DOCSLIB.ORG

Androgen Receptor Binding Sites Identified by a GREF GATA Model

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Androgen Receptor Binding Sites Identified by a GREF GATA Model doi:10.1016/j.jmb.2005.09.009 J. Mol. Biol. (2005) 353, 763–771 COMMUNICATION Androgen Receptor Binding Sites Identified by a GREF_GATA Model Katsuaki Masuda1, Thomas Werner2, Shilpi Maheshwari1 Matthias Frisch2, Soyon Oh1, Gyorgy Petrovics1, Klaus May2 Vasantha Srikantan1, Shiv Srivastava1 and Albert Dobi1* 1Center for Prostate Disease Changes in transcriptional regulation can be permissive for tumor Research, Department of progression by allowing for selective growth advantage of tumor cells. Surgery, Uniformed Services Tumor suppressors can effectively inhibit this process. The PMEPA1 gene, a University, Rockville, MD potent inhibitor of prostate cancer cell growth is an androgen-regulated 20852, USA gene. We addressed the question of whether or not androgen receptor can directly bind to specific PMEPA1 promoter upstream sequences. To test this 2Genomatix Software GmbH hypothesis we extended in silico prediction of androgen receptor binding D-80339 Munich, Germany sites by a modeling approach and verified the actual binding by in vivo chromatin immunoprecipitation assay. Promoter upstream sequences of highly androgen-inducible genes were examined from microarray data of prostate cancer cells for transcription factor binding sites (TFBSs). Results were analyzed to formulate a model for the description of specific androgen receptor binding site context in these sequences. In silico analysis and subsequent experimental verification of the selected sequences suggested that a model that combined a GREF and a GATA TFBS was sufficient for predicting a class of functional androgen receptor binding sites. The GREF matrix family represents androgen receptor, glucocorticoid receptor and progesterone receptor binding sites and the GATA matrix family represents GATA binding protein 1–6 binding sites. We assessed the regulatory sequences of the PMEPA1 gene by comparing our model-based GREF_GATA predictions to weight matrix-based predictions. Androgen receptor binding to predicted promoter upstream sequences of the PMEPA1 gene was confirmed by chromatin immunoprecipitation assay. Our results suggested that androgen receptor binding to cognate elements was consistent with the GREF_GATA model. In contrast, using only single GREF weight matrices resulted in additional matches, apparently false positives. Our findings indicate that complex models based on datasets selected by biological function can be superior predictors as they recognize TFBSs in their functional context. q 2005 Elsevier Ltd. All rights reserved. Keywords: model; androgen receptor; prostate cancer; promoter database; *Corresponding author GATA factor Alterations in transcription regulation are hall- expression of specific genes that control cell marks of cancer progression. Decreases in the proliferation may significantly contribute to malig- nant transformation. The PMEPA1 gene has been recognized as a potent cell growth inhibitor of Abbreviations used: TFBS, transcription factor binding 1 site; AR, androgen receptor; ChIP, chromatin immuno- prostate cancer cells, with decreased expression of precipitation; GREF, glucocorticoid responsive element PMEPA1 associated with higher pathologic stage in matrix family; ARE, androgen responsive element. prostate cancer specimens. Function for PMEPA1 in E-mail address of the corresponding author: the ubiquitin-proteasome pathway is suggested by [email protected] shared structural similarities between PMEPA1 and 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. 764 Predicting Androgen Receptor Binding Sites Table 1. Androgen-inducible gene training set Gene Accession Locus Contig Absolute positions (strand) KLK3 (PSA) HT2351 19q13.41 NM_145864 23,624,361–23,626,361 (C) NDRG1 D87953 8q24.3 NT_008046 47,529,674–47,527,674 (K) NKX3.1 U80669 8p21 NT_023666 1,916,763–1,914,763 (K) IHPK1 D87452 3p21.31 NT_022517 49,711,601–49,709,601 (K) Gene symbols, accession numbers and chromosomal locations (Homo sapiens NCBI Build 35, http://www.ncbi.nlm.nih.gov) are shown. Nedd4-binding proteins. Interestingly, PMEPA1 Defining a model for androgen receptor binding gene expression was shown to be regulated by in the context of prostate-specific hormone androgens in prostate cancer cells.2–4 In order to induction assess androgen receptor (AR) binding to regulatory elements of the PMEPA1 gene we Upstream sequences of highly androgen- integrated computation biology with wetlab experi- inducible genes were selected as a training set mentation. Computation molecular biology of from our previous oligonucleotide array experi- gene regulation is an emerging frontier of bio- ments in order to formulate a model that can predict 5 informatics. In the past, conclusive identification of functional androgen receptor binding sites in the functionally active transcription factor binding sites context of androgen-induced prostate cancer cells (TFBSs) relied mainly on laborious wetlab experi- (Table 1). Four genes were selected that stood out of mentations. However, recently chromatin immuno- the list of androgen-inducible genes both by the precipitation (ChIP)-based assays such as “ChIP- robustness of gene expression response (KLK3Z chip” and ChIP Display were shown to be robust 58! and NDRG1Z13!) and by kinetic consider- techniques to assess binding sites experimentally ations (IHPK1Z9! in 1 h and NKX3.1Z5! in 6,7 genome-wide. In support to these efforts, robust 6 h).4 platforms are currently available for in vitro Extraction of 50 upstream sequences relative to selection of single TFBSs to assess optimal cognate the transcription start sites of training set genes 8–10 sitesforvariouselements. Although bio- between positions K2000 and C1 was accom- informatic tools are available for the prediction of plished by the ElDorado system (Genomatix, 11 TFBSs, we preferred a weight matrix approach. Munich)† that is based on the genome assemblies However, the application of any weight matrix from NCBI (Homo sapiens NCBI Build 35‡). approach to long sequences is hampered by a large Sequences were verified by database searches in number of false positive predictions. Although the human genome. In this process upstream examining the evolutional conservation of tran- sequences of KLK3 and KLK2 genes were found to scription factor binding can enhance the specificity show high degrees of identity (approx. 80% within 12,13 of in silico predictions, this technique can be K1000;C1). Although, we previously found that confounded by species-specific genes. Notably, KLK2 was a highly androgen-inducible gene, KLK2 many human cancer-related genes as illustrated was excluded from the training set to avoid by prostate specific antigen (PSA, KLK3) barely sequence redundancy that may introduce bias into resemble their orthologs from other mammalian the model prediction. The training set was analyzed 14 species. Among mammals the pathogenesis of by the MatInspector20 to identify potential andro- prostate cancer (CaP) is unique to man. The only gen receptor binding sites. Androgen receptor can mammal known to develop CaP in a similar fashion recognize DNA sequences represented by the 15 is the canine. Fortunately, biological context glucocorticoid responsive element matrix family can significantly enhance the quality of TFBS (GREF) that includes matrices of androgen receptor, 16,17 models. Biologically linked sets of transcription glucocorticoid receptor and progesterone receptor factors observed in the combination of specific binding sites. Two GREFs in KLK3 were identified, 5 TFBSs can significantly improve prediction quality. one in NDRG1, two in NKX3.1 and one within the Consistent with this notion, we selected a training IHPK1 upstream sequences. In addition to the set of genes based on gene expression kinetics and GREF family the program found 27 other matrix dose–response of prostate cancer cells to androgen families to match within the sequences. The relation 4 hormone stimulus. Furthermore, we reasoned that of each predicted site was assessed relative to gene regulation by the combination of closely the GREF positions. The distance criterion for the situated binding sites is an essential feature of assessment was 20–33 base-pairs between the 18,19 functional transcription regulatory regions. centers of two adjacent sites (within 2–3 DNA Therefore, we formulated an androgen receptor- helical turn distance). Only the GATA matrix family binding model from the combination of adjacent matches showed distance correlation to the GREF matrices. We experimentally verified this model and went on to assess the utility of the model by testing upstream sequences of the androgen- † http://www.genomatix.de 2 inducible gene, PMEPA1. ‡ http://www.ncbi.nlm.nih.gov Predicting Androgen Receptor Binding Sites 765 Figure 1. Matrices within the training set sequences. (a) Position of matches of GREF (encircled filled arrows) and GATA (open arrowheads) matrix families within the training set of sequences. Arrowheads indicate the strand orientation. Adjacent GREF and GATA matrices (min. 20 and max. 33 bp interval between the centers of matrices) and the corresponding matrix anchor positions are boxed. (b) List of all matches of matrix families identified within the train- ing set sequences. Numbers indi- cate how often a given matrix matched within the pre-defined range of a GREF matrix match (20–33 bp). Only GATA matrix family matches fulfilled this con- dition in all four sequences. matrix family matches (Figure 1). Indeed, the Launcher13 and was successfully checked for identification of potential GATA binding
Load more

Recommended publications
  • Wo 2010/075007 A2
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 1 July 2010 (01.07.2010) WO 2010/075007 A2 (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12Q 1/68 (2006.01) G06F 19/00 (2006.01) kind of national protection available): AE, AG, AL, AM, C12N 15/12 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (21) International Application Number: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/US2009/067757 HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (22) International Filing Date: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, 11 December 2009 ( 11.12.2009) ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, (25) Filing Language: English SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, (26) Publication Language: English TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 12/3 16,877 16 December 2008 (16.12.2008) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, (71) Applicant (for all designated States except US): DODDS, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, W., Jean [US/US]; 938 Stanford Street, Santa Monica, TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, CA 90403 (US).
    [Show full text]
  • Genome-Wide Association Study of Multiplex Schizophrenia Pedigrees
    Article Genome-Wide Association Study of Multiplex Schizophrenia Pedigrees Douglas F. Levinson, M.D. Anthony O’Neill, M.D. in the primary European-ancestry analyses). Association was tested for single SNPs and Jianxin Shi, Ph.D. George N. Papadimitriou, M.D. genetic pathways. Polygenic scores based Kai Wang, Ph.D. Dimitris Dikeos, M.D. on family study results were used to predict case-control status in the Schizophrenia Sang Oh, M.Sc. Wolfgang Maier, M.D. Psychiatric GWAS Consortium (PGC) data set, and consistency of direction of effect Brien Riley, Ph.D. Bernard Lerer, M.D. with the family study was determined for Ann E. Pulver, Ph.D. Dominique Campion, M.D., top SNPs in the PGC GWAS analysis. Within- family segregation was examined for Dieter B. Wildenauer, Ph.D. Ph.D. schizophrenia-associated rare CNVs. Claudine Laurent, M.D., Ph.D. David Cohen, M.D., Ph.D. Results: No genome-wide significant asso- Maurice Jay, M.D. ciations were observed for single SNPs or for Bryan J. Mowry, M.D., pathways. PGC case and control subjects F.R.A.N.Z.C.P. Ayman Fanous, M.D. had significantly different genome-wide Pablo V. Gejman, M.D. Peter Eichhammer, M.D. polygenic scores (computed by weighting their genotypes by log-odds ratios from the 2 Michael J. Owen, Ph.D., Jeremy M. Silverman, Ph.D. family study) (best p=10 17, explaining F.R.C.Psych. 0.4% of the variance). Family study and Nadine Norton, Ph.D. PGC analyses had consistent directions for Kenneth S. Kendler, M.D.
    [Show full text]
  • Strand Breaks for P53 Exon 6 and 8 Among Different Time Course of Folate Depletion Or Repletion in the Rectosigmoid Mucosa
    SUPPLEMENTAL FIGURE COLON p53 EXONIC STRAND BREAKS DURING FOLATE DEPLETION-REPLETION INTERVENTION Supplemental Figure Legend Strand breaks for p53 exon 6 and 8 among different time course of folate depletion or repletion in the rectosigmoid mucosa. The input of DNA was controlled by GAPDH. The data is shown as ΔCt after normalized to GAPDH. The higher ΔCt the more strand breaks. The P value is shown in the figure. SUPPLEMENT S1 Genes that were significantly UPREGULATED after folate intervention (by unadjusted paired t-test), list is sorted by P value Gene Symbol Nucleotide P VALUE Description OLFM4 NM_006418 0.0000 Homo sapiens differentially expressed in hematopoietic lineages (GW112) mRNA. FMR1NB NM_152578 0.0000 Homo sapiens hypothetical protein FLJ25736 (FLJ25736) mRNA. IFI6 NM_002038 0.0001 Homo sapiens interferon alpha-inducible protein (clone IFI-6-16) (G1P3) transcript variant 1 mRNA. Homo sapiens UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 15 GALNTL5 NM_145292 0.0001 (GALNT15) mRNA. STIM2 NM_020860 0.0001 Homo sapiens stromal interaction molecule 2 (STIM2) mRNA. ZNF645 NM_152577 0.0002 Homo sapiens hypothetical protein FLJ25735 (FLJ25735) mRNA. ATP12A NM_001676 0.0002 Homo sapiens ATPase H+/K+ transporting nongastric alpha polypeptide (ATP12A) mRNA. U1SNRNPBP NM_007020 0.0003 Homo sapiens U1-snRNP binding protein homolog (U1SNRNPBP) transcript variant 1 mRNA. RNF125 NM_017831 0.0004 Homo sapiens ring finger protein 125 (RNF125) mRNA. FMNL1 NM_005892 0.0004 Homo sapiens formin-like (FMNL) mRNA. ISG15 NM_005101 0.0005 Homo sapiens interferon alpha-inducible protein (clone IFI-15K) (G1P2) mRNA. SLC6A14 NM_007231 0.0005 Homo sapiens solute carrier family 6 (neurotransmitter transporter) member 14 (SLC6A14) mRNA.
    [Show full text]
  • Molecular Cytogenetics Biomed Central
    Molecular Cytogenetics BioMed Central Research Open Access MODY-like diabetes associated with an apparently balanced translocation: possible involvement of MPP7 gene and cell polarity in the pathogenesis of diabetes Elizabeth J Bhoj1, Stefano Romeo1,2, Marco G Baroni2, Guy Bartov3, Roger A Schultz3,5 and Andrew R Zinn*1,4 Address: 1McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA, 2Department of Medical Sciences, Endocrinology, University of Cagliari, Cagliari, Italy, 3Department of Pathology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA , 4Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA and 5Signature Genomic Laboratories, LLC, Spokane, WA, USA Email: Elizabeth J Bhoj - [email protected]; Stefano Romeo - [email protected]; Marco G Baroni - [email protected]; Guy Bartov - [email protected]; Roger A Schultz - [email protected]; Andrew R Zinn* - [email protected] * Corresponding author Published: 13 February 2009 Received: 26 September 2008 Accepted: 13 February 2009 Molecular Cytogenetics 2009, 2:5 doi:10.1186/1755-8166-2-5 This article is available from: http://www.molecularcytogenetics.org/content/2/1/5 © 2009 Bhoj et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Characterization of disease-associated balanced translocations has led to the discovery of genes responsible for many disorders, including syndromes that include various forms of diabetes mellitus.
    [Show full text]
  • Microrna Regulation and Human Protein Kinase Genes
    MICRORNA REGULATION AND HUMAN PROTEIN KINASE GENES REQUIRED FOR INFLUENZA VIRUS REPLICATION by LAUREN ELIZABETH ANDERSEN (Under the Direction of Ralph A. Tripp) ABSTRACT Human protein kinases (HPKs) have profound effects on cellular responses. To better understand the role of HPKs and the signaling networks that influence influenza replication, a siRNA screen of 720 HPKs was performed. From the screen, 17 “hit” HPKs (NPR2, MAP3K1, DYRK3, EPHA6, TPK1, PDK2, EXOSC10, NEK8, PLK4, SGK3, NEK3, PANK4, ITPKB, CDC2L5, CALM2, PKN3, and HK2) were validated as important for A/WSN/33 influenza virus replication, and 6 HPKs (CDC2L5, HK2, NEK3, PANK4, PLK4 and SGK3) identified as important for A/New Caledonia/20/99 influenza virus replication. Meta-analysis of the hit HPK genes identified important for influenza virus replication showed a level of overlap, most notably with the p53/DNA damage pathway. In addition, microRNAs (miRNAs) predicted to target the validated HPK genes were determined based on miRNA seed site predictions from computational analysis and then validated using a panel of miRNA agonists and antagonists. The results identify miRNA regulation of hit HPK genes identified, specifically miR-148a by targeting CDC2L5 and miR-181b by targeting SGK3, and suggest these miRNAs also have a role in regulating influenza virus replication. Together these data advance our understanding of miRNA regulation of genes critical for virus replication and are important for development novel influenza intervention strategies. INDEX WORDS: Influenza virus,
    [Show full text]
  • Homologs of Genes Expressed in Caenorhabditis Elegans Gabaergic
    Hammock et al. Neural Development 2010, 5:32 http://www.neuraldevelopment.com/content/5/1/32 RESEARCH ARTICLE Open Access Homologs of genes expressed in Caenorhabditis elegans GABAergic neurons are also found in the developing mouse forebrain Elizabeth AD Hammock1,2*, Kathie L Eagleson3, Susan Barlow4,6, Laurie R Earls4,7, David M Miller III2,4,5, Pat Levitt3* Abstract Background: In an effort to identify genes that specify the mammalian forebrain, we used a comparative approach to identify mouse homologs of transcription factors expressed in developing Caenorhabditis elegans GABAergic neurons. A cell-specific microarray profiling study revealed a set of transcription factors that are highly expressed in embryonic C. elegans GABAergic neurons. Results: Bioinformatic analyses identified mouse protein homologs of these selected transcripts and their expression pattern was mapped in the mouse embryonic forebrain by in situ hybridization. A review of human homologs indicates several of these genes are potential candidates in neurodevelopmental disorders. Conclusions: Our comparative approach has revealed several novel candidates that may serve as future targets for studies of mammalian forebrain development. Background As with other cell types, the diversity of GABAergic Proper forebrain patterning and cell-fate specification neurons has its basis in different developmental origins, lay the foundation for complex behaviors. These neuro- with timing and location of birth playing key roles in developmental events in large part depend on a series of cell fate [1,6-8]. gene expression refinements (reviewed in [1]) that com- Despite the phenotypic variety of GABAergic neurons, mit cells to express certain phenotypic features that all use GABA as a neurotransmitter.
    [Show full text]
  • REPORT Familial Chilblain Lupus, a Monogenic Form of Cutaneous Lupus Erythematosus, Maps to Chromosome 3P
    REPORT Familial Chilblain Lupus, a Monogenic Form of Cutaneous Lupus Erythematosus, Maps to Chromosome 3p Min Ae Lee-Kirsch, Maolian Gong, Herbert Schulz, Franz Ru¨schendorf, Annette Stein, Christiane Pfeiffer, Annalisa Ballarini, Manfred Gahr, Norbert Hubner, and Maja Linne´ Systemic lupus erythematosus is a prototypic autoimmune disease. Apart from rare monogenic deficiencies of complement factors, where lupuslike disease may occur in association with other autoimmune diseases or high susceptibility to bacterial infections, its etiology is multifactorial in nature. Cutaneous findings are a hallmark of the disease and manifest either alone or in association with internal-organ disease. We describe a novel genodermatosis characterized by painful bluish- red inflammatory papular or nodular lesions in acral locations such as fingers, toes, nose, cheeks, and ears. The lesions sometimes appear plaquelike and tend to ulcerate. Manifestation usually begins in early childhood and is precipitated by cold and wet exposure. Apart from arthralgias, there is no evidence for internal-organ disease or an increased sus- ceptibility to infection. Histological findings include a deep inflammatory infiltrate with perivascular distribution and granular deposits of immunoglobulins and complement along the basement membrane. Some affected individuals show antinuclear antibodies or immune complex formation, whereas cryoglobulins or cold agglutinins are absent. Thus, the findings are consistent with chilblain lupus, a rare form of cutaneous lupus erythematosus. Investigation of a large German kindred with 18 affected members suggests a highly penetrant trait with autosomal dominant inheritance. By single-nucleotide-polymorphism–based genomewide linkage analysis, the locus was mapped to chromosome 3p. Hap- lotype analysis defined the locus to a 13.8-cM interval with a LOD score of 5.04.
    [Show full text]
  • Mycobacterium Tuberculosis Induced Transcription in Macrophages: the Role of TPL2/ERK Signalling in the Negative Regulation of T
    Mycobacterium tuberculosis induced transcription in macrophages: the role of TPL2/ERK signalling in the negative regulation of type I interferon production and implications for control of tuberculosis John Benson Ewbank August 2012 Division of Immunoregulation MRC National Institute for Medical Research The Ridgeway, Mill Hill London NW7 1AA Submitted to University College London for the Degree of Doctor of Philosophy I, John Benson Ewbank, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis Abstract Abstract Mycobacterium tuberculosis is an important global cause of mortality and morbidity. The major host cell of Mycobacterium tuberculosis is the macrophage, and Mycobacterium tuberculosis is able to subvert the macrophage response in order to survive and replicate. The majority of infected individuals mount an immune response capable of controlling Mycobacterium tuberculosis infection. This requires the cytokines IL-12, TNFα, IL-1 and IFNγ, which promote eradication or control of infection. However, other immune factors, including IL-10 and type I IFN, can inhibit this protective response. In this study we have used microarray analysis to study the temporal response of macrophages to Mycobacterium tuberculosis infection, in an unbiased fashion. In response to Mycobacterium tuberculosis infection, macrophages produced cytokines and chemokines, upregulated genes involved with major histocompatability class I antigen presentation, activated both pro- and anti-apoptotic genes and downregulated many genes involved in cell-division and metabolism. We also observed the early induction of genes regulated by the extracellular-regulated kinase (ERK) MAP kinase pathway, and the upregulation of genes known to be induced by type I IFN, leading us to further investigate the role of these pathways in the macrophage response to Mycobacterium tuberculosis.
    [Show full text]
  • Minimal Peroxide Exposure of Neuronal Cells Induces Multifaceted Adaptive Responses
    Minimal Peroxide Exposure of Neuronal Cells Induces Multifaceted Adaptive Responses Wayne Chadwick1, Yu Zhou1, Sung-Soo Park1, Liyun Wang1, Nicholas Mitchell2, Matthew D. Stone1, Kevin G. Becker3, Bronwen Martin4, Stuart Maudsley1* 1 Receptor Pharmacology Unit, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America, 2 Department of Biology, Saint Bonaventure University, Saint Bonaventure, New York, United States of America, 3 Gene Expression and Genomics Unit, Research Resources Branch, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America, 4 Metabolism Unit, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America Abstract Oxidative exposure of cells occurs naturally and may be associated with cellular damage and dysfunction. Protracted low level oxidative exposure can induce accumulated cell disruption, affecting multiple cellular functions. Accumulated oxidative exposure has also been proposed as one of the potential hallmarks of the physiological/pathophysiological aging process. We investigated the multifactorial effects of long-term minimal peroxide exposure upon SH-SY5Y neural cells to understand how they respond to the continued presence of oxidative stressors. We show that minimal protracted oxidative stresses induce complex molecular and physiological alterations in cell functionality. Upon chronic exposure to minimal doses of hydrogen peroxide, SH-SY5Y cells displayed a multifactorial response to the stressor. To fully appreciate the peroxide-mediated cellular effects, we assessed these adaptive effects at the genomic, proteomic and cellular signal processing level. Combined analyses of these multiple levels of investigation revealed a complex cellular adaptive response to the protracted peroxide exposure. This adaptive response involved changes in cytoskeletal structure, energy metabolic shifts towards glycolysis and selective alterations in transmembrane receptor activity.
    [Show full text]
  • Dissertation Submitted to the Combined Faculties for the Natural
    Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Diplom-Biochemiker Johannes Hermle born in: Offenbach a.M., Germany Oral-examination: July 26, 2017 siRNA SCREEN FOR IDENTIFICATION OF HUMAN KINASES INVOLVED IN ASSEMBLY AND RELEASE OF HIV-1 Referees: Prof. Dr. Hans-Georg Kräusslich Prof. Dr. Dirk Grimm ii Meiner Familie iii Summary Summary The replication of the human immunodeficiency virus type 1 (HIV-1) is as yet not fully understood. In particular the knowledge of interactions between viral and host cell proteins and the understanding of complete virus-host protein networks are still imprecise. An integral picture of the hijacked cellular machinery is essential for a better comprehension of the virus. And as a prerequisite, new tools are needed for this purpose. To create such a novel tool, a screening platform for host cell factors was established in this work. The screening assay serves as a powerful method to gain insights into virus-host-interactions. It was specifically tailored to addressing the stage of assembly and release of viral particles during the replication cycle of HIV-1. It was designed to be suitable for both RNAi and chemical compound screening. The first phase of this work comprised the setup and optimization of the assay. It was shown, that it was robust and reliable and delivered reproducible results. As a subsequent step, a siRNA library targeting 724 human kinases and accessory proteins was examined. After the evaluation of the complete siRNA library in a primary screen, all primary hits were validated in a second reconfirmation screen using different siRNAs.
    [Show full text]
  • Thesis Wagner 2019.P
    Identification of process-relevant kinases in human amniotic production cells Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Naturwissenschaften der Universität Ulm Vorgelegt von Andreas Wagner aus Bad Köstritz 2019 Die vorliegende Arbeit wurde im Institut für Angewandte Biotechnologie der Hochschule Biberach unter der Anleitung von Herr Prof. Dr. Jürgen Hannemann durchgeführt. Amtierender Dekan: Prof. Dr. Peter Dürre Erstgutachter: Prof. Dr. Jürgen Hannemann Zweitgutachter: Prof. Dr. Bernd Eikmanns Wahlprüfer: Prof. Kerstin Otte Wahlprüfer: Prof. Dierk Niessing Tag der Promotion: 17. Juni 2019 Diese Arbeit wurde durch ein Promotionsstipendium nach dem Landesgraduiertenförderungsgesetz (LGFG) des Landes Baden- Württemberg finanziert und im Rahmen des kooperativen Promotionskollegs (KPK) „Pharmazeutische Biotechnologie“ der Universität Ulm und der Hochschule Biberach durchgeführt. Teile dieser Arbeit wurden bereits in den folgenden Artikel publiziert: Fischer, Simon, Andreas Wagner, Aron Kos, Armaz Aschrafi, René Handrick, Juergen Hannemann, and Kerstin Otte. 2013. “Breaking Limitations of Complex Culture Media: Functional Non-Viral MiRNA Delivery into Pharmaceutical Production Cell Lines.” Journal of Biotechnology 168: 589–600. “It’s a dangerous business, Frodo, going out of your door,” he used to say. “You step into the Road, and if you don’t keep your feet, there is no knowing where you might be swept to” Frodo Baggins, citing his uncle Bilbo Baggins The Lord of the Rings – The Fellowship of the Ring J.R.R.
    [Show full text]
  • A Hidden Markov Model for Investigating Recent Positive Selection Through Haplotype Structure
    bioRxiv preprint doi: https://doi.org/10.1101/011247; this version posted November 8, 2014. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. A Hidden Markov Model for Investigating Recent Positive Selection through Haplotype Structure Hua Chena,b,∗, Jody Heyb, Montgomery Slatkinc aCenter for Computational Genomics, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China bCenter for Computational Genetics and Genomics, Temple University, Philadelphia PA 19122 cDepartment of Integrative Biology, University of California, Berkeley, CA 94720 Abstract Recent positive selection can increase the frequency of an advantageous mutant rapidly enough that a relatively long ancestral haplotype will be remained intact around it. We present a hidden Markov model (HMM) to identify such haplotype structures. With HMM identified haplotype structures, a population genetic model for the extent of ancestral haplotypes is then adopted for parameter inference of the selection intensity and the allele age. Simulations show that this method can detect selection under a wide range of conditions and has higher power than the existing frequency spectrum-based method. In addition, it provides good estimate of the selection coefficients and allele ages for strong selection. The method analyzes large data sets in a reasonable amount of running time. This method is applied to HapMap III data for a genome scan, and identifies a list of candidate regions putatively under recent positive selection. It is also applied to several genes known to be under recent positive selection, including the LCT, KITLG and TYRP1 genes in Northern Europeans, and OCA2 in East Asians, to estimate their allele ages and selection coefficients.
    [Show full text]