DOCSLIB.ORG

Light-Regulated Changes in Dnase I Hypersensitive Sites in the Rrna Genes of Pisum Sativum (Peas/Chromatin/Photoregulation/DNA/Nucleolar Organizer) LON S

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Light-Regulated Changes in Dnase I Hypersensitive Sites in the Rrna Genes of Pisum Sativum (Peas/Chromatin/Photoregulation/DNA/Nucleolar Organizer) LON S Proc. Natl. Acad. Sci. USA Vol. 84, pp. 1550-1554, March 1987 Botany Light-regulated changes in DNase I hypersensitive sites in the rRNA genes of Pisum sativum (peas/chromatin/photoregulation/DNA/nucleolar organizer) LON S. KAUFMAN*, JOHN C. WATSONt, AND WILLIAM F. THOMPSON: Carnegie Institution of Washington, 290 Panama Street, Stanford, CA 94305 Communicated by Winslow R. Briggs, November 10, 1986 (receivedfor review May 23, 1986) ABSTRACT We have examined the rDNA chromatin of transcribed genes (8), including the ribosomal RNA genes of Pisum sativum plants grown with or without exposure to light Tetrahymena (9-11), Xenopus (12), and Drosophila (13). for the presence of DNase I hypersensitive sites and possible However, DNase I hypersensitive sites have not yet been developmental changes in their distribution. Isolated nuclei reported in plant chromatin, and developmental changes in from pea seedlings were incubated with various concentrations the pattern of DNase I hypersensitivity have not been of DNase I. To visualize the hypersensitive sites, DNA purified reported for rDNA chromatin in any system. from these nuclei was restricted and analyzed by gel blot In the work reported here, we have examined the rDNA hybridization. We find that several sites exist in both the coding chromatin from pea buds for the presence of DNase I and noncoding regions of rDNA repeating units. Several of the hypersensitive sites. Using conditions that minimize the sites in the nontranscribed spacer region are present in the light activity of endogenous nucleases and proteases, we find that but are absent in the dark. Conversely, the hypersensitive sites DNase I hypersensitive sites exist in rDNA chromatin of within the mature rRNA coding regions are present in the dark isolated pea nuclei. When similar experiments are performed but absent in the light. There are two maojor'length variants of with nuclei isolated from the buds of dark-grown or light- the rRNA genes in P. sativum var. Alaska. The sites in the grown seedlings, we find several sites in the rRNA coding nontranscribed spacer region that appear during the light regions that are present in nuclei from dark-grown plants but treatment occur only in the shorter ofthese two length variants not in nuclei isolated from light-grown material. Conversely, in this cultivar. several DNase I hypersensitive sites in the nontranscribed spacers are present in nuclei from light-treated plants but are The cellular content of cytoplasmic rRNA in several plant absent from those of dark-grown seedlings. species is regulated by light (1), and light-regulated changes in the rate of nuclear rRNA gene transcription have been MATERIALS AND METHODS demonstrated in pea (2), Lemna (3), and barley (4). The overall transcriptional organization of rRNA genes is similar Seeds of Pisum sativum L. cv. Alaska (W. Altee Burpee, in plants and animals, but the plant genes tend to be more Werminster, PA) were imbibed for 5 hr and grown on two highly repeated than their animal counterparts (5). Ribosomal layers ofwater-soaked Kimpac (Kimberly-Clarke, Rosewell, RNA gene copy numbers can also vary substantially even GA) at 270C, 85% relative humidity, in absolute darkness among closely related genotypes within a species, indicating (14). Dark-grown plants were kept in these conditions for 7 that the number of nuclear rRNA genes in plants often days. Light-grown or light-treated plants were kept in the exceeds that needed to supply rRNA to the cytoplasmic dark for 4 days and then transferred to white light (102 ribosome pool (6). The presence of a large excess of gene ILmolmM2 sec-1; cool white fluorescent) for an additional 3 copies suggests that a mechanism must exist for determining days. The buds and hooks of the 7-day-old plants were which copies are transcriptionally active and which are silent. harvested into ice-cold containers under dim green light Regulation of rRNA gene expression by light may therefore (dark-grown plants) or in fluorescent room light (light-grown involve a recruitment ofmore genes into the transcriptionally plants). active pool. Nuclei were isolated as described by Watson and Thomp- The activity of individual rDNA loci (nucleolar organizers) son (15) except that the extraction and gradient buffers has been shown to be genetically determined (6, 7). For contained 5 mM sodium butyrate, 10 mM o-phenanthroline, example, the nucleolar organizer of the rDNA Aegilops 0.2% methylmethanethiosulfonate, and 0.1 mM phenylmeth- umbellulata chromosome 1 is dominant over all four Triticum ylsulfonyl fluoride. The final nuclear pellet was resuspended loci in chromosome addition lines containing this chromo- in DNase I buffer (250 mM sucrose/10 mM NaCl/10 mM some in a Triticum aestivum background (6). Since the rDNA Pipes, pH 7.0/3 mM MgCl2/3 mM CaCl2/5 mM sodium loci in these hybrids have rDNA repeats of different lengths butyrate/0.2% methylmethanethiosulfonate/0.1 mM phenyl- and distinguishable restriction patterns, molecular tests can methylsulfonyl fluoride/10 mM o-phenanthroline/5 mM 2- be designed to examine features of chromatin structure (e.g., mercaptoethanol) at a concentration of =250 jxg per ml of sensitivity to exogenous DNase I) and cytosine methylation DNA. ofthe DNA of each locus. Nucleolar dominance is correlated DNase I (Sigma) was added to aliquots of the nuclear with a more DNase I sensitive chromatin conformation and suspension to give final concentrations of 8.0, 2.4, 0.8, and a hypomethylation ofthe Aegilops rDNA (ref. 6; W.F.T. and R. B. Flavell, unpublished data). Abbreviations: NTS, nontranscribed spacer; L and S variants, longer It is well established in animal systems that DNase I and shorter variants of rDNA. hypersensitive sites are frequently associated with actively *Present address: Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60680. tPresent address: Department of Botany, University of Maryland, The publication costs of this article were defrayed in part by page charge College Park, MD 20742. payment. This article must therefore be hereby marked "advertisement" *Present address: Departments of Botany and Genetics, North in accordance with 18 U.S.C. §1734 solely to indicate this fact. Carolina State University, Raleigh, NC 27695. 1550 Botany: Kaufman et al. Proc. Natl. Acad. Sci. USA 84 (1987) 1551 0.2 units/ml. Incubation was for 10 min at 370C. Additional RESULTS aliquots ofnuclei were also incubated at 37TC with no enzyme (sham digest) and at 0C with no enzyme (control digest). In the garden pea, there are 3000-4000 copies of the rRNA After 10 min, the reaction was stopped by the addition of a genes (18). Most of these genes, together with spacer se- large volume of ice-cold DNase I buffer lacking both phen- quences between them, are present in large tandem arrays at ylmethylsulfonyl fluoride and methylmethanethiosulfonate. one of the two nucleolar organizer loci (19). The basic The nuclei were pelleted immediately and resuspended in a monomer repeating unit of -9 kilobases (kb) contains the small volume of the buffer. (Phenylmethylsulfonyl fluoride 18S, 5.8S, and 25S rRNA cistrons and a "nontranscribed" and methylmethanethiosulfonate were omitted at this stage to spacer (NTS) =3 kb long (Fig. 1). About one-third of each avoid interference with subsequent proteinase K treatment.) NTS is occupied by a tandem array of short (180 bp) repeated The resuspended nuclei were lysed by the addition of 1 vol of elements, called the subrepeat array, which occurs just 2x lysis buffer (200 mM Tris HCl, pH 8.0/50 mM EDTA/1 downstream from the 3' end of the 25S rRNA gene. M NaCl/2% Sarkosyl) and the lysate was treated with In many pea varieties, including cv. Alaska, two major proteinase K (0.5 mg/ml) at 550C for 1.5 hr. DNA was length classes of rDNA are observed (17, 20), and the purified by banding in cesium chloride/ethidium bromide different forms are thought to be located at different nucleolar gradients as described (15). Purified DNA was exhaustively organizers (20, 21). The number of elements in this subrepeat digested with either BamHI (Bethesda Research Laborato- array is variable both between cultivars and between loci ries) or EcoRI (Bethesda Research Laboratories) in the ionic within a cultivar. In P. sativum cv. Alaska, the longer (L) conditions recommended by the supplier. The DNA restric- variant contains nine elements per subrepeat array, while the tion fragments were size-fractionated on agarose gels, trans- shorter (S) variant subrepeat arrays contain seven elements ferred to either GeneScreen (New England Nuclear) or Nylon each. Although other variants exist (16), they are present in 66 (Schleicher & Schuell), and hybridized with the appropri- much lower copy numbers and do not contribute significantly ate DNA probe as described (15). to the data presented here. Restriction digests of genomic Bands in the hybridization profiles that occurred in either DNA indicate several differences in sequence between the L control or sham digests were discounted when mapping and S variants (17). For example, the L variant, but not the DNase I hypersensitive sites. These bands probably repre- S variant, contains two closely spaced EcoRI sites within the sent restriction fragments from minor length variants known subrepeat array. This difference in sequence and the differ- to be present at low copy number in the pea genome (16). ences in length between the two variants allow us to distin- Control experiments were conducted to be certain that the guish between them in gel blot hybridization studies. DNase I hypersensitive sites mapped reflect properties ofthe DNase I hypersensitive sites were mapped by using the chromatin complex rather than properties of naked DNA. method of indirect end-labeling (22, 23). Small hybridization Both dark-grown and light-grown seedling buds and hooks probes are used that hybridize only to one end ofthe genomic were used to prepare total cell DNA or DNA from isolated restriction fragment being mapped.
Load more

Recommended publications
  • Predicting Double-Strand DNA Breaks Using Epigenome Marks Or DNA at Kilobase Resolution
    bioRxiv preprint doi: https://doi.org/10.1101/149039; this version posted June 12, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Predicting double-strand DNA breaks using epigenome marks or DNA at kilobase resolution Rapha¨elMourad1 and Olivier Cuvier1 April 10, 2017 1 Laboratoire de Biologie Mol´eculaireEucaryote (LBME), CNRS, Universit´ePaul Sabatier (UPS), 31000 Toulouse, France Abstract Double-strand breaks (DSBs) result from the attack of both DNA strands by multiple sources, includ- ing exposure to ionizing radiation or reactive oxygen species. DSBs can cause abnormal chromosomal rearrangements which are linked to cancer development, and hence represent an important issue. Recent techniques allow the genome-wide mapping of DSBs at high resolution, enabling the comprehensive study of DSB origin. However these techniques are costly and challenging. Hence we devised a computational approach to predict DSBs using the epigenomic and chromatin context, for which public data are available from the ENCODE project. We achieved excellent prediction accuracy (AUC = 0:97) at high resolution (< 1 kb), and showed that only chromatin accessibility and H3K4me1 mark were sufficient for highly accurate prediction (AUC = 0:95). We also demonstrated the better sensitivity of DSB predictions com- pared to BLESS experiments. We identified chromatin accessibility, activity and long-range contacts as best predictors. In addition, our work represents the first step toward unveiling the "cis-DNA repairing" code underlying DSBs, paving the way for future studies of cis-elements involved in DNA damage and repair.
    [Show full text]
  • Npgrj Nmeth 890 511..518
    ARTICLES Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays s 1,6 1,2,6 1,2 2 2 2 d Peter J Sabo , Michael S Kuehn , Robert Thurman , Brett E Johnson , Ericka M Johnson , Hua Cao , o h 2 2 1 1 1 1 1 t Man Yu , Elizabeth Rosenzweig , Jeff Goldy , Andrew Haydock , Molly Weaver , Anthony Shafer , Kristin Lee , e 1 1 3 3 1 m Fidencio Neri , Richard Humbert , Michael A Singer , Todd A Richmond , Michael O Dorschner , e r u 4 5 3 2 1 t Michael McArthur , Michael Hawrylycz , Roland D Green , Patrick A Navas , William S Noble & a n 1 / John A Stamatoyannopoulos m o c . e r u Localized accessibility of critical DNA sequences to the entire genome and analysis of their relationship to the current t a n regulatory machinery is a key requirement for regulation of annotation of human genes. Comprehensive delineation of the . w human genes. Here we describe a high-resolution, genome-scale accessible chromatin compartment is expected to be of particular w w / approach for quantifying chromatin accessibility by measuring importance for identification of functional human genetic variants / : p t DNase I sensitivity as a continuous function of genome position that mediate individual variation in gene expression and physio- t h using tiling DNA microarrays (DNase-array). We demonstrate this logical phenotypes. On a broader level, human chromosomes p approach across 1% (B30 Mb) of the human genome, wherein have long been thought to be organized into discrete higher- u o r we localized 2,690 classical DNase I hypersensitive sites with order functional domains characterized by ‘open’ (active) G high sensitivity and specificity, and also mapped larger-scale and ‘closed’ (inactive) chromatin8–10.
    [Show full text]
  • Chromatin Accessibility and the Regulatory Epigenome
    REVIEWS EPIGENETICS Chromatin accessibility and the regulatory epigenome Sandy L. Klemm1,4, Zohar Shipony1,4 and William J. Greenleaf1,2,3* Abstract | Physical access to DNA is a highly dynamic property of chromatin that plays an essential role in establishing and maintaining cellular identity. The organization of accessible chromatin across the genome reflects a network of permissible physical interactions through which enhancers, promoters, insulators and chromatin-binding factors cooperatively regulate gene expression. This landscape of accessibility changes dynamically in response to both external stimuli and developmental cues, and emerging evidence suggests that homeostatic maintenance of accessibility is itself dynamically regulated through a competitive interplay between chromatin- binding factors and nucleosomes. In this Review , we examine how the accessible genome is measured and explore the role of transcription factors in initiating accessibility remodelling; our goal is to illustrate how chromatin accessibility defines regulatory elements within the genome and how these epigenetic features are dynamically established to control gene expression. Chromatin- binding factors Chromatin accessibility is the degree to which nuclear The accessible genome comprises ~2–3% of total Non- histone macromolecules macromolecules are able to physically contact chroma­ DNA sequence yet captures more than 90% of regions that bind either directly or tinized DNA and is determined by the occupancy and bound by TFs (the Encyclopedia of DNA elements indirectly to DNA. topological organization of nucleosomes as well as (ENCODE) project surveyed TFs for Tier 1 ENCODE chromatin- binding factors 13 Transcription factor other that occlude access to lines) . With the exception of a few TFs that are (TF). A non- histone protein that DNA.
    [Show full text]
  • Human Monocyte-To-Macrophage
    Dekkers et al. Epigenetics & Chromatin (2019) 12:34 https://doi.org/10.1186/s13072-019-0279-4 Epigenetics & Chromatin RESEARCH Open Access Human monocyte-to-macrophage diferentiation involves highly localized gain and loss of DNA methylation at transcription factor binding sites Koen F. Dekkers1†, Annette E. Neele2†, J. Wouter Jukema3, Bastiaan T. Heijmans1*‡ and Menno P. J. de Winther2,4*‡ Abstract Background: Macrophages and their precursors monocytes play a key role in infammation and chronic infamma- tory disorders. Monocyte-to-macrophage diferentiation and activation programs are accompanied by signifcant epigenetic remodeling where DNA methylation associates with cell identity. Here we show that DNA methylation changes characteristic for monocyte-to-macrophage diferentiation occur at transcription factor binding sites, and, in contrast to what was previously described, are generally highly localized and encompass both losses and gains of DNA methylation. Results: We compared genome-wide DNA methylation across 440,292 CpG sites between human monocytes, naïve macrophages and macrophages further activated toward a pro-infammatory state (using LPS/IFNγ), an anti-infam- matory state (IL-4) or foam cells (oxLDL and acLDL). Moreover, we integrated these data with public whole-genome sequencing data on monocytes and macrophages to demarcate diferentially methylated regions. Our analysis showed that diferential DNA methylation was most pronounced during monocyte-to-macrophage diferentiation, was typically restricted to single CpGs or very short regions, and co-localized with lineage-specifc enhancers irrespec- tive of whether it concerns gain or loss of methylation. Furthermore, diferentially methylated CpGs were located at sites characterized by increased binding of transcription factors known to be involved in monocyte-to-macrophage diferentiation including C/EBP and ETS for gain and AP-1 for loss of methylation.
    [Show full text]
  • In Vivo Analysis of the State of the Human Upa Enhancer Following Stimulation by TPA
    Oncogene (1999) 18, 2836 ± 2845 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc In vivo analysis of the state of the human uPA enhancer following stimulation by TPA Ine s IbanÄ ez-Tallon1,3, Giuseppina Caretti1,2, Francesco Blasi1,2 and Massimo P Crippa*,1 1Laboratory of Molecular Genetics, DIBIT - Ospedale. S. Raaele, Via Olgettina 58, 20132 Milano, Italy; 2Department of Genetics and Microbial Biology, University of Milano, Via Celoria 20, 20133 Milano, Italy We have analysed in vivo the 72.0 kb enhancer of the upstream combined PEA3/AP-1A and a downstream human urokinase-type plasminogen activator (uPA) gene AP-1B site (Berthelsen et al., 1996; De Cesare et al., in HepG2 cells, in which gene expression can be induced 1995; Nerlov et al., 1992). The AP-1 sites are separated by phorbol esters. The results reveal that, within the by a 74 bp `cooperation mediator' (COM) region, regulatory region, the enhancer, the silencer and the which contains the binding sites for several proteins, minimal promoter become hypersensitive to deoxyribo- named urokinase enhancer factors (UEF; Berthelsen et nuclease I (DNase I) upon induction of transcription. al., 1996; De Cesare et al., 1996; Nerlov et al., 1992). In The hypersensitivity of the enhancer can be reversed HeLa cells, the NF-kB site at 71865, downstream of after removal of the inducer. In vivo footprinting analysis AP-1B and in A549 cells the site at 71592 have also indicates that all the cis-acting elements of the enhancer, been reported to play a role in phorbol esters induction previously identi®ed in vitro, are occupied in vivo upon of uPA gene expression (Guerrini et al., 1996; Novak 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimula- et al., 1991).
    [Show full text]
  • The Mouse Albumin Promoter and a Distal Upstream Site Are Simultaneously Dnase I Hypersensitive in Liver Chromatin and Bind Similar Liver- Abundant Factors in Vitro
    Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press The mouse albumin promoter and a distal upstream site are simultaneously DNase I hypersensitive in liver chromatin and bind similar liver- abundant factors in vitro Jen-Kuei Liu, Yehudit Bergman,^ and Kenneth S. Zaret^ Section of Biochemistry, Brown University, Providence, Rhode Island 02912 USA In this paper we characterize the chromatin structure and nuclear proteins associated with different transcriptional states of the mouse serum albumin gene. We found the albumin gene to be transcribed in liver at rates 1000-fold or greater than in other tissues tested. We discovered seven DNase I hypersensitive sites encompassing the albumin gene only in liver chromatin, with strong hypersensitivity at the promoter and the enhancer, which is over 10 kb upstream. Using a gel retardation assay, we found a liver nuclear protein, or set of proteins, which binds specifically to DNA of a liver-specific hypersensitive site that maps 3.5 kb upstream, between the promoter and enhancer. Footprinting, heat insensitivity, and binding competition experiments indicate that the protein(s) have characteristics similar to a heat-stable, liver-abundant protein that binds to the albumin promoter and other enhancer and promoter sequences. Finally, we asked whether the liver-specific factors that cause DNase I hypersensitivity in vivo are present concurrently at the various sites in chromatin. We devised a simple new method to reveal that in liver, individual albumin genes are hypersensitive simultaneously at the promoter, the enhancer, and the - 3.5-kb site. Thus, transcriptionally active albumin genes appear to contain tissue-abundant factors that are present at three widely spaced points in chromatin, yet at the same point in time.
    [Show full text]
  • Torsional Stress Induces an S1 Nuclease-Hypersensitive Site Within
    Proc. Nati. Acad. Sci. USA Vol. 82, pp. 4018-4022, June 1985 Biochemistry Torsional stress induces an S1 nuclease-hypersensitive site within the promoter of the Xenopus laevis oocyte-type 5S RNA: gene : (gene expression/transcription factor/DNA-protein interaction) WANDA F. REYNOLDS AND JOEL M. GOTTESFELD Department of Molecular Biology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037 Communicated by James Bonner, February 22, 1985 ABSTRACT The internal promoter of the Xenopus laevis gene promoter adopts an S1 nuclease sensitive conformation oocyte-type 5S RNA gene is preferentially cleaved by S1 and in supercoiled DNA. This altered conformation may be Bal-31 nucleases in plasmid DNA. S1 nuclease sensitivity is similarly induced or stabilized by TFIIIA in linear DNA. largely dependent on supercoiling; however, Bal-31 cleaves These findings provide a clear correlation between an S1 within the 5S RNA gene in linear as well as in supercoiled DNA. nuclease-sensitive conformation and a promoter element. The S1 nuclease-hypersensitive site is centered at position +48-52 of the gene at the 5' boundary of the promoter. A METHODS DNase I-hypersensitive site is induced at this position upon DNAs. For DNase I "footprint" analysis, pXlo 3'A+56 binding of the transcription factor, TFIIIA, specific for the 5S was digested with EcoRI and end-labeled with polynucleotide RNA gene. The somatic-type 5S RNA gene promoter is not kinase (Bethesda Research Laboratories) and ['y-32P]ATP. preferentially cleaved by S1 nuclease or Bal-31 nuclease in The DNA was secondarily digested with HindIII and the supercoiled DNA, nor does TFIIIA induce a DNase I site at 530-bp fragment containing the gene was isolated by poly- position +50.
    [Show full text]
  • The Meiotic Recombination Hot Spot Created by the Single-Base Substitution Ade6-M26 Results in Remodeling of Chromatin Structure in Fission Yeast
    Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press The meiotic recombination hot spot created by the single-base substitution ade6-M26 results in remodeling of chromatin structure in fission yeast Ken-ichi Mizuno/'' Yukihiro Emura/'^* Michel Baur,^ Jiirg Kohli,^ Kunihiro Ohta/'^ and Takehiko Shibata^ ^Cellular and Molecular Biology Laboratory, The Institute of Physical and Chemical Research, Wako, Saitama 351-01, Japan; ^Applied Biology, Nihon University, Fujisawa, Japan; ^Institute of General Microbiology, University of Bern, Bern, Switzerland The G ^ T transversion mutation, ade6-M26, creates the heptanucleotide sequence ATGACTG, which lies close to the 5' end of the open reading frame of the ade6 gene in Schizosaccharomyces pombe. The mutation generates a meiosis-specific recombination hot spot and a binding site for the Mtsl/Mts2 protein. We examined the chromatin structure at the ade6 locus in the M26 strain and compared it to that of the wild-type and hot spot-negative control M375. Micrococcal nuclease (MNase) digestion and indirect end-labeling methods were applied. In the M26 strain, we detected a new MNase-hypersensitive site at the position of the M26 mutation and no longer observed the phasing of nucleosomes seen in the wild-type and the M375 strains. Quantitative comparison of MNase sensitivity of the chromatin in premeiotic and meiotic cultures revealed a small meiotic induction of MNase hypersensitivity in the ade6 promoter region of the wild-type and M375 strains. The meiotic induction of MNase hypersensitivity was enhanced significantly in the ade6 promoter region of the M26 strain and also occurred at the M26 mutation site.
    [Show full text]
  • Selective Loss of a Dnase I Hypersensitive Site Upstream of The
    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6540-6544, July 1992 Genetics Selective loss of a DNase I hypersensitive site upstream of the tyrosine aminotransferase gene in mice homozygous for lethal albino deletions (chromatin structure/gene regulation/liver differentiation) KENNETH S. ZARET*, PATRICE MILOS*, MARIE LIAt, DEEKSHA BALIt, AND SALOME GLUECKSOHN-WAELSCHt *Section of Biochemistry, Box G-J363, Brown University, Providence, RI 02912; and tpepartment of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 Contributed by Salome G. Waelsch, April 21, 1992 ABSTRACT Several overlapping chromosomal deletis eukaryotes, virtually all regulatory sequences-such as those sning the albino locus in the mouse cause perinatal lethality of promoters, enhancers, and locus control regions-show when homozygous and a block in the trnscriptional Induction DNase I hypersensitivity when the relevant elements are of various unlinked hepatocyte-specific genes. Studies of such active (10). Nuclease hypersensitivity is thought to be due to lethal albino deletion homozygotes in perinatal stages revealed a altered DNA structures or to specific DNA binding proteins deficiency in the transcriptional inducibility of the tyrosine that perturb or displace a nucleosome, rendering the adjacent aminotrnsferase (TAT) gene by glucocorticolds; yet, glucocor- sequences available for nuclease attack (11, 12). For exam- ticoid receptor and hormone levels were shown to be uected. ple, in vivo and in vitro footprinting studies have shown that To identify a molecular defect underlying thefailure ofi ble binding of the glucocorticoid receptor (13) and a liver- expression, we examine the chromatin structure of the TAT enriched transcription factor (14) disrupt a nucleosome (15) at gene.
    [Show full text]
  • Chromatin Accessibility: a Window Into the Genome Maria Tsompana1 and Michael J Buck1,2*
    Tsompana and Buck Epigenetics & Chromatin 2014, 7:33 http://www.epigeneticsandchromatin.com/content/7/1/33 REVIEW Open Access Chromatin accessibility: a window into the genome Maria Tsompana1 and Michael J Buck1,2* Abstract Transcriptional activation throughout the eukaryotic lineage has been tightly linked with disruption of nucleosome organization at promoters, enhancers, silencers, insulators and locus control regions due to transcription factor binding. Regulatory DNA thus coincides with open or accessible genomic sites of remodeled chromatin. Current chromatin accessibility assays are used to separate the genome by enzymatic or chemical means and isolate either the accessible or protected locations. The isolated DNA is then quantified using a next-generation sequencing platform. Wide application of these assays has recently focused on the identification of the instrumental epigenetic changes responsible for differential gene expression, cell proliferation, functional diversification and disease development. Here we discuss the limitations and advantages of current genome-wide chromatin accessibility assays with especial attention on experimental precautions and sequence data analysis. We conclude with our perspective on future improvements necessary for moving the field of chromatin profiling forward. Keywords: Chromatin, MNase, DNase, ATAC, FAIRE, Sequencing, Library, Epigenome, Histone, Nucleosome Introduction: chromatin accessibility eukaryotes [19,20]. Open or accessible regions of the Eukaryotic chromatin is tightly packaged
    [Show full text]
  • The Human B-Globin Locus Control Region a Center of Attraction
    Eur. J. Biochem. 269, 1589–1599 (2002) Ó FEBS 2002 REVIEW ARTICLE The human b-globin locus control region A center of attraction Padraic P. Levings and Jo¨ rg Bungert Department of Biochemistry and Molecular Biology, Gene Therapy Center, Center for Mammalian Genetics, College of Medicine, University of Florida, Gainesville, FL, USA The human b-globin gene locus is the subject of intense and capable of recruiting, with great efficiency, chromatin- study, and over the past two decades a wealth of information modifying, coactivator, and transcription complexes. These has accumulated on how tissue-specific and stage-specific complexes are used to establish accessible chromatin expression of its genes is achieved. The data are extensive and domains, allowing basal factors to be loaded on to specific it would be difficult, if not impossible, to formulate a com- globin gene promoters in a developmental stage-specific prehensive model integrating every aspect of what is cur- manner. We conceptually divide this process into four steps: rently known. In this review, we introduce the fundamental (a) generation of a highly accessible LCR holocomplex; characteristics of globin locus regulation as well as questions (b) recruitment of transcription and chromatin-modifying on which much of the current research is predicated. We then complexes to the LCR; (c) establishment of chromatin outline a hypothesis that encompasses more recent results, domains permissive for transcription; (d) transfer of tran- focusing on the modification of higher-order chromatin scription complexes to globin gene promoters. structure and recruitment of transcription complexes to the Keywords: chromatin domains; globin genes; intergenic globin locus.
    [Show full text]
  • Chromatin Fine Structure of the C-MYC Insulator Element/Dnase I-Hypersensitive Site I Is Not Preserved During Mitosis
    Chromatin fine structure of the c-MYC insulator element/DNase I-hypersensitive site I is not preserved during mitosis Jun-ichiro Komura*, Hironobu Ikehata, and Tetsuya Ono Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8575, Japan Edited by Arthur D. Riggs, Beckman Research Institute of the City of Hope, Duarte, CA, and approved August 3, 2007 (received for review March 14, 2007) During mitosis in higher eukaryotic cells, transcription is silenced mitotic chromatin (17). If the CTCF-associated local chromatin and transcription complexes are absent from promoters in the structure of insulator elements is preserved during mitosis, then condensed chromosomes; however, epigenetic information con- it appears possible that insulators may serve as components of cerning the pattern of expressed and silent genes must be pre- cellular mechanisms for the transfer of epigenetic information served. Recently, it has been reported that CTCF, a major protein in through the mitotic period and for the reestablishment of the vertebrate insulator elements, remains associated with mitotic domain structure of genes after this period. chromatin. If the structure of insulators is preserved during mitosis, An insulator element has been found Ϸ2 kb upstream of the then it is possible that insulators can function as components or major transcription start sites of the human c-MYC gene (18, 19). elements of the mechanism involved in the transfer of epigenetic The enhancer-blocking activity of this element has been dem- information through the mitotic phase and can help guide the onstrated by colony assays. The c-MYC insulator is located reconstitution of domain structure and nuclear organization after between a domain containing hyperacetylated histones and a the completion of this phase.
    [Show full text]