Predict AID Targeting in Non-Ig Genes Multiple Transcription Factor

Total Page:16

File Type:pdf, Size:1020Kb

Predict AID Targeting in Non-Ig Genes Multiple Transcription Factor Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021 is online at: average * The Journal of Immunology published online 20 March 2013 from submission to initial decision 4 weeks from acceptance to publication Multiple Transcription Factor Binding Sites Predict AID Targeting in Non-Ig Genes Jamie L. Duke, Man Liu, Gur Yaari, Ashraf M. Khalil, Mary M. Tomayko, Mark J. Shlomchik, David G. Schatz and Steven H. Kleinstein J Immunol http://www.jimmunol.org/content/early/2013/03/20/jimmun ol.1202547 Submit online. Every submission reviewed by practicing scientists ? is published twice each month by http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://www.jimmunol.org/content/suppl/2013/03/20/jimmunol.120254 7.DC1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 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 26, 2021. Published March 20, 2013, doi:10.4049/jimmunol.1202547 The Journal of Immunology Multiple Transcription Factor Binding Sites Predict AID Targeting in Non-Ig Genes Jamie L. Duke,* Man Liu,†,1 Gur Yaari,‡ Ashraf M. Khalil,x Mary M. Tomayko,{ Mark J. Shlomchik,†,x David G. Schatz,†,‖ and Steven H. Kleinstein*,‡ Aberrant targeting of the enzyme activation-induced cytidine deaminase (AID) results in the accumulation of somatic mutations in ∼25% of expressed genes in germinal center B cells. Observations in Ung2/2 Msh22/2 mice suggest that many other genes efficiently repair AID-induced lesions, so that up to 45% of genes may actually be targeted by AID. It is important to understand the mechanisms that recruit AID to certain genes, because this mistargeting represents an important risk for genome instability. We hypothesize that several mechanisms combine to target AID to each locus. To resolve which mechanisms affect AID targeting, we analyzed 7.3 Mb of sequence data, along with the regulatory context, from 83 genes in Ung2/2 Msh22/2 mice to identify common properties of AID targets. This analysis identifies three transcription factor binding sites (E-box motifs, along with YY1 Downloaded from and C/EBP-b binding sites) that may work together to recruit AID. Based on previous knowledge and these newly discovered features, a classification tree model was built to predict genome-wide AID targeting. Using this predictive model, we were able to identify a set of 101 high-interest genes that are likely targets of AID. The Journal of Immunology, 2013, 190: 000–000. omatic hypermutation (SHM) occurs in germinal center the enzyme that deaminates cytosines to initiate SHM, can act (GC) B cells, resulting in the introduction of point muta- outside of the Ig locus. In a previous sequencing study, we showed http://www.jimmunol.org/ tions into Ig genes. Although SHM provides an important that .45% of expressed genes in GC B cells are targeted by AID in S 2/2 2/2 source of genetic diversity, capable of producing specific Abs for Ung Msh2 double-knockout (dKO) mice, where the absence quickly evolving pathogens, the process also poses a severe threat of DNA repair reveals the “footprint” of AID. Even among genes to genomic stability. Activation-induced cytidine deaminase (AID), that were targeted by AID, this study revealed a wide range of mutation frequencies observed across 83 genes (1). In this study, we seek to address two basic questions that are raised by the former *Interdepartmental Program in Computational Biology and Bioinformatics, Yale study: Why are some genes targeted by AID, whereas others are University, New Haven, CT 06511; †Department of Immunobiology, Yale University ‡ not? and How do the genes targeted by AID accumulate different School of Medicine, New Haven, CT 06510; Department of Pathology, Yale Uni- by guest on September 26, 2021 versity School of Medicine, New Haven, CT 06510; xDepartment of Laboratory levels of mutation? The main hypothesis we pursue is that sequence Medicine, Yale University School of Medicine, New Haven, CT 06510; {Department features of each gene are responsible for this differential targeting. of Dermatology, Yale University School of Medicine, New Haven, CT 06510; and ‖ The current model of SHM proposes two phases (2). In the first Howard Hughes Medical Institute, New Haven, CT 06510 1 phase, AID converts a cytosine (C) residue to a uracil (U) in ssDNA Current address: Drinker Biddle & Reath LLP, Washington, D.C. created during the process of transcription, which, if left unrepaired, Received for publication September 26, 2012. Accepted for publication February 15, leads to a C to T (thymine) transition mutation when the DNA is 2013. replicated for cell division (3). The second phase of SHM begins J.L.D. was supported in part by the Pharmaceutical Research and Manufacturers of America Foundation and National Institutes of Health Grant T15 LM07056 from the when DNA repair mechanisms attempt to remove the uracil lesion National Library of Medicine. D.G.S. is an investigator of the Howard Hughes from the DNA. The repair of the uracil happens via two pathways: Medical Institute. Computational resources were provided by the Yale University base excision repair with UNG and mismatch repair facilitated by Biomedical High Performance Computing Center (National Institutes of Health Grant RR19895). the MSH2/MSH6 complex, both of which are capable of working J.L.D. and S.H.K. designed the analyses; M.L. and D.G.S. designed the RNA poly- in an error-prone fashion and contributing to the observed muta- merase II ChIP-Seq experiment; M.L. performed the ChIP portion of the experiment; tion frequency (4). In the dKO setting, the second phase of SHM M.M.T. and M.J.S. provided the microarray expression data; J.L.D. and G.Y. wrote is unavailable, thus revealing the underlying “footprint” of AID, software for the analyses; J.L.D. performed the analyses; and J.L.D., D.G.S., and S.H.K. wrote the manuscript. All authors commented on the manuscript. where the expectation is primarily C → T transition mutations. We The microarray data presented in this article have been submitted to the Gene Ex- previously sequenced 83 non-Ig genes from dKO mice on average pression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE44260) 70 times per gene over a 1-kb region downstream of the tran- under accession number GSE44260. scription start site (TSS) (1). Mutation frequencies varied widely, Address correspondence and reprint requests to Dr. Steven H. Kleinstein, Yale Uni- ranging from ,1 3 1025 to 116.1 3 1025 mutations/bp, but they versity School of Medicine, 300 George Street, Suite 505, New Haven, CT 06511. E-mail address: [email protected] were highly predictable for the same gene across samples from The online version of this article contains supplemental material. multiple mice. In the same system, sequencing of an IgH positive control, specifically the VhJ558-Jh4 intron 39 flanking region Abbreviations used in this article: AID, activation-induced cytidine deaminase; ChIP, chromatin immunoprecipitation; ChIP-Seq, chromatin immunoprecipitation followed (hereafter referred to as the Jh4 intron), found a mutation frequency by massively parallel sequencing; CSR, class switch recombination; dKO, double of 9.96 3 1023 mutations/bp. Each gene represents a unique ge- knockout; FDR, false-discovery rate; GC, germinal center; GSEA, gene set enrich- ment analysis; KO, knockout; KW, Kruskal–Wallis test; MWU, Mann–Whitney U nomic context in which to explore the various properties associated test; NB, negative binomial; RefSeq, National Center for Biotechnology Information with AID targeting. Reference Sequence; SHM, somatic hypermutation; TC-Seq, translocation-capture Differential AID activity in non-Ig genes may be influenced by sequencing; TSS, transcription start site; ZI-NB, zero-inflated negative binomial. multiple underlying mechanisms. A higher transcription rate may Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 be associated with an increased mutation frequency. Genes with www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202547 2 AID TARGETING IN NON-Ig GENES a higher mutation frequency may contain a large number of AID Dynabeads Protein A (Invitrogen) were incubated with RNA polymerase hotspots, such as WRC (W = A/T; R = A/G), and/or few AID II Ab N20 (Santa Cruz Biotechnologies) or normal rabbit serum. Excess Ab coldspots, such as SYC (S = C/G; Y = C/T), where the C is the was washed away. Then Ab-bound beads were incubated with chromatin from 20 million sorted spleen GC B cells (previously cross-linked with 1% mutated position (5, 6). Clonal recruitment of AID to certain HCHO and then sonicated to shear the DNA fragments to 100–300 bp) at 4˚C genes may lead to an increased mutation frequency (7). Finally, overnight. Beads were washed, chromatin was eluted, and the cross-linking the genes for which high mutation frequencies are observed may was reversed. DNA was purified, precipitated, and redissolved in TE buffer. share functional elements, like transcription factor binding sites, Precipitated DNA was quantified using a PicoGreen dsDNA quantification kit (Molecular Probe). A total of 200 ng chromatin immunoprecipitation which recruit AID to the locus for mutation. In this study, we first (ChIP) DNA (from 40 million cells) ends was repaired using polynucleotide examine each of the possible mechanisms independently and then kinase and Klenow enzyme, followed by treatment with Taq polymerase to develop an integrated model to predict targeting of AID in the generate a protruding 39 A base used for adaptor ligation.
Recommended publications
  • ARMCX3 (NM 177948) Human Untagged Clone – SC124834
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for SC124834 ARMCX3 (NM_177948) Human Untagged Clone Product data: Product Type: Expression Plasmids Product Name: ARMCX3 (NM_177948) Human Untagged Clone Tag: Tag Free Symbol: ARMCX3 Synonyms: ALEX3; dJ545K15.2; GASP6 Vector: pCMV6-XL4 E. coli Selection: Ampicillin (100 ug/mL) Cell Selection: None Fully Sequenced ORF: >NCBI ORF sequence for NM_177948, the custom clone sequence may differ by one or more nucleotides ATGGGCTACGCCAGGAAAGTAGGCTGGGTGACCGCAGGCCTGGTGATTGGGGCTGGCGCCTGCTATTGCA TTTATAGACTGACTAGGGGAAGAAAACAGAACAAGGAAAAAATGGCTGAGGGTGGATCTGGGGATGTGGA TGATGCTGGGGACTGTTCTGGGGCCAGGTATAATGACTGGTCTGATGATGATGATGACAGCAATGAGAGC AAGAGTATAGTATGGTACCCACCTTGGGCTCGGATTGGGACTGAAGCTGGAACCAGAGCTAGGGCCAGGG CAAGGGCCAGGGCTACCCGGGCACGTCGGGCTGTCCAGAAACGGGCTTCCCCCAATTCAGATGATACCGT TTTGTCCCCTCAAGAGCTACAAAAGGTTCTTTGCTTGGTTGAGATGTCTGAAAAGCCTTATATTCTTGAA GCAGCTTTAATTGCTCTGGGTAACAATGCTGCTTATGCATTTAACAGAGATATTATTCGTGATCTGGGTG GTCTCCCAATTGTCGCAAAGATTCTCAATACTCGGGATCCCATAGTTAAGGAAAAGGCTTTAATTGTCCT GAATAACTTGAGTGTGAATGCTGAAAATCAGCGCAGGCTTAAAGTATACATGAATCAAGTGTGTGATGAC ACAATCACTTCTCGCTTGAACTCATCTGTGCAGCTTGCTGGACTGAGATTGCTTACAAATATGACTGTTA CTAATGAGTATCAGCACATGCTTGCTAATTCCATTTCTGACTTTTTTCGTTTATTTTCAGCGGGAAATGA AGAAACCAAACTTCAGGTTCTGAAACTCCTTTTGAATTTGGCTGAAAATCCAGCCATGACTAGGGAACTG CTCAGGGCCCAAGTACCATCTTCACTGGGCTCCCTCTTTAATAAGAAGGAGAACAAAGAAGTTATTCTTA AACTTCTGGTCATATTTGAGAACATAAATGATAATTTCAAATGGGAAGAAAATGAACCTACTCAGAATCA
    [Show full text]
  • Anti-ATG9B Antibody (ARG59749)
    Product datasheet [email protected] ARG59749 Package: 50 μg anti-ATG9B antibody Store at: -20°C Summary Product Description Rabbit Polyclonal antibody recognizes ATG9B Tested Reactivity Hu Predict Reactivity Ms, Rat Tested Application ICC/IF, WB Host Rabbit Clonality Polyclonal Isotype IgG Target Name ATG9B Antigen Species Human Immunogen A 17 amino acid peptide within aa. 850 - 900 of Human ATG9B. Conjugation Un-conjugated Alternate Names APG9-like 2; SONE; NOS3AS; Autophagy-related protein 9B; Protein sONE; APG9L2; Nitric oxide synthase 3-overlapping antisense gene protein Application Instructions Application table Application Dilution ICC/IF 10 - 20 µg/ml WB 1 - 2 µg/ml Application Note * The dilutions indicate recommended starting dilutions and the optimal dilutions or concentrations should be determined by the scientist. Positive Control HeLa Calculated Mw 101 kDa Properties Form Liquid Purification Affinity purification with immunogen. Buffer PBS and 0.02% Sodium azide Preservative 0.02% Sodium azide Concentration 1 mg/ml Storage instruction For continuous use, store undiluted antibody at 2-8°C for up to a week. For long-term storage, aliquot and store at -20°C or below. Storage in frost free freezers is not recommended. Avoid repeated freeze/thaw cycles. Suggest spin the vial prior to opening. The antibody solution should be gently mixed before use. www.arigobio.com 1/3 Note For laboratory research only, not for drug, diagnostic or other use. Bioinformation Gene Symbol ATG9B Gene Full Name autophagy related 9B Background This gene functions in the regulation of autophagy, a lysosomal degradation pathway. This gene also functions as an antisense transcript in the posttranscriptional regulation of the endothelial nitric oxide synthase 3 gene, which has 3' overlap with this gene on the opposite strand.
    [Show full text]
  • Identification of the Binding Partners for Hspb2 and Cryab Reveals
    Brigham Young University BYU ScholarsArchive Theses and Dissertations 2013-12-12 Identification of the Binding arP tners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non- Redundant Roles for Small Heat Shock Proteins Kelsey Murphey Langston Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation Langston, Kelsey Murphey, "Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins" (2013). Theses and Dissertations. 3822. https://scholarsarchive.byu.edu/etd/3822 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Julianne H. Grose, Chair William R. McCleary Brian Poole Department of Microbiology and Molecular Biology Brigham Young University December 2013 Copyright © 2013 Kelsey Langston All Rights Reserved ABSTRACT Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactors and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston Department of Microbiology and Molecular Biology, BYU Master of Science Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity.
    [Show full text]
  • Supporting Information
    Supporting Information Mulders et al. 10.1073/pnas.0905780106 SI Materials and Methods in Opti-MEM (Invitrogen) to myoblasts, in both cases at a final Animals. Hemizygous DM500 mice, derived from the DM300– oligo concentration of 200 nM. Fresh medium was supplemented 328 line (1), express a transgenic human DM1 locus, which bears after 4 hours. After 24 h, medium was changed. RNA was a repeat segment that has expanded to Ϸ500 CTG triplets, isolated 48 h after transfection. because of intergenerational triplet-repeat instability. For the isolation of immortal DM500 myoblasts, DM500 mice were RNA Isolation. Typically, RNA from cultured cells was isolated crossed with H-2Kb-tsA58 transgenic mice (2). Homozygous using the Aurum Total RNA mini kit (guanidine-HCl/ HSALR20b mice express a (CUG)250 segment in the context of mercaptoethanol-based lysis, silica membrane binding; Bio- a human skeletal actin transcript (3). All animal experiments Rad), according to the manufacturer’s protocol. RNA from were approved by the Institutional Animal Care and Use Com- muscle tissue was isolated using TRIzol reagent (Invitrogen). mittees of the Radboud University Nijmegen and the University Alternative methods to isolate RNA from cultured cells in- of Rochester Medical Center. volved: (i) use of the TRIzol reagent, according to the manu- facturer’s protocol; (ii) an oligo(dT) affinity column (Nucleo- Cell Culture. An immortal mouse myoblast cell culture expressing Trap mRNA mini kit; Macherey-Nagel) for the isolation of hDMPK (CUG)500 transcripts was derived from GPS tissue poly(A) RNA; or (iii) a TRIzol procedure preceded by a isolated from DM500 mice additionally expressing 1 copy of the proteinase K digestion of the whole cell lysate (7): in short, cells H-2Kb-tsA58 allele (4).
    [Show full text]
  • Seq2pathway Vignette
    seq2pathway Vignette Bin Wang, Xinan Holly Yang, Arjun Kinstlick May 19, 2021 Contents 1 Abstract 1 2 Package Installation 2 3 runseq2pathway 2 4 Two main functions 3 4.1 seq2gene . .3 4.1.1 seq2gene flowchart . .3 4.1.2 runseq2gene inputs/parameters . .5 4.1.3 runseq2gene outputs . .8 4.2 gene2pathway . 10 4.2.1 gene2pathway flowchart . 11 4.2.2 gene2pathway test inputs/parameters . 11 4.2.3 gene2pathway test outputs . 12 5 Examples 13 5.1 ChIP-seq data analysis . 13 5.1.1 Map ChIP-seq enriched peaks to genes using runseq2gene .................... 13 5.1.2 Discover enriched GO terms using gene2pathway_test with gene scores . 15 5.1.3 Discover enriched GO terms using Fisher's Exact test without gene scores . 17 5.1.4 Add description for genes . 20 5.2 RNA-seq data analysis . 20 6 R environment session 23 1 Abstract Seq2pathway is a novel computational tool to analyze functional gene-sets (including signaling pathways) using variable next-generation sequencing data[1]. Integral to this tool are the \seq2gene" and \gene2pathway" components in series that infer a quantitative pathway-level profile for each sample. The seq2gene function assigns phenotype-associated significance of genomic regions to gene-level scores, where the significance could be p-values of SNPs or point mutations, protein-binding affinity, or transcriptional expression level. The seq2gene function has the feasibility to assign non-exon regions to a range of neighboring genes besides the nearest one, thus facilitating the study of functional non-coding elements[2]. Then the gene2pathway summarizes gene-level measurements to pathway-level scores, comparing the quantity of significance for gene members within a pathway with those outside a pathway.
    [Show full text]
  • Characterization of Genomic Copy Number Variation in Mus Musculus Associated with the Germline of Inbred and Wild Mouse Populations, Normal Development, and Cancer
    Western University Scholarship@Western Electronic Thesis and Dissertation Repository 4-18-2019 2:00 PM Characterization of genomic copy number variation in Mus musculus associated with the germline of inbred and wild mouse populations, normal development, and cancer Maja Milojevic The University of Western Ontario Supervisor Hill, Kathleen A. The University of Western Ontario Graduate Program in Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Maja Milojevic 2019 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Genetics and Genomics Commons Recommended Citation Milojevic, Maja, "Characterization of genomic copy number variation in Mus musculus associated with the germline of inbred and wild mouse populations, normal development, and cancer" (2019). Electronic Thesis and Dissertation Repository. 6146. https://ir.lib.uwo.ca/etd/6146 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract Mus musculus is a human commensal species and an important model of human development and disease with a need for approaches to determine the contribution of copy number variants (CNVs) to genetic variation in laboratory and wild mice, and arising with normal mouse development and disease. Here, the Mouse Diversity Genotyping array (MDGA)-approach to CNV detection is developed to characterize CNV differences between laboratory and wild mice, between multiple normal tissues of the same mouse, and between primary mammary gland tumours and metastatic lung tissue.
    [Show full text]
  • Wnt/Β-Catenin Signaling Pathway Induces Autophagy
    Yun et al. Cell Death and Disease (2020) 11:771 https://doi.org/10.1038/s41419-020-02988-8 Cell Death & Disease ARTICLE Open Access Wnt/β-catenin signaling pathway induces autophagy-mediated temozolomide-resistance in human glioblastoma Eun-Jin Yun 1,SangwooKim2,Jer-TsongHsieh3,4 and Seung Tae Baek 5,6 Abstract Temozolomide (TMZ) is widely used for treating glioblastoma multiforme (GBM), however, the treatment of such brain tumors remains a challenge due to the development of resistance. Increasing studies have found that TMZ treatment could induce autophagy that may link to therapeutic resistance in GBM, but, the precise mechanisms are not fully understood. Understanding the molecular mechanisms underlying the response of GBM to chemotherapy is paramount for developing improved cancer therapeutics. In this study, we demonstrated that the loss of DOC-2/DAB2 interacting protein (DAB2IP) is responsible for TMZ-resistance in GBM through ATG9B. DAB2IP sensitized GBM to TMZ and suppressed TMZ-induced autophagy by negatively regulating ATG9B expression. A higher level of ATG9B expression was associated with GBM compared to low-grade glioma. The knockdown of ATG9B expression in GBM cells suppressed TMZ-induced autophagy as well as TMZ-resistance. Furthermore, we showed that DAB2IP negatively regulated ATG9B expression by blocking the Wnt/β-catenin pathway. To enhance the benefit of TMZ and avoid therapeutic resistance, effective combination strategies were tested using a small molecule inhibitor blocking the Wnt/ β-catenin pathway in addition to TMZ. The combination treatment synergistically enhanced the efficacy of TMZ in GBM cells. In conclusion, the present study identified the mechanisms of TMZ-resistance of GBM mediated by DAB2IP 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; and ATG9B which provides insight into a potential strategy to overcome TMZ chemo-resistance.
    [Show full text]
  • 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.
    [Show full text]
  • The Inactive X Chromosome Is Epigenetically Unstable and Transcriptionally Labile in Breast Cancer
    Supplemental Information The inactive X chromosome is epigenetically unstable and transcriptionally labile in breast cancer Ronan Chaligné1,2,3,8, Tatiana Popova1,4, Marco-Antonio Mendoza-Parra5, Mohamed-Ashick M. Saleem5 , David Gentien1,6, Kristen Ban1,2,3,8, Tristan Piolot1,7, Olivier Leroy1,7, Odette Mariani6, Hinrich Gronemeyer*5, Anne Vincent-Salomon*1,4,6,8, Marc-Henri Stern*1,4,6 and Edith Heard*1,2,3,8 Extended Experimental Procedures Cell Culture Human Mammary Epithelial Cells (HMEC, Invitrogen) were grown in serum-free medium (HuMEC, Invitrogen). WI- 38, ZR-75-1, SK-BR-3 and MDA-MB-436 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) containing 10% fetal bovine serum (FBS). DNA Methylation analysis. We bisulfite-treated 2 µg of genomic DNA using Epitect bisulfite kit (Qiagen). Bisulfite converted DNA was amplified with bisulfite primers listed in Table S3. All primers incorporated a T7 promoter tag, and PCR conditions are available upon request. We analyzed PCR products by MALDI-TOF mass spectrometry after in vitro transcription and specific cleavage (EpiTYPER by Sequenom®). For each amplicon, we analyzed two independent DNA samples and several CG sites in the CpG Island. Design of primers and selection of best promoter region to assess (approx. 500 bp) were done by a combination of UCSC Genome Browser (http://genome.ucsc.edu) and MethPrimer (http://www.urogene.org). All the primers used are listed (Table S3). NB: MAGEC2 CpG analysis have been done with a combination of two CpG island identified in the gene core. Analysis of RNA allelic expression profiles (based on Human SNP Array 6.0) DNA and RNA hybridizations were normalized by Genotyping console.
    [Show full text]
  • BTG2: a Rising Star of Tumor Suppressors (Review)
    INTERNATIONAL JOURNAL OF ONCOLOGY 46: 459-464, 2015 BTG2: A rising star of tumor suppressors (Review) BIjING MAO1, ZHIMIN ZHANG1,2 and GE WANG1 1Cancer Center, Institute of Surgical Research, Daping Hospital, Third Military Medical University, Chongqing 400042; 2Department of Oncology, Wuhan General Hospital of Guangzhou Command, People's Liberation Army, Wuhan, Hubei 430070, P.R. China Received September 22, 2014; Accepted November 3, 2014 DOI: 10.3892/ijo.2014.2765 Abstract. B-cell translocation gene 2 (BTG2), the first 1. Discovery of BTG2 in TOB/BTG gene family gene identified in the BTG/TOB gene family, is involved in many biological activities in cancer cells acting as a tumor The TOB/BTG genes belong to the anti-proliferative gene suppressor. The BTG2 expression is downregulated in many family that includes six different genes in vertebrates: TOB1, human cancers. It is an instantaneous early response gene and TOB2, BTG1 BTG2/TIS21/PC3, BTG3 and BTG4 (Fig. 1). plays important roles in cell differentiation, proliferation, DNA The conserved domain of BTG N-terminal contains two damage repair, and apoptosis in cancer cells. Moreover, BTG2 regions, named box A and box B, which show a high level of is regulated by many factors involving different signal path- homology to the other domains (1-5). Box A has a major effect ways. However, the regulatory mechanism of BTG2 is largely on cell proliferation, while box B plays a role in combination unknown. Recently, the relationship between microRNAs and with many target molecules. Compared with other family BTG2 has attracted much attention. MicroRNA-21 (miR-21) members, BTG1 and BTG2 have an additional region named has been found to regulate BTG2 gene during carcinogenesis.
    [Show full text]
  • Actin Binding LIM 1 (Ablim1) Negatively Controls Osteoclastogenesis by Regulating Cell Migration and Fusion
    Received: 14 September 2017 | Accepted: 16 March 2018 DOI: 10.1002/jcp.26605 ORIGINAL RESEARCH ARTICLE Actin binding LIM 1 (abLIM1) negatively controls osteoclastogenesis by regulating cell migration and fusion Haruna Narahara1,2 | Eiko Sakai1 | Yu Yamaguchi1 | Shun Narahara1 | Mayumi Iwatake1,* | Kuniaki Okamoto1,† | Noriaki Yoshida2 | Takayuki Tsukuba1 1 Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Actin binding LIM 1 (abLIM1) is a cytoskeletal actin-binding protein that has been Nagasaki University, Nagasaki, Japan implicated in interactions between actin filaments and cytoplasmic targets. Previous 2 Department of Orthodontics and Dentofacial Orthopedics, Graduate School of Biomedical biochemical and cytochemical studies have shown that abLIM1 interacts and co- Sciences, Nagasaki University, Nagasaki, Japan localizes with F-actin in the retina and muscle. However, whether abLIM1 regulates Correspondence osteoclast differentiation has not yet been elucidated. In this study, we examined the Takayuki Tsukuba, Department of Dental role of abLIM1 in osteoclast differentiation and function. We found that abLIM1 Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852- expression was upregulated during receptor activator of nuclear factor kappa-B ligand 8588, Japan. (RANKL)-induced osteoclast differentiation, and that a novel transcript of abLIM1 was Email: [email protected] exclusively expressed in osteoclasts. Overexpression of abLIM1 in the murine Funding information monocytic cell line, RAW-D suppressed osteoclast differentiation and decreased JSPS KAKENHI, Grant numbers: 15H05298, 16K15790, 17H04379 expression of several osteoclast-marker genes. By contrast, small interfering RNA-induced knockdown of abLIM1 enhanced the formation of multinucleated osteoclasts and markedly increased the expression of the osteoclast-marker genes. Mechanistically, abLIM1 regulated the localization of tubulin, migration, and fusion in osteoclasts.
    [Show full text]
  • Supplemental Information
    Supplemental information Dissection of the genomic structure of the miR-183/96/182 gene. Previously, we showed that the miR-183/96/182 cluster is an intergenic miRNA cluster, located in a ~60-kb interval between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h) on mouse chr6qA3.3 (1). To start to uncover the genomic structure of the miR- 183/96/182 gene, we first studied genomic features around miR-183/96/182 in the UCSC genome browser (http://genome.UCSC.edu/), and identified two CpG islands 3.4-6.5 kb 5’ of pre-miR-183, the most 5’ miRNA of the cluster (Fig. 1A; Fig. S1 and Seq. S1). A cDNA clone, AK044220, located at 3.2-4.6 kb 5’ to pre-miR-183, encompasses the second CpG island (Fig. 1A; Fig. S1). We hypothesized that this cDNA clone was derived from 5’ exon(s) of the primary transcript of the miR-183/96/182 gene, as CpG islands are often associated with promoters (2). Supporting this hypothesis, multiple expressed sequences detected by gene-trap clones, including clone D016D06 (3, 4), were co-localized with the cDNA clone AK044220 (Fig. 1A; Fig. S1). Clone D016D06, deposited by the German GeneTrap Consortium (GGTC) (http://tikus.gsf.de) (3, 4), was derived from insertion of a retroviral construct, rFlpROSAβgeo in 129S2 ES cells (Fig. 1A and C). The rFlpROSAβgeo construct carries a promoterless reporter gene, the β−geo cassette - an in-frame fusion of the β-galactosidase and neomycin resistance (Neor) gene (5), with a splicing acceptor (SA) immediately upstream, and a polyA signal downstream of the β−geo cassette (Fig.
    [Show full text]