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Dominant Role of DNA Methylation Over H3k9me3 in ERV Silencing During

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Dominant Role of DNA Methylation Over H3k9me3 in ERV Silencing During Aus dem Adolf-Butenandt-Institut Lehrstuhl: Molekularbiologie Im Biomedizinischen Centrum der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. Peter B. Becker Arbeitsgruppe: Prof. Dr. Gunnar Schotta Dominant role of DNA methylation over H3K9me3 in ERV silencing during embryonic endoderm development Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München vorgelegt von Zeyang Wang aus Jilin, China München, 2020 Gedruckt mit Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians-Universität München Betreuer: Prof. Dr. rer. nat. Gunnar Schotta Zweitgutachter: Prof. Dr. Olivier Gires Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 15.04.2020 Eidesstattliche Versicherung Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema “Dominant role of DNA methylation over H3K9me3 in ERV silencing during embryonic endoderm development” selbständig verfasst, mich außer der angegebenen keiner weiteren Hilfsmittel bedient und alle Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstelle einzeln nachgewiesen habe. Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde. __Xiamen,_25,04,2020__ ______________________________________ Ort, Datum Unterschrift Zeyang Wang The work presented in this thesis is being assembled into a manuscript for publication in a peer-reviewed journal. Dominant role of DNA methylation over H3K9me3 in ERV silencing during embryonic endoderm development. Wang, Z.*, Fan, R.*, Cernilogar, F.M., Nuber A., Silvia Schirge, S., Lickert, H. and Schotta, G. * These authors contributed equally to this work. While carrying out my PhD thesis I collaborated with colleagues to support other scientific projects, which led to the following publications: Cernilogar, F.M., Hasenoder, S. *, Wang, Z. *, Scheibner, K. *, Burtscher, I., Sterr, M., Smialowski, P., Groh, S., Evenroed, I.M., Gilfillan, G.D., et al. Pre-marked chromatin and transcription factor co-binding shape the pioneering activity of Foxa2. Nucleic Acids Res. 2019 Jul 27. PMID: 31350899 * These authors contributed equally to this work I. Table of contents II. Abstract ............................................................................................................................................... 1 III. Zusammenfassung .............................................................................................................................. 3 1. Introduction ......................................................................................................................................... 5 1.1. Lineage decision during early embryonic development .......................................................... 5 1.2. Epigenetic modifications and gene expression ........................................................................ 7 1.3. Transposable elements ............................................................................................................ 8 1.3.1 Retrotransposons ........................................................................................................... 8 1.3.2 Endogenous retroviral elements .................................................................................... 8 1.3.3 Physiological roles of endogenous retrovirus ................................................................ 9 1.4 Silencing mechanisms of endogenous retroviral elements .................................................... 11 1.4.1 DNA methylation mediated ERV silencing ................................................................... 12 1.4.2 H3K9me3 mediated ERV silencing ................................................................................ 14 1.4.3 Crosstalk between Setdb1 mediated H3K9me3 and Dnmt1 mediated DNA methylation in ERV regulation ............................................................................................... 17 1.5 Setdb1 and Dnmt1 in cancer cells ........................................................................................... 19 1.6. Aim of the thesis ..................................................................................................................... 20 2. Results ............................................................................................................................................... 21 2.1 In vivo ERV expression analysis ............................................................................................... 21 2.2 Establishment of in vitro endoderm differentiation ............................................................... 23 2.2.1 In vitro XEN and DE differentiation .............................................................................. 23 2.2.2 Consistent IAP expression in vitro by immunostaining ................................................ 25 2.3 Characterization of in vitro differentiated cells....................................................................... 26 2.3.1 Fluorescence activated cell sorting indicates differentiation efficiency ...................... 26 2.3.2 Confirmation of Setdb1 deletion and endoderm marker gene expression ................. 27 2.4 ERV de-repression in Setdb1-deficient visceral endoderm progenitors in vitro ..................... 28 2.4.1 PCA analysis and endoderm marker gene expression ................................................. 28 2.4.2 Transcriptional effect of Setdb1 on gene regulation in differentiated endoderm cells ............................................................................................................................................... 30 2.4.3 Transcriptional effect of Setdb1 on repeat regulation in differentiated endoderm cells ............................................................................................................................................... 31 2.4.4 In vitro differentiation system reflects similar trend of IAP derepression in vivo ........ 33 2.4.5 Strong derepression of ERVs is associated with up-regulation of genes in vicinity ..... 34 2.5 Impaired repressive marks specifically in Setdb1END XEN cells ............................................. 36 2.5.1 Decrease of H3K9me3 at ERVs in Setdb1END XEN cells ............................................... 36 2.5.2 Derepressed ERVs lose H3K9me3 in Setdb1END XEN cells .......................................... 37 2.5.3 Loss of DNA methylation in Setdb1END XEN cells ....................................................... 40 2.6 Loss of Dnmt1 leads to ERV de-repression in both DE and XEN cells in presence of H3K9me3 ....................................................................................................................................................... 42 2.6.1 IAP derepression in both endoderm cells with ablation of DNMT methyltransferases ............................................................................................................................................... 42 2.6.2 No changes of H3K9me3 on ERVs in differentiated Dnmt1 knockout cells ................. 43 3. Discussion .......................................................................................................................................... 45 3.1 An in vitro differentiation system models definitive and visceral endoderm differentiation. 45 3.2 Transcriptional regulation of Setdb1 in two types of endoderm cells .................................... 48 3.3 Requirement of Setdb1 for the establishment of H3K9me3 ................................................... 48 3.4 Epigenetic control of repeats by H3K9me3 and DNA methylation ......................................... 49 3.4.1 Chromatin silencing pathways involved in retroelements regulation ......................... 49 3.4.2 A dominant role of DNA methylation through the crosstalk between H3K9me3 and DNA methylation in ERV regulation ...................................................................................... 51 3.5 Conclusions and prospects ...................................................................................................... 53 4. Material & Methods .......................................................................................................................... 55 4.1 Materials .......................................................................................................................................... 55 4.1.1Tissue culture reagents ......................................................................................................... 55 4.1.2 Cell lines ........................................................................................................................ 56 4.1.3 Kits ................................................................................................................................ 56 4.1.4 Antibodies ..................................................................................................................... 56 4.1.4.1 Primary antibodies ............................................................................................ 56 4.1.4.2 Secondary antibodies ........................................................................................ 57 4.1.5 Primers and plasmids ..................................................................................................
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