Characterization of the MYST histone acetyltransferase Mof2 ... ... Characterization of the MYST histone

  • View
    1

  • Download
    0

Embed Size (px)

Text of Characterization of the MYST histone acetyltransferase Mof2 ... ... Characterization of the MYST...

  • Characterization of the MYST histone acetyltransferase

    Mof2 in Drosophila melanogaster

    Inaugural-Dissertation

    zur

    Erlangung des Doktorgrades

    Dr. rer. nat.

    der Fakultät für

    Biologie

    an der

    Universität Duisburg-Essen

    vorgelegt von

    Christiane Heseding

    aus Wuppertal

    August 2014

  • List of figures................................................................................................................................................................ 3

    List of tables .................................................................................................................................................................. 4

    Abbrevations ................................................................................................................................................................ 4

    1 INTRODUCTION ................................................................................................................................. 8 1.1 ORGANISATION OF DNA IN THE EUKARYOTIC NUCLEUS ....................................................................... 8 1.2 HISTONE MODIFICATIONS ........................................................................................................................... 9 1.3 CHROMATIN STATES ................................................................................................................................. 11 1.4 INSULATOR PROTEINS .............................................................................................................................. 12 1.5 HISTONE ACETYLTRANSFERASE FAMILIES ............................................................................................ 13 1.6 MYST HISTONE ACETYLTRANSFERASES ............................................................................................... 14 1.7 MOF2 .......................................................................................................................................................... 17 1.8 DEVELOPMENT OF SPERM IN DROSOPHILA MELANOGASTER ............................................................ 17 1.9 FECUNDITY AND LONGEVITY ................................................................................................................... 19 1.10 THERMAL STRESS ..................................................................................................................................... 20 1.11 TELOMERES IN DROSOPHILA MELANOGASTER .................................................................................... 21 1.12 FUNCTION OF THE TRANSCRIPTION FACTOR DREF............................................................................ 23 1.13 FUNCTION OF THE CHROMATIN BINDING PROTEIN Z4/PUTZIG ....................................................... 24 1.14 OUTLINE OF THE THESIS .......................................................................................................................... 24

    2 MATERIAL AND METHODS .......................................................................................................... 26 2.1 ORGANISMS AND CELL LINES .................................................................................................................. 26

    2.1.1 E. coli strains.............................................................................................................................................. 26

    2.1.2 Drosophila stocks ..................................................................................................................................... 26

    2.1.3 Schneider cell line 2 ................................................................................................................................ 27

    2.1.4 Sf9 cells ......................................................................................................................................................... 28

    2.2 MEDIA AND GROWTH CONDITIONS ........................................................................................................ 28 2.2.1 E. coli media and growth conditions .............................................................................................. 28

    2.2.2 Drosophila husbandry ........................................................................................................................... 29

    2.3 TEST FOR LETHALITY OF THE HETEROZYGOUS PBAC(W+)CG1894F06204 STRAIN ......................... 29 2.4 MOLECULAR CLONING .............................................................................................................................. 31

    2.4.1 Vectors .......................................................................................................................................................... 31

    2.4.2 PCR ................................................................................................................................................................. 31

    2.4.3 Oligonucleotides ....................................................................................................................................... 31

    2.4.4 Plasmids ....................................................................................................................................................... 33

    2.5 DNA AND RNA ......................................................................................................................................... 35 2.5.1 Isolation of genomic DNA .................................................................................................................... 35

    2.5.2 ChIP-Seq ....................................................................................................................................................... 35

    2.5.3 RNA purification from fly tissue or cells ........................................................................................ 36

    2.5.4 cDNA synthesis ......................................................................................................................................... 36

    2.5.5 Real-time PCR ............................................................................................................................................ 36

    2.6 ANTIBODIES ............................................................................................................................................... 37 2.6.1 Antibody generation .............................................................................................................................. 37

    2.6.2 Affinity purification of antibodies ................................................................................................... 37

    2.6.3 Antibodies ................................................................................................................................................... 38

    2.7 PROTEIN EXPRESSION AND PURIFICATION ........................................................................................... 39 2.7.1 Protein expression and purification in E.coli ............................................................................. 39

    2.7.2 Integration of mof2 into the baculovirus genome and purification of genomic DNA

    from E. coli .................................................................................................................................................................. 39

    2.7.3 Protein expression in Sf9 cells ............................................................................................................ 40

    2.7.4 Protein expression test in Sf9 cells ................................................................................................... 41

    2.7.5 Protein purification from Sf9 cells ................................................................................................... 41

    2.7.6 SDS-PAGE and Western blot ................................................................................................................ 41

    2.7.7 Immunoprecipitation (IP) ................................................................................................................... 42

    2.8 CHROMATIN IMMUNOPRECIPITATION (CHIP) ..................................................................................... 43 2.9 HISTONE ACETYLATION ASSAY ................................................................................................................ 44

  • 2.10 IMMUNOHISTOCHEMISTRY ...................................................................................................................... 44 2.10.1 Preparation and fluorescent staining of polytene chromosome staining ................ 44

    2.10.2 Preparation and fluorescent staining of Drosophila embryos ...................................... 45

    2.10.3 Preparation and fluorescent steaining of Drosophila testes .......................................... 45

    2.11 KNOCK-DOWN OF MOF2 .......................................................................................................................... 46 2.11.1 Mof2 knock-down in S2 cells ......................................................................................................... 46

    2.11.2 Mof2 knock-down in flies................................................................................................................ 46

    2.12 BIOINFORMATICS ...................................................................................................................................... 47

    3 RESULTS