translating ribosome affinity purification for cell PDF
Preview translating ribosome affinity purification for cell
Aus dem Adolf-‐Butenandt-‐Institut Lehrstuhl Physiologische Chemie der Ludwig-‐Maximilians-‐Universität München Vorstand: Prof. Dr. Andreas Ladurner Cell-‐type-‐specific approaches for dissecting gene activity and chromatin structure within the Drosophila head Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften an der Medizinischen Fakultät der Ludwig-‐Maximilians-‐Universität vorgelegt von Tamás Schauer aus Mohács (Ungarn) 2013 Gedruckt mit Genehmigung der Medizinischen Fakultät der Ludwig-‐Maximilians-‐Universität München Betreuer: Prof. Dr. Andreas Ladurner Zweitgutachter: Prof. Dr. Ralph A.W. Rupp Dekan: Prof. Dr. med. Dr. h.c. M. Reiser, FACR, FRCR Tag der mündlichen Prüfung: 01.08.2014 2 Eidesstattliche Versicherung Schauer, Tamás Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema "Cell-‐type-‐specific approaches for dissecting gene activity and chromatin structure within the Drosophila head" 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 Doktorand 3 Acknowledgements Foremost I would like to thank Prof. Dr. Andreas Ladurner for giving me the opportunity to work on my PhD project in his group at EMBL and in his department at LMU. It would have been impossible to start, manage and finish my PhD work without his guidance and support. I am especially grateful to Dr. Carla Margulies for the successful co-‐work, the motivating discussions and the useful feedback that helped the progress of my PhD project. Thank you for the scientific and non-‐scientific input, the active co-‐writing of our papers and the careful reading of my thesis manuscript. My special thanks go to Dr. Petra Schwalie for the fruitful collaboration and discussions. Thank you for your enthusiastic work in analyzing my data what was crucial to write this manuscript. I wish to acknowledge Sandra Esser at LMU and Bianca Nijmejer at EMBL for the technical support. I am also grateful to Dr. Anton Eberharter and Dr. Corey Laverty for all the scientific and management-‐related help including reading this thesis. I also would to thank all members of the Ladurner group/department and Margulies group, especially Ava Handley for the discussions and helping each others´ work as well as Dr. Gyula Timinszky and Dr. Markus Hassler for the scientific advices. At last but not at least I thank my family, my parents, my friends in Hungary and in Germany as well as my life partner for supporting and motivating me in good and bad times. 4 Table of Contents Table of Contents 1 SUMMARY ................................................................................................................ 7 1.1 Summary (in English) ..................................................................................................................................... 7 1.2 Zusammenfassung ........................................................................................................................................... 9 2 INTRODUCTION ..................................................................................................... 13 2.1 Epigenetic landscape of development ................................................................................................... 14 2.1.1 Development of major cell-‐lineages in Drosophila ............................................................................. 15 2.1.2 Cell types of the fly head ..................................................................................................................................... 17 2.1.3 Gene regulatory networks ................................................................................................................................. 23 2.2 Regulation of gene expression ................................................................................................................. 25 2.2.1 RNA polymerase II-‐mediated transcription ............................................................................................ 26 2.2.2 Chromatin structure and function ................................................................................................................ 29 2.2.3 Post-‐transcriptional regulation ..................................................................................................................... 38 2.3 Cell-‐type-‐specific approaches to dissect gene activity ..................................................................... 41 2.3.1 General workflow ................................................................................................................................................... 41 2.3.2 Chromatin mapping-‐based methods ........................................................................................................... 44 2.3.3 RNA profiling-‐based methods ......................................................................................................................... 46 2.3.4 Data analysis ............................................................................................................................................................. 50 2.3.5 Validation of cell type specificity ................................................................................................................... 50 2.3.6 Perspectives ............................................................................................................................................................... 52 2.4 Aims of the thesis .......................................................................................................................................... 53 3 CAST-‐CHIP – CHROMATIN AFFINITY PURIFICATION FROM SPECIFIC CELL TYPES ........ 54 3.1 Summary .......................................................................................................................................................... 54 3.2 Introduction .................................................................................................................................................... 55 3.3 Methods ............................................................................................................................................................ 56 3.3.1 Experimental procedures .................................................................................................................................. 56 3.3.2 Data analysis ............................................................................................................................................................. 58 3.4 Results .............................................................................................................................................................. 60 3.4.1 Cell-‐type-‐specific gene activity maps using CAST-‐ChIP .................................................................... 60 3.4.2 Validation of CAST-‐ChIP ...................................................................................................................................... 67 3.5 Discussion ....................................................................................................................................................... 74 4 H2A.Z – AN EPIGENETIC MARK FOR UBIQUITOUS GENE ACTIVITY ............................. 76 4.1 Summary .......................................................................................................................................................... 76 4.2 Introduction .................................................................................................................................................... 77 4.3 Methods ............................................................................................................................................................ 78 5 Table of Contents 4.3.1 Experimental procedures .................................................................................................................................. 78 4.3.2 Data analysis ............................................................................................................................................................. 79 4.4 Results .............................................................................................................................................................. 82 4.4.1 H2A.Z is an active mark in differentiated cell types of the fly CNS ............................................ 82 4.4.2 H2A.Z associates with chromatin domains that display ubiquitous gene expression ... 94 4.5 Discussion ....................................................................................................................................................... 98 5 TRAP – TRANSLATING RIBOSOME AFFINITY PURIFICATION FOR CELL-‐TYPE-‐SPECIFIC TRANSLATOME PROFILING ........................................................................................ 101 5.1 Summary ........................................................................................................................................................ 101 5.2 Introduction .................................................................................................................................................. 102 5.3 Methods .......................................................................................................................................................... 103 5.3.1 Experimental procedures ............................................................................................................................... 103 5.3.2 Data analysis .......................................................................................................................................................... 105 5.4 Results ............................................................................................................................................................ 107 5.4.1 Establishing TRAP in Drosophila ................................................................................................................ 107 5.4.2 Genome-‐wide TRAP profiling ....................................................................................................................... 115 5.5 Discussion ..................................................................................................................................................... 128 6 DISCUSSION ............................................................................................................ 131 6.1 CAST-‐ChIP, a tool for cell-‐type-‐specific chromatin mapping ....................................................... 132 6.2 Ubiquitous genes, are they special? ...................................................................................................... 134 6.3 TRAP, profiling mRNA from cell types ................................................................................................. 137 6.4 Perspective .................................................................................................................................................... 138 7 LIST OF FIGURES AND TABLES ................................................................................. 141 8 ABBREVIATIONS ..................................................................................................... 143 9 APPENDIX ............................................................................................................... 144 9.1 CAST-‐ChIP protocol .................................................................................................................................... 144 9.1.1 Chromatin preparation .................................................................................................................................... 144 9.1.2 Chromatin Immunoprecipitation ............................................................................................................... 146 9.2 TRAP protocol .............................................................................................................................................. 147 9.2.1 TRAP procedure ................................................................................................................................................... 147 10 BIBLIOGRAPHY ..................................................................................................... 150 6 Summary 1 Summary 1.1 Summary (in English) Multicellular organisms develop from a single cell (the zygote) and each cell type inherits the same genetic material from the zygote. Adult organisms are composed of terminally differentiated cell populations that carry the same genome but differential epigenomes. The epigenome consists of modifications or marks of the genome that determine which genes are activated or repressed. The differential activity of genes in distinct cells maintains their phenotype, identity and function. However, there have been few tools available until recently that would allow us to profile gene activity at the level of specific cell types. The lack of easily applicable, efficient cell-‐type-‐specific tools prompted me to develop novel biochemical methods and to refine existing protocols for profiling transcription, chromatin and mRNA levels genome-‐wide. I used cephalic (head) cell types of the adult fruit fly (Drosophila melanogaster) as a model system to study differential gene activity in differentiated cell types including neurons, glia and the fat body (adipocytes). First, I developed a biochemical method, CAST-‐ChIP (Chromatin Affinity Purification from Specific cell Types), a combination of the UAS/Gal4 expression system and the affinity purification of tagged chromatin-‐bound reporters. To study transcription in distinct cell types, I expressed a tagged subunit of the RNA polymerase II complex in the cell type of interest and used the tag to generate cell-‐type-‐specific, genome-‐wide ChIP profiles. RNA polymerase II marks about 1500 genes unique to neurons or glia. Genes identified as neuronal share characteristic cellular function such as axon guidance of neurons and are expressed in other neuronal tissues, such as the larval central nervous system. Furthermore, I demonstrated that genomic regions marked by cell-‐type-‐specific RNA polymerase II show GFP-‐reporter activity localized within the labeled cell populations. This incidates that RNA polymerase II profiling is a suitable tool to distinguish gene activity in different cell types. Second, I applied CAST-‐ChIP to study chromatin structure of cell types by profiling the incorporation of the active histone variant (H2A.Z), as a proof-‐of-‐principle 7 Summary to study differences in chromatin structure between unrelated cell types. I found H2A.Z present at expressed genes and absent from inactive genes, as shown previously. However, H2A.Z-‐enriched regions do not completely overlap with RNA polymerase II regions. Interestingly, RNA polymerase II-‐bound genes lacking H2A.Z differ the most in their expression among dissected tissues. Therefore, I hypothesized that H2A.Z labels genes that are expressed in a cell-‐type-‐independent manner. To test this, I used CAST-‐ ChIP to compare the cell-‐type-‐specific incorporation of H2A.Z. Surprisingly, H2A.Z profiles are remarkably similar in neurons and glia, with only about hundred significant differences. In addition, H2A.Z is present at those regions which share RNA polymerase II in both cell types and is absent from cell-‐type-‐specific regions. ChIP analysis of the fat body, which is another head cell type with a different developmental origin, led to the same results. To validate these findings by comparing distinct developmental stages, I found only a few H2A.Z and thousands of RNA polymerase II regions that differ between the embryo and adult head tissues. Thus, CAST-‐ChIP revealed a novel function of H2A.Z in marking genes with ubiquitous, cell-‐type-‐invariant expression. Using this approach, I could distinguish between ubiquitous (house-‐keeping) and specifically regulated genes. Together with analyses conducted by other groups, I found that ubiquitous genes share common regulatory features including promoter structure and gene length, and they form clusters marked by insulator binding proteins. Third, I refined a fly RNA profiling approach (TRAP: Translating Ribosome Affinity Purification), first developed for the mouse, to obtain information about cell-‐ type-‐specific post-‐transcriptional processes that regulate cellular function downstream to transcription. TRAP measures the ribosome-‐bound fraction of RNA and therefore identifies genes that are not only transcribed but also translated (translatome). The dynamic range of TRAP was greater compared to the previous ChIP-‐based methods and I identified twice as many transcripts as RNA polymerase II-‐bound genes using CAST-‐ ChIP, indicating the greater resolution of the ribosome-‐tagging method. Using TRAP I uncovered transcripts carrying relevant neuronal functions that were hidden in the CAST-‐ChIP data lacking RNA polymerase II peaks. Several studies revealed that mild stress conditions induce changes only on the translational level; therefore, TRAP is a suitable tool to study such responses in various cell types. In summary, in my PhD thesis I present and compare cell-‐type-‐specific methods to profile gene activity in Drosophila differentiated cells. I developed a novel method 8 Summary (CAST-‐ChIP) and applied an existing method (TRAP) to map 1) transcription using RNA polymerase II CAST-‐ChIP; 2) chromatin structure using H2A.Z CAST-‐ChIP and 3) the translatome of ribosome-‐bound mRNA using TRAP. My results give useful, novel information for the scientific community: 1) the cell-‐type-‐specific profiles serve as a compendium of genes involved in the maintenance of cell identity and function; 2) using these approaches, I discovered a novel function of H2A.Z marking ubiquitous/ housekeeping genes, highlighting the differential regulation of cell-‐type-‐specific genes; 3) ChIP profiling does not identify all differences among cell types and therefore post-‐ transcriptional profiling has to be involved in the analysis. Cell-‐type-‐specific approaches presented in this thesis are promising tools that will allow us to describe cellular responses upon environmental perturbation, identifying differential responses to environmental change in distinct cell populations. 1.2 Zusammenfassung Mehrzellige Organismen entwickeln sich aus einer einzigen Zelle (der Zygote) und jede Zelle erbt das gleiche genetische Material aus der Zygote. Die adulten Organismen sind aus terminal differenzierten Zellpopulationen zusammengesetzt, die das gleiche Genom, aber unterschiedliche Epigenome tragen. Das Epigenom besteht aus Modifikationen oder Markierungen des Genoms. Diese bestimmen, welche Gene aktiviert oder reprimiert werden. Die unterschiedliche Aktivität von Genen in unterschiedlichen Zellen erhält deren Phänotyp, Identität und Funktion aufrecht. Der Mangel an leicht anwendbaren und effizienten Zelltyp-‐spezifischen Werkzeugen hat mich dazu veranlasst, neuartige biochemische Methoden zu entwickeln und bestehende Protokolle zu verfeinern. Dadurch kann die Transkription, die Chromatinstruktur und die Menge an mRNA Zelltyp-‐spezifisch, genomweit profiliert werden. Ich benutzte Zellen vom Kopf der adulten Fruchtfliege (Drosophila melanogaster) als Modellsystem um die Genaktivität der differenzierten Zelltypen wie Neuronen, Glia und Fettzellen zu untersuchen. Zuerst entwickelte ich ein biochemisches Verfahren, CAST-‐ChIP (Chromatin Affinitätsreinigung von spezifischen Zelltypen) genannt, welches eine Kombination aus dem UAS/Gal4 Expressionssystem und aus einer Affinitätsreinigung von einem 9 Summary markierten Chromatin-‐gebundenen Reporter darstellt. Um die Transkription in verschiedenen Zelltypen zu untersuchen, exprimierte ich eine markierte Untereinheit des RNA Polymerase II Komplex im Zelltyp von Interesse und erzeugte Zelltyp-‐ spezifische, genomweite ChIP Profile. RNA-‐Polymerase II markiert etwa 1500 Gene spezifisch für Neuronen oder Gliazellen. Gene, die als Neuron-‐spezifisch identifiziert wurden, haben charakteristische zelluläre Funktionen, wie zum Beispiel die Axon Führung von Neuronen. Sie werden auch in anderen neuronalen Geweben sowie im larvalen Zentralnervensystem exprimiert. Außerdem zeigte ich, dass genomische Regionen, die von Zelltyp-‐spezifischer RNA Polymerase II gebunden sind, GFP-‐ Reporter-‐Aktivität innerhalb der markierten Zellpopulationen zeigen, was darauf hindeutet, dass das RNA-‐Polymerase II "Profiling" eine geeignete Methode ist, um Zelltyp-‐spezifische Gen-‐Aktivitäten unterscheiden zu können. Zweitens, ich verwendete CAST-‐ChIP zur Untersuchung der Chromatin-‐Struktur verschiedener Zelltypen und erstellten Profile für den Einbau der aktiven Histon Variante H2A.Z ins Chromatin. Ich fand eine Inkorporation von H2A.Z bei exprimierten Genen und keinen H2A.Z Einbau bei inaktiven Genen, wie bereits gezeigt wurde. Allerdings überlappten die H2A.Z angereicherten Regionen nicht vollständig mit den RNA-‐Polymerase II Regionen. Interessanterweise unterschieden sich die Gene, die von RNA-‐Polymerase II jedoch nicht von H2A.Z gebunden wurden, in ihrer Expression zwischen sezierten Geweben. Daher stellte ich die Hypothese auf, dass die H2A.Z-‐ markierten Gene in einer Zelltyp-‐unabhängigen Weise exprimiert werden. Um dies zu testen, vergliche ich den Zelltyp-‐spezifischen Einbau von H2A.Z mit der CAST-‐ChIP Methode. Überraschenderweise sind die H2A.Z Profile bemerkenswert ähnlich in Neuronen und Gliazellen, mit nur etwa hundert signifikanten Unterschieden. Darüber hinaus ist H2A.Z anwesend in den Regionen, die auch RNA-‐Polymerase II in beiden Zelltypen (Neuronen und Gliazellen) aufweisen, fehlt jedoch in Zelltyp-‐spezifischen Regionen. ChIP "Profiling" in einem anderen Zelltyp mit einer unterschiedlichen Entwicklungsherkunft (z.B. Fettzellen im Kopf) ergab die gleichen Ergebnisse. Um die Resultate durch einen Vergleich verschiedener Entwicklungsstadien zu validieren, fand ich nur ein paar H2A.Z und Tausende von RNA Polymerase II unterschiedliche Regionen zwischen Embryos und Kopfgewebe im adulten Stadium. So ergab die Anwendung von CAST-‐ChIP eine neue Funktion von H2A.Z als eine bestimmte Markierung von Genen, die ubiquitär und Zelltyp-‐unabhängig exprimiert werden. Mit Hilfe dieser Methode 10
Description:The list of books you might like
