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Identification and Characterization of Hells-Cdca7, a Nucleosome

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Identification and Characterization of Hells-Cdca7, a Nucleosome Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2018 Identification and Characterization of Hells- CDCA7, a Nucleosome Remodeling Complex Required for Mitotic Chromosome Structure Christopher Jenness Follow this and additional works at: https://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons IDENTIFICATION AND CHARACTERIZATION OF HELLS-CDCA7, A NUCLEOSOME REMODELING COMPLEX REQUIRED FOR MITOTIC CHROMOSOME STRUCTURE A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Christopher Jenness June 2018 ã Copyright by Christopher Jenness 2018 IDENTIFICATION AND CHARACTERIZATION OF HELLS-CDCA7, A NUCLEOSOME REMODELING COMPLEX REQUIRED FOR MITOTIC CHROMOSOME STRUCTURE Christopher Jenness, Ph.D. The Rockefeller University 2018 The major components of mitotic chromosomes including histones, topoisomerase II and condensin are known to compact and shape the chromatin into rod-like chromatids. However, a complete picture of the proteins involved in shaping mitotic chromatin is unsettled. Here, I perform mass spectrometry on chromatin isolated from Xenopus egg extracts. By comparing interphase and M phase, I reveal how the chromatin proteome is affected by the cell cycle. I find that although topoisomerase II associates with chromatin throughout the cell cycle, its catalytic activity is greatly enhanced in mitosis where it can act on nucleosomal substrates. In contrast, condensin is specifically recruited to M phase chromatin and prefers a non-nucleosomal substrate. I show that nearly all proteins involved in nucleosome assembly and remodeling are evicted from mitotic chromosomes, concurrent with a reduction in mitotic nucleosome assembly and discuss a role for these phenomena in shaping mitotic chromosomes. In analyzing my mass spectrometry data, I noticed that subunits of stoichiometric chromatin-bound complexes behaved similarly across a variety of conditions. Using this principle, I identify a novel nucleosome remodeling complex comprising HELLS and CDCA7, two proteins known to be causative for Immunodeficiency, centromeric region instability, facial anomalies (ICF) syndrome, a rare immunodeficiency disease. Consistent with previous literature, HELLS alone fails to remodel nucleosomes, but the HELLS-CDCA7 complex possesses robust nucleosome remodeling activity. CDCA7 is essential for loading HELLS onto chromatin, and CDCA7 harboring patient ICF mutations fails to recruit the complex to chromatin. Finally, I show that the HELLS- CDCA7 complex is required for proper mitotic chromosome structure. Together, my study identifies a unique bipartite nucleosome remodeling complex where the functional remodeling activity is split between two proteins. These data lead me to a unifying model for ICF syndrome where all known ICF mutations converge on defective DNA methylation. I propose that HELLS-CDCA7 mediated remodeling of juxtacentromere heterochromatic nucleosomes facilitates DNMT3B-mediated methylation and discuss this model alongside alternative models for ICF syndrome. Finally, I consider the role of CDCA7 paralogs in differentially regulating HELLS, and present an outlook on the future of ICF syndrome research. ACKNOWLEDGEMENTS I would like to thank members of the Funabiki laboratory for their help during my time at The Rockefeller University. Christian Zierhut, Dave Wynne, Pavan Choppakatla, Michael Wheelock, Simona Giunta, Hironori Funabiki, David Allis, and Brian Chait provided valuable discussions, which helped the direction of the project. Additionally, I am grateful for Tom Muir accommodating me into his lab in September of 2014 where I learned a very different perspective on science under the mentorship of Manuel Muller. Many people provided reagents and technical advice including Geneviève Almouzni, Shawn Amed, Yoshiaki Azuma, Noah Dephoure, Joseph Fernandez, Rebecca Heald, Tatsuya Hirano, Nathan Gamarra, Hiroshi Kimura, Lance Langston, Geeta Narlikar, Atsuya Nishimaya, Miachel O’Donnell, Dirk Remus, Johannes Walter, Martin Whur, and Henry Zebroski. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS iii TABLE OF CONTENTS iv LIST OF FIGURES ix LIST OF TABLES xiii CHAPTER 1: INTRODUCTION 1 The cell cycle 1 The cell cycle 1 Mitosis 1 Chromatin throughout the cell cycle 3 Chromatin 5 The nucleosome 5 Forming a mitotic chromosome: Histones 6 Forming a mitotic chromosome: Non-histone proteins 8 Nucleosome remodeling complexes 13 Nucleosome remodeling 13 Nucleosome remodeling proteins 16 Regulation by accessory subunits 19 Regulation by H1 21 Mitotic regulation and function of nucleosome remodeling 22 complexes iv ICF syndrome 23 ICF patients 23 Genetic causes of ICF 25 Molecular causes of ICF 27 HELLS and CDCA7 31 HELLS 31 CDCA7 33 Open questions and significance 36 Cell cycle regulation of chromatin composition 36 Molecular basis of ICF syndrome 37 CHAPTER 2: CELL CYLE REGULATION OF CHROMATIN 38 COMPOSITION Results 38 An assay to systematically determine the cell cycle and 38 nucleosome regulation of chromatin composition Major and minor determinants of chromosome composition 42 Nucleosomal and cell cycle regulation of chromatin 47 Nucleosomal and cell cycle regulation of mitotic chromosome 47 structural proteins Cell cycle regulation of nucleosome remodeling and assembly 55 complexes Cell cycle regulation of nucleosome assembly 65 Discussion and perspective 77 The global landscape of cell cycle regulated chromatin proteins 77 v Mitotic eviction of nucleosome assembly and remodeling proteins 80 CHAPTER 3: HELLS-CDCA7, A BIPARTITIE NUCLEOSOME 84 REMODELING COMPLEX Results 84 Co-regulation of chromatin complexes 84 HELLS-CDCA7 are co-regulated on chromatin 91 HELLS-CDCA7 require nucleosomes for chromatin association 96 HELLS-CDCA7e form a stoichiometric complex on chromatin 100 HELLS-CDCA7e is a nucleosome remodeling complex 105 HELLS-CDCA7e is required for mitotic chromatin structure 110 ICF patient mutations reveal functions of CDCA7 117 Discussion and perspective 124 A streamlined comparative proteomics method for chromatin- 124 associated protein complex identification The role of HELLS-CDCA7 in shaping mitotic chromosomes 126 Nucleosome dependent DNA binding proteins 131 CHAPTER 4: DISCUSSION AND PERSPECTIVE 133 A unifying model for ICF syndrome 133 An alternative model for ICF syndrome 137 HELLS regulation by CDCA7, CDCA7L and CDCA7e 141 The similarities and differences between DDM1 and HELLS- 142 CDCA7 Outlook: Open Questions in ICF syndrome 149 vi CHAPTER 5: MATERIALS AND METHODS 152 Biochemistry 152 Cloning 152 Protein purification 152 Nucleosome array and mononucleosome purification 155 Antibodies 156 Immunoprecipitations 160 Chromatin interactions 160 ATPase assays 161 Nucleosome remodeling assays 162 Western blotting and immunofluorescence 163 Protein alignment 164 Xenopus egg extracts 165 Extract preparations and deletions 165 Analysis of chromatin associated proteins 166 Mass spectrometry 166 Immunoprecipitation 167 Nucleosome assembly on plasmids 168 Chromosome and spindle assembly 169 DNA methylation assays 170 Kinetoplast DNA decatenation assay 171 CHAPTER 6: APPENDIX 173 vii REFERENCES 199 viii LIST OF FIGURES Figure Title Page Figure 1-1 Factors influencing the compaction and shaping of 9 mitotic chromosomes Figure 1-2 Nucleosome remodeling activities. 15 Figure 1-3 Phylogeny of Snf2-like nucleosome remodeling proteins 18 Figure 1-4 Chromosome defects in ICF syndrome. 24 Figure 1-5 Mutational landscape of ICF syndrome 26 Figure 1-6 The understanding of the proteins involved in ICF 30 syndrome at the onset of this thesis Figure 1-7 HELLS possess a conserved ATPase domain, but lacks 32 chromatin binding domains common to nucleosome remodeling complexes Figure 1-8 The zinc finger domain of CDCA7 is conserved between 35 species and paralogs Figure 2-1 Schematic of mass spectrometry experiments to 40 determine the regulation of chromatin associated proteins Figure 2-2 Nucleosomes and the cell cycle are the major 44 determinants of chromosome composition Figure 2-3 Proteins with DNA binding activity require nucleosomes 49 to associate with physiological chromatin Figure 2-4 Summary of the cell cycle regulation of chromatin 53 associated proteins Figure 2-5 Differential regulation of condensin and topoisomerase 57 II Figure 2-6 Topoisomerase II decatenation activity is stimulated in 61 mitosis and can decatenate a nucleosomal substrate Figure 2-7 Condensin and topoisomerase II are largely unaffected 64 by the underlying chromatin ix Figure 2-8 Nucleosome remodeling and assembly proteins are 66 evicted from mitotic chromatin Figure 2-9 Nucleosome assembly is suppressed in mitosis 68 independent of the CPC Figure 2-10 Plx1 mediated eviction of HIRA from DNA beads in 70 mitosis Figure 2-11 Plx1 mediated eviction of HIRA from sperm chromatin in 72 mitosis Figure 2-12 HIRA harbors a Polo box binding motif and displays a 74 phospho-dependent gel shift in mitosis Figure 2-13 Plx1 mediated suppression of nucleosome assembly in 76 mitosis Figure 3-1 Subunits of 1:1 stoichiometric protein complexes are co- 85 regulated on chromatin Figure 3-2 Subunits of non-1:1 stoichiometric protein complexes 87 are co-regulated on chromatin Figure 3-3 Hierarchical clustering of mass spectrometry data 89 identifies known protein complexes Figure 3-4 HELLS and CDCA7e are co-regulated on chromatin 92 Figure 3-5 HELLS-CDCA7e is evicted from mitotic chromatin by 94 the CPC Figure 3-6 Histone
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