DOCSLIB.ORG

SMC6 Is an Essential Gene in Mice, but a Hypomorphic Mutant in The

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
SMC6 Is an Essential Gene in Mice, but a Hypomorphic Mutant in The G Model DNAREP-1762; No. of Pages 11 ARTICLE IN PRESS DNA Repair xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect DNA Repair jo urnal homepage: www.elsevier.com/locate/dnarepair SMC6 is an essential gene in mice, but a hypomorphic mutant in the ATPase domain has a mild phenotype with a range of subtle abnormalities a,1 a,1 a,2 b c Limei Ju , Jonathan Wing , Elaine Taylor , Renata Brandt , Predrag Slijepcevic , d d,e d,f d,g d Marion Horsch , Birgit Rathkolb , Ildikó Rácz , Lore Becker , Wolfgang Hans , d,h d,i,k d,j j e Thure Adler , Johannes Beckers , Jan Rozman , Martin Klingenspor , Eckhard Wolf f g,l h d,k , Andreas Zimmer , Thomas Klopstock , Dirk H. Busch , Valérie Gailus-Durner , d,k d,i,k,l b Helmut Fuchs , Martin Hrabeˇ de Angelis , Gilbertus van der Horst , a,∗ Alan R. Lehmann a Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, UK b Department of Cell Biology and Genetics, Erasmus university MC, Rotterdam, The Netherlands c Brunel Institute of Cancer Genetics and Pharmacogenomics, Division of Biosciences, School of Health Sciences & Social Care, Brunel University, Uxbridge, Middlesex, UB8 3PH, UK d German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany e Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian-Universität München, Genecenter, Feodor-Lynen-Str. 25, 81377 Munich, Germany f Institute of Molecular Psychiatry, University of Bonn, Sigmund-Freud-Straße 25, 53105 Bonn, Germany g Department of Neurology, Friedrich-Baur-Institut, Ludwig-Maximilians-Universität München, Ziemssenstraße 1, 80336 Munich, Germany h Institute for Medical Microbiology, Immunology, and Hygiene, Trogerstraße 30, Technische Universität München, 81675 Munich, Germany i Lehrstuhl für Experimentelle Genetik, Wissenschaftszentrum Weihenstephan, Technische Universität München, 85354 Freising, Germany j Molecular Nutritional Medicine, Else-Kröner Fresenius Center, Technische Universität München, 85350 Freising-Weihenstephan, Germany k Member of German Center for Diabetes Research (DZD), Ingolstädter Landstraße 1, 85764 Neuherberg, Germany l German Center for Vertigo and Balance Disorders, Marchioninistraße 15, 81377 Munich, Germany a r t i c l e i n f o a b s t r a c t Article history: Smc5-6 is a highly conserved protein complex related to cohesin and condensin involved in the struc- Received 29 August 2012 tural maintenance of chromosomes. In yeasts the Smc5-6 complex is essential for proliferation and is Received in revised form 5 February 2013 involved in DNA repair and homologous recombination. siRNA depletion of genes involved in the Smc5- Accepted 16 February 2013 6 complex in cultured mammalian cells results in sensitivity to some DNA damaging agents. In order Available online xxx to gain further insight into its role in mammals we have generated mice mutated in the Smc6 gene. A complete knockout resulted in early embryonic lethality, demonstrating that this gene is essential in Keywords: mammals. However, mutation of the highly conserved serine-994 to alanine in the ATP hydrolysis motif DNA repair in the SMC6 C-terminal domain, resulted in mice with a surprisingly mild phenotype. With the neo gene Mouse model selection marker in the intron following the mutation, resulting in reduced expression of the SMC6 gene, Sister-chromatid exchange SMC Protein the mice were reduced in size, but fertile and had normal lifespans. When the neo gene was removed, the mice had normal size, but detailed phenotypic analysis revealed minor abnormalities in glucose tol- erance, haematopoiesis, nociception and global gene expression patterns. Embryonic fibroblasts derived from the ser994 mutant mice were not sensitive to killing by a range of DNA damaging agents, but they were sensitive to the induction of sister chromatid exchanges induced by ultraviolet light or mitomycin C. They also accumulated more oxidative damage than wild-type cells. © 2013 Elsevier B.V. All rights reserved. Abbreviations: HR, homologous recombination; MEF, mouse embryonic fibroblast; MMC, mitomycin C; MMS, methyl methanesulfonate; SCE, sister chromatid exchange; SMC, structural maintenance of chromosomes; UV, ultraviolet light. ∗ Corresponding author. Tel.: +44 1273 678120; fax: +44 1273 678121. E-mail address: [email protected] (A.R. Lehmann). 1 These authors contributed equally. 2 Present address: Lancaster Medical School, Lancaster University, Bailrigg, Lancaster LA1 4YB, UK. 1568-7864/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.dnarep.2013.02.006 Please cite this article in press as: L. Ju, et al., SMC6 is an essential gene in mice, but a hypomorphic mutant in the ATPase domain has a mild phenotype with a range of subtle abnormalities, DNA Repair (2013), http://dx.doi.org/10.1016/j.dnarep.2013.02.006 G Model DNAREP-1762; No. of Pages 11 ARTICLE IN PRESS 2 L. Ju et al. / DNA Repair xxx (2013) xxx–xxx 1. Introduction N-terminal fragment of the protein was made. Mice homozygous for this insertion were embryonic lethal, and we were unable to The structural maintenance of chromosomes (SMC) proteins are establish MEFs from early embryos, suggesting that smc6 is an a family of related proteins that form the cores of three protein essential gene in mice. To generate a hypomorphic mouse with a complexes. Cohesin, containing Smc1 and 3, keeps sister chro- good chance of being viable, we made use of our previous exten- matids together after DNA replication, as well as having roles in sive analysis of S. pombe smc6 mutants. S. pombe smc6-S1045A is a gene expression and DNA repair [1]. Condensin contains Smc2 and separation-of-function mutation and cells with this mutation were 4 and is responsible for chromosome condensation at mitosis [2]. viable but sensitive to DNA damaging agents [25]. Importantly this The Smc5-6 complex is required for repair of DNA by homologous mutation is in the LSGG signature motif, which is conserved not recombination (HR) but its exact role is not fully understood [3,4]. only in all SMC proteins, but also in the ABC family of ATPases [26]. The SMC protein cores of these complexes all adopt a similar The Rad50 protein has a structure closely related to that of the SMC structure. The SMC proteins fold back on themselves via a hinge proteins. Mutation of the corresponding serine in the LSGG motif in the centre of the molecule, such that N and C terminal globular of Rad50 abolishes ATP hydrolysis [26], so we infer that the same domains are brought together to form an ATP binding and hydroly- is likely to be true in the SMC family. For these reasons, we chose sis “head domain”. The N and C terminal domains are joined to the to generate Smc6 mice with the corresponding mutation, namely hinge by long coiled coil domains. Each SMC protein interacts with S994A. Mice homozygous for SMC6-S994A were indeed viable. We its partner protein via their hinge domains, and the head domains describe the mild phenotypic alterations of this mutant mouse line of each partner are brought together to catalyse ATP hydrolysis. found in a comprehensive analysis of dysmorphology, behaviour, The head domains also bind non-SMC components, including a neurology, pain response, energy metabolism, clinical chemistry, so-called kleisin, which bridges the heads. steroid metabolism, immunological parameters, eye alterations, Apart from Smc5 and 6, the yeast Smc5-6 complexes have six cardiovascular and lung functions as well as RNA expression profil- additional components, Nse1-6 [5–7]. Nse1 is a RING finger protein, ing in liver, spleen and testis. We generated embryonic fibroblasts with associated E3 ubiquitin ligase activity [8]. Nse2/Mms21 is a (MEFs) from the mice and report on the DNA damage sensitivity of sumo ligase [6,9,10], which is bound to the Smc5 subunit [11] and these MEFs. sumoylates Smc6 in Schizosaccharomyces pombe [9] and Smc5 in Saccharomyces cerevisiae [6]. Nse3 is related to the MAGE protein family [12] and Nse4 is the kleisin component [13]. At least four 2. 2.Results of these Nse proteins (Nse1-4) have orthologs in human cells [12], whereas orthologs of the poorly conserved Nse5 and 6 have not yet 2.1. SMC6 knock-outs been identified [7,14]. In yeasts two different functions in HR have been delineated. The genes encoding the Smc5 and Smc6 proteins are essential An early function is a requirement for Smc5-6 to load Rad52 in both budding and fission yeast [16]. However, extensive stud- at sites of stalled replication forks [15], whereas the late func- ies using siRNA to deplete different components of the complex tion is an involvement in resolving recombination intermediates in cultured human cells in our and other labs showed minimal [16–19]. In addition Smc5-6 contributes to the regulation of cohesin effects on cell proliferation or viability. Furthermore it has been loading and unloading: (a) the formation of double-strand breaks shown recently that a SMC5 knockout is viable in chicken DT40 in S. cerevisiae triggers genome-wide loading of cohesin, which cells [27]. We therefore considered it worthwhile to examine if requires Smc6 [20]; (b) Smc5-6 is required for removal of cohesin the same was true in mice. We obtained from BayGenomics the at mitosis in S. pombe [21]; (c) Smc5-6 promotes cohesin loading RRT274 strain of mice, which contained an exon trap (consist- ␤ at double-strand breaks in human cells [22]. Together the defects ing of a splice acceptor site upstream of the geo fusion gene) in in homologous recombination and in cohesin regulation result in intron 6 of Smc6 (Fig. 1A), essentially rendering the Smc6 gene non- chromosome segregation defects. The Smc5-6 protein components functional.
Load more

Recommended publications
  • Understanding Molecular Functions of the SMC5/6 Complex
    G C A T T A C G G C A T genes Review Scaffolding for Repair: Understanding Molecular Functions of the SMC5/6 Complex Mariana Diaz 1,2 and Ales Pecinka 1,* ID 1 Institute of Experimental Botany of the Czech Academy of Sciences (IEB), Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelu˚ 31, 77900 Olomouc-Holice, Czech Republic 2 Max Planck Institute for Plant Breeding Research (MPIPZ), Carl-von-Linné-Weg 10, 50829 Cologne, Germany; [email protected] * Correspondence: [email protected]; Tel.: +420-585-238-709 Received: 15 November 2017; Accepted: 4 January 2018; Published: 12 January 2018 Abstract: Chromosome organization, dynamics and stability are required for successful passage through cellular generations and transmission of genetic information to offspring. The key components involved are Structural maintenance of chromosomes (SMC) complexes. Cohesin complex ensures proper chromatid alignment, condensin complex chromosome condensation and the SMC5/6 complex is specialized in the maintenance of genome stability. Here we summarize recent knowledge on the composition and molecular functions of SMC5/6 complex. SMC5/6 complex was originally identified based on the sensitivity of its mutants to genotoxic stress but there is increasing number of studies demonstrating its roles in the control of DNA replication, sister chromatid resolution and genomic location-dependent promotion or suppression of homologous recombination. Some of these functions appear to be due to a very dynamic interaction with cohesin or other repair complexes. Studies in Arabidopsis indicate that, besides its canonical function in repair of damaged DNA, the SMC5/6 complex plays important roles in regulating plant development, abiotic stress responses, suppression of autoimmune responses and sexual reproduction.
    [Show full text]
  • The Genome of Schmidtea Mediterranea and the Evolution Of
    OPEN ArtICLE doi:10.1038/nature25473 The genome of Schmidtea mediterranea and the evolution of core cellular mechanisms Markus Alexander Grohme1*, Siegfried Schloissnig2*, Andrei Rozanski1, Martin Pippel2, George Robert Young3, Sylke Winkler1, Holger Brandl1, Ian Henry1, Andreas Dahl4, Sean Powell2, Michael Hiller1,5, Eugene Myers1 & Jochen Christian Rink1 The planarian Schmidtea mediterranea is an important model for stem cell research and regeneration, but adequate genome resources for this species have been lacking. Here we report a highly contiguous genome assembly of S. mediterranea, using long-read sequencing and a de novo assembler (MARVEL) enhanced for low-complexity reads. The S. mediterranea genome is highly polymorphic and repetitive, and harbours a novel class of giant retroelements. Furthermore, the genome assembly lacks a number of highly conserved genes, including critical components of the mitotic spindle assembly checkpoint, but planarians maintain checkpoint function. Our genome assembly provides a key model system resource that will be useful for studying regeneration and the evolutionary plasticity of core cell biological mechanisms. Rapid regeneration from tiny pieces of tissue makes planarians a prime De novo long read assembly of the planarian genome model system for regeneration. Abundant adult pluripotent stem cells, In preparation for genome sequencing, we inbred the sexual strain termed neoblasts, power regeneration and the continuous turnover of S. mediterranea (Fig. 1a) for more than 17 successive sib- mating of all cell types1–3, and transplantation of a single neoblast can rescue generations in the hope of decreasing heterozygosity. We also developed a lethally irradiated animal4. Planarians therefore also constitute a a new DNA isolation protocol that meets the purity and high molecular prime model system for stem cell pluripotency and its evolutionary weight requirements of PacBio long-read sequencing12 (Extended Data underpinnings5.
    [Show full text]
  • PAX3-FOXO1 Candidate Interactors
    Supplementary Table S1: PAX3-FOXO1 candidate interactors Total number of proteins: 230 Nuclear proteins : 201 Exclusive unique peptide count RH4 RMS RMS RMS Protein name Gen name FLAG#1 FLAG#2 FLAG#3 FLAG#4 Chromatin regulating complexes Chromatin modifying complexes: 6 Proteins SIN 3 complex Histone deacetylase complex subunit SAP18 SAP18 2664 CoRESt complex REST corepressor 1 RCOR1 2223 PRC1 complex E3 ubiquitin-protein ligase RING2 RNF2/RING1B 1420 MLL1/MLL complex Isoform 14P-18B of Histone-lysine N-methyltransferase MLL MLL/KMT2A 0220 WD repeat-containing protein 5 WDR5 2460 Isoform 2 of Menin MEN1 3021 Chromatin remodelling complexes: 22 Proteins CHD4/NuRD complex Isoform 2 of Chromodomain-helicase-DNA-binding protein 4 CHD4 3 21 6 0 Isoform 2 of Lysine-specific histone demethylase 1A KDM1A/LSD1a 3568 Histone deacetylase 1 HDAC1b 3322 Histone deacetylase 2 HDAC2b 96710 Histone-binding protein RBBP4 RBBP4b 10 7 6 7 Histone-binding protein RBBP7 RBBP7b 2103 Transcriptional repressor p66-alpha GATAD2A 6204 Metastasis-associated protein MTA2 MTA2 8126 SWI/SNF complex BAF SMARCA4 isoform SMARCA4/BRG1 6 13 10 0 AT-rich interactive domain-containing protein 1A ARID1A/BAF250 2610 SWI/SNF complex subunit SMARCC1 SMARCC1/BAF155c 61180 SWI/SNF complex subunit SMARCC2 SMARCC2/BAF170c 2200 Isoform 2 of SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1 SMARCD1/BAF60ac 2004 Isoform 2 of SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 3 SMARCD3/BAF60cc 7209
    [Show full text]
  • Anti-SMC6 (S7822)
    Anti-SMC6 produced in rabbit, IgG fraction of antiserum Catalog Number S7822 Product Description Reagent Anti-SMC6 is produced in rabbit using as immunogen a Supplied as a solution in 0.01 M phosphate buffered synthetic peptide corresponding to amino acids 766-779 saline, pH 7.4, containing 15 mM sodium azide as a of human SMC6 (Gene ID: 79677) conjugated to KLH preservative. via an N-terminal cysteine residue. This sequence differs by one amino acid in mouse, and by two amino Precautions and Disclaimer acids in rat. Whole antiserum is fractionated and then This product is for R&D use only, not for drug, further purified by ion-exchange chromatography to household, or other uses. Please consult the Material provide the IgG fraction of antiserum that is essentially Safety Data Sheet for information regarding hazards free of other rabbit serum proteins. and safe handling practices. Anti-SMC6 (also known as SMC6L1) specifically Storage/Stability recognizes SMC6 by immunoblotting (126 kDa). For continuous use, store at 2-8 °C for up to one month. Staining of the SMC6 bands in immunoblotting is For extended storage, freeze in working aliquots. specifically inhibited by the immunizing peptide. Repeated freezing and thawing, or storage in “frost- free” freezers, is not recommended. If slight turbidity Proper cohesion of sister chromatids is a prerequisite occurs upon prolonged storage, clarify the solution by for the correct segregation of chromosomes during cell centrifugation before use. Working dilutions should be division. The cohesin chromosome complex is required discarded if not used within 12 hours. for sister chromatid cohesion.1 There are at least six SMC (Structural Maintenance of Chromosomes) family Product Profile members that form three heterodimers in specific Immunoblotting: a working dilution of 1:250-1:500 is combinations.
    [Show full text]
  • A Localized Nucleolar DNA Damage Response Facilitates Recruitment of the Homology-Directed Repair Machinery Independent of Cell Cycle Stage
    Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press A localized nucleolar DNA damage response facilitates recruitment of the homology-directed repair machinery independent of cell cycle stage Marjolein van Sluis and Brian McStay Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland, Galway, Ireland DNA double-strand breaks (DSBs) are repaired by two main pathways: nonhomologous end-joining and homologous recombination (HR). Repair pathway choice is thought to be determined by cell cycle timing and chromatin context. Nucleoli, prominent nuclear subdomains and sites of ribosome biogenesis, form around nucleolar organizer regions (NORs) that contain rDNA arrays located on human acrocentric chromosome p-arms. Actively transcribed rDNA repeats are positioned within the interior of the nucleolus, whereas sequences proximal and distal to NORs are packaged as heterochromatin located at the nucleolar periphery. NORs provide an opportunity to investigate the DSB response at highly transcribed, repetitive, and essential loci. Targeted introduction of DSBs into rDNA, but not abutting sequences, results in ATM-dependent inhibition of their transcription by RNA polymerase I. This is coupled with movement of rDNA from the nucleolar interior to anchoring points at the periphery. Reorganization renders rDNA accessible to repair factors normally excluded from nucleoli. Importantly, DSBs within rDNA recruit the HR machinery throughout the cell cycle. Additionally, unscheduled DNA synthesis, consistent with HR at damaged NORs, can be observed in G1 cells. These results suggest that HR can be templated in cis and suggest a role for chromosomal context in the maintenance of NOR genomic stability.
    [Show full text]
  • Functional Enrichments of Disease Variants Indicate Hundreds of Independent Loci Across Eight Diseases
    Functional enrichments of disease variants indicate hundreds of independent loci across eight diseases Abhishek K. Sarkar, Lucas D. Ward, & Manolis Kellis 1.00 0.75 Cohort correlation !"SS 0.50 #AN"$" %&!" N"!"C1 N"!"C2 Pearson 0.25 'verall )TC## 0.00 Hold-out -0.25 0 25000 50000 75000 100000 Top n SNPs (full meta-analysis) Supplementary Figure 1: Correlation between individual cohort 푧-scores and meta-analyzed 푧- scores of the remainder in a study of rheumatoid arthritis considering increasing number of SNPs. SNPs are ranked by 푝-value in the overall meta-analysis. Overall correlation is between sample-size weighted 푧-scores and published inverse-variance weighted 푧-scores. 1 15-state model, 5 marks, 127 epigenomes Cell type/ tissue group Epigenome name Addtl marks H3K4me1 H3K4me3 H3K36me3 H3K27me3 H3K9me3 H3K27ac H3K9ac DNase-Seq DNA methyl RNA-Seq EID states Chrom. E017 IMR90 fetal lung fibroblasts Cell Line 21 IMR90 E002 ES-WA7 Cell Line E008 H9 Cell Line 21 E001 ES-I3 Cell Line E015 HUES6 Cell Line ESC E014 HUES48 Cell Line E016 HUES64 Cell Line E003 H1 Cell Line 20 E024 ES-UCSF4 Cell Line E020 iPS-20b Cell Line E019 iPS-18 Cell Line iPSC E018 iPS-15b Cell Line E021 iPS DF 6.9 Cell Line E022 iPS DF 19.11 Cell Line E007 H1 Derived Neuronal Progenitor Cultured Cells 13 E009 H9 Derived Neuronal Progenitor Cultured Cells 1 E010 H9 Derived Neuron Cultured Cells 1 E013 hESC Derived CD56+ Mesoderm Cultured Cells ES-deriv E012 hESC Derived CD56+ Ectoderm Cultured Cells E011 hESC Derived CD184+ Endoderm Cultured Cells E004 H1 BMP4 Derived Mesendoderm Cultured Cells 11 E005 H1 BMP4 Derived Trophoblast Cultured Cells 15 E006 H1 Derived Mesenchymal Stem Cells 13 E062 Primary mononuclear cells from peripheral blood E034 Primary T cells from peripheral blood E045 Prim.
    [Show full text]
  • The Genetic Program of Pancreatic Beta-Cell Replication in Vivo
    Page 1 of 65 Diabetes The genetic program of pancreatic beta-cell replication in vivo Agnes Klochendler1, Inbal Caspi2, Noa Corem1, Maya Moran3, Oriel Friedlich1, Sharona Elgavish4, Yuval Nevo4, Aharon Helman1, Benjamin Glaser5, Amir Eden3, Shalev Itzkovitz2, Yuval Dor1,* 1Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel 2Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. 3Department of Cell and Developmental Biology, The Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel 4Info-CORE, Bioinformatics Unit of the I-CORE Computation Center, The Hebrew University and Hadassah, The Institute for Medical Research Israel- Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel 5Endocrinology and Metabolism Service, Department of Internal Medicine, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel *Correspondence: [email protected] Running title: The genetic program of pancreatic β-cell replication 1 Diabetes Publish Ahead of Print, published online March 18, 2016 Diabetes Page 2 of 65 Abstract The molecular program underlying infrequent replication of pancreatic beta- cells remains largely inaccessible. Using transgenic mice expressing GFP in cycling cells we sorted live, replicating beta-cells and determined their transcriptome. Replicating beta-cells upregulate hundreds of proliferation- related genes, along with many novel putative cell cycle components. Strikingly, genes involved in beta-cell functions, namely glucose sensing and insulin secretion were repressed. Further studies using single molecule RNA in situ hybridization revealed that in fact, replicating beta-cells double the amount of RNA for most genes, but this upregulation excludes genes involved in beta-cell function.
    [Show full text]
  • Supplemental Methods Definition of Clinical Outcomes and Statistics
    Supplemental methods Definition of clinical outcomes and statistics Overall survival was defined from time of diagnosis until death or last follow up. Time to locoregional recurrence (LRR) or distant metastasis (DM) was defined as time from diagnosis until either an event or last follow up. Clinical variables examined included tumor stage, nodal stage and primary tumor site. Univariate analysis was performed using Cox-regression (SPSS v25). Kaplan Meier curves were generated, and group comparisons were performed using log-rank statistics. In vivo shRNA screening Each library was cloned into the pRSI17 vector (Cellecta) and packed into lentivirus particles. HNSCC cell lines were infected in vitro through spinfection with virus containing the library at a low MOI (~20% infected cells as measured by flow cytometry) in order to minimize superinfection of cells. Cells were selected with puromycin for at least 2 days and grown in vitro for <3 population doublings prior to injection of 4 million cells subcutaneously in nude mouse flank. An additional 2 million cells from the day of injection were collected as a frozen reference cell pellet. Resulting xenografts were treated with 2 Gy/day of radiation once the tumor had reached approximately 100 mm3 to a total dose of 6-10 Gy depending upon the model. Following treatment the tumors were allowed to grow for approximately 2 weeks (volume ~ 500mm3). DNA was isolated from tumor and reference cells, amplified, and sequenced on Illumina sequencers as previously described 12. Hairpin counts were normalized to counts per million (CPM) per sample to enable comparison across samples. For each sample, (log2) fold-change of each hairpin in the tumor was calculated compared to the level in the reference pellet.
    [Show full text]
  • Supplemental Table 1A. Differential Gene Expression Profile of Adehcd40l and Adehnull Treated Cells Vs Untreated Cells
    Supplemental Table 1a. Differential Gene Expression Profile of AdEHCD40L and AdEHNull treated cells vs Untreated Cells Fold change Regulation Fold change Regulation ([AdEHCD40L] vs ([AdEHCD40L] ([AdEHNull] vs ([AdEHNull] vs Probe Set ID [Untreated]) vs [Untreated]) [Untreated]) [Untreated]) Gene Symbol Gene Title RefSeq Transcript ID NM_001039468 /// NM_001039469 /// NM_004954 /// 203942_s_at 2.02 down 1.00 down MARK2 MAP/microtubule affinity-regulating kinase 2 NM_017490 217985_s_at 2.09 down 1.00 down BAZ1A fibroblastbromodomain growth adjacent factor receptorto zinc finger 2 (bacteria-expressed domain, 1A kinase, keratinocyte NM_013448 /// NM_182648 growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer 203638_s_at 2.10 down 1.01 down FGFR2 syndrome, Jackson-Weiss syndrome) NM_000141 /// NM_022970 1570445_a_at 2.07 down 1.01 down LOC643201 hypothetical protein LOC643201 XM_001716444 /// XM_001717933 /// XM_932161 231763_at 3.05 down 1.02 down POLR3A polymerase (RNA) III (DNA directed) polypeptide A, 155kDa NM_007055 1555368_x_at 2.08 down 1.04 down ZNF479 zinc finger protein 479 NM_033273 /// XM_001714591 /// XM_001719979 241627_x_at 2.15 down 1.05 down FLJ10357 hypothetical protein FLJ10357 NM_018071 223208_at 2.17 down 1.06 down KCTD10 potassium channel tetramerisation domain containing 10 NM_031954 219923_at 2.09 down 1.07 down TRIM45 tripartite motif-containing 45 NM_025188 242772_x_at 2.03 down 1.07 down Transcribed locus 233019_at 2.19 down 1.08 down CNOT7 CCR4-NOT transcription complex, subunit 7 NM_013354
    [Show full text]
  • Integrative Analysis Reveals Unique Features of the Smc5/6 Complex
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.31.424863; this version posted January 1, 2021. 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. Integrative analysis reveals unique features of the Smc5/6 complex You Yu1*, Shibai Li2*, Zheng Ser3,4*, Tanmoy Sanyal5,6*, Koyi Choi2, Bingbing Wan2,#, Andrej Sali5,6,7, Alex Kentsis3, Dinshaw J. Patel1 and Xiaolan Zhao2 1, Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA 2, Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA 3, Molecular Pharmacology Program, Tow Center for Developmental Oncology, Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA 4, Tri-Institutional PhD Program in Chemical Biology, New York, NY 10065, USA 5, Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94158, USA 6, Quantitative Biosciences Institute, University of California San Francisco, San Francisco, CA, 94158, USA 7, Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA94158, USA. # Current address: Key Laboratory of Systems Biomedicine and Collaborative Innovation Center of Systems Biomedicine, Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China * These authors contributed equally to this work. Corresponding authors: [email protected] (XZ) and [email protected] (DJP) 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.31.424863; this version posted January 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.
    [Show full text]
  • Explore a Novel Function of Human Condensins in Cellular Senescence Hongzhen Wang1,2* , Xin Liu1 and Guiying Li2
    Wang et al. Cell Biosci (2020) 10:147 https://doi.org/10.1186/s13578-020-00512-1 Cell & Bioscience REVIEW Open Access Explore a novel function of human condensins in cellular senescence Hongzhen Wang1,2* , Xin Liu1 and Guiying Li2 Abstract There are two kinds of condensins in human cells, known as condensin I and condensin II. The canonical roles of condensins are participated in chromosome dynamics, including chromosome condensation and segregation during cell division. Recently, a novel function of human condensins has been found with increasing evidences that they play important roles in cellular senescence. This paper reviewed the research progress of human condensins involved in diferent types of cellular senescence, mainly oncogene-induced senescence (OIS) and replicative senescence (RS). The future perspectives of human condensins involved in cellular senescence are also discussed. Keywords: Human condensins, Cellular senescence, Oncogene-induced senescence, Replicative senescence Introduction Although both human condensins have similar com- Condensins were frstly identifed for their fundamen- ponents and alphabetic structure, they show diferent tal roles in establishment and maintenance of mitotic nuclear distribution, localization on chromosomes and chromosome condensation in cell-free system from play distinct roles in chromosome dynamics during mito- Xenopus laevis eggs [1, 2]. Until now, most multicellu- sis [9–13]. lar eukaryotes reported have two kinds of condensins, In detail, during interphase condensin I is present in
    [Show full text]
  • Nucleolar Organizer Regions: Genomic 'Dark Matter' Requiring Illumination
    Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Nucleolar organizer regions: genomic ‘dark matter’ requiring illumination Brian McStay Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland, Galway, Ireland Nucleoli form around tandem arrays of a ribosomal gene The relationship between nucleolar organizer regions repeat, termed nucleolar organizer regions (NORs). Dur- (NORs) and nucleoli was first established in the 1930s ing metaphase, active NORs adopt a characteristic under- (Heitz 1931; McClintock 1934), but, for decades, the con- condensed morphology. Recent evidence indicates that tent of the former and the role of the latter remained mys- the HMG-box-containing DNA-binding protein UBF (up- terious. The era of molecular and cellular biology revealed stream binding factor) is directly responsible for this mor- that NORs contain tandem arrays of ribosomal gene phology and provides a mitotic bookmark to ensure rapid (rDNA) repeats and that nucleoli are the sites of ribosome nucleolar formation beginning in telophase in human biogenesis. Biochemistry has revealed the inner workings cells. This is likely to be a widely employed strategy, as of the nucleolus and the complexity of ribosome biogene- UBF is present throughout metazoans. In higher eukary- sis (for review, see Pederson 2010). However, the genomic otes, NORs are typically located within regions of chro- architecture of NORs and the chromosomal context in mosomes that form perinucleolar heterochromatin which they lie remains undetermined for most eukary- during interphase. Typically, the genomic architecture otes. The resulting void has placed limitations on our un- of NORs and the chromosomal regions within which derstanding of the fundamental mechanisms by which they lie is very poorly described, yet recent evidence NORs orchestrate formation of the largest structure in points to a role for context in their function.
    [Show full text]