A novel role for the chromatin remodeling ATPase Brg1 during zygotic genome activation in Xenopus Dissertation Zur Erlangung des akademischen Grades Doctor rerum naturalis (Dr. rer.nat.) Vorgelegt der Fakultät für Biologie der Ludwigs-Maximilians-Universität zu München Gabriele Maria Wagner aus Heidelberg München 2014 Tag der Einreichung: 20. Mai 2014 Tag der mündlichen Prüfung: 31. Juli 2014 Erstgutachter: Prof. Dr. Peter Becker Zweitgutachter: Prof. Dr. Hans Straka Drittgutachter: Prof. Dr. Angelika Böttger Viertgutachter: Prof. Dr. John Parsch Eidesstattliche Erklärung Ich versichere hiermit an Eides statt, dass die vorgelegte Dissertation von mir selbständig und ohne unerlaubte Hilfe angefertigt ist. München, den …………………. ............................................................ -Unterschrift- Table of contents Zusammenfassung ............................................................................................................. I Summary ......................................................................................................................... III 1 Introduction ........................................................................................................... 1 1.1 Xenopus as a model organism ................................................................................................. 1 1.1.1 Life cycle of Xenopus ........................................................................................................... 1 1.1.2 The zygotic genome activation ............................................................................................ 2 1.1.3 Axial patterning during Xenopus development ................................................................... 4 1.2 Chromatin dynamics ................................................................................................................ 8 1.2.1 Chromatin structure ............................................................................................................ 8 1.2.2 Posttranslational modifications ......................................................................................... 10 1.2.3 Chromatin remodeling complexes .................................................................................... 12 1.3 The SWI/SNF complex in development ................................................................................. 13 1.4 Objectives .............................................................................................................................. 15 2 Materials and Methods ........................................................................................ 17 2.1 Materials ................................................................................................................................ 17 2.1.1 Animals .............................................................................................................................. 17 2.1.2 Bacteria .............................................................................................................................. 17 2.1.3 Enzymes ............................................................................................................................. 17 2.1.4 Chemicals ........................................................................................................................... 18 2.1.5 Antibodies.......................................................................................................................... 18 2.1.6 Oligonucleotides ................................................................................................................ 19 2.1.7 Plasmids ............................................................................................................................. 23 2.1.8 Equipment ......................................................................................................................... 24 2.1.9 Software ............................................................................................................................ 25 2.2 Methods ................................................................................................................................ 25 2.2.1 DNA standard methods ..................................................................................................... 25 2.2.2 RNA standard methods ..................................................................................................... 29 2.2.3 Protein analysis ................................................................................................................. 34 2.2.4 Embryological methods ..................................................................................................... 36 2.2.5 Embryological explantations ............................................................................................. 39 3 Results ................................................................................................................. 41 3.1 Comparison of morpholino oligonucleotides ........................................................................ 41 3.2 BMO1 reduces Brg1 protein level from MBT on ................................................................... 42 3.3 Morphological gain- and loss-of-function studies ................................................................. 44 3.3.1 Targeted loss-of-function analysis..................................................................................... 44 3.3.2 Targeted gain-of-function analysis .................................................................................... 45 3.4 Wnt signaling is not affected in Brg1 morphants .................................................................. 46 3.5 Axis patterning genes are downregulated in neurula stage ................................................. 48 3.6 Dorso-ventral patterning in gastrula embryos is perturbed ................................................. 50 3.7 Brg1 knockdown affects gene expression already at blastula .............................................. 52 3.8 ß-catenin target gene siamois is not changed upon loss of Brg1.......................................... 54 3.9 Exogenous chordin mRNA can compensate for the loss of Brg1 .......................................... 55 3.10 A Brg1 morphant BCNE causes loss of eye and brain structures .......................................... 57 3.11 Molecular analysis of BCNE transplants ................................................................................ 58 3.12 Brg1 knockdown impairs the transcriptional readout of wnt-and nodal inducers ............... 59 3.13 Genes outside the BCNE are affected by Brg1 knockdown................................................... 61 3.14 Brg1 knockdown in Xenopus tropicalis .................................................................................. 63 3.15 BMO1 and BMO2 reduce Brg1 protein level at blastula and cause embryonic lethality during gastrulation ................................................................................................................................ 64 3.16 Morphant embryos are not delayed in development and do not activate apoptosis .......... 66 3.17 Genome-wide transcriptome analysis of BMO1 morphant embryos ................................... 67 3.17.1 Genes related to “nervous system development” are affected in Brg1 morphants ........ 68 3.17.2 BMO1 affects a broad range of genes throughout all germ layers ................................... 69 3.18 Brg1 knockdown by BMO2 recapitulates the major changes in gene expression ................ 71 3.19 Global transcription changes at midblastula transition ........................................................ 73 3.20 Comparison of pre- and postMBT transcriptomes ................................................................ 74 3.21 Highly upregulated genes at MBT need Brg1 protein ........................................................... 75 4 Discussion ............................................................................................................ 79 4.1 Morpholino efficiency and targeting behavior ..................................................................... 80 4.2 Phenotypic analysis of Brg1 morphant embryos .................................................................. 81 4.2.1 Morpholino based knockdown analyses ........................................................................... 81 4.2.2 Overexpression of wild type Brg1 ..................................................................................... 83 4.3 Genome-wide transcriptome analysis of Brg1 morphant embryos ...................................... 84 4.4 Brg1 and its role in axes determination ................................................................................ 86 4.4.1 Antero-posterior axis ......................................................................................................... 86 4.4.2 Dorso-ventral patterning ................................................................................................... 87 4.5 Brg1 and its role in neurogenesis .......................................................................................... 90 4.6 Brg1 and its role during zygotic genome activation .............................................................. 92 4.7 Possible Brg1 mechanism at the burst of transcription ........................................................ 94 4.8 Conclusions and future directions ........................................................................................