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Regulation of Heterochromatic Gene Silencing in Mouse

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Regulation of Heterochromatic Gene Silencing in Mouse Aus dem Adolf-Butenandt-Institut der Ludwig-Maximilians-Universität München Lehrstuhl: Molekularbiologie Direktor: Prof. Dr. Peter B. Becker Arbeitsgruppe: Prof. Dr. Gunnar Schotta Regulation of heterochromatic gene silencing in mouse Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München vorgelegt von Dennis Šadić aus Daun München, 2014 2 Gedruckt mit Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians- Universität München Betreuer: Prof. Dr. Gunnar Schotta Zweitgutachter: Prof. Dr. Heiko Hermeking Dekan: Prof. Dr. Dr. h.c. Maximilian Reiser, FACR, FRCR Tag der mündlichen Prüfung: 19.09.2014 3 Eidesstattliche Versicherung Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema “Regulation of heterochromatic gene silencing in mouse” 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. ____________________ ______________________________________ Ort, Datum Unterschrift Dennis Šadić 4 The work of my thesis is under preparation for a publication in a peer-reviewed journal. During my PhD thesis I collaborated with others in the following scientific projects: Dambacher, S., Deng, W., Hahn, M., Sadic, D., Frohlich, J., Nuber, A., Hoischen, C., Diekmann, S., Leonhardt, H., and Schotta, G. (2012). CENP-C facilitates the recruitment of M18BP1 to centromeric chromatin. Nucleus 3, 101-110. Hahn, M., Dambacher, S., Dulev, S., Kuznetsova, A.Y., Eck, S., Worz, S., Sadic, D., Schulte, M., Mallm, J.P., Maiser, A., et al. (2013). Suv4-20h2 mediates chromatin compaction and is important for cohesin recruitment to heterochromatin. Genes & development 27, 859-872. Terrados, G., Finkernagel, F., Stielow, B., Sadic, D., Neubert, J., Herdt, O., Krause, M., Scharfe, M., Jarek, M., and Suske, G. (2012). Genome-wide localization and expression profiling establish Sp2 as a sequence-specific transcription factor regulating vitally important genes. Nucleic acids research 40, 7844-7857. I. Table of contents 5 I. Table of contents I. Table of contents ................................................................................................................ 5 II. Abstract ............................................................................................................................10 III. Zusammenfassung ..........................................................................................................11 1. Introduction ......................................................................................................................13 1.1. Heterochromatin regulation ........................................................................................13 1.1.1. DNA, chromatin and a definition of heterochromatin ............................................13 1.1.2. Regulation pathways at pericentric heterochromatin in mouse ............................14 1.2. Retrotransposon silencing ..........................................................................................16 1.2.1. LTR-retrotransposons in mammals ......................................................................17 1.2.2. Silencing of endogenous retroviruses by DNA methylation ..................................20 1.2.3. Repression of (endogenous) retroviruses by H3K9me3, Setdb1 and the corepressor Trim28 .......................................................................................................21 1.2.4. Repression of endogenous retroviruses independent of DNA methylation and the Trim28/Setdb1/H3K9me3-pathway ................................................................................23 1.2.5. Concluding remarks about retrotransposon silencing pathways ...........................24 1.3. Proteins involved in heterochromatic gene silencing in mouse ...................................25 1.3.1. KRAB zinc finger-dependent repression by the master regulator Trim28 .............25 1.3.2. The histone H3 lysine 9 methyltransferase Setdb1 (ESET) cooperates with Trim28 in heterochromatin formation and transcriptional repression ..........................................27 1.3.3. The chromatin-associated SNF2-type ATPase Atrx .............................................29 1.3.4. The role of Daxx in transcriptional repression and histone H3.3 incorporation .....32 1.4. Aim of the thesis ........................................................................................................37 2. Results .............................................................................................................................38 2.1. Investigation of a heterochromatic reporter gene locus. The pseudoautosomal exons of the midline1 gene are derepressed in Suv39h double knockout and Suv4-20h double knockout mouse embryonic stem cells ..............................................................................38 6 I. Table of contents 2.2. The pseudoautosomal midline1 transcript potentially originates from the Y-chromosome, is orientated in sense direction and originates from a complex genetic environment ......................................................................................................................40 2.3. A candidate luciferase reporter screen for heterochromatic gene regulators identifies Setdb1, Trim28, Prdm6 and Hp1 proteins as transcriptional repressors ............................42 2.4. EGFP is a suitable reporter gene for heterochromatic gene silencing ........................43 2.5. Generation and optimization of a second generation lentiviral packaging system pseudotyped with ecotropic envelope protein of M-MLV ...................................................45 2.6. Transrepressor binding sites cloned into lentiviral EGFP expression vectors are tools for monitoring transcriptional silencing kinetics .................................................................47 2.7. HeLa cells stably expressing Slc7a1 (mCAT-1) are susceptible to ecotropic M-MLV pseudotyped lentivirus transduction and can control for mouse- and cell-type-specific silencing activities .............................................................................................................49 2.8. The GAG-region of the mouse IAP-Ez retrotransposon subclass contains a mouse embryonic stem cell specific silencing sequence ..............................................................50 2.9. The minimal silencing element inside the IAP-GAG sequence is 160 bp long and requires the histone methyltransferases Setdb1 for its repressive activity. ........................52 2.10. The GAG2.22 sequence recruits Histone 3 Lysine 9 trimethylation when integrated into chromatin of mES cells ...............................................................................................54 2.11. The IAP-GAG silencing sequences are silenced in a Trim28-dependent manner .....55 2.12. Optimization of two genome-wide shRNA screening assays for factors involved in reporter gene-silencing .....................................................................................................57 2.13. Genome-wide screening for Zfp809 and IAP-GAG-dependent silencing identifies many potential genes involved in heterochromatic gene silencing ....................................59 2.14. A secondary genetic-screen identifies Atrx as a regulator of GAG-sequence silencing of IAP retrotransposon sequences ....................................................................................60 2.15. Atrx knockout cells show strongly retarded silencing kinetics of the GAG2.22-EGFP reporter .............................................................................................................................62 2.16. Atrx and H3K9me3 are enriched at endogenous IAP elements in mouse ES cells and are recruited to novel integrations of the GAG2.22 sequence ...........................................63 2.17. Atrx knockout cells have normal DNA methylation on IAP retrotransposon sequences and a normal cell cycle distribution ...................................................................................66 I. Table of contents 7 2.18. Daxx knockout cells phenocopy the impaired silencing kinetics of Atrx knockout cells .........................................................................................................................................67 2.19. H3.3 interaction mutants of Daxx and wild type Daxx protein rescue the silencing defect of Daxx knockout cells ............................................................................................69 2.20. Association of Daxx with H3.3 is dependent on arginine 257 and the Daxx C- terminus ............................................................................................................................70 2.21. H3.3 depleted cells show no change in GAG2.22 reporter gene silencing ................72 2.22. Atrx is important for efficient Trim28- and Setdb1-dependent silencing at endogenous IAP elements ....................................................................................................................74 2.23. Atrx depletion leads to a higher heterochromatin
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