A mitotic bookmark containing ubiquitin marks the promoters of active genes Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Alexander Etienne Hare, BS Biomedical Sciences Graduate Program The Ohio State University 2020 Dissertation Committee Jeffrey D. Parvin, adviser Michael Freitas Mark Parthun Qin Ma 1 Copyrighted by Alexander Etienne Hare 2020 2 Abstract When cells divide, their DNA condenses into tightly bound chromosomes. In doing so, they must erase some of the important epigenetic marks that instruct cells how to express their genes, such as post-translational modifications of histone tails. These marks are necessary for cellular differentiation, i.e. for a skin cell to remember it was a skin cell, and this cellular differentiation is created by specific patterns of transcription. Therefore, cells need a mechanism to “remember” transcriptional patterns across mitosis. We have discovered that the ubiquitin protein is found on chromosomes during cell division, which we call ubiquitin bookmarks. We hypothesize that an epigenetic pattern of ubiquitin bookmarking controls specific cellular processes or groups of genes. These bookmarks are found on the promoters of genes found to be transcriptionally active during interphase and their presence correlates with indicators of active transcription. We have identified that the ubiquitin ligase RING1A, a component of the polycomb repressive complex 1, is responsible for the creation of the ubiquitin bookmark. However, canonical PRC1 ubiquitin ligase RING1B is not necessary for the ubiquitin bookmark. We have shown that RING1A and RING1B exist in separable complexes, suggesting a novel RING1A-containing complex for bookmark ubiquitination. Through RING1A depletion, we can regulate production of this bookmark, allowing us to study the effects of the ubiquitin bookmark on multiple cellular functions. Using RT-qPCR and RNAseq, ii we have observed that transcription of bookmarked genes depends on the presence of the ubiquitin bookmark, and we have shown that this dependence is linked to genes with higher levels of transcription. Mass spectrometry analysis has shown dysregulation of histone modifications following disabled ubiquitin bookmarking. Understanding the scope and method by which the ubiquitin bookmark affects transcription is the next step in understanding the bookmark and being able to establish its importance in maintaining cellular differentiation. iii Dedication I dedicate this work to my parents, who have cultivated my love of science and discovery, and to Dr. Dora Ahmadi, who showed me what it was worth to push myself. iv Acknowledgments I acknowledge the ENCODE Consortium and the Bradley Bernstein lab, which provided the publicly available datasets used in this work. I acknowledge Amy Hite, Anjun Ma, and Li Yang, who each provided invaluable advice on bioinformatics analysis. Lastly, I acknowledge the OSUCCC Genomics Shared Resources, which is an NCI- subsidized Shared Resource supported by P30CA016058, for the sequencing experiments performed. v Vita May 2010…………………………………..Gatton Academy May 2014…………………...…….………..BS Mathematics, University of Kentucky May 2014……………………....…………..BS Biology, University of Kentucky June 2014 – Present ..………………..…….The Ohio State University Publications Hare, Alexander. & Rebaza, Jorge. (2015). Dynamics of predator-prey models with refuge, harvesting and dispersal. Quaestiones Mathematicae. 38. 1-15. 10.2989/16073606.2015.1026559. Hare, Janelle & Adhikari, Sabal & Lambert, Kasandra & Hare, Alexander & Grice, Alison. (2012). The Acinetobacter regulatory UmuDAb protein cleaves in response to DNA damage with chimeric LexA/UmuD characteristics. FEMS microbiology letters. 334. 10.1111/j.1574-6968.2012.02618.x. Hare AE, Parvin JD. Processes that Regulate the Ubiquitination of Chromatin and Chromatin-Associated Proteins in Ubiquitin Proteasome System - Current Insights into vi Mechanism Cellular Regulation and Disease, 978-1-83880-491-6, ed. Matthew Summers. DOI: 10.5772/intechopen.82567 Field of Study Major Field: Biomedical Sciences vii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments ............................................................................................................... v Vita ..................................................................................................................................... vi List of Figures .................................................................................................................... ix Conflicts of Interest ............................................................................................................. x Chapter 1: Processes that Regulate the Ubiquitination of Chromatin and Chromatin- Associated proteins ............................................................................................................. 1 Chapter 2: Rationale for Dissertation ............................................................................... 30 Chapter 3: A Mitotic Bookmark containing Ubiquitin Marks the Promoters of Active Genes ................................................................................................................................. 35 Chapter 4: Future Directions ............................................................................................. 87 References ......................................................................................................................... 92 Appendix A ..................................................................................................................... 111 viii List of Figures Figure 1-1 Ubiquitination of chromatin regulates DSB repair. .......................................... 8 Figure 1-2 The PAF1 complex ubiquitinates histone H2B during transcription elongation. ........................................................................................................................................... 16 Figure 1-3 Canonical PRC1 ubiquitinates H2AK119. ...................................................... 22 Figure 1-4 A mitotic bookmark containing ubiquitin is necessary for maintaining the active chromatin state after completion of mitosis. .......................................................... 28 Figure 3-1 Bookmark ubiquitination is dependent on RING1A ....................................... 44 Figure 3-2 RING1A exists in a separable complex from RING1B. ................................. 48 Figure 3-3 Ubiquitin is enriched on the promoters of active genes in multiple cell lines. 53 Figure 3-4 Bookmark ubiquitination is necessary for transcription of active genes. ....... 57 Figure 3-5 Ubiquitin marks correspond to genes downregulated following RING1A depletion ............................................................................................................................ 62 Figure 3-6 Epiprofile results from histone H3 modifications. .......................................... 65 Figure 3-7 Divergent Epiprofile results from histone H3. ................................................ 66 Figure 3-8 Select histone modifications from Epiprofile on histone H3. ......................... 67 Figure 3-9 H3K27ac-marked genes correspond to genes downregulated following RING1A depletion ............................................................................................................ 69 Figure 3-10 Distribution of H3K27ac-intersecting genes by number of overlaps ............ 70 Figure 3-11 Epiprofile results from histones H2A and H2B. ........................................... 72 Figure 3-12 Divergent Epiprofile results from histone H2A and H2B. ............................ 74 Figure 3-13 Epiprofile results from histones H1 and H4. ................................................. 76 Figure 3-14 Divergent Epiprofile results from histones H1 and H4. ................................ 78 Figure 3-15 Select histone modifications from Epiprofile from histones H1, H2A, H2B, and H4. .............................................................................................................................. 80 Figure 3-16 H4K20me1-marked genes correspond to genes downregulated following RING1A depletion ............................................................................................................ 82 ix Conflicts of Interest The author has no conflicts of interests to declare. x Chapter 1: Processes that Regulate the Ubiquitination of Chromatin and Chromatin- Associated proteins This chapter has been previously published as a book chapter (Hare and Parvin, 2019). 1 Introduction Ubiquitin is most clearly associated with the process of targeted protein degradation, but it is involved in many cellular processes such as chromatin regulation, immune response, and antigen processing1. Proteosomal degradation is mediated through polyubiquitin chains linked via lysine-48 (K48) on the ubiquitin chain, interacting with the proteasome. Other processes utilize monoubiquitination or polymerization of ubiquitin molecules via another lysine. The ubiquitination system in humans is incredibly complex, with over 1,000 known factors and over 10,000 known sites of ubiquitination, enabling its