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The Consequences of DNA Methylation Maintenance Deficiency in Human Embryonic Stem Cells

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The Consequences of DNA Methylation Maintenance Deficiency in Human Embryonic Stem Cells Inaugural Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) The consequences of DNA methylation maintenance deficiency in human Embryonic Stem Cells submitted to the Department of Biology, Chemistry, Pharmacy of Freie Universität Berlin by Aleksandra Alicja Sieńska 2020 The thesis summarizes the work was done from 10/2015 to 01/2020 at the Harvard Stem Cell Institute and Max Planck Institute for Molecular Genetics under the supervision of Prof. Dr. Alexander Meissner 1st Reviewer: Prof. Dr. Alexander Meissner 2nd Reviewer: Prof. Dr. Katja Nowick date of defense: 21.09.2020 I declare to Freie Universität Berlin that I have prepared this doctoral thesis independently and without using any other than the specified sources and aids. The present work is free from plagiarism. This thesis has not been submitted in the same or a similar form as an examination at any other university. ACKNOWLEDGEMENTS I would like to thank Prof. Dr. Alexander Meissner for giving me the opportunity to work in his laboratory. *** My kindest thanks to Toshiyuki Ushijima for performing the DNMT3A/B KO cell methylcytosine ELISA, Simon Lauer for kindly sharing his degron-DNMT1 tagged cell lines with me and performing the HEK293T cell western blot, and to Raha Weigert for performing the Scorecard qPCR. *** This work would not have been possible without the support of Dr. Andreas Gnirke, Dr. Hongcang Gu and Arman Mohammad, who performed the dual protocol single cell sequencing library preparation, as well as Dr. Martin Aryee, Dr. Ayush Raman and Caleb Laureau, who performed the Quality Control and genome alignment of the single-cell data. I would also like to thank Dr. Jocelyn Charlton and Dr. Sudhir Thakurela for their help with the analysis of scRRBS and scRNAseq data, respectively. *** I would like to thank the Joslin FACS Core and the HMS Imaging core for their support. *** A warm thank you to all the members of the Meissner Lab, former and present, for the amazing atmosphere and all the inspiring discussions on scientific and non-scientific topics alike. I am especially thankful to my students, whom I have learned from even more than they have learned from me. Working with you has been a privilege. *** Thank you to my family for their support and faith, I am truly blessed to be surrounded by so much kindness and love. *** I am eternally grateful to my husband, who supported me through thick and thin, from Boston to Berlin. It is no understatement to say that I would have never done this without you. Thank you for the long hours of scientific discussions, reading papers and always challenging me to think like a biologist. Table of contents ACKNOWLEDGEMENTS......................................................................................................... 3 1. ABSTRACT ......................................................................................................................... 8 2. ZUSAMMENFASSUNG .................................................................................................. 10 3. INTRODUCTION .............................................................................................................. 12 3.1 Regulation of gene expression ................................................................................ 12 3.2 The establishment of DNA methylation ................................................................. 13 3.3 Maintenance of DNA methylation ............................................................................ 14 3.4 Genome-wide distribution of DNA methylation ................................................... 15 DNA methylation controls gene expression on sex chromosomes. ................ 16 DNA methylation controls of gene expression on autosomes. ......................... 16 3.5 How DNA methylation exerts its function ............................................................. 17 3.6 Methylated cytosine as a mutagen ......................................................................... 18 3.7 The removal of DNA methylation ............................................................................ 18 3.8 DNA methylation is dynamic in the early development .................................... 19 3.9 The impact of loss of DNMT on embryonic development................................. 20 3.10 DNMT1 deficiency in embryonic stem cells ....................................................... 21 3.11 DNMT1 deficiency in somatic stem cells ............................................................ 22 3.12 DNMT1 deficiency in cancer cells ......................................................................... 24 4. AIMS OF THE STUDY .................................................................................................... 25 5. MATERIALS AND METHODS ....................................................................................... 26 Cell culture ........................................................................................................................... 26 Cell counting ....................................................................................................................... 26 Western Blot ........................................................................................................................ 27 Genomic DNA extraction .................................................................................................. 28 5mC ELISA ........................................................................................................................... 28 EdU stain .............................................................................................................................. 28 TUNEL stain ......................................................................................................................... 29 CFSE stain............................................................................................................................ 29 Hoechst 33342/DAPI stain ................................................................................................ 29 Immunofluorescence staining ........................................................................................ 29 Karyotyping ......................................................................................................................... 30 Cloning .................................................................................................................................. 31 Viral transduction ............................................................................................................... 32 Antibiotic selection ............................................................................................................ 32 Genotyping .......................................................................................................................... 32 RNA extraction .................................................................................................................... 33 Reverse transcription ........................................................................................................ 33 qRT-PCR ............................................................................................................................... 34 ScoreCard ............................................................................................................................ 35 NPC differentiation ............................................................................................................ 35 Small molecules, antibiotics, cytokines and morphogens ..................................... 35 Figures .................................................................................................................................. 36 Dual protocol scRNAseq+RRBS .................................................................................... 36 Quality control .................................................................................................................... 38 Bulk RNA-seq ...................................................................................................................... 39 6. RESULTS .......................................................................................................................... 40 6.1 Genetic approach to study the consequences of the disruption of the maintenance of DNA methylation. ................................................................................. 40 CHAPTER 1. Characterization of tools to acutely remove DNMT1. .......................... 41 6.2 DNMT1 depletion on the transcript level with tetracycline-inducible system ................................................................................................................................................. 41 6.3 DNMT1 depletion on the protein level with PROteolysis TArgeting Chimera (PROTAC) ............................................................................................................................. 43 6.4 Systematic analysis of downstream effects caused by the loss of DNMT1. 47 6.5 Cell cycle progression ............................................................................................... 48 6.6 Uncoupling DNA hypomethylation from the presence of DNMT1 .................. 51 6.7 Summary ........................................................................................................................ 53 CHAPTER 2. The impact of global DNA hypomethylation on genome stability. ... 54 6.8 DNA
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