Widespread regulation of gene expression in the Drosophila genome by the histone acetyltransferase dTip60 [Elektronische Ressource] / vorgelegt von Corinna Schirling

Corinna Schirling

Sujets

• Biologie

Informations

• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2008
• Nombre de lectures: 26
• Langue: English

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Widespread regulation of gene expression in the Drosophila genome by the histone acetyltransferase dTip60

  Inaugural-Dissertation zur Erlangung des Doktorgrades Dr. rer. nat.     des Fachbereichs Biologie und Geografie an der     Universität Duisburg-Essen   vorgelegt von Corinna Schirling   geboren in Heilbronn a. N.   Dezember 2008    

 

 

 

Die der vorliegenden Arbeit zugrunde liegenden Experimente wurden am Max-Planck- Institut für Molekulare Genetik in Berlin und am Ze ntrum für Medizinische Biotechnologie (ZMB), Arbeitsgruppe Genetik an der Universität Duisburg-Essen durchgeführt.        1. Gutachter: Prof. Dr. Ann Ehrenhofer-Murray        2. Gutachter: Prof. Dr. Bernhard Horsthemke        3. Gutachter: Prof. Dr. Harald Saumweber, HU Berlin        Vorsitzender des Prüfungsausschusses: Prof. Dr. Daniel Hoffmann        Tag der mündlichen Prüfung: 23. März 2009                  

   

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Table of contents Table of figures List of tables Abbrevations  _______________________________________________________ 1   Introduction 9   1.1   omatChrnagro ni_noitazi____________________9________________________   1.2   Histone modifications, histone variants and chromatin remodeling complexes 11   ________________ 1.3   The MYST family of HATs______ ____________________ 13   1.4    _______________ 14The MYST HAT dTip60 is part of a multiprotein complex   1.5   can have functions outside of the Tip60Some Tip60 complex components complex _____________ ___________________ 17   ________________________________ 1.6    dT p _______________________________________Geneti f c interactions o i 60 18   1.7   uFitcn sno Tof60ip_________18________________________________________   1.8   Aim of this work 20   __________________________________________________ ______________________________________________ 2   Material and Methods 22   2.1    22Organisms_______ _   ________________________________________________ 2.1.1   E. colistrains 22   ____________________________________________________________ 2.1.2    22Drosophila stocks_   ________________________________________________________ 2.2   Media and growth conditions ________________________________________ 23   2.2.1   Media and growth conditions forE. coli________3_2______________________________   2.2.2   Cultivation of Schneider SL2 Zellen 23   __________________________________________ 2.2.3   Drosophila husbandry _____________________________________________________ 23   2.3   Molecular cloning ___ 24   ______________________________________________ ___________________________________________ 2.4   P-element transformation 26   2.5   P-element mobilization 26   _____________________________________________ 2.6    _______________________Antibody generation 27   ________________________ 2.7    ________________ 28Preparation of cell extracts, SDS-PAGE and Western blot   ry_____________________________________________ 2.8   Immunohistochemist 28   2.8.1    yosWhole-mount fluorescent staining of Drosophila embr ________________________ 28   2.8.2   In situ staining of Drosophila embryos _____________ 29   ___________________________

   

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2.8.3    30Fluorescent staining of polytene chromosomes   _________________________________ 2.8.4   gnami anigininofg taS_____________________________________________ l wi discs 30   dT p _____________________________________ 2.9    31 knock-down in SL2 cellsi 60   10    _______________________________ gGenome-wide gene expression profi 2. lin 31   2.11   Quantitative real-time PCR 32   _________________________________________ 2.12   Chromatin Immunoprecipitation (ChIP) __ 34   ____________________________ ____________________________________________________ 2.13   Bioinformatics 34   3   Results 36   __________________________________________________________ 3.1    _______________________ 36Phenotypes of dTip60 knock-down in Drosophila   3.1.1   Use of the Gal4/UAS system for RNA interference in flies 36   ________________________ 3.1.2   Strong knock-down of dTip60 led to lethality, whereas moderate knock-down caused developmental defects in the wing___________________________________________________37   3.1.3   imaginal wing disc led to apoptosis and enrichment ofKnock-down of dTip60 in the H3K27me3 41   _____________________________________________________________________ 3.2   Generation of an antiserum specific for dTip60 _________________________ 42   3.3    43of dTip60 during embryonic development __________Nuclear localization   __ 3.4    45dTip60 localized to interbands on polytene chromosomes_________________   3.5   Genome-wide expression analysis revealed a major role for dTip60 in gene regulation_________________________________________48   ______________________ 3.6   dTip60-regulated genes were involved in chromatin-related processes ______ 50   3.7   dTip60 acted as a repressor at distinct g ___________________________ 51   enes 3.8   dTip60 was physical y located at repressed genes _______________________ l 55   3.9    ___________________ repCooperation of dTip60 and HDAC1 in g ene ression 56   4   Discussion 59   _______________________________________________________ 4.1    59 _______________________________dTip60 was essential for viability in flies   4.2   Effects of dTip60 on wing developmen ________________________________ 60   t 4.3   ip60 in transcriptional activation __________________________________ dT 62   4.4    63Function and localization of dTip60 on polytene chromosomes ____________   4.5    63dTip60 in transcriptional repression __________________________________   4.5.1    p on by deposition of H2Av _______________dTip60 might mediate its r e ressive functi 64   4.5.2   The repressive function of dTip60 might be independent of its HAT function_________ 65   4.5.3   dTip60 might cooperate with HDAC1 in gene regulation _________________________ 65   4

   

   4.5.4   the function of dTip60 might be context-dependent______________________________ 66   4.6   Suany armm________________ duoltoo_k___________6__8________________   _________________________________________________________ 5   Abstract 70   6    ____________Zusammenfassung ____________________________________ 71   7   References _______________________________________________________ 72   sagung _________________________________________ ____________ 8   Dank 80   Lebenslauf _______________________________________________________ 81   9       Table of figures   Figure 1 Chromatin forms higher structures. .............................................................10 Figure 2 Sites of histone acetylation .........................................................................11 Figure 3 Phylogenetic ..............................14 tree of MYST histone acetyltransferases. Figure 4 Components of transcription-associated complexes that contain Pontin and Reptin. ........................................................................................................................17 Figure 5 of purified recombinant fusion proteins dTip60-A and dTip60-B as Samples used for generation of a dTip60-specific antiserum. .....................................................27 Figure 6 .........37RNAi knock-down induced by the Gal4/UAS system. of  Mechanism Figure 7 of dsRNA constructs used for construction of the UAS-Gal4 RNAi Overview fly line (UAS sense and antisense) and RNAi in SL2 cells (ds1, ds2, ds3). .................38 Figure 8 in the Drosophila Downregulation of dTip60 by RNAi resulted in def ects wing. ...........................................................................................................................40 Figure 9 caused by dTip60- Position, eins length and frequency of ectopic wing v RNAi driven by T80 or en. ..........................................................................................40 Figure 10Effects of en-Gal4-driven dTip60 downregulation in the imaginal wing disc.  ....................................................................................................................................41  Figure 11 m raised against dTip60 Western blot analysis indicated that the antiseru recognized dTip60 protein.. .........................................................................................42 Figure 12 .......................44the dTip60 protein in Drosophila embryos. Localization of Figure 13 .................45Ubiquitous expression of dTip60 RNA during embryogenesis.  Figure 14 dTip60 localized to interbands of polytene chromosomes. .........................46 Figure 15 dTip60 is reduced on polytene chromosomes of dTip60-RNAi animals. . ..47

   

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Figure 16after 5 days of RNAi treatment with Proliferation of SL2 cells was reduced dsRNA against dTip60 (ds1-3) and Aur, but not with mock-treatment against eGFP. ..48 Figure 17 After five days of RNAi treatment with ds3, dTip6 0 mRNA was visibly reduced in comparison to Actin mRNA levels.............................................................49  Figure 18 Genome-wide expression analysis upon loss of dTip60..............................50 Figure 19 (eGFP-RNAi) Proliferation eated of treated (dTip60-RNAi) and mock-tr cells. . ..........................................................................................................................52 Figure 20 Upregulation of selected genes in SL2 cells after zero to five days of dTip60 RNAi treatment. ..........................................................................................................53 Figure 21 Downregulation of selected genes and dTip60 expression in SL2 cells after zero to five days of dTip60 RNAi treatment................................................................54  Figure 22 Localization of dTip60 protein at dTip60-regulated genes by ChIP analysis. ....................................................................................................................................55 

   

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  List of tables   Table 1 of the Tip60 complex in ComponentsH. sapiens,D. melanogasterandS. cerevisiae..... 61............................................................................................................... Table 2     E. colistrains used in this work ....................................................................22 Table 3 Drosophila lines used in this work................................................................22 Table 4 used in this work. ..........................................................................24 Plasmids Table 5 and generation of ng used for molecular cloning, taggi Oligonucleotides dsRNAs.......................................................................................................................25  Table 6 ................................................30 Primers used for generation of in situ probes Table 7 Primers used for quantitative PCR (qPCR) in the body of the gene ..............33 Table 8 used for qPCR at open reading frames (ORFs) ................................33 Primers Table 9 of dTip60 RNAi knock-down in flies.........................................39 Phenotypes Table 10GO category association of genes regulated by dTip60 ................................51 Table 11 Comparison of genes upregulated upon dTip60 depletion with TSA treatment and loss of HDAC1 or HDAC3 ..................................................................................57 Table 12 with TSA of genes downregulated upon dTip60 dep letion Comparison treatment and loss of HDAC1 or HDAC3....................................................................58      

   

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Abbreviations   aa bp ChIP HAT HDAC kDa Mbp ORF PAGE PCR RNAi rpm SDS SL2 wt    

   

                             

amino acid base pair Chromatin immunoprecipitation histone acetyltransferase histone deacetylase kilo dalton mega base pairs open reading frame polyacrylamide gel electrophoresis polymerase chain reaction RNA interference rounds per minute sodium dodecyl sulfate Schneider cells wild type

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