UNIVERSITY OF CALIFORNIA, SAN DIEGO The Role of Hepatocyte Nuclear Factors 1a and 4a (Hnf1a and Hnf4a) in the Specification and Transcriptional Regulation of the Kidney Proximal Tubule A dissertation submitted in partial satisfaction of the Requirements for the degree Doctor of Philosophy in Biomedical Sciences with a Specialization in Multi-Scale Biology by Gleb Nicolai Martovetsky Committee in charge: Professor Sanjay K. Nigam, Chair Professor Christopher K. Glass Professor Bing Ren Professor Scott C. Thomson Professor Karl Willert 2016 i Copyright Gleb Nicolai Martovetsky, 2016 All rights reserved ii The Dissertation of Gleb Nicolai Martovetsky is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2016 iii TABLE OF CONTENTS Signature Page ................................................................................................................... iii Table of Contents ............................................................................................................... iv List of Figures .................................................................................................................. viii List of Tables ...................................................................................................................... x Acknowledgments.............................................................................................................. xi Vita ................................................................................................................................... xiii Abstract of the Dissertation ............................................................................................. xiv CHAPTER 1: Introduction ................................................................................................. 1 1.1 THE PROXIMAL TUBULE .................................................................................... 1 1.1.1 Role in Solute Transport ..................................................................................... 1 1.1.2 Role in Drug Metabolism ................................................................................... 3 1.2 KIDNEY DEVELOPMENT ..................................................................................... 4 1.2.1 Nephrogenesis .................................................................................................... 4 1.2.2 Systems view of transcriptional regulation of embryonic and postnatal kidney development................................................................................................................. 4 1.3 TISSUE-SPECIFIC TRANSCRIPTIONAL REGULATION .................................. 7 1.3.1 Knockout mouse models relevant to the proximal tubule development ............ 7 1.3.2 Transcriptional regulation of mature proximal tubule function ......................... 9 1.3.3 Differentiation and Transdifferentiation ........................................................... 10 1.4 HEPATOCYTE NUCLEAR FACTORS 1A AND 4A (HNF1A AND HNF4A) .. 12 iv 1.4.1 Hnf1a ................................................................................................................ 13 1.4.2 Hnf4a ................................................................................................................ 13 1.4.3 Human relevance .............................................................................................. 15 1.5 CELLULAR STRATEGIES AIMED AT REPLACING KIDNEY FUNCTION . 15 1.6 SCOPE OF DISSERTATION ................................................................................. 16 CHAPTER 2: Hepatocyte Nuclear Factors 4a and 1a Regulate Kidney Developmental Expression of Drug-Metabolizing Enzymes and Drug Transporters ................................ 19 2.1 ABSTRACT ............................................................................................................ 19 2.2 INTRODUCTION ................................................................................................... 20 2.3 MATERIALS AND METHODS ............................................................................ 23 2.3.1 Microarray Expression Analysis ...................................................................... 23 2.3.2 Chromatin Immunoprecipitation ...................................................................... 24 2.3.3 Massively Parallel Sequencing and Analysis ................................................... 25 2.3.4 Organ and Cell Culture ..................................................................................... 26 2.3.5 Immunofluorescence ........................................................................................ 27 2.3.6 Lentiviral Transduction .................................................................................... 27 2.3.7 Real Time qPCR ............................................................................................... 28 2.3.8 6-Carboxyfluorescein Uptake Assay ................................................................ 29 2.4 RESULTS................................................................................................................ 29 2.4.1 Curating an extensive list of Phase I, II, and III genes ..................................... 29 2.4.2 Analysis of changes in DME expression during pre and postnatal kidney development identifies a set of potential transcriptional regulators .......................... 30 2.4.3 Analysis of the proximal tubule transcriptome during developmental time points reveals a dominant contribution of DMEs ...................................................... 31 v 2.4.4 Motif analysis of Ep300-bound cis-regulatory elements in the developing kidney suggests Hnf4a and Hnf1a as key regulators ................................................. 32 2.4.5 Transcriptomic analysis of microarray data from Hnf4a tissue-specific knockouts revealed that distinct subsets of Phase I, Phase II and Phase III genes were affected in developing liver, colon, intestine and pancreas ....................................... 34 2.4.6 Hepatocyte nuclear factor 4a plays a major role in establishing and maintaining transcriptional enhancer elements that regulate DME genes in the proximal tubule 36 2.4.7 Administration of an Hnf4a small molecule antagonist in an ex vivo kidney organ culture model markedly attenuated the expression of representative Phase I, II and III DMEs ............................................................................................................. 39 2.4.8 Overexpression of Hnf1a and Hnf4a in mouse embryonic fibroblasts induces expression of proximal tubule Phase I, II and III DMEs ........................................... 40 2.5 DISCUSSION ......................................................................................................... 41 2.6 ACKNOWLEDGEMENTS .................................................................................... 47 2.7 AUTHOR CONTRIBUTIONS ............................................................................... 47 CHAPTER 3: Transcriptional Switch Modulates Hnf1a and Hnf4a Specificity Toward Kidney Proximal Tubule-like or Hepatocyte-like Fate ..................................................... 57 3.1 ABSTRACT ............................................................................................................ 57 3.2 INTRODUCTION ................................................................................................... 58 3.3 EXPERIMENTAL PROCEDURES ....................................................................... 60 3.3.1 Cell Culture....................................................................................................... 60 3.3.2 Lentiviral Transduction .................................................................................... 60 3.3.3 Primary Proximal Tubule Cell Culture ............................................................. 61 3.3.4 Quantitative Reverse Transcription PCR ......................................................... 62 3.3.5 Immunohistochemistry/Microscopy ................................................................. 62 3.3.6 Microarray analysis .......................................................................................... 63 vi 3.3.7 ChIP-sequencing ............................................................................................... 64 3.4 RESULTS................................................................................................................ 64 3.4.1 Potential of Hnf1a and Hnf4a to induce a proximal tubule mRNA expression signature is dependent on cellular context ................................................................. 64 3.4.2 Transduction of Hnf1a and Hnf4a alters the morphology of MEFs and induces formation of tight junctions ....................................................................................... 67 3.4.3 More extensive characterization of the mRNA expression of transporters and junctional component genes reveals differential and synergistic regulation by Hnf1a and Hnf4a .................................................................................................................. 68 3.4.4 Liver lineage determining factors Gata4 and Foxa2/Foxa3 act as a transcriptional switch to redirect Hnf1a and Hnf4a-mediated transcriptional regulation from proximal tubule to hepatocyte cellular identity ............................... 70 3.5 DISCUSSION ........................................................................................................