Novel Approaches to the Visualization of Cell Specific Gene

NOVEL APPROACHES TO THE VISUALIZATION OF CELL SPECIFIC GENE EXPRESSION PATTERNS A DISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOENGINEERING AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Chuba Benson Oyolu December 2010 © 2011 by Chuba Benson Odimegwu Oyolu. All Rights Reserved. Re-distributed by Stanford University under license with the author. This dissertation is online at: http://purl.stanford.edu/gq062rg0666 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Julie Baker, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Russ Altman I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Karl Deisseroth Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii © Copyright by Chuba Benson Oyolu 2010 All Rights Reserved II I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy (Julie C. Baker PhD) Principal Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy (Russ B. Altman M.D. PhD) I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy (Karl Deisseroth M.D. PhD) Approved for the Stanford University Committee on Graduate Studies III ABSTRACT The fate of a cell is largely determined by the unique patterns of gene expression found within it. Complex biological machinery exists within each cell to manipulate chromatin state, and ultimately control gene expression. Developmental processes such as cellular differentiation require very specific chemical signals and environmental conditions. These serve as triggers to put the chromatin modification schemes that produce the resultant patterns of differential gene expression into action, leading to the formation of the cell type of interest. My thesis work is an in depth study of the link between chromatin modification, gene expression, and the unique genetic signatures that characterize distinct cells on unicellular and multi-cellular levels. On the multi-cellular level, I have examined histone modification patterns for their effects on gene activation and repression during human embryonic stem cell differentiation. On the unicellular level, I have worked with a variety of cell types to ascertain the degree of individuality that exists between single members of relatively homogenous cell groups while simultaneously looking for housekeeping gene expression signatures that can be used to classify each cell type into a unique group. To further elucidate the patterns of gene expression found within cell groups and the single cells that comprise them, I have worked to develop new computational methods that produce visual aids to elucidate gene expression signatures of single cells and cell groups. IV ACKNOWLEDGEMENTS I would first and foremost like to thank the creator for all the help and comfort that I received in dire moments without which I would never have come this far. To my parents Edith and Victor Oyolu, your advice and unconditional love gave me the confidence to persevere regardless of the circumstances and challenges I faced. I would like to thank the members of my thesis committee for the insightful and valuable advice they have given me throughout my career. I would like to especially thank Dr Julie Baker for excellent mentorship throughout my post-graduate degree, and all the members of the Baker lab for being so generous with their time and expertise. I would also like to thank my collaborators… especially those in the Quake and Sidow labs for excellent correspondence and remarkable technical work. The Genetics department not only welcomed me from Bioengineering with open arms, but also gave me the opportunity to do the work I enjoy. And for that, I will be eternally grateful. V TABLE OF CONTENTS Chapter 1 Introduction 1 Section I - Chromatin Modification Chapter 2 Nodal Signaling Refines Bivalent 3 Domains During Endoderm Formation in hESCs Chapter 3 Cell specific vector generated surface 56 plots “ChIPvect_gui” Section II - Single Cell Gene Expression Chapter 4 SC Express: A visual aid to uniquely identify 76 single cells Chapter 5 Analysis of Gene Expression Patterns 95 in Single Human Embryonic Stem Cells and Their Derivatives Allows for Cellular Classification Chapter 6 Outlook 124 Chapter 7 Archive: MATLAB code 128 References VI LIST OF FIGURES PAGE Figure 2.1 30 Figure 2.2 31 Figure 2.3 32 Figure 2.4 33 Figure 2.5 34 Figure S2.1 35 Figure S2.2 36 Figure S2.3 37 Figure S2.4 38 Figure S2.5 39 Figure S2.6 40 Figure S2.7 41 Figure S2.8 42 Figure S2.9 43 Figure 3.1 69 Figure 3.2 70 Figure 3.3 71 Figure 3.4 72 Figure 3.5 73 Figure 4.1 89 Figure 4.2 90 Figure 4.3 91 Figure 4.4 92 Figure 4.5 93 Figure 5.1 109 Figure 5.2 110 Figure 5.3 111 Figure 5.4 113 Figure 5.5 115 VII LIST OF TABLES PAGE Table 2.1 44 Table S2.1 45 Table S2.2 46 Table S2.3 48 Table 3.1 74 Table 3.2 75 Table 4.1 94 Table 5.1 116 Table 5.2 117 VIII CHAPTER 1 Introduction 1 Though a vast majority of cell types contain the same base genetic template, it is currently understood that the uniqueness of each cell is endowed through selective expression and repression of genes (Schnabel, Marlovits et al. 2002). Some of the factors internal to the cell that are known to influence gene expression include: histone modification, transcription factor binding, and DNA methylation (Jaenisch and Bird 2003; Brunner, Johnson et al. 2009). Environmental signals received by developing cells serve to trigger these mechanisms, leading to differential gene expression which eventually culminates in cell fate determination. The goal of my thesis is two-fold. First, to understand the epigenetic and transcriptional mechanisms that lead to differential gene expression on a multi-cellular level, and secondly, to determine the amount of genetic variation that exists between single members of the same cell group. Until relatively recently, differences in the sequence of DNA was assumed to be solely responsible for the morphological and functional differences between cells. Research in the past decade has shown that epigenetic mechanisms are in fact largely responsible for differential gene expression, and thus the functional and morphological differences between cells (Bernstein, Mikkelsen et al. 2006). Eukaryotic DNA in its native state is neatly packaged with histone proteins to form chromatin. Chromatin can take two forms: the heterochromatic (inactive) and euchromatic (active) form. It is currently held that the transition between these two forms of chromatin is 2 largely determined by modifications to the histone proteins that comprise the nucleosome (Bernstein, Mikkelsen et al. 2006). The advent of chromatin immunoprecipitation coupled with high- throughput sequencing (chip-seq) has provided a tool with which to monitor the effect of specific histone modifications on the control of gene expression (Johnson, Mortazavi et al. 2007). This method, coupled with expression profiling, has shown that in most cells, histone modifications on specific lysine residues, promote either activation or repression of genes. For example, it is held that tri-methylation of the fourth lysine residue (K4) on histone 3 (H3) is generally associated with the activation of gene expression (Shi, Hong et al. 2006). On the other side of the coin, tri-methylation of the twenty-seventh lysine residue (K27) of H3 is thought to be associated with gene repression (Viré, Brenner et al. 2006). Even with this good base of knowledge, many questions concerning the dynamics of histone modification during human embryonic stem cell (hESC) differentiation remain unanswered. hESCs have become one of the major tools in regenerative medicine and tissue engineering, making it imperative to understand the key mechanisms that govern their differentiation to more mature cell types. During development, the three primary germ layers that yield most all the cell types in the mature organism are specified: endoderm, mesoderm, and ectoderm (James M. Wells and Melton 2000). The primary germ layer known as 3 endoderm is of particular interest because it is the source of essential visceral organs such as the lung, liver, and pancreas (Kevin A D'Amour, Alan D Agulnick et al. 2005; Richard I. Sherwood, Cristian Jitianu et al. 2007). Though experimental protocols have been developed to effect the differentiation of hESCs to definitive endoderm, the dynamic changes in the state of chromatin that occur during this transition have not been well studied. Studying the effect of histone modifications on the activation of gene expression may yield valuable insight into the amount and type of genes actively involved in endoderm specification from hESCs. While methods such as chromatin immunoprecipitation and microarrays allow for the study of gene expression on a multi-cellular level, there is growing interest in the prospect of examining gene expression on the single cell level.

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