DOCSLIB.ORG

Identification of MAZ As a Novel Transcription Factor Regulating Erythropoiesis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

Identification of MAZ as a novel transcription factor regulating erythropoiesis Darya Deen1, Falk Butter2, Michelle L. Holland3, Vasiliki Samara4, Jacqueline A. Sloane-Stanley4, Helena Ayyub4, Matthias Mann5, David Garrick4,6,7 and Douglas Vernimmen1,7 1 The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, United Kingdom. 2 Institute of Molecular Biology (IMB), 55128 Mainz, Germany 3 Department of Medical and Molecular Genetics, School of Basic and Medical Biosciences, King's College London, London SE1 9RT, UK 4 MRC Molecular Haematology Unit, Weatherall Institute for Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom. 5 Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany. 6 Current address : INSERM UMRS-976, Institut de Recherche Saint Louis, Université de Paris, 75010 Paris, France. 7 These authors contributed equally Correspondence: [email protected], [email protected] ADDITIONAL MATERIAL Suppl Fig 1. Erythroid-specific hypersensitivity regions DNAse-Seq. (A) Structure of the α-globin locus. Chromosomal position (hg38 genome build) is shown above the genes. The locus consists of the embryonic ζ gene (HBZ), the duplicated foetal/adult α genes (HBA2 and HBA1) together with two flanking pseudogenes (HBM and HBQ1). The upstream, widely-expressed gene, NPRL3 is transcribed from the opposite strand to that of the HBA genes and contains the remote regulatory elements of the α-globin locus (MCS -R1 to -R3) (indicated by red dots). Below is shown the ATAC-seq signal in human erythroblasts (taken from 8) indicating open chromatin structure at promoter and distal regulatory elements (shaded regions). (B) Fine- mapping of the promoter region of the HBA genes. Nuclei from EBV and K562 were 1 exposed to the restriction enzymes BfaI, BsrFI, FokI, HinfI, EagI, NcoI, MseI and HindIII as indicated. DNA was subsequently purified, limit-digested with PstI and analysed by Southern blot using the 3’ HindIII/PstI segment of the HBA1 gene as a probe (blue rectangle). Suppl Figure 2. Both probes 11/12 and 13/14 bind the (d) complex. (A) Comparison between probe 11/12 and probe 13/14. The predicted core MAZ-binding motif is highlighted. (B) Gel-shift assays showing that both probes bind the (d) complex. The affinity of the (d) complex is stronger for the 13/14 probed as demonstrated by using unlabelled oligonucleotides as competitor (100-fold molar excess over radiolabelled probe). Suppl Figure 3. Characterisation of DNA binding of unknown protein by EMSA. (A) Increased concentration of EDTA abolished the protein/DNA complex for bands (a), (b), (c), and (d) with the probe 13/14, but not for the probe 7/8, which is bound by a non-Zinc finger protein, CBFβ (see Figure 1B). (B) A single nucleotide mutation (G- >T) abolishes binding of the (d) complex to probe 13/14. The sequence of the wild type and mutant (M3) probes are shown above, with the mutated nucleotide indicated in red. EMSA were carried out using K562 nuclear extracts and either wild type or mutant probes together with unlabelled competitor oligonucleotides (100-fold molar excess) as indicated. Suppl Figure 4. Dynamics of MAZ recruitment during erythroid differentiation. (A) Diagram representing the expression of the transferrin receptor (CD71) and glycophorin A (GPA) during erythroblast differentiation in Phase II culture conditions. (B) Flow cytometry analysis of CD71 and GPA during Phase II of a representative 2 primary erythroblast differentiation culture. Cells initially acquire expression of CD71 alone (d6) before becoming double positive for both CD71 and GPA (d9-12). At later stages in culture, expression of CD71 decreases (d15). (C) Quantitation of flow cytometry staining as demonstrated in (B). Shown is the mean and standard deviation from two independent differentiation cultures. (D) Real-time RT-PCR analysis of expression of α- (HBA2) and β- (HBB) globin at the indicated time points during Phase II of primary erythroid differentiation cultures. Expression (relative to GAPDH) shows the mean and range from two independent differentiation cultures. (E) ChIP-qPCR analysis of MAZ binding at the a-globin genes at the indicated time points during Phase II of primary erythroid differentiation cultures. The y axis represents enrichment over the input DNA, normalised to a control sequence in the human 18S gene. The x axis indicates the Taqman probes used. The positions of probes at the HBA Promoter/Exon1 and HBA Ex2, and probe 82 (used as a negative control) are indicated in Figure 2E. The promoter of the MYC gene, which has been previously shown to be bound by MAZ 57 was used as a positive control for the ChIP. Suppl Figure 5. MAZ ChIP-Seq quality control metrics. (A) Fingerprint of MAZ ChIP-Seq dataset compared to input showing an enrichment of DNA fragments for MAZ. (B) Cross-correlation plot profile for MAZ ChIP-Seq dataset indicating the normalized strand coefficient (NSC) and the ratio between the fragment-length peak and the read-length peak (relative strand correlation, RSC) for assessing signal-to- noise ratios. (C) Distribution of MAZ ChIP-seq signal relative to the called MAZ peaks. (D) The average relative MAZ signal around the MAZ peaks located at the promoters, intergenic and genic regions. 3 Suppl Figure 6. DNA binding specificities of MAZ. EMSA assays were carried out with the indicated probes and K562 nuclear extract. The consensus G3CG4 MAZ-binding motif is shown in bold. Variation from the wild type 13/14 probe in mutant probes is indicated in red. Variation from the G3CG4 consensus in probe 11/12 is indicated in blue. Suppl. Figure 7. MAZ occupies loci coding for erythroid transcription factors GATA1 and KLF1. MAZ ChIP-seq enrichment profile at GATA1 and KLF1. Table S1. Results of affinity-purification screen. Detected proteins with their gene names, description and unique identifiers (Uniprot and Ensembl). Pfam annotations (protein domains) were mapped to the protein entries. Table contains identified number of MS/MS spectra for proteins bound to WT and the M3 oligonucleotide (from two independent replicate experiments). Ratio for enrichment between the two oligonucleotide baits is calculated based on protein intensity and significance from MaxQuant analysis 44. Table S2. Erythroid-related traits used to search in the GeneATLAS database for MAZ variants. Table S3. Tissue specific MAZ peaks in ENCODE human cell lines and human primary erythroid cells. Table S4. Location of MAZ peaks within genomic loci associated with erythroid traits and erythropoiesis. Table S5. Sequences of oligonucleotides used in this study Table S6. Datasets used in the study 4 Deen et al., A B Pst fragmentI parent Suppl EBV-lymph K562 Figure 1 EBV-lymph K562 Bfa EBV-lymph I K562 HBA2 Bsr F I EBV-lymph K562 Fok EBV-lymph I K562 Hinf EBV-lymph HBA1 I K562 Eag EBV-lymph I K562 Nco EBV-lymph I K562 Mse I Hind III Deen et al., Suppl. Figure 3 A B 13/14 (WT) : 5 AGT CCC GGG CGG GGC CTG 13/14 (M3) : 5 AGT CCC GGG CTG GGC CTG EDTA a Nuclear extract: K562 K562 CBFb b Probe: 13/14 (WT) 13/14 (M3) c comp (100 X): none WT M3 none WT M3 d Sp1 Sp3 BKLF d 13/14 probe 7/8 probe K562 NE K562 NE Deen et al., Suppl Figure 4 A CMP CFUe Proerythroblast Early Intermediate Late (Early) Erythroblasts CD71 GPA d3 - d6 - d9 - d12 - d15 B Ph II : d0 d3 d6 d9 d12 d15 GPA CD71 C BFU-E CFU-E and Pro-eryrthroblast Intermediate erythroblast late erythroblast (CD71-GPA-) (CD71+GPAlo/int) (CD71+GPA+) (CD71-GPA+) 80 50 100 35 30 70 80 40 25 60 60 20 50 30 15 40 40 20 % of cells of% 30 % of cells of % % of cells of% 10 cells of % % of cells of % 20 20 5 10 10 0 0 0 0 0 3 6 9 12 15 0 3 6 9 12 15 0 3 6 9 12 15 0 3 6 9 12 15 Days in Ph II Days in Ph II Days in Ph II Days in Ph II D HBA2 HBB 20 40 35 16 30 12 25 20 8 15 10 4 5 Signal GAPDH to Signal relative Signal relative to GAPDHto Signal relative 0 0 0 3 6 9 12 15 0 3 6 9 12 15 Days in Ph II Days in Ph II E Deen et al., Suppl Figure 5 A B C D Deen et al., Suppl Figure 6 M1) Oligo Sequence Binding of MAZ Probe : 13/14 (WT)13/14 ( 13/14 (M2)13/14 (M3)13/14 (M4) 11/12 13/14 (WT) : 5 AGT CCC GGG CGG GGC CTG + 13/14 (M1) : 5 CAC ACC GGG CGG GGC CTG + 13/14 (M2) : 5 AGT CCC GGG CGT ATA CTG - 13/14 (M3) : 5 AGT CCC GGG CTG GGC CTG - (see also Sup. Fig.3B) 13/14 (M4) : 5 AGT CCC GTG CGG GGC CTG - 11/12 (WT): 5 GC CCG GGG CGC GGC CTG < K562 NE Gene Names Protein Names Mol. Weight [kDa] Uniprot Pfam Pfam Descriptions ENSEMBL Chrom Strand Position MS/MS Count neg1 MS/MS Count neg2 MS/MS Count pos1 MS/MS Count pos2 NEG/POS POS/NEG NEG/POS Significance Down FAM139A;FLJ40722;tcag7.730;FLJ00264 Uncharacterized protein FAM139A;Family with sequence similarity 139, member A;Putative uncharacterized100.9 protein FLJ40722;UncharacterizedA6NFQ2;Q17RQ4;Q14D25;Q8IWQ0;Q8N1I1;A6NGZ5;Q8NF84 protein ENSP00000291129 ENSG00000170379;ENSG000001598607 + 143028667 0 0 0 1 0.034891007 28.66067999 1.28E-08 GNL3;E2IG3;NS Guanine nucleotide-binding protein-like 3;Nucleolar GTP-binding protein 3;Nucleostemin;E2-induced gene62.0 3 protein;NovelQ9BVP2;Q5PU80 nucleolar protein 47;NNP47 PF08701;PF01926 GNL3L/Grn1 putative GTPase;GTPase of unknown functionENSG00000163938 3 + 52694976 0 0 0 7 0.03628214 27.56177032 1.86E-08 ANKK1;PKK2;SGK288 Ankyrin repeat and protein kinase domain-containing protein 1;Protein kinase PKK2;X-kinase;Sugen kinase84.632 288;SgK288Q8NFD2 PF00023;PF00069 Ankyrin repeat;Protein kinase domain
Recommended publications
  • Efficacy and Mechanistic Evaluation of Tic10, a Novel Antitumor Agent

    Efficacy and Mechanistic Evaluation of Tic10, a Novel Antitumor Agent

    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2012 Efficacy and Mechanisticv E aluation of Tic10, A Novel Antitumor Agent Joshua Edward Allen University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Oncology Commons Recommended Citation Allen, Joshua Edward, "Efficacy and Mechanisticv E aluation of Tic10, A Novel Antitumor Agent" (2012). Publicly Accessible Penn Dissertations. 488. https://repository.upenn.edu/edissertations/488 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/488 For more information, please contact [email protected]. Efficacy and Mechanisticv E aluation of Tic10, A Novel Antitumor Agent Abstract TNF-related apoptosis-inducing ligand (TRAIL; Apo2L) is an endogenous protein that selectively induces apoptosis in cancer cells and is a critical effector in the immune surveillance of cancer. Recombinant TRAIL and TRAIL-agonist antibodies are in clinical trials for the treatment of solid malignancies due to the cancer-specific cytotoxicity of TRAIL. Recombinant TRAIL has a short serum half-life and both recombinant TRAIL and TRAIL receptor agonist antibodies have a limited capacity to perfuse to tissue compartments such as the brain, limiting their efficacy in certain malignancies. To overcome such limitations, we searched for small molecules capable of inducing the TRAIL gene using a high throughput luciferase reporter gene assay. We selected TRAIL-inducing compound 10 (TIC10) for further study based on its induction of TRAIL at the cell surface and its promising therapeutic index. TIC10 is a potent, stable, and orally active antitumor agent that crosses the blood-brain barrier and transcriptionally induces TRAIL and TRAIL-mediated cell death in a p53-independent manner.
  • Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model

    Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model

    Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 T + is online at: average * The Journal of Immunology , 34 of which you can access for free at: 2016; 197:1477-1488; Prepublished online 1 July from submission to initial decision 4 weeks from acceptance to publication 2016; doi: 10.4049/jimmunol.1600589 http://www.jimmunol.org/content/197/4/1477 Molecular Profile of Tumor-Specific CD8 Cell Hypofunction in a Transplantable Murine Cancer Model Katherine A. Waugh, Sonia M. Leach, Brandon L. Moore, Tullia C. Bruno, Jonathan D. Buhrman and Jill E. Slansky J Immunol cites 95 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html http://www.jimmunol.org/content/suppl/2016/07/01/jimmunol.160058 9.DCSupplemental This article http://www.jimmunol.org/content/197/4/1477.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 25, 2021. The Journal of Immunology Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model Katherine A.
  • Allele-Specific Expression of Ribosomal Protein Genes in Interspecific Hybrid Catfish

    Allele-Specific Expression of Ribosomal Protein Genes in Interspecific Hybrid Catfish

    Allele-specific Expression of Ribosomal Protein Genes in Interspecific Hybrid Catfish by Ailu Chen A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama August 1, 2015 Keywords: catfish, interspecific hybrids, allele-specific expression, ribosomal protein Copyright 2015 by Ailu Chen Approved by Zhanjiang Liu, Chair, Professor, School of Fisheries, Aquaculture and Aquatic Sciences Nannan Liu, Professor, Entomology and Plant Pathology Eric Peatman, Associate Professor, School of Fisheries, Aquaculture and Aquatic Sciences Aaron M. Rashotte, Associate Professor, Biological Sciences Abstract Interspecific hybridization results in a vast reservoir of allelic variations, which may potentially contribute to phenotypical enhancement in the hybrids. Whether the allelic variations are related to the downstream phenotypic differences of interspecific hybrid is still an open question. The recently developed genome-wide allele-specific approaches that harness high- throughput sequencing technology allow direct quantification of allelic variations and gene expression patterns. In this work, I investigated allele-specific expression (ASE) pattern using RNA-Seq datasets generated from interspecific catfish hybrids. The objective of the study is to determine the ASE genes and pathways in which they are involved. Specifically, my study investigated ASE-SNPs, ASE-genes, parent-of-origins of ASE allele and how ASE would possibly contribute to heterosis. My data showed that ASE was operating in the interspecific catfish system. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5,420 (8.2%) and 13,390 (7.5%) SNPs were identified as significant ASE-SNPs, respectively.
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
  • Steroid-Dependent Regulation of the Oviduct: a Cross-Species Transcriptomal Analysis

    Steroid-Dependent Regulation of the Oviduct: a Cross-Species Transcriptomal Analysis

    University of Kentucky UKnowledge Theses and Dissertations--Animal and Food Sciences Animal and Food Sciences 2015 Steroid-dependent regulation of the oviduct: A cross-species transcriptomal analysis Katheryn L. Cerny University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Cerny, Katheryn L., "Steroid-dependent regulation of the oviduct: A cross-species transcriptomal analysis" (2015). Theses and Dissertations--Animal and Food Sciences. 49. https://uknowledge.uky.edu/animalsci_etds/49 This Doctoral Dissertation is brought to you for free and open access by the Animal and Food Sciences at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Animal and Food Sciences by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known.
  • Protein Identities in Evs Isolated from U87-MG GBM Cells As Determined by NG LC-MS/MS

    Protein Identities in Evs Isolated from U87-MG GBM Cells As Determined by NG LC-MS/MS

    Protein identities in EVs isolated from U87-MG GBM cells as determined by NG LC-MS/MS. No. Accession Description Σ Coverage Σ# Proteins Σ# Unique Peptides Σ# Peptides Σ# PSMs # AAs MW [kDa] calc. pI 1 A8MS94 Putative golgin subfamily A member 2-like protein 5 OS=Homo sapiens PE=5 SV=2 - [GG2L5_HUMAN] 100 1 1 7 88 110 12,03704523 5,681152344 2 P60660 Myosin light polypeptide 6 OS=Homo sapiens GN=MYL6 PE=1 SV=2 - [MYL6_HUMAN] 100 3 5 17 173 151 16,91913397 4,652832031 3 Q6ZYL4 General transcription factor IIH subunit 5 OS=Homo sapiens GN=GTF2H5 PE=1 SV=1 - [TF2H5_HUMAN] 98,59 1 1 4 13 71 8,048185945 4,652832031 4 P60709 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 SV=1 - [ACTB_HUMAN] 97,6 5 5 35 917 375 41,70973209 5,478027344 5 P13489 Ribonuclease inhibitor OS=Homo sapiens GN=RNH1 PE=1 SV=2 - [RINI_HUMAN] 96,75 1 12 37 173 461 49,94108966 4,817871094 6 P09382 Galectin-1 OS=Homo sapiens GN=LGALS1 PE=1 SV=2 - [LEG1_HUMAN] 96,3 1 7 14 283 135 14,70620005 5,503417969 7 P60174 Triosephosphate isomerase OS=Homo sapiens GN=TPI1 PE=1 SV=3 - [TPIS_HUMAN] 95,1 3 16 25 375 286 30,77169764 5,922363281 8 P04406 Glyceraldehyde-3-phosphate dehydrogenase OS=Homo sapiens GN=GAPDH PE=1 SV=3 - [G3P_HUMAN] 94,63 2 13 31 509 335 36,03039959 8,455566406 9 Q15185 Prostaglandin E synthase 3 OS=Homo sapiens GN=PTGES3 PE=1 SV=1 - [TEBP_HUMAN] 93,13 1 5 12 74 160 18,68541938 4,538574219 10 P09417 Dihydropteridine reductase OS=Homo sapiens GN=QDPR PE=1 SV=2 - [DHPR_HUMAN] 93,03 1 1 17 69 244 25,77302971 7,371582031 11 P01911 HLA class II histocompatibility antigen,
  • Targeting Tyro3 Ameliorates a Model of PGRN-Mutant FTLD-TDP Via Tau

    Targeting Tyro3 Ameliorates a Model of PGRN-Mutant FTLD-TDP Via Tau

    ARTICLE DOI: 10.1038/s41467-018-02821-z OPEN Targeting Tyro3 ameliorates a model of PGRN- mutant FTLD-TDP via tau-mediated synaptic pathology Kyota Fujita1, Xigui Chen1, Hidenori Homma1, Kazuhiko Tagawa1, Mutsuki Amano 2, Ayumu Saito3, Seiya Imoto 4, Hiroyasu Akatsu5, Yoshio Hashizume6, Kozo Kaibuchi2, Satoru Miyano3 & Hitoshi Okazawa1 PGRN 1234567890():,; Mutations in the progranulin ( ) gene cause a tau pathology-negative and TDP43 pathology-positive form of frontotemporal lobar degeneration (FTLD-TDP). We generated a knock-in mouse harboring the R504X mutation (PGRN-KI). Phosphoproteomic analysis of this model revealed activation of signaling pathways connecting PKC and MAPK to tau prior to TDP43 aggregation and cognitive impairments, and identified PKCα as the kinase responsible for the early-stage tau phosphorylation at Ser203. Disinhibition of Gas6 binding to Tyro3 due to PGRN reduction results in activation of PKCα via PLCγ, inducing tau phosphorylation at Ser203, mislocalization of tau to dendritic spines, and spine loss. Administration of a PKC inhibitor, B-Raf inhibitor, or knockdown of molecules in the Gas6-Tyro3-tau axis rescues spine loss and cognitive impairment of PGRN-KI mice. Collectively, these results suggest that targeting of early-stage and aggregation-independent tau signaling represents a promising therapeutic strategy for this disease. 1 Department of Neuropathology, Medical Research Institute and Center for Brain Integration Research, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. 2 Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, 65, Tsurumai, Showa, Nagoya, Aichi 466-8550, Japan. 3 Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108- 8639, Japan.
  • Download Download

    Download Download

    Supplementary Figure S1. Results of flow cytometry analysis, performed to estimate CD34 positivity, after immunomagnetic separation in two different experiments. As monoclonal antibody for labeling the sample, the fluorescein isothiocyanate (FITC)- conjugated mouse anti-human CD34 MoAb (Mylteni) was used. Briefly, cell samples were incubated in the presence of the indicated MoAbs, at the proper dilution, in PBS containing 5% FCS and 1% Fc receptor (FcR) blocking reagent (Miltenyi) for 30 min at 4 C. Cells were then washed twice, resuspended with PBS and analyzed by a Coulter Epics XL (Coulter Electronics Inc., Hialeah, FL, USA) flow cytometer. only use Non-commercial 1 Supplementary Table S1. Complete list of the datasets used in this study and their sources. GEO Total samples Geo selected GEO accession of used Platform Reference series in series samples samples GSM142565 GSM142566 GSM142567 GSM142568 GSE6146 HG-U133A 14 8 - GSM142569 GSM142571 GSM142572 GSM142574 GSM51391 GSM51392 GSE2666 HG-U133A 36 4 1 GSM51393 GSM51394 only GSM321583 GSE12803 HG-U133A 20 3 GSM321584 2 GSM321585 use Promyelocytes_1 Promyelocytes_2 Promyelocytes_3 Promyelocytes_4 HG-U133A 8 8 3 GSE64282 Promyelocytes_5 Promyelocytes_6 Promyelocytes_7 Promyelocytes_8 Non-commercial 2 Supplementary Table S2. Chromosomal regions up-regulated in CD34+ samples as identified by the LAP procedure with the two-class statistics coded in the PREDA R package and an FDR threshold of 0.5. Functional enrichment analysis has been performed using DAVID (http://david.abcc.ncifcrf.gov/)
  • Supplement 1 Microarray Studies

    Supplement 1 Microarray Studies

    EASE Categories Significantly Enriched in vs MG vs vs MGC4-2 Pt1-C vs C4-2 Pt1-C UP-Regulated Genes MG System Gene Category EASE Global MGRWV Pt1-N RWV Pt1-N Score FDR GO Molecular Extracellular matrix cellular construction 0.0008 0 110 genes up- Function Interpro EGF-like domain 0.0009 0 regulated GO Molecular Oxidoreductase activity\ acting on single dono 0.0015 0 Function GO Molecular Calcium ion binding 0.0018 0 Function Interpro Laminin-G domain 0.0025 0 GO Biological Process Cell Adhesion 0.0045 0 Interpro Collagen Triple helix repeat 0.0047 0 KEGG pathway Complement and coagulation cascades 0.0053 0 KEGG pathway Immune System – Homo sapiens 0.0053 0 Interpro Fibrillar collagen C-terminal domain 0.0062 0 Interpro Calcium-binding EGF-like domain 0.0077 0 GO Molecular Cell adhesion molecule activity 0.0105 0 Function EASE Categories Significantly Enriched in Down-Regulated Genes System Gene Category EASE Global Score FDR GO Biological Process Copper ion homeostasis 2.5E-09 0 Interpro Metallothionein 6.1E-08 0 Interpro Vertebrate metallothionein, Family 1 6.1E-08 0 GO Biological Process Transition metal ion homeostasis 8.5E-08 0 GO Biological Process Heavy metal sensitivity/resistance 1.9E-07 0 GO Biological Process Di-, tri-valent inorganic cation homeostasis 6.3E-07 0 GO Biological Process Metal ion homeostasis 6.3E-07 0 GO Biological Process Cation homeostasis 2.1E-06 0 GO Biological Process Cell ion homeostasis 2.1E-06 0 GO Biological Process Ion homeostasis 2.1E-06 0 GO Molecular Helicase activity 2.3E-06 0 Function GO Biological
  • Nucleolin and Its Role in Ribosomal Biogenesis

    Nucleolin and Its Role in Ribosomal Biogenesis

    NUCLEOLIN: A NUCLEOLAR RNA-BINDING PROTEIN INVOLVED IN RIBOSOME BIOGENESIS Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Julia Fremerey aus Hamburg Düsseldorf, April 2016 2 Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. A. Borkhardt Korreferent: Prof. Dr. H. Schwender Tag der mündlichen Prüfung: 20.07.2016 3 Die vorgelegte Arbeit wurde von Juli 2012 bis März 2016 in der Klinik für Kinder- Onkologie, -Hämatologie und Klinische Immunologie des Universitätsklinikums Düsseldorf unter Anleitung von Prof. Dr. A. Borkhardt und in Kooperation mit dem ‚Laboratory of RNA Molecular Biology‘ an der Rockefeller Universität unter Anleitung von Prof. Dr. T. Tuschl angefertigt. 4 Dedicated to my family TABLE OF CONTENTS 5 TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................... 5 LIST OF FIGURES ......................................................................................................10 LIST OF TABLES .......................................................................................................12 ABBREVIATION .........................................................................................................13 ABSTRACT ................................................................................................................19 ZUSAMMENFASSUNG
  • Differential Requirements for Tousled-Like Kinases 1 and 2 in Mammalian Development

    Differential Requirements for Tousled-Like Kinases 1 and 2 in Mammalian Development

    Cell Death and Differentiation (2017) 24, 1872–1885 & 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 1350-9047/17 www.nature.com/cdd Differential requirements for Tousled-like kinases 1 and 2 in mammalian development Sandra Segura-Bayona1,8, Philip A Knobel1,8, Helena González-Burón1,8, Sameh A Youssef2,3, Aida Peña-Blanco1, Étienne Coyaud4,5, Teresa López-Rovira1, Katrin Rein1, Lluís Palenzuela1, Julien Colombelli1, Stephen Forrow1, Brian Raught4,5, Anja Groth6, Alain de Bruin2,7 and Travis H Stracker*,1 The regulation of chromatin structure is critical for a wide range of essential cellular processes. The Tousled-like kinases, TLK1 and TLK2, regulate ASF1, a histone H3/H4 chaperone, and likely other substrates, and their activity has been implicated in transcription, DNA replication, DNA repair, RNA interference, cell cycle progression, viral latency, chromosome segregation and mitosis. However, little is known about the functions of TLK activity in vivo or the relative functions of the highly similar TLK1 and TLK2 in any cell type. To begin to address this, we have generated Tlk1- and Tlk2-deficient mice. We found that while TLK1 was dispensable for murine viability, TLK2 loss led to late embryonic lethality because of placental failure. TLK2 was required for normal trophoblast differentiation and the phosphorylation of ASF1 was reduced in placentas lacking TLK2. Conditional bypass of the placental phenotype allowed the generation of apparently healthy Tlk2-deficient mice, while only the depletion of both TLK1 and TLK2 led to extensive genomic instability, indicating that both activities contribute to genome maintenance. Our data identifies a specific role for TLK2 in placental function during mammalian development and suggests that TLK1 and TLK2 have largely redundant roles in genome maintenance.
  • Aneuploidy: Using Genetic Instability to Preserve a Haploid Genome?

    Aneuploidy: Using Genetic Instability to Preserve a Haploid Genome?

    Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Science (Cancer Biology) Aneuploidy: Using genetic instability to preserve a haploid genome? Submitted by: Ramona Ramdath In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Science Examination Committee Signature/Date Major Advisor: David Allison, M.D., Ph.D. Academic James Trempe, Ph.D. Advisory Committee: David Giovanucci, Ph.D. Randall Ruch, Ph.D. Ronald Mellgren, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: April 10, 2009 Aneuploidy: Using genetic instability to preserve a haploid genome? Ramona Ramdath University of Toledo, Health Science Campus 2009 Dedication I dedicate this dissertation to my grandfather who died of lung cancer two years ago, but who always instilled in us the value and importance of education. And to my mom and sister, both of whom have been pillars of support and stimulating conversations. To my sister, Rehanna, especially- I hope this inspires you to achieve all that you want to in life, academically and otherwise. ii Acknowledgements As we go through these academic journeys, there are so many along the way that make an impact not only on our work, but on our lives as well, and I would like to say a heartfelt thank you to all of those people: My Committee members- Dr. James Trempe, Dr. David Giovanucchi, Dr. Ronald Mellgren and Dr. Randall Ruch for their guidance, suggestions, support and confidence in me. My major advisor- Dr. David Allison, for his constructive criticism and positive reinforcement.