HOMOCYSTEINE-METHIONINE CYCLE IS A KEY METABOLIC SENSOR SYSTEM CONTROLLING METHYLATION-REGULATED PATHOLOGICAL SIGNALING - CD40 IS A PROTOTYPIC HOMOCYSTEINE-METHIONINE CYCLE REGULATED MASTER GENE A Thesis Submitted to the Temple University Graduate Board In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE by Chao Gao August 2019 Thesis Approvals: Dr. Hong Wang, Thesis Advisor, Center for Metabolic Disease Research Dr. Xiao-Feng Yang, co-Advisor, Centers for Inflammation, Translational & Clinical Lung Research, Metabolic Disease Research, and Cardiovascular Research Dr. Jun Yu, Center for Metabolic Disease Research Dr. Stefania Gallucci, Department of Microbiology and Immunology © Copyright 2019 by Chao Gao All Rights Reserved ii ABSTRACT Homocysteine-Methionine cycle is a key metabolic sensor system controlling methylation-regulated pathological signaling - CD40 is a prototypic homocysteine- methionine cycle regulated master Chao Gao Master of Science Temple University, 2019 Thesis Advisory Committee Chair: Hong Wang, MD, PhD Homocysteine-Methionine (HM) cycle produces a universal methyl group donor S-adenosylmethionine (SAM), a competitive methylation inhibitor S- adenosylhomocysteine (SAH), and an intermediate amino acid product homocysteine (Hcy). Elevated plasma levels of Hcy is termed as hyperhomocycteinemia (HHcy) which is an established risk factor for cardiovascular disease (CVD) and neural degenerative disease. We were the first to describe methylation inhibition as a mediating biochemical mechanism for endothelial injury and inflammatory monocyte differentiation in HHcy- related CVD and diabetes. We proposed metabolism-associated danger signal (MADS) recognition as a novel mechanism for metabolic risk factor-induced inflammatory responses, independent from pattern recognition receptor (PRR)-mediated pathogen- associated molecular pattern (PAMP)/danger-associated molecular pattern (DAMP) recognition. In this study, we examined the relationship of HM cycle gene expression with methylation regulation in human disease. We selected 115 genes in the extended HM iii cycle, including 31 metabolic enzymes and 84 methyltransferases (MT), examined their mRNA levels in 35 human disease conditions using a set of public databases. We discovered that: 1) HM cycle senses metabolic risk factor and controls SAM/SAH- dependent methylation. 2) Most of metabolic enzymes in HM cycle (8/11) are located in cytosol, while most of the SAM-dependent MTs (61/84) are located in the nucleus, and Hcy metabolism is absent in the nucleus. 3) 11 up-regulated, 3 down-regulated and 24 differentially regulated SAM/SAH-responsive signal pathways are involved in 7 human disease categories. 4) 8 SAM/SAH-responsive H3/H4 hypomethylation sites are identified in 8 disease conditions. We conclude that HM cycle is a key metabolic sensor system which mediates receptor-independent MADS recognition and modulates SAM/SAH-dependent methylation in human disease. We propose that HM metabolism takes place in cytosol and that nuclear methylation equilibration requires nuclear-cytosol transfer of SAM, SAH and Hcy. CD40 is a cell surface molecule which is expressed on antigen presenting cells such as monocyte, macrophage, dendritic cells and neutrophils. The costimulatory pair, CD40 and CD40L, enhances T cell activation and induce chronic inflammatory disease. Also, DNA hypomethylation on CD40 promotor induces inflammatory monocyte differentiation in chronic kidney disease. In order to figure out if CD40 is a prototypic HM cycle regulated master gene, RNA-seq analysis were performed for CD40+ and CD40- monocytes from mouse peripheral blood and 1,093 differentially expressed genes (DEGs) were selected from those two groups. All the DEGs modulate as much as 15 functional gene groups such as cytokines, enzymes and transcriptional factors. iv Furthermore, CD40+ monocytes activated trained immunity pathways especially in Acetyl-CoA generation and mevalonate pathway. In HM cycle, CD40 is a prototypic HM cycle regulated master gene to induce the most of the Hcy metabolic enzymes as well as MT, which can further modulate the methylation-regulated pathological signaling. The studies in this thesis were funded by the National Institutes of Health (NIH) grant to Dr. Hong Wang. v DEDICATION To my dearest family vi ACKNOWLEDGMENTS I would primarily like to thank my thesis advisor Dr. Hong Wang at Temple University Lewis Katz School of Medicine. As a great and dedicative scientist who devoted her life to the research work, she always encouraged me to find out the field I am really interested in and gave me all the freedom to pursue my research. Dr. Wang continuously supported my research and study with her patience, motivation, enthusiasm and immense knowledge. Her guidance helped me in all the time of research and writing of this thesis. Without her great support and encouragement, I cannot go through all the difficulties which I have met during my past two years and finally finish the project and have this thesis. Also, she was always very supportive so I could have opportunities to attend the different international conferences and present my work during past two years. Secondly, I would like to acknowledge Dr. Xiao-Feng Yang, who is my co- advisor and regularly provided scientific suggestions and insightful comments on my project. His hard questions incented me to widen my research from various perspectives. He always supported and motivated us to learn new techniques and build collaborations with other colleagues. Dr. Yang’s credible ideas have been great contributors in the completion of the thesis. Thirdly, I am very grateful to my committee members, Dr. Jun Yu and Dr. Stefania Gallucci, for their encouragement and positive critiques throughout my committee meetings. Without their passionate participation and input, the project could not have been successfully conducted and finished. I would also like to thank our collaborator Dr. Yan Zhou at Temple Health-Fox Chase Cancer Center for RNA-Seq analysis. vii As an international student who first came to the United States after college, I am so delighted to be accepted by the Biomedical Sciences program at Temple University Lewis Katz School of Medicine. It is really a happy time working with all co-workers and staffs in our school and in Dr. Wang and Dr. Yang’s labs. This lab is a sweet home and all our lab members treated me as their family member. I would like to especially thank Xiaohua Jiang for her consistent efforts on lab management. I would like to thank Dr. Pu Fang and Humin Shan for teaching me flow cytometry and single cell suspension. I would like to thank Dr. Hangfei Fu, Ying shao and Remon Cueto for sharing me the strategies on database mining and RNA-seq data analysis. I would also like to thank Jason Saredy for providing great suggestion on scientific writing. Last but not least, I would like to thank my lovely family. The reason why I have the courage to go abroad and pursue further study is their huge encouragement. They always told me to have a broad view and experience during my life and never give up my dream. Without their support, I could not have achieved such an accomplishment. Thanks all the people who have helped me during my study! viii TABLE OF CONTENTS PAGE ABSTRACT ................................................................................................................. iii DEDICATION ............................................................................................................. vi ACKNOWLEDGMENTS .......................................................................................... vii LIST OF FIGURES ..................................................................................................... xi LIST OF TABLES ...................................................................................................... xii LIST OF ABBREVIATIONS .................................................................................... xiii CHAPTER 1. INTRODUCTION .....................................................................................................1 1.1 Overview of Homocysteine and Hyperhomocysteinemia .................................1 1.2 Homocysteine-Methionine (HM) cycle .............................................................1 1.3 Methylation ........................................................................................................2 1.3.1 DNA methylation ......................................................................................4 1.3.2 Protein methylation ...................................................................................4 1.3.3 RNA methylation ......................................................................................5 1.4 Metabolism-associated danger signal (MADS) recognition ..............................6 1.5 CD40 and CD40L ..............................................................................................6 1.6 Hypothesis of Thesis ..........................................................................................7 2. MATERIALS AND METHODS ...............................................................................9 2.1 Selection of extended Homocysteine-Methionine (HM) cycle genes ...............9 2.2 Subcellular localization of extended HM cycle enzymes ..................................9 2.3 Expression profiles of 115 genes in extended HM cycle in 35 pathological ix conditions ..........................................................................................................9