University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2008-05-09 Epigenetic Determinants of Altered Gene Expression in Schizophrenia: a Dissertation Hsien-Sung Huang University of Massachusetts Medical School Follow this and additional works at:https://escholarship.umassmed.edu/gsbs_diss Part of theEnzymes and Coenzymes Commons,Genetic Phenomena Commons,Mental Disorders Commons,Nervous System Commons, and theTherapeutics Commons Repository Citation Huang, H. Epigenetic Determinants of Altered Gene Expression in Schizophrenia: a Dissertation. (2008). University of Massachusetts Medical School.GSBS Dissertations and Theses.Paper 365. DOI: 10.13028/h312-hs12. https://escholarship.umassmed.edu/ gsbs_diss/365 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. 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EPIGENETIC DETERMINANTS OF ALTERED GENE EXPRESSION IN SCHIZOPHRENIA A Dissertation Presented By Hsien-Sung Huang Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 9, 2008 Neuroscience EPIGENETIC DETERMINANTS OF ALTERED GENE EXPRESSION IN SCHIZOPHRENIA A Dissertation Presented By Hsien-Sung Huang Approved as to style and content by: Paul Gardner, Ph.D., Chair of Committee David Weaver, Ph.D., Member of Committee Job Dekker, Ph.D., Member of Committee Steven Grossman, M.D., Ph.D., Member of Committee Sabina Berretta, M.D., Member of Committee Schahram Akbarian, M.D., Ph.D., Thesis Advisor Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences Program in Neuroscience May 9, 2008 iii Dedicated to my parents Bi-Long Huang and Hsiu-Sa Huang Kao iv ACKNOWLEDGEMENTS Firstly, I would like to thank my mentor, Schahram Akbarian, for giving me this opportunity to work in his lab. He taught me a lot and I really learned a lot from him. He also recruited talented people and good fundings into his lab. This provides us excellent environment for doing research. I have to say that I’m really evolved during my years in Schahram lab. In other words, there are changes of histone methylation at good gene promoters in my prefrontal cortex. Secondly, I want to thank my chairman, Paul Gardner, for his help and support. Therefore, from my qualifier, each TRAC meeting to recent thesis defense can move so smoothly and on schedule. His humorous and witty character brought a lot of joys and laughters to my life. I would also like to thank David Weaver for his help and guidance when I started in UMMS. He is a very careful and critical scientist. That’s what a scientist should be. I want to thank Job Dekker for his positive inputs, suggestions and discussions in my each TRAC meeting. He always looked at my project in different angles and those help me a lot. I would also like to thank Steven Grossman for donating his time and talent to serve as a member of my committee. He always brought up the clinical views to my projects. I also thank Sabina Berretta for serving as an external member for my DEC. Thirdly, I would like to thank the technical support from Anouch Matevossian, Catheryne Whittle and Yin Guo. They are very professional and responsible. Without their helps, I think my project will definitely slow down. Finally, I want to thank fellow students in Schahram lab including Frederick (Al) Schroeder, Yan Jiang, Nikolaos Mellios, Caroline Connor and Rahul Bharadwaj for working and cooperating together. I especially want to thank Al for giving me non-scientific advice and Yan for providing me scientific advice. Those advices really help me a lot at certain critical time points of my life. I would also like to thank people who helped and guided me during my time in UMMS. v ABSTRACT Schizophrenia is a neurodevelopmental disorder affecting 1% of the general population. Dysfunction of the prefrontal cortex (PFC) is associated with the etiology of schizophrenia. Moreover, a substantial deficit of GAD1 mRNA in schizophrenic PFC has been reported by different groups. However, the underlying molecular mechanisms are still unclear. Interestingly, epigenetic factors such as histone modifications and DNA methylation could be involved in the pathogenesis of schizophrenia during the maturation of the PFC. In my work, I identified potential epigenetic changes in schizophrenic PFC and developmental changes of epigenetic marks in normal human PFC. Furthermore, mouse and neuronal precursor cell models were used to confirm and investigate the molecular mechanisms of the epigenetic changes in human PFC. My initial work examined whether chromatin immunoprecipitation can be applied to human postmortem brain. I used micrococcal nuclease (MNase)-digested chromatin instead of cross-linked and sonicated chromatin for further immunoprecipitation with specific anti-methyl histone antibodies. Surprisingly, the integrity of mono-nucleosomes was still maintained at least 30 vi hrs after death. Moreover, differences of histone methylation at different genomic loci were detectable and were preserved within a wide range of autolysis times and tissue pH values. Interestingly, MNase-treated chromatin is more efficient for subsequent immunoprecipitation than crosslinked and sonicated chromatin. During the second part of my dissertation work, I profiled histone methylation at GABAergic gene loci during human prefrontal development. Moreover, a microarray analysis was used to screen which histone methyltransferase (HMT) could be involved in histone methylation during human prefrontal development. Mixed-lineage leukemia 1 (MLL1), an HMT for methylation at histone H3 lysine 4 (H3K4), appears to be the best candidate after interpreting microarray results. Indeed, decreased methylation of histone H3 lysine 4 at a subset of GABAergic gene loci occurred in Mll1 mutant mice. Interestingly, clozapine, but not haloperidol, increased levels of trimethyl H3K4 (H3K4me3) and Mll1 occupancy at the Gad1 promoter. I profiled histone methylation and gene expression for GAD1 in schizophrenics and their matched controls. Interestingly, there are deficits of GAD1 mRNA levels and GAD1 H3K4me3 in female schizophrenics. Furthermore, I was also interested in whether the changes of GAD1 chromatin structure could vii contribute to cortical pathology in schizophrenics with GAD1 SNPs. Remarkably, homozygous risk alleles for schizophrenia at the 5’ end of the GAD1 gene are associated with a deficit of GAD1 mRNA levels together with decreased GAD1 H3K4me3 and increased GAD1 H3K27me3 in schizophrenics. Finally, I shifted focus on whether DNA methylation at the GAD1 promoter could contribute to a deficit of GAD1 mRNA in schizophrenia. However, no reproducible techniques are available for extracting genomic DNA specifically from GABAergic neurons in human brain. Therefore, I used an alternative approach that is based on immunoprecipitation of mononucleosomes with anti-methyl-histone antibodies differentiating between sites of active and silenced gene expression. The methylation frequencies of CpG dinucleotides at the GAD1 proximal promoter and intron 2 were determined from two chromatin fractions (H3K4me3 and H3K27me3) separately. Consistently, the proximal promoter region of GAD1 is more resistant to methylation in comparison to intron 2 of GAD1 in either open or repressive chromatin fractions. Interestingly, overall higher levels of DNA methylation were seen in repressive chromatin than in open chromatin. Surprisingly, schizophrenic subjects showed a significant decrease of DNA viii methylation at the GAD1 proximal promoter from repressive chromatin. Taken together, my work has advanced our knowledge of epigenetic mechanisms in human prefrontal development and the potential link to the etiology of schizophrenia. It could eventually provide a new approach for the treatment of schizophrenia, especially in the regulation of methylation at histone H3 lysine 4. ix TABLE OF CONTENTS TITLE PAGE i SIGNATURE PAGE ii DEDICATION iii ACKNOWLEDGEMENTS iv ABSTRACT v TABLE OF CONTENTS ix LIST OF TABLES xiv LIST OF FIGURES xv ABBREVIATIONS xvi COPYRIGHT NOTICE xviii CHAPTER I: INTRODUCTION Schizophrenia 1 Prefrontal cortex 7 GABAergic neurons 9