Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Summer 8-15-2015 Functional Identification and Characterization of cis-Regulatory Elements Christopher Michael Fiore Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Biology Commons Recommended Citation Fiore, Christopher Michael, "Functional Identification and Characterization of cis-Regulatory Elements" (2015). Arts & Sciences Electronic Theses and Dissertations. 579. https://openscholarship.wustl.edu/art_sci_etds/579 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Computational and Systems Biology Dissertation Examination Committee: Barak A. Cohen, Chair Joe Dougherty Justin Fay Gary Stormo Andrew Yoo Functional Identification and Characterization of cis-Regulatory Elements by Christopher M Fiore A dissertation presented to the Graduate School of Arts & Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2015 St. Louis, Missouri © 2015, Christopher M Fiore Table of Contents List of Figures ................................................................................................................................. iv List of Tables .................................................................................................................................. vi Acknowledgments ........................................................................................................................ vii Abstract ........................................................................................................................................... ix Chapter 1: Introduction .................................................................................................................... 1 Importance of transcriptional regulation ...................................................................................... 1 Mechanisms of transcriptional regulation .................................................................................... 2 Discovery and modeling of cis-regulatory elements ................................................................... 4 Regulation of Pluripotency .......................................................................................................... 9 High-throughput reporter assays and synthetic CREs ............................................................... 12 Focus of Dissertation ................................................................................................................. 12 Chapter 2: Interactions between transcription factors help specify cis-regulation in pluripotency ....................................................................................................................................................... 16 Introduction ................................................................................................................................ 17 Results ........................................................................................................................................ 19 Discussion .................................................................................................................................. 23 Materials and Methods ............................................................................................................... 25 Supplemental information .......................................................................................................... 33 Figures ........................................................................................................................................ 37 Supplemental Tables .................................................................................................................. 40 Supplemental Figures ................................................................................................................. 44 Chapter 3: Changes in cis-regulatory rules during differentiation ................................................ 51 Abstract ...................................................................................................................................... 51 Methods ...................................................................................................................................... 55 Figures ........................................................................................................................................ 57 Chapter 4: High-throughput functional testing of ENCODE segmentation predictions ............... 61 Introduction ................................................................................................................................ 63 Results ........................................................................................................................................ 64 Discussion .................................................................................................................................. 71 ii Methods ...................................................................................................................................... 73 Tables ......................................................................................................................................... 80 Figures ........................................................................................................................................ 81 Supplementary Figures and Tables ............................................................................................ 85 Chapter 5: Discussion .................................................................................................................. 101 Synthetic CREs and TF interactions ........................................................................................ 101 Predicting active genomic regulatory elements ....................................................................... 104 Future Directions ..................................................................................................................... 105 Conclusions .............................................................................................................................. 109 References ................................................................................................................................... 110 iii List of Figures Figure 2.1: Thermodynamic model of OSKE library…………………………………….…...…37 Figure 2.2: Expression by unique types of binding sites …………………………………..……38 Figure 2.3: Expression of CREs with only Klf4 binding sites…………………………………...39 Figure 2.S1: OSKE CRE-seq library…………………………………………………………….44 Figure 2.S2: Interaction rules in statistical thermodynamic model of transcription……………..45 Figure 2.S3: Enriched TF binding near interacting partners……………………………………..46 Figure 2.S4: Expression of CREs with two types of binding sites………………………………47 Figure 2.S5: Expression of Klf genes in overexpression conditions…………………………….48 Figure 2.S6: Predicted expression of KBS library……………………………………………….49 Figure 2.S7: Clusters of Klf4 binding sites function in the genome……………………………..50 Figure 3.1: Reproducibility of expression measurements in RA………………...………………57 Figure 3.2: Expression of CREs in LIF vs. RA media…………………………………………...58 Figure 3.3: Expression of CREs by unique sites…………………………………………………59 Figure 3.4: Thermodynamic model predictions of RA expression………………………………60 Figure 4.1: Reproducible expression measurements show differences in expression by segmentation class…………………..………………………………...……….…….81 Figure 4.2: Lower H3K27ac and H3K36me3 signal are associated with higher Weak Enhancer expression……...…………………………………………………...…….………….82 Figure 4.3: Chromatin features and sequence-specific binding identify active sequences………83 Figure 4.S1: Luciferase assay expression measurements…………………………………….….95 Figure 4.S2: Computing the fraction of active sequences does not depend on the method of choosing short segments………………………………………………….………..96 Figure 4.S3: Weak Enhancers control higher median expression than Enhancers….….………..97 Figure 4.S4: Expression and GC Fraction of Scrambled CREs………………………….………98 iv Figure 4.S5: Active Weak Enhancer and Enhancer CREs have lower levels of H3K36me3 and H3K27ac……………...………………………………………...……...…………..99 Figure 4.S6: Diagram of 200-mer Oligos Used to Construct the CRE-seq Library…..………..100 v List of Tables Table 2.S1: Fit parameter values from thermodynamic models. ................................................... 40 Table 2.S2: Sequences used………………………...……………………………………………41 Table 4.1: Percentage of active CREs by segmentation class…………………...……..………..80 Table 4.S1. Percentage of repressed CREs by segmentation class………………………………85 Table 4.S2: Predictive capability of single chromatin features………………………………….86 Table 4.S3: Enrichment of single chromatin features in subsets of CREs………..….………….87 Table 4.S4: Enriched Motifs in Activated Sequences………………………...…..……………..88 Table 4.S5: Logistic Regression