997-01-01

    The quality of sperm chromatin is an important factor in fertilization and is especially critical where one spermatozoon is artificially selected for fertilizing an egg (as in intracytoplasmic sperm injection). In this study, flow cytometry after staining of human spermatozoa with Acridine Orange was used to study chromatin structure. A method is described for estimating the percentage of cells in a human sperm sample that have completed epididymal maturation in regard to chromatin condensation. Of the 121 samples of the semen that were examined, nine contained a higher percentage of hypocondensed spermatozoa and six samples contained elevated amounts of hypercondensed spermatozoa. In addition to aberrancies in chromatin condensation other defects showed up as satellite populations of spermatozoa with higher than normal ratios of red/green fluorescence after Acridine Orange staining. Such defects were found in 15 semen samples. The use of swim-up and Percoll gradient centrifugation methods was shown to improve the percentage of spermatozoa with normal chromatin structure in some samples with poor initial quality.

  8. The CSB chromatin remodeler and CTCF architectural protein cooperate in response to oxidative stress

    PubMed Central

    Lake, Robert J.; Boetefuer, Erica L.; Won, Kyoung-Jae; Fan, Hua-Ying

    2016-01-01

    Cockayne syndrome is a premature aging disease associated with numerous developmental and neurological abnormalities, and elevated levels of reactive oxygen species have been found in cells derived from Cockayne syndrome patients. The majority of Cockayne syndrome cases contain mutations in the ATP-dependent chromatin remodeler CSB; however, how CSB protects cells from oxidative stress remains largely unclear. Here, we demonstrate that oxidative stress alters the genomic occupancy of the CSB protein and increases CSB occupancy at promoters. Additionally, we found that the long-range chromatin-structure regulator CTCF plays a pivotal role in regulating sites of genomic CSB occupancy upon oxidative stress. We show that CSB directly interacts with CTCF in vitro and that oxidative stress enhances the CSB-CTCF interaction in cells. Reciprocally, we demonstrate that CSB facilitates CTCF-DNA interactions in vitro and regulates CTCF-chromatin interactions in oxidatively stressed cells. Together, our results indicate that CSB and CTCF can regulate each other's chromatin association, thereby modulating chromatin structure and coordinating gene expression in response to oxidative stress. PMID:26578602

  9. Chromatin and RNA Maps Reveal Regulatory Long Noncoding RNAs in Mouse

    PubMed Central

    Vizán, Pedro; Stanton, Lawrence W.; Beato, Miguel; Di Croce, Luciano

    2015-01-01

    Discovering and classifying long noncoding RNAs (lncRNAs) across all mammalian tissues and cell lines remains a major challenge. Previously, mouse lncRNAs were identified using transcriptome sequencing (RNA-seq) data from a limited number of tissues or cell lines. Additionally, associating a few hundred lncRNA promoters with chromatin states in a single mouse cell line has identified two classes of chromatin-associated lncRNA. However, the discovery and classification of lncRNAs is still pending in many other tissues in mouse. To address this, we built a comprehensive catalog of lncRNAs by combining known lncRNAs with high-confidence novel lncRNAs identified by mapping and de novo assembling billions of RNA-seq reads from eight tissues and a primary cell line in mouse. Next, we integrated this catalog of lncRNAs with multiple genome-wide chromatin state maps and found two different classes of chromatin state-associated lncRNAs, including promoter-associated (plncRNAs) and enhancer-associated (elncRNAs) lncRNAs, across various tissues. Experimental knockdown of an elncRNA resulted in the downregulation of the neighboring protein-coding Kdm8 gene, encoding a histone demethylase. Our findings provide 2,803 novel lncRNAs and a comprehensive catalog of chromatin-associated lncRNAs across different tissues in mouse. PMID:26711262

  10. Chromatin structure of repeating CTG/CAG and CGG/CCG sequences in human disease.

    PubMed

    Wang, Yuh-Hwa

    2007-05-01

    In eukaryotic cells, chromatin structure organizes genomic DNA in a dynamic fashion, and results in regulation of many DNA metabolic processes. The CTG/CAG and CGG/CCG repeating sequences involved in several neuromuscular degenerative diseases display differential abilities for the binding of histone octamers. The effect of the repeating DNA on nucleosome assembly could be amplified as the number of repeats increases. Also, CpG methylation, and sequence interruptions within the triplet repeats exert an impact on the formation of nucleosomes along these repeating DNAs. The two most common triplet expansion human diseases, myotonic dystrophy 1 and fragile X syndrome, are caused by the expanded CTG/CAG and CGG/CCG repeats, respectively. In addition to the expanded repeats and CpG methylation, histone modifications, chromatin remodeling factors, and noncoding RNA have been shown to coordinate the chromatin structure at both myotonic dystrophy 1 and fragile X loci. Alterations in chromatin structure at these two loci can affect transcription of these disease-causing genes, leading to disease symptoms. These observations have brought a new appreciation that a full understanding of disease gene expression requires a knowledge of the structure of the chromatin domain within which the gene resides.

  11. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

    PubMed

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand. PMID:25916672

  12. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

    PubMed

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

  13. Mechanisms of ATP-Dependent Chromatin Remodeling Motors.

    PubMed

    Zhou, Coral Y; Johnson, Stephanie L; Gamarra, Nathan I; Narlikar, Geeta J

    2016-07-01

    Chromatin remodeling motors play essential roles in all DNA-based processes. These motors catalyze diverse outcomes ranging from sliding the smallest units of chromatin, known as nucleosomes, to completely disassembling chromatin. The broad range of actions carried out by these motors on the complex template presented by chromatin raises many stimulating mechanistic questions. Other well-studied nucleic acid motors provide examples of the depth of mechanistic understanding that is achievable from detailed biophysical studies. We use these studies as a guiding framework to discuss the current state of knowledge of chromatin remodeling mechanisms and highlight exciting open questions that would continue to benefit from biophysical analyses. PMID:27391925

  14. The SWI/SNF chromatin remodelling complex: Its role in maintaining genome stability and preventing tumourigenesis.

    PubMed

    Brownlee, Peter M; Meisenberg, Cornelia; Downs, Jessica A

    2015-08-01

    Genes encoding subunits of the two SWI/SNF chromatin remodelling complexes (BAF and PBAF) are mutated in almost 20% of all human cancers. In addition to a role in regulating transcription, recent work from our laboratory and others identified roles for both complexes in DNA damage responses and the maintenance of sister chromatid cohesion, which may have profound impacts on genome stability and contribute to its role as a tumour suppressor. Here, we review some of the transcription-independent functions of the SWI/SNF chromatin remodelling complex and discuss these in light of their potential relevance to tumourigenesis.

  15. A RSC/nucleosome complex determines chromatin architecture and facilitates activator binding.

    PubMed

    Floer, Monique; Wang, Xin; Prabhu, Vidya; Berrozpe, Georgina; Narayan, Santosh; Spagna, Dan; Alvarez, David; Kendall, Jude; Krasnitz, Alexander; Stepansky, Asya; Hicks, James; Bryant, Gene O; Ptashne, Mark

    2010-04-30

    How is chromatin architecture established and what role does it play in transcription? We show that the yeast regulatory locus UASg bears, in addition to binding sites for the activator Gal4, sites bound by the RSC complex. RSC positions a nucleosome, evidently partially unwound, in a structure that facilitates Gal4 binding to its sites. The complex comprises a barrier that imposes characteristic features of chromatin architecture. In the absence of RSC, ordinary nucleosomes encroach over the UASg and compete with Gal4 for binding. Taken with our previous work, the results show that both prior to and following induction, specific DNA-binding proteins are the predominant determinants of chromatin architecture at the GAL1/10 genes. RSC/nucleosome complexes are also found scattered around the yeast genome. Higher eukaryotic RSC lacks the specific DNA-binding determinants found on yeast RSC, and evidently Gal4 works in those organisms despite whatever obstacle broadly positioned nucleosomes present.

  16. The landscape of accessible chromatin in mammalian preimplantation embryos.

    PubMed

    Wu, Jingyi; Huang, Bo; Chen, He; Yin, Qiangzong; Liu, Yang; Xiang, Yunlong; Zhang, Bingjie; Liu, Bofeng; Wang, Qiujun; Xia, Weikun; Li, Wenzhi; Li, Yuanyuan; Ma, Jing; Peng, Xu; Zheng, Hui; Ming, Jia; Zhang, Wenhao; Zhang, Jing; Tian, Geng; Xu, Feng; Chang, Zai; Na, Jie; Yang, Xuerui; Xie, Wei

    2016-06-30

    In mammals, extensive chromatin reorganization is essential for reprogramming terminally committed gametes to a totipotent state during preimplantation development. However, the global chromatin landscape and its dynamics in this period remain unexplored. Here we report a genome-wide map of accessible chromatin in mouse preimplantation embryos using an improved assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) approach with CRISPR/Cas9-assisted mitochondrial DNA depletion. We show that despite extensive parental asymmetry in DNA methylomes, the chromatin accessibility between the parental genomes is globally comparable after major zygotic genome activation (ZGA). Accessible chromatin in early embryos is widely shaped by transposable elements and overlaps extensively with putative cis-regulatory sequences. Unexpectedly, accessible chromatin is also found near the transcription end sites of active genes. By integrating the maps of cis-regulatory elements and single-cell transcriptomes, we construct the regulatory network of early development, which helps to identify the key modulators for lineage specification. Finally, we find that the activities of cis-regulatory elements and their associated open chromatin diminished before major ZGA. Surprisingly, we observed many loci showing non-canonical, large open chromatin domains over the entire transcribed units in minor ZGA, supporting the presence of an unusually permissive chromatin state. Together, these data reveal a unique spatiotemporal chromatin configuration that accompanies early mammalian development. PMID:27309802

  17. ZmMBD101 is a DNA-binding protein that maintains Mutator elements chromatin in a repressive state in maize.

    PubMed

    Questa, Julia I; Rius, Sebastián P; Casadevall, Romina; Casati, Paula

    2016-01-01

    In maize (Zea mays), as well as in other crops, transposable elements (TEs) constitute a great proportion of the genome. Chromatin modifications play a vital role in establishing transposon silencing and perpetuating the acquired repressive state. Nucleosomes associated with TEs are enriched for dimethylation of histone H3 at lysine 9 and 27 (H3K9me2 and H3K27me2, respectively), signals of repressive chromatin. Here, we describe a chromatin protein, ZmMBD101, involved in the regulation of Mutator (Mu) genes in maize. ZmMBD101 is localized to the nucleus and contains a methyl-CpG-binding domain (MBD) and a zinc finger CW (CW) domain. Transgenic lines with reduced levels of ZmMBD101 transcript present enhanced induction of Mu genes when plants are irradiated with UV-B. Chromatin immunoprecipitation analysis with H3K9me2 and H3K27me2 antibodies indicated that ZmMBD101 is required to maintain the levels of these histone repressive marks at Mu terminal inverted repeats (TIRs) under UV-B conditions. Although Mutator inactivity is associated with DNA methylation, cytosine methylation at Mu TIRs is not affected in ZmMBD101 deficient plants. Several plant proteins are predicted to share the simple CW-MBD domain architecture present in ZmMBD101. We hypothesize that plant CW-MBD proteins may also function to protect plant genomes from deleterious transposition.

  18. Minireview: Conversing With Chromatin: The Language of Nuclear Receptors

    PubMed Central

    2014-01-01

    Nuclear receptors are transcription factors that are activated by physiological stimuli to bind DNA in the context of chromatin and regulate complex biological pathways. Major advances in nuclear receptor biology have been aided by genome scale examinations of receptor interactions with chromatin. In this review, we summarize the roles of the chromatin landscape in regulating nuclear receptor function. Chromatin acts as a central integrator in the nuclear receptor-signaling axis, operating in distinct temporal modalities. Chromatin effects nuclear receptor action by specifying its genomic localization and interactions with regulatory elements. On receptor binding, changes in chromatin operate as an effector of receptor signaling to modulate transcriptional events. Chromatin is therefore an integral component of the pathways that guide nuclear receptor action in cell-type-specific and cell state-dependent manners. PMID:24196351

  19. Quantification of chromatin condensation level by image processing.

    PubMed

    Irianto, Jerome; Lee, David A; Knight, Martin M

    2014-03-01

    The level of chromatin condensation is related to the silencing/activation of chromosomal territories and therefore impacts on gene expression. Chromatin condensation changes during cell cycle, progression and differentiation, and is influenced by various physicochemical and epigenetic factors. This study describes a validated experimental technique to quantify chromatin condensation. A novel image processing procedure is developed using Sobel edge detection to quantify the level of chromatin condensation from nuclei images taken by confocal microscopy. The algorithm was developed in MATLAB and used to quantify different levels of chromatin condensation in chondrocyte nuclei achieved through alteration in osmotic pressure. The resulting chromatin condensation parameter (CCP) is in good agreement with independent multi-observer qualitative visual assessment. This image processing technique thereby provides a validated unbiased parameter for rapid and highly reproducible quantification of the level of chromatin condensation.

  20. Minireview: Conversing with chromatin: the language of nuclear receptors.

    PubMed

    Biddie, Simon C; John, Sam

    2014-01-01

    Nuclear receptors are transcription factors that are activated by physiological stimuli to bind DNA in the context of chromatin and regulate complex biological pathways. Major advances in nuclear receptor biology have been aided by genome scale examinations of receptor interactions with chromatin. In this review, we summarize the roles of the chromatin landscape in regulating nuclear receptor function. Chromatin acts as a central integrator in the nuclear receptor-signaling axis, operating in distinct temporal modalities. Chromatin effects nuclear receptor action by specifying its genomic localization and interactions with regulatory elements. On receptor binding, changes in chromatin operate as an effector of receptor signaling to modulate transcriptional events. Chromatin is therefore an integral component of the pathways that guide nuclear receptor action in cell-type-specific and cell state-dependent manners. PMID:24196351

  1. Effect of rabbit age on sperm chromatin structure.

    PubMed

    Gogol, P; Bochenek, M; Smorag, Z

    2002-04-01

    The aim of this study was to determine the relationship between the age of male rabbits and the sperm chromatin structure. The studies involved the semen of New Zealand White rabbits between 5 and 28 months of age. A flow cytometry and sperm chromatin structure assay (SCSA) method was used to determine chromatin structure. The results of cytometric chromatin structure assay suggested a relatively high stability of sperm chromatin in the rabbit. Between 6 and 16 months of age, the mean percentage of sperm with damaged chromatin was the lowest and ranged from 1.7 to 2.4%. Decreased sperm chromatin stability was found in ejaculates taken from male rabbits less than 5 months and more than 20 months of age. PMID:11975746

  2. Calorie restriction and the exercise of chromatin

    PubMed Central

    Vaquero, Alejandro; Reinberg, Danny

    2009-01-01

    Since the earliest stages of evolution, organisms have faced the challenge of sensing and adapting to environmental changes for their survival under compromising conditions such as food depletion or stress. Implicit in these responses are mechanisms developed during evolution that include the targeting of chromatin to allow or prevent expression of fundamental genes and to protect genome integrity. Among the different approaches to study these mechanisms, the analysis of the response to a moderate reduction of energy intake, also known as calorie restriction (CR), has become one of the best sources of information regarding the factors and pathways involved in metabolic adaptation from lower to higher eukaryotes. Furthermore, responses to CR are involved in life span regulation—conserved from yeast to mammals—and therefore have garnered major research interest. Herein we review current knowledge of responses to CR at the molecular level and their functional link to chromatin. PMID:19608767

  3. Regulation of chromatin by histone modifications

    PubMed Central

    Bannister, Andrew J; Kouzarides, Tony

    2011-01-01

    Chromatin is not an inert structure, but rather an instructive DNA scaffold that can respond to external cues to regulate the many uses of DNA. A principle component of chromatin that plays a key role in this regulation is the modification of histones. There is an ever-growing list of these modifications and the complexity of their action is only just beginning to be understood. However, it is clear that histone modifications play fundamental roles in most biological processes that are involved in the manipulation and expression of DNA. Here, we describe the known histone modifications, define where they are found genomically and discuss some of their functional consequences, concentrating mostly on transcription where the majority of characterisation has taken place. PMID:21321607

  4. Perturbation of chromatin structure globally affects localization and recruitment of splicing factors.

    PubMed

    Schor, Ignacio E; Llères, David; Risso, Guillermo J; Pawellek, Andrea; Ule, Jernej; Lamond, Angus I; Kornblihtt, Alberto R

    2012-01-01

    Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3' splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin

  5. The polymorphisms of the chromatin fiber

    NASA Astrophysics Data System (ADS)

    Boulé, Jean-Baptiste; Mozziconacci, Julien; Lavelle, Christophe

    2015-01-01

    In eukaryotes, the genome is packed into chromosomes, each consisting of large polymeric fibers made of DNA bound with proteins (mainly histones) and RNA molecules. The nature and precise 3D organization of this fiber has been a matter of intense speculations and debates. In the emerging picture, the local chromatin state plays a critical role in all fundamental DNA transactions, such as transcriptional control, DNA replication or repair. However, the molecular and structural mechanisms involved remain elusive. The purpose of this review is to give an overview of the tremendous efforts that have been made for almost 40 years to build physiologically relevant models of chromatin structure. The motivation behind building such models was to shift our representation and understanding of DNA transactions from a too simplistic ‘naked DNA’ view to a more realistic ‘coated DNA’ view, as a step towards a better framework in which to interpret mechanistically the control of genetic expression and other DNA metabolic processes. The field has evolved from a speculative point of view towards in vitro biochemistry and in silico modeling, but is still longing for experimental in vivo validations of the proposed structures or even proof of concept experiments demonstrating a clear role of a given structure in a metabolic transaction. The mere existence of a chromatin fiber as a relevant biological entity in vivo has been put into serious questioning. Current research is suggesting a possible reconciliation between theoretical studies and experiments, pointing towards a view where the polymorphic and dynamic nature of the chromatin fiber is essential to support its function in genome metabolism.

  6. A Novel Toxoplasma gondii Nuclear Factor TgNF3 Is a Dynamic Chromatin-Associated Component, Modulator of Nucleolar Architecture and Parasite Virulence

    PubMed Central

    Olguin-Lamas, Alejandro; Madec, Edwige; Hovasse, Agnes; Werkmeister, Elisabeth; Callebaut, Isabelle; Slomianny, Christian; Delhaye, Stephane; Mouveaux, Thomas; Schaeffer-Reiss, Christine; Van Dorsselaer, Alain; Tomavo, Stanislas

    2011-01-01

    In Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome

  7. Characterization of a Novel Chromatin Sorting Tool Reveals Importance of Histone Variant H3.3 in Contextual Fear Memory and Motor Learning.

    PubMed

    McNally, Anna G; Poplawski, Shane G; Mayweather, Brittany A; White, Kyle M; Abel, Ted

    2016-01-01

    The consolidation of short-term labile memories for long-term storage requires transcription and there is growing interest in defining the epigenetic mechanisms regulating these transcriptional events. In particular, it has been hypothesized that combinations of histone post-translational modifications (PTMs) have the potential to store memory by dynamically defining the transcriptional status of any given gene loci. Studying epigenetic phenomena during long-term memory consolidation, however, is complicated by the complex cellular heterogeneity of the brain, in which epigenetic signal from memory-relevant cells can be obscured or diluted by the surrounding milieu. To address this issue, we have developed a transgenic mouse line expressing a tetO-regulated, hemagglutinin (HA)-tagged histone H3.3 exclusively in excitatory neurons of the forebrain. Unlike canonical histones, histone H3.3 is incorporated at promoter regions of transcriptionally active genes in a DNA replication-independent manner, stably "barcoding" active regions of the genome in post-mitotic cells. Immunoprecipitating H3.3-HA containing nucleosomes from the hippocampus will therefore enrich for memory-relevant chromatin by isolating actively transcribed regions of the excitatory neuron genome. To evaluate the validity of using H3.3 "barcoding" to sort chromatin, we performed a molecular and behavioral characterization of the H3.3-HA transgenic mouse line. Expectedly, we find that H3.3-HA is incorporated preferentially at promoter regions of actively-transcribed neuronal genes and that expression can be effectively regulated by doxycycline. Additionally, H3.3-HA overexpression does not adversely affect exploratory or anxiety-related behaviors, nor does it affect spatial memory. Transgenic animals do, however, exhibit deficits in contextual memory and motor learning, revealing the importance of this histone isoform in the brain. Future studies in the H3.3-HA transgenic mouse line will define the

  8. Characterization of a Novel Chromatin Sorting Tool Reveals Importance of Histone Variant H3.3 in Contextual Fear Memory and Motor Learning

    PubMed Central

    McNally, Anna G.; Poplawski, Shane G.; Mayweather, Brittany A.; White, Kyle M.; Abel, Ted

    2016-01-01

    The consolidation of short-term labile memories for long-term storage requires transcription and there is growing interest in defining the epigenetic mechanisms regulating these transcriptional events. In particular, it has been hypothesized that combinations of histone post-translational modifications (PTMs) have the potential to store memory by dynamically defining the transcriptional status of any given gene loci. Studying epigenetic phenomena during long-term memory consolidation, however, is complicated by the complex cellular heterogeneity of the brain, in which epigenetic signal from memory-relevant cells can be obscured or diluted by the surrounding milieu. To address this issue, we have developed a transgenic mouse line expressing a tetO-regulated, hemagglutinin (HA)-tagged histone H3.3 exclusively in excitatory neurons of the forebrain. Unlike canonical histones, histone H3.3 is incorporated at promoter regions of transcriptionally active genes in a DNA replication-independent manner, stably “barcoding” active regions of the genome in post-mitotic cells. Immunoprecipitating H3.3-HA containing nucleosomes from the hippocampus will therefore enrich for memory-relevant chromatin by isolating actively transcribed regions of the excitatory neuron genome. To evaluate the validity of using H3.3 “barcoding” to sort chromatin, we performed a molecular and behavioral characterization of the H3.3-HA transgenic mouse line. Expectedly, we find that H3.3-HA is incorporated preferentially at promoter regions of actively-transcribed neuronal genes and that expression can be effectively regulated by doxycycline. Additionally, H3.3-HA overexpression does not adversely affect exploratory or anxiety-related behaviors, nor does it affect spatial memory. Transgenic animals do, however, exhibit deficits in contextual memory and motor learning, revealing the importance of this histone isoform in the brain. Future studies in the H3.3-HA transgenic mouse line will define

  9. Premature chromatin condensation upon accumulation of NIMA.

    PubMed Central

    O'Connell, M J; Norbury, C; Nurse, P

    1994-01-01

    The NIMA protein kinase of Aspergillus nidulans is required for the G2/M transition of the cell cycle. Mutants lacking NIMA arrest without morphological characteristics of mitosis, but they do contain an activated p37nimX kinase (the Aspergillus homologue of p34cdc2). To gain a better understanding of NIMA function we have investigated the effects of expressing various NIMA constructs in Aspergillus, fission yeast and human cells. Our experiments have shown that the instability of the NIMA protein requires sequences in the non-catalytic C-terminus of the protein. Removal of this domain results in a stable protein that, once accumulated, promotes a lethal premature condensation of chromatin without any other aspects of mitosis. Similar effects were also observed in fission yeast and human cells accumulating Aspergillus NIMA. This phenotype is independent of cell cycle progression and does not require p34cdc2 kinase activity. As gain of NIMA function by accumulation results in premature chromatin condensation, and loss of NIMA function results in an inability to enter mitosis, we propose that NIMA functions in G2 to promote the condensation of chromatin normally associated with entry into mitosis. Images PMID:7957060

  10. Chromatin fiber allostery and the epigenetic code

    NASA Astrophysics Data System (ADS)

    Lesne, Annick; Foray, Nicolas; Cathala, Guy; Forné, Thierry; Wong, Hua; Victor, Jean-Marc

    2015-02-01

    The notion of allostery introduced for proteins about fifty years ago has been extended since then to DNA allostery, where a locally triggered DNA structural transition remotely controls other DNA-binding events. We further extend this notion and propose that chromatin fiber allosteric transitions, induced by histone-tail covalent modifications, may play a key role in transcriptional regulation. We present an integrated scenario articulating allosteric mechanisms at different scales: allosteric transitions of the condensed chromatin fiber induced by histone-tail acetylation modify the mechanical constraints experienced by the embedded DNA, thus possibly controlling DNA-binding of allosteric transcription factors or further allosteric mechanisms at the linker DNA level. At a higher scale, different epigenetic constraints delineate different statistically dominant subsets of accessible chromatin fiber conformations, which each favors the assembly of dedicated regulatory complexes, as detailed on the emblematic example of the mouse Igf2-H19 gene locus and its parental imprinting. This physical view offers a mechanistic and spatially structured explanation of the observed correlation between transcriptional activity and histone modifications. The evolutionary origin of allosteric control supports to speak of an ‘epigenetic code’, by which events involved in transcriptional regulation are encoded in histone modifications in a context-dependent way.

  11. Chromatin modifications remodel cardiac gene expression.

    PubMed

    Mathiyalagan, Prabhu; Keating, Samuel T; Du, Xiao-Jun; El-Osta, Assam

    2014-07-01

    Signalling and transcriptional control involve precise programmes of gene activation and suppression necessary for cardiovascular physiology. Deep sequencing of DNA-bound transcription factors reveals a remarkable complexity of co-activators or co-repressors that serve to alter chromatin modification and regulate gene expression. The regulated complexes characterized by genome-wide mapping implicate the recruitment and exchange of proteins with specific enzymatic activities that include roles for histone acetylation and methylation in key developmental programmes of the heart. As for transcriptional changes in response to pathological stress, co-regulatory complexes are also differentially utilized to regulate genes in cardiac disease. Members of the histone deacetylase (HDAC) family catalyse the removal of acetyl groups from proteins whose pharmacological inhibition has profound effects preventing heart failure. HDACs interact with a complex co-regulatory network of transcription factors, chromatin-remodelling complexes, and specific histone modifiers to regulate gene expression in the heart. For example, the histone methyltransferase (HMT), enhancer of zeste homolog 2 (Ezh2), is regulated by HDAC inhibition and associated with pathological cardiac hypertrophy. The challenge now is to target the activity of enzymes involved in protein modification to prevent or reverse the expression of genes implicated with cardiac hypertrophy. In this review, we discuss the role of HDACs and HMTs with a focus on chromatin modification and gene function as well as the clinical treatment of heart failure. PMID:24812277

  12. Molecular Architecture of  Yeast Chromatin Assembly Factor 1

    PubMed Central

    Kim, Daegeun; Setiaputra, Dheva; Jung, Taeyang; Chung, Jaehee; Leitner, Alexander; Yoon, Jungmin; Aebersold, Ruedi; Hebert, Hans; Yip, Calvin K.; Song, Ji-Joon

    2016-01-01

    Chromatin Assembly Complex 1 (CAF-1) is a major histone chaperone involved in deposition of histone H3 and H4 into nucleosome. CAF-1 is composed of three subunits; p150, p60 and p48 for human and Cac1, Cac2 and Cac3 for yeast. Despite of its central role in chromatin formation, structural features of the full CAF-1 in complex with histones and other chaperones have not been well characterized. Here, we dissect molecular architecture of yeast CAF-1 (yCAF-1) by cross-linking mass spectrometry (XL-MS) and negative stain single-particle electron microscopy (EM). Our work revealed that Cac1, the largest subunit of yCAF-1, might serve as a major histone binding platform linking Cac2 and Cac3. In addition, EM analysis showed that yCAF-1 adopts a bilobal shape and Cac1 connecting Cac2 and Cac3 to generate a platform for binding histones. This study provides the first structural glimpse of the full CAF-1 complex and a structural framework to understand histone chaperoning processes. PMID:27221973

  13. PARP1 enhances inflammatory cytokine expression by alteration of promoter chromatin structure in microglia

    PubMed Central

    Martínez-Zamudio, Ricardo Iván; Ha, Hyo Chol

    2014-01-01

    Background Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated enzyme that participates in processes such as transcription and DNA repair through the regulation of chromatin structure. Accumulating evidence suggests an important role for PARP1 enzymatic activity in promoting CNS inflammation by facilitating the expression of inflammatory cytokines in glial cells. However, the molecular mechanisms by which PARP1 enzymatic activity mediates this process are not well understood. In this report we sought to determine the molecular mechanisms by which PARP1 enzymatic activity facilitates the expression of Il1β and TNF in LPS-stimulated BV2 cells. Methods PARP1 enzymatic activity and histone ADP-ribosylation were measured in LPS-stimulated BV2 cells by radioactive labelling with 32P-NAD+. To assess the effect of histone ADP-ribosylation on nucleosome structure, in vitro nucleosome remodeling, nuclease accessibility and binding assays were performed. These studies were complemented by chromatin immunoprecipitation assays in resting and LPS-stimulated BV2 cells in order to determine the occupancy of PARP1, nucleosomes and the RelA subunit of NF-κB, as well as ADP-ribosylation, at the Il1β and Tnf promoters. Finally, we determined the effect of pharmacological inhibition of PARP1 enzymatic activity on the LPS stimulation-dependent induction of Il1β and Tnf mRNA. Results Our results indicate that LPS stimulation induces PARP1 enzymatic activity and histone ADP-ribosylation in the chromatin compartment of BV2 cells. In vitro studies show that nucleosome-bound PARP1 disrupts nucleosome structure histone ADP-ribosylation, increasing the accessibility of nucleosomal DNA. Consistent with this PARP1 is constitutively associated with at the Il1β and Tnf promoters in resting BV2 cells. Upon stimulation with LPS, ADP-ribosylation is observed at these promoters, and this is correlated with increased recruitment of the transcription factor NF-κB, resulting in robust

  14. A SWI/SNF Chromatin Remodelling Protein Controls Cytokinin Production through the Regulation of Chromatin Architecture

    PubMed Central

    Jégu, Teddy; Domenichini, Séverine; Blein, Thomas; Ariel, Federico; Christ, Aurélie; Kim, Soon-Kap; Crespi, Martin; Boutet-Mercey, Stéphanie; Mouille, Grégory; Bourge, Mickaël; Hirt, Heribert; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

    2015-01-01

    Chromatin architecture determines transcriptional accessibility to DNA and consequently gene expression levels in response to developmental and environmental stimuli. Recently, chromatin remodelers such as SWI/SNF complexes have been recognized as key regulators of chromatin architecture. To gain insight into the function of these complexes during root development, we have analyzed Arabidopsis knock-down lines for one sub-unit of SWI/SNF complexes: BAF60. Here, we show that BAF60 is a positive regulator of root development and cell cycle progression in the root meristem via its ability to down-regulate cytokinin production. By opposing both the deposition of active histone marks and the formation of a chromatin regulatory loop, BAF60 negatively regulates two crucial target genes for cytokinin biosynthesis (IPT3 and IPT7) and one cell cycle inhibitor (KRP7). Our results demonstrate that SWI/SNF complexes containing BAF60 are key factors governing the equilibrium between formation and dissociation of a chromatin loop controlling phytohormone production and cell cycle progression. PMID:26457678

  15. A SWI/SNF Chromatin Remodelling Protein Controls Cytokinin Production through the Regulation of Chromatin Architecture.

    PubMed

    Jégu, Teddy; Domenichini, Séverine; Blein, Thomas; Ariel, Federico; Christ, Aurélie; Kim, Soon-Kap; Crespi, Martin; Boutet-Mercey, Stéphanie; Mouille, Grégory; Bourge, Mickaël; Hirt, Heribert; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

    2015-01-01

    Chromatin architecture determines transcriptional accessibility to DNA and consequently gene expression levels in response to developmental and environmental stimuli. Recently, chromatin remodelers such as SWI/SNF complexes have been recognized as key regulators of chromatin architecture. To gain insight into the function of these complexes during root development, we have analyzed Arabidopsis knock-down lines for one sub-unit of SWI/SNF complexes: BAF60. Here, we show that BAF60 is a positive regulator of root development and cell cycle progression in the root meristem via its ability to down-regulate cytokinin production. By opposing both the deposition of active histone marks and the formation of a chromatin regulatory loop, BAF60 negatively regulates two crucial target genes for cytokinin biosynthesis (IPT3 and IPT7) and one cell cycle inhibitor (KRP7). Our results demonstrate that SWI/SNF complexes containing BAF60 are key factors governing the equilibrium between formation and dissociation of a chromatin loop controlling phytohormone production and cell cycle progression.

  16. The role of chromatin conformations in diffusional transport of chromatin-binding proteins: Cartesian lattice simulations

    NASA Astrophysics Data System (ADS)

    Wedemeier, Annika; Zhang, Ting; Merlitz, Holger; Wu, Chen-Xu; Langowski, Jörg

    2008-04-01

    In this paper, a lattice model for the diffusional transport of chromatin-binding particles in the interphase cell nucleus is proposed. Sliding effects are studied in dense networks of chromatin fibers created by three different methods: Randomly distributed, noninterconnected obstacles, a random walk chain model with an attractive step potential, and a self-avoiding random walk chain model with a hard repulsive core and attractive surroundings. By comparing a discrete and continuous version of the random walk chain model, we demonstrate that lattice discretization does not alter the diffusion of chromatin-binding particles. The influence of conformational properties of the fiber network on the particle sliding is investigated in detail while varying occupation volume, sliding probability, chain length, and persistence length. It is observed that adjacency of the monomers, the excluded volume effect incorporated in the self-avoiding random walk model, and the persistence length affect the chromatin-binding particle diffusion. It is demonstrated that sliding particles sense local chain structures. When plotting the diffusion coefficient as a function of the accessible volume for diffusing particles, the data fall onto master curves depending on the persistence length. However, once intersegment transfer is involved, chromatin-binding proteins no longer perceive local chain structures.

  17. Minor Loops in Major Folds: Enhancer-Promoter Looping, Chromatin Restructuring, and Their Association with Transcriptional Regulation and Disease.

    PubMed

    Matharu, Navneet; Ahituv, Nadav

    2015-12-01

    The organization and folding of chromatin within the nucleus can determine the outcome of gene expression. Recent technological advancements have enabled us to study chromatin interactions in a genome-wide manner at high resolution. These studies have increased our understanding of the hierarchy and dynamics of chromatin domains that facilitate cognate enhancer-promoter looping, defining the transcriptional program of different cell types. In this review, we focus on vertebrate chromatin long-range interactions as they relate to transcriptional regulation. In addition, we describe how the alteration of boundaries that mark discrete regions in the genome with high interaction frequencies within them, called topological associated domains (TADs), could lead to various phenotypes, including human diseases, which we term as "TADopathies." PMID:26632825

  18. The Methylated DNA Immunoprecipitation [MeDIP] to Investigate the Epigenetic Remodeling in Cell Fate Determination and Cancer Development.

    PubMed

    Masciarelli, Silvia; Bellissimo, Teresa; Iosue, Ilaria; Fazi, Francesco

    2016-01-01

    Epigenetic mechanisms such as DNA methylation, posttranslational modifications of histone proteins, remodeling of nucleosomes, and the expression of noncoding RNAs contribute to the regulation of gene expression for the cell fate determination and tissue development. The disruption of these epigenetic mechanisms, in conjunction with genetic alterations, is a decisive element for cancer development and progression. The cancer phenotype is characterized by global DNA hypomethylation and gene-specific hypermethylation. The methylated DNA immunoprecipitation [MeDIP] is a useful approach currently used to clarify the functional consequences of DNA methylation on cell fate determination and cancer development.

  19. Chromatin Remodeling Factors Isw2 and Ino80 Regulate Checkpoint Activity and Chromatin Structure in S Phase

    PubMed Central

    Lee, Laura; Rodriguez, Jairo; Tsukiyama, Toshio

    2015-01-01

    When cells undergo replication stress, proper checkpoint activation and deactivation are critical for genomic stability and cell survival and therefore must be highly regulated. Although mechanisms of checkpoint activation are well studied, mechanisms of checkpoint deactivation are far less understood. Previously, we reported that chromatin remodeling factors Isw2 and Ino80 attenuate the S-phase checkpoint activity in Saccharomyces cerevisiae, especially during recovery from hydroxyurea. In this study, we found that Isw2 and Ino80 have a more pronounced role in attenuating checkpoint activity during late S phase in the presence of methyl methanesulfonate (MMS). We therefore screened for checkpoint factors required for Isw2 and Ino80 checkpoint attenuation in the presence of MMS. Here we demonstrate that Isw2 and Ino80 antagonize checkpoint activators and attenuate checkpoint activity in S phase in MMS either through a currently unknown pathway or through RPA. Unexpectedly, we found that Isw2 and Ino80 increase chromatin accessibility around replicating regions in the presence of MMS through a novel mechanism. Furthermore, through growth assays, we provide additional evidence that Isw2 and Ino80 partially counteract checkpoint activators specifically in the presence of MMS. Based on these results, we propose that Isw2 and Ino80 attenuate S-phase checkpoint activity through a novel mechanism. PMID:25701287

  20. Capture of associated targets on chromatin links long-distance chromatin looping to transcriptional coordination.

    PubMed

    Bourgo, Ryan J; Singhal, Hari; Greene, Geoffrey L

    2016-01-01

    Here we describe a sensitive and novel method of identifying endogenous DNA-DNA interactions. Capture of Associated Targets on CHromatin (CATCH) uses efficient capture and enrichment of specific genomic loci of interest through hybridization and subsequent purification via complementary biotinylated oligonucleotide. The CATCH assay requires no enzymatic digestion or ligation, requires little starting material, provides high-quality data, has excellent reproducibility and is completed in less than 24 h. Efficacy is demonstrated through capture of three disparate loci, which demonstrate unique subsets of long-distance chromatin interactions enriched for both enhancer marks and oestrogen receptor-binding sites. In each experiment, CATCH-seq peaks representing long-distance chromatin interactions were centred near the TSS of genes, and, critically, the genes identified as physically interacting are shown to be transcriptionally coexpressed. These interactions could potentially create transcriptional hubs for the regulation of gene expression programmes. PMID:27634217

  1. Capture of associated targets on chromatin links long-distance chromatin looping to transcriptional coordination

    PubMed Central

    Bourgo, Ryan J.; Singhal, Hari; Greene, Geoffrey L.

    2016-01-01

    Here we describe a sensitive and novel method of identifying endogenous DNA–DNA interactions. Capture of Associated Targets on CHromatin (CATCH) uses efficient capture and enrichment of specific genomic loci of interest through hybridization and subsequent purification via complementary biotinylated oligonucleotide. The CATCH assay requires no enzymatic digestion or ligation, requires little starting material, provides high-quality data, has excellent reproducibility and is completed in less than 24 h. Efficacy is demonstrated through capture of three disparate loci, which demonstrate unique subsets of long-distance chromatin interactions enriched for both enhancer marks and oestrogen receptor-binding sites. In each experiment, CATCH-seq peaks representing long-distance chromatin interactions were centred near the TSS of genes, and, critically, the genes identified as physically interacting are shown to be transcriptionally coexpressed. These interactions could potentially create transcriptional hubs for the regulation of gene expression programmes. PMID:27634217

  2. Chromatin remodelers Isw1 and Chd1 maintain chromatin structure during transcription by preventing histone exchange

    PubMed Central

    Smolle, Michaela; Venkatesh, Swaminathan; Gogol, Madelaine M.; Li, Hua; Zhang, Ying; Florens, Laurence; Washburn, Michael P.; Workman, Jerry L.

    2012-01-01

    Set2-mediated methylation of histone H3 Lys36 (H3K36) is a mark associated with the coding sequences of actively transcribed genes, yet plays a negative role during transcription elongation. It prevents trans-histone exchange over coding regions and signals for histone deacetylation in the wake of RNA polymerase II (RNAPII) passage. We have found that in Saccharomyces cerevisiae the Isw1b chromatin-remodeling complex is specifically recruited to open reading frames (ORFs) by H3K36 methylation through the PWWP domain of its Ioc4 subunit in vivo and in vitro. Isw1b acts in conjunction with Chd1 to regulate chromatin structure by preventing trans-histone exchange from taking place over coding regions and thus maintains chromatin integrity during transcription elongation by RNA polymerase II. PMID:22922743

  3. Global Chromatin Domain Organization of the Drosophila Genome

    PubMed Central

    de Wit, Elzo; Braunschweig, Ulrich; Greil, Frauke; Bussemaker, Harmen J.; van Steensel, Bas

    2008-01-01

    In eukaryotes, neighboring genes can be packaged together in specific chromatin structures that ensure their coordinated expression. Examples of such multi-gene chromatin domains are well-documented, but a global view of the chromatin organization of eukaryotic genomes is lacking. To systematically identify multi-gene chromatin domains, we constructed a compendium of genome-scale binding maps for a broad panel of chromatin-associated proteins in Drosophila melanogaster. Next, we computationally analyzed this compendium for evidence of multi-gene chromatin domains using a novel statistical segmentation algorithm. We find that at least 50% of all fly genes are organized into chromatin domains, which often consist of dozens of genes. The domains are characterized by various known and novel combinations of chromatin proteins. The genes in many of the domains are coregulated during development and tend to have similar biological functions. Furthermore, during evolution fewer chromosomal rearrangements occur inside chromatin domains than outside domains. Our results indicate that a substantial portion of the Drosophila genome is packaged into functionally coherent, multi-gene chromatin domains. This has broad mechanistic implications for gene regulation and genome evolution. PMID:18369463

  4. Radiation Induced Chromatin Conformation Changes Analysed by Fluorescent Localization Microscopy, Statistical Physics, and Graph Theory

    PubMed Central

    Müller, Patrick; Hillebrandt, Sabina; Krufczik, Matthias; Bach, Margund; Kaufmann, Rainer; Hausmann, Michael; Heermann, Dieter W.

    2015-01-01

    It has been well established that the architecture of chromatin in cell nuclei is not random but functionally correlated. Chromatin damage caused by ionizing radiation raises complex repair machineries. This is accompanied by local chromatin rearrangements and structural changes which may for instance improve the accessibility of damaged sites for repair protein complexes. Using stably transfected HeLa cells expressing either green fluorescent protein (GFP) labelled histone H2B or yellow fluorescent protein (YFP) labelled histone H2A, we investigated the positioning of individual histone proteins in cell nuclei by means of high resolution localization microscopy (Spectral Position Determination Microscopy = SPDM). The cells were exposed to ionizing radiation of different doses and aliquots were fixed after different repair times for SPDM imaging. In addition to the repair dependent histone protein pattern, the positioning of antibodies specific for heterochromatin and euchromatin was separately recorded by SPDM. The present paper aims to provide a quantitative description of structural changes of chromatin after irradiation and during repair. It introduces a novel approach to analyse SPDM images by means of statistical physics and graph theory. The method is based on the calculation of the radial distribution functions as well as edge length distributions for graphs defined by a triangulation of the marker positions. The obtained results show that through the cell nucleus the different chromatin re-arrangements as detected by the fluorescent nucleosomal pattern average themselves. In contrast heterochromatic regions alone indicate a relaxation after radiation exposure and re-condensation during repair whereas euchromatin seemed to be unaffected or behave contrarily. SPDM in combination with the analysis techniques applied allows the systematic elucidation of chromatin re-arrangements after irradiation and during repair, if selected sub-regions of nuclei are

  5. Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells.

    PubMed

    Heo, Su-Jin; Han, Woojin M; Szczesny, Spencer E; Cosgrove, Brian D; Elliott, Dawn M; Lee, David A; Duncan, Randall L; Mauck, Robert L

    2016-08-23

    Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first 10-20 s), whereas changes in chromatin occurred at a later time point (∼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs.

  6. Repression of Germline RNAi Pathways in Somatic Cells by Retinoblastoma Pathway Chromatin Complexes

    PubMed Central

    Wu, Xiaoyun; Shi, Zhen; Cui, Mingxue; Han, Min; Ruvkun, Gary

    2012-01-01

    The retinoblastoma (Rb) tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene. PMID:22412383

  7. Radiation induced chromatin conformation changes analysed by fluorescent localization microscopy, statistical physics, and graph theory.

    PubMed

    Zhang, Yang; Máté, Gabriell; Müller, Patrick; Hillebrandt, Sabina; Krufczik, Matthias; Bach, Margund; Kaufmann, Rainer; Hausmann, Michael; Heermann, Dieter W

    2015-01-01

    It has been well established that the architecture of chromatin in cell nuclei is not random but functionally correlated. Chromatin damage caused by ionizing radiation raises complex repair machineries. This is accompanied by local chromatin rearrangements and structural changes which may for instance improve the accessibility of damaged sites for repair protein complexes. Using stably transfected HeLa cells expressing either green fluorescent protein (GFP) labelled histone H2B or yellow fluorescent protein (YFP) labelled histone H2A, we investigated the positioning of individual histone proteins in cell nuclei by means of high resolution localization microscopy (Spectral Position Determination Microscopy = SPDM). The cells were exposed to ionizing radiation of different doses and aliquots were fixed after different repair times for SPDM imaging. In addition to the repair dependent histone protein pattern, the positioning of antibodies specific for heterochromatin and euchromatin was separately recorded by SPDM. The present paper aims to provide a quantitative description of structural changes of chromatin after irradiation and during repair. It introduces a novel approach to analyse SPDM images by means of statistical physics and graph theory. The method is based on the calculation of the radial distribution functions as well as edge length distributions for graphs defined by a triangulation of the marker positions. The obtained results show that through the cell nucleus the different chromatin re-arrangements as detected by the fluorescent nucleosomal pattern average themselves. In contrast heterochromatic regions alone indicate a relaxation after radiation exposure and re-condensation during repair whereas euchromatin seemed to be unaffected or behave contrarily. SPDM in combination with the analysis techniques applied allows the systematic elucidation of chromatin re-arrangements after irradiation and during repair, if selected sub-regions of nuclei are

  8. Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells.

    PubMed

    Heo, Su-Jin; Han, Woojin M; Szczesny, Spencer E; Cosgrove, Brian D; Elliott, Dawn M; Lee, David A; Duncan, Randall L; Mauck, Robert L

    2016-08-23

    Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first 10-20 s), whereas changes in chromatin occurred at a later time point (∼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs. PMID:27558729

  9. Binding of 125I-labelled fibrin(ogen) fragments to platelets and to immunoprecipitated glycoprotein IIb-IIIa complex

    SciTech Connect

    Thorsen, L.I.; Brosstad, F.; Gogstad, G.; Sletten, K.; Solum, N.O.

    1986-06-01

    To further investigate which parts of the fibrinogen molecule that are responsible for its binding to the fibrinogen receptor on human platelets, the following approaches were made: The glycoprotein IIb-IIIa complex (the putative fibrinogen receptor) was immunoprecipitated in crossed immunoelectrophoresis of Triton X-100-extracts of platelets against antibodies to whole platelet proteins. Subsequently, the immunoplates were incubated with /sup 125/I-labelled, plasmin- or CNBr-cleaved fibrinogen fragments (pre-X,X,Y,D,Degta,Efg,N-DSK) or fibrin fragments (E1,N-dsk), characterized by partial sequenation. The immunoplates were exposed to X-ray films, and binding of the fragments to the glycoprotein IIb-IIIa complex was examined. The findings were compared to the results obtained from studies on binding of the same fragments to intact gel-filtered platelets after ADP-stimulation. The following conclusions were made: All fragments except Efg and Degta bound to the immunoprecipitated GPIIb-IIIa complex as well as to ADP-stimulated platelets suggesting that at least two sequences in the E domain and one in each of the D domains of fibrinogen are involved in binding to the platelet receptor. The GPIIb-IIIa complex is the only surface-located platelet antigen that binds fibrinogen and the aforementioned fragments. The binding of the fragments to the receptor is dependent on divalent cations.

  10. Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

    PubMed

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-05-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms--including humans--are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)(1) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method.

  11. An Overview of Chromatin-Regulating Proteins in Cells

    PubMed Central

    Zhang, Pingyu; Torres, Keila; Liu, Xiuping; Liu, Chang-gong; Pollock, Raphael E.

    2016-01-01

    In eukaryotic cells, gene expressions on chromosome DNA are orchestrated by a dynamic chromosome structure state that is largely controlled by chromatin-regulating proteins, which regulate chromatin structures, release DNA from the nucleosome, and activate or suppress gene expression by modifying nucleosome histones or mobilizing DNA-histone structure. The two classes of chromatin- regulating proteins are 1) enzymes that modify histones through methylation, acetylation, phosphorylation, adenosine diphosphate–ribosylation, glycosylation, sumoylation, or ubiquitylation and 2) enzymes that remodel DNA-histone structure with energy from ATP hydrolysis. Chromatin-regulating proteins, which modulate DNA-histone interaction, change chromatin conformation, and increase or decrease the binding of functional DNA-regulating protein complexes, have major functions in nuclear processes, including gene transcription and DNA replication, repair, and recombination. This review provides a general overview of chromatin-regulating proteins, including their classification, molecular functions, and interactions with the nucleosome in eukaryotic cells. PMID:26796306

  12. A role for chromatin topology in imprinted domain regulation.

    PubMed

    MacDonald, William A; Sachani, Saqib S; White, Carlee R; Mann, Mellissa R W

    2016-02-01

    Recently, many advancements in genome-wide chromatin topology and nuclear architecture have unveiled the complex and hidden world of the nucleus, where chromatin is organized into discrete neighbourhoods with coordinated gene expression. This includes the active and inactive X chromosomes. Using X chromosome inactivation as a working model, we utilized publicly available datasets together with a literature review to gain insight into topologically associated domains, lamin-associated domains, nucleolar-associating domains, scaffold/matrix attachment regions, and nucleoporin-associated chromatin and their role in regulating monoallelic expression. Furthermore, we comprehensively review for the first time the role of chromatin topology and nuclear architecture in the regulation of genomic imprinting. We propose that chromatin topology and nuclear architecture are important regulatory mechanisms for directing gene expression within imprinted domains. Furthermore, we predict that dynamic changes in chromatin topology and nuclear architecture play roles in tissue-specific imprint domain regulation during early development and differentiation.

  13. NET23/STING Promotes Chromatin Compaction from the Nuclear Envelope

    PubMed Central

    de las Heras, Jose I.; Saiz-Ros, Natalia; Makarov, Alexandr A.; Lazou, Vassiliki; Meinke, Peter; Waterfall, Martin; Kelly, David A.; Schirmer, Eric C.

    2014-01-01

    Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture. PMID:25386906

  14. Aging by epigenetics-A consequence of chromatin damage?

    SciTech Connect

    Sedivy, John M. Banumathy, Gowrishankar; Adams, Peter D.

    2008-06-10

    Chromatin structure is not fixed. Instead, chromatin is dynamic and is subject to extensive developmental and age-associated remodeling. In some cases, this remodeling appears to counter the aging and age-associated diseases, such as cancer, and extend organismal lifespan. However, stochastic non-deterministic changes in chromatin structure might, over time, also contribute to the break down of nuclear, cell and tissue function, and consequently aging and age-associated diseases.

  15. NET23/STING promotes chromatin compaction from the nuclear envelope.

    PubMed

    Malik, Poonam; Zuleger, Nikolaj; de las Heras, Jose I; Saiz-Ros, Natalia; Makarov, Alexandr A; Lazou, Vassiliki; Meinke, Peter; Waterfall, Martin; Kelly, David A; Schirmer, Eric C

    2014-01-01

    Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture. PMID:25386906

  16. NET23/STING promotes chromatin compaction from the nuclear envelope.

    PubMed

    Malik, Poonam; Zuleger, Nikolaj; de las Heras, Jose I; Saiz-Ros, Natalia; Makarov, Alexandr A; Lazou, Vassiliki; Meinke, Peter; Waterfall, Martin; Kelly, David A; Schirmer, Eric C

    2014-01-01

    Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture.

  17. Chromatin Dynamics in Vivo: A Game of Musical Chairs

    PubMed Central

    Melters, Daniël P.; Nye, Jonathan; Zhao, Haiqing; Dalal, Yamini

    2015-01-01

    Histones are a major component of chromatin, the nucleoprotein complex fundamental to regulating transcription, facilitating cell division, and maintaining genome integrity in almost all eukaryotes. In addition to canonical, replication-dependent histones, replication-independent histone variants exist in most eukaryotes. In recent years, steady progress has been made in understanding how histone variants assemble, their involvement in development, mitosis, transcription, and genome repair. In this review, we will focus on the localization of the major histone variants H3.3, CENP-A, H2A.Z, and macroH2A, as well as how these variants have evolved, their structural differences, and their functional significance in vivo. PMID:26262644

  18. Chromatin alterations in response to forced swimming underlie increased prodynorphin transcription.

    PubMed

    Reed, B; Fang, N; Mayer-Blackwell, B; Chen, S; Yuferov, V; Zhou, Y; Kreek, M J

    2012-09-18

    Antagonism of the kappa opioid receptor (KOR) has been reported to have anti-depressant-like properties. The dynorphin/KOR system is a crucial neurochemical substrate underlying the pathologies of addictive diseases, affective disorders and other disease states. However, the molecular underpinnings and neuroanatomical localization of the dysregulation of this system have not yet been fully elucidated. Utilizing the Porsolt Forced Swim Test (FST), an acute stressor commonly used as in rodent models measuring antidepressant efficacy, male Sprague-Dawley rats were subject to forced swimming for 15 min, treated 1h with vehicle or norbinaltorphimine (nor-BNI) (5 or 10mg/kg), and then 1 day later subject to FST for 5 min. In accordance with previous findings, nor-BNI dose dependently increased climbing time and reduced immobility. In comparison to control animals not exposed to FST, we observed a significant elevation in prodynorphin (pDyn) mRNA levels following FST using real-time optical polymerase chain reaction (PCR) in the caudate putamen but not in the nucleus accumbens, hypothalamus, amygdala, frontal cortex, or hippocampus. nor-BNI treatment did not affect pDyn mRNA levels in comparison to animals that received vehicle. The corresponding brain regions from the opposite hemisphere were analyzed for underlying chromatin modifications of the prodynorphin gene promoter region using chromatin immunoprecipitation with antibodies against specifically methylated histones H3K27Me2, H3K27Me3, H3K4Me2, and H3K4Me3, as well as CREB-1 and MeCP2. Significant alterations in proteins bound to DNA in the Cre-3, Cre-4, and Sp1 regions of the prodynorphin promoter were found in the caudate putamen of the FST saline-treated animals compared to control animals, with no changes observed in the hippocampus. Epigenetic changes resulting in elevated dynorphin levels specifically in the caudate putamen may in part underlie the enduring effects of stress.

  19. Chromatin Alterations in Response to Forced Swimming Underlie Increased Prodynorphin Transcription

    PubMed Central

    Reed, Brian; Fang, Nancy; Blackwell-Mayer, Brandan; Chen, Shasha; Yuferov, Vadim; Zhou, Yan; Kreek, Mary Jeanne

    2012-01-01

    Antagonism of the kappa opioid receptor (KOR) has been reported to have anti-depressant-like properties. The dynorphin/KOR system is a crucial neurochemical substrate underlying the pathologies of addictive diseases, affective disorders and other disease states. However, the molecular underpinnings and neuroanatomical localization of the dysregulation of this system have not yet been fully elucidated. Utilizing the Porsolt Forced Swim Test (FST), an acute stressor commonly used as in rodent models measuring antidepressant efficacy, male Sprague-Dawley rats were subject to forced swimming for 15 minutes, treated 1 hour with vehicle or nor-BNI (5 or 10 mg/kg), and then 1 day later subject to FST for five minutes. In accordance with previous findings, nor-BNI dose dependently increased climbing time and reduced immobility. In comparison to control animals not exposed to FST, we observed a significant elevation in prodynorphin (pDyn) mRNA levels following FST using real-time optical PCR in the caudate putamen but not in the nucleus accumbens, hypothalamus, amygdala, frontal cortex, or hippocampus. Nor-BNI treatment did not affect pDyn mRNA levels in comparison to animals that received vehicle. The corresponding brain regions from the opposite hemisphere were analyzed for underlying chromatin modifications of the prodynorphin gene promoter region using chromatin immunoprecipitation with antibodies against specifically methylated histones H3K27Me2, H3K27Me3, H3K4Me2, and H3K4Me3, as well as CREB-1 and MeCP2. Significant alterations in proteins bound to DNA in the Cre-3, Cre-4, and Sp1 regions of the prodynorphin promoter were found in the caudate putamen of the FST saline-treated animals compared to control animals, with no changes observed in the hippocampus. Epigenetic changes resulting in elevated dynorphin levels specifically in the caudate putamen may in part underlie the enduring effects of stress. PMID:22698692

  20. Formaldehyde crosslinking: a tool for the study of chromatin complexes.

    PubMed

    Hoffman, Elizabeth A; Frey, Brian L; Smith, Lloyd M; Auble, David T

    2015-10-30

    Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function.

  1. Interaction of sulfur mustard with rat liver salt fractionated chromatin.

    PubMed

    Jafari, Mahvash; Nateghi, M; Rabbani, A

    2010-01-01

    In this study, the interaction of an alkylating agent, sulfur mustard (SM) with rat liver active (S1 and S2) and inactive (P2) chromatin was investigated employing UV/vis spectroscopy and gel electrophoreses. The results show that SM affects the chromatin structure in a dose-dependent manner. The binding of SM to fractions is different. At lower concentrations (<500 microM), SM seems to unfold the structure and at higher concentrations, it induces aggregation and condensation of chromatin possibly via forming cross-links between the chromatin components. The extent of condensation in S2 is higher when compared to the P2 fraction.

  2. The centromere: chromatin foundation for the kinetochore machinery.

    PubMed

    Fukagawa, Tatsuo; Earnshaw, William C

    2014-09-01

    Since discovery of the centromere-specific histone H3 variant CENP-A, centromeres have come to be defined as chromatin structures that establish the assembly site for the complex kinetochore machinery. In most organisms, centromere activity is defined epigenetically, rather than by specific DNA sequences. In this review, we describe selected classic work and recent progress in studies of centromeric chromatin with a focus on vertebrates. We consider possible roles for repetitive DNA sequences found at most centromeres, chromatin factors and modifications that assemble and activate CENP-A chromatin for kinetochore assembly, plus the use of artificial chromosomes and kinetochores to study centromere function. PMID:25203206

  3. Formaldehyde crosslinking: a tool for the study of chromatin complexes.

    PubMed

    Hoffman, Elizabeth A; Frey, Brian L; Smith, Lloyd M; Auble, David T

    2015-10-30

    Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function. PMID:26354429

  4. Epigenetics: Beyond Chromatin Modifications and Complex Genetic Regulation1

    PubMed Central

    Eichten, Steven R.; Schmitz, Robert J.; Springer, Nathan M.

    2014-01-01

    Chromatin modifications and epigenetics may play important roles in many plant processes, including developmental regulation, responses to environmental stimuli, and local adaptation. Chromatin modifications describe biochemical changes to chromatin state, such as alterations in the specific type or placement of histones, modifications of DNA or histones, or changes in the specific proteins or RNAs that associate with a genomic region. The term epigenetic is often used to describe a variety of unexpected patterns of gene regulation or inheritance. Here, we specifically define epigenetics to include the key aspects of heritability (stable transmission of gene expression states through mitotic or meiotic cell divisions) and independence from DNA sequence changes. We argue against generically equating chromatin and epigenetics; although many examples of epigenetics involve chromatin changes, those chromatin changes are not always heritable or may be influenced by genetic changes. Careful use of the terms chromatin modifications and epigenetics can help separate the biochemical mechanisms of regulation from the inheritance patterns of altered chromatin states. Here, we also highlight examples in which chromatin modifications and epigenetics affect important plant processes. PMID:24872382

  5. Interphase Chromosome Conformation and Chromatin-chromatin Interactions in Human Epithelial Cells Cultured Under Different Gravity Conditions

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Hada, Megumi; Wu, Honglu

    2014-01-01

    On a multi-mega base pair scale of the DNA, the arrangement of chromatin is non-random. In M10 epithelial cells, both telomere regions tend to be located towards the exterior of the chromosome domain, whereas the rest p-arm of the chromatin region towards the interior. In contrast, most of the q-arm of the chromatin is found in the peripheral of the domain. In lymphocytes, the p-arm chromatin regions towards the interior in close proximity with each other, whereas two q-arm regions are nearness in space. It indicates that G0 lymphocytes may lack secondary 3D chromatin folding. There chromatin folding patterns are consistent with our previous finding of non-random distribution of intra-chromosomal exchanges. In simulated microgravity conditions, the chromosome conformation may be altered and new regions in close proximity, especially to region 2 are suggested.

  6. The Fun30 chromatin remodeler Fft3 controls nuclear organization and chromatin structure of insulators and subtelomeres in fission yeast.

    PubMed

    Steglich, Babett; Strålfors, Annelie; Khorosjutina, Olga; Persson, Jenna; Smialowska, Agata; Javerzat, Jean-Paul; Ekwall, Karl

    2015-03-01

    In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3∆ cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and the nuclear envelope.

  7. Facioscapulohumeral muscular dystrophy: consequences of chromatin relaxation

    PubMed Central

    van der Maarel, Silvère M.; Miller, Daniel G.; Tawil, Rabi; Filippova, Galina N.; Tapscott, Stephen J.

    2013-01-01

    Purpose of review In recent years we have seen remarkable progress in our understanding of the disease mechanism underlying facioscapulohumeral muscular dystrophy (FSHD). The purpose of this review is to provide a comprehensive overview of our current understanding of the disease mechanism and to discuss the observations supporting the possibility of a developmental defect in this disorder. Recent findings In the majority of cases FSHD is caused by contraction of the D4Z4 repeat array (FSHD1). This results in local chromatin relaxation and stable expression of the DUX4 retrogene in skeletal muscle, but only when a polymorphic DUX4 polyadenylation signal is present. In some cases (FSHD2), D4Z4 chromatin relaxation and stable DUX4 expression occurs in the absence of D4Z4 array contraction. DUX4 is a germline transcription factor and its expression in skeletal muscle leads to activation of early stem cell and germline programs and transcriptional activation of retroelements. Summary Recent studies have provided a plausible disease mechanism for FSHD where FSHD results from inappropriate expression of the germline transcription factor DUX4. The genes regulated by DUX4 suggest several mechanisms of muscle damage, and provide potential biomarkers and therapeutic targets that should be investigated in future studies. PMID:22892954

  8. Detection of genes expressed in Bordetella bronchiseptica colonizing rat trachea by in vivo expressed-tag immunoprecipitation method.

    PubMed

    Abe, Hiroyuki; Kamitani, Shigeki; Fukui-Miyazaki, Aya; Shinzawa, Naoaki; Nakamura, Keiji; Horiguchi, Yasuhiko

    2015-05-01

    Analyses of bacterial genes expressed in response to the host environment provide clues to understanding the host-pathogen interactions that lead to the establishment of infection. In this study, a novel method named In Vivo Expressed-Tag ImmunoPrecipitation (IVET-PI) was developed for detecting genes expressed in bacteria that are recovered in a small numbers from host tissues. IVET-IP was designed to overcome some drawbacks of previous similar methods. We applied IVET-IP to Bordetella bronchiseptica colonizing rat trachea and identified 173 genes that were expressed in the bacteria over the entire course of an infection. These gene products included two transcriptional factors that are involved in the expression of filamentous hemagglutinin, adenylate cyclase toxin, and major virulence factors for the bordetellae. We consider that this method might provide novel insight into the course of Bordetella infection. PMID:25683445

  9. The brain as immunoprecipitator of serum autoantibodies against N-Methyl-D-aspartate receptor subunit NR1.

    PubMed

    Castillo-Gomez, Esther; Kästner, Anne; Steiner, Johann; Schneider, Anja; Hettling, Bilke; Poggi, Giulia; Ostehr, Kristin; Uhr, Manfred; Asif, Abdul R; Matzke, Mike; Schmidt, Ulrike; Pfander, Viktoria; Hammer, Christian; Schulz, Thomas F; Binder, Lutz; Stöcker, Winfried; Weber, Frank; Ehrenreich, Hannelore

    2016-01-01

    Autoantibodies (AB) against N-methyl-D-aspartate receptor subunit NR1 (NMDAR1) are highly seroprevalent in health and disease. Symptomatic relevance may arise upon compromised blood-brain barrier (BBB). However, it remained unknown whether circulating NMDAR1 AB appear in the cerebrospinal fluid (CSF). Of n = 271 subjects with CSF-serum pairs, 26 were NMDAR1 AB seropositive, but only 1 was CSF positive. Contrariwise, tetanus AB (non-brain-binding) were present in serum and CSF of all subjects, with CSF levels higher upon BBB dysfunction. Translational mouse experiments proved the hypothesis that the brain acts as an 'immunoprecipitator'; simultaneous injection of NMDAR1 AB and the non-brain-binding green fluorescent protein AB resulted in high detectability of the former in brain and the latter in CSF. PMID:26505629

  10. Detection of genes expressed in Bordetella bronchiseptica colonizing rat trachea by in vivo expressed-tag immunoprecipitation method.

    PubMed

    Abe, Hiroyuki; Kamitani, Shigeki; Fukui-Miyazaki, Aya; Shinzawa, Naoaki; Nakamura, Keiji; Horiguchi, Yasuhiko

    2015-05-01

    Analyses of bacterial genes expressed in response to the host environment provide clues to understanding the host-pathogen interactions that lead to the establishment of infection. In this study, a novel method named In Vivo Expressed-Tag ImmunoPrecipitation (IVET-PI) was developed for detecting genes expressed in bacteria that are recovered in a small numbers from host tissues. IVET-IP was designed to overcome some drawbacks of previous similar methods. We applied IVET-IP to Bordetella bronchiseptica colonizing rat trachea and identified 173 genes that were expressed in the bacteria over the entire course of an infection. These gene products included two transcriptional factors that are involved in the expression of filamentous hemagglutinin, adenylate cyclase toxin, and major virulence factors for the bordetellae. We consider that this method might provide novel insight into the course of Bordetella infection.

  11. Development of a Luciferase Immunoprecipitation System Assay To Detect IgG Antibodies against Human Respiratory Syncytial Virus Nucleoprotein

    PubMed Central

    Kumari, Sangeeta; Crim, Roberta Lynne; Kulkarni, Ashwin; Audet, Susette A.; Mdluli, Thembi; Murata, Haruhiko

    2014-01-01

    The nucleoprotein of respiratory syncytial virus (RSV-N) is immunogenic and elicits an IgG response following infection. The RSV-N gene was cloned into a mammalian expression vector, pREN2, and the expressed luciferase-tagged protein (Ruc-N) detected anti-RSV-N-specific IgG antibodies using a high-throughput immunoprecipitation method (the luciferase immunoprecipitation system [LIPS]-NRSV assay). The specificity of the assay was evaluated using monoclonal antibodies (MAbs) and monospecific pre- and postimmunization rabbit antisera. Blood serum samples from chimpanzees and humans with proven/probable RSV infection were also tested. The pre- and postimmunization serum samples from rabbits given human metapneumovirus (HMPV) or measles virus were negative when tested by the LIPS-NRSV assay, while antisera obtained after immunization with either the RSV-A or RSV-B strain gave positive signals in a dose-dependent manner. RSV-N MAb 858-3 gave a positive signal in the LIPS-NRSV assay, while MAbs against other paramyxovirus nucleoproteins or RSV-F or RSV-G did not. Serum samples from chimpanzees simultaneously immunized with vaccinia-RSV-F and vaccinia-RSV-G recombinant viruses were negative in the LIPS-NRSV assay; however, anti-RSV-N IgG responses were detected following subsequent RSV challenge. Seven of the 12 infants who were seronegative at 9 months of age had detectable anti-RSV-N antibodies when they were retested at 15 to 18 months of age. The LIPS-NRSV assay detects specific anti-RSV-N IgG responses that may be used as a biomarker of RSV infection. PMID:24403526

  12. CHD5, a brain-specific paralog of Mi2 chromatin remodeling enzymes, regulates expression of neuronal genes.

    PubMed

    Potts, Rebecca Casaday; Zhang, Peisu; Wurster, Andrea L; Precht, Patricia; Mughal, Mohamed R; Wood, William H; Zhang, Yonqing; Becker, Kevin G; Mattson, Mark P; Pazin, Michael J

    2011-01-01

    CHD5 is frequently deleted in neuroblastoma and is a tumor suppressor gene. However, little is known about the role of CHD5 other than it is homologous to chromatin remodeling ATPases. We found CHD5 mRNA was restricted to the brain; by contrast, most remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66ß, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Expression of gene sets linked to aging and Alzheimer's disease were strongly altered by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 protein is found in a NuRD-like multi-protein complex. CHD5 expression is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of genes implicated in aging and Alzheimer's disease.

  13. CHD5, a Brain-Specific Paralog of Mi2 Chromatin Remodeling Enzymes, Regulates Expression of Neuronal Genes

    PubMed Central

    Potts, Rebecca Casaday; Zhang, Peisu; Wurster, Andrea L.; Precht, Patricia; Mughal, Mohamed R.; Wood, William H.; Zhang, Yonqing; Becker, Kevin G.; Mattson, Mark P.; Pazin, Michael J.

    2011-01-01

    CHD5 is frequently deleted in neuroblastoma and is a tumor suppressor gene. However, little is known about the role of CHD5 other than it is homologous to chromatin remodeling ATPases. We found CHD5 mRNA was restricted to the brain; by contrast, most remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66ß, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Expression of gene sets linked to aging and Alzheimer's disease were strongly altered by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 protein is found in a NuRD-like multi-protein complex. CHD5 expression is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of genes implicated in aging and Alzheimer's disease. PMID:21931736

  14. SWI/SNF chromatin remodeling complex is critical for the expression of microphthalmia-associated transcription factor in melanoma cells

    SciTech Connect

    Vachtenheim, Jiri; Ondrusova, Lubica; Borovansky, Jan

    2010-02-12

    The microphthalmia-associated transcription factor (MITF) is required for melanocyte development, maintenance of the melanocyte-specific transcription, and survival of melanoma cells. MITF positively regulates expression of more than 25 genes in pigment cells. Recently, it has been demonstrated that expression of several MITF downstream targets requires the SWI/SNF chromatin remodeling complex, which contains one of the two catalytic subunits, Brm or Brg1. Here we show that the expression of MITF itself critically requires active SWI/SNF. In several Brm/Brg1-expressing melanoma cell lines, knockdown of Brg1 severely compromised MITF expression with a concomitant dowregulation of MITF targets and decreased cell proliferation. Although Brm was able to substitute for Brg1 in maintaining MITF expression and melanoma cell proliferation, sequential knockdown of both Brm and Brg1 in 501mel cells abolished proliferation. In Brg1-null SK-MEL-5 melanoma cells, depletion of Brm alone was sufficient to abrogate MITF expression and cell proliferation. Chromatin immunoprecipitation confirmed the binding of Brg1 or Brm to the promoter of MITF. Together these results demonstrate the essential role of SWI/SNF for expression of MITF and suggest that SWI/SNF may be a promissing target in melanoma therapy.

  15. Global chromatin fibre compaction in response to DNA damage

    SciTech Connect

    Hamilton, Charlotte; Hayward, Richard L.; Gilbert, Nick

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. Black-Right-Pointing-Pointer DNA repair foci are found in soluble chromatin. Black-Right-Pointing-Pointer Biophysical analysis reveals global chromatin fibre compaction after DNA damage. Black-Right-Pointing-Pointer DNA damage is accompanied by rapid linker histone dephosphorylation. -- Abstract: DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation ({gamma}H2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and {gamma}H2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by

  16. A dynamic CTCF chromatin binding landscape promotes DNA hydroxymethylation and transcriptional induction of adipocyte differentiation

    PubMed Central

    Dubois-Chevalier, Julie; Oger, Frédérik; Dehondt, Hélène; Firmin, François F.; Gheeraert, Céline; Staels, Bart; Lefebvre, Philippe; Eeckhoute, Jérôme

    2014-01-01

    CCCTC-binding factor (CTCF) is a ubiquitously expressed multifunctional transcription factor characterized by chromatin binding patterns often described as largely invariant. In this context, how CTCF chromatin recruitment and functionalities are used to promote cell type-specific gene expression remains poorly defined. Here, we show that, in addition to constitutively bound CTCF binding sites (CTS), the CTCF cistrome comprises a large proportion of sites showing highly dynamic binding patterns during the course of adipogenesis. Interestingly, dynamic CTCF chromatin binding is positively linked with changes in expression of genes involved in biological functions defining the different stages of adipogenesis. Importantly, a subset of these dynamic CTS are gained at cell type-specific regulatory regions, in line with a requirement for CTCF in transcriptional induction of adipocyte differentiation. This relates to, at least in part, CTCF requirement for transcriptional activation of both the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARG) and its target genes. Functionally, we show that CTCF interacts with TET methylcytosine dioxygenase (TET) enzymes and promotes adipogenic transcriptional enhancer DNA hydroxymethylation. Our study reveals a dynamic CTCF chromatin binding landscape required for epigenomic remodeling of enhancers and transcriptional activation driving cell differentiation. PMID:25183525

  17. Different conformations of ribosomal DNA in active and inactive chromatin in Xenopus laevis.

    PubMed

    Spadafora, C; Riccardi, P

    1985-12-20

    The chromatin structure of the ribosomal DNA in Xenopus laevis was studied by micrococcal nuclease digestions of blood, liver and embryonic cell nuclei. We have found that BglI-restricted DNA from micrococcal nuclease-digested blood cell nuclei has an increased electrophoretic mobility compared to the undigested control. Micrococcal nuclease digestion of liver cell nuclei causes a very slight shift in mobility, only in the region of the spacer containing the "Bam Islands". In contrast, the mobility of ribosomal DNA in chromatin of embryonic cells, under identical digestion conditions, remains unaffected by the nuclease activity. Denaturing gels or ligase action on the nuclease-treated DNA abolishes the differences in the electrophoretic mobility. Ionic strength and ethidium bromide influence the relative electrophoretic migration of the two DNA fragment populations, suggesting that secondary structure may play an important role in the observed phenomena. In addition, restriction analysis under native electrophoretic conditions of DNA prepared from blood, liver and embryonic cells shows that blood cell DNA restriction fragments always have a faster mobility than the corresponding fragments of liver and embryo cell DNA. We therefore propose that nicking activity by micrococcal nuclease modifies the electrophoretic mobility of an unusual DNA conformation, present in blood cell, and to a lesser extent, in liver cell ribosomal chromatin. A possible function for these structures is discussed. The differences of the ribosomal chromatin structures in adult and embryonic tissues may reflect the potential of the genes to be expressed.

  18. Chromatin Modulation of Herpesvirus Lytic Gene Expression: Managing Nucleosome Density and Heterochromatic Histone Modifications.

    PubMed

    Kristie, Thomas M

    2016-01-01

    Like their cellular hosts, herpesviruses are subject to the regulatory impacts of chromatin assembled on their genomes. Upon infection, these viruses are assembled into domains of chromatin with heterochromatic signatures that suppress viral gene expression or euchromatic characteristics that promote gene expression. The organization and modulation of these chromatin domains appear to be intimately linked to the coordinated expression of the different classes of viral genes and thus ultimately play an important role in the progression of productive infection or the establishment and maintenance of viral latency. A recent report from the Knipe laboratory (J. S. Lee, P. Raja, and D. M. Knipe, mBio 7:e02007-15, 2016) contributes to the understanding of the dynamic modulation of chromatin assembled on the herpes simplex virus genome by monitoring the levels of characteristic heterochromatic histone modifications (histone H3 lysine 9 and 27 methylation) associated with a model viral early gene during the progression of lytic infection. Additionally, this study builds upon previous observations that the viral immediate-early protein ICP0 plays a role in reducing the levels of heterochromatin associated with the early genes. PMID:26884430

  19. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters.

    PubMed

    Lavender, Christopher A; Cannady, Kimberly R; Hoffman, Jackson A; Trotter, Kevin W; Gilchrist, Daniel A; Bennett, Brian D; Burkholder, Adam B; Burd, Craig J; Fargo, David C; Archer, Trevor K

    2016-08-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  20. Characterization of an alternative chromatin remodeling to parasperm in a cottid fish, Hemilepidotus gilberti.

    PubMed

    Nakauchi, Yuni; Hayakawa, Youichi; Fujinoki, Masakatsu; Yamamura, Orio; Kobayashi, Makito; Watanabe, Akihiko

    2011-06-01

    The dimorphic sperm of Hemilepidotus gilberti, i.e., haploid eusperm and diploid parasperm, have different morphologies corresponding to their own roles in fertilization. To estimate how these specific sperm morphologies were established, we focused on the nuclear morphologies and examined their changing processes in dimorphic spermiogenesis. Electron microscopic observation revealed that, in euspermatids, chromatin condensation first appeared as a mosaic pattern of moderate electrodense material in the peripheral region of the round nucleus. Those materials spread across the whole area to form a uniformly condensed nucleus. Chromatin condensation began similarly in paraspermatids to that in euspermatids. These became localized to one side of a nucleus and further condensed to form strong electrodense chromatin clusters, which are a specific feature of parasperm. From the remodeled nuclei of eusperm and parasperm, we found five and three kinds of sperm-specific basic proteins (SBPs), respectively, substituted to histones. The N-terminus amino acid sequences of the SBPs suggest that, in parasperm, one major SBP and two minor ones were distinct from each other. In eusperm nuclei, two kinds of specific SBPs were detected in addition to the homologs of parasperm SBPs. The specific SBPs had homologous amino acid sequences with huge arginine clusters, and one of them was most dominant among the five kinds of SBPs. The different combinations of SBPs in the eusperm and parasperm may cause a specific pattern of chromatin condensation in the dimorphic sperm nuclei of H. gilberti.

  1. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters

    PubMed Central

    Lavender, Christopher A.; Hoffman, Jackson A.; Trotter, Kevin W.; Gilchrist, Daniel A.; Bennett, Brian D.; Burkholder, Adam B.; Fargo, David C.; Archer, Trevor K.

    2016-01-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  2. The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae.

    PubMed

    Gartenberg, Marc R; Smith, Jeffrey S

    2016-08-01

    Transcriptional silencing in Saccharomyces cerevisiae occurs at several genomic sites including the silent mating-type loci, telomeres, and the ribosomal DNA (rDNA) tandem array. Epigenetic silencing at each of these domains is characterized by the absence of nearly all histone modifications, including most prominently the lack of histone H4 lysine 16 acetylation. In all cases, silencing requires Sir2, a highly-conserved NAD(+)-dependent histone deacetylase. At locations other than the rDNA, silencing also requires additional Sir proteins, Sir1, Sir3, and Sir4 that together form a repressive heterochromatin-like structure termed silent chromatin. The mechanisms of silent chromatin establishment, maintenance, and inheritance have been investigated extensively over the last 25 years, and these studies have revealed numerous paradigms for transcriptional repression, chromatin organization, and epigenetic gene regulation. Studies of Sir2-dependent silencing at the rDNA have also contributed to understanding the mechanisms for maintaining the stability of repetitive DNA and regulating replicative cell aging. The goal of this comprehensive review is to distill a wide array of biochemical, molecular genetic, cell biological, and genomics studies down to the "nuts and bolts" of silent chromatin and the processes that yield transcriptional silencing. PMID:27516616

  3. Repair of DNA lesions in chromosomal DNA impact of chromatin structure and Cockayne syndrome proteins.

    PubMed

    Fousteri, Maria; van Hoffen, Anneke; Vargova, Hana; Mullenders, Leon H F

    2005-07-28

    Decondensation of chromatin is essential to facilitate access to DNA metabolizing processes such as transcription and DNA repair. Disruption of histone-DNA contacts by histone modification or by ATP dependent chromatin remodelling allows DNA-binding proteins to compete with histones for DNA. The efficiency of global genome nucleotide excision repair (GGR) that removes a variety of helix distorting DNA lesions is known to be affected by chromatin structure most notably demonstrated by the slow repair of heterochromatin. In addition, the efficiency of GGR to repair lesions in transcriptionally active genes requires functional CSA and B proteins. We found that repair of UV-photolesions in both strands of the active adenosine deaminase gene was delayed in CS cells when compared to normal human fibroblasts. We suggest that the lack of transcription recovery characteristic for CS cells exposed to DNA damaging agents, might lead to changes in the chromatin structure of active genes, causing less efficient repair of lesions in these genes when compared to normal cells. PMID:15961352

  4. Integrating and mining the chromatin landscape of cell-type specificity using self-organizing maps.

    PubMed

    Mortazavi, Ali; Pepke, Shirley; Jansen, Camden; Marinov, Georgi K; Ernst, Jason; Kellis, Manolis; Hardison, Ross C; Myers, Richard M; Wold, Barbara J

    2013-12-01

    We tested whether self-organizing maps (SOMs) could be used to effectively integrate, visualize, and mine diverse genomics data types, including complex chromatin signatures. A fine-grained SOM was trained on 72 ChIP-seq histone modifications and DNase-seq data sets from six biologically diverse cell lines studied by The ENCODE Project Consortium. We mined the resulting SOM to identify chromatin signatures related to sequence-specific transcription factor occupancy, sequence motif enrichment, and biological functions. To highlight clusters enriched for specific functions such as transcriptional promoters or enhancers, we overlaid onto the map additional data sets not used during training, such as ChIP-seq, RNA-seq, CAGE, and information on cis-acting regulatory modules from the literature. We used the SOM to parse known transcriptional enhancers according to the cell-type-specific chromatin signature, and we further corroborated this pattern on the map by EP300 (also known as p300) occupancy. New candidate cell-type-specific enhancers were identified for multiple ENCODE cell types in this way, along with new candidates for ubiquitous enhancer activity. An interactive web interface was developed to allow users to visualize and custom-mine the ENCODE SOM. We conclude that large SOMs trained on chromatin data from multiple cell types provide a powerful way to identify complex relationships in genomic data at user-selected levels of granularity.

  5. Histone H3 phosphorylation – A versatile chromatin modification for different occasions

    PubMed Central

    Sawicka, Anna; Seiser, Christian

    2012-01-01

    Post-translation modifications of histones modulate the accessibility and transcriptional competence of specific chromatin regions within the eukaryotic genome. Phosphorylation of histone H3 is unique in the sense that it associates on one hand with open chromatin during gene activation and marks on the other hand highly condensed chromatin during mitosis. Phosphorylation of serine residues at histone H3 is a highly dynamic process that creates together with acetylation and methylation marks at neighboring lysine residues specific combinatorial patterns that are read by specific detector proteins. In this review we describe the importance of different histone H3 phosphorylation marks for chromatin condensation during mitosis. In addition, we review the signals that trigger histone H3 phosphorylation and the factors that control this reversible modification during interphase and mediate the biological readout of the signal. Finally, we discuss different models describing the role of histone H3 phosphorylation in the activation of transcription of poised genes or by transient derepression of epigenetically silenced genes. We propose that histone H3 phosphorylation in the context with lysine methylation might temporarily relieve the silencing of specific genes without affecting the epigenetic memory. PMID:22564826

  6. Recognition of chromatin by the plant alkaloid, ellipticine as a dual binder

    SciTech Connect

    Banerjee, Amrita; Sanyal, Sulagna; Majumder, Parijat; Chakraborty, Payal; Jana, Kuladip; Dasgupta, Dipak

    2015-07-10

    Recognition of core histone components of chromatin along with chromosomal DNA by a class of small molecule modulators is worth examining to evaluate their intracellular mode of action. A plant alkaloid ellipticine (ELP) which is a putative anticancer agent has so far been reported to function via DNA intercalation, association with topoisomerase II and binding to telomere region. However, its effect upon the potential intracellular target, chromatin is hitherto unreported. Here we have characterized the biomolecular recognition between ELP and different hierarchical levels of chromatin. The significant result is that in addition to DNA, it binds to core histone(s) and can be categorized as a ‘dual binder’. As a sequel to binding with histone(s) and core octamer, it alters post-translational histone acetylation marks. We have further demonstrated that it has the potential to modulate gene expression thereby regulating several key biological processes such as nuclear organization, transcription, translation and histone modifications. - Highlights: • Ellipticine acts a dual binder binding to both DNA and core histone(s). • It induces structural perturbations in chromatin, chromatosome and histone octamer. • It alters histones acetylation and affects global gene expression.

  7. New insights into protamine-like component organization in Mytilus galloprovincialis' sperm chromatin.

    PubMed

    Vassalli, Quirino Attilio; Caccavale, Filomena; Avagnano, Stefano; Murolo, Alessandra; Guerriero, Giulia; Fucci, Laura; Ausió, Juan; Piscopo, Marina

    2015-03-01

    We have analyzed Mytilus galloprovincialis' sperm chromatin, which consists of three protamine-like proteins, PL-II, PL-III, and PL-IV, in addition to a residual amount of the four core histones. We have probed the structure of this sperm chromatin through digestion with micrococcal nuclease (MNase) in combination with salt fractionation. Furthermore, we used the electrophoretic mobility shift assay to define DNA-binding mode of PL-II and PL-III and turbidimetric assays to determine their self-association ability in the presence of sodium phosphate. Although in literature it is reported that M. galloprovincialis' sperm chromatin lacks nucleosomal organization, our results obtained by MNase digestion suggest the existence of a likely unusual organization, in which there would be a more accessible location of PL-II/PL-IV when compared with PL-III and core histones. So, we hypothesize that in M. galloprovincialis' sperm chromatin organization DNA is wrapped around a PL-III protein core and core histones and PL-II and PL-IV are bound to the flanking DNA regions (similarly to somatic histone H1). Furthermore, we propose that PL's K/R ratio affects their DNA-binding mode and self-association ability as reported previously for somatic and sperm H1 histones.

  8. Basal chromatin modification at the IL-4 gene in helper T cells

    SciTech Connect

    Grogan, Jane L.; Wang, Zhi-En; Stanley, Sarah; Harmon, Brian; Loots, Gaby G.; Rubin, Edward M.; Locksley, Richard M.

    2003-04-15

    Chromatin immunoprecipitations in naive CD4, but not CD8, T cells, demonstrated association of the IL-4 promoter with acetylated histone. Histone modifications and rapid IL-4 transcription were absent in conserved noncoding sequence 1 (CNS-1){sup -/-} cells lacking an 8-kb-distant enhancer in the IL-4/IL-13 intergenic region, but also in CD4{sup -/-} and Itk{sup -/-} cells, which have similar Th2 deficiencies. Histones associated with the IL-13 promoter were not similarly acetylated in naive T cells, but became acetylated in differentiated Th2 cells. Conversely, Th1 differentiation induced histone methylation at the type 2 cytokine locus. Like CD4{sup -/-} and Itk{sup -/-} mice, CNS-1{sup -/-} BALB/c mice were highly resistant to the Th2-inducing protozoan, Leishmania major. CNS-1 deficiency led to failure of IL-4 gene repositioning to heterochromatin after Th1 polarization, possibly related to the presence of reiterative Ikaros binding sites in the intergenic element. Hyperacetylation of nonexpressed genes may serve to mark lineage-specific loci for rapid expression and further modification.

  9. Novel RNA Duplex Locks HIV-1 in a Latent State via Chromatin-mediated Transcriptional Silencing

    PubMed Central

    Ahlenstiel, Chantelle; Mendez, Catalina; Lim, Steven T H; Marks, Katherine; Turville, Stuart; Cooper, David A; Kelleher, Anthony D; Suzuki, Kazuo

    2015-01-01

    Transcriptional gene silencing (TGS) of mammalian genes can be induced by short interfering RNA (siRNA) targeting promoter regions. We previously reported potent TGS of HIV-1 by siRNA (PromA), which targets tandem NF-κB motifs within the viral 5′LTR. In this study, we screened a siRNA panel with the aim of identifying novel 5′LTR targets, to provide multiplexing potential with enhanced viral silencing and application toward developing alternate therapeutic strategies. Systematic examination identified a novel siRNA target, si143, confirmed to induce TGS as the silencing mechanism. TGS was prolonged with virus suppression >12 days, despite a limited ability to induce post- TGS. Epigenetic changes associated with silencing were suggested by partial reversal by histone deacetylase inhibitors and confirmed by chromatin immunoprecipitation analyses, which showed induction of H3K27me3 and H3K9me3, reduction in H3K9Ac, and recruitment of argonaute-1, all characteristic marks of heterochromatin and TGS. Together, these epigenetic changes mimic those associated with HIV-1 latency. Further, robust resistance to reactivation was observed in the J-Lat 9.2 cell latency model, when transduced with shPromA and/or sh143. These data support si/shRNA-mediated TGS approaches to HIV-1 and provide alternate targets to pursue a functional cure, whereby the viral reservoir is locked in latency following antiretroviral therapy cessation. PMID:26506039

  10. Sensitive detection of chromatin-altering polymorphisms reveals autoimmune disease mechanisms.

    PubMed

    del Rosario, Ricardo Cruz-Herrera; Poschmann, Jeremie; Rouam, Sigrid Laure; Png, Eileen; Khor, Chiea Chuen; Hibberd, Martin Lloyd; Prabhakar, Shyam

    2015-05-01

    Most disease associations detected by genome-wide association studies (GWAS) lie outside coding genes, but very few have been mapped to causal regulatory variants. Here, we present a method for detecting regulatory quantitative trait loci (QTLs) that does not require genotyping or whole-genome sequencing. The method combines deep, long-read chromatin immunoprecipitation-sequencing (ChIP-seq) with a statistical test that simultaneously scores peak height correlation and allelic imbalance: the genotype-independent signal correlation and imbalance (G-SCI) test. We performed histone acetylation ChIP-seq on 57 human lymphoblastoid cell lines and used the resulting reads to call 500,066 single-nucleotide polymorphisms de novo within regulatory elements. The G-SCI test annotated 8,764 of these as histone acetylation QTLs (haQTLs)—an order of magnitude larger than the set of candidates detected by expression QTL analysis. Lymphoblastoid haQTLs were highly predictive of autoimmune disease mechanisms. Thus, our method facilitates large-scale regulatory variant detection in any moderately sized cohort for which functional profiling data can be generated, thereby simplifying identification of causal variants within GWAS loci. PMID:25799442

  11. Chromatin Regulators as a Guide for Cancer Treatment Choice.

    PubMed

    Gurard-Levin, Zachary A; Wilson, Laurence O W; Pancaldi, Vera; Postel-Vinay, Sophie; Sousa, Fabricio G; Reyes, Cecile; Marangoni, Elisabetta; Gentien, David; Valencia, Alfonso; Pommier, Yves; Cottu, Paul; Almouzni, Geneviève

    2016-07-01

    The limited capacity to predict a patient's response to distinct chemotherapeutic agents is a major hurdle in cancer management. The efficiency of a large fraction of current cancer therapeutics (radio- and chemotherapies) is influenced by chromatin structure. Reciprocally, alterations in chromatin organization may affect resistance mechanisms. Here, we explore how the misexpression of chromatin regulators-factors involved in the establishment and maintenance of functional chromatin domains-can inform about the extent of docetaxel response. We exploit Affymetrix and NanoString gene expression data for a set of chromatin regulators generated from breast cancer patient-derived xenograft models and patient samples treated with docetaxel. Random Forest classification reveals specific panels of chromatin regulators, including key components of the SWI/SNF chromatin remodeler, which readily distinguish docetaxel high-responders and poor-responders. Further exploration of SWI/SNF components in the comprehensive NCI-60 dataset reveals that the expression inversely correlates with docetaxel sensitivity. Finally, we show that loss of the SWI/SNF subunit BRG1 (SMARCA4) in a model cell line leads to enhanced docetaxel sensitivity. Altogether, our findings point toward chromatin regulators as biomarkers for drug response as well as therapeutic targets to sensitize patients toward docetaxel and combat drug resistance. Mol Cancer Ther; 15(7); 1768-77. ©2016 AACR. PMID:27196757

  12. Brd4 shields chromatin from ATM kinase signaling storms.

    PubMed

    Choi, Serah; Bakkenist, Christopher J

    2013-09-17

    Upon activation, ataxia telangiectasia mutated (ATM) kinase rapidly phosphorylates hundreds of proteins, setting off chaotic signaling storms from areas of damaged chromatin. Recent work by Kaidi and Jackson and Floyd et al. advance our knowledge of the mechanisms that initiate or limit ATM kinase signaling storms at chromatin. PMID:24045152

  13. A Broad Set of Chromatin Factors Influences Splicing

    PubMed Central

    Allemand, Eric; Myers, Michael P.; Garcia-Bernardo, Jose; Harel-Bellan, Annick; Krainer, Adrian R.; Muchardt, Christian

    2016-01-01

    Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing. PMID:27662573

  14. Biochemical analysis of chromatin containing recombinant Drosophila core histones.

    PubMed

    Levenstein, Mark E; Kadonaga, James T

    2002-03-01

    To investigate the effects of histone modifications upon chromatin structure and function, we studied the assembly and properties of chromatin that contains unmodified recombinant core histones. To this end, we synthesized the Drosophila core histones in Escherichia coli. The purified histones were lacking covalent modifications as well as their N-terminal initiating methionine residues. The recombinant histones were efficiently assembled into periodic nucleosome arrays in a completely purified recombinant system with Drosophila ATP-utilizing chromatin assembly and remodeling factor (ACF), Drosophila nucleosome assembly protein-1, plasmid DNA, and ATP. With the Gal4-VP16 activator and a crude transcription extract, we found that the transcriptional properties of ACF-assembled chromatin containing unmodified histones were similar to those of chromatin containing native histones. We then examined ACF-catalyzed chromatin remodeling with completely purified factors and chromatin consisting of unmodified histones. In these experiments, we observed promoter-specific disruption of the regularity of nucleosome arrays upon binding of Gal4-VP16 as well as nucleosome positioning by R3 Lac repressor and subsequent nucleosome remobilization upon isopropyl-beta-D-thiogalactopyranoside-induced dissociation of R3 from the template. Thus, chromatin assembly and remodeling by ACF can occur in the absence of histone modifications.

  15. Brd4 Shields Chromatin from ATM Kinase Signaling Storms

    PubMed Central

    Choi, Serah; Bakkenist, Christopher J.

    2014-01-01

    Upon activation, ataxia telangiectasia mutated (ATM) kinase rapidly phosphorylates hundreds of proteins, setting off chaotic signaling storms from areas of damaged chromatin. Recent work by Kaidi and Jackson and Floyd et al. advance our knowledge of the mechanisms that initiate or limit ATM kinase signaling storms at chromatin. PMID:24045152

  16. CYTOKINE-INDUCED CHROMATIN MODIFICATIONS OF THE TYPE I COLLAGEN ALPHA 2 GENE DURING INTESTINAL ENDOTHELIAL-TO-MESENCHYMAL TRANSITION

    PubMed Central

    Sadler, Tammy; Scarpa, Melania; Rieder, Florian; West, Gail; Stylianou, Eleni

    2013-01-01

    Background Fibrosis of the intestine is currently an irreversible complication of Inflammatory Bowel Disease yet little is understood of the underlying pathogenesis and anti-fibrotic strategies remain elusive. To develop effective therapies, knowledge of the mechanism of transcription and excessive deposition of type I collagen - a hallmark of fibrosis, is needed. We have shown previously that endothelial-to-mesenchymal transition (EndoMT) contributes to the pool of intestinal fibrotic cells and that a cytokine cocktail (IL1-β, TNF-α and TGF-β) induces Collagen I alpha 2 (COL1A2) mRNA and protein. Methods Chromatin immunoprecipitation assays on pure cultures of human intestinal mucosal endothelial cells undergoing EndoMT were performed with antibodies to specific histone modifications and RNA polymerase II. RT-PCR was used to quantify the levels of Col1A2 and endothelial specific von Willebrand factor (vWF) mRNA. Results We show that cytokines induce selective chromatin modifications (histone 4 hyperacetylation and hypermethylation of histone 3) and phosphorylated RNA polymerase II at the COL1A2 promoter. Hypoacetylated and hypomethylated histone 3 was detected on the repressed vWF gene. Prolonged exposure to cytokines (16 days) retained hyperacetylation of select lysines in H4 on the COL1A2 promoter. Removal of cytokines after 16 days and continued culture for 10 days, showed persistent hyperacetylation at lysine 16 in histone H4. Conclusion This is the first study to show that COL1A2 gene expression is associated with cytokine-induced, temporally ordered and persistent chromatin modifications and suggests that these are important determinants of gene expression in EndoMT and intestinal fibrosis. PMID:23635716

  17. Nonhistone Proteins Control Gene Expression in Reconstituted Chromatin

    PubMed Central

    Barrett, T.; Maryanka, D.; Hamlyn, P. H.; Gould, H. J.

    1974-01-01

    Chromatin was reconstituted from the purified DNA and histones of chicken erythrocytes and the nonhistone proteins of either chicken reticulocytes or chicken liver. Reconstituted chromatins, native chicken reticulocyte chromatin, and free DNA were transcribed with Escherichia coli RNA polymerase and the concentrations of globin-specific sequences in the RNA products were measured by hybridization with [3H]DNA complementary to chicken globin messenger RNA. Reticulocyte, but not liver, nonhistone proteins were shown to activate the globin genes in reconstituted erythrocyte chromatin. The transcripts of native and reconstituted chromatins were indistinguishable in respect of both the total yield of the RNA and the fractional yield of globin-specific sequences. Images PMID:4140516

  18. Silent chromatin at the middle and ends: lessons from yeasts

    PubMed Central

    Bühler, Marc; Gasser, Susan M

    2009-01-01

    Eukaryotic centromeres and telomeres are specialized chromosomal regions that share one common characteristic: their underlying DNA sequences are assembled into heritably repressed chromatin. Silent chromatin in budding and fission yeast is composed of fundamentally divergent proteins tat assemble very different chromatin structures. However, the ultimate behaviour of silent chromatin and the pathways that assemble it seem strikingly similar among Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe) and other eukaryotes. Thus, studies in both yeasts have been instrumental in dissecting the mechanisms that establish and maintain silent chromatin in eukaryotes, contributing substantially to our understanding of epigenetic processes. In this review, we discuss current models for the generation of heterochromatic domains at centromeres and telomeres in the two yeast species. PMID:19629038

  19. Data on the kinetics of in vitro assembled chromatin.

    PubMed

    Völker-Albert, Moritz Carl; Pusch, Miriam Caroline; Schmidt, Andreas; Imhof, Axel

    2016-09-01

    Here, we use LC-MS/MS and SWATH-MS to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos (DREX). This system allows easy manipulation of distinct aspects of chromatin assembly such as post-translational histone modifications, the levels of histone chaperones and the concentration of distinct DNA binding factors. In total, 480 proteins have been quantified as chromatin enriched factors and their binding kinetics have been monitored in the time course of 15 min, 1 h and 4 h of chromatin assembly. The data accompanying the manuscript on this approach, Völker-Albert et al., 2016 "A quantitative proteomic analysis of in vitro assembled chromatin" [1], has been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with the dataset identifier submission number PRIDE: PXD002537 and PRIDE: PXD003445. PMID:27331114

  20. HAMLET interacts with histones and chromatin in tumor cell nuclei.

    PubMed

    Düringer, Caroline; Hamiche, Ali; Gustafsson, Lotta; Kimura, Hiroshi; Svanborg, Catharina

    2003-10-24

    HAMLET is a folding variant of human alpha-lactalbumin in an active complex with oleic acid. HAMLET selectively enters tumor cells, accumulates in their nuclei and induces apoptosis-like cell death. This study examined the interactions of HAMLET with nuclear constituents and identified histones as targets. HAMLET was found to bind histone H3 strongly and to lesser extent histones H4 and H2B. The specificity of these interactions was confirmed using BIAcore technology and chromatin assembly assays. In vivo in tumor cells, HAMLET co-localized with histones and perturbed the chromatin structure; HAMLET was found associated with chromatin in an insoluble nuclear fraction resistant to salt extraction. In vitro, HAMLET bound strongly to histones and impaired their deposition on DNA. We conclude that HAMLET interacts with histones and chromatin in tumor cell nuclei and propose that this interaction locks the cells into the death pathway by irreversibly disrupting chromatin organization.

  1. DNA Damage Repair in the Context of Plant Chromatin1

    PubMed Central

    Donà, Mattia; Mittelsten Scheid, Ortrun

    2015-01-01

    The integrity of DNA molecules is constantly challenged. All organisms have developed mechanisms to detect and repair multiple types of DNA lesions. The basic principles of DNA damage repair (DDR) in prokaryotes and unicellular and multicellular eukaryotes are similar, but the association of DNA with nucleosomes in eukaryotic chromatin requires mechanisms that allow access of repair enzymes to the lesions. This is achieved by chromatin-remodeling factors, and their necessity for efficient DDR has recently been demonstrated for several organisms and repair pathways. Plants share many features of chromatin organization and DNA repair with fungi and animals, but they differ in other, important details, which are both interesting and relevant for our understanding of genome stability and genetic diversity. In this Update, we compare the knowledge of the role of chromatin and chromatin-modifying factors during DDR in plants with equivalent systems in yeast and humans. We emphasize plant-specific elements and discuss possible implications. PMID:26089404

  2. Differential expression of thrombospondin (THBS1) in tumorigenic and nontumorigenic prostate epithelial cells in response to a chromatin-binding soy peptide.

    PubMed

    Galvez, Alfredo F; Huang, Liping; Magbanua, Mark M J; Dawson, Kevin; Rodriguez, Raymond L

    2011-01-01

    The chemopreventive properties of the chromatin-binding soy peptide, lunasin, are well documented, but its mechanism of action is unclear. To elucidate the mechanism by which lunasin reduces tumor foci formation in cultured mammalian cells, nontumorigenic (RWPE-1) and tumorigenic (RWPE-2) human prostate epithelial cells were treated with lunasin followed by gene expression profiling and characterization of the chromatin acetylation status for certain chemopreventive genes. The genes HIF1A, PRKAR1A, TOB1, and THBS1 were upregulated by lunasin in RWPE-1 but not in RWPE-2 cells. Using histone acetyltransferase (HAT) assays with acid-extracted histones as templates, we showed that lunasin specifically inhibited H4K8 acetylation while enhanced H4K16 acetylation catalyzed by HAT enzymes p300, PCAF, and HAT1A. These results suggest a novel mechanism for lunasin-dependent upregulation of gene expression. Chromatin immunoprecipitation (ChIP) revealed hypoacetylation of H4K16 in RWPE-2 cells, specifically at the 5' end of THBS1 containing a CpG island. Moreover, bisulfite PCR (BSP) and subsequent DNA sequencing indicated that this CpG island was hypomethylated in RWPE-1 but hypermethylated in RWPE-2 cells. Histone hypoacetylation and DNA hypermethylation in the 5' region of THBS1 may explain the inability of lunasin to upregulate this gene in RWPE-2 cells. PMID:21526452

  3. BRG1, the ATPase subunit of SWI/SNF chromatin remodeling complex, interacts with HDAC2 to modulate telomerase expression in human cancer cells

    PubMed Central

    Wu, Shu; Ge, Yuanlong; Huang, Laiqiang; Liu, Haiying; Xue, Yong; Zhao, Yong

    2014-01-01

    Telomerase is often upregulated during initiation and/or progression of human tumors, suggesting that repression of telomerase might inhibit cancer growth or progression. Here, we report that BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex, is a general suppressor of hTERT transcription in human cancer cells. While overexpression of BRG1 inhibits hTERT transcription, depletion of BRG1 stimulates transcription of hTERT, leading to higher telomerase activity and longer telomeres. Chromatin-immunoprecipitation assays revealed that BRG1 binds to the transcription start site (TSS) of the hTERT promoter and forms a ternary complex with histone deacetylase 2 (HDAC2). BRG1 remodels chromatin structure to facilitate the action of HDAC2, leading to deacetylation of H3K9ac and H4ac at the TSS and suppression of hTERT transcription. On the other hand, β-catenin binds to the TSS and stimulates hTERT transcription. Thus, BRG1/HDAC2 and β-catenin constitute a manipulative apparatus at the TSS to play opposite but complementary roles in regulating hTERT expression. These results uncover a yin-yang mechanism in modulating hTERT transcription and provide explanation for limited transcription of hTERT in human cancer cells. BRG1/HDAC2 may have a potential as an anti-cancer therapeutic and/or for reactivating cellular proliferative capacity in the context of in vitro tissue engineering. PMID:25486475

  4. Ligand Binding Shifts Highly Mobile Retinoid X Receptor to the Chromatin-Bound State in a Coactivator-Dependent Manner, as Revealed by Single-Cell Imaging

    PubMed Central

    Brazda, Peter; Krieger, Jan; Daniel, Bence; Jonas, David; Szekeres, Tibor; Langowski, Jörg; Tóth, Katalin; Vámosi, György

    2014-01-01

    Retinoid X receptor (RXR) is a promiscuous nuclear receptor forming heterodimers with several other receptors, which activate different sets of genes. Upon agonist treatment, the occupancy of its genomic binding regions increased, but only a modest change in the number of sites was revealed by chromatin immunoprecipitation followed by sequencing, suggesting a rather static behavior. However, such genome-wide and biochemical approaches do not take into account the dynamic behavior of a transcription factor. Therefore, we characterized the nuclear dynamics of RXR during activation in single cells on the subsecond scale using live-cell imaging. By applying fluorescence recovery after photobleaching and fluorescence correlation spectroscopy (FCS), techniques with different temporal and spatial resolutions, a highly dynamic behavior could be uncovered which is best described by a two-state model (slow and fast) of receptor mobility. In the unliganded state, most RXRs belonged to the fast population, leaving ∼15% for the slow, chromatin-bound fraction. Upon agonist treatment, this ratio increased to ∼43% as a result of an immediate and reversible redistribution. Coactivator binding appears to be indispensable for redistribution and has a major contribution to chromatin association. A nuclear mobility map recorded by light sheet microscopy-FCS shows that the ligand-induced transition from the fast to the slow population occurs throughout the nucleus. Our results support a model in which RXR has a distinct, highly dynamic nuclear behavior and follows hit-and-run kinetics upon activation. PMID:24449763

  5. Single and combinatorial chromatin coupling events underlies the function of transcript factor krüppel-like factor 11 in the regulation of gene networks

    PubMed Central

    2014-01-01

    Background Krüppel-like factors (KLFs) are a group of master regulators of gene expression conserved from flies to human. However, scant information is available on either the mechanisms or functional impact of the coupling of KLF proteins to chromatin remodeling machines, a deterministic step in transcriptional regulation. Results and discussion In the current study, we use genome-wide analyses of chromatin immunoprecipitation (ChIP-on-Chip) and Affymetrix-based expression profiling to gain insight into how KLF11, a human transcription factor involved in tumor suppression and metabolic diseases, works by coupling to three co-factor groups: the Sin3-histone deacetylase system, WD40-domain containing proteins, and the HP1-histone methyltransferase system. Our results reveal that KLF11 regulates distinct gene networks involved in metabolism and growth by using single or combinatorial coupling events. Conclusion This study, the first of its type for any KLF protein, reveals that interactions with multiple chromatin systems are required for the full gene regulatory function of these proteins. PMID:24885560

  6. Chromatin insulators: lessons from the fly.

    PubMed

    Gurudatta, B V; Corces, Victor G

    2009-07-01

    Chromatin insulators are DNA-protein complexes with broad functions in nuclear biology. Drosophila has at least five different types of insulators; recent results suggest that these different insulators share some components that may allow them to function through common mechanisms. Data from genome-wide localization studies of insulator proteins indicate a possible functional specialization, with different insulators playing distinct roles in nuclear biology. Cells have developed mechanisms to control insulator activity by recruiting specialized proteins or by covalent modification of core components. Current results suggest that insulators set up cell-specific blueprints of nuclear organization that may contribute to the establishment of different patterns of gene expression during cell differentiation and development.

  7. On the mechanochemical machinery underlying chromatin remodeling

    NASA Astrophysics Data System (ADS)

    Yusufaly, Tahir I.

    This dissertation discuss two recent efforts, via a unique combination of structural bioinformatics and density functional theory, to unravel some of the details concerning how molecular machinery within the eukaryotic cell nucleus controls chromatin architecture. The first, a study of the 5-methylation of cytosine in 5'-CG-3' : 5'-CG-3' base-pair steps, reveals that the methyl groups roughen the local elastic energy landscape of the DNA. This enhances the probability of the canonical B-DNA structure transitioning into the undertwisted A-like and overtwisted C-like forms seen in nucleosomes, or looped segments of DNA bound to histones. The second part focuses on the formation of salt bridges between arginine residues in histones and phosphate groups on the DNA backbone. The arginine residues are ob- served to apply a tunable mechanical load to the backbone, enabling precision-controlled activation of DNA deformations.

  8. Visualizing Long Noncoding RNAs on Chromatin

    PubMed Central

    Hinten, Michael; Maclary, Emily; Gayen, Srimonta; Harris, Clair; Kalantry, Sundeep

    2016-01-01

    Fluorescence in situ hybridization (FISH) enables the detection of specific nucleic acid sequences within single cells. For example, RNA FISH provides information on both the expression level and localization of RNA transcripts and, when combined with detection of associated proteins and chromatin modifications, can lend essential insights into long noncoding RNA (lncRNA) function. Epigenetic effects have been postulated for many lncRNAs, but shown for only a few. Advances in in situ techniques and microscopy, however, now allow for visualization of lncRNAs that are expressed at very low levels or are not very stable. FISH-based detections of RNA and DNA coupled with immunological staining of proteins/histone modifications offer the possibility to connect lncRNAs to epigenetic effects. Here, we describe an integrated set of protocols to detect, individually or in combination, specific RNAs, DNAs, proteins, and histone modifications in single cells at a high level of sensitivity using conventional fluorescence microscopy. PMID:26721489

  9. De novo deciphering three-dimensional chromatin interaction and topological domains by wavelet transformation of epigenetic profiles.

    PubMed

    Chen, Yong; Wang, Yunfei; Xuan, Zhenyu; Chen, Min; Zhang, Michael Q

    2016-06-20

    Defining chromatin interaction frequencies and topological domains is a great challenge for the annotations of genome structures. Although the chromosome conformation capture (3C) and its derivative methods have been developed for exploring the global interactome, they are limited by high experimental complexity and costs. Here we describe a novel computational method, called CITD, for de novo prediction of the chromatin interaction map by integrating histone modification data. We used the public epigenomic data from human fibroblast IMR90 cell and embryonic stem cell (H1) to develop and test CITD, which can not only successfully reconstruct the chromatin interaction frequencies discovered by the Hi-C technology, but also provide additional novel details of chromosomal organizations. We predicted the chromatin interaction frequencies, topological domains and their states (e.g. active or repressive) for 98 additional cell types from Roadmap Epigenomics and ENCODE projects. A total of 131 protein-coding genes located near 78 preserved boundaries among 100 cell types are found to be significantly enriched in functional categories of the nucleosome organization and chromatin assembly. CITD and its predicted results can be used for complementing the topological domains derived from limited Hi-C data and facilitating the understanding of spatial principles underlying the chromosomal organization. PMID:27060148

  10. De novo deciphering three-dimensional chromatin interaction and topological domains by wavelet transformation of epigenetic profiles

    PubMed Central

    Chen, Yong; Wang, Yunfei; Xuan, Zhenyu; Chen, Min; Zhang, Michael Q.

    2016-01-01

    Defining chromatin interaction frequencies and topological domains is a great challenge for the annotations of genome structures. Although the chromosome conformation capture (3C) and its derivative methods have been developed for exploring the global interactome, they are limited by high experimental complexity and costs. Here we describe a novel computational method, called CITD, for de novo prediction of the chromatin interaction map by integrating histone modification data. We used the public epigenomic data from human fibroblast IMR90 cell and embryonic stem cell (H1) to develop and test CITD, which can not only successfully reconstruct the chromatin interaction frequencies discovered by the Hi-C technology, but also provide additional novel details of chromosomal organizations. We predicted the chromatin interaction frequencies, topological domains and their states (e.g. active or repressive) for 98 additional cell types from Roadmap Epigenomics and ENCODE projects. A total of 131 protein-coding genes located near 78 preserved boundaries among 100 cell types are found to be significantly enriched in functional categories of the nucleosome organization and chromatin assembly. CITD and its predicted results can be used for complementing the topological domains derived from limited Hi-C data and facilitating the understanding of spatial principles underlying the chromosomal organization. PMID:27060148

  11. BioTAP-XL - crosslinking/tandem affinity purification to study DNA targets, RNA and protein components of chromatin associated complexes

    PubMed Central

    Alekseyenko, Artyom A.; McElroy, Kyle A.; Kang, Hyuckjoon; Zee, Barry M.; Kharchenko, Peter V.; Kuroda, Mitzi I.

    2015-01-01

    In order to understand how chromatin complexes function in the nucleus, it is important to obtain a comprehensive picture of their protein, DNA, and RNA components and their mutual interactions. Here we present a chromatin cross-linking approach (BioTAP-XL) which utilizes mass-spectrometry to identify protein complex components, together with high-throughput sequencing to identify RNA components and DNA binding sites. We describe full protocols for Drosophila cells and for human cells in culture, along with an additional protocol for Drosophila embryos as the source material. A key element of our approach in all cases is the generation of control data from input chromatin samples. PMID:25559106

  12. Chromatin changes predict recurrence after radical prostatectomy

    PubMed Central

    Hveem, Tarjei S; Kleppe, Andreas; Vlatkovic, Ljiljana; Ersvær, Elin; Wæhre, Håkon; Nielsen, Birgitte; Kjær, Marte Avranden; Pradhan, Manohar; Syvertsen, Rolf Anders; Nesheim, John Arne; Liestøl, Knut; Albregtsen, Fritz; Danielsen, Håvard E

    2016-01-01

    Background: Pathological evaluations give the best prognostic markers for prostate cancer patients after radical prostatectomy, but the observer variance is substantial. These risk assessments should be supported and supplemented by objective methods for identifying patients at increased risk of recurrence. Markers of epigenetic aberrations have shown promising results in several cancer types and can be assessed by automatic analysis of chromatin organisation in tumour cell nuclei. Methods: A consecutive series of 317 prostate cancer patients treated with radical prostatectomy at a national hospital between 1987 and 2005 were followed for a median of 10 years (interquartile range, 7–14). On average three tumour block samples from each patient were included to account for tumour heterogeneity. We developed a novel marker, termed Nucleotyping, based on automatic assessment of disordered chromatin organisation, and validated its ability to predict recurrence after radical prostatectomy. Results: Nucleotyping predicted recurrence with a hazard ratio (HR) of 3.3 (95% confidence interval (CI), 2.1–5.1). With adjustment for clinical and pathological characteristics, the HR was 2.5 (95% CI, 1.5–4.1). An updated stratification into three risk groups significantly improved the concordance with patient outcome compared with a state-of-the-art risk-stratification tool (P<0.001). The prognostic impact was most evident for the patients who were high-risk by clinical and pathological characteristics and for patients with Gleason score 7. Conclusion: A novel assessment of epigenetic aberrations was capable of improving risk stratification after radical prostatectomy. PMID:27124335

  13. Engineering chromatin states: chemical and synthetic biology approaches to investigate histone modification function.

    PubMed

    Pick, Horst; Kilic, Sinan; Fierz, Beat

    2014-08-01

    Patterns of histone post-translational modifications (PTMs) and DNA modifications establish a landscape of chromatin states with regulatory impact on gene expression, cell differentiation and development. These diverse modifications are read out by effector protein complexes, which ultimately determine their functional outcome by modulating the activity state of underlying genes. From genome-wide studies employing high-throughput ChIP-Seq methods as well as proteomic mass spectrometry studies, a large number of PTMs are known and their coexistence patterns and associations with genomic regions have been mapped in a large number of different cell types. Conversely, the molecular interplay between chromatin effector proteins and modified chromatin regions as well as their resulting biological output is less well understood on a molecular level. Within the last decade a host of chemical approaches has been developed with the goal to produce synthetic chromatin with a defined arrangement of PTMs. These methods now permit systematic functional studies of individual histone and DNA modifications, and additionally provide a discovery platform to identify further interacting nuclear proteins. Complementary chemical- and synthetic-biology methods have emerged to directly observe and modulate the modification landscape in living cells and to readily probe the effect of altered PTM patterns on biological processes. Herein, we review current methodologies allowing chemical and synthetic biological engineering of distinct chromatin states in vitro and in vivo with the aim of obtaining a molecular understanding of histone and DNA modification function. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.

  14. Anti-chromatin antibodies in systemic lupus erythematosus: a useful marker for lupus nephropathy

    PubMed Central

    Cervera, R; Vinas, O; Ramos-Casals, M; Font, J; Garcia-Carrasco, M; Siso, A; Ramirez, F; Machuca, Y; Vives, J; Ingelmo, M; Burlingame, R

    2003-01-01

    Background: Anti-chromatin antibodies have recently been described in patients with systemic lupus erythematosus (SLE) and it has been suggested that their presence is associated with lupus nephritis. Objective: To assess the prevalence and clinical associations of these antibodies in SLE. Methods: The presence of anti-chromatin antibodies in 100 patients with SLE was investigated by an enzyme linked immunosorbent assay (ELISA). To determine the specificity of these antibodies, 100 patients with primary Sjögren's syndrome, 30 with primary antiphospholipid syndrome (APS), 10 with systemic sclerosis, and 100 normal controls were also tested. Results: Positive levels were detected in 69/100 (69%) patients with SLE. In contrast, they were found in only 8/100 (8%) of those with primary Sjögren's syndrome, in 1/10 (10%) with systemic sclerosis, in 2/30 (7%) with primary APS, and in none of the 100 healthy controls. Patients with anti-chromatin antibodies had a twofold higher prevalence of lupus nephropathy than those without these antibodies (58% v 29%, p<0.01). A significant correlation was found between the levels of anti-chromatin antibodies and disease activity score as measured by the European Consensus Lupus Activity Measurement (ECLAM; p=0.011). Conclusions: The measurement of anti-chromatin antibodies appears to be a useful addition to the laboratory tests that can help in the diagnosis and treatment of SLE. These antibodies are both sensitive and specific for SLE, and are a useful marker for an increased risk of lupus nephritis. PMID:12695155

  15. Diazinon alters sperm chromatin structure in mice by phosphorylating nuclear protamines

    SciTech Connect

    Pina-Guzman, B.; Solis-Heredia, M.J.; Quintanilla-Vega, B. . E-mail: mquintan@mail.cinvestav.mx

    2005-01-15

    Organophosphorus (OP) pesticides, widely used in agriculture and pest control, are associated with male reproductive effects, including sperm chromatin alterations, but the mechanisms underlying these effects are unknown. The main toxic action of OP is related to phosphorylation of proteins. Chemical alterations in sperm nuclear proteins (protamines), which pack DNA during the last steps of spermatogenesis, contribute to male reproductive toxicity. Therefore, in the present study, we tested the ability of diazinon (DZN), an OP compound, to alter sperm chromatin by phosphorylating nuclear protamines. Mice were injected with a single dose of DZN (8.12 mg/kg, i.p.), and killed 8 and 15 days after treatment. Quality of sperm from epididymis and vas deferens was evaluated through standard methods and chromatin condensation by flow cytometry (DNA Fragmented Index parameters: DFI and DFI%) and fluorescence microscopy using chromomycin-A{sub 3} (CMA{sub 3}). Increases in DFI (15%), DFI% (4.5-fold), and CMA{sub 3} (2-fold) were observed only at 8 days post-treatment, indicating an alteration in sperm chromatin condensation and DNA damage during late spermatid differentiation. In addition, an increase of phosphorous content (approximately 50%) in protamines, especially in the phosphoserine content (approximately 73%), was found at 8 days post-treatment. Sperm viability, motility, and morphology showed significant alterations at this time. These data strongly suggest that spermatozoa exposed during the late steps of maturation were the targets of DZN exposure. The correlation observed between the phosphorous content in nuclear protamines with DFI%, DFI, and CMA{sub 3} provides evidence that phosphorylation of nuclear protamines is involved in the OP effects on sperm chromatin.

  16. Regulatory motifs on ISWI chromatin remodelers: molecular mechanisms and kinetic proofreading

    NASA Astrophysics Data System (ADS)

    Brysbaert, Guillaume; Lensink, Marc F.; Blossey, Ralf

    2015-02-01

    Recently, kinetic proofreading scenarios have been proposed for the regulation of chromatin remodeling, first on purely theoretical grounds (Blossey and Schiessel 2008 HFSP J. 2 167-70) and deduced from experiments on the ISWI/ACF system (Narlikar 2010 Curr. Opin. Chem. Biol. 14 660). In the kinetic proofreading scenario of chromatin remodeling, the combination of the recognition of a histone tail state and ATP-hydrolysis in the remodeler motor act together to select (i.e. proofread) a nucleosomal substrate. ISWI remodelers have recently been shown to have an additional level of regulation as they contain auto-inhibitory motifs which need to be inactivated through an interaction with the nucleosome. In this paper we show that the auto-regulatory effect enhances substrate recognition in kinetic proofreading. We further report some suggestive additional insights into the molecular mechanism underlying ISWI-autoregulation.

  17. Regulatory motifs on ISWI chromatin remodelers: molecular mechanisms and kinetic proofreading.

    PubMed

    Brysbaert, Guillaume; Lensink, Marc F; Blossey, Ralf

    2015-02-18

    Recently, kinetic proofreading scenarios have been proposed for the regulation of chromatin remodeling, first on purely theoretical grounds (Blossey and Schiessel 2008 HFSP J. 2 167-70) and deduced from experiments on the ISWI/ACF system (Narlikar 2010 Curr. Opin. Chem. Biol. 14 660). In the kinetic proofreading scenario of chromatin remodeling, the combination of the recognition of a histone tail state and ATP-hydrolysis in the remodeler motor act together to select (i.e. proofread) a nucleosomal substrate. ISWI remodelers have recently been shown to have an additional level of regulation as they contain auto-inhibitory motifs which need to be inactivated through an interaction with the nucleosome. In this paper we show that the auto-regulatory effect enhances substrate recognition in kinetic proofreading. We further report some suggestive additional insights into the molecular mechanism underlying ISWI-autoregulation. PMID:25563573

  18. Three-Dimensional, Live-Cell Imaging of Chromatin Dynamics in Plant Nuclei Using Chromatin Tagging Systems.

    PubMed

    Hirakawa, Takeshi; Matsunaga, Sachihiro

    2016-01-01

    In plants, chromatin dynamics spatiotemporally change in response to various environmental stimuli. However, little is known about chromatin dynamics in the nuclei of plants. Here, we introduce a three-dimensional, live-cell imaging method that can monitor chromatin dynamics in nuclei via a chromatin tagging system that can visualize specific genomic loci in living plant cells. The chromatin tagging system is based on a bacterial operator/repressor system in which the repressor is fused to fluorescent proteins. A recent refinement of promoters for the system solved the problem of gene silencing and abnormal pairing frequencies between operators. Using this system, we can detect the spatiotemporal dynamics of two homologous loci as two fluorescent signals within a nucleus and monitor the distance between homologous loci. These live-cell imaging methods will provide new insights into genome organization, development processes, and subnuclear responses to environmental stimuli in plants. PMID:27557696

  19. Fractal Characterization of Chromatin Decompaction in Live Cells.

    PubMed

    Yi, Ji; Stypula-Cyrus, Yolanda; Blaha, Catherine S; Roy, Hemant K; Backman, Vadim

    2015-12-01

    Chromatin organization has a fundamental impact on the whole spectrum of genomic functions. Quantitative characterization of the chromatin structure, particularly at submicron length scales where chromatin fractal globules are formed, is critical to understanding this structure-function relationship. Such analysis is currently challenging due to the diffraction-limited resolution of conventional light microscopy. We herein present an optical approach termed inverse spectroscopic optical coherence tomography to characterize the mass density fractality of chromatin, and we apply the technique to observe chromatin decompaction in live cells. The technique makes it possible for the first time, to our knowledge, to sense intracellular morphology with length-scale sensitivity from ∼30 to 450 nm, thus primarily probing the higher-order chromatin structure, without resolving the actual structures. We used chromatin decompaction due to inhibition of histone deacytelases and measured the subsequent changes in the fractal dimension of the intracellular structure. The results were confirmed by transmission electron microscopy and confocal fluorescence microscopy.

  20. Effective Identification of Akt Interacting Proteins by Two-Step Chemical Crosslinking, Co-Immunoprecipitation and Mass Spectrometry

    PubMed Central

    Huang, Bill X.; Kim, Hee-Yong

    2013-01-01

    Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis. An intrinsic problem of the method is loss of interacting proteins during procedures to remove non-specific proteins. Moreover, antibody contamination often interferes with the detection of less abundant proteins. Here, we developed a novel two-step chemical crosslinking strategy to overcome these problems which resulted in a dramatic improvement in identifying Akt interacting partners. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, dithiobis[succinimidylpropionate], a cleavable crosslinker, was introduced to produce stable complexes between Akt and binding partners prior to the SDS-PAGE and nanoLC-MS/MS analysis. This approach enabled identification of ten Akt partners from cell lysates containing as low as 1.5 mg proteins, including two new potential Akt interacting partners. None of these but one protein was detectable without crosslinking procedures. The present method provides a sensitive and effective tool to probe Akt-interacting proteins. This strategy should also prove useful for other protein interactions, particularly those involving less abundant or weakly associating partners. PMID:23613850

  1. Specific detection of peste des petits ruminants virus antibodies in sheep and goat sera by the luciferase immunoprecipitation system.

    PubMed

    Berguido, Francisco J; Bodjo, Sanne Charles; Loitsch, Angelika; Diallo, Adama

    2016-01-01

    Peste des petits ruminants (PPR) is a contagious and often fatal transboundary animal disease affecting mostly sheep, goats and wild small ruminants. This disease is endemic in most of Africa, the Middle, Near East, and large parts of Asia. The causal agent is peste des petits ruminants virus (PPRV), which belongs to the genus Morbillivirus in the family Paramyxoviridae. This genus also includes measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). All are closely related viruses with serological cross reactivity. In this study, we have developed a Luciferase Immunoprecipitation System (LIPS) for the rapid detection of antibodies against PPRV in serum samples and for specific differentiation from antibodies against RPV. PPR and rinderpest (RP) serum samples were assayed by PPR-LIPS and two commercially available PPR cELISA tests. The PPR-LIPS showed high sensitivity and specificity for the samples tested and showed no cross reactivity with RPV unlike the commercial PPR cELISA tests which did cross react with RPV. Based on the results shown in this study, PPR-LIPS is presented as a good candidate for the specific serosurveillance of PPR.

  2. Specific detection of peste des petits ruminants virus antibodies in sheep and goat sera by the luciferase immunoprecipitation system.

    PubMed

    Berguido, Francisco J; Bodjo, Sanne Charles; Loitsch, Angelika; Diallo, Adama

    2016-01-01

    Peste des petits ruminants (PPR) is a contagious and often fatal transboundary animal disease affecting mostly sheep, goats and wild small ruminants. This disease is endemic in most of Africa, the Middle, Near East, and large parts of Asia. The causal agent is peste des petits ruminants virus (PPRV), which belongs to the genus Morbillivirus in the family Paramyxoviridae. This genus also includes measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). All are closely related viruses with serological cross reactivity. In this study, we have developed a Luciferase Immunoprecipitation System (LIPS) for the rapid detection of antibodies against PPRV in serum samples and for specific differentiation from antibodies against RPV. PPR and rinderpest (RP) serum samples were assayed by PPR-LIPS and two commercially available PPR cELISA tests. The PPR-LIPS showed high sensitivity and specificity for the samples tested and showed no cross reactivity with RPV unlike the commercial PPR cELISA tests which did cross react with RPV. Based on the results shown in this study, PPR-LIPS is presented as a good candidate for the specific serosurveillance of PPR. PMID:26506137

  3. Identification of cross-reactive promastigote cell surface antigens of some leishmanial stocks by 125I labeling and immunoprecipitation.

    PubMed Central

    Gardiner, P R; Jaffe, C L; Dwyer, D M

    1984-01-01

    Externally oriented surface membrane constituents of promastigotes from several Leishmania species were radiolabeled with 125I. Autoradiographs of cell surface-labeled and sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the stocks revealed distinctive patterns of bands in the molecular weight range of 6,000 to 240,000. Immunoprecipitation of detergent extracts of the labeled promastigote stocks with anti-Leishmania donovani membrane serum demonstrated that each of the stocks contained some antigenically cross-reactive determinants. The electrophoretic patterns of these determinants serve both to distinguish the parasite stocks (by unique, species-specific patterns) and to indicate antigenic similarities in stocks thought to be different by other biochemical criteria. At least 12 cross-reactive cell surface antigens in two New World leishmanias are recognized by polyvalent anti-L. donovani serum, suggesting that these common leishmanial antigens may account for the documented serological cross-reactivities among various Leishmania species. In all stocks tested, an iodinated protein was identified which had a relative molecular weight of 65,000 under reducing conditions but which demonstrated an increase in relative mobility in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Distinctive patterns of the antigens common to the several stocks were also demonstrated with the use of monoclonal antibodies. Images PMID:6363295

  4. Spatially confined folding of chromatin in the interphase nucleus

    PubMed Central

    Mateos-Langerak, Julio; Bohn, Manfred; de Leeuw, Wim; Giromus, Osdilly; Manders, Erik M. M.; Verschure, Pernette J.; Indemans, Mireille H. G.; Gierman, Hinco J.; Heermann, Dieter W.; van Driel, Roel; Goetze, Sandra

    2009-01-01

    Genome function in higher eukaryotes involves major changes in the spatial organization of the chromatin fiber. Nevertheless, our understanding of chromatin folding is remarkably limited. Polymer models have been used to describe chromatin folding. However, none of the proposed models gives a satisfactory explanation of experimental data. In particularly, they ignore that each chromosome occupies a confined space, i.e., the chromosome territory. Here, we present a polymer model that is able to describe key properties of chromatin over length scales ranging from 0.5 to 75 Mb. This random loop (RL) model assumes a self-avoiding random walk folding of the polymer backbone and defines a probability P for 2 monomers to interact, creating loops of a broad size range. Model predictions are compared with systematic measurements of chromatin folding of the q-arms of chromosomes 1 and 11. The RL model can explain our observed data and suggests that on the tens-of-megabases length scale P is small, i.e., 10–30 loops per 100 Mb. This is sufficient to enforce folding inside the confined space of a chromosome territory. On the 0.5- to 3-Mb length scale chromatin compaction differs in different subchromosomal domains. This aspect of chromatin structure is incorporated in the RL model by introducing heterogeneity along the fiber contour length due to different local looping probabilities. The RL model creates a quantitative and predictive framework for the identification of nuclear components that are responsible for chromatin–chromatin interactions and determine the 3-dimensional organization of the chromatin fiber. PMID:19234129

  5. Chromatin binding and polymerization of the endogenous Xenopus egg lamins: the opposing effects of glycogen and ATP.

    PubMed

    Lourim, D; Krohne, G

    1998-12-18

    We have previously identified and quantitated three B-type lamin isoforms present in the nuclei of mature Xenopus laevis oocytes, and in cell-free egg extracts. As Xenopus egg extracts are frequently used to analyze nuclear envelope assembly and lamina functions, we felt it was imperative that the polymerization and chromatin-binding properties of the endogenous B-type egg lamins be investigated. While we have demonstrated that soluble B-type lamins bind to chromatin, we have also observed that the polymerization of egg lamins does not require membranes or chromatin. Lamin assembly is enhanced by the addition of glycogen/glucose, or by the depletion of ATP from the extract. Moreover, the polymerization of egg cytosol lamins and their binding to demembranated sperm or chromatin assembled from naked lambda-DNA is inhibited by an ATP regeneration system. These ATP-dependent inhibitory activities can be overcome by the coaddition of glycogen to egg cytosol. We have observed that glycogen does not alter ATP levels during cytosol incubation, but rather, as glycogen-enhanced lamin polymerization is inhibited by okadaic acid, we conclude that glycogen activates protein phosphatases. Because protein phosphatase 1 (PP1) is the only phosphatase known to be specifically regulated by glycogen our data indicate that PP1 is involved in lamin polymerization. Our results show that ATP and glycogen effect lamin polymerization and chromatin binding by separate and opposing mechanisms. PMID:9819358

  6. Gain-of-function mutations in chromatin regulators as an oncogenic mechanism and opportunity for drug intervention

    PubMed Central

    Shen, Chen

    2015-01-01

    Purpose of review Somatic gain-of-function mutations that drive cancer pathogenesis are well-established opportunities for therapeutic intervention, as demonstrated by the clinical efficacy of kinase inhibitors in kinase-mutant malignancies. Here, we discuss recently discovered gain-of-function mutations in chromatin regulatory machineries that promote the pathogenesis of cancer. The current understanding of underlying molecular mechanisms and the therapeutic potential for direct chemical inhibition will be reviewed. Recent findings Point mutations that increase the catalytic activity of EZH2 and NSD2 histone methyltransferases are found in distinct subsets of B cell neoplasms, which promote cell transformation by elevating the global level of H3K27 tri-methylation or H3K36 di-methylation, respectively. In addition, mutations in histone H3 have been identified in certain pediatric cancers which cause reprogramming of H3K27 and H3K36 methylation by dominantly interfering with histone methyltransferase activity. Finally, chromosomal translocations involving chromatin regulator genes can lead to the formation of fusion oncoproteins that directly modify chromatin as their mechanism of action. Summary While relatively rare in aggregate, gain-of-function mutations in chromatin regulators represent compelling therapeutic targets in genetically-defined subsets of cancer patients. However, a broader clinical impact for epigenetic therapies in oncology will require an increased understanding of how non-mutated chromatin regulators function as cancer-specific dependencies. PMID:25402979

  7. Nanoscale squeezing in elastomeric nanochannels for single chromatin linearization

    PubMed Central

    Matsuoka, Toshiki; Kim, Byoung Choul; Huang, Jiexi; Douville, Nicholas Joseph; Thouless, M.D.; Takayama, Shuichi

    2012-01-01

    This paper describes a novel nanofluidic phenomenon where untethered DNA and chromatin are linearized by rapidly narrowing an elastomeric nanochannel filled with solutions of the biopolymers. This nanoscale squeezing procedure generates hydrodynamic flows while also confining the biopolymers into smaller and smaller volumes. The unique features of this technique enable full linearization then trapping of biopolymers such as DNA. The versatility of the method is also demonstrated by analysis of chromatin stretchability and mapping of histone states using single strands of chromatin. PMID:23186544

  8. Methods for the analysis of protein-chromatin interactions.

    PubMed

    Brickwood, Sarah J; Myers, Fiona A; Chandler, Simon P

    2002-01-01

    The analysis of protein interactions with chromatin is vital for the understanding of DNA sequence recognition in vivo. Chromatin binding requires the interaction of proteins with DNA lying on the macromolecular protein surface of nucleosomes, a situation that can alter factor binding characteristics substantially when compared with naked DNA. It is therefore important to study these protein-DNA interactions in the context of a chromatin substrate, the more physiologically relevant binding situation. In this article we review techniques used in the investigation of protein interactions with defined nucleosomal templates. PMID:11876294

  9. Stress-induced structural changes in plant chromatin.

    PubMed

    Probst, Aline V; Mittelsten Scheid, Ortrun

    2015-10-01

    Stress defense in plants is elaborated at the level of protection and adaptation. Dynamic changes in sophisticated chromatin substructures and concomitant transcriptional changes play an important role in response to stress, as illustrated by the transient rearrangement of compact heterochromatin structures or the modulation of chromatin composition and modification upon stress exposure. To connect cytological, developmental, and molecular data around stress and chromatin is currently an interesting, multifaceted, and sometimes controversial field of research. This review highlights some of the most recent findings on nuclear reorganization, histone variants, histone chaperones, DNA- and histone modifications, and somatic and meiotic heritability in connection with stress.

  10. Chromatin regulation at the frontier of synthetic biology

    PubMed Central

    Keung, Albert J.; Joung, J. Keith; Khalil, Ahmad S.; Collins, James J.

    2016-01-01

    As synthetic biology approaches are extended to diverse applications throughout medicine, biotechnology and basic biological research, there is an increasing need to engineer yeast, plant and mammalian cells. Eukaryotic genomes are regulated by the diverse biochemical and biophysical states of chromatin, which brings distinct challenges, as well as opportunities, over applications in bacteria. Recent synthetic approaches, including `epigenome editing', have allowed the direct and functional dissection of many aspects of physiological chromatin regulation. These studies lay the foundation for biomedical and biotechnological engineering applications that could take advantage of the unique combinatorial and spatiotemporal layers of chromatin regulation to create synthetic systems of unprecedented sophistication. PMID:25668787

  11. Clinical and public health research using methylated DNA Immunoprecipitation (MeDIP): A comparison of commercially available kits to examine differential DNA methylation across the genome

    PubMed Central

    Brebi-Mieville, Priscilla; Ili-Gangas, Carmen; Leal-Rojas, Pamela; Noordhuis, Maartje; Soudry, Ethan; Perez, Jimena; Roa, Juan Carlos; Sidransky, David; Guerrero-Preston, Rafael

    2012-01-01

    The methylated DNA immunoprecipitation method (MeDIP) is a genome-wide, high-resolution approach that detects DNA methylation with oligonucleotide tiling arrays or high throughput sequencing platforms. A simplified high-throughput MeDIP assay will enable translational research studies in clinics and populations, which will greatly enhance our understanding of the human methylome. We compared three commercial kits, MagMeDIP Kit TM (Diagenode), Methylated-DNA IP Kit (Zymo Research) and Methylamp™ Methylated DNA Capture Kit (Epigentek), in order to identify which one has better reliability and sensitivity for genomic DNA enrichment. Each kit was used to enrich two samples, one from fresh tissue and one from a cell line, with two different DNA amounts. The enrichment efficiency of each kit was evaluated by agarose gel band intensity after Nco I digestion and by reaction yield of methylated DNA. A successful enrichment is expected to have a 1:4 to 10:1 conversion ratio and a yield of 80% or higher. We also evaluated the hybridization efficiency to genome-wide methylation arrays in a separate cohort of tissue samples. We observed that the MagMeDIP kit had the highest yield for the two DNA amounts and for both the tissue and cell line samples, as well as for the positive control. In addition, the DNA was successfully enriched from a 1:4 to 10:1 ratio. Therefore, the MagMeDIP kit is a useful research tool that will enable clinical and public health genome-wide DNA methylation studies. PMID:22207357

  12. Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

    PubMed

    Breitkopf, Susanne B; Yuan, Min; Pihan, German A; Asara, John M

    2012-10-01

    Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

  13. Chromatin architecture near a potential 3' end of the igh locus involves modular regulation of histone modifications during B-Cell development and in vivo occupancy at CTCF sites.

    PubMed

    Garrett, Francine E; Emelyanov, Alexander V; Sepulveda, Manuel A; Flanagan, Patrick; Volpi, Sabrina; Li, Fubin; Loukinov, Dmitry; Eckhardt, Laurel A; Lobanenkov, Victor V; Birshtein, Barbara K

    2005-02-01

    The murine Igh locus has a 3' regulatory region (3' RR) containing four enhancers (hs3A, hs1,2, hs3B, and hs4) at DNase I-hypersensitive sites. The 3' RR exerts long-range effects on class switch recombination (CSR) to several isotypes through its control of germ line transcription. By measuring levels of acetylated histones H3 and H4 and of dimethylated H3 (K4) with chromatin immunoprecipitation assays, we found that early in B-cell development, chromatin encompassing the enhancers of the 3' RR began to attain stepwise modifications typical of an open conformation. The hs4 enhancer was associated with active chromatin initially in pro- and pre-B cells and then together with hs3A, hs1,2, and hs3B in B and plasma cells. Histone modifications were similar in resting splenic B cells and in splenic B cells induced by lipopolysaccharide to undergo CSR. From the pro-B-cell stage onward, the approximately 11-kb region immediately downstream of hs4 displayed H3 and H4 modifications indicative of open chromatin. This region contained newly identified DNase I-hypersensitive sites and several CTCF target sites, some of which were occupied in vivo in a developmentally regulated manner. The open chromatin environment of the extended 3' RR in mature B cells was flanked by regions associated with dimethylated K9 of histone H3. Together, these data suggest that 3' RR elements are located within a specific chromatin subdomain that contains CTCF binding sites and developmentally regulated modules.

  14. Nucleosomal regulation of chromatin composition and nuclear assembly revealed by histone depletion

    PubMed Central

    Zierhut, Christian; Jenness, Christopher; Kimura, Hiroshi; Funabiki, Hironori

    2014-01-01

    Nucleosomes are the fundamental unit of chromatin, but the analysis of transcription-independent nucleosome functions has been thwarted by the confounding gene expression changes resultant of histone manipulation. Here we solve this dilemma by developing Xenopus laevis egg extracts deficient for nucleosome formation, and analyze the proteomic landscape and behavior of nucleosomal chromatin and nucleosome-free DNA. We show that while nucleosome-free DNA can recruit nuclear envelope membranes, nucleosomes are required for spindle assembly, lamina and nuclear pore complex (NPC) formation. In addition to RCC1, we reveal that ELYS, the initiator of NPC formation, fails to associate with naked DNA, but directly binds histones H2A–H2B and nucleosomes. Tethering ELYS and RCC1 to DNA bypassed the requirement for nucleosomes in NPC formation in a synergistic manner. Thus, the minimal essential function of nucleosomes in NPC formation is to recruit RCC1 and ELYS. PMID:24952593

  15. Valproate and Amitriptyline Exert Common and Divergent Influences on Global and Gene Promoter-Specific Chromatin Modifications in Rat Primary Astrocytes

    PubMed Central

    Perisic, Tatjana; Zimmermann, Nicole; Kirmeier, Thomas; Asmus, Maria; Tuorto, Francesca; Uhr, Manfred; Holsboer, Florian; Rein, Theo; Zschocke, Jürgen

    2010-01-01

    Aberrant biochemical processes in the brain frequently go along with subtle shifts of the cellular epigenetic profile that might support the pathogenic progression of psychiatric disorders. Although recent reports have implied the ability of certain antidepressants and mood stabilizers to modulate epigenetic parameters, studies comparing the actions of these compounds under the same conditions are lacking. In this study, we screened amitriptyline (AMI), venlafaxine, citalopram, as well as valproic acid (VPA), carbamazepine, and lamotrigine for their potential actions on global and local epigenetic modifications in rat primary astrocytes. Among all drugs, VPA exposure evoked the strongest global chromatin modifications, including histone H3/H4 hyperacetylation, 2MeH3K9 hypomethylation, and DNA demethylation, as determined by western blot and luminometric methylation analysis, respectively. CpG demethylation occurred independently of DNA methyltransferase (DNMT) suppression. Strikingly, AMI also induced slight cytosine demethylation, paralleled by the reduction in DNMT enzymatic activity, without affecting the global histone acetylation status. Locally, VPA-induced chromatin modifications were reflected at the glutamate transporter (GLT-1) promoter as shown by bisulfite sequencing and acetylated histone H4 chromatin immunoprecipitation analysis. Distinct CpG sites in the distal part of the GLT-1 promoter were demethylated and enriched in acetylated histone H4 in response to VPA. For the first time, we could show that these changes were associated with an enhanced transcription of this astrocyte-specific gene. In contrast, AMI failed to stimulate GLT-1 transcription and to alter promoter methylation levels. In conclusion, VPA and AMI globally exerted chromatin-modulating activities using different mechanisms that divergently precipitated at an astroglial gene locus. PMID:19924110

  16. A tale of tails: How histone tails mediate chromatin compaction in different salt and linker histone environments

    PubMed Central

    Arya, Gaurav; Schlick, Tamar

    2009-01-01

    To elucidate the role of the histone tails in chromatin compaction and in higher-order folding of chromatin under physiological conditions, we extend a mesoscale model of chromatin [Arya, Zhang, and Schlick, Biophys. J. 91, 133 (2006); Arya and Schlick, Proc. Natl. Acad. Sci. USA 103, 16236 (2006)] to account for divalent cations (Mg2+) and linker histones. Configurations of 24-nucleosome oligonucleosomes in different salt environments and in the presence and absence of linker histones are sampled by a mixture of local and global Monte Carlo methods. Analyses of the resulting ensembles reveals a dynamic synergism between the histone tails, linker histones, and physiological ions in forming compact higher-order structures of chromatin. In the presence of monovalent salt alone, oligonucleosomes remain relatively unfolded and the histone tails do not mediate many internucleosomal interactions. Upon the addition of linker histones and divalent cations, the oligonucleosomes undergo a significant compaction triggered by: a dramatic increase in the internucleosomal interactions mediated by the histone tails; formation of a rigid linker DNA “stem” around the linker histones' C-terminal domains; and reducion in the electrostatic repulsion between linker DNAs via sharp bending in some linker DNAs caused by the divalent cations. Among all histone tails, the H4 tails mediate the most internucleosomal interactions, consistent with experimental observations, followed by the H3, H2A, and H2B tails in decreasing order. Apart from mediating internucleosomal interactions, the H3 tails also contribute to chromatin compaction by attaching to the entering and exiting linker DNA to screen electrotatic repulsion among the linker DNAs. This tendency of the H3 tails to attach to linker DNA, however, decreases significantly upon the addition of linker histones due to competition effects. The H2A and H2B tails do not mediate significant internucleosomal interactions but are important for

  17. Ancestral Chromatin Configuration Constrains Chromatin Evolution on Differentiating Sex Chromosomes in Drosophila

    PubMed Central

    Zhou, Qi; Bachtrog, Doris

    2015-01-01

    Sex chromosomes evolve distinctive types of chromatin from a pair of ancestral autosomes that are usually euchromatic. In Drosophila, the dosage-compensated X becomes enriched for hyperactive chromatin in males (mediated by H4K16ac), while the Y chromosome acquires silencing heterochromatin (enriched for H3K9me2/3). Drosophila autosomes are typically mostly euchromatic but the small dot chromosome has evolved a heterochromatin-like milieu (enriched for H3K9me2/3) that permits the normal expression of dot-linked genes, but which is different from typical pericentric heterochromatin. In Drosophila busckii, the dot chromosomes have fused to the ancestral sex chromosomes, creating a pair of ‘neo-sex’ chromosomes. Here we collect genomic, transcriptomic and epigenomic data from D. busckii, to investigate the evolutionary trajectory of sex chromosomes from a largely heterochromatic ancestor. We show that the neo-sex chromosomes formed <1 million years ago, but nearly 60% of neo-Y linked genes have already become non-functional. Expression levels are generally lower for the neo-Y alleles relative to their neo-X homologs, and the silencing heterochromatin mark H3K9me2, but not H3K9me3, is significantly enriched on silenced neo-Y genes. Despite rampant neo-Y degeneration, we find that the neo-X is deficient for the canonical histone modification mark of dosage compensation (H4K16ac), relative to autosomes or the compensated ancestral X chromosome, possibly reflecting constraints imposed on evolving hyperactive chromatin in an originally heterochromatic environment. Yet, neo-X genes are transcriptionally more active in males, relative to females, suggesting the evolution of incipient dosage compensation on the neo-X. Our data show that Y degeneration proceeds quickly after sex chromosomes become established through genomic and epigenetic changes, and are consistent with the idea that the evolution of sex-linked chromatin is influenced by its ancestral configuration. PMID

  18. Ancestral Chromatin Configuration Constrains Chromatin Evolution on Differentiating Sex Chromosomes in Drosophila.

    PubMed

    Zhou, Qi; Bachtrog, Doris

    2015-06-01

    Sex chromosomes evolve distinctive types of chromatin from a pair of ancestral autosomes that are usually euchromatic. In Drosophila, the dosage-compensated X becomes enriched for hyperactive chromatin in males (mediated by H4K16ac), while the Y chromosome acquires silencing heterochromatin (enriched for H3K9me2/3). Drosophila autosomes are typically mostly euchromatic but the small dot chromosome has evolved a heterochromatin-like milieu (enriched for H3K9me2/3) that permits the normal expression of dot-linked genes, but which is different from typical pericentric heterochromatin. In Drosophila busckii, the dot chromosomes have fused to the ancestral sex chromosomes, creating a pair of 'neo-sex' chromosomes. Here we collect genomic, transcriptomic and epigenomic data from D. busckii, to investigate the evolutionary trajectory of sex chromosomes from a largely heterochromatic ancestor. We show that the neo-sex chromosomes formed <1 million years ago, but nearly 60% of neo-Y linked genes have already become non-functional. Expression levels are generally lower for the neo-Y alleles relative to their neo-X homologs, and the silencing heterochromatin mark H3K9me2, but not H3K9me3, is significantly enriched on silenced neo-Y genes. Despite rampant neo-Y degeneration, we find that the neo-X is deficient for the canonical histone modification mark of dosage compensation (H4K16ac), relative to autosomes or the compensated ancestral X chromosome, possibly reflecting constraints imposed on evolving hyperactive chromatin in an originally heterochromatic environment. Yet, neo-X genes are transcriptionally more active in males, relative to females, suggesting the evolution of incipient dosage compensation on the neo-X. Our data show that Y degeneration proceeds quickly after sex chromosomes become established through genomic and epigenetic changes, and are consistent with the idea that the evolution of sex-linked chromatin is influenced by its ancestral configuration.

  19. Polymer Physics of the Large-Scale Structure of Chromatin.

    PubMed

    Bianco, Simona; Chiariello, Andrea Maria; Annunziatella, Carlo; Esposito, Andrea; Nicodemi, Mario

    2016-01-01

    We summarize the picture emerging from recently proposed models of polymer physics describing the general features of chromatin large scale spatial architecture, as revealed by microscopy and Hi-C experiments. PMID:27659986

  20. Control of chromatin structure by long noncoding RNA

    PubMed Central

    Böhmdorfer, Gudrun; Wierzbicki, Andrzej T.

    2015-01-01

    Long noncoding RNA (lncRNA) is a pivotal factor regulating various aspects of genome activity. Genome regulation via DNA methylation and posttranslational histone modifications is a well-documented function of lncRNA in plants, fungi, and animals. Here, we summarize evidence showing that lncRNA also controls chromatin structure including nucleosome positioning and chromosome looping. We focus on data from plant experimental systems, discussed in the context of other eukaryotes. We explain the mechanisms of lncRNA-controlled chromatin remodeling and the implications of the functional interplay between noncoding transcription and several different chromatin remodelers. We propose that the unique properties of RNA make it suitable for controlling chromatin modifications and structure. PMID:26410408

  1. HACking the centromere chromatin code: insights from human artificial chromosomes.

    PubMed

    Bergmann, Jan H; Martins, Nuno M C; Larionov, Vladimir; Masumoto, Hiroshi; Earnshaw, William C

    2012-07-01

    The centromere is a specialized chromosomal region that serves as the assembly site of the kinetochore. At the centromere, CENP-A nucleosomes form part of a chromatin landscape termed centrochromatin. This chromatin environment conveys epigenetic marks regulating kinetochore formation. Recent work sheds light on the intricate relationship between centrochromatin state, the CENP-A assembly pathway and the maintenance of centromere function. Here, we review the emerging picture of how chromatin affects mammalian kinetochore formation. We place particular emphasis on data obtained from Human Artificial Chromosome (HAC) biology and the targeted engineering of centrochromatin using synthetic HACs. We discuss implications of these findings, which indicate that a delicate balance of histone modifications and chromatin state dictates both de novo centromere formation and the maintenance of centromere identity in dividing cell populations. PMID:22825423

  2. Neutron scattering studies on chromatin higher-order structure

    SciTech Connect

    Graziano, V.; Gerchman, S.E.; Schneider, D.K.; Ramakrishnan, V.

    1994-12-31

    We have been engaged in studies of the structure and condensation of chromatin into the 30nm filament using small-angle neutron scattering. We have also used deuterated histone H1 to determine its location in the chromatin 30nm filament. Our studies indicate that chromatin condenses with increasing ionic strength to a limiting structure that has a mass per unit length of 6-7 nucleosomes/11 nm. They also show that the linker histone H1/H5 is located in the interior of the chromatin filament, in a position compatible with its binding to the inner face of the nucleosome. Analysis of the mass per unit length as a function of H5 stoichiometry suggests that 5-7 contiguous nucleosomes need to have H5 bound before a stable higher order structure can exist.

  3. In vitro binding of nitracrine to DNA in chromatin.

    PubMed

    Wilmańska, D; Szmigiero, L; Gniazdowski, M

    1989-01-01

    In the presence of sulfhydryl compounds nitracrine, an anticancer drug, binds covalently to DNA. The accessibility of DNA in chromatin both to nitracrine and to 8-methoxypsoralen, which was used as a reference compound in this study, when assayed in NaCl concentrations from 0 to 2 M show similar characteristics. The initial decrease reaches a minimum at 0.15 M NaCl above which dissociation of non-histone proteins and histones at higher ionic strengths is demonstrated by an increase in accessible sites. The relative accessibility of DNA in chromatin to nitracrine is, however, lower than that found for 8-methoxypsoralen. Partial dissociation of chromatin with 0.7 M NaCl increases the accessibility of DNA in chromatin when assayed in the absence of NaCl but has no apparent influence when estimated at ionic strength close to physiological conditions. PMID:2742691

  4. HACking the centromere chromatin code: insights from human artificial chromosomes.

    PubMed

    Bergmann, Jan H; Martins, Nuno M C; Larionov, Vladimir; Masumoto, Hiroshi; Earnshaw, William C

    2012-07-01

    The centromere is a specialized chromosomal region that serves as the assembly site of the kinetochore. At the centromere, CENP-A nucleosomes form part of a chromatin landscape termed centrochromatin. This chromatin environment conveys epigenetic marks regulating kinetochore formation. Recent work sheds light on the intricate relationship between centrochromatin state, the CENP-A assembly pathway and the maintenance of centromere function. Here, we review the emerging picture of how chromatin affects mammalian kinetochore formation. We place particular emphasis on data obtained from Human Artificial Chromosome (HAC) biology and the targeted engineering of centrochromatin using synthetic HACs. We discuss implications of these findings, which indicate that a delicate balance of histone modifications and chromatin state dictates both de novo centromere formation and the maintenance of centromere identity in dividing cell populations.

  5. The chromatin landscape of Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Toth, Zsolt; Brulois, Kevin; Jung, Jae U

    2013-05-01

    Kaposi's sarcoma-associated herpesvirus is an oncogenic γ-herpesvirus that causes latent infection in humans. In cells, the viral genome adopts a highly organized chromatin structure, which is controlled by a wide variety of cellular and viral chromatin regulatory factors. In the past few years, interrogation of the chromatinized KSHV genome by whole genome-analyzing tools revealed that the complex chromatin landscape spanning the viral genome in infected cells has important regulatory roles during the viral life cycle. This review summarizes the most recent findings regarding the role of histone modifications, histone modifying enzymes, DNA methylation, microRNAs, non-coding RNAs and the nuclear organization of the KSHV epigenome in the regulation of latent and lytic viral gene expression programs as well as their connection to KSHV-associated pathogenesis. PMID:23698402

  6. Insights into Chromatin Structure and Dynamics in Plants

    PubMed Central

    Rosa, Stefanie; Shaw, Peter

    2013-01-01

    The packaging of chromatin into the nucleus of a eukaryotic cell requires an extraordinary degree of compaction and physical organization. In recent years, it has been shown that this organization is dynamically orchestrated to regulate responses to exogenous stimuli as well as to guide complex cell-type-specific developmental programs. Gene expression is regulated by the compartmentalization of functional domains within the nucleus, by distinct nucleosome compositions accomplished via differential modifications on the histone tails and through the replacement of core histones by histone variants. In this review, we focus on these aspects of chromatin organization and discuss novel approaches such as live cell imaging and photobleaching as important tools likely to give significant insights into our understanding of the very dynamic nature of chromatin and chromatin regulatory processes. We highlight the contribution plant studies have made in this area showing the potential advantages of plants as models in understanding this fundamental aspect of biology. PMID:24833230

  7. Polymer Physics of the Large-Scale Structure of Chromatin.

    PubMed

    Bianco, Simona; Chiariello, Andrea Maria; Annunziatella, Carlo; Esposito, Andrea; Nicodemi, Mario

    2016-01-01

    We summarize the picture emerging from recently proposed models of polymer physics describing the general features of chromatin large scale spatial architecture, as revealed by microscopy and Hi-C experiments.

  8. ATP-dependent chromatin remodeling shapes the DNA replication landscape

    PubMed Central

    Vincent, Jack A.; Kwong, Tracey J.; Tsukiyama, Toshio

    2009-01-01

    Summary The eukaryotic DNA replication machinery must traverse every nucleosome in the genome during S phase. As nucleosomes are generally inhibitory to DNA-dependent processes, chromatin structure must undergo extensive reorganization to facilitate DNA synthesis. However, the identity of chromatin-remodeling factors involved in replication and how they affect DNA synthesis is largely unknown. Here we show that two highly conserved ATP-dependent chromatin-remodeling complexes in Saccharomyces cerevisiae, Isw2 and Ino80, function in parallel to promote replication fork progression. As a result, Isw2 and Ino80 play especially important roles for replication of late-replicating regions during periods of replication stress. Both Isw2 and Ino80 complexes are enriched at sites of replication, suggesting that these complexes act directly to promote fork progression. These findings identify ATP-dependent chromatin-remodeling complexes promoting DNA replication, and define a specific stage of replication that requires remodeling for normal function. PMID:18408730

  9. Chromatin mechanisms in the developmental control of imprinted gene expression.

    PubMed

    Sanli, Ildem; Feil, Robert

    2015-10-01

    Hundreds of protein-coding genes and regulatory non-coding RNAs (ncRNAs) are subject to genomic imprinting. The mono-allelic DNA methylation marks that control imprinted gene expression are somatically maintained throughout development, and this process is linked to specific chromatin features. Yet, at many imprinted genes, the mono-allelic expression is lineage or tissue-specific. Recent studies provide mechanistic insights into the developmentally-restricted action of the 'imprinting control regions' (ICRs). At several imprinted domains, the ICR expresses a long ncRNA that mediates chromatin repression in cis (and probably in trans as well). ICRs at other imprinted domains mediate higher-order chromatin structuration that enhances, or prevents, transcription of close-by genes. Here, we present how chromatin and ncRNAs contribute to developmental control of imprinted gene expression and discuss implications for disease. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

  10. ISWI chromatin remodeling complexes in the DNA damage response

    PubMed Central

    Aydin, Özge Z; Vermeulen, Wim; Lans, Hannes

    2014-01-01

    Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA. PMID:25486562

  11. The Regulation of Chromatin by Dynamic SUMO Modifications.

    PubMed

    Wilson, Nicole R; Hochstrasser, Mark

    2016-01-01

    Protein modification by the small ubiquitin-related modifier (SUMO) protein regulates numerous cellular pathways and mounting evidence reveals a critical role for SUMO in modulating gene expression. Dynamic sumoylation of transcription factors, chromatin-modifying enzymes, histones, and other chromatin-associated factors significantly affects the transcriptional status of the eukaryotic genome. Recent studies have employed high-throughput ChIP-Seq analyses to gain clues regarding the role of the SUMO pathway in regulating chromatin-based transactions. Indeed, the global distribution of SUMO across chromatin reveals an important function for SUMO in controlling transcription, particularly of genes involved in protein synthesis. These newly appreciated patterns of genome-wide sumoylation will inform more directed studies aimed at analyzing how the dynamics of gene expression are controlled by posttranslational SUMO modification. PMID:27631795

  12. ISWI chromatin remodeling complexes in the DNA damage response.

    PubMed

    Aydin, Özge Z; Vermeulen, Wim; Lans, Hannes

    2014-01-01

    Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA.

  13. Rad51–Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

    PubMed Central

    Mitra, Sreyoshi; Gómez-Raja, Jonathan; Larriba, Germán; Dubey, Dharani Dhar; Sanyal, Kaustuv

    2014-01-01

    Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-ACaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-ACaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain

  14. A systematic immunoprecipitation approach reinforces the concept of common conformational alterations in amyotrophic lateral sclerosis-linked SOD1 mutants.

    PubMed

    Fujisawa, Takao; Yamaguchi, Namiko; Kadowaki, Hisae; Tsukamoto, Yuka; Tsuburaya, Naomi; Tsubota, Atsushi; Takahashi, Hiromitsu; Naguro, Isao; Takahashi, Yuji; Goto, Jun; Tsuji, Shoji; Nishitoh, Hideki; Homma, Kengo; Ichijo, Hidenori

    2015-10-01

    Mutations in the Cu, Zn superoxide dismutase (SOD1) gene are one of the causative agents of amyotrophic lateral sclerosis (ALS). Although more than 100 different mutations in SOD1 have been identified, it is unclear whether all the mutations are pathogenic or just single nucleotide polymorphisms (SNPs) unrelated to the disease. Our previous systematic analysis found that all pathogenic SOD1 mutants (SOD1(mut)) have a common property, namely, an association with Derlin-1, a component of the endoplasmic reticulum-associated degradation machinery. For the proposed mechanism, we found that most pathogenic SOD1(mut) have a constitutively exposed Derlin-1-binding region (DBR), which is concealed in wild-type SOD1 (SOD1(WT)). Moreover, we generated MS785, a monoclonal antibody against DBR. MS785 distinguished most ALS-causative SOD1(mut) from both SOD1(WT) and non-toxic SOD1(mut). However, MS785 could not recognize SOD1(mut) that has mutations in the MS785 epitope region. Here, we developed a new diagnostic antibody, which could compensate for this shortcoming of MS785. We hypothesized that in ALS-causative SOD1(mut), the DBR-neighboring region [SOD1(30-40)] may also be exposed. We then generated MS27, a monoclonal antibody against SOD1(30-40). We found that MS27 could distinguish SOD1(WT) from the pathogenic SOD1(mut), which has mutations in the MS785 epitope region. Moreover, all pathogenic SOD1(mut), without exception, were immunoprecipitated with a combination of MS785 and MS27. The MS785-MS27 combination could be developed as a novel mechanism-based biomarker for the diagnosis of ALS.

  15. Chromatin topology is coupled to Polycomb group protein subnuclear organization

    PubMed Central

    Wani, Ajazul H.; Boettiger, Alistair N.; Schorderet, Patrick; Ergun, Ayla; Münger, Christine; Sadreyev, Ruslan I.; Zhuang, Xiaowei; Kingston, Robert E.; Francis, Nicole J.

    2016-01-01

    The genomes of metazoa are organized at multiple scales. Many proteins that regulate genome architecture, including Polycomb group (PcG) proteins, form subnuclear structures. Deciphering mechanistic links between protein organization and chromatin architecture requires precise description and mechanistic perturbations of both. Using super-resolution microscopy, here we show that PcG proteins are organized into hundreds of nanoscale protein clusters. We manipulated PcG clusters by disrupting the polymerization activity of the sterile alpha motif (SAM) of the PcG protein Polyhomeotic (Ph) or by increasing Ph levels. Ph with mutant SAM disrupts clustering of endogenous PcG complexes and chromatin interactions while elevating Ph level increases cluster number and chromatin interactions. These effects can be captured by molecular simulations based on a previously described chromatin polymer model. Both perturbations also alter gene expression. Organization of PcG proteins into small, abundant clusters on chromatin through Ph SAM polymerization activity may shape genome architecture through chromatin interactions. PMID:26759081

  16. Epigenetic regulation of open chromatin in pluripotent stem cells.

    PubMed

    Kobayashi, Hiroshi; Kikyo, Nobuaki

    2015-01-01

    The recent progress in pluripotent stem cell research has opened new avenues of disease modeling, drug screening, and transplantation of patient-specific tissues unimaginable until a decade ago. The central mechanism underlying pluripotency is epigenetic gene regulation; the majority of cell signaling pathways, both extracellular and cytoplasmic, alter, eventually, the epigenetic status of their target genes during the process of activating or suppressing the genes to acquire or maintain pluripotency. It has long been thought that the chromatin of pluripotent stem cells is open globally to enable the timely activation of essentially all genes in the genome during differentiation into multiple lineages. The current article reviews descriptive observations and the epigenetic machinery relevant to what is supposed to be globally open chromatin in pluripotent stem cells, including microscopic appearance, permissive gene transcription, chromatin remodeling complexes, histone modifications, DNA methylation, noncoding RNAs, dynamic movement of chromatin proteins, nucleosome accessibility and positioning, and long-range chromosomal interactions. Detailed analyses of each element, however, have revealed that the globally open chromatin hypothesis is not necessarily supported by some of the critical experimental evidence, such as genomewide nucleosome accessibility and nucleosome positioning. Greater understanding of epigenetic gene regulation is expected to determine the true nature of the so-called globally open chromatin in pluripotent stem cells.

  17. Minor Groove Binder Distamycin Remodels Chromatin but Inhibits Transcription

    PubMed Central

    Majumder, Parijat; Banerjee, Amrita; Shandilya, Jayasha; Senapati, Parijat; Chatterjee, Snehajyoti; Kundu, Tapas K.; Dasgupta, Dipak

    2013-01-01

    The condensed structure of chromatin limits access of cellular machinery towards template DNA. This in turn represses essential processes like transcription, replication, repair and recombination. The repression is alleviated by a variety of energy dependent processes, collectively known as “chromatin remodeling”. In a eukaryotic cell, a fine balance between condensed and de-condensed states of chromatin helps to maintain an optimum level of gene expression. DNA binding small molecules have the potential to perturb such equilibrium. We present herein the study of an oligopeptide antibiotic distamycin, which binds to the minor groove of B-DNA. Chromatin mobility assays and circular dichroism spectroscopy have been employed to study the effect of distamycin on chromatosomes, isolated from the liver of Sprague-Dawley rats. Our results show that distamycin is capable of remodeling both chromatosomes and reconstituted nucleosomes, and the remodeling takes place in an ATP-independent manner. Binding of distamycin to the linker and nucleosomal DNA culminates in eviction of the linker histone and the formation of a population of off-centered nucleosomes. This hints at a possible corkscrew type motion of the DNA with respect to the histone octamer. Our results indicate that distamycin in spite of remodeling chromatin, inhibits transcription from both DNA and chromatin templates. Therefore, the DNA that is made accessible due to remodeling is either structurally incompetent for transcription, or bound distamycin poses a roadblock for the transcription machinery to advance. PMID:23460895

  18. Integrative annotation of chromatin elements from ENCODE data

    PubMed Central

    Hoffman, Michael M.; Ernst, Jason; Wilder, Steven P.; Kundaje, Anshul; Harris, Robert S.; Libbrecht, Max; Giardine, Belinda; Ellenbogen, Paul M.; Bilmes, Jeffrey A.; Birney, Ewan; Hardison, Ross C.; Dunham, Ian; Kellis, Manolis; Noble, William Stafford

    2013-01-01

    The ENCODE Project has generated a wealth of experimental information mapping diverse chromatin properties in several human cell lines. Although each such data track is independently informative toward the annotation of regulatory elements, their interrelations contain much richer information for the systematic annotation of regulatory elements. To uncover these interrelations and to generate an interpretable summary of the massive datasets of the ENCODE Project, we apply unsupervised learning methodologies, converting dozens of chromatin datasets into discrete annotation maps of regulatory regions and other chromatin elements across the human genome. These methods rediscover and summarize diverse aspects of chromatin architecture, elucidate the interplay between chromatin activity and RNA transcription, and reveal that a large proportion of the genome lies in a quiescent state, even across multiple cell types. The resulting annotation of non-coding regulatory elements correlate strongly with mammalian evolutionary constraint, and provide an unbiased approach for evaluating metrics of evolutionary constraint in human. Lastly, we use the regulatory annotations to revisit previously uncharacterized disease-associated loci, resulting in focused, testable hypotheses through the lens of the chromatin landscape. PMID:23221638

  19. Isolation and Proteomics Analysis of Barley Centromeric Chromatin Using PICh.

    PubMed

    Zeng, Zixian; Jiang, Jiming

    2016-06-01

    Identification of proteins that are directly or indirectly associated with a specific DNA sequence is often an important goal in molecular biology research. Proteomics of isolated chromatin fragments (PICh) is a technique used to isolate chromatin that contains homologous DNA sequence to a specific nucleic acid probe. All proteins directly and indirectly associated with the DNA sequences that hybridize to the probe are then identified by proteomics.1 We used the PICh technique to isolate chromatin associated with the centromeres of barley (Hordeum vulgare) by using a 2'-deoxy-2'fluoro-ribonucleotides (2'-F RNA) probe that is homologous to the AGGGAG satellite DNA specific to barley centromeres. Proteins associated with the barley centromeric chromatin were then isolated and identified by mass spectrometry. Both alpha-cenH3 and beta-cenH3, the two centromeric histone H3 variants associated with barley centromeres, were positively identified. Interestingly, several different H2A and H2B variants were recovered in the PIChed chromatin. The limitations and future potential of PICh in plant chromatin research are discussed. PMID:27142171

  20. Compact structure of ribosomal chromatin in Xenopus laevis.

    PubMed Central

    Spadafora, C; Crippa, M

    1984-01-01

    Micrococcal nuclease digestion was used as a tool to study the organization of the ribosomal chromatin in liver, blood and embryo cells of X. laevis. It was found that in liver and blood cells, ribosomal DNA is efficiently protected from nuclease attack in comparison to bulk chromatin. Although ribosomal chromatin is fragmented in a typical nucleosomal pattern, a considerable portion of ribosomal DNA retains a high molecular weight even after extensive digestion. A greater accessibility of the coding region in comparison to the non-coding spacer was found. In embryos, when ribosomal DNA is fully transcribed, these genes are even more highly protected than in adult tissues: in fact, the nucleosomal ladder can hardly be detected and rDNA is preserved in high molecular weight. Treatment of chromatin with 0.8 M NaCl abolishes the specific resistance of the ribosomal chromatin to digestion. The ribosomal chromatin, particularly in its active state, seems to be therefore tightly complexed with chromosomal proteins which protect its DNA from nuclease degradation. Images PMID:6709502

  1. Ectopically tethered CP190 induces large-scale chromatin decondensation

    NASA Astrophysics Data System (ADS)

    Ahanger, Sajad H.; Günther, Katharina; Weth, Oliver; Bartkuhn, Marek; Bhonde, Ramesh R.; Shouche, Yogesh S.; Renkawitz, Rainer

    2014-01-01

    Insulator mediated alteration in higher-order chromatin and/or nucleosome organization is an important aspect of epigenetic gene regulation. Recent studies have suggested a key role for CP190 in such processes. In this study, we analysed the effects of ectopically tethered insulator factors on chromatin structure and found that CP190 induces large-scale decondensation when targeted to a condensed lacO array in mammalian and Drosophila cells. In contrast, dCTCF alone, is unable to cause such a decondensation, however, when CP190 is present, dCTCF recruits it to the lacO array and mediates chromatin unfolding. The CP190 induced opening of chromatin may not be correlated with transcriptional activation, as binding of CP190 does not enhance luciferase activity in reporter assays. We propose that CP190 may mediate histone modification and chromatin remodelling activity to induce an open chromatin state by its direct recruitment or targeting by a DNA binding factor such as dCTCF.

  2. Environmental-stress-induced Chromatin Regulation and its Heritability

    PubMed Central

    Fang, Lei; Wuptra, Kenly; Chen, Danqi; Li, Hongjie; Huang, Shau-Ku; Jin, Chunyuan; Yokoyama, Kazunari K

    2014-01-01

    Chromatin is subject to proofreading and repair mechanisms during the process of DNA replication, as well as repair to maintain genetic and epigenetic information and genome stability. The dynamic structure of chromatin modulates various nuclear processes, including transcription and replication, by altering the accessibility of the DNA to regulatory factors. Structural changes in chromatin are affected by the chemical modification of histone proteins and DNA, remodeling of nucleosomes, incorporation of variant histones, noncoding RNAs, and nonhistone DNA-binding proteins. Phenotypic diversity and fidelity can be balanced by controlling stochastic switching of chromatin structure and dynamics in response to the environmental disruptors and endogenous stresses. The dynamic chromatin remodeling can, therefore, serve as a sensor, through which environmental and/or metabolic agents can alter gene expression, leading to global cellular changes involving multiple interactive networks. Furthermore its recent evidence also suggests that the epigenetic changes are heritable during the development. This review will discuss the environmental sensing system for chromatin regulation and genetic and epigenetic controls from developmental perspectives. PMID:25045581

  3. Environmental-stress-induced Chromatin Regulation and its Heritability.

    PubMed

    Fang, Lei; Wuptra, Kenly; Chen, Danqi; Li, Hongjie; Huang, Shau-Ku; Jin, Chunyuan; Yokoyama, Kazunari K

    2014-01-15

    Chromatin is subject to proofreading and repair mechanisms during the process of DNA replication, as well as repair to maintain genetic and epigenetic information and genome stability. The dynamic structure of chromatin modulates various nuclear processes, including transcription and replication, by altering the accessibility of the DNA to regulatory factors. Structural changes in chromatin are affected by the chemical modification of histone proteins and DNA, remodeling of nucleosomes, incorporation of variant histones, noncoding RNAs, and nonhistone DNA-binding proteins. Phenotypic diversity and fidelity can be balanced by controlling stochastic switching of chromatin structure and dynamics in response to the environmental disruptors and endogenous stresses. The dynamic chromatin remodeling can, therefore, serve as a sensor, through which environmental and/or metabolic agents can alter gene expression, leading to global cellular changes involving multiple interactive networks. Furthermore its recent evidence also suggests that the epigenetic changes are heritable during the development. This review will discuss the environmental sensing system for chromatin regulation and genetic and epigenetic controls from developmental perspectives.

  4. Atomic force microscope imaging of chromatin assembled in Xenopus laevis egg extract.

    PubMed

    Fu, Hongxia; Freedman, Benjamin S; Lim, Chwee Teck; Heald, Rebecca; Yan, Jie

    2011-06-01

    Gaps persist in our understanding of chromatin lower- and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatin assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures. PMID:21369955

  5. DNA-protein interactions in nucleosomes and in chromatin. Structural studies of chromatin stabilized by ultraviolet-light induced crosslinking.

    PubMed

    Mandel, R; Kolomijtseva, G; Brahms, J G

    1979-05-15

    Crosslinking induced by ultraviolet light irradiation at 254 nm has been utilized to investigate the structure of chromatin and isolated nucleosomes. The results presented here imply that the four core histones, as well as histone H1, have reactive groups within a bond length of the DNA bases. In nucleosomes depleted of H1, all of the core histones react similarly with the DNA and form crosslinks. In chromatin, the rate of crosslinking of all histones to DNA is essentially similar. Comparison of mononucleosomes, dinucleosomes and whole chromatin shows that the rate of crosslinking increases significantly with increasing number of connected nucleosomes. These differences in the rate of crosslinking are interpreted in terms of interactions between neighbouring nucleosomes on the chromatin fiber, which are absent in an isolated mononucleosome.

  6. The proto-chromatosome: A fundamental subunit of chromatin?

    PubMed

    Ocampo, Josefina; Cui, Feng; Zhurkin, Victor B; Clark, David J

    2016-07-01

    Eukaryotic DNA is packaged into regularly spaced nucleosomes, resembling beads on a string. Each bead contains ∼147 bp wrapped around a core histone octamer. Linker histone (H1) binds to the linker DNA to drive chromatin folding. Micrococcal nuclease (MNase) digestion studies reveal 2 mono-nucleosomal intermediates: the core particle (∼147 bp) and the chromatosome (∼160 bp; a core particle with additional DNA protected by H1). We have recently developed an improved method for mapping nucleosomes, using exonuclease III to remove residual linker (MNase-Exo-seq). (1) We discovered 2 new intermediate particles corresponding to core particles with ∼7 bp of linker protruding from one side (∼154 bp) or both sides (∼161 bp), which are formed in the absence of H1. We propose that these "proto-chromatosomes" are stabilized by core histone-DNA contacts in the linker, ∼7 bp from the nucleosome boundaries. These contacts may determine the topography of the H1 binding site. PMID:27645053

  7. DAnCER: Disease-Annotated Chromatin Epigenetics Resource

    PubMed Central

    Turinsky, Andrei L.; Turner, Brian; Borja, Rosanne C.; Gleeson, James A.; Heath, Michael; Pu, Shuye; Switzer, Thomas; Dong, Dong; Gong, Yunchen; On, Tuan; Xiong, Xuejian; Emili, Andrew; Greenblatt, Jack; Parkinson, John; Zhang, Zhaolei; Wodak, Shoshana J.

    2011-01-01

    Chromatin modification (CM) is a set of epigenetic processes that govern many aspects of DNA replication, transcription and repair. CM is carried out by groups of physically interacting proteins, and their disruption has been linked to a number of complex human diseases. CM remains largely unexplored, however, especially in higher eukaryotes such as human. Here we present the DAnCER resource, which integrates information on genes with CM function from five model organisms, including human. Currently integrated are gene functional annotations, Pfam domain architecture, protein interaction networks and associated human diseases. Additional supporting evidence includes orthology relationships across organisms, membership in protein complexes, and information on protein 3D structure. These data are available for 962 experimentally confirmed and manually curated CM genes and for over 5000 genes with predicted CM function on the basis of orthology and domain composition. DAnCER allows visual explorations of the integrated data and flexible query capabilities using a variety of data filters. In particular, disease information and functional annotations are mapped onto the protein interaction networks, enabling the user to formulate new hypotheses on the function and disease associations of a given gene based on those of its interaction partners. DAnCER is freely available at http://wodaklab.org/dancer/. PMID:20876685

  8. Interphase Chromosome Conformation and Chromatin-Chromatin Interactions in Human Epithelial Cells Cultured Under Different Gravity Conditions

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Wong, Michael; Hada, Megumi; Wu, Honglu

    2015-01-01

    Microgravity has been shown to alter global gene expression patterns and protein levels both in cultured cells and animal models. It has been suggested that the packaging of chromatin fibers in the interphase nucleus is closely related to genome function, and the changes in transcriptional activity are tightly correlated with changes in chromatin folding. This study explores the changes of chromatin conformation and chromatin-chromatin interactions in the simulated microgravity environment, and investigates their correlation to the expression of genes located at different regions of the chromosome. To investigate the folding of chromatin in interphase under various culture conditions, human epithelial cells, fibroblasts, and lymphocytes were fixed in the G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome as separate colors. After images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multi-mega base pair scale. In order to determine the effects of microgravity on chromosome conformation and orientation, measures such as distance between homologous pairs, relative orientation of chromosome arms about a shared midpoint, and orientation of arms within individual chromosomes were all considered as potentially impacted by simulated microgravity conditions. The studies revealed non-random folding of chromatin in interphase, and suggested an association of interphase chromatin folding with radiation-induced chromosome aberration hotspots. Interestingly, the distributions of genes with expression changes over chromosome 3 in cells cultured under microgravity environment are apparently clustered on specific loci and chromosomes. This data provides important insights into how mammalian cells respond to microgravity at molecular level.

  9. Visualization of chromatin domains created by the gypsy insulator of Drosophila.

    PubMed

    Byrd, Keith; Corces, Victor G

    2003-08-18

    Insulators might regulate gene expression by establishing and maintaining the organization of the chromatin fiber within the nucleus. Biochemical fractionation and in situ high salt extraction of lysed cells show that two known protein components of the gypsy insulator are present in the nuclear matrix. Using FISH with DNA probes located between two endogenous Su(Hw) binding sites, we show that the intervening DNA is arranged in a loop, with the two insulators located at the base. Mutations in insulator proteins, subjecting the cells to a brief heat shock, or destruction of the nuclear matrix lead to disruption of the loop. Insertion of an additional gypsy insulator in the center of the loop results in the formation of paired loops through the attachment of the inserted sequences to the nuclear matrix. These results suggest that the gypsy insulator might establish higher-order domains of chromatin structure and regulate nuclear organization by tethering the DNA to the nuclear matrix and creating chromatin loops. PMID:12925706

  10. The Chd1 chromatin remodeler can sense both entry and exit sides of the nucleosome

    PubMed Central

    Nodelman, Ilana M.; Horvath, Kyle C.; Levendosky, Robert F.; Winger, Jessica; Ren, Ren; Patel, Ashok; Li, Ming; Wang, Michelle D.; Roberts, Elijah; Bowman, Gregory D.

    2016-01-01

    Chromatin remodelers are essential for establishing and maintaining the placement of nucleosomes along genomic DNA. Yet how chromatin remodelers recognize and respond to distinct chromatin environments surrounding nucleosomes is poorly understood. Here, we use Lac repressor as a tool to probe how a DNA-bound factor influences action of the Chd1 remodeler. We show that Chd1 preferentially shifts nucleosomes away from Lac repressor, demonstrating that a DNA-bound factor defines a barrier for nucleosome positioning. Rather than an absolute block in sliding, the barrier effect was achieved by altered rates of nucleosome sliding that biased redistribution of nucleosomes away from the bound Lac repressor site. Remarkably, in addition to slower sliding toward the LacO site, the presence of Lac repressor also stimulated sliding in the opposite direction. These experiments therefore demonstrate that Chd1 responds to the presence of a bound protein on both entry and exit sides of the nucleosome. This sensitivity to both sides of the nucleosome allows for a faster and sharper response than would be possible by responding to only the entry side, and we speculate that dual entry/exit sensitivity is also important for regularly spaced nucleosome arrays generated by Chd1 and the related ISWI remodelers. PMID:27174939

  11. Chromatin dynamics during cellular differentiation in the female reproductive lineage of flowering plants.

    PubMed

    Baroux, Célia; Autran, Daphné

    2015-07-01

    Sexual reproduction in flowering plants offers a number of remarkable aspects to developmental biologists. First, the spore mother cells - precursors of the plant reproductive lineage - are specified late in development, as opposed to precocious germline isolation during embryogenesis in most animals. Second, unlike in most animals where meiosis directly produces gametes, plant meiosis entails the differentiation of a multicellular, haploid gametophyte, within which gametic as well as non-gametic accessory cells are formed. These observations raise the question of the factors inducing and modus operandi of cell fate transitions that originate in floral tissues and gametophytes, respectively. Cell fate transitions in the reproductive lineage imply cellular reprogramming operating at the physiological, cytological and transcriptome level, but also at the chromatin level. A number of observations point to large-scale chromatin reorganization events associated with cellular differentiation of the female spore mother cells and of the female gametes. These include a reorganization of the heterochromatin compartment, the genome-wide alteration of the histone modification landscape, and the remodeling of nucleosome composition. The dynamic expression of DNA methyltransferases and actors of small RNA pathways also suggest additional, global epigenetic alterations that remain to be characterized. Are these events a cause or a consequence of cellular differentiation, and how do they contribute to cell fate transition? Does chromatin dynamics induce competence for immediate cellular functions (meiosis, fertilization), or does it also contribute long-term effects in cellular identity and developmental competence of the reproductive lineage? This review attempts to review these fascinating questions. PMID:26031902

  12. Impaired methylation modifications of FZD3 alter chromatin accessibility and are involved in congenital hydrocephalus pathogenesis.

    PubMed

    Wang, Li; Shangguan, Shaofang; Chang, Shaoyan; Wang, Zhen; Lu, Xiaolin; Wu, Lihua; Li, Rui; Bao, Yihua; Qiu, Zhiyong; Niu, Bo; Zhang, Ting

    2014-06-20

    Congenital hydrocephalus is heterogeneous in its etiology, and in addition to a genetic component, has been shown to be caused by environmental factors. Until now, however, no methylation alterations of target genes have been connected with congenital hydrocephalus in humans. Frizzled 3(FZD3) is a planar cell polarity (PCP) gene required for PCP signaling. Partial restoration of frizzled 3 activities in FZD3 mutant mice results in hydrocephalus. To analyze the possible roles of epigenetic modifications of the FZD3 gene in congenital hydrocephalus pathogenesis, DNA methylation in the promoter region of FZD3 was assayed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Gene expression and chromatin accessibility were also determined to assess the role of methylation alterations. Our study found methylation levels of the FZD3 gene were increased in congenital hydrocephalus, especially in males (10.57 ± 3.90 vs. 7.08 ± 0.94, p=0.001). Hypermethylation of FZD3 increased congenital hydrocephalus risk, with an odds ratio of 10.125 (p=0.003). Aberrant methylation modification of FZD3 altered both chromatin structure in this region and FZD3 expression levels. Totally, aberrant methylation modification of the FZD3 gene increases the risk of congenital hydrocephalus by altering chromatin structure and disturbing gene expression.

  13. SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

    PubMed Central

    Mund, Andreas; Schubert, Tobias; Staege, Hannah; Kinkley, Sarah; Reumann, Kerstin; Kriegs, Malte; Fritsch, Lauriane; Battisti, Valentine; Ait-Si-Ali, Slimane; Hoffbeck, Anne-Sophie; Soutoglou, Evi; Will, Hans

    2012-01-01

    Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure. PMID:23034801

  14. FOXA1 repression is associated with loss of BRCA1 and increased promoter methylation and chromatin silencing in breast cancer.

    PubMed

    Gong, C; Fujino, K; Monteiro, L J; Gomes, A R; Drost, R; Davidson-Smith, H; Takeda, S; Khoo, U S; Jonkers, J; Sproul, D; Lam, E W-F

    2015-09-24

    FOXA1 expression correlates with the breast cancer luminal subtype and patient survival. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with the downregulation of FOXA1 expression. Knockdown of BRCA1 resulted in the downregulation of FOXA1 expression and enhancement of FOXA1 promoter methylation in MCF-7 breast cancer cells, whereas the reconstitution of BRCA1 in Brca1-deficent mouse mammary epithelial cells (MMECs) promoted Foxa1 expression and methylation. These data suggest that BRCA1 suppresses FOXA1 hypermethylation and silencing. Consistently, the treatment of MMECs with the DNA methylation inhibitor 5-aza-2'-deoxycitydine induced Foxa1 mRNA expression. Furthermore, treatment with GSK126, an inhibitor of EZH2 methyltransferase activity, induced FOXA1 expression in BRCA1-deficient but not in BRCA1-reconstituted MMECs. Likewise, the depletion of EZH2 by small interfering RNA enhanced FOXA1 mRNA expression. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNA methyltransferases (DNMT)1/3a/3b and H3K27me3 are recruited to the endogenous FOXA1 promoter, further supporting the hypothesis that these proteins interact to modulate FOXA1 methylation and repression. Further co-immunoprecipitation and ChIP analysis showed that both BRCA1 and DNMT3b form complexes with EZH2 but not with each other, consistent with the notion that BRCA1 binds to EZH2 and negatively regulates its methyltransferase activity. We also found that EZH2 promotes and BRCA1 impairs the deposit of the gene silencing histone mark H3K27me3 on the FOXA1 promoter. These associations were validated in a familial breast cancer patient cohort. Integrated analysis of the global gene methylation and expression profiles of a set of 33 familial breast tumours revealed that FOXA1 promoter methylation is inversely correlated with the transcriptional expression of FOXA1 and that BRCA1 mutation breast cancer is significantly

  15. Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain.

    PubMed Central

    Kalos, M; Fournier, R E

    1995-01-01

    The human apolipoprotein B (apoB) gene resides within a 47.5-kb chromatin domain that is flanked by sequences that bind to the nuclear matrix. These matrix attachment regions (MARs) are boundaries between nuclease-sensitive and -resistant chromatin. As domain boundaries are thought to function as insulator elements, shielding sequences between them from effects of neighboring chromatin, this raised the possibility that the apoB MARs have functions that could be assayed by transfection. To test this possibility, we examined effects of the apoB MARs on transgene expression in transiently and stably transfected rat and human hepatoma cells. The apoB MARs had no effects on expression of transiently transfected reporters, but they altered expression of stably integrated transgenes in dramatic and reproducible ways. Single integrated copies of transgenes that contained the apoB promoter and second intron enhancer, which are sufficient for high-level expression in transient assays, were expressed at low and variable levels in stable transfectant clones. In contrast, transgenes containing the apoB 5' and 3' MARs were expressed at levels nearly 200-fold higher than levels of the minimal reporters in stable transfectants, and expression was position independent. Transgenes that contained the apoB MARs and an additional 3.3 kb of apoB 5' flanking sequence were also expressed in an elevated, position-independent manner. Surprisingly, tandem transgene arrays in multicopy transfectants were transcriptionally inactive. These observations suggest that the apoB MARs function as insulator elements, shielding transgene expression from effects of neighboring chromatin domains. PMID:7799927

  16. Chromatin perturbations during the DNA damage response in higher eukaryotes.

    PubMed

    Bakkenist, Christopher J; Kastan, Michael B

    2015-12-01

    The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response.

  17. Structural Fluctuations of the Chromatin Fiber within Topologically Associating Domains.

    PubMed

    Tiana, Guido; Amitai, Assaf; Pollex, Tim; Piolot, Tristan; Holcman, David; Heard, Edith; Giorgetti, Luca

    2016-03-29

    Experiments based on chromosome conformation capture have shown that mammalian genomes are partitioned into topologically associating domains (TADs), within which the chromatin fiber preferentially interacts. TADs may provide three-dimensional scaffolds allowing genes to contact their appropriate distal regulatory DNA sequences (e.g., enhancers) and thus to be properly regulated. Understanding the cell-to-cell and temporal variability of the chromatin fiber within TADs, and what determines them, is thus of great importance to better understand transcriptional regulation. We recently described an equilibrium polymer model that can accurately predict cell-to-cell variation of chromosome conformation within single TADs, from chromosome conformation capture-based data. Here we further analyze the conformational and energetic properties of our model. We show that the chromatin fiber within TADs can easily fluctuate between several conformational states, which are hierarchically organized and are not separated by important free energy barriers, and that this is facilitated by the fact that the chromatin fiber within TADs is close to the onset of the coil-globule transition. We further show that in this dynamic state the properties of the chromatin fiber, and its contact probabilities in particular, are determined in a nontrivial manner not only by site-specific interactions between strongly interacting loci along the fiber, but also by nonlocal correlations between pairs of contacts. Finally, we use live-cell experiments to measure the dynamics of the chromatin fiber in mouse embryonic stem cells, in combination with dynamical simulations, and predict that conformational changes within one TAD are likely to occur on timescales that are much shorter than the duration of one cell cycle. This suggests that genes and their regulatory elements may come together and disassociate several times during a cell cycle. These results have important implications for transcriptional

  18. Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity

    PubMed Central

    Kim, Tae-Hee; Li, Fugen; Ferreiro-Neira, Isabel; Ho, Li-Lun; Luyten, Annouck; Nalapareddy, Kodandaramireddy; Long, Henry; Verzi, Michael; Shivdasani, Ramesh A.

    2014-01-01

    Cells differentiate when transcription factors (TFs) bind accessible cis-regulatory elements to establish specific gene expression programs. In differentiating embryonic stem (ES) cells, chromatin at lineage-restricted genes becomes sequentially accessible1-4, probably by virtue of “pioneer” TF activity5, but tissues may utilize other strategies in vivo. Lateral inhibition is a pervasive process in which one cell forces a different identity on its neighbors6, and it is unclear how chromatin in equipotent progenitors undergoing lateral inhibition quickly enables distinct, transiently reversible cell fates. Here we report the chromatin and transcriptional underpinnings of differentiation in mouse small intestine crypts, where Notch signaling mediates lateral inhibition to assign progenitor cells into absorptive or secretory lineages7-9. Transcript profiles in isolated LGR5+ intestinal stem cells (ISC)10 and secretory and absorptive progenitors indicated that each cell population was distinct and the progenitors specified. Nevertheless, secretory and absorptive progenitors showed comparable levels of H3K4me2 and H3K27ac histone marks and DNaseI hypersensitivity - signifying accessible, permissive chromatin - at most of the same cis-elements. Enhancers acting uniquely in progenitors were well-demarcated in LGR5+ ISC, revealing early priming of chromatin for divergent transcriptional programs, and retained active marks well after lineages were specified. On this chromatin background, ATOH1, a secretory-specific TF, controls lateral inhibition through Delta-like Notch ligand genes and also drives numerous secretory lineage genes. Depletion of ATOH1 from specified secretory cells converted them into functional enterocytes, indicating prolonged responsiveness of marked enhancers to presence or absence of a key TF. Thus, lateral inhibition and intestinal crypt lineage plasticity involve interaction of a lineage-restricted TF with broadly permissive chromatin established

  19. Chromatin perturbations during the DNA damage response in higher eukaryotes

    PubMed Central

    Bakkenist, Christopher J.; Kastan, Michael B.

    2016-01-01

    The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response

  20. The Chromatin Regulator CHD8 Is a Context-Dependent Mediator of Cell Survival in Murine Hematopoietic Malignancies

    PubMed Central

    Shingleton, Jennifer R.; Hemann, Michael T.

    2015-01-01

    Aberrant chromatin regulation is a frequent driver of leukemogenesis. Mutations in chromatin regulators often result in more stem-like cells that seed a bulk leukemic population. Inhibitors targeting these proteins represent an emerging class of therapeutics, and identifying further chromatin regulators that promote disease progression may result in additional drug targets. We identified the chromatin-modifying protein CHD8 as necessary for cell survival in a mouse model of BCR-Abl+ B-cell acute lymphoblastic leukemia. This disease has a poor prognosis despite treatment with kinase inhibitors targeting BCR-Abl. Although implicated as a risk factor in autism spectrum disorder and a tumor suppressor in prostate and lung cancer, the mechanism of CHD8’s activity is still unclear and has never been studied in the context of hematopoietic malignancies. Here we demonstrate that depletion of CHD8 in B-ALL cells leads to cell death. While multiple B cell malignancies were dependent on CHD8 expression for survival, T cell malignancies displayed milder phenotypes upon CHD8 knockdown. In addition, ectopic expression of the Notch1 intracellular domain in a T cell malignancy partially alleviated the detrimental effect of CHD8 depletion. Our results demonstrate that CHD8 has a context-dependent role in cell survival, and its inhibition may be an effective treatment for B lymphoid malignancies. PMID:26588464

  1. Fetal iron deficiency induces chromatin remodeling at the Bdnf locus in adult rat hippocampus.

    PubMed

    Tran, Phu V; Kennedy, Bruce C; Lien, Yu-Chin; Simmons, Rebecca A; Georgieff, Michael K

    2015-02-15

    Fetal and subsequent early postnatal iron deficiency causes persistent impairments in cognitive and affective behaviors despite prompt postnatal iron repletion. The long-term cognitive impacts are accompanied by persistent downregulation of brain-derived neurotrophic factor (BDNF), a factor critical for hippocampal plasticity across the life span. This study determined whether early-life iron deficiency epigenetically modifies the Bdnf locus and whether dietary choline supplementation during late gestation reverses these modifications. DNA methylation and histone modifications were assessed at the Bdnf-IV promoter in the hippocampus of rats [at postnatal day (PND) 65] that were iron-deficient (ID) during the fetal-neonatal period. Iron deficiency was induced in rat pups by providing pregnant and nursing dams an ID diet (4 mg/kg Fe) from gestational day (G) 2 through PND7, after which iron deficiency was treated with an iron-sufficient (IS) diet (200 mg/kg Fe). This paradigm resulted in about 60% hippocampal iron loss on PND15 with complete recovery by PND65. For choline supplementation, pregnant rat dams were given dietary choline (5 g/kg) from G11 through G18. DNA methylation was determined by quantitative sequencing of bisulfite-treated DNA, revealing a small alteration at the Bdnf-IV promoter. Chromatin immunoprecipitation analysis showed increased HDAC1 binding accompanied by reduced binding of RNA polymerase II and USF1 at the Bdnf-IV promoter in formerly ID rats. These changes were correlated with altered histone methylations. Prenatal choline supplementation reverses these epigenetic modifications. Collectively, the findings identify epigenetic modifications as a potential mechanism to explicate the long-term repression of Bdnf following fetal and early postnatal iron deficiency.

  2. Plc1p is required for proper chromatin structure and activity of the kinetochore in Saccharomyces cerevisiae by facilitating recruitment of the RSC complex.

    PubMed

    Desai, Parima; Guha, Nilanjan; Galdieri, Luciano; Hadi, Sara; Vancura, Ales

    2009-05-01

    High-fidelity chromosome segregation during mitosis requires kinetochores, protein complexes that assemble on centromeric DNA and mediate chromosome attachment to spindle microtubules. In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in alterations in chromatin structure of centromeres, reduced binding of microtubules to minichromosomes, and a higher frequency of chromosome loss. The mechanism of Plc1p's involvement in kinetochore activity was not initially obvious; however, a testable hypothesis emerged with the discovery of the role of inositol polyphosphates (InsPs), produced by a Plc1p-dependent pathway, in the regulation of chromatin-remodeling complexes. In addition, the remodels structure of chromatin (RSC) chromatin-remodeling complex was found to associate with kinetochores and to affect centromeric chromatin structure. We report here that Plc1p and InsPs are required for recruitment of the RSC complex to kinetochores, which is important for establishing proper chromatin structure of centromeres and centromere proximal regions. Mutations in PLC1 and components of the RSC complex exhibit strong genetic interactions and display synthetic growth defect, altered nuclear morphology, and higher frequency of minichromosome loss. The results thus provide a mechanistic explanation for the previously elusive role of Plc1p and InsPs in kinetochore function.

  3. Discovery of transcription factors and regulatory regions driving in vivo tumor development by ATAC-seq and FAIRE-seq open chromatin profiling.

    PubMed

    Davie, Kristofer; Jacobs, Jelle; Atkins, Mardelle; Potier, Delphine; Christiaens, Valerie; Halder, Georg; Aerts, Stein

    2015-02-01

    Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif

  4. Poly(ADP-ribose) Polymerase-1 (PARP-1) Contributes to the Barrier Function of a Vertebrate Chromatin Insulator*

    PubMed Central

    Aker, Mari; Bomsztyk, Karol; Emery, David W.

    2010-01-01

    The prototypic chromatin insulator cHS4 has proven effective in reducing silencing chromosomal position effects in a variety of settings. Most of this barrier insulator activity has been mapped to a 250-bp core region, as well as to several proteins that bind this region. However, recent studies from our laboratory demonstrated that an extended 400-bp core region of the cHS4 element is necessary to achieve full barrier insulator activity when used as a single copy in the context of recombinant gammaretroviral and lentiviral vectors. In this study, electrophoretic gel mobility shift assays revealed specific DNA-protein binding activities associated with the distal portion of this extended core region. Affinity purification and tandem mass spectrometry studies led to the identification of one of these proteins as poly(ADP-ribose) polymerase-1 (PARP-1). The identity of this binding activity as PARP-1 was subsequently verified by a variety of biochemical studies in vitro and by chromatin immunoprecipitation studies in vivo. Functional studies with gammaretroviral reporter vectors in cell lines and primary mouse bone marrow progenitor cultures showed that cHS4 barrier activity was abrogated upon mutation of the putative PARP-1-binding site or upon treatment with a PARP inhibitor, respectively. The barrier activity of the cHS4 element was also found to be abrogated in studies using bone marrow from Parp1-null mice. Taken together, this study demonstrates that binding of PARP-1 plays a key functional role in the barrier activity of the extended cHS4 insulator core element. PMID:20876582

  5. Histone modifications and chromatin dynamics: a focus on filamentous fungi

    PubMed Central

    Brosch, Gerald; Loidl, Peter; Graessle, Stefan

    2008-01-01

    The readout of the genetic information of eukaryotic organisms is significantly regulated by modifications of DNA and chromatin proteins. Chromatin alterations induce genome-wide and local changes in gene expression and affect a variety of processes in response to internal and external signals during growth, differentiation, development, in metabolic processes, diseases, and abiotic and biotic stresses. This review aims at summarizing the roles of histone H1 and the acetylation and methylation of histones in filamentous fungi and links this knowledge to the huge body of data from other systems. Filamentous fungi show a wide range of morphologies and have developed a complex network of genes that enables them to use a great variety of substrates. This fact, together with the possibility of simple and quick genetic manipulation, highlights these organisms as model systems for the investigation of gene regulation. However, little is still known about regulation at the chromatin level in filamentous fungi. Understanding the role of chromatin in transcriptional regulation would be of utmost importance with respect to the impact of filamentous fungi in human diseases and agriculture. The synthesis of compounds (antibiotics, immunosuppressants, toxins, and compounds with adverse effects) is also likely to be regulated at the chromatin level. PMID:18221488

  6. Chromatin remodeling facilitates DNA incision in UV-damaged nucleosomes.

    PubMed

    Lee, Kyungeun; Kim, Deok Ryong; Ahn, Byungchan

    2004-08-31

    The DNA repair machinery must locate and repair DNA damage all over the genome. As nucleosomes inhibit DNA repair in vitro, it has been suggested that chromatin remodeling might be required for efficient repair in vivo. To investigate a possible contribution of nucleosome dynamics and chromatin remodeling to the repair of UV-photoproducts in nucleosomes, we examined the effect of a chromatin remodeling complex on the repair of UV-lesions by Micrococcus luteus UV endonuclease (ML-UV endo) and T4-endonuclease V (T4-endoV) in reconstituted mononucleosomes positioned at one end of a 175-bp long DNA fragment. Repair by ML-UV endo and T4-endoV was inefficient in mononucleosomes compared with naked DNA. However, the human nucleosome remodeling complex, hSWI/SNF, promoted more homogeneous repair by ML-UV endo and T4-endo V in reconstituted nucleosomes. This result suggests that recognition of DNA damage could be facilitated by a fluid state of the chromatin resulting from chromatin remodeling activities. PMID:15359130

  7. Changes in chromatin structure associated with Alzheimer's disease.

    PubMed

    Lewis, P N; Lukiw, W J; De Boni, U; McLachlan, D R

    1981-11-01

    The enzyme micrococcal nuclease was used to examine the accessibility of chromatin extracted from brains of 13 patients with senile and presenile dementia of the Alzheimer type. Compared with chromatin extracted from brains of 8 patients without neurological signs or brain pathology and brains of 7 patients with nonAlzheimer dementia, Alzheimer chromatin was less accessible to this enzyme. Reduced accessibility was reflected by a reduced yield of mononucleosomes in comparison with dinucleosomes and larger oligomers. Both neuronal and glial chromatin were found to be similarly affected. The reduced yield of mononucleosomes from Alzheimer chromatin is not due to their increased breakdown, but is probably related to protein associated with the internucleosomal linker region that retards nuclease action. Dinucleosomes isolated from control and Alzheimer nuclease digests were examined for their protein complement. Three perchloric acid-soluble proteins situated in the histone H1 region of sodium dodecyl sulfate (SDS) gels were present in elevated levels in Alzheimer dinucleosomes. These results represent the first example of altered chromosomal proteins associated with a diseased state of the brain.

  8. Forced unraveling of chromatin fibers with nonuniform linker DNA lengths

    PubMed Central

    Ozer, Gungor; Collepardo-Guevara, Rosana; Schlick, Tamar

    2015-01-01

    The chromatin fiber undergoes significant structural changes during the cell’s life cycle to modulate DNA accessibility. Detailed mechanisms of such structural transformations of chromatin fibers as affected by various internal and external conditions such as the ionic conditions of the medium, the linker DNA length, and the presence of linker histones, constitute an open challenge. Here we utilize Monte Carlo (MC) simulations of a coarse grained model of chromatin with nonuniform linker DNA lengths as found in vivo to help explain some aspects of this challenge. We investigate the unfolding mechanisms of chromatin fibers with alternating linker lengths of 26-62 bp and 44-79 bp using a series of end-to-end stretching trajectories with and without linker histones and compare results to uniform-linker-length fibers. We find that linker histones increase overall resistance of nonuniform fibers and lead to fiber unfolding with superbeads-on-a-string cluster transitions. Chromatin fibers with nonuniform linker DNA lengths display a more complex, multi-step yet smoother process of unfolding compared to their uniform counterparts, likely due to the existence of a more continuous range of nucleosome-nucleosome interactions. This finding echoes the theme that some heterogeneity in fiber component is biologically advantageous. PMID:25564319

  9. Circadian rhythms and memory formation: regulation by chromatin remodeling.

    PubMed

    Sahar, Saurabh; Sassone-Corsi, Paolo

    2012-01-01

    Epigenetic changes, such as DNA methylation or histone modification, can remodel the chromatin and regulate gene expression. Remodeling of chromatin provides an efficient mechanism of transducing signals, such as light or nutrient availability, to regulate gene expression. CLOCK:BMAL1 mediated activation of clock-controlled genes (CCGs) is coupled to circadian changes in histone modification at their promoters. Several chromatin modifiers, such as the deacetylases SIRT1 and HDAC3 or methyltransferase MLL1, have been shown to be recruited to the promoters of the CCGs in a circadian manner. Interestingly, the central element of the core clock machinery, the transcription factor CLOCK, also possesses histone acetyltransferase activity. Rhythmic expression of the CCGs is abolished in the absence of these chromatin modifiers. Recent research has demonstrated that chromatin remodeling is at the cross-roads of circadian rhythms and regulation of metabolism and aging. It would be of interest to identify if similar pathways exist in the epigenetic regulation of memory formation. PMID:22470318

  10. Chromatin is an ancient innovation conserved between Archaea and Eukarya.

    PubMed

    Ammar, Ron; Torti, Dax; Tsui, Kyle; Gebbia, Marinella; Durbic, Tanja; Bader, Gary D; Giaever, Guri; Nislow, Corey

    2012-12-13

    The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ∼147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved -1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient.DOI:http://dx.doi.org/10.7554/eLife.00078.001.

  11. Chromatin modifications and DNA repair: beyond double-strand breaks

    PubMed Central

    House, Nealia C. M.; Koch, Melissa R.; Freudenreich, Catherine H.

    2014-01-01

    DNA repair must take place in the context of chromatin, and chromatin modifications and DNA repair are intimately linked. The study of double-strand break repair has revealed numerous histone modifications that occur after induction of a DSB, and modification of the repair factors themselves can also occur. In some cases the function of the modification is at least partially understood, but in many cases it is not yet clear. Although DSB repair is a crucial activity for cell survival, DSBs account for only a small percentage of the DNA lesions that occur over the lifetime of a cell. Repair of single-strand gaps, nicks, stalled forks, alternative DNA structures, and base lesions must also occur in a chromatin context. There is increasing evidence that these repair pathways are also regulated by histone modifications and chromatin remodeling. In this review, we will summarize the current state of knowledge of chromatin modifications that occur during non-DSB repair, highlighting similarities and differences to DSB repair as well as remaining questions. PMID:25250043

  12. Deciphering Noncoding RNA and Chromatin Interactions: Multiplex Chromatin Interaction Analysis by Paired-End Tag Sequencing (mChIA-PET).

    PubMed

    Choy, Jocelyn; Fullwood, Melissa J

    2017-01-01

    Genomic DNA is dynamically associated with protein factors and folded to form chromatin fibers. The 3-dimensional (3D) configuration of the chromatin will enable the distal genetic elements to come into close proximity, allowing transcriptional regulation. Noncoding RNA can mediate the 3D structure of chromatin. Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a valuable and powerful technique in molecular biology which allows the study of unbiased, genome-wide de novo chromatin interactions with paired-end tags. Here, we describe the standard version of ChIA-PET and a Multiplex ChIA-PET version. PMID:27662871

  13. TALE proteins bind to both active and inactive chromatin.

    PubMed

    Scott, James N F; Kupinski, Adam P; Kirkham, Christopher M; Tuma, Roman; Boyes, Joan

    2014-02-15

    TALE (transcription activator-like effector) proteins can be tailored to bind to any DNA sequence of choice and thus are of immense utility for genome editing and the specific delivery of transcription activators. However, to perform these functions, they need to occupy their sites in chromatin. In the present study, we have systematically assessed TALE binding to chromatin substrates and find that in vitro TALEs bind to their target site on nucleosomes at the more accessible entry/exit sites, but not at the nucleosome dyad. We show further that in vivo TALEs bind to transcriptionally repressed chromatin and that transcription increases binding by only 2-fold. These data therefore imply that TALEs are likely to bind to their target in vivo even at inactive loci.

  14. Systematic identification of protein combinations mediating chromatin looping

    PubMed Central

    Zhang, Kai; Li, Nan; Ainsworth, Richard I.; Wang, Wei

    2016-01-01

    Chromatin looping plays a pivotal role in gene expression and other biological processes through bringing distal regulatory elements into spatial proximity. The formation of chromatin loops is mainly mediated by DNA-binding proteins (DBPs) that bind to the interacting sites and form complexes in three-dimensional (3D) space. Previously, identification of DBP cooperation has been limited to those binding to neighbouring regions in the proximal linear genome (1D cooperation). Here we present the first study that integrates protein ChIP-seq and Hi-C data to systematically identify both the 1D- and 3D-cooperation between DBPs. We develop a new network model that allows identification of cooperation between multiple DBPs and reveals cell-type-specific and -independent regulations. Using this framework, we retrieve many known and previously unknown 3D-cooperations between DBPs in chromosomal loops that may be a key factor in influencing the 3D organization of chromatin. PMID:27461729

  15. Absence of canonical active chromatin marks in developmentally regulated genes

    PubMed Central

    Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-01-01

    The interplay of active and repressive histone modifications is assumed to play a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated to stable production of RNA, while unmarked chromatin would permit rapid gene activation and de-activation during development. In this case, regulation by transcription factors would play a comparatively more important regulatory role. PMID:26280901

  16. Dynamical DNA accessibility induced by chromatin remodeling and protein binding

    NASA Astrophysics Data System (ADS)

    Montel, F.; Faivre-Moskalenko, C.; Castelnovo, M.

    2014-11-01

    Chromatin remodeling factors are enzymes being able to alter locally chromatin structure at the nucleosomal level and they actively participate in the regulation of gene expression. Using simple rules for individual nucleosome motion induced by a remodeling factor, we designed simulations of the remodeling of oligomeric chromatin, in order to address quantitatively collective effects in DNA accessibility upon nucleosome mobilization. Our results suggest that accessibility profiles are inhomogeneous thanks to borders effects like protein binding. Remarkably, we show that the accessibility lifetime of DNA sequence is roughly doubled in the vicinity of borders as compared to its value in bulk regions far from the borders. These results are quantitatively interpreted as resulting from the confined diffusion of a large nucleosome depleted region.

  17. Spatial organization of chromatin domains and compartments in single chromosomes.

    PubMed

    Wang, Siyuan; Su, Jun-Han; Beliveau, Brian J; Bintu, Bogdan; Moffitt, Jeffrey R; Wu, Chao-ting; Zhuang, Xiaowei

    2016-08-01

    The spatial organization of chromatin critically affects genome function. Recent chromosome-conformation-capture studies have revealed topologically associating domains (TADs) as a conserved feature of chromatin organization, but how TADs are spatially organized in individual chromosomes remains unknown. Here, we developed an imaging method for mapping the spatial positions of numerous genomic regions along individual chromosomes and traced the positions of TADs in human interphase autosomes and X chromosomes. We observed that chromosome folding deviates from the ideal fractal-globule model at large length scales and that TADs are largely organized into two compartments spatially arranged in a polarized manner in individual chromosomes. Active and inactive X chromosomes adopt different folding and compartmentalization configurations. These results suggest that the spatial organization of chromatin domains can change in response to regulation. PMID:27445307

  18. Higher-order structure of Saccharomyces cerevisiae chromatin

    SciTech Connect

    Lowary, P.T.; Widom, J. )

    1989-11-01

    We have developed a method for partially purifying chromatin from Saccharomyces cerevisiae (baker's yeast) to a level suitable for studies of its higher-order folding. This has required the use of yeast strains that are free of the ubiquitous yeast killer virus. Results from dynamic light scattering, electron microscopy, and x-ray diffraction show that the yeast chromatin undergoes a cation-dependent folding into 30-nm filaments that resemble those characteristic of higher-cell chromatin; moreover, the packing of nucleosomes within the yeast 30-nm filaments is similar to that of higher cells. These results imply that yeast has a protein or protein domain that serves the role of the histone H 1 found in higher cells; physical and genetic studies of the yeast activity could help elucidate the structure and function of H 1. Images of the yeast 30-nm filaments can be used to test crossed-linker models for 30-nm filament structure.

  19. Human pescadillo induces large-scale chromatin unfolding.

    PubMed

    Zhang, Hao; Fang, Yan; Huang, Cuifen; Yang, Xiao; Ye, Qinong

    2005-06-01

    The human pescadillo gene encodes a protein with a BRCT domain. Pescadillo plays an important role in DNA synthesis, cell proliferation and transformation. Since BRCT domains have been shown to induce chromatin large-scale unfolding, we tested the role of Pescadillo in regulation of large-scale chromatin unfolding. To this end, we isolated the coding region of Pescadillo from human mammary MCF10A cells. Compared with the reported sequence, the isolated Pescadillo contains in-frame deletion from amino acid 580 to 582. Targeting the Pescadillo to an amplified, lac operator-containing chromosome region in the mammalian genome results in large-scale chromatin decondensation. This unfolding activity maps to the BRCT domain of Pescadillo. These data provide a new clue to understanding the vital role of Pescadillo.

  20. HMGA proteins as modulators of chromatin structure during transcriptional activation

    PubMed Central

    Ozturk, Nihan; Singh, Indrabahadur; Mehta, Aditi; Braun, Thomas; Barreto, Guillermo

    2013-01-01

    High mobility group (HMG) proteins are the most abundant non-histone chromatin associated proteins. HMG proteins bind to DNA and nucleosome and alter the structure of chromatin locally and globally. Accessibility to DNA within chromatin is a central factor that affects DNA-dependent nuclear processes, such as transcription, replication, recombination, and repair. HMG proteins associate with different multi-protein complexes to regulate these processes by mediating accessibility to DNA. HMG proteins can be subdivided into three families: HMGA, HMGB, and HMGN. In this review, we will focus on recent advances in understanding the function of HMGA family members, specifically their role in gene transcription regulation during development and cancer. PMID:25364713

  1. Spatial organization of chromatin domains and compartments in single chromosomes.

    PubMed

    Wang, Siyuan; Su, Jun-Han; Beliveau, Brian J; Bintu, Bogdan; Moffitt, Jeffrey R; Wu, Chao-ting; Zhuang, Xiaowei

    2016-08-01

    The spatial organization of chromatin critically affects genome function. Recent chromosome-conformation-capture studies have revealed topologically associating domains (TADs) as a conserved feature of chromatin organization, but how TADs are spatially organized in individual chromosomes remains unknown. Here, we developed an imaging method for mapping the spatial positions of numerous genomic regions along individual chromosomes and traced the positions of TADs in human interphase autosomes and X chromosomes. We observed that chromosome folding deviates from the ideal fractal-globule model at large length scales and that TADs are largely organized into two compartments spatially arranged in a polarized manner in individual chromosomes. Active and inactive X chromosomes adopt different folding and compartmentalization configurations. These results suggest that the spatial organization of chromatin domains can change in response to regulation.

  2. The integrity of sperm chromatin in young tropical composite bulls.

    PubMed

    Fortes, M R S; Holroyd, R G; Reverter, A; Venus, B K; Satake, N; Boe-Hansen, G B

    2012-07-15

    Sperm chromatin fragmentation is associated with subfertility, but its relationship with age progression in young bulls is poorly understood. The objective was to assess sperm chromatin fragmentation during the early post-pubertal development of 20 tropical composite bulls, using a sperm chromatin structure assay (SCSA) and sperm-bos-halomax (SBH). Bulls were subjected to bull breeding soundness evaluation (BBSE) at mean ages of 13, 18, and 24 mo. Traits measured included liveweight (WT), body condition score (BCS) and scrotal circumference (SC). Semen samples were collected by electroejaculation and assessed for mass activity (MA), motility (Mot), concentration (conc), sperm morphology and chromatin fragmentation. Concentration (r=0.34, P=0.0076), Mot (r=0.36, P=0.0041) and percentage of morphologic normal sperm (percent normal sperm (PNS); r=0.31, P=0.0132) were positively correlated with age. The percentage of sperm with proximal droplets (PD) was negatively correlated with age (r=-0.28, P=0.0348), whereas neither SCSA nor SBH results were significantly correlated with age. The percentage of sperm with chromatin fragmentation using SCSA was correlated with PNS (r=-0.53, P<0.0001), the percentage of sperm with head abnormalities (r=0.68, P<0.0001) and the percentage of intact sperm (Int) with SBH (r=-0.26, P=0.0456). In summary, for assessment of sperm chromatin fragmentation, samples could be equally collected at 13, 18 or 24 mo of age, as results did not vary with age. PMID:22494672

  3. N-Butyrate alters chromatin accessibility to DNA repair enzymes

    SciTech Connect

    Smith, P.J.

    1986-03-01

    Current evidence suggests that the complex nature of mammalian chromatin can result in the concealment of DNA damage from repair enzymes and their co-factors. Recently it has been proposed that the acetylation of histone proteins in chromatin may provide a surveillance system whereby damaged regions of DNA become exposed due to changes in chromatin accessibility. This hypothesis has been tested by: (i) using n-butyrate to induce hyperacetylation in human adenocarcinoma (HT29) cells; (ii) monitoring the enzymatic accessibility of chromatin in permeabilised cells; (iii) measuring u.v. repair-associated nicking of DNA in intact cells and (iv) determining the effects of n-butyrate on cellular sensitivity to DNA damaging agents. The results indicate that the accessibility of chromatin to Micrococcus luteus u.v. endonuclease is enhanced by greater than 2-fold in n-butyrate-treated cells and that there is a corresponding increase in u.v. repair incision rates in intact cells exposed to the drug. Non-toxic levels of n-butyrate induce a block to G1 phase transit and there is a significant growth delay on removal of the drug. Resistance of HT29 cells to u.v.-radiation and adriamycin is enhanced in n-butyrate-treated cells whereas X-ray sensitivity is increased. Although changes in the responses of cells to DNA damaging agents must be considered in relation to the effects of n-butyrate on growth rate and cell-cycle distribution, the results are not inconsistent with the proposal that increased enzymatic-accessibility/repair is biologically favourable for the resistance of cells to u.v.-radiation damage. Overall the results support the suggested operation of a histone acetylation-based chromatin surveillance system in human cells.

  4. Chromatin dynamics during interphase explored by single-particle tracking.

    PubMed

    Levi, Valeria; Gratton, Enrico

    2008-01-01

    Our view of the structure and function of the interphase nucleus has changed drastically in recent years. It is now widely accepted that the nucleus is a well organized and highly compartmentalized organelle and that this organization is intimately related to nuclear function. In this context, chromatin-initially considered a randomly entangled polymer-has also been shown to be structurally organized in interphase and its organization was found to be very important to gene regulation. Relevant and not completely answered questions are how chromatin organization is achieved and what mechanisms are responsible for changes in the positions of chromatin loci in the nucleus. A significant advance in the field resulted from tagging chromosome sites with bacterial operator sequences, and visualizing these tags using green fluorescent protein fused with the appropriate repressor protein. Simultaneously, fluorescence imaging techniques evolved significantly during recent years, allowing observation of the time evolution of processes in living specimens. In this context, the motion of the tagged locus was observed and analyzed to extract quantitative information regarding its dynamics. This review focuses on recent advances in our understanding of chromatin dynamics in interphase with the emphasis placed on the information obtained from single-particle tracking (SPT) experiments. We introduce the basis of SPT methods and trajectory analysis, and summarize what has been learnt by using this new technology in the context of chromatin dynamics. Finally, we briefly describe a method of SPT in a two-photon excitation microscope that has several advantages over methods based on conventional microscopy and review the information obtained using this novel approach to study chromatin dynamics. PMID:18461483

  5. Chromatin dynamics during interphase explored by single particle tracking

    PubMed Central

    Levi, Valeria; Gratton, Enrico

    2009-01-01

    Our view of the structure and function of the interphase nucleus has drastically changed in the last years. It is now widely accepted that the nucleus is a well organized and highly compartmentalized organelle and that this organization is intimately related to nuclear function. In this context, chromatin -initially considered a randomly entangled polymer- has also been shown to be structurally organized in interphase and its organization was found to be very important to gene regulation. Relevant and not completely answered questions are how chromatin organization is achieved and what mechanisms are responsible for changes in the positions of chromatin loci in the nucleus. A significant advance in the field resulted from tagging chromosome sites with bacterial operator sequences, and visualizing these tags using green fluorescent protein fused with the appropriate repressor protein. Simultaneously, fluorescence imaging techniques significantly evolved during the last years allowing the observation of the time evolution of processes in living specimens. In this context, the motion of the tagged locus was observed and analyzed to extract quantitative information regarding its dynamics. This review focuses on recent advances in our understanding of chromatin dynamics in interphase with the emphasis placed on the information obtained from single particle tracking (SPT) experiments. We introduce the basis of SPT methods and trajectories analysis, and summarize what has been learnt by using this new technology in the context of chromatin dynamics. Finally, we briefly describe a method of SPT in a two-photon excitation microscope that has several advantages over methods based on conventional microscopy and review the information obtained by using this novel approach to study chromatin dynamics. PMID:18461483

  6. Measurement of local chromatin compaction by spectral precision distance microscopy

    NASA Astrophysics Data System (ADS)

    Rauch, Joachim; Hausmann, Michael; Solovei, Irina; Horsthemke, Bernhard; Cremer, Thomas; Cremer, Christoph G.

    2000-12-01

    Fluorescence in situ hybridization (FISH) offers an appropriate technique to specifically label any given chromatin region by multi spectrally labeled, specific DNA probes. Using confocal laser scanning microscopy, quantitative measurements on the spatial distribution of labeling sites can be performed in 3D conserved cell nuclei. Recently, 'Spectral Precision Distance Microscopy' has been developed that allows 3D distance measurements between point-like fluorescence objects of different spectral signatures far beyond the diffraction limited resolution. In a well characterized and sequenced DNA region, the Prader- Willi/Angelman region q11-13 on chromosome 15, geometric distances between the fluorescence intensity bary centers of four different 'point-like' labeling sites were measured. More than 300 cell nuclei were evaluated with a 3D resolution equivalent better than 100 nm. The geometric bary center distances in nanometers were compared with the genomic bary center distance in kilobases (kb). A direct correlation, for instance linear correlation between geometric and genomic distances was not observed. From the measured values, a local compaction factor for the high order chromatin folding in the analyzed genome region was calculated. Along the 1000 kb chromatin segment analyzed, which spans nearly the compete Prader-Willi/Angelman region, different compaction factors were found. The compaction factor 40 typical for a straight 30 nm chromatin fiber was not observed. This shows that chromatin folding and compaction in intact nuclei may be more complex. With SPDM, however, a microscopical technique is available that can sensitively analyze chromatin organization in the 100 nm range in 3D conserved cell nuclei.

  7. Statistical physics of nucleosome positioning and chromatin structure

    NASA Astrophysics Data System (ADS)

    Morozov, Alexandre

    2012-02-01

    Genomic DNA is packaged into chromatin in eukaryotic cells. The fundamental building block of chromatin is the nucleosome, a 147 bp-long DNA molecule wrapped around the surface of a histone octamer. Arrays of nucleosomes are positioned along DNA according to their sequence preferences and folded into higher-order chromatin fibers whose structure is poorly understood. We have developed a framework for predicting sequence-specific histone-DNA interactions and the effective two-body potential responsible for ordering nucleosomes into regular higher-order structures. Our approach is based on the analogy between nucleosomal arrays and a one-dimensional fluid of finite-size particles with nearest-neighbor interactions. We derive simple rules which allow us to predict nucleosome occupancy solely from the dinucleotide content of the underlying DNA sequences.Dinucleotide content determines the degree of stiffness of the DNA polymer and thus defines its ability to bend into the nucleosomal superhelix. As expected, the nucleosome positioning rules are universal for chromatin assembled in vitro on genomic DNA from baker's yeast and from the nematode worm C.elegans, where nucleosome placement follows intrinsic sequence preferences and steric exclusion. However, the positioning rules inferred from in vivo C.elegans chromatin are affected by global nucleosome depletion from chromosome arms relative to central domains, likely caused by the attachment of the chromosome arms to the nuclear membrane. Furthermore, intrinsic nucleosome positioning rules are overwritten in transcribed regions, indicating that chromatin organization is actively managed by the transcriptional and splicing machinery.

  8. Rapid genome-scale mapping of chromatin accessibility in tissue

    PubMed Central

    2012-01-01

    Background The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples. Results Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh. Conclusion The TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied across a broad range of

  9. A Direct Intersection between p53 and Transforming Growth Factor β Pathways Targets Chromatin Modification and Transcription Repression of the α-Fetoprotein Gene†

    PubMed Central

    Wilkinson, Deepti S.; Ogden, Stacey K.; Stratton, Sabrina A.; Piechan, Julie L.; Nguyen, Thi T.; Smulian, George A.; Barton, Michelle Craig

    2005-01-01

    We purified the oncoprotein SnoN and found that it functions as a corepressor of the tumor suppressor p53 in the regulation of the hepatic α-fetoprotein (AFP) tumor marker gene. p53 promotes SnoN and histone deacetylase interaction at an overlapping Smad binding, p53 regulatory element (SBE/p53RE) in AFP. Comparison of wild-type and p53-null mouse liver tissue by using chromatin immunoprecipitation (ChIP) reveals that the absence of p53 protein correlates with the disappearance of SnoN at the SBE/p53RE and loss of AFP developmental repression. Treatment of AFP-expressing hepatoma cells with transforming growth factor-β1 (TGF-β1) induced SnoN transcription and Smad2 activation, concomitant with AFP repression. ChIP assays show that TGF-β1 stimulates p53, Smad4, P-Smad2 binding, and histone H3K9 deacetylation and methylation, at the SBE/p53RE. Depletion, by small interfering RNA, of SnoN and/or p53 in hepatoma cells disrupted repression of AFP transcription. These findings support a model of cooperativity between p53 and TGF-β effectors in chromatin modification and transcription repression of an oncodevelopmental tumor marker gene. PMID:15657445

  10. Tumor promoter arsenite stimulates histone H3 phosphoacetylation of proto-oncogenes c-fos and c-jun chromatin in human diploid fibroblasts.

    PubMed

    Li, Ji; Gorospe, Myriam; Barnes, Janice; Liu, Yusen

    2003-04-11

    Although epidemiological studies have long established that inorganic arsenic is a potent human carcinogen, the underlying mechanisms are still poorly understood. Recent studies suggest that inorganic arsenic may act as a tumor promoter by perturbing key signaling transduction pathways. We have shown previously that arsenite can potently activate the mitogen-activated protein kinase cascades and induce the expression of proliferation-associated genes, including proto-oncogenes c-jun and c-fos. In order to elucidate further the molecular mechanisms underlying its tumor-promoting properties, we investigated the signaling events involved in arsenite-mediated induction of c-fos and c-jun. We found that induction of both c-fos and c-jun by arsenite can be substantially inhibited by the MEK- selective inhibitor U0126, suggesting that the ERK pathway is critically involved in their up-regulation. Interestingly, arsenite dramatically induced the phosphorylation and acetylation of histone H3 preceding the induction of mRNAs encoding c-fos and c-jun. Finally, chromatin immunoprecipitation assays revealed that arsenite treatment markedly induced the phosphorylation/acetylation of histone H3 associated with the c-fos and c-jun genes through an ERK-dependent pathway. Our results strongly suggest that arsenic-triggered alterations in chromatin structure perturb specific gene transcription, including that of proto-oncogenes c-jun and c-fos, and may thereby contribute to the carcinogenic process.

  11. CDC-48/p97 coordinates CDT-1 degradation with GINS chromatin dissociation to ensure faithful DNA replication.

    PubMed

    Franz, André; Orth, Michael; Pirson, Paul A; Sonneville, Remi; Blow, J Julian; Gartner, Anton; Stemmann, Olaf; Hoppe, Thorsten

    2011-10-01

    Faithful transmission of genomic information requires tight spatiotemporal regulation of DNA replication factors. In the licensing step of DNA replication, CDT-1 is loaded onto chromatin to subsequently promote the recruitment of additional replication factors, including CDC-45 and GINS. During the elongation step, the CDC-45/GINS complex moves with the replication fork; however, it is largely unknown how its chromatin association is regulated. Here, we show that the chaperone-like ATPase CDC-48/p97 coordinates degradation of CDT-1 with release of the CDC-45/GINS complex. C. elegans embryos lacking CDC-48 or its cofactors UFD-1/NPL-4 accumulate CDT-1 on mitotic chromatin, indicating a critical role of CDC-48 in CDT-1 turnover. Strikingly, CDC-48(UFD-1/NPL-4)-deficient embryos show persistent chromatin association of CDC-45/GINS, which is a consequence of CDT-1 stabilization. Moreover, our data confirmed a similar regulation in Xenopus egg extracts, emphasizing a conserved coordination of licensing and elongation events during eukaryotic DNA replication by CDC-48/p97.

  12. Expanding the phenotype associated with FOXG1 mutations and in vivo FoxG1 chromatin-binding dynamics.

    PubMed

    De Filippis, R; Pancrazi, L; Bjørgo, K; Rosseto, A; Kleefstra, T; Grillo, E; Panighini, A; Cardarelli, F; Meloni, I; Ariani, F; Mencarelli, M A; Hayek, J; Renieri, A; Costa, M; Mari, F

    2012-10-01

    Mutations in the Forkhead box G1 (FOXG1) gene, a brain specific transcriptional factor, are responsible for the congenital variant of Rett syndrome. Until now FOXG1 point mutations have been reported in 12 Rett patients. Recently seven additional patients have been reported with a quite homogeneous severe phenotype designated as the FOXG1 syndrome. Here we describe two unrelated patients with a de novo FOXG1 point mutation, p.Gln46X and p.Tyr400X, respectively, having a milder phenotype and sharing a distinctive facial appearance. Although FoxG1 action depends critically on its binding to chromatin, very little is known about the dynamics of this process. Using fluorescence recovery after photobleaching, we showed that most of the GFP-FoxG1 fusion protein associates reversibly to chromatin whereas the remaining fraction is bound irreversibly. Furthermore, we showed that the two pathologic derivatives of FoxG1 described in this paper present a dramatic alteration in chromatin affinity and irreversibly bound fraction in comparison with Ser323fsX325 mutant (associated with a severe phenotype) and wild type Foxg1 protein. Our observations suggest that alterations in the kinetics of FoxG1 binding to chromatin might contribute to the pathological effects of FOXG1 mutations.

  13. Clks 1, 2 and 4 prevent