THE CILIATED CELL TRANSCRIPTOME A DISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL SCIENCES AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Ramona Hoh March 2010 ! © 2010 by Ramona Amy Hoh. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/sk794dv5857 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. Timothy Stearns, 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. Mark Krasnow 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. Maxence Nachury 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. William Nelson 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 Abstract Multiciliated cells of the respiratory epithelium are unique in that they generate hundreds of modified centrioles called basal bodies per cell. Each basal body anchors a motile cilium at the cell apical surface, and coordinated beating of motile cilia is vital for protecting from airway infection and for respiratory function. We used mice expressing GFP from the promoter of a ciliated cell-specific gene, FOXJ1, to obtain sorted populations of ciliating cells for transcriptional analysis. In addition to successfully identifying candidates found in other proteomics and genomics studies of motile and nonmotile cilia, approximately half of the significantly upregulated genes identified here have not yet been linked to cilia, and of those a third of are currently uncharacterized. We identified several genes associated with human diseases. These include FTO, which has been linked to human obesity, and DYX1C1, which is a candidate gene for developmental dyslexia. Interestingly, FTO localizes to cilia and Dyx1c1-GFP localizes to cilia and centrosomes, establishing novel links between cilia and two genetic diseases with poorly understood cellular and molecular etiology. Finally, we identified a number of transcription factors that are differentially expressed in ciliating mouse tracheal epithelial cells, including the proto-oncogene c- myb. We show that C-myb is expressed specifically in ciliating cells, and that this expression is temporally restricted to early in the differentiation process. These results suggest a role for the leukemogenic transcription factor C-myb in ciliated cell differentiation. ! iv Acknowledgments Thanks to Tim Stearns for his mentorship and support, and for giving me the freedom to try some pretty ridiculous things. Maybe some day we will find RNA at the centrosome. The Stearns lab was a fantastic group of people to learn science from. Special thanks to Anna Ballew for being a wonderful baymate and for all our insightful discussions about science, to Tim Stowe for working with me to publish the microarray data, and to Eszter Vladar, for MTEC protocols and advice. Thanks to Dan Van de Mark for his work on the promoter project during his rotation. Thanks to my committee members for all of their helpful advice, and to Fraser Tan, Adam Adler and Matt Saunders, my collaborators at Stanford and elsewhere. Special thanks to Bernie Daigle for his expertise on the design and analysis of microarray experiments, and to Allyson O’Donnell and Clara Bermejo, two ass-kicking postdocs who are also the most wonderful of friends. Thanks to Bryan Guillemette, for help with dataset annotation and for being so very dear. Finally, thanks to my parents, who always encouraged me to think for myself and make my own decisions, and who supported me lovingly in all of them. ! v Table of Contents Chapter 1: Introduction 1 References 10 Chapter 2: Transcriptional profiling of ciliogenesis 14 Abstract 15 Introduction 15 Results 17 Discussion 22 Figures 24 Methods and Materials 37 References 40 Chapter 3: Novel localization of candidate genes 42 Abstract 43 Results 43 Discussion 56 Figures 64 Methods and Materials 81 References 83 Chapter 4: Transcriptional Regulation of Ciliogenesis 92 Abstract 93 Introduction 93 Results 95 Discussion 98 Figures 100 Methods and Materials 105 References 106 ! vi List of Illustrations Chapter 1: Introduction Chapter 2: Transcriptional profiling of ciliogenesis Figure 1: Microarray analysis of ciliating mouse tracheal epithelial cells 24 Figure 2: Microarray hybridizations and design matrices for differential expression analysis 25 Figure 3: Differentially expressed genes in MTECs, and the effect of subtractive analysis 26 Figure 4: Identification of known and putative ciliary and basal body genes in the ALI+4 transcriptome 27 Figure 5: Identification of known and putative ciliary and basal body genes in the ALI+12 transcriptome 32 Figure 6: Identification of putative and validated FoxJ1 target genes in the MTEC transcriptome 35 Figure 7: GO Term analysis of genes upregulated during ciliogenesis 36 Chapter 3: Novel localization of candidate genes Figure 1: Expression of centrosome and centriole related genes in ciliated MTECs 64 Figure 2: Expression of genes encoding candidate proteins in ciliated MTECs 65 Figure 3: Mdm1-GFP localizes to the centrosome and the primary cilium 66 Figure 4: Dyx1c1-GFP localizes to the centrosome in NIH-3T3 cells 67 Figure 5: KIAA0319-GFP localizes to the centrosome in NIH-3T3 cells 68 Figure 6: Genes associated with neuronal migration and developmental dyslexia are upregulated in ciliated MTECs 69 Figure 7: Expression of genes encoding heterotrimeric G-proteins and associated molecules in MTECs 70 Figure 8: Gnb4 is a cilium associated protein 71 Figure 9: Mlf1 localizes to motile cilia 72 ! vii Figure 10: Fatso is a cilium associated protein 73 Figure 11: Fatso is a microtubule associated protein 74 Figure 12: Whsc1 expression in ciliated cells 75 Figure 13: Expression of human disease gene homologs during ciliogenesis in MTECs 76 Figure 14: Expression of small GTPases and modifying proteins during ciliogenesis 77 Figure 15: Upregulated taste receptor genes in ciliated MTECS 78 Figure 16: Expression of olfactory receptor genes in ciliated MTECs 79 Figure 17: Expression of vomeronasal receptors in ciliated MTECs 80 Chapter 4: Transcriptional Regulation of Ciliogenesis Figure 1:Temporal expression profiles of genes encoding regulators of DNA dependent transcription during ciliogenesis 100 Figure 2: C-myb expression before and during ciliogenesis 101 Figure 3: Restriction of c-myb expression during ciliogenesis 102 Figure 4: Promoter analysis of differentially expressed genes during early ciliogenesis 103 Figure 5: Experimental flowchart for characterizing c-myb knockdown phenotype 104 ! viii Chapter 1: Introduction 1 Introduction Cilia structure and function Cilia and flagella are microtubule-based structures that project outward from the cell surface and have important sensory and motile functions. They are composed of a microtubule-based axoneme that projects from a modified centriole called a basal body. The axoneme is surrounded by a specialized plasma membrane that is continuous with the plasma membrane of the cell body. At the distal end of the basal body is the transition zone, where the triplet microtubule basal body extends into the doublet microtubule axoneme. Transition fibers link the transition zone of the centriole to a specialized region of the plasma membrane called the “ciliary necklace” (Gilula and Satir, 1972; Sorokin, 1962). It has been proposed that this region acts akin to the nuclear pore complexes of the nuclear envelope to selectively allow transport in and out of the ciliary compartment (Deane et al., 2001; Rosenbaum and Witman, 2002). Cilia are commonly grouped into two broad classifications. Motile cilia generally have a “9+2” structure, which refers to the nine doublet microtubules and a central pair that make the axoneme. The nine outer doublets are composed of A and B subfibers that are connected by nexin linkages. Motility is conferred by the sliding of doublet microtubules relative to each other and this requires the central pair microtubules, outer and inner dynein arms that project from the A tubule and reversibly bind the neighboring B-tubule, and by radial spokes that connect the A tubule of the outer doublet to the central pair. In mammals, motile cilia are found on cells of the airway epithelium, brain ventricles, oviduct, sperm and embryonic node. Each of these, with the exception of sperm and the ciliated cells of the embryonic node, possesses hundreds of motile cilia, and their coordinated beating is vital for movement of fluid and particles over the surface of the epithelium. In the node, motile cilia are unique in that they lack a central pair but are able to rotate due to the activity of ciliary dynein, and this movement generates nodal flow that it critical for the establishment of left-right asymmetry (Hirokawa et al., 2006). The archetypal “9+0” primary cilium is distinct from the motile cilium in that it lacks a central pair and the 2 dynein arms required for ciliary motility, and only one is found per cell. Primary cilia have been found on almost every cell examined in the human body with the exception of the myeloid and lymphoid lineages. Once thought to be vestigial, primary cilia are now known to be important for detecting chemical and mechanical stimuli.