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1 Understanding the Role of the FACT Complex During Development in C. Elegans Dissertation Presented in Partial Fulfillment of T Understanding the role of the FACT complex during development in C. elegans Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Brittany Zaneta Suggs Graduate Program in Molecular Genetics The Ohio State University 2017 Dissertation Committee Dr. Helen Chamberlin, Advisor Dr. Sharon Amacher Dr. Christin Burd Dr. Robin Wharton 1 Copyrighted by Brittany Zaneta Suggs 2017 2 Abstract The maternal to zygotic transition is a developmental phase shared by all multicellular organisms. It is during this time that control of the developing embryo is transferred from maternally provided mRNAs and proteins to the zygotic genome. In order for the zygotic genome to be transcribed, transcription factors must be able to access the promoters of their target genes. Prior to this point, the chromatin has been held in a transcriptionally silent state which must be alleviated before transcription can occur. There are several mechanisms by which chromatin can be rearranged into a more transcriptionally competent state. In this work, I focus on the nucleosome reorganizing FACT complex. FACT is a heterodimeric complex composed of SSRP1 and SPT16 and is known to be involved in various functions such as transcription, DNA replication, and DNA repair. Both SSRP1 and SPT16 have been found to be essential for embryonic development, and in their absence, lethality occurs early. While much is known regarding the functions of the FACT complex at the level of the cell, the requirement for FACT during embryogenesis has been a significant impediment to understanding the role of FACT in development on an organismal scale. In this study, I have evaluated the developmental consequence of FACT depletion during the embryonic development of the nematode Caenorhabditis elegans using RNAi ii and genetic mutants. I provide evidence for a requirement for FACT in the formation of the anterior pharynx and in the maintenance of cell cycle length and progression. Additionally, I investigate the relationship between FACT and APX-1, a well described component of the pharyngeal development pathway, and, from those data, propose two possible models for how FACT may be involved in pharyngeal development. Together, these data begin to show how the cellular functions of FACT impacts the development of the organism as a whole. iii Acknowledgments First, I give honor to God without whom this journey would have been impossible. For this, You receive all glory and praise. I am deeply grateful for the mentorship of Dr. Helen Chamberlin. Her endless patience and constant encouragement and support have been invaluable through the years. I truly cannot thank you enough. Thank you to my committee, Dr. Sharon Amacher, Dr. Christin Burd, and Dr. Robin Wharton, for your suggestions, counsel, and even the difficult questions through the development of this project. To Karley Mahalak, Allison Webb, Marcos Corchado, Kristin Balmert and Leann Kelley, my amazing worm guys, I cannot express how grateful I am that I was on this journey with you. Your support—in all its forms—during the tough times and the laughter during the good times will forever be cherished memories. I look forward to our continued friendships. Thank you to my KCC family. Your prayers were greatly appreciated. Lastly, but most certainly not least, I would like to thank my family. Mom, dad, Treena, Rayna, Wayne: We did it! And ‘we’ it most certainly was. Without you, your love, encouragement and continued support, I would not have made it. I love you all more than I could ever express. Thank you. iv Vita February 4, 1989…………………………........Born-Rison, AR 2007-2011…………………………………….B.S. Biology, Henderson State University 2011-2017……………………………………Graduate Teaching and Research Associate, The Ohio State University Fields of Study Major Field: Molecular Genetics v Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv Vita ...................................................................................................................................... v List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix Chapter 1. Introduction ....................................................................................................... 1 1.1 The Maternal to Zygotic Transition .......................................................................... 1 1.2 Destabilization and degradation of maternal components ........................................ 2 1.2.1 Functions of maternal clearance and the consequences of clearance failure ..... 2 1.2.2 Mechanisms of maternal clearance .................................................................... 3 1.3 Zygotic Genome Activation (ZGA) .......................................................................... 5 1.3.1 Functions of the ZGA and the consequences of activation failure .................... 5 1.3.2 Mechanisms of ZGA .......................................................................................... 6 1.4 Chromatin reorganization ......................................................................................... 8 1.5 FACT in transcription ............................................................................................. 12 1.6 FACT and Chromatin Maintenance: ....................................................................... 13 1.7 FACT and differentiation ........................................................................................ 15 1.8 Research question and chapter previews ................................................................ 18 Chapter 2: FACT complex genes have critical maternal and zygotic functions in C. elegans............................................................................................................................... 20 2.1 Introduction ............................................................................................................. 20 2.2 Results ..................................................................................................................... 21 2.2.1 FACT complex genes in C. elegans include two SSRP1 orthologs ................ 21 2.2.2 SSRP1 orthologs hmg-3 and hmg-4 have redundant functions in embryonic development .............................................................................................................. 23 vi 2.2.3 Anterior pharynx development is defective in hmg-3; hmg-4- or spt-16- depleted embryos ...................................................................................................... 24 2.2.4 HMG-3, HMG-4 and SPT-16 are present across developmental time ............ 25 2.2.5 The embryonic functions of hmg-3 and hmg-4 have a maternal contribution 25 2.2.6 FACT complex genes have required larval functions ...................................... 27 2.2.7 Depletion of FACT components alters the embryonic cell cycle .................... 28 2.3 Discussion ............................................................................................................... 30 2. 4 Experimental Procedures ....................................................................................... 33 2.4.1 C. elegans strains, maintenance and construction ............................................ 33 2.4.2 Creation of GFP-tagged alleles ........................................................................ 34 2.4.3 RNA Interference ............................................................................................. 35 2.4.4 Fluorescent Microscopy ................................................................................... 36 2.4.5 Time course confocal microscopy and embryonic lineage analysis ................ 37 Chapter 3: Epistasis analysis proposes multiple models of FACT function ..................... 52 3.1 Introduction ............................................................................................................. 52 3.2 Results ..................................................................................................................... 54 3.2.1 FACT knockdown reduces area of pharyngeal markers in apx-1 mutants ...... 54 3.2.2 Knockdown of FACT components causes a delay in expression of PHA- 4::GFP ....................................................................................................................... 55 3.3 Discussion ............................................................................................................... 56 3.4 Materials and Methods ............................................................................................ 60 3.4.1 C. elegans strains, maintenance and construction ............................................ 60 3.4.2 RNA Interference ............................................................................................. 61 Chapter 4: Discussion ....................................................................................................... 69 4.1 Results overview ..................................................................................................... 69 4.2 Retention of SSRP1 duplicates
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