FACT Is Recruited to the +1 Nucleosome of Transcribed Genes and Spreads in a Chd1-Dependent Manner
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H2A Ubiquitylated Mononucleosomes Next-Generation Substrates for Deubiquitylation Enzyme (DUB) Assays
H2A Ubiquitylated Mononucleosomes Next-Generation Substrates for Deubiquitylation Enzyme (DUB) Assays Next-Generation DUB Assay Substrates are here. Get results that matter. • Enabling access to DUB targets that require nucleosome substrates in vitro • Proper substrates for DUB inhibitor development • Unmatched quality control for results you can trust Histone monoubiquitylation (ub1) acts as a critical signaling center that regulates cascades of downstream epigenetic enzymes to modify gene transcription. The physiological substrate for chromatin-targeting DUBs is the nucleosome (Nuc), the basic repeating unit of chromatin (comprised of histone proteins wrapped by DNA). Current high-throughput screening (HTS) DUB assays use unnatural modified or diubiquitin conjugates as substrates, which poorly mimic endogenous targets in vivo. In collaboration with Boston Biochem, EpiCypher is delivering ubiquitylated nucleosome substrates for drug screening and chromatin biology research. FIGURE 1 Ub Ub Schematic representation of mononucleosoms assembled from recombinant human histones Ub expressed in E. coli (two each of histones H2A, H2B, H3 and H4). H2A H2A Approximately 50% of the nucleosomes are monoubiquitylated on histone H2A lysine 118, while the other 50% are monoubiquitylated on both histone H2A lysine 118 and histone H2A lysine 119 (multi-mono- ubiquitylated). Next Generation Deubiquitylation Enzyme (DUB) Assay Substrates EpiCypher has developed recombinant mononucleosomes carrying monoubiquitylation on H2A. These ubiquitylated nucleosomes are generated enzymatically using the RING1B/BMI1 ubiquitin ligase complex. The resulting product is highly pure (>95% of nucleosomes are ubiquitylated) and consists of nucleosomes monoubiquitylated at H2A lysine 118/119 (Figure 1; the physiological target of RING1B/BMI1 in vivo). FIGURE 2 Deubiquitylation Assay Data: Mononucleosomes H2A Ubiquityl, Recombinant Human, Biotinylated (1 μg) were employed in a deubiquitylation (DUB) assay using no enzyme (Lane 1), USP5 (Lane 2) or USP16 (Lane 3) and run on an SDS PAGE gel. -
Watanabe S, Resch M, Lilyestrom W, Clark N
NIH Public Access Author Manuscript Biochim Biophys Acta. Author manuscript; available in PMC 2010 November 1. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Biochim Biophys Acta. 2010 ; 1799(5-6): 480±486. doi:10.1016/j.bbagrm.2010.01.009. Structural characterization of H3K56Q nucleosomes and nucleosomal arrays Shinya Watanabe1,*, Michael Resch2,*, Wayne Lilyestrom2, Nicholas Clark2, Jeffrey C. Hansen2, Craig Peterson1, and Karolin Luger2,3 1 Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St.; Worcester, Massachusetts 01605 2 Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870 3 Howard Hughes Medical Institute Abstract The posttranslational modification of histones is a key mechanism for the modulation of DNA accessibility. Acetylated lysine 56 in histone H3 is associated with nucleosome assembly during replication and DNA repair, and is thus likely to predominate in regions of chromatin containing nucleosome free regions. Here we show by x-ray crystallography that mutation of H3 lysine 56 to glutamine (to mimic acetylation) or glutamate (to cause a charge reversal) has no detectable effects on the structure of the nucleosome. At the level of higher order chromatin structure, the K to Q substitution has no effect on the folding of model nucleosomal arrays in cis, regardless of the degree of nucleosome density. In contrast, defects in array-array interactions in trans (‘oligomerization’) are selectively observed for mutant H3 lysine 56 arrays that contain nucleosome free regions. Our data suggests that H3K56 acetylation is one of the molecular mechanisms employed to keep chromatin with nucleosome free regions accessible to the DNA replication and repair machinery. -
DNA Condensation and Packaging
DNA condensation and packaging October 13, 2009 Professor Wilma K. Olson Viral DNA - chain molecules in confined spaces Viruses come in all shapes and sizes Clockwise: Human immuno deficiency virus (HIV); Aeromonas virus 31, Influenza virus, Orf virus, Herpes simplex virus (HSV), Small pox virus Image from U Wisconsin Microbial World website: http://bioinfo.bact.wisc.edu DNA packaging pathway of T3 and T7 bacteriophages • In vivo pathway - solid arrows Fang et al. (2008) “Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo.” J. Mol. Biol. 384, 1384-1399 Cryo EM images of T3 capsids with 10.6 kbp packaged DNA • Labels mark particles representative of different types of capsids • Arrows point to tails on capsids Fang et al. (2008) “Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo.”” J. Mol. Biol. 384, 1384-1399 Cryo EM images of representative particles • (b) 10.6 kbp DNA • (c) 22 kbp DNA • (d) bacteriophage T3 Fang et al. (2008) “Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo.” J. Mol. Biol. 384, 1384-1399 3D icosohedral reconstructions of cryo-EM-imaged particles Threefold surface views and central cross sections • (b) 10.6 kbp DNA • (c) 22 kbp DNA • (d) bacteriophage T3 Fang et al. (2008) “Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo.” J. Mol. Biol. 384, 1384-1399 Top-down views of λ phage DNA toroids captured in cryo-EM micrographs Note the circumferential winding of DNA found in collapsed toroidal particles produced in the presence of multi-valent cations. Hud & Vilfan (2005) “Toroidal DNA condensates: unraveling the fine structure and the role of nucleation in determining size.” Ann. -
Investigating the Epigenetic Mechanisms of Trophoblast Giant Cells
Biology ︱ Assistant Professor Koji Hayakawa NUCLEOSOME STRUCTURE OF TROPHOBLAST GIANT CELL (TGC) Diploid TSC Polyploid TGC H2A H2B H2AX/ Investigating the H2AZ epigenetic mechanisms Entry into DNA Endocycle H3 H4 H3.3 of trophoblast giant cells TGCs possess a loose chromatin structure owing to alterations in the histone composition of the nucleosomes, which involves the replacement of canonical histones with histone variants such as H2AX, H2AZ, and H3.3 during differentiation. Trophoblast giant cells (TGCs) ucleosome is a large molecule in the placenta of rodents, are a unique days, and that certain histone variants Many polyploid cells identified in plants are found in the placental in the cell which is primarily cell type that replicate their DNA until were associated with differentiated walls of rodents and play a Nmade up of DNA and proteins. the cell contains thousands of copies, cells. Overall, there was much less and animals appear to have a secretory role in maintaining pregnancy. The major protein in nucleosome is unlike most cells which normally contain variation in TGCs compared to the In contrast to most cell types called a histone, around which DNA two sets of chromosomes (diploid cells). undifferentiated, diploid cells. They or nutritive function. which contain two copies of wraps. These proteins are classified into The reasons for this condition are not found the histone profile to be very from undifferentiated cells showed distinct concentrations of salt buffer to disrupt each chromosome (diploid), canonical histones and non-canonical clear. However, it has been suggested similar in differentiated TSCs at day six bands when digested, demonstrating that DNA-protein bonds. -
Condensed DNA: Condensing the Concepts
Progress in Biophysics and Molecular Biology 105 (2011) 208e222 Contents lists available at ScienceDirect Progress in Biophysics and Molecular Biology journal homepage: www.elsevier.com/locate/pbiomolbio Review Condensed DNA: Condensing the concepts Vladimir B. Teif a,b,*, Klemen Bohinc c,d a BioQuant and German Cancer Research Center, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany b Institute of Bioorganic Chemistry, Belarus National Academy of Sciences, Kuprevich 5/2, 220141, Minsk, Belarus c Faculty of Health Sciences, Zdravstvena pot 5, 1000 Ljubljana, Slovenia d Faculty of Electrical Engineering, University of Ljubljana, Trzaska 25, 1000 Ljubljana, Slovenia article info abstract Article history: DNA is stored in vivo in a highly compact, so-called condensed phase, where gene regulatory processes Available online 16 July 2010 are governed by the intricate interplay between different states of DNA compaction. These systems often have surprising properties, which one would not predict from classical concepts of dilute solutions. The Keywords: mechanistic details of DNA packing are essential for its functioning, as revealed by the recent devel- DNA condensation opments coming from biochemistry, electrostatics, statistical mechanics, and molecular and cell biology. Ligand binding Different aspects of condensed DNA behavior are linked to each other, but the links are often hidden in Counterion correlations the bulk of experimental and theoretical details. Here we try to condense some of these concepts and Macromolecular crowding fi Chromatin provide interconnections between the different elds. After a brief description of main experimental Gene regulation features of DNA condensation inside viruses, bacteria, eukaryotes and the test tube, main theoretical approaches for the description of these systems are presented. -
What to Do About Those Immunoprecipitation Blues Chip-Seq, DIP-Seq and Related Techniques Are Informative Genome-Wide Assays, but They Don’T Always Work As Planned
technology feature What to do about those immunoprecipitation blues ChIP-seq, DIP-seq and related techniques are informative genome-wide assays, but they don’t always work as planned. Vivien Marx hen the chromatin immunoprecipitation (ChIP) Wor DNA immunoprecipitation (DIP) blues hit, it’s good to realize you’re not alone. ChIP is part of the winding path of characterizing gene function. To find where transcription factors (TFs) or histone marks bind to genomic loci of interest, labs might choose ChIP-seq, which involves cross-linking, then shearing chromatin, immunoprecipitation with an antibody that recognizes a TF or histone mark of interest, followed by sequencing. DIP plus sequencing (DIP-seq) can be used to locate DNA changes such as methylation. ChIP-seq and DIP-seq have been the “cornerstone of epigenetics research” since the field moved from gene-specific epigenetics to the genome- wide approaches of epigenomics, says Colm Nestor, a researcher at Linköping University. Both techniques are cheap, relatively easy to set up, and widely used “because they work very well—when well controlled, that is,” he says. With ChIP-seq, antibodies are sometimes Immunoprecipitation can be used to locate DNA changes such as methylation or places where transcription not as specific as a lab might have first factors bind, and can involve the struggle with artifacts. Credit: A. Lentini, Linköping University assumed1. Some genomic loci can be ‘sticky’ in too many wrong ways, or masked protein epitopes are not ‘sticky’ when they should be. As Dana Farber Cancer Institute researchers differential binding. His former PhD student sharply defined peaks in open chromatin Xiaole Shirley Liu and Clifford Meyer point Dhawal Jain, now a postdoctoral fellow in regions, where nucleosomes are usually out, some of ChIP-seq’s lurking bias issues the lab of Harvard University researcher lacking. -
Control of Eukaryotic DNA Replication Initiation—Mechanisms to Ensure Smooth Transitions
G C A T T A C G G C A T genes Review Control of Eukaryotic DNA Replication Initiation—Mechanisms to Ensure Smooth Transitions Karl-Uwe Reusswig and Boris Pfander * Max Planck Institute of Biochemistry, DNA Replication and Genome Integrity, 82152 Martinsried, Germany; [email protected] * Correspondence: [email protected] Received: 31 December 2018; Accepted: 25 January 2019; Published: 29 January 2019 Abstract: DNA replication differs from most other processes in biology in that any error will irreversibly change the nature of the cellular progeny. DNA replication initiation, therefore, is exquisitely controlled. Deregulation of this control can result in over-replication characterized by repeated initiation events at the same replication origin. Over-replication induces DNA damage and causes genomic instability. The principal mechanism counteracting over-replication in eukaryotes is a division of replication initiation into two steps—licensing and firing—which are temporally separated and occur at distinct cell cycle phases. Here, we review this temporal replication control with a specific focus on mechanisms ensuring the faultless transition between licensing and firing phases. Keywords: DNA replication; DNA replication initiation; cell cycle; post-translational protein modification; protein degradation; cell cycle transitions 1. Introduction DNA replication control occurs with exceptional accuracy to keep genetic information stable over as many as 1016 cell divisions (estimations based on [1]) during, for example, an average human lifespan. A fundamental part of the DNA replication control system is dedicated to ensure that the genome is replicated exactly once per cell cycle. If this control falters, deregulated replication initiation occurs, which leads to parts of the genome becoming replicated more than once per cell cycle (reviewed in [2–4]). -
The General Transcription Factors of RNA Polymerase II
Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW The general transcription factors of RNA polymerase II George Orphanides, Thierry Lagrange, and Danny Reinberg 1 Howard Hughes Medical Institute, Department of Biochemistry, Division of Nucleic Acid Enzymology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854-5635 USA Messenger RNA (mRNA) synthesis occurs in distinct unique functions and the observation that they can as- mechanistic phases, beginning with the binding of a semble at a promoter in a specific order in vitro sug- DNA-dependent RNA polymerase to the promoter re- gested that a preinitiation complex must be built in a gion of a gene and culminating in the formation of an stepwise fashion, with the binding of each factor promot- RNA transcript. The initiation of mRNA transcription is ing association of the next. The concept of ordered as- a key stage in the regulation of gene expression. In eu- sembly recently has been challenged, however, with the karyotes, genes encoding mRNAs and certain small nu- discovery that a subset of the GTFs exists in a large com- clear RNAs are transcribed by RNA polymerase II (pol II). plex with pol II and other novel transcription factors. However, early attempts to reproduce mRNA transcrip- The existence of this pol II holoenzyme suggests an al- tion in vitro established that purified pol II alone was not ternative to the paradigm of sequential GTF assembly capable of specific initiation (Roeder 1976; Weil et al. (for review, see Koleske and Young 1995). -
The FACT Spt16 ''Peptidase'' Domain Is a Histone H3–H4 Binding Module
The FACT Spt16 ‘‘peptidase’’ domain is a histone H3–H4 binding module Tobias Stuwe, Michael Hothorn*, Erwan Lejeune, Vladimir Rybin, Miriam Bortfeld, Klaus Scheffzek, and Andreas G. Ladurner† European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Edited by Alan R. Fersht, University of Cambridge, Cambridge, United Kingdom, and approved March 27, 2008 (received for review December 28, 2007) The FACT complex is a conserved cofactor for RNA polymerase II scription and replication by removing one H2A–H2B dimer from elongation through nucleosomes. FACT bears histone chaperone nucleosomes, thus relieving the barrier to polymerase progres- activity and contributes to chromatin integrity. However, the sion. After Pol II passage, FACT may restore the proper molecular mechanisms behind FACT function remain elusive. Here chromatin state (14, 20). we report biochemical, structural, and mutational analyses that There is little mechanistic insight into how FACT may be able identify the peptidase homology domain of the Schizosaccharo- to perform its chaperoning functions. In particular, it is unclear myces pombe FACT large subunit Spt16 (Spt16-N) as a binding how it interacts with histones. To start dissecting the structure module for histones H3 and H4. The 2.1-Å crystal structure of and function of the essential Spt16 subunit of FACT, we sought Spt16-N reveals an aminopeptidase P fold whose enzymatic activ- to identify the molecular functions inherent to this Ͼ100-kDa ity has been lost. Instead, the highly conserved fold directly binds multidomain protein. Structural information on FACT exists for histones H3–H4 through a tight interaction with their globular core the HMG-like module Nhp6A from S. -
Nucleosome Loss Leads to Global Transcriptional Up-Regulation and Genomic Instability During Yeast Aging
Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Nucleosome loss leads to global transcriptional up-regulation and genomic instability during yeast aging Zheng Hu,1,6 Kaifu Chen,2,3,6 Zheng Xia,2,3 Myrriah Chavez,1,4 Sangita Pal,1,5 Ja-Hwan Seol,1 Chin-Chuan Chen,1 Wei Li,2,7,8 and Jessica K. Tyler1,7,8 1Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA; 2Dan L. Duncan Cancer Center, 3Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA; 4Molecular Biology Graduate Program, University of Colorado School of Medicine, Denver, Colorado 80010, USA; 5Genes and Development Graduate Program, The University of Texas Graduate School of Biomedical Sciences, Houston, Texas 77030, USA All eukaryotic cells divide a finite number of times, although the mechanistic basis of this replicative aging remains unclear. Replicative aging is accompanied by a reduction in histone protein levels, and this is a cause of aging in budding yeast. Here we show that nucleosome occupancy decreased by 50% across the whole genome during replicative aging using spike-in controlled micrococcal nuclease digestion followed by sequencing. Furthermore, nucleosomes became less well positioned or moved to sequences predicted to better accommodate histone octamers. The loss of histones during aging led to transcriptional induction of all yeast genes. Genes that are normally repressed by promoter nucleosomes were most induced, accompanied by preferential nucleosome loss from their promoters. We also found elevated levels of DNA strand breaks, mitochondrial DNA transfer to the nuclear genome, large-scale chromosomal alterations, translocations, and retrotransposition during aging. -
REVIEW Chromatin Immunoprecipitation: Advancing
R113 REVIEW Chromatin immunoprecipitation: advancing analysis of nuclear hormone signaling Aurimas Vinckevicius and Debabrata Chakravarti Division of Reproductive Biology Research, Department of Obstetrics and Gynecology, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, 303 East Superior Street, Lurie 4-119, Chicago, Illinois 60611, USA (Correspondence should be addressed to D Chakravarti; Email: [email protected]) Abstract Recent decades have been filled with groundbreaking research in the field of endocrine hormone signaling. Pivotal events like the isolation and purification of the estrogen receptor, the cloning of glucocorticoid receptor cDNA, or dissemination of nuclear hormone receptor (NHR) DNA binding sequences are well recognized for their contributions. However, the novel genome-wide and gene-specific information obtained over the last decade describing NHR association with chromatin, cofactors, and epigenetic modifications, as well as their role in gene regulation, has been largely facilitated by the adaptation of the chromatin immunoprecipitation (ChIP) technique. Use of ChIP-based technologies has taken the field of hormone signaling from speculating about the transcription-enabling properties of acetylated chromatin and putative transcription (co-)factor genomic occupancy to demonstrating the detailed, stepwise mechanisms of factor binding and transcriptional initiation; from treating hormone-induced transcription as a steady-state event to understanding its dynamic and cyclic nature; from looking at the DNA sequences recognized by various DNA- binding domains in vitro to analyzing the cell-specific genome-wide pattern of nuclear receptor binding and interpreting its physiological implications. Not only have these events propelled hormone research, but, as some of the pioneering studies, have also contributed tremendously to the field of molecular endocrinology as a whole. -
The Interactions of the Largest Subunit of RNA Polymerase II with Other Cellular Proteins: a Bioinformatic Approach
Curr. Issues Mol. Biol. 11 (Suppl. 1) i65–71. Online journal at www.cimb.org The Interactions of the Largest Subunit of RNA Polymerase II with Other Cellular Proteins: a Bioinformatic Approach Abhijit Shukla1, Aparna Natarajan1, Sukesh protein-protein interactions. Based on these experimental Bhaumik1, Hany A. El-Shemy2 and David studies, several bioinformatic tools have been developed Lightfoot1* to comprehensively analyze protein interaction networks of different cellular proteins. Here, we have used the 1Southern Illinois University School of Medicine, Cytoscape software (Zhang et al., 2007) and Biogrid/ Department of Biochemistry and Molecular Biology, Biomart database to identify the interactions of the Carbondale, IL 62901, USA largest subunit of RNA Polymerase II (RNAPII), Rpb1, 2Faculty of Agriculture Research Park (FARP) and with other proteins in yeast. Such analysis has revealed Biochemistry Department, Faculty of Agriculture, a large number of primary interactions of Rpb1p with University of Cairo, 12613 Giza, Egypt many proteins involved in the regulation of transcription, chromatin structure, DNA repair, and other cellular events Received 5 August 2008 as discussed below. Revised 27 September 2008 Accepted 8 October 2008 Rpb1 and its interactions with other RNAPII subunits The protein coding genes are transcribed into mRNA by RNAPII that is highly conserved from yeast to human. Abstract RNAPII is composed of 12 different subunits. These subunits are Rpb1, Rpb2, Rpb3, Rpb4, Rpb5, Rpb6, The function of a protein is governed by its interaction Rpb7, Rpb8, Rpb9, Rpb10, Rpb11, and Rpb12. Rpb1 with other proteins inside a cell. Therefore, it is important is the largest subunit, and is essential to maintain the to identify the interacting partners of a particular structural integrity of RNAPII.