Insights into the comparative biological roles of S. cerevisiae nucleoplasmin-like FKBPs Fpr3 and Fpr4 by Neda Savic B.Sc. Portland State University, 2012 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY in the Department of Biochemistry and Microbiology © Neda Savic, 2019 University of Victoria All rights reserved. This dissertation may not be reproduced in whole or in part, by photocopy or other means, without the written permission of the author. ii Supervisory Committee Insights into the comparative biological roles of S. cerevisiae nucleoplasmin-like FKBPs Fpr3 and Fpr4 by Neda Savic B.Sc. Portland State University, 2012 Supervisory Committee Dr. Christopher J. Nelson, Supervisor Department of Biochemistry and Microbiology Dr. Juan Ausio, Departmental Member Department of Biochemistry and Microbiology Dr. Caren C. Helbing, Departmental Member Department of Biochemistry and Microbiology Dr. Peter C. Constabel, Outside Member Department of Biology iii Abstract The nucleoplasmin (NPM) family of acidic histone chaperones and the FK506-binding (FKBP) peptidyl proline isomerases are both linked to chromatin regulation. In vertebrates, NPM and FKBP domains are found on separate proteins. In fungi, NPM-like and FKBP domains are expressed as a single polypeptide in nucleoplasmin-like FKBP (NPL-FKBP) histone chaperones. Saccharomyces cerevisiae has two NPL-FKBPs: Fpr3 and Fpr4. These paralogs are 72% similar and are clearly derived from a common ancestral gene. This suggests that they may have redundant functions. Their retention over millions of years of evolution also implies that each must contribute non-redundantly to organism fitness. The redundant and separate biological functions of these chromatin regulators have not been studied. In this dissertation I take a systems biology approach to fill this knowledge gap. First, I refine the powerful synthetic genetic array (SGA) method of annotating gene-gene interactions, making it amenable for the analyses of paralogous genes. Using these ‘paralog-SGA’ screens I define distinct genetic interactions unique to either Fpr3 or Fpr4, shared genetic interactions common to both paralogs, and masked genetic interactions which are direct evidence for processes where these enzymes are functionally redundant. I provide transcriptomic evidence that Fpr3 and Fpr4 cooperate to regulate genes involved in polyphosphate metabolism and ribosome biogenesis. I identify an important role for Fpr4 at the 5’ ends of protein coding genes and the non-transcribed spacers of ribosomal DNA. Finally, I show that yeast lacking Fpr4 exhibit a genome instability phenotype at rDNA, implying that this histone chaperone regulates chromatin structure and DNA access at this locus. Collectively, these data demonstrate that Fpr3 and Fpr4 operate separately, cooperatively and redundantly to regulate a variety of chromatin environments. This work is the first comprehensive and comparative study of NPL-FKBP chaperones and as such represents a significant contribution to our understanding of their biological functions. iv Table of Contents Supervisory Committee................................................................................................................. ii Abstract ......................................................................................................................................... iii Table of Contents .......................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures .............................................................................................................................. vii List of Abbreviations .................................................................................................................... ix Acknowledgements ..................................................................................................................... xiv Dedication..................................................................................................................................... xv Chapter 1 Introduction ......................................................................................................... 1 1.1 General Introduction ............................................................................................................. 1 1.2 Chromatin and its modifications ........................................................................................... 2 1.2.1 The ADA histone acetyltransferase complex ................................................................ 3 1.2.2 The Set1/COMPASS histone methyltransferase complex ............................................. 5 1.2.3 The SWI/SNF nucleosome remodeling complex .......................................................... 6 1.3 The nucleoplasmin (NPM) family of histone chaperones ..................................................... 6 1.3.1 NPM1 ............................................................................................................................. 8 1.3.2 NPM2 and NPM3 .......................................................................................................... 9 1.3.3 NPM family histone chaperones in disease ................................................................... 9 1.4 Prolyl isomerization ............................................................................................................ 10 1.4.1 Peptidyl-prolyl isomerases ........................................................................................... 11 1.4.2 Prolyl-isomerases as a molecular switch: the CYP33-MLL1 case study .................... 14 1.4.3 Yeast nuclear FKBPs target histones ........................................................................... 15 1.4.4 Vertebrate nuclear FKBPs ........................................................................................... 16 1.5 The Nucleoplasmin-like FKBPs ......................................................................................... 17 1.5.1 Nucleoplasmin-like FKBPs in plants and insects ........................................................ 17 1.5.2 Nucleoplasmin-like FKBPs in fungi ............................................................................ 19 1.5.3 Gene duplication events ............................................................................................... 19 1.6 Yeast Fpr3 and Fpr4 ............................................................................................................ 21 1.6.1 Protein features of Fpr3 and Fpr4 ................................................................................ 21 1.6.2 Evidence for Fpr3 and Fpr4 functional similarity........................................................ 23 1.6.3 Evidence for Fpr3 and Fpr4 functional divergence ..................................................... 24 1.7 Ribosome Biogenesis .......................................................................................................... 24 1.7.1 The nucleolus and rDNA chromatin regulation ........................................................... 25 1.7.2 rRNA processing and quality control .......................................................................... 27 1.8 Research Objectives ............................................................................................................ 29 Chapter 2 Genetic interactions reveal comparative functions of Fpr3 and Fpr4 .......... 31 2.1 Introduction ......................................................................................................................... 31 2.2 Results ................................................................................................................................. 33 2.2.1 Paralog-SGA reveals distinct genetic interaction fingerprints for duplicated genes ... 33 2.2.2 FPR3 and FPR4 have separate, shared, and redundant genetic interactions including with genes involved in chromatin biology ............................................................................ 38 2.2.3 The TRAMP5 nuclear exosome is a masked genetic interactor of FPR3 and FPR4 .. 41 v 2.2.4 Suppressor genetic interactors support chromatin-centric functions for Fpr3 and Fpr4 .............................................................................................................................................. 45 2.3 Discussion ........................................................................................................................... 50 2.4 Materials and Methods ........................................................................................................ 54 Chapter 3 Fpr3 and Fpr4 regulate transcription from multiple genomic loci .............. 58 3.1 Introduction ......................................................................................................................... 58 3.2 Results ................................................................................................................................. 59 3.2.1 Fpr3 and Fpr4 regulate the expression of separate and common genes ....................... 59 3.2.2 The TRAMP5 RNA exosome masks the impact of Fpr4 on transcription .................. 63 3.2.3 Evidence for Fpr4 action at the 5’ end of the transcription unit .................................. 64 3.2.4 Fpr3