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Sample records for addition chromatin immunoprecipitation

  1. Chromatin Immunoprecipitation for Human Monocyte Derived Macrophages

    PubMed Central

    Wooden, Jessica; Ciborowski, Pawel

    2014-01-01

    The importance of Chromatin Immunoprecipitation (ChIP) technology has grown exponentially along with an increased interest in epigenetic regulation. The correlation of transcription factors with histone marks is now well established as the center of epigenetic studies; therefore, precise knowledge about histone marks is critical to unravel their molecular function and to understand their role in biological systems. This knowledge constantly accumulates and is provided openly in the expanding hubs of information such as the USCS Genome Browser. Nevertheless, as we gain more knowledge, we realize that the DNA-protein interactions are not driven by a “one size fits all” rule. Also, the diversity of interactions between DNA, histones, and transcriptional regulators is much bigger than previously considered. Besides a detailed protocol of sample preparation for the ChIP assay from primary human monocyte-derived macrophages (MDM)a, we show that differences between various types of cells exist. Furthermore, we can postulate that such variations exist between transformed macrophage-like cell lines and primary macrophages obtained from healthy volunteers. We found that the most efficient fixation time for MDM is 10 minutes. Finally, to perform multiple analytical assays, we showed that even with thorough methodology, the yield of material obtained from primary cells is the major challenge. PMID:25220915

  2. Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses.

    PubMed

    Teste, Bruno; Champ, Jerome; Londono-Vallejo, Arturo; Descroix, Stéphanie; Malaquin, Laurent; Viovy, Jean-Louis; Draskovic, Irena; Mottet, Guillaume

    2017-01-31

    Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.

  3. 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

  4. Microfluidics Technologies for Low Cell Number Chromatin Immunoprecipitation.

    PubMed

    Wu, Angela R; Quake, Stephen R

    2016-04-01

    Protein-DNA interactions are responsible for numerous critical cellular events: For example, gene expression and silencing are mediated by transcription factor protein binding and histone protein modifications, and DNA replication and repair rely on site-specific protein binding. Chromatin immunoprecipitation (ChIP) is the only molecular assay that directly determines, in a living cell, the binding association between a protein of interest and specific genomic loci. It is an indispensible tool in the biologist's toolbox, but the many limitations of this technique prevent broad adoption of ChIP in biological studies. The typical ChIP assay can take up to 1 wk to complete, and the process is technically tricky, yet tedious. The ChIP assay yields are also low, thus requiring on the order of millions to billions of cells as starting material, which makes the assay unfeasible for studies using rare or precious samples. For example, fluorescence-activated cell sorting (FACS) of cancer stem cells (CSCs) obtained from primary tumors, rarely yields more than ~100,000 CSCs per tumor. This protocol describes a microfluidics-based strategy for performing ChIP, which uses automation and scalability to reduce both total and hands-on assay time, and improve throughput. It allows whole fixed cells as input, and enables automated ChIP from as few as 2000 cells.

  5. Allele-specific chromatin immunoprecipitation studies show genetic influence on chromatin state in human genome.

    PubMed

    Kadota, Mitsutaka; Yang, Howard H; Hu, Nan; Wang, Chaoyu; Hu, Ying; Taylor, Philip R; Buetow, Kenneth H; Lee, Maxwell P

    2007-05-18

    Several recent studies have shown a genetic influence on gene expression variation, including variation between the two chromosomes within an individual and variation between individuals at the population level. We hypothesized that genetic inheritance may also affect variation in chromatin states. To test this hypothesis, we analyzed chromatin states in 12 lymphoblastoid cells derived from two Centre d'Etude du Polymorphisme Humain families using an allele-specific chromatin immunoprecipitation (ChIP-on-chip) assay with Affymetrix 10K SNP chip. We performed the allele-specific ChIP-on-chip assays for the 12 lymphoblastoid cells using antibodies targeting at RNA polymerase II and five post-translation modified forms of the histone H3 protein. The use of multiple cell lines from the Centre d'Etude du Polymorphisme Humain families allowed us to evaluate variation of chromatin states across pedigrees. These studies demonstrated that chromatin state clustered by family. Our results support the idea that genetic inheritance can determine the epigenetic state of the chromatin as shown previously in model organisms. To our knowledge, this is the first demonstration in humans that genetics may be an important factor that influences global chromatin state mediated by histone modification, the hallmark of the epigenetic phenomena.

  6. 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.

  7. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes.

    PubMed

    Mohammed, Hisham; Taylor, Christopher; Brown, Gordon D; Papachristou, Evaggelia K; Carroll, Jason S; D'Santos, Clive S

    2016-02-01

    Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) is a method that allows the study of protein complexes, in particular chromatin and transcription factor complexes, in a rapid and robust manner by mass spectrometry (MS). The method can be used in parallel with chromatin immunoprecipitation-sequencing (ChIP-seq) experiments to provide information on both the cistrome and interactome for a given protein. The method uses formaldehyde fixation to stabilize protein complexes. By using antibodies against the endogenous target, the cross-linked complex is immunoprecipitated, rigorously washed, and then digested into peptides while avoiding antibody contamination (on-bead digestion). By using this method, MS identification of the target protein and several dozen interacting proteins is possible using a 100-min LC-MS/MS run. The protocol does not require substantial proteomics expertise, and it typically takes 2-3 d from the collection of material to results.

  8. DNA-Binding Factor Target Identification by Chromatin Immunoprecipitation (ChIP) in Plants.

    PubMed

    Posé, David; Yant, Levi

    2016-01-01

    Chromatin immunoprecipitation (ChIP) allows the precise identification of genomic loci that physically interact with a protein of interest, whether that protein is a transcription factor, a core polymerase, a histone, or other chromatin-associated protein. In short, tissue is first cross-linked to freeze a population of DNA-protein interactions at a stage of interest. Chromatin is then extracted, fragmented, and incubated with a specific antibody against the protein of interest. Next, the resultant DNA-protein complexes are immunoprecipitated and captured using beads that bind to the antibody constant region. Samples are finally reverse cross-linked to separate the bound fragments and the DNA is purified. This DNA is analyzed by quantitative PCR for enrichment of genomic regions expected to be bound by the protein under study. The protocol detailed in this chapter has been successfully applied in the identification of target genes for seven transcriptional regulators of diverse classes involved in Arabidopsis thaliana floral transition.

  9. 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

  10. Mapping genomic targets of DNA helicases by chromatin immunoprecipitation in Saccharomyces cerevisiae.

    PubMed

    Cobb, Jennifer; van Attikum, Haico

    2010-01-01

    DNA helicases utilize the energy of nucleotide hydrolysis to unwind the two annealed strands of the DNA helix and are involved in many aspects of DNA metabolism such as replication, recombination, and repair. Chromatin immunoprecipitation (ChIP) has been instrumental in determining the genomic targets of many DNA helicases and DNA helicase-containing complexes including the minichromosome maintenance (Mcm) proteins 2-7, the RecQ helicase Sgs1 as well as the Rvb1 and Rvb2 helicase-containing INO80 and SWR1 chromatin remodeling complexes. Here we describe a ChIP method that has been successfully used to map these proteins at chromosomal double-strand breaks and replication forks in the model organism Saccharomyces cerevisiae.

  11. Chromatin immunoprecipitation microarrays for identification of genes silenced by histone H3 lysine 9 methylation.

    PubMed

    Kondo, Yutaka; Shen, Lanlan; Yan, Pearlly S; Huang, Tim Hui-Ming; Issa, Jean-Pierre J

    2004-05-11

    Switching from acetylation to methylation at histone H3 lysine 9 (K9) has recently been shown to contribute to euchromatin gene silencing. To identify genes silenced by K9 modifications, we probed a human CpG island microarray with DNA obtained by chromatin immunoprecipitation (ChIP) in a cancer cell line using an anti-H3-K9 methylated antibody or an anti-H3-K9 acetylated antibody. Of the 27 clones with the highest signal ratio of K9 methylation over acetylation (Me/Ac), 13 contained repetitive sequences. Among 14 nonrepetitive clones, we identified 11 genes (seven known and four previously undescribed), one EST, and two unknown fragments. Using ChIP-PCR, all 18 examined clones showed higher ratios of H3-K9 Me/Ac than the active gene control, P21, thus confirming the microarray data. In addition, we found a strong correlation between the K9 Me/Ac ratio and CpG island DNA methylation (R = 0.92, P < 0.01), and five of seven genes examined (megalin, thrombospondin-4, KR18, latrophilin-3, and phosphatidylinositol-3-OH kinase P101 subunit) showed lack of expression by RT-PCR and reactivation by DNA methylation and/or histone deacetylase inhibition, suggesting that these genes are true targets of silencing through histone modifications. All five genes also showed significant DNA methylation in a cell line panel and in primary colon cancers. Our data suggest that CpG island microarray coupled with ChIP can identify novel targets of gene silencing in cancer. This unbiased approach confirms the tight coupling between DNA methylation and histone modifications in cancer and could be used to probe gene silencing in nonneoplastic conditions as well.

  12. Direct targets of the tomato-ripening regulator RIN identified by transcriptome and chromatin immunoprecipitation analyses.

    PubMed

    Fujisawa, Masaki; Shima, Yoko; Higuchi, Naoki; Nakano, Toshitsugu; Koyama, Yoshiyuki; Kasumi, Takafumi; Ito, Yasuhiro

    2012-06-01

    The physiological and biochemical changes in fruit ripening produce key attributes of fruit quality including color, taste, aroma and texture. These changes are driven by the highly regulated and synchronized activation of a huge number of ripening-associated genes. In tomato (Solanum lycopersicum), a typical climacteric fruit, the MADS-box transcription factor RIN is one of the earliest-acting ripening regulators, required for both ethylene-dependent and ethylene-independent pathways. Although we previously identified several direct RIN targets, many additional targets remain unidentified, likely including key ripening-associated genes. Here, we report the identification of novel RIN targets by transcriptome and chromatin immunoprecipitation (ChIP) analyses. Transcriptome comparisons by microarray of wild-type and rin mutant tomatoes identified 342 positively regulated genes and 473 negatively regulated genes by RIN during ripening. Most of the positively regulated genes contained possible RIN-binding (CArG-box) sequences in their promoters. Subsequently, we selected six genes from the positively regulated genes and a ripening regulator gene, CNR, and assayed their promoters by quantitative ChIP-PCR to examine RIN binding. All of the seven genes, which are involved in cell wall modification, aroma and flavor development, pathogen defense and transcriptional regulation during ripening, are targets of RIN, suggesting that RIN may control multiple diverse ripening processes. In particular, RIN directly regulates the expression of the ripening-associated transcription factors, CNR, TDR4 and a GRAS family gene, providing an important clue to elucidate the complicated transcriptional cascade for fruit ripening.

  13. Protocol: Chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species

    PubMed Central

    2012-01-01

    In this report we describe a chromatin immunoprecipitation (ChIP) protocol for two fully sequenced model diatom species Phaeodactylum tricornutum and Thalassiosira pseudonana. This protocol allows the extraction of satisfactory amounts of chromatin and gives reproducible results. We coupled the ChIP assay with real time quantitative PCR. Our results reveal that the two major histone marks H3K4me2 and H3K9me2 exist in P. tricornutum and T. pseudonana. As in other eukaryotes, H3K4me2 marks active genes whereas H3K9me2 marks transcriptionally inactive transposable elements. Unexpectedly however, T. pseudonana housekeeping genes also show a relative enrichment of H3K9me2. We also discuss optimization of the procedure, including growth conditions, cross linking and sonication. Validation of the protocol provides a set of genes and transposable elements that can be used as controls for studies using ChIP in each diatom species. This protocol can be easily adapted to other diatoms and eukaryotic phytoplankton species for genetic and biochemical studies. PMID:23217141

  14. Features of Mammalian microRNA Promoters Emerge from Polymerase II Chromatin Immunoprecipitation Data

    PubMed Central

    Gordon, Ben; Bhattacharjee, Arindam; Kaminski, Naftali; Benos, Panayiotis V.

    2009-01-01

    Background MicroRNAs (miRNAs) are short, non-coding RNA regulators of protein coding genes. miRNAs play a very important role in diverse biological processes and various diseases. Many algorithms are able to predict miRNA genes and their targets, but their transcription regulation is still under investigation. It is generally believed that intragenic miRNAs (located in introns or exons of protein coding genes) are co-transcribed with their host genes and most intergenic miRNAs transcribed from their own RNA polymerase II (Pol II) promoter. However, the length of the primary transcripts and promoter organization is currently unknown. Methodology We performed Pol II chromatin immunoprecipitation (ChIP)-chip using a custom array surrounding regions of known miRNA genes. To identify the true core transcription start sites of the miRNA genes we developed a new tool (CPPP). We showed that miRNA genes can be transcribed from promoters located several kilobases away and that their promoters share the same general features as those of protein coding genes. Finally, we found evidence that as many as 26% of the intragenic miRNAs may be transcribed from their own unique promoters. Conclusion miRNA promoters have similar features to those of protein coding genes, but miRNA transcript organization is more complex. PMID:19390574

  15. Chromatin Immunoprecipitation Assay to Identify Genomic Binding Sites of Regulatory Factors.

    PubMed

    Wagner, Meike; Jung, Johannes; Koslowski, Michael; Türeci, Özlem; Tiwari, Vijay K; Sahin, Ugur

    2016-01-01

    DNA-protein interactions are vital to fundamental cellular events including transcription, replication, DNA repair, and recombination. Thus, their study holds the key to our understanding of mechanisms underlying normal development and homeostasis as well as disease. Transcriptional regulation is a highly complex process that involves recruitment of numerous factors resulting in formation of multi-protein complexes at gene promoters to regulate gene expression. The studied proteins can be, for example, transcription factors, epigenetic regulators, co-activators, co-repressors, or ligand-activated nuclear receptors as estrogen receptor-α (ERα) bound either directly to the DNA or indirectly by interaction with other DNA-bound factors. Chromatin immunoprecipitation (ChIP) assay is a powerful method to study interactions of proteins and a specific genomic DNA region. Recruitment of ERα to promoters of estrogen-dependent genes is a common mechanism to activate or enhance gene transcription in breast cancer thus promoting tumor progression. In this chapter, we demonstrate a stepwise protocol for ChIP assay using binding of ERα to its genomic targets after stimulation with 17β-estradiol (E2) in breast cancer cells as an example.

  16. Immediate chromatin immunoprecipitation and on-bead quantitative PCR analysis: a versatile and rapid ChIP procedure.

    PubMed

    Harmeyer, Kayla M; South, Paul F; Bishop, Brett; Ogas, Joe; Briggs, Scott D

    2015-03-31

    Genome-wide chromatin immunoprecipitation (ChIP) studies have brought significant insight into the genomic localization of chromatin-associated proteins and histone modifications. The large amount of data generated by these analyses, however, require approaches that enable rapid validation and analysis of biological relevance. Furthermore, there are still protein and modification targets that are difficult to detect using standard ChIP methods. To address these issues, we developed an immediate chromatin immunoprecipitation procedure which we call ZipChip. ZipChip significantly reduces the time and increases sensitivity allowing for rapid screening of multiple loci. Here we describe how ZipChIP enables detection of histone modifications (H3K4 mono- and trimethylation) and two yeast histone demethylases, Jhd2 and Rph1, which were previously difficult to detect using standard methods. Furthermore, we demonstrate the versatility of ZipChIP by analyzing the enrichment of the histone deacetylase Sir2 at heterochromatin in yeast and enrichment of the chromatin remodeler, PICKLE, at euchromatin in Arabidopsis thaliana.

  17. Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors.

    PubMed

    Heisig, Julia; Weber, David; Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

    2012-01-01

    HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.

  18. Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA

    PubMed Central

    Statham, Aaron L.; Robinson, Mark D.; Song, Jenny Z.; Coolen, Marcel W.; Stirzaker, Clare; Clark, Susan J.

    2012-01-01

    The complex relationship between DNA methylation, chromatin modification, and underlying DNA sequence is often difficult to unravel with existing technologies. Here, we describe a novel technique based on high-throughput sequencing of bisulfite-treated chromatin immunoprecipitated DNA (BisChIP-seq), which can directly interrogate genetic and epigenetic processes that occur in normal and diseased cells. Unlike most previous reports based on correlative techniques, we found using direct bisulfite sequencing of Polycomb H3K27me3-enriched DNA from normal and prostate cancer cells that DNA methylation and H3K27me3-marked histones are not always mutually exclusive, but can co-occur in a genomic region-dependent manner. Notably, in cancer, the co-dependency of marks is largely redistributed with an increase of the dual repressive marks at CpG islands and transcription start sites of silent genes. In contrast, there is a loss of DNA methylation in intergenic H3K27me3-marked regions. Allele-specific methylation status derived from the BisChIP-seq data clearly showed that both methylated and unmethylated alleles can simultaneously be associated with H3K27me3 histones, highlighting that DNA methylation status in these regions is not dependent on Polycomb chromatin status. BisChIP-seq is a novel approach that can be widely applied to directly interrogate the genomic relationship between allele-specific DNA methylation, histone modification, or other important epigenetic regulators. PMID:22466171

  19. 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

  20. 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

  1. A comparative analysis of shotgun-cloning and tagged-random amplification-cloning of chromatin immunoprecipitation-isolated genome fragments.

    PubMed

    White, Robert B; Ziman, Melanie R

    2006-07-28

    The cloning of transcription factor antibody-immunoprecipitated genomic fragments from chromatin immunoprecipitation (ChIP) experiments is a technically challenging procedure, especially when the input genomic DNA is isolated from whole tissues (in vivo) rather than cultured cells. Here we adapt a technique known as Tagged-Random PCR (T-PCR) to amplify ChIP-immunoprecipitated DNA from mouse embryonic tissue prior to cloning. Importantly, we then compare this technique with tandem shotgun-cloning experiments in terms of its capacity to identify target genes. We find that T-PCR dramatically increases the efficiency of cloning ChIP fragments without distortion of the relative location of cloned fragments to putative target genes. Thus, T-PCR is a simple procedure which greatly enhances the efficiency of cloning tissue-derived ChIP fragments.

  2. Chromatin immunoprecipitation scanning identifies glucocorticoid receptor binding regions in the proximal promoter of a ubiquitously expressed glucocorticoid target gene in brain.

    PubMed

    van der Laan, Siem; Sarabdjitsingh, R Angela; Van Batenburg, Marcel F; Lachize, Servane B; Li, Hualing; Dijkmans, Thomas F; Vreugdenhil, Erno; de Kloet, E Ron; Meijer, Onno C

    2008-09-01

    While the actions of glucocorticoids on brain functions have been comprehensively studied, the underlying genomic mechanisms are poorly understood. In this study, we show that glucocorticoid-induced leucine zipper (GILZ) mRNA is strongly and ubiquitously induced in rat brain. To decipher the molecular mechanisms underlying these genomic effects, it is of interest to identify the regulatory sites in the promoter region. Alignment of the rat GILZ promoter with the well-characterized human promoter resulted in poor sequence homology. Consequently, we analyzed the rat 5' flanking sequence by Matrix REDUCE and identified two high-affinity glucocorticoid response elements (GRE) located 2 kb upstream of the transcription start site. These findings were corroborated using the glucocorticoid receptor (GR) expressing Ns-1 PC12 rat cell-line. In these cells, dexamethasone treatment leads to a progressive increase of GILZ mRNA expression levels via a GR-dependent mechanism. Subsequently, using chromatin immunoprecipitation assays we show that the two high-affinity GREs are located within the GR-binding regions. Lastly, we demonstrate using multiple tissue in situ hybridization a marked increase in mRNA expression levels in spleen, thymus, heart, lung, liver, muscle, testis, kidney, colon, ileum, as well as in brain and conclude that the GILZ gene can be used to study glucocorticoid effects in many additional rodent tissues.

  3. Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules.

    PubMed

    Peach, Sally E; Rudomin, Emily L; Udeshi, Namrata D; Carr, Steven A; Jaffe, Jacob D

    2012-05-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.

  4. Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules*

    PubMed Central

    Peach, Sally E.; Rudomin, Emily L.; Udeshi, Namrata D.; Carr, Steven A.; Jaffe, Jacob D.

    2012-01-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the “histone code.” We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to “canonical” chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living

  5. Binding of lac repressor-GFP fusion protein to lac operator sites inserted in the tobacco chloroplast genome examined by chromatin immunoprecipitation.

    PubMed

    Newell, Christine A; Gray, John C

    2010-08-01

    Chromatin immunoprecipitation (ChIP) has been used to detect binding of DNA-binding proteins to sites in nuclear and mitochondrial genomes. Here, we describe a method for detecting protein-binding sites on chloroplast DNA, using modifications to the nuclear ChIP procedures. The method was developed using the lac operator (lacO)/lac repressor (LacI) system from Escherichia coli. The lacO sequences were integrated into a single site between the rbcL and accD genes in tobacco plastid DNA and homoplasmic transplastomic plants were crossed with transgenic tobacco plants expressing a nuclear-encoded plastid-targeted GFP-LacI fusion protein. In the progeny, the GFP-LacI fusion protein could be visualized in living tissues using confocal microscopy, and was found to co-localize with plastid nucleoids. Isolated chloroplasts from the lacO/GFP-LacI plants were lysed, treated with micrococcal nuclease to digest the DNA to fragments of approximately 600 bp and incubated with antibodies to GFP and protein A-Sepharose. PCR analysis on DNA extracted from the immunoprecipitate demonstrated IPTG (isopropylthiogalactoside)-sensitive binding of GFP-LacI to lacO. Binding of GFP-LacI to endogenous sites in plastid DNA showing sequence similarity to lacO was also detected, but required reversible cross-linking with formaldehyde. This may provide a general method for the detection of binding sites on plastid DNA for specific proteins.

  6. 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).

  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 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

  9. 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

  10. 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

  11. RNA immunoprecipitation for determining RNA-protein associations in vivo.

    PubMed

    Gilbert, Chris; Svejstrup, Jesper Q

    2006-08-01

    Similar to chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP) can be used to detect the association of individual proteins with specific nucleic acid regions, in this case on RNA. Live cells are treated with formaldehyde to generate protein-RNA cross-links between molecules that are in close proximity in vivo. RNA sequences that cross-link with a given protein are isolated by immunoprecipitation of the protein, and reversal of the formaldehyde cross-linking permits recovery and quantitative analysis of the immunoprecipitated RNA by reverse transcription PCR. The basics of RIP are very similar to those of ChIP, but with some important caveats. This unit describes the RIP procedure for Saccharomyces cerevisiae. Although the corresponding steps for metazoan cells have not yet been worked out, it is likely that the yeast procedure can easily be adapted for use in other organisms.

  12. Chromatin enrichment for proteomics

    PubMed Central

    Kustatscher, Georg; Wills, Karen L. H.; Furlan, Cristina; Rappsilber, Juri

    2015-01-01

    During interphase, chromatin hosts fundamental cellular processes, such as gene expression, DNA replication and DNA damage repair. To analyze chromatin on a proteomic scale, we have developed chromatin enrichment for proteomics (ChEP), which is a simple biochemical procedure that enriches interphase chromatin in all its complexity. It enables researchers to take a ‘snapshot’ of chromatin and to isolate and identify even transiently bound factors. In ChEP, cells are fixed with formaldehyde; subsequently, DNA together with all cross-linked proteins is isolated by centrifugation under denaturing conditions. This approach enables the analysis of global chromatin composition and its changes, which is in contrast with existing chromatin enrichment procedures, which either focus on specific chromatin loci (e.g., affinity purification) or are limited in specificity, such as the analysis of the chromatin pellet (i.e., analysis of all insoluble nuclear material). ChEP takes half a day to complete and requires no specialized laboratory skills or equipment. ChEP enables the characterization of chromatin response to drug treatment or physiological processes. Beyond proteomics, ChEP may preclear chromatin for chromatin immunoprecipitation (ChIP) analyses. PMID:25101823

  13. Chromatin endogenous cleavage and psoralen crosslinking assays to analyze rRNA gene chromatin in vivo.

    PubMed

    Griesenbeck, Joachim; Wittner, Manuel; Charton, Romain; Conconi, Antonio

    2012-01-01

    In eukaryotes, multiple copies of ribosomal RNA (rRNA) genes co-exist in two different chromatin states: actively transcribed (nucleosome depleted) chromatin, and nontranscribed (nucleosomal) chromatin. The presence of two rRNA gene populations compromises the interpretation of analyses obtained by the standard biochemical methods that are used to study chromatin structure (e.g., nuclease digestion and chromatin immunoprecipitation). Here, we provide a protocol to investigate the specific association of proteins with the two rRNA gene chromatin populations in vivo, using Saccharomyces cerevisiae as a model eukaryote.

  14. Immunoprecipitation of SV40 replicating minichromosomes complexed with bacteriophage T4 gene 32 protein.

    PubMed Central

    Miranda, E I; Garrido-Guerrero, E; Garcia-Carranca, A; Gariglio, P

    1992-01-01

    Simian Virus 40 (SV40) DNA replication is a useful model to study eukaryotic cell DNA replication because it encodes only one replication protein and its genome has a nucleoprotein structure ('minichromosome') indistinguishable from cellular chromatin. Late after infection SV40 replicating DNA molecules represent about 5% of total viral minichromosomes. Since gene 32 protein (P32) from bacteriophage T4 interacts with single-stranded DNA and SV40 replication complexes are expected to contain single-stranded regions at the replication forks, we asked whether P32 might be used to isolate replicating SV40 minichromosomes. When nuclear extracts from SV40 infected cells were treated sequentially with P32 and anti-P32 antibodies, pulse-labeled minichromosomes were selectively immunoprecipitated. Agarose gel electrophoresis analysis confirmed that immunoprecipitated material corresponded to SV40 replicative intermediates. Protein analysis of the pelleted material revealed several proteins of viral and cellular origin. Among them, T antigen and histones were found to be complexed with at least other three proteins from cellular origin, to the replicative complexes. Additionally, anti-P32 antibodies were able to detect three cellular proteins of approximately 70, 32 and 13 kDa in western blots. These proteins could correspond to those found as part of an eukaryotic multisubunit single-stranded DNA binding protein. The use of P32 and anti-P32 antibodies thus allows the separation of replicating from mature SV40 minichromosomes and can constitute a novel method to enrich and to study replicative active chromatin. Images PMID:1311833

  15. Chromatin Dynamics during Lytic Infection with Herpes Simplex Virus 1

    PubMed Central

    Conn, Kristen L.; Schang, Luis M.

    2013-01-01

    Latent HSV-1 genomes are chromatinized with silencing marks. Since 2004, however, there has been an apparent inconsistency in the studies of the chromatinization of the HSV-1 genomes in lytically infected cells. Nuclease protection and chromatin immunoprecipitation assays suggested that the genomes were not regularly chromatinized, having only low histone occupancy. However, the chromatin modifications associated with transcribed and non-transcribed HSV-1 genes were those associated with active or repressed transcription, respectively. Moreover, the three critical HSV-1 transcriptional activators all had the capability to induce chromatin remodelling, and interacted with critical chromatin modifying enzymes. Depletion or overexpression of some, but not all, chromatin modifying proteins affected HSV-1 transcription, but often in unexpected manners. Since 2010, it has become clear that both cellular and HSV-1 chromatins are highly dynamic in infected cells. These dynamics reconcile the weak interactions between HSV-1 genomes and chromatin proteins, detected by nuclease protection and chromatin immunoprecipitation, with the proposed regulation of HSV-1 gene expression by chromatin, supported by the marks in the chromatin in the viral genomes and the abilities of the HSV-1 transcription activators to modulate chromatin. It also explains the sometimes unexpected results of interventions to modulate chromatin remodelling activities in infected cells. PMID:23863878

  16. 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

  17. Chromatin hydrodynamics.

    PubMed

    Bruinsma, Robijn; Grosberg, Alexander Y; Rabin, Yitzhak; Zidovska, Alexandra

    2014-05-06

    Following recent observations of large scale correlated motion of chromatin inside the nuclei of live differentiated cells, we present a hydrodynamic theory-the two-fluid model-in which the content of a nucleus is described as a chromatin solution with the nucleoplasm playing the role of the solvent and the chromatin fiber that of a solute. This system is subject to both passive thermal fluctuations and active scalar and vector events that are associated with free energy consumption, such as ATP hydrolysis. Scalar events drive the longitudinal viscoelastic modes (where the chromatin fiber moves relative to the solvent) while vector events generate the transverse modes (where the chromatin fiber moves together with the solvent). Using linear response methods, we derive explicit expressions for the response functions that connect the chromatin density and velocity correlation functions to the corresponding correlation functions of the active sources and the complex viscoelastic moduli of the chromatin solution. We then derive general expressions for the flow spectral density of the chromatin velocity field. We use the theory to analyze experimental results recently obtained by one of the present authors and her co-workers. We find that the time dependence of the experimental data for both native and ATP-depleted chromatin can be well-fitted using a simple model-the Maxwell fluid-for the complex modulus, although there is some discrepancy in terms of the wavevector dependence. Thermal fluctuations of ATP-depleted cells are predominantly longitudinal. ATP-active cells exhibit intense transverse long wavelength velocity fluctuations driven by force dipoles. Fluctuations with wavenumbers larger than a few inverse microns are dominated by concentration fluctuations with the same spectrum as thermal fluctuations but with increased intensity.

  18. Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres

    PubMed Central

    Alonso, Alicia; Fritz, Björn; Hasson, Dan; Abrusan, György; Cheung, Fanny; Yoda, Kinya; Radlwimmer, Bernhard; Ladurner, Andreas G; Warburton, Peter E

    2007-01-01

    Background Mammalian centromere formation is dependent on chromatin that contains centromere protein (CENP)-A, which is the centromere-specific histone H3 variant. Human neocentromeres have acquired CENP-A chromatin epigenetically in ectopic chromosomal locations on low-copy complex DNA. Neocentromeres permit detailed investigation of centromeric chromatin organization that is not possible in the highly repetitive alpha satellite DNA present at endogenous centromeres. Results We have examined the distribution of CENP-A, as well as two additional centromeric chromatin-associated proteins (CENP-C and CENP-H), across neocentromeric DNA using chromatin immunoprecipitation (ChIP) on CHIP assays on custom genomic microarrays at three different resolutions. Analysis of two neocentromeres using a contiguous bacterial artificial chromosome (BAC) microarray spanning bands 13q31.3 to 13q33.1 shows that both CENP-C and CENP-H co-localize to the CENP-A chromatin domain. Using a higher resolution polymerase chain reaction (PCR)-amplicon microarray spanning the neocentromere, we find that the CENP-A chromatin is discontinuous, consisting of a major domain of about 87.8 kilobases (kb) and a minor domain of about 13.2 kb, separated by an approximately 158 kb region devoid of CENPs. Both CENP-A domains exhibit co-localization of CENP-C and CENP-H, defining a distinct inner kinetochore chromatin structure that is consistent with higher order chromatin looping models at centromeres. The PCR microarray data suggested varying density of CENP-A nucleosomes across the major domain, which was confirmed using a higher resolution oligo-based microarray. Conclusion Centromeric chromatin consists of several CENP-A subdomains with highly discontinuous CENP-A chromatin at both the level of individual nucleosomes and at higher order chromatin levels, raising questions regarding the overall structure of centromeric chromatin. PMID:17651496

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

    PubMed Central

    2016-01-01

    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 106–107 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. PMID:26745449

  20. Chromatin Dynamics

    PubMed Central

    Hübner, Michael R.; Spector, David L.

    2010-01-01

    The expression patterns of many protein-coding genes are orchestrated in response to exogenous stimuli, as well as cell-type-specific developmental programs. In recent years, researchers have shown that dynamic chromatin movements and interactions in the nucleus play a crucial role in gene regulation. In this review, we highlight our current understanding of the organization of chromatin in the interphase nucleus and the impact of chromatin dynamics on gene expression. We also discuss the current state of knowledge with regard to the localization of active and inactive genes within the three-dimensional nuclear space. Furthermore, we address recent findings that demonstrate the movements of chromosomal regions and genomic loci in association with changes in transcriptional activity. Finally, we discuss the role of intra-and interchromosomal interactions in the control of coregulated genes. PMID:20462379

  1. Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

    PubMed

    Marcon, Edyta; Jain, Harshika; Bhattacharya, Anandi; Guo, Hongbo; Phanse, Sadhna; Pu, Shuye; Byram, Gregory; Collins, Ben C; Dowdell, Evan; Fenner, Maria; Guo, Xinghua; Hutchinson, Ashley; Kennedy, Jacob J; Krastins, Bryan; Larsen, Brett; Lin, Zhen-Yuan; Lopez, Mary F; Loppnau, Peter; Miersch, Shane; Nguyen, Tin; Olsen, Jonathan B; Paduch, Marcin; Ravichandran, Mani; Seitova, Alma; Vadali, Gouri; Vogelsang, Maryann S; Whiteaker, Jeffrey R; Zhong, Guoqing; Zhong, Nan; Zhao, Lei; Aebersold, Ruedi; Arrowsmith, Cheryl H; Emili, Andrew; Frappier, Lori; Gingras, Anne-Claude; Gstaiger, Matthias; Paulovich, Amanda G; Koide, Shohei; Kossiakoff, Anthony A; Sidhu, Sachdev S; Wodak, Shoshana J; Gräslund, Susanne; Greenblatt, Jack F; Edwards, Aled M

    2015-08-01

    Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

  2. Sequential Immunoprecipitation of Secretory Vesicle Proteins from Biosynthetically Labelled Cells.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabelling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labelled proteins can then be immunoprecipitated and analysed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labelling and immunoprecipitation of pancreatic islet proteins which are known to be affected in psychiatric disorders such as schizophrenia.

  3. 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…

  4. Chromatin and DNA replication.

    PubMed

    MacAlpine, David M; Almouzni, Geneviève

    2013-08-01

    The size of a eukaryotic genome presents a unique challenge to the cell: package and organize the DNA to fit within the confines of the nucleus while at the same time ensuring sufficient dynamics to allow access to specific sequences and features such as genes and regulatory elements. This is achieved via the dynamic nucleoprotein organization of eukaryotic DNA into chromatin. The basic unit of chromatin, the nucleosome, comprises a core particle with 147 bp of DNA wrapped 1.7 times around an octamer of histones. The nucleosome is a highly versatile and modular structure, both in its composition, with the existence of various histone variants, and through the addition of a series of posttranslational modifications on the histones. This versatility allows for both short-term regulatory responses to external signaling, as well as the long-term and multigenerational definition of large functional chromosomal domains within the nucleus, such as the centromere. Chromatin organization and its dynamics participate in essentially all DNA-templated processes, including transcription, replication, recombination, and repair. Here we will focus mainly on nucleosomal organization and describe the pathways and mechanisms that contribute to assembly of this organization and the role of chromatin in regulating the DNA replication program.

  5. 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.

  6. Taenia taeniaeformis: immunoprecipitation analysis of the protein antigens of oncospheres and larvae.

    PubMed

    Bowtell, D D; Mitchell, G F; Anders, R F; Lightowlers, M W; Rickard, M D

    1983-12-01

    Biosynthetically or exogenously labeled proteins and immunoprecipitated protein antigens of established 28-day-old larvae of Taenia taeniaeformis were compared with proteins and antigens of infective oncospheres using single and two-dimensional gel electrophoresis. Immunoprecipitation was carried out using sera from infected mice and mouse antisera raised to larvae or oncospheres, and emphasis was placed on identifying antigens common to both oncospheres and larvae. Two major larval antigens of Mr 40,000 and 200,000, designated Tt40 and Tt200, are common to somatic larval preparations and oncospheres. Additionally, two major oncosphere antigens of Mr 55,000 and 60,000, designated Tt55 and Tt60, are also present in larval excretory and secretory (i.e., ES or exoantigen) products. Information obtained from these immunoprecipitation analyses will facilitate isolation and production of common as well as stage-specific protein antigens in the development of defined-antigen vaccines in this model system of cysticercosis.

  7. Teaching resources. Chromatin remodeling.

    PubMed

    Lue, Neal F

    2005-07-26

    This Teaching Resource provides lecture notes and slides for a class covering chromatin remodeling mechanisms and is part of the course "Cell Signaling Systems: a Course for Graduate Students." The lecture begins with a discussion of chromatin organization and then proceeds to describe the process of chromatin remodeling through a review of chromatin remodeling complexes and methods used to study their function.

  8. 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

  9. The Ino80 chromatin-remodeling complex restores chromatin structure during UV DNA damage repair

    PubMed Central

    Sarkar, Sovan; Kiely, Rhian

    2010-01-01

    Chromatin structure is modulated during deoxyribonucleic acid excision repair, but how this is achieved is unclear. Loss of the yeast Ino80 chromatin-remodeling complex (Ino80-C) moderately sensitizes cells to ultraviolet (UV) light. In this paper, we show that INO80 acts in the same genetic pathway as nucleotide excision repair (NER) and that the Ino80-C contributes to efficient UV photoproduct removal in a region of high nucleosome occupancy. Moreover, Ino80 interacts with the early NER damage recognition complex Rad4–Rad23 and is recruited to chromatin by Rad4 in a UV damage–dependent manner. Using a modified chromatin immunoprecipitation assay, we find that chromatin disruption during UV lesion repair is normal, whereas the restoration of nucleosome structure is defective in ino80 mutant cells. Collectively, our work suggests that Ino80 is recruited to sites of UV lesion repair through interactions with the NER apparatus and is required for the restoration of chromatin structure after repair. PMID:21135142

  10. Chromatin signatures of the Drosophila replication program.

    PubMed

    Eaton, Matthew L; Prinz, Joseph A; MacAlpine, Heather K; Tretyakov, George; Kharchenko, Peter V; MacAlpine, David M

    2011-02-01

    DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events.

  11. A multiplexed system for quantitative comparisons of chromatin landscapes

    PubMed Central

    van Galen, Peter; Viny, Aaron D.; Ram, Oren; Ryan, Russell J.H.; Cotton, Matthew J.; Donohue, Laura; Sievers, Cem; Drier, Yotam; Liau, Brian B.; Gillespie, Shawn M.; Carroll, Kaitlin M.; Cross, Michael B.; Levine, Ross L.; Bernstein, Bradley E.

    2015-01-01

    Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of P300, EZH2 or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions and drug treatments. PMID:26687680

  12. A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes.

    PubMed

    van Galen, Peter; Viny, Aaron D; Ram, Oren; Ryan, Russell J H; Cotton, Matthew J; Donohue, Laura; Sievers, Cem; Drier, Yotam; Liau, Brian B; Gillespie, Shawn M; Carroll, Kaitlin M; Cross, Michael B; Levine, Ross L; Bernstein, Bradley E

    2016-01-07

    Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments.

  13. Chromatin modification in zebrafish development.

    PubMed

    Cayuso Mas, Jordi; Noël, Emily S; Ober, Elke A

    2011-01-01

    The generation of complex organisms requires that an initial population of cells with identical gene expression profiles can adopt different cell fates during development by progressively diverging transcriptional programs. These programs depend on the binding of transcritional regulators to specific genomic sites, which in turn is controlled by modifications of the chromatin. Chromatin modifications may occur directly upon DNA by methylation of specific nucleotides, or may involve post-translational modification of histones. Local regulation of histone post-translational modifications regionalizes the genome into euchromatic regions, which are more accessible to DNA-binding factors, and condensed heterochromatic regions, inhibiting the binding of such factors. In addition, these modifications may be required in a genome-wide fashion for processes such as DNA replication or chromosome condensation. From an embryologist's point of view chromatin modifications are intensively studied in the context of imprinting and have more recently received increasing attention in understanding the basis of pluripotency and cellular differentiation. Here, we describe recently uncovered roles of chromatin modifications in zebrafish development and regeneration, as well as available resources and commonly used techniques. We provide a general introduction into chromatin modifications and their respective functions with a focus on gene transcription, as well as key aspects of their roles in the early zebrafish embryo, neural development, formation of the digestive system and tissue regeneration.

  14. Genome-Wide Views of Chromatin Structure

    PubMed Central

    Rando, Oliver J.; Chang, Howard Y.

    2010-01-01

    Eukaryotic genomes are packaged into a nucleoprotein complex known as chromatin, which affects most processes that occur on DNA. Along with genetic and biochemical studies of resident chromatin proteins and their modifying enzymes, mapping of chromatin structure in vivo is one of the main pillars in our understanding of how chromatin relates to cellular processes. In this review, we discuss the use of genomic technologies to characterize chromatin structure in vivo, with a focus on data from budding yeast and humans. The picture emerging from these studies is the detailed chromatin structure of a typical gene, where the typical behavior gives insight into the mechanisms and deep rules that establish chromatin structure. Important deviation from the archetype is also observed, usually as a consequence of unique regulatory mechanisms at special genomic loci. Chromatin structure shows substantial conservation from yeast to humans, but mammalian chromatin has additional layers of complexity that likely relate to the requirements of multicellularity such as the need to establish faithful gene regulatory mechanisms for cell differentiation. PMID:19317649

  15. The Pu.1 locus is differentially regulated at the level of chromatin structure and noncoding transcription by alternate mechanisms at distinct developmental stages of hematopoiesis.

    PubMed

    Hoogenkamp, Maarten; Krysinska, Hanna; Ingram, Richard; Huang, Gang; Barlow, Rachael; Clarke, Deborah; Ebralidze, Alexander; Zhang, Pu; Tagoh, Hiromi; Cockerill, Peter N; Tenen, Daniel G; Bonifer, Constanze

    2007-11-01

    The Ets family transcription factor PU.1 is crucial for the regulation of hematopoietic development. Pu.1 is activated in hematopoietic stem cells and is expressed in mast cells, B cells, granulocytes, and macrophages but is switched off in T cells. Many of the transcription factors regulating Pu.1 have been identified, but little is known about how they organize Pu.1 chromatin in development. We analyzed the Pu.1 promoter and the upstream regulatory element (URE) using in vivo footprinting and chromatin immunoprecipitation assays. In B cells, Pu.1 was bound by a set of transcription factors different from that in myeloid cells and adopted alternative chromatin architectures. In T cells, Pu.1 chromatin at the URE was open and the same transcription factor binding sites were occupied as in B cells. The transcription factor RUNX1 was bound to the URE in precursor cells, but binding was down-regulated in maturing cells. In PU.1 knockout precursor cells, the Ets factor Fli-1 compensated for the lack of PU.1, and both proteins could occupy a subset of Pu.1 cis elements in PU.1-expressing cells. In addition, we identified novel URE-derived noncoding transcripts subject to tissue-specific regulation. Our results provide important insights into how overlapping, but different, sets of transcription factors program tissue-specific chromatin structures in the hematopoietic system.

  16. 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.

  17. Long range chromatin organization

    PubMed Central

    Acuña, Luciana I Gómez; Kornblihtt, Alberto R

    2014-01-01

    Splicing is a predominantly co-transcriptional process that has been shown to be tightly coupled to transcription. Chromatin structure is a key factor that mediates this functional coupling. In light of recent evidence that shows the importance of higher order chromatin organization in the coordination and regulation of gene expression, we discuss here the possible roles of long-range chromatin organization in splicing and alternative splicing regulation. PMID:25764333

  18. Direct Evidence for Pitavastatin Induced Chromatin Structure Change in the KLF4 Gene in Endothelial Cells

    PubMed Central

    Kanki, Yasuharu; Kohro, Takahide; Li, Guoliang; Ohta, Yoshihiro; Kimura, Hiroshi; Kobayashi, Mika; Taguchi, Akashi; Tsutsumi, Shuichi; Iwanari, Hiroko; Yamamoto, Shogo; Aruga, Hirofumi; Dong, Shoulian; Stevens, Junko F.; Poh, Huay Mei; Yamamoto, Kazuki; Kawamura, Takeshi; Mimura, Imari; Suehiro, Jun-ichi; Sugiyama, Akira; Kaneki, Kiyomi; Shibata, Haruki; Yoshinaka, Yasunobu; Doi, Takeshi; Asanuma, Akimune; Tanabe, Sohei; Tanaka, Toshiya; Minami, Takashi; Hamakubo, Takao; Sakai, Juro; Nozaki, Naohito; Aburatani, Hiroyuki; Nangaku, Masaomi; Ruan, Xiaoan; Tanabe, Hideyuki; Ruan, Yijun; Ihara, Sigeo; Endo, Akira; Kodama, Tatsuhiko; Wada, Youichiro

    2014-01-01

    Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells. PMID:24797675

  19. 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

  20. Alternative epigenetic chromatin states of polycomb target genes.

    PubMed

    Schwartz, Yuri B; Kahn, Tatyana G; Stenberg, Per; Ohno, Katsuhito; Bourgon, Richard; Pirrotta, Vincenzo

    2010-01-01

    Polycomb (PcG) regulation has been thought to produce stable long-term gene silencing. Genomic analyses in Drosophila and mammals, however, have shown that it targets many genes, which can switch state during development. Genetic evidence indicates that critical for the active state of PcG target genes are the histone methyltransferases Trithorax (TRX) and ASH1. Here we analyze the repertoire of alternative states in which PcG target genes are found in different Drosophila cell lines and the role of PcG proteins TRX and ASH1 in controlling these states. Using extensive genome-wide chromatin immunoprecipitation analysis, RNAi knockdowns, and quantitative RT-PCR, we show that, in addition to the known repressed state, PcG targets can reside in a transcriptionally active state characterized by formation of an extended domain enriched in ASH1, the N-terminal, but not C-terminal moiety of TRX and H3K27ac. ASH1/TRX N-ter domains and transcription are not incompatible with repressive marks, sometimes resulting in a "balanced" state modulated by both repressors and activators. Often however, loss of PcG repression results instead in a "void" state, lacking transcription, H3K27ac, or binding of TRX or ASH1. We conclude that PcG repression is dynamic, not static, and that the propensity of a target gene to switch states depends on relative levels of PcG, TRX, and activators. N-ter TRX plays a remarkable role that antagonizes PcG repression and preempts H3K27 methylation by acetylation. This role is distinct from that usually attributed to TRX/MLL proteins at the promoter. These results have important implications for Polycomb gene regulation, the "bivalent" chromatin state of embryonic stem cells, and gene expression in development.

  1. 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

  2. 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.

  3. On the physical and chemical dynamics of chromatin

    NASA Astrophysics Data System (ADS)

    Apratim, Manjul

    The research performed leading to this dissertation is an endeavor to explore two broad classes of developmental phenomena in the chromatin complex in eukaryotic cells---physical, for instance, long range interactions between enhancers and promoters, and chemical, such as epigenetic chromatin silencing. I begin by introducing the reader to both types of phenomena, and then set the stage for our strategy in the exploration of the physical side of these processes by creating a new machinery from existing pieces of polymer physics. I then make a brief foray into theoretical realms in an attempt to answer the question of what kinds of conformations of polymers dominate in what regimes. Subsequently, I proceed to consider the problem of analyzing and interpreting data from a major technique of probing the behavior of the chromatin complex in vivo --- Chromosome Conformation Capture --- towards which end we have developed and implemented a new and robust algorithm called 'G.R.O.M.A.T.I.N.'. Subsequently, I explore how similar ideas may be invoked in the analysis of direct microscopic observations of native chromatin structure via Fluorescence in situ Hybridization. Following this, I look at the problems of epigenetic chromatin silencing domain formation and stability in the presence of titration feedback and of stochastic noise, and demonstrate how the widely accepted polymerization model of silencing is consistent with Chromatin Immunoprecipitation data from silencing domains in budding yeast. I finally conclude with musings on recent evidence pinpointing the need to unify the physical and chemical pictures into one grand formulation.

  4. Methylation profiling using methylated DNA immunoprecipitation and tiling array hybridization.

    PubMed

    Cheung, Hoi-Hung; Lee, Tin-Lap; Rennert, Owen M; Chan, Wai-Yee

    2012-01-01

    DNA methylation is an important epigenetic modification that regulates development and plays a role in the pathophysiology of many diseases. It is dynamically changed during germline development. Methylated DNA immunoprecipitation (MeDIP) is an efficient, cost-effective method for locus-specific and genome-wide analysis. Methylated DNA fragments are enriched by a 5-methylcytidine-recognizing antibody, therefore allowing the analysis of both CpG and non-CpG methylation. The enriched DNA fragments can be amplified and hybridized to tiling arrays covering CpG islands, promoters, or the entire genome. Comparison of different methylomes permits the discovery of differentially methylated regions that might be important in disease- or tissue-specific expression. Here, we describe an established MeDIP protocol and tiling array hybridization method for profiling methylation of testicular germ cells.

  5. Chromatin Topological Transitions

    NASA Astrophysics Data System (ADS)

    Lavelle, C.; Bancaud, A.; Recouvreux, P.; Barbi, M.; Victor, J.; Viovy, J.

    DNA transaction events occurring during a cell cycle (transcription,repair, replication) are always associated with severe topological constraints on the double helix. However, since nuclear DNA is bound to various proteins (including histones) that control its accessibility and 3D organization, these topological constraints propagate or accumulate on a chromatin substrate. This paper focuses on chromatin fiber response to physiological mechanical constraints expected to occur during transcription elongation. We will show in particular how recent single molecule techniques help us to understand how chromatin conformational dynamics could manage harsh DNA supercoiling changes.

  6. A chromatin-associated and transcriptionally inactive p53-Mdm2 complex occurs in mdm2 SNP309 homozygous cells.

    PubMed

    Arva, Nicoleta C; Gopen, Tamara R; Talbott, Kathryn E; Campbell, Latoya E; Chicas, Agustin; White, David E; Bond, Gareth L; Levine, Arnold J; Bargonetti, Jill

    2005-07-22

    In cancer cells, the function of the tumor suppressor protein p53 is usually blocked. Impairment of the p53 pathway results in tumor cells with endogenous overexpression of Mdm2 via a naturally occurring single nucleotide polymorphism (SNP) in the mdm2 gene at position 309. Here we report that in mdm2 SNP309 cells, inactivation of p53 results in a chromatin-associated Mdm2-p53 complex without clearance of p53 by protein degradation. Nuclear accumulation of p53 protein in mdm2 SNP309 cells results after 6 h of camptothecin, etoposide, or mitomycin C treatment, with the p53 protein phosphorylated at Ser15. Chromatin immunoprecipitation demonstrated p53 and Mdm2 bound to p53 responsive elements. Interestingly, although the p53 protein was able to bind to DNA, quantitative PCR showed compromised transcription of endogenous target genes. Additionally, exogenously introduced p53 was incapable of activating transcription from p53 responsive elements in SNP309 cells, confirming the trans-acting nature of the inhibitor. Inhibition of Mdm2 by siRNA resulted in transcriptional activation of these p53 targets. Our data suggest that overproduction of Mdm2, resulting from a naturally occurring SNP, inhibits chromatin-bound p53 from activating the transcription of its target genes.

  7. 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.

  8. High-resolution, genome-wide mapping of chromatin modifications by GMAT.

    PubMed

    Roh, Tae-Young; Zhao, Keji

    2008-01-01

    One major postgenomic challenge is to characterize the epigenomes that control genome functions. The epigenomes are mainly defined by the specific association of nonhistone proteins with chromatin and the covalent modifications of chromatin, including DNA methylation and posttranslational histone modifications. The in vivo protein-binding and chromatin-modification patterns can be revealed by the chromatin immunoprecipitation assay (ChIP). By combining the ChIP assays and the serial analysis of gene expression (SAGE) protocols, we have developed an unbiased and high-resolution genome-wide mapping technique (GMAT) to determine the genome-wide protein-targeting and chromatin-modification patterns. GMAT has been successfully applied to mapping the target sites of the histone acetyltransferase, Gcn5p, in yeast and to the discovery of the histone acetylation islands as an epigenetic mark for functional regulatory elements in the human genome.

  9. High resolution genome-wide mapping of the primary structure of chromatin

    PubMed Central

    Zhang, Zhenhai; Pugh, B. Franklin

    2011-01-01

    The genomic organization of chromatin is increasingly recognized as a key regulator of cell behavior, but deciphering its regulation mechanisms requires detailed knowledge of chromatin’s primary structure - the assembly of nucleosomes throughout the genome. This Primer explains the principles for mapping and analyzing the primary organization of chromatin on a genomic scale. After introducing chromatin organization and its impact on gene regulation and human health, we then describe methods that detect nucleosome positioning and occupancy levels using chromatin-immunoprecipitation in combination with deep sequencing (ChIP-Seq), a strategy that is now straightforward and cost-efficient. We then explore current strategies for converting the sequence information into knowledge about chromatin, an exciting challenge for biologists and bioinformaticians. PMID:21241889

  10. Whole genome methylation profiling by immunoprecipitation of methylated DNA.

    PubMed

    Sharp, Andrew J

    2012-01-01

    I provide a protocol for DNA methylation profiling based on immunoprecipitation of methylated DNA using commercially available monoclonal antibodies that specifically recognize 5-methylcytosine. Quantification of the level of enrichment of the resulting DNA enables DNA methylation to be assayed for any genomic locus, including entire chromosomes or genomes if appropriate microarray or high-throughput sequencing platforms are used. In previous studies (1, 2), I have used hybridization to oligonucleotide arrays from Roche Nimblegen Inc, which allow any genomic region of interest to be interrogated, dependent on the array design. For example, using modern tiling arrays comprising millions of oligonucleotide probes, several complete human chromosomes can be assayed at densities of one probe per 100 bp or greater, sufficient to yield high-quality data. However, other methods such as quantitative real-time PCR or high-throughput sequencing can be used, giving either measurement of methylation at a single locus or across the entire genome, respectively. While the data produced by single locus assays is relatively simple to analyze and interpret, global assays such as microarrays or high-throughput sequencing require more complex statistical approaches in order to effectively identify regions of differential methylation, and a brief outline of some approaches is given.

  11. RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases.

    PubMed

    Halász, László; Karányi, Zsolt; Boros-Oláh, Beáta; Rózsa, Tímea; Sipos, Éva; Nagy, Éva; Mosolygó-L, Ágnes; Mázló, Anett; Rajnavölgyi, Éva; Halmos, Gábor; Székvölgyi, Lóránt

    2017-03-24

    The impact of R-loops on the physiology and pathology of chromosomes has been demonstrated extensively by chromatin biology research. The progress in this field has been driven by technological advancement of R-loop mapping methods that largely relied on a single approach, DNA-RNA immunoprecipitation (DRIP). Most of the DRIP protocols use the experimental design that was developed by a few laboratories, without paying attention to the potential caveats that might affect the outcome of RNA-DNA hybrid mapping. To assess the accuracy and utility of this technology, we pursued an analytical approach to estimate inherent biases and errors in the DRIP protocol. By performing DRIP-sequencing, qPCR and receiver operator characteristic (ROC) analysis, we tested the effect of formaldehyde fixation, cell lysis temperature, mode of genome fragmentation, and removal of free RNA on the efficacy of RNA-DNA hybrid detection, and implemented workflows that were able to distinguish complex and weak DRIP signals in a noisy background with high confidence. We also show that some of the workflows perform poorly and generate random answers. Furthermore, we found that the most commonly used genome fragmentation method (restriction enzyme digestion) led to the overrepresentation of lengthy DRIP fragments over coding ORFs, and this bias was enhanced at the first exons. Biased genome sampling severely compromised the mapping resolution and prevented the assignment of precise biological function to a significant fraction of R-loops. The revised workflow presented herein is established and optimized using objective ROC analyses and provides reproducible and highly specific RNA-DNA hybrid detection.

  12. Chromatin poises miRNA- and protein-coding genes for expression.

    PubMed

    Barski, Artem; Jothi, Raja; Cuddapah, Suresh; Cui, Kairong; Roh, Tae-Young; Schones, Dustin E; Zhao, Keji

    2009-10-01

    Chromatin modifications have been implicated in the regulation of gene expression. While association of certain modifications with expressed or silent genes has been established, it remains unclear how changes in chromatin environment relate to changes in gene expression. In this article, we used ChIP-seq (chromatin immunoprecipitation with massively parallel sequencing) to analyze the genome-wide changes in chromatin modifications during activation of total human CD4(+) T cells by T-cell receptor (TCR) signaling. Surprisingly, we found that the chromatin modification patterns at many induced and silenced genes are relatively stable during the short-term activation of resting T cells. Active chromatin modifications were already in place for a majority of inducible protein-coding genes, even while the genes were silent in resting cells. Similarly, genes that were silenced upon T-cell activation retained positive chromatin modifications even after being silenced. To investigate if these observations are also valid for miRNA-coding genes, we systematically identified promoters for known miRNA genes using epigenetic marks and profiled their expression patterns using deep sequencing. We found that chromatin modifications can poise miRNA-coding genes as well. Our data suggest that miRNA- and protein-coding genes share similar mechanisms of regulation by chromatin modifications, which poise inducible genes for activation in response to environmental stimuli.

  13. The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components*

    PubMed Central

    Soldi, Monica; Bonaldi, Tiziana

    2013-01-01

    Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational modifications, histone variants, and DNA methylation. Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. Here we describe a modified protocol for chromatin immunoprecipitation combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH, both enriched in silent domains. Chromatin proteomics expands the arsenal of tools for deciphering how all the distinct protein components act together to enforce a given region-specific chromatin status. PMID:23319141

  14. Fragment complementation and co-immunoprecipitation assays for understanding R protein structure and function.

    PubMed

    Moffett, Peter

    2011-01-01

    Plant disease resistance (R) proteins confer protection against specific pathogens or pathogen isolates. R proteins function by recognizing pathogen-encoded avirulence (Avr) proteins and translating this recognition event into an initiation of downstream signaling pathways. Key to understanding this process is the study of the protein-protein interactions involving R proteins. Recognition and signaling mechanisms are mediated by both intramolecular interactions that take place between different domains of R proteins as well as intermolecular interactions between R proteins and additional plant proteins. These processes have been studied in part by using Agrobacterium-mediated transient expression of R protein fragments in Nicotiana benthamiana which allows for the rapid assessment of functionality. Furthermore, pairs of proteins or protein fragments can be transiently expressed as fusions with different epitope tags. One putative protein partner is subjected to immunoprecipitation. Subsequent immunoblotting is performed to determine whether the second protein has remained associated (or co-immunoprecipitated) with the first, indicating a protein-protein interaction. This technique has contributed substantially to structure-function analyses of R proteins and to the characterization of interactions between R proteins and other plant proteins.

  15. Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi.

    PubMed

    Leandro de Jesus, Teresa Cristina; Calderano, Simone Guedes; Vitorino, Francisca Nathalia de Luna; Llanos, Ricardo Pariona; Lopes, Mariana de Camargo; de Araújo, Christiane Bezerra; Thiemann, Otavio Henrique; Reis, Marcelo da Silva; Elias, Maria Carolina; Chagas da Cunha, Julia Pinheiro

    2017-01-01

    Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosoma cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and nonreplicative forms accompanied by a shift on global transcription levels and by changes in its chromatin architecture. Here, we investigated the T. cruzi chromatin proteome using three different protocols and compared it between replicative (epimastigote) and nonreplicative (trypomastigote) forms by high-resolution mass spectrometry. More than 2000 proteins were identified and quantified both in chromatin and nonchromatin extracts. Besides histones and other known nuclear proteins, trypanosomes chromatin also contains metabolic (mainly from carbohydrate pathway), cytoskeleton and many other proteins with unknown functions. Strikingly, the two parasite forms differ greatly regarding their chromatin-associated factors composition and amount. Although the nucleosome content is the same for both life forms (as seen by MNase digestion), the remaining proteins were much less detected in nonreplicative forms, suggesting that they have a naked chromatin. Proteins associated to DNA proliferation, such as PCNA, RPA, and DNA topoisomerases were exclusively found in the chromatin of replicative stages. On the other hand, the nonreplicative stages have an enrichment of a histone H2B variant. Furthermore, almost 20% of replicative stages chromatin-associated proteins are expressed in nonreplicative forms, but located at nonchromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and nonchromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. We confirmed their location in chromatin and their differential expression, using Western blotting assays and chromatin immunoprecipitation (ChIP). Our results indicate that the

  16. Dietary control of chromatin

    PubMed Central

    Huang, Zhiguang; Cai, Ling; Tu, Benjamin P

    2015-01-01

    Organisms must be able to rapidly alter gene expression in response to changes in their nutrient environment. This review summarizes evidence that epigenetic modifications of chromatin depend on particular metabolites of intermediary metabolism, enabling the facile regulation of gene expression in tune with metabolic state. Nutritional or dietary control of chromatin is an often-overlooked, yet fundamental regulatory mechanism directly linked to human physiology. Nutrient-sensitive epigenetic marks are dynamic, suggesting rapid turnover, and may have functions beyond the regulation of gene transcription, including pH regulation and as carbon sources in cancer cells. PMID:26094239

  17. Reciprocal nuclear shuttling of two antagonizing Zn finger proteins modulates Tup family corepressor function to repress chromatin remodeling.

    PubMed

    Hirota, Kouji; Hoffman, Charles S; Ohta, Kunihiro

    2006-12-01

    The Schizosaccharomyces pombe global corepressors Tup11 and Tup12, which are orthologs of Saccharomyces cerevisiae Tup1, are involved in glucose-dependent transcriptional repression and chromatin alteration of the fbp1+ gene. The fbp1+ promoter contains two regulatory elements, UAS1 and UAS2, one of which (UAS2) serves as a binding site for two antagonizing C2H2 Zn finger transcription factors, the Rst2 activator and the Scr1 repressor. In this study, we analyzed the role of Tup proteins and Scr1 in chromatin remodeling at fbp1+ during glucose repression. We found that Scr1, cooperating with Tup11 and Tup12, functions to maintain the chromatin of the fbp1+ promoter in a transcriptionally inactive state under glucose-rich conditions. Consistent with this notion, Scr1 is quickly exported from the nucleus to the cytoplasm at the initial stage of derepression, immediately after glucose starvation, at which time Rst2 is known to be imported into the nucleus. In addition, chromatin immunoprecipitation assays revealed a switching of Scr1 to Rst2 bound at UAS2 during glucose derepression. On the other hand, Tup11 and Tup12 persist in the nucleus and bind to the fbp1+ promoter under both derepressed and repressed conditions. These observations suggest that Tup1-like proteins recruited to the fbp1+ promoter are controlled by either of two antagonizing C2H2 Zn finger proteins. We propose that the actions of Tup11 and Tup12 are regulated by reciprocal nuclear shuttling of the two antagonizing Zn finger proteins in response to the extracellular glucose concentration. This notion provides new insights into the molecular mechanisms of the Tup family corepressors in gene regulation.

  18. Chromatin organization and dynamics in double-strand break repair.

    PubMed

    Seeber, Andrew; Gasser, Susan M

    2016-10-31

    Chromatin is organized and segmented into a landscape of domains that serve multiple purposes. In contrast to transcription, which is controlled by defined sequences at distinct sites, DNA damage can occur anywhere. Repair accordingly must occur everywhere, yet it is inevitably affected by its chromatin environment. In this review, we summarize recent work investigating how changes in chromatin organization facilitate and/or guide DNA double-strand break repair. In addition, we examine new live cell studies on the dynamics of chromatin and the mechanisms that regulate its movement.

  19. Archaeal chromatin proteins.

    PubMed

    Zhang, ZhenFeng; Guo, Li; Huang, Li

    2012-05-01

    Archaea, along with Bacteria and Eukarya, are the three domains of life. In all living cells, chromatin proteins serve a crucial role in maintaining the integrity of the structure and function of the genome. An array of small, abundant and basic DNA-binding proteins, considered candidates for chromatin proteins, has been isolated from the Euryarchaeota and the Crenarchaeota, the two major phyla in Archaea. While most euryarchaea encode proteins resembling eukaryotic histones, crenarchaea appear to synthesize a number of unique DNA-binding proteins likely involved in chromosomal organization. Several of these proteins (e.g., archaeal histones, Sac10b homologs, Sul7d, Cren7, CC1, etc.) have been extensively studied. However, whether they are chromatin proteins and how they function in vivo remain to be fully understood. Future investigation of archaeal chromatin proteins will lead to a better understanding of chromosomal organization and gene expression in Archaea and provide valuable information on the evolution of DNA packaging in cellular life.

  20. 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,…

  1. 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

  2. Differential association of chromatin proteins identifies BAF60a/SMARCD1 as a regulator of embryonic stem cell differentiation.

    PubMed

    Alajem, Adi; Biran, Alva; Harikumar, Arigela; Sailaja, Badi Sri; Aaronson, Yair; Livyatan, Ilana; Nissim-Rafinia, Malka; Sommer, Andreia Gianotti; Mostoslavsky, Gustavo; Gerbasi, Vincent R; Golden, Daniel E; Datta, Arnab; Sze, Siu Kwan; Meshorer, Eran

    2015-03-31

    Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation.

  3. 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

  4. Imprinting at the PLAGL1 domain is contained within a 70-kb CTCF/cohesin-mediated non-allelic chromatin loop

    PubMed Central

    Iglesias-Platas, Isabel; Court, Franck; Camprubi, Cristina; Sparago, Angela; Guillaumet-Adkins, Amy; Martin-Trujillo, Alex; Riccio, Andrea; Moore, Gudrun E.; Monk, David

    2013-01-01

    Paternal duplications of chromosome 6q24, a region that contains the imprinted PLAGL1 and HYMAI transcripts, are associated with transient neonatal diabetes mellitus. A common feature of imprinted genes is that they tend to cluster together, presumably as a result of sharing common cis-acting regulatory elements. To determine the extent of this imprinted cluster in human and mouse, we have undertaken a systematic analysis of allelic expression and DNA methylation of the genes mapping within an ∼1.4-Mb region flanking PLAGL1/Plagl1. We confirm that all nine neighbouring genes are biallelically expressed in both species. In human we identify two novel paternally expressed PLAGL1 coding transcripts that originate from unique promoter regions. Chromatin immunoprecipitation for CTCF and the cohesin subunits RAD21 and SMC3 reveals evolutionarily conserved binding sites within unmethylated regions ∼5 kb downstream of the PLAGL1 differentially methylated region and within the PLAGL1 3′ untranslated region (UTR). Higher-order chromatin looping occurs between these regions in both expressing and non-expressing tissues, forming a non-allelic chromatin loop around the PLAGL1/Plagl1 gene. In placenta and brain tissues, we identify an additional interaction between the PLAGL1 P3/P4 promoters and the unmethylated element downstream of the PLAGL1 differentially methylated region that we propose facilitates imprinted expression of these alternative isoforms. PMID:23295672

  5. Human sperm chromatin stabilization: a proposed model including zinc bridges.

    PubMed

    Björndahl, Lars; Kvist, Ulrik

    2010-01-01

    The primary focus of this review is to challenge the current concepts on sperm chromatin stability. The observations (i) that zinc depletion at ejaculation allows a rapid and total sperm chromatin decondensation without the addition of exogenous disulfide cleaving agents and (ii) that the human sperm chromatin contains one zinc for every protamine for every turn of the DNA helix suggest an alternative model for sperm chromatin structure may be plausible. An alternative model is therefore proposed, that the human spermatozoon could at ejaculation have a rapidly reversible zinc dependent chromatin stability: Zn(2+) stabilizes the structure and prevents the formation of excess disulfide bridges by a single mechanism, the formation of zinc bridges with protamine thiols of cysteine and potentially imidazole groups of histidine. Extraction of zinc enables two biologically totally different outcomes: immediate decondensation if chromatin fibers are concomitantly induced to repel (e.g. by phosphorylation in the ooplasm); otherwise freed thiols become committed into disulfide bridges creating a superstabilized chromatin. Spermatozoa in the zinc rich prostatic fluid (normally the first expelled ejaculate fraction) represent the physiological situation. Extraction of chromatin zinc can be accomplished by the seminal vesicular fluid. Collection of the ejaculate in one single container causes abnormal contact between spermatozoa and seminal vesicular fluid affecting the sperm chromatin stability. There are men in infertile couples with low content of sperm chromatin zinc due to loss of zinc during ejaculation and liquefaction. Tests for sperm DNA integrity may give false negative results due to decreased access for the assay to the DNA in superstabilized chromatin.

  6. ATP dependent chromatin remodeling enzymes in embryonic stem cells.

    PubMed

    Saladi, Srinivas Vinod; de la Serna, Ivana L

    2010-03-01

    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.

  7. Chromatin modifications that support acetylcholine receptor gene activation are established during muscle cell determination and differentiation.

    PubMed

    Herndon, Carter A; Snell, Jeff; Fromm, Larry

    2011-02-01

    Localization of acetylcholine receptors (AChRs) to the postsynaptic region of muscle is mediated in part by transcriptional mechanisms. An important way of regulating transcription is through targeting histone modifications on chromatin to distinct gene loci. Using chromatin immunoprecipitation, we examined the developmental regulation of certain histone modifications at the AChR epsilon subunit locus, including methylations at lysine residues K4 and K27 and acetylations at K9 and K14. We modeled various stages of muscle development in cell culture, including pre-determined cells, committed but undifferentiated myoblasts, and differentiated myotubes, and modeled synaptic myotube nuclei by stimulating myotubes with neuregulin (NRG) 1. We found that a pattern of histone modifications associated with transcriptional activation is targeted to the AChR epsilon subunit locus in myotubes prior to stimulation with NRG1 and does not change upon addition of NRG1. Instead, we found that during muscle cell determination and differentiation, specific histone modifications are targeted to the AChR epsilon subunit locus. Within the gene, at K4, dimethylation is induced during muscle cell determination, while trimethylation is induced during differentiation. At K27, loss of trimethylation and appearance of monomethylation occurs during determination and differentiation. In addition, in a region upstream of the gene, K4 di- and trimethylation, and K9/14 acetylation are induced in a distinct developmental pattern, which may reflect a functional regulatory element. These results suggest synaptic signaling does not directly target histone modifications but rather the histone modification pattern necessary for transcriptional activation is previously established in a series of steps during muscle development.

  8. Cas9 Functionally Opens Chromatin

    PubMed Central

    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

  9. Molecular Toxicology of Chromatin

    DTIC Science & Technology

    1992-01-01

    FINAL 01 Jan 89 TO 31 Dec 91 4. ITL ANO SUS Y, L RE %UMAS MOLECULAR TOXICOLOGY OF CHROMATIN AFOSR-89-0231 PE - 61102F AUT PR - 2312 TA - A5 Dr Ernest Kun...Waterbury, CT), 2-mercaptoethanol, NAD+, NADPH, nucleo- tides, sodium tungstate , hydrogen peroxide, Tris and MES buffers from Sigma (St. Louis, MO...ml) with sodium tungstate (5.93 g, in 20 ml H20) for 1.5 h followed by extraction of the green product into ethyl acetate, washing with 0.1 N HCl, and

  10. BIOCHEMICAL ANALYSES OF TRANSCRIPTIONAL REGULATORY MECHANISMS IN A CHROMATIN CONTEXT

    PubMed Central

    KONESKY, KASEY L.; LAYBOURN, PAUL J.

    2007-01-01

    We have optimized a recombinant chromatin assembly system that properly incorporates core histones and histone H1 into a chromatin template containing a natural promoter sequence. This article provides a step-by-step procedure for expression and purification of the proteins required for assembling well-defined chromatin templates. We describe how the degree of chromatin assembly in the absence and presence of histone H1 is measured using topological analysis and the use of micrococcal nuclease digestion performed to confirm H1 incorporation and determine the quality of in vitro chromatin templates. Further we describe the use sucrose gradient ultracentrifugation to verify that no unincorporated H1 remains as a second means for deciding on the proper H1 to core histone ratio during assembly. Additionally, we discuss the use of both yeast and Drosophila NAP-1 (yNAP-1 and dNAP-1, respectively) in the assembly of H1-containing chromatin. Finally, we provide detailed description of functional assays for investigating the mechanism of transcriptional regulation in a chromatin context (transcription, histone acetyltransferase activity, and protein association with promoter-bound complexes using immobilized chromatin templates). PMID:17309835

  11. Chromatin dynamics: Interplay between remodeling enzymes and histone modifications

    PubMed Central

    Swygert, Sarah G.; Peterson, Craig L.

    2014-01-01

    Chromatin dynamics play an essential role in regulating the accessibility of genomic DNA for a variety of nuclear processes, including gene transcription and DNA repair. The posttranslational modification of the core histones and the action of ATP-dependent chromatin remodeling enzymes represent two primary mechanisms by which chromatin dynamics are controlled and linked to nuclear events. Although there are examples in which a histone modification or a remodeling enzyme may be sufficient to drive a chromatin transition, these mechanisms typically work in concert to integrate regulatory inputs, leading to a coordinated alteration in chromatin structure and function. Indeed, site-specific histone modifications can facilitate the recruitment of chromatin remodeling enzymes to particular genomic regions, or they can regulate the efficiency or the outcome of a chromatin remodeling reaction. Conversely, chromatin remodeling enzymes can also influence, and sometimes directly modulate, the modification state of histones. These functional interactions are generally complex, frequently transient, and often require the association of myriad additional factors. PMID:24583555

  12. The nucleosome: orchestrating DNA damage signaling and repair within chromatin.

    PubMed

    Agarwal, Poonam; Miller, Kyle M

    2016-10-01

    DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.

  13. Mapping chromatin modifications in nanochannels

    NASA Astrophysics Data System (ADS)

    Lim, Shuang Fang; Karpusenko, Alena; Riehn, Robert

    2013-03-01

    DNA and chromatin are elongated to a fixed fraction of their contour length when introduced into quasi-1d nanochannels. Because single molecules are analyzed, their hold great potential for the analysis for the genetic analysis of material from single cells. In this study, we have reconstituted chromatin with histones from a variety of sources, and mapped the modification profile of the chromatin. We monitored methylation and acetylation patterns of the histone tail protein residues using fluorescently labelled antibodies. Using those, we distinguished chromatin reconstituted from chicken erythrocytes, calf thymus, and HeLa cells. We discuss prospects for profiling histone modifications for whole chromosomes from single cells.

  14. Chromatin structure in barley nuclei.

    PubMed

    Mithieux, G; Roux, B

    1983-10-03

    In order to study the chromatin structure of a higher plant we used a high-yield method, which allows one to obtain up to 10(9) nuclei/kg fresh barley leaves. Significant amounts of low-ionic-strength-soluble chromatin can be extracted from these nuclei. Physicochemical properties were examined and discussed. Electric birefringence allowed us to observe the same transition in electro-optical properties as has been observed for animal chromatin, and suggested the existence of a symetrical structure occurring for approximately six nucleosomes. Circular dichroism showed that barley oligonucleosomes exhibit a higher molar ellipticity at 282 nm than total soluble chromatin and than their animal counterparts.

  15. Multiplex Sequential Immunoprecipitation of Insulin Secretory Granule Proteins from Radiolabeled Pancreatic Islets.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Specific proteins can then be immunoprecipitated and analyzed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labeling of pancreatic islets with (35)S-methionine, followed by multiplex sequential immunoprecipitation of insulin and three other secretory granule accessory proteins. This provided a means of distinguishing those pancreatic islet proteins with different biosynthetic rates in response to the media glucose concentrations.

  16. 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.

  17. Chromatinization of the KSHV Genome During the KSHV Life Cycle

    PubMed Central

    Uppal, Timsy; Jha, Hem C.; Verma, Subhash C.; Robertson, Erle S.

    2015-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) belongs to the gamma herpesvirus family and is the causative agent of various lymphoproliferative diseases in humans. KSHV, like other herpesviruses, establishes life-long latent infection with the expression of a limited number of viral genes. Expression of these genes is tightly regulated by both the viral and cellular factors. Recent advancements in identifying the expression profiles of viral transcripts, using tilling arrays and next generation sequencing have identified additional coding and non-coding transcripts in the KSHV genome. Determining the functions of these transcripts will provide a better understanding of the mechanisms utilized by KSHV in altering cellular pathways involved in promoting cell growth and tumorigenesis. Replication of the viral genome is critical in maintaining the existing copies of the viral episomes during both latent and lytic phases of the viral life cycle. The replication of the viral episome is facilitated by viral components responsible for recruiting chromatin modifying enzymes and replication factors for altering the chromatin complexity and replication initiation functions, respectively. Importantly, chromatin modification of the viral genome plays a crucial role in determining whether the viral genome will persist as latent episome or undergo lytic reactivation. Additionally, chromatinization of the incoming virion DNA, which lacks chromatin structure, in the target cells during primary infection, helps in establishing latent infection. Here, we discuss the recent advancements on our understating of KSHV genome chromatinization and the consequences of chromatin modifications on viral life cycle. PMID:25594667

  18. Keratin proteins in human lung carcinomas. Combined use of morphology, keratin immunocytochemistry, and keratin immunoprecipitation.

    PubMed Central

    Banks-Schlegel, S. P.; McDowell, E. M.; Wilson, T. S.; Trump, B. F.; Harris, C. C.

    1984-01-01

    Light-microscopic immunocytochemistry and electron microscopy demonstrated that adenocarcinomas (AC) and squamous cell (epidermoid) carcinomas (SCCs) of human lung contained keratin proteins in the form of tonofilament bundles. However, moderately differentiated (md) SCCs contained abundant keratin, whereas poorly differentiated (pd) SCCs and all ACs contained lesser amounts. Lung tumors with the diagnosis of AC or SCC, as defined by WHO criteria, were also analyzed by immunoprecipitation techniques for the presence of keratin proteins. Regardless of the degree of tumor differentiation, SCCs contained a 44 kd keratin which was lacking in ACs. Interestingly, normal bronchial epithelium also contained the same 44 kd keratin. In addition, as SCCs became more differentiated, they exhibited even greater differences in the profile of synthesized keratins. Specifically, the relative abundance of the intermediate-sized keratins (57 and 59 kd) was increased in the md SCCs. Although keratin protein patterns appear to be a valuable adjunct in distinguishing AC from SCC, their usefulness as a diagnostic tool will require survey of a larger number of poorly differentiated tumors. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:6198920

  19. Chromatin condensation during terminal erythropoiesis.

    PubMed

    Zhao, Baobing; Yang, Jing; Ji, Peng

    2016-09-02

    Mammalian terminal erythropoiesis involves gradual but dramatic chromatin condensation steps that are essential for cell differentiation. Chromatin and nuclear condensation is followed by a unique enucleation process, which is believed to liberate more spaces for hemoglobin enrichment and enable the generation of a physically flexible mature red blood cell. Although these processes have been known for decades, the mechanisms are still unclear. Our recent study reveals an unexpected nuclear opening formation during mouse terminal erythropoiesis that requires caspase-3 activity. Major histones, except H2AZ, are partially released from the opening, which is important for chromatin condensation. Block of the nuclear opening through caspase inhibitor or knockdown of caspase-3 inhibits chromatin condensation and enucleation. We also demonstrate that nuclear opening and histone release are cell cycle regulated. These studies reveal a novel mechanism for chromatin condensation in mammalia terminal erythropoiesis.

  20. 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.

  1. Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks.

    PubMed

    Aymard, François; Bugler, Beatrix; Schmidt, Christine K; Guillou, Emmanuelle; Caron, Pierre; Briois, Sébastien; Iacovoni, Jason S; Daburon, Virginie; Miller, Kyle M; Jackson, Stephen P; Legube, Gaëlle

    2014-04-01

    Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.

  2. Defining the chromatin signature of inducible genes in T cells

    PubMed Central

    2009-01-01

    Background Specific chromatin characteristics, especially the modification status of the core histone proteins, are associated with active and inactive genes. There is growing evidence that genes that respond to environmental or developmental signals may possess distinct chromatin marks. Using a T cell model and both genome-wide and gene-focused approaches, we examined the chromatin characteristics of genes that respond to T cell activation. Results To facilitate comparison of genes with similar basal expression levels, we used expression-profiling data to bin genes according to their basal expression levels. We found that inducible genes in the lower basal expression bins, especially rapidly induced primary response genes, were more likely than their non-responsive counterparts to display the histone modifications of active genes, have RNA polymerase II (Pol II) at their promoters and show evidence of ongoing basal elongation. There was little or no evidence for the presence of active chromatin marks in the absence of promoter Pol II on these inducible genes. In addition, we identified a subgroup of genes with active promoter chromatin marks and promoter Pol II but no evidence of elongation. Following T cell activation, we find little evidence for a major shift in the active chromatin signature around inducible gene promoters but many genes recruit more Pol II and show increased evidence of elongation. Conclusions These results suggest that the majority of inducible genes are primed for activation by having an active chromatin signature and promoter Pol II with or without ongoing elongation. PMID:19807913

  3. A synergistic DNA logic predicts genome-wide chromatin accessibility

    PubMed Central

    Hashimoto, Tatsunori; Sherwood, Richard I.; Kang, Daniel D.; Rajagopal, Nisha; Barkal, Amira A.; Zeng, Haoyang; Emons, Bart J.M.; Srinivasan, Sharanya; Jaakkola, Tommi; Gifford, David K.

    2016-01-01

    Enhancers and promoters commonly occur in accessible chromatin characterized by depleted nucleosome contact; however, it is unclear how chromatin accessibility is governed. We show that log-additive cis-acting DNA sequence features can predict chromatin accessibility at high spatial resolution. We develop a new type of high-dimensional machine learning model, the Synergistic Chromatin Model (SCM), which when trained with DNase-seq data for a cell type is capable of predicting expected read counts of genome-wide chromatin accessibility at every base from DNA sequence alone, with the highest accuracy at hypersensitive sites shared across cell types. We confirm that a SCM accurately predicts chromatin accessibility for thousands of synthetic DNA sequences using a novel CRISPR-based method of highly efficient site-specific DNA library integration. SCMs are directly interpretable and reveal that a logic based on local, nonspecific synergistic effects, largely among pioneer TFs, is sufficient to predict a large fraction of cellular chromatin accessibility in a wide variety of cell types. PMID:27456004

  4. Determinants of Sir2-Mediated, Silent Chromatin Cohesion.

    PubMed

    Chen, Yu-Fan; Chou, Chia-Ching; Gartenberg, Marc R

    2016-08-01

    Cohesin associates with distinct sites on chromosomes to mediate sister chromatid cohesion. Single cohesin complexes are thought to bind by encircling both sister chromatids in a topological embrace. Transcriptionally repressed chromosomal domains in the yeast Saccharomyces cerevisiae represent specialized sites of cohesion where cohesin binds silent chromatin in a Sir2-dependent fashion. In this study, we investigated the molecular basis for Sir2-mediated cohesion. We identified a cluster of charged surface residues of Sir2, collectively termed the EKDK motif, that are required for cohesin function. In addition, we demonstrated that Esc8, a Sir2-interacting factor, is also required for silent chromatin cohesion. Esc8 was previously shown to associate with Isw1, the enzymatic core of ISW1 chromatin remodelers, to form a variant of the ISW1a chromatin remodeling complex. When ESC8 was deleted or the EKDK motif was mutated, cohesin binding at silenced chromatin domains persisted but cohesion of the domains was abolished. The data are not consistent with cohesin embracing both sister chromatids within silent chromatin domains. Transcriptional silencing remains largely intact in strains lacking ESC8 or bearing EKDK mutations, indicating that silencing and cohesion are separable functions of Sir2 and silent chromatin.

  5. Structure of chromatin in spermatozoa.

    PubMed

    Björndahl, Lars; Kvist, Ulrik

    2014-01-01

    The specialized structure of the sperm chromatin has a dual function - first to protect the DNA from damage during storage and transport to the oocyte, and then to enable a rapid and complete unpacking of the undamaged paternal genome in the ooplasm. It is evident that zinc has a pivotal role in maintaining the structural stability and in enabling a rapid decondensation at the appropriate time. It is important for the sperm chromatin structure that the spermatozoa are ejaculated together with the zinc-rich prostatic secretion. Early exposure to zinc-binding seminal vesicular fluid can deplete the sperm chromatin of zinc and most likely induce surplus formation of disulfide bridges, likely to cause incomplete and delayed decondensation of the sperm chromatin in the oocyte. A premature decrease in sperm chromatin structure stability is likely to increase the risk for damage to the DNA due to increased access to the genome for DNA damaging compounds. The status of the sperm chromatin structure can vary in vitro depending on the exposure to zinc-depleting conditions when spermatozoa are stored in semen after ejaculation. When sperm DNA damage tests are evaluated and validated, it is therefore essential to also take into account the dynamics of zinc-dependent and zinc-independent sperm chromatin stability.

  6. 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.

  7. Chromatin and Transcription in Yeast

    PubMed Central

    Rando, Oliver J.; Winston, Fred

    2012-01-01

    Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field. PMID:22345607

  8. Mesoscale Modeling of Chromatin Folding

    NASA Astrophysics Data System (ADS)

    Schlick, Tamar

    2009-03-01

    Eukaryotic chromatin is the fundamental protein/nucleic acid unit that stores the genetic material. Understanding how chromatin fibers fold and unfold in physiological conditions is important for interpreting fundamental biological processes like DNA replication and transcription regulation. Using a mesoscopic model of oligonucleosome chains and tailored sampling protocols, we elucidate the energetics of oligonucleosome folding/unfolding and the role of each histone tail, linker histones, and divalent ions in regulating chromatin structure. The resulting compact topologies reconcile features of the zigzag model with straight linker DNAs with the solenoid model with bent linker DNAs for optimal fiber organization and reveal dynamic and energetic aspects involved.

  9. A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues.

    PubMed

    Baszczynski, C L

    1990-06-01

    Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.

  10. 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

  11. 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

  12. Chromatin condensation of Xist genomic loci during oogenesis in mice.

    PubMed

    Fukuda, Atsushi; Mitani, Atsushi; Miyashita, Toshiyuki; Umezawa, Akihiro; Akutsu, Hidenori

    2015-12-01

    Repression of maternal Xist (Xm-Xist) during preimplantation in mouse embryos is essential for establishing imprinted X chromosome inactivation. Nuclear transplantation (NT) studies using nuclei derived from non-growing (ng) and full-grown (fg) oocytes have indicated that maternal-specific repressive modifications are imposed on Xm-Xist during oogenesis, as well as on autosomal imprinted genes. Recent studies have revealed that histone H3 lysine 9 trimethylation (H3K9me3) enrichments on Xm-Xist promoter regions are involved in silencing at the preimplantation stages. However, whether H3K9me3 is imposed on Xm-Xist during oogenesis is not known. Here, we dissected the chromatin states in ng and fg oocytes and early preimplantation stage embryos. Chromatin immunoprecipitation experiments against H3K9me3 revealed that there was no significant enrichment within the Xm-Xist region during oogenesis. However, NT embryos with ng nuclei (ngNT) showed extensive Xm-Xist derepression and H3K9me3 hypomethylation of the promoter region at the 4-cell stage, which corresponds to the onset of paternal Xist expression. We also found that the chromatin state at the Xist genomic locus became markedly condensed as oocyte growth proceeded. Although the condensed Xm-Xist genomic locus relaxed during early preimplantation phases, the extent of the relaxation across Xm-Xist loci derived from normally developed oocytes was significantly smaller than those of paternal-Xist and ngNT-Xist genomic loci. Furthermore, Xm-Xist from 2-cell metaphase nuclei became derepressed following NT. We propose that chromatin condensation is associated with imprinted Xist repression and that skipping of the condensation step by NT leads to Xist activation during the early preimplantation phase.

  13. Unusual chromatin in human telomeres.

    PubMed Central

    Tommerup, H; Dousmanis, A; de Lange, T

    1994-01-01

    We report that human telomeres have an unusual chromatin structure characterized by diffuse micrococcal nuclease patterns. The altered chromatin manifested itself only in human telomeres that are relatively short (2 to 7 kb). In contrast, human and mouse telomeres with telomeric repeat arrays of 14 to 150 kb displayed a more canonical chromatin structure with extensive arrays of tightly packed nucleosomes. All telomeric nucleosomes showed a shorter repeat size than bulk nucleosomes, and telomeric mononucleosomal particles were found to be hypersensitive to micrococcal nuclease. However, telomeric nucleosomes were similar to bulk nucleosomes in the rate at which they sedimented through sucrose gradients. We speculate that mammalian telomeres have a bipartite structure with unusual chromatin near the telomere terminus and a more canonical nucleosomal organization in the proximal part of the telomere. Images PMID:8065312

  14. Regulation of cellular chromatin state

    PubMed Central

    Mishra, Rakesh K; Dhawan, Jyotsna

    2010-01-01

    The identity and functionality of eukaryotic cells is defined not just by their genomic sequence which remains constant between cell types, but by their gene expression profiles governed by epigenetic mechanisms. Epigenetic controls maintain and change the chromatin state throughout development, as exemplified by the setting up of cellular memory for the regulation and maintenance of homeotic genes in proliferating progenitors during embryonic development. Higher order chromatin structure in reversibly arrested adult stem cells also involves epigenetic regulation and in this review we highlight common trends governing chromatin states, focusing on quiescence and differentiation during myogenesis. Together, these diverse developmental modules reveal the dynamic nature of chromatin regulation providing fresh insights into the role of epigenetic mechanisms in potentiating development and differentiation. PMID:20592864

  15. Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

    PubMed Central

    Poh, Huay Mei; Peh, Su Qin; Ong, Chin Thing; Zhang, Jingyao; Ruan, Xiaoan; Ruan, Yijun

    2012-01-01

    Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video. PMID:22564980

  16. Linker DNA destabilizes condensed chromatin.

    PubMed

    Green, G R; Ferlita, R R; Walkenhorst, W F; Poccia, D L

    2001-01-01

    The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.

  17. Centromeric chromatin in fission yeast.

    PubMed

    Partridge, Janet F

    2008-05-01

    A fundamental requirement for life is the ability of cells to divide properly and to pass on to their daughters a full complement of genetic material. The centromere of the chromosome is essential for this process, as it provides the DNA sequences on which the kinetochore (the proteinaceous structure that links centromeric DNA to the spindle microtubules) assembles to allow segregation of the chromosomes during mitosis. It has long been recognized that kinetochore assembly is subject to epigenetic control, and deciphering how centromeres promote faithful chromosome segregation provides a fascinating intellectual challenge. This challenge is made more difficult by the scale and complexity of DNA sequences in metazoan centromeres, thus much research has focused on dissecting centromere function in the single celled eukaryotic yeasts. Interestingly, in spite of similarities in the genome size of budding and fission yeasts, they seem to have adopted some striking differences in their strategy for passing on their chromosomes. Budding yeast have "point" centromeres, where a 125 base sequence is sufficient for mitotic propagation, whereas fission yeast centromeres are more reminiscent of the large repetitive centromeres of metazoans. In addition, the centromeric heterochromatin which coats centromeric domains of fission yeast and metazoan centromeres and is critical for their function, is largely absent from budding yeast centromeres. This review focuses on the assembly and maintenance of centromeric chromatin in the fission yeast.

  18. Titration and hysteresis in epigenetic chromatin silencing

    NASA Astrophysics Data System (ADS)

    Dayarian, Adel; Sengupta, Anirvan M.

    2013-06-01

    Epigenetic mechanisms of silencing via heritable chromatin modifications play a major role in gene regulation and cell fate specification. We consider a model of epigenetic chromatin silencing in budding yeast and study the bifurcation diagram and characterize the bistable and the monostable regimes. The main focus of this paper is to examine how the perturbations altering the activity of histone modifying enzymes affect the epigenetic states. We analyze the implications of having the total number of silencing proteins, given by the sum of proteins bound to the nucleosomes and the ones available in the ambient, to be constant. This constraint couples different regions of chromatin through the shared reservoir of ambient silencing proteins. We show that the response of the system to perturbations depends dramatically on the titration effect caused by the above constraint. In particular, for a certain range of overall abundance of silencing proteins, the hysteresis loop changes qualitatively with certain jump replaced by continuous merger of different states. In addition, we find a nonmonotonic dependence of gene expression on the rate of histone deacetylation activity of Sir2. We discuss how these qualitative predictions of our model could be compared with experimental studies of the yeast system under anti-silencing drugs.

  19. The SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA Represses Abscisic Acid Responses in the Absence of the Stress Stimulus in Arabidopsis[W

    PubMed Central

    Han, Soon-Ki; Sang, Yi; Rodrigues, Americo; Wu, Miin-Feng; Rodriguez, Pedro L.; Wagner, Doris

    2012-01-01

    The survival of plants as sessile organisms depends on their ability to cope with environmental challenges. Of key importance in this regard is the phytohormone abscisic acid (ABA). ABA not only promotes seed dormancy but also triggers growth arrest in postgermination embryos that encounter water stress. This is accompanied by increased desiccation tolerance. Postgermination ABA responses in Arabidopsis thaliana are mediated in large part by the ABA-induced basic domain/leucine zipper transcription factor ABA INSENSITIVE5 (ABI5). Here, we show that loss of function of the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) causes ABA hypersensitivity during postgermination growth arrest. ABI5 expression was derepressed in brm mutants in the absence of exogenous ABA and accumulated to high levels upon ABA sensing. This effect was likely direct; chromatin immunoprecipitation revealed BRM binding to the ABI5 locus. Moreover, loss of BRM activity led to destabilization of a nucleosome likely to repress ABI5 transcription. Finally, the abi5 null mutant was epistatic to BRM in postgermination growth arrest. In addition, vegetative growth defects typical of brm mutants in the absence of ABA treatment could be partially overcome by reduction of ABA responses, and brm mutants displayed increased drought tolerance. We propose a role for BRM in the balance between growth or stress responses. PMID:23209114

  20. Dietary phenolic acids attenuate multiple stages of protein glycation and high-glucose-stimulated proinflammatory IL-1beta activation by interfering with chromatin remodeling and transcription in monocytes.

    PubMed

    Wu, Chi-Hao; Yeh, Chi-Tai; Shih, Ping-Hsiao; Yen, Gow-Chin

    2010-07-01

    This study examined the effects of dietary phenolic acids on individual stages of protein glycation and utilized monocyte cultures to assess whether these phytochemicals modulate the activation of proinflammatory cytokine under high glucose (HG, 15 mmol/L) conditions mimicking diabetes. In vitro glycation assays showed that a number of phenolic acids exerted inhibitory effects on the glycation reaction and its subsequent crosslinking. Phenolic acids, especially methoxyphenolic acids, prevented increase in both levels of the interleukin-1beta (IL-1beta) and oxidative stress caused by HG. The effect appeared to be mediated by modulation of the protein kinase C/nuclear factor-kappaB axis. Chromatin immunoprecipitation demonstrated for the first time that HG increased the recruitment of nuclear factor-kappaB p65 and CREB-binding protein to the IL-1beta promoter. Interestingly, HG also increased histone acetylation and methylation within the IL-1beta promoter and decreased histone deacetylase activities in monocytes, thus facilitating chromatin remodeling and transcription. Such inappropriate inflammatory responses were found to be controlled effectively by treatment with methoxyphenolic compounds. In conclusion, this study suggests that phenolic acids could exert their anti-inflammatory activities as antiglycation agents and as modifiers of signaling pathways. It provides evidence for a novel mechanism by which phenolics supplementation might have additional protective effects against diabetic complications.

  1. B23/nucleophosmin is involved in regulation of adenovirus chromatin structure at late infection stages, but not in virus replication and transcription.

    PubMed

    Samad, Mohammad Abdus; Komatsu, Tetsuro; Okuwaki, Mitsuru; Nagata, Kyosuke

    2012-06-01

    B23/nucleophosmin has been identified in vitro as a stimulatory factor for replication of adenovirus DNA complexed with viral basic core proteins. In the present study, the in vivo function of B23 in the adenovirus life cycle was studied. It was found that both the expression of a decoy mutant derived from adenovirus core protein V that tightly associates with B23 and small interfering RNA-mediated depletion of B23 impeded the production of progeny virions. However, B23 depletion did not significantly affect the replication and transcription of the virus genome. Chromatin immunoprecipitation analyses revealed that B23 depletion significantly increased the association of viral DNA with viral core proteins and cellular histones. These results suggest that B23 is involved in the regulation of association and/or dissociation of core proteins and cellular histones with the virus genome. In addition, these results suggest that proper viral chromatin assembly, regulated in part by B23, is crucial for the maturation of infectious virus particles.

  2. CAST-ChIP maps cell-type-specific chromatin states in the Drosophila central nervous system.

    PubMed

    Schauer, Tamás; Schwalie, Petra C; Handley, Ava; Margulies, Carla E; Flicek, Paul; Ladurner, Andreas G

    2013-10-17

    Chromatin organization and gene activity are responsive to developmental and environmental cues. Although many genes are transcribed throughout development and across cell types, much of gene regulation is highly cell-type specific. To readily track chromatin features at the resolution of cell types within complex tissues, we developed and validated chromatin affinity purification from specific cell types by chromatin immunoprecipitation (CAST-ChIP), a broadly applicable biochemical procedure. RNA polymerase II (Pol II) CAST-ChIP identifies ~1,500 neuronal and glia-specific genes in differentiated cells within the adult Drosophila brain. In contrast, the histone H2A.Z is distributed similarly across cell types and throughout development, marking cell-type-invariant Pol II-bound regions. Our study identifies H2A.Z as an active chromatin signature that is refractory to changes across cell fates. Thus, CAST-ChIP powerfully identifies cell-type-specific as well as cell-type-invariant chromatin states, enabling the systematic dissection of chromatin structure and gene regulation within complex tissues such as the brain.

  3. 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

  4. ERECTA signaling controls Arabidopsis inflorescence architecture through chromatin-mediated activation of PRE1 expression.

    PubMed

    Cai, Hanyang; Zhao, Lihua; Wang, Lulu; Zhang, Man; Su, Zhenxia; Cheng, Yan; Zhao, Heming; Qin, Yuan

    2017-03-13

    Flowering plants display a remarkable diversity in inflorescence architecture, and pedicel length is one of the key contributors to this diversity. In Arabidopsis thaliana, the receptor-like kinase ERECTA (ER) mediated signaling pathway plays important roles in regulating inflorescence architecture by promoting cell proliferation. However, the regulating mechanism remains elusive in the pedicel. Genetic interactions between ERECTA signaling and the chromatin remodeling complex SWR1 in the control of inflorescence architecture were studied. Comparative transcriptome analysis was applied to identify downstream components. Chromatin immunoprecipitation and nucleosome occupancy was further investigated. The results indicated that the chromatin remodeler SWR1 coordinates with ERECTA signaling in regulating inflorescence architecture by activating the expression of PRE1 family genes and promoting pedicel elongation. It was found that SWR1 is required for the incorporation of the H2A.Z histone variant into nucleosomes of the whole PRE1 gene family and the ERECTA controlled expression of PRE1 gene family through regulating nucleosome dynamics. We propose that utilization of a chromatin remodeling complex to regulate gene expression is a common theme in developmental control across kingdoms. These findings shed light on the mechanisms through which chromatin remodelers orchestrate complex transcriptional regulation of gene expression in coordination with a developmental cue.

  5. Fragmentation of chromatin with 125I radioactive disintegrations.

    PubMed Central

    Turner, G N; Nobis, P; Dewey, W C

    1976-01-01

    The DNA in Chinese hamster cells was labeled first for 3 h with [3H]TdR and then for 3 h with [125I]UdR. Chromatin was extracted, frozen, and stored at -30 degrees C until 1.0 X 10(17) and 1.25 X 10(17) disintegrations/g of labeled DNA occurred for 125I and 3H respectively. Velocity sedimentation of chromatin (DNA with associated chromosomal proteins) in neutral sucrose gradients indicated that the localized energy from the 125I disintegrations, which gave about 1 double-strand break/disintegration plus an additional 1.3 single strand breaks, selectively fragmented the [125I] chromatin into pieces smaller than the [3H] chromatin. In other words, 125I disintegrations caused much more localized damage in the chromatin labeled with 125I than in the chromatin labeled with 3H, and fragments induced in DNA by 125I disintegrations were not held together by the associated chromosomal proteins. Use of this 125I technique for studying chromosomal proteins associated with different regions in the cellular DNA is discussed. For these studies, the number of disintegrations required for fragmenting DNA molecules of different sizes is illustrated. PMID:963201

  6. Alteration of Large-Scale Chromatin Structure by Estrogen Receptor

    PubMed Central

    Nye, Anne C.; Rajendran, Ramji R.; Stenoien, David L.; Mancini, Michael A.; Katzenellenbogen, Benita S.; Belmont, Andrew S.

    2002-01-01

    The estrogen receptor (ER), a member of the nuclear hormone receptor superfamily important in human physiology and disease, recruits coactivators which modify local chromatin structure. Here we describe effects of ER on large-scale chromatin structure as visualized in live cells. We targeted ER to gene-amplified chromosome arms containing large numbers of lac operator sites either directly, through a lac repressor-ER fusion protein (lac rep-ER), or indirectly, by fusing lac repressor with the ER interaction domain of the coactivator steroid receptor coactivator 1. Significant decondensation of large-scale chromatin structure, comparable to that produced by the ∼150-fold-stronger viral protein 16 (VP16) transcriptional activator, was produced by ER in the absence of estradiol using both approaches. Addition of estradiol induced a partial reversal of this unfolding by green fluorescent protein-lac rep-ER but not by wild-type ER recruited by a lac repressor-SRC570-780 fusion protein. The chromatin decondensation activity did not require transcriptional activation by ER nor did it require ligand-induced coactivator interactions, and unfolding did not correlate with histone hyperacetylation. Ligand-induced coactivator interactions with helix 12 of ER were necessary for the partial refolding of chromatin in response to estradiol using the lac rep-ER tethering system. This work demonstrates that when tethered or recruited to DNA, ER possesses a novel large-scale chromatin unfolding activity. PMID:11971975

  7. Bivalent chromatin marks developmental regulatory genes in the mouse embryonic germline in vivo.

    PubMed

    Sachs, Michael; Onodera, Courtney; Blaschke, Kathryn; Ebata, Kevin T; Song, Jun S; Ramalho-Santos, Miguel

    2013-06-27

    Developmental regulatory genes have both activating (H3K4me3) and repressive (H3K27me3) histone modifications in embryonic stem cells (ESCs). This bivalent configuration is thought to maintain lineage commitment programs in a poised state. However, establishing physiological relevance has been complicated by the high number of cells required for chromatin immunoprecipitation (ChIP). We developed a low-cell-number chromatin immunoprecipitation (low-cell ChIP) protocol to investigate the chromatin of mouse primordial germ cells (PGCs). Genome-wide analysis of embryonic day 11.5 (E11.5) PGCs revealed H3K4me3/H3K27me3 bivalent domains highly enriched at developmental regulatory genes in a manner remarkably similar to ESCs. Developmental regulators remain bivalent and transcriptionally silent through the initiation of sexual differentiation at E13.5. We also identified >2,500 "orphan" bivalent domains that are distal to known genes and expressed in a tissue-specific manner but silent in PGCs. Our results demonstrate the existence of bivalent domains in the germline and raise the possibility that the somatic program is continuously maintained as bivalent, potentially imparting transgenerational epigenetic inheritance.

  8. Chromatin structure and DNA damage

    SciTech Connect

    Gale, J.M.

    1987-01-01

    This dissertation examines the structure and structural transitions of chromatin in relation to DNA damage. The ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro was examined following DNA photodamage introduced by two different agents. (1) 254-nm UV radiation and (2) trimethylpsoralen (plus near-UV radiation). Both agents are highly specific for DNA and form adducts predicted to cause different degrees of distortion in the DNA helix. The salt-induced structural transitions of intact and histone H1 depleted chromatin fibers were monitored by both analytical ultracentrifugation and light scattering. Our results show that even in the presence of extremely large, nonphysiological amounts of photodamage by either agent the ability of chromatin to fold into higher ordered structures is not affected. The compact, 30 nm fiber must therefore be able to accommodate a large amount of DNA damage without any measurable changes in the overall size or degree of compaction of this structure. The distribution of pyrimidine dimers was mapped at the single nucleotide level in nucleosome core DNA from UV-irradiated mononucleosomes, chromatin fibers, and human cells in culture using the 3' ..-->.. 5' exonuclease activity of T4 DNA polymerase.

  9. Chromatin shapes the mitotic spindle.

    PubMed

    Dinarina, Ana; Pugieux, Céline; Corral, Maria Mora; Loose, Martin; Spatz, Joachim; Karsenti, Eric; Nédélec, François

    2009-08-07

    In animal and plant cells, mitotic chromatin locally generates microtubules that self-organize into a mitotic spindle, and its dimensions and bipolar symmetry are essential for accurate chromosome segregation. By immobilizing microscopic chromatin-coated beads on slide surfaces using a microprinting technique, we have examined the effect of chromatin on the dimensions and symmetry of spindles in Xenopus laevis cytoplasmic extracts. While circular spots with diameters around 14-18 microm trigger bipolar spindle formation, larger spots generate an incorrect number of poles. We also examined lines of chromatin with various dimensions. Their length determined the number of poles that formed, with a 6 x 18 microm rectangular patch generating normal spindle morphology. Around longer lines, multiple poles formed and the structures were disorganized. While lines thinner than 10 mum generated symmetric structures, thicker lines induced the formation of asymmetric structures where all microtubules are on the same side of the line. Our results show that chromatin defines spindle shape and orientation. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data.

  10. 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.

  11. Immunoprecipitation of native botulinum neurotoxin complexes from Clostridium botulinum subtype A strains.

    PubMed

    Lin, Guangyun; Tepp, William H; Bradshaw, Marite; Fredrick, Chase M; Johnson, Eric A

    2015-01-01

    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.

  12. Chromatin Remodeling and Plant Immunity.

    PubMed

    Chen, W; Zhu, Q; Liu, Y; Zhang, Q

    2017-01-01

    Chromatin remodeling, an important facet of the regulation of gene expression in eukaryotes, is performed by two major types of multisubunit complexes, covalent histone- or DNA-modifying complexes, and ATP-dependent chromosome remodeling complexes. Snf2 family DNA-dependent ATPases constitute the catalytic subunits of ATP-dependent chromosome remodeling complexes, which accounts for energy supply during chromatin remodeling. Increasing evidence indicates a critical role of chromatin remodeling in the establishment of long-lasting, even transgenerational immune memory in plants, which is supported by the findings that DNA methylation, histone deacetylation, and histone methylation can prime the promoters of immune-related genes required for disease defense. So what are the links between Snf2-mediated ATP-dependent chromosome remodeling and plant immunity, and what mechanisms might support its involvement in disease resistance?

  13. Identification of protein interaction partners in mammalian cells using SILAC-immunoprecipitation quantitative proteomics.

    PubMed

    Emmott, Edward; Goodfellow, Ian

    2014-07-06

    Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

  14. Yo antibodies in ovarian and breast cancer patients detected by a sensitive immunoprecipitation technique.

    PubMed

    Monstad, S E; Storstein, A; Dørum, A; Knudsen, A; Lønning, P E; Salvesen, H B; Aarseth, J H; Vedeler, C A

    2006-04-01

    Onconeural antibodies are found in patients with cancer and are associated with paraneoplastic neurological syndromes (PNS). The objective of the present study was to assess the frequency of Yo antibodies in ovarian and breast cancer using a sensitive immunoprecipitation technique, and to look for any association of Yo antibodies with neurological symptoms and prognostic factors. A multiwell adapted fluid-phase immunoassay using radiolabelled recombinant cerebellar degeneration related protein (cdr2), produced by coupled in vitro transcription/translation was used for the detection of Yo antibodies. This technique combines high specificity and sensitivity with high sample analysing capacity for the antibody in question. Sera or EDTA-blood from 810 ovarian (n = 557) and breast cancer (n = 253) patients were analysed for Yo antibodies by immunoprecipitation, as well as immunofluorescence and immune blots. Two hundred healthy blood donors and sera from 17 patients with paraneoplastic cerebellar degeneration and Yo antibodies served as controls. Immunoprecipitation was more sensitive in detecting Yo antibodies than immunofluorescence and immune blots. The prevalence of Yo antibodies was 13/557 (2.3%) in ovarian cancer and 4/253 (1.6%) in breast cancer using immunoprecipitation. Yo antibodies were not correlated with specific histological subgroups. The Yo index of ovarian cancer patients in FIGO stage IV was higher compared to FIGO stage I-III. The prevalence of Yo antibodies was 3 times higher in patients with stage III breast cancer than in stage I and II. Only 2/17 (11.8%) patients with Yo antibodies detected during the screen of 810 cancer patients had PNS. The results show that the prevalence of Yo antibodies is low in ovarian and breast cancer. Yo antibodies may be associated with advanced cancer, but less often with PNS.

  15. Probing activation/deactivation of the BRASSINOSTEROID INSENSITIVE1 receptor kinase by immunoprecipitation

    PubMed Central

    Martins, Sara; Vert, Grégory; Jaillais, Yvon

    2017-01-01

    Summary Brassinosteroids are plant sterol-derived hormones that control plant growth and development. The BR receptor complex is encoded by the BRASSINOSTEROID INSENSITIVE1 (BRI1) and members of the SOMATIC EMBRYOGENESIS RECEPTOR KINASE family. BR receptor complex activation and deactivation uses different post-translational modifications and recruitment of partner proteins. In this chapter, we describe optimized immunoprecipitation protocols and variants for biochemical analyses of BRI1 post-translational modification and protein-protein interaction. PMID:28124254

  16. Complexity of chromatin folding is captured by the strings and binders switch model.

    PubMed

    Barbieri, Mariano; Chotalia, Mita; Fraser, James; Lavitas, Liron-Mark; Dostie, Josée; Pombo, Ana; Nicodemi, Mario

    2012-10-02

    Chromatin has a complex spatial organization in the cell nucleus that serves vital functional purposes. A variety of chromatin folding conformations has been detected by single-cell imaging and chromosome conformation capture-based approaches. However, a unified quantitative framework describing spatial chromatin organization is still lacking. Here, we explore the "strings and binders switch" model to explain the origin and variety of chromatin behaviors that coexist and dynamically change within living cells. This simple polymer model recapitulates the scaling properties of chromatin folding reported experimentally in different cellular systems, the fractal state of chromatin, the processes of domain formation, and looping out. Additionally, the strings and binders switch model reproduces the recently proposed "fractal-globule" model, but only as one of many possible transient conformations.

  17. The RIPper case: identification of RNA-binding protein targets by RNA immunoprecipitation.

    PubMed

    Köster, Tino; Haas, Meike; Staiger, Dorothee

    2014-01-01

    Control at the posttranscriptional level emerges as an important layer of regulation in the circadian timing system. RNA-binding proteins that specifically interact with cis-regulatory motifs within pre-mRNAs are key elements of this regulation. While the ability to interact with RNA in vitro has been demonstrated for numerous Arabidopsis RNA-binding proteins, a full understanding of posttranscriptional networks controlled by an RNA-binding protein requires the identification of its immediate in vivo targets. Here we describe differential RNA immunoprecipitation in transgenic Arabidopsis thaliana plants expressing RNA-binding protein variants epitope-tagged with green fluorescent protein. To control for RNAs that nonspecifically co-purify with the RNA-binding protein, transgenic plants are generated with a mutated version of the RNA-binding protein that is not capable of binding to its target RNAs. The RNA-binding protein variants are expressed under the control of their authentic promoter and cis-regulatory motifs. Incubation of the plants with formaldehyde in vivo cross-links the proteins to their RNA targets. A whole-cell extract is then prepared and subjected to immunoprecipitation with an antibody against the GFP tag and to mock precipitation with an antibody against the unrelated red fluorescent protein. The RNAs coprecipitating with the proteins are eluted from the immunoprecipitate and identified via reverse transcription-PCR.

  18. 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.

  19. Genome Wide Analysis of Chromatin Regulation by Cocaine Reveals a Novel Role for Sirtuins

    PubMed Central

    Renthal, William; Kumar, Arvind; Xiao, Guanghua; Wilkinson, Matthew; Covington, Herbert E.; Maze, Ian; Sikder, Devanjan; Robison, Alfred J.; LaPlant, Quincey; Dietz, David M.; Russo, Scott J.; Vialou, Vincent; Chakravarty, Sumana; Kodadek, Thomas J.; Stack, Ashley; Kabbaj, Mohammed; Nestler, Eric J.

    2009-01-01

    Summary Changes in gene expression contribute to the long-lasting regulation of the brain’s reward circuitry seen in drug addiction, however, the specific genes regulated and the transcriptional mechanisms underlying such regulation remain poorly understood. Here, we used chromatin immunoprecipitation coupled with promoter microarray analysis to characterize genome-wide chromatin changes in the mouse nucleus accumbens, a crucial brain reward region, after repeated cocaine administration. Our findings reveal several interesting principles of gene regulation by cocaine and of the role of ΔFosB and CREB, two prominent cocaine-induced transcription factors, in this brain region. The findings also provide novel and comprehensive insight into the molecular pathways regulated by cocaine – including a new role for sirtuins (Sirt1 and Sirt2) –which are induced in the nucleus accumbens by cocaine and, in turn, dramatically enhance the behavioral effects of the drug. PMID:19447090

  20. Identification and validation of regulatory SNPs that modulate transcription factor chromatin binding and gene expression in prostate cancer

    PubMed Central

    Jin, Hong-Jian; Jung, Segun; DebRoy, Auditi R.; Davuluri, Ramana V.

    2016-01-01

    Prostate cancer (PCa) is the second most common solid tumor for cancer related deaths in American men. Genome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with the increased risk of PCa. Because most of the susceptibility SNPs are located in noncoding regions, little is known about their functional mechanisms. We hypothesize that functional SNPs reside in cell type-specific regulatory elements that mediate the binding of critical transcription factors (TFs), which in turn result in changes in target gene expression. Using PCa-specific functional genomics data, here we identify 38 regulatory candidate SNPs and their target genes in PCa. Through risk analysis by incorporating gene expression and clinical data, we identify 6 target genes (ZG16B, ANKRD5, RERE, FAM96B, NAALADL2 and GTPBP10) as significant predictors of PCa biochemical recurrence. In addition, 5 SNPs (rs2659051, rs10936845, rs9925556, rs6057110 and rs2742624) are selected for experimental validation using Chromatin immunoprecipitation (ChIP), dual-luciferase reporter assay in LNCaP cells, showing allele-specific enhancer activity. Furthermore, we delete the rs2742624-containing region using CRISPR/Cas9 genome editing and observe the drastic downregulation of its target gene UPK3A. Taken together, our results illustrate that this new methodology can be applied to identify regulatory SNPs and their target genes that likely impact PCa risk. We suggest that similar studies can be performed to characterize regulatory variants in other diseases. PMID:27409348

  1. 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.

  2. 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

  3. Multiple modes of chromatin configuration at natural meiotic recombination hot spots in fission yeast.

    PubMed

    Hirota, Kouji; Steiner, Walter W; Shibata, Takehiko; Ohta, Kunihiro

    2007-11-01

    The ade6-M26 meiotic recombination hot spot of fission yeast is defined by a cyclic AMP-responsive element (CRE)-like heptanucleotide sequence, 5'-ATGACGT-3', which acts as a binding site for the Atf1/Pcr1 heterodimeric transcription factor required for hot spot activation. We previously demonstrated that the local chromatin around the M26 sequence motif alters to exhibit higher sensitivity to micrococcal nuclease before the initiation of meiotic recombination. In this study, we have examined whether or not such alterations in chromatin occur at natural meiotic DNA double-strand break (DSB) sites in Schizosaccharomyces pombe. At one of the most prominent DSB sites, mbs1 (meiotic break site 1), the chromatin structure has a constitutively accessible configuration at or near the DSB sites. The establishment of the open chromatin state and DSB formation are independent of the CRE-binding transcription factor, Atf1. Analysis of the chromatin configuration at CRE-dependent DSB sites revealed both differences from and similarities to mbs1. For example, the tdh1+ locus, which harbors a CRE consensus sequence near the DSB site, shows a meiotically induced open chromatin configuration, similar to ade6-M26. In contrast, the cds1+ locus is similar to mbs1 in that it exhibits a constitutive open configuration. Importantly, Atf1 is required for the open chromatin formation in both tdh1+ and cds1+. These results suggest that CRE-dependent meiotic chromatin changes are intrinsic processes related to DSB formation in fission yeast meiosis. In addition, the results suggest that the chromatin configuration in natural meiotic recombination hot spots can be classified into at least three distinct categories: (i) an Atf1-CRE-independent constitutively open chromatin configuration, (ii) an Atf1-CRE-dependent meiotically induced open chromatin configuration, and (iii) an Atf1-CRE-dependent constitutively open chromatin configuration.

  4. Remodeling of chromatin under low intensity diffuse ultrasound.

    PubMed

    Noriega, Sandra; Budhiraja, Gaurav; Subramanian, Anuradha

    2012-08-01

    A variety of mechanotransduction pathways mediate the response of fibroblasts or chondrocytes to ultrasound stimulation. In addition, regulatory pathways that co-ordinate stimulus-specific cellular responses are likely to exist. In this study, analysis was confined to the hypothesis that ultrasound stimulation (US) influences the chromatin structure, and that these changes may reflect a regulatory pathway that connects nuclear architecture, chromatin structure and gene expression. Murine fibroblasts seeded on tissue culture plates were stimulated with US (5.0 MHz (14 kPa), 51-s per application) and the thermal denaturation profiles of nuclei isolated from fibroblasts were assessed by dynamic scanning calorimetry (DSC). When compared to the thermal profiles obtained from the nuclei of non-stimulated cells, the nuclei obtained from stimulated cells showed a change in peak profiles and peak areas, which is indicative of chromatin remodeling. Independently, US was also observed to impact the histone (H1):chromatin association as measured indirectly by DAPI staining. Based on our work, it appears plausible that US can produce a remodeling of chromatin, thus triggering signal cascade and other intracellular mechanisms.

  5. The Circadian NAD+ Metabolism: Impact on Chromatin Remodeling and Aging

    PubMed Central

    Bessho, Yasumasa

    2016-01-01

    Gene expression is known to be a stochastic phenomenon. The stochastic gene expression rate is thought to be altered by topological change of chromosome and/or by chromatin modifications such as acetylation and methylation. Changes in mechanical properties of chromosome/chromatin by soluble factors, mechanical stresses from the environment, or metabolites determine cell fate, regulate cellular functions, or maintain cellular homeostasis. Circadian clock, which drives the expression of thousands of genes with 24-hour rhythmicity, has been known to be indispensable for maintaining cellular functions/homeostasis. During the last decade, it has been demonstrated that chromatin also undergoes modifications with 24-hour rhythmicity and facilitates the fine-tuning of circadian gene expression patterns. In this review, we cover data which suggests that chromatin structure changes in a circadian manner and that NAD+ is the key metabolite for circadian chromatin remodeling. Furthermore, we discuss the relationship among circadian clock, NAD+ metabolism, and aging/age-related diseases. In addition, the interventions of NAD+ metabolism for the prevention and treatment of aging and age-related diseases are also discussed. PMID:28050554

  6. [Resolution of spatial constraints during replication of peripheral chromatin].

    PubMed

    Zhironkina, O A; Kurchashova, S Yu; Bratseva, A L; Cherepanynets, V D; Strelkova, O S; Belmont, A S; Kireev, I I

    2014-01-01

    Tight association of peripheral chromatin with nuclear lamina unavoidably creates topological constraints during replication. Additional complications are associated with high stability of lamina meshwork, which may hinder an access of replication factors to the sites of DNA synthesis in highly condensed template with limited mobility. In the current work we studied structural organization and dynamics of lamina as a function of replicative status of associated peripheral heterochromatin. The studies of molecular mobility of laminas at various stages of S-phase in vivo and using super-resolution microscopy showed no correlation between lamina dynamics and replicative status of attached heterochromatin. These data support the hypothesis that lamina-chromatin interactions during S-phase are regulated at the level of adapter proteins. Ultrastructural studies have demonstrated that temporal break of lamina-chromatin connections during replication does not cause noticeable spatial separation of replicating domains from nuclear periphery.

  7. Interplay between modifications of chromatin and meiotic recombination hotspots.

    PubMed

    Brachet, Elsa; Sommermeyer, Vérane; Borde, Valérie

    2012-02-01

    Meiotic recombination lies at the heart of sexual reproduction. It is essential for producing viable gametes with a normal haploid genomic content and its dysfunctions can be at the source of aneuploidies, such as the Down syndrome, or many genetic disorders. Meiotic recombination also generates genetic diversity that is transmitted to progeny by shuffling maternal and paternal alleles along chromosomes. Recombination takes place at non-random chromosomal sites called 'hotspots'. Recent evidence has shown that their location is influenced by properties of chromatin. In addition, many studies in somatic cells have highlighted the need for changes in chromatin dynamics to allow the process of recombination. In this review, we discuss how changes in the chromatin landscape may influence the recombination map, and reciprocally, how recombination events may lead to epigenetic modifications at sites of recombination, which could be transmitted to progeny.

  8. Identifying different types of chromatin using Giemsa staining.

    PubMed

    Stockert, Juan C; Blázquez-Castro, Alfonso; Horobin, Richard W

    2014-01-01

    Mixtures of polychrome methylene blue-eosin Y (i.e., Giemsa stain) are widely used in biological staining. They induce a striking purple coloration of chromatin DNA (the Romanowsky-Giemsa effect), which contrasts with the blue-stained RNA-containing cytoplasm and nucleoli. After specific prestaining treatments that induce chromatin disorganization (giving banded or harlequin chromosomes), Giemsa staining produces a differential coloration, with C- and G-bands appearing in purple whereas remaining chromosome regions are blue. Unsubstituted (TT) and bromo-substituted (BT) DNAs also appear purple and blue, respectively. The same occurs in the case of BT and BB chromatids.In addition to discussing the use of Giemsa stain as a suitable method to reveal specific features of chromosome structure, some molecular processes and models are also described to explain Giemsa staining mechanisms of chromatin.

  9. Epigenetic chromatin states uniquely define the developmental plasticity of murine hematopoietic stem cells.

    PubMed

    Weishaupt, Holger; Sigvardsson, Mikael; Attema, Joanne L

    2010-01-14

    Heritable epigenetic signatures are proposed to serve as an important regulatory mechanism in lineage fate determination. To investigate this, we profiled chromatin modifications in murine hematopoietic stem cells, lineage-restricted progenitors, and CD4(+) T cells using modified genome-scale mini-chromatin immunoprecipitation technology. We show that genes involved in mature hematopoietic cell function associate with distinct chromatin states in stem and progenitor cells, before their activation or silencing upon cellular maturation. Many lineage-restricted promoters are associated with bivalent histone methylation and highly combinatorial histone modification patterns, which may determine their selective priming of gene expression during lineage commitment. These bivalent chromatin states are conserved in mammalian evolution, with a particular overrepresentation of promoters encoding key regulators of hematopoiesis. After differentiation into progenitors and T cells, activating histone modifications persist at transcriptionally repressed promoters, suggesting that these transcriptional programs might be reactivated after lineage restriction. Collectively, our data reveal the epigenetic framework that underlies the cell fate options of hematopoietic stem cells.

  10. Regulation of oxidized base damage repair by chromatin assembly factor 1 subunit A

    PubMed Central

    Yang, Chunying; Sengupta, Shiladitya; Hegde, Pavana M.; Mitra, Joy; Jiang, Shuai; Holey, Brooke; Sarker, Altaf H.; Tsai, Miaw-Sheue; Hegde, Muralidhar L.; Mitra, Sankar

    2017-01-01

    Reactive oxygen species (ROS), generated both endogenously and in response to exogenous stress, induce point mutations by mis-replication of oxidized bases and other lesions in the genome. Repair of these lesions via base excision repair (BER) pathway maintains genomic fidelity. Regulation of the BER pathway for mutagenic oxidized bases, initiated by NEIL1 and other DNA glycosylases at the chromatin level remains unexplored. Whether single nucleotide (SN)-BER of a damaged base requires histone deposition or nucleosome remodeling is unknown, unlike nucleosome reassembly which is shown to be required for other DNA repair processes. Here we show that chromatin assembly factor (CAF)-1 subunit A (CHAF1A), the p150 subunit of the histone H3/H4 chaperone, and its partner anti-silencing function protein 1A (ASF1A), which we identified in human NEIL1 immunoprecipitation complex, transiently dissociate from chromatin bound NEIL1 complex in G1 cells after induction of oxidative base damage. CHAF1A inhibits NEIL1 initiated repair in vitro. Subsequent restoration of the chaperone-BER complex in cell, presumably after completion of repair, suggests that histone chaperones sequester the repair complex for oxidized bases in non-replicating chromatin, and allow repair when oxidized bases are induced in the genome. PMID:27794043

  11. The Fanconi anemia proteins FANCD2 and FANCJ interact and regulate each other's chromatin localization.

    PubMed

    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

    2014-09-12

    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.

  12. A Polycomb Group Protein Is Retained at Specific Sites on Chromatin in Mitosis

    PubMed Central

    Follmer, Nicole E.; Wani, Ajazul H.; Francis, Nicole J.

    2012-01-01

    Epigenetic regulation of gene expression, including by Polycomb Group (PcG) proteins, may depend on heritable chromatin states, but how these states can be propagated through mitosis is unclear. Using immunofluorescence and biochemical fractionation, we find PcG proteins associated with mitotic chromosomes in Drosophila S2 cells. Genome-wide sequencing of chromatin immunoprecipitations (ChIP–SEQ) from mitotic cells indicates that Posterior Sex Combs (PSC) is not present at well-characterized PcG targets including Hox genes in mitosis, but does remain at a subset of interphase sites. Many of these persistent sites overlap with chromatin domain borders described by Sexton et al. (2012), which are genomic regions characterized by low levels of long range contacts. Persistent PSC binding sites flank both Hox gene clusters. We hypothesize that disruption of long-range chromatin contacts in mitosis contributes to PcG protein release from most sites, while persistent binding at sites with minimal long-range contacts may nucleate re-establishment of PcG binding and chromosome organization after mitosis. PMID:23284300

  13. TGF-β signaling to chromatin: how Smads regulate transcription during self-renewal and differentiation.

    PubMed

    Gaarenstroom, Tessa; Hill, Caroline S

    2014-08-01

    Ligands of the TGF-β superfamily (including the TGF-βs, Nodal and BMPs) play instructive roles during embryonic development. This is achieved by regulation of genes important for both maintaining pluripotency and germ layer specification and differentiation. Here we review how the TGF-β superfamily ligands signal to the chromatin to regulate transcription during development. The effectors of the pathway, the Smad transcription factors, are regulated in a combinatorial and spatiotemporal manner. This occurs via post-translational modifications affecting stability, localization and activity, as well as through interactions with other transcription factors and chromatin modifying enzymes, which occur on DNA. Expression profiling and Chromatin Immunoprecipitation have defined Smad target genes and binding sites on a genome-wide scale, which vary between cell types and differentiation stages. This has led to the insight that Smad-mediated transcriptional responses are influenced by the presence of master transcription factors, such as OCT4, SOX2 and NANOG in embryonic stem cells, interaction with other signal-induced factors, as well as by the general chromatin remodeling machinery. Interplay with transcriptional repressors and the polycomb group proteins also regulates the balance between expression of self-renewal and mesendoderm-specific genes in embryonic stem cells and during early development.

  14. Sequential Co-immunoprecipitation and Immunoblot Approach to Determine Oligomerisation of G-Protein-Coupled Receptors.

    PubMed

    Guest, Paul C

    2017-01-01

    G-protein-coupled receptors (GPCRs) play a major role in psychiatric disorders and are the targets of several current therapeutic approaches in this field. A number of studies have now shown that GPCRs can assemble as high molecular weight homo- and hetero-oligomers, which could affect ligand binding, intracellular signalling or trafficking. This information could be critical in design of new drugs to treat neurological and psychiatric disorders. This chapter describes a sequential co-immunoprecipitation and immunoblot protocol for determining oligomerisation of the 5-hydroxytryptamine (HT)1A receptor with other GPCRs in co-transfected HEK-293 cells.

  15. Bioorthogonal click chemistry to assay mu-opioid receptor palmitoylation using 15-hexadecynoic acid and immunoprecipitation

    PubMed Central

    Ebersole, Brittany; Petko, Jessica; Levenson, Robert

    2014-01-01

    We have developed a modification of bioorthogonal click chemistry to assay the palmitoylation of cellular proteins. This assay utilizes 15-hexadecynoic acid (15-HDYA) as a chemical probe in combination with protein immunoprecipitation using magnetic beads in order to detect S-palmitoylation of proteins of interest. Here we demonstrate the utility of this approach for the mu-opioid receptor (MOR), a GPCR responsible for mediating the analgesic and addictive properties of most clinically relevant opioid agonist drugs. This technique provides a rapid, non-isotopic, and efficient method to assay the palmitoylation status of a variety of cellular proteins including most GPCRs. PMID:24463015

  16. Histone lysine methylation and chromatin replication.

    PubMed

    Rivera, Carlos; Gurard-Levin, Zachary A; Almouzni, Geneviève; Loyola, Alejandra

    2014-12-01

    In eukaryotic organisms, the replication of the DNA sequence and its organization into chromatin are critical to maintain genome integrity. Chromatin components, such as histone variants and histone post-translational modifications, along with the higher-order chromatin structure, impact several DNA metabolic processes, including replication, transcription, and repair. In this review we focus on lysine methylation and the relationships between this histone mark and chromatin replication. We first describe studies implicating lysine methylation in regulating early steps in the replication process. We then discuss chromatin reassembly following replication fork passage, where the incorporation of a combination of newly synthesized histones and parental histones can impact the inheritance of lysine methylation marks on the daughter strands. Finally, we elaborate on how the inheritance of lysine methylation can impact maintenance of the chromatin landscape, using heterochromatin as a model chromatin domain, and we discuss the potential mechanisms involved in this process.

  17. 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

  18. 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

  19. Immunoprecipitation of Serum Albumin with Protein A-Sepharose: A Biochemistry Laboratory Experiment

    NASA Astrophysics Data System (ADS)

    Bohinski, Robert C.

    2000-11-01

    An exercise has been designed and optimized to acquaint students with the simple yet powerful technique of immunoprecipitation. Protein A-Sepharose (PA-S) is used as a solid-phase precipitant to recover bovine serum albumin (BSA, the antigen) recognized by anti-BSA antibody (Ab). The high degree of binding specificity between antigen and antibody is illustrated by recovery of BSA from a complex mixture of proteins obtained from wheat germ and chicken breast. Various controls are included for a thorough data analysis. The solid phase of Ag/Ab/PA-S is recovered by centrifugation, thoroughly washed, and treated to dissociate the BSA antigen. Samples are examined by discontinuous denaturing gel electrophoresis (SDS-PAGE) with Coomassie blue staining. The supernatants, containing proteins that are not precipitated, are also analyzed. Antigenic cross-reactivity, ranging from strong to none, is demonstrated in a second part by using serum albumins from seven different sources. Systems can be set up, shaken, and prepared for electrophoresis in a single lab period with time for laboratory lecture and discussion about antibody structure and function, antibody-based methods in general, and immunoprecipitation in particular.

  20. Chromatin fiber polymorphism triggered by variations of DNA linker lengths.

    PubMed

    Collepardo-Guevara, Rosana; Schlick, Tamar

    2014-06-03

    Deciphering the factors that control chromatin fiber structure is key to understanding fundamental chromosomal processes. Although details remain unknown, it is becoming clear that chromatin is polymorphic depending on internal and external factors. In particular, different lengths of the linker DNAs joining successive nucleosomes (measured in nucleosome-repeat lengths or NRLs) that characterize different cell types and cell cycle stages produce different structures. NRL is also nonuniform within single fibers, but how this diversity affects chromatin fiber structure is not clear. Here we perform Monte Carlo simulations of a coarse-grained oligonucleosome model to help interpret fiber structure subject to intrafiber NRL variations, as relevant to proliferating cells of interphase chromatin, fibers subject to remodeling factors, and regulatory DNA sequences. We find that intrafiber NRL variations have a profound impact on chromatin structure, with a wide range of different architectures emerging (highly bent narrow forms, canonical and irregular zigzag fibers, and polymorphic conformations), depending on the NRLs mixed. This stabilization of a wide range of fiber forms might allow NRL variations to regulate both fiber compaction and selective DNA exposure. The polymorphic forms spanning canonical to sharply bent structures, like hairpins and loops, arise from large NRL variations and are surprisingly more compact than uniform NRL structures. They are distinguished by tail-mediated far-nucleosome interactions, in addition to the near-nucleosome interactions of canonical 30-nm fibers. Polymorphism is consistent with chromatin's diverse biological functions and heterogeneous constituents. Intrafiber NRL variations, in particular, may contribute to fiber bending and looping and thus to distant communication in associated regulatory processes.

  1. 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

  2. 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

  3. Histone Deacetylase Activity Represses Gamma Interferon-Inducible HLA-DR Gene Expression following the Establishment of a DNase I-Hypersensitive Chromatin Conformation

    PubMed Central

    Osborne, Aaron; Zhang, Hongquan; Yang, Wen-Ming; Seto, Edward; Blanck, George

    2001-01-01

    Expression of the retinoblastoma tumor suppressor protein (Rb) is required for gamma interferon (IFN-γ)-inducible major histocompatibility complex class II gene expression and transcriptionally productive HLA-DRA promoter occupancy in several human tumor cell lines. Treatment of these Rb-defective tumor cell lines with histone deacetylase (HDAC) inhibitors rescued IFN-γ-inducible HLA-DRA and -DRB mRNA and cell surface protein expression, demonstrating repression of these genes by endogenous cellular HDAC activity. Additionally, Rb-defective, transcriptionally incompetent tumor cells retained the HLA-DRA promoter DNase I-hypersensitive site. Thus, HDAC-mediated repression of the HLA-DRA promoter occurs following the establishment of an apparent nucleosome-free promoter region and before transcriptionally productive occupancy of the promoter by the required transactivators. Repression of HLA-DRA promoter activation by HDAC activity likely involves a YY1 binding element located in the first exon of the HLA-DRA gene. Chromatin immunoprecipitation experiments localized YY1 to the HLA-DRA gene in Rb-defective tumor cells. Additionally, mutation of the YY1 binding site prevented repression of the promoter by HDAC1 and partially prevented activation of the promoter by trichostatin A. Mutation of the octamer element also significantly reduced the ability of HDAC1 to confer repression of inducible HLA-DRA promoter activation. Treatment of Rb-defective tumor cells with HDAC inhibitors greatly reduced the DNA binding activity of Oct-1, a repressor of inducible HLA-DRA promoter activation. These findings represent the first evidence that HDAC activity can repress IFN-γ-inducible HLA class II gene expression and also demonstrate that HDAC activity can contribute to promoter repression following the establishment of a DNase I-hypersensitive chromatin conformation. PMID:11533238

  4. HDACi--targets beyond chromatin.

    PubMed

    Buchwald, Marc; Krämer, Oliver H; Heinzel, Thorsten

    2009-08-08

    Histone deacetylases (HDACs) play an important role in gene regulation. Inhibitors of HDACs (HDACi) are novel anti-cancer drugs, which induce histone (hyper-) acetylation and counteract aberrant gene repression. On the other hand, HDACi treatment can also result in decreased gene expression, and targeting HDACs affects more than chromatin. Recently, HDACi were shown to evoke non-histone protein acetylation, which can alter signaling networks relevant for tumorgenesis. Furthermore, HDACi can promote the degradation of (proto-) oncoproteins. Here, we summarize these findings and discuss how these substances could be beneficial for the treatment and prevention of human ailments, such as cancer and unbalanced immune functions.

  5. 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

  6. Mechanism of the interaction of plant alkaloid vincristine with DNA and chromatin: spectroscopic study.

    PubMed

    Mohammadgholi, Azadeh; Rabbani-Chadegani, Azra; Fallah, Sodabeh

    2013-05-01

    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.

  7. 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.

  8. Characterization of a Protein Interactome by Co-Immunoprecipitation and Shotgun Mass Spectrometry.

    PubMed

    Maccarrone, Giuseppina; Bonfiglio, Juan Jose; Silberstein, Susana; Turck, Christoph W; Martins-de-Souza, Daniel

    2017-01-01

    Identifying the partners of a given protein (the interactome) may provide leads about the protein's function and the molecular mechanisms in which it is involved. One of the alternative strategies used to characterize protein interactomes consists of co-immunoprecipitation (co-IP) followed by shotgun mass spectrometry. This enables the isolation and identification of a protein target in its native state and its interactome from cells or tissue lysates under physiological conditions. In this chapter, we describe a co-IP protocol for interactome studies that uses an antibody against a protein of interest bound to protein A/G plus agarose beads to isolate a protein complex. The interacting proteins may be further fractionated by SDS-PAGE, followed by in-gel tryptic digestion and nano liquid chromatography high-resolution tandem mass spectrometry (nLC ESI-MS/MS) for identification purposes. The computational tools, strategy for protein identification, and use of interactome databases also will be described.

  9. The Chd Family of Chromatin Remodelers

    PubMed Central

    Marfella, Concetta G.A.; Imbalzano, Anthony N.

    2007-01-01

    Chromatin remodeling enzymes contribute to the dynamic changes that occur in chromatin structure during cellular processes such as transcription, recombination, repair, and replication. Members of the chromodomain helicase DNA-binding (Chd) family of enzymes belong to the SNF2 superfamily of ATP-dependent chromatin remodelers. The Chd proteins are distinguished by the presence of two N-terminal chromodomains that function as interaction surfaces for a variety of chromatin components. Genetic, biochemical, and structural studies demonstrate that Chd proteins are important regulators of transcription and play critical roles during developmental processes. Numerous Chd proteins are also implicated in human disease. PMID:17350655

  10. Development of microLIPS (Luciferase Immunoprecipitation Systems): a novel microfluidic assay for rapid serum antibody detection

    NASA Astrophysics Data System (ADS)

    Chandrangsu, Matt; Burbelo, Peter D.; Iadarola, Michael J.; Smith, Paul D.; Morgan, Nicole Y.

    2012-06-01

    There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this technology in the field: first, simplifying assay operation through the automation of flow control using power-free means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding capacity, in order to move towards portable battery powered electronics.

  11. Measuring NLR Oligomerization I: Size Exclusion Chromatography, Co-immunoprecipitation, and Cross-Linking.

    PubMed

    Khare, Sonal; Radian, Alexander D; Dorfleutner, Andrea; Stehlik, Christian

    2016-01-01

    Oligomerization of nod-like receptors (NLRs) can be detected by several biochemical techniques dependent on the stringency of protein-protein interactions. Some of these biochemical methods can be combined with functional assays, such as caspase-1 activity assay. Size exclusion chromatography (SEC) allows separation of native protein lysates into different sized complexes by fast protein liquid chromatography (FPLC) for follow-up analysis. Using co-immunoprecipitation (co-IP), combined with SEC or on its own, enables subsequent antibody-based purification of NLR complexes and associated proteins, which can then be analyzed by immunoblot and/or subjected to functional caspase-1 activity assay. Chemical cross-linking covalently joins two or more molecules, thus capturing the oligomeric state with high sensitivity and stability. Apoptosis-associated speck-like protein containing a caspase activation domain (ASC) oligomerization has been successfully used as readout for NLR or AIM2-like receptor (ALR) inflammasome activation in response to various pathogen- or damage-associated molecular patterns (PAMPs or DAMPs) in human and mouse macrophages and THP-1 cells. Here, we provide a detailed description of the methods used for NLRP7 oligomerization in response to infection with Staphylococcus aureus (S. aureus) in primary human macrophages, co-immunoprecipitation and immunoblot analysis of NLRP7 and NLRP3 inflammasome complexes, as well as caspase-1 activity assays. Also, ASC oligomerization is shown in response to dsDNA, LPS/ATP, and LPS/nigericin in mouse bone marrow-derived macrophages (BMDMs) and/or THP-1 cells or human primary macrophages.

  12. Identification of Methylated Deoxyadenosines in Genomic DNA by dA6m DNA Immunoprecipitation

    PubMed Central

    Koziol, Magdalena J.; Bradshaw, Charles R.; Allen, George E.; Costa, Ana S.H.; Frezza, Christian

    2017-01-01

    dA6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N6-methyldeoxyadenosine (dA6m). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start sites and excluded from exons, suggesting a role in transcriptional regulation (Koziol et al., 2015). Importantly, its existence suggests that DNA might be more diverse than previously believed, as further DNA modifications might exist in eukaryotic DNA (Koziol et al., 2015). This protocol describes the method to perform dA6m DNA immunoprecipitation (DIP), as was applied to characterize the first dA6m methylome analysis in higher eukaryotes (Koziol et al., 2015). In this protocol, we describe how genomic DNA is isolated, fragmented and then DNA containing dA6m is pulled down with an antibody that recognizes dA6m in genomic DNA. After subsequent washes, DNA fragments that do not contain dA6m are eliminated, and the dA6m containing fragments are eluted from the antibody in order to be processed further for subsequent analyses. Background This protocol was developed in order to identify regions in the genome that contain dA6m. It can be used to detect dA6m in different genomes. As a guideline, this protocol was established from existing approaches used to detect adenosine methylation in RNA (Dominissini et al., 2013). We developed this protocol and adapted it for the detection of dA6m in DNA, rather than detecting adenosine methylation RNA. This was required, as no protocol was available at that time to allow the genome-wide identification of dA6m in eukaryotic DNA. PMID:28180135

  13. Nuclear Phosphoinositide Regulation of Chromatin.

    PubMed

    Hamann, Bree L; Blind, Raymond D

    2017-03-03

    Phospholipid signaling has clear connections to a wide array of cellular processes, particularly in gene expression and in controlling the chromatin biology of cells. However, most of the work elucidating how phospholipid signaling pathways contribute to cellular physiology have studied cytoplasmic membranes, while relatively little attention has been paid to the role of phospholipid signaling in the nucleus. Recent work from several labs has shown that nuclear phospholipid signaling can have important roles that are specific to this cellular compartment. This review focuses on the nuclear phospholipid functions and the activities of phospholipid signaling enzymes that regulate metazoan chromatin and gene expression. In particular, we highlight the roles that nuclear phosphoinositides play in several nuclear-driven physiological processes, such as differentiation, proliferation, and gene expression. Taken together, the recent discovery of several specifically nuclear phospholipid functions could have dramatic impact on our understanding of the fundamental mechanisms that enable tight control of cellular physiology. This article is protected by copyright. All rights reserved.

  14. 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

  15. Systematic Mapping of RNA-Chromatin Interactions In Vivo.

    PubMed

    Sridhar, Bharat; Rivas-Astroza, Marcelo; Nguyen, Tri C; Chen, Weizhong; Yan, Zhangming; Cao, Xiaoyi; Hebert, Lucie; Zhong, Sheng

    2017-02-20

    RNA molecules can attach to chromatin. It remains difficult to know what RNAs are associated with chromatin and where the genomic target loci of these RNAs are. Here, we present MARGI (mapping RNA-genome interactions), a technology to massively reveal native RNA-chromatin interactions from unperturbed cells. The gist of this technology is to ligate chromatin-associated RNAs (caRNAs) with their target genomic sequences by proximity ligation, forming RNA-DNA chimeric sequences, which are converted to a sequencing library for paired-end sequencing. Using MARGI, we produced RNA-genome interaction maps for human embryonic stem cells (ESCs) and human embryonic kidney (HEK) cells. MARGI revealed hundreds of caRNAs, including previously known XIST, SNHG1, NEAT1, and MALAT1, as well as each caRNA's genomic interaction loci. Using a cross-species experiment, we estimated that approximately 2.2% of MARGI-identified interactions were false positives. In ESCs and HEK cells, the RNA ends of more than 5% of MARGI read pairs were mapped to distal or inter-chromosomal locations as compared to the locations of their corresponding DNA ends. The majority of transcription start sites are associated with distal or inter-chromosomal caRNAs. Chromatin-immunoprecipitation-sequencing (ChIP-seq)-reported H3K27ac and H3K4me3 levels are positively correlated, while H3K9me3 is negatively correlated, with MARGI-reported RNA attachment levels. The MARGI technology should facilitate revealing novel RNA functions and their genomic target regions.

  16. Identification of lamin B–regulated chromatin regions based on chromatin landscapes

    PubMed Central

    Zheng, Xiaobin; Kim, Youngjo; Zheng, Yixian

    2015-01-01

    Lamins, the major structural components of the nuclear lamina (NL) found beneath the nuclear envelope, are known to interact with most of the nuclear peripheral chromatin in metazoan cells. Although NL–chromatin associations correlate with a repressive chromatin state, the role of lamins in tethering chromatin to NL and how such tether influences gene expression have remained challenging to decipher. Studies suggest that NL proteins regulate chromatin in a context-dependent manner. Therefore understanding the context of chromatin states based on genomic features, including chromatin–NL interactions, is important to the study of lamins and other NL proteins. By modeling genome organization based on combinatorial patterns of chromatin association with lamin B1, core histone modification, and core and linker histone occupancy, we report six distinct large chromatin landscapes, referred to as histone lamin landscapes (HiLands)-red (R), -orange (O), -yellow (Y), -green (G), -blue (B), and -purple (P), in mouse embryonic stem cells (mESCs). This HiLands model demarcates the previously mapped lamin-associated chromatin domains (LADs) into two HiLands, HiLands-B and HiLands-P, which are similar to facultative and constitutive heterochromatins, respectively. Deletion of B-type lamins in mESCs caused a reduced interaction between regions of HiLands-B and NL as measured by emerin–chromatin interaction. Our findings reveal the importance of analyzing specific chromatin types when studying the function of NL proteins in chromatin tether and regulation. PMID:25995381

  17. Generation of bivalent chromatin domains during cell fate decisions

    PubMed Central

    2011-01-01

    Background In self-renewing, pluripotent cells, bivalent chromatin modification is thought to silence (H3K27me3) lineage control genes while 'poising' (H3K4me3) them for subsequent activation during differentiation, implying an important role for epigenetic modification in directing cell fate decisions. However, rather than representing an equivalently balanced epigenetic mark, the patterns and levels of histone modifications at bivalent genes can vary widely and the criteria for identifying this chromatin signature are poorly defined. Results Here, we initially show how chromatin status alters during lineage commitment and differentiation at a single well characterised bivalent locus. In addition we have determined how chromatin modifications at this locus change with gene expression in both ensemble and single cell analyses. We also show, on a global scale, how mRNA expression may be reflected in the ratio of H3K4me3/H3K27me3. Conclusions While truly 'poised' bivalently modified genes may exist, the original hypothesis that all bivalent genes are epigenetically premarked for subsequent expression might be oversimplistic. In fact, from the data presented in the present work, it is equally possible that many genes that appear to be bivalent in pluripotent and multipotent cells may simply be stochastically expressed at low levels in the process of multilineage priming. Although both situations could be considered to be forms of 'poising', the underlying mechanisms and the associated implications are clearly different. PMID:21645363

  18. Chromatin immunoselection defines a TAL-1 target gene.

    PubMed Central

    Cohen-Kaminsky, S; Maouche-Chrétien, L; Vitelli, L; Vinit, M A; Blanchard, I; Yamamoto, M; Peschle, C; Roméo, P H

    1998-01-01

    Despite the major functions of the basic helix-loop-helix transcription factor TAL-1 in hematopoiesis and T-cell leukemogenesis, no TAL-1 target gene has been identified. Using immunoprecipitation of genomic fragments bound to TAL-1 in the chromatin of murine erythro-leukemia (MEL) cells, we found that 10% of the immunoselected fragments contained a CAGATG or a CAGGTG E-box, followed by a GATA site. We studied one of these fragments containing two E-boxes, CAGATG and CAGGTC, followed by a GATA motif, and showed that TAL-1 binds to the CAGGTG E-box with an affinity modulated by the CAGATG or the GATA site, and that the CAGGTG-GATA motif exhibits positive transcriptional activity in MEL but not in HeLa cells. This immunoselected sequence is located within an intron of a new gene co-expressed with TAL-1 in endothelial and erythroid cells, but not expressed in fibroblasts or adult liver where no TAL-1 mRNA was detected. Finally, in vitro differentiation of embryonic stem cells towards the erythro/megakaryocytic pathways showed that the TAL-1 target gene expression followed TAL-1 and GATA-1 expression. These results establish that TAL-1 is likely to activate its target genes through a complex that binds an E-box-GATA motif and define the first gene regulated by TAL-1. PMID:9724651

  19. [Comparative investigation of the non-histone proteins of chromatin from pigeon erythroblasts and erythrocytes].

    PubMed

    Fedina, A B; Gazarian, G G

    1976-01-01

    Chromosomal non-histone proteins are obtained from nuclei of two types of pigeon erythroid cells: erythroblasts (cells active in RNA synthesis) and erythrocytes (cells with repressed RNA synthesis). They are well soluble in solutions of low ionic strength. Electrophoretic separation of the obtained non-histone proteins in polyacrylamide gels with urea and SDS shows the presence of qualitative differences in the pattern of non-histone proteins of chromatine from erythroblasts and erythrocytes. By electrophoresis in urea some protein bands of non-histone proteins of chromatine from erythroblasts were found which disappear with the aging of cells. At the same time two protein fractions were observed in chromatine from erythrocytes which were absent in that of erythroblasts. Disappearance of some high molecular weight protein fractions from erythrocyte chromatine as compared to erythroblasts was observed by separation of the non-histone proteins in the presence of SDS. These fractions of the non-histone proteins disappearing during aging of cells are well extractable from erythroblast chromatine by 0.35 M NaCl solution. In the in vitro system with E. coli RNA polymerase addition of non-histone proteins of chromatine from erythroblasts to chromatine from erythrocytes increases RNA synthesis 2--3 times. At the same time addition of non-histone proteins from erythrocytes is either without any influence on this process or somewhat inhibiting.

  20. 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...

  1. Chromatin remodeling: nucleosomes bulging at the seams.

    PubMed

    Peterson, Craig L

    2002-04-02

    ATP-dependent chromatin remodeling enzymes, such as SWI/SNF, hydrolyze thousands of ATPs to regulate gene expression on chromatin fibers. Recent mechanistic studies suggest that these enzymes generate localized changes in DNA topology that drive formation of multiple, remodeled nucleosomal states.

  2. Chromatin roadblocks to reprogramming 50 years on.

    PubMed

    Skene, Peter J; Henikoff, Steven

    2012-10-29

    A half century after John Gurdon demonstrated nuclear reprogramming, for which he was awarded the 2012 Nobel Prize in Physiology or Medicine, his group provides insights into the molecular mechanisms whereby chromatin remodeling is required for nuclear reprogramming. Among the issues addressed in Gurdon's latest work are the chromatin impediments to artificially induced reprogramming, discovered by Shinya Yamanaka, who shared the award with Gurdon.

  3. 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.

  4. TOPICAL REVIEW: The physics of chromatin

    NASA Astrophysics Data System (ADS)

    Schiessel, Helmut

    2003-05-01

    Recent progress has been made in the understanding of the physical properties of chromatin - the dense complex of DNA and histone proteins that occupies the nuclei of plant and animal cells. Here I will focus on the two lowest levels of the hierarchy of DNA folding into the chromatin complex. (i) The nucleosome, the chromatin repeating unit consisting of a globular aggregate of eight histone proteins with the DNA wrapped around it: its overcharging, the DNA unwrapping transition, the 'sliding' of the octamer along the DNA. (ii) The 30 nm chromatin fibre, the necklace-like structure of nucleosomes connected via linker DNA: its geometry, its mechanical properties under stretching and its response to changing ionic conditions. I will stress that chromatin combines two seemingly contradictory features: (1) high compaction of DNA within the nuclear envelope and, at the same time, (2) accessibility to genes, promoter regions and gene regulatory sequences.

  5. [Chromatin morphology and cytokinesis in pleurocapsalean cyanobacteria].

    PubMed

    Pinevich, A V; Gavrilova, O V; Averina, S G

    2007-01-01

    By means of differential interference contrast (DIC) and fluorescence microscopy, chromatin morphology and cytokinesis have been described in the cyanobacterium Pleurocapsa sp. CALU 1126 capable of multiple fission (multiple reproduction of the mother cell, the macrocyte, with formation of unique reproductive cells, the baeocytes). Two kinds of chromatin behavior have been revealed in the cell cycle: 1) the formation of numerous chromatin areas before their compartmentalization by multiple fission; 2) chromatin condensation in the phase of binary fission, and chromatin decondensation in growth period. The cytokinetic essence of multiple fission has been shown to consist of successive binary fissions of the macrocyte, while in between the mother cells (pre-baeocytes) do not grow.

  6. Interactions of transcription factors with chromatin.

    PubMed

    van Bakel, Harm

    2011-01-01

    Sequence-specific transcription factors (TFs) play a central role in regulating transcription initiation by directing the recruitment and activity of the general transcription machinery and accessory factors. It is now well established that many of the effects exerted by TFs in eukaryotes are mediated through interactions with a host of coregulators that modify the chromatin state, resulting in a more open (in case of activation) or closed conformation (in case of repression). The relationship between TFs and chromatin is a two-way street, however, as chromatin can in turn influence the recognition and binding of target sequences by TFs. The aim of this chapter is to highlight how this dynamic interplay between TF-directed remodelling of chromatin and chromatin-adjusted targeting of TF binding determines where and how transcription is initiated, and to what degree it is productive.

  7. Initiation of meiotic recombination in chromatin structure.

    PubMed

    Yamada, Takatomi; Ohta, Kunihiro

    2013-08-01

    Meiotic homologous recombination is markedly activated during meiotic prophase to play central roles in faithful chromosome segregation and conferring genetic diversity to gametes. It is initiated by programmed DNA double-strand breaks (DSBs) by the conserved protein Spo11, and preferentially occurs at discrete sites called hotspots. Since the functions of Spo11 are influenced by both of local chromatin at hotspots and higher-order chromosome structures, formation of meiotic DSBs is under regulation of chromatin structure. Therefore, investigating features and roles of meiotic chromatin is crucial to elucidate the in vivo mechanism of meiotic recombination initiation. Recent progress in genome-wide chromatin analyses tremendously improved our understanding on this point, but many critical questions are left unaddressed. In this review, we summarize current knowledge in the field, and also discuss the future problems that must be solved to understand the role of chromatin structure in meiotic recombination.

  8. The Rb1 tumour suppressor gene modifies telomeric chromatin architecture by regulating TERRA expression

    PubMed Central

    Gonzalez-Vasconcellos, I.; Schneider, R.; Anastasov, N.; Alonso-Rodriguez, S.; Sanli-Bonazzi, B.; Fernández, J. L.; Atkinson, M. J.

    2017-01-01

    The tumour suppressor gene (Rb1) is necessary for the maintenance of telomere integrity in osteoblastic cells. We now show that the compaction of telomeric chromatin and the appropriate histone modifications of telomeric DNA are both dependent upon Rb1-mediated transcription of the telomere-derived long non-coding RNA TERRA. Expression of TERRA was reduced in Rb1 haploinsufficient cells, and further decreased by shRNA-mediated reduction of residual Rb1 expression. Restoration of Rb1 levels through lentiviral transduction was sufficient to reestablish both transcription of TERRA and condensation of telomeric chromatin. The human chromosome 15q TERRA promoter contains predicted retinoblastoma control elements, and was able to confer Rb1-dependent transcription upon a promoterless reporter gene. Chromatin immunoprecipitation revealed preferential binding of phosphorylated over non-phosphorylated Rb1 at the TERRA promoter. As Rb1-deficient cells show increased genomic instability we suggest that this novel non-canonical action of Rb1 may contribute to the tumour suppressive actions of Rb1. PMID:28169375

  9. Comparative analysis of chromatin binding by Sex Comb on Midleg (SCM) and other polycomb group repressors at a Drosophila Hox gene.

    PubMed

    Wang, Liangjun; Jahren, Neal; Miller, Ellen L; Ketel, Carrie S; Mallin, Daniel R; Simon, Jeffrey A

    2010-06-01

    Sex Comb on Midleg (SCM) is a transcriptional repressor in the Polycomb group (PcG), but its molecular role in PcG silencing is not known. Although SCM can interact with Polycomb repressive complex 1 (PRC1) in vitro, biochemical studies have indicated that SCM is not a core constituent of PRC1 or PRC2. Nevertheless, SCM is just as critical for Drosophila Hox gene silencing as canonical subunits of these well-characterized PcG complexes. To address functional relationships between SCM and other PcG components, we have performed chromatin immunoprecipitation studies using cultured Drosophila Schneider line 2 (S2) cells and larval imaginal discs. We find that SCM associates with a Polycomb response element (PRE) upstream of the Ubx gene which also binds PRC1, PRC2, and the DNA-binding PcG protein Pleiohomeotic (PHO). However, SCM is retained at this Ubx PRE despite genetic disruption or knockdown of PHO, PRC1, or PRC2, suggesting that SCM chromatin targeting does not require prior association of these other PcG components. Chromatin immunoprecipitations (IPs) to test the consequences of SCM genetic disruption or knockdown revealed that PHO association is unaffected, but reduced levels of PRE-bound PRC2 and PRC1 were observed. We discuss these results in light of current models for recruitment of PcG complexes to chromatin targets.

  10. Overlapping chromatin-remodeling systems collaborate genome wide at dynamic chromatin transitions.

    PubMed

    Morris, Stephanie A; Baek, Songjoon; Sung, Myong-Hee; John, Sam; Wiench, Malgorzata; Johnson, Thomas A; Schiltz, R Louis; Hager, Gordon L

    2014-01-01

    ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4 and Snf2h. The localization patterns of all three proteins substantially overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome wide and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Many regions require the activity of more than one remodeler to regulate accessibility. These findings provide a dynamic view of chromatin organization and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes.

  11. Chromatin Dynamics of Circadian Transcription

    PubMed Central

    Aguilar-Arnal, Lorena; Sassone-Corsi, Paolo

    2015-01-01

    The molecular circadian clock orchestrates the daily cyclical expression of thousands of genes. Disruption of this transcriptional program leads to a variety of pathologies, including insomnia, depression and metabolic disorders. Circadian rhythms in gene expression rely on specific chromatin transitions which are ultimately coordinated by the molecular clock. As a consequence, a highly plastic and dynamic circadian epigenome can be delineated across different tissues and cell types. Intriguingly, genome topology appears to coordinate cyclic transcription at circadian interactomes, in which circadian genes are in physical contact within the cell nucleus in a time-specific manner. Moreover, the clock machinery shows functional interplays with key metabolic regulators, thereby connecting the circadian epigenome to cellular metabolism. Unraveling the molecular aspects of such interplays is likely to reveal new therapeutic strategies towards the treatment of metabolic disorders. PMID:27014564

  12. 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.

  13. Alterations in chromatin structure during early sea urchin embryogenesis.

    PubMed Central

    Savić, A; Richman, P; Williamson, P; Poccia, D

    1981-01-01

    Sea urchin sperm before fertilization possess the longest nucleosome repeat length yet determined for any chromatin. By the time the fertilized egg gives rise to a blastula or gastrula embryo, the chromatin has a considerably shorter repeat length and, in addition, a sequence of different histone variants of H1, H2A, and H2B has appeared. We have investigated the relationship between these variations in histone composition and concomitant alterations in chromatin structure during the earliest stages of embryogenesis in two species of sea urchin. In contrast to the long repeat distance in sperm, chromatin loaded with cleavage stage histones has a much smaller repeat. Later stages containing predominantly alpha histones display an intermediate spacing. More detailed analysis of the events in the first cell cycle was carried out with polyspermically fertilized eggs. During the first 30 min after fertilization, in which sperm-specific H1 is completely replaced by cleavage-stage H1, the male pronuclear repeat remains unchanged. The decrease toward the repeat length of cleavage stages begins at about the time of DNA synthesis. Higher degrees of polyspermy extend the length of the cell cycle, including the duration of S phase and the length of time to reach the first chromosome condensation. At these higher degrees of polyspermy, the decrease in repeat length is also slowed. We conclude that the adjustment of the arrangement of nucleosomes in embryonic chromatin from that found in sperm can occur within the first cell cycle and that its timing is cell-cycle dependent. The adjustment is separable from a corresponding change in H1 composition. Images PMID:6943576

  14. Ago2 Immunoprecipitation Identifies Predicted MicroRNAs in Human Embryonic Stem Cells and Neural Precursors

    PubMed Central

    Swerdel, Mavis R.; Moore, Jennifer C.; Cohen, Rick I.; Wu, Hao; Sun, Yi E.; Hart, Ronald P.

    2009-01-01

    Background MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation. Methodology/Principal Findings SOLiD ultra-deep sequencing identified >107 unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs. Conclusions/Significance Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation. PMID:19784364

  15. Searching for Biomarkers: Humoral Response Profiling with Luciferase Immunoprecipitation Systems (LIPS)

    PubMed Central

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

    2013-01-01

    B-cell mediated humoral responses are triggered in many human diseases including autoimmune, cancer, 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 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 provide a framework for incorporating LIPS antibody profiling into global health initiatives and disease monitoring. PMID:21679112

  16. SWI/SNF Protein Component BAF250a Regulates Cardiac Progenitor Cell Differentiation by Modulating Chromatin Accessibility during Second Heart Field Development*

    PubMed Central

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

    2012-01-01

    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. PMID:22621927

  17. Direct screening for chromatin status on DNA barcodes in yeast delineates the regulome of H3K79 methylation by Dot1

    PubMed Central

    Vlaming, Hanneke; Molenaar, Thom M; van Welsem, Tibor; Poramba-Liyanage, Deepani W; Smith, Desiree E; Velds, Arno; Hoekman, Liesbeth; Korthout, Tessy; Hendriks, Sjoerd; Maarten Altelaar, AF; van Leeuwen, Fred

    2016-01-01

    Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants. In Epi-ID, chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing, allowing for quantitative comparison of many mutants in parallel. Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes. These include histone deposition, homologous recombination, and adenosine kinase, which influences the methionine cycle. Gcn5, the acetyltransferase within the SAGA complex, was found to regulate histone methylation and H2B ubiquitination. The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features. DOI: http://dx.doi.org/10.7554/eLife.18919.001 PMID:27922451

  18. SIR-nucleosome interactions: structure-function relationships in yeast silent chromatin.

    PubMed

    Oppikofer, Mariano; Kueng, Stephanie; Gasser, Susan M

    2013-09-15

    Discrete regions of the eukaryotic genome assume a heritable chromatin structure that is refractory to gene expression, referred to as heterochromatin or "silent" chromatin. Constitutively silent chromatin is found in subtelomeric domains in a number of species, ranging from yeast to man. In addition, chromatin-dependent repression of mating type loci occurs in both budding and fission yeasts, to enable sexual reproduction. The silencing of chromatin in budding yeast is characterized by an assembly of Silent Information Regulatory (SIR) proteins-Sir2, Sir3 and Sir4-with unmodified nucleosomes. Silencing requires the lysine deacetylase activity of Sir2, extensive contacts between Sir3 and the nucleosome, as well as interactions among the SIR proteins, to generate the Sir2-3-4 or SIR complex. Results from recent structural and reconstitution studies suggest an updated model for the ordered assembly and organization of SIR-dependent silent chromatin in yeast. Moreover, studies of subtelomeric gene expression reveal the importance of subtelomeric silent chromatin in the regulation of genes other than the silent mating type loci. This review covers recent advances in this field.

  19. Impact of Chromatin Structure on PR Signaling: Transition from Local to Global Analysis

    PubMed Central

    Grøntved, Lars; Hager, Gordon L

    2011-01-01

    The progesterone receptor (PR) interacts with chromatin in a highly dynamic manner that requires ongoing chromatin remodeling, interaction with chaparones and activity of the proteasome. Here we discuss dynamic interaction of steroid receptor with chromatin, with special attention not only to PR but also to the glucocorticoid receptor (GR), as these receptors share many similarities regarding interaction with, and remodeling of, chromatin. Both receptors can bind nucleosomal DNA and have accordingly been described as pioneering factors. However recent genomic approaches (ChIP-seq and DHS-seq) show that a large fraction of receptor binding events occur at pre-accessible chromatin. Thus factors which generate and maintain accessible chromatin during development, and in fully differentiated tissue, contribute a major fraction of receptor tissue specificity. In addition, chromosome conformation capture techniques suggest that steroid receptors preferentially sequester within distinct nuclear hubs. We will integrate dynamic studies from single cells and genomic studies from cell populations, and discuss how genomic approaches have reshaped our current understanding of mechanisms that control steroid receptor interaction with chromatin. PMID:21958695

  20. Effect of hyperthermia on replicating chromatin

    SciTech Connect

    Warters, R.L.; Roti Roti, J.L.

    1981-10-01

    The extent of heat-induced structural alterations in chromatin containing nascent (pulse-labeled) DNA was assayed using the enzyme micrococcal nuclease. The basic nucleosome structure in nascent and mature chromatin of S-phase cells appeared unaltered for up to 16 hr after exposure to hyperthermic temperatures as high as 48/sup 0/C for 15 min. However, the rate of nuclease digestion of DNA in both nascent and mature chromatin is inhibited following exposure to hyperthermic temperatures. In unheated cells, pulse-labeled nascent DNA matured into mature chromatin structure with a half-time of 2.5 min. The half-time for the maturation of pulse-labeled DNA from nascent into mature chromatin increased in a linear manner as a function of increasing temperature of exposure with constant heating time at temperatures above 43/sup 0/C. Both the reduced nuclease digestibility of nascent DNA and the increased time for chromatin structural changes could be due to the increased protein mass of chromatin following hyperthermia.

  1. The Chromatin Regulator DMAP1 Modulates Activity of the Nuclear Factor κB (NF-κB) Transcription Factor Relish in the Drosophila Innate Immune Response*

    PubMed Central

    Goto, Akira; Fukuyama, Hidehiro; Imler, Jean-Luc; Hoffmann, Jules A.

    2014-01-01

    The host defense of the model organism Drosophila is under the control of two major signaling cascades controlling transcription factors of the NF-κB family, the Toll and the immune deficiency (IMD) pathways. The latter shares extensive similarities with the mammalian TNF-R pathway and was initially discovered for its role in anti-Gram-negative bacterial reactions. A previous interactome study from this laboratory reported that an unexpectedly large number of proteins are binding to the canonical components of the IMD pathway. Here, we focus on DNA methyltransferase-associated protein 1 (DMAP1), which this study identified as an interactant of Relish, a Drosophila transcription factor reminiscent of the mammalian p105 NF-κB protein. We show that silencing of DMAP1 expression both in S2 cells and in flies results in a significant reduction of Escherichia coli-induced expression of antimicrobial peptides. Epistatic analysis indicates that DMAP1 acts in parallel or downstream of Relish. Co-immunoprecipitation experiments further reveal that, in addition to Relish, DMAP1 also interacts with Akirin and the Brahma-associated protein 55 kDa (BAP55). Taken together, these results reveal that DMAP1 is a novel nuclear modulator of the IMD pathway, possibly acting at the level of chromatin remodeling. PMID:24947515

  2. Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins.

    PubMed

    Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

    2016-04-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first showed that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we showed that this technology can be used to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis.

  3. Activation of DNA damage response signaling by condensed chromatin.

    PubMed

    Burgess, Rebecca C; Burman, Bharat; Kruhlak, Michael J; Misteli, Tom

    2014-12-11

    The DNA damage response (DDR) occurs in the context of chromatin, and architectural features of chromatin have been implicated in DNA damage signaling and repair. Whereas a role of chromatin decondensation in the DDR is well established, we show here that chromatin condensation is integral to DDR signaling. We find that, in response to DNA damage chromatin regions transiently expand before undergoing extensive compaction. Using a protein-chromatin-tethering system to create defined chromatin domains, we show that interference with chromatin condensation results in failure to fully activate DDR. Conversely, forced induction of local chromatin condensation promotes ataxia telangiectasia mutated (ATM)- and ATR-dependent activation of upstream DDR signaling in a break-independent manner. Whereas persistent chromatin compaction enhanced upstream DDR signaling from irradiation-induced breaks, it reduced recovery and survival after damage. Our results demonstrate that chromatin condensation is sufficient for activation of DDR signaling and is an integral part of physiological DDR signaling.

  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. Nucleosome structure in chromatin from heated cells

    SciTech Connect

    Warters, R.L.; Roti Roti, J.L.; Winward, R.T.

    1980-12-01

    The effect of hyperthermia (40 to 80/sup 0/C) on the nucleosome structure of mammalian chromatin was determined using the enzyme micrococcal nuclease. At equivalent fractional DNA digestion it was found that neither the size of DNA nor the total fraction of cellular DNA associated with nucleosome structure is altered by heat exposure up to 48/sup 0/C for 30 min. It is proposed that this heat-induced reduction in the accessibility to nuclease attack of DNA in chromatin from heated cells is due to the increased protein mass associated with chromatin.

  6. reSETting chromatin during transcription elongation

    PubMed Central

    Smolle, Michaela; Workman, Jerry L.; Venkatesh, Swaminathan

    2013-01-01

    Maintenance of ordered chromatin structure over the body of genes is vital for the regulation of transcription. Increased access to the underlying DNA sequence results in the recruitment of RNA polymerase II to inappropriate, promoter-like sites within genes, resulting in unfettered transcription. Two new papers show how the Set2-mediated methylation of histone H3 on Lys36 (H3K36me) maintains chromatin structure by limiting histone dynamics over gene bodies, either by recruiting chromatin remodelers that preserve ordered nucleosomal distribution or by lowering the binding affinity of histone chaperones for histones, preventing their removal. PMID:23257840

  7. 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

  8. Chromatin targeting drugs in cancer and immunity.

    PubMed

    Prinjha, Rab; Tarakhovsky, Alexander

    2013-08-15

    Recent advances in the enzymology of transcription and chromatin regulation have led to the discovery of proteins that play a prominent role in cell differentiation and the maintenance of specialized cell functions. Knowledge about post-synthetic DNA and histone modifications as well as information about the rules that guide the formation of multimolecular chromatin-bound complexes have helped to delineate gene-regulating pathways and describe how these pathways are altered in various pathological conditions. The present review focuses on the emerging area of therapeutic interference with chromatin function for the purpose of cancer treatment and immunomodulation.

  9. SH2B1 modulates chromatin state and MyoD occupancy to enhance expressions of myogenic genes.

    PubMed

    Chen, Kuan-Wei; Chang, Yu-Jung; Yeh, Chia-Ming; Lian, Yen-Ling; Chan, Michael W Y; Kao, Cheng-Fu; Chen, Linyi

    2017-02-01

    As mesoderm-derived cell lineage commits to myogenesis, a spectrum of signaling molecules, including insulin growth factor (IGF), activate signaling pathways and ultimately instruct chromatin remodeling and the transcription of myogenic genes. MyoD is a key transcription factor during myogenesis. In this study, we have identified and characterized a novel myogenic regulator, SH2B1. Knocking down SH2B1 delays global chromatin condensation and decreases the formation of myotubes. SH2B1 interacts with histone H1 and is required for the removal of histone H1 from active transcription sites, allowing for the expressions of myogenic genes, IGF2 and MYOG. Chromatin immunoprecipitation assays suggest the requirement of SH2B1 for the induction of histone H3 lysine 4 trimethylation as well as the reduction of histone H3 lysine 9 trimethylation at the promoters and/or enhancers of IGF2 and MYOG genes during myogenesis. Furthermore, SH2B1 is required for the transcriptional activity of MyoD and MyoD occupancy at the enhancer/promoter regions of IGF2 and MYOG during myogenesis. Together, this study demonstrates that SH2B1 fine-tunes global-local chromatin states, expressions of myogenic genes and ultimately promotes myogenesis.

  10. Chromatin association of the SMC5/6 complex is dependent on binding of its NSE3 subunit to DNA

    PubMed Central

    Zabrady, Katerina; Adamus, Marek; Vondrova, Lucie; Liao, Chunyan; Skoupilova, Hana; Novakova, Marketa; Jurcisinova, Lenka; Alt, Aaron; Oliver, Antony W.; Lehmann, Alan R.; Palecek, Jan J.

    2016-01-01

    SMC5/6 is a highly conserved protein complex related to cohesin and condensin, which are the key components of higher-order chromatin structures. The SMC5/6 complex is essential for proliferation in yeast and is involved in replication fork stability and processing. However, the precise mechanism of action of SMC5/6 is not known. Here we present evidence that the NSE1/NSE3/NSE4 sub-complex of SMC5/6 binds to double-stranded DNA without any preference for DNA-replication/recombination intermediates. Mutations of key basic residues within the NSE1/NSE3/NSE4 DNA-binding surface reduce binding to DNA in vitro. Their introduction into the Schizosaccharomyces pombe genome results in cell death or hypersensitivity to DNA damaging agents. Chromatin immunoprecipitation analysis of the hypomorphic nse3 DNA-binding mutant shows a reduced association of fission yeast SMC5/6 with chromatin. Based on our results, we propose a model for loading of the SMC5/6 complex onto the chromatin. PMID:26446992

  11. Dicentric chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast.

    PubMed

    Thrower, D A; Bloom, K

    2001-09-01

    We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional dicentric chromosome in budding yeast cells and made visible by expression of a lac repressor-green fluorescent fusion protein. Centromeres on the same chromatid of a dicentric chromosome attach to opposite poles approximately 50% of the time, resulting in chromosome bridges during anaphase. In cells deleted for yKU70, yKU80, or SIR2, a 10-kb region of the dicentric chromosome stretched along the spindle axis to a length of 6 microm during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the dicentric chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.

  12. The yeast and human FACT chromatin-reorganizing complexes solve R-loop-mediated transcription–replication conflicts

    PubMed Central

    Herrera-Moyano, Emilia; Mergui, Xénia; García-Rubio, María L.; Barroso, Sonia; Aguilera, Andrés

    2014-01-01

    FACT (facilitates chromatin transcription) is a chromatin-reorganizing complex that swaps nucleosomes around the RNA polymerase during transcription elongation and has a role in replication that is not fully understood yet. Here we show that recombination factors are required for the survival of yeast FACT mutants, consistent with an accumulation of DNA breaks that we detected by Rad52 foci and transcription-dependent hyperrecombination. Breaks also accumulate in FACT-depleted human cells, as shown by γH2AX foci and single-cell electrophoresis. Furthermore, FACT-deficient yeast and human cells show replication impairment, which in yeast we demonstrate by ChIP–chip (chromatin immunoprecipitation [ChIP] coupled with microarray analysis) of Rrm3 to occur genome-wide but preferentially at highly transcribed regions. Strikingly, in yeast FACT mutants, high levels of Rad52 foci are suppressed by RNH1 overexpression; R loops accumulate at high levels, and replication becomes normal when global RNA synthesis is inhibited in FACT-depleted human cells. The results demonstrate a key function of FACT in the resolution of R-loop-mediated transcription–replication conflicts, likely associated with a specific chromatin organization. PMID:24636987

  13. Chromatin dynamics during DNA replication

    PubMed Central

    Bar-Ziv, Raz; Voichek, Yoav; Barkai, Naama

    2016-01-01

    Chromatin is composed of DNA and histones, which provide a unified platform for regulating DNA-related processes, mostly through their post-translational modification. During DNA replication, histone arrangement is perturbed, first to allow progression of DNA polymerase and then during repackaging of the replicated DNA. To study how DNA replication influences the pattern of histone modification, we followed the cell-cycle dynamics of 10 histone marks in budding yeast. We find that histones deposited on newly replicated DNA are modified at different rates: While some marks appear immediately upon replication (e.g., H4K16ac, H3K4me1), others increase with transcription-dependent delays (e.g., H3K4me3, H3K36me3). Notably, H3K9ac was deposited as a wave preceding the replication fork by ∼5–6 kb. This replication-guided H3K9ac was fully dependent on the acetyltransferase Rtt109, while expression-guided H3K9ac was deposited by Gcn5. Further, topoisomerase depletion intensified H3K9ac in front of the replication fork and in sites where RNA polymerase II was trapped, suggesting supercoiling stresses trigger H3K9 acetylation. Our results assign complementary roles for DNA replication and gene expression in defining the pattern of histone modification. PMID:27225843

  14. Transcription of nucleosomes from human chromatin.

    PubMed Central

    Shaw, P A; Sahasrabuddhe, C G; Hodo, H G; Saunders, G F

    1978-01-01

    Nucleosomes (chromatin subunits) prepared by micrococcal nuclease digestion of human nuclei are similar in histone content but substantially reduced in non-histone proteins as compared to undigested chromatin. Chromatin transcription experiments indicate that the DNA in the nucleosomes is accessible to DNA-dependent RNA polymerase in vitro. The template capacities of chromatin and nucleosomes are 1.5 and 10%, respectively, relative to high molecular weight DNA, with intermediate values for oligonucleosomes. Three distinct sizes of transcripts, 150, 120 and 95 nucleotides in length, are obtained when nucleosomes are used as templates. However, when nucleosomal DNA is used as a template, the predominant size of transcripts is 150 nucleotides. When oligonucleosomes are used as templates longer transcripts are obtained. This indicates that RNA polymerase can transcribe the DNA contained in the nucleosomes. PMID:693325

  15. Bioactive Dietary Supplements Reactivate ER Expression in ER-Negative Breast Cancer Cells by Active Chromatin Modifications

    PubMed Central

    Meeran, Syed M.; Patel, Shweta N.; Li, Yuanyuan; Shukla, Samriddhi; Tollefsbol, Trygve O.

    2012-01-01

    Breast cancer is the most common cancer and the leading cause of cancer death in women. Although tamoxifen therapy is successful for some patients, it does not provide adequate benefit for those who have estrogen receptor (ER)-negative cancers. Therefore, we approached novel treatment strategies by combining two potential bioactive dietary supplements for the reactivation of ERα expression for effective treatment of ERα-negative breast cancer with tamoxifen. Bioactive dietary supplements such as green tea polyphenols (GTPs) and sulforaphane (SFN) inhibit DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), respectively, which are of central importance to cancer prevention. In the present study, we have observed that treatment of ERα-negative breast cancer cells with GTPs and SFN alone or in combination leads to the reactivation of ERα expression. The combination of 20 µg/mL GTPs and 5 µM SFN was found to be the optimal dose of ERα-reactivation at 3 days in MDA-MB-231 cells. The reactivation of ERα expression was consistently correlated with ERα promoter hypomethylation and hyperacetylation. Chromatin immunoprecipitation (ChIP) analysis of the ERα promoter revealed that GTPs and SFN altered the binding of ERα-transcriptional co-repressor complex thereby contributing to ERα-reactivation. In addition, treatment with tamoxifen in combination with GTPs and SFN significantly increased both cell death and inhibition of cellular proliferation in MDA-MB-231 cells in comparison to treatment with tamoxifen alone. Collectively, our findings suggest that a novel combination of bioactive-HDAC inhibitors with bioactive-demethylating agents is a promising strategy for the effective treatment of hormonal refractory breast cancer with available anti-estrogens. PMID:22662208

  16. Nucleosome repeat lengths and columnar chromatin structure.

    PubMed

    Trifonov, Edward N

    2016-06-01

    Thorough quantitative study of nucleosome repeat length (NRL) distributions, conducted in 1992 by J. Widom, resulted in a striking observation that the linker lengths between the nucleosomes are quantized. Comparison of the NRL average values with the MNase cut distances predicted from the hypothetical columnar structure of chromatin (this work) shows a close correspondence between the two. This strongly suggests that the NRL distribution, actually, reflects the dominant role of columnar chromatin structure common for all eukaryotes.

  17. Chromatin fiber functional organization: Some plausible models

    NASA Astrophysics Data System (ADS)

    Lesne, A.; Victor, J.-M.

    2006-03-01

    We here present a modeling study of the chromatin fiber functional organization. Multi-scale modeling is required to unravel the complex interplay between the fiber and the DNA levels. It suggests plausible scenarios, including both physical and biological aspects, for fiber condensation, its targeted decompaction, and transcription regulation. We conclude that a major role of the chromatin fiber structure might be to endow DNA with allosteric potentialities and to control DNA transactions by an epigenetic tuning of its mechanical and topological constraints.

  18. Recruitment of Phosphorylated Chromatin Assembly Factor 1 to Chromatin after UV Irradiation of Human Cells

    PubMed Central

    Martini, Emmanuelle; Roche, Danièle M.J.; Marheineke, Kathrin; Verreault, Alain; Almouzni, Geneviève

    1998-01-01

    The subcellular distribution and posttranslational modification of human chromatin assembly factor 1 (CAF-1) have been investigated after UV irradiation of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-associated fraction. This fraction is most abundant during S phase in nonirradiated cells and is much reduced in G2 cells. After UV irradiation, the chromatin-associated form of CAF-1 dramatically increased in all cells irrespective of their position in the cell cycle. Such chromatin recruitment resembles that seen for PCNA, a DNA replication and repair factor. The chromatin-associated fraction of p60 was predominantly hypophosphorylated in nonirradiated G2 cells. UV irradiation resulted in the rapid recruitment to chromatin of phosphorylated forms of the p60 subunit. Furthermore, the amount of the p60 and p150 subunits of CAF-1 associated with chromatin was a function of the dose of UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of lesions and the phosphorylation state of CAF-1. The recruitment of CAF-1 to chromatin in response to UV irradiation of human cells described here supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair. PMID:9813080

  19. Transcriptional Coactivator PC4, a Chromatin-Associated Protein, Induces Chromatin Condensation▿ †

    PubMed Central

    Das, Chandrima; Hizume, Kohji; Batta, Kiran; Kumar, B. R. Prashanth; Gadad, Shrikanth S.; Ganguly, Semanti; Lorain, Stephanie; Verreault, Alain; Sadhale, Parag P.; Takeyasu, Kunio; Kundu, Tapas K.

    2006-01-01

    Human transcriptional coactivator PC4 is a highly abundant multifunctional protein which plays diverse important roles in cellular processes, including transcription, replication, and repair. It is also a unique activator of p53 function. Here we report that PC4 is a bona fide component of chromatin with distinct chromatin organization ability. PC4 is predominantly associated with the chromatin throughout the stages of cell cycle and is broadly distributed on the mitotic chromosome arms in a punctate manner except for the centromere. It selectively interacts with core histones H3 and H2B; this interaction is essential for PC4-mediated chromatin condensation, as demonstrated by micrococcal nuclease (MNase) accessibility assays, circular dichroism spectroscopy, and atomic force microscopy (AFM). The AFM images show that PC4 compacts the 100-kb reconstituted chromatin distinctly compared to the results seen with the linker histone H1. Silencing of PC4 expression in HeLa cells results in chromatin decompaction, as evidenced by the increase in MNase accessibility. Knocking down of PC4 up-regulates several genes, leading to the G2/M checkpoint arrest of cell cycle, which suggests its physiological role as a chromatin-compacting protein. These results establish PC4 as a new member of chromatin-associated protein family, which plays an important role in chromatin organization. PMID:16982701

  20. 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

  1. 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.

  2. Programming smooth muscle plasticity with chromatin dynamics.

    PubMed

    McDonald, Oliver G; Owens, Gary K

    2007-05-25

    Smooth muscle cells (SMCs) possess remarkable phenotypic plasticity that allows rapid adaptation to fluctuating environmental cues. For example, vascular SMCs undergo profound changes in their phenotype during neointimal formation in response to vessel injury or within atherosclerotic plaques. Recent studies have shown that interaction of serum response factor (SRF) and its numerous accessory cofactors with CArG box DNA sequences within promoter chromatin of SMC genes is a nexus for integrating signals that influence SMC differentiation in development and disease. During development, SMC-restricted sets of posttranslational histone modifications are acquired within the CArG box chromatin of SMC genes. These modifications in turn control the chromatin-binding properties of SRF. The histone modifications appear to encode a SMC-specific epigenetic program that is used by extracellular cues to influence SMC differentiation, by regulating binding of SRF and its partners to the chromatin template. Thus, SMC differentiation is dynamically regulated by the interplay between SRF accessory cofactors, the SRF-CArG interaction, and the underlying histone modification program. As such, the inherent plasticity of the SMC lineage offers unique glimpses into how cellular differentiation is dynamically controlled at the level of chromatin within the context of changing microenvironments. Further elucidation of how chromatin regulates SMC differentiation will undoubtedly yield valuable insights into both normal developmental processes and the pathogenesis of several vascular diseases that display detrimental SMC phenotypic behavior.

  3. Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq

    PubMed Central

    Adli, Mazhar; Bernstein, Bradley E.

    2015-01-01

    Chromatin immunoprecipitation (ChIP) combined with high-throughput sequencing (ChIP-seq) has become the gold standard for whole-genome mapping of protein-DNA interactions. However, conventional ChIP protocols necessitate the use of large numbers of cells, and library preparation steps associated with current high-throughput sequencing platforms require substantial amounts of DNA; both of these factors preclude the application of ChIP-seq technology to many biologically important but rare cell types. Here we describe a nano-ChIP-seq protocol that combines a high-sensitivity small-scale ChIP assay and a tailored procedure for generating high-throughput sequencing libraries from scarce amounts of ChIP DNA. In terms of the numbers of cells required, the method provides two to three orders of magnitude of improvement over the conventional ChIP-seq method and the entire procedure can be completed within 4 d. PMID:21959244

  4. Induction of p53-dependent activation of the human proliferating cell nuclear antigen gene in chromatin by ionizing radiation.

    PubMed

    Shan, Bin; Xu, Jin; Zhuo, Ying; Morris, Cindy A; Morris, Gilbert F

    2003-11-07

    A human fibroblast cell line with conditional p53 expression displayed a p53-dependent increase in both the protein and mRNA levels of proliferating cell nuclear antigen (PCNA) after exposure to ionizing radiation (IR). The combination of p53 induction and IR cooperated to activate a transiently expressed human PCNA promoter-reporter gene via a p53-responsive element. Chromatin immunoprecipitation assays with antibodies specific for p53 or p300/CREB-binding protein revealed specific p53-dependent enrichment of PCNA promoter sequences in immunoprecipitates of sheared chromatin prepared from irradiated cells. Maximal and specific association of acetylated histone H4 with the PCNA promoter also depended on p53 induction and exposure to IR. These data demonstrate p53 binding to a target site in the PCNA promoter, recruitment of p300/CREB-binding protein, and localized acetylation of histone H4 in an IR-dependent manner. These molecular events are likely to play a role in mediating activation of PCNA gene expression by p53 during the cellular response to DNA damage. The analyses indicate that the combination of p53 induction and IR activate the PCNA gene via mechanisms similar to that of p21/wild-type p53-activated factor but to a lesser extent. This differential regulation of PCNA and p21/wild-type p53-activated factor may establish the proper ratio of the two proteins to coordinate DNA repair with cell cycle arrest.

  5. Varied interactions between proviruses and adjacent host chromatin.

    PubMed Central

    Conklin, K F; Groudine, M

    1986-01-01

    Retroviruses integrated at unique locations in the host genome can be expressed at different levels. We have analyzed the preintegration sites of three transcriptionally competent avian endogenous proviruses (evs) to determine whether the various levels of provirus expression correlate with their location in active or inactive regions of chromatin. Our results show that in three of four cell types, the chromatin conformation (as defined by relative nuclease sensitivity) of virus preintegration sites correlates with the level of expression of the resident provirus in ev+ cells: two inactive proviruses (ev-1 and ev-2) reside in nuclease-resistant chromatin domains and one active provirus (ev-3) resides in a nuclease-sensitive domain. Nuclear runoff transcription assays reveal that the preintegration sites of the active and inactive viruses are not transcribed. However, in erythrocytes of 15-day-old chicken embryos (15d RBCs), the structure and activity of the ev-3 provirus is independent of the conformation of its preintegration site. In this cell type, the ev-3 preintegration site is organized in a nuclease-resistant conformation, while the ev-3 provirus is in a nuclease-sensitive conformation and is transcribed. In addition, the nuclease sensitivity of host sequences adjacent to ev-3 is altered in ev-3+ 15d RBCs relative to that found in 15d RBCs that lack ev-3. These data suggest that the relationship between preintegration site structure and retrovirus expression is more complex than previously described. Images PMID:3025623

  6. 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.

  7. Role of Rb family in the epigenetic definition of chromatin.

    PubMed

    Gonzalo, Susana; Blasco, María A

    2005-06-01

    Epigenetic changes can influence a variety of cellular processes from regulation of gene transcription to proper chromosome segregation. The molecular activities that dictate the assembly, maintenance and regulation of chromatin structure are beginning to be identified. A recent study demonstrates that the Rb family of tumor suppressors plays a major role in global chromatin structure. In addition to the well-known function of Rb family inducing a repressive chromatin state around euchromatic promoters, Rb proteins have a direct role in the assembly of pericentric and telomeric heterochromatin domains. In particular, the Rb family maintains histone 4 lysine 20 tri-methylation (H4K20) at these constitutive heterochromatin domains. Lack of the Rb family results in decreased H4K20 tri-methylation, coincidental with chromosome segregation defects and abnormal telomere elongation, two processes frequently altered in human cancer. Maintenance of heterochromatic domains, such as those of centromeres and telomeres, may represent a novel tumor suppressor function for the Rb family by ensuing genomic stability.

  8. Topological interactions between ring polymers: Implications for chromatin loops

    NASA Astrophysics Data System (ADS)

    Bohn, Manfred; Heermann, Dieter W.

    2010-01-01

    Chromatin looping is a major epigenetic regulatory mechanism in higher eukaryotes. Besides its role in transcriptional regulation, chromatin loops have been proposed to play a pivotal role in the segregation of entire chromosomes. The detailed topological and entropic forces between loops still remain elusive. Here, we quantitatively determine the potential of mean force between the centers of mass of two ring polymers, i.e., loops. We find that the transition from a linear to a ring polymer induces a strong increase in the entropic repulsion between these two polymers. On top, topological interactions such as the noncatenation constraint further reduce the number of accessible conformations of close-by ring polymers by about 50%, resulting in an additional effective repulsion. Furthermore, the transition from linear to ring polymers displays changes in the conformational and structural properties of the system. In fact, ring polymers adopt a markedly more ordered and aligned state than linear ones. The forces and accompanying changes in shape and alignment between ring polymers suggest an important regulatory function of such a topology in biopolymers. We conjecture that dynamic loop formation in chromatin might act as a versatile control mechanism regulating and maintaining different local states of compaction and order.

  9. Chromatin modification by PSC occurs at one PSC per nucleosome and does not require the acidic patch of histone H2A.

    PubMed

    Lo, Stanley M; McElroy, Kyle A; Francis, Nicole J

    2012-01-01

    Chromatin architecture is regulated through both enzymatic and non-enzymatic activities. For example, the Polycomb Group (PcG) proteins maintain developmental gene silencing using an array of chromatin-based mechanisms. The essential Drosophila PcG protein, Posterior Sex Combs (PSC), compacts chromatin and inhibits chromatin remodeling and transcription through a non-enzymatic mechanism involving nucleosome bridging. Nucleosome bridging is achieved through a combination of nucleosome binding and self-interaction. Precisely how PSC interacts with chromatin to bridge nucleosomes is not known and is the subject of this work. We determine the stoichiometry of PSC-chromatin interactions in compact chromatin (in which nucleosomes are bridged) using Scanning Transmission Electron Microscopy (STEM). We find that full compaction occurs with one PSC per nucleosome. In addition to compacting chromatin, we show that PSC oligomerizes nucleosome arrays. PSC-mediated oligomerization of chromatin occurs at similar stoichiometry as compaction suggesting it may also involve nucleosome bridging. Interactions between the tail of histone H4 and the acidic patch of histone H2A are important for chromatin folding and oligomerization, and several chromatin proteins bind the histone H2A acidic patch. However, mutation of the acidic patch of histone H2A does not affect PSC's ability to inhibit chromatin remodeling or bridge nucleosomes. In fact, PSC does not require nucleosomes for bridging activity but can bridge naked DNA segments. PSC clusters nucleosomes on sparsely assembled templates, suggesting it interacts preferentially with nucleosomes over bare DNA. This may be due to the ability of PSC to bind free histones. Our data are consistent with a model in which each PSC binds a nucleosome and at least one other PSC to directly bridge nucleosomes and compact chromatin, but also suggest that naked DNA can be included in compacted structures. We discuss how our data highlight the diversity

  10. β-Catenin and peroxisome proliferator-activated receptor-δ coordinate dynamic chromatin loops for the transcription of vascular endothelial growth factor A gene in colon cancer cells.

    PubMed

    Hwang, Injoo; Kim, Jeeho; Jeong, Sunjoo

    2012-11-30

    Vascular endothelial growth factor A (VEGFA) mRNA is regulated by β-catenin and peroxisome proliferator activated receptor δ (PPAR-δ) activation in colon cancer cells, but the detailed mechanism remains to be elucidated. As chromatin loops are generally hubs for transcription factors, we tested here whether β-catenin could modulate chromatin looping near the VEGFA gene and play any important role for PPAR-δ activated VEGFA transcription. First, we identified the far upstream site as an important site for VEGFA transcription by luciferase assay and chromatin immunoprecipitation in colorectal carcinoma HCT116 cells. Chromatin conformation capture analysis also revealed the chromatin loops formed by the β-catenin bindings on these sites near the VEGFA gene. Dynamic association and dissociation of β-catenin/TCF-4/PPAR-δ on the far upstream site and β-catenin/NF-κB p65 on the downstream site were also detected depending on PPAR-δ activation. Interestingly, β-catenin-mediated chromatin loops were relieved by PPAR-δ activation, suggesting a regulatory role of β-catenin for VEGFA transcription. Based on these data, we propose a model for PPAR-δ-activated VEGFA transcription that relies on β-catenin-mediated chromatin looping as a prerequisite for the activation. Our findings could extend to other β-catenin regulated target genes and could provide a general mechanism and novel paradigm for β-catenin-mediated oncogenesis.

  11. Changing chromatin fiber conformation by nucleosome repositioning.

    PubMed

    Müller, Oliver; Kepper, Nick; Schöpflin, Robert; Ettig, Ramona; Rippe, Karsten; Wedemann, Gero

    2014-11-04

    Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ?10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.

  12. Changing Chromatin Fiber Conformation by Nucleosome Repositioning

    PubMed Central

    Müller, Oliver; Kepper, Nick; Schöpflin, Robert; Ettig, Ramona; Rippe, Karsten; Wedemann, Gero

    2014-01-01

    Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell. PMID:25418099

  13. A computer lab exploring evolutionary aspects of chromatin structure and dynamics for an undergraduate chromatin course*.

    PubMed

    Eirín-López, José 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 vast body of knowledge to the classroom. With this aim, the present work describes a multidisciplinary computer lab designed to introduce undergraduate students to the dynamic nature of chromatin, within the context of the one semester course "Chromatin: Structure, Function and Evolution." This exercise is organized in three parts including (a) molecular evolutionary biology of histone families (using the H1 family as example), (b) histone structure and variation across different animal groups, and (c) effect of histone diversity on nucleosome structure and chromatin dynamics. By using freely available bioinformatic tools that can be run on common computers, the concept of chromatin dynamics is interactively illustrated from a comparative/evolutionary perspective. At the end of this computer lab, students are able to translate the bioinformatic information into a biochemical context in which the relevance of histone primary structure on chromatin dynamics is exposed. During the last 8 years this exercise has proven to be a powerful approach for teaching chromatin structure and dynamics, allowing students a higher degree of independence during the processes of learning and self-assessment.

  14. 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…

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

    PubMed Central

    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

  16. 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.

  17. Toward multiscale modeling of the chromatin fiber: a coarse grain model for DNA

    NASA Astrophysics Data System (ADS)

    Savelyev, Alexey; Papoian, Garegin

    2008-03-01

    In eukaryotic cells DNA is compacted a million-fold into a chromatin. Understanding the mechanism of chromatin folding is of great biological importance. All-atom Molecular Dynamics (MD) simulations could provide crucial insights into the electrostatic and structural mechanisms of chromatin folding. However, because of the enormous size of even short chromatin fiber segment and long folding time-scales, atomistic simulations are computationally impractical. Our long-term aim is to build an accurate coarse-grain (CG) model of the chromatin, derived systematically from all-atom simulations of its smaller parts. Here we report the development of the CG model for a linear DNA chain, playing the role of a linker DNA segment in the chromatin. We derived CG inter-DNA electrostatic potential from atomistic simulations with explicit solvent and mobile ions, instead of relying on the standard models of continuum electrostatics, which are inadequate at small intermolecular distances. In addition, we used the ideas of renormalization group theory to construct an optimization scheme for parameterizing the CG force field. This novel approach is designed to accurately reproduce correlations among various CG degrees of freedom. The implementation of these correlations was left as an open question in the prior studies of CG polymer models.

  18. 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

  19. 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%.

  20. Improvements in immunoprecipitation of specific messenger RNA. Isolation of highly purified conalbumin mRNA in high yield.

    PubMed

    Payvar, F; Schimke, R T

    1979-11-01

    We have described previously procedures for the isolation of specific mRNA employing immunoprecipitation of polysomes. In spite of our success with ovalbumin mRNA in the chicken oviduct, we have had considerable difficulties in applying these same published techniques to the immunopurification of conalbumin mRNA, despite the fact that the chicken oviduct synthesizes up to 10% of protein as conalbumin. Here we describe a number of modifications and refinements which have proved essential in obtaining intact conalbumin mRNA in high purity and high yields. These refinements include: (a) improved purification of conalbumin in order to remove contaminating proteins that result in impure antibodies; (b) improved isolation of specific conalbumin antibody in high yields; (c) improved methods for reducing contamination by non-specific polysomes; (d) improved techniques for isolation of RNA from immunoprecipitates resulting in less degradation and higher recovery of conalbumin mRNA; (E) improved techniques for efficient translation of conalbumin mRNA involving treatment of the RNA with methylmercury prior to translation. We conclude that problems involved in the immunoprecipitation of different mRNAs may differ, and that various refinements in techniques may be required for obtaining highly purified preparations of intact mRNA in high yields.

  1. Elucidate Chromatin Folding at the Mesoscale

    NASA Astrophysics Data System (ADS)

    Qiu, Xiangyun

    Knowledge of the three-dimensional structure of chromatin, an active participant of all gene-directed processes, is required to decode its (epi)genetics-structure-function relationships. Albeit often simplified as ``beads-on-a-string'', chromatin possesses daunting complexity in its intricate intra- and inter-nucleosome interactions, as well as the myriad types of molecules acting on it. On the other hand, the folding of chromatin from an extended chain of nucleosomes is highly constrained, e.g., by rather bulky nucleosomes and semi-rigid linker dsDNAs. Further given the well-defined nucleosome and dsDNA structures at the nanometer scale, this creates an opportunity for low-resolution structural methods such as small angle scattering to obtain mesoscale structures of chromatin, which can be further refined computationally to yield atomistic structures of chromatin. Here we present results from our recent studies of recombinant nucleosome arrays with solution small angle x-ray scattering (SAXS) and ensemble structure modeling.

  2. Toxicants and human sperm chromatin integrity.

    PubMed

    Delbès, Geraldine; Hales, Barbara F; Robaire, Bernard

    2010-01-01

    The integrity of the paternal genome is essential as the spermatozoon can bring genetic damage into the oocyte at fertilization and contribute to the development of abnormal pregnancy outcome. During the past two decades, many assays have been developed to measure sperm DNA strand breaks, chromatin structure and compaction and assess the proteins associated with the DNA, as well as epigenetic modifications. Using these assays, it has been shown that exposure to physical agents or chemicals, including therapeutic drugs and environmental toxicants, can affect the integrity of sperm chromatin, inducing structural, genetic and/or epigenetic abnormalities. The mechanisms by which such damage is triggered are still largely unresolved and the susceptibility of each individual will depend on their genetic background, lifestyle and exposure to various insults. Depending on the nature of the chemicals, they may directly target the DNA, induce an oxidative stress, or modify the epigenetic elements. The significance of measuring the sperm chromatin integrity comes from the fact that this end-point correlates well with the low IVF and ICSI outcomes, and idiopathic infertility. Nevertheless, it is hard to establish a direct link between the paternal sperm chromatin integrity and the health of the future generations. Thus, it seems essential to undertake studies that will resolve the impact of chemical and environmental factors on chromatin structure and epigenetic components of human spermatozoa and to elucidate what sperm nuclear end-points are predictors of the quality of progeny outcome.

  3. Chromatin associations in Arabidopsis interphase nuclei.

    PubMed

    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.

  4. Chromatin Proteins: Key Responders to Stress

    PubMed Central

    Smith, Karen T.; Workman, Jerry L.

    2012-01-01

    Environments can be ever-changing and stresses are commonplace. In order for organisms to survive, they need to be able to respond to change and adapt to new conditions. Fortunately, many organisms have systems in place that enable dynamic adaptation to immediate stresses and changes within the environment. Much of this cellular response is coordinated by modulating the structure and accessibility of the genome. In eukaryotic cells, the genome is packaged and rolled up by histone proteins to create a series of DNA/histone core structures known as nucleosomes; these are further condensed into chromatin. The degree and nature of the condensation can in turn determine which genes are transcribed. Histones can be modified chemically by a large number of proteins that are thereby responsible for dynamic changes in gene expression. In this Primer we discuss findings from a study published in this issue of PLoS Biology by Weiner et al. that highlight how chromatin structure and chromatin binding proteins alter transcription in response to environmental changes and stresses. Their study reveals the importance of chromatin in mediating the speed and amplitude of stress responses in cells and suggests that chromatin is a critically important component of the cellular response to stress. PMID:22859908

  5. Reshaping chromatin after DNA damage: the choreography of histone proteins.

    PubMed

    Polo, Sophie E

    2015-02-13

    DNA damage signaling and repair machineries operate in a nuclear environment where DNA is wrapped around histone proteins and packaged into chromatin. Understanding how chromatin structure is restored together with the DNA sequence during DNA damage repair has been a topic of intense research. Indeed, chromatin integrity is central to cell functions and identity. However, chromatin shows remarkable plasticity in response to DNA damage. This review presents our current knowledge of chromatin dynamics in the mammalian cell nucleus in response to DNA double strand breaks and UV lesions. I provide an overview of the key players involved in regulating histone dynamics in damaged chromatin regions, focusing on histone chaperones and their concerted action with histone modifiers, chromatin remodelers and repair factors. I also discuss how these dynamics contribute to reshaping chromatin and, by altering the chromatin landscape, may affect the maintenance of epigenetic information.

  6. 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

  7. The forkhead transcription factor FoxI1 remains bound to condensed mitotic chromosomes and stably remodels chromatin structure.

    PubMed

    Yan, Jizhou; Xu, Lisha; Crawford, Gregory; Wang, Zenfeng; Burgess, Shawn M

    2006-01-01

    All forkhead (Fox) proteins contain a highly conserved DNA binding domain whose structure is remarkably similar to the winged-helix structures of histones H1 and H5. Little is known about Fox protein binding in the context of higher-order chromatin structure in living cells. We created a stable cell line expressing FoxI1-green fluorescent protein (GFP) or FoxI1-V5 fusion proteins under control of the reverse tetracycline-controlled transactivator doxycycline inducible system and found that unlike most transcription factors, FoxI1 remains bound to the condensed chromosomes during mitosis. To isolate DNA fragments directly bound by the FoxI1 protein within living cells, we performed chromatin immunoprecipitation assays (ChIPs) with antibodies to either enhanced GFP or the V5 epitope and subcloned the FoxI1-enriched DNA fragments. Sequence analyses indicated that 88% (106/121) of ChIP sequences contain the consensus binding sites for all Fox proteins. Testing ChIP sequences with a quantitative DNase I hypersensitivity assay showed that FoxI1 created stable DNase I sensitivity changes in condensed chromosomes. The majority of ChIP targets and random targets increased in resistance to DNase I in FoxI1-expressing cells, but a small number of targets became more accessible to DNase I. Consistently, the accessibility of micrococcal nuclease to chromatin was generally inhibited. Micrococcal nuclease partial digestion generated a ladder in which all oligonucleosomes were slightly longer than those observed with the controls. On the basis of these findings, we propose that FoxI1 is capable of remodeling chromatin higher-order structure and can stably create site-specific changes in chromatin to either stably create or remove DNase I hypersensitive sites.

  8. Chromatin status of apoptosis genes correlates with sensitivity to chemo-, immune- and radiation therapy in colorectal cancer cell lines.

    PubMed

    Benard, Anne; Janssen, Connie M; van den Elsen, Peter J; van Eggermond, Marja C J A; Hoon, Dave S B; van de Velde, Cornelis J H; Kuppen, Peter J K

    2014-12-01

    The apoptosis pathway of programmed cell death is frequently deregulated in cancer. An intact apoptosis pathway is required for proper response to anti-cancer treatment. We investigated the chromatin status of key apoptosis genes in the apoptosis pathway in colorectal cancer cell lines in relation to apoptosis induced by chemo-, immune- or radiation therapy. Using chromatin immunoprecipitation (ChIP), we measured the presence of transcription-activating histone modifications H3Ac and H3K4me3 and silencing modifications H3K9me3 and H3K27me3 at the gene promoter regions of key apoptosis genes Bax, Bcl2, Caspase-9, Fas (CD95) and p53. Cell lines DLD1, SW620, Colo320, Caco2, Lovo and HT29 were treated with cisplatin, anti-Fas or radiation. The apoptotic response was measured by flow cytometry using propidium iodide and annexin V-FITC. The chromatin status of the apoptosis genes reflected the activation status of the intrinsic (Bax, Bcl2, Caspase-9 and p53) and extrinsic (Fas) pathways. An active intrinsic apoptotic pathway corresponded to sensitivity to cisplatin and radiation treatment of cell lines DLD1, SW620 and Colo320. An active Fas promoter corresponded to an active extrinsic apoptotic pathway in cell line DLD1. mRNA expression data correlated with the chromatin status of the apoptosis genes as measured by ChIP. In conclusion, the results presented in this study indicate that the balance between activating and silencing histone modifications, reflecting the chromatin status of apoptosis genes, can be used to predict the response of tumor cells to different anti-cancer therapies and could provide a novel target to sensitize tumors to obtain adequate treatment responses.

  9. The Groucho Co-repressor Is Primarily Recruited to Local Target Sites in Active Chromatin to Attenuate Transcription

    PubMed Central

    Jennings, Barbara H.

    2014-01-01

    Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE) proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling), and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. We have used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) to profile Gro recruitment in two Drosophila cell lines. We find that Gro predominantly binds at discrete peaks (<1 kilobase). We also demonstrate that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in “active” chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. We also find that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. Our results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone deacetylation

  10. Predictive chromatin signatures in the mammalian genome

    PubMed Central

    Hon, Gary C.; Hawkins, R. David; Ren, Bing

    2009-01-01

    The DNA sequence of an organism is a blueprint of life: it harbors not only the information about proteins and other molecules produced in each cell, but also instructions on when and where such molecules are made. Chromatin, the structure of histone and DNA that has co-evolved with eukaryotic genome, also contains information that indicates the function and activity of the underlying DNA sequences. Such information exists in the form of covalent modifications to the histone proteins that comprise the nucleosome. Thanks to the development of high throughput technologies such as DNA microarrays and next generation DNA sequencing, we have begun to associate the various combinations of chromatin modification patterns with functional sequences in the human genome. Here, we review the rapid progress from descriptive observations of histone modification profiles to highly predictive models enabling use of chromatin signatures to enumerate novel functional sequences in mammalian genomes that have escaped previous detection. PMID:19808796

  11. Nucleosome dynamics during chromatin remodeling in vivo.

    PubMed

    Ramachandran, Srinivas; Henikoff, Steven

    2016-01-01

    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.

  12. 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

  13. 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

  14. 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

  15. 4D chromatin dynamics in cycling cells

    PubMed Central

    Strickfaden, Hilmar; Zunhammer, Andreas; van Koningsbruggen, Silvana; Köhler, Daniela

    2010-01-01

    This live cell study of chromatin dynamics in four dimensions (space and time) in cycling human cells provides direct evidence for three hypotheses first proposed by Theodor Boveri in seminal studies of fixed blastomeres from Parascaris equorum embryos: (I) Chromosome territory (CT) arrangements are stably maintained during interphase. (II) Chromosome proximity patterns change profoundly during prometaphase. (III) Similar CT proximity patterns in pairs of daughter nuclei reflect symmetrical chromosomal movements during anaphase and telophase, but differ substantially from the arrangement in mother cell nucleus. Hypothesis I could be confirmed for the majority of interphase cells. A minority, however, showed complex, rotational movements of CT assemblies with large-scale changes of CT proximity patterns, while radial nuclear arrangements were maintained. A new model of chromatin dynamics is proposed. It suggests that long-range DNA-DNA interactions in cell nuclei may depend on a combination of rotational CT movements and locally constrained chromatin movements. PMID:21327076

  16. PPARβ Interprets a Chromatin Signature of Pluripotency to Promote Embryonic Differentiation at Gastrulation

    PubMed Central

    Rotman, Nicolas; Guex, Nicolas; Gouranton, Erwan; Wahli, Walter

    2013-01-01

    Epigenetic post-transcriptional modifications of histone tails are thought to help in coordinating gene expression during development. An epigenetic signature is set in pluripotent cells and interpreted later at the onset of differentiation. In pluripotent cells, epigenetic marks normally associated with active genes (H3K4me3) and with silent genes (H3K27me3) atypically co-occupy chromatin regions surrounding the promoters of important developmental genes. However, it is unclear how these epigenetic marks are recognized when cell differentiation starts and what precise role they play. Here, we report the essential role of the nuclear receptor peroxisome proliferator-activated receptor β (PPARβ, NR1C2) in Xenopus laevis early development. By combining loss-of-function approaches, large throughput transcript expression analysis by the mean of RNA-seq and intensive chromatin immunoprecipitation experiments, we unveil an important cooperation between epigenetic marks and PPARβ. During Xenopus laevis gastrulation PPARβ recognizes H3K27me3 marks that have been deposited earlier at the pluripotent stage to activate early differentiation genes. Thus, PPARβis the first identified transcription factor that interprets an epigenetic signature of pluripotency, in vivo, during embryonic development. This work paves the way for a better mechanistic understanding of how the activation of hundreds of genes is coordinated during early development. PMID:24367589

  17. Dynamic transition of transcription and chromatin landscape during fission yeast adaptation to glucose starvation.

    PubMed

    Oda, Arisa; Takemata, Naomichi; Hirata, Yoshito; Miyoshi, Tomoichiro; Suzuki, Yutaka; Sugano, Sumio; Ohta, Kunihiro

    2015-05-01

    Shortage of glucose, the primary energy source for all organisms, is one of the most critical stresses influencing cell viability. Glucose starvation promptly induces changes in mRNA and noncoding RNA (ncRNA) transcription. We previously reported that glucose starvation induces long ncRNA (lncRNA) transcription in the 5' segment of a fission yeast gluconeogenesis gene (fbp1+), which leads to stepwise chromatin alteration around the fbp1+ promoter and to subsequent robust gene activation. Here, we analyzed genomewide transcription by strand-specific RNA sequencing, together with chromatin landscape by immunoprecipitation sequencing (ChIP-seq). Clustering analysis showed that distinct mRNAs and ncRNAs are induced at the early, middle and later stages of cellular response to glucose starvation. The starvation-induced transcription depends substantially on the stress-responsive transcription factor Atf1. Using a new computer program that examines dynamic changes in expression patterns, we identified ncRNAs with similar behavior to the fbp1+ lncRNA. We confirmed that there are continuous lncRNAs associated with local reduction of histone density. Overlapping with the regions for transcription of these lncRNAs, antisense RNAs are antagonistically transcribed under glucose-rich conditions. These results suggest that Atf1-dependent integrated networks of mRNA and lncRNA govern drastic changes in cell physiology in response to glucose starvation.

  18. Nitric oxide deficiency determines global chromatin changes in Duchenne muscular dystrophy.

    PubMed

    Colussi, Claudia; Gurtner, Aymone; Rosati, Jessica; Illi, Barbara; Ragone, Gianluca; Piaggio, Giulia; Moggio, Maurizio; Lamperti, Costanza; D'Angelo, Grazia; Clementi, Emilio; Minetti, Giulia; Mozzetta, Chiara; Antonini, Annalisa; Capogrossi, Maurizio C; Puri, Pier Lorenzo; Gaetano, Carlo

    2009-07-01

    The present study provides evidence that abnormal patterns of global histone modification are present in the skeletal muscle nuclei of mdx mice and Duchenne muscular dystrophy (DMD) patients. A combination of specific histone H3 modifications, including Ser-10 phosphorylation, acetylation of Lys 9 and 14, and Lys 79 methylation, were found enriched in muscle biopsies from human patients affected by DMD and in late-term fetuses, early postnatal pups, or adult mdx mice. In this context, chromatin immunoprecipitation experiments showed an enrichment of these modifications at the loci of genes involved in proliferation or inflammation, suggesting a regulatory effect on gene expression. Remarkably, the reexpression of dystrophin induced by gentamicin treatment or the administration of nitric oxide (NO) donors reversed the abnormal pattern of H3 histone modifications. These findings suggest an unanticipated link between the dystrophin-activated NO signaling and the remodeling of chromatin. In this context, the regulation of class IIa histone deacetylases (HDACs) 4 and 5 was found altered as a consequence of the reduced NO-dependent protein phosphatase 2A activity, indicating that both NO and class IIa HDACs are important for satellite cell differentiation and gene expression in mdx mice. In conclusion, this work provides the first evidence of a role for NO as an epigenetic regulator in DMD.

  19. 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

  20. Actin Family Proteins in the Human INO80 Chromatin Remodeling Complex Exhibit Functional Roles in the Induction of Heme Oxygenase-1 with Hemin.

    PubMed

    Takahashi, Yuichiro; Murakami, Hirokazu; Akiyama, Yusuke; Katoh, Yasutake; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-Ichi; Igarashi, Kazuhiko; Harata, Masahiko

    2017-01-01

    Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex.

  1. Actin Family Proteins in the Human INO80 Chromatin Remodeling Complex Exhibit Functional Roles in the Induction of Heme Oxygenase-1 with Hemin

    PubMed Central

    Takahashi, Yuichiro; Murakami, Hirokazu; Akiyama, Yusuke; Katoh, Yasutake; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-ichi; Igarashi, Kazuhiko; Harata, Masahiko

    2017-01-01

    Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex. PMID:28270832

  2. 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.

  3. Spindle assembly on immobilized chromatin micropatterns.

    PubMed

    Pugieux, Céline; Dmitrieff, Serge; Tarnawska, Katarzyna; Nédélec, François

    2014-01-01

    We describe a method to assemble meiotic spindles on immobilized micropatterns of chromatin built on a first layer of biotinylated BSA deposited by microcontact printing. Such chromatin patterns routinely produce bipolar spindles with a yield of 60%, and offer the possibility to follow spindle assembly dynamics, from the onset of nucleation to the establishment of a quasi steady state. Hundreds of spindles can be recorded in parallel for different experimental conditions. We also describe the semi-automated image analysis pipeline, which is used to analyze the assembly kinetics of spindle arrays, or the final morphological diversity of the spindles.

  4. Dynamics of Histone Tails within Chromatin

    NASA Astrophysics Data System (ADS)

    Bernier, Morgan; North, Justin; Page, Michael; Jaroniec, Christopher; Hammel, Christopher; Poirier, Michael

    2012-02-01

    Genetic information in humans is encoded within DNA molecules that is wrapped around histone octamer proteins and compacted into a highly conserved structural polymer, chromatin. The physical and material properties of chromatin appear to influence gene expression by altering the accessibility of proteins to the DNA. The tails of the histones are flexible domains that are thought to play a role in regulating DNA accessibility and compaction; however the molecular mechanisms for these phenomena are not understood. I will present CW-EPR studies on site directed spin labeled nucleosomes that probe the structure and dynamics of these histone tails within nucleosomes.

  5. Modification of Chromatin Structure by the Thyroid Hormone Receptor.

    PubMed

    Li; Sachs; Shi; Wolffe

    1999-05-01

    Pioneering experiments and recent observations have established the thyroid hormone receptor as a master manipulator of the chromosomal environment in targeting the activation and repression of transcription. Here we review how the thyroid hormone receptor is assembled into chromatin, where in the absence of thyroid hormone the receptor recruits histone deacetylase to silence transcription. On addition of hormone, the receptor undergoes a conformational change that leads to the release of deacetylase, while facilitating the recruitment of transcriptional coactivators that act as histone acetyltransferases. We discuss the biological importance of these observations for gene control by the thyroid hormone receptor and for oncogenic transformation by the mutated thyroid hormone receptor, v-ErbA.

  6. Kinetic Control of Nucleosome Displacement by ISWI/ACF Chromatin Remodelers

    NASA Astrophysics Data System (ADS)

    Florescu, Ana-Maria; Schiessel, Helmut; Blossey, Ralf

    2012-09-01

    Chromatin structure is dynamically organized by chromatin remodelers, motor protein complexes which move and remove nucleosomes. The regulation of remodeler action has recently been proposed to underlie a kinetic proofreading scheme which combines the recognition of histone-tail states and the ATP-dependent loosening of DNA around nucleosomes. Members of the ISWI-family of remodelers additionally recognize linker length between nucleosomes. Here, we show that the additional proofreading step involving linker length alone is sufficient to promote the formation of regular arrays of nucleosomes. ATP-dependent remodeling by bidirectional motors is shown to reinforce positioning as compared to statistical positioning.

  7. 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

  8. Allelic Imbalance in Regulation of ANRIL through Chromatin Interaction at 9p21 Endometriosis Risk Locus

    PubMed Central

    Nakaoka, Hirofumi; Gurumurthy, Aishwarya; Hayano, Takahide; Ahmadloo, Somayeh; Omer, Waleed H; Yoshihara, Kosuke; Yamamoto, Akihito; Kurose, Keisuke; Enomoto, Takayuki; Akira, Shigeo; Hosomichi, Kazuyoshi; Inoue, Ituro

    2016-01-01

    Genome-wide association studies (GWASs) have discovered numerous single nucleotide polymorphisms (SNPs) associated with human complex disorders. However, functional characterization of the disease-associated SNPs remains a formidable challenge. Here we explored regulatory mechanism of a SNP on chromosome 9p21 associated with endometriosis by leveraging “allele-specific” functional genomic approaches. By re-sequencing 1.29 Mb of 9p21 region and scrutinizing DNase-seq data from the ENCODE project, we prioritized rs17761446 as a candidate functional variant that was in perfect linkage disequilibrium with the original GWAS SNP (rs10965235) and located on DNase I hypersensitive site. Chromosome conformation capture followed by high-throughput sequencing revealed that the protective G allele of rs17761446 exerted stronger chromatin interaction with ANRIL promoter. We demonstrated that the protective allele exhibited preferential binding affinities to TCF7L2 and EP300 by bioinformatics and chromatin immunoprecipitation (ChIP) analyses. ChIP assays for histone H3 lysine 27 acetylation and RNA polymerase II reinforced the enhancer activity of the SNP site. The allele specific expression analysis for eutopic endometrial tissues and endometrial carcinoma cell lines showed that rs17761446 was a cis-regulatory variant where G allele was associated with increased ANRIL expression. Our work illuminates the allelic imbalances in a series of transcriptional regulation from factor binding to gene expression mediated by chromatin interaction underlie the molecular mechanism of 9p21 endometriosis risk locus. Functional genomics on common disease will unlock functional aspect of genotype-phenotype correlations in the post-GWAS stage. PMID:27055116

  9. 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

  10. Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila

    PubMed Central

    Pindyurin, Alexey V.; Pagie, Ludo; Kozhevnikova, Elena N.; van Arensbergen, Joris; van Steensel, Bas

    2016-01-01

    Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity, it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila. Here, we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress possible toxic effects of Dam. In addition, we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. These new DamID tools will be valuable for the mapping of binding patterns of chromatin proteins in Drosophila tissues and especially in cell lineages. PMID:27001518

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

    PubMed Central

    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. PMID:26863565

  12. A High-Resolution Whole-Genome Map of Key Chromatin Modifications in the Adult Drosophila melanogaster

    PubMed Central

    Yin, Hang; Sweeney, Sarah; Raha, Debasish; Snyder, Michael; Lin, Haifan

    2011-01-01

    Epigenetic research has been focused on cell-type-specific regulation; less is known about common features of epigenetic programming shared by diverse cell types within an organism. Here, we report a modified method for chromatin immunoprecipitation and deep sequencing (ChIP–Seq) and its use to construct a high-resolution map of the Drosophila melanogaster key histone marks, heterochromatin protein 1a (HP1a) and RNA polymerase II (polII). These factors are mapped at 50-bp resolution genome-wide and at 5-bp resolution for regulatory sequences of genes, which reveals fundamental features of chromatin modification landscape shared by major adult Drosophila cell types: the enrichment of both heterochromatic and euchromatic marks in transposons and repetitive sequences, the accumulation of HP1a at transcription start sites with stalled polII, the signatures of histone code and polII level/position around the transcriptional start sites that predict both the mRNA level and functionality of genes, and the enrichment of elongating polII within exons at splicing junctions. These features, likely conserved among diverse epigenomes, reveal general strategies for chromatin modifications. PMID:22194694

  13. 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

    2016-01-20

    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.

  14. A high-resolution whole-genome map of key chromatin modifications in the adult Drosophila melanogaster.

    PubMed

    Yin, Hang; Sweeney, Sarah; Raha, Debasish; Snyder, Michael; Lin, Haifan

    2011-12-01

    Epigenetic research has been focused on cell-type-specific regulation; less is known about common features of epigenetic programming shared by diverse cell types within an organism. Here, we report a modified method for chromatin immunoprecipitation and deep sequencing (ChIP-Seq) and its use to construct a high-resolution map of the Drosophila melanogaster key histone marks, heterochromatin protein 1a (HP1a) and RNA polymerase II (polII). These factors are mapped at 50-bp resolution genome-wide and at 5-bp resolution for regulatory sequences of genes, which reveals fundamental features of chromatin modification landscape shared by major adult Drosophila cell types: the enrichment of both heterochromatic and euchromatic marks in transposons and repetitive sequences, the accumulation of HP1a at transcription start sites with stalled polII, the signatures of histone code and polII level/position around the transcriptional start sites that predict both the mRNA level and functionality of genes, and the enrichment of elongating polII within exons at splicing junctions. These features, likely conserved among diverse epigenomes, reveal general strategies for chromatin modifications.

  15. Chromatin as an expansive canvas for chemical biology.

    PubMed

    Fierz, Beat; Muir, Tom W

    2012-04-17

    Chromatin is extensively chemically modified and thereby acts as a dynamic signaling platform controlling gene function. Chromatin regulation is integral to cell differentiation, lineage commitment and organism development, whereas chromatin dysregulation can lead to age-related and neurodegenerative disorders as well as cancer. Investigating chromatin biology presents a unique challenge, as the issue spans many disciplines, including cell and systems biology, biochemistry and molecular biophysics. In recent years, the application of chemical biology methods for investigating chromatin processes has gained considerable traction. Indeed, chemical biologists now have at their disposal powerful chemical tools that allow chromatin biology to be scrutinized at the level of the cell all the way down to the single chromatin fiber. Here we present recent examples of how this rapidly expanding palette of chemical tools is being used to paint a detailed picture of chromatin function in organism development and disease.

  16. 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.

  17. Monte Carlo simulation of chromatin stretching

    NASA Astrophysics Data System (ADS)

    Aumann, Frank; Lankas, Filip; Caudron, Maïwen; Langowski, Jörg

    2006-04-01

    We present Monte Carlo (MC) simulations of the stretching of a single 30nm chromatin fiber. The model approximates the DNA by a flexible polymer chain with Debye-Hückel electrostatics and uses a two-angle zigzag model for the geometry of the linker DNA connecting the nucleosomes. The latter are represented by flat disks interacting via an attractive Gay-Berne potential. Our results show that the stiffness of the chromatin fiber strongly depends on the linker DNA length. Furthermore, changing the twisting angle between nucleosomes from 90° to 130° increases the stiffness significantly. An increase in the opening angle from 22° to 34° leads to softer fibers for small linker lengths. We observe that fibers containing a linker histone at each nucleosome are stiffer compared to those without the linker histone. The simulated persistence lengths and elastic moduli agree with experimental data. Finally, we show that the chromatin fiber does not behave as an isotropic elastic rod, but its rigidity depends on the direction of deformation: Chromatin is much more resistant to stretching than to bending.

  18. An Isochore Framework Underlies Chromatin Architecture

    PubMed Central

    Jabbari, Kamel; Bernardi, Giorgio

    2017-01-01

    A recent investigation showed the existence of correlations between the architectural features of mammalian interphase chromosomes and the compositional properties of isochores. This result prompted us to compare maps of the Topologically Associating Domains (TADs) and of the Lamina Associated Domains (LADs) with the corresponding isochore maps of mouse and human chromosomes. This approach revealed that: 1) TADs and LADs correspond to isochores, i.e., isochores are the genomic units that underlie chromatin domains; 2) the conservation of TADs and LADs in mammalian genomes is explained by the evolutionary conservation of isochores; 3) chromatin domains corresponding to GC-poor isochores (e.g., LADs) show not only self-interactions but also intrachromosomal interactions with other domains also corresponding to GC-poor isochores even if located far away; in contrast, chromatin domains corresponding to GC-rich isochores (e.g., TADs) show more localized chromosomal interactions, many of which are inter-chromosomal. In conclusion, this investigation establishes a link between DNA sequences and chromatin architecture, explains the evolutionary conservation of TADs and LADs and provides new information on the spatial distribution of GC-poor/gene-poor and GC-rich/gene-rich chromosomal regions in the interphase nucleus. PMID:28060840

  19. 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.

  20. 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.

  1. RNA-Seq and RNA Immunoprecipitation Analyses of the Transcriptome of Streptomyces coelicolor Identify Substrates for RNase III

    PubMed Central

    Gatewood, Marcha L.; Bralley, Patricia; Weil, M. Ryan

    2012-01-01

    RNase III is a key enzyme in the pathways of RNA degradation and processing in bacteria and has been suggested as a global regulator of antibiotic production in Streptomyces coelicolor. Using RNA-Seq, we have examined the transcriptomes of S. coelicolor M145 and an RNase III (rnc)-null mutant of that strain. RNA preparations with reduced levels of structural RNAs were prepared by subtractive hybridization prior to RNA-Seq analysis. We initially identified 7,800 transcripts of known and putative protein-coding genes in M145 and the null mutant, JSE1880, along with transcripts of 21 rRNA genes and 65 tRNA genes. Approximately 3,100 of the protein-coding transcripts were categorized as low-abundance transcripts. For further analysis, we selected those transcripts of known and putative protein-coding genes whose levels changed by ≥2-fold between the two S. coelicolor strains and organized those transcripts into 16 functional categories. We refined our analysis by performing RNA immunoprecipitation of the mRNA preparation from JSE1880 using a mutant RNase III protein that binds to transcripts but does not cleave them. This analysis identified ca. 800 transcripts that were enriched in the RNA immunoprecipitates, including 28 transcripts whose levels also changed by ≥2-fold in the RNA-Seq analysis. We compare our results with those obtained by microarray analysis of the S. coelicolor transcriptome and with studies describing the characterization of small noncoding RNAs. We have also used the RNA immunoprecipitation results to identify new substrates for RNase III cleavage. PMID:22389483

  2. Inferential modeling of 3D chromatin structure.

    PubMed

    Wang, Siyu; Xu, Jinbo; Zeng, Jianyang

    2015-04-30

    For eukaryotic cells, the biological processes involving regulatory DNA elements play an important role in cell cycle. Understanding 3D spatial arrangements of chromosomes and revealing long-range chromatin interactions are critical to decipher these biological processes. In recent years, chromosome conformation capture (3C) related techniques have been developed to measure the interaction frequencies between long-range genome loci, which have provided a great opportunity to decode the 3D organization of the genome. In this paper, we develop a new Bayesian framework to derive the 3D architecture of a chromosome from 3C-based data. By modeling each chromosome as a polymer chain, we define the conformational energy based on our current knowledge on polymer physics and use it as prior information in the Bayesian framework. We also propose an expectation-maximization (EM) based algorithm to estimate the unknown parameters of the Bayesian model and infer an ensemble of chromatin structures based on interaction frequency data. We have validated our Bayesian inference approach through cross-validation and verified the computed chromatin conformations using the geometric constraints derived from fluorescence in situ hybridization (FISH) experiments. We have further confirmed the inferred chromatin structures using the known genetic interactions derived from other studies in the literature. Our test results have indicated that our Bayesian framework can compute an accurate ensemble of 3D chromatin conformations that best interpret the distance constraints derived from 3C-based data and also agree with other sources of geometric constraints derived from experimental evidence in the previous studies. The source code of our approach can be found in https://github.com/wangsy11/InfMod3DGen.

  3. MPE-seq, a new method for the genome-wide analysis of chromatin structure

    PubMed Central

    Ishii, Haruhiko; Kadonaga, James T.; Ren, Bing

    2015-01-01

    The analysis of chromatin structure is essential for the understanding of transcriptional regulation in eukaryotes. Here we describe methidiumpropyl-EDTA sequencing (MPE-seq), a method for the genome-wide characterization of chromatin that involves the digestion of nuclei withMPE-Fe(II) followed by massively parallel sequencing. Like micrococcal nuclease (MNase), MPE-Fe(II) preferentially cleaves the linker DNA between nucleosomes. However, there are differences in the cleavage of nuclear chromatin by MPE-Fe(II) relative to MNase. Most notably, immediately upstream of the transcription start site of active promoters, we frequently observed nucleosome-sized (141–190 bp) and subnucleosome-sized (such as 101–140 bp) peaks of digested chromatin fragments with MPE-seq but not with MNase-seq. These peaks also correlate with the presence of core histones and could thus be due, at least in part, to noncanonical chromatin structures such as labile nucleosome-like particles that have been observed in other contexts. The subnucleosome-sized MPE-seq peaks exhibit a particularly distinct association with active promoters. In addition, unlike MNase, MPE-Fe(II) cleaves nuclear DNA with little sequence bias. In this regard, we found that DNA sequences at RNA splice sites are hypersensitive to digestion by MNase but not by MPE-Fe(II). This phenomenon may have affected the analysis of nucleosome occupancy over exons. These findings collectively indicate that MPE-seq provides a unique and straightforward means for the genome-wide analysis of chromatin structure with minimal DNA sequence bias. In particular, the combined use of MPE-seq and MNase-seq enables the identification of noncanonical chromatin structures that are likely to be important for the regulation of gene expression. PMID:26080409

  4. 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

  5. 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

  6. 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.

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

    PubMed Central

    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

  8. Coupling Circadian Rhythms of Metabolism and Chromatin Remodeling

    PubMed Central

    Masri, Selma; Orozco-Solis, Ricardo; Aguilar-Arnal, Lorena; Cervantes, Marlene; Sassone-Corsi, Paolo

    2015-01-01

    The circadian clock controls a large variety of neuronal, endocrine, behavioral and physiological responses in mammals. This control is exerted in large part at the transcriptional level on genes expressed in a cyclic manner. A highly specialized transcriptional machinery based on clock regulatory factors organized in feedback autoregulatory loops governs a significant portion of the genome. These oscillations in gene expression are paralleled by critical events of chromatin remodeling that appear to provide plasticity to circadian regulation. Specifically, the NAD+-dependent deacetylases SIRT1 and SIRT6 have been linked to circadian control of gene expression. This, and additional accumulating evidence, shows that the circadian epigenome appears to share intimate links with cellular metabolic processes and has remarkable plasticity showing reprogramming in response to nutritional challenges. In addition to SIRT1 and SIRT6, a number of chromatin remodelers have been implicated in clock control, including the histone H3K4 tri-methyltransferase MLL1. Deciphering the molecular mechanisms that link metabolism, epigenetic control and circadian responses will provide valauble insights towards innovative strategies of therapeutic intervention. PMID:26332964

  9. 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.

  10. Urb-RIP – An Adaptable and Efficient Approach for Immunoprecipitation of RNAs and Associated RNAs/Proteins

    PubMed Central

    Cottrell, Kyle A.; Djuranovic, Sergej

    2016-01-01

    Post-transcriptional regulation of gene expression is an important process that is mediated by interactions between mRNAs and RNA binding proteins (RBP), non-coding RNAs (ncRNA) or ribonucleoproteins (RNP). Key to the study of post-transcriptional regulation of mRNAs and the function of ncRNAs such as long non-coding RNAs (lncRNAs) is an understanding of what factors are interacting with these transcripts. While several techniques exist for the enrichment of a transcript whether it is an mRNA or an ncRNA, many of these techniques are cumbersome or limited in their application. Here we present a novel method for the immunoprecipitation of mRNAs and ncRNAs, Urb—RNA immunoprecipitation (Urb-RIP). This method employs the RRM1 domain of the “resurrected” snRNA-binding protein Urb to enrich messages containing a stem-loop tag. Unlike techniques which employ the MS2 protein, which require large repeats of the MS2 binding element, Urb-RIP requires only one stem-loop. This method routinely provides over ~100-fold enrichment of tagged messages. Using this technique we have shown enrichment of tagged mRNAs and lncRNAs as well as miRNAs and RNA-binding proteins bound to those messages. We have confirmed, using Urb-RIP, interaction between RNA PolIII transcribed lncRNA BC200 and polyA binding protein. PMID:27930710

  11. Identification of novel 14-3-3ζ interacting proteins by quantitative immunoprecipitation combined with knockdown (QUICK).

    PubMed

    Ge, Feng; Li, Wen-Liang; Bi, Li-Jun; Tao, Sheng-Ce; Zhang, Zhi-Ping; Zhang, Xian-En

    2010-11-05

    The family of 14-3-3 proteins has emerged as critical regulators of diverse cellular responses under both physiological and pathological conditions. To gain insight into the molecular action of 14-3-3ζ in multiple myeloma (MM), we performed a systematic proteomic analysis of 14-3-3ζ-associated proteins. This analysis, recently developed by Matthias Mann, termed quantitative immunoprecipitation combined with knockdown (QUICK), integrates RNAi, SILAC, immunoprecipitation, and quantitative MS technologies. Quantitative mass spectrometry analysis allowed us to distinguish 14-3-3ζ-interacting proteins from background proteins, resulting in the identification of 292 proteins in total with 95 novel interactions. Three 14-3-3ζ-interacting proteins-BAX, HSP70, and BAG3-were further confirmed by reciprocal coimmunoprecipitations and colocalization analysis. Our results therefore not only uncover a large number of novel 14-3-3ζ-associated proteins that possess a variety of cellular functions, but also provide new research directions for the study of the functions of 14-3-3ζ. This study also demonstrated that QUICK is a useful approach to detect specific protein-protein interactions with very high confidence and may have a wide range of applications in the investigation of protein complex interaction networks.

  12. HP1 Binding to Chromatin Methylated at H3K9 Is Enhanced by Auxiliary Factors▿

    PubMed Central

    Eskeland, Ragnhild; Eberharter, Anton; Imhof, Axel

    2007-01-01

    A large portion of the eukaryotic genome is packaged into transcriptionally silent heterochromatin. Several factors that play important roles during the establishment and maintenance of this condensed form have been identified. Methylation of lysine 9 within histone H3 and the subsequent binding of the chromodomain protein heterochromatin protein 1 (HP1) are thought to initiate heterochromatin formation in vivo and to propagate a heterochromatic state lasting through several cell divisions. For the present study we analyzed the binding of HP1 to methylated chromatin in a fully reconstituted system. In contrast to its strong binding to methylated peptides, HP1 binds only weakly to methylated chromatin. However, the addition of recombinant SU(VAR) protein, such as ACF1 or SU(VAR)3-9, facilitates HP1 binding to chromatin methylated at lysine 9 within the H3 N terminus (H3K9). We propose that HP1 has multiple target sites that contribute to its recognition of chromatin, only one of them being methylated at H3K9. These findings have implications for the mechanisms of recognition of specific chromatin modifications in vivo. PMID:17101786

  13. 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

    2015-04-28

    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.

  14. Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction.

    PubMed

    Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H

    2017-01-09

    The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively.

  15. Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction

    PubMed Central

    Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K.; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G.; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H.

    2017-01-01

    The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively. PMID:27899623

  16. The influence of chromatin structure on the frequency of radiation-induced DNA strand breaks: a study using nuclear and nucleoid monolayers

    SciTech Connect

    Ljungman, M. )

    1991-04-01

    To assess the influence of chromatin structure on the frequency of radiation-induced DNA strand breaks, the alkaline unwinding technique was applied to nuclear and nucleoid monolayers. These chromatin substrates were prepared by treating human fibroblasts grown as monolayers with the nonionic detergent Triton X-100 and varying concentrations of cations. The chromatin structure was modified either by a stepwise removal of DNA-bound proteins by extraction in increasing concentrations of monovalent salt, or by the addition or deletion of mono- and divalent cations to condense or decondense the chromatin, respectively. It was found that the stepwise removal of DNA-bound proteins from the chromatin dramatically increased the frequency of radiation-induced DNA strand breaks. The DNA-bound proteins showed a qualitative difference in their ability to protect the DNA where proteins removed by salt concentrations above 1.0 M exerted the greatest protection. Furthermore, the frequency of radiation-induced DNA strand breaks was found to be 6 times lower in condensed chromatin than in decondensed chromatin and about 80 times lower than in protein-depleted chromatin. It is concluded that the presence of DNA-bound proteins and the folding of the chromatin into higher-order structures protect the DNA against radiation-induced strand breaks.

  17. Mechanisms of ATP-Dependent Chromatin Remodeling Motors.

    PubMed

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

    2016-07-05

    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.

  18. E2F1 Coregulates Cell Cycle Genes and Chromatin Components during the Transition of Oligodendrocyte Progenitors from Proliferation to Differentiation

    PubMed Central

    Magri, Laura; Swiss, Victoria A.; Jablonska, Beata; Lei, Liang; Pedre, Xiomara; Walsh, Martin; Zhang, Weijia; Gallo, Vittorio; Canoll, Peter

    2014-01-01

    Cell cycle exit is an obligatory step for the differentiation of oligodendrocyte progenitor cells (OPCs) into myelinating cells. A key regulator of the transition from proliferation to quiescence is the E2F/Rb pathway, whose activity is highly regulated in physiological conditions and deregulated in tumors. In this paper we report a lineage-specific decline of nuclear E2F1 during differentiation of rodent OPC into oligodendrocytes (OLs) in developing white matter tracts and in cultured cells. Using chromatin immunoprecipitation (ChIP) and deep-sequencing in mouse and rat OPCs, we identified cell cycle genes (i.e., Cdc2) and chromatin components (i.e., Hmgn1, Hmgn2), including those modulating DNA methylation (i.e., Uhrf1), as E2F1 targets. Binding of E2F1 to chromatin on the gene targets was validated and their expression assessed in developing white matter tracts and cultured OPCs. Increased expression of E2F1 gene targets was also detected in mouse gliomas (that were induced by retroviral transformation of OPCs) compared with normal brain. Together, these data identify E2F1 as a key transcription factor modulating the expression of chromatin components in OPC during the transition from proliferation to differentiation. PMID:24453336

  19. A role for the Drosophila SU(VAR)3-9 protein in chromatin organization at the histone gene cluster and in suppression of position-effect variegation.

    PubMed Central

    Ner, Sarbjit S; Harrington, Michael J; Grigliatti, Thomas A

    2002-01-01

    Mutations in the gene for Su(var)3-9 are dominant suppressors of position-effect variegation (PEV). We show that SU(VAR)3-9 is a chromatin-associated protein and identify the large multicopy histone gene cluster (HIS-C) as one of its target loci. The organization of nucleosomes over the entire HIS-C region is altered in Su(var)3-9 mutants and there is a concomitant increase in expression of the histone genes. SU(VAR)3-9 is a histone H3 methyltransferase and, using chromatin immunoprecipitation, we show that SU(VAR)3-9 is present at the HIS-C locus and that the histone H3 at the HIS-C locus is methylated. We propose that SU(VAR)3-9 is involved in packaging HIS-C into a distinct chromatin domain that has some of the characteristics of beta-heterochromatin. We suggest that methylation of histone H3 is important for the chromatin structure at HIS-C. The chromosomal deficiency for the HIS-C is also a suppressor of PEV. In contrast to what might be expected, we show that hemizygosity for the HIS-C locus leads to a substantial increase in the histone transcripts. PMID:12524347

  20. 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.

  1. Physical properties of unacetylated chromatin as examined by magnetic tweezers

    NASA Astrophysics Data System (ADS)

    McGill, Kerry; Dunlap, David; Lucchesi, John

    2011-10-01

    As the source of genetic material, DNA is involved in a variety of biological processes like transcription, cell replication, and more. In these processes, DNA is manipulated into different structures and is subjected to different levels of physical force on a molecular scale. When tension is applied to one hierarchical structure called chromatin, it appears to behave like a Hookian spring. The base component of chromatin is a nucleosome, which is constructed when DNA coils around octamers of histone proteins. The histones can become acetylated---a chemical process in which an acetyl functional group attaches to amino acids of the histones, often lysines. Acetylation may loosen chromatin's coils and therefore lower the amount of tension required to stretch the chromatin. Comparing the levels of tension required to stretch acetylated chromatin could reveal, directly, physical differences in the chromatin fiber that bear ion the function of the DNA molecule. Work presented will be the investigation of unacetylated chromatin.

  2. Solenoidal model for superstructure in chromatin.

    PubMed Central

    Finch, J T; Klug, A

    1976-01-01

    Chromatin prepared by brief digestion of nuclei with micrococcal nuclease, and extracted in 0.2 mM EDTA, appears in the electron microscope as filaments of about 100 A diameter which coil loosely. In 0.2 mM Mg++ these "nucleofilaments" condense into a supercoil or solenoidal structure of pitch about 110 A corresponding to the diameter of a nucleofilament. It is proposed that the x-ray reflections at orders of 110 A observed in chromatin originate in the spacing between turns of the solenoid rather than that between nucleosomes along the nucleofilament. The solenoidal structure appears to need histone H1 for its stabilization. Under certain conditions, isolated nucleosomes can also aggregate into a similar structure. The solenoidal structure can be correlated with the "thread" of diameter about 300 A observed by other workers in nuclei. Images PMID:1064861

  3. Chromatin remodelling during male gametophyte development.

    PubMed

    Borg, Michael; Berger, Frédéric

    2015-07-01

    The plant life cycle alternates between a diploid sporophytic phase and haploid gametophytic phase, with the latter giving rise to the gametes. Male gametophyte development encompasses two mitotic divisions that results in a simple three-celled structure knows as the pollen grain, in which two sperm cells are encased within a larger vegetative cell. Both cell types exhibit a very different type of chromatin organization - highly condensed in sperm cell nuclei and highly diffuse in the vegetative cell. Distinct classes of histone variants have dynamic and differential expression in the two cell lineages of the male gametophyte. Here we review how the dynamics of histone variants are linked to reprogramming of chromatin activities in the male gametophyte, compaction of the sperm cell genome and zygotic transitions post-fertilization.

  4. Mechanobiology of Chromatin and the Nuclear Interior.

    PubMed

    Spagnol, Stephen T; Armiger, Travis J; Dahl, Kris Noel

    2016-06-01

    The view of the cell nucleus has evolved from an isolated, static organelle to a dynamic structure integrated with other mechanical elements of the cell. Both dynamics and integration appear to contribute to a mechanical regulation of genome expression. Here, we review physical structures inside the nucleus at different length scales and the dynamic reorganization modulated by cellular forces. First, we discuss nuclear organization focusing on self-assembly and disassembly of DNA structures and various nuclear bodies. We then discuss the importance of connections from the chromatin fiber through the nuclear envelope to the rest of the cell as they relate to mechanobiology. Finally, we discuss how cell stimulation, both chemical and physical, can alter nuclear structures and ultimately cellular function in healthy cells and in some model diseases. The view of chromatin and nuclear bodies as mechanical entities integrated with force generation from the cytoskeleton combines polymer physics with cell biology and medicine.

  5. 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

  6. 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

  7. Perturbation of Chromatin Structure Globally Affects Localization and Recruitment of Splicing Factors

    PubMed Central

    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

  8. Transcription, chromatin condensation, and gene migration

    PubMed Central

    2009-01-01

    The binding of fluorescently tagged proteins to tandem DNA arrays has been instrumental in understanding nuclear organization and function. Through the use of more natural tandem DNA arrays, Hu et al. (Hu, Y., I. Kireev, M. Plutz, N. Ashourian, and A.S. Belmont. 2009. J. Cell Biol. 185:87–100) gain new insights into chromatin organization and dynamics, and into the association of splicing factors with active genes. PMID:19349577

  9. Reproducibility of 3D chromatin configuration reconstructions

    PubMed Central

    Segal, Mark R.; Xiong, Hao; Capurso, Daniel; Vazquez, Mariel; Arsuaga, Javier

    2014-01-01

    It is widely recognized that the three-dimensional (3D) architecture of eukaryotic chromatin plays an important role in processes such as gene regulation and cancer-driving gene fusions. Observing or inferring this 3D structure at even modest resolutions had been problematic, since genomes are highly condensed and traditional assays are coarse. However, recently devised high-throughput molecular techniques have changed this situation. Notably, the development of a suite of chromatin conformation capture (CCC) assays has enabled elicitation of contacts—spatially close chromosomal loci—which have provided insights into chromatin architecture. Most analysis of CCC data has focused on the contact level, with less effort directed toward obtaining 3D reconstructions and evaluating the accuracy and reproducibility thereof. While questions of accuracy must be addressed experimentally, questions of reproducibility can be addressed statistically—the purpose of this paper. We use a constrained optimization technique to reconstruct chromatin configurations for a number of closely related yeast datasets and assess reproducibility using four metrics that measure the distance between 3D configurations. The first of these, Procrustes fitting, measures configuration closeness after applying reflection, rotation, translation, and scaling-based alignment of the structures. The others base comparisons on the within-configuration inter-point distance matrix. Inferential results for these metrics rely on suitable permutation approaches. Results indicate that distance matrix-based approaches are preferable to Procrustes analysis, not because of the metrics per se but rather on account of the ability to customize permutation schemes to handle within-chromosome contiguity. It has recently been emphasized that the use of constrained optimization approaches to 3D architecture reconstruction are prone to being trapped in local minima. Our methods of reproducibility assessment provide a

  10. Dynamic chromatin regulation at Notch target genes

    PubMed Central

    Giaimo, Benedetto Daniele; Oswald, Franz; Borggrefe, Tilman

    2017-01-01

    ABSTRACT RBPJ is the central transcription factor that controls the Notch-dependent transcriptional response by coordinating repressing histone H3K27 deacetylation and activating histone H3K4 methylation. Here, we discuss the molecular mechanisms how RBPJ interacts with opposing NCoR/HDAC-corepressing or KMT2D/UTX-coactivating complexes and how this is controlled by phosphorylation of chromatin modifiers. PMID:28027012

  11. 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.

  12. [The biological aspects of chromatin diminution].

    PubMed

    Akif'ev, A P; Grishanin, A K

    1993-01-01

    The chromatine diminution (CD), first discovered by Boveri (1887) in ascarids, represents programmed elimination of a part of genetic material in the nuclei of the somatic cells in cyclops and ascarids, and in the protist macronuclei. The CD can be considered as a macromutation sharply changing chromosomal structure, though minimally effecting the phenotype. The analysis of CD is of significance for discussing mechanisms of origin of chromosomal organization, transformation of genome molecular structure in eucaryote evolution, role of the extra DNA.

  13. Polyamine analogs modulate gene expression by inhibiting lysine-specific demethylase 1 (LSD1) and altering chromatin structure in human breast cancer cells.

    PubMed

    Zhu, Qingsong; Huang, Yi; Marton, Laurence J; Woster, Patrick M; Davidson, Nancy E; Casero, Robert A

    2012-02-01

    Aberrant epigenetic repression of gene expression has been implicated in most cancers, including breast cancer. The nuclear amine oxidase, lysine-specific demethylase 1 (LSD1) has the ability to broadly repress gene expression by removing the activating mono- and di-methylation marks at the lysine 4 residue of histone 3 (H3K4me1 and me2). Additionally, LSD1 is highly expressed in estrogen receptor α negative (ER-) breast cancer cells. Since epigenetic marks are reversible, they make attractive therapeutic targets. Here we examine the effects of polyamine analog inhibitors of LSD1 on gene expression, with the goal of targeting LSD1 as a therapeutic modality in the treatment of breast cancer. Exposure of the ER-negative human breast cancer cells, MDA-MB-231 to the LSD1 inhibitors, 2d or PG11144, significantly increases global H3K4me1 and H3K4me2, and alters gene expression. Array analysis indicated that 98 (75 up and 23 down) and 477 (237 up and 240 down) genes changed expression by at least 1.5-fold or greater after treatment with 2d and PG11144, respectively. The expression of 12 up-regulated genes by 2d and 14 up-regulated genes by PG11144 was validated by quantitative RT-PCR. Quantitative chromatin immunoprecipitation (ChIP) analysis demonstrated that up-regulated gene expression by polyamine analogs is associated with increase of the active histone marks H3K4me1, H3K4me2 and H3K9act, and decrease of the repressive histone marks H3K9me2 and H3K27me3, in the promoter regions of the relevant target genes. These data indicate that the pharmacologic inhibition of LSD1 can effectively alter gene expression and that this therapeutic strategy has potential.

  14. 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

  15. 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.

  16. Dynamic chromatin boundaries delineate a latency control region of Epstein-Barr virus.

    PubMed

    Chau, Charles M; Lieberman, Paul M

    2004-11-01

    The oncogenic potential of latent Epstein-Barr virus (EBV) can be regulated by epigenetic factors controlling LMP1 and EBNA2 gene transcription. The EBV latency control region (LCR) constitutes approximately 12 kb of viral sequence spanning the divergent promoters of LMP1 and EBNA2 and encompasses the EBV latent replication origin OriP and RNA polymerase III-transcribed EBV-encoded RNA genes. We have used the chromatin immunoprecipitation assay to examine the chromatin architecture of the LCR in different types of EBV latency programs. We have found that histone H3 K4 methylation (H3mK4) was enriched throughout a large domain that extended from internal repeat 1 (IR1) to the terminal repeat in type III latency where EBNA2 and LMP1 genes are expressed. In type I latency where EBNA2 and LMP1 genes are transcriptionally silent, the H3mK4 domain contracts and does not enter the EBNA2 or LMP1 promoters. In contrast, histone H3 K9 methylation (H3mK9), associated with silent heterochromatin, was enriched in the EBNA2 and LMP1 upstream control regions in type I but not type III cells. MTA [5'-deoxy-5'(methylthio)adenosine], a pharmacological inhibitor of protein methylation, globally reduced histone H3mK4 and inhibited EBNA2 transcription in type III cells. 5'-Azacytidine, an inhibitor of DNA methylation that derepresses EBNA2 transcription in type I latency, caused H3mK4 expansion and a corresponding loss of H3mK9 at IR1. The chromatin boundary protein and transcription repressor CCCTC-binding factor was enriched at the EBNA2 transcription control region in type I but not type III cells. We also present evidence that OriP binding factors EBNA1 and ORC2 can interact with sequences outside of OriP including a region within IR1 that may influence EBNA2 transcription status. These results indicate that types I and III latency programs have distinct histone methylation patterns in the LCR and suggest that chromatin architecture coordinates gene expression of LMP1 and EBNA2.

  17. Nucleosome acetylation sequencing to study the establishment of chromatin acetylation.

    PubMed

    Mittal, Chitvan; Blacketer, Melissa J; Shogren-Knaak, Michael A

    2014-07-15

    The establishment of posttranslational chromatin modifications is a major mechanism for regulating how genomic DNA is utilized. However, current in vitro chromatin assays do not monitor histone modifications at individual nucleosomes. Here we describe a strategy, nucleosome acetylation sequencing, that allows us to read the amount of modification at each nucleosome. In this approach, a bead-bound trinucleosome substrate is enzymatically acetylated with radiolabeled acetyl CoA by the SAGA complex from Saccharomyces cerevisae. The product is digested by restriction enzymes that cut at unique sites between the nucleosomes and then counted to quantify the extent of acetylation at each nucleosomal site. We find that we can sensitively, specifically, and reproducibly follow enzyme-mediated nucleosome acetylation. Applying this strategy, when acetylation proceeds extensively, its distribution across nucleosomes is relatively uniform. However, when substrates are used that contain nucleosomes mutated at the major sites of SAGA-mediated acetylation, or that are studied under initial rate conditions, changes in the acetylation distribution can be observed. Nucleosome acetylation sequencing should be applicable to analyzing a wide range of modifications. Additionally, because our trinucleosomes synthesis strategy is highly modular and efficient, it can be used to generate nucleosomal systems in which nucleosome composition differs across the array.

  18. Probing the impact of chromatin conformation on genome editing tools

    PubMed Central

    Chen, Xiaoyu; Rinsma, Marrit; Janssen, Josephine M.; Liu, Jin; Maggio, Ignazio; Gonçalves, Manuel A.F.V.

    2016-01-01

    Transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases derived from clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 systems have become ubiquitous genome editing tools. Despite this, the impact that distinct high-order chromatin conformations have on these sequence-specific designer nucleases is, presently, ill-defined. The same applies to the relative performance of TALENs and CRISPR/Cas9 nucleases at isogenic target sequences subjected to different epigenetic modifications. Here, to address these gaps in our knowledge, we have implemented quantitative cellular systems based on genetic reporters in which the euchromatic and heterochromatic statuses of designer nuclease target sites are stringently controlled by small-molecule drug availability. By using these systems, we demonstrate that TALENs and CRISPR/Cas9 nucleases are both significantly affected by the high-order epigenetic context of their target sequences. In addition, this outcome could also be ascertained for S. pyogenes CRISPR/Cas9 complexes harbouring Cas9 variants whose DNA cleaving specificities are superior to that of the wild-type Cas9 protein. Thus, the herein investigated cellular models will serve as valuable functional readouts for screening and assessing the role of chromatin on designer nucleases based on different platforms or with different architectures or compositions. PMID:27280977

  19. 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

  20. 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

  1. Plant chromatin warms up in Madrid: meeting summary of the 3rd European Workshop on Plant Chromatin 2013, Madrid, Spain.

    PubMed

    Jarillo, José A; Gaudin, Valérie; Hennig, Lars; Köhler, Claudia; Piñeiro, Manuel

    2014-04-01

    The 3rd European Workshop on Plant Chromatin (EWPC) was held on August 2013 in Madrid, Spain. A number of different topics on plant chromatin were presented during the meeting, including new factors mediating Polycomb Group protein function in plants, chromatin-mediated reprogramming in plant developmental transitions, the role of histone variants, and newly identified chromatin remodeling factors. The function of interactions between chromatin and transcription factors in the modulation of gene expression, the role of chromatin dynamics in the control of nuclear processes and the influence of environmental factors on chromatin organization were also reported. In this report, we highlight some of the new insights emerging in this growing area of research, presented at the 3rd EWPC.

  2. Influence of a CTCF-Dependent Insulator on Multiple Aspects of Enhancer-Mediated Chromatin Organization.

    PubMed

    Varma, Garima; Rawat, Pratishtha; Jalan, Manisha; Vinayak, Manjula; Srivastava, Madhulika

    2015-10-01

    Developmental stage-specific enhancer-promoter-insulator interactions regulate the chromatin configuration necessary for transcription at various loci and additionally for VDJ recombination at antigen receptor loci that encode immunoglobulins and T-cell receptors. To investigate these regulatory interactions, we analyzed the epigenetic landscape of the murine T-cell receptor β (TCRβ) locus in the presence and absence of an ectopic CTCF-dependent enhancer-blocking insulator, H19-ICR, in genetically manipulated mice. Our analysis demonstrated the ability of the H19-ICR insulator to restrict several aspects of enhancer-based chromatin alterations that are observed during activation of the TCRβ locus for transcription and recombination. The H19-ICR insulator abrogated enhancer-promoter contact-dependent chromatin alterations and additionally prevented Eβ-mediated histone modifications that have been suggested to be independent of enhancer-promoter interaction. Observed enhancer-promoter-insulator interactions, in conjunction with the chromatin structure of the Eβ-regulated domain at the nucleosomal level, provide useful insights regarding the activity of the regulatory elements in addition to supporting the accessibility hypothesis of VDJ recombination. Analysis of H19-ICR in the heterologous context of the developmentally regulated TCRβ locus suggests that different mechanisms proposed for CTCF-dependent insulator action might be manifested simultaneously or selectively depending on the genomic context and the nature of enhancer activity being curtailed.

  3. 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.

  4. Polysome immunoprecipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA.

    PubMed Central

    Robson, K J; Chandra, T; MacGillivray, R T; Woo, S L

    1982-01-01

    The mRNA for phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been purified from total rat liver mRNAs, of which it constitutes less than 0.25%, to greater than 10% purity in a single step by specific polysome immunoprecipitation. The purified mRNA was used for synthesis and cloning of its cDNA. Recombinant colonies containing phenylalanine hydroxylase DNA sequences were identified by differential hybridization, hybrid-selected translation, and blot hybridization analysis. The rat cDNA clone was capable of hybridizing with human phenylalanine hydroxylase mRNA, which will permit the isolation of the corresponding human gene for analysis of phenylketonuria, a hereditary disorder in phenylalanine metabolism that causes permanent mental retardation in humans. Images PMID:6750607

  5. Genome-Wide Association between Transcription Factor Expression and Chromatin Accessibility Reveals Regulators of Chromatin Accessibility

    PubMed Central

    Rueedi, Rico

    2017-01-01

    To better understand genome regulation, it is important to uncover the role of transcription factors in the process of chromatin structure establishment and maintenance. Here we present a data-driven approach to systematically characterise transcription factors that are relevant for this process. Our method uses a linear mixed modelling approach to combine datasets of transcription factor binding motif enrichments in open chromatin and gene expression across the same set of cell lines. Applying this approach to the ENCODE dataset, we confirm already known and imply numerous novel transcription factors that play a role in the establishment or maintenance of open chromatin. In particular, our approach rediscovers many factors that have been annotated as pioneer factors. PMID:28118358

  6. Genome-Wide Association between Transcription Factor Expression and Chromatin Accessibility Reveals Regulators of Chromatin Accessibility.

    PubMed

    Lamparter, David; Marbach, Daniel; Rueedi, Rico; Bergmann, Sven; Kutalik, Zoltán

    2017-01-01

    To better understand genome regulation, it is important to uncover the role of transcription factors in the process of chromatin structure establishment and maintenance. Here we present a data-driven approach to systematically characterise transcription factors that are relevant for this process. Our method uses a linear mixed modelling approach to combine datasets of transcription factor binding motif enrichments in open chromatin and gene expression across the same set of cell lines. Applying this approach to the ENCODE dataset, we confirm already known and imply numerous novel transcription factors that play a role in the establishment or maintenance of open chromatin. In particular, our approach rediscovers many factors that have been annotated as pioneer factors.

  7. 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

  8. 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

  9. Eubacterial components similar to small nuclear ribonucleoproteins: identification of immunoprecipitable proteins and capped RNAs in a cyanobacterium and a gram-positive eubacterium.

    PubMed Central

    Kovacs, S A; O'Neil, J; Watcharapijarn, J; Moe-Kirvan, C; Vijay, S; Silva, V

    1993-01-01

    Small nuclear ribonucleoprotein (snRNP) particles play an important role in the processing of pre-mRNA. snRNPs have been identified immunologically in a variety of cells, but none have ever been observed in prokaryotic systems. This report provides the first evidence for the presence of snRNP-like components in two types of prokaryotic cells: those of the cyanobacterium Synechococcus leopoliensis and those of the gram-positive eubacterium Bacillus subtilis. These components consist of snRNP-immunoreactive proteins and RNAs, including some with the snRNP-unique 5' m2,2,7G (m3G) cap. Immunoreactivity was determined by immunoprecipitation procedures, with either antinuclear-antibody-positive (RNP- and Sm-monospecific) patient sera or a m3G monoclonal antibody, with radiolabelled cell extracts that were preadsorbed with antinuclear-antibody-negative sera. S. leopoliensis immunoprecipitates showed the presence of high-molecular-mass proteins (14 to 70 kDa) and RNAs (138 to 243 nucleotides) that are analogous in size to proteins and RNAs found in human (HEp-2) cell immunoprecipitates but absent in Escherichia coli immunoprecipitates. Thin-layer chromatography of S. leopoliensis immunoprecipitates confirmed the presence of a capped nucleotide similar to a capped nucleotide in HEp-2 immunoprecipitates; no such nucleotide was observed in E. coli immunoprecipitates. Immunoreactive RNAs (117-170 nucleotides) were identified in a second eubacterium, B. subtilis, as well. This work suggests that snRNPs or their evolutionary predecessors predate the emergence of eukaryotic cells. Images PMID:8458830

  10. 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

  11. 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.

  12. Time-resolved global and chromatin proteomics during herpes simplex virus (HSV-1) infection.

    PubMed

    Kulej, Katarzyna; Avgousti, Daphne C; Sidoli, Simone; Herrmann, Christin; Della Fera, Ashley N; Kim, Eui Tae; Garcia, Benjamin A; Weitzman, Matthew D

    2017-02-08

    Herpes simplex virus (HSV-1) lytic infection results in global changes to the host cell proteome and the proteins associated with host chromatin. We present a system level characterization of proteome dynamics during infection by performing a multi-dimensional analysis during HSV-1 lytic infection of human foreskin fibroblast (HFF) cells. Our study includes identification and quantification of the host and viral proteomes, phosphoproteomes, chromatin bound proteomes and post-translational modifications (PTMs) on cellular histones during infection. We analyzed proteomes across six time points of virus infection (0, 3, 6, 9, 12 and 15 hours post-infection) and clustered trends in abundance using fuzzy c-means. Globally, we accurately quantified more than 4,000 proteins, 200 differently modified histone peptides and 9,000 phosphorylation sites on cellular proteins. In addition, we identified 67 viral proteins and quantified 571 phosphorylation events (465 with high confidence site localization) on viral proteins, which is currently the most comprehensive map of HSV-1 phosphoproteome. We investigated chromatin bound proteins by proteomic analysis of the high-salt chromatin fraction and identified 510 proteins that were significantly different in abundance during infection. We found 53 histone marks significantly regulated during virus infection, including a steady increase of histone H3 acetylation (H3K9ac and H3K14ac). Our data provide a resource of unprecedented depth for human and viral proteome dynamics during infection. Collectively, our results indicate that the proteome composition of the chromatin of HFF cells is highly affected during HSV-1 infection, and that phosphorylation events are abundant on viral proteins. We propose that our epi-proteomics approach will prove to be important in the characterization of other model infectious systems that involve changes to chromatin composition.

  13. MRN1 Implicates Chromatin Remodeling Complexes and Architectural Factors in mRNA Maturation

    PubMed Central

    Düring, Louis; Thorsen, Michael; Petersen, Darima Sophia Njama; Køster, Brian; Jensen, Torben Heick; Holmberg, Steen

    2012-01-01

    A functional relationship between chromatin structure and mRNA processing events has been suggested, however, so far only a few involved factors have been characterized. Here we show that rsc nhp6ΔΔ mutants, deficient for the function of the chromatin remodeling factor RSC and the chromatin architectural proteins Nhp6A/Nhp6B, accumulate intron-containing pre-mRNA at the restrictive temperature. In addition, we demonstrate that rsc8-ts16 nhp6ΔΔ cells contain low levels of U6 snRNA and U4/U6 di-snRNA that is further exacerbated after two hours growth at the restrictive temperature. This change in U6 snRNA and U4/U6 di-snRNA levels in rsc8-ts16 nhp6ΔΔ cells is indicative of splicing deficient conditions. We identify MRN1 (multi-copy suppressor of rsc nhp6ΔΔ) as a growth suppressor of rsc nhp6ΔΔ synthetic sickness. Mrn1 is an RNA binding protein that localizes both to the nucleus and cytoplasm. Genetic interactions are observed between 2 µm-MRN1 and the splicing deficient mutants snt309Δ, prp3, prp4, and prp22, and additional genetic analyses link MRN1, SNT309, NHP6A/B, SWI/SNF, and RSC supporting the notion of a role of chromatin structure in mRNA processing. PMID:23028530

  14. Drugging Chromatin in Cancer: Recent Advances and Novel Approaches

    PubMed Central

    Cai, Sheng F.; Chen, Chun-Wei; Armstrong, Scott A.

    2015-01-01

    Chromatin regulatory mechanisms play a major role in the control of gene expression programs during normal development and are disrupted in specific disease states, particularly in cancer. Important mediators of chromatin regulatory processes can broadly be classified into writers, erasers, and readers of covalent chromatin modifications that modulate eukaryotic gene transcription and maintain the integrity of the genome. The reversibility and disease-specific nature of these chromatin states make these regulators attractive therapeutic targets. As such, there is an ever-increasing number of candidate therapies aimed at targeting cancer-associated chromatin states that are in various stages of preclinical and clinical development. In this review, we discuss recent advances that have been made in the rational therapeutic targeting of chromatin regulatory mechanisms and highlight certain cancers where there is a specific rationale to assess these therapeutic approaches. PMID:26590715

  15. Roles of chromatin remodellers in DNA double strand break repair.

    PubMed

    Jeggo, Penny A; Downs, Jessica A

    2014-11-15

    Now that we have a good understanding of the DNA double strand break (DSB) repair mechanisms and DSB-induced damage signalling, attention is focusing on the changes to the chromatin environment needed for efficient DSB repair. Mutations in chromatin remodelling complexes have been identified in cancers, making it important to evaluate how they impact upon genomic stability. Our current understanding of the DSB repair pathways suggests that each one has distinct requirements for chromatin remodelling. Moreover, restricting the extent of chromatin modifications could be a significant factor regulating the decision of pathway usage. In this review, we evaluate the distinct DSB repair pathways for their potential need for chromatin remodelling and review the roles of ATP-driven chromatin remodellers in the pathways.

  16. 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

  17. In vitro Chromatin Assembly - Strategies and Quality Control

    PubMed Central

    Muthurajan, Uma; Mattiroli, Francesca; Bergeron, Serge; Zhou, Keda; Gu, Yajie; Chakravarthy, Srinivas; Dyer, Pamela; Irving, Thomas; Luger, Karolin

    2016-01-01

    Chromatin accessibility is modulated by structural transitions that provide timely access to the genetic and epigenetic information during many essential nuclear processes. These transitions are orchestrated by regulatory proteins that coordinate intricate structural modifications and signalling pathways. In vitro reconstituted chromatin samples from defined components are instrumental in defining the mechanistic details of such processes. The bottleneck to appropriate in vitro analysis is the production of high quality, and quality-controlled, chromatin substrates. In this chapter we describe methods for in vitro chromatin reconstitution and quality control. We highlight the strengths and weaknesses of various approaches, and emphasize quality control steps that ensure reconstitution of a bona fide homogenous chromatin preparation. This is essential for optimal reproducibility and reliability of ensuing experiments using chromatin substrates. PMID:27372747

  18. 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.

  19. 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

  20. Prolonged TNFα primes fibroblast-like synoviocytes in a gene-specific manner by altering chromatin

    PubMed Central

    Sohn, Christopher; Lee, Angela; Qiao, Yu; Loupasakis, Konstantinos; Ivashkiv, Lionel B.; Kalliolias, George D.

    2015-01-01

    Objective During the course of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) are chronically exposed to an inflammatory milieu. In the current study we test the hypothesis that chronic exposure of FLS to TNFα augments inflammatory responses to secondary stimuli (priming effect). Methods FLS obtained from RA patients were chronically exposed to TNFα (3 days) and then were stimulated with interferons (IFNs). Expression of IFN-target genes was measured by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Total STAT1 protein and IFN-mediated STAT1 activation were evaluated by Western blotting. Total histone levels, histone acetylation, NF-κB p65 and RNA polymerase II (pol II) recruitment were measured at the promoter of CXCL10 (encodes IP-10) by chromatin immunoprecipitation assays. Results Prolonged pre-exposure of FLS to TNFα enhanced the magnitude and extended the kinetics of CXCL10/IP-10, CXCL9/MIG and CXCL11/ITAC production upon subsequent IFN stimulation. This phenotype was retained over a period of days even after the removal of TNFα. Prolonged TNFα decreased histone levels, increased acetylation of the remaining histones, and heightened recruitment of NF-κB p65 and pol II to the CXCL10 promoter. In parallel, an increase in intracellular STAT1 led to amplification of IFN-induced STAT1 activation. Conclusions Our study reveals a novel pathogenic function of TNFα, namely prolonged and gene-specific priming of FLS for enhanced transcription of inflammatory chemokine genes due to priming of chromatin, sustained activation of NF-κB, and amplification of STAT1 activation downstream of IFNs. These data also suggest that FLS gain an “inflammatory memory” upon chronic exposure to TNFα. PMID:25199798

  1. 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

  2. The E3 ubiquitin ligase RAD18 regulates ubiquitylation and chromatin loading of FANCD2 and FANCI.

    PubMed

    Williams, Stacy A; Longerich, Simonne; Sung, Patrick; Vaziri, Cyrus; Kupfer, Gary M

    2011-05-12

    Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand cross-links. Central to this pathway is the monoubiquitylation and chromatin localization of 2 FA proteins, FA complementation group D2 (FANCD2) and FANCI. In the present study, we show that RAD18 binds FANCD2 and is required for efficient monoubiquitylation and chromatin localization of both FANCD2 and FANCI. Human RAD18-knockout cells display increased sensitivity to mitomycin C and a delay in FANCD2 foci formation compared with their wild-type counterparts. In addition, RAD18-knockout cells display a unique lack of FANCD2 and FANCI localization to chromatin in exponentially growing cells. FANCD2 ubiquitylation is normal in cells containing a ubiquitylation-resistant form of proliferating cell nuclear antigen, and chromatin loading of FA core complex proteins appears normal in RAD18-knockout cells. Mutation of the RING domain of RAD18 ablates the interaction with and chromatin loading of FANCD2. These data suggest a key role for the E3 ligase activity of RAD18 in the recruitment of FANCD2 and FANCI to chromatin and the events leading to their ubiquitylation during S phase.

  3. Constitutive oligomerization of human D2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells. Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer.

    PubMed

    Gazi, Lucien; López-Giménez, Juan F; Rüdiger, Martin P; Strange, Philip G

    2003-10-01

    Human D2Long (D2L) and D2Short (D2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [3H]Spiperone labelled D2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D2L or D2S receptors, with a pKd value of approximately 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D2L and D2S receptors in Sf9 cells. When the FLAG-tagged D2S and HIV-tagged D2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D2 receptors. In both cases, constitutive homo-oligomers were revealed for D2L and D2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D2S receptors. The D2 receptor ligands dopamine, R-(-)propylnorapomorphine, and raclopride did not affect oligomerization of D2L and D2S in Sf9 and HEK293 cells. Human D2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D2 oligomerization in these cells is not regulated by ligands.

  4. 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.

  5. 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.

  6. 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

  7. Histone chaperone networks shaping chromatin function.

    PubMed

    Hammond, Colin M; Strømme, Caroline B; Huang, Hongda; Patel, Dinshaw J; Groth, Anja

    2017-03-01

    The association of histones with specific chaperone complexes is important for their folding, oligomerization, post-translational modification, nuclear import, stability, assembly and genomic localization. In this way, the chaperoning of soluble histones is a key determinant of histone availability and fate, which affects all chromosomal processes, including gene expression, chromosome segregation and genome replication and repair. Here, we review the distinct structural and functional properties of the expanding network of histone chaperones. We emphasize how chaperones cooperate in the histone chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin.

  8. Chromatin, DNA structure and alternative splicing.

    PubMed

    Nieto Moreno, Nicolás; Giono, Luciana E; Cambindo Botto, Adrián E; Muñoz, Manuel J; Kornblihtt, Alberto R

    2015-11-14

    Coupling of transcription and alternative splicing via regulation of the transcriptional elongation rate is a well-studied phenomenon. Template features that act as roadblocks for the progression of RNA polymerase II comprise histone modifications and variants, DNA-interacting proteins and chromatin compaction. These may affect alternative splicing decisions by inducing pauses or decreasing elongation rate that change the time-window for splicing regulatory sequences to be recognized. Herein we discuss the evidence supporting the influence of template structural modifications on transcription and splicing, and provide insights about possible roles of non-B DNA conformations on the regulation of alternative splicing.

  9. Mechanisms of ATP Dependent Chromatin Remodeling

    PubMed Central

    Gangaraju, Vamsi K.; Bartholomew, Blaine

    2007-01-01

    The inter-relationship between DNA repair and ATP dependent chromatin remodeling has begun to become very apparent with recent discoveries. ATP dependent remodeling complexes mobilize nucleosomes along DNA, promote the exchange of histones, or completely displace nucleosomes from DNA. These remodeling complexes are often categorized based on the domain organization of their catalytic subunit. The biochemical properties and structural information of several of these remodeling complexes are reviewed. The different models for how these complexes are able to mobilize nucleosomes and alter nucleosome structure are presented incorporating several recent findings. Finally the role of histone tails and their respective modifications in ATP-dependent remodeling are discussed. PMID:17306844

  10. Radiation-induced thymine base damage in replicating chromatin

    SciTech Connect

    Warters, R.L.; Childers, T.J.

    1982-06-01

    The efficiency of radiation-induced production of 5',6'-dihydroxydihydrothymine (t/sup ..gamma../)-type damage was determined in nascent and mature chromatin DNA for the dose range of 50 to 150 krad. These large doses affected neither the total fraction of nuclear DNA in chromatin subunits nor the nucleosome subunit repeat length. The DNA in nascent chromatin, however, was found to be 3.3 times more sensitive than mature chromatin DNA to ..gamma..-ray (/sup 137/Cs)-induced t/sup ..gamma../-type damage, while thymine damage of this type was uniformly distributed in the nucleosomal DNA of mature chromatin (i.e., in the nucleosome core and spacer DNA). The half-time for the transition of nascent DNA sensitivity to mature chromatin DNA sensitivity levels was the same as the half-time at 37/sup 0/C for the maturation of nascent into mature chromatin structure. The rate at which nascent chromatin matured was unaffected by radiation doses as large as 150 krad. The most logical explanation for the greater sensitivity of nascent DNA to radiation is the decreased concentration of histone chromosomal proteins in nascent chromatin.

  11. Regulation of chromatin structure in the cardiovascular system.

    PubMed

    Rosa-Garrido, Manuel; Karbassi, Elaheh; Monte, Emma; Vondriska, Thomas M

    2013-01-01

    It has been appreciated for some time that cardiovascular disease involves large-scale transcriptional changes in various cell types. What has become increasingly clear only in the past few years, however, is the role of chromatin remodeling in cardiovascular phenotypes in normal physiology, as well as in development and disease. This review summarizes the state of the chromatin field in terms of distinct mechanisms to regulate chromatin structure in vivo, identifying when these modes of regulation have been demonstrated in cardiovascular tissues. We describe areas in which a better understanding of chromatin structure is leading to new insights into the fundamental biology of cardiovascular disease. 

  12. Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes*

    PubMed Central

    Hoffman, Elizabeth A.; Frey, Brian L.; Smith, Lloyd M.; Auble, David T.

    2015-01-01

    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

  13. 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.

  14. The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage

    PubMed Central

    Sellou, Hafida; Lebeaupin, Théo; Chapuis, Catherine; Smith, Rebecca; Hegele, Anna; Singh, Hari R.; Kozlowski, Marek; Bultmann, Sebastian; Ladurner, Andreas G.; Timinszky, Gyula; Huet, Sébastien

    2016-01-01

    Chromatin relaxation is one of the earliest cellular responses to DNA damage. However, what determines these structural changes, including their ATP requirement, is not well understood. Using live-cell imaging and laser microirradiation to induce DNA lesions, we show that the local chromatin relaxation at DNA damage sites is regulated by PARP1 enzymatic activity. We also report that H1 is mobilized at DNA damage sites, but, since this mobilization is largely independent of poly(ADP-ribosyl)ation, it cannot solely explain the chromatin relaxation. Finally, we demonstrate the involvement of Alc1, a poly(ADP-ribose)- and ATP-dependent remodeler, in the chromatin-relaxation process. Deletion of Alc1 impairs chromatin relaxation after DNA damage, while its overexpression strongly enhances relaxation. Altogether our results identify Alc1 as an important player in the fast kinetics of the NAD+- and ATP-dependent chromatin relaxation upon DNA damage in vivo. PMID:27733626

  15. 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.

  16. The relative infrequency and low levels of neutralising and immunoprecipitating antibody to herpes simplex viruses types 1 and 2 in patients with a history of recurrent herpes genitalis.

    PubMed

    Woodman, C B; Stocker, D; Sugrue, D; Desberbasques, M; Hartley, C E; Fuller, A; Buchan, A; Skinner, G R

    1983-01-01

    Twenty-seven per cent of 70 patients with a history of recurrent herpes genitalis but no concomitant history of recurrent oral or peri-genital disease, had no detectable neutralising antibody against type 1 or type 2 herpes simplex virus; the prevalence and levels of neutralising antibody were similar to 53 patients with no history of herpetic disease and significantly lower than 67 patients with a history of recurrent herpes genitalis in association with oral or peri-genital disease all of whom had neutralising antibody against both virus types. There were similar differences between groups for immunoprecipitating antibody where 80% of patients were herpes genitalis alone had no detectable immunoprecipitating antibody. The results indicate that the failure to detect immunising and immunoprecipitating antibody in an individual's serum is compatible with a long and even severe history of recurrent herpes genitalis and consequently that the development of neutralising antibody does not necessarily indicate an episode of primary herpetic disease.

  17. Identification of a novel distal control region upstream of the human steroidogenic acute regulatory protein (StAR) gene that participates in SF-1-dependent chromatin architecture.

    PubMed

    Mizutani, Tetsuya; Yazawa, Takashi; Ju, Yunfeng; Imamichi, Yoshitaka; Uesaka, Miki; Inaoka, Yoshihiko; Matsuura, Kaoru; Kamiki, Yasue; Oki, Masaya; Umezawa, Akihiro; Miyamoto, Kaoru

    2010-09-03

    StAR (steroidogenic acute regulatory protein) mediates the transport of cholesterol from the outer to the inner mitochondrial membrane, the process of which is the rate-limiting step for steroidogenesis. Transcriptional regulation of the proximal promoter of the human StAR gene has been well characterized, whereas analysis of its distal control region has not. Recently, we found that SF-1 (steroidogenic factor 1) induced the differentiation of mesenchymal stem cells (MSCs) into steroidogenic cells with the concomitant strong induction of StAR expression. Here, we show, using differentiated MSCs, that StAR expression is regulated by a novel distal control region. Using electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays, we identified novel SF-1 binding sites between 3,000 and 3,400 bp upstream of StAR. A luciferase reporter assay revealed that the region worked as a strong regulator to exert maximal transcription of StAR. ChIP analysis of histone H3 revealed that upon SF-1 expression, nucleosome eviction took place at the SF-1 binding sites, not only in the promoter but also in the distal SF-1 binding sites. Chromosome conformation capture analysis revealed that the region upstream of StAR formed a chromatin loop both in the differentiated MSCs and in KGN cells, a human granulosa cell tumor cell line, where SF-1 is endogenously expressed. Finally, SF-1 knockdown resulted in disrupted formation of this chromatin loop in KGN cells. These results indicate that the novel distal control region participate in StAR activation through SF-1 dependent alterations of chromatin structure, including histone eviction and chromatin loop formation.

  18. 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.

  19. Chromatin structure determines accessibility of a hairpin polyamide-chlorambucil conjugate at histone H4 genes in pancreatic cancer cells.

    PubMed

    Jespersen, Christine; Soragni, Elisabetta; James Chou, C; Arora, Paramjit S; Dervan, Peter B; Gottesfeld, Joel M

    2012-06-15

    We have shown that a specific pyrrole-imidazole polyamide-DNA alkylator (chlorambucil) conjugate, 1R-Chl, alters the growth characteristics of various cancer cell lines in culture, and causes these cells to arrest in the G2/M stage of the cell cycle, without apparent cytotoxicity. This molecule has also shown efficacy in several mouse xenograft models, preventing tumor growth. Previous microarray studies have suggested that members of the histone H4 gene family, H4c and H4j/k, are the primary targets of this molecule, leading to reduced histone mRNA synthesis and growth arrest in cancer cells. In the present study, we examine the effects of 1R-Chl on transcription of other members of the H4 gene family, with the result that mRNA transcription of most genomic copies of H4 are down-regulated by 1R-Chl in a human pancreatic cancer cell line (MIA PaCa-2), but not in a cell line of non-cancerous origin (HEK293 cells). The basis for this differential effect is likely an open chromatin conformation within the H4 genes in cancer cells. Chromatin immunoprecipitation experiments show increased histone acetylation on the histone H4 genes in cancer cells, compared to HEK293 cells, explaining the differential activity of this molecule in cancer versus non-cancer cells.

  20. Trout red blood cell arrestin (TRCarr), a novel member of the arrestin family: cloning, immunoprecipitation and expression of recombinant TRCarr.

    PubMed

    Jahns, R; Borgese, F; Lindenthal, S; Straub, A; Motais, R; Fiévet, B

    1996-06-01

    Arrestins are cytosolic proteins involved in the desensitization of G-protein-coupled receptors. We report the cloning of trout red blood cell arrestin which shows 76, 82 and 52% identity with bovine beta-arrestin1, beta-arrestin2 and retinal arrestin respectively. Antibodies were generated against the C-terminus of trout red blood cell arrestin. These antibodies detected arrestin in erythrocyte cytosol and were able to precipitate the native protein. The Na+/H+ antiporter of trout red blood cell is activated by beta-adrenergic stimulation and is then desensitized whereas the transmembrane signalling pathway is not. To investigate the subcellular distribution of arrestin on beta-adrenergic activation and desensitization of the antiporter, precipitation experiments were carried out on trout erythrocytes. A desensitization-dependent shift in cytosolic arrestin to the membranes could not be detected using the immunoprecipitation technique but we cannot exclude the possibility that a small number of cytosolic arrestins might be involved in the regulation of membrane proteins in trout erythrocyte. Recombinant trout arrestin was produced in a protease-deficient Escherichia coli strain and its functionality was tested in a reconstituted rhodopsin assay. The recombinant protein provides a suitable tool for investigating the target for arrestin in trout red blood cell, which still remains to be identified.

  1. Multifunctionalization of cetuximab with bioorthogonal chemistries and parallel EGFR profiling of cell-lines using imaging, FACS and immunoprecipitation approaches.

    PubMed

    Reschke, Melanie L; Uprety, Rajendra; Bodhinayake, Imithri; Banu, Matei; Boockvar, John A; Sauve, Anthony A

    2014-12-01

    The ability to derivatize antibodies is currently limited by the chemical structure of antibodies as polypeptides. Modern methods of bioorthogonal and biocompatible chemical modifications could make antibody functionalization more predictable and easier, without compromising the functions of the antibody. To explore this concept, we modified the well-known anti-epidermal growth factor receptor (EGFR) drug, cetuximab (Erbitux®), with 5-azido-2-nitro-benzoyl (ANB) modifications by optimization of an acylation protocol. We then show that the resulting ANB-cetuximab can be reliably modified with dyes (TAMRA and carboxyrhodamine) or a novel synthesized cyclooctyne modified biotin. The resulting dye- and biotin-modified cetuximabs were then tested across several assay platforms with several cell lines including U87, LN229, F98EGFR, F98WT and HEK293 cells. The assay platforms included fluorescence microscopy, FACS and biotin-avidin based immunoprecipitation methods. The modified antibody performs consistently in all of these assay platforms, reliably determining relative abundances of EGFR expression on EGFR expressing cells (LN229 and F98EGFR) and failing to cross react with weak to negative EGFR expressing cells (U87, F98WT and HEK293). The ease of achieving diverse and assay relevant functionalizations as well as the consequent rapid construction of highly correlated antigen expression data sets highlights the power of bioorthogonal and biocompatible methods to conjugate macromolecules. These data provide a proof of concept for a multifunctionalization strategy that leverages the biochemical versatility and antigen specificity of antibodies.

  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.

  3. 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

  4. CTCF modulates Estrogen Receptor function through specific chromatin and nuclear matrix interactions

    PubMed Central

    Fiorito, Elisa; Sharma, Yogita; Gilfillan, Siv; Wang, Shixiong; Singh, Sachin Kumar; Satheesh, Somisetty V.; Katika, Madhumohan R.; Urbanucci, Alfonso; Thiede, Bernd; Mills, Ian G.; Hurtado, Antoni

    2016-01-01

    Enhancer regions and transcription start sites of estrogen-target regulated genes are connected by means of Estrogen Receptor long-range chromatin interactions. Yet, the complete molecular mechanisms controlling the transcriptional output of engaged enhancers and subsequent activation of coding genes remain elusive. Here, we report that CTCF binding to enhancer RNAs is enriched when breast cancer cells are stimulated with estrogen. CTCF binding to enhancer regions results in modulation of estrogen-induced gene transcription by preventing Estrogen Receptor chromatin binding and by hindering the formation of additional enhancer-promoter ER looping. Furthermore, the depletion of CTCF facilitates the expression of target genes associated with cell division and increases the rate of breast cancer cell proliferation. We have also uncovered a genomic network connecting loci enriched in cell cycle regulator genes to nuclear lamina that mediates the CTCF function. The nuclear lamina and chromatin interactions are regulated by estrogen-ER. We have observed that the chromatin loops formed when cells are treated with estrogen establish contacts with the nuclear lamina. Once there, the portion of CTCF associated with the nuclear lamina interacts with enhancer regions, limiting the formation of ER loops and the induction of genes present in the loop. Collectively, our results reveal an important, unanticipated interplay between CTCF and nuclear lamina to control the transcription of ER target genes, which has great implications in the rate of growth of breast cancer cells. PMID:27638884

  5. Changes in cyst's nuclear chromatin resulting after experimental manipulation of Taenia crassiceps mice infections: biological implications.

    PubMed

    Fragoso, Gladis; Bobes, Raul J; Espinoza, Bertha; Martínez, María Luisa; Pérez-Morales, Deyanira; Rosas, Gabriela; Sciutto, Edda; Laclette, Juan Pedro

    2012-04-01

    During some estimations of the nuclear DNA content, based on determinations propidium iodide (PI) binding through fluorocytometry for Taenia crassiceps cysticerci, significant variation in the results were found. This initial observation led to a series of experiments designed to explain the variation. These changes could be induced by the diameter of the needles in the syringes used for the mouse to mouse transfer of the cysts. Nuclei from cysts transferred through 27-gauge needles showed 30% less PI staining than those transferred through 21 gauge needles, after 2 months infections. Reduction in PI capture induced by 27-gauge needle was reversible when the cysts were maintained in their mice hosts during 5 months. Moreover, variation in PI binding to cysticercal DNA was also found when comparing parasites grown in male versus female mice. The use of agents that homogenize the chromatin structure during PI staining, allowed demonstrating that variation were entirely due to differences in the chromatin relaxation/compaction. Additional experiments demonstrated that the higher compaction is accompanied by a reduced ability of cysts to grow in the peritoneal cavity of BALB/cAnN mice. Furthermore, proteomic analysis also showed that these changes in chromatin relaxation/compaction resulted in different levels and patterns of protein expression. Our results strongly suggest that chromatin is involved in several well characterized phenomena of the T. crassiceps murine model, and open new avenues for a detailed approach to understand such a complex host-parasite relationship.

  6. Integrating and mining the chromatin landscape of cell-type specificity using self-organizing maps

    PubMed Central

    Mortazavi, Ali; Pepke, Shirley; Jansen, Camden; Marinov, Georgi K.; Ernst, Jason; Kellis, Manolis; Hardison, Ross C.; Myers, Richard M.; Wold, Barbara J.

    2013-01-01

    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. PMID:24170599

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. Chromatin-dependent regulation of RNA polymerases II and III activity throughout the transcription cycle.

    PubMed

    Jordán-Pla, Antonio; Gupta, Ishaan; de Miguel-Jiménez, Lola; Steinmetz, Lars M; Chávez, Sebastián; Pelechano, Vicent; Pérez-Ortín, José E

    2015-01-01

    The particular behaviour of eukaryotic RNA polymerases along different gene regions and amongst distinct gene functional groups is not totally understood. To cast light onto the alternative active or backtracking states of RNA polymerase II, we have quantitatively mapped active RNA polymerases at a high resolution following a new biotin-based genomic run-on (BioGRO) technique. Compared with conventional profiling with chromatin immunoprecipitation, the analysis of the BioGRO profiles in Saccharomyces cerevisiae shows that RNA polymerase II has unique activity profiles at both gene ends, which are highly dependent on positioned nucleosomes. This is the first demonstration of the in vivo influence of positioned nucleosomes on transcription elongation. The particular features at the 5' end and around the polyadenylation site indicate that this polymerase undergoes extensive specific-activity regulation in the initial and final transcription elongation phases. The genes encoding for ribosomal proteins show distinctive features at both ends. BioGRO also provides the first nascentome analysis for RNA polymerase III, which indicates that transcription of tRNA genes is poorly regulated at the individual copy level. The present study provides a novel perspective of the transcription cycle that incorporates inactivation/reactivation as an important aspect of RNA polymerase dynamics.

  13. Discovering hotspots in functional genomic data superposed on 3D chromatin configuration reconstructions

    PubMed Central

    Capurso, Daniel; Bengtsson, Henrik; Segal, Mark R.

    2016-01-01

    The spatial organization of the genome influences cellular function, notably gene regulation. Recent studies have assessed the three-dimensional (3D) co-localization of functional annotations (e.g. centromeres, long terminal repeats) using 3D genome reconstructions from Hi-C (genome-wide chromosome conformation capture) data; however, corresponding assessments for continuous functional genomic data (e.g. chromatin immunoprecipitation-sequencing (ChIP-seq) peak height) are lacking. Here, we demonstrate that applying bump hunting via the patient rule induction method (PRIM) to ChIP-seq data superposed on a Saccharomyces cerevisiae 3D genome reconstruction can discover ‘functional 3D hotspots’, regions in 3-space for which the mean ChIP-seq peak height is significantly elevated. For the transcription factor Swi6, the top hotspot by P-value contains MSB2 and ERG11 – known Swi6 target genes on different chromosomes. We verify this finding in a number of ways. First, this top hotspot is relatively stable under PRIM across parameter settings. Second, this hotspot is among the top hotspots by mean outcome identified by an alternative algorithm, k-Nearest Neighbor (k-NN) regression. Third, the distance between MSB2 and ERG11 is smaller than expected (by resampling) in two other 3D reconstructions generated via different normalization and reconstruction algorithms. This analytic approach can discover functional 3D hotspots and potentially reveal novel regulatory interactions. PMID:26869583

  14. A model for the importance of zinc in the dynamics of human sperm chromatin stabilization after ejaculation in relation to sperm DNA vulnerability.

    PubMed

    Björndahl, Lars; Kvist, Ulrik

    2011-02-01

    The focus of this review is the dual functions of the sperm chromatin stabilization and how external factors can interfere with these functions. Zinc depletion after ejaculation allows for rapid and total sperm chromatin decondensation without addition of exogenous disulfide cleaving agents. Zinc depletion without concomitant repulsion of chromatin fibers induces another type of stability that requires exogenous disulfide cleaving agents to allow decondensation. It is essential to extend the present concept, that the sperm chromatin stability is based on disulfide bridges only, to include also the functions of Zn(2+). It is suggested that the chromatin stability of the ejaculated human spermatozoon is rapidly reversible due to the dual function of Zn(2+) that stabilizes the structure and prevents the formation of excess disulfide bridges by a single mechanism: the formation of zinc bridges involving protamine thiols of cysteine and potentially also imidazole groups of histidine. Extraction of zinc from the freshly ejaculated spermatozoon allows two totally different biological results: (1) immediate decondensation if chromatin fibers concomitantly are induced to repel (e.g., through phosphorylation in the ooplasm) and (2) thiols freed from Zn(2+) are available to form disulfide bridges creating a superstabilized chromatin. Spermatozoa in the zinc rich prostatic fluid (in first ejaculated fraction) represent physiology. Extraction of chromatin zinc can be caused by unphysiological exposure of spermatozoa to the zinc chelating and oxidative seminal vesicular fluid, a situation common to most assisted reproductive techniques (ART) laboratories where the entire ejaculate is collected into a single container in which spermatozoa and secretions are mixed during at least 30 min. Some men in infertile couples have low content of sperm chromatin zinc due to loss of zinc during ejaculation and liquefaction. Tests for sperm DNA integrity may give false negative results due to

  15. 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

  16. Multiple modes of chromatin remodeling by Forkhead box proteins.

    PubMed

    Lalmansingh, Avin S; Karmakar, Sudipan; Jin, Yetao; Nagaich, Akhilesh K

    2012-07-01

    Forkhead box (FOX) proteins represent a large family of transcriptional regulators unified by their DNA binding domain (DBD) known as a 'forkhead' or 'winged helix' domain. Over 40 FOX genes have been identified in the mammalian genome. FOX proteins share significant sequence similarities in the DBD which allow them to bind to a consensus DNA response element. However, their modes of action are quite diverse as they regulate gene expression by acting as pioneer factors, transcription factors, or both. This review focuses on the mechanisms of chromatin remodeling with an emphasis on three sub-classes-FOXA, FOXO, and FOXP members. FOXA proteins serve as pioneer factors to open up local chromatin structure and thereby increase accessibility of chromatin to factors regulating transcription. FOXP proteins, in contrast, function as classic transcription factors to recruit a variety of chromatin modifying enzymes to regulate gene expression. FOXO proteins represent a hybrid subclass having dual roles as pioneering factors and transcription factors. A subset of FOX proteins interacts with condensed mitotic chromatin and may function as 'bookmarking' agents to maintain transcriptional competence at specific genomic sites. The overall diversity in chromatin remodeling function by FOX proteins is related to unique structural motifs present within the DBD flanking regions that govern selective interactions with core histones and/or chromatin coregulatory proteins. This article is part of a Special Issue entitled: Chromatin in time and space.

  17. A Quantitative Proteomic Analysis of In Vitro Assembled Chromatin*

    PubMed Central

    Völker-Albert, Moritz Carl; Pusch, Miriam Caroline; Fedisch, Andreas; Schilcher, Pierre; Schmidt, Andreas; Imhof, Axel

    2016-01-01

    The structure of chromatin is critical for many aspects of cellular physiology and is considered to be the primary medium to store epigenetic information. It is defined by the histone molecules that constitute the nucleosome, the positioning of the nucleosomes along the DNA and the non-histone proteins that associate with it. These factors help to establish and maintain a largely DNA sequence-independent but surprisingly stable structure. Chromatin is extensively disassembled and reassembled during DNA replication, repair, recombination or transcription in order to allow the necessary factors to gain access to their substrate. Despite such constant interference with chromatin structure, the epigenetic information is generally well maintained. Surprisingly, the mechanisms that coordinate chromatin assembly and ensure proper assembly are not particularly well understood. Here, we use label free quantitative mass spectrometry to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos. The use of a data independent acquisition method for proteome wide quantitation allows a time resolved comparison of in vitro chromatin assembly. A comparison of our in vitro data with proteomic studies of replicative chromatin assembly in vivo reveals an extensive overlap showing that the in vitro system can be used for investigating the kinetics of chromatin assembly in a proteome-wide manner. PMID:26811354

  18. 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

  19. Discovery of MLL1 binding units, their localization to CpG Islands, and their potential function in mitotic chromatin

    PubMed Central

    2013-01-01

    Background Mixed Lineage Leukemia 1 (MLL1) is a mammalian ortholog of the Drosophila Trithorax. In Drosophila, Trithorax complexes transmit the memory of active genes to daughter cells through interactions with Trithorax Response Elements (TREs). However, despite their functional importance, nothing is known about sequence features that may act as TREs in mammalian genomic DNA. Results By analyzing results of reported DNA binding assays, we identified several CpG rich motifs as potential MLL1 binding units (defined as morphemes). We find that these morphemes are dispersed within a relatively large collection of human promoter sequences and appear densely packed near transcription start sites of protein-coding genes. Genome wide analyses localized frequent morpheme occurrences to CpG islands. In the human HOX loci, the morphemes are spread across CpG islands and in some cases tail into the surrounding shores and shelves of the islands. By analyzing results of chromatin immunoprecipitation assays, we found a connection between morpheme occurrences, CpG islands, and chromatin segments reported to be associated with MLL1. Furthermore, we found a correspondence of reported MLL1-driven “bookmarked” regions in chromatin to frequent occurrences of MLL1 morphemes in CpG islands. Conclusion Our results implicate the MLL1 morphemes in sequence-features that define the mammalian TREs and provide a novel function for CpG islands. Apparently, our findings offer the first evidence for existence of potential TREs in mammalian genomic DNA and the first evidence for a connection between CpG islands and gene-bookmarking by MLL1 to transmit the memory of highly active genes during mitosis. Our results further suggest a role for overlapping morphemes in producing closely packed and multiple MLL1 binding events in genomic DNA so that MLL1 molecules could interact and reside simultaneously on extended potential transcriptional maintenance elements in human chromosomes to transmit the

  20. The chromatin regulatory code: Beyond a histone code

    NASA Astrophysics Data System (ADS)

    Lesne, A.

    2006-03-01

    In this commentary on the contribution by Arndt Benecke in this issue, I discuss why the notion of “chromatin code” introduced and elaborated in this paper is to be preferred to that of “histone code”. Speaking of a code as regards nucleosome conformation and histone tail post-translational modifications only makes sense within the chromatin fiber, where their physico-chemical features can be translated into regulatory programs at the genome level, by means of a complex, multi-level interplay with the fiber architecture and dynamics settled in the course of Evolution. In particular, this chromatin code presumably exploits allosteric transitions of the chromatin fiber. The chromatin structure dependence of its translation suggests two alternative modes of transcription initiation regulation, also proposed in the paper by A. Benecke in this issue for interpreting strikingly bimodal micro-array data.

  1. Transcription upregulation via force-induced direct stretching of chromatin

    NASA Astrophysics Data System (ADS)

    Tajik, Arash; Zhang, Yuejin; Wei, Fuxiang; Sun, Jian; Jia, Qiong; Zhou, Wenwen; Singh, Rishi; Khanna, Nimish; Belmont, Andrew S.; Wang, Ning

    2016-12-01

    Mechanical forces play critical roles in the function of living cells. However, the underlying mechanisms of how forces influence nuclear events remain elusive. Here, we show that chromatin deformation as well as force-induced transcription of a green fluorescent protein (GFP)-tagged bacterial-chromosome dihydrofolate reductase (DHFR) transgene can be visualized in a living cell by using three-dimensional magnetic twisting cytometry to apply local stresses on the cell surface via an Arg-Gly-Asp-coated magnetic bead. Chromatin stretching depended on loading direction. DHFR transcription upregulation was sensitive to load direction and proportional to the magnitude of chromatin stretching. Disrupting filamentous actin or inhibiting actomyosin contraction abrogated or attenuated force-induced DHFR transcription, whereas activating endogenous contraction upregulated force-induced DHFR transcription. Our findings suggest that local stresses applied to integrins propagate from the tensed actin cytoskeleton to the LINC complex and then through lamina-chromatin interactions to directly stretch chromatin and upregulate transcription.

  2. Chromatin dynamics during herpes simplex virus-1 lytic infection.

    PubMed

    Placek, Brandon J; Berger, Shelley L

    2010-01-01

    Herpes simplex virus type 1 is a DNA virus that can establish lytic infections in epithelial cells and latent infections in sensory neurons. Upon entry into the nucleus the genome of HSV-1 rapidly associates with histone proteins. Similar to the genomes of the cellular host, HSV-1 is subject to chromatin-based regulation of transcription and replication. However, unlike the host genome, nucleosomes appear to be underrepresented on the HSV genome. During lytic infection, when the genome is transcribed, the HSV-1 chromatin structure appears to be disorganized, and characterized by histone variant sub-types and post-translational modifications representative of active chromatin. In contrast, during latency, when the majority of the viral genome is transcriptionally silent, the chromatin is compacted into a regularly repeating, compact heterochromatic structure. Here we discuss recent studies that underscore the importance of chromatin regulation during the lytic phase of the HSV-1 life-cycle.

  3. 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.

  4. Diverse lamin-dependent mechanisms interact to control chromatin dynamics

    PubMed Central

    Camozzi, Daria; Capanni, Cristina; Cenni, Vittoria; Mattioli, Elisabetta; Columbaro, Marta; Squarzoni, Stefano; Lattanzi, Giovanna

    2014-01-01

    Interconnected functional strategies govern chromatin dynamics in eukaryotic cells. In this context, A and B type lamins, the nuclear intermediate filaments, act on diverse platforms involved in tissue homeostasis. On the nuclear side, lamins elicit large scale or fine chromatin conformational changes, affect DNA damage response factors and transcription factor shuttling. On the cytoplasmic side, bridging-molecules, the LINC complex, associate with lamins to coordinate chromatin dynamics with cytoskeleton and extra-cellular signals.   Consistent with such a fine tuning, lamin mutations and/or defects in their expression or post-translational processing, as well as mutations in lamin partner genes, cause a heterogeneous group of diseases known as laminopathies. They include muscular dystrophies, cardiomyopathy, lipodystrophies, neuropathies, and progeroid syndromes. The study of chromatin dynamics under pathological conditions, which is summarized in this review, is shedding light on the complex and fascinating role of the nuclear lamina in chromatin regulation. PMID:25482195

  5. DNA Damage Repair in the Context of Plant Chromatin1

    PubMed Central

    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

  6. 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.

  7. Chromatin insulators: lessons from the fly

    PubMed Central

    Gurudatta, B. V.

    2009-01-01

    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. PMID:19752045

  8. Chromatin dynamics in kidney development and function.

    PubMed

    Bechtel-Walz, Wibke; Huber, Tobias B

    2014-06-01

    Epigenetic mechanisms are fundamental key features of developing cells connecting developmental regulatory factors to chromatin modification. Changes in the environment during renal development can have long-lasting effects on the permanent tissue structure and the level of expression of important functional genes. These changes are believed to contribute to kidney disease occurrence and progression. Although the mechanisms of early patterning and cell fate have been well described for renal development, little is known about associated epigenetic modifications and their impact on how genes interact to specify the renal epithelial cells of nephrons and how this specification is relevant to maintaining normal renal function. A better understanding of the renal cell-specific epigenetic modifications and the interaction of different cell types to form this highly complex organ will not only help to better understand developmental defects and early loss of kidney function in children, but also help to understand and improve chronic disease progression, cell regeneration and renal aging.

  9. 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.

  10. Chromatin remodeling: from transcription to cancer.

    PubMed

    Yaniv, Moshe

    2014-09-01

    In this short review article, I have tried to trace the path that led my laboratory from the early studies of the structure of papova minichromosomes and transcription control to the investigation of chromatin remodeling complexes of the SWI/SNF family. I discuss briefly the genetic and biochemical studies that lead to the discovery of the SWI/SNF complex in yeast and drosophila and summarize some of the studies on the developmental role of the murine complex. The discovery of the tumor suppressor function of the SNF5/INI1/SMARCB1 gene in humans and the identification of frequent mutations in other subunits of this complex in different human tumors opened a fascinating field of research on this epigenetic regulator. The hope is to better understand tumor development and to develop novel treatments.

  11. 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

  12. Baseline Chromatin Modification Levels May Predict ...

    EPA Pesticide Factsheets

    Traditional toxicological paradigms have relied on factors such as age, genotype, and disease status to explain variability in responsiveness to toxicant exposure; however, these are neither sufficient to faithfully identify differentially responsive individuals nor are they modifiable factors that can be leveraged to mitigate the exposure effects. Unlike these factors, the epigenome is dynamic and shaped by an individual's environment. We sought to determine whether baseline levels of specific chromatin modifications correlated with the interindividual variability in their ozone (03)-mediated induction in an air-liquid interface model using primary human bronchial epithelial cells from a panel of 11 donors. We characterized the relationship between the baseline abundance of 6 epigenetic markers with established roles as key regulators of gene expression-histone H3 lysine 4 trimethylation (H3K4me3), H3K27 acetylation (H3K27ac), pan­acetyl H4 (H4ac), histone H3K27 di/trimethylation (H3K27me2/3), unmodified H3, and5-hydroxymethylcytosine (5-hmC)-and the variability in the 03-induced expression of IL-8, IL-6, COX2, and HMOX1. Baseline levels of H3K4me3, H3K27me2/3, and 5-hmC, but not H3K27ac, H4ac, and total H3, correlated with the interindividual variability in 03-mediated induction of HMOX1 and COX2. In contrast, none of the chromatin modifications that we examined correlated with the induction of IL-8 and IL-6. From these findings, we propose an "epigenetic see

  13. 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.

  14. Nanoscale changes in chromatin organization represent the initial steps of tumorigenesis: a transmission electron microscopy study

    PubMed Central

    2014-01-01

    Background Nuclear alterations are a well-known manifestation of cancer. However, little is known about the early, microscopically-undetectable stages of malignant transformation. Based on the phenomenon of field cancerization, the tissue in the field of a tumor can be used to identify and study the initiating events of carcinogenesis. Morphological changes in nuclear organization have been implicated in the field of colorectal cancer (CRC), and we hypothesize that characterization of chromatin alterations in the early stages of CRC will provide insight into cancer progression, as well as serve as a biomarker for early detection, risk stratification and prevention. Methods For this study we used transmission electron microscopy (TEM) images of nuclei harboring pre-neoplastic CRC alterations in two models: a carcinogen-treated animal model of early CRC, and microscopically normal-appearing tissue in the field of human CRC. We quantify the chromatin arrangement using approaches with two levels of complexity: 1) binary, where chromatin is separated into areas of dense heterochromatin and loose euchromatin, and 2) grey-scale, where the statistics of continuous mass-density distribution within the nucleus is quantified by its spatial correlation function. Results We established an increase in heterochromatin content and clump size, as well as a loss of its characteristic peripheral positioning in microscopically normal pre-neoplastic cell nuclei. Additionally, the analysis of chromatin density showed that its spatial distribution is altered from a fractal to a stretched exponential. Conclusions We characterize quantitatively and qualitatively the nanoscale structural alterations preceding cancer development, which may allow for the establishment of promising new biomarkers for cancer risk stratification and diagnosis. The findings of this study confirm that ultrastructural changes of chromatin in field carcinogenesis represent early neoplastic events leading to the

  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. Structure and Dynamic Properties of a Glucocorticoid Receptor-Induced Chromatin Transition

    PubMed Central

    Fletcher, Terace M.; Ryu, Byung-Woo; Baumann, Christopher T.; Warren, Barbour S.; Fragoso, Gilberto; John, Sam; Hager, Gordon L.

    2000-01-01

    Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). Recent evidence indicates that this transition extends upstream of the B nucleosome, encompassing a region larger than a single nucleosome (G. Fragoso, W. D. Pennie, S. John, and G. L. Hager, Mol. Cell. Biol. 18:3633–3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process; in the absence of ATP, GR binds to multiple sites on the chromatin array and prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a “hit-and-run” mechanism for GR action in living cells (J. G. McNally, W. G. Müller, D. Walker, and G. L. Hager, Science 287:1262–1264, 2000). PMID:10938123

  17. Effects of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions.

    PubMed Central

    Ahmed, K; Wilson, M J; Goueli, S A; Williams-Ashman, H G

    1978-01-01

    Studies are presented on the influence of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions involving both exogenous and endogenous substrates. The activities toward the model acidic protein substrate, dephosphophosvitin, were maximal at 160--200mM-NaCl (or -KCl or -NH4Cl). Under these conditions, spermidine and spermine added in concentrations up to 2mM were essentially without effect. However, without addition of NaCl to the medium, marked stimulation of these reactions was elicited by these polyamines at 1--2mM concentrations. The stimulatory effects were not due to non-specific changes in the ionic strength or to substitution of spermine for Mg2+, as maximal stimulation by 1 mM-spermine was observed only at optimal (2--4mM) Mg2+ concentrations. Qualitatively similar effects of polyamines were observed with enzyme preparations from the prostates of castrated rats, and with chromatin and non-histone-protein preparations from other tissues besides ventral prostate. When phosphorylation of endogenous non-histone proteins of the chromatin was measured, spermine stimulated both the initial rates and the final extent of transphosphorylation, even in the presence of optimal concentration of NaCl. By contrast, spermine or spermidine had no effect on the chromatin- and non-histone-protein-associated protein kinase reactions determined with lysine-rich histones as substrates. Chemically NN-dimethylated dephosphophosvitin was a less active substrate for the chromatin-associated protein kinase, but its phosphorylation was more markedly stimulated by spermine in comparison with unmodified dephosphophosvitin. These observations hint that the polyamine stimulations of the various protein kinase reactions may be due to effects on the conformations of the non-histone protein substrates rather than on the kinases themselves. PMID:747650

  18. Analysis of antibody responses to Hymenolepis nana infection in mice by the enzyme-linked immunosorbent assay and immunoprecipitation.

    PubMed

    Ito, A; Honey, R D; Scanlon, T; Lightowlers, M W; Rickard, M D

    1988-05-01

    Serum antibody responses in two strains of mice infected with embryonated eggs of Hymenolepis nana were analysed by the enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) using sodium deoxycholate (DOC)-solubilized antigens prepared from embryonated eggs (eggs), mouse-derived cysticercoids (cysts) and adult tapeworms with immature segments only (adults). Highly susceptible dd mice, which harbour mature tapeworms for a long period (greater than 70 days), produced high levels of antibodies to all three different stages of H. nana. BALB/c mice, almost all of which expel adult tapeworms by 30 days after infection, produced high levels of antibody against egg antigens only. The high antibody titres to cyst and adult antigens in dd mice did not lead to expulsion of the worms. However, worms are rejected early in BALB/c mice when there is little or no detectable serum antibody. The antibody responses to eggs seen in BALB/c mice which had long since shed their adult worms were probably due to ingestion of eggs from faeces of other infected mice. Antibodies to eggs were not detected in BALB/c mice which were initially inoculated with eggs (day 0) and then treated with praziquantel on day 6 after the tissue phase of infection only. The different antibody responses to egg antigens and the other two antigens (cyst and adult) in BALB/c mice suggest a difference in antigen specificity between eggs and both cysts and adults. A major antigen component with Mr 32,000 appears to be specific to the egg (or oncosphere) stage of H. nana. Antibody to this major component of eggs was absorbed only with intact eggs, but not with intact cysts nor adults with immature segments only, so that the antigen appears to be on the surface of the oncosphere.

  19. 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.

  20. Defining the multivalent functions of CTCF from chromatin state and three-dimensional chromatin interactions.

    PubMed

    Lu, Yiming; Shan, Guangyu; Xue, Jiguo; Chen, Changsheng; Zhang, Chenggang

    2016-07-27

    CCCTC-binding factor (CTCF) is a multi-functional protein that is assigned various, even contradictory roles in the genome. High-throughput sequencing-based technologies such as ChIP-seq and Hi-C provided us the opportunity to assess the multivalent functions of CTCF in the human genome. The location of CTCF-binding sites with respect to genomic features provides insights into the possible roles of this protein. Here we present the first genome-wide survey and characterization of three important functions of CTCF: enhancer insulator, chromatin barrier and enhancer linker. We developed a novel computational framework to discover the multivalent functions of CTCF based on chromatin state and three-dimensional chromatin architecture. We applied our method to five human cell lines and identified ∼46 000 non-redundant CTCF sites related to the three functions. Disparate effects of these functions on gene expression were found and distinct genomic features of these CTCF sites were characterized in GM12878 cells. Finally, we investigated the cell-type specificities of CTCF sites related to these functions across five cell types. Our study provides new insights into the multivalent functions of CTCF in the human genome.

  1. Persistent Chromatin Modifications Induced by High Fat Diet.

    PubMed

    Leung, Amy; Trac, Candi; Du, Juan; Natarajan, Rama; Schones, Dustin E

    2016-05-13

    Obesity is a highly heritable complex disease that results from the interaction of multiple genetic and environmental factors. Formerly obese individuals are susceptible to metabolic disorders later in life, even after lifestyle changes are made to mitigate the obese state. This is reminiscent of the metabolic memory phenomenon originally observed for persistent complications in diabetic patients, despite subsequent glycemic control. Epigenetic modifications represent a potential mediator of this observed memory. We previously demonstrated that a high fat diet leads to changes in chromatin accessibility in the mouse liver. The regions of greatest chromatin changes in accessibility are largely strain-dependent, indicating a genetic component in diet-induced chromatin alterations. We have now examined the persistence of diet-induced chromatin accessibility changes upon diet reversal in two strains of mice. We find that a substantial fraction of loci that undergo chromatin accessibility changes with a high fat diet remains in the remodeled state after diet reversal in C57BL/6J mice. In contrast, the vast majority of diet-induced chromatin accessibility changes in A/J mice are transient. Our data also indicate that the persistent chromatin accessibility changes observed in C57BL/6J mice are associated with specific transcription factors and histone post-translational modifications. The persistent loci identified here are likely to be contributing to the overall phenotype and are attractive targets for therapeutic intervention.

  2. Fractal Characterization of Chromatin Decompaction in Live Cells

    PubMed Central

    Yi, Ji; Stypula-Cyrus, Yolanda; Blaha, Catherine S.; Roy, Hemant K.; Backman, Vadim

    2015-01-01

    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. PMID:26636933

  3. Distal chromatin structure influences local nucleosome positions and gene expression.

    PubMed

    Jansen, An; van der Zande, Elisa; Meert, Wim; Fink, Gerald R; Verstrepen, Kevin J

    2012-05-01

    The positions of nucleosomes across the genome influence several cellular processes, including gene transcription. However, our understanding of the factors dictating where nucleosomes are located and how this affects gene regulation is still limited. Here, we perform an extensive in vivo study to investigate the influence of the neighboring chromatin structure on local nucleosome positioning and gene expression. Using truncated versions of the Saccharomyces cerevisiae URA3 gene, we show that nucleosome positions in the URA3 promoter are at least partly determined by the local DNA sequence, with so-called 'anti-nucleosomal elements' like poly(dA:dT) tracts being key determinants of nucleosome positions. In addition, we show that changes in the nucleosome positions in the URA3 promoter strongly affect the promoter activity. Most interestingly, in addition to demonstrating the effect of the local DNA sequence, our study provides novel in vivo evidence that nucleosome positions are also affected by the position of neighboring nucleosomes. Nucleosome structure may therefore be an important selective force for conservation of gene order on a chromosome, because relocating a gene to another genomic position (where the positions of neighboring nucleosomes are different from the original locus) can have dramatic consequences for the gene's nucleosome structure and thus its expression.

  4. 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.

  5. DNA packing in chromatine, a manifestation of the Bonnet transformation.

    PubMed

    Blum, Z; Lidin, S

    1988-08-01

    The packing of DNA is described using the formalism of differential geometry. Winding of the DNA double helix around the histone 2-5 octamer forming a nucleosome and the condensation of the so-formed bead-on-a-string chromatine aided by histone 1 is interpreted as two consecutive isometric, i.e. Bonnet, transformations. The DNA double helix can be approximated to a helicoid which can be transformed isometrically to a catenoid, an approximation of the nucleosome. Owing to the organization of the histone octamer the extended chromatine takes a helicoidal shape allowing a second Bonnet transformation to consummate the condensation into a chromatine fibre.

  6. Post-translational modifications of the intrinsically disordered terminal domains of histone H1: effects on secondary structure and chromatin dynamics.

    PubMed

    Roque, A; Ponte, I; Suau, P

    2016-04-21

    H1 linker histones are involved both in the maintenance of chromatin higher-order structure and in gene regulation. H1 binds to linker DNA regions on the surface of the nucleosome. In higher eukaryotes, H1 contains three distinct domains: a short N-terminal domain (NTD), a central globular domain, and a long C-terminal domain (CTD). Terminal domains determine subtype specificity and to a large extent the linker DNA binding and chromatin condensing properties of histone H1. This review is focused on the recent numerous studies that have provided insights in the role of H1 terminal domains in chromatin dynamics. The N- and C-terminal domains behave as intrinsically disordered proteins with coupled binding and folding. We examine the potential kinetic advantages of intrinsic disorder in the recognition of the specific H1 binding sites in chromatin. As typical intrinsically disordered regions, H1 terminal domains are post-translationally modified. Post-translational modifications in the NTD determine the interaction of histone H1 with other proteins involved in heterochromatin formation and transcriptional regulation, while phosphorylation by cyclin-dependent kinases modulates the secondary structure of the CTD and chromatin condensation. We review the arguments in favor of the involvement of H1 hyperphosphorylation in metaphase chromatin condensation and of partial phosphorylation in interphase chromatin relaxation. In addition, the interplay of histone H1 and other chromatin architectural proteins, such as proteins of the high-mobility group, protamines, and MeCP2, is associated with changes in chromatin structure.

  7. Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder

    PubMed Central

    Gui, Yaoting; Guo, Guangwu; Huang, Yi; Hu, Xueda; Tang, Aifa; Gao, Shengjie; Wu, Renhua; Chen, Chao; Li, Xianxin; Zhou, Liang; He, Minghui; Li, Zesong; Sun, Xiaojuan; Jia, Wenlong; Chen, Jinnong; Yang, Shangming; Zhou, Fangjian; Zhao, Xiaokun; Wan, Shengqing; Ye, Rui; Liang, Chaozhao; Liu, Zhisheng; Huang, Peide; Liu, Chunxiao; Jiang, Hui; Wang, Yong; Zheng, Hancheng; Sun, Liang; Liu, Xingwang; Jiang, Zhimao; Feng, Dafei; Chen, Jing; Wu, Song; Zou, Jing; Zhang, Zhongfu; Yang, Ruilin; Zhao, Jun; Xu, Congjie; Yin, Weihua; Guan, Zhichen; Ye, Jiongxian; Zhang, Hong; Li, Jingxiang; Kristiansen, Karsten; Nickerson, Michael L; Theodorescu, Dan; Li, Yingrui; Zhang, Xiuqing; Li, Songgang; Wang, Jian; Yang, Huanming; Wang, Jun; Cai, Zhiming

    2017-01-01

    Transitional cell carcinoma (TCC) is the most common type of bladder cancer. Here we sequenced the exomes of nine individuals with TCC and screened all the somatically mutated genes in a prevalence set of 88 additional individuals with TCC with different tumor stages and grades. In our study, we discovered a variety of genes previously unknown to be mutated in TCC. Notably, we identified genetic aberrations of the chromatin remodeling genes (UTX, MLL-MLL3, CREBBP-EP300, NCOR1, ARID1A and CHD6) in 59% of our 97 subjects with TCC. Of these genes, we showed UTX to be altered substantially more frequently in tumors of low stages and grades, highlighting its potential role in the classification and diagnosis of bladder cancer. Our results provide an overview of the genetic basis of TCC and suggest that aberration of chromatin regulation might be a hallmark of bladder cancer. PMID:21822268

  8. Nucleosome arrangement in alpha-satellite chromatin of African green monkey cells.

    PubMed Central

    Smith, M R; Lieberman, M W

    1984-01-01

    By analyzing the accessibility of restriction endonuclease sites in African green monkey alpha-satellite chromatin, we demonstrate the absence of a unique phase relationship between nucleosomes and alpha-satellite DNA. The data indicate a minimum of three different positions for nucleosome cores relative to the alpha-satellite sequence and suggest a random distribution in at least some regions. In addition, while we confirm published reports that staphylococcal nuclease cuts the alpha-satellite sequence in chromatin at a highly preferred site, two-dimensional gel electrophoresis of nuclear digests demonstrates that this site is preferentially cut by staphylococcal nuclease even when it is within the nucleosome core. These data indicate that staphylococcal nuclease is not useful for determining nucleosome positions on alpha-satellite DNA, and perhaps on other specific DNA sequences as well. Images PMID:6089117

  9. 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

  10. Long range epigenetic silencing is a trans-species mechanism that results in cancer specific deregulation by overriding the chromatin domains of normal cells.

    PubMed

    Forn, Marta; Muñoz, Mar; Tauriello, Daniele V F; Merlos-Suárez, Anna; Rodilla, Verónica; Bigas, Anna; Batlle, Eduard; Jordà, Mireia; Peinado, Miguel A

    2013-12-01

    DNA methylation and chromatin remodeling are frequently implicated in the silencing of genes involved in carcinogenesis. Long Range Epigenetic Silencing (LRES) is a mechanism of gene inactivation that affects multiple contiguous CpG islands and has been described in different human cancer types. However, it is unknown whether there is a coordinated regulation of the genes embedded in these regions in normal cells and in early stages of tumor progression. To better characterize the molecular events associated with the regulation and remodeling of these regions we analyzed two regions undergoing LRES in human colon cancer in the mouse model. We demonstrate that LRES also occurs in murine cancer in vivo and mimics the molecular features of the human phenomenon, namely, downregulation of gene expression, acquisition of inactive histone marks, and DNA hypermethylation of specific CpG islands. The genes embedded in these regions showed a dynamic and autonomous regulation during mouse intestinal cell differentiation, indicating that, in the framework considered here, the coordinated regulation in LRES is restricted to cancer. Unexpectedly, benign adenomas in Apc(Min/+) mice showed overexpression of most of the genes affected by LRES in cancer, which suggests that the repressive remodeling of the region is a late event. Chromatin immunoprecipitation analysis of the transcriptional insulator CTCF in mouse colon cancer cells revealed disrupted chromatin domain boundaries as compared with normal cells. Malignant regression of cancer cells by in vitro differentiation resulted in partial reversion of LRES and gain of CTCF binding. We conclude that genes in LRES regions are plastically regulated in cell differentiation and hyperproliferation, but are constrained to a coordinated repression by abolishing boundaries and the autonomous regulation of chromatin domains in cancer cells.

  11. 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.

  12. Replication forks, chromatin loops and dormant replication origins.

    PubMed

    Blow, J Julian; Ge, Xin Quan

    2008-01-01

    When DNA replication is slowed down, normally dormant replication origins are activated. Recent work demonstrates that cells adapt by changing the organization of chromatin loops and maintaining the new pattern of origin use in subsequent cell cycles.

  13. Chromatin remodeling in DNA double-strand break repair.

    PubMed

    Bao, Yunhe; Shen, Xuetong

    2007-04-01

    ATP-dependent chromatin remodeling complexes use ATP hydrolysis to remodel nucleosomes and have well-established functions in transcription. However, emerging lines of evidence suggest that chromatin remodeling complexes are important players in DNA double-strand break (DSB) repair as well. The INO80 and SWI2 subfamilies of chromatin remodeling complexes have been found to be recruited to the double-strand lesions and to function directly in both homologous recombination and non-homologous end-joining, the two major conserved DSB repair pathways. Improperly repaired DSBs are implicated in cancer development in higher organisms. Understanding how chromatin remodeling complexes contribute to DSB repair should provide new insights into the mechanisms of carcinogenesis and might suggest new targets for cancer treatment.

  14. 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.

  15. Histone variants and chromatin assembly in plant abiotic stress responses.

    PubMed

    Zhu, Yan; Dong, Aiwu; Shen, Wen-Hui

    2013-01-01

    Genome organization into nucleosomes and higher-order chromatin structures has profound implications for the regulation of gene expression, DNA replication and repair. The structure of chromatin can be remodeled by several mechanisms; among others, nucleosome assembly/disassembly and replacement of canonical histones with histone variants constitute important ones. In this review, we provide a brief description on the current knowledge about histone chaperones involved in nucleosome assembly/disassembly and histone variants in Arabidopsis thaliana. We discuss recent advances in revealing crucial functions of histone chaperones, nucleosome assembly/disassembly and histone variants in plant response to abiotic stresses. It appears that chromatin structure remodeling may provide a flexible, global and stable means for the regulation of gene transcription to help plants more effectively cope with environmental stresses. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.

  16. Cohesin organizes chromatin loops at DNA replication factories

    PubMed Central

    Guillou, Emmanuelle; Ibarra, Arkaitz; Coulon, Vincent; Casado-Vela, Juan; Rico, Daniel; Casal, Ignacio; Schwob, Etienne; Losada, Ana; Méndez, Juan

    2010-01-01

    Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication. PMID:21159821

  17. Regulation of meiotic chromatin loop size by chromosomal position.

    PubMed Central

    Heng, H H; Chamberlain, J W; Shi, X M; Spyropoulos, B; Tsui, L C; Moens, P B

    1996-01-01

    At meiotic prophase, chromatin loops around a proteinaceous core, with the sizes of these loops varying between species. Comparison of the morphology of sequence-related inserts at different sites in transgenic mice demonstrates that loop size also varies with chromosomal geography. Similarly, chromatin loop lengths differ dramatically for interstitially and terminally located hamster telomeric sequences. Sequences, telomeric or otherwise, located at chromosome termini, closely associate with the meiotic proteinaceous core, forming shorter loops than identical interstitial sequences. Thus, we present evidence that different chromatin packaging mechanisms exist for interstitial versus terminal chromosomal regions, which act separately from those operating at the level of the DNA sequence. Chromosomal position plays the dominant role in chromatin packaging. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8610120

  18. Nuclear morphometry and chromatin textural characteristics of basal cell carcinoma.

    PubMed

    Mendaçolli, Paola Jung; Brianezi, Gabrielli; Schmitt, Juliano Vilaverde; Marques, Mariângela Esther Alencar; Miot, Hélio Amante

    2015-01-01

    Histological subtypes of basal cell carcinoma have biological, evolutionary and distinct prognostic behavior. The analysis of characteristics of the nucleus can provide data on their cellular physiology and behavior. The authors of this study evaluated nuclear morphological parameters and textural patterns of chromatin from different subtypes of basal cell carcinoma: nodular (n=37), superficial (n=28) and sclerodermiform (n=28). The parameters were compared between neoplasms' subtypes and with unaffected adjacent basal epithelium. Nuclear area and diameter of sclerodermiform neoplasms were superior to the other subtypes. Chromatin's color intensity and fractal dimension were less intense in superficial subtypes. Nuclear roundness and chromatin's entropy presented lower values in tumors than in normal epithelium. There was significant correlation between morphological and textural variables of normal skin and tumors. Morphometric elements and textural chromatin's homogeneity of basal cell carcinomas may be related to evolutionary, biological and behavior particularities related to each histotype.

  19. Roles and activities of chromatin remodeling ATPases in plants.

    PubMed

    Han, Soon-Ki; Wu, Miin-Feng; Cui, Sujuan; Wagner, Doris

    2015-07-01

    Chromatin remodeling ATPases and their associated complexes can alter the accessibility of the genome in the context of chromatin by using energy derived from the hydrolysis of ATP to change the positioning, occupancy and composition of nucleosomes. In animals and plants, these remodelers have been implicated in diverse processes ranging from stem cell maintenance and differentiation to developmental phase transitions and stress responses. Detailed investigation of their roles in individual processes has suggested a higher level of selectivity of chromatin remodeling ATPase activity than previously anticipated, and diverse mechanisms have been uncovered that can contribute to the selectivity. This review summarizes recent advances in understanding the roles and activities of chromatin remodeling ATPases in plants.

  20. 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.

  1. Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src.

    PubMed Central

    Modderman, P W; von dem Borne, A E; Sonnenberg, A

    1994-01-01

    P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well. However, tyrosine phosphorylation of P-selectin was greatly stimulated by treatment with the permeating phosphatase inhibitor, pervanadate. When P-selectin immunoprecipitates were incubated with [gamma-32P]ATP (in vitro kinase assay), a fraction of P-selectin was phosphorylated on its tyrosine residues by a co-precipitated kinase. P-selectin phosphorylated in vitro co-migrated with 140 kDa surface-labelled 125I-P-selectin during SDS/PAGE under reducing conditions. Under non-reducing conditions, however, phosphorylated P-selectin was disulphide-linked to unknown protein(s) in a 205 kDa complex. In vitro kinase assays of the most abundant platelet tyrosine kinase, pp60c-src, demonstrated the presence of similar 140 and 205 kDa phosphorylated proteins in SDS/PAGE under reducing and non-reducing conditions respectively. Extraction and reprecipitation studies with proteins phosphorylated in vitro indicated that P-selectin and pp60c-src form a 205 kDa 1:1 disulphide-linked complex. In the complex, pp60c-src autophosphorylation is inhibited and P-selectin is phosphorylated on tyrosine residues. As protein disulphides in the cytoplasm of intact cells are extremely rare, our results suggest that P-selectin and pp60c-src, which co-localize in platelet dense granules, may be non-covalently associated and spontaneously form disulphide bridges during lysis. In addition, the observed tyrosine phosphorylation of P-selectin in intact platelets suggests that its function might be regulated by phosphorylation by pp60c-src. Images Figure 1 Figure 2 Figure 3

  2. HMG Nuclear Proteins: Linking Chromatin Structure to Cellular Phenotype

    PubMed Central

    Reeves, Raymond

    2009-01-01

    I. Summary Although the three families of mammalian HMG proteins (HMGA, HMGB and HMGN) participate in many of the same nuclear processes, each family plays its own unique role in modulating chromatin structure and regulating genomic function. This review focuses on the similarities and differences in the mechanisms by which the different HMG families impact chromatin structure and influence cellular phenotype. The biological implications of having three architectural transcription factor families with complementary, but partially overlapping, nuclear functions are discussed. PMID:19748605

  3. Chromatin Insulators: Linking genome organization to cellular function

    PubMed Central

    Phillips-Cremins, Jennifer E.; Corces, Victor G.

    2013-01-01

    A growing body of evidence suggests that insulators have a primary role in orchestrating the topological arrangement of higher-order chromatin architecture. Insulator-mediated long-range interactions can influence the epigenetic status of the genome and, in certain contexts, may have important effects on gene expression. Here we discuss higher-order chromatin organization as a unifying mechanism for diverse insulator actions across the genome. PMID:23706817

  4. Osterix and NO66 histone demethylase control the chromatin of Osterix target genes during osteoblast differentiation.

    PubMed

    Sinha, Krishna M; Yasuda, Hideyo; Zhou, Xin; deCrombrugghe, Benoit

    2014-04-01

    Commitment of Runx2-expressing precursor osteoblasts to functional osteoblasts and then to osteocytes is triggered by Osterix (Osx), which activates its target genes in those cells during bone formation. It is not yet known whether Osx has a role in remodeling the chromatin architecture of its target genes during the transition from preosteoblast to osteoblast. In testing the hypothesis that Osx is indispensable for active chromatin architecture, we first showed that in Osx-null calvarial cells occupancy of the transcriptional activators, including lysine 4 methyl transferase (Wdr5), c-Myc, and H2A.Z, at the Osx target gene Bsp was very markedly decreased. The levels of methylation of lysines 4 and 36 and acetylation of histone H3, markers for active chromatin, were also reduced at the Bsp gene in these cells. In contrast, occupancy of the transcriptional repressors HP1 and the nucleolar protein 66 (NO66), a histone demethylase previously identified as an Osx-interacting protein, was increased at the Bsp gene in Osx-null calvarial cells. Furthermore, the Bsp promoter was hypermethylated in embryonic stem (ES) cells and in embryonic day 9.5 (E9.5) embryos but was markedly hypomethylated in the calvaria of E18.5 embryos, coinciding with robust Bsp expression. In contrast, CpG methylation in the Bsp promoter remained high in Osx-null calvaria compared to Osx-wild-type calvaria. Our data also revealed that NO66 interacted with DNA Methyltransferase 1A (DNMT1A), histone deacetylase 1A (HDAC1A), and HP1, which are known to control histone and DNA methylation. In addition, HP1 stimulated the demethylase activity of NO66 for its substrates "trimethylation of histone H3 at lysine 4" (H3K4me3) and "trimethylation of histone H3 at lysine 36" (H3K36me3). Our findings strongly suggest that in the absence of Osx, the chromatin of Osx target genes is transcriptionally inactive. We propose that Osx is a molecular switch for the formation of an active chromatin state during

  5. Higher chromatin mobility supports totipotency and precedes pluripotency in vivo.

    PubMed

    Bošković, Ana; Eid, André; Pontabry, Julien; Ishiuchi, Takashi; Spiegelhalter, Coralie; Raghu Ram, Edupuganti V S; Meshorer, Eran; Torres-Padilla, Maria-Elena

    2014-05-15

    The fusion of the gametes upon fertilization results in the formation of a totipotent cell. Embryonic chromatin is expected to be able to support a large degree of plasticity. However, whether this plasticity relies on a particular conformation of the embryonic chromatin is unknown. Moreover, whether chromatin plasticity is functionally linked to cellular potency has not been addressed. Here, we adapted fluorescence recovery after photobleaching (FRAP) in the developing mouse embryo and show that mobility of the core histones H2A, H3.1, and H3.2 is unusually high in two-cell stage embryos and decreases as development proceeds. The transition toward pluripotency is accompanied by a decrease in histone mobility, and, upon lineage allocation, pluripotent cells retain higher mobility than the differentiated trophectoderm. Importantly, totipotent two-cell-like embryonic stem cells also display high core histone mobility, implying that reprogramming toward totipotency entails changes in chromatin mobility. Our data suggest that changes in chromatin dynamics underlie the transitions in cellular plasticity and that higher chromatin mobility is at the nuclear foundations of totipotency.

  6. A map of open chromatin in human pancreatic islets

    PubMed Central

    Gaulton, Kyle J.; Nammo, Takao; Pasquali, Lorenzo; Simon, Jeremy M.; Giresi, Paul G.; Fogarty, Marie P.; Panhuis, Tami M.; Mieczkowski, Piotr; Secchi, Antonio; Bosco, Domenico; Berney, Thierry; Montanya, Eduard; Mohlke, Karen L.; Lieb, Jason D.; Ferrer, Jorge

    2010-01-01

    Tissue-specific transcriptional regulation is central to human disease1. To identify regulatory DNA active in human pancreatic islets, we profiled chromatin by FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements)2–4 coupled with high-throughput sequencing. We identified ~80,000 open chromatin sites. Comparison of islet FAIRE-seq to five non-islet cell lines revealed ~3,300 physically linked clusters of islet-selective open chromatin sites, which typically encompassed single genes exhibiting islet-specific expression. We mapped sequence variants to open chromatin sites and found that rs7903146, a TCF7L2 intronic variant strongly associated with type 2 diabetes (T2D)5, is located in islet-selective open chromatin. We show that rs7903146 heterozygotes exhibit allelic imbalance in islet FAIRE signal, and that the variant alters enhancer activity, indicating that genetic variation at this locus acts in cis with local chromatin and regulatory changes. These findings illuminate the tissue-specific organization of cis-regulatory elements, and show that FAIRE-seq can guide identification of regulatory variants important for disease. PMID:20118932

  7. Isolation and Proteomics Analysis of Barley Centromeric Chromatin Using PICh.

    PubMed

    Zeng, Zixian; Jiang, Jiming

    2016-06-03

    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.

  8. A WD-Repeat Protein Stabilizes ORC Binding to Chromatin

    PubMed Central

    Shen, Zhen; Sathyan, Kizhakke M.; Geng, Yijie; Zheng, Ruiping; Chakraborty, Arindam; Freeman, Brian; Wang, Fei; Prasanth, Kannanganattu V.; Prasanth, Supriya G.

    2015-01-01

    SUMMARY Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells. PMID:20932478

  9. Brain neuronal chromatin responses in acute soman intoxicated rats.

    PubMed

    Martin, L J; Doebler, J A; Wall, T J; Shih, T M; Anthony, A

    1986-08-01

    Male Sprague-Dawley rats (200 g) were injected subcutaneously with soman, a potent neuronal acetylcholinesterase (AChE) inhibitor, at doses of 0.5, 0.8 and 1.0 LD50 (1 LD50 = 135 micrograms/kg) before decapitation at 1 and 24 h post-exposure. Correlative data were obtained on the severity of brain AChE inactivation and physicochemical changes in nuclear chromatin of cerebrocortical (layer V) and striatal neurons using Feulgen-DNA (F-DNA) cytophotometry and ocular filar micrometry. Decreased lability of neurons to F-DNA acid hydrolysis (reduced F-DNA yield), nuclear shrinkage and chromatin aggregation (decreased chromophore area) were used as indices of suppression of genomic template activity; conversely, increases in F-DNA yield and chromophore area signify enhanced neuroexcitation. At 1 hr post-soman there was a dose-dependent inactivation of AChE with a moderate increase in chromatin activation, i.e., nuclear hypertrophy and chromatin dispersion. At 24 hr post-soman there was a partial restoration of AChE activity, notably in striatal neurons, with a suppression in chromatin template activity. These data indicate that actions of soman on neuronal functioning are time-dependent. The absence of any dose-related neuronal chromatin changes may signify existence of non-cholinergic mediated events.

  10. 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.

  11. 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

  12. Chd5 orchestrates chromatin remodeling during sperm development

    PubMed Central

    Li, Wangzhi; Wu, Jie; Kim, Sang-Yong; Zhao, Ming; Hearn, Stephen A.; Zhang, Michael Q.; Meistrich, Marvin L.

    2014-01-01

    One of the most remarkable chromatin remodeling processes occurs during spermiogenesis, the post-meiotic phase of sperm development during which histones are replaced with sperm-specific protamines to repackage the genome into the highly compact chromatin structure of mature sperm. Here we identify Chromodomain helicase DNA binding protein 5 (Chd5) as a master regulator of the histone-to-protamine chromatin remodeling process. Chd5 deficiency leads to defective sperm chromatin compaction and male infertility in mice, mirroring the observation of low CHD5 expression in testes of infertile men. Chd5 orchestrates a cascade of molecular events required for histone removal and replacement, including histone 4 (H4) hyperacetylation, histone variant expression, nucleosome eviction, and DNA damage repair. Chd5 deficiency also perturbs expression of transition proteins (Tnp1/Tnp2) and protamines (Prm1/2). These findings define Chd5 as a multi-faceted mediator of histone-to-protamine replacement and depict the cascade of molecular events underlying chromatin remodeling during this process of extensive chromatin remodeling. PMID:24818823

  13. Chromatin associated SETD3 negatively regulates VEGF expression

    PubMed Central

    Cohn, Ofir; Feldman, Michal; Weil, Lital; Kublanovsky, Margarita; Levy, Dan

    2016-01-01

    SETD3 is a member of the protein lysine methyltransferase (PKMT) family, which catalyzes the addition of methyl group to lysine residues. Accumulating data suggest that PKMTs are involved in the regulation of a broad spectrum of biological processes by targeting histone and non-histone proteins. Using a proteomic approach, we have identified 172 new SETD3 interacting proteins. We show that SETD3 binds and methylates the transcription factor FoxM1, which has been previously shown to be associated with the regulation of VEGF expression. We further demonstrate that under hypoxic conditions SETD3 is down-regulated. Mechanistically, we find that under basal conditions, SETD3 and FoxM1 are enriched on the VEGF promoter. Dissociation of both SETD3 and FoxM1 from the VEGF promoter under hypoxia correlates with elevated expression of VEGF. Taken together, our data reveal a new SETD3-dependent methylation-based signaling pathway at chromatin that regulates VEGF expression under normoxic and hypoxic conditions. PMID:27845446

  14. DAnCER: disease-annotated chromatin epigenetics resource.

    PubMed

    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/.

  15. ChIP-seq profiling of the active chromatin marker H3K4me3 and PPARγ, CEBPα and LXR target genes in human SGBS adipocytes

    PubMed Central

    Galhardo, Mafalda; Sinkkonen, Lasse; Berninger, Philipp; Lin, Jake; Sauter, Thomas; Heinäniemi, Merja

    2014-01-01

    Transcription factors (TFs) represent key factors to establish a cellular phenotype. It is known that several TFs could play a role in disease, yet less is known so far how their targets overlap. We focused here on identifying the most highly induced TFs and their putative targets during human adipogenesis. Applying chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) in the human SGBS pre-adipocyte cell line, we identified genes with binding sites in their vicinity for the three TFs studied, PPARγ, CEBPα and LXR. Here we describe the experimental design and quality controls in detail for the deep sequencing data and related results published by Galhardo et al. in Nucleic Acids Research 2014 [1] associated with the data uploaded to NCBI Gene Expression Omnibus (GSE41578). PMID:26484099

  16. Structural differences in the chromatin from compartmentalized cells of the sea urchin embryo: differential nuclease accessibility of micromere chromatin.

    PubMed Central

    Cognetti, G; Shaw, B R

    1981-01-01

    The chromatin structure of three cell types isolated from the 16-cell stage sea urchin embryo has been probed with micrococcal nuclease. In micromeres, the four small cells at the vegetal pole, the chromatin is found to be considerably more resistant to degradation by micrococcal nuclease than chromatin in the larger mesomere and macromere cells which undergo more cellular divisions and are committed to different developmental fates. The micromeres show an order of magnitude decrease in the initial digestion rate and a limit digest value which is one third that of the larger blastomeres; both observations are suggestive of the formation of a more condensed chromatin structure during the process of commitment, or as the rate of cell division decreases. The decreased sensitivity to nuclease for micromeres is similar to results reported for sperm and larval stages of development. Images PMID:7312627

  17. 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.

  18. The tumour suppressor CHD5 forms a NuRD-type chromatin remodelling complex.

    PubMed

    Kolla, Venkatadri; Naraparaju, Koumudi; Zhuang, Tiangang; Higashi, Mayumi; Kolla, Sriharsha; Blobel, Gerd A; Brodeur, Garrett M

    2015-06-01

    Eukaryotic gene expression is developmentally regulated, in part by chromatin remodelling, and its dysregulation has been linked to cancer. CHD5 (chromodomain helicase DNA-binding protein 5) is a tumour suppressor gene (TSG) that maps to a region of consistent deletion on 1p36.31 in neuroblastomas (NBs) and other tumour types. CHD5 encodes a protein with chromatin remodelling, helicase and DNA-binding motifs that is preferentially expressed in neural and testicular tissues. CHD5 is highly homologous to CHD3 and CHD4, which are the core subunits of nucleosome remodelling and deacetylation (NuRD) complexes. To determine if CHD5 forms a similar complex, we performed studies on nuclear extracts from NBLS, SY5Y (both with endogenous CHD5 expression), NLF (CHD5 null) and NLF cells stably transfected with CHD5 cDNA (wild-type and V5-histidine-tagged). Immunoprecipitation (IP) was performed with either CHD5 antibody or antibody to V5/histidine-tagged protein. We identified NuRD components both by GST-FOG1 (Friend Of GATA1) pull-down and by IP. We also performed MS/MS analysis to confirm the presence of CHD5 or other protein components of the NuRD complex, as well as to identify other novel proteins. CHD5 was clearly associated with all canonical NuRD components, including metastasis-associated protein (MTA)1/2, GATA zinc finger domain containing 2A (GATAD2A), histone deacetylase (HDAC)1/2, retinoblastoma-binding protein (RBBP)4/7 and methyl DNA-binding domain protein (MBD)2/3, as determined by Western blotting and MS/MS. Our data suggest CHD5 forms a NuRD complex similar to CHD4. However, CHD5-NuRD may also have unique protein associations that confer functional specificity and may contribute to normal development and to tumour suppression in NB and other cancers.

  19. Small Molecules Modulate Chromatin Accessibility to Promote NEUROG2-Mediated Fibroblast-to-Neuron Reprogramming.

    PubMed

    Smith, Derek K; Yang, Jianjing; Liu, Meng-Lu; Zhang, Chun-Li

    2016-11-08

    Pro-neural transcription factors and small molecules can induce the reprogramming of fibroblasts into functional neurons; however, the immediate-early molecular events that catalyze this conversion have not been well defined. We previously demonstrated that neurogenin 2 (NEUROG2), forskolin (F), and dorsomorphin (D) can reprogram fibroblasts into functional neurons with high efficiency. Here, we used this model to define the genetic and epigenetic events that initiate an acquisition of neuronal identity. We demonstrate that NEUROG2 is a pioneer factor, FD enhances chromatin accessibility and H3K27 acetylation, and synergistic transcription activated by these factors is essential to successful reprogramming. CREB1 promotes neuron survival and acts with NEUROG2 to upregulate SOX4, which co-activates NEUROD1 and NEUROD4. In addition, SOX4 targets SWI/SNF subunits and SOX4 knockdown results in extensive loss of open chromatin and abolishes reprogramming. Applying these insights, adult human glioblastoma cell and skin fibroblast reprogramming can be improved using SOX4 or chromatin-modifying chemicals.

  20. The chromatin structure of Saccharomyces cerevisiae autonomously replicating sequences changes during the cell division cycle.

    PubMed Central

    Brown, J A; Holmes, S G; Smith, M M

    1991-01-01

    The chromatin structures of two well-characterized autonomously replicating sequence (ARS) elements were examined at their chromosomal sites during the cell division cycle in Saccharomyces cerevisiae. The H4 ARS is located near one of the duplicate nonallelic histone H4 genes, while ARS1 is present near the TRP1 gene. Cells blocked in G1 either by alpha-factor arrest or by nitrogen starvation had two DNase I-hypersensitive sites of about equal intensity in the ARS element. This pattern of DNase I-hypersensitive sites was altered in synchronous cultures allowed to proceed into S phase. In addition to a general increase in DNase I sensitivity around the core consensus sequence, the DNase I-hypersensitive site closest to the core consensus became more nuclease sensitive than the distal site. This change in chromatin structure was restricted to the ARS region and depended on replication since cdc7 cells blocked near the time of replication initiation did not undergo the transition. Subsequent release of arrested cdc7 cells restored entry into S phase and was accompanied by the characteristic change in ARS chromatin structure. Images PMID:1922046

  1. Widespread Collaboration of Isw2 and Sin3-Rpd3 Chromatin Remodeling Complexes in Transcriptional Repression

    PubMed Central

    Fazzio, Thomas G.; Kooperberg, Charles; Goldmark, Jesse P.; Neal, Cassandra; Basom, Ryan; Delrow, Jeffrey; Tsukiyama, Toshio

    2001-01-01

    The yeast Isw2 chromatin remodeling complex functions in parallel with the Sin3-Rpd3 histone deacetylase complex to repress early meiotic genes upon recruitment by Ume6p. For many of these genes, the effect of an isw2 mutation is partially masked by a functional Sin3-Rpd3 complex. To identify the full range of genes repressed or activated by these factors and uncover hidden targets of Isw2-dependent regulation, we performed full genome expression analyses using cDNA microarrays. We find that the Isw2 complex functions mainly in repression of transcription in a parallel pathway with the Sin3-Rpd3 complex. In addition to Ume6 target genes, we find that many Ume6-independent genes are derepressed in mutants lacking functional Isw2 and Sin3-Rpd3 complexes. Conversely, we find that ume6 mutants, but not isw2 sin3 or isw2 rpd3 double mutants, have reduced fidelity of mitotic chromosome segregation, suggesting that one or more functions of Ume6p are independent of Sin3-Rpd3 and Isw2 complexes. Chromatin structure analyses of two nonmeiotic genes reveals increased DNase I sensitivity within their regulatory regions in an isw2 mutant, as seen previously for one meiotic locus. These data suggest that the Isw2 complex functions at Ume6-dependent and -independent loci to create DNase I-inaccessible chromatin structure by regulating the positioning or placement of nucleosomes. PMID:11533234

  2. 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.

  3. Large-scale chromatin decondensation and recondensation regulated by transcription from a natural promoter

    PubMed Central

    Müller, Waltraud G.; Walker, Dawn; Hager, Gordon L.; McNally, James G.

    2001-01-01

    We have examined the relationship between transcription and chromatin structure using a tandem array of the mouse mammary tumor virus (MMTV) promoter driving a ras reporter. The array was visualized as a distinctive fluorescent structure in live cells stably transformed with a green fluorescent protein (GFP)-tagged glucocorticoid receptor (GR), which localizes to the repeated MMTV elements after steroid hormone treatment. Also found at the array by immunofluorescence were two different steroid receptor coactivators (SRC1 and CBP) with acetyltransferase activity, a chromatin remodeler (BRG1), and two transcription factors (NFI and AP-2). Within 3 h after hormone addition, arrays visualized by GFP-GR or DNA fluorescent in situ hybridization (FISH) decondensed to varying degrees, in the most pronounced cases from a ∼0.5-μm spot to form a fiber 1–10 μm long. Arrays later recondensed by 3–8 h of hormone treatment. The degree of decondensation was proportional to the amount of transcript produced by the array as detected by RNA FISH. Decondensation was blocked by two different drugs that inhibit polymerase II, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) and α-amanitin. These observations demonstrate a role for polymerase in producing and maintaining decondensed chromatin. They also support fiber-packing models of higher order structure and suggest that transcription from a natural promoter may occur at much higher DNA-packing densities than reported previously. PMID:11448988

  4. 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

  5. Genetic modifiers of chromatin acetylation antagonize the reprogramming of epi-polymorphisms.

    PubMed

    Abraham, Anne-Laure; Nagarajan, Muniyandi; Veyrieras, Jean-Baptiste; Bottin, Hélène; Steinmetz, Lars M; Yvert, Gaël

    2012-09-01

    Natural populations are known to differ not only in DNA but also in their chromatin-associated epigenetic marks. When such inter-individual epigenomic differences (or "epi-polymorphisms") are observed, their stability is usually not known: they may or may not be reprogrammed over time or upon environmental changes. In addition, their origin may be purely epigenetic, or they may result from regulatory variation encoded in the DNA. Studying epi-polymorphisms requires, therefore, an assessment of their nature and stability. Here we estimate the stability of yeast epi-polymorphisms of chromatin acetylation, and we provide a genome-by-epigenome map of their genetic control. A transient epi-drug treatment was able to reprogram acetylation variation at more than one thousand nucleosomes, whereas a similar amount of variation persisted, distinguishing "labile" from "persistent" epi-polymorphisms. Hundreds of genetic loci underlied acetylation variation at 2,418 nucleosomes either locally (in cis) or distantly (in trans), and this genetic control overlapped only partially with the genetic control of gene expression. Trans-acting regulators were not necessarily associated with genes coding for chromatin modifying enzymes. Strikingly, "labile" and "persistent" epi-polymorphisms were associated with poor and strong genetic control, respectively, showing that genetic modifiers contribute to persistence. These results estimate the amount of natural epigenomic variation that can be lost after transient environmental exposures, and they reveal the complex genetic architecture of the DNA-encoded determinism of chromatin epi-polymorphisms. Our observations provide a basis for the development of population epigenetics.

  6. Meiotic behavior of aneuploid chromatin in mouse models of Down syndrome.

    PubMed

    Reinholdt, Laura G; Czechanski, Anne; Kamdar, Sonya; King, Benjamin L; Sun, Fengyun; Handel, Mary Ann

    2009-12-01

    Aneuploidy, which leads to unpaired chromosomal axes during meiosis, is frequently accompanied by infertility. We previously showed, using three mouse models of Down syndrome, that it is an extra chromosome, but not extra gene dose, that is associated with male infertility and virtual absence of post-meiotic gem cells. Here, we test the hypothesis that aneuploid segments are differentially modified and expressed during meiosis, depending on whether they are present as an extra chromosome or not. In all three models examined, the trisomic region lacks a pairing partner, but in one case, spermatocytes have an extra (and unpaired) chromosome, while the two other models involve translocation of the trisomic region rather than an extra chromosome. An extra unpaired chromosome was always modified by phosphorylation of histone H2AX and lacked RNA PolII. But in the case of trisomic regions attached to a paired chromosome, assembly of these protein modifications was affected by the position of a trisomic region relative to a centromere and the physical extent of the unpaired chromatin. Analysis of gene expression in testes revealed that extra copy number alone was not sufficient for meiotic upregulation of genes in the trisomic interval. Additionally and unexpectedly, presence of meiotic gene silencing chromatin modifications was not sufficient for downregulation of genes in unpaired trisomic chromatin. Thus, the meiotic chromatin modifications that are cytologically visible are unlikely to be directly involved in sterility versus fertility of DS models. Finally, the presence of an extra unpaired chromosome, but not the presence of extra (trisomic) genes, caused global deregulation of transcription in spermatocytes. These results reveal mechanisms by which an extra chromosome, but not trisomic gene dose, impact on meiotic progress and infertility.

  7. Chromosome-wide analysis of parental allele-specific chromatin and DNA methylation.

    PubMed

    Singh, Purnima; Wu, Xiwei; Lee, Dong-Hoon; Li, Arthur X; Rauch, Tibor A; Pfeifer, Gerd P; Mann, Jeffrey R; Szabó, Piroska E

    2011-04-01

    To reveal the extent of domain-wide epigenetic features at imprinted gene clusters, we performed a high-resolution allele-specific chromatin analysis of over 100 megabases along the maternally or paternally duplicated distal chromosome 7 (Chr7) and Chr15 in mouse embryo fibroblasts (MEFs). We found that reciprocal allele-specific features are limited to imprinted genes and their differentially methylated regions (DMRs), whereas broad local enrichment of H3K27me3 (BLOC) is a domain-wide feature at imprinted clusters. We uncovered novel allele-specific features of BLOCs. A maternally biased BLOC was found along the H19-Igf2 domain. A paternal allele-specific gap was found along Kcnq1ot1, interrupting a biallelic BLOC in the Kcnq1-Cdkn1c domain. We report novel allele-specific chromatin marks at the Peg13 and Slc38a4 DMRs, Cdkn1c upstream region, and Inpp5f_v2 DMR and paternal allele-specific CTCF binding at the Peg13 DMR. Additionally, we derived an imprinted gene predictor algorithm based on our allele-specific chromatin mapping data. The binary predictor H3K9ac and CTCF or H3K4me3 in one allele and H3K9me3 in the reciprocal allele, using a sliding-window approach, recognized with precision the parental allele specificity of known imprinted genes, H19, Igf2, Igf2as, Cdkn1c, Kcnq1ot1, and Inpp5f_v2 on Chr7 and Peg13 and Slc38a4 on Chr15. Chromatin features, therefore, can unequivocally identify genes with imprinted expression.

  8. Promoter chromatin remodeling of immediate-early genes is mediated through H3 phosphorylation at either serine 28 or 10 by the MSK1 multi-protein complex

    PubMed Central

    Drobic, Bojan; Pérez-Cadahía, Beatriz; Yu, Jenny; Kung, Sam Kam-Pun; Davie, James R.

    2010-01-01

    Upon activation of the ERK and p38 MAPK pathways, the MSK1/2-mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early (IE) gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and sequential chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 the ATPase subunit of the SWI/SNF remodeler, is recruited to the promoter of target genes by transcription factors such as Elk-1 or NF-κB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of IE genes enabling the binding of transcription factors like JUN and the onset of transcription. PMID:20129940

  9. 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.

  10. Micronucleus formation during chromatin condensation and under apoptotic conditions.

    PubMed

    Kiraly, Gabor; Simonyi, Athene S; Turani, Melinda; Juhasz, Istvan; Nagy, Gabor; Banfalvi, Gaspar

    2017-02-01

    In early S phase the newly replicated DNA is folded back to increasingly compact structures. The process of chromatin condensation inside the nucleus starts with the formation of a micronucleus observed in five established cell lines (K562, CHO, Indian muntjac, murine preB and SCC). Supercoiling of chromatin generates a polarized end-plate region extruded from the nucleus. The extruded chromatin is turned around itself forming the head portion (micronucleus) visible by fluorescence microscopy until the middle of S phase when chromatin structures are succeeded by distinguishable early forms of chromosomes. The generation of micronuclei upon apoptotic treatment was achieved by the methotrexate (MTX) treatment of cells. A close correlation was found between the frequency of micronucleus and MTX concentration, with low frequency at low (0.1 µM) and increasingly higher frequency between 1 and 100 µM concentrations. Characteristic deformation and shrinkage of nuclei indicated apoptosis. High MTX concentration (100 µM) caused the enlargement and necrotic disruption of nuclei. Inhibition of DNA synthesis during replicative DNA synthesis by biotinylated nucleotide prevented the formation of metaphase chromosomes and elevated the frequency of early intermediates of chromosome condensation including micronucleus formation. Based on these observations the micronucleus is regarded as: (a) a regularly occuring element of early chromatin condensation and (b) a typical sign of nuclear membrane damage under toxic conditions. Explanation is given why the micronucleus is hidden in nuclei under normal chromatin condensation and why chromatin motifs including micronuclei become visible upon cellular damage.

  11. The Chromatin Regulator CHD8 Is a Context-Dependent Mediator of Cell Survival in Murine Hematopoietic Malignancies.

    PubMed

    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.

  12. Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling

    PubMed Central

    Valiuliene, Giedre; Stirblyte, Ieva; Cicenaite, Dovile; Kaupinis, Algirdas; Valius, Mindaugas; Navakauskiene, Ruta

    2015-01-01

    Epigenetic changes play a significant role in leukaemia pathogenesis, therefore histone deacetylases (HDACis) are widely accepted as an attractive strategy for acute promyelocytic leukaemia (APL) treatment. Belinostat (Bel, PXD101), a hydroxamate-type HDACi, has proved to be a promising cure in clinical trials for solid tumours and haematological malignancies. However, insight into molecular effects of Bel on APL, is still lacking. In this study, we investigated the effect of Bel alone and in combination with differentiation inducer retinoic acid (RA) on human promyelocytic leukaemia NB4 and HL-60 cells. We found that treatment with Bel, depending on the dosage used, inhibits cell proliferation, whereas in combination with RA enhances and accelerates granulocytic leukaemia cell differentiation. We also evaluated the effect of used treatments with Bel and RA on certain epigenetic modifiers (HDAC1, HDAC2, PCAF) as well as cell cycle regulators (p27) gene expression and protein level modulation. We showed that Bel in combination with RA up-regulates basal histone H4 hyperacetylation level more strongly compared to Bel or RA alone. Furthermore, chromatin immunoprecipitation assay indicated that Bel induces the accumulation of hyperacetylated histone H4 at the p27 promoter region. Mass spectrometry analysis revealed that in control NB4 cells, hyperacetylated histone H4 is mainly found in association with proteins involved in DNA replication and transcription, whereas after Bel treatment it is found with proteins implicated in pro-apoptotic processes, in defence against oxidative stress and tumour suppression. Summarizing, our study provides some novel insights into the molecular mechanisms of HDACi Bel action on APL cells. PMID:25864732

  13. Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling.

    PubMed

    Valiuliene, Giedre; Stirblyte, Ieva; Cicenaite, Dovile; Kaupinis, Algirdas; Valius, Mindaugas; Navakauskiene, Ruta

    2015-07-01

    Epigenetic changes play a significant role in leukaemia pathogenesis, therefore histone deacetylases (HDACis) are widely accepted as an attractive strategy for acute promyelocytic leukaemia (APL) treatment. Belinostat (Bel, PXD101), a hydroxamate-type HDACi, has proved to be a promising cure in clinical trials for solid tumours and haematological malignancies. However, insight into molecular effects of Bel on APL, is still lacking. In this study, we investigated the effect of Bel alone and in combination with differentiation inducer retinoic acid (RA) on human promyelocytic leukaemia NB4 and HL-60 cells. We found that treatment with Bel, depending on the dosage used, inhibits cell proliferation, whereas in combination with RA enhances and accelerates granulocytic leukaemia cell differentiation. We also evaluated the effect of used treatments with Bel and RA on certain epigenetic modifiers (HDAC1, HDAC2, PCAF) as well as cell cycle regulators (p27) gene expression and protein level modulation. We showed that Bel in combination with RA up-regulates basal histone H4 hyperacetylation level more strongly compared to Bel or RA alone. Furthermore, chromatin immunoprecipitation assay indicated that Bel induces the accumulation of hyperacetylated histone H4 at the p27 promoter region. Mass spectrometry analysis revealed that in control NB4 cells, hyperacetylated histone H4 is mainly found in association with proteins involved in DNA replication and transcription, whereas after Bel treatment it is found with proteins implicated in pro-apoptotic processes, in defence against oxidative stress and tumour suppression. Summarizing, our study provides some novel insights into the molecular mechanisms of HDACi Bel action on APL cells.

  14. Changes in chromatin accessibility across the GM-CSF promoter upon T cell activation are dependent on nuclear factor kappaB proteins.

    PubMed

    Holloway, Adele F; Rao, Sudha; Chen, Xinxin; Shannon, M Frances

    2003-02-17

    Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a key cytokine in myelopoiesis and aberrant expression is associated with chronic inflammatory disease and myeloid leukemias. This aberrant expression is often associated with constitutive nuclear factor (NF)-kappaB activation. To investigate the relationship between NF-kappaB and GM-CSF transcription in a chromatin context, we analyzed the chromatin structure of the GM-CSF gene in T cells and the role of NF-kappaB proteins in chromatin remodeling. We show here that chromatin remodeling occurs across a region of the GM-CSF gene between -174 and +24 upon T cell activation, suggesting that remodeling is limited to a single nucleosome encompassing the proximal promoter. Nuclear NF-kappaB levels appear to play a critical role in this process. In addition, using an immobilized template assay we found that the ATPase component of the SWI/SNF chromatin remodeling complex, brg1, is recruited to the GM-CSF proximal promoter in an NF-kappaB-dependent manner in vitro. These results suggest that chromatin remodeling across the GM-CSF promoter in T cells is a result of recruitment of SWI/SNF type remodeling complexes by NF-kappaB proteins binding to the CD28 response region of the promoter.

  15. 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

  16. Food additives

    PubMed Central

    Spencer, Michael

    1974-01-01

    Food additives are discussed from the food technology point of view. The reasons for their use are summarized: (1) to protect food from chemical and microbiological attack; (2) to even out seasonal supplies; (3) to improve their eating quality; (4) to improve their nutritional value. The various types of food additives are considered, e.g. colours, flavours, emulsifiers, bread and flour additives, preservatives, and nutritional additives. The paper concludes with consideration of those circumstances in which the use of additives is (a) justified and (b) unjustified. PMID:4467857

  17. Chromatin remodeling and bivalent histone modifications in embryonic stem cells.

    PubMed

    Harikumar, Arigela; Meshorer, Eran

    2015-12-01

    Pluripotent embryonic stem cells (ESCs) are characterized by distinct epigenetic features including a relative enrichment of histone modifications related to active chromatin. Among these is tri-methylation of lysine 4 on histone H3 (H3K4me3). Several thousands of the H3K4me3-enriched promoters in pluripotent cells also contain a repressive histone mark, namely H3K27me3, a situation referred to as "bivalency". While bivalent promoters are not unique to pluripotent cells, they are relatively enriched in these cell types, largely marking developmental and lineage-specific genes which are silent but poised for immediate action. The H3K4me3 and H3K27me3 modifications are catalyzed by lysine methyltransferases which are usually found within, although not entirely limited to, the Trithorax group (TrxG) and Polycomb group (PcG) protein complexes, respectively, but these do not provide selective bivalent specificity. Recent studies highlight the family of ATP-dependent chromatin remodeling proteins as regulators of bivalent domains. Here, we discuss bivalency in general, describe the machineries that catalyze bivalent chromatin domains, and portray the emerging connection between bivalency and the action of different families of chromatin remodelers, namely INO80, esBAF, and NuRD, in pluripotent cells. We posit that chromatin remodeling proteins may enable "bivalent specificity", often selectively acting on, or selectively depleted from, bivalent domains.

  18. Forced unraveling of chromatin fibers with nonuniform linker DNA lengths

    NASA Astrophysics Data System (ADS)

    Ozer, Gungor; Collepardo-Guevara, Rosana; Schlick, Tamar

    2015-02-01

    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.

  19. Extended chromatin fibers: evidence from scanning force microscopy studies

    NASA Astrophysics Data System (ADS)

    Leuba, Sanford S.; Yang, Guoliang; Robert, Charles; van Holde, Kensal; Zlatanova, Jordanka; Bustamante, Carlos J.

    1995-03-01

    Unfixed chicken erythrocyte fibers in very low salt have been imaged using the scanning force microscope (SFM) operating in the tapping mode in air at ambient humidity. These images reveal a 3D organization of the fibers. The planar 'zig-zag' conformation is rare, and extended 'beads- on-a-string' fibers are seen only in chromatin depleted of H1 and H5. Glutaraldehyde fixation reveals very similar structures. Fibers fixed in 10 mM salt appear somewhat more compacted. These results, when compared with modeling studies indicate that chromatin fibers may exist as irregular 3D arrays of nucleosomes even at low ionic strength. The basic subunit of chromatin, the nucleosome, is composed of a core particle of 146 bp of DNA wrapping 1.75 left-handed superhelical turns around an octamer of core histones and of DNA connecting consecutive core particles. The linker of lysine-rich histones (H1 family) bind the DNA entering and exiting the nucleosome core particle. We suggest that by binding the entry/exit DNA, histone H1 may fix the entry/exit DNA angle. The fixed entry/exit angle, the rigidity of the linker DNA at low ionic strength, and the natural variability of the linker DNA length determine an irregular 3D fiber of chromatin. Our results emphasize the role of H1 in determining the entry/exit DNA angle, which further helps determine the mutual disposition of adjacent nucleosomes an the packing of the chromatin fiber.

  20. Micron-scale coherence in interphase chromatin dynamics

    PubMed Central

    Zidovska, Alexandra; Weitz, David A.; Mitchison, Timothy J.

    2013-01-01

    Chromatin structure and dynamics control all aspects of DNA biology yet are poorly understood, especially at large length scales. We developed an approach, displacement correlation spectroscopy based on time-resolved image correlation analysis, to map chromatin dynamics simultaneously across the whole nucleus in cultured human cells. This method revealed that chromatin movement was coherent across large regions (4–5 µm) for several seconds. Regions of coherent motion extended beyond the boundaries of single-chromosome territories, suggesting elastic coupling of motion over length scales much larger than those of genes. These large-scale, coupled motions were ATP dependent and unidirectional for several seconds, perhaps accounting for ATP-dependent directed movement of single genes. Perturbation of major nuclear ATPases such as DNA polymerase, RNA polymerase II, and topoisomerase II eliminated micron-scale coherence, while causing rapid, local movement to increase; i.e., local motions accelerated but became uncoupled from their neighbors. We observe similar trends in chromatin dynamics upon inducing a direct DNA damage; thus we hypothesize that this may be due to DNA damage responses that physically relax chromatin and block long-distance communication of forces. PMID:24019504

  1. The role of noncoding RNAs in chromatin regulation during differentiation.

    PubMed

    Nahkuri, Satu; Paro, Renato

    2012-01-01

    A myriad of nuclear noncoding RNAs (ncRNAs) have been discovered since the paradigm of RNAs as plain conveyors of protein translation was discarded. There is increasing evidence that at vital intersections of developmental pathways, ncRNAs target the chromatin modulating machinery to its site of action. However, the mechanistic details of processes involved are still largely unclear, and well-characterized metazoan ncRNA species implicated in chromatin regulation during differentiation remain few. Nevertheless, four major categories are slowly emerging: cis-acting antisense ncRNAs that flag the neighboring genes for the propagation of chromatin marks; allele-specific ncRNAs that perform similar tasks, but target larger loci that typically vary in size from hundreds of thousands of base pairs to a whole chromosome; structural ncRNAs proposed to act as scaffolds that couple chromatin shaping complexes of distinct functionalities; and cofactor ncRNAs with a capacity to inhibit or activate essential components of the intertwined chromatin and transcription apparatuses.

  2. Chromatin is an ancient innovation conserved between Archaea and Eukarya

    PubMed Central

    Ammar, Ron; Torti, Dax; Tsui, Kyle; Gebbia, Marinella; Durbic, Tanja; Bader, Gary D; Giaever, Guri; Nislow, Corey

    2012-01-01

    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 PMID:23240084

  3. Topological constraints strongly affect chromatin reconstitution in silico

    PubMed Central

    Brackley, C.A.; Allan, J.; Keszenman-Pereyra, D.; Marenduzzo, D.

    2015-01-01

    The fundamental building block of chromatin, and of chromosomes, is the nucleosome, a composite material made up from DNA wrapped around a histone octamer. In this study we provide the first computer simulations of chromatin self-assembly, starting from DNA and histone proteins, and use these to understand the constraints which are imposed by the topology of DNA molecules on the creation of a polynucleosome chain. We take inspiration from the in vitro chromatin reconstitution protocols which are used in many experimental studies. Our simulations indicate that during self-assembly, nucleosomes can fall into a number of topological traps (or local folding defects), and this may eventually lead to the formation of disordered structures, characterised by nucleosome clustering. Remarkably though, by introducing the action of topological enzymes such as type I and II topoisomerase, most of these defects can be avoided and the result is an ordered 10-nm chromatin fibre. These findings provide new insight into the biophysics of chromatin formation, both in the context of reconstitution in vitro and in terms of the topological constraints which must be overcome during de novo nucleosome formation in vivo, e.g. following DNA replication or repair. PMID:25432958

  4. Transcription upregulation via force-induced direct stretching of chromatin

    PubMed Central

    Tajik, Arash; Zhang, Yuejin; Wei, Fuxiang; Sun, Jian; Jia, Qiong; Zhou, Wenwen; Singh, Rishi; Khanna, Nimish; Belmont, Andrew S.; Wang, Ning

    2016-01-01

    Mechanical forces play critical roles in the function of living cells. However, the underlying mechanisms of how forces influence nuclear events remain elusive. Here, we show that chromatin deformation as well as force-induced transcription of a green-fluorescent-protein (GFP) tagged bacterial-chromosome dihydrofolate reductase (DHFR) transgene can be visualized in a living cell by using three-dimensional magnetic twisting cytometry to apply local stresses on the cell surface via an Arg-Gly-Asp-coated magnetic bead. Chromatin stretching depended on loading direction. DHFR transcription upregulation was sensitive to load direction and proportional to the magnitude of chromatin stretching. Disrupting filamentous actin or inhibiting actomyosin contraction abrogated or attenuated force-induced DHFR transcription, whereas activating endogenous contraction upregulated force-induced DHFR transcription. Our findings suggest that local stresses applied to integrins propagate from the tensed actin cytoskeleton to the LINC complex and then through lamina-chromatin interactions to directly stretch chromatin and upregulate transcription. PMID:27548707

  5. Co-immunoprecipitation with Tau Isoform-specific Antibodies Reveals Distinct Protein Interactions and Highlights a Putative Role for 2N Tau in Disease*

    PubMed Central

    Liu, Chang; Song, Xiaomin; Nisbet, Rebecca

    2016-01-01

    Alternative splicing generates multiple isoforms of the microtubule-associated protein Tau, but little is known about their specific function. In the adult mouse brain, three Tau isoforms are expressed that contain either 0, 1, or 2 N-terminal inserts (0N, 1N, and 2N). We generated Tau isoform-specific antibodies and performed co-immunoprecipitations followed by tandem mass tag multiplexed quantitative mass spectrometry. We identified novel Tau-interacting proteins of which one-half comprised membrane-bound proteins, localized to the plasma membrane, mitochondria, and other organelles. Tau was also found to interact with proteins involved in presynaptic signal transduction. MetaCore analysis revealed one major Tau interaction cluster that contained 33 Tau pulldown proteins. To explore the pathways in which these proteins are involved, we conducted an ingenuity pathway analysis that revealed two significant overlapping pathways, “cell-to-cell signaling and interaction” and “neurological disease.” The functional enrichment tool DAVID showed that in particular the 2N Tau-interacting proteins were specifically associated with neurological disease. Finally, for a subset of Tau interactions (apolipoprotein A1 (apoA1), apoE, mitochondrial creatine kinase U-type, β-synuclein, synaptogyrin-3, synaptophysin, syntaxin 1B, synaptotagmin, and synapsin 1), we performed reverse co-immunoprecipitations, confirming the preferential interaction of specific isoforms. For example, apoA1 displayed a 5-fold preference for the interaction with 2N, whereas β-synuclein showed preference for 0N. Remarkably, a reverse immunoprecipitation with apoA1 detected only the 2N isoform. This highlights distinct protein interactions of the different Tau isoforms, suggesting that they execute different functions in brain tissue. PMID:26861879

  6. 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.

  7. 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

  8. Extension of chromatin accessibility by nuclear matrix attachment regions

    NASA Astrophysics Data System (ADS)

    Jenuwein, Thomas; Forrester, William C.; Fernández-Herrero, Luis A.; Laible, Götz; Dull, Maude; Grosschedl, Rudolf

    1997-01-01

    Transcription of the variable region of the rearranged immunoglobulin μ gene is dependent on an enhancer sequence situated within one of the introns of the gene. Experiments with transgenic mice have shown that activation of the promoter controlling this transcription also requires the matrix-attachment regions (MARs) that flank the intronic enhancer1. As this μ gene enhancer can establish local areas of accessible chromatin2, we investigated whether the MARs can extend accessibility to more distal positions. We eliminated interactions between enhancer- and promoter-bound factors by linking μ enhancer/MAR fragments to the binding sites for bacteriophage RNA polymerases that were either close to or one kilobase distal to the enhancer. The μ enhancer alone mediated chromatin accessibility at the proximal site but required a flanking MAR to confer accessibility upon the distal promoter. This long-range accessibilty correlates with extended demethylation of the geμ enhancer to generate an extended domain of accessible chromatin.

  9. Modification of enhancer chromatin: what, how and why?

    PubMed Central

    Calo, Eliezer; Wysocka, Joanna

    2013-01-01

    Emergence of form and function during embryogenesis arises in large part through cell type- and cell state- specific variation in gene expression patterns, mediated by specialized cis-regulatory elements called enhancers. Recent large-scale epigenomic mapping revealed unexpected complexity and dynamics of enhancer utilization patterns, with 400,000 putative human enhancers annotated by the ENCODE project alone. These large-scale efforts were largely enabled through understanding that enhancers share certain stereotypical chromatin features. However, an important question still lingers: What is the functional significance of enhancer chromatin modification? Here we give an overview of enhancer-associated modifications of histones and DNA, and discuss enzymatic activities involved in their dynamic deposition and removal. We describe potential downstream effectors of these marks and propose models for exploring functions of chromatin modification in regulating enhancer activity during development. PMID:23473601

  10. 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

    2016-08-05

    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.

  11. 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

  12. Balancing chromatin remodeling and histone modifications in transcription

    PubMed Central

    Petty, Emily; Pillus, Lorraine

    2013-01-01

    Chromatin remodelers use the energy of ATP hydrolysis to reposition or evict nucleosomes or to replace canonical histones with histone variants. By regulating nucleosome dynamics, remodelers gate access to the underlying DNA for replication, repair, and transcription. Nucleosomes are subject to extensive post-translational modifications that can recruit regulatory proteins or alter the local chromatin structure. Just as extensive cross-talk has been observed between different histone post-translational modifications, there is growing evidence for both coordinated and antagonistic functional relationships between nucleosome remodeling and modifying machineries. Defining the combined functions of the complexes that alter nucleosome interactions, position, and stability is key to understanding processes that require access to DNA, particularly with growing appreciation of their contributions to human health and disease. Here, we highlight recent advances in the interactions between histone modifications and the ISWI and CHD1 chromatin remodelers from studies in budding yeast, fission yeast, flies, and mammalian cells, with a focus on yeast. PMID:23870137

  13. PARP1 orchestrates epigenetic events setting up chromatin domains.

    PubMed

    Ciccarone, Fabio; Zampieri, Michele; Caiafa, Paola

    2017-03-01

    Epigenetic events include reversible modifications of DNA and histone tails driving chromatin organization and thus transcription. The epigenetic regulation is a highly integrated process underlying the plasticity of the genomic information both in the context of complex physiological and pathological processes. The global regulatory aspects of epigenetic events are largely unknown. PARylation and PARP1 are recently emerging as multi-level regulatory effectors that modulate the topology of chromatin by orchestrating very different processes. This review focuses in particular on the role of PARP1 in epigenetics, trying to build a comprehensive perspective of its involvement in the regulation of epigenetic modifications of histones and DNA, contextualizing it in the global organization of chromatin domains in the nucleus.

  14. H4K44 Acetylation Facilitates Chromatin Accessibility during Meiosis.

    PubMed

    Hu, Jialei; Donahue, Greg; Dorsey, Jean; Govin, Jérôme; Yuan, Zuofei; Garcia, Benjamin A; Shah, Parisha P; Berger, Shelley L

    2015-12-01

    Meiotic recombination hotspots are associated with histone post-translational modifications and open chromatin. However, it remains unclear how histone modifications and chromatin structure regulate meiotic recombination. Here, we identify acetylation of histone H4 at Lys44 (H4K44ac) occurring on the nucleosomal lateral surface. We show that H4K44 is acetylated at pre-meiosis and meiosis and displays genome-wide enrichment at recombination hotspots in meiosis. Acetylation at H4K44 is required for normal meiotic recombination, normal levels of double-strand breaks (DSBs) during meiosis, and optimal sporulation. Non-modifiable H4K44R results in increased nucleosomal occupancy around DSB hotspots. Our results indicate that H4K44ac functions to facilitate chromatin accessibility favorable for normal DSB formation and meiotic recombination.

  15. 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

  16. Performance of Competitive and Indirect Enzyme-Linked Immunosorbent Assays, Gel Immunoprecipitation with Native Hapten Polysaccharide, and Standard Serological Tests in Diagnosis of Sheep Brucellosis

    PubMed Central

    Marín, C. M.; Moreno, E.; Moriyón, I.; Díaz, R.; Blasco, J. M.

    1999-01-01

    Competitive and standard enzyme-linked immunosorbent assays (ELISAs), rose bengal (RB), complement fixation, and agar gel immunoprecipitation with native hapten (AGID-NH) were compared by using sera from Brucella-free, Brucella melitensis-infected, and B. melitensis Rev1-vaccinated sheep. The most sensitive tests were indirect ELISA and RB, and the most specific tests were AGID-NH and competitive ELISA. We show that RB followed by AGID-NH is a simple and effective system for diagnosing sheep brucellosis. PMID:10066666

  17. Integration of Elf-4 into stem/progenitor and erythroid regulatory networks through locus-wide chromatin studies coupled with in vivo functional validation.

    PubMed

    Smith, Aileen M; Calero-Nieto, Fernando J; Schütte, Judith; Kinston, Sarah; Timms, Richard T; Wilson, Nicola K; Hannah, Rebecca L; Landry, Josette-Renee; Göttgens, Berthold

    2012-02-01

    The ETS transcription factor Elf-4 is an important regulator of hematopoietic stem cell (HSC) and T cell homeostasis. To gain insights into the transcriptional circuitry within which Elf-4 operates, we used comparative sequence analysis coupled with chromatin immunoprecipitation (ChIP) with microarray technology (ChIP-chip) assays for specific chromatin marks to identify three promoters and two enhancers active in hematopoietic and endothelial cell lines. Comprehensive functional validation of each of these regulatory regions in transgenic mouse embryos identified a tissue-specific enhancer (-10E) that displayed activity in fetal liver, dorsal aorta, vitelline vessels, yolk sac, and heart. Integration of a ChIP-sequencing (ChIP-Seq) data set for 10 key stem cell transcription factors showed Pu.1, Fli-1, and Erg were bound to the -10E element, and mutation of three highly conserved ETS sites within the enhancer abolished its activity. Finally, the transcriptional repressor Gfi1b was found to bind to and repress one of the Elf-4 promoters (-30P), and we show that this repression of Elf-4 is important for the maturation of primary fetal liver erythroid cells. Taken together, our results provide a comprehensive overview of the transcriptional control of Elf-4 within the hematopoietic system and, thus, integrate Elf-4 into the wider transcriptional regulatory networks that govern hematopoietic development.

  18. Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18.

    PubMed

    Bao, Baolong; Pestinger, Valerie; Hassan, Yousef I; Borgstahl, Gloria E O; Kolar, Carol; Zempleni, Janos

    2011-05-01

    Holocarboxylase synthetase (HCS) mediates the binding of biotin to lysine (K) residues in histones H2A, H3 and H4; HCS knockdown disturbs gene regulation and decreases stress resistance and lifespan in eukaryotes. We tested the hypothesis that HCS interacts physically with histone H3 for subsequent biotinylation. Co-immunoprecipitation experiments were conducted and provided evidence that HCS co-localizes with histone H3 in human cells; physical interactions between HCS and H3 were confirmed using limited proteolysis assays. Yeast two-hybrid (Y2H) studies revealed that the N-terminal and C-terminal domains in HCS participate in H3 binding. Recombinant human HCS was produced and exhibited biological activity, as evidenced by biotinylation of its known substrate, recombinant p67. Recombinant histone H3.2 and synthetic H3-based peptides were also good targets for biotinylation by recombinant HCS (rHCS) in vitro, based on tracing histone-bound biotin with [(3)H]biotin, streptavidin and anti-biotin antibody. Biotinylation site-specific antibodies were generated and revealed that both K9 and K18 in H3 were biotinylated by HCS. Collectively, these studies provide conclusive evidence that HCS interacts directly with histone H3, causing biotinylation of K9 and K18. We speculate that the targeting of HCS to distinct regions in human chromatin is mediated by DNA sequence, biotin, RNA, epigenetic marks or chromatin proteins.

  19. NUP98-PHF23 is a chromatin modifying oncoprotein that causes a wide array of leukemias sensitive to inhibition of PHD domain histone reader function

    PubMed Central

    Gough, Sheryl M; Lee, Fan; Yang, Fan; Walker, Robert L; Zhu, Yeulin J; Pineda, Marbin; Onozawa, Masahiro; Chung, Yang Jo; Bilke, Sven; Wagner, Elise K; Denu, John M; Ning, Yi; Xu, Bowen; Wang, Gang Greg; Meltzer, Paul S; Aplan, Peter D

    2014-01-01

    In this report, we show that expression of a NUP98-PHF23 (NP23) fusion, associated with acute myeloid leukemia (AML) in humans, leads to myeloid, erythroid, T-cell, and B-cell leukemia in mice. The leukemic and pre-leukemic tissues display a stem cell-like expression signature including Hoxa, Hoxb, and Meis1 genes. The PHF23 PHD domain is known to bind H3K4me3 residues, and chromatin immunoprecipitation experiments demonstrated that the NP23 protein bound chromatin at a specific subset of H3K4me3 sites, including Hoxa, Hoxb, and Meis1. Treatment of NP23 cells with disulfiram, which inhibits the binding of PHD domains to H3K4me3 residues, rapidly and selectively killed NP23 myeloblasts; cell death was preceded by decreased expression of Hoxa, Hoxb, and Meis1. Furthermore, AML driven by a related fusion gene, NUP98-JARID1A (NJL), was also sensitive to disulfiram. Thus, the NP23 mouse provides a platform to evaluate compounds that disrupt binding of oncogenic PHD proteins to H3K4me3. PMID:24535671

  20. The Interplay of Chromatin Landscape and DNA-Binding Context Suggests Distinct Modes of EIN3 Regulation in Arabid