Pioneering function of Isl1 in epigenetic regulation during second heart field development Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften - Doctor rerum naturalium - (Dr. rer. nat.) eingereicht am Fachbereich Biologie und Chemie der Justus-Liebig-Universität Gießen von Rui Gao Aus Shandong, China 2019 angefertigt am Max-Planck-Institut für Herz- und Lungenforschung, Bad Nauheim und Medizinische Fakultät Mannheim - Universität Heidelberg, Mannheim 1. Gutachter: Prof. Dr. Katja Sträßer Institut für Biochemie, Justus-Liebig-Universität Gießen 35392 Gießen/ Germany 2. Gutachter: Prof. Dr. Gergana Dobreva Medizinische Fakultät Mannheim, Universität Heidelberg 68167 Mannheim/ Germany Disputation am 31.10.2019 EIDESSTATTLICHE ERKLÄRUNG „Ich erkläre: Ich habe die vorgelegte Dissertation selbständig und ohne unerlaubte fremde Hilfe und nur mit den Hilfen angefertigt, die ich in der Dissertation angegeben habe. Alle Textstellen, die wörtlich oder sinngemäß aus veröffentlichten Schriften entnommen sind, und alle Angaben, die auf mündlichen Auskünften beruhen, sind als solche kenntlich gemacht. Bei den von mir durchgeführten und in der Dissertation erwähnten Untersuchungen habe ich die Grundsätze guter wissenschaftlicher Praxis, wie sie in der „Satzung der Justus-Liebig-Universität Gießen zur Sicherung guter wissenschaftlicher Praxis“ niedergelegt sind, eingehalten.“ Mannheim, den Rui Gao Table of Contents List of Figures ................................................................................................. 5 List of Tables .................................................................................................. 7 Zusammenfassung ......................................................................................... 8 Abstract ......................................................................................................... 9 1. Introduction .............................................................................................. 10 1.1 Heart development ................................................................................................ 10 1.1.1 Mouse heart development ...................................................................................... 10 1.1.2 Congenital heart disease .......................................................................................... 14 1.1.3 Cell types and cardiac lineages in mammalian heart .............................................. 17 1.1.4 Signaling pathways in cardiac development ............................................................ 19 1.1.5 Transcriptional factors involved in heart development .......................................... 25 1.2 Isl1 ........................................................................................................................ 31 1.2.1 Protein structure of ISL1 .......................................................................................... 31 1.2.2 Expression and function of Isl1 during development .............................................. 32 1.2.3 Expression and function of Isl1 during heart development .................................... 33 1.2.4 ISL1 and CHD ............................................................................................................ 36 1.3 Pioneer transcription factors .................................................................................. 36 1.4 Epigenetic regulation ............................................................................................. 40 1.4.1 Chromatin structure ................................................................................................. 40 1.4.2 Epigenetic control mechanisms ............................................................................... 41 2. Objectives ................................................................................................. 51 3. Results ...................................................................................................... 52 3.1 Isl1 regulates cardiogenesis in CPCs by directly targeting a large group of downstream targets .................................................................................................... 52 3.1.1 Generation of Isl1 KO ESC line ................................................................................. 52 3.1.2 Establishing in vitro mESC-based cardiac differentiation protocol ......................... 53 3.1.3 Isl1 KO ESC lines can differentiate into CPCs ........................................................... 54 3.1.4 Isl1 loss-of-function results in downregulation of a large number of genes critical for cardiogenesis in CPCs .................................................................................................. 55 3.1.5 Isl1 directly regulates a large amount of genes in CPCs .......................................... 57 1 3.2 Isl1 is essential in regulating second heart field development in mouse embryos .... 58 3.2.1 Isl1 knockout leads to significant gene expression changes at early stage of mouse embryonic development ................................................................................................... 58 3.2.2 Reduced level of Isl1 results in cardiac defects and deregulation of target genes in E10.5 Isl1 hypomorphic embryos ...................................................................................... 60 3.2.3 ChIP-Seq analysis identified Isl1 primary downstream targets in vivo .................... 62 3.3 Isl1 orchestrates a regulatory network driving cardiogenesis .................................. 63 3.4 Baf60c is a key Isl1 downstream target ................................................................... 66 3.4.1 Baf60c is directly regulated by Isl1 .......................................................................... 66 3.4.2 RNA-Seq analysis identified Baf60c regulated genes in CPCs .................................. 67 3.4.3 Isl1 and Baf60c share common targets .................................................................... 69 3.5 Isl1 works in concert with the Brg1-based SWI/SNF complex to regulate its target genes .......................................................................................................................... 70 3.5.1 Isl1 interacts with Brg1 and Baf60c ......................................................................... 70 3.5.2 The interaction of Isl1 with Brg1 does not depend on Baf60c ................................ 71 3.5.3 Co-occupancy of Isl1 and Brg1 in CPCs .................................................................... 72 3.5.4 Isl1 works cooperatively with Brg1 to regulate its target gene expression ............ 74 3.5.5 Isl1 recruits the Brg1 complex to its target sequences ............................................ 75 3.6 Isl1 acts as a pioneer factor in cardiogenesis .......................................................... 76 3.6.1 Isl1 binding correlates with sites of open chromatin .............................................. 76 3.6.2 Isl1 and Brg1/Baf60c complex work synergistically to open the chromatin during SHF development .............................................................................................................. 77 3.6.3 Isl1 plays a pioneering function in all three cardiovascular lineages ...................... 80 3.6.4 Isl1 acts as a pioneer factor independently of Gata4 .............................................. 81 3.7 Working model ...................................................................................................... 83 4. Discussion ................................................................................................. 84 4.1 Isl1 is critical for SHF development and depletion of Isl1 leads to cardiac defects as seen in human CHD patients ........................................................................................ 85 4.2 Isl1 organizes a regulatory gene network driving second heart field development .. 87 4.3 Baf60c is a key downstream target of Isl1 ............................................................... 89 4.4 Isl1 works together with Brg1-Baf60c-based SWI/SNF complex to regulate gene expression program during cardiogenesis .................................................................... 90 4.5 Isl1 functions as a pioneer factor during cardiogenesis ........................................... 91 2 4.6 Isl1 plays a pioneering function independently of Gata4 ......................................... 93 4.7 Conclusions and perspectives ................................................................................. 95 5. Materials and Methods ............................................................................. 97 5.1 Chemicals used in this study .................................................................................. 97 5.2 Kits used in this study ............................................................................................ 98 5.3 Cell culture medium and supplements ................................................................... 99 5.4 Further materials .................................................................................................. 100 5.5 Equipment used in this study ................................................................................ 101 5.6 RNA extraction, cDNA synthesis and qPCR ............................................................ 102 5.6.1 RNA extraction ....................................................................................................... 102 5.6.2 cDNA synthesis