Farshad Hassanzadeh Niri
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Identification of CECR2-containing chromatin-remodeling complexes and their Chromatin-binding sites in mice by Farshad Hassanzadeh Niri A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular Biology and Genetics Department of Biological Sciences University of Alberta © Farshad Hassanzadeh Niri, 2017 Abstract Eukaryotic nuclear DNA is packaged into chromatin, a complex nucleoprotein structure. This has functional consequences by controlling the accessibility of DNA to binding factors responsible for many important cellular processes such as transcription, DNA replication and DNA repair. ATP-dependent chromatin remodeling complexes such as the ISWI family can regulate these cellular processes by altering the chromatin structure. CECR2 is a chromatin remodeling factor that forms a complex with ISWI proteins SNF2H and SNF2L. Loss-of- function mutations in Cecr2 result in the perinatal lethal neural tube defect, exencephaly. Nonpenetrant animals that survive to adulthood exhibit subfertility. CECR2 loss affects transcription of multiple genes and is also involved in γ-H2AX formation and DSB repair. The mutant phenotypes indicate that CECR2 plays an important role in neural tube development and reproduction, but the mechanism of its function is not known. I therefore have investigated the components of the CECR2 complex and its chromatin binding sites in ES cells and testis. I hypothesized that the CECR2 complexes contain tissue-specific components that may correspond to tissue-specific functions in ES cells and testis. I also hypothesized that the CECR2 containing complexes occupy different chromatin binding sites. This work first required the development of a highly specific CECR2-specific antibody. I confirmed that CECR2 forms a complex with SNF2H and SNF2L both in mouse ES cells and in testes. I showed that the CECR2-containing complex in mouse ES cells and testes has a size of approximately 2 MDa, suggesting the presence of additional components in the complex. Mass spectrometric analysis of CECR2-containing complexes revealed novel binding partners of CECR2 in ES cells and adult testis. I identified CCAR2 as a new member of the CECR2-containing complex in ES cells and possibly testis. CCAR2 has been shown to be ii involved in DNA damage response, which is also a known function of CECR2. Strikingly, there is a difference in the composition of complexes isolated from ES cells and adult testis. LUZP1 (leucine zipper protein 1) was confirmed to be a binding partner of CECR2 only in mouse ES cells and not in testes. Luzp1 mutant mice display exencephaly in 42% of embryos, indicating its role in neural tube closure. LUZP1 appears to play a role in stabilizing the CECR2 complex. I showed that the CECR2 complexes have different components, which opens the door toward understanding the multiple functions of CECR2. Disruption of Cecr2 results in dysregulation of expression of many genes in mouse embryos, however the direct transcriptional targets of the CECR2 complexes are unknown. Therefore, to find the direct binding sites of the CECR2 complex, chromatin immunoprecipitation followed by sequencing (ChIP-seq) analysis was performed in ES cells and adult testis. Looking at the overlapping binding sites of CECR2 and SNF2H allowed for a more powerful analysis and investigating the additional overlap with the binding sites of LUZP1 in ES cells allowed me to look for the ES cell-specific binding sites. Little overlap of CECR2 binding sites between ES cells and testes was observed, suggesting tissue-specific transcriptional regulation. Analysis of the ChIP-seq data revealed that the CECR2 complex occupies the promoter and cis-regulatory regions of many genes. The identified genes in ES cells are involved in different aspects of embryonic development including brain, heart and kidney development. The Cecr2 mutants exhibit abnormalities in these three organs. Gene ontology (GO) analyses of the genes associated with the binding sites of the CECR2 complex in ES cells showed that this complex modulates important molecular pathways including Shh and Wnt signaling. The genes identified in testes are involved in different aspects of reproduction and development. I identified many candidate iii genes that can be used to investigate the CECR2 function in neurulation and fertility. Hsd17b2, Lpar1, Nf, Lrp6 and Phactr4 are possibly directly regulated by the CECR2 complex, all of which cause exencephaly when mutated. Elmo1, Fgfr4, Ggt1, Insr, Itgb3 and Schip1 from the ES cell dataset and Cdc14b, Nfia, Pcsk1 and Styx from the testis dataset are candidate genes involved in reproduction. My findings revealed that there are ES and testis-specific CECR2 complexes in mice. I showed that these complexes have different compositions and chromatin binding sites, which will facilitate understanding the multiple functions of CECR2 during development and reproduction. iv Preface The research project involving mice, of which this thesis is a part, received research ethics approval from the Animal Care and Use Committee of the University of Alberta, University of Alberta AUP 00000094. Some of the research for this thesis has been completed in collaboration. Section 2.21.1: Aligning ChIP-seq reads and detecting peaks were performed in collaboration with Dr. Paul Stothard. Figure 3-12: Kacie Norton (graduate student) performed the IF staining and contributed the data to my thesis. Figures 4-1 & 4-2: Alaina Tripstra (graduate student) performed IF and contributed the results as the figures to my thesis. Section 3.10.1.3: Nhu Trieu (undergraduate student under my supervision) performed this part of the experiments and contributed the data to my thesis. Section 3.10.4.1 Kenji Rowel Lim (undergraduate student under my supervision) performed part of the experiments in this section and contributed the data to my thesis. Figure 3-39: Justin Elliott (undergraduate student under my supervision) performed part of the apoptosis experiment and contributed the figure to my thesis. v Acknowledgements First and foremost, I am deeply grateful to my supervisor, Dr. Heather McDermid, for her great supervision, patience and intellectual and financial support, which made the completion of this research possible. She provided a creative and open environment for us to work in. She is not only a remarkable scientist but also a great role model to us. I would also like to thank the rest of my thesis advisory committee, Dr. Kirst King- Jones and Dr. Roseline Godbout, for their guidance and help. I am also grateful to the present and former members of the McDermid lab. They are all my friends and I enjoyed working with them. Thank to Renee Leduc, Kacie Norton, Alaina Trepstra and Parmveer Singh for being such great colleagues. Thanks to all undergraduate students who worked with me in the lab during my PhD program. I also thank my friends Dr. Mohammad Shahrooie and Dr. Dhamo Neel for their helpful advice. I would also like to thank Ehsan Misaghi for helping me in computational analyses. Finally, I would like to dedicate this thesis to my family. They always support me with their endless love. vi Table of Contents 1. INTRODUCTION 1 1.1. CHROMATIN STRUCTURE 1 1.2. CHROMATIN STRUCTURE AND GENE REGULATION 2 1.3. CHROMATIN REMODELING 2 1.4. ATP-DEPENDENT CHROMATIN REMODELING 4 1.5. THE ISWI FAMILY OF NUCLEOSOME REMODELING COMPLEXES 5 1.5.1. THE MAMMALIAN ISWI COMPLEXES 6 1.5.2. FUNCTIONS OF ISWI COMPLEXES 9 1.6. ISWI TARGETING MECHANISM 15 1.6.1. PROTEIN DOMAINS AND TARGETING ISWI COMPLEXES TO CHROMATIN 15 1.6.2. TARGETING BY SEQUENCE SPECIFIC BINDING PROTEINS 22 1.6.3. TARGETING TO THE BINDING SITES BY RNA 22 1.7. DEVELOPMENTAL IMPORTANCE OF BAZ-LIKE PROTEINS 23 1.8. BIOLOGICAL AND FUNCTIONAL ROLES OF CECR2 25 1.8.1. CECR2 MUTATIONS 25 1.8.2. CECR2 MUTANT PHENOTYPES 27 1.8.3. CECR2 EXPRESSION PATTERN 28 1.9. CECR2-CONTAINING COMPLEXES 29 1.10. GENOME-WIDE ANALYSIS OF THE ATP-DEPENDENT CHROMATIN REMODELLERS 30 1.11. RATIONALE: 33 2. MATERIALS AND METHODS 35 2.1. MAINTAINING THE MOUSE COLONY 35 2.1.1. BREEDING 35 2.1.2. EUTHANASIA 35 2.2. GENOTYPING CECR2 MUTATIONS 36 2.2.1. GENOMIC DNA EXTRACTION 36 2.2.2. CECR2TM1.1HEMC GENOTYPING 36 2.2.3. CECR2GT45BIC GENOTYPING 37 2.2.4. AGAROSE GEL ELCTROPHORESIS 37 2.3. AFFINITY PURIFICATION OF CHICKEN POLYCLONAL ANTIBODIES TO MOUSE CECR2 38 2.4. PRODUCTION OF ANTIBODIES DIRECTED AGAINST MOUSE CECR2 IN RABBIT 40 2.4.1. TOTAL RNA EXTRACTION 40 2.4.2. CDNA SYNTHESIS 40 2.4.3. AMPLIFICATION OF HISTIDINE-TAGGED CECR2 FRAGMENTS 40 2.4.4. A-TAILING AND LIGATION 41 2.4.5. PREPARATION OF COMPETENT E. COLI (DH5 ALPHA) 41 2.4.6. CLONING THE LIGATED-CECR2-FRAGMENTS 41 2.4.7. SUBCLONING INTO THE EXPRESSION VECTOR 42 2.4.8. EXPRESSION AND PURIFICATION OF RECOMBINANT POLYPEPTIDE (B5P44) 42 2.4.9. IMMUNIZATION AND PRODUCTION OF POLYCLONAL ANTIBODIES 42 2.5. MOUSE EMBRYONIC STEM (ES) CELL CULTURE 45 2.6. PREPARATION OF NUCLEAR EXTRACTS FROM ES CELLS 46 2.7. PREPARATION OF NUCLEAR EXTRACTS FROM TISSUES 46 2.8. PREPARATION OF WHOLE CELL LYSATES FROM ES CELLS 47 vii 2.9. PREPARATION OF WHOLE CELL LYSATES FROM TISSUES 48 2.10. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) 48 2.11. COOMASSIE BLUE STAINING OF POLYACRYLAMIDE GELS 49 2.12. WESTERN BLOT ANALYSIS 49 2.13. IMMUNOPRECIPITATION 50 2.14. GEL FILTRATION 51 2.15. TCA PRECIPITATION 53 2.16. ETHIDIUM BROMIDE AND RNASE A TREATMENT OF PROTEIN EXTRACTS 53 2.17. MASS SPECTROMETRY 53 2.17.1. CROSSLINKING ANTIBODY TO DYNABEADS 53 2.17.2. LARGE SCALE IP USING ES CELLS 54 2.17.3. LARGE SCALE IP USING TESTIS 55 2.17.4. IN-GEL TRYPTIC DIGESTION 55 2.17.5. PURIFICATION OF PEPTIDES AFTER IN-GEL DIGESTION AND LC-MS/MS 56 2.17.6.