HMGA2-mediated epigenetic regulation of Gata6 controls epithelial canonical WNT signaling during lung development and homeostasis INAUGURALDISSERTATION zur Erlangung des Doktorgrades der Naturwissenschaften - Doctor rerum naturalium - (Dr. rer. nat.) eingereicht am Fachbereich Biologie und Chemie der Justus-Liebig-Universität Giessen vorgelegt von Indrabahadur Singh Bad Nauheim, 2014 Die vorliegende Arbeit wurde am Max-Planck-Institut für Herz- und Lungenforschung in der Abteilung "Epigenetik des Lungenkrebs" unter der Leitung von Herrn Dr. Guillermo Barreto angefertigt. Erstgutachter: Prof. Dr. Dr. Thomas Braun Abteilung Entwicklung und Umbau des Herzens Max-Planck-Institut für Herz- und Lungenforschung Ludwigstraße 43, 61231 Bad Nauheim Zweitgutachter: Prof. Dr. Rainer Renkawitz Institut für Genetik Justus-Liebig-Universität Giessen Heinrich-Buff-Ring 58, 35392 Giessen Disputation am 24-09-2014 DEDICATED TO MY PARENTS & GRANDPARENTS Whose perpetual affection and blessing always Inspired me for higher ambition in life TABLE OF CONTENTS Table of Contents Table of Contents ....................................................................................................................... VIII List of Figures and Tables............................................................................................................. XI ZUSAMMENFASSUNG ......................................................................................................... XIV ABSTRACT ................................................................................................................................... 1 Chapter 1: INTRODUCTION ..................................................................................................... 2 1.1 Lung Homeostasis ................................................................................................................ 2 1.2 Lung Development .................................................................................................................... 4 1.2.1 WNT signaling during lung development .......................................................................... 5 1.2.2 GATA6 function during lung development ........................................................................ 6 1.3 HMG proteins ........................................................................................................................... 7 1.3.1 HMGA ................................................................................................................................ 8 1.3.2 HMGA2 .............................................................................................................................. 9 1.3.3 HMGA and Transcription ................................................................................................. 11 1.3.4 HMGA and Chromatin ..................................................................................................... 12 1.4 ATM and H2AXS139ph ......................................................................................................... 14 Chapter 2: AIMS......................................................................................................................... 15 Chapter 3: MATERIALS & METHODS ................................................................................. 16 3.1 Molecular Biology Methods ................................................................................................... 16 3.1.1 RNA isolation and reverse transcription .......................................................................... 16 3.1.2 Quantitative PCR .............................................................................................................. 16 3.1.3 Primer efficiency determination ....................................................................................... 17 3.1.4 Affymetrix Microarrays .................................................................................................... 18 3.1.5 Semiquantitative PCR....................................................................................................... 18 3.1.6 DNA elution from agarose gel .......................................................................................... 19 3.1.7 Nucleic acids concentration determination ....................................................................... 19 3.1.8 Cloning ............................................................................................................................. 19 3.1.9 Preparation of competent E.coli cells ............................................................................... 20 3.1.10 Transformation of E. coli competent cells ...................................................................... 21 3.1.11 Plasmid DNA isolation ................................................................................................... 21 VIII TABLE OF CONTENTS 3.1.12 In situ hybridization of whole mount mouse embryonic lungs ...................................... 21 3.1.13 Chromatin immunoprecipitation..................................................................................... 22 3.1.14 Luciferase Reporter Assay .............................................................................................. 22 3.2 Cell biological methods .......................................................................................................... 22 3.2.1 Cell culture ....................................................................................................................... 22 3.2.2 Cryopreservation and thawing of cells ............................................................................. 23 3.2.3 Transient transfection ....................................................................................................... 23 3.2.4 Transient transfection efficiency assessment ................................................................... 24 3.2.5 Cell treatment ................................................................................................................... 24 3.2.6 Lung explants culture methods ......................................................................................... 24 3.3 Animal experiments ................................................................................................................ 25 3.3.1 Mice .................................................................................................................................. 25 3.3.2 Compliance Measurement ................................................................................................ 25 3.4 Histology and Immunohistochemistry .................................................................................... 26 3.4.1 H&E staining .................................................................................................................... 26 3.4.2 Immunostaining ................................................................................................................ 26 3.4.4 β-Gal staining ................................................................................................................... 27 3.4.5 Flow Cytometry ................................................................................................................ 28 3.5 Biochemical methods .............................................................................................................. 28 3.5.1 In vitro chromatin reconstitution and Histone eviction assays ......................................... 28 3.5.2 Affinity-precipitation and Immunoprecipitation .............................................................. 28 3.5.3 Mass spectrometry: sample preparation, methods and data analysis ............................... 29 3.5.6 Western blot ...................................................................................................................... 30 3.6 Statistical analysis ................................................................................................................... 30 Chapter 4: RESULTS ................................................................................................................. 31 4.1 Hmga2 is required for proper distal epithelial cell differentiation during lung development and homeostasis ............................................................................................................................ 31 4.1.1 Hmga2 is expressed in the mouse embryonic lung at the distal airways ......................... 31 4.1.2 Hmga2 is required for proper differentiation of the distal epithelium during lung development............................................................................................................................... 32 IX TABLE OF CONTENTS 4.1.3 Hmga2 knockout compromised adult lung function due to epithelial cell differentiation defect ......................................................................................................................................... 37 4.2 Hmga2 modulates Canonical WNT signaling during lung development and homeostasis .... 39 4.2.1 Hmga2 knockout led to enhanced canonical WNT signaling during lung development . 39 4.2.2 Hmga2 knockout showed enhanced canonical WNT signaling in adult lung .................. 43 4.2.3 HMGA2 directly activates Gata6 expression and thereby modulates canonical WNT signaling..................................................................................................................................... 46 4.3 HMGA2 mediated Gata6 transcriptional