Mad2l2 as a safeguard for open chromatin in embryonic stem cells Dissertation for the award of the degree “Doctor rerum naturalium.” Of the Georg-August-Universität Göttingen within the doctoral program (Genes and Development) of the Georg-August University School of Science (GAUSS) Submitted by: Ali Rahjouei From: Iran Göttingen, 2016 1 Members of the Thesis Committee Reviewer 1: Professor Dr. Michael Kessel Developmental Biology Group, Max Planck Institute for Biophysical Chemistry Reviewer 2: Professor Dr. Wolfgang Fischle Biological and Environmental Sciences & Engineering Division, King Abdullah University of Science and Technology Professor Dr. Tomas Pieler Department of Developmental Biochemistry, University Medical Center Göttingen Members of the Extended Thesis Committee Professor Dr. Ahmed Mansouri Molecular Cell Differentiation Group, Max Planck Institute for Biophysical Chemistry Professor Dr. Detlef Doenecke Department of Molecular Biology, University Medical Center Göttingen Dr. Halyna Shcherbata Gene expression and signaling Biology Group, Max Planck Institute for Biophysical Chemistry Date of the oral June 2016 2 Affirmation: Here I declare that my doctoral thesis entitled “Mad2l2 as a safeguard for open chromatin in embryonic stem cells” has been written independently with no other sources and aids than quoted. Ali Rahjouei, Goettingen, April 2016 3 Contents Acknowledgment ...................................................................................................................................... 6 Summary .................................................................................................................................................. 7 1. Introduction .......................................................................................................................................... 8 1.1. Pluripotency in peri-implantation embryos .................................................................................. 8 1.2. Embryonic stem cells................................................................................................................... 10 1.2.1. Naive embryonic stem cells ..................................................................................................... 10 1.2.2. Metastable or transient ESCs ................................................................................................ 11 1.2.3. Primed ESCs ........................................................................................................................... 12 1.3. Primordial germ cell-like cells ..................................................................................................... 12 1.4. Double strand break (DSB) repair ............................................................................................... 14 1.4.1. Non-homologous end joining ................................................................................................ 14 1.4.2. Homologous recombination.................................................................................................. 15 1.5. The Mitotic arrest deficient 2-like protein 2 (Mad2l2) ............................................................... 16 1.6. Aim/objective of the thesis ......................................................................................................... 17 2. Materials and methods ...................................................................................................................... 18 2.1. Polymerase chain reaction (PCR) ................................................................................................ 18 2.2. Reverse transcriptase – polymerase chain reaction (RT‐PCR) .................................................... 18 2.3. Quantitative PCR (qPCR) ............................................................................................................. 19 2.4.1- MEF (isolation, expansion, freezing, thawing) ..................................................................... 21 2.4.2. Embryonic stem cells ........................................................................................................... 21 2.5. Differentiation of ESCs ................................................................................................................ 22 2.5.1. EpiLCs ................................................................................................................................... 22 2.5.2. PGCLCs ................................................................................................................................. 22 2.6. Cell manipulation ........................................................................................................................ 23 2.6.1. Inhibitor and cisplatin experiments ..................................................................................... 23 2.6.2. Cell transfection ................................................................................................................... 23 2.7. ABC staining ................................................................................................................................ 23 2.8. Immunofluorescence staining ..................................................................................................... 24 2.9. TUNEL assay ................................................................................................................................ 26 2.10. Metaphase chromosome spreads ............................................................................................. 26 2.11. Dot blot ..................................................................................................................................... 27 2.12. Western blotting ....................................................................................................................... 28 2.13. Cryosectioning ........................................................................................................................... 28 2.14. STED microscopy ....................................................................................................................... 29 2.15. Immune precipitation ............................................................................................................... 29 2.16. Cell fractionation ....................................................................................................................... 30 2.17. Flow cytometry ......................................................................................................................... 31 2.18.1. Chromatin immunoprecipitation ........................................................................................... 31 2.19. Bioinformatic analysis ............................................................................................................... 31 2.19.1. For RNA-seq data reanalysis workflow .............................................................................. 31 4 2.19.2. ChIP-seq ............................................................................................................................. 32 3. Results ................................................................................................................................................ 33 3.1. The in vitro differentiation of ESCs towards germ cells .............................................................. 33 3.1.1 Mad2l2-deficient EpiLCs resemble wild-type EpiLCs ............................................................. 35 3.1.2 The differentiation of ESCs under PGCLC-inducing conditions in the presence or absence of Mad2l2 ............................................................................................................................................... 37 3.2. DDR in Mad2l2-deficient murine embryonic fibroblasts............................................................. 46 3.2.1. Mad2l2-deficiency leads to activation of a DDR. ................................................................. 46 3.2.2. Cell proliferation is decreased in Mad2l2-deficient MEFs .................................................... 47 3.2.3. Histone modifications were misregulated in Mad2l2-deficient MEFs after DNA damage. .. 48 3.3. Characterization of Wild-type and Mad2l2-deficient ESCs ......................................................... 49 3.3.1. Mad2l2 is highly expressed in the euchromatin of ESCs ....................................................... 49 3.3.2. Comparison histone 3 modifications in wild-type and mutant ESCs ..................................... 51 3.3.3. No evidence for DNA damage in Mad2l2-deficient ESCs ...................................................... 53 3.3.4. Comparison of transcriptomes from wild-type and Mad2l2-deficient ESCs ......................... 54 3.3.5. Dppa3 is downregulated in Mad2l2-deficient ESCs ............................................................... 57 3.3.6. Increase of DNA methylation in Mad2l2-deficient ESCs ........................................................ 60 3.4. Mechanistic aspects of Mad2l2 function .................................................................................... 63 3.4.1. Absence of Mad2l2 from meiotic testicular cells .................................................................. 63 3.4.2. Mad2l2 interacts with DDR factors directly .......................................................................... 64 3.4.3. Inhibition of histone or DNA modifications by small inhibitors in Mad2l2-deficient