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Sample records for activity chromatin immunoprecipitation

  1. Mapping Recombination Initiation Sites Using Chromatin Immunoprecipitation.

    PubMed

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

    2016-01-01

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

  2. Chromatin immunoprecipitation and an open chromatin assay in zebrafish erythrocytes.

    PubMed

    Yang, S; Ott, C J; Rossmann, M P; Superdock, M; Zon, L I; Zhou, Y

    2016-01-01

    Zebrafish is an excellent genetic and developmental model for the study of vertebrate development and disease. Its ability to produce an abundance of transparent, externally developed embryos has facilitated large-scale genetic and chemical screens for the identification of critical genes and chemical factors that modulate developmental pathways. These studies can have profound implications for the diagnosis and treatment of a variety of human diseases. Recent advancements in molecular and genomic studies have provided valuable tools and resources for comprehensive and high-resolution analysis of epigenomes during cell specification and lineage differentiation throughout development. In this chapter, we describe two simple methods to evaluate protein-DNA interaction and chromatin architecture in erythrocytes from adult zebrafish. These are chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) and an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). These techniques, together with gene expression profiling, are useful for analyzing epigenomic regulation of cell specification, differentiation, and function during zebrafish development in both normal and disease models. PMID:27443937

  3. PROTOCOLS: Chromatin Immunoprecipitation from Arabidopsis Tissues

    PubMed Central

    Yamaguchi, Nobutoshi; Winter, Cara M.; Wu, Miin-Feng; Kwon, Chang Seob; William, Dilusha A.; Wagner, Doris

    2014-01-01

    The ability of proteins to associate with genomic DNA in the context of chromatin is critical for many nuclear processes including transcription, replication, recombination, and DNA repair. Chromatin immunoprecipication (ChIP) is a practical and useful technique for characterizing protein / DNA association in vivo. The procedure generally includes six steps: (1) crosslinking the protein to the DNA; (2) isolating the chromatin; (3) chromatin fragmentation; (4) imunoprecipitation with antibodies against the protein of interest; (5) DNA recovery; and (6) PCR identification of factor associated DNA sequences. In this protocol, we describe guidelines, experimental setup, and conditions for ChIP in intact Arabidopsis tissues. This protocol has been used to study association of histone modifications, of chromatin remodeling ATPases, as well as of sequence-specific transcription factors with the genomic DNA in various Arabidopsis thaliana tissues. The protocol described focuses on ChIP-qPCR, but can readily be adapted for use in ChIP-chip or ChIP-seq experiments. The entire procedure can be completed within 3 days. PMID:24653666

  4. Mapping regulatory factors by immunoprecipitation from native chromatin

    PubMed Central

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

    2015-01-01

    Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin (ORGANIC) is a high-resolution method that can be used to quantitatively map protein-DNA interactions with high specificity and sensitivity. This method uses micrococcal nuclease (MNase) digestion of chromatin and low-salt solubilization to preserve protein-DNA complexes followed by immunoprecipitation and paired-end sequencing for genome-wide mapping of binding sites. In this Unit, we describe methods for isolation of nuclei and MNase digestion of unfixed chromatin, immunoprecipitation of protein-DNA complexes, and high-throughput sequencing to map sites of bound factors. PMID:25827087

  5. Microfluidics Technologies for Low Cell Number Chromatin Immunoprecipitation.

    PubMed

    Wu, Angela R; Quake, Stephen R

    2016-01-01

    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. PMID:26700100

  6. Chromatin Immunoprecipitation to Detect DNA Replication and Repair Factors

    PubMed Central

    Gadaleta, Mariana C.; Iwasaki, Osamu; Noguchi, Chiaki; Noma, Ken-Ichi; Noguchi, Eishi

    2015-01-01

    DNA replication is tightly coupled with DNA repair processes in order to preserve genomic integrity. During DNA replication, the replication fork encounters a variety of obstacles including DNA damage/adducts, secondary structures, and programmed fork-blocking sites, which are all difficult to replicate. The replication fork also collides with the transcription machinery, which shares the template DNA with the replisome complex. Under these conditions, replication forks stall, causing replication stress and/or fork collapse, ultimately leading to genomic instability. The mechanisms to overcome these replication problems remain elusive. Therefore, it is important to investigate how DNA repair and replication factors are recruited and coordinated at chromosomal regions that are difficult to replicate. In this chapter, we describe a chromatin immunoprecipitation method to locate proteins required for DNA repair during DNA replication in the fission yeast Schizosaccharomyces pombe. This method can also easily be adapted to study replisome components or chromatin-associated factors. PMID:25916713

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-11-20

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

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

    PubMed

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Cheaib, Miriam; Simon, Martin

    2013-03-01

    We report the detailed evaluation of crucial parameters for chromatin immunoprecipitation (ChIP) of macronuclear DNA in the unicellular eukaryote Paramecium tetraurelia. Optimized parameters include crosslinking conditions, chromatin sonication and antibody titration thus providing a detailed protocol for successful ChIP in P. tetraurelia. As this ciliate is bacterivorous and RNAi by feeding represents a powerful tool for analysis of gene function, we moreover determined the effects of ingested nucleic acids by food bacteria. Feasibility of our protocol is demonstrated by characterisation of chromatin remodelling at promoters of cytosolic HSP70 isoforms during transcriptional activation under heat shock conditions by analyzing RNA abundance, nucleosome occupancy and levels of H3 lysine 9 acetylation. PMID:23385475

  14. Proteins that bind regulatory regions identified by histone modification chromatin immunoprecipitations and mass spectrometry

    PubMed Central

    Engelen, Erik; Brandsma, Johannes H.; Moen, Maaike J.; Signorile, Luca; Dekkers, Dick H. W.; Demmers, Jeroen; Kockx, Christel E. M.; Ozgür, Zehila; van IJcken, Wilfred F. J.; van den Berg, Debbie L. C.; Poot, Raymond A.

    2015-01-01

    The locations of transcriptional enhancers and promoters were recently mapped in many mammalian cell types. Proteins that bind those regulatory regions can determine cell identity but have not been systematically identified. Here we purify native enhancers, promoters or heterochromatin from embryonic stem cells by chromatin immunoprecipitations (ChIP) for characteristic histone modifications and identify associated proteins using mass spectrometry (MS). 239 factors are identified and predicted to bind enhancers or promoters with different levels of activity, or heterochromatin. Published genome-wide data indicate a high accuracy of location prediction by ChIP-MS. A quarter of the identified factors are important for pluripotency and includes Oct4, Esrrb, Klf5, Mycn and Dppa2, factors that drive reprogramming to pluripotent stem cells. We determined the genome-wide binding sites of Dppa2 and find that Dppa2 operates outside the classical pluripotency network. Our ChIP-MS method provides a detailed read-out of the transcriptional landscape representative of the investigated cell type. PMID:25990348

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

    PubMed Central

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

    2013-01-01

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

  16. Important biological information uncovered in previously unaligned reads from chromatin immunoprecipitation experiments (ChIP-Seq).

    PubMed

    Ouma, Wilberforce Zachary; Mejia-Guerra, Maria Katherine; Yilmaz, Alper; Pareja-Tobes, Pablo; Li, Wei; Doseff, Andrea I; Grotewold, Erich

    2015-01-01

    Establishing the architecture of gene regulatory networks (GRNs) relies on chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) methods that provide genome-wide transcription factor binding sites (TFBSs). ChIP-Seq furnishes millions of short reads that, after alignment, describe the genome-wide binding sites of a particular TF. However, in all organisms investigated an average of 40% of reads fail to align to the corresponding genome, with some datasets having as much as 80% of reads failing to align. We describe here the provenance of previously unaligned reads in ChIP-Seq experiments from animals and plants. We show that a substantial portion corresponds to sequences of bacterial and metazoan origin, irrespective of the ChIP-Seq chromatin source. Unforeseen was the finding that 30%-40% of unaligned reads were actually alignable. To validate these observations, we investigated the characteristics of the previously unaligned reads corresponding to TAL1, a human TF involved in lineage specification of hemopoietic cells. We show that, while unmapped ChIP-Seq read datasets contain foreign DNA sequences, additional TFBSs can be identified from the previously unaligned ChIP-Seq reads. Our results indicate that the re-evaluation of previously unaligned reads from ChIP-Seq experiments will significantly contribute to TF target identification and determination of emerging properties of GRNs. PMID:25727450

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

    PubMed

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

    2014-01-01

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

  18. Prenucleosomes and Active Chromatin

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Birkaya, Barbara; Ortt, Kori; Sinha, Satrajit

    2007-01-01

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

  20. Chromatin immunoprecipitation reveals that the 180-bp satellite repeat is the key functional DNA element of Arabidopsis thaliana centromeres.

    PubMed Central

    Nagaki, Kiyotaka; Talbert, Paul B; Zhong, Cathy Xiaoyan; Dawe, R Kelly; Henikoff, Steven; Jiang, Jiming

    2003-01-01

    The centromeres of Arabidopsis thaliana chromosomes contain megabases of complex DNA consisting of numerous types of repetitive DNA elements. We developed a chromatin immunoprecipitation (ChIP) technique using an antibody against the centromeric H3 histone, HTR12, in Arabidopsis. ChIP assays showed that the 180-bp centromeric satellite repeat was precipitated with the antibody, suggesting that this repeat is the key component of the centromere/kinetochore complex in Arabidopsis. PMID:12663558

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

    PubMed

    Burlibașa, Liliana; Suciu, Ilinca

    2015-12-01

    Oogenesis is a critical event in the formation of female gamete, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the presence of H4 acetylation of the oocyte and somatic 5S rRNA genes in Triturus cristatus, using chromatin immunoprecipitation assay (ChIP). Our findings suggest that some epigenetic mechanisms such as histone acetylation could be involved in the transcriptional regulation of 5S rRNA gene families. PMID:25315165

  2. ChIPseq in Yeast Species: From Chromatin Immunoprecipitation to High-Throughput Sequencing and Bioinformatics Data Analyses.

    PubMed

    Lelandais, Gaëlle; Blugeon, Corinne; Merhej, Jawad

    2016-01-01

    Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIPseq) is a powerful technique for the genome-wide location of protein DNA-binding sites. The ChIP experiment consists in treating living cells with a cross-linking agent to bind proteins to their DNA substrates. After fragmentation of DNA, specific fractions associated with a particular protein of interest are purified by immunoaffinity. They are next sequenced and identified on the reference genome using dedicated bioinformatics programs. Several technical aspects are important to obtain high-quality ChIPseq results. This includes the quality of antibodies, the sequencing protocols, the use of accurate controls and the careful choice of bioinformatics tools. We present here a general protocol to perform ChIPseq analyses in yeast species. This protocol has been optimized to identify target genes of specific transcription factors but can be used for any other DNA binding proteins. PMID:26483023

  3. Inference of RNA Polymerase II Transcription Dynamics from Chromatin Immunoprecipitation Time Course Data

    PubMed Central

    wa Maina, Ciira; Honkela, Antti; Matarese, Filomena; Grote, Korbinian; Stunnenberg, Hendrik G.; Reid, George; Lawrence, Neil D.; Rattray, Magnus

    2014-01-01

    Gene transcription mediated by RNA polymerase II (pol-II) is a key step in gene expression. The dynamics of pol-II moving along the transcribed region influence the rate and timing of gene expression. In this work, we present a probabilistic model of transcription dynamics which is fitted to pol-II occupancy time course data measured using ChIP-Seq. The model can be used to estimate transcription speed and to infer the temporal pol-II activity profile at the gene promoter. Model parameters are estimated using either maximum likelihood estimation or via Bayesian inference using Markov chain Monte Carlo sampling. The Bayesian approach provides confidence intervals for parameter estimates and allows the use of priors that capture domain knowledge, e.g. the expected range of transcription speeds, based on previous experiments. The model describes the movement of pol-II down the gene body and can be used to identify the time of induction for transcriptionally engaged genes. By clustering the inferred promoter activity time profiles, we are able to determine which genes respond quickly to stimuli and group genes that share activity profiles and may therefore be co-regulated. We apply our methodology to biological data obtained using ChIP-seq to measure pol-II occupancy genome-wide when MCF-7 human breast cancer cells are treated with estradiol (E2). The transcription speeds we obtain agree with those obtained previously for smaller numbers of genes with the advantage that our approach can be applied genome-wide. We validate the biological significance of the pol-II promoter activity clusters by investigating cluster-specific transcription factor binding patterns and determining canonical pathway enrichment. We find that rapidly induced genes are enriched for both estrogen receptor alpha (ER) and FOXA1 binding in their proximal promoter regions. PMID:24830797

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Lau, On Sun; Bergmann, Dominique C

    2015-10-01

    Cell type-specific transcriptional regulators play critical roles in the generation and maintenance of multicellularity. As they are often expressed at low levels, in vivo DNA-binding studies of these regulators by standard chromatin immunoprecipitation (ChIP) assays are technically challenging. We describe here an optimized ChIP protocol termed Maximized Objects for Better Enrichment (MOBE)-ChIP, which enhances the sensitivity of ChIP assays for detecting cell type-specific signals. The protocol, which is based on the disproportional increase of target signals over background at higher scales, uses substantially greater volume of starting materials than conventional ChIPs to achieve high signal enrichment. This technique can capture weak binding events that are ambiguous in standard ChIP assays, and is useful both in gene-specific and whole-genome analysis. This protocol has been optimized for Arabidopsis, but should be applicable to other model systems with minor modifications. The full procedure can be completed within 3 days. PMID:26332947

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

    PubMed Central

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

    2015-01-01

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

  7. Deregulated Direct Targets of the Hepatitis B Virus (HBV) Protein, HBx, Identified through Chromatin Immunoprecipitation and Expression Microarray Profiling*

    PubMed Central

    Sung, Wing-Kin; Lu, Yiwei; Lee, Charlie W. H.; Zhang, Dongwei; Ronaghi, Mostafa; Lee, Caroline G. L.

    2009-01-01

    The hepatitis B-X (HBx) protein is strongly associated with hepatocellular carcinoma. It is implicated not to directly cause cancer but to play a role in hepatocellular carcinoma as a co-factor. The oncogenic potential of HBx primarily lies in its interaction with transcriptional regulators resulting in aberrant gene expression and deregulated cellular pathways. Utilizing ultraviolet irradiation to simulate a tumor-initiating event, we integrated chip-based chromatin immunoprecipitation (ChIP-chip) with expression microarray profiling and identified 184 gene targets directly deregulated by HBx. One-hundred forty-four transcription factors interacting with HBx were computationally inferred. We experimentally validated that HBx interacts with some of the predicted transcription factors (pTF) as well as the promoters of the deregulated target genes of these pTFs. Significantly, we demonstrated that the pTF interacts with the promoters of the deregulated HBx target genes and that deregulation by HBx of these HBx target genes carrying the pTF consensus sequences can be reversed using pTF small interfering RNAs. The roles of these deregulated direct HBx target genes and their relevance in cancer was inferred via querying against biogroup/cancer-related microarray databases using web-based NextBioTM software. Six pathways, including the Jak-STAT pathway, were predicted to be significantly deregulated when HBx binds indirectly to direct target gene promoters. In conclusion, this study represents the first ever demonstration of the utilization of ChIP-chip to identify deregulated direct gene targets from indirect protein-DNA binding as well as transcriptional factors directly interacting with HBx. Increased knowledge of the gene/transcriptional factor targets of HBx will enhance our understanding of the role of HBx in hepatocellular carcinogenesis and facilitate the design of better strategies in combating hepatitis B virus-associated hepatocellular carcinoma. PMID:19439406

  8. Deregulated direct targets of the hepatitis B virus (HBV) protein, HBx, identified through chromatin immunoprecipitation and expression microarray profiling.

    PubMed

    Sung, Wing-Kin; Lu, Yiwei; Lee, Charlie W H; Zhang, Dongwei; Ronaghi, Mostafa; Lee, Caroline G L

    2009-08-14

    The hepatitis B-X (HBx) protein is strongly associated with hepatocellular carcinoma. It is implicated not to directly cause cancer but to play a role in hepatocellular carcinoma as a co-factor. The oncogenic potential of HBx primarily lies in its interaction with transcriptional regulators resulting in aberrant gene expression and deregulated cellular pathways. Utilizing ultraviolet irradiation to simulate a tumor-initiating event, we integrated chip-based chromatin immunoprecipitation (ChIP-chip) with expression microarray profiling and identified 184 gene targets directly deregulated by HBx. One-hundred forty-four transcription factors interacting with HBx were computationally inferred. We experimentally validated that HBx interacts with some of the predicted transcription factors (pTF) as well as the promoters of the deregulated target genes of these pTFs. Significantly, we demonstrated that the pTF interacts with the promoters of the deregulated HBx target genes and that deregulation by HBx of these HBx target genes carrying the pTF consensus sequences can be reversed using pTF small interfering RNAs. The roles of these deregulated direct HBx target genes and their relevance in cancer was inferred via querying against biogroup/cancer-related microarray databases using web-based NextBio(TM) software. Six pathways, including the Jak-STAT pathway, were predicted to be significantly deregulated when HBx binds indirectly to direct target gene promoters. In conclusion, this study represents the first ever demonstration of the utilization of ChIP-chip to identify deregulated direct gene targets from indirect protein-DNA binding as well as transcriptional factors directly interacting with HBx. Increased knowledge of the gene/transcriptional factor targets of HBx will enhance our understanding of the role of HBx in hepatocellular carcinogenesis and facilitate the design of better strategies in combating hepatitis B virus-associated hepatocellular carcinoma. PMID

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    SciTech Connect

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

    2009-10-01

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

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

    PubMed Central

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

    2013-01-01

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

  13. 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

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

    PubMed Central

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

    2014-01-01

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

  15. 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). PMID:25089258

  16. Gene expression profiling and chromatin immunoprecipitation identify DBN1, SETMAR and HIG2 as direct targets of SOX11 in mantle cell lymphoma.

    PubMed

    Wang, Xiao; Björklund, Stefan; Wasik, Agata M; Grandien, Alf; Andersson, Patrik; Kimby, Eva; Dahlman-Wright, Karin; Zhao, Chunyan; Christensson, Birger; Sander, Birgitta

    2010-01-01

    The SRY (sex determining region Y)-box 11 (SOX11) gene, located on chromosome 2p25, encodes for a transcription factor that is involved in tissue remodeling during embryogenesis and is crucial for neurogenesis. The role for SOX11 in hematopoiesis has not yet been defined. Two genes under direct control of SOX11 are the class- III β-tubulin gene (TUBB3) in neural cells and the transcription factor TEA domain family member 2 (TEAD2) in neural and mesenchymal progenitor cells. Normal, mature lymphocytes lack SOX11 but express SOX4, another member of the same group of SOX transcription factors. We and others recently identified SOX11 as aberrantly expressed in mantle cell lymphoma (MCL). Since SOX11 is variably expressed in MCL it may not be essential for tumorigenesis, but may carry prognostic information. Currently, no specific functional effects have been linked to SOX11 expression in MCL and it is not known which genes are under influence of SOX11 in lymphoma. In this study we found variable expression of SOX11, SOX4 and SOX12 mRNA in mantle cell lymphoma cell lines. Downregulation of SOX11 expression by siRNA verified that SOX11 controlled the expression of the gene TUBB3 in the MCL cell line Granta 519. Furthermore we identified, by global gene expression analysis, 26 new target genes influenced by siRNA SOX11 downmodulation. Among these genes, DBN1, SETMAR and HIG2 were found to be significantly correlated to SOX11 expression in two cohorts of primary mantle cell lymphomas. Chromatin immunoprecipitation (ChIP) analysis showed that these genes are direct targets of the SOX11 protein. In spite of almost complete downregulation of the SOX11 protein no significant effects on Granta 519 cell proliferation or survival in short term in vitro experiments was found. In summary we have identified a number of genes influenced by SOX11 expression in MCL cell lines and primary MCL. Among these genes, DBN1, SETMAR and HIG2 are direct transcriptional targets of the SOX11

  17. Chromatin remodeling effects on enhancer activity.

    PubMed

    García-González, Estela; Escamilla-Del-Arenal, Martín; Arzate-Mejía, Rodrigo; Recillas-Targa, Félix

    2016-08-01

    During organism development, a diversity of cell types emerges with disparate, yet stable profiles of gene expression with distinctive cellular functions. In addition to gene promoters, the genome contains enhancer regulatory sequences, which are implicated in cellular specialization by facilitating cell-type and tissue-specific gene expression. Enhancers are DNA binding elements characterized by highly sophisticated and various mechanisms of action allowing for the specific interaction of general and tissue-specific transcription factors (TFs). However, eukaryotic organisms package their genetic material into chromatin, generating a physical barrier for TFs to interact with their cognate sequences. The ability of TFs to bind DNA regulatory elements is also modulated by changes in the chromatin structure, including histone modifications, histone variants, ATP-dependent chromatin remodeling, and the methylation status of DNA. Furthermore, it has recently been revealed that enhancer sequences are also transcribed into a set of enhancer RNAs with regulatory potential. These interdependent processes act in the context of a complex network of chromatin interactions, which together contributes to a renewed vision of how gene activation is coordinated in a cell-type-dependent manner. In this review, we describe the interplay between genetic and epigenetic aspects associated with enhancers and discuss their possible roles on enhancer function. PMID:27026300

  18. The Ig kappa 3' enhancer is activated by gradients of chromatin accessibility and protein association.

    PubMed

    McDevit, Daniel C; Perkins, Leslie; Atchison, Michael L; Nikolajczyk, Barbara S

    2005-03-01

    The Igkappa locus is recombined following initiation of a signaling cascade during the early pre-B stage of B cell development. The Ig kappa3' enhancer plays an important role in normal B cell development by regulating kappa locus activation. Quantitative analyses of kappa3' enhancer chromatin structure by restriction endonuclease accessibility and protein association by chromatin immunoprecipitation in a developmental series of primary murine B cells and murine B cell lines demonstrate that the enhancer is activated progressively through multiple steps as cells mature. Moderate kappa3' chromatin accessibility and low levels of protein association in pro-B cells are increased substantially as the cells progress from pro- to pre-B, then eventually mature B cell stages. Chromatin immunoprecipitation assays suggest transcriptional regulators of the kappa3' enhancer, specifically PU.1 and IFN regulatory factor-4, exploit enhanced accessibility by increasing association as cells mature. Characterization of histone acetylation patterns at the kappa3' enhancer and experimental inhibition of histone deacetylation suggest changes therein may determine changes in enzyme and transcription factor accessibility. This analysis demonstrates kappa activation is a multistep process initiated in early B cell precursors before Igmu recombination and finalized only after the pre-B cell stage. PMID:15728493

  19. High-throughput evaluation of aryl hydrocarbon receptor-binding sites selected via chromatin immunoprecipitation-based screening in Hepa-1c1c7 cells stimulated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

    PubMed

    Kinehara, Masaki; Fukuda, Itsuko; Yoshida, Ken-Ichi; Ashida, Hitoshi

    2008-12-01

    Upon binding to ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) is activated to form a heterodimer with an aryl hydrocarbon receptor nuclear translocator (Arnt) and binds to DNA. It has been shown that the binding of AhR to DNA depends on the dioxin response element (DRE) and controls xenobiotic-response genes. AhR-binding DNA fragments from mouse hepatoma Hepa-1c1c7 cells stimulated with TCDD were once enriched in a chromatin immunoprecipitation (ChIP) DNA library and screened through a high-throughput southwestern chemistry-based enzyme-linked immunosorbent assay (SW-ELISA). After screening 1700 fragments, the ChIP-SW-ELISA screening strategy allowed us to isolate 77 fragments tightly interacting with AhR in the presence of TCDD. Only 39 of the 77 fragments appeared to contain a typical DRE, indicating that in some cases the DRE was dispensable for AhR-binding, while 75 fragments were located within promoter-distal regions. Genomic mapping of the 77 fragments enabled us to estimate 121 potential AhR targets including known targets such as Cyp1A1 and Cyp1B1, but only a limited number exhibited an altered expression dependent on TCDD. This study revealed the fact that TCDD-activated AhR frequently binds to promoter-distal regions even without a DRE and is not always involved in transcriptional regulation, suggesting that within the genome DNA-binding of AhR could take place often in many regions without cis-regulatory elements and might not be a key determinant to establish its regulatory function. PMID:19282623

  20. Active establishment of centromeric CENP-A chromatin by RSF complex

    PubMed Central

    Perpelescu, Marinela; Nozaki, Naohito; Obuse, Chikashi; Yang, Hua

    2009-01-01

    Centromeres are chromosomal structures required for equal DNA segregation to daughter cells, comprising specialized nucleosomes containing centromere protein A (CENP-A) histone, which provide the basis for centromeric chromatin assembly. Discovery of centromere protein components is progressing, but knowledge related to their establishment and maintenance remains limited. Previously, using anti-CENP-A native chromatin immunoprecipitation, we isolated the interphase–centromere complex (ICEN). Among ICEN components, subunits of the remodeling and spacing factor (RSF) complex, Rsf-1 and SNF2h proteins, were found. This paper describes the relationship of the RSF complex to centromere structure and function, demonstrating its requirement for maintenance of CENP-A at the centromeric core chromatin in HeLa cells. The RSF complex interacted with CENP-A chromatin in mid-G1. Rsf-1 depletion induced loss of centromeric CENP-A, and purified RSF complex reconstituted and spaced CENP-A nucleosomes in vitro. From these data, we propose the RSF complex as a new factor actively supporting the assembly of CENP-A chromatin. PMID:19398759

  1. 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

  2. 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

  3. High resolution Chromatin Immunoprecipitation (ChIP) sequencing reveals novel bindings targets and prognostic role for SOX11 in Mantle cell lymphoma

    PubMed Central

    Kuo, Pei-Yu; Leshchenko, Violetta V.; Fazzari, Melissa J.; Perumal, Deepak; Gellen, Tobias; He, Tianfang; Iqbal, Javeed; Baumgartner-Wennerholm, Stefanie; Nygren, Lina; Zhang, Fan; Zhang, Weijia; Suh, K. Stephen; Goy, Andre; Yang, David T.; Chan, Wing-Chung; Kahl, Brad S.; Verma, Amit K.; Gascoyne, Randy D.; Kimby, Eva; Sander, Birgitta; Ye, B. Hilda; Melnick, Ari M.; Parekh, Samir

    2015-01-01

    SOX11 (Sex determining region Y-box 11) expression is specific for MCL as compared to other Non-Hodgkin's lymphomas. However, the function and direct binding targets of SOX11 in MCL are largely unknown. We used high-resolution ChIP-Seq to identify the direct target genes of SOX11 in a genome-wide, unbiased manner and elucidate its functional significance. Pathway analysis identified WNT, PKA and TGF-beta signaling pathways as significantly enriched by SOX11 target genes. qCHIP and promoter reporter assays confirmed that SOX11 directly binds to individual genes and modulates their transcription activities in these pathways in MCL. Functional studies using RNA interference demonstrate that SOX11 directly regulates WNT in MCL. We analyzed SOX11 expression in three independent well-annotated tissue microarrays from the University of Wisconsin (UW), Karolinska Institute and British Columbia Cancer Agency (BCCA). Our findings suggest that high SOX11 expression is associated with improved survival in a subset of MCL patients, particularly those treated with intensive chemotherapy. Transcriptional regulation of WNT and other biological pathways affected by SOX11 target genes may help explain the impact of SOX11 expression on patient outcomes. PMID:24681958

  4. Assays to Measure PTEN Lipid Phosphatase Activity In Vitro from Purified Enzyme or Immunoprecipitates.

    PubMed

    Spinelli, Laura; Leslie, Nicholas R

    2016-01-01

    PTEN is a one of the most frequently mutated tumor suppressors in human cancers. It is essential for regulating diverse biological processes and through its lipid phosphatase activity regulates the PI 3-Kinase signaling pathway. Sensitive phosphatase assays are employed to study the catalytic activity of PTEN against phospholipid substrates. Here we describe protocols to assay PTEN lipid phosphatase activity using either purified enzyme (purified PTEN lipid phosphatase assay) or PTEN immunopurified from tissues or cultured cells (cellular IP PTEN lipid phosphatase assay) against vesicles containing radiolabeled PIP3 substrate. PMID:27514802

  5. 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

  6. 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

  7. The Sch9 kinase is a chromatin-associated transcriptional activator of osmostress-responsive genes

    PubMed Central

    Pascual-Ahuir, Amparo; Proft, Markus

    2007-01-01

    The yeast Sch9 kinase has been implicated in the cellular adjustment to nutrient availability and in the regulation of aging. Here, we define a novel role for Sch9 in the transcriptional activation of osmostress inducible genes. Loss-of-function mutants sch9 are sensitive to hyperosmotic stress and show an impaired transcriptional response upon osmotic shock of several defense genes. We show that Sch9 is required for gene expression regulated by Sko1, a transcription factor, which is directly targeted by the Hog1 MAP kinase. Sch9 interacts in vitro with both Sko1 and Hog1. Additionally, Sch9 phosphorylates Sko1 in vitro. When artificially tethered to promoter DNA, Sch9 strongly activates transcription independently of osmotic stress. Using in vivo chromatin immunoprecipitation, we demonstrate that Sch9 is recruited to the GRE2 and CTT1 genes exclusively under osmostress conditions, and that this recruitment is dependent on Hog1 and Sko1. Furthermore, Sch9 is required for the proper recruitment of Hog1 at the same genes. Our data reveal the complexity of stress-induced transcription by the regulated association of signaling kinases to chromatin. PMID:17568771

  8. 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

  9. Immunoprecipitation of ROR1

    PubMed Central

    Bicocca, Vincent T.; Tyner, Jeffrey W.

    2016-01-01

    ROR1 is a receptor tyrosine kinase family member studied for its roles in development and cancer. Here we describe a protocol for immunoprecipitation of endogenous ROR1 from t(1;19) (a disease subtype categorized by its chromosome translocation) acute lymphoblastic leukemia immortalized cell lines.

  10. Chromatin remodeling during Saccharomyces cerevisiae ADH2 gene activation.

    PubMed

    Verdone, L; Camilloni, G; Di Mauro, E; Caserta, M

    1996-05-01

    We have analyzed at both low and high resolution the distribution of nucleosomes over the Saccharomyces cerevisiae ADH2 promoter region in its chromosomal location, both under repressing (high-glucose) conditions and during derepression. Enzymatic treatments (micrococcal nuclease and restriction endonucleases) were used to probe the in vivo chromatin structure during ADH2 gene activation. Under glucose-repressed conditions, the ADH2 promoter was bound by a precise array of nucleosomes, the principal ones positioned at the RNA initiation sites (nucleosome +1), at the TATA box (nucleosome -1), and upstream of the ADR1-binding site (UAS1) (nucleosome -2). The UAS1 sequence and the adjacent UAS2 sequence constituted a nucleosome-free region. Nucleosomes -1 and +1 were destabilized soon after depletion of glucose and had become so before the appearance of ADH2 mRNA. When the transcription rate was high, nucleosomes -2 and +2 also underwent rearrangement. When spheroplasts were prepared from cells grown in minimal medium, detection of this chromatin remodeling required the addition of a small amount of glucose. Cells lacking the ADR1 protein did not display any of these chromatin modifications upon glucose depletion. Since the UAS1 sequence to which Adr1p binds is located immediately upstream of nucleosome -1, Adr1p is presumably required for destabilization of this nucleosome and for aiding the TATA-box accessibility to the transcription machinery. PMID:8628264

  11. Senataxin controls meiotic silencing through ATR activation and chromatin remodeling

    PubMed Central

    Yeo, Abrey J; Becherel, Olivier J; Luff, John E; Graham, Mark E; Richard, Derek; Lavin, Martin F

    2015-01-01

    Senataxin, defective in ataxia oculomotor apraxia type 2, protects the genome by facilitating the resolution of RNA–DNA hybrids (R-loops) and other aspects of RNA processing. Disruption of this gene in mice causes failure of meiotic recombination and defective meiotic sex chromosome inactivation, leading to male infertility. Here we provide evidence that the disruption of Setx leads to reduced SUMOylation and disruption of protein localization across the XY body during meiosis. We demonstrate that senataxin and other DNA damage repair proteins, including ataxia telangiectasia and Rad3-related protein-interacting partner, are SUMOylated, and a marked downregulation of both ataxia telangiectasia and Rad3-related protein-interacting partner and TopBP1 leading to defective activation and signaling through ataxia telangiectasia and Rad3-related protein occurs in the absence of senataxin. Furthermore, chromodomain helicase DNA-binding protein 4, a component of the nucleosome remodeling and deacetylase chromatin remodeler that interacts with both ataxia telangiectasia and Rad3-related protein and senataxin was not recruited efficiently to the XY body, triggering altered histone acetylation and chromatin conformation in Setx−/− pachytene-staged spermatocytes. These results demonstrate that senataxin has a critical role in ataxia telangiectasia and Rad3-related protein- and chromodomain helicase DNA-binding protein 4-mediated transcriptional silencing and chromatin remodeling during meiosis providing greater insight into its critical role in gene regulation to protect against neurodegeneration. PMID:27462424

  12. 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

  13. An Essential Viral Transcription Activator Modulates Chromatin Dynamics

    PubMed Central

    Gibeault, Rebecca L.; Bildersheim, Michael D.

    2016-01-01

    Although ICP4 is the only essential transcription activator of herpes simplex virus 1 (HSV-1), its mechanisms of action are still only partially understood. We and others propose a model in which HSV-1 genomes are chromatinized as a cellular defense to inhibit HSV-1 transcription. To counteract silencing, HSV-1 would have evolved proteins that prevent or destabilize chromatinization to activate transcription. These proteins should act as HSV-1 transcription activators. We have shown that HSV-1 genomes are organized in highly dynamic nucleosomes and that histone dynamics increase in cells infected with wild type HSV-1. We now show that whereas HSV-1 mutants encoding no functional ICP0 or VP16 partially enhanced histone dynamics, mutants encoding no functional ICP4 did so only minimally. Transient expression of ICP4 was sufficient to enhance histone dynamics in the absence of other HSV-1 proteins or HSV-1 DNA. The dynamics of H3.1 were increased in cells expressing ICP4 to a greater extent than those of H3.3. The dynamics of H2B were increased in cells expressing ICP4, whereas those of canonical H2A were not. ICP4 preferentially targets silencing H3.1 and may also target the silencing H2A variants. In infected cells, histone dynamics were increased in the viral replication compartments, where ICP4 localizes. These results suggest a mechanism whereby ICP4 activates transcription by disrupting, or preventing the formation of, stable silencing nucleosomes on HSV-1 genomes. PMID:27575707

  14. Chromatin dynamics associated with HIV-1 Tat activated transcription

    PubMed Central

    Easley, Rebecca; Van Duyne, Rachel; Coley, Will; Guendel, Irene; Dadgar, Sherry; Kehn-Hall, Kylene; Kashanchi, Fatah

    2009-01-01

    Summary Chromatin remodeling is an essential event for HIV-1 transcription. Over the last two decades this field of research has come to the forefront, as silencing of the HIV-1 provirus through chromatin modifications has been linked to latency. Here, we focus on chromatin remodeling, especially in relation to the transactivator Tat, and review the most important and newly emerging studies that investigate remodeling mechanisms. We begin by discussing covalent modifications that can alter chromatin structure including acetylation, deacetylation, and methylation, as well as topics addressing the interplay between chromatin remodeling and splicing. Next, we focus on complexes that use the energy of ATP to remove or secure nucleosomes and can additionally act to control HIV-1 transcription. Finally, we cover recent literature on viral microRNAs which have been shown to alter chromatin structure by inducing methylation or even by remodeling nucleosomes. PMID:19716452

  15. Chromatin regulates origin activity in Drosophila follicle cells.

    PubMed

    Aggarwal, Bhagwan D; Calvi, Brian R

    2004-07-15

    It is widely believed that DNA replication in multicellular animals (metazoa) begins at specific origins to which a pre-replicative complex (pre-RC) binds. Nevertheless, a consensus sequence for origins has yet to be identified in metazoa. Origin identity can change during development, suggesting that there are epigenetic influences. A notable example of developmental specificity occurs in Drosophila, where somatic follicle cells of the ovary transition from genomic replication to exclusive re-replication at origins that control amplification of the eggshell (chorion) protein genes. Here we show that chromatin acetylation is critical for this developmental transition in origin specificity. We find that histones at the active origins are hyperacetylated, coincident with binding of the origin recognition complex (ORC). Mutation of the histone deacetylase (HDAC) Rpd3 induced genome-wide hyperacetylation, genomic replication and a redistribution of the origin-binding protein ORC2 in amplification-stage cells, independent of effects on transcription. Tethering Rpd3 or Polycomb proteins to the origin decreased its activity, whereas tethering the Chameau acetyltransferase increased origin activity. These results suggest that nucleosome acetylation and other epigenetic changes are important modulators of origin activity in metazoa. PMID:15254542

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

    PubMed Central

    Lee, Laura; Rodriguez, Jairo; Tsukiyama, Toshio

    2015-01-01

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

  17. Coordinated Regulation of PPARγ Expression and Activity through Control of Chromatin Structure in Adipogenesis and Obesity

    PubMed Central

    Eeckhoute, Jérôme; Oger, Frédérik; Staels, Bart; Lefebvre, Philippe

    2012-01-01

    The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is required for differentiation and function of mature adipocytes. Its expression is induced during adipogenesis where it plays a key role in establishing the transcriptome of terminally differentiated white fat cells. Here, we review findings indicating that PPARγ expression and activity are intricately regulated through control of chromatin structure. Hierarchical and combinatorial activation of transcription factors, noncoding RNAs, and chromatin remodelers allows for temporally controlled expression of PPARγ and its target genes through sequential chromatin remodelling. In obesity, these regulatory pathways may be altered and lead to modified PPARγ activity. PMID:22991504

  18. 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

  19. Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

    PubMed Central

    Chen, Lu; Ooi, Soon-Keat; Conaway, Joan W.; Conaway, Ronald C.

    2014-01-01

    Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes. PMID:25407555

  20. Activation Domain-Mediated Enhancement of Activator Binding to Chromatin in Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Bunker, Christopher A.; Kingston, Robert E.

    1996-10-01

    DNA binding by transcriptional activators is typically an obligatory step in the activation of gene expression. Activator binding and subsequent steps in transcription are repressed by genomic chromatin. Studies in vitro have suggested that overcoming this repression is an important function of some activation domains. Here we provide quantitative in vivo evidence that the activation domain of GAL4-VP16 can increase the affinity of GAL4 for its binding site on genomic DNA in mammalian cells. Moreover, the VP16 activation domain has a much greater stimulatory effect on expression from a genomic reporter gene than on a transiently transfected reporter gene, where factor binding is more permissive. We found that not all activation domains showed a greater activation potential in a genomic context, suggesting that only some activation domains can function in vivo to alleviate the repressive effects of chromatin. These data demonstrate the importance of activation domains in relieving chromatin-mediated repression in vivo and suggest that one way they function is to increase binding of the activator itself.

  1. Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains

    PubMed Central

    Ulianov, Sergey V.; Khrameeva, Ekaterina E.; Gavrilov, Alexey A.; Flyamer, Ilya M.; Kos, Pavel; Mikhaleva, Elena A.; Penin, Aleksey A.; Logacheva, Maria D.; Imakaev, Maxim V.; Chertovich, Alexander; Gelfand, Mikhail S.; Shevelyov, Yuri Y.; Razin, Sergey V.

    2016-01-01

    Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C and poly(A)+ RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin. PMID:26518482

  2. Nuclear Fractionation Reveals Thousands of Chromatin-Tethered Noncoding RNAs Adjacent to Active Genes.

    PubMed

    Werner, Michael S; Ruthenburg, Alexander J

    2015-08-18

    A number of long noncoding RNAs (lncRNAs) have been reported to regulate transcription via recruitment of chromatin modifiers or bridging distal enhancer elements to gene promoters. However, the generality of these modes of regulation and the mechanisms of chromatin attachment for thousands of unstudied human lncRNAs remain unclear. To address these questions, we performed stringent nuclear fractionation coupled to RNA sequencing. We provide genome-wide identification of human chromatin-associated lncRNAs and demonstrate tethering of RNA to chromatin by RNAPII is a pervasive mechanism of attachment. We also uncovered thousands of chromatin-enriched RNAs (cheRNAs) that share molecular properties with known lncRNAs. Although distinct from eRNAs derived from active prototypical enhancers, the production of cheRNAs is strongly correlated with the expression of neighboring protein-coding genes. This work provides an updated framework for nuclear RNA organization that includes a large chromatin-associated transcript population correlated with active genes and may prove useful in de novo enhancer annotation. PMID:26257179

  3. Diverse Activities of Histone Acylations Connect Metabolism to Chromatin Function.

    PubMed

    Dutta, Arnob; Abmayr, Susan M; Workman, Jerry L

    2016-08-18

    Modifications of histones play important roles in balancing transcriptional output. The discovery of acyl marks, besides histone acetylation, has added to the functional diversity of histone modifications. Since all modifications use metabolic intermediates as substrates for chromatin-modifying enzymes, the prevalent landscape of histone modifications in any cell type is a snapshot of its metabolic status. Here, we review some of the current findings of how differential use of histone acylations regulates gene expression as response to metabolic changes and differentiation programs. PMID:27540855

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

    PubMed

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

    2015-01-01

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

  5. Characteristic low density and shear sensitivity of cross-linked chromatin containing polycomb complexes.

    PubMed

    Schwartz, Yuri B; Kahn, Tatyana G; Pirrotta, Vincenzo

    2005-01-01

    Chromatin cross-linking is widely used for mapping the distribution of chromosomal proteins by immunoprecipitation, but our knowledge of the physical properties of chromatin complexes remains rudimentary. Density gradients have been long used to separate fragments of cross-linked chromatin with their bound proteins from free protein or free DNA. We find that the association of DNA fragments with very-high-molecular-weight protein complexes shifts their buoyant density to values much lower then that of bulk chromatin. We show that in a CsCl gradient, Polycomb response elements, promoters of active genes, and insulator or boundary elements are found at buoyant densities similar to those of free protein and are depleted from the bulk chromatin fractions. In these regions, the low density is associated with the presence of large protein complexes and with high sensitivity to sonication. Our results suggest that separation of different chromatin regions according to their buoyant density may bias chromatin immunoprecipitation results. Density centrifugation of cross-linked chromatin may provide a simple approach to investigate the properties of large chromatin complexes in vivo. PMID:15601863

  6. Xenobiotic activity in serum and sperm chromatin integrity in European and inuit populations.

    PubMed

    Krüger, Tanja; Spanò, Marcello; Long, Manhai; Eleuteri, Patrizia; Rescia, Michele; Hjelmborg, Philip S; Manicardi, Gian-Carlo; Bizzaro, Davide; Giwercman, Alexander; Toft, Gunnar; Bonde, Jens Peter; Bonefeld-Jorgensen, Eva C

    2008-04-01

    Lipophilic persistent organic pollutants (POPs) are ubiquitous in the environment and suspected to interfere with hormone activities and reproduction. In previous studies we demonstrated that POP exposure can affect sperm DNA integrity and differences between Inuits and Europeans in sperm DNA integrity and xenobiotic activity were observed. The aim of this study was to investigate possible relations between human sperm chromatin integrity and the xenobiotic serum activity of lipophilic POPs assessed as effects on the estrogen (ER), androgen (AR), and/or aryl hydrocarbon (AhR) receptors. Human sperm chromatin integrity was assessed as DNA fragmentation index (%DFI) and high DNA stainability (%HDS) using the flow cytometric sperm chromatin structure assay (SCSA). Xenobiotic receptor activities were determined using chemically activated luciferase gene expression (CALUX) assay. The study included 53 Greenlandic Inuits and 247 Europeans (Sweden, Warsaw (Poland) and Kharkiv (Ukraine)). A heterogeneous pattern of correlations was found. For Inuits, ER and AhR activities and %DFI were inversely correlated, whereas a positive correlation between AR activity and %DFI was found for Europeans. In contrast, no correlation between receptor activities and %HDS was observed for Inuits but for Europeans positive and negative correlations were observed between ER and AR activities and %HDS, respectively. We suggest that the different patterns of xenobiotic serum activities, in combination with diet associated factors and/or genetics, might be connected to the observed differences in sperm chromatin integrity between the Inuits and Europeans. PMID:18076054

  7. 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

  8. Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity.

    PubMed Central

    Almouzni, G; Méchali, M

    1988-01-01

    Undiluted extracts from eggs or oocytes of Xenopus laevis support the assembly of chromatin with physiologically spaced nucleosomes. Micrococcal nuclease and DNase I digestion experiments show that nucleosome formation as well as supercoiling of circular DNA concomitant to assembly do not require ATP or Mg2+. However these factors are essential for the stability and the physiological spacing of the assembled chromatin. gamma-S-ATP can substitute for ATP in this process. With topoisomers of defined linking number topological interconversions proceed by steps of unity, both in vitro as well as in vivo, indicating that topoisomerase I is dominantly acting in this process. Novobiocin sensitivity occurred only with diluted extracts and was unrelated to an inhibition of topoisomerase II. Finally, nucleosome assembly occurs efficiently on linear DNA although the assembled DNA is less stable than with circular DNA. From these results we propose that mature chromatin is formed in a two-step reaction. In the first step, nucleosome deposition occurs independently of ATP and Mg2+. Thus, nucleosome formation can be uncoupled from their spacing. In this step, topoisomerase activity is involved in the relaxation of the topological constraints generated by chromatin assembly rather than in the process of assembly itself. The second step, requiring ATP and Mg2+, generates properly spaced chromatin. Images PMID:2854062

  9. 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

    2016-01-01

    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

  10. Different functional modes of p300 in activation of RNA polymerase III transcription from chromatin templates.

    PubMed

    Mertens, Claudia; Roeder, Robert G

    2008-09-01

    Transcriptional coactivators that regulate the activity of human RNA polymerase III (Pol III) in the context of chromatin have not been reported. Here, we describe a completely defined in vitro system for transcription of a human tRNA gene assembled into a chromatin template. Transcriptional activation and histone acetylation in this system depend on recruitment of p300 by general initiation factor TFIIIC, thus providing a new paradigm for recruitment of histone-modifying coactivators. Beyond its role as a chromatin-modifying factor, p300 displays an acetyltransferase-independent function at the level of preinitiation complex assembly. Thus, direct interaction of p300 with TFIIIC stabilizes binding of TFIIIC to core promoter elements and results in enhanced transcriptional activity on histone-free templates. Additional studies show that p300 is recruited to the promoters of actively transcribed tRNA and U6 snRNA genes in vivo. These studies identify TFIIIC as a recruitment factor for p300 and thus may have important implications for the emerging concept that tRNA genes or TFIIIC binding sites act as chromatin barriers to prohibit spreading of silenced heterochromatin domains. PMID:18644873

  11. Different Functional Modes of p300 in Activation of RNA Polymerase III Transcription from Chromatin Templates▿

    PubMed Central

    Mertens, Claudia; Roeder, Robert G.

    2008-01-01

    Transcriptional coactivators that regulate the activity of human RNA polymerase III (Pol III) in the context of chromatin have not been reported. Here, we describe a completely defined in vitro system for transcription of a human tRNA gene assembled into a chromatin template. Transcriptional activation and histone acetylation in this system depend on recruitment of p300 by general initiation factor TFIIIC, thus providing a new paradigm for recruitment of histone-modifying coactivators. Beyond its role as a chromatin-modifying factor, p300 displays an acetyltransferase-independent function at the level of preinitiation complex assembly. Thus, direct interaction of p300 with TFIIIC stabilizes binding of TFIIIC to core promoter elements and results in enhanced transcriptional activity on histone-free templates. Additional studies show that p300 is recruited to the promoters of actively transcribed tRNA and U6 snRNA genes in vivo. These studies identify TFIIIC as a recruitment factor for p300 and thus may have important implications for the emerging concept that tRNA genes or TFIIIC binding sites act as chromatin barriers to prohibit spreading of silenced heterochromatin domains. PMID:18644873

  12. Nuclear reorganisation and chromatin decondensation are conserved, but distinct, mechanisms linked to Hox gene activation.

    PubMed

    Morey, Céline; Da Silva, Nelly R; Perry, Paul; Bickmore, Wendy A

    2007-03-01

    The relocalisation of some genes to positions outside chromosome territories, and the visible decondensation or unfolding of interphase chromatin, are two striking facets of nuclear reorganisation linked to gene activation that have been assumed to be related to each other. Here, in a study of nuclear reorganisation around the Hoxd cluster, we suggest that this may not be the case. Despite its very different genomic environment from Hoxb, Hoxd also loops out from its chromosome territory, and unfolds, upon activation in differentiating embryonic stem (ES) cells and in the tailbud of the embryo. However, looping out and decondensation are not simply two different manifestations of the same underlying change in chromatin structure. We show that, in the limb bud of the embryonic day 9.5 embryo, where Hoxd is also activated, there is visible decondensation of chromatin but no detectable movement of the region out from the chromosome territory. During ES cell differentiation, decondensed alleles can also be found inside of chromosome territories, and loci that have looped out of the territories can appear to still be condensed. We conclude that evolutionarily conserved chromosome remodelling mechanisms, predating the duplication of mammalian Hox loci, underlie Hox regulation along the rostrocaudal embryonic axis. However, we suggest that separate modes of regulation can modify Hoxd chromatin in different ways in different developmental contexts. PMID:17251268

  13. Opposing ISWI- and CHD-class chromatin remodeling activities orchestrate heterochromatic DNA repair

    PubMed Central

    Klement, Karolin; Luijsterburg, Martijn S.; Pinder, Jordan B.; Cena, Chad S.; Del Nero, Victor; Wintersinger, Christopher M.; Dellaire, Graham; van Attikum, Haico

    2014-01-01

    Heterochromatin is a barrier to DNA repair that correlates strongly with elevated somatic mutation in cancer. CHD class II nucleosome remodeling activity (specifically CHD3.1) retained by KAP-1 increases heterochromatin compaction and impedes DNA double-strand break (DSB) repair requiring Artemis. This obstruction is alleviated by chromatin relaxation via ATM-dependent KAP-1S824 phosphorylation (pKAP-1) and CHD3.1 dispersal from heterochromatic DSBs; however, how heterochromatin compaction is actually adjusted after CHD3.1 dispersal is unknown. In this paper, we demonstrate that Artemis-dependent DSB repair in heterochromatin requires ISWI (imitation switch)-class ACF1–SNF2H nucleosome remodeling. Compacted chromatin generated by CHD3.1 after DNA replication necessitates ACF1–SNF2H–mediated relaxation for DSB repair. ACF1–SNF2H requires RNF20 to bind heterochromatic DSBs, underlies RNF20-mediated chromatin relaxation, and functions downstream of pKAP-1–mediated CHD3.1 dispersal to enable DSB repair. CHD3.1 and ACF1–SNF2H display counteractive activities but similar histone affinities (via the plant homeodomains of CHD3.1 and ACF1), which we suggest necessitates a two-step dispersal and recruitment system regulating these opposing chromatin remodeling activities during DSB repair. PMID:25533843

  14. Opposing ISWI- and CHD-class chromatin remodeling activities orchestrate heterochromatic DNA repair.

    PubMed

    Klement, Karolin; Luijsterburg, Martijn S; Pinder, Jordan B; Cena, Chad S; Del Nero, Victor; Wintersinger, Christopher M; Dellaire, Graham; van Attikum, Haico; Goodarzi, Aaron A

    2014-12-22

    Heterochromatin is a barrier to DNA repair that correlates strongly with elevated somatic mutation in cancer. CHD class II nucleosome remodeling activity (specifically CHD3.1) retained by KAP-1 increases heterochromatin compaction and impedes DNA double-strand break (DSB) repair requiring Artemis. This obstruction is alleviated by chromatin relaxation via ATM-dependent KAP-1S824 phosphorylation (pKAP-1) and CHD3.1 dispersal from heterochromatic DSBs; however, how heterochromatin compaction is actually adjusted after CHD3.1 dispersal is unknown. In this paper, we demonstrate that Artemis-dependent DSB repair in heterochromatin requires ISWI (imitation switch)-class ACF1-SNF2H nucleosome remodeling. Compacted chromatin generated by CHD3.1 after DNA replication necessitates ACF1-SNF2H-mediated relaxation for DSB repair. ACF1-SNF2H requires RNF20 to bind heterochromatic DSBs, underlies RNF20-mediated chromatin relaxation, and functions downstream of pKAP-1-mediated CHD3.1 dispersal to enable DSB repair. CHD3.1 and ACF1-SNF2H display counteractive activities but similar histone affinities (via the plant homeodomains of CHD3.1 and ACF1), which we suggest necessitates a two-step dispersal and recruitment system regulating these opposing chromatin remodeling activities during DSB repair. PMID:25533843

  15. Decarbamoyl mitomycin C (DMC) activates p53-independent ataxia telangiectasia and rad3 related protein (ATR) chromatin eviction

    PubMed Central

    Xiao, Gu; Kue, Pao; Bhosle, Rahul; Bargonetti, Jill

    2015-01-01

    Interstrand crosslinks induce DNA replication fork stalling that in turn activates the ATR-dependent checkpoint and DNA repair on nuclear chromatin. Mitomycin C (MC) and Decarbamoyl Mitomycin C (DMC) induce different types of DNA crosslinks with DMC being a more cytotoxic agent. We previously reported that the novel DMC induced β–interstrand DNA crosslinks induce a p53-independent form of cell death. The p53-independent DMC cytotoxicity associates with the activation, and subsequent depletion, of Chk1. In this study we further dissect the novel DMC signal transduction pathway and asked how it influences chromatin-associated proteins. We found that treatment with DMC, but not MC, stimulated the disassociation of ATR from chromatin and re-localization of ATR to the cytoplasm. The chromatin eviction of ATR was coupled with the formation of nuclear Rad51 foci and the phosphorylation of Chk1. Furthermore, DMC but not MC, activated expression of gadd45α mRNA. Importantly, knocking down p53 via shRNA did not inhibit the DMC-induced disassociation of ATR from chromatin or reduce the activation of transcription of gadd45α. Our results suggest that DMC induces a p53-independent disassociation of ATR from chromatin that facilitates Chk1 checkpoint activation and Rad51 chromatin recruitment. Our findings provide evidence that ATR chromatin eviction in breast cancer cells is an area of study that should be focused on for inducing p53-independent cell death. PMID:25565400

  16. GATA-1 modulates the chromatin structure and activity of the chicken alpha-globin 3' enhancer.

    PubMed

    Escamilla-Del-Arenal, Martín; Recillas-Targa, Félix

    2008-01-01

    Long-distance regulatory elements and local chromatin structure are critical for proper regulation of gene expression. Here we characterize the chromatin conformation of the chicken alpha-globin silencer-enhancer elements located 3' of the domain. We found a characteristic and erythrocyte-specific structure between the previously defined silencer and the enhancer, defined by two nuclease hypersensitive sites, which appear when the enhancer is active during erythroid differentiation. Fine mapping of these sites demonstrates the absence of a positioned nucleosome and the association of GATA-1. Functional analyses of episomal vectors, as well as stably integrated constructs, revealed that GATA-1 plays a major role in defining both the chromatin structure and the enhancer activity. We detected a progressive enrichment of histone acetylation on critical enhancer nuclear factor binding sites, in correlation with the formation of an apparent nucleosome-free region. On the basis of these results, we propose that the local chromatin structure of the chicken alpha-globin enhancer plays a central role in its capacity to differentially regulate alpha-globin gene expression during erythroid differentiation and development. PMID:17984219

  17. Chromatin Remodeling Inactivates Activity Genes and Regulates Neural Coding

    PubMed Central

    Hill, Kelly K.; Hemberg, Martin; Reddy, Naveen C.; Cho, Ha Y.; Guthrie, Arden N.; Oldenborg, Anna; Heiney, Shane A.; Ohmae, Shogo; Medina, Javier F.; Holy, Timothy E.; Bonni, Azad

    2016-01-01

    Activity-dependent transcription influences neuronal connectivity, but the roles and mechanisms of inactivation of activity-dependent genes have remained poorly understood. Genome-wide analyses in the mouse cerebellum revealed that the nucleosome remodeling and deacetylase (NuRD) complex deposits the histone variant H2A.z at promoters of activity-dependent genes, thereby triggering their inactivation. Purification of translating mRNAs from synchronously developing granule neurons (Sync-TRAP) showed that conditional knockout of the core NuRD subunit Chd4 impairs inactivation of activity-dependent genes when neurons undergo dendrite pruning. Chd4 knockout or expression of NuRD-regulated activity genes impairs dendrite pruning. Imaging of behaving mice revealed hyperresponsivity of granule neurons to sensorimotor stimuli upon Chd4 knockout. Our findings define an epigenetic mechanism that inactivates activity-dependent transcription and regulates dendrite patterning and sensorimotor encoding in the brain. PMID:27418512

  18. Mutations in CHD2 cause defective association with active chromatin in chronic lymphocytic leukemia.

    PubMed

    Rodríguez, David; Bretones, Gabriel; Quesada, Víctor; Villamor, Neus; Arango, Javier R; López-Guillermo, Armando; Ramsay, Andrew J; Baumann, Tycho; Quirós, Pedro M; Navarro, Alba; Royo, Cristina; Martín-Subero, José I; Campo, Elías; López-Otín, Carlos

    2015-07-01

    Great progress has recently been achieved in the understanding of the genomic alterations driving chronic lymphocytic leukemia (CLL). Nevertheless, the specific molecular mechanisms governing chromatin remodeling in CLL are unknown. Here we report the genetic and functional characterization of somatic mutations affecting the chromatin remodeler CHD2, one of the most frequently mutated genes in CLL (5.3%) and in monoclonal B lymphocytosis (MBL, 7%), a B-cell expansion that can evolve to CLL. Most of the mutations affecting CHD2, identified by whole-exome sequencing of 456 CLL and 43 MBL patients, are either truncating or affect conserved residues in functional domains, thus supporting a putative role for CHD2 as a tumor suppressor gene. CHD2 mutants show altered nuclear distribution, and a chromodomain helicase DNA binding protein 2 (CHD2) mutant affected in its DNA-binding domain exhibits defective association with active chromatin. Clinicobiological analyses show that most CLL patients carrying CHD2 mutations also present mutated immunoglobulin heavy chain variable region genes (IGHVs), being the most frequently mutated gene in this prognostic subgroup. This is the first study providing functional evidence supporting CHD2 as a cancer driver and opens the way to further studies of the role of this chromatin remodeler in CLL. PMID:26031915

  19. Human BRCA1-BARD1 ubiquitin ligase activity counteracts chromatin barriers to DNA resection.

    PubMed

    Densham, Ruth M; Garvin, Alexander J; Stone, Helen R; Strachan, Joanna; Baldock, Robert A; Daza-Martin, Manuel; Fletcher, Alice; Blair-Reid, Sarah; Beesley, James; Johal, Balraj; Pearl, Laurence H; Neely, Robert; Keep, Nicholas H; Watts, Felicity Z; Morris, Joanna R

    2016-07-01

    The opposing activities of 53BP1 and BRCA1 influence pathway choice in DNA double-strand-break repair. How BRCA1 counteracts the inhibitory effect of 53BP1 on DNA resection and homologous recombination is unknown. Here we identify the site of BRCA1-BARD1 required for priming ubiquitin transfer from E2∼ubiquitin and demonstrate that BRCA1-BARD1's ubiquitin ligase activity is required for repositioning 53BP1 on damaged chromatin. We confirm H2A ubiquitination by BRCA1-BARD1 and show that an H2A-ubiquitin fusion protein promotes DNA resection and repair in BARD1-deficient cells. BRCA1-BARD1's function in homologous recombination requires the chromatin remodeler SMARCAD1. SMARCAD1 binding to H2A-ubiquitin and optimal localization to sites of damage and activity in DNA repair requires its ubiquitin-binding CUE domains. SMARCAD1 is required for 53BP1 repositioning, and the need for SMARCAD1 in olaparib or camptothecin resistance is alleviated by 53BP1 loss. Thus, BRCA1-BARD1 ligase activity and subsequent SMARCAD1-dependent chromatin remodeling are critical regulators of DNA repair. PMID:27239795

  20. HOS1-mediated activation of FLC via chromatin remodeling under cold stress.

    PubMed

    Jung, Jae-Hoon; Park, Chung-Mo

    2013-01-01

    The Arabidopsis E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1) has been shown to act as a negative regulator of cold responses by degrading the INDUCER OF CBF EXPRESSION 1 (ICE1) transcription factor through the ubiquitin/proteasome pathway. Notably, loss-of-function hos1 mutants exhibit early flowering, and the transcript level of the floral repressor FLOWERING LOCUS C (FLC) is downregulated in the mutants. However, it is largely unknown how HOS1 regulates FLC transcription. We found that HOS1 activates FLC transcription by inhibiting the activity of histone deacetylase 6 (HDA6) under cold stress. Cold temperatures induce the binding of HOS1 to FLC chromatin in an FVE-dependent manner. Cold-activated HOS1 promotes the dissociation of HDA6 from FLC chromatin, and the cold effects disappear in both hos1 and fve mutants. It is therefore clear that HOS1 regulates FLC transcription via chromatin remodeling, providing new insights into the signaling crosstalks between cold response and flowering time control. PMID:24390058

  1. The Costimulatory Receptor OX40 Inhibits Interleukin-17 Expression through Activation of Repressive Chromatin Remodeling Pathways.

    PubMed

    Xiao, Xiang; Shi, Xiaomin; Fan, Yihui; Wu, Chenglin; Zhang, Xiaolong; Minze, Laurie; Liu, Wentao; Ghobrial, Rafik M; Lan, Peixiang; Li, Xian Chang

    2016-06-21

    T helper 17 (Th17) cells are prominently featured in multiple autoimmune diseases, but the regulatory mechanisms that control Th17 cell responses are poorly defined. Here we found that stimulation of OX40 triggered a robust chromatin remodeling response and produced a "closed" chromatin structure at interleukin-17 (IL-17) locus to inhibit Th17 cell function. OX40 activated the NF-κB family member RelB, and RelB recruited the histone methyltransferases G9a and SETDB1 to the Il17 locus to deposit "repressive" chromatin marks at H3K9 sites, and consequently repressing IL-17 expression. Unlike its transcriptional activities, RelB acted independently of both p52 and p50 in the suppression of IL-17. In an experimental autoimmune encephalomyelitis (EAE) disease model, we found that OX40 stimulation inhibited IL-17 and reduced EAE. Conversely, RelB-deficient CD4(+) T cells showed enhanced IL-17 induction and exacerbated the disease. Our data uncover a mechanism in the control of Th17 cells that might have important clinic implications. PMID:27317259

  2. RING1A and BMI1 bookmark active genes via ubiquitination of chromatin-associated proteins

    PubMed Central

    Arora, Mansi; Packard, Colin Z.; Banerjee, Tapahsama; Parvin, Jeffrey D.

    2016-01-01

    During mitosis the chromatin undergoes dramatic architectural changes with the halting of the transcriptional processes and evacuation of nearly all transcription associated machinery from genes and promoters. Molecular bookmarking of genes during mitosis is a mechanism of faithfully transmitting cell-specific transcription patterns through cell division. We previously discovered chromatin ubiquitination at active promoters as a potential mitotic bookmark. In this study, we identify the enzymes involved in the deposition of ubiquitin before mitosis. We find that the polycomb complex proteins BMI1 and RING1A regulate the ubiquitination of chromatin associated proteins bound to promoters, and this modification is necessary for the expression of marked genes once the cells enter G1. Depletion of RING1A, and thus inactivation of mitotic bookmarking by ubiquitination, is deleterious to progression through G1, cell survival and proliferation. Though the polycomb complex proteins are thought to primarily regulate gene expression by transcriptional repression, in this study, we discover that these two polycomb proteins regulate the transcription of active genes during the mitosis to G1 transition. PMID:26578590

  3. Chromatin remodelling and autocrine TNFα are required for optimal interleukin-6 expression in activated human neutrophils.

    PubMed

    Zimmermann, Maili; Aguilera, Francisco Bianchetto; Castellucci, Monica; Rossato, Marzia; Costa, Sara; Lunardi, Claudio; Ostuni, Renato; Girolomoni, Giampiero; Natoli, Gioacchino; Bazzoni, Flavia; Tamassia, Nicola; Cassatella, Marco A

    2015-01-01

    Controversy currently exists about the ability of human neutrophils to produce IL-6. Here, we show that the chromatin organization of the IL-6 genomic locus in human neutrophils is constitutively kept in an inactive configuration. However, we also show that upon exposure to stimuli that trigger chromatin remodelling at the IL-6 locus, such as ligands for TLR8 or, less efficiently, TLR4, highly purified neutrophils express and secrete IL-6. In TLR8-activated neutrophils, but not monocytes, IL-6 expression is preceded by the induction of a latent enhancer located 14 kb upstream of the IL-6 transcriptional start site. In addition, IL-6 induction is potentiated by endogenous TNFα, which prolongs the synthesis of the IκBζ co-activator and sustains C/EBPβ recruitment and histone acetylation at IL-6 regulatory regions. Altogether, these data clarify controversial literature on the ability of human neutrophils to generate IL-6 and uncover chromatin-dependent layers of regulation of IL-6 in these cells. PMID:25616107

  4. A systematic method to identify modulation of transcriptional regulation via chromatin activity reveals regulatory network during mESC differentiation.

    PubMed

    Duren, Zhana; Wang, Yong

    2016-01-01

    Chromatin regulators (CRs) are crucial for connecting the chromatin level and transcriptome level by modulating chromatin structures, establishing, and maintaining epigenetic modifications. We present a systematic method to identify MOdulation of transcriptional regulation via CHromatin Activity (MOCHA) from gene expression data and demonstrate its advantage in associating CRs to their chromatin localization and understand CRs' function. We first re-construct the CRs modulation network by integrating the correlation and conditional correlation concepts. Then we quantify the chromatin activity as hidden variable in network by integrating the upstream and downstream information. We applied MOCHA to systematically explore the interplay of CRs, TFs, and target genes in mouse embryonic stem cells (ESC). As a result, MOCHA identified 420 chromatin regulators with modulation preference, including Pou5f1 and Eed. We found that BAF complex, NuRD complex, and polycomb-group proteins, regulate the delicate balance between pluripotency and differentiation by modulating key TFs including Klf4, Tcf3, and Max; NuRD complex members Mbd3 and Hdac1 may modulate Klf4 to achieve its dual functional roles in pluripotent and differentiation stages;Imprinted gene H19 and Igf2 are modulated by DNA methylation, histone acetylation, and insulator CTCF. Finally, we analyzed CR's combinational modulation pattern by constructing a CR-CR interaction network. PMID:26949222

  5. A systematic method to identify modulation of transcriptional regulation via chromatin activity reveals regulatory network during mESC differentiation

    PubMed Central

    Duren, Zhana; Wang, Yong

    2016-01-01

    Chromatin regulators (CRs) are crucial for connecting the chromatin level and transcriptome level by modulating chromatin structures, establishing, and maintaining epigenetic modifications. We present a systematic method to identify MOdulation of transcriptional regulation via CHromatin Activity (MOCHA) from gene expression data and demonstrate its advantage in associating CRs to their chromatin localization and understand CRs’ function. We first re-construct the CRs modulation network by integrating the correlation and conditional correlation concepts. Then we quantify the chromatin activity as hidden variable in network by integrating the upstream and downstream information. We applied MOCHA to systematically explore the interplay of CRs, TFs, and target genes in mouse embryonic stem cells (ESC). As a result, MOCHA identified 420 chromatin regulators with modulation preference, including Pou5f1 and Eed. We found that BAF complex, NuRD complex, and polycomb-group proteins, regulate the delicate balance between pluripotency and differentiation by modulating key TFs including Klf4, Tcf3, and Max; NuRD complex members Mbd3 and Hdac1 may modulate Klf4 to achieve its dual functional roles in pluripotent and differentiation stages;Imprinted gene H19 and Igf2 are modulated by DNA methylation, histone acetylation, and insulator CTCF. Finally, we analyzed CR’s combinational modulation pattern by constructing a CR-CR interaction network. PMID:26949222

  6. Isolation of an activator-dependent, promoter-specific chromatin remodeling factor

    PubMed Central

    Ehrensberger, Andreas H.; Kornberg, Roger D.

    2011-01-01

    Repressed PHO5 gene chromatin, isolated from yeast in the native state, was remodeled by yeast extract in a gene activator-dependent, ATP-dependent manner. The product of the reaction bore the hallmark of the process in vivo, the selective removal of promoter nucleosomes, without effect on open reading frame nucleosomes. Fractionation of the extract identified a single protein, chromodomain helicase DNA binding protein 1 (Chd1), capable of the remodeling activity. Deletion of the CHD1 gene in an isw1Δ pho80Δ strain abolished PHO5 gene expression, demonstrating the relevance of the remodeling reaction in vitro to the process in vivo. PMID:21646535

  7. Isolation of an activator-dependent, promoter-specific chromatin remodeling factor.

    PubMed

    Ehrensberger, Andreas H; Kornberg, Roger D

    2011-06-21

    Repressed PHO5 gene chromatin, isolated from yeast in the native state, was remodeled by yeast extract in a gene activator-dependent, ATP-dependent manner. The product of the reaction bore the hallmark of the process in vivo, the selective removal of promoter nucleosomes, without effect on open reading frame nucleosomes. Fractionation of the extract identified a single protein, chromodomain helicase DNA binding protein 1 (Chd1), capable of the remodeling activity. Deletion of the CHD1 gene in an isw1Δ pho80Δ strain abolished PHO5 gene expression, demonstrating the relevance of the remodeling reaction in vitro to the process in vivo. PMID:21646535

  8. The Arabidopsis SWI2/SNF2 chromatin Remodeler BRAHMA regulates polycomb function during vegetative development and directly activates the flowering repressor gene SVP.

    PubMed

    Li, Chenlong; Chen, Chen; Gao, Lei; Yang, Songguang; Nguyen, Vi; Shi, Xuejiang; Siminovitch, Katherine; Kohalmi, Susanne E; Huang, Shangzhi; Wu, Keqiang; Chen, Xuemei; Cui, Yuhai

    2015-01-01

    The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development. PMID:25615622

  9. The chromatin remodelling factor Brg-1 interacts with β-catenin to promote target gene activation

    PubMed Central

    Barker, Nick; Hurlstone, Adam; Musisi, Hannah; Miles, Antony; Bienz, Mariann; Clevers, Hans

    2001-01-01

    Wnt-induced formation of nuclear Tcf–β-catenin complexes promotes transcriptional activation of target genes involved in cell fate decisions. Inappropriate expression of Tcf target genes resulting from mutational activation of this pathway is also implicated in tumorigenesis. The C-terminus of β-catenin is indispensable for the transactivation function, which probably reflects the presence of binding sites for essential transcriptional coactivators such as p300/CBP. However, the precise mechanism of transactivation remains unclear. Here we demonstrate an interaction between β-catenin and Brg-1, a component of mammalian SWI/SNF and Rsc chromatin-remodelling complexes. A functional consequence of reintroduction of Brg-1 into Brg-1-deficient cells is enhanced activity of a Tcf-responsive reporter gene. Consistent with this, stable expression of inactive forms of Brg-1 in colon carcinoma cell lines specifically inhibits expression of endogenous Tcf target genes. In addition, we observe genetic interactions between the Brg-1 and β-catenin homologues in flies. We conclude that β-catenin recruits Brg-1 to Tcf target gene promoters, facilitating chromatin remodelling as a prerequisite for transcriptional activation. PMID:11532957

  10. Pioneering Activity of the C-Terminal Domain of EBF1 Shapes the Chromatin Landscape for B Cell Programming.

    PubMed

    Boller, Sören; Ramamoorthy, Senthilkumar; Akbas, Duygu; Nechanitzky, Robert; Burger, Lukas; Murr, Rabih; Schübeler, Dirk; Grosschedl, Rudolf

    2016-03-15

    Lymphopoiesis requires the activation of lineage-specific genes embedded in naive, inaccessible chromatin or in primed, accessible chromatin. The mechanisms responsible for de novo gain of chromatin accessibility, known as "pioneer" function, remain poorly defined. Here, we showed that the EBF1 C-terminal domain (CTD) is required for the regulation of a specific gene set involved in B cell fate decision and differentiation, independently of activation and repression functions. Using genome-wide analysis of DNaseI hypersensitivity and DNA methylation in multipotent Ebf1(-/-) progenitors and derivative EBF1wt- or EBF1ΔC-expressing cells, we found that the CTD promoted chromatin accessibility and DNA demethylation in previously naive chromatin. The CTD allowed EBF1 to bind at inaccessible genomic regions that offer limited co-occupancy by other transcription factors, whereas the CTD was dispensable for EBF1 binding at regions that are occupied by multiple transcription factors. Thus, the CTD enables EBF1 to confer permissive lineage-specific changes in progenitor chromatin landscape. PMID:26982363

  11. HMGN proteins modulate chromatin regulatory sites and gene expression during activation of naïve B cells

    PubMed Central

    Zhang, Shaofei; Zhu, Iris; Deng, Tao; Furusawa, Takashi; Rochman, Mark; Vacchio, Melanie S.; Bosselut, Remy; Yamane, Arito; Casellas, Rafael; Landsman, David; Bustin, Michael

    2016-01-01

    The activation of naïve B lymphocyte involves rapid and major changes in chromatin organization and gene expression; however, the complete repertoire of nuclear factors affecting these genomic changes is not known. We report that HMGN proteins, which bind to nucleosomes and affect chromatin structure and function, co-localize with, and maintain the intensity of DNase I hypersensitive sites genome wide, in resting but not in activated B cells. Transcription analyses of resting and activated B cells from wild-type and Hmgn−/− mice, show that loss of HMGNs dampens the magnitude of the transcriptional response and alters the pattern of gene expression during the course of B-cell activation; defense response genes are most affected at the onset of activation. Our study provides insights into the biological function of the ubiquitous HMGN chromatin binding proteins and into epigenetic processes that affect the fidelity of the transcriptional response during the activation of B cell lymphocytes. PMID:27112571

  12. The Chromatin Remodeler CHD8 Is Required for Activation of Progesterone Receptor-Dependent Enhancers

    PubMed Central

    Giannopoulou, Eugenia G.; Soronellas, Daniel; Vázquez-Chávez, Elena; Vicent, Guillermo P.; Elemento, Olivier; Beato, Miguel; Reyes, José C.

    2015-01-01

    While the importance of gene enhancers in transcriptional regulation is well established, the mechanisms and the protein factors that determine enhancers activity have only recently begun to be unravelled. Recent studies have shown that progesterone receptor (PR) binds regions that display typical features of gene enhancers. Here, we show by ChIP-seq experiments that the chromatin remodeler CHD8 mostly binds promoters under proliferation conditions. However, upon progestin stimulation, CHD8 re-localizes to PR enhancers also enriched in p300 and H3K4me1. Consistently, CHD8 depletion severely impairs progestin-dependent gene regulation. CHD8 binding is PR-dependent but independent of the pioneering factor FOXA1. The SWI/SNF chromatin-remodelling complex is required for PR-dependent gene activation. Interestingly, we show that CHD8 interacts with the SWI/SNF complex and that depletion of BRG1 and BRM, the ATPases of SWI/SNF complex, impairs CHD8 recruitment. We also show that CHD8 is not required for H3K27 acetylation, but contributes to increase accessibility of the enhancer to DNaseI. Furthermore, CHD8 was required for RNAPII recruiting to the enhancers and for transcription of enhancer-derived RNAs (eRNAs). Taken together our data demonstrate that CHD8 is involved in late stages of PR enhancers activation. PMID:25894978

  13. CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene

    PubMed Central

    Liu, Yu-Chin; Chang, Pu-Yuan; Chao, Chuck C.-K.

    2015-01-01

    In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy. PMID:26384430

  14. CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene.

    PubMed

    Liu, Yu-Chin; Chang, Pu-Yuan; Chao, Chuck C-K

    2015-12-15

    In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy. PMID:26384430

  15. Mutation of the TERT promoter, switch to active chromatin, and monoallelic TERT expression in multiple cancers.

    PubMed

    Stern, Josh Lewis; Theodorescu, Dan; Vogelstein, Bert; Papadopoulos, Nickolas; Cech, Thomas R

    2015-11-01

    Somatic mutations in the promoter of the gene for telomerase reverse transcriptase (TERT) are the most common noncoding mutations in cancer. They are thought to activate telomerase, contributing to proliferative immortality, but the molecular events driving TERT activation are largely unknown. We observed in multiple cancer cell lines that mutant TERT promoters exhibit the H3K4me2/3 mark of active chromatin and recruit the GABPA/B1 transcription factor, while the wild-type allele retains the H3K27me3 mark of epigenetic silencing; only the mutant promoters are transcriptionally active. These results suggest how a single-base-pair mutation can cause a dramatic epigenetic switch and monoallelic expression. PMID:26515115

  16. Mutation of the TERT promoter, switch to active chromatin, and monoallelic TERT expression in multiple cancers

    PubMed Central

    Stern, Josh Lewis; Theodorescu, Dan; Vogelstein, Bert; Papadopoulos, Nickolas; Cech, Thomas R.

    2015-01-01

    Somatic mutations in the promoter of the gene for telomerase reverse transcriptase (TERT) are the most common noncoding mutations in cancer. They are thought to activate telomerase, contributing to proliferative immortality, but the molecular events driving TERT activation are largely unknown. We observed in multiple cancer cell lines that mutant TERT promoters exhibit the H3K4me2/3 mark of active chromatin and recruit the GABPA/B1 transcription factor, while the wild-type allele retains the H3K27me3 mark of epigenetic silencing; only the mutant promoters are transcriptionally active. These results suggest how a single-base-pair mutation can cause a dramatic epigenetic switch and monoallelic expression. PMID:26515115

  17. Inhibitors of Histone Deacetylase and DNA Methyltransferase Synergistically Activate the Methylated Metallothionein I Promoter by Activating the Transcription Factor MTF-1 and Forming an Open Chromatin Structure

    PubMed Central

    Ghoshal, Kalpana; Datta, Jharna; Majumder, Sarmila; Bai, Shoumei; Dong, Xiaocheng; Parthun, Mark; Jacob, Samson T.

    2002-01-01

    Inhibitors of DNA methyltransferase (Dnmt) and histone deacetylases (HDAC) synergistically activate the methylated metallothionein I gene (MT-I) promoter in mouse lymphosarcoma cells. The cooperative effect of these two classes of inhibitors on MT-I promoter activity was robust following demethylation of only a few CpG dinucleotides by brief exposure to 5-azacytidine (5-AzaC) but persisted even after prolonged treatment with the nucleoside analog. HDAC inhibitors (trichostatin A [TSA] and depsipeptide) either alone or in combination with 5-AzaC did not facilitate demethylation of the MT-I promoter. Treatment of cells with HDAC inhibitors increased accumulation of multiply acetylated forms of H3 and H4 histones that remained unaffected after treatment with 5-AzaC. Chromatin immunoprecipitation (ChIP) assay showed increased association of acetylated histone H4 and lysine 9 (K9)-acetyl H3 with the MT-I promoter after treatment with TSA, which was not affected following treatment with 5-AzaC. In contrast, the association of K9-methyl histone H3 with the MT-I promoter decreased significantly after treatment with 5-AzaC and TSA. ChIP assay with antibodies specific for methyl-CpG binding proteins (MBDs) demonstrated that only methyl-CpG binding protein 2 (MeCP2) was associated with the MT-I promoter, which was significantly enhanced after TSA treatment. Association of histone deacetylase 1 (HDAC1) with the promoter decreased after treatment with TSA or 5-AzaC and was abolished after treatment with both inhibitors. Among the DNA methyltransferases, both Dnmt1 and Dnmt3a were associated with the MT-I promoter in the lymphosarcoma cells, and association of Dnmt1 decreased with time after treatment with 5-AzaC. Treatment of these cells with HDAC inhibitors also increased expression of the MTF-1 (metal transcription factor-1) gene as well as its DNA binding activity. In vivo genomic footprinting studies demonstrated increased occupancy of MTF-1 to metal response elements of

  18. In vivo haematopoietic activity is induced in neurosphere cells by chromatin-modifying agents

    PubMed Central

    Schmittwolf, Carolin; Kirchhof, Nicole; Jauch, Anna; Dürr, Michael; Harder, Friedrich; Zenke, Martin; Müller, Albrecht M

    2005-01-01

    Modifications of DNA and chromatin are fundamental for the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. To test whether the developmental potential of fetal brain-derived cells that form floating sphere colonies (neurospheres) can be modified by destabilizing their epigenotype, neurosphere cells were treated with chemical compounds that alter the acetylation and methylation patterns of chromatin and DNA. Intravenous infusion of bulk or clonally derived neurosphere cells treated with a combination of trichostatin A (TSA) plus 5-aza-2′-deoxycytidine (AzaC) (TSA/AzaC neurosphere cells) yielded long-term, multilineage and transplantable neurosphere-derived haematopoietic repopulation. Untreated neurosphere cells exhibited no haematopoietic repopulation activity. The neurosphere-derived haematopoietic cells showed a diploid karyotype, indicating that they are unlikely to be products of cell fusion events, a conclusion strengthened by multicolour fluorescence in situ hybridization. Our results indicate that altering the epigenotype of neurosphere cells followed by transplantation enables the generation of neurosphere-derived haematopoietic cells. PMID:15660132

  19. Control of α-herpesvirus IE gene expression by HCF-1 coupled chromatin modification activities

    PubMed Central

    Kristie, Thomas M.; Liang, Yu; Vogel, Jodi L.

    2009-01-01

    Summary The immediate early genes of the α-herpesviruses HSV and VZV are transcriptionally regulated by viral and cellular factors in a complex combinatorial manner. Despite this complexity and the apparent redundancy of activators, the expression of the viral IE genes is critically dependent upon the cellular transcriptional coactivator HCF-1. Although the role of HCF-1 had remained elusive, recent studies have demonstrated that the protein is a component of multiple chromatin modification complexes including the Set1/MLL1 histone H3K4 methyltransferases. Studies using model viral promoter-reporter systems as well as analyses of components recruited to the viral genome during the initiation of infection have elucidated the significance of HCF-1 chromatin modification complexes in contributing to the final state of modified histones assembled on the viral IE promoters. Strikingly, the absence of HCF-1 results in the accumulation of nucleosomes bearing repressive marks on the viral IE promoters and silencing of viral gene expression. PMID:19682612

  20. TSH stimulates 32P-labeling of thyroid nuclear HMG 14, a protein associated with actively transcribed chromatin

    SciTech Connect

    Cooper, E.; Palmer, R.J.; Spaulding, S.W.

    1982-04-01

    Thyroid slices were incubated with 32P with or without TSH. 32P-labeling of acid-soluble nuclear proteins was then examined by two-dimensional polyacrylamide gel electrophoresis and autoradiography. We found that TSH enhanced the labeling of the high mobility group protein HMG 14, a protein that is preferentially associated with actively transcribed chromatin. This observation suggests that changes in HMG 14 phosphorylation may be involved in mediating TSH-induced effects on the structure and function of active chromatin.

  1. Butyrate Induced IGF2 Activation Correlated with Distinct Chromatin Signatures Due to Histone Modification.

    PubMed

    Shin, Joo Heon; Li, Robert W; Gao, Yuan; Bickhart, Derek M; Liu, George E; Li, Weizhong; Wu, Sitao; Li, Cong-Jun

    2013-01-01

    Histone modification has emerged as a very important mechanism regulating the transcriptional status of the genome. Insulin-like growth factor 2 (IGF2) is a peptide hormone controlling various cellular processes, including proliferation and apoptosis. H19 gene is closely linked to IGF2 gene, and IGF2 and H19 are reciprocally regulated imprinted genes. The epigenetic signature of H19 promoter (hypermethylation) on the paternal allele plays a vital role in allowing the expression of the paternal allele of IGF2.46 Our previous studies demonstrate that butyrate regulates the expression of IGF2 as well as genes encoding IGF Binding proteins. To obtain further understanding of histone modification and its regulatory potentials in controlling IGF2/H19 gene expression, we investigated the histone modification status of some key histones associated with the expression of IGF2/H19 genes in bovine cells using RNA-seq in combination with Chip-seq technology. A high-resolution map of the major chromatin modification at the IGF2/H19 locus induced by butyrate was constructed to illustrate the fundamental association of the chromatin modification landscape that may play a role in the activation of the IGF2 gene. High-definition epigenomic landscape mapping revealed that IGF2 and H19 have distinct chromatin modification patterns at their coding and promoter regions, such as TSSs and TTSs. Moreover, the correlation between the differentially methylated regions (DMRs) of IGF2/H19 locus and histone modification (acetylation and methylation) indicated that epigenetic signatures/markers of DNA methylation, histone methylation and histone acetylation were differentially distributed on the expressed IGF2 and silenced H19 genes. Our evidence also suggests that butyrate-induced regional changes of histone acetylation statusin the upstream regulation domain of H19 may be related to the reduced expression of H19 and strong activation of IGF2. Our results provided insights into the mechanism

  2. Analysis of Active and Inactive X Chromosome Architecture Reveals the Independent Organization of 30 nm and Large-Scale Chromatin Structures

    PubMed Central

    Naughton, Catherine; Sproul, Duncan; Hamilton, Charlotte; Gilbert, Nick

    2010-01-01

    Summary Using a genetic model, we present a high-resolution chromatin fiber analysis of transcriptionally active (Xa) and inactive (Xi) X chromosomes packaged into euchromatin and facultative heterochromatin. Our results show that gene promoters have an open chromatin structure that is enhanced upon transcriptional activation but the Xa and the Xi have similar overall 30 nm chromatin fiber structures. Therefore, the formation of facultative heterochromatin is dependent on factors that act at a level above the 30 nm fiber and transcription does not alter bulk chromatin fiber structures. However, large-scale chromatin structures on Xa are decondensed compared with the Xi and transcription inhibition is sufficient to promote large-scale chromatin compaction. We show a link between transcription and large-scale chromatin packaging independent of the bulk 30 nm chromatin fiber and propose that transcription, not the global compaction of 30 nm chromatin fibers, determines the cytological appearance of large-scale chromatin structures. PMID:21070966

  3. Biochemical Analysis of Distinct Activation Functions in p300 That Enhance Transcription Initiation with Chromatin Templates

    PubMed Central

    Kraus, W. Lee; Manning, E. Tory; Kadonaga, James T.

    1999-01-01

    To investigate the mechanisms of transcriptional enhancement by the p300 coactivator, we analyzed wild-type and mutant versions of p300 with a chromatin transcription system in vitro. Estrogen receptor, NF-κB p65 plus Sp1, and Gal4-VP16 were used as different sequence-specific activators. The CH3 domain (or E1A-binding region) was found to be essential for the function of each of the activators tested. The bromodomain was also observed to be generally important for p300 coactivator activity, though to a lesser extent than the CH3 domain/E1A-binding region. The acetyltransferase activity and the C-terminal region (containing the steroid receptor coactivator/p160-binding region and the glutamine-rich region) were each found to be important for activation by estrogen receptor but not for that by Gal4-VP16. The N-terminal region of p300, which had been previously found to interact with nuclear hormone receptors, was not seen to be required for any of the activators, including estrogen receptor. Single-round transcription experiments revealed that the functionally important subregions of p300 contribute to its ability to promote the assembly of transcription initiation complexes. In addition, the acetyltransferase activity of p300 was observed to be distinct from the broadly essential activation function of the CH3 domain/E1A-binding region. These results indicate that specific regions of p300 possess distinct activation functions that are differentially required to enhance the assembly of transcription initiation complexes. Interestingly, with the estrogen receptor, four distinct regions of p300 each have an essential role in the transcription activation process. These data exemplify a situation in which a network of multiple activation functions is required to achieve gene transcription. PMID:10567538

  4. Butyrate-induced changes in nuclease sensitivity of chromatin cannot be correlated with transcriptional activation

    SciTech Connect

    Birren, B.W.; Taplitz, S.J.; Herschman, H.R.

    1987-11-01

    The authors examined in the H4IIE rat heptoma cell line the relationship between butyrate-induced changes in the nuclease sensitivity of chromatin and changes in transcriptional activity of specific genes. The butyrate-inducible metallothionein I (MT-I) gene underwent a dramatic increase in DNase I sensitivity after 3 h of butyrate treatment. However, genes not transcribed in H4IIE cells underwent the same changes in DNase I sensitivity. Thus, butyrate-induced increases in DNase I sensitivity are not sufficient for the transcriptional activation of a gene. Butyrate treatment has also been reported to alter the sensitivity of sequence to micrococcal nuclease (MNase) in a manner reflecting their tissue-specific expression. Butyrate exposure caused increased digestion of the MT-I gene by MNase. However, butyrate-induced MNase sensitivity also occurred for genes which are neither transcribed in untreated cells nor butyrate inducible. Moreover, cadmium, a potent transcriptional activator of the MT-I gene, does not alter the sensitivity of the MT-I gene to MNase. Thus, the butyrate-induced alterations in MNase sensitivity are neither sufficient for, necessary for, nor indicative of transcriptional activation.

  5. Membrane-associated glucocorticoid activity is necessary for modulation of long-term memory via chromatin modification

    PubMed Central

    Roozendaal, Benno; Hernandez, Angelina; Cabrera, Sara M.; Hagewoud, Roelina; Malvaez, Melissa; Stefanko, Daniel P.; Haettig, Jakob; Wood, Marcelo A.

    2010-01-01

    Glucocorticoid hormones enhance the consolidation of long-term memory of emotionally arousing training experiences. This memory enhancement requires activation of the cAMP-dependent kinase pathway and the subsequent phosphorylation of cAMP response-element binding (CREB) protein. Here, we demonstrate that glucocorticoids enhance the consolidation of hippocampus-dependent and hippocampus-independent aspects of object recognition memory via chromatin modification. More specifically, systemic corticosterone increases histone acetylation, a form of chromatin modification, in both the hippocampus and insular cortex following training on an object recognition task. This led us to examine whether increasing histone acetylation via histone deacetylase (HDAC) inhibition enhances memory in a similar manner as corticosterone. We found a double dissociation between posttraining HDAC inhibitor infusion into the insular cortex and hippocampus on the enhancement of object recognition and object location memory, respectively. In determining the molecular pathway upstream of glucocorticoids’ effects on chromatin modification, we found that activation of membrane-associated glucocorticoid receptors (GRs) and the subsequent interaction between phospho-CREB and CREB-binding protein (CBP) appear to be necessary for glucocorticoids to enhance memory consolidation via chromatin modification. In contrast, mineralocorticoid receptors (MRs) do not appear to be involved. The findings also indicate that glucocorticoid activity has differential influences on hippocampus-dependent and hippocampus-independent components of memory for objects. PMID:20371824

  6. Chromatin Computation

    PubMed Central

    Bryant, Barbara

    2012-01-01

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

  7. Upstream activation sequence-dependent alteration of chromatin structure and transcription activation of the yeast GAL1-GAL10 genes.

    PubMed Central

    Fedor, M J; Kornberg, R D

    1989-01-01

    Conversion of the positioned nucleosome array characteristic of the repressed GAL1-GAL10 promoter region to the more accessible conformation of the induced state was found to depend on the upstream activation sequence, GAL4 protein, a positive regulator of transcription, and galactose, the inducing agent. The effect of the GAL4 protein-upstream activation sequence complex on the structure of adjacent chromatin required no other promoter sequences. Although sequences protected by histones in the repressed state became more accessible to micrococcal nuclease and (methidiumpropyl-EDTA)iron(II) cleavage following induction of transcription, DNA-protein particles containing these sequences retained the electrophoretic mobility of nucleosomes, indicating that the promoter region can be associated with nucleosomes under conditions of transcription activation. Images PMID:2657404

  8. TOUSLED Kinase Activity Oscillates during the Cell Cycle and Interacts with Chromatin Regulators1

    PubMed Central

    Ehsan, Hashimul; Reichheld, Jean-Philippe; Durfee, Tim; Roe, Judith L.

    2004-01-01

    The TOUSLED (TSL)-like nuclear protein kinase family is highly conserved in plants and animals. tsl loss of function mutations cause pleiotropic defects in both leaf and flower development, and growth and initiation of floral organ primordia is abnormal, suggesting that basic cellular processes are affected. TSL is more highly expressed in exponentially growing Arabidopsis culture cells than in stationary, nondividing cells. While its expression remains constant throughout the cell cycle in dividing cells, TSL kinase activity is higher in enriched late G2/M-phase and G1-phase populations of Arabidopsis suspension culture cells compared to those in S-phase. tsl mutants also display an aberrant pattern and increased expression levels of the mitotic cyclin gene CycB1;1, suggesting that TSL represses CycB1;1 expression at certain times during development or that cells are delayed in mitosis. TSL interacts with and phosphorylates one of two Arabidopsis homologs of the nucleosome assembly/silencing protein Asf1 and histone H3, as in humans, and a novel plant SANT/myb-domain protein, TKI1, suggesting that TSL plays a role in chromatin metabolism. PMID:15047893

  9. Chromatin transitions during activation and repression of galactose-regulated genes in yeast.

    PubMed Central

    Cavalli, G; Thoma, F

    1993-01-01

    To study the fate of nucleosomes during transcription, a yeast gene 'GAL-URARIB' was constructed which is tightly regulated by the GAL1 promoter and shows in its inactive state a series of positioned nucleosomes that are sensitive for monitoring structural changes by micrococcal nuclease. Upon transcriptional activation, nucleosome positions were lost, but a residual nucleosomal repeat with an altered repeat length and no changes in psoralen accessibility measured by a band shift assay indicated that nucleosomes were present but rearranged on the transcribed gene. When chromatin was prepared 10 or 50 min after glucose repression, nucleosomes were repositioned in a large fraction of the population by a rapid process which most likely did not depend on histone synthesis or DNA replication. However, complete regeneration of the inactive structure and repeat length was observed after one cell generation (2.5 h) suggesting that in this step some missing histones were replaced. The results are consistent with a local dissociation of nucleosomes at the site of the polymerase followed by a rapid reassembly into nucleosomes behind it. The data are further supported by analysis of the chromosomal GAL1, GAL7 and GAL10 genes. Images PMID:8223470

  10. Identification of Arabidopsis SUMO-interacting proteins that regulate chromatin activity and developmental transitions

    PubMed Central

    Elrouby, Nabil; Bonequi, Mitzi Villajuana; Porri, Aimone; Coupland, George

    2013-01-01

    Posttranslational modification of proteins by small ubiquitin-like modifier (SUMO) plays essential roles in eukaryotic growth and development. Many covalently modified SUMO targets have been identified; however, the extent and significance of noncovalent interactions of SUMO with cellular proteins is poorly understood. Here, large-scale yeast two-hybrid screens repeatedly identified a surprisingly small number of proteins that interacted with three Arabidopsis SUMO isoforms. These SUMO-interacting proteins are nuclear and fall into two main categories: six histone or DNA methyltransferses or demethylases and six proteins that we show to be the evolutionary and functional homologs of SUMO-targeted ubiquitin ligases (STUbLs). The selectivity of the screen for several methylases and demethylases suggests that SUMO interaction with these proteins has a significant impact on chromatin methylation. Furthermore, the Arabidopsis STUbLs (AT-STUbLs) complemented to varying degrees the growth defects of the Schizosaccharomyces pombe STUbL mutant rfp1/rfp2, and three of them also complemented the genome integrity defects of this mutant, demonstrating that these proteins show STUbL activity. We show that one of the AT-STUbLs least related to the S. pombe protein, AT-STUbL4, has acquired a plant-specific function in the floral transition. It reduces protein levels of CYCLING DOF FACTOR 2, hence increasing transcript levels of CONSTANS and promoting flowering through the photoperiodic pathway. PMID:24255109

  11. Chromatin, TAFs, and a novel multiprotein coactivator are required for synergistic activation by Sp1 and SREBP-1a in vitro.

    PubMed

    Näär, A M; Beaurang, P A; Robinson, K M; Oliner, J D; Avizonis, D; Scheek, S; Zwicker, J; Kadonaga, J T; Tjian, R

    1998-10-01

    The promoter selectivity factor Sp1 often cooperates with other enhancer-binding proteins to activate transcription. To study the molecular underpinnings of these regulatory events, we have reconstituted in vitro the synergy observed in vivo between Sp1 and the sterol-regulated factor SREBP-1a at the low density lipoprotein receptor (LDLR) promoter. Using a highly purified human transcription system, we found that chromatin, TAFs, and a novel SREBP-binding coactivator activity, which includes CBP, are all required to mediate full synergistic activation by Sp1 and SREBP-1a. The SREBP-binding domain of CBP inhibits activation by SREBP-1a and Sp1 in a dominant-negative fashion that is both chromatin- and activator-specific. Whereas recombinant CBP alone is not sufficient to mediate activation, a human cellular fraction containing CBP can support high levels of chromatin-dependent synergistic activation. Purification of this activity to near homogeneity resulted in the identification of a multiprotein coactivator, including CBP, that selectively binds to the SREBP-1a activation domain and is capable of mediating high levels of synergistic activation by SREBP/Sp1 on chromatin templates. The development of a reconstituted chromatin transcription system has allowed us to isolate a novel coactivator that is recruited by the SREBP-1a activation domain and that functions in concert with TFIID to coordinate the action of multiple activators at complex promoters in the context of chromatin. PMID:9765204

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

    PubMed

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

    2016-02-01

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

  13. Transcription Factor AP1 Potentiates Chromatin Accessibility and Glucocorticoid Receptor Binding

    PubMed Central

    Biddie, Simon C.; John, Sam; Sabo, Pete J.; Thurman, Robert E.; Johnson, Thomas A.; Schiltz, R. Louis; Miranda, Tina B.; Sung, Myong-Hee; Trump, Saskia; Lightman, Stafford L.; Vinson, Charles; Stamatoyannopoulos, John A.; Hager, Gordon L.

    2011-01-01

    Summary Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with co-regulators. The role of these interactions in determining selective binding of GR to regulatory elements remains unclear. Recent findings indicate a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing, we identify the activator protein 1 (AP1) as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. We propose a model where the basal occupancy of transcription factors act to prime chromatin and direct inducible transcription factors to select regions in the genome. PMID:21726817

  14. SATB1 Packages Densely Looped, Transcriptionally Active Chromatin for Coordinated Expression of Cytokine Genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    SATB1 (special AT-rich sequence binding protein 1) organizes cell type–specific nuclear architecture by anchoring specialized DNA sequences and recruiting chromatin remodeling factors to control gene transcription. We studied the role of SATB1 in regulating the coordinated expression of Il5, Il4 and...

  15. Cas9 Functionally Opens Chromatin.

    PubMed

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

    2016-01-01

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

  16. 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

  17. A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression1,2,3

    PubMed Central

    Oey, Nicodemus E.; Leung, How Wing; Ezhilarasan, Rajaram; Zhou, Lei; Beuerman, Roger W.; VanDongen, Hendrika M.A.

    2015-01-01

    Abstract Chromatin modification is an important epigenetic mechanism underlying neuroplasticity. Histone methylation and acetylation have both been shown to modulate gene expression, but the machinery responsible for mediating these changes in neurons has remained elusive. Here we identify a chromatin-modifying complex containing the histone demethylase PHF8 and the acetyltransferase TIP60 as a key regulator of the activity-induced expression of Arc, an important mediator of synaptic plasticity. Clinically, mutations in PHF8 cause X-linked mental retardation while TIP60 has been implicated in the pathogenesis of Alzheimer’s disease. Within minutes of increased synaptic activity, this dual function complex is rapidly recruited to the Arc promoter, where it specifically counteracts the transcriptionally repressive histone mark H3K9me2 to facilitate the formation of the transcriptionally permissive H3K9acS10P, thereby favoring transcriptional activation. Consequently, gain-of-function of the PHF8−TIP60 complex in primary rat hippocampal neurons has a positive effect on early activity-induced Arc gene expression, whereas interfering with the function of this complex abrogates it. A global proteomics screen revealed that the majority of common interactors of PHF8 and TIP60 were involved in mRNA processing, including PSF, an important molecule involved in neuronal gene regulation. Finally, we proceeded to show, using super-resolution microscopy, that PHF8 and TIP60 interact at the single molecule level with PSF, thereby situating this chromatin modifying complex at the crossroads of transcriptional activation. These findings point toward a mechanism by which an epigenetic pathway can regulate neuronal activity-dependent gene transcription, which has implications in the development of novel therapeutics for disorders of learning and memory. PMID:26464965

  18. Dynamics of the Ternary Complex Formed by c-Myc Interactor JPO2, Transcriptional Co-activator LEDGF/p75, and Chromatin*

    PubMed Central

    Hendrix, Jelle; van Heertum, Bart; Vanstreels, Els; Daelemans, Dirk; De Rijck, Jan

    2014-01-01

    Lens epithelium-derived growth factor (LEDGF/p75) is a transcriptional co-activator involved in targeting human immunodeficiency virus (HIV) integration and the development of MLL fusion-mediated acute leukemia. A previous study revealed that LEDGF/p75 dynamically scans the chromatin, and upon interaction with HIV-1 integrase, their complex is locked on chromatin. At present, it is not known whether LEDGF/p75-mediated chromatin locking is typical for interacting proteins. Here, we employed continuous photobleaching and fluorescence correlation and cross-correlation spectroscopy to investigate in vivo chromatin binding of JPO2, a LEDGF/p75- and c-Myc-interacting protein involved in transcriptional regulation. In the absence of LEDGF/p75, JPO2 performs chromatin scanning inherent to transcription factors. However, whereas the dynamics of JPO2 chromatin binding are decelerated upon interaction with LEDGF/p75, very strong locking of their complex onto chromatin is absent. Similar results were obtained with the domesticated transposase PogZ, another cellular interaction partner of LEDGF/p75. We furthermore show that diffusive JPO2 can oligomerize; that JPO2 and LEDGF/p75 interact directly and specifically in vivo through the specific interaction domain of JPO2 and the C-terminal domain of LEDGF/p75, comprising the integrase-binding domain; and that modulation of JPO2 dynamics requires a functional PWWP domain in LEDGF/p75. Our results suggest that the dynamics of the LEDGF/p75-chromatin interaction depend on the specific partner and that strong chromatin locking is not a property of all LEDGF/p75-binding proteins. PMID:24634210

  19. Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

    SciTech Connect

    Xu, Ren; Nelson, Celeste M.; Muschler, John L.; Veiseh, Mandana; Vonderhaar, Barbara K.; Bissell, Mina J.

    2009-06-03

    Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We show that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and {beta}-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.

  20. Multiple single-stranded cis elements are associated with activated chromatin of the human c-myc gene in vivo.

    PubMed Central

    Michelotti, G A; Michelotti, E F; Pullner, A; Duncan, R C; Eick, D; Levens, D

    1996-01-01

    Transcription activation and repression of eukaryotic genes are associated with conformational and topological changes of the DNA and chromatin, altering the spectrum of proteins associated with an active gene. Segments of the human c-myc gene possessing non-B structure in vivo located with enzymatic and chemical probes. Sites hypertensive to cleavage with single-strand-specific S1 nuclease or the single-strand-selective agent potassium permanganate included the major promoters P1 and P2 as well as the far upstream sequence element (FUSE) and CT elements, which bind, respectively, the single-strand-specific factors FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K in vitro. Active and inactive c-myc genes yielded different patterns of S1 nuclease and permanganate sensitivity, indicating alternative chromatin configurations of active and silent genes. The melting of specific cis elements of active c-myc genes in vivo suggested that transcriptionally associated torsional strain might assist strand separation and facilitate factor binding. Therefore, the interaction of FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K with supercoiled DNA was studied. Remarkably, both proteins recognize their respective elements torsionally strained but not as liner duplexes. Single-strand- or supercoil-dependent gene regulatory proteins may directly link alterations in DNA conformation and topology with changes in gene expression. PMID:8649373

  1. The Histone Demethylase KDM5 Activates Gene Expression by Recognizing Chromatin Context through Its PHD Reader Motif.

    PubMed

    Liu, Xingyin; Secombe, Julie

    2015-12-15

    KDM5 family proteins are critically important transcriptional regulators whose physiological functions in the context of a whole animal remain largely unknown. Using genome-wide gene expression and binding analyses in Drosophila adults, we demonstrate that KDM5 (Lid) is a direct regulator of genes required for mitochondrial structure and function. Significantly, this occurs independently of KDM5's well-described JmjC domain-encoded histone demethylase activity. Instead, it requires the PHD motif of KDM5 that binds to histone H3 that is di- or trimethylated on lysine 4 (H3K4me2/3). Genome-wide, KDM5 binding overlaps with the active chromatin mark H3K4me3, and a fly strain specifically lacking H3K4me2/3 binding shows defective KDM5 promoter recruitment and gene activation. KDM5 therefore plays a central role in regulating mitochondrial function by utilizing its ability to recognize specific chromatin contexts. Importantly, KDM5-mediated regulation of mitochondrial activity is likely to be key in human diseases caused by dysfunction of this family of proteins. PMID:26673323

  2. Micro-phase assembly of active sites in a coarse-grained model of chromatin by Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Zhang, Yang; Heermann, Dieter; Farmer, Barry; Pandey, Ras

    2011-03-01

    A coarse-grained model is used to study the self-assembly of active sites in a DNA (chromatin) chain. The chromosome is described by a bond-fluctuating chain of two types of nodes A (interacting) and B (non-interacting), distributed randomly with concentration C and 1 - C respectively. Active nodes interact with a Lennard-Jones (LJ) potential and execute their stochastic motion with the Metropolis algorithm. The depth of the LJ potential (f) , a measure of interaction strength and the concentration (C) of the active sites are varied. A number of local and global physical quantities are studied such as mobility (Mn) profile of each node, their local structural profile, root mean square (RMS) displacement (R) , radius of gyration (Rg) , and structure factor S (q) . We find that the chain segments assemble into microphase of blobs which requires higher concentration of active sites at weaker interaction. These findings are consistent with that of a dynamic loop model of chromatin on global (large) scale but differ at small scales. This work is supported in part by the Alexander von Humboldt foundation and AFRL.

  3. Nucleoporins and chromatin metabolism.

    PubMed

    Ptak, Christopher; Wozniak, Richard W

    2016-06-01

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

  4. The Chromatin-Modifying Enzyme Ezh2 Is Critical for the Maintenance of Regulatory T Cell Identity after Activation

    PubMed Central

    DuPage, Michel; Chopra, Gaurav; Quiros, Jason; Rosenthal, Wendy L.; Morar, Malika M.; Holohan, Dan; Zhang, Ruan; Turka, Laurence; Marson, Alexander; Bluestone, Jeffrey A.

    2015-01-01

    SUMMARY Regulatory T cells (Treg cells) are required for immune homeostasis. Chromatin remodeling is essential for establishing diverse cellular identities, but how the epigenetic program in Treg cells is maintained throughout the dynamic activation process remains unclear. Here we have shown that CD28 co-stimulation, an extracellular cue intrinsically required for Treg cell maintenance, induced the chromatin-modifying enzyme, Ezh2. Treg-specific ablation of Ezh2 resulted in spontaneous autoimmunity with reduced Foxp3+ cells in non-lymphoid tissues and impaired resolution of experimental autoimmune encephalomyelitis. Utilizing a model designed to selectively deplete wild-type Treg cells in adult mice co-populated with Ezh2-deficient Treg cells, Ezh2-deficient cells were destabilized and failed to prevent autoimmunity. After activation, the transcriptome of Ezh2-deficient Treg cells was disrupted, with altered expression of Treg cell lineage genes in a pattern similar to Foxp3-deficient Treg cells. These studies reveal a critical role for Ezh2 in the maintenance of Treg cell identity during cellular activation. PMID:25680271

  5. A unique nucleosome arrangement, maintained actively by chromatin remodelers facilitates transcription of yeast tRNA genes

    PubMed Central

    2013-01-01

    Background RNA polymerase (pol) III transcribes a unique class of genes with intra-genic promoters and high transcriptional activity. The major contributors to the pol III transcriptome, tRNAs genes are found scattered on all chromosomes of yeast. A prototype tDNA of <150 bp length, is generally considered nucleosome-free while some pol III-transcribed genes have been shown to have nucleosome-positioning properties. Results Using high resolution ChIP-chip and ChIP-seq methods, we found several unique features associated with nucleosome profiles on all tRNA genes of budding yeast, not seen on nucleosome-dense counterparts in fission yeast and resting human CD4+ T cells. The nucleosome-free region (NFR) on all but three yeast tDNAs is found bordered by an upstream (US) nucleosome strongly positioned at −140 bp position and a downstream (DS) nucleosome at variable positions with respect to the gene terminator. Perturbation in this nucleosomal arrangement interferes with the tRNA production. Three different chromatin remodelers generate and maintain the NFR by targeting different gene regions. Isw1 localizes to the gene body and makes it nucleosome-depleted, Isw2 maintains periodicity in the upstream nucleosomal array, while RSC targets the downstream nucleosome. Direct communication of pol III with RSC serves as a stress-sensory mechanism for these genes. In its absence, the downstream nucleosome moves towards the gene terminator. Levels of tRNAs from different families are found to vary considerably as different pol III levels are seen even on isogenes within a family. Pol III levels show negative correlation with the nucleosome occupancies on different genes. Conclusions Budding yeast tRNA genes maintain an open chromatin structure, which is not due to sequence-directed nucleosome positioning or high transcription activity of genes. Unlike 5′ NFR on pol II-transcribed genes, the tDNA NFR, which facilitates tDNA transcription, results from action of chromatin

  6. Active and Repressive Chromatin Are Interspersed without Spreading in an Imprinted Gene Cluster in the Mammalian Genome

    PubMed Central

    Regha, Kakkad; Sloane, Mathew A.; Huang, Ru; Pauler, Florian M.; Warczok, Katarzyna E.; Melikant, Balázs; Radolf, Martin; Martens, Joost H.A.; Schotta, Gunnar; Jenuwein, Thomas; Barlow, Denise P.

    2010-01-01

    SUMMARY The Igf2r imprinted cluster is an epigenetic silencing model in which expression of a ncRNA silences multiple genes in cis. Here, we map a 250 kb region in mouse embryonic fibroblast cells to show that histone modifications associated with expressed and silent genes are mutually exclusive and localized to discrete regions. Expressed genes were modified at promoter regions by H3K4me3 + H3K4me2 + H3K9Ac and on putative regulatory elements flanking active promoters by H3K4me2 + H3K9Ac. Silent genes showed two types of nonoverlapping profile. One type spread over large domains of tissue-specific silent genes and contained H3K27me3 alone. A second type formed localized foci on silent imprinted gene promoters and a nonexpressed pseudogene and contained H3K9me3 + H4K20me3 ± HP1. Thus, mammalian chromosome arms contain active chromatin interspersed with repressive chromatin resembling the type of heterochromatin previously considered a feature of centromeres, telomeres, and the inactive X chromosome. PMID:17679087

  7. Low levels of cadmium chloride alter the immunoprecipitation of corneal cadherin-complex proteins.

    PubMed

    Weidner, W J; Waddell, D S; Sillman, A J

    2000-12-01

    The effect of cadmium chloride on the immunoprecipitation of cadherin and the associated adherens junctional proteins, alpha- and beta-catenin, was examined in isolated bullfrog (Rana catesbeiana) corneas utilizing Western blot and enhanced chemoluminescent techniques. Application of either 1.0 microM or 75.0 microM CdCl2 to the corneal endothelium for 2 h markedly decreased the immunoprecipitation of cadherins as compared to paired control corneas. Immunoprecipitation of alpha-catenin was increased in response to both doses of CdCl2, while the immunoprecipitation of beta-catenin was little changed by either cadmium dose. There is accumulating evidence that cadmium may increase epithelial paracellular permeability by interfering with cadherin complex activity at intercellular junctions. The present study suggests that inorganic cadmium in low micromolar concentrations may decrease the integrity of the corneal endothelium, at least in part through a similar mechanism involving disruption of junctional cadherin complex function. PMID:11201663

  8. Modulating chromatin structure and DNA accessibility by deacetylase inhibition enhances the anti-cancer activity of silver nanoparticles.

    PubMed

    Igaz, Nóra; Kovács, Dávid; Rázga, Zsolt; Kónya, Zoltán; Boros, Imre M; Kiricsi, Mónika

    2016-10-01

    Histone deacetylase (HDAC) inhibitors are considered as novel therapeutic agents inducing cell cycle arrest and apoptotic cell death in various cancer cells. Inhibition of deacetylase activity results in a relaxed chromatin structure thereby rendering the genetic material more vulnerable to DNA targeting agents that could be exploited by combinational cancer therapy. The unique potential of silver nanoparticles (AgNPs) in tumor therapy relies on the generation of reactive radicals which trigger oxidative stress, DNA damage and apoptosis in cancer cells. The revolutionary application of AgNPs as chemotherapeutical drugs seems very promising, nevertheless the exact molecular mechanisms of AgNP action in combination with other anti-cancer agents have yet to be elucidated in details before clinical administrations. As a step towards this we investigated the combinational effect of HDAC inhibition and AgNP administration in HeLa cervical cancer cells. We identified synergistic inhibition of cancer cell growth and migration upon combinational treatments. Here we report that the HDAC inhibitor Trichostatin A enhances the DNA targeting capacity and apoptosis inducing efficacy of AgNPs most probably due to its effect on chromatin condensation. These results point to the potential benefits of combinational application of HDAC inhibitors and AgNPs in novel cancer medication protocols. PMID:27434153

  9. Immunoprecipitation of the parathyroid hormone receptor

    SciTech Connect

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

    1987-01-01

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

  10. Noradrenergic activation of the basolateral amygdala enhances object recognition memory and induces chromatin remodeling in the insular cortex

    PubMed Central

    Beldjoud, Hassiba; Barsegyan, Areg; Roozendaal, Benno

    2015-01-01

    It is well established that arousal-induced memory enhancement requires noradrenergic activation of the basolateral complex of the amygdala (BLA) and modulatory influences on information storage processes in its many target regions. While this concept is well accepted, the molecular basis of such BLA effects on neural plasticity changes within other brain regions remains to be elucidated. The present study investigated whether noradrenergic activation of the BLA after object recognition training induces chromatin remodeling through histone post-translational modifications in the insular cortex (IC), a brain region that is importantly involved in object recognition memory. Male Sprague—Dawley rats were trained on an object recognition task, followed immediately by bilateral microinfusions of norepinephrine (1.0 μg) or saline administered into the BLA. Saline-treated control rats exhibited poor 24-h retention, whereas norepinephrine treatment induced robust 24-h object recognition memory. Most importantly, this memory-enhancing dose of norepinephrine induced a global reduction in the acetylation levels of histone H3 at lysine 14, H2B and H4 in the IC 1 h later, whereas it had no effect on the phosphorylation of histone H3 at serine 10 or tri-methylation of histone H3 at lysine 27. Norepinephrine administered into the BLA of non-trained control rats did not induce any changes in the histone marks investigated in this study. These findings indicate that noradrenergic activation of the BLA induces training-specific effects on chromatin remodeling mechanisms, and presumably gene transcription, in its target regions, which may contribute to the understanding of the molecular mechanisms of stress and emotional arousal effects on memory consolidation. PMID:25972794

  11. 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.

  12. Virogenic BrdU and BrdU-sensitive DNA sequences are disproportionately concentrated in the template-active chromatin of rat embryo cells

    PubMed Central

    Schwartz, Stephen A.

    1979-01-01

    In order to characterize the molecular mechanism responsible for the BrdU-mediated activation of endogenous retrovirus from normal rat embryo cells, the previously reported selective distribution of bromouracil in DNA was correlated with the corresponding organization of the nucleo-protein complex in regard to nucleosome structure and template - active and -inactive chromatin. Following micrococcal nuclease digestion of chromatin labeled with either [3H]thymidine or [3H]BrdU, the amount and specific activities of the respective nucleosomal DNA were indistinguishable. Comparable findings were obtained following direct examination of the nuclease-sensitive, “spacer” DNA. However, when each chromatin type was fractionated into template-active and -inactive components, it was evident that [3H]bromouracil was nonrandomly more concentrated in the template-active portion in comparison to the random distribution of [3H]thymine moieties. Furthermore, it was apparent that the template-active chromatin fraction was substantially enriched in the nucleotide sequences of rat DNA known to be sensitive to the virogenic action of BrdU. PMID:424312

  13. 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. PMID:26459223

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

    PubMed Central

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

    2015-01-01

    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. PMID:26459223

  15. 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…

  16. The Chromatin Regulator BRPF3 Preferentially Activates the HBO1 Acetyltransferase but Is Dispensable for Mouse Development and Survival.

    PubMed

    Yan, Kezhi; You, Linya; Degerny, Cindy; Ghorbani, Mohammad; Liu, Xin; Chen, Lulu; Li, Lin; Miao, Dengshun; Yang, Xiang-Jiao

    2016-02-01

    To interpret epigenetic information, chromatin readers utilize various protein domains for recognition of DNA and histone modifications. Some readers possess multidomains for modification recognition and are thus multivalent. Bromodomain- and plant homeodomain-linked finger-containing protein 3 (BRPF3) is such a chromatin reader, containing two plant homeodomain-linked fingers, one bromodomain and a PWWP domain. However, its molecular and biological functions remain to be investigated. Here, we report that endogenous BRPF3 preferentially forms a tetrameric complex with HBO1 (also known as KAT7) and two other subunits but not with related acetyltransferases such as MOZ, MORF, TIP60, and MOF (also known as KAT6A, KAT6B, KAT5, and KAT8, respectively). We have also characterized a mutant mouse strain with a lacZ reporter inserted at the Brpf3 locus. Systematic analysis of β-galactosidase activity revealed dynamic spatiotemporal expression of Brpf3 during mouse embryogenesis and high expression in the adult brain and testis. Brpf3 disruption, however, resulted in no obvious gross phenotypes. This is in stark contrast to Brpf1 and Brpf2, whose loss causes lethality at E9.5 and E15.5, respectively. In Brpf3-null mice and embryonic fibroblasts, RT-quantitative PCR uncovered no changes in levels of Brpf1 and Brpf2 transcripts, confirming no compensation from them. These results indicate that BRPF3 forms a functional tetrameric complex with HBO1 but is not required for mouse development and survival, thereby distinguishing BRPF3 from its paralogs, BRPF1 and BRPF2. PMID:26677226

  17. PAK1 and CtBP1 Regulate the Coupling of Neuronal Activity to Muscle Chromatin and Gene Expression

    PubMed Central

    Thomas, Jean-Luc; Ravel-Chapuis, Aymeric; Valente, Carmen; Corda, Daniela; Méjat, Alexandre

    2015-01-01

    Acetylcholine receptor (AChR) expression in innervated muscle is limited to the synaptic region. Neuron-induced electrical activity participates in this compartmentalization by promoting the repression of AChR expression in the extrasynaptic regions. Here, we show that the corepressor CtBP1 (C-terminal binding protein 1) is present on the myogenin promoter together with repressive histone marks. shRNA-mediated downregulation of CtBP1 expression is sufficient to derepress myogenin and AChR expression in innervated muscle. Upon denervation, CtBP1 is displaced from the myogenin promoter and relocates to the cytoplasm, while repressive histone marks are replaced by activating ones concomitantly to the activation of myogenin expression. We also observed that upon denervation the p21-activated kinase 1 (PAK1) expression is upregulated, suggesting that phosphorylation by PAK1 may be involved in the relocation of CtBP1. Indeed, preventing CtBP1 Ser158 phosphorylation induces CtBP1 accumulation in the nuclei and abrogates the activation of myogenin and AChR expression. Altogether, these findings reveal a molecular mechanism to account for the coordinated control of chromatin modifications and muscle gene expression by presynaptic neurons via a PAK1/CtBP1 pathway. PMID:26416879

  18. PAK1 and CtBP1 Regulate the Coupling of Neuronal Activity to Muscle Chromatin and Gene Expression.

    PubMed

    Thomas, Jean-Luc; Moncollin, Vincent; Ravel-Chapuis, Aymeric; Valente, Carmen; Corda, Daniela; Méjat, Alexandre; Schaeffer, Laurent

    2015-12-01

    Acetylcholine receptor (AChR) expression in innervated muscle is limited to the synaptic region. Neuron-induced electrical activity participates in this compartmentalization by promoting the repression of AChR expression in the extrasynaptic regions. Here, we show that the corepressor CtBP1 (C-terminal binding protein 1) is present on the myogenin promoter together with repressive histone marks. shRNA-mediated downregulation of CtBP1 expression is sufficient to derepress myogenin and AChR expression in innervated muscle. Upon denervation, CtBP1 is displaced from the myogenin promoter and relocates to the cytoplasm, while repressive histone marks are replaced by activating ones concomitantly to the activation of myogenin expression. We also observed that upon denervation the p21-activated kinase 1 (PAK1) expression is upregulated, suggesting that phosphorylation by PAK1 may be involved in the relocation of CtBP1. Indeed, preventing CtBP1 Ser158 phosphorylation induces CtBP1 accumulation in the nuclei and abrogates the activation of myogenin and AChR expression. Altogether, these findings reveal a molecular mechanism to account for the coordinated control of chromatin modifications and muscle gene expression by presynaptic neurons via a PAK1/CtBP1 pathway. PMID:26416879

  19. Sperm Chromatin-Induced Ectopic Polar Body Extrusion in Mouse Eggs after ICSI and Delayed Egg Activation

    PubMed Central

    Deng, Manqi; Li, Rong

    2009-01-01

    Meiotic chromosomes in an oocyte are not only a maternal genome carrier but also provide a positional signal to induce cortical polarization and define asymmetric meiotic division of the oocyte, resulting in polar body extrusion and haploidization of the maternal genome. The meiotic chromosomes play dual function in determination of meiosis: 1) organizing a bipolar spindle formation and 2) inducing cortical polarization and assembly of a distinct cortical cytoskeleton structure in the overlying cortex for polar body extrusion. At fertilization, a sperm brings exogenous paternal chromatin into the egg, which induces ectopic cortical polarization at the sperm entry site and leads to a cone formation, known as fertilization cone. Here we show that the sperm chromatin-induced fertilization cone formation is an abortive polar body extrusion due to lack of spindle induction by the sperm chromatin during fertilization. If experimentally manipulating the fertilization process to allow sperm chromatin to induce both cortical polarization and spindle formation, the fertilization cone can be converted into polar body extrusion. This suggests that sperm chromatin is also able to induce polar body extrusion, like its maternal counterpart. The usually observed cone formation instead of ectopic polar body extrusion induced by sperm chromatin during fertilization is due to special sperm chromatin compaction which restrains it from rapid spindle induction and therefore provides a protective mechanism to prevent a possible paternal genome loss during ectopic polar body extrusion. PMID:19787051

  20. The Chromatin Remodeling Protein Bptf Promotes Posterior Neuroectodermal Fate by Enhancing Smad2-Activated wnt8a Expression.

    PubMed

    Ma, Yuanqing; Liu, Xiuli; Liu, Zhaoting; Wei, Shi; Shang, Hanqiao; Xue, Yu; Cao, Yu; Meng, Anming; Wang, Qiang

    2015-06-01

    During vertebrate embryogenesis, the neuroectoderm is induced from dorsal ectoderm and then partitioned into anterior and posterior neuroectodermal domains by posteriorizing signals, such as Wnt and fibroblast growth factor. However, little is known about epigenetic regulation of posteriorizing gene expression. Here, we report a requirement of the chromatin remodeling protein Bptf for neuroectodermal posteriorization in zebrafish embryos. Knockdown of bptf leads to an expansion of the anterior neuroectoderm at the expense of the posterior ectoderm. Bptf functionally and physically interacts with p-Smad2, which is activated by non-Nodal TGF-β signaling, to promote the expression of wnt8a, a critical gene for neural posteriorization. Bptf and Smad2 directly bind to and activate the wnt8a promoter through recruiting NURF remodeling complex. When bptf function or TGF-β signal transduction is inhibited, the nucleosome density on the wnt8a promoter is increased. We propose that Bptf and TGF-β/Smad2 mediate nucleosome remodeling to regulate wnt8a expression and hence neural posteriorization. PMID:26041917

  1. Interstitial chromatin alteration causes persistent p53 activation involved in the radiation-induced senescence-like growth arrest

    SciTech Connect

    Suzuki, Masatoshi; Suzuki, Keiji; Kodama, Seiji; Watanabe, Masami . E-mail: nabe@rri.kyoto-u.ac.jp

    2006-02-03

    Various stresses including ionizing radiation give normal human fibroblasts a phenotype of senescence-like growth arrest (SLGA), manifested by p53-dependent irreversible G1 arrest. To determine the mechanism of persistent activation of p53, we examined phosphorylated Ataxia telangiectasia mutated (ATM) and phosphorylated histone H2AX foci formation after X-irradiation. Although the multiple tiny foci, detected soon after (<30 min) irradiation, gradually disappeared, some of these foci changed to large foci and persisted for 5 days. Large foci containing phosphorylated ATM and {gamma}-H2AX co-localized and foci with p53 phosphorylated at serine 15 also showed the same distribution. Interestingly, the signals obtained by telomere fluorescence in situ hybridization (FISH) assay did not co-localize with 90% of the large foci. Our results indicate that chromatin alteration in interstitial chromosomal regions is the most likely cause of continuous activation of p53, which results in the induction of SLGA by ionizing radiation.

  2. Interstitial chromatin alteration causes persistent p53 activation involved in the radiation-induced senescence-like growth arrest.

    PubMed

    Suzuki, Masatoshi; Suzuki, Keiji; Kodama, Seiji; Watanabe, Masami

    2006-02-01

    Various stresses including ionizing radiation give normal human fibroblasts a phenotype of senescence-like growth arrest (SLGA), manifested by p53-dependent irreversible G1 arrest. To determine the mechanism of persistent activation of p53, we examined phosphorylated Ataxia telangiectasia mutated (ATM) and phosphorylated histone H2AX foci formation after X-irradiation. Although the multiple tiny foci, detected soon after (<30 min) irradiation, gradually disappeared, some of these foci changed to large foci and persisted for 5 days. Large foci containing phosphorylated ATM and gamma-H2AX co-localized and foci with p53 phosphorylated at serine 15 also showed the same distribution. Interestingly, the signals obtained by telomere fluorescence in situ hybridization (FISH) assay did not co-localize with 90% of the large foci. Our results indicate that chromatin alteration in interstitial chromosomal regions is the most likely cause of continuous activation of p53, which results in the induction of SLGA by ionizing radiation. PMID:16360120

  3. Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility

    SciTech Connect

    Dogan, Nergiz; Wu, Weisheng; Morrissey, Christapher S.; Chen, Kuan-Bei; Stonestrom, Aaron; Long, Maria; Keller, Cheryl A.; Cheng, Yong; Jain, Deepti; Visel, Axel; Pennacchio, Len A.; Weiss, Mitchell J.; Blobel, Gerd A.; Hardison, Ross C.

    2015-04-23

    Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the results of these and other studies. Results: TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included. Conclusions: Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.

  4. Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility

    DOE PAGESBeta

    Dogan, Nergiz; Wu, Weisheng; Morrissey, Christapher S.; Chen, Kuan-Bei; Stonestrom, Aaron; Long, Maria; Keller, Cheryl A.; Cheng, Yong; Jain, Deepti; Visel, Axel; et al

    2015-04-23

    Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the resultsmore » of these and other studies. Results: TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included. Conclusions: Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.« less

  5. 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

  6. The chromatin regulator DMAP1 modulates activity of the nuclear factor B (NF-B) transcription factor Relish in the Drosophila innate immune response.

    PubMed

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

    2014-07-25

    The host defense of the model organism Drosophila is under the control of two major signaling cascades controlling transcription factors of the NF-B 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-B 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

  7. A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin.

    PubMed

    Sowd, Gregory A; Serrao, Erik; Wang, Hao; Wang, Weifeng; Fadel, Hind J; Poeschla, Eric M; Engelman, Alan N

    2016-02-23

    Integration is vital to retroviral replication and influences the establishment of the latent HIV reservoir. HIV-1 integration favors active genes, which is in part determined by the interaction between integrase and lens epithelium-derived growth factor (LEDGF)/p75. Because gene targeting remains significantly enriched, relative to random in LEDGF/p75 deficient cells, other host factors likely contribute to gene-tropic integration. Nucleoporins 153 and 358, which bind HIV-1 capsid, play comparatively minor roles in integration targeting, but the influence of another capsid binding protein, cleavage and polyadenylation specificity factor 6 (CPSF6), has not been reported. In this study we knocked down or knocked out CPSF6 in parallel or in tandem with LEDGF/p75. CPSF6 knockout changed viral infectivity kinetics, decreased proviral formation, and preferentially decreased integration into transcriptionally active genes, spliced genes, and regions of chromatin enriched in genes and activating histone modifications. LEDGF/p75 depletion by contrast preferentially altered positional integration targeting within gene bodies. Dual factor knockout reduced integration into genes to below the levels observed with either single knockout and revealed that CPSF6 played a more dominant role than LEDGF/p75 in directing integration to euchromatin. CPSF6 complementation rescued HIV-1 integration site distribution in CPSF6 knockout cells, but complementation with a capsid binding mutant of CPSF6 did not. We conclude that integration targeting proceeds via two distinct mechanisms: capsid-CPSF6 binding directs HIV-1 to actively transcribed euchromatin, where the integrase-LEDGF/p75 interaction drives integration into gene bodies. PMID:26858452

  8. Distinct structural transitions of chromatin topological domains correlate with coordinated hormone-induced gene regulation

    PubMed Central

    Le Dily, François; Baù, Davide; Pohl, Andy; Vicent, Guillermo P.; Serra, François; Soronellas, Daniel; Castellano, Giancarlo; Wright, Roni H.G.; Ballare, Cecilia; Filion, Guillaume; Marti-Renom, Marc A.

    2014-01-01

    The human genome is segmented into topologically associating domains (TADs), but the role of this conserved organization during transient changes in gene expression is not known. Here we describe the distribution of progestin-induced chromatin modifications and changes in transcriptional activity over TADs in T47D breast cancer cells. Using ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing), Hi-C (chromosome capture followed by high-throughput sequencing), and three-dimensional (3D) modeling techniques, we found that the borders of the ∼2000 TADs in these cells are largely maintained after hormone treatment and that up to 20% of the TADs could be considered as discrete regulatory units where the majority of the genes are either transcriptionally activated or repressed in a coordinated fashion. The epigenetic signatures of the TADs are homogeneously modified by hormones in correlation with the transcriptional changes. Hormone-induced changes in gene activity and chromatin remodeling are accompanied by differential structural changes for activated and repressed TADs, as reflected by specific and opposite changes in the strength of intra-TAD interactions within responsive TADs. Indeed, 3D modeling of the Hi-C data suggested that the structure of TADs was modified upon treatment. The differential responses of TADs to progestins and estrogens suggest that TADs could function as “regulons” to enable spatially proximal genes to be coordinately transcribed in response to hormones. PMID:25274727

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

    PubMed Central

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

    2002-01-01

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

  10. Chemically induced enucleation of activated bovine oocytes: chromatin and microtubule organization and production of viable cytoplasts.

    PubMed

    Saraiva, Naiara Zoccal; Oliveira, Clara Slade; Leal, Cláudia Lima Verde; de Lima, Marina Ragagnin; Del Collado, Maite; Vantini, Roberta; Monteiro, Fabio Morato; Niciura, Simone Cristina Méo; Garcia, Joaquim Mansano

    2015-12-01

    As the standard enucleation method in mammalian nuclear transfer is invasive and damaging to cytoplast spatial organization, alternative procedures have been developed over recent years. Among these techniques, chemically induced enucleation (IE) is especially interesting because it does not employ ultraviolet light and reduces the amount of cytoplasm eliminated during the procedure. The objective of this study was to optimize the culture conditions with demecolcine of pre-activated bovine oocytes for chemically IE, and to evaluate nuclear and microtubule organization in cytoplasts obtained by this technique and their viability. In the first experiment, a negative effect on oocyte activation was verified when demecolcine was added at the beginning of the process, reducing activation rates by approximately 30%. This effect was not observed when demecolcine was added to the medium after 1.5 h of activation. In the second experiment, although a reduction in the number of microtubules was observed in most oocytes, these structures did not disappear completely during assessment. Approximately 50% of treated oocytes presented microtubule reduction at the end of the evaluation period, while 23% of oocytes were observed to exhibit the complete disappearance of these structures and 28% exhibited visible microtubules. These findings indicated the lack of immediate microtubule repolymerization after culture in demecolcine-free medium, a fact that may negatively influence embryonic development. However, cleavage rates of 63.6-70.0% and blastocyst yield of 15.5-24.2% were obtained in the final experiment, without significant differences between techniques, indicating that chemically induced enucleation produces normal embryos. PMID:25318529

  11. Insights into GATA-1 Mediated Gene Activation versus Repression via Genome-wide Chromatin Occupancy Analysis

    PubMed Central

    Yu, Ming; Riva, Laura; Xie, Huafeng; Schindler, Yocheved; Moran, Tyler B.; Cheng, Yong; Yu, Duonan; Hardison, Ross; Weiss, Mitchell J; Orkin, Stuart H.; Bernstein, Bradley E.; Fraenkel, Ernest; Cantor, Alan B.

    2009-01-01

    Summary The transcription factor GATA-1 is required for terminal erythroid maturation and functions as an activator or repressor depending on gene context. Yet its in vivo site selectivity and ability to distinguish between activated versus repressed genes remain incompletely understood. In this study, we performed GATA-1 ChIP-seq in erythroid cells and compared it to GATA-1 induced gene expression changes. Bound and differentially expressed genes contain a greater number of GATA binding motifs, a higher frequency of palindromic GATA sites, and closer occupancy to the transcriptional start site versus non-differentially expressed genes. Moreover, we show that the transcription factor Zbtb7a occupies GATA-1 bound regions of some direct GATA-1 target genes, that the presence of SCL/TAL1 helps distinguish transcriptional activation versus repression, and that Polycomb Repressive Complex 2 (PRC2) is involved in epigenetic silencing of a subset of GATA-1 repressed genes. These data provide insights into GATA-1 mediated gene regulation in vivo. PMID:19941827

  12. Extended chromatin and DNA fibers from active plant nuclei for high-resolution FISH.

    PubMed

    Lavania, U C; Yamamoto, M; Mukai, Y

    2003-10-01

    The conventional protocol for isolation of cell wall free nuclei for release of DNA fibers for plants involves mechanical removal of the cell wall and separation of debris by sieve filtration. The mechanical grinding pressure applied during the process leaves only the more tolerant G(1) nuclei intact, and all other states of active nuclei that may be present in the target tissues (e.g., leaf) are simply crushed/disrupted during the isolation process. Here we describe an alternative enzymatic protocol for isolation of nuclei from root tip tissue. Cell wall free nuclei at a given stage of cell cycle, free of any cell debris, could be realized in suspension that are fit for preparation of extended fibers suitable for fiber FISH applications. The protocol utilizes selective harvest of active nuclei from root tip tissue in liquid suspension under the influence of cell wall-degrading enzymes, and provides opportunities to target cell cycle-specific nuclei from interphase through division phase for the release of extended DNA fibers. Availability of cell cycle-specific fibers may have added value in transcriptional analysis, DNA:RNA hybridization, visualization of DNA replication and replication forks, and improved FISH efficiency. PMID:14500692

  13. Open chromatin in pluripotency and reprogramming

    PubMed Central

    Meshorer, Eran; Ramalho-Santos, Miguel

    2013-01-01

    Pluripotent stem cells can be derived from embryos or induced from adult cells by reprogramming. They are unique from any other stem cell in that they can give rise to all cell types of the body. Recent findings indicate that a particularly open chromatin state contributes to maintenance of pluripotency. Two emerging principles are that: specific factors maintain a globally open chromatin state that is accessible for transcriptional activation; and other chromatin regulators contribute locally to the silencing of lineage-specific genes until differentiation is triggered. These same principles may apply during reacquisition of an open chromatin state upon reprogramming to pluripotency, and during de-differentiation in cancer. PMID:21179060

  14. Intergenic Locations of Rice Centromeric Chromatin

    PubMed Central

    Yan, Huihuang; Talbert, Paul B; Lee, Hye-Ran; Jett, Jamie; Henikoff, Steven; Chen, Feng; Jiang, Jiming

    2008-01-01

    Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. Plant and animal centromeres are typically located in megabase-sized arrays of tandem satellite repeats, making their precise mapping difficult. However, some rice centromeres are largely embedded in nonsatellite DNA, providing an excellent model to study centromere structure and evolution. We used chromatin immunoprecipitation and 454 sequencing to define the boundaries of nine of the 12 centromeres of rice. Centromere regions from chromosomes 8 and 9 were found to share synteny, most likely reflecting an ancient genome duplication. For four centromeres, we mapped discrete subdomains of binding by the centromeric histone variant CENH3. These subdomains were depleted in both intact and nonfunctional genes relative to interspersed subdomains lacking CENH3. The intergenic location of rice centromeric chromatin resembles the situation for human neocentromeres and supports a model of the evolution of centromeres from gene-poor regions. PMID:19067486

  15. Active promoters give rise to false positive ‘Phantom Peaks’ in ChIP-seq experiments

    PubMed Central

    Jain, Dhawal; Baldi, Sandro; Zabel, Angelika; Straub, Tobias; Becker, Peter B.

    2015-01-01

    Chromatin immunoprecipitation (ChIP) is widely used to identify chromosomal binding sites. Chromatin proteins are cross-linked to their target sequences in living cells. The purified chromatin is sheared and the relevant protein is enriched by immunoprecipitation with specific antibodies. The co-purifying genomic DNA is then determined by massive parallel sequencing (ChIP-seq). We applied ChIP-seq to map the chromosomal binding sites for two ISWI-containing nucleosome remodeling factors, ACF and RSF, in Drosophila embryos. Employing several polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF-1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters. Further validation included controls using chromatin of mutant embryos that do not express ACF1 or RSF-1. Surprisingly, the ChIP-seq profiles were unchanged, suggesting that they were not due to specific immunoprecipitation. Conservative analysis lists about 3000 chromosomal loci, mostly active promoters that are prone to non-specific enrichment in ChIP and appear as ‘Phantom Peaks’. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin. Mining the modENCODE ChIP-seq profiles identifies potential Phantom Peaks in many profiles of epigenetic regulators. These profiles and other ChIP-seq data featuring prominent Phantom Peaks must be validated with chromatin from cells in which the protein of interest has been depleted. PMID:26117547

  16. The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state.

    PubMed

    Alarcon, Valentina; Hernández, Sergio; Rubio, Lorena; Alvarez, Francisca; Flores, Yvo; Varas-Godoy, Manuel; De Ferrari, Giancarlo V; Kann, Michael; Villanueva, Rodrigo A; Loyola, Alejandra

    2016-01-01

    With about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients. PMID:27174370

  17. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators

    PubMed Central

    Polstein, Lauren R.; Perez-Pinera, Pablo; Kocak, D. Dewran; Vockley, Christopher M.; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E.; Reddy, Timothy E.; Gersbach, Charles A.

    2015-01-01

    Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. PMID:26025803

  18. The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state

    PubMed Central

    Alarcon, Valentina; Hernández, Sergio; Rubio, Lorena; Alvarez, Francisca; Flores, Yvo; Varas-Godoy, Manuel; De Ferrari, Giancarlo V.; Kann, Michael; Villanueva, Rodrigo A.; Loyola, Alejandra

    2016-01-01

    With about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients. PMID:27174370

  19. Variable protection by OH scavengers against radiation-induced inactivation of isolated transcriptionally active chromatin: the influence of secondary radicals

    SciTech Connect

    Herskind, C.; Westergaard, O.

    1988-04-01

    Isolated r-chromatin, the chromatin form of the extrachromosomal gene coding for the rRNA precursor in Tetrahymena, has been used to study radiation-induced inactivation in vitro in the presence of the OH radical scavengers, t-butanol, formate ions, and methanol. Induction of biologically important DNA lesions was detected by the effect on transcription by endogenous RNA polymerases associated with the isolated r-chromatin. The OH scavengers were found to give strong protection in the presence of oxygen as anticipated from previous results obtained with this system. By contrast, only a modest protection was observed under 100% N/sub 2/ or 100% N/sub 2/O, and the level of protection was different for each scavenger. The data suggest that secondary radicals may inactivate r-chromatin under anoxia. In the presence of oxygen, the secondary radicals react with O/sub 2/ to form organic peroxy radicals (or O/sub 2/-) which seem to be less reactive. Since the protective effect of the OH scavengers varies with the gassing conditions, the dose modifying effects of O/sub 2/ and N/sub 2/O relative to N/sub 2/ depend on the identity and concentration of OH scavenger. The implications for radiation-chemical studies on DNA and living cells are discussed.

  20. Single cell correlation fractal dimension of chromatin

    PubMed Central

    Récamier, Vincent; Izeddin, Ignacio; Bosanac, Lana; Dahan, Maxime; Proux, Florence; Darzacq, Xavier

    2014-01-01

    Chromatin is a major nuclear component, and it is an active matter of debate to understand its different levels of spatial organization, as well as its implication in gene regulation. Measurements of nuclear chromatin compaction were recently used to understand how DNA is folded inside the nucleus and to detect cellular dysfunctions such as cancer. Super-resolution imaging opens new possibilities to measure chromatin organization in situ. Here, we performed a direct measure of chromatin compaction at the single cell level. We used histone H2B, one of the 4 core histone proteins forming the nucleosome, as a chromatin density marker. Using photoactivation localization microscopy (PALM) and adaptive optics, we measured the three-dimensional distribution of H2B with nanometric resolution. We computed the distribution of distances between every two points of the chromatin structure, namely the Ripley K(r) distribution. We found that the K(r) distribution of H2B followed a power law, leading to a precise measurement of the correlation fractal dimension of chromatin of 2.7. Moreover, using photoactivable GFP fused to H2B, we observed dynamic evolution of chromatin sub-regions compaction. As a result, the correlation fractal dimension of chromatin reported here can be interpreted as a dynamically maintained non-equilibrium state. PMID:24637833

  1. 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.

  2. Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase.

    PubMed

    Peace, Jared M; Villwock, Sandra K; Zeytounian, John L; Gan, Yan; Aparicio, Oscar M

    2016-03-01

    The Saccharomyces cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. We hypothesized that, as stimulators of early origin activation, Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, are not well-suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advances the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase. PMID:26728715

  3. The Forkhead Transcription Factor FOXM1 Controls Cell Cycle-Dependent Gene Expression through an Atypical Chromatin Binding Mechanism

    PubMed Central

    Chen, Xi; Müller, Gerd A.; Quaas, Marianne; Fischer, Martin; Han, Namshik; Stutchbury, Benjamin; Engeland, Kurt

    2013-01-01

    There are nearly 50 forkhead (FOX) transcription factors encoded in the human genome and, due to sharing a common DNA binding domain, they are all thought to bind to similar DNA sequences. It is therefore unclear how these transcription factors are targeted to specific chromatin regions to elicit specific biological effects. Here, we used chromatin immunoprecipitation followed by sequencing (ChIP-seq) to investigate the genome-wide chromatin binding mechanisms used by the forkhead transcription factor FOXM1. In keeping with its previous association with cell cycle control, we demonstrate that FOXM1 binds and regulates a group of genes which are mainly involved in controlling late cell cycle events in the G2 and M phases. However, rather than being recruited through canonical RYAAAYA forkhead binding motifs, FOXM1 binding is directed via CHR (cell cycle genes homology region) elements. FOXM1 binds these elements through protein-protein interactions with the MMB transcriptional activator complex. Thus, we have uncovered a novel and unexpected mode of chromatin binding of a FOX transcription factor that allows it to specifically control cell cycle-dependent gene expression. PMID:23109430

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

    PubMed Central

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

    2014-01-01

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

  5. Dynamic Activation and Repression of the Plasmodium falciparum rif Gene Family and Their Relation to Chromatin Modification

    PubMed Central

    Cabral, Fernanda J.; Fotoran, Wesley L.; Wunderlich, Gerhard

    2012-01-01

    The regulation of variant gene expression in Plasmodium falciparum is still only partially understood. Regulation of var genes, the most studied gene family involved in antigenic variation, is orchestrated by a dynamic pattern of inherited chromatin states. Although recent evidence pointed to epigenetic regulation of transcribed and repressed rif loci, little is known about specific on/off associated histone modifications of individual rif genes. To investigate the chromatin marks for transcribed and repressed rif loci, we cultivated parasites and evaluated the transcriptional status of chosen rif targets by qRT-PCR and performed ChIP assays using H3K9ac and H3K9me3 antibodies. We then monitored changes in the epigenetic patterns in parasites after several reinvasions and also evaluated the “poised” mark in trophozoites and schizonts of the same erythrocytic cycle by ChIP using H3K4me2 specific antibodies. Our results show that H3K9 is acetylated in transcribed rif loci and trimethylated or even unmodified in repressed rif loci. These transcriptional and epigenetic states are inherited after several reinvasions. The poised modification H3K4me2 showed a tendency to be more present in loci in trophozoites that upon progression to schizonts strongly transcribe the respective locus. However, this effect was not consistently observed for all monitored loci. While our data show important similarities to var transcription-associated chromatin modifications, the observed swiftly occurring modifications at rif loci and the absence of H3K9 modification point to a different dynamic of recruitment of chromatin modifying enzymes. PMID:22235345

  6. NF-E2, FLI1 and RUNX1 collaborate at areas of dynamic chromatin to activate transcription in mature mouse megakaryocytes

    PubMed Central

    Zang, Chongzhi; Luyten, Annouck; Chen, Justina; Liu, X. Shirley; Shivdasani, Ramesh A.

    2016-01-01

    Mutations in mouse and human Nfe2, Fli1 and Runx1 cause thrombocytopenia. We applied genome-wide chromatin dynamics and ChIP-seq to determine these transcription factors’ (TFs) activities in terminal megakaryocyte (MK) maturation. Enhancers with H3K4me2-marked nucleosome pairs were most enriched for NF-E2, FLI and RUNX sequence motifs, suggesting that this TF triad controls much of the late MK program. ChIP-seq revealed NF-E2 occupancy near previously implicated target genes, whose expression is compromised in Nfe2-null cells, and many other genes that become active late in MK differentiation. FLI and RUNX were also the motifs most enriched near NF-E2 binding sites and ChIP-seq implicated FLI1 and RUNX1 in activation of late MK, including NF-E2-dependent, genes. Histones showed limited activation in regions of single TF binding, while enhancers that bind NF-E2 and either RUNX1, FLI1 or both TFs gave the highest signals for TF occupancy and H3K4me2; these enhancers associated best with genes activated late in MK maturation. Thus, three essential TFs co-occupy late-acting cis-elements and show evidence for additive activity at genes responsible for platelet assembly and release. These findings provide a rich dataset of TF and chromatin dynamics in primary MK and explain why individual TF losses cause thrombopocytopenia. PMID:27457419

  7. NF-E2, FLI1 and RUNX1 collaborate at areas of dynamic chromatin to activate transcription in mature mouse megakaryocytes.

    PubMed

    Zang, Chongzhi; Luyten, Annouck; Chen, Justina; Liu, X Shirley; Shivdasani, Ramesh A

    2016-01-01

    Mutations in mouse and human Nfe2, Fli1 and Runx1 cause thrombocytopenia. We applied genome-wide chromatin dynamics and ChIP-seq to determine these transcription factors' (TFs) activities in terminal megakaryocyte (MK) maturation. Enhancers with H3K4me2-marked nucleosome pairs were most enriched for NF-E2, FLI and RUNX sequence motifs, suggesting that this TF triad controls much of the late MK program. ChIP-seq revealed NF-E2 occupancy near previously implicated target genes, whose expression is compromised in Nfe2-null cells, and many other genes that become active late in MK differentiation. FLI and RUNX were also the motifs most enriched near NF-E2 binding sites and ChIP-seq implicated FLI1 and RUNX1 in activation of late MK, including NF-E2-dependent, genes. Histones showed limited activation in regions of single TF binding, while enhancers that bind NF-E2 and either RUNX1, FLI1 or both TFs gave the highest signals for TF occupancy and H3K4me2; these enhancers associated best with genes activated late in MK maturation. Thus, three essential TFs co-occupy late-acting cis-elements and show evidence for additive activity at genes responsible for platelet assembly and release. These findings provide a rich dataset of TF and chromatin dynamics in primary MK and explain why individual TF losses cause thrombopocytopenia. PMID:27457419

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

    PubMed Central

    Zhou, Jian; Troyanskaya, Olga G.

    2016-01-01

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

  9. Highly condensed chromatins are formed adjacent to subtelomeric and decondensed silent chromatin in fission yeast

    PubMed Central

    Matsuda, Atsushi; Chikashige, Yuji; Ding, Da-Qiao; Ohtsuki, Chizuru; Mori, Chie; Asakawa, Haruhiko; Kimura, Hiroshi; Haraguchi, Tokuko; Hiraoka, Yasushi

    2015-01-01

    It is generally believed that silent chromatin is condensed and transcriptionally active chromatin is decondensed. However, little is known about the relationship between the condensation levels and gene expression. Here we report the condensation levels of interphase chromatin in the fission yeast Schizosaccharomyces pombe examined by super-resolution fluorescence microscopy. Unexpectedly, silent chromatin is less condensed than the euchromatin. Furthermore, the telomeric silent regions are flanked by highly condensed chromatin bodies, or ‘knobs'. Knob regions span ∼50 kb of sequence devoid of methylated histones. Knob condensation is independent of HP1 homologue Swi6 and other gene silencing factors. Disruption of methylation at lysine 36 of histone H3 (H3K36) eliminates knob formation and gene repression at the subtelomeric and adjacent knob regions. Thus, epigenetic marks at H3K36 play crucial roles in the formation of a unique chromatin structure and in gene regulation at those regions in S. pombe. PMID:26205977

  10. Methylated DNA Immunoprecipitation Analysis of Mammalian Endogenous Retroviruses.

    PubMed

    Rebollo, Rita; Mager, Dixie L

    2016-01-01

    Endogenous retroviruses are repetitive sequences found abundantly in mammalian genomes which are capable of modulating host gene expression. Nevertheless, most endogenous retrovirus copies are under tight epigenetic control via histone-repressive modifications and DNA methylation. Here we describe a common method used in our laboratory to detect, quantify, and compare mammalian endogenous retrovirus DNA methylation. More specifically we describe methylated DNA immunoprecipitation (MeDIP) followed by quantitative PCR. PMID:26895065

  11. The NuRD Chromatin-Remodeling Enzyme CHD4 Promotes Embryonic Vascular Integrity by Transcriptionally Regulating Extracellular Matrix Proteolysis

    PubMed Central

    Ingram, Kyle G.; Curtis, Carol D.; Silasi-Mansat, Robert; Lupu, Florea; Griffin, Courtney T.

    2013-01-01

    The extracellular matrix (ECM) supports vascular integrity during embryonic development. Proteolytic degradation of ECM components is required for angiogenesis, but excessive ECM proteolysis causes blood vessel fragility and hemorrhage. Little is understood about how ECM proteolysis is transcriptionally regulated during embryonic vascular development. We now show that the NuRD ATP-dependent chromatin-remodeling complex promotes vascular integrity by preventing excessive ECM proteolysis in vivo. Mice lacking endothelial CHD4—a catalytic subunit of NuRD complexes—died at midgestation from vascular rupture. ECM components surrounding rupture-prone vessels in Chd4 mutants were significantly downregulated prior to embryonic lethality. Using qPCR arrays, we found two critical mediators of ECM stability misregulated in mutant endothelial cells: the urokinase-type plasminogen activator receptor (uPAR or Plaur) was upregulated, and thrombospondin-1 (Thbs1) was downregulated. Chromatin immunoprecipitation assays showed that CHD4-containing NuRD complexes directly bound the promoters of these genes in endothelial cells. uPAR and THBS1 respectively promote and inhibit activation of the potent ECM protease plasmin, and we detected increased plasmin activity around rupture-prone vessels in Chd4 mutants. We rescued ECM components and vascular rupture in Chd4 mutants by genetically reducing urokinase (uPA or Plau), which cooperates with uPAR to activate plasmin. Our findings provide a novel mechanism by which a chromatin-remodeling enzyme regulates ECM stability to maintain vascular integrity during embryonic development. PMID:24348274

  12. Chromatin Structure Regulates Gene Conversion

    PubMed Central

    Cummings, W. Jason; Yabuki, Munehisa; Ordinario, Ellen C; Bednarski, David W; Quay, Simon; Maizels, Nancy

    2007-01-01

    Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors. We found that the immunoglobulin Vλ pseudogene array is characterized by histone modifications associated with active chromatin. We directly demonstrated the importance of chromatin structure for gene conversion, using a regulatable experimental system in which the heterochromatin protein HP1 (Drosophila melanogaster Su[var]205), expressed as a fusion to Escherichia coli lactose repressor, is tethered to polymerized lactose operators integrated within the pseudo-Vλ donor array. Tethered HP1 diminished histone acetylation within the pseudo-Vλ array, and altered the outcome of Vλ diversification, so that nontemplated mutations rather than templated mutations predominated. Thus, chromatin structure regulates homology-directed repair. These results suggest that histone modifications may contribute to maintaining genomic stability by preventing recombination between repetitive sequences. PMID:17880262

  13. Gearing up chromatin

    PubMed Central

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

    2014-01-01

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

  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. Differential Acetylation of Histone H3 at the Regulatory Region of OsDREB1b Promoter Facilitates Chromatin Remodelling and Transcription Activation during Cold Stress

    PubMed Central

    Roy, Dipan; Paul, Amit; Roy, Adrita; Ghosh, Ritesh; Ganguly, Payel; Chaudhuri, Shubho

    2014-01-01

    The rice ortholog of DREB1, OsDREB1b, is transcriptionally induced by cold stress and over-expression of OsDREB1b results in increase tolerance towards high salt and freezing stress. This spatio-temporal expression of OsDREB1b is preceded by the change in chromatin structure at the promoter and the upstream region for gene activation. The promoter and the upstream region of OsDREB1b genes appear to be arranged into a nucleosome array. Nucleosome mapping of ∼700bp upstream region of OsDREB1b shows two positioned nucleosomes between −610 to −258 and a weakly positioned nucleosome at the core promoter and the TSS. Upon cold stress, there is a significant change in the nucleosome arrangement at the upstream region with increase in DNaseI hypersensitivity or MNase digestion in the vicinity of cis elements and TATA box at the core promoter. ChIP assays shows hyper-acetylation of histone H3K9 throughout the locus whereas region specific increase was observed in H3K14ac and H3K27ac. Moreover, there is an enrichment of RNA PolII occupancy at the promoter region during transcription activation. There is no significant change in the H3 occupancy in OsDREB1b locus negating the possibility of nucleosome loss during cold stress. Interestingly, cold induced enhanced transcript level of OsDREB1b as well as histone H3 acetylation at the upstream region was found to diminish when stressed plants were returned to normal temperature. The result indicates absolute necessity of changes in chromatin conformation for the transcription up-regulation of OsDREB1b gene in response to cold stress. The combined results show the existence of closed chromatin conformation at the upstream and promoter region of OsDREB1b in the transcription “off” state. During cold stress, changes in region specific histone modification marks promote the alteration of chromatin structure to facilitate the binding of transcription machinery for proper gene expression. PMID:24940877

  16. Inhibition of DNA Methylation Alters Chromatin Organization, Nuclear Positioning and Activity of 45S rDNA Loci in Cycling Cells of Q. robur

    PubMed Central

    Horvat, Tomislav; Maglica, Željka; Vojta, Aleksandar; Zoldoš, Vlatka

    2014-01-01

    Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are organized into two rDNA loci, the major (NOR-1) and the minor (NOR-2) locus. We present the first cytogenetic evidence indicating that the NOR-1 represents the active nucleolar organizer responsible for rRNA synthesis, while the NOR-2 probably stays transcriptionally silent and does not participate in the formation of the nucleolus in Q. robur, which is a situation resembling the well-known phenomenon of nucleolar dominance. rDNA chromatin topology analyses in cycling root tip cells by light and electron microscopy revealed the minor locus to be highly condensed and located away from the nucleolus, while the major locus was consistently associated with the nucleolus and often exhibited different levels of condensation. In addition, silver precipitation was confined exclusively to the NOR-1 locus. Also, NOR-2 was highly methylated at cytosines and rDNA chromatin was marked with histone modifications characteristic for repressive state. After treatment of the root cells with the methylation inhibitor 5-aza-2′-deoxycytidine, we observed an increase in the total level of rRNA transcripts and a decrease in DNA methylation level at the NOR-2 locus. Also, NOR-2 sites relocalized with respect to the nuclear periphery/nucleolus, however, the relocation did not affect the contribution of this locus to nucleolar formation, nor did it affect rDNA chromatin decondensation, strongly suggesting that NOR-2 has lost the function of rRNA synthesis and nucleolar organization. PMID:25093501

  17. Inhibition of DNA methylation alters chromatin organization, nuclear positioning and activity of 45S rDNA loci in cycling cells of Q. robur.

    PubMed

    Bočkor, Vedrana Vičić; Barišić, Darko; Horvat, Tomislav; Maglica, Željka; Vojta, Aleksandar; Zoldoš, Vlatka

    2014-01-01

    Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are organized into two rDNA loci, the major (NOR-1) and the minor (NOR-2) locus. We present the first cytogenetic evidence indicating that the NOR-1 represents the active nucleolar organizer responsible for rRNA synthesis, while the NOR-2 probably stays transcriptionally silent and does not participate in the formation of the nucleolus in Q. robur, which is a situation resembling the well-known phenomenon of nucleolar dominance. rDNA chromatin topology analyses in cycling root tip cells by light and electron microscopy revealed the minor locus to be highly condensed and located away from the nucleolus, while the major locus was consistently associated with the nucleolus and often exhibited different levels of condensation. In addition, silver precipitation was confined exclusively to the NOR-1 locus. Also, NOR-2 was highly methylated at cytosines and rDNA chromatin was marked with histone modifications characteristic for repressive state. After treatment of the root cells with the methylation inhibitor 5-aza-2'-deoxycytidine, we observed an increase in the total level of rRNA transcripts and a decrease in DNA methylation level at the NOR-2 locus. Also, NOR-2 sites relocalized with respect to the nuclear periphery/nucleolus, however, the relocation did not affect the contribution of this locus to nucleolar formation, nor did it affect rDNA chromatin decondensation, strongly suggesting that NOR-2 has lost the function of rRNA synthesis and nucleolar organization. PMID:25093501

  18. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    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

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

    PubMed

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

    2016-06-30

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

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

    PubMed

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

    2014-02-01

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

  3. The chromatin scaffold protein SAFB1 renders chromatin permissive for DNA damage signaling.

    PubMed

    Altmeyer, Matthias; Toledo, Luis; Gudjonsson, Thorkell; Grøfte, Merete; Rask, Maj-Britt; Lukas, Claudia; Akimov, Vyacheslav; Blagoev, Blagoy; Bartek, Jiri; Lukas, Jiri

    2013-10-24

    Although the general relevance of chromatin modifications for genotoxic stress signaling, cell-cycle checkpoint activation, and DNA repair is well established, how these modifications reach initial thresholds in order to trigger robust responses remains largely unexplored. Here, we identify the chromatin-associated scaffold attachment factor SAFB1 as a component of the DNA damage response and show that SAFB1 cooperates with histone acetylation to allow for efficient γH2AX spreading and genotoxic stress signaling. SAFB1 undergoes a highly dynamic exchange at damaged chromatin in a poly(ADP-ribose)-polymerase 1- and poly(ADP-ribose)-dependent manner and is required for unperturbed cell-cycle checkpoint activation and guarding cells against replicative stress. Altogether, our data reveal that transient recruitment of an architectural chromatin component is required in order to overcome physiological barriers by making chromatin permissive for DNA damage signaling, whereas the ensuing exclusion of SAFB1 may help prevent excessive signaling. PMID:24055346

  4. The Tudor protein survival motor neuron (SMN) is a chromatin-binding protein that interacts with methylated lysine 79 of histone H3.

    PubMed

    Sabra, Mirna; Texier, Pascale; El Maalouf, Jhony; Lomonte, Patrick

    2013-08-15

    Spinal muscular atrophy (SMA) is a muscular disease characterized by the death of motoneurons, and is a major genetic cause of infant mortality. Mutations in the SMN1 gene, which encodes the protein survival motor neuron (SMN), are responsible for the disease. SMN belongs to the Tudor domain protein family, whose members are known to interact with methylated arginine (R) or lysine (K) residues. SMN has well-defined roles in the metabolism of small non-coding ribonucleoproteins (snRNPs) and spliceosome activity. We previously showed that SMN relocated to damaged interphase centromeres, together with the Cajal-body-associated proteins coilin and fibrillarin, during the so-called interphase centromere damage response (iCDR). Here we reveal that SMN is a chromatin-binding protein that specifically interacts with methylated histone H3K79, a gene expression- and splicing-associated histone modification. SMN relocation to damaged centromeres requires its functional Tudor domain and activity of the H3K79 methyltransferase DOT1L. In vitro pulldown assays showed that SMN interacts with H3K79me1,2 at its functional Tudor domain. Chromatin immunoprecipitation confirmed that SMN binds to H3K79me1,2-containing chromatin in iCDR-induced cells. These data reveal a novel SMN property in the detection of specific chromatin modifications, and shed new light on the involvement of a putative epigenetic dimension to the occurrence of SMA. PMID:23750013

  5. Purification of RNA Polymerase I-Associated Chromatin from Yeast Cells.

    PubMed

    Bruckmann, Astrid; Linnemann, Jan; Perez-Fernandez, Jorge

    2016-01-01

    The native template of all eukaryotic nuclear RNA polymerases is chromatin. To understand how transcription occurs in vivo, it is important to define the chromatin environment of transcribing RNA Pols. Here, we describe a method used to characterize the distribution and the protein environment of RNA Pol I on ribosomal DNA during transcription in the yeast S. cerevisiae. The method is based on conventional chromatin immunoprecipitation and we propose quality control analyses at different steps of the procedure. Finally, the obtained samples are a useful source for downstream analyses by semiquantitative mass spectrometry or quantitative PCR. PMID:27576721

  6. High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei.

    PubMed

    Juodka, B; Spiess, E; Angiolillo, A; Joswig, G; Rothbarth, K; Werner, D

    1995-04-25

    Cell lysis in presence of SDS and proteinase K followed by salting-out of residual polypeptides by dehydration and precipitation with saturated sodium chloride solution [Miller, S.A., Dykes, D.D. and Polesky, H.F., Nucleic Acids Res., 16, 1215, 1988] efficiently resolves deproteinized DNA. However, this DNA is still associated with prominent polypeptides which remain stably attached to DNA during further treatments, e.g. during repeated salting-out steps, prolonged incubation of DNA in 1% SDS or 4 M urea at 56 degrees C and ethanol precipitation. The persistent polypeptides (62, 52 and 40 kDa) released from Ehrlich ascites cell DNA were further characterized. Microsequencing indicates that the DNA binding polypeptides are not yet characterized at the sequence level. Nuclease digestion of the DNA releases stable DNA-protein complexes with the shape of globular particles (12.8 +/- 0.8 nm) and their larger aggregates in which DNA remains protected from nuclease digestion. The isolated DNA-polypeptide complexes show ATPase (Km = 7.4 x 10(-4) M) and protein kinase activity. Antibodies reveal a parallel distribution of the complexes with chromatin, however, the complexes are retained in chromatin-depleted nuclei. PMID:7753627

  7. The iab-7 polycomb response element maps to a nucleosome-free region of chromatin and requires both GAGA and pleiohomeotic for silencing activity.

    PubMed

    Mishra, R K; Mihaly, J; Barges, S; Spierer, A; Karch, F; Hagstrom, K; Schweinsberg, S E; Schedl, P

    2001-02-01

    In the work reported here we have undertaken a functional dissection of a Polycomb response element (PRE) from the iab-7 cis-regulatory domain of the Drosophila melanogaster bithorax complex (BX-C). Previous studies mapped the iab-7 PRE to an 860-bp fragment located just distal to the Fab-7 boundary. Located within this fragment is an approximately 230-bp chromatin-specific nuclease-hypersensitive region called HS3. We have shown that HS3 is capable of functioning as a Polycomb-dependent silencer in vivo, inducing pairing-dependent silencing of a mini-white reporter. The HS3 sequence contains consensus binding sites for the GAGA factor, a protein implicated in the formation of nucleosome-free regions of chromatin, and Pleiohomeotic (Pho), a Polycomb group protein that is related to the mammalian transcription factor YY1. We show that GAGA and Pho interact with these sequences in vitro and that the consensus binding sites for the two proteins are critical for the silencing activity of the iab-7 PRE in vivo. PMID:11158316

  8. Chromatin remodeling by nucleosome disassembly in vitro.

    PubMed

    Lorch, Yahli; Maier-Davis, Barbara; Kornberg, Roger D

    2006-02-28

    The RSC chromatin-remodeling complex completely disassembles a nucleosome in the presence of the histone chaperone Nap1 and ATP. Disassembly occurs in a stepwise manner, with the removal of H2A/H2B dimers, followed by the rest of the histones and the release of naked DNA. RSC and related chromatin-remodeling complexes may be responsible for the removal of promoter nucleosomes during transcriptional activation in vivo. PMID:16492771

  9. Effect of growth hormone on ribonucleic acid metabolism. The template activity of the chromatin and molecular species of ribonucleic acid synthesized after treatment with the hormone

    PubMed Central

    Gupta, S. L.; Talwar, G. P.

    1968-01-01

    Growth hormone stimulates the synthesis of RNA in hypophysectomized rat liver. The question whether the hormonal stimulation of RNA synthesis is due to the activation of repressed cistrons or to other factors was studied. Nuclear RNA from the livers of adult female hypophysectomized and growth-hormone-treated rats was examined for molecular homology by hybridization techniques: no new species of RNA were detected after hormone treatment. The template activity of the chromatin for RNA synthesis is also not increased by the action of growth hormone. Short- and long-pulse-labelling experiments demonstrate that the hormonal stimulation of RNA synthesis is most marked in experiments where the period of incorporation of radioactive precursors is limited to 1–2hr. It is concluded that the hormone influences essentially the rate of RNA synthesis in these tissues. PMID:5701666

  10. Partial activation of gene activity and chromatin remodeling of the human 14q32.1 serpin gene cluster by HNF-1 alpha and HNF-4 in fibroblast microcell hybrids.

    PubMed

    Rollini, P; Xu, L; Fournier, R E

    1999-07-01

    The genes encoding alpha 1-antitrypsin (alpha 1AT, gene symbol P I) and corticosteroid-binding globulin (CBG) are part of a cluster of serine protease inhibitor (serpin) genes on human chromosome 14q32.1. Both genes are highly expressed in the liver and in cultured hepatoma cells, and the approximately 100-kb region around these genes contains an extensive array of expression-associated DNase I-hypersensitive sites (DHSs). Activation of human alpha 1AT and CBG transcription occurred when human chromosome 14 was transferred from nonexpressing cells to rat hepatoma cells. This activation event was accompanied by long-range chromatin reorganization of the entire region and the de novo formation of 17 expression-associated DHSs. Both gene activation and chromatin remodeling in hepatic cells required the liver-enriched transactivators hepatocyte nuclear factors-1 alpha and -4 (HNF-1 alpha and HNF-4). In this study, we tested whether ectopic expression of HNF-1 alpha and HNF-4 in nonexpressing cells could activate alpha 1AT and/or CBG transcription, and we monitored the chromatin structure of the locus in stably transfected fibroblasts. We report that both alpha 1AT and CBG mRNAs were expressed in fibroblast transfectants that stably expressed HNF-1 alpha and HNF-4, but expression was only approximately 1-10% of that observed in hepatic cells. Gene activation in these cells was accompanied by partial chromatin remodeling, as 6 of 17 expression-associated DHSs were formed. The potential implications of these results are discussed. PMID:11586788

  11. Chromatin deregulation in disease.

    PubMed

    Mirabella, Anne C; Foster, Benjamin M; Bartke, Till

    2016-03-01

    The regulation of chromatin by epigenetic mechanisms plays a central role in gene expression and is essential for development and maintenance of cell identity and function. Aberrant chromatin regulation is observed in many diseases where it leads to defects in epigenetic gene regulation resulting in pathological gene expression programmes. These defects are caused by inherited or acquired mutations in genes encoding enzymes that deposit or remove DNA and histone modifications and that shape chromatin architecture. Chromatin deregulation often results in neurodevelopmental disorders and intellectual disabilities, frequently linked to physical and developmental abnormalities, but can also cause neurodegenerative diseases, immunodeficiency, or muscle wasting syndromes. Epigenetic diseases can either be of monogenic origin or manifest themselves as complex multifactorial diseases such as in congenital heart disease, autism spectrum disorders, or cancer in which mutations in chromatin regulators are contributing factors. The environment directly influences the epigenome and can induce changes that cause or predispose to diseases through risk factors such as stress, malnutrition or exposure to harmful chemicals. The plasticity of chromatin regulation makes targeting the enzymatic machinery an attractive strategy for therapeutic intervention and an increasing number of small molecule inhibitors against a variety of epigenetic regulators are in clinical use or under development. In this review, we will give an overview of the molecular lesions that underlie epigenetic diseases, and we will discuss the impact of the environment and prospects for epigenetic therapies. PMID:26188466

  12. E2F1 coregulates cell cycle genes and chromatin components during the transition of oligodendrocyte progenitors from proliferation to differentiation.

    PubMed

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

    2014-01-22

    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

  13. 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. PMID:19264835

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

    PubMed

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

    2015-01-01

    Modification of DNA resulting in 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5hmC) has been shown to influence the local chromatin environment and affect transcription. Although recent advances in next generation sequencing technology allow researchers to map epigenetic modifications across the genome, such experiments are often time-consuming and cost prohibitive. Here we present a rapid and cost effective method of generating genome wide DNA modification maps utilising commercially available semiconductor based technology (DNA immunoprecipitation semiconductor sequencing; "DIP-SC-seq") on the Ion Proton sequencer. Focussing on the 5hmC mark we demonstrate, by directly comparing with alternative sequencing strategies, that this platform can successfully generate genome wide 5hmC patterns from as little as 500 ng of genomic DNA in less than 4 days. Such a method can therefore facilitate the rapid generation of multiple genome wide epigenetic datasets. PMID:25985418

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

    PubMed Central

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

    2015-01-01

    Modification of DNA resulting in 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5hmC) has been shown to influence the local chromatin environment and affect transcription. Although recent advances in next generation sequencing technology allow researchers to map epigenetic modifications across the genome, such experiments are often time-consuming and cost prohibitive. Here we present a rapid and cost effective method of generating genome wide DNA modification maps utilising commercially available semiconductor based technology (DNA immunoprecipitation semiconductor sequencing; “DIP-SC-seq”) on the Ion Proton sequencer. Focussing on the 5hmC mark we demonstrate, by directly comparing with alternative sequencing strategies, that this platform can successfully generate genome wide 5hmC patterns from as little as 500 ng of genomic DNA in less than 4 days. Such a method can therefore facilitate the rapid generation of multiple genome wide epigenetic datasets. PMID:25985418

  16. The D-isoAsp-25 variant of histone H2B is highly enriched in active chromatin: potential role in the regulation of gene expression?

    PubMed

    Qin, Zhenxia; Zhu, Jeff X; Aswad, Dana W

    2016-02-01

    Approximately 12 % of histone H2B in mammalian brain contains an unusual D-aspartate residue in its N-terminal tail. Most of this D-aspartate is linked to the C-flanking glycine via an isopeptide bond. To explore the possible significance of these modifications, we generated an antibody to the D-isoaspartyl form of H2B, and used it to assess its levels in H2B associated with "active" vs. "silent" chromatin. We found that the D-isoaspartyl form of H2B appears to be highly enriched in the former. This irreversible modification could serve a novel regulatory function in gene expression. PMID:26666674

  17. [Corrigendum] Chromatin immunoprecipitation-sequencing predicts p300 binding sites in the MCF7 human breast cancer cell line.

    PubMed

    Wang, Xiemei; Li, Shaolin

    2016-08-01

    After the publication of the article, the authors noted that the name of the first author had been spelt incorrectly. The name of the first author should be correctly written as XIUMEI WANG and not as XIEMEI WANG. This had occurred due to a typing error on behalf of the authors upon submission. [the original article was published in the International Journal of Molecular Medicine 35: 973-978, 2015; DOI: 10.3892/ijmm.2015.2081]. PMID:27315489

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

    PubMed Central

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

    2014-01-01

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

  19. The molecular topography of silenced chromatin in Saccharomyces cerevisiae

    PubMed Central

    Thurtle, Deborah M.; Rine, Jasper

    2014-01-01

    Heterochromatin imparts regional, promoter-independent repression of genes and is epigenetically heritable. Understanding how silencing achieves this regional repression is a fundamental problem in genetics and development. Current models of yeast silencing posit that Sir proteins, recruited by transcription factors bound to the silencers, spread throughout the silenced region. To test this model directly at high resolution, we probed the silenced chromatin architecture by chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) of Sir proteins, histones, and a key histone modification, H4K16-acetyl. These analyses revealed that Sir proteins are strikingly concentrated at and immediately adjacent to the silencers, with lower levels of enrichment over the promoters at HML and HMR, the critical targets for transcriptional repression. The telomeres also showed discrete peaks of Sir enrichment yet a continuous domain of hypoacetylated histone H4K16. Surprisingly, ChIP-seq of cross-linked chromatin revealed a distribution of nucleosomes at silenced loci that was similar to Sir proteins, whereas native nucleosome maps showed a regular distribution throughout silenced loci, indicating that cross-linking captured a specialized chromatin organization imposed by Sir proteins. This specialized chromatin architecture observed in yeast informs the importance of a steric contribution to regional repression in other organisms. PMID:24493645

  20. An essential role for the intra-oocyte MAPK activity in the NSN-to-SN transition of germinal vesicle chromatin configuration in porcine oocytes.

    PubMed

    Sun, Ming-Ju; Zhu, Shuai; Li, You-Wei; Lin, Juan; Gong, Shuai; Jiao, Guang-Zhong; Chen, Fei; Tan, Jing-He

    2016-01-01

    The mechanisms for the transition from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) chromatin configuration during oocyte growth/maturation are unclear. By manipulating enzyme activities and measuring important molecules using small-follicle pig oocytes with a high proportion of NSN configuration and an extended germinal vesicle stage in vitro, this study has the first time up-to-date established the essential role for intra-oocyte mitogen-activated protein kinase (MAPK) in the NSN-to-SN transition. Within the oocyte in 1-2 mm follicles, a cAMP decline activates MAPK, which prevents the NSN-to-SN transition by activating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) while inhibiting histone deacetylase (HDAC). In cumulus cells of 1-2 mm follicles, a lower level of estradiol and oocyte-derived paracrine factor (ODPF) reduces natriuretic peptide receptor 2 (NPR2) while enhancing FSH and cAMP actions. FSH elevates cAMP levels, which decreases NPR2 while activating MAPK. MAPK closes the gap junctions, which, together with the NPR2 decrease, reduces cyclic guanosine monophosphate (cGMP) delivery leading to the cAMP decline within oocytes. In 3-6 mm follicles, a higher level of estradiol and ODPF and a FSH shortage initiate a reversion of the above events leading to MAPK inactivation and NSN-to-SN transition within oocytes. PMID:27009903

  1. An essential role for the intra-oocyte MAPK activity in the NSN-to-SN transition of germinal vesicle chromatin configuration in porcine oocytes

    PubMed Central

    Sun, Ming-Ju; Zhu, Shuai; Li, You-Wei; Lin, Juan; Gong, Shuai; Jiao, Guang-Zhong; Chen, Fei; Tan, Jing-He

    2016-01-01

    The mechanisms for the transition from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) chromatin configuration during oocyte growth/maturation are unclear. By manipulating enzyme activities and measuring important molecules using small-follicle pig oocytes with a high proportion of NSN configuration and an extended germinal vesicle stage in vitro, this study has the first time up-to-date established the essential role for intra-oocyte mitogen-activated protein kinase (MAPK) in the NSN-to-SN transition. Within the oocyte in 1–2 mm follicles, a cAMP decline activates MAPK, which prevents the NSN-to-SN transition by activating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) while inhibiting histone deacetylase (HDAC). In cumulus cells of 1–2 mm follicles, a lower level of estradiol and oocyte-derived paracrine factor (ODPF) reduces natriuretic peptide receptor 2 (NPR2) while enhancing FSH and cAMP actions. FSH elevates cAMP levels, which decreases NPR2 while activating MAPK. MAPK closes the gap junctions, which, together with the NPR2 decrease, reduces cyclic guanosine monophosphate (cGMP) delivery leading to the cAMP decline within oocytes. In 3–6 mm follicles, a higher level of estradiol and ODPF and a FSH shortage initiate a reversion of the above events leading to MAPK inactivation and NSN-to-SN transition within oocytes. PMID:27009903

  2. Epigenomic regulation of oncogenesis by chromatin remodeling.

    PubMed

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

    2016-08-25

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

  3. 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. PMID:25728061

  4. The Centromere: Chromatin Foundation for the Kinetochore Machinery

    PubMed Central

    Fukagawa, Tatsuo; Earnshaw, William C.

    2014-01-01

    Since discovery of the centromere-specific histone H3 variant CENP-A, centromeres have come to be defined as chromatin structures that establish the assembly site for the complex kinetochore machinery. In most organisms, centromere activity is defined epigenetically, rather than by specific DNA sequences. In this review, we describe selected classic work and recent progress in studies of centromeric chromatin with a focus on vertebrates. We consider possible roles for repetitive DNA sequences found at most centromeres, chromatin factors and modifications that assemble and activate CENP-A chromatin for kinetochore assembly, plus the use of artificial chromosomes and kinetochores to study centromere function. PMID:25203206

  5. The centromere: chromatin foundation for the kinetochore machinery.

    PubMed

    Fukagawa, Tatsuo; Earnshaw, William C

    2014-09-01

    Since discovery of the centromere-specific histone H3 variant CENP-A, centromeres have come to be defined as chromatin structures that establish the assembly site for the complex kinetochore machinery. In most organisms, centromere activity is defined epigenetically, rather than by specific DNA sequences. In this review, we describe selected classic work and recent progress in studies of centromeric chromatin with a focus on vertebrates. We consider possible roles for repetitive DNA sequences found at most centromeres, chromatin factors and modifications that assemble and activate CENP-A chromatin for kinetochore assembly, plus the use of artificial chromosomes and kinetochores to study centromere function. PMID:25203206

  6. Pioneer transcription factors, chromatin dynamics, and cell fate control.

    PubMed

    Zaret, Kenneth S; Mango, Susan E

    2016-04-01

    Among the diverse transcription factors that are necessary to elicit changes in cell fate, both in embryonic development and in cellular reprogramming, a subset of factors are capable of binding to their target sequences on nucleosomal DNA and initiating regulatory events in silent chromatin. Such 'pioneer transcription factors' initiate cooperative interactions with other regulatory proteins to elicit changes in local chromatin structure. As a consequence of pioneer factor binding, the local chromatin can either become open and competent for activation, closed and repressed, or transcriptionally active. Understanding how pioneer factors initiate chromatin dynamics and how such can be blocked at heterochromatic sites provides insights into controlling cell fate transitions at will. PMID:26826681

  7. Chromatin signatures of cancer

    PubMed Central

    Morgan, Marc A.; Shilatifard, Ali

    2015-01-01

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

  8. The Cold Signaling Attenuator HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 Activates FLOWERING LOCUS C Transcription via Chromatin Remodeling under Short-Term Cold Stress in Arabidopsis[C][W

    PubMed Central

    Jung, Jae-Hoon; Park, Ju-Hyung; Lee, Sangmin; To, Taiko Kim; Kim, Jong-Myong; Seki, Motoaki; Park, Chung-Mo

    2013-01-01

    Exposure to short-term cold stress delays flowering by activating the floral repressor FLOWERING LOCUS C (FLC) in Arabidopsis thaliana. The cold signaling attenuator HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 (HOS1) negatively regulates cold responses. Notably, HOS1-deficient mutants exhibit early flowering, and FLC expression is suppressed in the mutants. However, it remains unknown how HOS1 regulates FLC expression. Here, we show that HOS1 induces FLC expression by antagonizing the actions of FVE and its interacting partner histone deacetylase 6 (HDA6) under short-term cold stress. HOS1 binds to FLC chromatin in an FVE-dependent manner, and FVE is essential for the HOS1-mediated activation of FLC transcription. HOS1 also interacts with HDA6 and inhibits the binding of HDA6 to FLC chromatin. Intermittent cold treatments induce FLC expression by activating HOS1, which attenuates the activity of HDA6 in silencing FLC chromatin, and the effects of intermittent cold are diminished in hos1 and fve mutants. These observations indicate that HOS1 acts as a chromatin remodeling factor for FLC regulation under short-term cold stress. PMID:24220632

  9. Studies on chromatin. II. Isolation and characterization of chromatin subunits.

    PubMed Central

    Bakayev, V V; Melnickov, A A; Osicka, V D; Varshausky, A J

    1975-01-01

    Earlier findings /1-10/ bearing on a subunit organization of chromatin were confirmed and in some points detailed. Besides this, a large-scale isolation of chromatin subunits; their protein composition, electron microscopic appearance and CsCl banding pattern are described. Although the purified chromatin subunit contains all five histones, the relative content of histone H1 i in it is two times lower than that in the original chromatin. tit is shown that a mild digestion of chromatin with staphylococcal nuclease produced not only separate chromatin subunits and their "oligomers' but also deoxyribonucleoprotein particles which sediment more slowly than subunits. It appears that these particles and subunits are produced from different initial structures in the chromatin. Finally, a crystallization of the purified chromatin subunit as a cetyltrimethyl ammonium salt is described. Images PMID:1178523

  10. TRIC: Capturing the direct cellular targets of promoter-bound transcriptional activators.

    PubMed

    Dugan, Amanda; Pricer, Rachel; Katz, Micah; Mapp, Anna K

    2016-08-01

    Transcriptional activators coordinate the dynamic assembly of multiprotein coactivator complexes required for gene expression to occur. Here we combine the power of in vivo covalent chemical capture with p-benzoyl-L-phenylalanine (Bpa), a genetically incorporated photo-crosslinking amino acid, and chromatin immunoprecipitation (ChIP) to capture the direct protein interactions of the transcriptional activator VP16 with the general transcription factor TBP at the GAL1 promoter in live yeast. PMID:27213278

  11. Co-Immunoprecipitation of Membrane-Bound Receptors

    PubMed Central

    Avila, Julian R.; Lee, Jin Suk; Torii, Keiko U.

    2015-01-01

    The study of cell-surface receptor dynamics is critical for understanding how cells sense and respond to changing environments. Therefore, elucidating the mechanisms by which signals are perceived and communicated into the cell is necessary to understand immunity, development, and stress. Challenges in testing interactions of membrane-bound proteins include their dynamic nature, their abundance, and the complex dual environment (lipid/soluble) in which they reside. Co-Immunoprecipitation (Co-IP) of tagged membrane proteins is a widely used approach to test protein-protein interaction in vivo. In this protocol we present a method to perform Co-IP using enriched membrane proteins in isolated microsomal fractions. The different variations of this protocol are highlighted, including recommendations and troubleshooting guides in order to optimize its application. This Co-IP protocol has been developed to test the interaction of receptor-like kinases, their interacting partners, and peptide ligands in stable Arabidopsis thaliana lines, but can be modified to test interactions in transiently expressed proteins in tobacco, and potentially in other plant models, or scaled for large-scale protein-protein interactions at the membrane. PMID:26097438

  12. Optimized method for methylated DNA immuno-precipitation

    PubMed Central

    Guerrero-Bosagna, Carlos; Jensen, Per

    2015-01-01

    Methylated DNA immunoprecipitation (MeDIP) is one of the most widely used methods to evaluate DNA methylation on a whole genome scale, and involves the capture of the methylated fraction of the DNA by an antibody specific to methyl-cytosine. MeDIP was initially coupled with microarray hybridization to detect local DNA methylation enrichments along the genome. More recently, MeDIP has been coupled with next generation sequencing, which highlights its current and future applicability. In previous studies in which MeDIP was applied, the protocol took around 3 days to be performed. Given the importance of MeDIP for studies involving DNA methylation, it was important to optimize the method in order to deliver faster turnouts. The present article describes optimization steps of the MeDIP method. The length of the procedure was reduced in half without compromising the quality of the results. This was achieved by:•Reduction of the number of washes in different stages of the protocol, after a careful evaluation of the number of indispensable washes.•Reduction of reaction times for detaching methylated DNA fragments from the complex agarose beads:antibody.•Modification of the methods to purify methylated DNA, which incorporates new devices and procedures, and eliminates a lengthy phenol and chloroform:isoamyl alcohol extraction. PMID:26740923

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

    PubMed

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

    2011-06-01

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

  14. 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,…

  15. 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. PMID:23751185

  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. 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

  18. Hydrogen sulfide attenuates cytokine production through the modulation of chromatin remodeling

    PubMed Central

    RIOS, ESTER C.S.; SZCZESNY, BARTOSZ; SORIANO, FRANCISCO G.; OLAH, GABOR; SZABO, CSABA

    2015-01-01

    Hydrogen sulfide (H2S) is an endogenous gaseous biological mediator, which regulates, among others, the oxidative balance of cells under normal physiological conditions, as well as in various diseases. Several previous studies have reported that H2S attenuates inflammatory mediator production. In this study, we investigated the role of H2S in chromatin modulation in an in vitro model of lipopolysaccharide (LPS)-induced inflammation and evaluated its effects on inflammatory cytokine production. Tamm-Horsfall protein 1 (THP-1) differentiated macrophages were pre-treated with sodium hydrosulfide (NaHS) (an H2S donor) at 0.01, 0.1, 0.5 or 1 mM for 30 min. To stimulate cytokine production, the cells were challenged with bacterial LPS (1 μg/ml) for 1, 4, 8 or 24 h. Histone H3 acetylation was analyzed by chromatin immunoprecipitation (ChIP), cytokine production was measured by ELISA and histone deacetylase (HDAC) activity was analyzed using a standard biochemical assay. H2S inhibited the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in a concentration-dependent manner; it was most effective at the two highest concentrations used. This effect was associated with a decrease in histone H3 acetylation at the IL-6 and TNF-α promoters in the cells exposed to H2S or H2S + LPS. The findings of the present study suggest that H2S suppresses histone acetylation, which, in turn, inhibits chromatin openness, leading to a decrease in the gene transcription of various pro-inflammatory cytokines. Therefore, this mechanism may contribute to the previously demonstrated anti-inflammatory effects of H2S and various H2S donors. PMID:25873160

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

    PubMed

    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

    2016-04-01

    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

  20. The Fun30 Chromatin Remodeler Fft3 Controls Nuclear Organization and Chromatin Structure of Insulators and Subtelomeres in Fission Yeast

    PubMed Central

    Khorosjutina, Olga; Persson, Jenna; Smialowska, Agata; Javerzat, Jean-Paul; Ekwall, Karl

    2015-01-01

    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. PMID:25798942

  1. The E2F functional analogue SBF recruits the Rpd3(L) HDAC, via Whi5 and Stb1, and the FACT chromatin reorganizer, to yeast G1 cyclin promoters.

    PubMed

    Takahata, Shinya; Yu, Yaxin; Stillman, David J

    2009-11-01

    Regulation of the CLN1 and CLN2 G1 cyclin genes controls cell cycle progression. The SBF activator binds to these promoters but is kept inactive by the Whi5 and Stb1 inhibitors. The Cdc28 cyclin-dependent kinase phosphorylates Whi5, ending the inhibition. Our chromatin immunoprecipitation (ChIP) experiments show that SBF, Whi5 and Stb1 recruit both Cdc28 and the Rpd3(L) histone deacetylase to CLN promoters, extending the analogy with mammalian G1 cyclin promoters in which Rb recruits histone deacetylases. Finally, we show that the SBF subunit Swi6 recruits the FACT chromatin reorganizer to SBF- and MBF-regulated genes. Mutations affecting FACT reduce the transient nucleosome eviction seen at these promoters during a normal cell cycle and also reduce expression. Temperature-sensitive mutations affecting FACT and Cdc28 can be suppressed by disruption of STB1 and WHI5, suggesting that one critical function of FACT and Cdc28 is overcoming chromatin repression at G1 cyclin promoters. Thus, SBF recruits complexes to promoters that either enhance (FACT) or repress (Rpd3L) accessibility to chromatin, and also recruits the kinase that activates START. PMID:19745812

  2. A conserved acidic patch in the Myb domain is required for activation of an endogenous target gene and for chromatin binding

    PubMed Central

    Ko, Emily Ray; Ko, Dennis; Chen, Carolyn; Lipsick, Joseph S

    2008-01-01

    The c-Myb protein is a transcriptional regulator initially identified by homology to the v-Myb oncoprotein, and has since been implicated in human cancer. The most highly conserved portion of the c-Myb protein is the DNA-binding domain which consists of three imperfect repeats. Many other proteins contain one or more Myb-related domains, including a number of proteins that do not bind directly to DNA. We performed a phylogenetic analysis of diverse classes of Myb-related domains and discovered a highly conserved patch of acidic residues common to all Myb-related domains. These acidic residues are positioned in the first of three alpha-helices within each of the three repeats that comprise the c-Myb DNA-binding domain. Interestingly, these conserved acidic residues are present on a surface of the protein which is distinct from that which binds to DNA. Alanine mutagenesis revealed that the acidic patch of the third c-Myb repeat is essential for transcriptional activity, but neither for nuclear localization nor DNA-binding. Instead, these acidic residues are required for efficient chromatin binding and interaction with the histone H4 N-terminal tail. PMID:18840288

  3. Bacterial chromatin: converging views at different scales.

    PubMed

    Dame, Remus T; Tark-Dame, Mariliis

    2016-06-01

    Bacterial genomes are functionally organized and compactly folded into a structure referred to as bacterial chromatin or the nucleoid. An important role in genome folding is attributed to Nucleoid-Associated Proteins, also referred to as bacterial chromatin proteins. Although a lot of molecular insight in the mechanisms of operation of these proteins has been generated in the test tube, knowledge on genome organization in the cellular context is still lagging behind severely. Here, we discuss important advances in the understanding of three-dimensional genome organization due to the application of Chromosome Conformation Capture and super-resolution microscopy techniques. We focus on bacterial chromatin proteins whose proposed role in genome organization is supported by these approaches. Moreover, we discuss recent insights into the interrelationship between genome organization and genome activity/stability in bacteria. PMID:26942688

  4. 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

  5. Open chromatin reveals the functional maize genome.

    PubMed

    Rodgers-Melnick, Eli; Vera, Daniel L; Bass, Hank W; Buckler, Edward S

    2016-05-31

    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

  6. Nuclease digestion studies of chromatin structure

    SciTech Connect

    Deutsch, S.M.

    1987-01-01

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

  7. The IKAROS Interaction with a Complex Including Chromatin Remodeling and Transcription Elongation Activities Is Required for Hematopoiesis

    PubMed Central

    Bottardi, Stefania; Mavoungou, Lionel; Pak, Helen; Daou, Salima; Bourgoin, Vincent; Lakehal, Yahia A.; Affar, El Bachir; Milot, Eric

    2014-01-01

    IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of IkNULL hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation. PMID:25474253

  8. Inactive X chromosome-specific histone H3 modifications and CpG hypomethylation flank a chromatin boundary between an X-inactivated and an escape gene

    PubMed Central

    Goto, Yuji; Kimura, Hiroshi

    2009-01-01

    In mammals, the dosage compensation of sex chromosomes between males and females is achieved by transcriptional inactivation of one of the two X chromosomes in females. However, a number of genes escape X-inactivation in humans. It remains poorly understood how the transcriptional activity of these ‘escape genes’ is maintained despite the chromosome-wide heterochromatin formation. To address this question, we analyzed a putative chromatin boundary between the inactivated RBM10 and an escape gene, UBA1/UBE1. Chromatin immunoprecipitation revealed that trimethylated histone H3 lysine 9 and H4 lysine 20 were enriched in the last exon through the proximal downstream region of RBM10, but were remarkably diminished at ∼2 kb upstream of the UBA1 transcription start site. Whereas RNA polymerase II was not loaded onto the intergenic region, CTCF (CCCTC binding factor) was enriched around the boundary, where some CpG sites were hypomethylated specifically on inactive X. These findings suggest that local DNA hypomethylation and CTCF binding are involved in the formation of a chromatin boundary, which protects the UBA1 escape gene against the chromosome-wide transcriptional silencing. PMID:19843608

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

    PubMed

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

    2015-06-01

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

  10. 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

  11. 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

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

    PubMed

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

    2015-01-01

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

  13. Mechanism of chromatin remodeling.

    PubMed

    Lorch, Yahli; Maier-Davis, Barbara; Kornberg, Roger D

    2010-02-23

    Results from biochemical and structural studies of the RSC chromatin-remodeling complex prompt a proposal for the remodeling mechanism: RSC binding to the nucleosome releases the DNA from the histone surface and initiates DNA translocation (through one or a small number of DNA base pairs); ATP binding completes translocation, and ATP hydrolysis resets the system. Binding energy thus plays a central role in the remodeling process. RSC may disrupt histone-DNA contacts by affecting histone octamer conformation and through extensive interaction with the DNA. Bulging of the DNA from the octamer surface is possible, and twisting is unavoidable, but neither is the basis of remodeling. PMID:20142505

  14. 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.

  15. Genome maintenance in the context of 4D chromatin condensation.

    PubMed

    Yu, Sonia; Yang, Fan; Shen, Wen H

    2016-08-01

    The eukaryotic genome is packaged in the three-dimensional nuclear space by forming loops, domains, and compartments in a hierarchical manner. However, when duplicated genomes prepare for segregation, mitotic cells eliminate topologically associating domains and abandon the compartmentalized structure. Alongside chromatin architecture reorganization during the transition from interphase to mitosis, cells halt most DNA-templated processes such as transcription and repair. The intrinsically condensed chromatin serves as a sophisticated signaling module subjected to selective relaxation for programmed genomic activities. To understand the elaborate genome-epigenome interplay during cell cycle progression, the steady three-dimensional genome requires a time scale to form a dynamic four-dimensional and a more comprehensive portrait. In this review, we will dissect the functions of critical chromatin architectural components in constructing and maintaining an orderly packaged chromatin environment. We will also highlight the importance of the spatially and temporally conscious orchestration of chromatin remodeling to ensure high-fidelity genetic transmission. PMID:27098512

  16. Chromatin Isolation by RNA Purification (ChIRP)

    PubMed Central

    Chu, Ci; Quinn, Jeffrey; Chang, Howard Y.

    2012-01-01

    Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13. Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification

  17. 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.

  18. EBV latency types adopt alternative chromatin conformations.

    PubMed

    Tempera, Italo; Klichinsky, Michael; Lieberman, Paul M

    2011-07-01

    Epstein-Barr Virus (EBV) can establish latent infections with distinct gene expression patterns referred to as latency types. These different latency types are epigenetically stable and correspond to different promoter utilization. Here we explore the three-dimensional conformations of the EBV genome in different latency types. We employed Chromosome Conformation Capture (3C) assay to investigate chromatin loop formation between the OriP enhancer and the promoters that determine type I (Qp) or type III (Cp) gene expression. We show that OriP is in close physical proximity to Qp in type I latency, and to Cp in type III latency. The cellular chromatin insulator and boundary factor CTCF was implicated in EBV chromatin loop formation. Combining 3C and ChIP assays we found that CTCF is physically associated with OriP-Qp loop formation in type I and OriP-Cp loop formation in type III latency. Mutations in the CTCF binding site located at Qp disrupt loop formation between Qp and OriP, and lead to the activation of Cp transcription. Mutation of the CTCF binding site at Cp, as well as siRNA depletion of CTCF eliminates both OriP-associated loops, indicating that CTCF plays an integral role in loop formation. These data indicate that epigenetically stable EBV latency types adopt distinct chromatin architectures that depend on CTCF and mediate alternative promoter targeting by the OriP enhancer. PMID:21829357

  19. 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

  20. Combinatorial epigenetic patterns as quantitative predictors of chromatin biology

    PubMed Central

    2014-01-01

    Background Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is the most widely used method for characterizing the epigenetic states of chromatin on a genomic scale. With the recent availability of large genome-wide data sets, often comprising several epigenetic marks, novel approaches are required to explore functionally relevant interactions between histone modifications. Computational discovery of "chromatin states" defined by such combinatorial interactions enabled descriptive annotations of genomes, but more quantitative approaches are needed to progress towards predictive models. Results We propose non-negative matrix factorization (NMF) as a new unsupervised method to discover combinatorial patterns of epigenetic marks that frequently co-occur in subsets of genomic regions. We show that this small set of combinatorial "codes" can be effectively displayed and interpreted. NMF codes enable dimensionality reduction and have desirable statistical properties for regression and classification tasks. We demonstrate the utility of codes in the quantitative prediction of Pol2-binding and the discrimination between Pol2-bound promoters and enhancers. Finally, we show that specific codes can be linked to molecular pathways and targets of pluripotency genes during differentiation. Conclusions We have introduced and evaluated a new computational approach to represent combinatorial patterns of epigenetic marks as quantitative variables suitable for predictive modeling and supervised machine learning. To foster widespread adoption of this method we make it available as an open-source software-package – epicode at https://github.com/mcieslik-mctp/epicode. PMID:24472558

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

    PubMed

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

    2016-01-01

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

  2. Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the epsilon-globin gene in vivo by 5' hypersensitive site 2 of the beta-globin locus control region.

    PubMed Central

    Gong, Q H; McDowell, J C; Dean, A

    1996-01-01

    Much of our understanding of the process by which enhancers activate transcription has been gained from transient-transfection studies in which the DNA is not assembled with histones and other chromatin proteins as it is in the cell nucleus. To study the activation of a mammalian gene in a natural chromatin context in vivo, we constructed a minichromosome containing the human epsilon-globin gene and portions of the beta-globin locus control region (LCR). The minichromosomes replicate and are maintained at stable copy number in human erythroid cells. Expression of the minichromosomal epsilon-globin gene requires the presence of beta-globin LCR elements in cis, as is the case for the chromosomal gene. We determined the chromatin structure of the epsilon-globin gene in both the active and inactive states. The transcriptionally inactive locus is covered by an array of positioned nucleosomes extending over 1,400 bp. In minichromosomes with a (mu)LCR or DNase I-hypersensitive site 2 (HS2) which actively transcribe the epsilon-globin gene, the nucleosome at the promoter is altered or disrupted while positioning of nucleosomes in the rest of the locus is retained. All or virtually all minichromosomes are simultaneously hypersensitive to DNase I both at the promoter and at HS2. Transcriptional activation and promoter remodeling, as well as formation of the HS2 structure itself, depended on the presence of the NF-E2 binding motif in HS2. The nucleosome at the promoter which is altered upon activation is positioned over the transcriptional elements of the epsilon-globin gene, i.e., the TATA, CCAAT, and CACCC elements, and the GATA-1 site at -165. The simple availability of erythroid transcription factors that recognize these motifs is insufficient to allow expression. As in the chromosomal globin locus, regulation also occurs at the level of chromatin structure. These observations are consistent with the idea that one role of the beta-globin LCR is to maintain promoters free

  3. Perturbation of chromatin structure in the region of the adult beta-globin gene in chicken erythrocyte chromatin.

    PubMed

    Caplan, A; Kimura, T; Gould, H; Allan, J

    1987-01-01

    An EcoRI chromatin fragment containing the adult beta-globin gene and flanking sequences, isolated from chicken erythrocyte nuclei, sediments at a reduced rate relative to bulk chromatin fragments of the same size. We show that the specific retardation cannot be reversed by adding extra linker histones to native chromatin. When the chromatin fragments are unfolded either by removing linker histones or lowering the ionic strength, the difference between globin and bulk chromatin fragments is no longer seen. The refolded chromatin obtained by restoring the linker histones to the depleted chromatin, however, exhibits the original sedimentation difference. This difference is therefore due to a special property of the histone octamers on the active gene that determines the extent of its folding into higher-order structure. That it is not due to the differential binding of linker histones in vitro is shown by measurements of the protein to DNA ratios using CsCl density-gradients. Both before and after selective removal of the linker histones, the globin gene fragment and bulk chromatin fragments exhibit only a marginal difference in buoyant density. In addition, we show that cleavage of the EcoRI fragment by digestion at the 5' and 3' nuclease hypersensitive sites flanking the globin gene liberates a fragment from between these sites that sediments normally. We conclude that the hypersensitive sites per se are responsible for the reduction in sedimentation rate. The non-nucleosomal DNA segments appear to be too long to be incorporated into the chromatin solenoid and thus create spacers between separate solenoidal elements in the chromatin, which can account for its hydrodynamic behaviour. PMID:3586025

  4. The Emerging Roles of ATP-Dependent Chromatin Remodeling Enzymes in Nucleotide Excision Repair

    PubMed Central

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

    2012-01-01

    DNA repair in eukaryotic cells takes place in the context of chromatin, where DNA, including damaged DNA, is tightly packed into nucleosomes and higher order chromatin structures. Chromatin intrinsically restricts accessibility of DNA repair proteins to the damaged DNA and impacts upon the overall rate of DNA repair. Chromatin is highly responsive to DNA damage and undergoes specific remodeling to facilitate DNA repair. How damaged DNA is accessed, repaired and restored to the original chromatin state, and how chromatin remodeling coordinates these processes in vivo, remains largely unknown. ATP-dependent chromatin remodelers (ACRs) are the master regulators of chromatin structure and dynamics. Conserved from yeast to humans, ACRs utilize the energy of ATP to reorganize packing of chromatin and control DNA accessibility by sliding, ejecting or restructuring nucleosomes. Several studies have demonstrated that ATP-dependent remodeling activity of ACRs plays important roles in coordination of spatio-temporal steps of different DNA repair pathways in chromatin. This review focuses on the role of ACRs in regulation of various aspects of nucleotide excision repair (NER) in the context of chromatin. We discuss current understanding of ATP-dependent chromatin remodeling by various subfamilies of remodelers and regulation of the NER pathway in vivo. PMID:23109894

  5. Chromatin folding and DNA replication inhibition mediated by a highly antitumor-active tetrazolato-bridged dinuclear platinum(II) complex

    PubMed Central

    Imai, Ryosuke; Komeda, Seiji; Shimura, Mari; Tamura, Sachiko; Matsuyama, Satoshi; Nishimura, Kohei; Rogge, Ryan; Matsunaga, Akihiro; Hiratani, Ichiro; Takata, Hideaki; Uemura, Masako; Iida, Yutaka; Yoshikawa, Yuko; Hansen, Jeffrey C.; Yamauchi, Kazuto; Kanemaki, Masato T.; Maeshima, Kazuhiro

    2016-01-01

    Chromatin DNA must be read out for various cellular functions, and copied for the next cell division. These processes are targets of many anticancer agents. Platinum-based drugs, such as cisplatin, have been used extensively in cancer chemotherapy. The drug–DNA interaction causes DNA crosslinks and subsequent cytotoxicity. Recently, it was reported that an azolato-bridged dinuclear platinum(II) complex, 5-H-Y, exhibits a different anticancer spectrum from cisplatin. Here, using an interdisciplinary approach, we reveal that the cytotoxic mechanism of 5-H-Y is distinct from that of cisplatin. 5-H-Y inhibits DNA replication and also RNA transcription, arresting cells in the S/G2 phase, and are effective against cisplatin-resistant cancer cells. Moreover, it causes much less DNA crosslinking than cisplatin, and induces chromatin folding. 5-H-Y will expand the clinical applications for the treatment of chemotherapy-insensitive cancers. PMID:27094881

  6. Chromatin folding and DNA replication inhibition mediated by a highly antitumor-active tetrazolato-bridged dinuclear platinum(II) complex.

    PubMed

    Imai, Ryosuke; Komeda, Seiji; Shimura, Mari; Tamura, Sachiko; Matsuyama, Satoshi; Nishimura, Kohei; Rogge, Ryan; Matsunaga, Akihiro; Hiratani, Ichiro; Takata, Hideaki; Uemura, Masako; Iida, Yutaka; Yoshikawa, Yuko; Hansen, Jeffrey C; Yamauchi, Kazuto; Kanemaki, Masato T; Maeshima, Kazuhiro

    2016-01-01

    Chromatin DNA must be read out for various cellular functions, and copied for the next cell division. These processes are targets of many anticancer agents. Platinum-based drugs, such as cisplatin, have been used extensively in cancer chemotherapy. The drug-DNA interaction causes DNA crosslinks and subsequent cytotoxicity. Recently, it was reported that an azolato-bridged dinuclear platinum(II) complex, 5-H-Y, exhibits a different anticancer spectrum from cisplatin. Here, using an interdisciplinary approach, we reveal that the cytotoxic mechanism of 5-H-Y is distinct from that of cisplatin. 5-H-Y inhibits DNA replication and also RNA transcription, arresting cells in the S/G2 phase, and are effective against cisplatin-resistant cancer cells. Moreover, it causes much less DNA crosslinking than cisplatin, and induces chromatin folding. 5-H-Y will expand the clinical applications for the treatment of chemotherapy-insensitive cancers. PMID:27094881

  7. Aging-related chromatin defects via loss of the NURD complex

    PubMed Central

    Pegoraro, Gianluca; Kubben, Nard; Wickert, Ute; Göhler, Heike; Hoffmann, Katrin; Misteli, Tom

    2009-01-01

    Physiological and premature aging are characterized by multiple defects in chromatin structure and accumulation of persistent DNA damage. Here we identify the NURD remodeling complex as a key modulator of these aging-associated chromatin defects. We demonstrate loss of several NURD components during premature and normal aging and we find aging-associated reduction of HDAC1 activity. Silencing of individual NURD subunits recapitulates some chromatin defects associated with aging and we provide evidence that structural chromatin defects precede DNA damage accumulation. These results outline a molecular mechanism for chromatin defects during aging. PMID:19734887

  8. Super-resolution microscopy reveals decondensed chromatin structure at transcription sites

    NASA Astrophysics Data System (ADS)

    Wang, Yejun; Maharana, Shovamayee; Wang, Michelle D.; Shivashankar, G. V.

    2014-03-01

    Remodeling of the local chromatin structure is essential for the regulation of gene expression. While a number of biochemical and bioimaging experiments suggest decondensed chromatin structures are associated with transcription, a direct visualization of DNA and transcriptionally active RNA polymerase II (RNA pol II) at super-resolution is still lacking. Here we investigate the structure of chromatin isolated from HeLa cells using binding activatable localization microscopy (BALM). The sample preparation method preserved the structural integrity of chromatin. Interestingly, BALM imaging of the chromatin spreads revealed the presence of decondensed chromatin as gap structures along the spreads. These gaps were enriched with phosphorylated S5 RNA pol II, and were sensitive to the cellular transcriptional state. Taken together, we could visualize the decondensed chromatin regions together with active RNA pol II for the first time using super-resolution microscopy.

  9. Super-resolution microscopy reveals decondensed chromatin structure at transcription sites.

    PubMed

    Wang, Yejun; Maharana, Shovamayee; Wang, Michelle D; Shivashankar, G V

    2014-01-01

    Remodeling of the local chromatin structure is essential for the regulation of gene expression. While a number of biochemical and bioimaging experiments suggest decondensed chromatin structures are associated with transcription, a direct visualization of DNA and transcriptionally active RNA polymerase II (RNA pol II) at super-resolution is still lacking. Here we investigate the structure of chromatin isolated from HeLa cells using binding activatable localization microscopy (BALM). The sample preparation method preserved the structural integrity of chromatin. Interestingly, BALM imaging of the chromatin spreads revealed the presence of decondensed chromatin as gap structures along the spreads. These gaps were enriched with phosphorylated S5 RNA pol II, and were sensitive to the cellular transcriptional state. Taken together, we could visualize the decondensed chromatin regions together with active RNA pol II for the first time using super-resolution microscopy. PMID:24667378

  10. Super-resolution microscopy reveals decondensed chromatin structure at transcription sites

    PubMed Central

    Wang, Yejun; Maharana, Shovamayee; Wang, Michelle D.; Shivashankar, G. V.

    2014-01-01

    Remodeling of the local chromatin structure is essential for the regulation of gene expression. While a number of biochemical and bioimaging experiments suggest decondensed chromatin structures are associated with transcription, a direct visualization of DNA and transcriptionally active RNA polymerase II (RNA pol II) at super-resolution is still lacking. Here we investigate the structure of chromatin isolated from HeLa cells using binding activatable localization microscopy (BALM). The sample preparation method preserved the structural integrity of chromatin. Interestingly, BALM imaging of the chromatin spreads revealed the presence of decondensed chromatin as gap structures along the spreads. These gaps were enriched with phosphorylated S5 RNA pol II, and were sensitive to the cellular transcriptional state. Taken together, we could visualize the decondensed chromatin regions together with active RNA pol II for the first time using super-resolution microscopy. PMID:24667378

  11. Studying Protein-Protein Interactions in Budding Yeast Using Co-immunoprecipitation.

    PubMed

    Foltman, Magdalena; Sanchez-Diaz, Alberto

    2016-01-01

    Understanding protein-protein interactions and the architecture of protein complexes in which they work is essential to identify their biological role. Protein co-immunoprecipitation (co-IP) is an invaluable technique used in biochemistry allowing the identification of protein interactors. Here, we describe in detail an immunoaffinity purification protocol as a one-step or two-step immunoprecipitation from budding yeast Saccharomyces cerevisiae cells to subsequently detect interactions between proteins involved in the same biological process. PMID:26519317

  12. 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.

  13. Ultrastructure of bovine sperm chromatin.

    PubMed

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

    2015-12-01

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

  14. Regulation of DNA transposition by CpG methylation and chromatin structure in human cells

    PubMed Central

    2013-01-01

    Background The activity of transposable elements can be regulated by different means. DNA CpG methylation is known to decrease or inhibit transpositional activity of diverse transposons. However, very surprisingly, it was previously shown that CpG methylation of the Sleeping Beauty (SB) transposon significantly enhanced transposition in mouse embryonic stem cells. Results In order to investigate the unexpected response of SB transposition to CpG methylation, related transposons from the Tc1/mariner superfamily, that is, Tc1, Himar1, Hsmar1, Frog Prince (FP) and Minos were tested to see how transposition was affected by CpG methylation. A significant increase of >20-fold in transposition of SB, FP and Minos was seen, whereas Tc1, Himar1 and Hsmar1 showed no difference in transposition upon CpG-methylation. The terminal inverted repeats (TIRs) of the SB, FP and Minos elements share a common structure, in which each TIR contains two functionally important binding sites for the transposase (termed the IR/DR structure). The group of IR/DR elements showed increased excision after CpG methylation compared to untreated transposon donor plasmids. We found that de novo CpG methylation is not required for transposition. A mutated FP donor plasmid with depleted CpG sites in both TIRs was as efficient in transposition as the wild-type transposon, indicating that CpG sites inside the TIRs are not responsible for altered binding of factors potentially modulating transposition. By using an in vivo one-hybrid DNA-binding assay in cultured human cells we found that CpG methylation had no appreciable effect on the affinity of SB transposase to its binding sites. However, chromatin immunoprecipitation indicated that CpG-methylated transposon donor plasmids are associated with a condensed chromatin structure characterized by trimethylated histone H3K9. Finally, DNA compaction by protamine was found to enhance SB transposition. Conclusions We have shown that DNA CpG methylation

  15. Loss of Interdependent Binding by the FoxO1 and FoxA1/A2 Forkhead Transcription Factors Culminates in Perturbation of Active Chromatin Marks and Binding of Transcriptional Regulators at Insulin-sensitive Genes.

    PubMed

    Yalley, Akua; Schill, Daniel; Hatta, Mitsutoki; Johnson, Nicole; Cirillo, Lisa Ann

    2016-04-15

    FoxO1 binds to insulin response elements located in the promoters of insulin-like growth factor-binding protein 1 (IGFBP1) and glucose-6-phosphatase (G6Pase), activating their expression. Insulin-mediated phosphorylation of FoxO1 promotes cytoplasmic translocation, inhibiting FoxO1-mediated transactivation. We have previously demonstrated that FoxO1 opens and remodels chromatin assembled from the IGFBP1 promoter via a highly conserved winged helix motif. This finding, which established FoxO1 as a "pioneer" factor, suggested a model whereby FoxO1 chromatin remodeling at regulatory targets facilitates binding and recruitment of additional regulatory factors. However, the impact of FoxO1 phosphorylation on its ability to bind chromatin and the effect of FoxO1 loss on recruitment of neighboring transcription factors at its regulatory targets in liver chromatin is unknown. In this study, we demonstrate that an amino acid substitution that mimics insulin-mediated phosphorylation of a serine in the winged helix DNA binding motif curtails FoxO1 nucleosome binding. We also demonstrate that shRNA-mediated loss of FoxO1 binding to the IGFBP1 and G6Pase promoters in HepG2 cells significantly reduces binding of RNA polymerase II and the pioneer factors FoxA1/A2. Knockdown of FoxA1 similarly reduced binding of RNA polymerase II and FoxO1. Reduction in acetylation of histone H3 Lys-27 accompanies loss of FoxO1 and FoxA1/A2 binding. Interdependent binding of FoxO1 and FoxA1/A2 possibly entails cooperative binding because FoxO1 and FoxA1/A2 facilitate one another's binding to IGFPB1 promoter DNA. These results illustrate how transcription factors can nucleate transcriptional events in chromatin in response to signaling events and suggest a model for regulation of hepatic glucose metabolism through interdependent FoxO/FoxA binding. PMID:26929406

  16. Chromatin, epigenetics and stem cells.

    PubMed

    Roloff, Tim C; Nuber, Ulrike A

    2005-03-01

    Epigenetics is a term that has changed its meaning with the increasing biological knowledge on developmental processes. However, its current application to stem cell biology is often imprecise and is conceptually problematic. This article addresses two different subjects, the definition of epigenetics and chromatin states of stem and differentiated cells. We describe mechanisms that regulate chromatin changes and provide an overview of chromatin states of stem and differentiated cells. Moreover, a modification of the current epigenetics definition is proposed that is not restricted by the heritability of gene expression throughout cell divisions and excludes translational gene expression control. PMID:15819395

  17. Chromatin organization: form to function.

    PubMed

    de Graaf, Carolyn A; van Steensel, Bas

    2013-04-01

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

  18. CTCF-Mediated Functional Chromatin Interactome in Pluripotent Cells

    PubMed Central

    Handoko, Lusy; Xu, Han; Li, Guoliang; Ngan, Chew Yee; Chew, Elaine; Schnapp, Marie; Lee, Charlie Wah Heng; Ye, Chaopeng; Ping, Joanne Lim Hui; Mulawadi, Fabianus; Wong, Eleanor; Sheng, Jianpeng; Zhang, Yubo; Poh, Thompson; Chan, Chee Seng; Kunarso, Galih; Shahab, Atif; Bourque, Guillaume; Cacheux-Rataboul, Valere; Sung, Wing-Kin; Ruan, Yijun; Wei, Chia-Lin

    2011-01-01

    Mammalian genomes are viewed as functional organizations that orchestrate spatial and temporal gene regulation. CTCF, the most characterized insulator-binding protein, has been implicated as a key genome organizer. Yet, little is known about CTCF-associated higher order chromatin structures at a global scale. Here, we applied Chromatin Interaction Analysis by Paired-End-Tag sequencing to elucidate the CTCF-chromatin interactome in pluripotent cells. From this analysis, 1,480 cis and 336 trans interacting loci were identified with high reproducibility and precision. Associating these chromatin interaction loci with their underlying epigenetic states, promoter activities, enhancer binding and nuclear lamina occupancy, we uncovered five distinct chromatin domains that suggest potential new models of CTCF function in chromatin organization and transcriptional control. Specifically, CTCF interactions demarcate chromatin-nuclear membrane attachments and influence proper gene expression through extensive crosstalk between promoters and regulatory elements. This highly complex nuclear organization offers insights towards the unifying principles governing genome plasticity and function. PMID:21685913

  19. 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

  20. 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

  1. Towards an immuno-precipitated neurodevelopmental animal model of schizophrenia.

    PubMed

    Meyer, Urs; Feldon, Joram; Schedlowski, Manfred; Yee, Benjamin K

    2005-01-01

    Epidemiological studies have indicated an association between maternal bacterial and viral infections during pregnancy and the higher incidence of schizophrenia in the resultant offspring post-puberty. One hypothesis asserts that the reported epidemiological link is mediated by prenatal activation of the foetal immune system in response to the elevation of maternal cytokine level due to infection. Here, we report that pregnant mouse dams receiving a single exposure to the cytokine-releasing agent, polyriboinosinic-polyribocytidilic acid (PolyI:C; at 2.5, 5.0, or 10.0 mg/kg) on gestation day 9 produced offspring that subsequently exhibited multiple schizophrenia-related behavioural deficits in adulthood, in comparison to offspring from vehicle injected or non-injected control dams. The efficacy of the PolyI:C challenge to induce cytokine responses in naïve non-pregnant adult female mice and in foetal brain tissue when injected to pregnant mice were further ascertained in separate subjects: (i) a dose-dependent elevation of interleukin-10 was detected in the adult female mice at 1 and 6h post-injection, (ii) 12 h following prenatal PolyI:C challenge, the foetal levels of interleukin-1beta were elevated. The spectrum of abnormalities included impairments in exploratory behaviour, prepulse inhibition, latent inhibition, the US-pre-exposure effect, spatial working memory; and enhancement in the locomotor response to systemic amphetamine (2.5 mg/kg, i.p.) as well as in discrimination reversal learning. The neuropsychological parallels between prenatal PolyI:C treatment in mice and psychosis in humans, demonstrated here, leads us to conclude that prenatal PolyI:C treatment represents one of the most powerful environmental-developmental models of schizophrenia to date. The uniqueness of this model lies in its epidemiological and immunological relevance. It is, sui generis, ideally suited for the investigation of the neuropsychoimmunological mechanisms implicated in the

  2. 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

  3. 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

  4. Painting a Clearer Picture of Chromatin.

    PubMed

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

    2016-02-22

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

  5. 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.

  6. Ectopically tethered CP190 induces large-scale chromatin decondensation

    PubMed Central

    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. PMID:24472778

  7. High Mobility Group Protein N5 (HMGN5) and Lamina-associated Polypeptide 2α (LAP2α) Interact and Reciprocally Affect Their Genome-wide Chromatin Organization*

    PubMed Central

    Zhang, Shaofei; Schones, Dustin E.; Malicet, Cedric; Rochman, Mark; Zhou, Ming; Foisner, Roland; Bustin, Michael

    2013-01-01

    The interactions of nuclear lamins with the chromatin fiber play an important role in regulating nuclear architecture and chromatin function; however, the full spectrum of these interactions is not known. We report that the N-terminal domain of the nucleosome-binding protein HMGN5 interacts with the C-terminal domain of the lamin-binding protein LAP2α and that these proteins reciprocally alter their interaction with chromatin. Chromatin immunoprecipitation analysis of cells lacking either HMGN5 or LAP2α reveals that loss of either protein affects the genome-wide distribution of the remaining partner. Our study identifies a new functional link between chromatin-binding and lamin-binding proteins. PMID:23673662

  8. 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.

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

    PubMed Central

    Gerdes, J; Stein, H

    1980-01-01

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

  10. A Cbx8-containing polycomb complex facilitates the transition to gene activation during ES cell differentiation.

    PubMed

    Creppe, Catherine; Palau, Anna; Malinverni, Roberto; Valero, Vanesa; Buschbeck, Marcus

    2014-12-01

    Polycomb proteins play an essential role in maintaining the repression of developmental genes in self-renewing embryonic stem cells. The exact mechanism allowing the derepression of polycomb target genes during cell differentiation remains unclear. Our project aimed to identify Cbx8 binding sites in differentiating mouse embryonic stem cells. Therefore, we used a genome-wide chromatin immunoprecipitation of endogenous Cbx8 coupled to direct massive parallel sequencing (ChIP-Seq). Our analysis identified 171 high confidence peaks. By crossing our data with previously published microarray analysis, we show that several differentiation genes transiently recruit Cbx8 during their early activation. Depletion of Cbx8 partially impairs the transcriptional activation of these genes. Both interaction analysis, as well as chromatin immunoprecipitation experiments support the idea that activating Cbx8 acts in the context of an intact PRC1 complex. Prolonged gene activation results in eviction of PRC1 despite persisting H3K27me3 and H2A ubiquitination. The composition of PRC1 is highly modular and changes when embryonic stem cells commit to differentiation. We further demonstrate that the exchange of Cbx7 for Cbx8 is required for the effective activation of differentiation genes. Taken together, our results establish a function for a Cbx8-containing complex in facilitating the transition from a Polycomb-repressed chromatin state to an active state. As this affects several key regulatory differentiation genes this mechanism is likely to contribute to the robust execution of differentiation programs. PMID:25500566

  11. Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin.

    PubMed

    Bandaria, Jigar N; Qin, Peiwu; Berk, Veysel; Chu, Steven; Yildiz, Ahmet

    2016-02-11

    Telomeres, repetitive DNA sequences at chromosome ends, are shielded against the DNA damage response (DDR) by the shelterin complex. To understand how shelterin protects telomere ends, we investigated the structural organization of telomeric chromatin in human cells using super-resolution microscopy. We found that telomeres form compact globular structures through a complex network of interactions between shelterin subunits and telomeric DNA, but not by DNA methylation, histone deacetylation, or histone trimethylation at telomeres and subtelomeric regions. Mutations that abrogate shelterin assembly or removal of individual subunits from telomeres cause up to a 10-fold increase in telomere volume. Decompacted telomeres accumulate DDR signals and become more accessible to telomere-associated proteins. Recompaction of telomeric chromatin using an orthogonal method displaces DDR signals from telomeres. These results reveal the chromatin remodeling activity of shelterin and demonstrate that shelterin-mediated compaction of telomeric chromatin provides robust protection of chromosome ends against the DDR machinery. PMID:26871633

  12. Probing Chromatin-modifying Enzymes with Chemical Tools.

    PubMed

    Fischle, Wolfgang; Schwarzer, Dirk

    2016-03-18

    Chromatin is the universal template of genetic information in all eukaryotic organisms. Chemical modifications of the DNA-packaging histone proteins and the DNA bases are crucial signaling events in directing the use and readout of eukaryotic genomes. The enzymes that install and remove these chromatin modifications as well as the proteins that bind these marks govern information that goes beyond the sequence of DNA. Therefore, these so-called epigenetic regulators are intensively studied and represent promising drug targets in modern medicine. We summarize and discuss recent advances in the field of chemical biology that have provided chromatin research with sophisticated tools for investigating the composition, activity, and target sites of chromatin modifying enzymes and reader proteins. PMID:26845102

  13. ISWI chromatin remodeling complexes in the DNA damage response

    PubMed Central

    Aydin, Özge Z; Vermeulen, Wim; Lans, Hannes

    2014-01-01

    Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA. PMID:25486562

  14. Establishing Chromatin Regulatory Landscape during Mouse Preimplantation Development.

    PubMed

    Lu, Falong; Liu, Yuting; Inoue, Azusa; Suzuki, Tsukasa; Zhao, Keji; Zhang, Yi

    2016-06-01

    How the chromatin regulatory landscape in the inner cell mass cells is established from differentially packaged sperm and egg genomes during preimplantation development is unknown. Here, we develop a low-input DNase I sequencing (liDNase-seq) method that allows us to generate maps of DNase I-hypersensitive site (DHS) of mouse preimplantation embryos from 1-cell to morula stage. The DHS landscape is progressively established with a drastic increase at the 8-cell stage. Paternal chromatin accessibility is quickly reprogrammed after fertilization to the level similar to maternal chromatin, while imprinted genes exhibit allelic accessibility bias. We demonstrate that transcription factor Nfya contributes to zygotic genome activation and DHS formation at the 2-cell stage and that Oct4 contributes to the DHSs gained at the 8-cell stage. Our study reveals the dynamic chromatin regulatory landscape during early development and identifies key transcription factors important for DHS establishment in mammalian embryos. PMID:27259149

  15. Effect of irradiation and endogenous nucleases on rat liver chromatin

    SciTech Connect

    Gelderblom, D.; Smit, B.J.; Boehm, L.

    1984-08-01

    The assessment of the consequences of irradiation on chromatin is complicated by endogenous nucleases. Isolation and prolonged storage of rat liver nuclei in buffers containing divalent metal ions activates these enzymes and promotes the degradation of chromatin. Irradiation of rat liver nuclei to dose levels of 20,000 rad under conditions in which endogenous nucleases are inhibited and analysis of the irradiated chromatin by sucrose density gradient centrifugation gave no evidence for monosomes or oligosomes. When chromatin from irradiated nuclei was digested with micrococcal nuclease, the levels of monosomes and oligosomes were identical to those of micrococcal nuclease digests of unirradiated control nuclei. These results suggest that irradiation results in neither a direct fragmentation of linkers nor the sensitization of linkers for subsequent cleavage by micrococcal nuclease.

  16. Facilitation of base excision repair by chromatin remodeling.

    PubMed

    Hinz, John M; Czaja, Wioletta

    2015-12-01

    Base Excision Repair (BER) is a conserved, intracellular DNA repair system that recognizes and removes chemically modified bases to insure genomic integrity and prevent mutagenesis. Aberrant BER has been tightly linked with a broad spectrum of human pathologies, such as several types of cancer, neurological degeneration, developmental abnormalities, immune dysfunction and aging. In the cell, BER must recognize and remove DNA lesions from the tightly condensed, protein-coated chromatin. Because chromatin is necessarily refractory to DNA metabolic processes, like transcription and replication, the compaction of the genomic material is also inhibitory to the repair systems necessary for its upkeep. Multiple ATP-dependent chromatin remodelling (ACR) complexes play essential roles in modulating the protein-DNA interactions within chromatin, regulating transcription and promoting activities of some DNA repair systems, including double-strand break repair and nucleotide excision repair. However, it remains unclear how BER operates in the context of chromatin, and if the chromatin remodelling processes that govern transcription and replication also actively regulate the efficiency of BER. In this review we highlight the emerging role of ACR in regulation of BER. PMID:26422134

  17. Chromatin topology is coupled to Polycomb group protein subnuclear organization.

    PubMed

    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

  18. 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

  19. Characterization of Novel Paternal ncRNAs at the Plagl1 Locus, Including Hymai, Predicted to Interact with Regulators of Active Chromatin

    PubMed Central

    Cirillo, Davide; Court, Franck; Guillaumet-Adkins, Amy; Camprubi, Cristina; Bourc’his, Deborah; Hata, Kenichiro; Feil, Robert; Tartaglia, Gian; Arnaud, Philippe; Monk, David

    2012-01-01

    Genomic imprinting is a complex epigenetic mechanism of transcriptional control that utilizes DNA methylation and histone modifications to bring about parent-of-origin specific monoallelic expression in mammals. Genes subject to imprinting are often organised in clusters associated with large non-coding RNAs (ncRNAs), some of which have cis-regulatory functions. Here we have undertaken a detailed allelic expression analysis of an imprinted domain on mouse proximal chromosome 10 comprising the paternally expressed Plagl1 gene. We identified three novel Plagl1 transcripts, only one of which contains protein-coding exons. In addition, we characterised two unspliced ncRNAs, Hymai, the mouse orthologue of HYMAI, and Plagl1it (Plagl1 intronic transcript), a transcript located in intron 5 of Plagl1. Imprinted expression of these novel ncRNAs requires DNMT3L-mediated maternal DNA methylation, which is also indispensable for establishing the correct chromatin profile at the Plagl1 DMR. Significantly, the two ncRNAs are retained in the nucleus, consistent with a potential regulatory function at the imprinted domain. Analysis with catRAPID, a protein-ncRNA association prediction algorithm, suggests that Hymai and Plagl1it RNAs both have potentially high affinity for Trithorax chromatin regulators. The two ncRNAs could therefore help to protect the paternal allele from DNA methylation by attracting Trithorax proteins that mediate H3 lysine-4 methylation. Submitted GenBank nucleotides sequences: Plagl1it: JN595789 Hymai: JN595790 PMID:22723905

  20. Chromatin perturbations during the DNA damage response in higher eukaryotes

    PubMed Central

    Bakkenist, Christopher J.; Kastan, Michael B.

    2016-01-01

    The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response

  1. 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

  2. Mammalian sperm chromatin as a model for chromatin function in DNA degradation and DNA replication.

    PubMed

    Ortega, Michael A; Sil, Payel; Ward, W Steven

    2011-02-01

    Reproductive biology is considered a specialty field, however, an argument can be made that it is instead generally applicable to many fields of biology. The one-cell embryo is presented here as a model system for the study of eukaryotic DNA replication, apoptotic DNA degradation, and signaling mechanisms between the cytoplasm and nucleus. Two unique aspects of this system combine to make it particularly useful for the study of chromatin function. First, the evolutionary pressure that lead to the extreme condensation of mammalian sperm DNA resulted in a cell with virtually inert chromatin, no DNA replication or transcription ongoing in the sperm cell, and all of the cells in a G(0) state. This chromatin is suddenly transformed into actively transcribing and replicating DNA upon fertilization. Therefore, the sperm chromatin is poised to become active but does not yet possess sufficient components present in somatic chromatin structure for all these processes. The second unique aspect of this system is that the one cell embryo houses two distinct nuclei, termed pronuclei, through the first round of DNA synthesis. This means the sperm cell can be experimentally manipulated to test the affects of the various treatments on the biological functions of interest. Experimental manipulations of the system have already revealed a certain level of plasticity in the coordination of both the timing of DNA synthesis in the two pronuclei and in the response to cellular signals by each pronucleus involved with the progression through the G1/S checkpoint, including the degradation of DNA in the paternal pronucleus. The fact that two nuclei in the same cytoplasm can undergo different responses infers a level of autonomy in the nuclear control of the cell cycle. Thus, the features of mammalian fertilization can provide unique insights for the normal biology of the cell cycle in somatic cells. PMID:21204750

  3. 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.

  4. Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein.

    PubMed

    Verhelst, Judith; De Vlieger, Dorien; Saelens, Xavier

    2015-01-01

    Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP-Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually. PMID:25938860

  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. PMID:26577786

  6. 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

  7. p300 Forms a Stable, Template-Committed Complex with Chromatin: Role for the Bromodomain

    PubMed Central

    Manning, E. Tory; Ikehara, Tsuyoshi; Ito, Takashi; Kadonaga, James T.; Kraus, W. Lee

    2001-01-01

    The nature of the interaction of coactivator proteins with transcriptionally active promoters in chromatin is a fundamental question in transcriptional regulation by RNA polymerase II. In this study, we used a biochemical approach to examine the functional association of the coactivator p300 with chromatin templates. Using in vitro transcription template competition assays, we observed that p300 forms a stable, template-committed complex with chromatin during the transcription process. The template commitment is dependent on the time of incubation of p300 with the chromatin template and occurs independently of the presence of a transcriptional activator protein. In studies examining interactions between p300 and chromatin, we found that p300 binds directly to chromatin and that the binding requires the p300 bromodomain, a conserved 110-amino-acid sequence found in many chromatin-associated proteins. Furthermore, we observed that the isolated p300 bromodomain binds directly to histones, preferentially to histone H3. However, the isolated p300 bromodomain does not bind to nucleosomal histones under the same assay conditions, suggesting that free histones and nucleosomal histones are not equivalent as binding substrates. Collectively, our results suggest that the stable association of p300 with chromatin is mediated, at least in part, by the bromodomain and is critically important for p300 function. Furthermore, our results suggest a model for p300 function that involves distinct activator-dependent targeting and activator-independent chromatin binding activities. PMID:11359896

  8. Chromatin insulators: regulatory mechanisms and epigenetic inheritance

    PubMed Central

    Bushey, Ashley M.; Dorman, Elizabeth R.; Corces, Victor G.

    2008-01-01

    Enhancer-blocking insulators are DNA elements that disrupt the communication between a regulatory sequence, such as an enhancer or a silencer, and a promoter. Insulators participate in both transcriptional regulation and global nuclear organization, two features of chromatin that are thought to be maintained from one generation to the next through epigenetic mechanisms. Furthermore, there are many regulatory mechanisms in place that enhance or hinder insulator activity. These modes of regulation could be used to establish cell-type specific insulator activity that is epigenetically inherited along a cell and/or organismal lineage. This review will discuss the evidence for epigenetic inheritance and regulation of insulator function. PMID:18851828

  9. Integrative annotation of chromatin elements from ENCODE data

    PubMed Central

    Hoffman, Michael M.; Ernst, Jason; Wilder, Steven P.; Kundaje, Anshul; Harris, Robert S.; Libbrecht, Max; Giardine, Belinda; Ellenbogen, Paul M.; Bilmes, Jeffrey A.; Birney, Ewan; Hardison, Ross C.; Dunham, Ian; Kellis, Manolis; Noble, William Stafford

    2013-01-01

    The ENCODE Project has generated a wealth of experimental information mapping diverse chromatin properties in several human cell lines. Although each such data track is independently informative toward the annotation of regulatory elements, their interrelations contain much richer information for the systematic annotation of regulatory elements. To uncover these interrelations and to generate an interpretable summary of the massive datasets of the ENCODE Project, we apply unsupervised learning methodologies, converting dozens of chromatin datasets into discrete annotation maps of regulatory regions and other chromatin elements across the human genome. These methods rediscover and summarize diverse aspects of chromatin architecture, elucidate the interplay between chromatin activity and RNA transcription, and reveal that a large proportion of the genome lies in a quiescent state, even across multiple cell types. The resulting annotation of non-coding regulatory elements correlate strongly with mammalian evolutionary constraint, and provide an unbiased approach for evaluating metrics of evolutionary constraint in human. Lastly, we use the regulatory annotations to revisit previously uncharacterized disease-associated loci, resulting in focused, testable hypotheses through the lens of the chromatin landscape. PMID:23221638

  10. Linker histone variants control chromatin dynamics during early embryogenesis

    PubMed Central

    Saeki, Hideaki; Ohsumi, Keita; Aihara, Hitoshi; Ito, Takashi; Hirose, Susumu; Ura, Kiyoe; Kaneda, Yasufumi

    2005-01-01

    Complex transitions in chromatin structure produce changes in genome function during development in metazoa. Linker histones, the last component of nucleosomes to be assembled into chromatin, comprise considerably divergent subtypes as compared with core histones. In all metazoa studied, their composition changes dramatically during early embryogenesis concomitant with zygotic gene activation, leading to distinct functional changes that are still poorly understood. Here, we show that early embryonic linker histone B4, which is maternally expressed, is functionally different from somatic histone H1 in influencing chromatin structure and dynamics. We developed a chromatin assembly system with nucleosome assembly protein-1 as a linker histone chaperone. This assay system revealed that maternal histone B4 allows chromatin to be remodeled by ATP-dependent chromatin remodeling factor, whereas somatic histone H1 prevents this remodeling. Structural analysis shows that histone B4 does not significantly restrict the accessibility of linker DNA. These findings define the functional significance of developmental changes in linker histone variants. We propose a model that holds that maternally expressed linker histones are key molecules specifying nuclear dynamics with respect to embryonic totipotency. PMID:15821029

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

    PubMed Central

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

    2015-01-01

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

  12. Minireview: role of kinases and chromatin remodeling in progesterone signaling to chromatin.

    PubMed

    Vicent, Guillermo P; Nacht, A Silvina; Zaurín, Roser; Ballaré, Cecilia; Clausell, Jaime; Beato, Miguel

    2010-11-01

    Steroid hormones regulate gene expression by interaction of their receptors with hormone-responsive elements on DNA or with other transcription factors, but they can also activate cytoplasmic signaling cascades. Rapid activation of Erk by progestins via an interaction of the progesterone receptor (PR) with the estrogen receptor is critical for transcriptional activation of the mouse mammary tumor virus (MMTV) promoter and other progesterone target genes. Erk activation leads to the phosphorylation of PR, activation of mitogen- and stress-activated protein kinase 1, and the recruitment of a complex of the three activated proteins and of P300/CBP-associated factor (PCAF) to a single nucleosome, resulting in the phosphoacetylation of histone H3 and the displacement of heterochromatin protein 1γ. Hormone-dependent gene expression requires ATP-dependent chromatin remodeling complexes. Two switch/sucrose nonfermentable-like complexes, Brahma-related gene 1-associated factor (BAF) and polybromo-BAF are present in breast cancer cells, but only BAF is recruited to the MMTV promoter and cooperates with PCAF during activation of hormone-responsive promoters. PCAF acetylates histone H3 at K14, an epigenetic mark recognized by BAF subunits, thus anchoring the complex to chromatin. BAF catalyzes localized displacement of histones H2A and H2B, facilitating access of nuclear factor 1 and additional PR complexes to the hidden hormone-responsive elements on the MMTV promoter. The linker histone H1 is a structural component of chromatin generally regarded as a general repressor of transcription. However, it contributes to a better regulation of the MMTV promoter by favoring a more homogeneous nucleosome positioning, thus reducing basal transcription and actually enhancing hormone induced transcription. During transcriptional activation, H1 is phosphorylated and displaced from the promoter. The kinase cyclin-dependent kinase 2 is activated after progesterone treatment and could

  13. Snapshots: Chromatin Control of Viral Infection

    PubMed Central

    Knipe, David M.; Lieberman, Paul M.; Jung, Jae U.; McBride, Alison A.; Morris, Kevin V.; Ott, Melanie; Margolis, David; Nieto, Amelia; Nevels, Michael; Parks, Robin J.; Kristie, Thomas M.

    2012-01-01

    Like their cellular host counterparts, many invading viral pathogens must contend with, modulate, and utilize the host cell’s chromatin machinery to promote efficient lytic infection or control persistent-latent states. While not intended to be comprehensive, this review represents a compilation of conceptual snapshots of the dynamic interplay of viruses with the chromatin environment. Contributions focus on chromatin dynamics during infection, viral circumvention of cellular chromatin repression, chromatin organization of large DNA viruses, tethering and persistence, viral interactions with cellular chromatin modulation machinery, and control of viral latency-reactivation cycles. PMID:23217624

  14. 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.

  15. Chromatin Assembly at Kinetochores Is Uncoupled from DNA Replication

    PubMed Central

    Shelby, Richard D.; Monier, Karine; Sullivan, Kevin F.

    2000-01-01

    The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric “state” on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [3H]thymidine we demonstrate that CENP-A–associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation. PMID:11086012

  16. Tandem immunoprecipitation approach to identify HIV-1 Gag associated host factors.

    PubMed

    Gao, Wei; Li, Min; Zhang, Jingxin

    2014-07-01

    HIV-1 Gag by itself is able to assemble and release from host cells and thus serves as a simplified model to identify host factors involved in this stage of the HIV-1 life cycle. In this study, a tandem immunoprecipitation approach is taken to immunoprecipitate Gag-interacting host proteins from transfected 293T cells. It is demonstrated that with the tandem immunoprecipitation method Gag-interacting host factors can be precipitated more efficiently than by single-step immunoprecipitation. Gag proteins are found to interact with multiple RNA-binding proteins such as hnRNPs, nucleolin, EF1a and ribosomal proteins. Such interactions are mediated by cellular RNAs and the Gag Nuclear Capsid (NC) domain. Deletion of the NC domain results in removal of most of the RNA-binding proteins, as well as a reduction of the Gag releasing capability, which can be restored by replacing the deleted NC domain with another multimerization motif. Importantly, interactions between Gag and host factors are relevant functionally, as evidenced by significantly increased nucleolin protein in the cytoplasm where it is recruited into the Gag complex, and enhanced Gag release when nucleolin is over-expressed. PMID:24690621

  17. Global quantitative modeling of chromatin factor interactions.

    PubMed

    Zhou, Jian; Troyanskaya, Olga G

    2014-03-01

    Chromatin is the driver of gene regulation, yet understanding the molecular interactions underlying chromatin factor combinatorial patterns (or the "chromatin codes") remains a fundamental challenge in chromatin biology. Here we developed a global modeling framework that leverages chromatin profiling data to produce a systems-level view of the macromolecular complex of chromatin. Our model ultilizes maximum entropy modeling with regularization-based structure learning to statistically dissect dependencies between chromatin factors and produce an accurate probability distribution of chromatin code. Our unsupervised quantitative model, trained on genome-wide chromatin profiles of 73 histone marks and chromatin proteins from modENCODE, enabled making various data-driven inferences about chromatin profiles and interactions. We provided a highly accurate predictor of chromatin factor pairwise interactions validated by known experimental evidence, and for the first time enabled higher-order interaction prediction. Our predictions can thus help guide future experimental studies. The model can also serve as an inference engine for predicting unknown chromatin profiles--we demonstrated that with this approach we can leverage data from well-characterized cell types to help understand less-studied cell type or conditions. PMID:24675896

  18. Thioredoxin interacting protein (TXNIP) induces inflammation through chromatin modification in retinal capillary endothelial cells under diabetic conditions.

    PubMed

    Perrone, Lorena; Devi, Takhellambam S; Hosoya, Ken-ichi; Terasaki, Tetsuya; Singh, Lalit P

    2009-10-01

    Chronic hyperglycemia and activation of receptor for advanced glycation end products (RAGE) are known risk factors for microvascular disease development in diabetic retinopathy. Thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of antioxidant thioredoxin (TRX), plays a causative role in diabetes and its vascular complications. Herein we investigate whether HG and RAGE induce inflammation in rat retinal endothelial cells (EC) under diabetic conditions in culture through TXNIP activation and whether epigenetic mechanisms play a role in inflammatory gene expression. We show that RAGE activation by its ligand S100B or HG treatment of retinal EC induces the expression of TXNIP and inflammatory genes such as Cox2, VEGF-A, and ICAM1. TXNIP silencing by siRNA impedes RAGE and HG effects while stable over-expression of a cDNA for human TXNIP in EC elevates inflammation. p38 MAPK-NF-kappaB signaling pathway and histone H3 lysine (K) nine modifications are involved in TXNIP-induced inflammation. Chromatin immunoprecipitation (ChIP) assays reveal that TXNIP over-expression in EC abolishes H3K9 tri-methylation, a marker for gene inactivation, and increases H3K9 acetylation, an indicator of gene induction, at proximal Cox2 promoter bearing the NF-kappaB-binding site. These findings have important implications toward understanding the molecular mechanisms of ocular inflammation and endothelial dysfunction in diabetic retinopathy. PMID:19562690

  19. Chromatin states reveal functional associations for globally defined transcription start sites in four human cell lines

    PubMed Central

    2014-01-01

    Background Deciphering the most common modes by which chromatin regulates transcription, and how this is related to cellular status and processes is an important task for improving our understanding of human cellular biology. The FANTOM5 and ENCODE projects represent two independent large scale efforts to map regulatory and transcriptional features to the human genome. Here we investigate chromatin features around a comprehensive set of transcription start sites in four cell lines by integrating data from these two projects. Results Transcription start sites can be distinguished by chromatin states defined by specific combinations of both chromatin mark enrichment and the profile shapes of these chromatin marks. The observed patterns can be associated with cellular functions and processes, and they also show association with expression level, location relative to nearby genes, and CpG content. In particular we find a substantial number of repressed inter- and intra-genic transcription start sites enriched for active chromatin marks and Pol II, and these sites are strongly associated with immediate-early response processes and cell signaling. Associations between start sites with similar chromatin patterns are validated by significant correlations in their global expression profiles. Conclusions The results confirm the link between chromatin state and cellular function for expressed transcripts, and also indicate that active chromatin states at repressed transcripts may poise transcripts for rapid activation during immune response. PMID:24669905

  20. Human THAP7 is a chromatin-associated, histone tail-binding protein that represses transcription via recruitment of HDAC3 and nuclear hormone receptor corepressor.

    PubMed

    Macfarlan, Todd; Kutney, Sara; Altman, Brian; Montross, Rebecca; Yu, Jiujiu; Chakravarti, Debabrata

    2005-02-25

    The identities of signal transducer proteins that integrate histone hypoacetylation and transcriptional repression are largely unknown. Here we demonstrate that THAP7, an uncharacterized member of the recently identified THAP (Thanatos-associated protein) family of proteins, is ubiquitously expressed, associates with chromatin, and represses transcription. THAP7 binds preferentially to hypoacetylated (un-, mono-, and diacetylated) histone H4 tails in vitro via its C-terminal 77 amino acids. Deletion of this domain, or treatment of cells with the histone deacetylase inhibitor TSA, which leads to histone hyperacetylation, partially disrupts THAP7/chromatin association in living cells. THAP7 coimmunoprecipitates with histone deacetylase 3 (HDAC3) and the nuclear hormone receptor corepressor (NCoR) and represses transcription as a Gal4 fusion protein. Chromatin immunoprecipitation assays demonstrate that these corepressors are recruited to promoters in a THAP7 dependent manner and promote histone H3 hypoacetylation. The conserved THAP domain is a key determinant for full HDAC3 association in vitro, and both the THAP domain and the histone interaction domain are important for the repressive properties of THAP7. Full repression mediated by THAP7 is also dependent on NCoR expression. We hypothesize that THAP7 is a dual function repressor protein that actively targets deacetylation of histone H3 necessary to establish transcriptional repression and functions as a signal transducer of the repressive mark of hypoacetylated histone H4. This is the first demonstration of the transcriptional regulatory properties of a human THAP domain protein, and a critical identification of a potential transducer of the repressive signal of hypoacetylated histone H4 in higher eukaryotes. PMID:15561719

  1. Extinction of Oct-3/4 gene expression in embryonal carcinoma [times] fibroblast somatic cell hybrids is accompanied by changes in the methylation status, chromatin structure, and transcriptional activity of the Oct-3/4 upstream region

    SciTech Connect

    Ben-Shushan, E.; Pikarsky, E.; Klar, A.; Bergman, Y. )

    1993-02-01

    The OCT-3/4 gene provides an excellent model system with which to study the extinction phenomenon in somatic cell hybrids. The molecular mechanism that underlies the extinction of a tissue-specific transcription factor in somatic cell hybrides is evaluated and compared with its down-regulation in retinoic acid treated embryonal carcinoma cells. This study draws a connection between the shutdown of OCT-3/4 expression in retinoic acid (RA)-differentiated embryonal carcinoma (EC) cells and its extinction in hybrid cells. This repression of OCT-3/4 expression is achieved through changes in the methylation status, chromatin structure, and transcriptional activity of the OCT-3/4 upstream regulatory region. 59 refs.

  2. Recruitment, Duplex Unwinding and Protein-Mediated Inhibition of the Dead-Box RNA Helicase Dbp2 at Actively Transcribed Chromatin.

    PubMed

    Ma, Wai Kit; Paudel, Bishnu P; Xing, Zheng; Sabath, Ivan G; Rueda, David; Tran, Elizabeth J

    2016-03-27

    RNA helicases play fundamental roles in modulating RNA structures and facilitating RNA-protein (RNP) complex assembly in vivo. Previously, our laboratory demonstrated that the DEAD-box RNA helicase Dbp2 in Saccharomyces cerevisiae is required to promote efficient assembly of the co-transcriptionally associated mRNA-binding proteins Yra1, Nab2, and Mex67 onto poly(A)(+)RNA. We also found that Yra1 associates directly with Dbp2 and functions as an inhibitor of Dbp2-dependent duplex unwinding, suggestive of a cycle of unwinding and inhibition by Dbp2. To test this, we undertook a series of experiments to shed light on the order of events for Dbp2 in co-transcriptional mRNP assembly. We now show that Dbp2 is recruited to chromatin via RNA and forms a large, RNA-dependent complex with Yra1 and Mex67. Moreover, single-molecule fluorescence resonance energy transfer and bulk biochemical assays show that Yra1 inhibits unwinding in a concentration-dependent manner by preventing the association of Dbp2 with single-stranded RNA. This inhibition prevents over-accumulation of Dbp2 on mRNA and stabilization of a subset of RNA polymerase II transcripts. We propose a model whereby Yra1 terminates a cycle of mRNP assembly by Dbp2. PMID:26876600

  3. Rules of Engagement for Base Excision Repair in Chromatin

    PubMed Central

    Odell, Ian D.; Wallace, Susan S.; Pederson, David S.

    2012-01-01

    Most of the DNA in eukaryotes is packaged in tandemly arrayed nucleosomes that, together with numerous DNA- and nucleosome-associated enzymes and regulatory factors, make up chromatin. Chromatin modifying and remodeling agents help regulate access to selected DNA segments in chromatin, thereby facilitating transcription and DNA replication and repair. Studies of nucleotide excision repair (NER), single strand break repair (SSBR), and the homology-directed (HDR) and non-homologous end-joining (NHEJ) double strand break repair pathways have led to an ‘access-repair-restore’ paradigm, in which chromatin in the vicinity of damaged DNA is disrupted, thereby enabling efficient repair and the subsequent repackaging of DNA into nucleosomes. When damage is extensive, these repair processes are accompanied by cell cycle checkpoint activation, which provides cells with sufficient time to either complete the repair or initiate apoptosis. It is not clear, however, if base excision repair (BER) of the ~20,000 or more oxidative DNA damages that occur daily in each nucleated human cell can be viewed through this same lens. Until recently, we did not know if BER requires or is accompanied by nucleosome disruption, and it is not yet clear that anything short of overwhelming oxidative damage (resulting in the shunting of DNA substrates into other repair pathways) results in checkpoint activation. This review highlights studies of how oxidatively damaged DNA in nucleosomes is discovered and repaired, and offers a working model of events associated with BER in chromatin that we hope will have heuristic value. PMID:22718094

  4. Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

    PubMed Central

    Emmott, Edward; Goodfellow, Ian

    2014-01-01

    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. PMID:25046639

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

    PubMed

    Conrad, Nicholas K

    2008-01-01

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

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

    PubMed

    Franciosini, Anna; Serino, Giovanna

    2016-01-01

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

  7. A conformational study of the binding of a high mobility group protein with chromatin.

    PubMed

    Sasi, R; Hüvös, P E; Fasman, G D

    1982-10-10

    The nature of the binding of a high mobility group protein (HMG 17) to native and H1-H5-depleted chicken erythrocyte chromatin was studied, as a function of ionic strength, using circular dichroism and thermal denaturation techniques. The circular dichroism properties of the HMG 17-reconstituted whole chromatin and H1-H5-depleted chromatin demonstrated that a condensation of chromatin structure occurred upon HMG 17 binding at low ionic strength (1 mM Na phosphate, 0.25 mM EDTA, pH 7.0). Thermal denaturation profiles confirmed this change in the structure of chromatin induced by HMG 17. Thermal denaturation profiles were resolved into three-component transitions. In general, a shift in the temperature of maximum dh/dT for each transition (Tm) was observed for all transitions upon HMG 17 binding. DNA melting in the first transition, originating from linker regions of whole chromatin, was nearly totally depleted and was distributed mainly into the highest melting transition. The same trends were also observed in H1-H5-depleted chromatin. These results indicate that the binding sites of HMG 17 are situated in the linker regions immediately adjacent to the core. The nature of the interaction of HMG 17 at higher ionic strength (50 mM NaCl, 1 mM Na phosphate, 0.25 mM EDTA, pH 7.0) with whole chromatin and H1-H5-depleted chromatin was found to be different but a decrease in [theta] values was found in both chromatins. These observations suggest that HMG 17 does not loosen chromatin structure but produces an overall stabilization and condensation of structure. The implications of these results to the currently accepted models of transcriptionally active chromatin are discussed. PMID:6214552

  8. A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis.

    PubMed

    Down, Thomas A; Rakyan, Vardhman K; Turner, Daniel J; Flicek, Paul; Li, Heng; Kulesha, Eugene; Gräf, Stefan; Johnson, Nathan; Herrero, Javier; Tomazou, Eleni M; Thorne, Natalie P; Bäckdahl, Liselotte; Herberth, Marlis; Howe, Kevin L; Jackson, David K; Miretti, Marcos M; Marioni, John C; Birney, Ewan; Hubbard, Tim J P; Durbin, Richard; Tavaré, Simon; Beck, Stephan

    2008-07-01

    DNA methylation is an indispensible epigenetic modification required for regulating the expression of mammalian genomes. Immunoprecipitation-based methods for DNA methylome analysis are rapidly shifting the bottleneck in this field from data generation to data analysis, necessitating the development of better analytical tools. In particular, an inability to estimate absolute methylation levels remains a major analytical difficulty associated with immunoprecipitation-based DNA methylation profiling. To address this issue, we developed a cross-platform algorithm-Bayesian tool for methylation analysis (Batman)-for analyzing methylated DNA immunoprecipitation (MeDIP) profiles generated using oligonucleotide arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). We developed the latter approach to provide a high-resolution whole-genome DNA methylation profile (DNA methylome) of a mammalian genome. Strong correlation of our data, obtained using mature human spermatozoa, with those obtained using bisulfite sequencing suggest that combining MeDIP-seq or MeDIP-chip with Batman provides a robust, quantitative and cost-effective functional genomic strategy for elucidating the function of DNA methylation. PMID:18612301

  9. ARTEMIS nuclease facilitates apoptotic chromatin cleavage.

    PubMed

    Britton, Sébastien; Frit, Philippe; Biard, Denis; Salles, Bernard; Calsou, Patrick

    2009-10-15

    One hallmark of apoptosis is DNA degradation that first appears as high molecular weight fragments followed by extensive internucleosomal fragmentation. During apoptosis, the DNA-dependent protein kinase (DNA-PK) is activated. DNA-PK is involved in the repair of DNA double-strand breaks (DSB) and its catalytic subunit is associated with the nuclease ARTEMIS. Here, we report that, on initiation of apoptosis in human cells by agents causing DNA DSB or by staurosporine or other agents, ARTEMIS binds to apoptotic chromatin together with DNA-PK and other DSB repair proteins. ARTEMIS recruitment to chromatin showed a time and dose dependency. It required DNA-PK protein kinase activity and was blocked by antagonizing the onset of apoptosis with a pan-caspase inhibitor or on overexpression of the antiapoptotic BCL2 protein. In the absence of ARTEMIS, no defect in caspase-3, poly(ADP-ribose) polymerase-1, and XRCC4 cleavage or in H2AX phosphorylation was observed and DNA-PK catalytic subunit was still phosphorylated on S2056 in response to staurosporine. However, DNA fragmentation including high molecular weight fragmentation was delayed in ARTEMIS-deficient cells compared with cells expressing ARTEMIS. In addition, ARTEMIS enhanced the kinetics of MLL gene cleavage at a breakage cluster breakpoint that is frequently translocated in acute or therapy-related leukemias. These results show a facilitating role for ARTEMIS at least in early, site-specific chromosome breakage during apoptosis. PMID:19808974

  10. Facioscapulohumeral muscular dystrophy: consequences of chromatin relaxation

    PubMed Central

    van der Maarel, Silvère M.; Miller, Daniel G.; Tawil, Rabi; Filippova, Galina N.; Tapscott, Stephen J.

    2013-01-01

    Purpose of review In recent years we have seen remarkable progress in our understanding of the disease mechanism underlying facioscapulohumeral muscular dystrophy (FSHD). The purpose of this review is to provide a comprehensive overview of our current understanding of the disease mechanism and to discuss the observations supporting the possibility of a developmental defect in this disorder. Recent findings In the majority of cases FSHD is caused by contraction of the D4Z4 repeat array (FSHD1). This results in local chromatin relaxation and stable expression of the DUX4 retrogene in skeletal muscle, but only when a polymorphic DUX4 polyadenylation signal is present. In some cases (FSHD2), D4Z4 chromatin relaxation and stable DUX4 expression occurs in the absence of D4Z4 array contraction. DUX4 is a germline transcription factor and its expression in skeletal muscle leads to activation of early stem cell and germline programs and transcriptional activation of retroelements. Summary Recent studies have provided a plausible disease mechanism for FSHD where FSHD results from inappropriate expression of the germline transcription factor DUX4. The genes regulated by DUX4 suggest several mechanisms of muscle damage, and provide potential biomarkers and therapeutic targets that should be investigated in future studies. PMID:22892954

  11. Glycolytic metabolism influences global chromatin structure

    PubMed Central

    Liu, Xue-Song; Little, John B.; Yuan, Zhi-Min

    2015-01-01

    Metabolic rewiring, specifically elevated glycolytic metabolism is a hallmark of cancer. Global chromatin structure regulates gene expression, DNA repair, and also affects cancer progression. But the interrelationship between tumor metabolism and chromatin architecture remain unclear. Here we show that increased glycolysis in cancer cells promotes an open chromatin configuration. Using complementary methods including Micrococcal nuclease (MNase) digestion assay, electron microscope and immunofluorescence staining, we demonstrate that glycolysis inhibition by pharmacological and genetic approaches was associated with induction of compacted chromatin structure. This condensed chromatin status appeared to result chiefly from histone hypoacetylation as restoration of histone acetylation with an HDAC inhibitor reversed the compacted chromatin state. Interestingly, glycolysis inhibition-induced chromatin condensation impeded DNA repair efficiency leading to increased sensitivity of cancer cells to DNA damage drugs, which may represent a novel molecular mechanism that can be exploited for cancer therapy. PMID:25784656

  12. Looking at plant cell cycle from the chromatin window

    PubMed Central

    Desvoyes, Bénédicte; Fernández-Marcos, María; Sequeira-Mendes, Joana; Otero, Sofía; Vergara, Zaida; Gutierrez, Crisanto

    2014-01-01

    The cell cycle is defined by a series of complex events, finely coordinated through hormonal, developmental and environmental signals, which occur in a unidirectional manner and end up in producing two daughter cells. Accumulating evidence reveals that chromatin is not a static entity throughout the cell cycle. In fact, there are many changes that include nucleosome remodeling, histone modifications, deposition and exchange, among others. Interestingly, it is possible to correlate the occurrence of several of these chromatin-related events with specific processes necessary for cell cycle progression, e.g., licensing of DNA replication origins, the E2F-dependent transcriptional wave in G1, the activation of replication origins in S-phase, the G2-specific transcription of genes required for mitosis or the chromatin packaging occurring in mitosis. Therefore, an emerging view is that chromatin dynamics must be considered as an intrinsic part of cell cycle regulation. In this article, we review the main features of several key chromatin events that occur at defined times throughout the cell cycle and discuss whether they are actually controlling the transit through specific cell cycle stages. PMID:25120553

  13. Chromatin modifications and DNA repair: beyond double-strand breaks

    PubMed Central

    House, Nealia C. M.; Koch, Melissa R.; Freudenreich, Catherine H.

    2014-01-01

    DNA repair must take place in the context of chromatin, and chromatin modifications and DNA repair are intimately linked. The study of double-strand break repair has revealed numerous histone modifications that occur after induction of a DSB, and modification of the repair factors themselves can also occur. In some cases the function of the modification is at least partially understood, but in many cases it is not yet clear. Although DSB repair is a crucial activity for cell survival, DSBs account for only a small percentage of the DNA lesions that occur over the lifetime of a cell. Repair of single-strand gaps, nicks, stalled forks, alternative DNA structures, and base lesions must also occur in a chromatin context. There is increasing evidence that these repair pathways are also regulated by histone modifications and chromatin remodeling. In this review, we will summarize the current state of knowledge of chromatin modifications that occur during non-DSB repair, highlighting similarities and differences to DSB repair as well as remaining questions. PMID:25250043

  14. 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

  15. A chromatin perspective of adipogenesis

    PubMed Central

    Musri, Melina M; Gomis, Ramon

    2010-01-01

    The transcriptional cascade governing adipogenesis has been thoroughly examined throughout the years. Transcription factors PPARγ and C/EBPα are universally recognized as the master regulators of adipocyte differentiation and together they direct the establishment of the gene expression pattern of mature adipose cells. However, this familiar landscape has been considerably broadened in recent years by the identification of novel factors that participate in the regulation of adipogenesis, either favoring or inhibiting it, through their effects on chromatin. Epigenetic signals and chromatin-modifying proteins contribute to adipogenesis and, through regulation of the phenotypic maintenance of the mature adipocytes, to the control of metabolism. In this review we intend to summarize the recently described epigenetic events that participate in adipogenesis and their connections with the main factors that constitute the classical transcriptional cascade. PMID:20592861

  16. Super-resolution imaging reveals distinct chromatin folding for different epigenetic states.

    PubMed

    Boettiger, Alistair N; Bintu, Bogdan; Moffitt, Jeffrey R; Wang, Siyuan; Beliveau, Brian J; Fudenberg, Geoffrey; Imakaev, Maxim; Mirny, Leonid A; Wu, Chao-ting; Zhuang, Xiaowei

    2016-01-21

    Metazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression. Here we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive or Polycomb-repressed states, and observed distinct chromatin organizations for each state. All three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed domains show the densest packing and most intriguing chromatin folding behaviour, in which chromatin packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins play an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that is directly

  17. Hydrogen peroxide mediates higher order chromatin degradation.

    PubMed

    Bai, H; Konat, G W

    2003-01-01

    Although a large body of evidence supports a causative link between oxidative stress and neurodegeneration, the mechanisms are still elusive. We have recently demonstrated that hydrogen peroxide (H(2)O(2)), the major mediator of oxidative stress triggers higher order chromatin degradation (HOCD), i.e. excision of chromatin loops at the matrix attachment regions (MARs). The present study was designed to determine the specificity of H(2)O(2) in respect to HOCD induction. Rat glioma C6 cells were exposed to H(2)O(2) and other oxidants, and the fragmentation of genomic DNA was assessed by field inversion gel electrophoresis (FIGE). S1 digestion before FIGE was used to detect single strand fragmentation. The exposure of C6 cells to H(2)O(2) induced a rapid and extensive HOCD. Thus, within 30 min, total chromatin was single strandedly digested into 50 kb fragments. Evident HOCD was elicited by H(2)O(2) at concentrations as low as 5 micro M. HOCD was mostly reversible during 4-8h following the removal of H(2)O(2) from the medium indicating an efficient relegation of the chromatin fragments. No HOCD was induced by H(2)O(2) in isolated nuclei indicating that HOCD-endonuclease is activated indirectly by cytoplasmic signal pathways triggered by H(2)O(2). The exposure of cells to a synthetic peroxide, i.e. tert-butyrylhydroperoxide (tBH) also induced HOCD, but to a lesser extent than H(2)O(2). Contrary to the peroxides, the exposure of cells to equitoxic concentration of hypochlorite and spermine NONOate, a nitric oxide generator, failed to induce rapid HOCD. These results indicate that rapid HOCD is not a result of oxidative stress per se, but is rather triggered by signaling cascades initiated specifically by H(2)O(2). Furthermore, the rapid and extensive HOCD was observed in several rat and human cell lines challenged with H(2)O(2), indicating that the process is not restricted to glial cells, but rather represents a general response of cells to H(2)O(2). PMID:12421592

  18. Cell-Specific Determinants of Peroxisome Proliferator-Activated Receptor γ Function in Adipocytes and Macrophages ▿ §

    PubMed Central

    Lefterova, Martina I.; Steger, David J.; Zhuo, David; Qatanani, Mohammed; Mullican, Shannon E.; Tuteja, Geetu; Manduchi, Elisabetta; Grant, Gregory R.; Lazar, Mitchell A.

    2010-01-01

    The nuclear receptor peroxisome proliferator activator receptor γ (PPARγ) is the target of antidiabetic thiazolidinedione drugs, which improve insulin resistance but have side effects that limit widespread use. PPARγ is required for adipocyte differentiation, but it is also expressed in other cell types, notably macrophages, where it influences atherosclerosis, insulin resistance, and inflammation. A central question is whether PPARγ binding in macrophages occurs at genomic locations the same as or different from those in adipocytes. Here, utilizing chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we demonstrate that PPARγ cistromes in mouse adipocytes and macrophages are predominantly cell type specific. In thioglycolate-elicited macrophages, PPARγ colocalizes with the hematopoietic transcription factor PU.1 in areas of open chromatin and histone acetylation, near a distinct set of immune genes in addition to a number of metabolic genes shared with adipocytes. In adipocytes, the macrophage-unique binding regions are marked with repressive histone modifications, typically associated with local chromatin compaction and gene silencing. PPARγ, when introduced into preadipocytes, bound only to regions depleted of repressive histone modifications, where it increased DNA accessibility, enhanced histone acetylation, and induced gene expression. Thus, the cell specificity of PPARγ function is regulated by cell-specific transcription factors, chromatin accessibility, and histone marks. Our data support the existence of an epigenomic hierarchy in which PPARγ binding to cell-specific sites not marked by repressive marks opens chromatin and leads to local activation marks, including histone acetylation. PMID:20176806

  19. Spatial organization of chromatin domains and compartments in single chromosomes.

    PubMed

    Wang, Siyuan; Su, Jun-Han; Beliveau, Brian J; Bintu, Bogdan; Moffitt, Jeffrey R; Wu, Chao-ting; Zhuang, Xiaowei

    2016-08-01

    The spatial organization of chromatin critically affects genome function. Recent chromosome-conformation-capture studies have revealed topologically associating domains (TADs) as a conserved feature of chromatin organization, but how TADs are spatially organized in individual chromosomes remains unknown. Here, we developed an imaging method for mapping the spatial positions of numerous genomic regions along individual chromosomes and traced the positions of TADs in human interphase autosomes and X chromosomes. We observed that chromosome folding deviates from the ideal fractal-globule model at large length scales and that TADs are largely organized into two compartments spatially arranged in a polarized manner in individual chromosomes. Active and inactive X chromosomes adopt different folding and compartmentalization configurations. These results suggest that the spatial organization of chromatin domains can change in response to regulation. PMID:27445307

  20. Chromatin Dynamics in Lineage Commitment and Cellular Reprogramming

    PubMed Central

    Shchuka, Virlana M.; Malek-Gilani, Nakisa; Singh, Gurdeep; Langroudi, Lida; Dhaliwal, Navroop K.; Moorthy, Sakthi D.; Davidson, Scott; Macpherson, Neil N.; Mitchell, Jennifer A.

    2015-01-01

    Dynamic structural properties of chromatin play an essential role in defining cell identity and function. Transcription factors and chromatin modifiers establish and maintain cell states through alteration of DNA accessibility and histone modifications. This activity is focused at both gene-proximal promoter regions and distally located regulatory elements. In the three-dimensional space of the nucleus, distal elements are localized in close physical proximity to the gene-proximal regulatory sequences through the formation of chromatin loops. These looping features in the genome are highly dynamic as embryonic stem cells differentiate and commit to specific lineages, and throughout reprogramming as differentiated cells reacquire pluripotency. Identifying these functional distal regulatory regions in the genome provides insight into the regulatory processes governing early mammalian development and guidance for improving the protocols that generate induced pluripotent cells. PMID:26193323

  1. Chromatin Dynamics in Lineage Commitment and Cellular Reprogramming.

    PubMed

    Shchuka, Virlana M; Malek-Gilani, Nakisa; Singh, Gurdeep; Langroudi, Lida; Dhaliwal, Navroop K; Moorthy, Sakthi D; Davidson, Scott; Macpherson, Neil N; Mitchell, Jennifer A

    2015-01-01

    Dynamic structural properties of chromatin play an essential role in defining cell identity and function. Transcription factors and chromatin modifiers establish and maintain cell states through alteration of DNA accessibility and histone modifications. This activity is focused at both gene-proximal promoter regions and distally located regulatory elements. In the three-dimensional space of the nucleus, distal elements are localized in close physical proximity to the gene-proximal regulatory sequences through the formation of chromatin loops. These looping features in the genome are highly dynamic as embryonic stem cells differentiate and commit to specific lineages, and throughout reprogramming as differentiated cells reacquire pluripotency. Identifying these functional distal regulatory regions in the genome provides insight into the regulatory processes governing early mammalian development and guidance for improving the protocols that generate induced pluripotent cells. PMID:26193323

  2. Higher-order structure of Saccharomyces cerevisiae chromatin

    SciTech Connect

    Lowary, P.T.; Widom, J. )

    1989-11-01

    We have developed a method for partially purifying chromatin from Saccharomyces cerevisiae (baker's yeast) to a level suitable for studies of its higher-order folding. This has required the use of yeast strains that are free of the ubiquitous yeast killer virus. Results from dynamic light scattering, electron microscopy, and x-ray diffraction show that the yeast chromatin undergoes a cation-dependent folding into 30-nm filaments that resemble those characteristic of higher-cell chromatin; moreover, the packing of nucleosomes within the yeast 30-nm filaments is similar to that of higher cells. These results imply that yeast has a protein or protein domain that serves the role of the histone H 1 found in higher cells; physical and genetic studies of the yeast activity could help elucidate the structure and function of H 1. Images of the yeast 30-nm filaments can be used to test crossed-linker models for 30-nm filament structure.

  3. 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.

  4. Insulation of the Chicken β-Globin Chromosomal Domain from a Chromatin-Condensing Protein, MENT

    PubMed Central

    Istomina, Natalia E.; Shushanov, Sain S.; Springhetti, Evelyn M.; Karpov, Vadim L.; A. Krasheninnikov, Igor; Stevens, Kimberly; Zaret, Kenneth S.; Singh, Prim B.; Grigoryev, Sergei A.

    2003-01-01

    Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken β-globin domain. We observed two sharp transitions of MENT concentration coinciding with the β-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation. PMID:12944473

  5. 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.

  6. The condensed chromatin fiber: an allosteric chemo-mechanical machine for signal transduction and genome processing

    NASA Astrophysics Data System (ADS)

    Lesne, Annick; Bécavin, Christophe; Victor, Jean–Marc

    2012-02-01

    Allostery is a key concept of molecular biology which refers to the control of an enzyme activity by an effector molecule binding the enzyme at another site rather than the active site (allos = other in Greek). We revisit here allostery in the context of chromatin and argue that allosteric principles underlie and explain the functional architecture required for spacetime coordination of gene expression at all scales from DNA to the whole chromosome. We further suggest that this functional architecture is provided by the chromatin fiber itself. The structural, mechanical and topological features of the chromatin fiber endow chromosomes with a tunable signal transduction from specific (or nonspecific) effectors to specific (or nonspecific) active sites. Mechanical constraints can travel along the fiber all the better since the fiber is more compact and regular, which speaks in favor of the actual existence of the (so-called 30 nm) chromatin fiber. Chromatin fiber allostery reconciles both the physical and biochemical approaches of chromatin. We illustrate this view with two supporting specific examples. Moreover, from a methodological point of view, we suggest that the notion of chromatin fiber allostery is particularly relevant for systemic approaches. Finally we discuss the evolutionary power of allostery in the context of chromatin and its relation to modularity.

  7. Rearrangement of chromatin domains during development in Xenopus.

    PubMed

    Vassetzky, Y; Hair, A; Méchali, M

    2000-06-15

    A dynamic change in the organization of different gene domains transcribed by RNA polymerase I, II, or III occurs during the progression from quiescent [pre-midblastula transition (pre-MBT)] to active (post-MBT) embryos during Xenopus development. In the rDNA, c-myc, and somatic 5S gene domains, a transition from random to specific anchorage to the nuclear matrix occurs when chromatin domains become active. The keratin gene domain was also randomly associated to the nuclear matrix before MBT, whereas a defined attachment site was found in keratinocytes. In agreement with this specification, ligation-mediated (LM)-PCR genomic footprinting carried out on the subpopulation of 5S domains specifically attached to the matrix reveals the hallmarks of determined chromatin after the midblastula transition. In contrast, the same analysis performed on the total 5S gene population does not reveal specific chromatin organization, validating the use of nuclear matrix fractionation to unveil active chromatin domains. These data provide a means for the determination of active chromosomal territories in the embryo and emphasize the role of nuclear architecture in regulated gene expression during development. PMID:10859171

  8. Rearrangement of chromatin domains during development in Xenopus

    PubMed Central

    Vassetzky, Yegor; Hair, Alan; Méchali, Marcel

    2000-01-01

    A dynamic change in the organization of different gene domains transcribed by RNA polymerase I, II, or III occurs during the progression from quiescent [pre-midblastula transition (pre-MBT)] to active (post-MBT) embryos during Xenopus development. In the rDNA, c-myc, and somatic 5S gene domains, a transition from random to specific anchorage to the nuclear matrix occurs when chromatin domains become active. The keratin gene domain was also randomly associated to the nuclear matrix before MBT, whereas a defined attachment site was found in keratinocytes. In agreement with this specification, ligation-mediated (LM)-PCR genomic footprinting carried out on the subpopulation of 5S domains specifically attached to the matrix reveals the hallmarks of determined chromatin after the midblastula transition. In contrast, the same analysis performed on the total 5S gene population does not reveal specific chromatin organization, validating the use of nuclear matrix fractionation to unveil active chromatin domains. These data provide a means for the determination of active chromosomal territories in the embryo and emphasize the role of nuclear architecture in regulated gene expression during development. PMID:10859171

  9. Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription.

    PubMed

    Chambeyron, Séverine; Bickmore, Wendy A

    2004-05-15

    The colinearity of genes in Hox clusters suggests a role for chromosome structure in gene regulation. We reveal programmed changes in chromatin structure and nuclear organization upon induction of Hoxb expression by retinoic acid. There is an early increase in the histone modifications that are marks of active chromatin at both the early expressed gene Hoxb1, and also at Hoxb9 that is not expressed until much later. There is also a visible decondensation of the chromatin between Hoxb1 and Hoxb9 at this early stage. However, a further change in higher-order chromatin structure, looping out of genes from the chromosome territory, occurs in synchrony with the execution of the gene expression program. We suggest that higher-order chromatin structure regulates the expression of the HoxB cluster at several levels. Locus-wide changes in chromatin structure (histone modification and chromatin decondensation) may establish a transcriptionally poised state but are not sufficient for the temporal program of gene expression. The choreographed looping out of decondensed chromatin from chromosome territories may then allow for activation of high levels of transcription from the sequence of genes along the cluster. PMID:15155579

  10. Identification of lamin B-regulated chromatin regions based on chromatin landscapes.

    PubMed

    Zheng, Xiaobin; Kim, Youngjo; Zheng, Yixian

    2015-07-15

    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

  11. Statistical physics of nucleosome positioning and chromatin structure

    NASA Astrophysics Data System (ADS)

    Morozov, Alexandre

    2012-02-01

    Genomic DNA is packaged into chromatin in eukaryotic cells. The fundamental building block of chromatin is the nucleosome, a 147 bp-long DNA molecule wrapped around the surface of a histone octamer. Arrays of nucleosomes are positioned along DNA according to their sequence preferences and folded into higher-order chromatin fibers whose structure is poorly understood. We have developed a framework for predicting sequence-specific histone-DNA interactions and the effective two-body potential responsible for ordering nucleosomes into regular higher-order structures. Our approach is based on the analogy between nucleosomal arrays and a one-dimensional fluid of finite-size particles with nearest-neighbor interactions. We derive simple rules which allow us to predict nucleosome occupancy solely from the dinucleotide content of the underlying DNA sequences.Dinucleotide content determines the degree of stiffness of the DNA polymer and thus defines its ability to bend into the nucleosomal superhelix. As expected, the nucleosome positioning rules are universal for chromatin assembled in vitro on genomic DNA from baker's yeast and from the nematode worm C.elegans, where nucleosome placement follows intrinsic sequence preferences and steric exclusion. However, the positioning rules inferred from in vivo C.elegans chromatin are affected by global nucleosome depletion from chromosome arms relative to central domains, likely caused by the attachment of the chromosome arms to the nuclear membrane. Furthermore, intrinsic nucleosome positioning rules are overwritten in transcribed regions, indicating that chromatin organization is actively managed by the transcriptional and splicing machinery.

  12. Efficient cell migration requires global chromatin condensation

    PubMed Central

    Gerlitz, Gabi; Bustin, Michael

    2010-01-01

    Cell migration is a fundamental process that is necessary for the development and survival of multicellular organisms. Here, we show that cell migration is contingent on global condensation of the chromatin fiber. Induction of directed cell migration by the scratch-wound assay leads to decreased DNaseI sensitivity, alterations in the chromatin binding of architectural proteins and elevated levels of H4K20me1, H3K27me3 and methylated DNA. All these global changes are indicative of increased chromatin condensation in response to induction of directed cell migration. Conversely, chromatin decondensation inhibited the rate of cell migration, in a transcription-independent manner. We suggest that global chromatin condensation facilitates nuclear movement and reshaping, which are important for cell migration. Our results support a role for the chromatin fiber that is distinct from its known functions in genetic processes. PMID:20530575

  13. Super-resolution imaging reveals distinct chromatin folding for different epigenetic states

    PubMed Central

    Boettiger, Alistair N.; Bintu, Bogdan; Moffitt, Jeffrey R.; Wang, Siyuan; Beliveau, Brian J.; Fudenberg, Geoffrey; Imakaev, Maxim; Mirny, Leonid A.; Wu, Chao-ting; Zhuang, Xiaowei

    2015-01-01

    Metazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories1–5. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms2–4,6–8, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression9–11. Here, we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive, or Polycomb-repressed states and observed distinct chromatin organizations for each state. Remarkably, all three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed chromatin shows the densest packing and most intriguing folding behaviour in which packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins plays an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    1991-10-01

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

  16. Preparation of Chromatin Templates to Study RNA Polymerase I Transcription In Vitro.

    PubMed

    Längst, Gernot

    2016-01-01

    Cellular DNA is packaged into chromatin, which is the substrate of all DNA-dependent processes in eukaryotes. The regulation of chromatin requires specialized enzyme activities to allow the access of sequence-specific binding proteins and RNA polymerases. In order to dissect chromatin-dependent features of transcription regulation in detail, in vitro systems to generate defined chromatin templates for transcription are required. I present a protocol that allows the assembly of nucleosomes on ribosomal RNA (rRNA) minigenes by salt gradient dialysis and subsequent sucrose gradient centrifugation. This procedure yields high nucleosome occupancy and high dynamic response in subsequent transcriptional analysis. It provides an invaluable tool to study rRNA gene transcription, as transcription on free DNA is clearly different from the more in vivo-like transcription on reconstituted chromatin templates. PMID:27576714

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

    SciTech Connect

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

    1981-12-01

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

  18. 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

  19. The interaction of PRC2 with RNA or chromatin is mutually antagonistic.

    PubMed

    Beltran, Manuel; Yates, Christopher M; Skalska, Lenka; Dawson, Marcus; Reis, Filipa P; Viiri, Keijo; Fisher, Cynthia L; Sibley, Christopher R; Foster, Benjamin M; Bartke, Till; Ule, Jernej; Jenner, Richard G

    2016-07-01

    Polycomb repressive complex 2 (PRC2) modifies chromatin to maintain genes in a repressed state during development. PRC2 is primarily associated with CpG islands at repressed genes and also possesses RNA binding activity. However, the RNAs that bind PRC2 in cells, the subunits that mediate these interactions, and the role of RNA in PRC2 recruitment to chromatin all remain unclear. By performing iCLIP for PRC2 in comparison with other RNA binding proteins, we show here that PRC2 binds nascent RNA at essentially all active genes. Although interacting with RNA promiscuously, PRC2 binding is enriched at specific locations within RNAs, primarily exon-intron boundaries and the 3' UTR. Deletion of other PRC2 subunits reveals that SUZ12 is sufficient to establish this RNA binding profile. Contrary to prevailing models, we also demonstrate that the interaction of PRC2 with RNA or chromatin is mutually antagonistic in cells and in vitro. RNA degradation in cells triggers PRC2 recruitment to CpG islands at active genes. Correspondingly, the release of PRC2 from chromatin in cells increases RNA binding. Consistent with this, RNA and nucleosomes compete for PRC2 binding in vitro. We propose that RNA prevents PRC2 recruitment to chromatin at active genes and that mutual antagonism between RNA and chromatin underlies the pattern of PRC2 chromatin association across the genome. PMID:27197219

  20. 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.

  1. Single-molecule and population probing of chromatin structure using DNA methyltransferases

    PubMed Central

    Kilgore, Jessica A.; Hoose, Scott A.; Gustafson, Tanya L.; Porter, Weston; Kladde, Michael P.

    2007-01-01

    Probing chromatin structure with DNA methyltransferases offers advantages over more commonly used nuclease-based and chromatin immunoprecipitation methods for detection of nucleosomes and non-histone protein-DNA interactions. Here we describe two related methods in which the readout of MTase accessibility is obtained by assaying 5-methylcytosine in DNA through the PCR-based technique of bisulfite genomic sequencing. The methyltransferase accessibility protocol (MAP) determines the relative frequency at which the enzyme accesses each of its target sites over an entire population of PCR amplified product. While MAP yields much quantitative information about relative accessibility of a region of chromatin, a complementary single-molecule view of methyltransferase accessibility, termed MAP for individual templates (MAP-IT), is provided by analysis of cloned PCR products. Absolute rather than relative methylation frequencies in a region are obtained by summing the methylation status at each site over a cohort of clones. Moreover, as the integrity of individual molecules is maintained in MAP-IT, unique information about the distribution of multiple footprints along continuous regions is gleaned. In principle, the population MAP and single-molecule MAP-IT strategies can be used to analyze chromatin structure in a variety of model systems. Here we describe the application of MAP in living S. cerevisiae cells and MAP-IT in the analysis of a mammalian tumor suppressor gene in nuclei. This application of MAP-IT provides the first means to simultaneously determine CpG methylation of mammalian genes and their overlying chromatin structure in the same single DNA molecule. PMID:17309843

  2. Sulforaphane modulates telomerase activity via epigenetic regulation in prostate cancer cell lines.

    PubMed

    Abbas, Ata; Hall, J Adam; Patterson, William L; Ho, Emily; Hsu, Anna; Al-Mulla, Fahd; Georgel, Philippe T

    2016-02-01

    Epidemiologic studies have revealed that diets rich in sulforaphane (SFN), an isothiocyanate present in cruciferous vegetables, are associated with a marked decrease in prostate cancer incidence. The chemo-preventive role of SFN is associated with its histone de-acetylase inhibitor activity. However, the effect of SFN on chromatin composition and dynamic folding, especially in relation to HDAC inhibitor activity, remains poorly understood. In this study, we found that SFN can inhibit the expression and activity of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, in 2 prostate cancer cell lines. This decrease in gene expression is correlated with SFN-induced changes in chromatin structure and composition. The SFN-mediated changes in levels of histone post-translational modifications, more specifically acetylation of histone H3 lysine 18 and di-methylation of histone H3 lysine 4, 2 modifications linked with high risk of prostate cancer recurrence, were associated with regulatory elements within the hTERT promoter region. Chromatin condensation may also play a role in SFN-mediated hTERT repression, since expression and recruitment of MeCP2, a known chromatin compactor, were altered in SFN treated prostate cancer cells. Chromatin immuno-precipitation (ChIP) of MeCP2 showed enrichment over regions of the hTERT promoter with increased nucleosome density. These combined results strongly support a role for SFN in the mediation of epigenetic events leading to the repression of hTERT in prostate cancer cells. This ability of SFN to modify chromatin composition and structure associated with target gene expression provides a new model by which dietary phytochemicals may exert their chemoprevention activity. PMID:26458818

  3. Four enzymes cooperate to displace histone H1 during the first minute of hormonal gene activation

    PubMed Central

    Vicent, Guillermo Pablo; Nacht, A. Silvina; Font-Mateu, Jofre; Castellano, Giancarlo; Gaveglia, Laura; Ballaré, Cecilia; Beato, Miguel

    2011-01-01

    Gene regulation by external signals requires access of transcription factors to DNA sequences of target genes, which is limited by the compaction of DNA in chromatin. Although we have gained insight into how core histones and their modifications influence this process, the role of linker histones remains unclear. Here we show that, within the first minute of progesterone action, a complex cooperation between different enzymes acting on chromatin mediates histone H1 displacement as a requisite for gene induction and cell proliferation. First, activated progesterone receptor (PR) recruits the chromatin remodeling complexes NURF and ASCOM (ASC-2 [activating signal cointegrator-2] complex) to hormone target genes. The trimethylation of histone H3 at Lys 4 by the MLL2/MLL3 subunits of ASCOM, enhanced by the hormone-induced displacement of the H3K4 demethylase KDM5B, stabilizes NURF binding. NURF facilitates the PR-mediated recruitment of Cdk2/CyclinA, which is required for histone H1 displacement. Cooperation of ATP-dependent remodeling, histone methylation, and kinase activation, followed by H1 displacement, is a prerequisite for the subsequent displacement of histone H2A/H2B catalyzed by PCAF and BAF. Chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) and expression arrays show that H1 displacement is required for hormone induction of most hormone target genes, some of which are involved in cell proliferation. PMID:21447625

  4. Computational strategies to address chromatin structure problems.

    PubMed

    Perišić, Ognjen; Schlick, Tamar

    2016-01-01

    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. PMID:27345617

  5. Regeneration of chromatin-bound and membrane lipids from liver and thymus of V-irradiated rats

    SciTech Connect

    Kaznacheev, Yu.S.; Kolomiitseva, I.K.; Kulagina, T.P.; Markevich, L.N.

    1985-06-20

    This paper compares the regeneration of nuclear and chromatin lipids from the liver and thymus of control and irradiated rats according to the criterion of the incorporation of (/sup 14/C) acetate. The chromatin-bound lipids were found to have high metabolic activity, which was sharply pronounced in thymus cells. The corresponding lipids of intact nuclei suggests that the chromatin lipids are structurally separate from the rest of the nuclear lipids.

  6. Developmental changes in DNA methylation and covalent histone modifications of chromatin associated with the epsilon-, gamma-, and beta-globin gene promoters in Papio anubis.

    PubMed

    Lavelle, Donald; Vaitkus, Kestis; Hankewych, Maria; Singh, Mahipal; DeSimone, Joseph

    2006-01-01

    The baboon is a suitable and relevant animal model to study the mechanism of human globin gene switching. This investigation addresses the role of DNA methylation and histone coding in globin gene switching in the baboon, Papio anubis. Bisulfite sequencing and chromatin immunoprecipitation studies were performed in erythroid cells purified from fetuses of varying gestational ages and from adult bone marrow to analyze the manner that changes in DNA methylation of the epsilon-, gamma-, and beta-globin promoters and association of ac-H3, ac-H4, H3-dimeK4, H3-dimeK36, and H3-dimeK79 with the epsilon-, gamma-, and beta-globin promoters occur during development. Changes in DNA methylation of the epsilon- and gamma-globin gene promoters during transitional stages of globin gene switching were consistent with the stochastic model of methylation and a role of DNA methylation in gene silencing. Enrichment of ac-H3, ac-H4, and pol II at the promoters of developmentally active genes was observed, while the pattern of distribution of H3-dimeK4 and H3-dimeK79 suggests that these modifications are found near both currently and formerly active promoters. Enrichment of H3-dimeK36 at the silenced epsilon-globin gene promoter was observed. These studies demonstrate that coordinated epigenetic modifications in the chromatin structure of the beta-like globin gene promoters accompany the highly regulated changes in expression patterns of these genes during development. PMID:16527500

  7. Id2 leaves the chromatin of the E2F4–p130-controlled c-myc promoter during hepatocyte priming for liver regeneration

    PubMed Central

    Rodríguez, José L.; Sandoval, Juan; Serviddio, Gaetano; Sastre, Juan; Morante, María; Perrelli, Maria-Giulia; Martínez-Chantar, María L.; Viña, José; Viña, Juan R.; Mato, José M.; Ávila, Matías A.; Franco, Luis; López-Rodas, Gerardo; Torres, Luis

    2006-01-01

    The Id (inhibitor of DNA binding or inhibitor of differentiation) helix–loop–helix proteins are involved in the regulation of cell growth, differentiation and cancer. The fact that the molecular mechanisms of liver regeneration are not completely understood prompted us to study the fate of Id2 in proliferating liver. Id2 increases in liver regeneration after partial hepatectomy, following the early induction of its gene. Co-immunoprecipitation shows that Id2 forms a complex with E2F4, p130 and mSin3A in quiescent liver and all these components are present at the c-myc promoter as shown using ChIP (chromatin immunoprecipitation). Activation of c-myc during hepatocyte priming (G0–G1 transition) correlates with the dissociation of Id2 and HDAC (histone deacetylase), albeit p130 remains bound at least until 6 h. Moreover, as the G0–G1 transition progresses, Id2 and HDAC again bind the c-myc promoter concomitantly with the repression of this gene. The time course of c-myc binding to the Id2 promoter, as determined by ChIP assays is compatible with a role of the oncoprotein as a transcriptional inducer of Id2 in liver regeneration. Immunohistochemical analysis shows that Id2 also increases in proliferating hepatocytes after bile duct ligation. In this case, the pattern of Id2 presence in the c-myc promoter parallels that found in regenerating liver. Our results may suggest a control role for Id2 in hepatocyte priming, through a p130 dissociation-independent regulation of c-myc. PMID:16776654

  8. Structure, Assembly and Reading of Centromeric Chromatin

    PubMed Central

    Maddox, Paul S; Corbett, Kevin D; Desai, Arshad

    2011-01-01

    Centromeres are epigenetically defined chromatin domains marked by the presence of the histone H3 variant CENP-A. Here we review recent structural and biochemical work on CENP-A, and advances in understanding the mechanisms that propagate and read centromeric chromatin domains. PMID:22178421

  9. Chromatin Remodelers: From Function to Dysfunction

    PubMed Central

    Längst, Gernot; Manelyte, Laura

    2015-01-01

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

  10. Connecting the dots: chromatin and alternative splicing in EMT

    PubMed Central

    Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

    2015-01-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

  11. In vitro chromatin templates to study nucleotide excision repair.

    PubMed

    Liu, Xiaoqi

    2015-12-01

    In eukaryotic cells, DNA associates with histones and exists in the form of a chromatin hierarchy. Thus, it is generally believed that many eukaryotic cellular DNA processing events such as replication, transcription, recombination and DNA repair are influenced by the packaging of DNA into chromatin. This mini-review covers the current knowledge of DNA damage and repair in chromatin based on in vitro studies. Specifically, nucleosome assembly affects DNA damage formation in both random sequences and sequences with strong nucleosome-positioning signals such as 5S rDNA. At least three systems have been used to analyze the effect of nucleosome folding on nucleotide excision repair (NER) in vitro: (a) human cell extracts that have to rely on labeling of repair synthesis to monitor DNA repair, due to very low repair efficacy; (b) Xenopus oocyte nuclear extracts, that have very robust DNA repair efficacy, have been utilized to follow direct removal of DNA damage; (c) six purified human DNA repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1) that have been used to reconstitute excision repair in vitro. In general, the results have shown that nucleosome folding inhibits NER and, therefore, its activity must be enhanced by chromatin remodeling factors like SWI/SNF. In addition, binding of transcription factors such as TFIIIA to the 5S rDNA promoter also modulates NER efficacy. PMID:26531320

  12. Citrullination regulates pluripotency and histone H1 binding to chromatin

    NASA Astrophysics Data System (ADS)

    Christophorou, Maria A.; Castelo-Branco, Gonçalo; Halley-Stott, Richard P.; Oliveira, Clara Slade; Loos, Remco; Radzisheuskaya, Aliaksandra; Mowen, Kerri A.; Bertone, Paul; Silva, José C. R.; Zernicka-Goetz, Magdalena; Nielsen, Michael L.; Gurdon, John B.; Kouzarides, Tony

    2014-03-01

    Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

  13. Human Genome Replication Proceeds through Four Chromatin States

    PubMed Central

    Julienne, Hanna; Zoufir, Azedine; Audit, Benjamin; Arneodo, Alain

    2013-01-01

    Advances in genomic studies have led to significant progress in understanding the epigenetically controlled interplay between chromatin structure and nuclear functions. Epigenetic modifications were shown to play a key role in transcription regulation and genome activity during development and differentiation or in response to the environment. Paradoxically, the molecular mechanisms that regulate the initiation and the maintenance of the spatio-temporal replication program in higher eukaryotes, and in particular their links to epigenetic modifications, still remain elusive. By integrative analysis of the genome-wide distributions of thirteen epigenetic marks in the human cell line K562, at the 100 kb resolution of corresponding mean replication timing (MRT) data, we identify four major groups of chromatin marks with shared features. These states have different MRT, namely from early to late replicating, replication proceeds though a transcriptionally active euchromatin state (C1), a repressive type of chromatin (C2) associated with polycomb complexes, a silent state (C3) not enriched in any available marks, and a gene poor HP1-associated heterochromatin state (C4). When mapping these chromatin states inside the megabase-sized U-domains (U-shaped MRT profile) covering about 50% of the human genome, we reveal that the associated replication fork polarity gradient corresponds to a directional path across the four chromatin states, from C1 at U-domains borders followed by C2, C3 and C4 at centers. Analysis of the other genome half is consistent with early and late replication loci occurring in separate compartments, the former correspond to gene-rich, high-GC domains of intermingled chromatin states C1 and C2, whereas the latter correspond to gene-poor, low-GC domains of alternating chromatin states C3 and C4 or long C4 domains. This new segmentation sheds a new light on the epigenetic regulation of the spatio-temporal replication program in human and provides a

  14. Organization of spacer DNA in chromatin.

    PubMed Central

    Lohr, D; Van Holde, K E

    1979-01-01

    Detailed analysis of the DNA fragment patterns produced by DNase I digestion of yeast, HeLa, and chicken erythrocyte nuclei reveals surprising features of nucleosome phasing. First, the spacer regions in phased yeast chromatin must be of lengths (10m + 5) base pairs, where m = 0, 1, 2,....This feature is not seen in parallel studies of chicken erythrocyte chromatin. The 5-base pair increment in the yeast spacer imposes interesting restraints on the higher order structure of yeast chromatin. Second, we have been able to simulate the DNase I cutting patterns and get good agreement with the observed yeast patterns. Third, three different chromatins show a long range periodicity in the DNase I digest pattern, with a period half that of the staphylococcal nuclease repeat. These results suggest that the amount of chromatin observed in discrete extended-ladder bands is a minimum estimate of phasing and in fact phasing may be a more general feature. Images PMID:392519

  15. Nitric oxide modulates chromatin folding in human endothelial cells via protein phosphatase 2A activation and class II histone deacetylases nuclear shuttling.

    PubMed

    Illi, Barbara; Dello Russo, Claudio; Colussi, Claudia; Rosati, Jessica; Pallaoro, Michele; Spallotta, Francesco; Rotili, Dante; Valente, Sergio; Ragone, Gianluca; Martelli, Fabio; Biglioli, Paolo; Steinkuhler, Christian; Gallinari, Paola; Mai, Antonello; Capogrossi, Maurizio C; Gaetano, Carlo

    2008-01-01

    Nitric oxide (NO) modulates important endothelial cell (EC) functions and gene expression by a molecular mechanism which is still poorly characterized. Here we show that in human umbilical vein ECs (HUVECs) NO inhibited serum-induced histone acetylation and enhanced histone deacetylase (HDAC) activity. By immunofluorescence and Western blot analyses it was found that NO induced class II HDAC4 and 5 nuclear shuttling and that class II HDACs selective inhibitor MC1568 rescued serum-dependent histone acetylation above control level in NO-treated HUVECs. In contrast, class I HDACs inhibitor MS27-275 had no effect, indicating a specific role for class II HDACs in NO-dependent histone deacetylation. In addition, it was found that NO ability to induce HDAC4 and HDAC5 nuclear shuttling involved the activation of the protein phosphatase 2A (PP2A). In fact, HDAC4 nuclear translocation was impaired in ECs expressing small-t antigen and exposed to NO. Finally, in cells engineered to express a HDAC4-Flag fusion protein, NO induced the formation of a macromolecular complex including HDAC4, HDAC3, HDAC5, and an active PP2A. The present results show that NO-dependent PP2A activation plays a key role in class II HDACs nuclear translocation. PMID:17975112

  16. 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.

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

    PubMed Central

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

    2015-01-01

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

  18. Myc-binding-site recognition in the human genome is determined by chromatin context.

    PubMed

    Guccione, Ernesto; Martinato, Francesca; Finocchiaro, Giacomo; Luzi, Lucilla; Tizzoni, Laura; Dall' Olio, Valentina; Zardo, Giuseppe; Nervi, Clara; Bernard, Loris; Amati, Bruno

    2006-07-01

    Large-scale chromatin immunoprecipitation (ChIP) studies have been effective in unravelling the distribution of DNA-binding transcription factors along eukaryotic genomes, but specificity determinants remain elusive. Gene-regulatory regions display distinct histone variants and modifications (or marks). An attractive hypothesis is that these marks modulate protein recognition, but whether or not this applies to transcription factors remains unknown. Based on large-scale datasets and quantitative ChIP, we dissect the correlations between 35 histone marks and genomic binding by the transcription factor Myc. Our data reveal a relatively simple combinatorial organization of histone marks in human cells, with a few main groups of marks clustering on distinct promoter populations. A stretch of chromatin bearing high H3 K4/K79 methylation and H3 acetylation (or 'euchromatic island'), which is generally associated with a pre-engaged basal transcription machinery, is a strict pre-requisite for recognition of any target site by Myc (whether the consensus CACGTG or an alternative sequence). These data imply that tethering of a transcription factor to restricted chromatin domains is rate-limiting for sequence-specific DNA binding in vivo. PMID:16767079

  19. Interplay of RNA Pol IV and ROS1 during post-embryonic 5S rDNA chromatin remodeling.

    PubMed

    Douet, Julien; Blanchard, Bertrand; Cuvillier, Claudine; Tourmente, Sylvette

    2008-12-01

    We have investigated the chromatin structure of 5S rDNA, a heterochromatic pericentromeric tandemly repeated family, at 2, 3, 4 and 5 days post-germination. Our results revealed a large-scale reorganization of 5S rDNA chromatin that occurs during the first days of development. Unexpectedly, there is a decondensation followed by a 're'condensation of 5S rDNA chromatin, to obtain almost mature nuclei 5 d post-germination. The reorganization of 5S rDNA chromatin is accompanied by a rapid and active demethylation of 5S rDNA mediated by the ROS1 (repressor of silencing 1) demethylase, whereas the plant-specific RNA polymerase IV (Pol IV) is essential to the 5S chromatin 're'condensation. In conclusion, Pol IV and ROS1 collaborate to unlock the 5S rDNA chromatin inherited from the seed, and establish adult features. PMID:18845569

  20. A Generic Tool for Transcription Factor Target Gene Discovery in Arabidopsis Cell Suspension Cultures Based on Tandem Chromatin Affinity Purification1[W][OPEN

    PubMed Central

    Verkest, Aurine; Abeel, Thomas; Heyndrickx, Ken S.; Van Leene, Jelle; Lanz, Christa; Van De Slijke, Eveline; De Winne, Nancy; Eeckhout, Dominique; Persiau, Geert; Van Breusegem, Frank; Inzé, Dirk; Vandepoele, Klaas; De Jaeger, Geert

    2014-01-01

    Genome-wide identification of transcription factor (TF) binding sites is pivotal to our understanding of gene expression regulation. Although much progress has been made in the determination of potential binding regions of proteins by chromatin immunoprecipitation, this method has some inherent limitations regarding DNA enrichment efficiency and antibody necessity. Here, we report an alternative strategy for assaying in vivo TF-DNA binding in Arabidopsis (Arabidopsis thaliana) cells by tandem chromatin affinity purification (TChAP). Evaluation of TChAP using the E2Fa TF and comparison with traditional chromatin immunoprecipitation and single chromatin affinity purification illustrates the suitability of TChAP and provides a resource for exploring the E2Fa transcriptional network. Integration with transcriptome, cis-regulatory element, functional enrichment, and coexpression network analyses demonstrates the quality of the E2Fa TChAP sequencing data and validates the identification of new direct E2Fa targets. TChAP enhances both TF target mapping throughput, by circumventing issues related to antibody availability, and output, by improving DNA enrichment efficiency. PMID:24453163

  1. Structural and functional genome analysis using extended chromatin

    SciTech Connect

    Heaf, T.; Ward, D.C.

    1994-09-01

    Highly extended linear chromatin fibers (ECFs) produced by detergent and high-salt lysis and stretching of nuclear chromatin across the surface of a glass slide can by hybridized over physical distances of at least several Mb. This allows long-range FISH analysis of the human genome with excellent DNA resolution (<10 kb/{mu}m). The insertion of Alu elements which are more than 50-fold underrepresented in centromeres can be seen within and near long tandem arrays of alpha-satellite DNA. Long tracts of trinucleotide repeats, i.e. (CCA){sub n}, can be localized within larger genomic regions. The combined application of BrdU incorporation and ECFs allows one to study the spatio-temporal distribution of DNA replication sites in finer detail. DNA synthesis occurs at multiple discrete sites within Mb arrays of alpha-satellite. Replicating DNA is tightly associated with the nuclear matrix and highly resistant to stretching out, while ECFs containing newly replicated DNA are easily released. Asynchrony in replication timing is accompanied by differences in condensation of homologous DNA segments. Extended chromatin reveals differential packaging of active and inactive DNA. Upon transcriptional inactivation by AMD, the normally compact rRNA genes become much more susceptible to decondensation procedures. By extending the chromatin from pachytene spermatocytes, meiotic pairing and genetic exchange between homologs can be visualized directly. Histone depletion by high salt and detergent produces loop chromatin surrounding the nuclear matrix in a halo-like fashion. DNA halos can be used to map nuclear matrix attachment sites in somatic cells and in mature sperm. Alpha-satellite containing DNA loops appear to be attached to the sperm-cell matrix by CENP-B boxes, short 17 bp sequences found in a subset of alpha satellite monomers. Sperm telomeres almost always appear as hybridization doublets, suggesting the presence of already replicated chromosome ends.

  2. A duplicated NUCLEOLIN gene with antagonistic activity is required for chromatin organization of silent 45S rDNA in Arabidopsis.

    PubMed

    Durut, Nathalie; Abou-Ellail, Mohamed; Pontvianne, Frédéric; Das, Sadhan; Kojima, Hisae; Ukai, Seiko; de Bures, Anne; Comella, Pascale; Nidelet, Sabine; Rialle, Stéphanie; Merret, Remy; Echeverria, Manuel; Bouvet, Philippe; Nakamura, Kenzo; Sáez-Vásquez, Julio

    2014-03-01

    In plants as well as in animals, hundreds to thousands of 45S rRNA gene copies localize in Nucleolus Organizer Regions (NORs), and the activation or repression of specific sets of rDNA depends on epigenetic mechanisms. Previously, we reported that the Arabidopsis thaliana nucleolin protein NUC1, an abundant and evolutionarily conserved nucleolar protein in eukaryotic organisms, is required for maintaining DNA methylation levels and for controlling the expression of specific rDNA variants in Arabidopsis. Interestingly, in contrast with animal or yeast cells, plants contain a second nucleolin gene. Here, we report that Arabidopsis NUC1 and NUC2 nucleolin genes are both required for plant growth and survival and that NUC2 disruption represses flowering. However, these genes seem to be functionally antagonistic. In contrast with NUC1, disruption of NUC2 induces CG hypermethylation of rDNA and NOR association with the nucleolus. Moreover, NUC2 loss of function triggers major changes in rDNA spatial organization, expression, and transgenerational stability. Our analyses indicate that silencing of specific rRNA genes is mostly determined by the active or repressed state of the NORs and that nucleolin proteins play a key role in the developmental control of this process. PMID:24668745

  3. Transcriptional repressors, corepressors and chromatin modifying enzymes in T cell development

    PubMed Central

    Shapiro, Michael J.; Shapiro, Virginia Smith

    2010-01-01

    Gene expression is regulated by the combined action of transcriptional activators and transcriptional repressors. Transcriptional repressors function by recruiting corepressor complexes containing histone-modifying enzymes to specific sites within DNA. Chromatin modifying complexes are subsequently recruited, either directly by transcriptional repressors, or indirectly via corepressor complexes and/or histone modifications, to remodel chromatin into either a transcription-friendly ‘open’ form or an inhibitory ‘closed’ form. Transcriptional repressors, corepressors and chromatin modifying complexes play critical roles throughout T cell development. Here, we highlight those genes that function to repress transcription and that have been shown to be required for T cell development. PMID:21163671

  4. Effective Identification of Akt Interacting Proteins by Two-Step Chemical Crosslinking, Co-Immunoprecipitation and Mass Spectrometry

    PubMed Central

    Huang, Bill X.; Kim, Hee-Yong

    2013-01-01

    Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis. An intrinsic problem of the method is loss of interacting proteins during procedures to remove non-specific proteins. Moreover, antibody contamination often interferes with the detection of less abundant proteins. Here, we developed a novel two-step chemical crosslinking strategy to overcome these problems which resulted in a dramatic improvement in identifying Akt interacting partners. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, dithiobis[succinimidylpropionate], a cleavable crosslinker, was introduced to produce stable complexes between Akt and binding partners prior to the SDS-PAGE and nanoLC-MS/MS analysis. This approach enabled identification of ten Akt partners from cell lysates containing as low as 1.5 mg proteins, including two new potential Akt interacting partners. None of these but one protein was detectable without crosslinking procedures. The present method provides a sensitive and effective tool to probe Akt-interacting proteins. This strategy should also prove useful for other protein interactions, particularly those involving less abundant or weakly associating partners. PMID:23613850

  5. Methods to Identify Chromatin-Bound Protein Complexes: From Genome-Wide to Locus-Specific Approaches.

    PubMed

    Massie, Charles E

    2016-01-01

    High-throughput sequencing approaches coupled with functional genomics experiments have facilitated a rapid growth in our understanding of chromatin biology, from genome-wide maps of transcription factor binding and histone modifications to insights into higher order chromatin organization under specific cellular conditions. However in most cases these methods require a prior knowledge of the system of interest (e.g., targets for immunoprecipitation or modulation) and therefore are limited in their utility to identify novel components of pathways or for the study of uncharacterized pathways. Several orthologous proteomics approaches have been developed recently that bridge this gap, allowing the identification of protein complexes globally or at specific genomic loci. In this chapter the relative advantages of each approach will be explored and a detailed protocol given for DNA pull-down of a specific androgen receptor (AR) genomic target. PMID:27246338

  6. ATM and KAT5 safeguard replicating chromatin against formaldehyde damage

    PubMed Central

    Ortega-Atienza, Sara; Wong, Victor C.; DeLoughery, Zachary; Luczak, Michal W.; Zhitkovich, Anatoly

    2016-01-01

    Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA. PMID:26420831

  7. 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.

  8. Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells

    PubMed Central

    Jullien, Denis; Vignard, Julien; Fedor, Yoann; Béry, Nicolas; Olichon, Aurélien; Crozatier, Michèle; Erard, Monique; Cassard, Hervé; Ducommun, Bernard; Salles, Bernard

    2016-01-01

    ABSTRACT Chromatin function is involved in many cellular processes, its visualization or modification being essential in many developmental or cellular studies. Here, we present the characterization of chromatibody, a chromatin-binding single-domain, and explore its use in living cells. This non-intercalating tool specifically binds the heterodimer of H2A–H2B histones and displays a versatile reactivity, specifically labeling chromatin from yeast to mammals. We show that this genetically encoded probe, when fused to fluorescent proteins, allows non-invasive real-time chromatin imaging. Chromatibody is a dynamic chromatin probe that can be modulated. Finally, chromatibody is an efficient tool to target an enzymatic activity to the nucleosome, such as the DNA damage-dependent H2A ubiquitylation, which can modify this epigenetic mark at the scale of the genome and result in DNA damage signaling and repair defects. Taken together, these results identify chromatibody as a universal non-invasive tool for either in vivo chromatin imaging or to manipulate the chromatin landscape. PMID:27206857

  9. hiHMM: Bayesian non-parametric joint inference of chromatin state maps

    PubMed Central

    Sohn, Kyung-Ah; Ho, Joshua W. K.; Djordjevic, Djordje; Jeong, Hyun-hwan; Park, Peter J.; Kim, Ju Han

    2015-01-01

    Motivation: Genome-wide mapping of chromatin states is essential for defining regulatory elements and inferring their activities in eukaryotic genomes. A number of hidden Markov model (HMM)-based methods have been developed to infer chromatin state maps from genome-wide histone modification data for an individual genome. To perform a principled comparison of evolutionarily distant epigenomes, we must consider species-specific biases such as differences in genome size, strength of signal enrichment and co-occurrence patterns of histone modifications. Results: Here, we present a new Bayesian non-parametric method called hierarchically linked infinite HMM (hiHMM) to jointly infer chromatin state maps in multiple genomes (different species, cell types and developmental stages) using genome-wide histone modification data. This flexible framework provides a new way to learn a consistent definition of chromatin states across multiple genomes, thus facilitating a direct comparison among them. We demonstrate the utility of this method using synthetic data as well as multiple modENCODE ChIP-seq datasets. Conclusion: The hierarchical and Bayesian non-parametric formulation in our approach is an important extension to the current set of methodologies for comparative chromatin landscape analysis. Availability and implementation: Source codes are available at https://github.com/kasohn/hiHMM. Chromatin data are available at http://encode-x.med.harvard.edu/data_sets/chromatin/. Contact: peter_park@harvard.edu or juhan@snu.ac.kr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25725496

  10. How the cell cycle impacts chromatin architecture and influences cell fate

    PubMed Central

    Ma, Yiqin; Kanakousaki, Kiriaki; Buttitta, Laura

    2015-01-01

    Since the earliest observations of cells undergoing mitosis, it has been clear that there is an intimate relationship between the cell cycle and nuclear chromatin architecture. The nuclear envelope and chromatin undergo robust assembly and disassembly during the cell cycle, and transcriptional and post-transcriptional regulation of histone biogenesis and chromatin modification is controlled in a cell cycle-dependent manner. Chromatin binding proteins and chromatin modifications in turn influence the expression of critical cell cycle regulators, the accessibility of origins for DNA replication, DNA repair, and cell fate. In this review we aim to provide an integrated discussion of how the cell cycle machinery impacts nuclear architecture and vice-versa. We highlight recent advances in understanding cell cycle-dependent histone biogenesis and histone modification deposition, how cell cycle regulators control histone modifier activities, the contribution of chromatin modifications to origin firing for DNA replication, and newly identified roles for nucleoporins in regulating cell cycle gene expression, gene expression memory and differentiation. We close with a discussion of how cell cycle status may impact chromatin to influence cell fate decisions, under normal contexts of differentiation as well as in instances of cell fate reprogramming. PMID:25691891

  11. Non-Coding RNA: Sequence-Specific Guide for Chromatin Modification and DNA Damage Signaling

    PubMed Central

    Francia, Sofia

    2015-01-01

    Chromatin conformation shapes the environment in which our genome is transcribed into RNA. Transcription is a source of DNA damage, thus it often occurs concomitantly to DNA damage signaling. Growing amounts of evidence suggest that different types of RNAs can, independently from their protein-coding properties, directly affect chromatin conformation, transcription and splicing, as well as promote the activation of the DNA damage response (DDR) and DNA repair. Therefore, transcription paradoxically functions to both threaten and safeguard genome integrity. On the other hand, DNA damage signaling is known to modulate chromatin to suppress transcription of the surrounding genetic unit. It is thus intriguing to understand how transcription can modulate DDR signaling while, in turn, DDR signaling represses transcription of chromatin around the DNA lesion. An unexpected player in this field is the RNA interference (RNAi) machinery, which play roles in transcription, splicing and chromatin modulation in several organisms. Non-coding RNAs (ncRNAs) and several protein factors involved in the RNAi pathway are well known master regulators of chromatin while only recent reports show their involvement in DDR. Here, we discuss the experimental evidence supporting the idea that ncRNAs act at the genomic loci from which they are transcribed to modulate chromatin, DDR signaling and DNA repair. PMID:26617633

  12. Correlated Spatio-Temporal Fluctuations in Chromatin Compaction States Characterize Stem Cells

    PubMed Central

    Talwar, Shefali; Kumar, Abhishek; Rao, Madan; Menon, Gautam I.; Shivashankar, G.V.

    2013-01-01

    Stem cells integrate signals from the microenvironment to generate lineage-specific gene expression programs upon differentiation. Undifferentiated cell nuclei are easily deformable, with an active transcriptome, whereas differentiated cells have stiffer nuclei and condensed chromatin. Chromatin organization in the stem cell state is known to be highly dynamic but quantitative characterizations of its plasticity are lacking. Using fluorescence imaging, we study the spatio-temporal dynamics of nuclear architecture and chromatin compaction in mouse embryonic stem (ES) cells and differentiated states. Individual ES cells exhibit a relatively narrow variation in chromatin compaction, whereas primary mouse embryonic fibroblasts (PMEF) show broad distributions. However, spatial correlations in chromatin compaction exhibit an emergent length scale in PMEFs, although they are unstructured and longer ranged in ES cells. We provide evidence for correlated fluctuations with large amplitude and long intrinsic timescales, including an oscillatory component, in both chromatin compaction and nuclear area in ES cells. Such fluctuations are largely frozen in PMEF. The role of actin and Lamin A/C in modulating these fluctuations is described. A simple theoretical formulation reproduces the observed dynamics. Our results suggest that, in addition to nuclear plasticity, correlated spatio-temporal structural fluctuations of chromatin in undifferentiated cells characterize the stem cell state. PMID:23442906

  13. Regulation of oncogene-induced cell cycle exit and senescence by chromatin modifiers

    PubMed Central

    David, Gregory

    2012-01-01

    Oncogene activation leads to dramatic changes in numerous biological pathways controlling cellular division, and results in the initiation of a transcriptional program that promotes transformation. Conversely, it also triggers an irreversible cell cycle exit called cellular senescence, which allows the organism to counteract the potentially detrimental uncontrolled proliferation of damaged cells. Therefore, a tight transcriptional control is required at the onset of oncogenic signal, coordinating both positive and negative regulation of gene expression. Not surprisingly, numerous chromatin modifiers contribute to the cellular response to oncogenic stress. While these chromatin modifiers were initially thought of as mere mediators of the cellular response to oncogenic stress, recent studies have uncovered a direct and specific regulation of chromatin modifiers by oncogenic signals. We review here the diverse functions of chromatin modifiers in the cellular response to oncogenic stress, and discuss the implications of these findings on the regulation of cell cycle progression and proliferation by activated oncogenes. PMID:22825329

  14. Discovery and Characterization of Chromatin States for Systematic Annotation of the Human Genome

    NASA Astrophysics Data System (ADS)

    Ernst, Jason; Kellis, Manolis

    A plethora of epigenetic modifications have been described in the human genome and shown to play diverse roles in gene regulation, cellular differentiation and the onset of disease. Although individual modifications have been linked to the activity levels of various genetic functional elements, their combinatorial patterns are still unresolved and their potential for systematic de novo genome annotation remains untapped. Here, we use a multivariate Hidden Markov Model to reveal chromatin states in human T cells, based on recurrent and spatially coherent combinations of chromatin marks.We define 51 distinct chromatin states, including promoter-associated, transcription-associated, active intergenic, largescale repressed and repeat-associated states. Each chromatin state shows specific enrichments in functional annotations, sequence motifs and specific experimentally observed characteristics, suggesting distinct biological roles. This approach provides a complementary functional annotation of the human genome that reveals the genome-wide locations of diverse classes of epigenetic function.

  15. Chromatin Heterogeneity and Distribution of Regulatory Elements in the Late-Replicating Intercalary Heterochromatin Domains of Drosophila melanogaster Chromosomes

    PubMed Central

    Khoroshko, Varvara A.; Levitsky, Viktor G.; Zykova, Tatyana Yu.; Antonenko, Oksana V.; Belyaeva, Elena S.; Zhimulev, Igor F.

    2016-01-01

    Late-replicating domains (intercalary heterochromatin) in the Drosophila genome display a number of features suggesting their organization is quite unique. Typically, they are quite large and encompass clusters of functionally unrelated tissue-specific genes. They correspond to the topologically associating domains and conserved microsynteny blocks. Our study aims at exploring further details of molecular organization of intercalary heterochromatin and has uncovered surprising heterogeneity of chromatin composition in these regions. Using the 4HMM model developed in our group earlier, intercalary heterochromatin regions were found to host chromatin fragments with a particular epigenetic profile. Aquamarine chromatin fragments (spanning 0.67% of late-replicating regions) are characterized as a class of sequences that appear heterogeneous in terms of their decompactization. These fragments are enriched with enhancer sequences and binding sites for insulator proteins. They likely mark the chromatin state that is related to the binding of cis-regulatory proteins. Malachite chromatin fragments (11% of late-replicating regions) appear to function as universal transitional regions between two contrasting chromatin states. Namely, they invariably delimit intercalary heterochromatin regions from the adjacent active chromatin of interbands. Malachite fragments also flank aquamarine fragments embedded in the repressed chromatin of late-replicating regions. Significant enrichment of insulator proteins CP190, SU(HW), and MOD2.2 was observed in malachite chromatin. Neither aquamarine nor malachite chromatin types appear to correlate with the positions of highly conserved non-coding elements (HCNE) that are typically replete in intercalary heterochromatin. Malachite chromatin found on the flanks of intercalary heterochromatin regions tends to replicate earlier than the malachite chromatin embedded in intercalary heterochromatin. In other words, there exists a gradient of

  16. Chromatin Heterogeneity and Distribution of Regulatory Elements in the Late-Replicating Intercalary Heterochromatin Domains of Drosophila melanogaster Chromosomes.

    PubMed

    Khoroshko, Varvara A; Levitsky, Viktor G; Zykova, Tatyana Yu; Antonenko, Oksana V; Belyaeva, Elena S; Zhimulev, Igor F

    2016-01-01

    Late-replicating domains (intercalary heterochromatin) in the Drosophila genome display a number of features suggesting their organization is quite unique. Typically, they are quite large and encompass clusters of functionally unrelated tissue-specific genes. They correspond to the topologically associating domains and conserved microsynteny blocks. Our study aims at exploring further details of molecular organization of intercalary heterochromatin and has uncovered surprising heterogeneity of chromatin composition in these regions. Using the 4HMM model developed in our group earlier, intercalary heterochromatin regions were found to host chromatin fragments with a particular epigenetic profile. Aquamarine chromatin fragments (spanning 0.67% of late-replicating regions) are characterized as a class of sequences that appear heterogeneous in terms of their decompactization. These fragments are enriched with enhancer sequences and binding sites for insulator proteins. They likely mark the chromatin state that is related to the binding of cis-regulatory proteins. Malachite chromatin fragments (11% of late-replicating regions) appear to function as universal transitional regions between two contrasting chromatin states. Namely, they invariably delimit intercalary heterochromatin regions from the adjacent active chromatin of interbands. Malachite fragments also flank aquamarine fragments embedded in the repressed chromatin of late-replicating regions. Significant enrichment of insulator proteins CP190, SU(HW), and MOD2.2 was observed in malachite chromatin. Neither aquamarine nor malachite chromatin types appear to correlate with the positions of highly conserved non-coding elements (HCNE) that are typically replete in intercalary heterochromatin. Malachite chromatin found on the flanks of intercalary heterochromatin regions tends to replicate earlier than the malachite chromatin embedded in intercalary heterochromatin. In other words, there exists a gradient of

  17. Cytomixis doesn't induce obvious changes in chromatin modifications and programmed cell death in tobacco male meiocytes.

    PubMed

    Mursalimov, Sergey; Permyakova, Natalya; Deineko, Elena; Houben, Andreas; Demidov, Dmitri

    2015-01-01

    Cytomixis is a poorly studied process of nuclear migration between plant cells. It is so far unknown what drives cytomixis and what is the functional state of the chromatin migrating between cells. Using immunostaining, we have analyzed the distribution of posttranslational histone modifications (methylation, acetylation, and phosphorylation) that reflect the functional state of chromatin in the tobacco microsporocytes involved in cytomixis. We demonstrate that the chromatin in the cytomictic cells does not differ from the chromatin in intact microsporocytes according to all 14 analyzed histone modification types. We have also for the first time demonstrated that the migrating chromatin contains normal structures of the synaptonemal complex (SC) and lacks any signs of apoptosis. As has been shown, the chromatin migrating between cells in cytomixis is neither selectively heterochromatized nor degraded both before its migration to another cell and after it enters a recipient cell as micronuclei. We also showed that cytomictic chromatin contains marks typical for transcriptionally active chromatin as well as heterochromatin. Moreover, marks typical for chromosome condensation, SC formation and key proteins required for the formation of bivalents were also detected at migrated chromatin. PMID:26528310

  18. Histone H3 Acetylation and H3 K4 Methylation Define Distinct Chromatin Regions Permissive for Transgene Expression

    PubMed Central

    Yan, Chunhong; Boyd, Douglas D.

    2006-01-01

    Histone modifications are associated with distinct transcription states and serve as heritable epigenetic markers for chromatin structure and function. While H3 K9 methylation defines condensed heterochromatin that is able to silence a nearby gene, how gene silencing within euchromatin regions is achieved remains elusive. We report here that histone H3 K4 methylation or K9/K14 acetylation defines distinct chromatin regions permissive or nonpermissive for transgene expression. A permissive chromatin region is enriched in H3 K4 methylation and H3 acetylation, while a nonpermissive region is poor in or depleted of these two histone modifications. The histone modification states of the permissive chromatin can spread to transgenic promoters. However, de novo histone H3 acetylation and H3 K4 methylation at a transgenic promoter in a nonpermissive chromatin region are stochastic, leading to variegated transgene expression. Moreover, nonpermissive chromatin progressively silences a transgene, an event that is accompanied by the reduction of H3 K4 methylation and H3 acetylation levels at the transgenic promoter. These repressive effects of nonpermissive chromatin cannot be completely countered by strong transcription activators, indicating the dominance of the chromatin effects. We therefore propose a model in which histone H3 acetylation and H3 K4 methylation localized to discrete sites in the mammalian genome mark distinct chromatin functions that dictate transgene expression or silencing. PMID:16914722

  19. Cytomixis doesn’t induce obvious changes in chromatin modifications and programmed cell death in tobacco male meiocytes

    PubMed Central

    Mursalimov, Sergey; Permyakova, Natalya; Deineko, Elena; Houben, Andreas; Demidov, Dmitri

    2015-01-01

    Cytomixis is a poorly studied process of nuclear migration between plant cells. It is so far unknown what drives cytomixis and what is the functional state of the chromatin migrating between cells. Using immunostaining, we have analyzed the distribution of posttranslational histone modifications (methylation, acetylation, and phosphorylation) that reflect the functional state of chromatin in the tobacco microsporocytes involved in cytomixis. We demonstrate that the chromatin in the cytomictic cells does not differ from the chromatin in intact microsporocytes according to all 14 analyzed histone modification types. We have also for the first time demonstrated that the migrating chromatin contains normal structures of the synaptonemal complex (SC) and lacks any signs of apoptosis. As has been shown, the chromatin migrating between cells in cytomixis is neither selectively heterochromatized nor degraded both before its migration to another cell and after it enters a recipient cell as micronuclei. We also showed that cytomictic chromatin contains marks typical for transcriptionally active chromatin as well as heterochromatin. Moreover, marks typical for chromosome condensation, SC formation and key proteins required for the formation of bivalents were also detected at migrated chromatin. PMID:26528310

  20. 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

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

    PubMed

    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

    2016-02-18

    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

  2. Barrier-to-Autointegration Factor Proteome Reveals Chromatin-Regulatory Partners

    PubMed Central

    Montes de Oca, Rocío; Shoemaker, Christopher J.; Gucek, Marjan; Cole, Robert N.; Wilson, Katherine L.

    2009-01-01

    Nuclear lamin filaments and associated proteins form a nucleoskeletal (“lamina”) network required for transcription, replication, chromatin organization and epigenetic regulation in metazoans. Lamina defects cause human disease (“laminopathies”) and are linked to aging. Barrier-to-autointegration factor (BAF) is a mobile and essential component of the nuclear lamina that binds directly to histones, lamins and LEM-domain proteins, including the inner nuclear membrane protein emerin, and has roles in chromatin structure, mitosis and gene regulation. To understand BAF's mechanisms of action, BAF associated proteins were affinity-purified from HeLa cell nuclear lysates using BAF-conjugated beads, and identified by tandem mass spectrometry or independently identified and quantified using the iTRAQ method. We recovered A- and B-type lamins and core histones, all known to bind BAF directly, plus four human transcription factors (Requiem, NonO, p15, LEDGF), disease-linked proteins (e.g., Huntingtin, Treacle) and several proteins and enzymes that regulate chromatin. Association with endogenous BAF was independently validated by co-immunoprecipitation from HeLa cells for seven candidates including Requiem, poly(ADP-ribose) polymerase 1 (PARP1), retinoblastoma binding protein 4 (RBBP4), damage-specific DNA binding protein 1 (DDB1) and DDB2. Interestingly, endogenous BAF and emerin each associated with DDB2 and CUL4A in a UV- and time-dependent manner, suggesting BAF and emerin have dynamic roles in genome integrity and might help couple DNA damage responses to the nuclear lamina network. We conclude this proteome is a rich source of candidate partners for BAF and potentially also A- and B-type lamins, which may reveal how chromatin regulation and genome integrity are linked to nuclear structure. PMID:19759913

  3. 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.

  4. Pulling chromatin apart: Unstacking or Unwrapping?

    PubMed Central

    2012-01-01

    Background Understanding the mechanical properties of chromatin is an essential step towards deciphering the physical rules of gene regulation. In the past ten years, many single molecule experiments have been carried out, and high resolution measurements of the chromatin fiber stiffness are now available. Simulations have been used in order to link those measurements with structural cues, but so far no clear agreement among different groups has been reached. Results We revisit here some of the most precise experimental results obtained with carefully reconstituted fibers. Conclusions We show that the mechanical properties of the chromatin fiber can be quantitatively accounted for by the stiffness of the DNA molecule and the 3D structure of the chromatin fiber. PMID:23186373

  5. Engineered apoptotic nucleases for chromatin research.

    PubMed

    Xiao, Fei; Widlak, Piotr; Garrard, William T

    2007-01-01

    We have created new genomics tools for chromatin research by genetically engineering the human and mouse major apoptotic nucleases that are responsible for internucleosomal DNA cleavage, DNA fragmentation factor (DFF). Normally, in its inactive form, DFF is a heterodimer composed of a 45-kDa chaperone inhibitor subunit (DFF45 or ICAD), and a 40-kDa latent endonuclease subunit (DFF40 or CAD). Upon caspase-3 cleavage of DFF45, DFF40 forms active endonuclease homo-oligomers. Although Saccharomyces cerevisiae lacks DFF, expression of caspase-3 is lethal in this organism, but expression of the highly sequence-specific tobacco etch virus protease (TEVP) is harmless. Therefore, we inserted TEVP cleavage sites immediately downstream of the two caspase-3 cleavage sites within DFF45, generating a novel form of DFF (DFF-T) whose nuclease activity proved to be exclusively under the control of TEVP. We demonstrate that co-expression of TEVP and DFF-T under galactose control results in nucleosomal DNA laddering and cell death in S. cerevisiae. We also created synthetic DFF genes with optimized codons for high-level expression in Eschericia coli or S. cerevisiae. We further demonstrate the excellence of the synthetic gene products for in vitro mapping of the nucleosome positions and hypersensitive sites in specific genes such as the yeast PHO5. PMID:17626049

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

    PubMed Central

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

    2014-01-01

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

  7. 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

  8. 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. PMID:26208520

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

    PubMed Central

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

    2012-01-01

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

  10. 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

  11. Studying G protein-coupled receptors: immunoblotting, immunoprecipitation, phosphorylation, surface labeling, and cross-linking protocols.

    PubMed

    Pal, Kasturi; Badgandi, Hemant; Mukhopadhyay, Saikat

    2015-01-01

    Primary cilia are signaling organelles that have been shown to coordinate cellular responses to extracellular cues during physiological processes ranging from organ patterning to cell cycle regulation. A variety of receptors, including G protein-coupled receptors (GPCRs), downstream effectors (adenylyl cyclases), and second messengers, such as calcium, accumulate in the ciliary compartment. Isolation of GPCRs is essential for studying posttranslational modifications, intracellular trafficking, and protein-protein interactions that are important in downstream signaling. However, the presence of multiple hydrophobic transmembrane domains, and the inherent conformational flexibility of GPCRs make their extraction from membranes and solubilization particularly challenging. Here, we describe detailed methods for immunoblotting and immunoprecipitation of GPCRs from whole cell extracts. These methods are applicable for studying other multipass transmembrane proteins (such as adenylyl cyclases). We also describe methods for determining GPCR phosphorylation, surface labeling by biotinylation, and cross-linking to detect transient interactions with other proteins. These methods are amenable for studying both ciliary and nonciliary GPCRs in the context of cellular signaling pathways. PMID:25837398

  12. Early Aberrations in Chromatin Dynamics in Embryos Produced Under In Vitro Conditions

    PubMed Central

    Deshmukh, Rahul S.; Strejcek, Frantisek; Vejlsted, Morten; Lucas-Hahn, Andrea; Petersen, Bjorn; Li, Juan; Callesen, Henrik; Niemann, Heiner; Hyttel, Poul

    2012-01-01

    Abstract In vitro production of porcine embryos by means of in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) is limited by great inefficienciy. The present study investigated chromatin and nucleolar dynamics in porcine embryos developed in vivo (IV) and compared this physiological standard to that of embryos produced by IVF, parthenogenetic activation (PA), or SCNT. In contrast to IV embryos, chromatin spatial and temporal dynamics in PA, IVF, and SCNT embryos were altered; starting with aberrant chromatin–nuclear envelope interactions at the two-cell stage, delayed chromatin decondensation and nucleolar development at the four-cell stage, and ultimately culminating in failure of proper first lineage segregation at the blastocyst stage, demonstrated by poorly defined inner cell mass. Interestingly, in vitro produced (IVP) embryos also lacked a heterochromatin halo around nucleolar precursors, indicating imperfections in global chromatin remodeling after fertilization/activation. Porcine IV-produced zygotes and embryos display a well-synchronized pattern of chromatin dynamics compatible with genome activation and regular nucleolar formation at the four-cell stage. Production of porcine embryos under in vitro conditions by IVF, PA, or SCNT is associated with altered chromatin remodeling, delayed nucleolar formation, and poorly defined lineage segregation at the blastocyst stage, which in turn may impair their developmental capacity. PMID:22468997

  13. Effects of thyrotropin on the phosphorylation of histones and nonhistone phosphoproteins in micrococcal nuclease-sensitive and resistant thyroid chromatin

    SciTech Connect

    Cooper, E.; Spaulding, S.W.

    1983-05-01

    Actively transcribed regions of chromatin are more susceptible than bulk chromatin to digestion by nucleases, and useful information about the composition and structure of active chromatin may be obtained by studying the chromatin fragments released from nuclei by limited nuclease digestion. In the present study, we have used micrococcal nuclease to investigate the effects of TSH on protein phosphorylation in nuclease-sensitive fractions of calf thyroid chromatin. Batches of calf thyroid slices were incubated for 2 h with /sup 32/Pi, with or without 50 mU/ml TSH. Nuclei were then prepared and the distribution of /sup 32/P-labeled histones, high mobility group (HMG) proteins, and other acid-soluble phosphoproteins between micrococcal nuclease-sensitive and resistant fractions of chromatin was examined. TSH increased the amount of /sup 32/P incorporated into HMG 14 and the histones H1 and H3. Hormone-dependent increases in the /sup 32/P-labeling of H1 and H3 were not selectively associated with micrococcal nuclease-sensitive chromatin. In contrast, (/sup 32/P) HMG-14 was preferentially solubilized from nuclei by micrococcal nuclease. This lends support to the view that TSH-induced effects on the structure and function of transcriptionally active chromatin may be mediated in part by phosphorylation of HMG 14.

  14. Epigenetic regulation of centromeric chromatin: old dogs, new tricks?

    PubMed Central

    Allshire, Robin C.; Karpen, Gary H.

    2008-01-01

    The assembly of just a single kinetochore at the centromere of each sister chromatid is essential for accurate chromosome segregation during cell division. Surprisingly, despite their vital function, centromeres show considerable plasticity with respect to their chromosomal locations and activity. The establishment and maintenance of centromeric chromatin, and therefore the location of kinetochores, is epigenetically regulated. The histone H3 variant CENP-A is the key determinant of centromere identity and kinetochore assembly. Recent studies have identified many factors that affect CENP-A localization, but their precise roles in this process are unknown. We build on these advances and on new information about the timing of CENP-A assembly during the cell cycle to propose new models for how centromeric chromatin is established and propagated. PMID:19002142

  15. At High Levels, Constitutively Activated STAT3 Induces Apoptosis of Chronic Lymphocytic Leukemia Cells.

    PubMed

    Rozovski, Uri; Harris, David M; Li, Ping; Liu, Zhiming; Wu, Ji Yuan; Grgurevic, Srdana; Faderl, Stefan; Ferrajoli, Alessandra; Wierda, William G; Martinez, Matthew; Verstovsek, Srdan; Keating, Michael J; Estrov, Zeev

    2016-05-15

    In chronic lymphocytic leukemia (CLL), the increment in PBLs is slower than the expected increment calculated from the cells' proliferation rate, suggesting that cellular proliferation and apoptosis are concurrent. Exploring this phenomenon, we found overexpression of caspase-3, higher cleaved poly (ADP-ribose) polymerase levels (p < 0.007), and a higher apoptosis rate in cells from patients with high counts compared with cells from patients with low counts. Although we previously found that STAT3 protects CLL cells from apoptosis, STAT3 levels were significantly higher in cells from patients with high counts than in cells from patients with low counts. Furthermore, overexpression of STAT3 did not protect the cells. Rather, it upregulated caspase-3 and induced apoptosis. Remarkably, putative STAT3 binding sites were identified in the caspase-3 promoter, and a luciferase assay, chromatin immunoprecipitation, and an EMSA revealed that STAT3 activated caspase-3 However, caspase-3 levels increased only when STAT3 levels were sufficiently high. Using chromatin immunoprecipitation and EMSA, we found that STAT3 binds with low affinity to the caspase-3 promoter, suggesting that at high levels, STAT3 activates proapoptotic mechanisms and induces apoptosis in CLL cells. PMID:27076684

  16. OPERating ON chromatin, a colorful language where context matters

    PubMed Central

    Gardner, Kathryn E.; Allis, C. David; Strahl, Brian D.

    2011-01-01

    Histones, the fundamental packaging elements of eukaryotic DNA, are highly decorated with a diverse set of post-translational modifications (PTMs) that are recognized to govern the structure and function of chromatin. Ten years ago, we put forward the histone code hypothesis, which provided a model to explain how single and/or combinatorial PTMs on histones regulate the diverse activities associated with chromatin (e.g. gene transcription). At that time, there was a limited understanding of both the number of PTMs that occur on histones as well as the proteins that place, remove and interpret them. Since the conception of this hypothesis, the field has witnessed an unprecedented advance in our understanding of the enzymes that contribute to the establishment of histone PTMs, as well as the diverse effector proteins that bind them. While debate continues as to whether histone PTMs truly constitute a strict “code”, it is becoming clear that PTMs on histone proteins function in elaborate combinations to regulate the many activities associated with chromatin. In this special issue, we celebrate the 50th anniversary of the landmark publication of the lac operon with a review that provides a current view of the histone code hypothesis, the lessons we have learned over the last decade, and the technologies that will drive our understanding of histone PTMs forward in the future. PMID:21272588

  17. The insulation of genes from external enhancers and silencing chromatin

    PubMed Central

    Burgess-Beusse, Bonnie; Farrell, Catherine; Gaszner, Miklos; Litt, Michael; Mutskov, Vesco; Recillas-Targa, Felix; Simpson, Melanie; West, Adam; Felsenfeld, Gary

    2002-01-01

    Insulators are DNA sequence elements that can serve in some cases as barriers to protect a gene against the encroachment of adjacent inactive condensed chromatin. Some insulators also can act as blocking elements to protect against the activating influence of distal enhancers associated with other genes. Although most of the insulators identified so far derive from Drosophila, they also are found in vertebrates. An insulator at the 5′ end of the chicken β-globin locus marks a boundary between an open chromatin domain and a region of constitutively condensed chromatin. Detailed analysis of this element shows that it possesses both enhancer blocking activity and the ability to screen reporter genes against position effects. Enhancer blocking is associated with binding of the protein CTCF; sites that bind CTCF are found at other critical points in the genome. Protection against position effects involves other properties that appear to be associated with control of histone acetylation and methylation. Insulators thus are complex elements that can help to preserve the independent function of genes embedded in a genome in which they are surrounded by regulatory signals they must ignore. PMID:12154228

  18. Nucleolin: dual roles in rDNA chromatin transcription.

    PubMed

    Durut, Nathalie; Sáez-Vásquez, Julio

    2015-02-01

    Nucleolin is a major nucleolar protein conserved in all eukaryotic organisms. It is a multifunctional protein involved in different cellular aspects like chromatin organization and stability, DNA and RNA metabolism, assembly of ribonucleoprotein complexes, cytokinesis, cell proliferation and stress response. The multifunctionality of nucleolin is linked to its tripartite structure, post-translational modifications and its ability of shuttling from and to the nucleolus/nucleoplasm and cytoplasm. Nucleolin has been now studied for many years and its activities and properties have been described in a number of excellent reviews. Here, we overview the role of nucleolin in RNA polymerase I (RNAPI) transcription and describe recent results concerning its functional interaction with rDNA chromatin organization. For a long time, nucleolin has been associated with rRNA gene expression and pre-rRNA processing. However, the functional connection between nucleolin and active versus inactive rRNA genes is still not fully understood. Novel evidence indicates that the nucleolin protein might be required for controlling the transcriptional ON/OFF states of rDNA chromatin in both mammals and plants. PMID:25225127

  19. Identification of noncoding transcripts from within CENP-A chromatin at fission yeast centromeres.

    PubMed

    Choi, Eun Shik; Strålfors, Annelie; Castillo, Araceli G; Durand-Dubief, Mickaël; Ekwall, Karl; Allshire, Robin C

    2011-07-01

    The histone H3 variant CENP-A is the most favored candidate for an epigenetic mark that specifies the centromere. In fission yeast, adjacent heterochromatin can direct CENP-A(Cnp1) chromatin establishment, but the underlying features governing where CENP-A(Cnp1) chromatin assembles are unknown. We show that, in addition to centromeric regions, a low level of CENP-A(Cnp1) associates with gene promoters where histone H3 is depleted by the activity of the Hrp1(Chd1) chromatin-remodeling factor. Moreover, we demonstrate that noncoding RNAs are transcribed by RNA polymerase II (RNAPII) from CENP-A(Cnp1) chromatin at centromeres. These analyses reveal a similarity between centromeres and a subset of RNAPII genes and suggest a role for remodeling at RNAPII promoters within centromeres that influences the replacement of histone H3 with CENP-A(Cnp1). PMID:21531710

  20. Identification of Noncoding Transcripts from within CENP-A Chromatin at Fission Yeast Centromeres*

    PubMed Central

    Choi, Eun Shik; Strålfors, Annelie; Castillo, Araceli G.; Durand-Dubief, Mickaël; Ekwall, Karl; Allshire, Robin C.

    2011-01-01

    The histone H3 variant CENP-A is the most favored candidate for an epigenetic mark that specifies the centromere. In fission yeast, adjacent heterochromatin can direct CENP-ACnp1 chromatin establishment, but the underlying features governing where CENP-ACnp1 chromatin assembles are unknown. We show that, in addition to centromeric regions, a low level of CENP-ACnp1 associates with gene promoters where histone H3 is depleted by the activity of the Hrp1Chd1 chromatin-remodeling factor. Moreover, we demonstrate that noncoding RNAs are transcribed by RNA polymerase II (RNAPII) from CENP-ACnp1 chromatin at centromeres. These analyses reveal a similarity between centromeres and a subset of RNAPII genes and suggest a role for remodeling at RNAPII promoters within centromeres that influences the replacement of histone H3 with CENP-ACnp1. PMID:21531710

  1. Application of the Protein Semisynthesis Strategy to the Generation of Modified Chromatin

    PubMed Central

    Holt, Matthew; Muir, Tom

    2016-01-01

    Histone proteins are subject to a host of posttranslational modifications (PTMs) that modulate chromatin structure and function. Such control is achieved by the direct alteration of the intrinsic physical properties of the chromatin fiber or by regulating the recruitment and activity of a host of trans-acting nuclear factors. The sheer number of histone PTMs presents a formidable barrier to understanding the molecular mechanisms at the heart of epigenetic regulation of eukaryotic genomes. One aspect of this multifarious problem, namely how to access homogeneously modified chromatin for biochemical studies, is well suited to the sensibilities of the organic chemist. Indeed, recent years have witnessed a critical role for synthetic protein chemistry methods in generating the raw materials needed for studying how histone PTMs regulate chromatin biochemistry. This review focuses on what is arguably the most powerful, and widely employed, of these chemical strategies, namely histone semisynthesis via the chemical ligation of peptide fragments. PMID:25784050

  2. Chromatin Structure and Dynamics in Hot Environments: Architectural Proteins and DNA Topoisomerases of Thermophilic Archaea

    PubMed Central

    Visone, Valeria; Vettone, Antonella; Serpe, Mario; Valenti, Anna; Perugino, Giuseppe; Rossi, Mosè; Ciaramella, Maria

    2014-01-01

    In all organisms of the three living domains (Bacteria, Archaea, Eucarya) chromosome-associated proteins play a key role in genome functional organization. They not only compact and shape the genome structure, but also regulate its dynamics, which is essential to allow complex genome functions. Elucidation of chromatin composition and regulation is a critical issue in biology, because of the intimate connection of chromatin with all the essential information processes (transcription, replication, recombination, and repair). Chromatin proteins include architectural proteins and DNA topoisomerases, which regulate genome structure and remodelling at two hierarchical levels. This review is focussed on architectural proteins and topoisomerases from hyperthermophilic Archaea. In these organisms, which live at high environmental temperature (>80 °C <113 °C), chromatin proteins and modulation of the DNA secondary structure are concerned with the problem of DNA stabilization against heat denaturation while maintaining its metabolic activity. PMID:25257534

  3. From Structural Variation of Gene Molecules to Chromatin Dynamics and Transcriptional Bursting

    PubMed Central

    Boeger, Hinrich; Shelansky, Robert; Patel, Heta; Brown, Christopher R.

    2015-01-01

    Transcriptional activation of eukaryotic genes is accompanied, in general, by a change in the sensitivity of promoter chromatin to endonucleases. The structural basis of this alteration has remained elusive for decades; but the change has been viewed as a transformation of one structure into another, from “closed” to “open” chromatin. In contradistinction to this static and deterministic view of the problem, a dynamical and probabilistic theory of promoter chromatin has emerged as its solution. This theory, which we review here, explains observed variation in promoter chromatin structure at the level of single gene molecules and provides a molecular basis for random bursting in transcription—the conjecture that promoters stochastically transition between transcriptionally conducive and inconducive states. The mechanism of transcriptional regulation may be understood only in probabilistic terms. PMID:26136240

  4. The cellular bromodomain protein Brd4 has multiple functions in E2-mediated papillomavirus transcription activation.

    PubMed

    Helfer, Christine M; Yan, Junpeng; You, Jianxin

    2014-08-01

    The cellular bromodomain protein Brd4 functions in multiple processes of the papillomavirus life cycle, including viral replication, genome maintenance, and gene transcription through its interaction with the viral protein, E2. However, the mechanisms by which E2 and Brd4 activate viral transcription are still not completely understood. In this study, we show that recruitment of positive transcription elongation factor b (P-TEFb), a functional interaction partner of Brd4 in transcription activation, is important for E2's transcription activation activity. Furthermore, chromatin immunoprecipitation (ChIP) analyses demonstrate that P-TEFb is recruited to the actual papillomavirus episomes. We also show that E2's interaction with cellular chromatin through Brd4 correlates with its papillomavirus transcription activation function since JQ1(+), a bromodomain inhibitor that efficiently dissociates E2-Brd4 complexes from chromatin, potently reduces papillomavirus transcription. Our study identifies a specific function of Brd4 in papillomavirus gene transcription and highlights the potential use of bromodomain inhibitors as a method to disrupt the human papillomavirus (HPV) life cycle. PMID:25140737

  5. Chromatin remodeller SMARCA4 recruits topoisomerase 1 and suppresses transcription-associated genomic instability

    PubMed Central

    Husain, Afzal; Begum, Nasim A.; Taniguchi, Takako; Taniguchi, Hisaaki; Kobayashi, Maki; Honjo, Tasuku

    2016-01-01

    Topoisomerase 1, an enzyme that relieves superhelical tension, is implicated in transcription-associated mutagenesis and genome instability-associated with neurodegenerative diseases as well as activation-induced cytidine deaminase. From proteomic analysis of TOP1-associated proteins, we identify SMARCA4, an ATP-dependent chromatin remodeller; FACT, a histone chaperone; and H3K4me3, a transcriptionally active chromatin marker. Here we show that SMARCA4 knockdown in a B-cell line decreases TOP1 recruitment to chromatin, and leads to increases in Igh/c-Myc chromosomal translocations, variable and switch region mutations and negative superhelicity, all of which are also observed in response to TOP1 knockdown. In contrast, FACT knockdown inhibits association of TOP1 with H3K4me3, and severely reduces DNA cleavage and Igh/c-Myc translocations, without significant effect on TOP1 recruitment to chromatin. We thus propose that SMARCA4 is involved in the TOP1 recruitment to general chromatin, whereas FACT is required for TOP1 binding to H3K4me3 at non-B DNA containing chromatin for the site-specific cleavage. PMID:26842758

  6. Sperm cryopreservation: effects on chromatin structure.

    PubMed

    Paoli, Donatella; Lombardo, Francesco; Lenzi, Andrea; Gandini, Loredana

    2014-01-01

    Cryopreservation is a technique that can keep sperm alive indefinitely, enabling the conservation of male fertility. It involves the cooling of semen samples and their storage at -196°C in liquid nitrogen. At this temperature all metabolic processes are arrested. Sperm cryopreservation is of fundamental importance for patients undergoing medical or surgical treatments that could induce sterility, such as cancer patients about to undergo genotoxic chemotherapy or radiotherapy, as it offers these patients not only the hope of future fertility but also psychological support in dealing with the various stages of the treatment protocols.Despite its importance for assisted reproduction technology (ART) and its success in terms of babies born, this procedure can cause cell damage and impaired sperm function. Various studies have evaluated the impact of cryopreservation on chromatin structure, albeit with contradictory results. Some, but not all, authors found significant sperm DNA damage after cryopreservation. However, studies attempting to explain the mechanisms involved in the aetiology of cryopreservation-induced DNA damage are still limited. Some reported an increase in sperm with activated caspases after cryopreservation, while others found an increase in the percentage of oxidative DNA damage. There is still little - and contradictory - information on the mechanism of the generation of DNA fragmentation after cryopreservation. More studies are needed to establish the true importance of such damage, especially to improve the results of ART. PMID:23955677

  7. Macronuclear chromatin structure dynamics in Colpoda inflata (Protista, Ciliophora) resting encystment.

    PubMed

    Tiano, L; Chessa, M G; Carrara, S; Tagliafierro, G; Delmonte Corrado, M U

    1999-01-01

    The chromatin structure dynamics of the Colpoda inflata macronucleus have been investigated in relation to its functional condition, concerning chromatin body extrusion regulating activity. Samples of 2- and 25-day-old resting cysts derived from a standard culture, and of 1-year-old resting cysts derived from a senescent culture, were examined by means of histogram analysis performed on acquired optical microscopy images. Three groups of histograms were detected in each sample. Histogram classification, clustering and matching were assessed in order to obtain the mean histogram of each group. Comparative analysis of the mean histogram showed a similarity in the grey level range of 25-day- and 1-year-old cysts, unlike the wider grey level range found in 2-day-old cysts. Moreover, the respective mean histograms of the three cyst samples appeared rather similar in shape. All this implies that macronuclear chromatin structural features of 1-year-old cysts are common to both cyst standard cultures. The evaluation of the acquired images and their respective histograms evidenced a dynamic state of the macronuclear chromatin, appearing differently condensed in relation to the chromatin body extrusion regulating activity of the macronucleus. The coexistence of a chromatin-decondensed macronucleus with a pycnotic extrusion body suggests that chromatin unable to decondense, thus inactive, is extruded. This finding, along with the presence of chromatin structural features common to standard and senescent cyst populations, supports the occurrence of 'rejuvenated' cell lines from 1-year-old encysted senescent cells, a phenomenon which could be a result of accomplished macronuclear renewal. PMID:10439214

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

    PubMed Central

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

    2011-01-01

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

  9. 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

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

    PubMed

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

    2016-08-01

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

  11. Chromatin signaling in muscle stem cells: interpreting the regenerative microenvironment

    PubMed Central

    Brancaccio, Arianna; Palacios, Daniela

    2015-01-01

    Muscle regeneration in the adult occurs in response to damage at expenses of a population of adult stem cells, the satellite cells. Upon injury, either physical or genetic, signals released within the satellite cell niche lead to the commitment, expansion and differentiation of the pool of muscle progenitors to repair damaged muscle. To achieve this goal satellite cells undergo a dramatic transcriptional reprogramming to coordinately activate and repress specific subset of genes. Although the epigenetics of muscle regeneration has been extensively discussed, less emphasis has been put on how extra-cellular cues are translated into the specific chromatin reorganization necessary for progression through the myogenic program. In this review we will focus on how satellite cells sense the regenerative microenvironment in physiological and pathological circumstances, paying particular attention to the mechanism through which the external stimuli are transduced to the nucleus to modulate chromatin structure and gene expression. We will discuss the pathways involved and how alterations in this chromatin signaling may contribute to satellite cells dysfunction during aging and disease. PMID:25904863

  12. Wavelet transform analysis of chromatin texture changes during heat shock.

    PubMed

    Herbomel, G; Grichine, A; Fertin, A; Delon, A; Vourc'h, C; Souchier, C; Usson, Y

    2016-06-01

    Texture analysis can be a useful tool to investigate the organization of chromatin. Approaches based on multiscale analysis and in particular the 'à trou' wavelet analysis has already been used for microscopy (Olivo Marin). In order to analyse texture changes, the statistical properties of the wavelet coefficient images were summarized by the first four statistical orders: mean, standard deviation, skewness and kurtosis of the coefficient image histogram. The 'à trou' transform provided a representation of the wavelet coefficients and texture parameters with the same statistical robustness throughout the scale spaces. It was applied for quantifying chromatin texture and heat-induced chromatin changes in living cells. We investigated the changes by both laser scanning and spinning disk confocal microscopies and compared the texture parameters before and after increasing duration of heat shock exposure (15 min, 30 min and 1 h). Furthermore, as activation of the heat shock response also correlates with a rapid localization of HSF1 within a few nuclear structures termed nuclear stress bodies (nSBs), we compared the dynamics of nSBs formation with that of textural changes during 1 h of continuous heat shock. Next, we studied the recovery phase following a 1-h heat shock. Significant differences were observed, particularly affecting the perinucleolar region, even for the shortest heat shock time affecting mostly the skewness and standard deviation. Furthermore, progressive changes could be observed according to the duration of heat shock, mostly affecting fine details (pixel-wise changes) as revealed by the parameters, obtained from the first- and second-order wavelet coefficients. 'A trou' wavelet texture analysis provided a sensitive and efficient tool to investigate minute changes of chromatin. PMID:26694695

  13. Targeting chromatin to improve radiation response

    PubMed Central

    Olcina, M M; O'Dell, S

    2015-01-01

    Chromatin, the structure formed by the wrapping of approximately 146 base pairs of DNA around an octamer of histones, has a profound impact on numerous DNA-based processes. Chromatin modifications and chromatin remodellers have recently been implicated in important aspects of the DNA damage response including facilitating the initial sensing of the damage as well as subsequent recruitment of repair factors. Radiation is an effective cancer therapy for a large number of tumours, and there is considerable interest in finding approaches that might further increase the efficacy of radiotherapy. The use of radiation leads to the generation of DNA damage and, therefore, agents that can affect the sensing and repair of DNA damage may have an impact on overall radiation efficacy. The chromatin modifications as well as chromatin modifiers that have been associated with the DNA damage response will be summarized in this review. An emphasis will be placed on those processes that can be pharmacologically manipulated with currently available inhibitors. The rationale for the use of these inhibitors in combination with radiation will also be described. PMID:25513745

  14. 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

  15. Chromatin remodeling in cardiovascular development and physiology

    PubMed Central

    Han, Pei; Hang, Calvin T.; Yang, Jin; Chang, Ching-Pin

    2010-01-01

    Chromatin regulation provides an important means of controlling cardiac gene expression under different physiological and pathological conditions. Processes that direct the development of normal embryonic hearts and pathology of stressed adult hearts may share general mechanisms that govern cardiac gene expression by chromatin-regulating factors. These common mechanisms may provide a framework for us to investigate the interactions among diverse chromatin remodelers/modifiers and various transcription factors in the fine regulation of gene expression, essential for all aspects of cardiovascular biology. Aberrant cardiac gene expression, triggered by a variety of pathological insults, can cause heart diseases in both animals and humans. The severity of cardiomyopathy and heart failure correlates strongly with abnormal cardiac gene expression. Therefore, controlling cardiac gene expression presents a promising approach to the treatment of human cardiomyopathy. This review focuses on the roles of ATP-dependent chromatin-remodeling factors and chromatin-modifying enzymes in the control of gene expression during cardiovascular development and disease. PMID:21293009

  16. Chromatin assembly during S phase: contributions from histone deposition, DNA replication and the cell division cycle.

    PubMed

    Krude, T; Keller, C

    2001-05-01

    During S phase of the eukaryotic cell division cycle, newly replicated DNA is rapidly assembled into chromatin. Newly synthesised histones form complexes with chromatin assembly factors, mediating their deposition onto nascent DNA and their assembly into nucleosomes. Chromatin assembly factor 1, CAF-1, is a specialised assembly factor that targets these histones to replicating DNA by association with the replication fork associated protein, proliferating cell nuclear antigen, PCNA. Nucleosomes are further organised into ordered arrays along the DNA by the activity of ATP-dependent chromatin assembly and spacing factors such as ATP-utilising chromatin assembly and remodelling factor ACE An additional level of controlling chromatin assembly pathways has become apparent by the observation of functional requirements for cyclin-dependent protein kinases, casein kinase II and protein phosphatases. In this review, we will discuss replication-associated histone deposition and nucleosome assembly pathways, and we will focus in particular on how nucleosome assembly is linked to DNA replication and how it may be regulated by the cell cycle control machinery. PMID:11437228

  17. HIRA orchestrates a dynamic chromatin landscape in senescence and is required for suppression of neoplasia.

    PubMed

    Rai, Taranjit Singh; Cole, John J; Nelson, David M; Dikovskaya, Dina; Faller, William J; Vizioli, Maria Grazia; Hewitt, Rachael N; Anannya, Orchi; McBryan, Tony; Manoharan, Indrani; van Tuyn, John; Morrice, Nicholas; Pchelintsev, Nikolay A; Ivanov, Andre; Brock, Claire; Drotar, Mark E; Nixon, Colin; Clark, William; Sansom, Owen J; Anderson, Kurt I; King, Ayala; Blyth, Karen; Adams, Peter D

    2014-12-15

    Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression. PMID:25512559

  18. Biophysical Regulation of Chromatin Architecture Instills a Mechanical Memory in Mesenchymal Stem Cells

    PubMed Central

    Heo, Su-Jin; Thorpe, Stephen D.; Driscoll, Tristan P.; Duncan, Randall L.; Lee, David A.; Mauck, Robert L.

    2015-01-01

    Mechanical cues direct the lineage commitment of mesenchymal stem cells (MSCs). In this study, we identified the operative molecular mechanisms through which dynamic tensile loading (DL) regulates changes in chromatin organization and nuclear mechanics in MSCs. Our data show that, in the absence of exogenous differentiation factors, short term DL elicits a rapid increase in chromatin condensation, mediated by acto-myosin based cellular contractility and the activity of the histone-lysine N-methyltransferase EZH2. The resulting change in chromatin condensation stiffened the MSC nucleus, making it less deformable when stretch was applied to the cell. We also identified stretch induced ATP release and purinergic calcium signaling as a central mediator of this chromatin condensation process. Further, we showed that DL, through differential stabilization of the condensed chromatin state, established a ‘mechanical memory’ in these cells. That is, increasing strain levels and number of loading events led to a greater degree of chromatin condensation that persisted for longer periods of time after the cessation of loading. These data indicate that, with mechanical perturbation, MSCs develop a mechanical memory encoded in structural changes in the nucleus which may sensitize them to future mechanical loading events and define the trajectory and persistence of their lineage specification. PMID:26592929

  19. HIRA orchestrates a dynamic chromatin landscape in senescence and is required for suppression of neoplasia

    PubMed Central

    Cole, John J.; Nelson, David M.; Dikovskaya, Dina; Faller, William J.; Vizioli, Maria Grazia; Hewitt, Rachael N.; Anannya, Orchi; McBryan, Tony; Manoharan, Indrani; van Tuyn, John; Morrice, Nicholas; Pchelintsev, Nikolay A.; Ivanov, Andre; Brock, Claire; Drotar, Mark E.; Nixon, Colin; Clark, William; Sansom, Owen J.; Anderson, Kurt I.; King, Ayala; Blyth, Karen

    2014-01-01

    Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression. PMID:25512559

  20. Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks.

    PubMed

    Kruhlak, Michael J; Celeste, Arkady; Dellaire, Graham; Fernandez-Capetillo, Oscar; Müller, Waltraud G; McNally, James G; Bazett-Jones, David P; Nussenzweig, André

    2006-03-13

    The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion. The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated. However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30-40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate-dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair. PMID:16520385

  1. MRG15 activates the cdc2 promoter via histone acetylation in human cells

    SciTech Connect

    Pena, AndreAna N.; Tominaga, Kaoru; Pereira-Smith, Olivia M.

    2011-07-01

    Chromatin remodeling is required for transcriptional activation and repression. MRG15 (MORF4L1), a chromatin modulator, is a highly conserved protein and is present in complexes containing histone acetyltransferases (HATs) as well as histone deacetylases (HDACs). Loss of expression of MRG15 in mice and Drosophila results in embryonic lethality and fibroblast and neural stem/progenitor cells cultured from Mrg15 null mouse embryos exhibit marked proliferative defects when compared with wild type cells. To determine the role of MRG15 in cell cycle progression we performed chromatin immunoprecipitation with an antibody to MRG15 on normal human fibroblasts as they entered the cell cycle from a quiescent state, and analyzed various cell cycle gene promoters. The results demonstrated a 3-fold increase in MRG15 occupancy at the cdc2 promoter during S phase of the cell cycle and a concomitant increase in acetylated histone H4. H4 lysine 12 was acetylated at 24 h post-serum stimulation while there was no change in acetylation of lysine 16. HDAC1 and 2 were decreased at this promoter during cell cycle progression. Over-expression of MRG15 in HeLa cells activated a cdc2 promoter-reporter construct in a dose-dependent manner, whereas knockdown of MRG15 resulted in decreased promoter activity. In order to implicate HAT activity, we treated cells with the HAT inhibitor anacardic acid and determined that HAT inhibition results in loss of expression of cdc2 mRNA. Further, chromatin immunoprecipitation with Tip60 localizes the protein to the same 110 bp stretch of the cdc2 promoter pulled down by MRG15. Additionally, we determined that cotransfection of MRG15 with the known associated HAT Tip60 had a cooperative effect in activating the cdc2 promoter. These results suggest that MRG15 is acting in a HAT complex involving Tip60 to modify chromatin via acetylation of histone H4 at the cdc2 promoter to activate transcription.

  2. Nucleosome dynamics during chromatin remodeling in vivo

    PubMed Central

    Ramachandran, Srinivas; Henikoff, Steven

    2016-01-01

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

  3. 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

  4. 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

  5. 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

  6. [Comparative characteristics of chromatin endonuclease fragments].

    PubMed

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

    1977-05-01

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

  7. 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

  8. Linker Histones Incorporation Maintains Chromatin Fiber Plasticity

    PubMed Central

    Recouvreux, Pierre; Lavelle, Christophe; Barbi, Maria; Conde e Silva, Natalia; Le Cam, Eric; Victor, Jean-Marc; Viovy, Jean-Louis

    2011-01-01

    Genomic DNA in eukaryotic cells is organized in supercoiled chromatin fibers, which undergo dynamic changes during such DNA metabolic processes as transcription or replication. Indeed, DNA-translocating enzymes like polymerases produce physical constraints in vivo. We used single-molecule micromanipulation by magnetic tweezers to study the response of chromatin to mechanical constraints in the same range as those encountered in vivo. We had previously shown that under positive torsional constraints, nucleosomes can undergo a reversible chiral transition toward a state of positive topology. We demonstrate here that chromatin fibers comprising linker histones present a torsional plasticity similar to that of naked nucleosome arrays. Chromatosomes can undergo a reversible chiral transition toward a state of positive torsion (reverse chromatosome) without loss of linker histones. PMID:21641318

  9. 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. PMID:26933790

  10. GW8510 Increases Insulin Expression in Pancreatic Alpha Cells through Activation of p53 Transcriptional Activity

    PubMed Central

    Fomina-Yadlin, Dina; Kubicek, Stefan; Vetere, Amedeo; He, Kaihui Hu; Schreiber, Stuart L.; Wagner, Bridget K.

    2012-01-01

    Background Expression of insulin in terminally differentiated non-beta cell types in the pancreas could be important to treating type-1 diabetes. Previous findings led us to hypothesize involvement of kinase inhibition in induction of insulin expression in pancreatic alpha cells. Methodology/Principal Findings Alpha (αTC1.6) cells and human islets were treated with GW8510 and other small-molecule inhibitors for up to 5 days. Alpha cells were assessed for gene- and protein-expression levels, cell-cycle status, promoter occupancy status by chromatin immunoprecipitation (ChIP), and p53-dependent transcriptional activity. GW8510, a putative CDK2 inhibitor, up-regulated insulin expression in mouse alpha cells and enhanced insulin secretion in dissociated human islets. Gene-expression profiling and gene-set enrichment analysis of GW8510-treated alpha cells suggested up-regulation of the p53 pathway. Accordingly, the compound increased p53 transcriptional activity and expression levels of p53 transcriptional targets. A predicted p53 response element in the promoter region of the mouse Ins2 gene was verified by chromatin immunoprecipitation (ChIP). Further, inhibition of Jun N-terminal kinase (JNK) and p38 kinase activities suppressed insulin induction by GW8510. Conclusions/Significance The induction of Ins2 by GW8510 occurred through p53 in a JNK- and p38-dependent manner. These results implicate p53 activity in modulation of Ins2 expression levels in pancreatic alpha cells, and point to a potential approach toward using small molecules to generate insulin in an alternative cell type. PMID:22242153

  11. Transcriptome regulation and chromatin occupancy by E2F3 and MYC in mice

    PubMed Central

    Tang, Xing; Liu, Huayang; Srivastava, Arunima; Pécot, Thierry; Chen, Zhong; Wang, Qianben; Huang, Kun; Sáenz-Robles, Maria Teresa; Cantalupo, Paul; Pipas, James; Leone, Gustavo

    2016-01-01

    E2F3 and MYC are transcription factors that control cellular proliferation. To study their mechanism of action in the context of a regenerating tissue, we isolated both proliferating (crypts) and non-dividing (villi) cells from wild-type and Rb depleted small intestines of mice and performed ChIP-exo-seq (chromatin immunoprecipitation combined with lambda exonuclease digestion followed by high-throughput sequencing). The genome-wide chromatin occupancy of E2F3 and MYC was determined by mapping sequence reads to the genome and predicting preferred binding sites (peaks). Binding sites could be accurately identified within small regions of only 24 bp-28 bp long, highlighting the precision to which binding peaks can be identified by ChIP-exo-seq. Forty randomly selected E2F3- and MYC-specific binding sites were validated by ChIP-PCR. In addition, we also presented gene expression data sets from wild type, Rb-, E2f3- and Myc-depleted crypts and villi within this manuscript. These represent comprehensive and validated datasets that can be integrated to identify putative direct targets of E2F3 and MYC involved in the control of cellular proliferation in normal and Rb-deficient small intestines. PMID:26881867

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-24

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

  14. Affinity purification of specific chromatin segments from chromosomal loci in yeast.

    PubMed

    Griesenbeck, Joachim; Boeger, Hinrich; Strattan, J Seth; Kornberg, Roger D

    2003-12-01

    Single-copy gene and promoter regions have been excised from yeast chromosomes and have been purified as chromatin by conventional and affinity methods. Promoter regions isolated in transcriptionally repressed and activated states maintain their characteristic chromatin structures. Gel filtration analysis establishes the uniformity of the transcriptionally activated state. Activator proteins interact in the manner anticipated from previous studies in vivo. This work opens the way to the direct study of specific gene regions of eukaryotic chromosomes in diverse functional and structural states. PMID:14645537

  15. 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

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

    PubMed Central

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

    2007-01-01

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

  17. Identification and validation of protein-protein interactions by combining co-immunoprecipitation, antigen competition, and stable isotope labeling.

    PubMed

    Sommer, Frederik; Mühlhaus, Timo; Hemme, Dorothea; Veyel, Daniel; Schroda, Michael

    2014-01-01

    Co-immunoprecipitation (coIP) in combination with mass spectrometry (MS) is a powerful tool to identify potential protein-protein interactions. However, unspecifically precipitated proteins usually result in large numbers of false-positive identifications. Here we describe a detailed protocol particularly useful in plant sciences that is based on (15)N stable isotope labeling of cells, (14)N antigen titration, and coIP/MS to distinguish true from false protein-protein interactions. PMID:25059616

  18. CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation

    PubMed Central

    Narendra, Varun; Rocha, Pedro P.; An, Disi; Raviram, Ramya; Skok, Jane A.; Mazzoni, Esteban O.; Reinberg, Danny

    2015-01-01

    Polycomb and trithorax group proteins encode the epigenetic memory of cellular positional identity by establishing inheritable domains of repressive and active chromatin within the Hox clusters. Here, we demonstrate that the CCCTC-binding factor (CTCF) functions to insulate these adjacent yet antagonistic chromatin domains during embryonic stem cell differentiation into cervical motor neurons. Deletion of CTCF binding sites within the Hox clusters results in the expansion of active chromatin into the repressive domain. CTCF functions as an insulator by organizing Hox clusters into spatially disjoint domains. Ablation of CTCF binding disrupts topological boundaries such that caudal Hox genes leave the repressed domain and become subject to transcriptional activation. Hence, CTCF is required to insulate facultative heterochromatin from impinging euchromatin to produce discrete positional identities. PMID:25722416

  19. CDK2-dependent activation of PARP-1 is required for hormonal gene regulation in breast cancer cells

    PubMed Central

    Wright, Roni H.G.; Castellano, Giancarlo; Bonet, Jaume; Le Dily, Francois; Font-Mateu, Jofre; Ballaré, Cecilia; Nacht, A. Silvina; Soronellas, Daniel; Oliva, Baldo; Beato, Miguel

    2012-01-01

    Eukaryotic gene regulation implies that transcription factors gain access to genomic information via poorly understood processes involving activation and targeting of kinases, histone-modifying enzymes, and chromatin remodelers to chromatin. Here we report that progestin gene regulation in breast cancer cells requires a rapid and transient increase in poly-(ADP)-ribose (PAR), accompanied by a dramatic decrease of cellular NAD that could have broad implications in cell physiology. This rapid increase in nuclear PARylation is mediated by activation of PAR polymerase PARP-1 as a result of phosphorylation by cyclin-dependent kinase CDK2. Hormone-dependent phosphorylation of PARP-1 by CDK2, within the catalytic domain, enhances its enzymatic capabilities. Activated PARP-1 contributes to the displacement of histone H1 and is essential for regulation of the majority of hormone-responsive genes and for the effect of progestins on cell cycle progression. Both global chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) and gene expression analysis show a strong overlap between PARP-1 and CDK2. Thus, progestin gene regulation involves a novel signaling pathway that connects CDK2-dependent activation of PARP-1 with histone H1 displacement. Given the multiplicity of PARP targets, this new pathway could be used for the pharmacological management of breast cancer. PMID:22948662

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

    PubMed Central

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

    2016-01-01

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

  1. Chromatin Compaction Protects Genomic DNA from Radiation Damage

    PubMed Central

    Takata, Hideaki; Hanafusa, Tomo; Mori, Toshiaki; Shimura, Mari; Iida, Yutaka; Ishikawa, Kenichi; Yoshikawa, Kenichi; Yoshikawa, Yuko; Maeshima, Kazuhiro

    2013-01-01

    Genomic DNA is organized three-dimensionally in the nucleus, and is thought to form compact chromatin domains. Although chromatin compaction is known to be essential for mitosis, whether it confers other advantages, particularly in interphase cells, remains unknown. Here, we report that chromatin compaction protects genomic DNA from radiation damage. Using a newly developed solid-phase system, we found that the frequency of double-strand breaks (DSBs) in compact chromatin after ionizing irradiation was 5–50-fold lower than in decondensed chromatin. Since radical scavengers inhibited DSB induction in decondensed chromatin, condensed chromatin had a lower level of reactive radical generation after ionizing irradiation. We also found that chromatin compaction protects DNA from attack by chemical agents. Our findings suggest that genomic DNA compaction plays an important role in maintaining genomic integrity. PMID:24130727

  2. Chromatin higher-order structures and gene regulation

    PubMed Central

    Li, Guohong

    2011-01-01

    Genomic DNA in the eukaryotic nucleus is hierarchically packaged by histones into chromatin to fit inside the nucleus. The dynamics of higher-order chromatin compaction play a critical role in transcription and other biological processes inherent to DNA. Many factors, including histone variants, histone modifications, DNA methylation and the binding of non-histone architectural proteins regulate the structure of chromatin. Although the structure of nucleosomes, the fundamental repeating unit of chromatin, is clear, there is still much discussion on the higher-order levels of chromatin structure. In this review, we focus on the recent progress in elucidating the structure of the 30-nm chromatin fiber. We also discuss the structural plasticity/dynamics and epigenetic inheritance of higher-order chromatin and the roles of chromatin higher-order organization in eukaryotic gene regulation. PMID:21342762

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

    PubMed

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

    2015-07-01

    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. 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.

  5. Chromosomes without a 30-nm chromatin fiber

    PubMed Central

    Joti, Yasumasa; Hikima, Takaaki; Nishino, Yoshinori; Kamada, Fukumi; Hihara, Saera; Takata, Hideaki; Ishikawa, Tetsuya; Maeshima, Kazuhiro

    2012-01-01

    How is a long strand of genomic DNA packaged into a mitotic chromosome or nucleus? The nucleosome fiber (beads-on-a-string), in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fiber, and a further helically folded larger fiber. However, when frozen hydrated human mitotic cells were observed using cryoelectron microscopy, no higher-order structures that included 30-nm chromatin fibers were found. To investigate the bulk structure of mitotic chromosomes further, we performed small-angle X-ray scattering (SAXS), which can detect periodic structures in noncrystalline materials in solution. The results were striking: no structural feature larger than 11 nm was detected, even at a chromosome-diameter scale (~1 μm). We also found a similar scattering pattern in interphase nuclei of HeLa cells in the range up to ~275 nm. Our findings suggest a common structural feature in interphase and mitotic chromatins: compact and irregular folding of nucleosome fibers occurs without a 30-nm chromatin structure. PMID:22825571

  6. Chromatin-modifying and -remodeling complexes.

    PubMed

    Kornberg, R D; Lorch, Y

    1999-04-01

    Nucleosomes have long been known to inhibit DNA transactions on chromosomes and a remarkable abundance of multiprotein complexes that either enhance or relieve this inhibition have been described. Most is known about chromatin-remodeling complexes that perturb nucleosome structure. PMID:10322131

  7. Monte Carlo simulation of chromatin stretching.

    PubMed

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

    2006-04-01

    We present Monte Carlo (MC) simulations of the stretching of a single 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 degrees to 130 degrees increases the stiffness significantly. An increase in the opening angle from 22 degrees to 34 degrees 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. PMID:16711856

  8. 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.

  9. Chromatin organization of gammaherpesvirus latent genomes.

    PubMed

    Tempera, Italo; Lieberman, Paul M

    2010-01-01

    The gammaherpesviruses are a subclass of the herpesvirus family that establish stable latent infections in proliferating lymphoid and epithelial cells. The latent genomes are maintained as multicopy chromatinized episomes that replicate in synchrony with the cellular genome. Importantly, most of the episomes do not integrate into the host chromosome. Therefore, it is essential that the viral "minichromosome" establish a chromatin structure that is suitable for gene expression, DNA replication, and chromosome segregation. Evidence suggests that chromatin organization is important for each of these functions and plays a regulatory role in the establishment and maintenance of latent infection. Here, we review recent studies on the chromatin organization of the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). We discuss the potential role of viral origins of DNA replication and viral encoded origin-binding proteins like EBNA1 and LANA in establishment of viral chromosome organization during latent infection. We also discuss the roles of host cell factors, like CTCF and cohesins, that contribute to higher-order chromosome structures that may be important for stable gene expression programs during latent infection in proliferating cells. PMID:19853673

  10. 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.

  11. Histone octamer transfer by a chromatin-remodeling complex.

    PubMed

    Lorch, Y; Zhang, M; Kornberg, R D

    1999-02-01

    RSC, an abundant, essential chromatin-remodeling complex related to SWI/SNF complex, catalyzes the transfer of a histone octamer from a nucleosome core particle to naked DNA. The newly formed octamer-DNA complex is identical with a nucleosome in all respects. The reaction requires ATP and involves an activated RSC-nucleosome intermediate. The mechanism may entail formation of a duplex displacement loop on the nucleosome, facilitating the entry of exogeneous DNA and the release of the endogenous molecule. PMID:10025404

  12. Xenobiotic- and vitamin D-responsive induction of the steroid/bile acid-sulfotransferase Sult2A1 in young and old mice: the role of a gene enhancer in the liver chromatin.

    PubMed

    Seo, Young-Kyo; Chung, Yoon-Tae; Kim, Soyoung; Echchgadda, Ibtissam; Song, Chung S; Chatterjee, Bandana

    2007-01-15

    The xenobiotic-activated nuclear receptors PXR (pregnane X receptor) and CAR (constitutive androstane receptor) and the vitamin D(3)-activated nuclear receptor VDR regulate steroid and xenobiotic metabolism by inducing the phase I cytochrome P450 monooxygenases, phase II conjugating transferases, and the phase III transporters, which mediate the efflux of water-soluble lipid metabolites from cells. Metabolic stress due to the deviant expression of steroid- and xenobiotic-metabolizing enzymes is known to have severe health consequences including accelerated aging, and increased expression of these enzymes is associated with extended longevity [Gachon, F, Olela, FF, Schaad, O, Descombes, P and Schibler, U, 2006. The circadian PAR-domain basic leucine zipper transcription factors DBP, TEF, and HLF modulate basal and inducible xenobiotic detoxification. 4, 25-36.; McElwee, JJ, Schuster, E, Blanc, E, Thomas, JH and Gems, D, 2004. Shared Transcriptional Signature in Caenorhabditis elegans Dauer Larvae and Long-lived daf-2 Mutants Implicates Detoxification System in Longevity Assurance. J. Biol. Chem., 279, 44533-43.]. Information on the similarities and dissimilarities in drug metabolism between the young and old, as may be uncovered by studying aging regulation of the genes relevant to steroid and xenobiotic metabolism, is likely to have clinical significance. In this report, we examined the VDR- and PXR-mediated gene induction of the phase II sulfotransferase Sult2A1 in the livers of 4-month- and 20-month-old mice. Sult2A1 converts bile acids, steroids and a number of drugs to the corresponding sulfated metabolites, which are readily eliminated from the body due to increased water solubility. In RT-PCR assay, aging did not change the induction of Sult2A1 mRNAs by the hormonally active vitamin D(3) and the catatoxic synthetic steroid PCN (pregnenolone-16alpha-carbonitrile). Chromatin immunoprecipitation (ChIP) from liver nuclei showed that aging had no effect on the

  13. Inhibition of chromatin remodeling by Polycomb Group protein Posterior Sex Combs is mechanistically distinct from nucleosome binding1

    PubMed Central

    Lo, Stanley M.; Francis, Nicole J.

    2010-01-01

    Polycomb Group (PcG) proteins are essential regulators of development that maintain gene silencing in Drosophila and mammals through alterations of chromatin structure. One key PcG protein, Posterior Sex Combs (PSC), is part of at least two complexes: Polycomb Repressive Complex 1 (PRC1) and dRING Associated Factors (dRAF). PRC1-class complexes compact chromatin and inhibit chromatin remodeling, while dRAF has E3 ligase activity for ubiquitylation of histone H2A; activities of both complexes can inhibit transcription. The noncovalent effects of PRC1-class complexes on chromatin can be recapitulated by PSC alone, and the region of PSC required for these activities is essential for PSC function in vivo. To understand how PSC interacts with chromatin to exert its repressive effects, we compared the ability of PSC to bind to and inhibit remodeling of various nucleosomal templates, and determined which regions of PSC are required for mononucleosome binding and inhibition of chromatin remodeling. We find that PSC binds mononucleosome templates but inhibits their remodeling poorly. Addition of linker DNA to mononucleosomes allows their remodeling to be inhibited, although higher concentrations of PSC are required than for inhibition of multi-nucleosome templates. The C-terminal region of PSC (aa 456-1603) is important for inhibition of chromatin remodeling, and we identified aa 456-909 as sufficient for stable nucleosome binding but not for inhibition of chromatin remodeling. Our data suggest distinct mechanistic steps between nucleosome binding and inhibition of chromatin remodeling. PMID:20873869

  14. Sequence Features and Transcriptional Stalling within Centromere DNA Promote Establishment of CENP-A Chromatin

    PubMed Central

    Catania, Sandra; Pidoux, Alison L.; Allshire, Robin C.

    2015-01-01

    Centromere sequences are not conserved between species, and there is compelling evidence for epigenetic regulation of centromere identity, with location being dictated by the presence of chromatin containing the histone H3 variant CENP-A. Paradoxically, in most organisms CENP-A chromatin generally occurs on particular sequences. To investigate the contribution of primary DNA sequence to establishment of CENP-A chromatin in vivo, we utilised the fission yeast Schizosaccharomyces pombe. CENP-ACnp1 chromatin is normally assembled on ∼10 kb of central domain DNA within these regional centromeres. We demonstrate that overproduction of S. pombe CENP-ACnp1 bypasses the usual requirement for adjacent heterochromatin in establishing CENP-ACnp1 chromatin, and show that central domain DNA is a preferred substrate for de novo establishment of CENP-ACnp1 chromatin. When multimerised, a 2 kb sub-region can establish CENP-ACnp1 chromatin and form functional centromeres. Randomization of the 2 kb sequence to generate a sequence that maintains AT content and predicted nucleosome positioning is unable to establish CENP-ACnp1 chromatin. These analyses indicate that central domain DNA from fission yeast centromeres contains specific information that promotes CENP-ACnp1 incorporation into chromatin. Numerous transcriptional start sites were detected on the forward and reverse strands within the functional 2 kb sub-region and active promoters were identified. RNAPII is enriched on central domain DNA in wild-type cells, but only low levels of transcripts are detected, consistent with RNAPII stalling during transcription of centromeric DNA. Cells lacking factors involved in restarting transcription—TFIIS and Ubp3—assemble CENP-ACnp1 on central domain DNA when CENP-ACnp1 is at wild-type levels, suggesting that persistent stalling of RNAPII on centromere DNA triggers chromatin remodelling events that deposit CENP-ACnp1. Thus, sequence-encoded features of centromeric DNA create an

  15. Inferential modeling of 3D chromatin structure

    PubMed Central

    Wang, Siyu; Xu, Jinbo; Zeng, Jianyang

    2015-01-01

    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. PMID:25690896

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

    PubMed Central

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

    2011-01-01

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

  17. Discovery and characterization of chromatin states for systematic annotation of the human genome

    PubMed Central

    Ernst, Jason; Kellis, Manolis

    2010-01-01

    A plethora of epigenetic modifications have been described in the human genome and shown to play diverse roles in gene regulation, cellular differentiation, and the onset of disease. While some modifications have been linked with activity levels of different functional elements, their combinatorial patterns remain unresolved, and their potential for systematic de novo genome annotation remains untapped. In this paper, we systematically discover and characterize recurrent spatially-coherent and biologically-meaningful chromatin mark combinations, or chromatin states, in human T-cells. We describe 51 distinct chromatin states, including promoter-associated, transcription-associated, active intergenic, large-scale repressed and repeat-associated states. Each chromatin state shows specific functional, experimental, conservation, annotation, and sequence-motif enrichments, revealing their distinct candidate biological roles. Overall, our work provides a complementary functional annotation of the human genome revealing the genome-wide locations of diverse classes of epigenetic functions, including previously-unsuspected chromatin states enriched in transcription end sites, distinct repeat families, and disease-SNP-associated states. PMID:20657582

  18. Akirin Links Twist-Regulated Transcription with the Brahma Chromatin Remodeling Complex during Embryogenesis

    PubMed Central

    Nowak, Scott J.; Aihara, Hitoshi; Gonzalez, Katie; Nibu, Yutaka; Baylies, Mary K.

    2012-01-01

    The activities of developmentally critical transcription factors are regulated via interactions with cofactors. Such interactions influence transcription factor activity either directly through protein–protein interactions or indirectly by altering the local chromatin environment. Using a yeast double-interaction screen, we identified a highly conserved nuclear protein, Akirin, as a novel cofactor of the key Drosophila melanogaster mesoderm and muscle transcription factor Twist. We find that Akirin interacts genetically and physically with Twist to facilitate expression of some, but not all, Twist-regulated genes during embryonic myogenesis. akirin mutant embryos have muscle defects consistent with altered regulation of a subset of Twist-regulated genes. To regulate transcription, Akirin colocalizes and genetically interacts with subunits of the Brahma SWI/SNF-class chromatin remodeling complex. Our results suggest that, mechanistically, Akirin mediates a novel connection between Twist and a chromatin remodeling complex to facilitate changes in the chromatin environment, leading to the optimal expression of some Twist-regulated genes during Drosophila myogenesis. We propose that this Akirin-mediated link between transcription factors and the Brahma complex represents a novel paradigm for providing tissue and target specificity for transcription factor interactions with the chromatin remodeling machinery. PMID:22396663

  19. Akirin links twist-regulated transcription with the Brahma chromatin remodeling complex during embryogenesis.

    PubMed

    Nowak, Scott J; Aihara, Hitoshi; Gonzalez, Katie; Nibu, Yutaka; Baylies, Mary K

    2012-01-01

    The activities of developmentally critical transcription factors are regulated via interactions with cofactors. Such interactions influence transcription factor activity either directly through protein-protein interactions or indirectly by altering the local chromatin environment. Using a yeast double-interaction screen, we identified a highly conserved nuclear protein, Akirin, as a novel cofactor of the key Drosophila melanogaster mesoderm and muscle transcription factor Twist. We find that Akirin interacts genetically and physically with Twist to facilitate expression of some, but not all, Twist-regulated genes during embryonic myogenesis. akirin mutant embryos have muscle defects consistent with altered regulation of a subset of Twist-regulated genes. To regulate transcription, Akirin colocalizes and genetically interacts with subunits of the Brahma SWI/SNF-class chromatin remodeling complex. Our results suggest that, mechanistically, Akirin mediates a novel connection between Twist and a chromatin remodeling complex to facilitate changes in the chromatin environment, leading to the optimal expression of some Twist-regulated genes during Drosophila myogenesis. We propose that this Akirin-mediated link between transcription factors and the Brahma complex represents a novel paradigm for providing tissue and target specificity for transcription factor interactions with the chromatin remodeling machinery. PMID:22396663

  20. Systematic dissection of roles for chromatin regulators in a yeast stress response.

    PubMed

    Weiner, Assaf; Chen, Hsiuyi V; Liu, Chih Long; Rahat, Ayelet; Klien, Avital; Soares, Luis; Gudipati, Mohanram; Pfeffner, Jenna; Regev, Aviv; Buratowski, Stephen; Pleiss, Jeffrey A; Friedman, Nir; Rando, Oliver J

    2012-01-01

    Packaging of eukaryotic genomes into chromatin has wide-ranging effects on gene transcription. Curiously, it is commonly observed that deletion of a global chromatin regulator affects expression of only a limited subset of genes bound to or modified by the regulator in question. However, in many single-gene studies it has become clear that chromatin regulators often do not affect steady-state transcription, but instead are required for normal transcriptional reprogramming by environmental cues. We therefore have systematically investigated the effects of 83 histone mutants, and 119 gene deletion mutants, on induction/repression dynamics of 170 transcripts in response to diamide stress in yeast. Importantly, we find that chromatin regulators play far more pronounced roles during gene induction/repression than they do in steady-state expression. Furthermore, by jointly analyzing the substrates (histone mutants) and enzymes (chromatin modifier deletions) we identify specific interactions between histone modifications and their regulators. Combining these functional results with genome-wide mapping of several histone marks in the same time course, we systematically investigated the correspondence between histone modification occurrence and function. We followed up on one pathway, finding that Set1-dependent H3K4 methylation primarily acts as a gene repressor during multiple stresses, specifically at genes involved in ribosome biosynthesis. Set1-dependent repression of ribosomal genes occurs via distinct pathways for ribosomal protein genes and ribosomal biogenesis genes, which can be separated based on genetic requirements for repression and based on chromatin changes during gene repression. Together, our dynamic studies provide a rich resource for investigating chromatin regulation, and identify a significant role for the "activating" mark H3K4me3 in gene repression. PMID:22912562

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-08-26

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

  3. Chromatin remodeling by curcumin alters endogenous aryl hydrocarbon receptor signaling.

    PubMed

    Mohammadi-Bardbori, Afshin; Akbarizadeh, Amin Reza; Delju, Fatemeh; Rannug, Agneta

    2016-05-25

    The aim of this study was to gain more information about the mechanisms that regulate expression of the aryl hydrocarbon receptor (AHR) target gene CYP1A1. Human hepatoma cells (HepG2 and Huh7) and human immortalized keratinocytes (HaCaT) were treated with different concentrations of the dietary polyphenolic compound curcumin (CUR) alone or in combination with the natural AHR agonist 6-formylindolo[3,2-b]carbazole (FICZ). In an earlier study, we described that CUR can activate the AHR indirectly by inhibiting metabolic clearance of FICZ. Here, we measured cell viability, activation of AHR signaling, oxidative stress and histone modifying activities in response to CUR at concentrations ranging from 0.1 to 50 μM. We observed apparent non-linear responses on cell viability and activation of AHR signaling. The CYP1A1 expression and the CYP1A1 enzyme activity in the presence of CUR reflected the histone acetylation efficiency observed in nuclear extracts. At the lowest concentration, CUR significantly decreased histone deacetylase activity and increased the FICZ-induced CYP1A1 activity. In contrast, at the highest concentration, CUR increased the formation of reactive oxygen species, significantly inhibited histone acetylation, and temporally decreased FICZ-induced CYP1A1 activity. The results suggest that CUR can both increase and decrease the accessibility of DNA and thereby influence transcriptional responses to the ligand-activated AHR. This suggestion was supported by the fact that chromatin remodeling treatments with trichostatin A, p300, or 5-aza-dC increased CYP1A1 transcription. We conclude that the AHR-dependent transcriptional efficiency is modified by factors that influence the cellular redox status and the chromatin structure. PMID:27041069

  4. Analysis of Mcm2-7 chromatin binding during anaphase and in the transition to quiescence in fission yeast

    SciTech Connect

    Namdar, Mandana; Kearsey, Stephen E. . E-mail: stephen.kearsey@zoo.ox.ac.uk

    2006-10-15

    Mcm2-7 proteins are generally considered to function as a heterohexameric complex, providing helicase activity for the elongation step of DNA replication. These proteins are loaded onto replication origins in M-G1 phase in a process termed licensing or pre-replicative complex formation. It is likely that Mcm2-7 proteins are loaded onto chromatin simultaneously as a pre-formed hexamer although some studies suggest that subcomplexes are recruited sequentially. To analyze this process in fission yeast, we have compared the levels and chromatin binding of Mcm2-7 proteins during the fission yeast cell cycle. Mcm subunits are present at approximately 1 x 10{sup 4} molecules/cell and are bound with approximately equal stoichiometry on chromatin in G1/S phase cells. Using a single cell assay, we have correlated the timing of chromatin association of individual Mcm subunits with progression through mitosis. This showed that Mcm2, 4 and 7 associate with chromatin at about the same stage of anaphase, suggesting that licensing involves the simultaneous binding of these subunits. We also examined Mcm2-7 chromatin association when cells enter a G0-like quiescent state. Chromatin binding is lost in this transition in a process that does not require DNA replication or the selective degradation of specific subunits.

  5. Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure.

    PubMed

    Becker, Annette; Zhang, Peng; Allmann, Lena; Meilinger, Daniela; Bertulat, Bianca; Eck, Daniel; Hofstaetter, Maria; Bartolomei, Giody; Hottiger, Michael O; Schreiber, Valérie; Leonhardt, Heinrich; Cardoso, M Cristina

    2016-03-01

    The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1(-/-) compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function. PMID:26772194

  6. Targeted Histone Peptides: Insights into the Spatial Regulation of the Methyltransferase PRC2 using a Surrogate of Heterotypic Chromatin***

    PubMed Central

    Brown, Zachary Z.; Müller, Manuel M.; Kong, Ha Eun; Lewis, Peter W.

    2015-01-01

    Eukaryotic genomes are dynamically regulated through a host of epigenetic stimuli. The substrate for these epigenetic transactions, chromatin, is a polymer of nucleosome building blocks. In native (i.e. cellular) chromatin, each nucleosome can differ from its neighbors through the localized installation of covalent modifications to both the genomic DNA and the histone packaging proteins. The heterotypic nature of chromatin presents a formidable obstacle to biochemical studies seeking to understand the role of context on epigenetic regulation and that, as a consequence, must employ compositionally defined chromatin substrates. Here, we introduce a chemical approach to the production of heterotypic ‘designer’ chromatin that can be used in such studies. Our method involves attachment of a user-defined modified histone peptide to a designated nucleosome within the polymer by using a Peptide Nucleic Acid (PNA) targeting compound. We apply this strategy to dissect the role of chromatin context on both the activation and inhibition of the histone methyltransferase, PRC2, which methylates Lys 27 of histone H3 (H3K27). Our studies show that PRC2 can be stimulated to produce de novo H3K27 methylation from a defined nucleation site. More generally, this technology promises to facilitate biochemical studies that require the use of heterotypic chromatin substrates. PMID:25873363

  7. VRK1 chromatin kinase phosphorylates H2AX and is required for foci formation induced by DNA damage.

    PubMed

    Salzano, Marcella; Sanz-García, Marta; Monsalve, Diana M; Moura, David S; Lazo, Pedro A

    2015-01-01

    All types of DNA damage cause a local alteration and relaxation of chromatin structure. Sensing and reacting to this initial chromatin alteration is a necessary trigger for any type of DNA damage response (DDR). In this context, chromatin kinases are likely candidates to participate in detection and reaction to a locally altered chromatin as a consequence of DNA damage and, thus, initiate the appropriate cellular response. In this work, we demonstrate that VRK1 is a nucleosomal chromatin kinase and that its depletion causes loss of histones H3 and H4 acetylation, which are required for chromatin relaxation, both in basal conditions and after DNA damage, independently of ATM. Moreover, VRK1 directly and stably interacts with histones H2AX and H3 in basal conditions. In response to DNA damage induced by ionizing radiation, histone H2AX is phosphorylated in Ser139 by VRK1. The phosphorylation of H2AX and the formation of γH2AX foci induced by ionizing radiation (IR), are prevented by VRK1 depletion and are rescued by kinase-active, but not kinase-dead, VRK1. In conclusion, we found that VRK1 is a novel chromatin component that reacts to its alterations and participates very early in DDR, functioning by itself or in cooperation with ATM. PMID:25923214

  8. Mechanisms of ATP-Dependent Chromatin Remodeling Motors.

    PubMed

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

    2016-07-01

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

  9. Histone acetylation: a switch between repressive and permissive chromatin

    PubMed Central

    Eberharter, Anton; Becker, Peter B.

    2002-01-01

    The organization of eukaryotic chromatin has a major impact on all nuclear processes involving DNA substrates. Gene expression is affected by the positioning of individual nucleosomes relative to regulatory sequence elements, by the folding of the nucleosomal fiber into higher-order structures and by the compartmentalization of functional domains within the nucleus. Because site-specific acetylation of nucleosomal histones influences all three aspects of chromatin organization, it is central to the switch between permissive and repressive chromatin structure. The targeting of enzymes that modulate the histone acetylation status of chromatin, in synergy with the effects mediated by other chromatin remodeling factors, is central to gene regulation. PMID:11882541

  10. ChromoShake: a chromosome dynamics simulator reveals that chromatin loops stiffen centromeric chromatin

    PubMed Central

    Lawrimore, Josh; Aicher, Joseph K.; Hahn, Patrick; Fulp, Alyona; Kompa, Ben; Vicci, Leandra; Falvo, Michael; Taylor, Russell M.; Bloom, Kerry

    2016-01-01

    ChromoShake is a three-dimensional simulator designed to find the thermodynamically favored states for given chromosome geometries. The simulator has been applied to a geometric model based on experimentally determined positions and fluctuations of DNA and the distribution of cohesin and condensin in the budding yeast centromere. Simulations of chromatin in differing initial configurations reveal novel principles for understanding the structure and function of a eukaryotic centromere. The entropic position of DNA loops mirrors their experimental position, consistent with their radial displacement from the spindle axis. The barrel-like distribution of cohesin complexes surrounding the central spindle in metaphase is a consequence of the size of the DNA loops within the pericentromere to which cohesin is bound. Linkage between DNA loops of different centromeres is requisite to recapitulate experimentally determined correlations in DNA motion. The consequences of radial loops and cohesin and condensin binding are to stiffen the DNA along the spindle axis, imparting an active function to the centromere in mitosis. PMID:26538024

  11. Promoter of CaZF, a Chickpea Gene That Positively Regulates Growth and Stress Tolerance, Is Activated by an AP2-Family Transcription Factor CAP2

    PubMed Central

    Jain, Deepti; Chattopadhyay, Debasis

    2013-01-01

    Plants respond to different forms of stresses by inducing transcription of a common and distinct set of genes by concerted actions of a cascade of transcription regulators. We previously reported that a gene, CaZF encoding a C2H2-zinc finger family protein from chickpea (Cicer arietinum) imparted high salinity tolerance when expressed in tobacco plants. We report here that in addition to promoting tolerance against dehydration, salinity and high temperature, the CaZF overexpressing plants exhibited similar phenotype of growth and development like the plants overexpressing CAP2, encoding an AP2-family transcription factor from chickpea. To investigate any relationship between these two genes, we performed gene expression analysis in the overexpressing plants, promoter-reporter analysis and chromatin immunoprecipitation. A number of transcripts that exhibited enhanced accumulation upon expression of CAP2 or CaZF in tobacco plants were found common. Transient expression of CAP2 in chickpea leaves resulted in increased accumulation of CaZF transcript. Gel mobility shift and transient promoter-reporter assays suggested that CAP2 activates CaZF promoter by interacting with C-repeat elements (CRTs) in CaZF promoter. Chromatin immunoprecipitation (ChIP) assay demonstrated an in vivo interaction of CAP2 protein with CaZF promoter. PMID:23418595

  12. Kinase-mediated changes in nucleosome conformation trigger chromatin decondensation via poly-ADP-ribosylation

    PubMed Central

    Thomas, Colin J.; Kotova, Elena; Andrake, Mark; Adolf-Bryfogle, Jared; Glaser, Robert; Regnard, Catherine; Tulin, Alexei V.

    2014-01-01

    SUMMARY Dynamically controlled post-translational modifications of nucleosomal histones alter chromatin condensation to regulate transcriptional activation. We report that a nuclear tandem kinase, JIL-1, controls gene expression by activating Poly(ADP-ribose) Polymerase 1 (PARP-1). JIL-1 phosphorylates the C-terminus of the H2Av histone variant, which stimulates PARP-1 enzymatic activity in the surrounding chromatin, leading to further modification of histones and chromatin loosening. The H2Av nucleosome has a higher surface representation of PARP-1 binding patch consisting of H3 and H4 epitopes. Phosphorylation of H2Av by JIL-1 restructures this surface patch leading to activation of PARP-1. Exposure of Val61 and Leu23 of the H4 histone is critical for PARP-1 binding on nucleosome and PARP-1 activation following H2Av phosphorylation. We propose that chromatin loosening and associated initiation of gene expression is activated by phosphorylation of H2Av in a nucleosome positioned in promoter regions of PARP-1 dependent genes. PMID:24508391

  13. Chromatin signature discovery via histone modification profile alignments

    PubMed Central

    Wang, Jianrong; Lunyak, Victoria V.; Jordan, I. King

    2012-01-01

    We report on the development of an unsupervised algorithm for the genome-wide discovery and analysis of chromatin signatures. Our Chromatin-profile Alignment followed by Tree-clustering algorithm (ChAT) employs dynamic programming of combinatorial histone modification profiles to identify locally similar chromatin sub-regions and provides complementary utility with respect to existing methods. We applied ChAT to genomic maps of 39 histone modifications in human CD4+ T cells to identify both known and novel chromatin signatures. ChAT was able to detect chromatin signatures previously associated with transcription start sites and enhancers as well as novel signatures associated with a variety of regulatory elements. Promoter-associated signatures discovered with ChAT indicate that complex chromatin signatures, made up of numerous co-located histone modifications, facilitate cell-type specific gene expression. The discovery of novel L1 retrotransposon-associated bivalent chromatin signatures suggests that these elements influence the mono-allelic expression of human genes by shaping the chromatin environment of imprinted genomic regions. Analysis of long gene-associated chromatin signatures point to a role for the H4K20me1 and H3K79me3 histone modifications in transcriptional pause release. The novel chromatin signatures and functional associations uncovered by ChAT underscore the ability of the algorithm to yield novel insight on chromatin-based regulatory mechanisms. PMID:22989711

  14. Transcription in the maintenance of centromere chromatin identity

    PubMed Central

    Chan, F. Lyn; Wong, Lee H.

    2012-01-01

    Recent evidence has shown that transcription is permissible through the purportedly repressive centromere domain, and that this transcriptional activity is of functional consequence. The best-studied example is transcription of the pericentric DNA repeats in the generation of siRNAs required for pericentric heterochromatin assembly in yeast. However, non-siRNA transcripts emanating from both pericentric and centromere core domains have also been detected in a cell cycle and cellular differentiation-dependent manner. Elevated levels of centromeric transcripts have also been detected in some cancers; however, it is still unclear how high levels of centromere transcripts may contribute towards disease progression. More recent studies have demonstrated that careful regulation of the histone modifications and transcription level at the centromere is vital for the recruitment of key centromere proteins and assembly of CENP-A domain. Here, we compare the transcriptional dynamics and function of various transcripts derived from pericentromeric and centromere core regions. We also propose a model in which the chromatin remodelling activity of transcription, and the resultant transcripts, contribute synergistically to perpetuate centromere chromatin identity. PMID:23066104

  15. 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.

  16. Population Variation and Genetic Control of Modular Chromatin Architecture in Humans.

    PubMed

    Waszak, Sebastian M; Delaneau, Olivier; Gschwind, Andreas R; Kilpinen, Helena; Raghav, Sunil K; Witwicki, Robert M; Orioli, Andrea; Wiederkehr, Michael; Panousis, Nikolaos I; Yurovsky, Alisa; Romano-Palumbo, Luciana; Planchon, Alexandra; Bielser, Deborah; Padioleau, Ismael; Udin, Gilles; Thurnheer, Sarah; Hacker, David; Hernandez, Nouria; Reymond, Alexandre; Deplancke, Bart; Dermitzakis, Emmanouil T

    2015-08-27

    Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain. PMID:26300124

  17. A Chromatin-Focused siRNA Screen for Regulators of p53-Dependent Transcription

    PubMed Central

    Sammons, Morgan A.; Zhu, Jiajun; Berger, Shelley L.

    2016-01-01

    The protein product of the Homo sapiens TP53 gene is a transcription factor (p53) that regulates the expression of genes critical for the response to DNA damage and tumor suppression, including genes involved in cell cycle arrest, apoptosis, DNA repair, metabolism, and a number of other tumorigenesis-related pathways. Differential transcriptional regulation of these genes is believed to alter the balance between two p53-dependent cell fates: cell cycle arrest or apoptosis. A number of previously identified p53 cofactors covalently modify and alter the function of both the p53 protein and histone proteins. Both gain- and loss-of-function mutations in chromatin modifiers have been strongly implicated in cancer development; thus, we sought to identify novel chromatin regulatory proteins that affect p53-dependent transcription and the balance between the expression of pro-cell cycle arrest and proapoptotic genes. We utilized an siRNA library designed against predicted chromatin regulatory proteins, and identified known and novel chromatin-related factors that affect both global p53-dependent transcription and gene-specific regulators of p53 transcriptional activation. The results from this screen will serve as a comprehensive resource for those interested in further characterizing chromatin and epigenetic factors that regulate p53 transcription. PMID:27334938

  18. The effect of vinca alkaloid anticancer drug, vinorelbine, on chromatin and histone proteins in solution.

    PubMed

    Rabbani-Chadegani, Azra; Chamani, Elham; Hajihassan, Zahra

    2009-06-24

    Vinorelbine (navelbin) belongs to vinca alkaloid anticancer drugs family with a broad spectrum of selective activity against mitotic microtubules. The present study is the first report demonstrating chromatin components as a novel target for vinorelbine in hepatocytes. The interaction was carried out in solution, employing fluorescence, UV spectroscopy and thermal denaturation techniques. Fluorescence emission spectra represented quenching of DNA chromospheres with drug and decreased fluorescence emission intensity in a dose-dependent manner. Binding of vinorelbine to chromatin induced very high hypochromicity and shifted DNA melting temperature to lower Tm. Vinorelbine binds to histone proteins with very high affinity when compared with the interaction of DNA intercalator anticancer drug, daunomycin, and the globular domain of the histones is considered as a main drug binding site. The results also showed that in the presence of vinorelbine, the absorbance of chromatin at 260 nm was decreased and the binding pattern was similar to daunomycin-chromatin complex. The results for the first time suggest that apart from tubulins, chromatin components can also be considered as a new target for this anticancer drug. PMID:19394329

  19. High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility.

    PubMed

    Pattenden, Samantha G; Simon, Jeremy M; Wali, Aminah; Jayakody, Chatura N; Troutman, Jacob; McFadden, Andrew W; Wooten, Joshua; Wood, Cameron C; Frye, Stephen V; Janzen, William P; Davis, Ian J

    2016-03-15

    Mutations in chromatin-modifying proteins and transcription factors are commonly associated with a wide variety of cancers. Through gain- or loss-of-function, these mutations may result in characteristic alterations of accessible chromatin, indicative of shifts in the landscape of regulatory elements genome-wide.