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Defining and Exploiting the Cytosine Deaminase Activity of Apobec3a on the Extended Epigenome

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Defining and Exploiting the Cytosine Deaminase Activity of Apobec3a on the Extended Epigenome University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2018 Defining And Exploiting The Cytosine Deaminase Activity Of Apobec3a On The Extended Epigenome Emily Katharine Schutsky University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, and the Genetics Commons Recommended Citation Schutsky, Emily Katharine, "Defining And Exploiting The Cytosine Deaminase Activity Of Apobec3a On The Extended Epigenome" (2018). Publicly Accessible Penn Dissertations. 2794. https://repository.upenn.edu/edissertations/2794 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2794 For more information, please contact [email protected]. Defining And Exploiting The Cytosine Deaminase Activity Of Apobec3a On The Extended Epigenome Abstract AID/APOBEC family cytosine deaminases canonically play crucial roles in immunity by converting cytosine to uracil in single-stranded DNA (ssDNA). Outside of this established physiological role, AID/ APOBEC enzymes have also been implicated in the poorly-understood process of DNA demethylation through their proposed deamination of epigenetically-modified cytosine bases like 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC). However, there has been no thorough biochemical characterization of AID/APOBEC activity on these substrates, or on the recently-discovered 5-formylcytosine (fC) and 5-carboxylcytosine (caC) to inform this proposed role. Here, we provide the first steady-state kinetic measurements of the most active family member–APOBEC3A(A3A)–against various natural and unnatural modified substrates using a novel, restriction enzyme-based deamination assay. We determined that A3A has poor activity against bulkier oxidized cytosines, such as hmC, fC, and caC, and therefore likely does not contribute substantially to active DNA demethylation via deamination of these bases. By contrast, A3A efficiently deaminates mC in a manner not fully explained by the steric discrimination observed with other AID/APOBEC family members. Because A3A exhibits enzymatic proficiency for C and mC but potently discriminates against x-mCso in vitro, we hypothesized that we could leverage A3A’s biochemical properties to efficiently localize ox-mCs in the genome. We developed APOBEC-Coupled Epigenetic Sequencing (ACE-Seq) – a nondestructive alternative to bisulfite-based methods for base resolution localization of hmC. We first alidatedv this A3A- based method on differentially-modified phage genomes ot demonstrate efficient conversion of C/mC and protection of hmC on a genome scale. We next applied ACE-Seq to mouse embryonic stem cell DNA and found a striking correlation with published hmC data from bisulfite-based methods. eW also sequenced sorted mouse glutamatergic neurons and, using >100-fold less, saw significant correlation with previously published TAB-Seq data from unsorted mouse cortex DNA. Finally, we demonstrate the applicability of nondestructive ACE-Seq to locus-specific analysis with longer (>1 kb) amplicons, which could permit clustering analyses on single reads from larger regions such as enhancers. Altogether, we propose ACE-Seq as a facile alternative to bisulfite-based hmC localization methods, especially for analyzing limiting DNA samples, such as those from small or transient cell populations. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor Rahul M. Kohli Keywords 5-hydroxymethylcytosine, ACE-Seq, AID/APOBEC, bisulfite-free, deamination, epigenetics Subject Categories Biochemistry | Genetics This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2794 DEFINING AND EXPLOITING THE CYTOSINE DEAMINASE ACTIVITY OF APOBEC3A ON THE EXTENDED EPIGENOME Emily K. Schutsky A DISSERTATION in Biochemistry and Molecular Biophysics Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2018 Supervisor of Dissertation Signature: ____________________________________ Rahul M. Kohli, M.D., Ph.D., Assistant Professor of Medicine Graduate Group Chairperson Signature: _____________________________________ Kim A. Sharp, Ph.D., Associate Professor of Biochemistry and Biophysics Dissertation Committee Ronen Marmorstein, Ph.D., George W. Raiziss Professor Gregory D. Van Duyne, Ph.D., Jacob Gershon-Cohen Professor Of Medical Science Matthew D. Weitzman, Ph.D., Associate Professor of Pathology and Laboratory Medicine Hongjun Song, Ph.D., Perelman Professor of Neuroscience To my Shepherd, in whom I lack nothing ii ACKNOWLEDGMENT In no way was this work a single-handed effort. Every day, I was physically, emotionally, intellectually, and spiritually supported by so many people who made this culmination possible. All of you believed in me especially in moments when I couldn’t. This is for you. To my labmates, past and present, who individually and uniquely filled each of my days with laughter, resilience, and hope for the next. To my advisor, mentor and friend – Rahul Kohli – who made me feel wholly appreciated, supported, and cared for during each high and low of my graduate career. To my thesis committee – Ronen Marmorstein, Gregory Van Duyne, and Matthew Weitzman – whose advice and suggestions gave me this amazing story to tell. To my collaborators in the Wu lab – Emily Fabyanic, Peng Hu, and Hao Wu – who were a constant source of help and support in making ACE-Seq possible. To the students and faculty in the BMB Graduate Group, and in the BGS graduate program, who created an amazing community of passionate intellectual pursuit. To the city of Philadelphia, who will always hold the dearest place in my heart as a home I didn’t expect, and as a source of brotherly love that has permeated my soul. To even better friends than I could ever have asked for, whose love for me has encouraged me to stand firm in each struggle, big and small. To Ace, the namesake of ACE-Seq and my favorite [canine] nephew, whose love is unconditional as only a dog’s can be. To my family, who has been unceasingly supportive since day one, and who will continue to love and support me no matter where life may lead me. To the God who never fails, and who loves me beyond measure. Thank you. iii ABSTRACT DEFINING AND EXPLOITING THE CYTOSINE DEAMINASE ACTIVITY OF APOBEC3A ON THE EXTENDED EPIGENOME Emily K. Schutsky Rahul M. Kohli, M.D., Ph.D. AID/APOBEC family cytosine deaminases canonically play crucial roles in immunity by converting cytosine to uracil in single-stranded DNA (ssDNA). Outside of this established physiological role, AID/APOBEC enzymes have also been implicated in the poorly-understood process of DNA demethylation through their proposed deamination of epigenetically-modified cytosine bases like 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC). However, there has been no thorough biochemical characterization of AID/APOBEC activity on these substrates, or on the recently-discovered 5-formylcytosine (fC) and 5-carboxylcytosine (caC) to inform this proposed role. Here, we provide the first steady-state kinetic measurements of the most active family member–APOBEC3A(A3A)–against various natural and unnatural modified substrates using a novel, restriction enzyme-based deamination assay. We determined that A3A has poor activity against bulkier oxidized cytosines, such as hmC, fC, and caC, and therefore likely does not contribute substantially to active DNA demethylation via deamination of these bases. By contrast, A3A efficiently deaminates mC in a manner not fully explained by the steric discrimination observed with other AID/APOBEC family members. Because A3A exhibits enzymatic proficiency for C and mC but potently discriminates against ox-mCs in vitro, we hypothesized that we could leverage A3A’s biochemical properties to efficiently localize ox-mCs in the genome. We developed APOBEC-Coupled Epigenetic Sequencing (ACE-Seq) – a nondestructive alternative to bisulfite-based methods for base resolution localization of hmC. We first validated this A3A-based method on differentially- iv modified phage genomes to demonstrate efficient conversion of C/mC and protection of hmC on a genome scale. We next applied ACE-Seq to mouse embryonic stem cell DNA and found a striking correlation with published hmC data from bisulfite-based methods. We also sequenced sorted mouse glutamatergic neurons and, using >100-fold less input DNA, saw significant correlation with previously published TAB-Seq data from unsorted mouse cortex DNA. Finally, we demonstrate the applicability of nondestructive ACE-Seq to locus-specific analysis with longer (>1 kb) amplicons, which could permit clustering analyses on single reads from larger regions such as enhancers. Altogether, we propose ACE-Seq as a facile alternative to bisulfite- based hmC localization methods, especially for analyzing limiting DNA samples, such as those from small or transient cell populations. v TABLE OF CONTENTS ACKNOWLEDGMENT .............................................................................................................. iii ABSTRACT ................................................................................................................................... iv LIST OF TABLES ...................................................................................................................... viii LIST OF
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