STUDIES OF ZINC FINGER PROTEINS IN EPIGENETIC GENE REGULATION NAN LIU MÜNCHEN 2014 STUDIES OF ZINC FINGER PROTEINS IN EPIGENETIC GENE REGULATION NAN LIU Dissertation an der Fakultät für Biologie der Ludwig‐Maximilians‐Universität München vorgelegt von Nan Liu aus Yushu, China München, den 2014 Erstgutachter: Prof. Dr. Heinrich Leonhardt Zweitgutachter: PD Dr. Anna Friedl Tag der mündlichen Prüfung: 04.12.2014 Contents Content Summary ...................................................................................................... i 1 Introduction .......................................................................................... 1 1.1 Epigenetic regulation ............................................................................ 2 1.1.1 DNA methylation ................................................................................................... 2 1.1.2 Histone modifications ............................................................................................ 4 1.2 Zinc finger proteins (ZFPs) and zinc finger motifs................................... 7 1.2.1 C2H2 type zinc fingers............................................................................................ 8 1.2.2 CXXC type zinc fingers .......................................................................................... 10 1.2.3 Treble clef type zinc fingers ................................................................................. 12 1.3 ZFPs act as modifiers in the epigenetic regulatory system .................... 17 1.3.1 Writers of DNA modifications .............................................................................. 17 1.3.2 Readers of DNA modifications ............................................................................. 20 1.3.3 Erasers of DNA modifications .............................................................................. 25 1.3.4 Histone modifiers ................................................................................................. 28 1.4 Applications of ZFPs ............................................................................. 30 1.4.1 Genome modification with engineered zinc finger nucleases ............................ 30 1.4.2 Manipulating gene expression with engineered zinc finger transcription factors32 1.4.3 Therapeutic applications of engineered ZFPs ...................................................... 34 1.5 Aims of this work ................................................................................. 36 2 Materials and Methods ....................................................................... 37 2.1 Materials ............................................................................................. 38 2.1.1 Technical devices ................................................................................................. 38 2.1.2 Consumables ........................................................................................................ 40 2.1.3 Reagents and kits ................................................................................................. 41 2.1.4 Cell lines ............................................................................................................... 45 ii Content 2.1.5 Plasmid constructs ............................................................................................... 46 2.1.6 Primer sequences ................................................................................................. 49 2.2 Methods .............................................................................................. 54 2.2.1 Expression constructs .......................................................................................... 54 2.2.2 Antibodies ............................................................................................................ 55 2.2.3 Cell culture and transfection ............................................................................... 55 2.2.4 Generation of stable cell lines and DNA methylation analyses ........................... 56 2.2.5 Cell inhibitor treatment ....................................................................................... 56 2.2.6 Coimmunoprecipitation (Co‐IP) and western blot .............................................. 56 2.2.7 Western blot quantification ................................................................................. 57 2.2.8 Immunofluorescence staining ............................................................................. 57 2.2.9 Protein production and purification .................................................................... 57 2.2.10 RNA isolation and Real‐time PCR ......................................................................... 58 2.2.11 Northern blot ....................................................................................................... 58 2.2.12 5’ RACE ................................................................................................................. 59 2.2.13 F3H assay ............................................................................................................. 59 2.2.14 Determination of global genomic 5hmC levels ................................................... 60 2.2.15 In vitro DNA binding assay ................................................................................... 61 2.2.16 Statistical analysis ................................................................................................ 61 3 Results................................................................................................. 63 3.1 Intrinsic and extrinsic connections of TET3 dioxygenase with CXXC modules............................................................................................... 64 3.1.1 Identification and expression pattern of mouse tet3 transcripts encoding a CXXC domain ................................................................................................................. 64 3.1.2 CXXC4 interacts with TET3 in vivo and is expressed in the adult brain .............. 70 3.1.3 The CXXC domains of TET1, TET3CXXC, CXXC4 and CXXC5 bind CpG containing DNA substrates .................................................................................................... 72 iii Content 3.1.4 TET3CXXC oxidizes genomic 5mC in vivo and shows slightly lower mobility than the TET3 isoform lacking the CXXC domain ........................................................ 75 3.1.5 Detection of TET3CXXC at the protein level ........................................................... 76 3.2 DNMT1 ubiquitin interacting motif UIM is required for maintenance of DNA methylation by binding to UHRF1‐dependent H3 ubiquitination .. 78 3.2.1 UHRF1 ubiquitinates histone H3 on K18 and K23 residues in mammalian cells . 78 3.2.2 The DNMT1 UIM mediated binding to ubiquitinated H3 is required for methylation activity in vivo ................................................................................. 79 3.3 UHRF1 and USP7 control stability of heterochromatin protein CBX1 by polyubiquitination ............................................................................... 82 3.3.1 UHRF1 associates and colocalizes with all three CBXs ........................................ 82 3.3.2 UHRF1 is the E3 ligase for ubiquitination of CBX1 ............................................... 83 3.3.3 Association between UHRF1 and CBX1 is independent of PxVxV motif ............. 86 3.3.4 USP7 stabilizes CBX1 by deubiquitination ........................................................... 88 4 Discussion ........................................................................................... 89 4.1 Regulation of TET enzymes via CXXC domains in cis and in trans .......... 90 4.1.1 Regulation of TET proteins via CXXC domains in cis ............................................ 90 4.1.2 Regulation of TET proteins via CXXC domains in trans ........................................ 93 4.2 Functions of UHRF1 in targeting DNMT1 to hemimethylated DNA substrates ............................................................................................ 96 4.2.1 Direct targeting mechanism mediated by the SRA domain ................................ 96 4.2.2 Indirect targeting mechanism coordinated by RING and PHD domains ............. 98 4.2.3 Chromatin association of DNMT1 is regulated by dynamic posttranslational modifications ..................................................................................................... 100 4.3 Potential roles of UHRF2 in epigenetics .............................................. 103 4.4 Regulatory role of UHRF1 in chromatin dynamics .............................. 106 iv Content 4.4.1 A novel approach to detect ubiquitination in vitro and in living cells ............... 106 4.4.2 Chromatin structure and heterochromatin protein HP1 .................................. 107 5 Annex ................................................................................................ 109 5.1 References ......................................................................................... 110 5.2 Abbreviations .................................................................................... 132 5.3 Declaration ........................................................................................ 137 5.4 Acknowledgements ........................................................................... 139 6 Curriculum Vitae................................................................................ 141 7 Publications 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