James Madison University JMU Scholarly Commons Masters Theses The Graduate School Spring 2018 Characterizing epigenetic regulation in the developing chicken retina Bejan Abbas Rasoul James Madison University Follow this and additional works at: https://commons.lib.jmu.edu/master201019 Part of the Computational Biology Commons, Developmental Biology Commons, Genomics Commons, and the Molecular Genetics Commons Recommended Citation Rasoul, Bejan Abbas, "Characterizing epigenetic regulation in the developing chicken retina" (2018). Masters Theses. 569. https://commons.lib.jmu.edu/master201019/569 This Thesis is brought to you for free and open access by the The Graduate School at JMU Scholarly Commons. It has been accepted for inclusion in Masters Theses by an authorized administrator of JMU Scholarly Commons. For more information, please contact [email protected]. Characterizing Epigenetic Regulation in the Developing Chicken Retina Bejan Abbas Rasoul A thesis submitted to the Graduate Faculty of JAMES MADISON UNIVERSITY In Partial Fulfillment of the Requirements for the degree of Master of Science Department of Biology May 2018 FACULTY COMMITTEE: Committee Chair: Dr. Raymond A. Enke Committee Members/ Readers: Dr. Steven G. Cresawn Dr. Kimberly H. Slekar DEDICATION For my father, who has always put the education of everyone above all else. ii ACKNOWLEDGMENTS I thank my advisor, Dr. Ray Enke, first, for accepting me as his graduate student and second, for provided significant support, advice and time to aid in the competition of my project and thesis. I would also like to thank my committee members not just for their role in this process but for being members of the JMU Biology Department ready to give any help they can. In addition, I would like to thank the JMU Biology Department for guiding me through the process of becoming a better student, teacher, researcher, and person. I would like to thank Dr. Winston Timp and Isac Lee at Johns Hopkins University Department of Biomedical Engineering for the help they provided with analyzing and visualizing the whole genome bisulfite sequencing data. The DMRs they provided were used in my intersect analysis. In addition, Isac was extremely helpful in producing the plots seen in Figures 5 and 6 and providing the methylation plots used in the top panels of Figures 7 and 12. I would also like to thank members of the Enke Lab including the undergraduate students who told me all they knew. I would like to thank Ashton Holub for smoothing my R learning process. I would also like to thank Jeremy Bravo for providing the histology used for this thesis and shown in Figure 9. Lastly, I would like to thank all my family for their support and encouragement. I would not be here today without my sister’s advice, my husband’s unconditional support, and the words from my father that inspired me to aim high. iii TABLE OF CONTENTS Dedication ........................................................................................................................... ii Acknowledgements ............................................................................................................ iii List of Tables .......................................................................................................................v List of Figures .................................................................................................................... vi Abstract ............................................................................................................................. vii Introduction ..........................................................................................................................1 I. The Vertebrate Eye ...............................................................................................1 a. The Retina ................................................................................................1 b. Photoreceptors..........................................................................................3 c. Phototransduction .....................................................................................3 d. Vertebrate Retinal Development ..............................................................5 II. Gene Regulation ..................................................................................................7 III. Types of Epigenetic Modifications ....................................................................7 a. Histone Modifications ..............................................................................7 b. DNA Methylation ....................................................................................8 IV. DNA Methylation and Disease in the Retina ..................................................11 V. Specific Purpose an Goals of the Study ............................................................13 V. The Chicken as a Model for Studying the Vertebrate Retina ...........................14 Methods..............................................................................................................................17 I. Computational Methods (Bioinformatics) ..........................................................17 a. RNA sequencing mRNA Transcriptome Analysis ................................17 b. Whole Genome Bisulfite Sequencing Analysis .....................................18 c. Intersection between DEGs and DMRs .................................................19 II. Experimental Methods ......................................................................................20 a. Embryos .................................................................................................20 b. Tissue Processing, Imaging, and Histology ...........................................20 c. Nucleic Acid Extraction .........................................................................21 d. DNA Methylation Analysis (Bisulfite Sequencing) ..............................22 Results ................................................................................................................................23 I. Development of the Chicken Retina ...................................................................23 II. Global DNA Methylation Analysis of the Developing Chicken Retina, Brain, and Cornea .............................................................................................................24 III. Differential Methylation of Photoreceptor-specific Genes ..............................30 IV. Using WGBS to detect novel DMRs around Candidate Genes .......................48 Discussion ..........................................................................................................................55 Appendix A ........................................................................................................................62 Appendix B ........................................................................................................................65 Appendix C ........................................................................................................................74 Appendix D ........................................................................................................................79 References ..........................................................................................................................83 iv LIST OF TABLES Table 1. Protein function and associated retinal diseases of candidate genes...................32 Table 2. Chicken (G. gallus) PCR and Bisulfite Sequencing primers ..............................34 Table 3. Gene Ontology Analysis of E8 retina v E18 retina DEGs with proximal DMRs ............................................................................................................................................53 Table A1. Comparison of up regulated and down regulated genes between samples ......62 Table A2. Comparison of hypermethylated and hypomethylated regions between samples ............................................................................................................................................62 Table B1. Bisulfite-PCR and sequencing primers ............................................................65 Table B2: Sample key for DNA used in Bisulfite PCR ....................................................67 Table D1. Kruskal-Wallis Rank Sum Test for differences between E8 retina and E18 retina stages ........................................................................................................................79 Table D2. Kruskal-Wallis Rank Sum Test for differences between E18 retina and E18 cornea tissues .....................................................................................................................79 Table D3. Kruskal-Wallis Rank Sum Test for differences in average methylation levels of E6-E20 developmental stages ........................................................................................80 Table D4. Pairwise comparison done with the Wilcoxon Rank Sum Test on average methylation values in E6-E20 developmental stages .........................................................80 Table D5. Kruskal-Wallis Rank Sum Test for differences between E18 retina, E18 cornea, and E18 brain tissues .............................................................................................82 Table D6. Pairwise comparison done with the Wilcoxon Rank Sum Test on average methylation values in E18 retina, cornea, and brain tissues ..............................................82