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Characterization of Higher Order Chromatin Structures and Chromatin States in Cell Models of Human Herpesvirus Infection

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University of Vermont UVM ScholarWorks Graduate College Dissertations and Theses Dissertations and Theses 2021 Characterization Of Higher Order Chromatin Structures And Chromatin States In Cell Models Of Human Herpesvirus Infection Michael Mariani University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Biology Commons, Genetics and Genomics Commons, and the Virology Commons Recommended Citation Mariani, Michael, "Characterization Of Higher Order Chromatin Structures And Chromatin States In Cell Models Of Human Herpesvirus Infection" (2021). Graduate College Dissertations and Theses. 1424. https://scholarworks.uvm.edu/graddis/1424 This Dissertation is brought to you for free and open access by the Dissertations and Theses at UVM ScholarWorks. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of UVM ScholarWorks. For more information, please contact [email protected]. CHARACTERIZATION OF HIGHER ORDER CHROMATIN STRUCTURES AND CHROMATIN STATES IN CELL MODELS OF HUMAN HERPESVIRUS INFECTION A Dissertation Presented by Michael Mariani to The Faculty of the Graduate College of The University of Vermont In Partial Fulfilment of the Requirements For the Degree of Doctor of Philosophy Specializing in Cellular, Molecular, and Biomedical Sciences May, 2021 Defense Date: March 24, 2021 Dissertation Examination Committee: Seth Frietze, Ph.D., Advisor Matthew Poynter, Ph.D., Chairperson David Pederson Ph.D. Stephen Keller Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College COPYRIGHT © Copyright by Michael Mariani May 2021 ABSTRACT Human herpesviruses are ubiquitous pathogens worldwide with 90% of the global population infected with one or more Human herpesviruses (HHV’s) by adulthood. All herpesviruses have three unique life cycle stages. Upon resolution of a primary acute stage infection, they can establish a latent stage infection within the host cell nucleus. This stage is characterized primarily by transcriptional quiescence of the viral genome. Specific physiological conditions (e.g., cell stress) can cause the latent virus to enter the reactivation stage, often many years after resolution of the acute infection, in which the virus becomes replicationally active again. HHV’s are known to cause disease in humans in all three stages of their lifecycle and chronic infection is becoming increasingly associated with a wide range of human morbidities. The field has been met with challenges in establishing tractable cell models to study HHV infection in vitro and the exact mechanisms regulating the maintenance of, and transition between, the life cycle stages are largely undefined; however, chromatin structure and function are known to play a role. In this dissertation I employ both molecular and computational approaches to study the higher order chromatin structures and chromatin states in cell models of infection for two Human herpesviruses at specific stages in their life cycles. In the first part of this dissertation, I successfully demonstrate the establishment of a latent Human betaherpesvirus 6A (HHV-6A) infection, which requires integration of its genome into the host cell genome, in an in vitro-derived HEK-293 cell model. I then employ unbiased epigenomic methods along with bioinformatics techniques to identify three-dimensional virus-host contact regions and characterize the chromatin states of the latent virus. I then demonstrate the above in both a patient-derived cell model and in infected primary cells. Finally, I developed a novel and innovative computational approach to identify the sites of HHV-6A integration in host cell chromosomes using next generation sequencing data: to my knowledge, a first in the field. In the second part of this work, I employ a human fetal lung fibroblast (HFL) cell model of acute infection to study Varicella Zoster Virus (VZV) chromatin biology - specifically with regards to the transcription factor CTCF. CTCF is a key organizer of chromatin three-dimensional structure and plays a role in chromatin organization and transcriptional regulation in certain HHV’s. Herein, I present evidence that CTCF is likely involved in virus-host structural interactions – an important finding that is countervailing to the current suppositions of the Human alphaherpesvirus field. Overall, the work presented herein offers novel insight into the complex and dynamic relationship between HHV’s and host cell chromatin. With a more comprehensive characterization of the higher order chromatin structures and chromatin states that define this relationship, we can better define the HHV life cycle. Such knowledge will allow for the development of improved treatments against these insidious infections. CITATIONS Material from this thesis (or dissertation) has been published in the following form: Mariani, M., Zimmerman, C., Rodriguez P., Hasenohr, E., Aimola, G., Gerrard, D.L., Richman, A., Dest, A., Flamand, L., Kaufer, B., Frietze, S.. (2021). Higher-Order Chromatin Structures of Chromosomally Integrated HHV-6A Predict Integration Sites. Frontiers in Cellular and Infection Microbiology, 11, 67. ii DEDICATION I dedicate this dissertation to my family: Mom, Dad and Rob. If it is said that I have accomplished anything in this life, it is only by dint of your unwavering love and support. iii ACKNOWLEDGEMENTS I would like to acknowledge my advisor, Seth Frietze, for giving me the opportunity to work and study in his laboratory, as well as for providing me with mentorship and guidance during my tenure as a graduate student; the other members of my thesis committee: Matthew Poynter, David Pederson, and Stephen Keller, for their ongoing support and feedback; Randall Cohrs for his mentorship and guidance; Richard Single for his academic guidance early in my graduate student career; my lab mates Princess Rodriguez and Diana Gerrard for their compassion, friendship, and feedback. My former lab mates Arvis Sulovari, Xun Chen, and Jason Kost for their friendship, mentorship, and support; Andrew Bubak and Sara Bustos - my other lab mates across the country with whom it has been a pleasure to work; Joe Boyd for his scientific and bioinformatic feedback; all the past and present members of the Frietze laboratory for their passion for scientific endeavor and academic inquisitiveness; the members of the CMB class of 2015 for their friendliness, tenacity, perspicacity, and humanity; the CMB staff and faculty for keeping the whole show running smoothly; all my family, friends, relatives, mentors, teachers and health professionals, both past and present: the magnitude of my gratitude is not quantifiable. iv iv TABLE OF CONTENTS CITATIONS..................................................................................................................ii DEDICATION.............................................................................................................. iii ACKNOWLEDGEMENTS ..........................................................................................iv CHAPTER 1 .................................................................................................................1 Human herpesviruses ................................................................................................1 Health effects acute and chronic ................................................................................2 HHV virology ............................................................................................................4 Chromatin biology of the host ..................................................................................9 The transcription factor CTCF .................................................................................11 Genomics methodology ............................................................................................12 Human gammaherpesviruses and host chromatin (EBV) .........................................17 Human alphaherpesviruses and host chromatin (HSV-1) ......................................... 21 Human betaherpesviruses and host chromatin (HHV-6A)....................................... 27 VZV and host chromatin biology............................................................................. 31 Concluding remarks ................................................................................................. 39 CHAPTER 2 ................................................................................................................ 42 Title .......................................................................................................................... 42 Authors ..................................................................................................................... 42 Affiliations ............................................................................................................... 42 Correspondence ........................................................................................................ 43 Abstract ..................................................................................................................... 43 Introduction ............................................................................................................... 44 Materials and Methods .............................................................................................. 46 Ethics..................................................................................................................... 46
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