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Investigating the Cytoplasmic Role of E2F4 in Multiciliogenesis

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Investigating the cytoplasmic role of E2F4 in multiciliogenesis by Renin Hazan M.S. Molecular Biology and Genetics, Bogazici University, 2009 SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY MAY 2020 ©2020 Massachusetts Institute of Technology. All Rights Reserved. Signature of Author:……………...…………………………………………................................. Department of Biology May 14, 2020 Certified by:..…………………………………………………….………………………………. Jacqueline A. Lees Virginia and D.K. Ludwig Professor of Cancer Research Thesis Supervisor Accepted by:……………………………………..………………………….…………………… Stephen Bell Uncas and Helen Whitaker Professor of Biology Co-Director, Biology Graduate Committe Investigating the cytoplasmic role of E2F4 in multiciliogenesis by Renin Hazan Submitted to the Department of Biology on May 14, 2020 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology Abstract The E2F family of transcription factors plays essential transcriptional roles in many cellular processes including proliferation and terminal differentiation. Transgenic mouse models have established that E2F4 is necessary for multiciliogenesis in the airway epithelia. Here we show that E2F4 plays two distinct roles in multiciliogenesis. In early stages, it functions in the nucleus to transcriptionally activate centriole biogenesis genes required for cilia formation. Subsequently, E2F4 locates to the cytoplasm and colocalizes with the early components of deuterosome complexes, which enable the large-scale amplification of basal bodies, from which cilia are assembled. Reconstitution experiments using E2f4-deficient tracheal precursor cells in an in vitro differentiation assay, showed that both nuclear and cytoplasmic forms of E2F4 are essential for multiciliogenesis. Our biochemical analyses showed that E2F4 associates with two distinct components of the deuterosome complex, Deup1 and SAS6. We found that these proteins use distinct motifs to interact with E2F4, a coiled coil domain in Deup1 and the pisa domain/motif II in SAS6. However, the same amino terminal region of E2F4, residues 1-197, is necessary and sufficient to bind both Deup1 and SAS6. Importantly, in vitro reconstitution and differentiation experiments showed that E2F41-197 is sufficient to perform E2F4’s cytoplasmic role in multiciliogenesis. The previously reported redundancy between E2F4 and E2F5 in multiciliogenesis led us to investigate whether other E2Fs associate with the deuterosome components. This showed that Deup1 and SAS6 also associate with E2F5, but not E2F1. Guided by the crystal structure of E2F4 and protein sequence comparison, we narrowed down the Deup1 and SAS6 interaction domains within E2F4. This identified residues 48-53 of E2F4 as being of central importance in both Deup1 and SAS6 binding but not required for E2F4’s interaction with its classic dimerization partner, DP1, arguing that they contribute to a specific Deup1 and SAS6 interaction motif, rather than affecting structural integrity. Taken together, these data identify a novel cytoplasmic role for E2F4 and E2F5 in the differentiation of multiciliated cells, which likely reflects interaction with core components of the deuterosome complex to enable the amplification of basal bodies, from which cilia are assembled. Thesis Supervisor: Jacqueline A. Lees Title: Professor of Biology 2 Acknowledgements Firstly, I would like to express my sincere gratitude to my thesis advisor and mentor Prof. Jacqueline Lees. Her guidance and support have been a light on my path as I worked towards completing my Ph.D. degree. Our meetings, where we discussed ways to make progress had a major impact on my development as a scientist and I greatly appreciated her input on my scientific presentations and papers. It has been a great privilege to be part of her lab. I would like to thank research scientist, Paul Danielian from our lab who helped me master many laboratory skills from my first day in the lab. He has been a compassionate guide throughout my Ph.D. and I feel deeply grateful for his day-to-day guidance and support. I would also like to thank all the current and previous members of Lees lab. It has been scientifically enriching and pleasant environment in which to work. Secondly, I would like to acknowledge my committee members, Prof. Michael Yaffe and Prof. Michael Hemann. Their input and suggestions were invaluable to the development of my project. Additionally, I want to acknowledge our collaborators, research scientist Munemasa Mori and Prof. Wellington Cardoso at Columbia University who contributed significantly to this project. Lastly, I would like to thank to my family. I feel deeply grateful to my parents for their unconditional love and guidance and my sister Selin for her love, friendship and support. With the deep love and belief of my family, I found the courage to pursue my dream of completing my Ph.D. in a place far from my home. 3 Table of Contents Abstract .......................................................................................................................................... 2 Acknowledgements ....................................................................................................................... 3 Chapter one: INTRODUCTION ................................................................................................ 7 Part 1. Overview of the pocket protein family and the E2F family of transcription factors ......................................................................................................................................... 8 A. The pocket protein family .................................................................................................. 8 a. Discovery of the retinoblastoma protein ............................................................................ 8 b. Function of the retinoblastoma protein .............................................................................. 9 i. The retinoblastoma gene and the cell cycle regulation ................................................. 11 ii. Structure and post-modifications of the retinoblastoma protein.................................. 13 c. The pocket protein family members, p107 and p130 ....................................................... 15 B. E2F Family of Transcription Factors .............................................................................. 17 a. Discovery of E2F activity ................................................................................................ 17 b. Classification of the E2F family ...................................................................................... 18 i. The activating E2Fs; E2F1, E2F2 and E2F3 ................................................................ 20 1. Discovery and structure ........................................................................................... 20 2. The roles of activating E2F’s in cell cycle progression ........................................... 20 3. The roles of activating E2Fs in apoptosis ................................................................ 23 4. The roles of activating E2Fs in differentiation and development ............................ 23 ii. E2F4 and E2F5 ............................................................................................................ 24 1. Discovery and structure ........................................................................................... 24 2. The roles of repressive E2Fs in cell cycle ............................................................... 26 3. The roles of repressive E2Fs in differentiation and development ........................... 28 iii. E2F6, E2F7 and E2F8 ................................................................................................ 30 1. Discovery and structure ........................................................................................... 30 2. The roles of E2F6-8 in cell cycle and differentiation .............................................. 31 Part 2. Multiciliogenesis and the canonical suppressive E2Fs, E2F4 and E2F5 .............. 33 A. Primary cilia versus cilia in multiciliated cells ............................................................... 33 a. Differences in structure and function ............................................................................... 33 4 b. Multiciliated cell fate determination ................................................................................ 36 B. Centriole biogenesis and function .................................................................................... 41 a. Structure and distinct roles of centrioles .......................................................................... 41 b. Mother-centriole-dependent centriole biogenesis in proliferating cells .......................... 41 c. De novo centriole biogenesis in multiciliated cells .......................................................... 45 d. Maturation of nascent centrioles ...................................................................................... 48 e. Regulation of multiciliogenesis by cyclin/CDK complexes ............................................ 48 C. Role of suppressive E2Fs, E2F4 and E2F5 in multiciliogenesis .................................... 50 References ................................................................................................................................ 52 Chapter two ................................................................................................................................
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