Aus dem Adolf-Butenandt-Institut der Ludwig-Maximilians-Universität München Lehrstuhl: Molekularbiologie Direktor: Prof. Dr. Peter B. Becker Arbeitsgruppe: Prof. Dr. Gunnar Schotta Deletion of Setdb1 in Sox17 lineage cells impairs early embryonic development in the mouse Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften (Dr. rer. Nat.) an der Medizinischen Fakultät Der Ludwig-Maximilians-Universität München Vorgelegt von Rui Fan Aus Shi Jia Zhuang, China 2015 Gedruckt mit Genehmigung der Medizinschen Fakultät der Ludwig Maximilians-Universität München Betreuer: Prof. Dr. Gunnar Schotta Zweitgutachter: Prof. Dr. Axel Imhof Dekan: Prof. Dr. med. Dr. h. c. Maximilian Reiser, FACR, FRCR Tag der mündlichen Prüfung: 24. 02. 2015 Eidesstattliche Versicherung Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema “Deletion of Setdb1 in Sox17 lineage cells impairs early embryonic development in the 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 Rui Fan TABLE OF CONTENTS 4 TABLE OF CONTENTS………………………………………….…………...4 ABSTRACT……………………….……………………………………………6 1. INTRODUCTION……………………………………………………...…....8 1.1 Chromatin structure…………..………….…………………………………………………8 1.2 Epigenetic mechanism regulates gene transcription………………….................................9 1.2.1 Histone modification ...….….…………..………...…………………………….………..9 1.2.2 Chromatin remodelers .…...….………..……………...………………………………...10 1.2.3 Histone variants ……...……….……...………………………..………………………..11 1.3 H3K9-specific methyltransferase………………..………………………………………..11 1.3.1 Suv39h1 and Suv39h2 ...…..…………….……………...…...………………………….12 1.3.2 G9a and Glp1……………..….….…..….……………………..………………………...13 1.3.3 Setdb1….....…..……...….……………………………………..……………..………....14 1.4 Lineage decisions during early embryonic development………………..….…………….15 1.5 Wnt and Nodal signaling pathways regulate endoderm differentiation during early embryonic development .……………..…………………………………...17 1.6 Epigenetic mechanism regulation is connected to ES cell pluripotency and cell lineage commitment…………..………………………..………..19 1.7 The Functions of Setdb1 during development…………………………………..…..........20 AIM OF THE THESIS………………………………………………...……..22 2. RESULTS…………………………………………………...………………23 2.1 Establishment of Setdb1 conditional knockout mice………………………...…………...23 2.1.1 Genomic locus of Setdb1 and knockout strategy……...………….……...…..………....23 2.1.2 Generation of Setdb1 conditional knockout MEF and ES lines......…...…..............…....24 2.1.3 Generation of Setdb1 knock-in cell lines…......……..…………….………………….....30 2.2 Phenotypic defects of mutant embryos upon deletion of Setdb1 in endoderm……..……31 2.2.1 Expression profile of Setdb1 during mouse development……..……………..………....31 2.2.2 Early lethality of mouse embryos upon deletion of Setdb1 in endoderm…….…….…...34 2.2.3 Sox17-mediated deletion of Setdb1 leads to two different phenotypes………….….......35 2.2.4 Structural changes of the hindgut region in Setdb1END mutant embryos…….…….….36 2.3 Molecular changes during embryonic development upon deletion of Setdb1………........42 2.3.1 Gene expression changes upon deletion of Setdb1 in endoderm at E7.75…...…42 5 TABLE OF CONTENTS 2.3.2 Endoderm specific transcription factors are reduced in the Posterior region of the embryos………..……..…………………………………………..46 2.3.3 Increased apoptosis in the posterior part of the mutant embryos upon deletion of Setdb1……………………………………………….…...…..……47 2.3.4 Aberrant coverage of the node region in Setdb1END embryos…. ……......……..50 2.4 Characterization of in vitro differentiated Setdb1 mutant endoderm cells…………….…53 2.4.1 Establishment of an in vitro endoderm differentiation system…...……..…...…53 2.4.2 No alteration in histone modifications upon deletion of Setdb1 in endoderm cells…………………….………………………………………..…..55 2.4.3 Molecular changes of the in vitro differentiated Setdb1 mutant cells….…...…..58 3. DISCUSSION……………………………………………………………….64 3.1 Setdb1 is implicated in the endoderm specification………………………………...…….64 3.2 Setdb1 regulates gene expression in different biological processes………………….......67 3.2.1 Setdb1 regulates gene expression during early embryonic development……..… .…...67 3.2.2 Setdb1 regulates the expression of imprinted genes.….….….………….......….….....69 3.2.3 IAP elements are derepressed upon deletion of Setdb1 during early embryonic development………..…………………….…………………………….70 3.3 Loss of Setdb1 affects the properties of endoderm cells in vitro……….…………...…....71 4. MATERIAL AND METHOD……..…………………………………...….74 4.1 Cell biology methods………………………………………………………………..……74 4.2 Molecular biology methods………………………………………………………..…......75 4.3 Biochemical Methods……………………………………………………………..……...75 4.4 Embryology methods………………………………………………………………..……77 4.5 Mouse methods………………………………………………………………………..….79 ABBREVIATIONS………..……………………………………………….....85 ACKNOWLEDGEMENTS……………...…………………………….….….87 CURRICULUM VITEA……………………………………………….…..…88 APPENDIX…………………………………………………….................89 Table 1. Affymetrix array data list of Setdb1END embryos………………………..….....….89 Table 2. Mass spec list of Setdb1 Flag IPs from two repeats (MaxQuant analysis)............. 103 Table 3. List of dysregulated genes from Setdb1 mutant endoderm cells…..…..…...……105 REFERENCES………………………………………………….……..…….125 ABSTRACT 6 ABSTRACT Over the last decades, epigenetic mechanisms have been related to severe diseases (Dambacher et al. 2010) and developmental defects (Hahn et al. 2010). For my thesis, I mainly focused on the functional study of Setdb1, which is a histone methyltransferase that mediates H3K9 methylation, during endoderm development. Previous studies have already shown that Setdb1 could di- and tri-methylate H3K9 in euchromatin (Wang et al. 2003) and deletion of Setdb1 results in the lethality of embryos at a very early embryonic stage (Dodge et al. 2004). Additionally, Chip-Seq data showed that Setdb1 binds to pluripotency-associated genes and development regulators in ES cells (Bilodeau et al. 2009, Yuan et al. 2009). These results indicate the potential functions of Setdb1 in regulating mouse development. During early embryonic development, three germ layers start to be formed at gastrulation stage (Takaoka et al. 2012). In order to study the spatial and temporal function of Setdb1 in endoderm development we specifically deleted Setdb1 in Sox17 lineage cells. In order to specifically test the function of Setdb1 in definitive endoderm, we additionally introduced a GFP-reporter gene. GFP is activated by the expression of Cre recombinase. Thus, we assume that GFP positive cells have also deleted Setdb1. By monitoring the Setdb1 deleted cells, we found that mutant embryos which showed a broad GFP signal even outside endoderm were severely developmentally retarded. In contrast, a specific GFP signal in the endoderm region correlated with milder endoderm-related defects. In the subsequent studies I focused on embryos with specific deletion of Setdb1 in the endoderm. In order to further characterize the phenotype we performed immunohistochemistry to check for structural changes. Setdb1 mutant embryos cannot complete axis rotation and show progressive deterioration of the hindgut region. In mutant embryos the hindgut diverticulum is formed but loses the contact with splanchnopleura. Moreover the size of the dorsal aorta is much bigger in mutant embryos. During development the diverticulum cannot further develop resulting in perturbed mesoderm development. Whole mount embryo immunostaining revealed an increase in apoptotic cells in the posterior part of mutant embryos and the reduced expression of endoderm genes in the primitive streak region. Interestingly we found that Setdb1 mutant cells display altered morphogenesis and cover part of the node region. We assume that impaired signal transmission through the cilia on the surface of the node contributes to the developmental defect of Setdb1 mutant embryos. To identify genes which are regulated by Setdb1, mutant and control embryos at E7.75 were collected for microarray analysis. Several genes belonging to different biological processes were found to be dysregulated. AFP, which is a visceral endoderm gene, is upregulated in Setdb1 mutant embryos. However, through the whole mount embryo immunostaining, AFP positive cells are mainly restricted to the extra-embryonic part indicating that the loss of Setdb1 in visceral endoderm does not affect the development of the embryonic part. We also generated Setdb1flox/flox; Sox17-cre; GFP-reporter ES cells and induced differentiation of these cells into endoderm cells. RNA-seq analysis revealed that endoderm commitment genes are upregulated in the mutant cells. We therefore assume that Setdb1 balances the expression of the endoderm differentiation program. 7 ABSTRACT ABSTRACT Epigenetische Mechanismen haben eine grosse Bedeutung bei der Krankheitsentstehung und für die Embryonalentwicklung. In meiner Arbeit habe ich mich auf die Funktionen der Histon- Methyltransferase Setdb1 bei der Endodermentwicklung konzentriert. Voherige Studien konnten zeigen, dass Setdb1 essentiell für die Embryonalentwicklung ist und Histon H3 an Lysin