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FUNCTIONAL CHARACTERIZATION of SCHIZOPHRENIA-ASSOCIATED VARIATION in CACNA1C by Nicole Eckart a Dissertation Submitted to Johns

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FUNCTIONAL CHARACTERIZATION of SCHIZOPHRENIA-ASSOCIATED VARIATION in CACNA1C by Nicole Eckart a Dissertation Submitted to Johns FUNCTIONAL CHARACTERIZATION OF SCHIZOPHRENIA-ASSOCIATED VARIATION IN CACNA1C by Nicole Eckart A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy in Human Genetics Baltimore, Maryland March, 2016 Abstract Background Schizophrenia is a complex psychiatric disorder with heritability estimated to be around 80%. Genetic studies have identified over 100 schizophrenia risk loci. These risk loci most often lie in non-coding sequences and are enriched for expression quantitative trait loci (eQTLs), suggesting that dysregulation of transcriptional control plays a role in complex disease pathogenesis. Of particular interest are non-coding variants in calcium channel subunits, which have been associated with multiple psychiatric disorders. Specifically, genome wide association studies (GWAS) have repeatedly identified the single nucleotide polymorphism (SNP) rs1006737 in the third intron CACNA1C to be strongly associated with schizophrenia. Methods We genotyped schizophrenia-associated variants and measured gene expression by qPCR in human post mortem brain samples. We looked for statistically significant genotype-expression correlations to identify eQTLs. We further investigated the putative eQTLs with dual luciferase reporter assays by transfecting reporter constructs into relevant cell lines for all variants in high linkage disequilibrium (LD) with the schizophrenia-associated variant. Furthermore, we investigated allele-specific protein binding of variant sequences in the putative eQTL through electrophoretic mobility shift assays (EMSAs) with nuclear extract from two cell lines is incubated with radiolabeled DNA probes, and a larger molecular weight band is produced if the probe binds to a protein or protein complex from the nuclear extract. With protein microarrays, proteins are immobilized on a glass ii slide and fluorescently labeled DNA probes are hybridized to the microarray, producing a signal when they bind a protein, we identified specific DNA-protein interactions. Lastly, we identified potential regulatory elements that interact with the promoters of our genes of interest through circular chromatin conformation capture with next generation sequencing (4C-seq). In this assay, cells were cross-linked to capture protein- mediated DNA-DNA interactions. After digestion, interacting DNA fragments were ligated together to form small hybrid circular molecules. Then, for a known viewpoint of interest, the unknown interacting fragments were identified through next generation sequencing. Results and Conclusions Here, we showed that rs1006737 marks an eQTL for CACNA1C transcript levels in human post mortem brain tissue. We tested 16 SNPs in high LD with rs1007637 and found that one, rs4765905, consistently showed allele-dependent regulatory function in reporter assays. We found allele-specific protein binding for 13 SNPs including rs4765905 and using protein microarrays, we identified several proteins binding more than three SNPs, but not control sequences, suggesting possible functional interactions and combinatorial haplotype effects. Finally, using 4C-seq, we showed interaction of the CACNA1C promoter with the eQTL and other potential regulatory regions. Our results elucidate the pathogenic relevance of one of the best-supported risk loci for schizophrenia. Advisor: Dimitrios Avramopoulos Reader: Andrew S. McCallion iii Acknowledgements First, I’d like to thank my thesis advisor, Dimitri Avramopoulos, for his guidance and support. I have learned so much about genetics and genomics and have become a better scientist from working with you. I’d also like to thank the members of my thesis committee, Andy McCallion, Dave Valle, Kathy Burns, and Ann Pulver, for your feedback and many suggestions as this research project progressed. This work wouldn’t have been possible without the support of my lab mates, past and present. Ruihua Wang extracted much of the DNA and RNA needed for the eQTL study and always was a valuable resource for reagents, protocols, and encouragement. Megan Szymanski Pierce also extracted DNA and RNA for the eQTL study and helped me settle into the lab before she graduated. Rebecca Yang, a summer student, performed some of the protein microarrays. Thank you to Mariela Zeledon for teaching me the dual luciferase assay and EMSA protocols, Cassandra Obie for teaching me everything I need to know about cell culture, and Gary Steele for helping me troubleshoot PCR and cloning experiments. I would also like to thank Xuan Pham for sharing a bay with me and making my time in lab so enjoyable. I will miss all of our conversations about science and life! And thank you to all of the many other members of the Avramopoulos, Valle, and Vernon labs for your constant support and intellectual contributions. Our collaborators in Heng Zhu’s lab enabled us to conduct the protein microarray study. Thank you especially to Qifeng Song, who manufactured the microarrays and taught me the protocol, and to Cedric Moore, who helped me with the analysis. Thank you, also, to our collaborators in Andy McCallion’s lab. Maggie Baker provided lots of support and advice as we optimized the 4C-seq protocol together. Sarah McClymont and iv Xylena Reed helped me with next-gen sequencing. Xylena and Dave Gorkin also offered their insight on the experimental design of the dual luciferase assays. Thank you to the Human Genetics program for giving me this incredible opportunity. Dave Valle, Kirby Smith, Andy McCallion, and Sandy Muscelli do so much to run the program and support the students’ education. Thank you to the many faculty and staff who have been so influential by sharing their personal stories and offering their insights on how to become a successful scientist. I would also like to thank all of my classmates for always being there to encourage each other. You have challenged me to become a better student, presenter, and scientist. Thank you to all of the Human Genetics students for being mentors, role models, and friends. And a special thanks to Foram Ashar, Shannon Ellis, and Courtney Woods for not only being wonderful colleagues, but also the greatest of friends. Thank you to my amazing family and friends for supporting me throughout this entire process. I am grateful for your encouragement and proud to share these accomplishments with you. To my in-laws, who love me as one of their own, I’m so happy to be a part of your family. Thank you to my parents for all of your love and support, for the many opportunities you gave me, and for teaching me the virtues of hard work, integrity, and compassion. And finally, I am thankful for my husband, Tim, and his unwavering support. Thank you for all of the sacrifices you’ve made, your patience, and your sense of humor. Without all of you, none of this would have been possible. v Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iv Table of Contents ............................................................................................................... vi List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii Chapter 1: Introduction ....................................................................................................... 1 Chapter 2: Schizophrenia Risk Variants as Potential eQTLs ............................................. 7 2.1 Introduction ............................................................................................................... 7 2.2 Methods................................................................................................................... 13 2.3 Results ..................................................................................................................... 16 2.4 Discussion ............................................................................................................... 18 2.5 Chapter 2 Tables ..................................................................................................... 21 2.6 Chapter 2 Figures .................................................................................................... 24 Chapter 3: Enhancer Reporter Assays .............................................................................. 29 3.1 Introduction ............................................................................................................. 29 3.2 Methods................................................................................................................... 31 3.3 Results ..................................................................................................................... 36 3.4 Discussion ............................................................................................................... 37 3.5 Chapter 3 Tables ..................................................................................................... 40 3.6 Chapter 3 Figures .................................................................................................... 41 Chapter 4: Protein Binding Assays ..................................................................................
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