QUANTITATIVE STUDY ON REGULATORY MECHANISMS OF CELL PHENOTYPE TRANSITION by Jingyu Zhang B.S., Shandong University, 2006 M.S., Ocean University of China, 2010 M.S., Virginia Polytechnology and State University, 2013 Submitted to the Graduate Faculty of School of Medicine in partial fulfillment of the requirements for the degree of University of Pittsburgh 2018 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented By Jingyu Zhang It was defended on June 6, 2018 and approved by Ipsita Banerjee, PhD, Associate Professor, Department of Bioengineering Robin E.C. Lee, PhD, Assistant Professor, Department of Computational and Systems Biology Jian Ma, PhD, Associate Professor, CMU Department of Computational Biology Dissertation Director: Jianhua Xing, PhD, Associate Professor, Department of Computational and Systems Biology ii Copyright © by Jingyu Zhang 2018 iii QUANTITATIVE STUDY ON REGULATORY MECHANISMS OF CELL PHENOTYPE TRANSITION Jingyu Zhang, PhD University of Pittsburgh, 2018 Cells in a multicellular organism share the same set of genome and can assume multiple phenotypes. Uncovering mechanisms of regulating cell phenotype changes has become an important and active research area. This dissertation presents a collection of my combined computational and experimental efforts on studying cell phenotype transitions. Chapter I gives a literature overview on cell phenotype conversion and regulation. One can identify four generic modules that function coordinately to regulate cell phenotypes. The whole system forms a highly interconnected network and involves a large number of molecular species for epigenetic, transcriptional and translational regulations. Chapter II addresses how a cell interprets temporal and strength information of signals and makes cell fate decision. I performed an integrated quantitative and computational analysis on how extracellular TGF-β signal is transmitted intracellularly to activate SNAIL1 expression. I demonstrated how quantitative information of TGF-β is distributed through upstream divergent pathways then crosstalk at various places and converge on to SNAIL1. This crosstalk network interprets the duration of TGF-β signal and is robust against stochastic fluctuations. iv Chapter III and IV focus on co-regulation of multiple genes that orchestrate cell functionand phenotype changes. In eukaryotic cells, the expression level of a gene is determined by both transcription factors and the local environment, such as histone modifications and three- dimensional chromosome structure. I used EMT in human cell line and neural cell differentiation process in mouse as model systems, respectively. Through performing combined analysis of data of gene expression, epigenetic modification and chromosome conformation, I examined how the local environment and transcriptional factor regulation is coupled. I discovered that genes co- regulated by a common transcription factor (TF) has tendency to be close both sequentially and spatially. The local spatial organization bridged by TFs is cell type specific. Reorganization of DNA local conformation has impact on gene co-regulation during cell phenotype transition. The final chapter gives a brief summary of the conclusion from my computational and experimental researches in this dissertation. In this chapter, I also introduced the future work in investigating the relationship between chromosome conformation and gene regulation during cell type transition. v TABLE OF CONTENTS PREFACE ................................................................................................................................... xiii 1.0 INTRODUCTION ..................................................................................................................1 1.1 CELL PHENOTYPE TRANSITION PLAYS KEY ROLE IN LIFE OF MULTICELLULAR ORGANISMS...............................................................................1 1.2 SIGNAL TRANSDUCTION PATHWAYS OF EMT INDUCED BY TGF-β, SHH, AND WNT AND THEIR CROSSTALKS ....................................................................4 1.2.1 Multiple signal transduction pathway and their crosstalks. .....................................6 1.2.1.1 TGF-β pathway..............................................................................................7 1.2.1.2 SHH pathway engages in EMT and crosstalks to TGF-β ...........................13 1.2.1.3 WNT pathway in cancer progress and EMT ................................................16 1.2.1 Systems biology in signaling crosstalk and drug discovery ...................................18 1.3 GENE REGULATION IS MUTUALLY DEPENDENT ON CHROMOSOME STRUCTURE DYNAMICS DURING CELL TYPE TRANSITION ........................22 1.3.1 Regulation of gene expression takes place at three distinct layers .........................23 1.3.1.1 Conformational change of chromosomes coordinates global gene expression. ..................................................................................................24 1.3.1.2 Epigenetic modification affects local accessibility of chromosomes .........25 1.3.2 Transcription factors determine expression levels of individual genes .................30 vi 2.0 PATHWAY CROSSTALK ENABLES CELLS TO INTERPRET TGF-β DURATION ...33 2.1 INTRODUCTION .........................................................................................................34 2.2 RESULTS ......................................................................................................................35 2.2.1 Network analysis reveals a highly connected TGF-β signaling network ...............35 2.2.2 The canonical TGF-β/SMAD pathway initializes a transient wave of SNAIL1 expression .............................................................................................................39 2.2.3 GLI1 contributes to activating the second wave of SNAIL1 .................................41 2.2.4 GSK3 in a phosphorylation form with augmented enzymatic activity accumulates at endoplasmic reticulum and Golgi apparatus .....................................................46 2.2.5 A temporal and compartment switch from active to inhibitory GSK3 phosphorylation smoothens the SMAD-GLI1 relay and reduces cell-to-cell heterogeneity on GLI1 activation. ........................................................................48 2.2.6 The SMAD-GLI1 relay increases the network information capacity and leads to differential response to TGF-β duration ..............................................................50 2.3 DISCUSSION ................................................................................................................54 2.3.1 pSMADs are major inducers for the first wave of SNAIL1 expression ................54 2.3.2 GLI1 is a signaling hub for multiple pathways and temporal checkpoint for activating second-wave of sustained SNAIL1 expression. ...................................54 2.3.3 GSK3 fine-tunes the threshold of the GLI1 checkpoint and synchronizes responses of a population of cells .........................................................................57 2.3.4 Cells use TOSS formed by a composite network to increase information transfer capacity. ................................................................................................................58 2.4 METHODS ....................................................................................................................59 vii 2.5 SUPPLEMENTARY MATERIALS AND FIGURES ..................................................65 2.5.1 Mathematical modeling ..........................................................................................65 2.5.2 Supplementary figures ............................................................................................73 3.0 SPATIAL CLUSTERING AND COMMON REGULATORY ELEMENTS CORRELATE WITH TGF-Β INDUCED CONCERTED GENE EXPRESSION .......... 82 3.1 INTRODUCTION .........................................................................................................83 3.2 RESULTS ......................................................................................................................85 3.2.1 Gene expression change reflects cell phenotype transition in response to TGF-β...85 3.2.2 Genes classes based on both expression pattern and upstream regulators have more common characters ...............................................................................................86 3.2.3 Genes sharing common regulators have higher probability to be spatially close ...91 3.2.4 Temporal switching between AP1 hetero- and homo-dimers fine tunes local chromosome structures and leads to different expression patterns of downstream genes .....................................................................................................................93 3.3 DISCUSSION ...............................................................................................................97 3.4 MATERIALS AND METHODS ..................................................................................99 3.5 SUPPLEMENTARY TABLES AND FIGURES .........................................................103 4.0 CELLS REORGANIZE CHROMOSOME STRUCTURE FOR COORDINATED HISTONE MODIFICATION AND GENE EXPRESSION DURING CELL DIFFERENTIATION .......................................................................................................109 4.1 INTRODUCTION ......................................................................................................110 4.2 RESULTS ...................................................................................................................113