UNIVERSITY OF CALIFORNIA, MERCED High-throughput Analysis of WNT Signaling Pathway in Osteoblasts A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Quantitative and Systems Biology by Aimy Sebastian Committee in charge: Dr. Suzanne S. Sindi, Chair Dr. David H. Ardell Dr. Gabriela G. Loots Dr. Jennifer O. Manilay 2016 Copyright Aimy Sebastian, 2016 All rights reserved The Dissertation of Aimy Sebastian is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Gabriela G. Loots Jennifer O. Manilay David H. Ardell Suzanne S. Sindi, Chair Date University of California, Merced 2016 iii Table of Contents Signature Page ..................................................................................................................... iii Table of Contents ................................................................................................................. iv List of Figures ...................................................................................................................... vi List of Tables ..................................................................................................................... viii Acknowledgements .............................................................................................................. ix VITA ..................................................................................................................................... x Abstract .............................................................................................................................. xiii Chapter 1: Introduction ........................................................................................................ 1 1.1. WNT proteins ................................................................................................................. 1 1.2. WNT signaling pathways ............................................................................................... 2 1.3. Overview of bone development and metabolism ........................................................... 5 1.4. WNT signaling in bone development and homeostasis ............................................... 10 1.5. Objectives of the study ................................................................................................. 23 Chapter 2: High-throughput Sequencing to Study Gene Expression and Gene Regulation ............................................................................................................................ 27 2.1. Introduction .................................................................................................................. 27 2.2 High-throughput sequencing technologies ................................................................... 27 2.3. Profiling gene expression changes with RNA-seq ...................................................... 28 2.4. Studying transcription regulation with ChIP-seq ........................................................ 37 2.5. Summary ...................................................................................................................... 41 Chapter 3: WNT Co-receptors LRP5 and LRP6 Differentially Mediate WNT3A Signaling in Osteoblasts ........................................................................................ 43 3.1. Introduction .................................................................................................................. 43 3.2. Methods ........................................................................................................................ 43 3.3. Results ......................................................................................................................... 46 3.4. Discussion ................................................................................................................... 57 iv Chapter 4: Integrative Analysis of ChIP-seq and RNA-seq Experiments Identifies WNT3A Inducible Regulatory Elements in Osteoblasts ...................................97 4.1. Introduction ................................................................................................................97 4.2. Methods ......................................................................................................................98 4.3. Results ......................................................................................................................100 4.4. Discussion ................................................................................................................106 Chapter 5: WNT16 Regulates the Expression of Canonical and Non-canonical WNT Targets in Osteoblasts ............................................................................................121 5.1. Introduction ..............................................................................................................121 5.2. Methods ....................................................................................................................122 5.3. Results ......................................................................................................................123 5.4. Discussion ................................................................................................................129 Chapter 6: Conclusion......................................................................................................148 References .......................................................................................................................151 v List of Figures 1. Figure 1.1: Dendrogram of mouse WNT proteins ...................................................1 2. Figure 1.2: Canonical WNT signaling ....................................................................3 3. Figure 1.3: Non-canonical WNT pathways .............................................................4 4. Figure 1.4: Structure of a long bone ........................................................................6 5. Figure 1.5: Osteoblast differentiation ......................................................................7 6. Figure 1.6: Bone formation ......................................................................................8 7. Figure 1.7: Bone remodeling ...................................................................................9 8. Figure 1.8: Osteoporotic bone ................................................................................10 9. Figure 1.9: Cell type specific promoters commonly used to drive transgene or Cre-recombinase expression in rodent animal models ......................................11 10. Figure 2.1: Fastq file format ..................................................................................29 11. Figure 2.2: RNA-seq data analysis workflow ........................................................30 12. Figure 2.3: Low and high quality RNA-seq data ...................................................31 13. Figure 2.4: Read mapping ......................................................................................33 14. Figure 2.5: RNA-seq data before and after filtering ..............................................34 15. Figure 2.6: RNA-seq data before and after normalization .....................................35 16. Figure 2.7: Heatmap representation of differentially expressed genes ..................36 17. Figure 2.8: ChIP-seq experimental workflow ........................................................38 18. Figure 2.9: Enhancers ............................................................................................39 19. Figure 2.10: Osteoblast enhancers .........................................................................41 20. Figure 3.1: Experimental design ............................................................................47 21. Figure 3.2: qPCR analysis of known WNT targets................................................48 22. Figure 3.3: Enriched GO terms associated with WNT3A targets ..........................49 23. Figure 3.4: Expression profiles of WNT3A targets during the differentiation of early pre-osteoblast to mature osteoblasts (Day 2 to Day 18) ...........................51 24. Figure 3.5: Osteoblast proliferation assay .............................................................52 25. Figure 3.6: Calvarial injection ...............................................................................53 26. Figure 3.7: Histology and µ-CT data .....................................................................54 27. Figure 3.8: Venn diagrams of sets of genes differentially expressed in Lrp5-/-, Lrp6-/- and Lrp5-/-;Lrp6-/- osteoblasts compared to respective controls .....56 28. Figure 3.9: Venn diagrams of significantly differentially expressed genes in WNT3A-treated WT, Lrp5-/-, Lrp6-/- and Lrp5-/-;Lrp6-/- osteoblasts. ................57 29. Figure 4.1: TCF/LEF binding sites in Axin2 promoter .......................................101 30. Figure 4.2: Distribution of overlapping H3K4me1 and H3K27ac peaks ............102 31. Figure 4.3: Overlapping H3K4me1 and H3K27ac peaks ....................................102 32. Figure 4.4: Enriched ontology terms associated with osteoblast enhancers ........103 33. Figure 4.5: Putative WNT inducible enhancer in Ptch1 intronic region ..............104 34. Figure 4.6: Enhancer validation ...........................................................................105 vi 35. Figure 4.7: AP-1 binding motif ............................................................................105