Investigating the cytoplasmic role of 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 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 biogenesis 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 use distinct motifs to interact with E2F4, a 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 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 . Guided by the crystal structure of E2F4 and 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 and the E2F family of transcription factors ...... 8 A. The pocket protein family ...... 8 a. Discovery of the ...... 8 b. Function of the retinoblastoma protein ...... 9 i. The retinoblastoma and the 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, and ...... 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 ...... 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 ...... 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 ...... 71 Cytoplasmic E2F4 forms organizing centres for initiation of centriole amplification during multiciliogenesis...... 71 Abstract ...... 72 Introduction ...... 73 Results ...... 76 E2F4 undergoes a nuclear-cytoplasmic shift during multiciliogenesis ...... 76 Localization of cytoplasmic E2F4 to areas of procentriolar biogenesis ...... 78 Cytoplasmic E2F4 and PCM1 form organizing centres for the centriole biogenesis ...... 78 Deuterosomes assemble in cytoplasmic E2F4-PCM1 granules...... 80 E2F4 binds to early components of centriole biogenesis ...... 82 The conserved role of E2F4 in transcriptional regulation of centriole biogenesis genes ..... 86 E2F4’s both nuclear and cytoplasmic roles are necessary for multiciliogenesis ...... 88 Discussion...... 93 Materials and Method ...... 96 References ...... 101 Chapter three ...... 104 Biochemical analysis of complexes composed of E2F4 and the early components of centriole biogenesis during multiciliogenesis...... 104 Abstract ...... 105 Introduction ...... 106 Results ...... 109

5 E2F4 binds to the deuterosome specific protein, Deup1 ...... 109 The Deup1 paralogue, Cep63 shows no interaction with E2F4...... 109 E2F4 also binds to the deuterosome complex component, SAS6 ...... 111 N-terminal regions of Deup1 and SAS6 are necessary for interaction with E2F4 ...... 114 Partially overlapping domains of E2F4 mediate its association with Deup1 and SAS6 .... 118 E2F4 binding to Deup1 leads to post-translational modifications in both proteins...... 127 E2F4 1-197ΔDBD is sufficient for the cytoplasmic function of E2F4 in multiciliogenesis. .... 129 Deup1 binds to specifically to E2F4 and E2F5, but not E2F1...... 131 Residues 48-53 of E2F4 is necessary for its binding to Deup1 and SAS6 ...... 134 Discussion...... 137 Materials and Method ...... 141 References ...... 147 Chapter four: ...... 149 DISCUSSION ...... 149 The transcriptional role of E2F4 in the centriole biogenesis ...... 150 The roles of E2F4 and E2F5 in centriole biogenesis and multiciliogenesis are highly conserved ...... 152 The roles of cytoplasmic E2F4 in multiciliogenesis ...... 153 E2F4’s interactions with Deup1 and SAS6 ...... 154 E2F41-197 is sufficient to bind to Deup1 and SAS6, and also enable multiciliogenesis ...... 156 The residues 48-53 of E2F4 are necessary for binding to Deup1 and SAS6 ...... 157 Post-translational modification in both E2F4 and Deup1 ...... 158 Direct versus indirect interaction of E2F4 with Deup1 and SAS6 ...... 160 E2F4 binds to Deup1 but not Cep63 ...... 161 E2F4 and E2F5 function redundantly in multiciliogenesis ...... 162 E2F4 and pRB show partial redundancy in multiciliogenesis ...... 163 An evolutionary analysis of the nuclear export of E2F4/5 ...... 164 The origin and function of deuterosome complex in multiciliogenesis and the role of E2F4 ...... 166 References ...... 168 Biographical Note...... 172

6

Chapter one: INTRODUCTION

7 Part 1. Overview of the pocket protein family and the E2F family of transcription factors

Since the initial discovery of E2F activity through its involvement in adenovirus mediated transformation, eight genes encoding at least ten distinct E2F proteins have been identified in mammals and analyzed for their distinct functions. Early in vitro studies focused on understanding how distinct E2F proteins are expressed, their functions in the cell cycle and how they are regulated by their interaction with members of the pocket protein family. This analysis identified both overlapping and unique roles for the E2F family members and lead to the categorization of E2Fs as activators, classical repressors and pocket protein independent repressors. Single and double knockout mice models uncovered E2Fs context-dependent functions in proliferation, apoptosis and differentiation. This thesis focuses on the role of E2F4 in multiciliogenesis. The first part of this chapter introduces the pocket protein and E2F families with a particular emphasis on the canonical and non-canonical roles of the repressor E2Fs, E2F4 and E2F5. The second part explains our current molecular understanding of multiciliogenesis, including its transcriptional regulation and the process of centriole biogenesis.

A. The pocket protein family a. Discovery of the retinoblastoma protein

Retinoblastoma is a rare childhood tumor that is lethal unless it is treated. The tumor occurs sporadically in some of patients, but is inherited in others. The unique genetics of retinoblastoma cancer lead to Knudson’s “two hits” hypothesis that suggests the necessity of two separate allele mutations for disease (Knudson, 1971). In its inherited form, one mutation is inherited through germline and the mutation of second allele occurs somatically. In its sporadic

8 form, the two mutations or “hits” occur somatically (Knudson, 1971). This model suggested that the Retinoblastoma (RB-1) gene belongs to a class of human genes called “tumor suppressors” in which the loss of activity of both alleles is associated with tumorigenesis. Cloning of the human

RB-1 gene in region 13q14 (Friend et al., 1986) and identification of biallelic RB-1 mutations in retinoblastoma validated its tumor suppressor function (Fung et al., 1987; Lee et al.,

1987d). Loss of function mutations in RB-1 are observed in around one-third of human tumors and are most common in retinoblastoma, osteosarcoma and small cell lung carcinoma (SCLC)

(Chauveinc et al., 2001; Kaye & Harbour, 2004; Weinberg, 1992). Additionally, mutations in the upstream components of pRB pathway that lead to the deregulation or inactivation of pRB are frequently detected in human cancers, where RB-1 gene is unaltered (Sherr & McCormick,

2002). For example, mutations or amplifications that result in constitutively active cyclin/CDK complexes or cause loss of function of the cyclin/CDK inhibitor are common (Figure 1). b. Function of the retinoblastoma protein

RB-1 encodes a 928 amino acid nuclear phosphoprotein, approximately 110 kilodaltons in size (Lee et al., 1987c). A major advance in the search for pRB function was the discovery of the link between pRB and the oncoproteins produced by the small DNA tumor viruses, like simian virus 40 (SV40) large T-antigen, adenovirus early region 1A (E1A) and human papillomavirus (HPV) type 16 E7. The virus encoded oncoproteins disrupt the control of the cellular proliferation and promotes cellular transformation. Based on antagonistic roles of viral versus the pRB , it was proposed that the viral oncogenes might work by binding and inhibiting pRB. This hypothesis was confirmed when one of the

9 RB-E2F pathway Alterations in human tumors components

INK4A melanoma, mesothelioma,colon, biliary tract, p16 esophageal and pancreatic carcinoma, others

CycD1 melanoma, sarcoma, breast, colon, others

CDK4 melanoma, sarcoma, gliomas, others

pRB retinoblastoma, small-cell lung cancer, osteosarcoma, bladder cancer, others

E2F3 bladder and

Figure 1. Mutations in the pRB pathway in human cancers. Tumorigenic events that result in the overexpression of cyclin D1 and CDK4 or lead to the loss of function of the cyclin dependent kinase (CDK) inhibitor, p16INK4A induce the formation of constitutively phosphorylated pRB leading to uncontrolled cell proliferation. Mutations that occur at the pRB pathway are generally mutually exclusive. Additionally, amplifications of E2F3 have been observed in bladder and prostate cancers. (Derived from Dimova & Dyson, 2005).

10 proteins that viral oncoproteins interact was identified as pRB (DeCaprio et al., 1988; Dyson et al., 1989; Münger et al., 1989; Whyte et al., 1988). Further studies showed that viral oncoproteins bind preferentially to pRB in its unphosphorylated state (Ludlow et al., 1989). A highly conserved LxCxE motif in the viral oncoproteins was found to be necessary for this interaction (Jones et al.,1990). Thus, it was proposed that viral oncoproteins promoted proliferation and cellular transformation in part by inactivating the unphosphorylated form of pRB. i. The retinoblastoma gene and the cell cycle regulation

Subsequent studies showed that phosphorylation of pRB occurs in a cell cycle-dependent manner (Figure 2). While pRB protein is hypophosphorylated during G1 phase of cycling cells as well as in quiescent and terminally differentiated cells, multiple phosphorylated forms exist during S and G2/M phases of cycling cells (Buchkovich et al., 1989; Chen et al., 1989; DeCaprio et al., 1989; Mihara et al., 1989). Further studies showed that mitogenic signals promote accumulation of the cyclin D/CDK4 and cyclin D/CDK6 complexes that phosphorylate and inactivate pRB in late G1 phase and thus allow the passage of cells to S phase (Ewen et al., 1993;

Kato et al., 1993). Subsequently, pRB is phosphorylated by cyclin E/CDK2 and kept inactive through the rest of cell cycle (Hinds et al., 1992). pRB gets dephosphorylated by protein phosphatase type 1 (PP1) in the late stages of mitosis (Ludlow et al., 1993). The tumor suppressor p16 was identified as an upstream regulator of the pRB pathway and it binds to

CDK4 and inhibits the catalytic activity of cyclin D/CDK4 complexes (Serrano et al., 1993).

The hypophosphorylated form of pRB that is present early to mid G1 stage was shown to suppress the cell cycle progression in the absence of mitotic signals (Weinberg, 1992).

11

P P P P P P PP1 P RB RB

G0 P P P P P P M P RB RB Mitogen mid G1 G2 G1 E2F Growth Stimuli

P P P P P P CyclinD P RB S Cdk4/6 mid G1 E2F

Cell cycle progression

P P P P P P P RB Cyclin E late G1 Cdk2

Figure 2. pRB’s cell cycle dependent phosphorylation regulates G1/S transition. In G0 and G1 stages of the cell cycle, pRB binds to E2F proteins to suppress genes necessary for S phase entry. Phosphorylation of pRB by cyclin D/CDK4 and cyclin D/CDK6 complexes at mid G1 and cyclin E/CDK2 complexes at late G1 leads to its inactivation allowing cells to enter to S phase. At the late stage of mitosis, pRB gets dephosphorylated by protein phosphatase type 1 (PP1) facilitating the repression of E2F activity and cell cycle exit. (Derived from Knudson & Knudson, 2008).

12 Constitutively active pRB protein, with regards to cell cycle regulation, can be generated by mutation of 11 of 16 putative CDK phosphorylation sites (Knudsen & Wang, 1997).

pRB suppresses passage through the cell cycle by physically associating with members of the E2F family of transcription factors and thereby preventing E2F dependent activation of genes necessary for DNA synthesis and cell cycle progression (Nevins, 1992). The E2F family of transcription factors will be explained in more detail in the next section. We note here that pRB interacts with the transactivation domain of E2Fs through the interface surface generated between A and B subdomains (Lee et al., 2002; Xiao et al, 2003). The separation of the E2F binding site from the LxCxE binding cleft allows pRB to recruit chromatin regulators to pRB-

E2F complexes. In summary, the tumor suppressor pRB is involved in regulating entry into the cell cycle and can be inactivated via mutation, interaction with oncoproteins or phosphorylation. ii. Structure and post-modifications of the retinoblastoma protein

According to detailed biochemical analysis, pRB is composed of three structural domains; the amino-terminal, small pocket and carboxy-terminal domains (Figure 3A). The small pocket was initially identified as the smallest region necessary to bind viral oncoproteins.

It forms a globular structure through the interaction between its two subdomains; A and B domains that are separated by a linker region denoted as “spacer”. Viral oncoproteins recognize the LxCxE binding cleft of pRB present at the B-box while an A-box portion is required for stable folding of B-box (Lee et al., 1998a). As indicated above, studies showed that the LxCxE binding cleft also mediates interaction with many cellular proteins that have the LxCxE motif such as chromatin remodeling enzymes, histone modifying enzymes and the condensing II

13 A) Large Pocket N-terminus Small Pocket C-terminus Spacer pocket proteins A B LXCXE interaction E2F binding

B) Cyclin A/cdk2 Cyclin E/cdk2 PP1 binding

pRB A B

p107 A B

p130 A B

Kinase inhibitor cycA/Cdk2 B domain function cycA/Cdk2 insertion binding

Figure 3. Domain structure of pocket protein family members. (A) Schematic diagrams showing domain structures and location of common protein interaction sites in the pocket proteins. They all have a homologous central domain named “small pocket” that is necessary and sufficient for their binding to viral oncogenes. The small pocket is composed of two subdomains, A and B, each resembling cyclin folds with three additional helices that are separated by an unstructured flexible linker region denoted as “spacer”. The amino-terminal domain structurally resembles the small pocket domain with small differences yet this region does not interact with many proteins and thus, is much less essential for pRB’s main functions. Several regions in pocket proteins like the C-terminal region and linker domain are intrinsically disordered. The linker domain contains CDK dependent phosphorylation sites that play a major role in inactivation of pocket proteins. (B) Comparison of the pocket protein family members. p107 and p130 include an extra kinase inhibitor site as well as cyclin/CDK binding sites. Additionally there are insertions in the B domains of p107 and p130. (Derived from Henley & Dick, 2012).

14 complex, CDH1 (reviewed in Dick & Rubin, 2013). The linker sequence contains cyclin dependent kinase (CDK) phosphorylation sites that play an important role in inactivation of pRB. pRB’s bipartite nuclear localization signal is present at the carboxy-terminal region.

Additionally, the carboxy-terminus carries overlapping docking sites for kinases such as cyclin

A/CDK2, cyclin D/CDK4 and cyclin D/CDK6, and phosphatases such as protein phosphatase 1

(PP1) (Hirschi et al., 2010). The small pocket domain and carboxy-terminus are together referred to as the “large pocket domain”. Studies have shown this domain is both necessary and sufficient for the tumor suppressive functions of pRB (Qin et al., 1992; Yang et al., 2002). c. The pocket protein family members, p107 and p130

After the cloning of RB-1, two other proteins, p107 (RBL1) and p130 (RBL2) were identified through their association with viral oncoprotein E1A (Ewen et al., 1991; Li et al.,

1993). p107 and p130 show a higher degree of homology towards each other (54% identity) than to pRB (25% identity). These three proteins; pRB, p107 and p130 are found to share secondary and tertiary structural similarities in their amino-terminal, “pocket” and carboxy- terminal domains and their binding to viral oncoproteins and a partially overlapping set of cellular proteins all require the shared LxCxE binding cleft region (Figure 3B). As a consequence pRB, p107 and p130 are collectively known as the “pocket protein” family. Studies identified a number of molecular functions that the pocket protein family members share. In particular, they all regulate proliferation and passage through cell cycle by transcriptional suppression and their phosphorylation by cyclin/CDKs leads to their inactivation.

Genetic and cellular analyses showed that the pocket protein family members have both distinct and redundant functions in development and tumorigenesis. As the higher level of

15 homology between p107 and p130 suggested, these two proteins carry several shared functions that are absent in pRB. Yet there are other roles of p107 and/or p130 where pRB shows partial redundancy. While the RB-1 gene is mutated in many human cancer types, p107 and p130 are rarely mutated. Homozygous inactivation of Rb leads to embryonic lethality in mice, and Rb heterozygosity results in pituitary and thyroid tumors with high penetrance (Jacks et al., 1992).

In contrast, p107-/- or p130-/- mice do not show any overt phenotypes or enhanced tumorigenesis in a mixed genetic background (Cobrinik et al., 1996; Lee et al., 1996b). In other genetic backgrounds, loss of p107 or p130 can yield some gene specific developmental defects; p107-/- mice developed myeloproliferative disorder as well as defects in white adipocyte development

(LeCouter, et al., 1998a) and p130-/- mice exhibited defects in neural, muscle and heart development that result in lethality after e13.5 (LeCouter et al., 1998b).

Additional studies determined p107 and p130 have overlapping roles in the differentiation of long bones. p107 -/-;p130-/- mice showed enhanced chondrocyte growth and defects in the endochondral bone development resulting in formation of shorter limbs and neonatal lethality (Cobrinik et al., 1996). These defects were linked to enhanced cell cycle and the absence of the Runx2 expression necessary for bone development

(Cobrinik et al., 1996; Rossi et al., 2002). Additionally, p107-/-;p130-/- mice showed epidermal defects underscoring overlapping role in keratinocyte differentiation and hair follicles development (Ruiz et al., 2003). Similarly loss of p107 in pRbF19/F19;K14cre mice expanded the defects in keratinocytes (Ruiz et al., 2004). Another example of redundancy is the broad spectrum of tumors present in chimeras of Rb+/-;p107-/-, Rb+/-;p130-/- and Rb-/-;p130-/- but not in pure Rb+/- or chimeric Rb-/- mice, supporting the tumor suppressive roles for p107 and p130.

16 B. E2F Family of Transcription Factors

The pocket proteins suppress cell cycle progression and proliferation through their interactions with the E2F family of transcription factors. Since the initial discovery of E2F proteins as cell cycle regulators, they were implicated in regulating a large variety of biological activities including development, apoptosis and the DNA damage response. a. Discovery of E2F activity

E2F was discovered as a cellular activity necessary for the transcriptional activation of the viral gene E2 by the early region 1A (E1A) transforming protein of adenovirus (Kovesdi et al., 1986; Yee et al., 1989). Further studies showed that E2F regulates the transcription of many cellular genes including cell cycle regulators, such as cyclin E, cyclin A, cdc2, Cdc25A, enzymes that function in nucleotide biosynthesis, such as dihydrofolate reductase, thymidylate synthetase and thymidine kinase, and the main components of DNA-replication machinery, such as Cdc6,

ORC1, MCM (Dyson et al., 1989; Trimarchi & Lees, 2002). These data showed that, in addition to its necessity for adenoviral transformation, E2F has cellular transcriptional roles in the cell cycle and DNA synthesis. Additional experiments demonstrated that heterodimerization of E2Fs with DP proteins is important for their high affinity DNA binding and transcriptional activation as well as their regulation by pocket proteins (Bandara et al., 1993; Helin et al., 1993b).

As indicated before, experiments that focused on identifying proteins associated with pRB resulted in the cloning of the first E2F transcription factor (Helin et al., 1992a; Kaelin et al.,

1992; Shan et al., 1992). E2F was shown to interact with hypophosphorylated pRB and the complex was disrupted by the adenovirus E1A protein (Bagchi et al., 1991; Chellappan et al.,

1991). Studies using pRB-binding mutant forms of E2Fs identified how pRB-E2F complexes

17 inhibit the G1/S transition (Helin et al., 1993c). Firstly, pRB binds to the transactivation domain of E2Fs and blocks E2F’s ability to recruit transcriptional activator machinery to the promoters of genes necessary for cell cycle and DNA synthesis. Secondly, suppressive E2F-RB complexes recruit chromatin modifiers, like histone deacetylases (HDACs) and Brahma-related gene 1

(BRG1) resulting in formation of more condensed chromatin at gene promoters. b. Classification of the E2F family

Homology mapping shows the E2F family of proteins has been highly conserved through evolution. In Drosophila melanogaster, there is one activator form (dE2F1) and one repressor form (dE2F2) (Duronio et al., 1995; Sawado et al., 1998) and three E2F like proteins (EFL-1,

EFL-2 and EFL-3) are present in Caenorhabditis elegans (Ceol & Horvitz, 2001; Winn et al.,

2011). During evolution, an increase in the number of E2F genes occurred (Cao et al., 2010).

For example, in Arabidopsis thaliana there are two activator and four repressor forms of E2Fs

(Mariconti et al., 2002). In rest of this section, I will give an overall summary of the mammalian

E2F family of proteins with a focus on two repressive E2Fs, E2F4 and E2F5 that are the center of this thesis.

In mammals, eight genes encoding at least ten distinct E2F proteins have been identified based on their one or two winged-helix DNA binding domain (DBD) that allows E2Fs to bind to their targets’ promoters (reviewed in Attwooll et al., 2004; Dimova & Dyson, 2005). E2Fs are grouped as activators or repressors based on structural and functional differences in vitro (Figure

4). The high level of redundancy observed between the E2Fs has shown that E2Fs have overlapping functions in many cases and also play roles different from the original simple classification.

18

h e t S E2F1 1 437 e r o R d D O i m 1 437 N

T E2F2 A e A r

b i z V i I a E2F3a 1 465 n t d T i o i n C n g

w

A 1 334

E2F3b r e i t q h u

D i

1 r E2F4 410 P e

S s p

r R E2F5 1 346 o t O e i n S s

S E2F6 1 281 E

R E2F7 1 911 P E

R E2F8 1 867

DNA binding domain Cyclin A/CDK binding site Dimerization domain Nuclear localization signals (NLS) Transactivation domain Nuclear export signals (NES)

Pocket proteins binding site

Figure 4. The E2F family of transcription factors. E2Fs were subdivided as activators or repressors based on their structures and functions. All E2F members contain homologous winged helix DNA binding domain. The activating E2Fs, E2F1, E2F2, E2F3a and E2F3b, and classic E2F repressors, E2F4 and E2F5, each possess dimerization domains (to dimerize with DP family members) and overlapping transactivation and pocket protein binding domains. While the activating E2Fs carry nuclear localization signal (NLS) and cyclin A/CDK binding motifs, E2F4 and E2F5 possess nuclear export signals (NES) causing distinct subcellular localization patterns for each subfamily. E2F6, E2F7 and E2F8 lack the domain necessary for transactivation and binding to the pocket proteins, thus are known as pocket protein independent repressors. E2F7 and E2F8 carry a duplication of DNA binding domain in place of dimerization domain, therefore they bind to DNA as homodimers or heterodimers with each other. (Derived from Bracken et al., 2004).

19 i. The activating E2Fs; E2F1, E2F2 and E2F3

1. Discovery and structure

The first member of E2F family, E2F1 was cloned based on its ability to interact with pRB (Helin et al., 1992; Kaelin et al., 1992). Using low stringency hybridization, cDNA clones that two additional E2F-like proteins E2F2 and E2F3 were identified at the 1p36 and

6q22 loci, respectively (Ivey-Hoyle et al., 1993; Lees et al., 1993). All the activating E2Fs have highly homologous domain structures and include domains for DNA binding, dimerization, transactivation and pocket protein binding (Figure 4). Activating E2Fs predominantly show nuclear localization due to the presence of nuclear localization signals at their amino-terminal regions (Lindeman et al., 1997; Magae et al., 1996). Further studies discovered that the E2F3 expresses an additional short variant from a previously unidentified promoter located at the first intron (Leone et al., 2000). The initially identified, long form is called E2F3a and the shorter form that lacks the amino-terminal domain E2F3b.

2. The roles of activating E2F’s in cell cycle progression

Early studies identified that the G1/S transition and passage through cell cycle depends on the transcriptional activity of the activating E2Fs, whose expression peaks during G1/S and are found predominantly in nucleus. The association of the activating E2Fs with the pocket protein pRB suppresses their transcriptional activity by blocking the transactivation domain during early G1 and prevents passage through cell cycle. Mitogenic signals through the activity of cyclin/CDK complexes lead to the phosphorylation and subsequent inactivation of pRB. Thus the activating E2Fs that are released can activate the E2F-responsive genes that drive the G/S transition (Figure 5). Chromatin immunoprecipitation experiments also verified during early G1

20

pRB P P P P E2F p16 pRB P P P 1,2,3 P P p107 P

p130 P P p107 HDAC cdk4/6 p130 cdk2 CycD CycE E2F E2F 4,5 1,2,3

G0 - G1 G1/S transition

Figure 5. Roles of E2Fs and the pocket proteins in the G1/S transition. In the G0/G1 cell cycle stages, repressive E2Fs, such as E2F4 and E2F5, occupy the promoters of E2F-responsive cell cycle genes together with pocket proteins, p130 and p107. They are able to suppress transcription by preventing the binding of the activating E2Fs, such as E2F1, 2 and 3, as well as recruiting chromatin remodeling complexes like histone deacetylases (HDACs). Similar functions may be played by E2F6, 7 and 8 in a non-pocket protein dependent manner. The interaction of activating E2Fs with pRB also inhibits their transcriptional activation at G0/G1 stage. Following mitogenic signaling, cyclin/CDK complexes get activated and phosphorylate the pocket protein members. Phosphorylation leads to the disruption of E2F- pocket protein complexes. E2F4 and E2F5 get exported from nucleus and concomitantly, free activating E2Fs bind to the promoters of cell cycle genes and activate their transcription, thus allowing cell cycle progression and proliferation. pRB remains phosphorylated until the end of cell cycle, where protein phosphatase 1 (PP1) dephosphorylates pRB facilitating cell cycle exit.

21 stage repressor E2F occupy at the promoters of cell cycle genes and they are replaced by E2F1,

E2F2 and E2F3 as cells enter mid-to-late G1 (Takahashi et al., 2000).

There is conflicting data on whether the activating E2Fs function by activating transcription of genes through their transactivation domain in S phase or by preventing the binding of suppressive E2F-pocket protein complexes to promoters in G0/G1. An E2F1 mutant lacking the transactivation domain but retaining the DNA binding domain behaves similar to wildtype E2F1, suggesting that displacement of suppressive complexes may be sufficient for activation of some promoters (Cao et al., 2011).

E2F1, E2F2 and E2F3 mainly function as activators of genes responsible from proliferation. Thus, overexpression of any of these members is sufficient to initiate S-phase entry in quiescent (G0) cells and this function depends on transcriptional activity of these proteins (Johnson et al., 1993). Mouse embryo fibroblasts (MEFs) lacking E2F3 exhibit reduced proliferation (Humbert et al., 2000a). MEFs null for all three activating E2Fs undergo cell cycle arrest due to -p21Cip pathway induction and the presence of E2F4-p130 repressive complexes on promoters of the E2F responsive genes (Wu et al., 2001). Further studies showed that the role of activating E2Fs in proliferation might be dispensable in certain contexts. As an example, embryonic stem cells (ES) lacking all the activating E2Fs proliferate in vitro and in vivo and generate mesoderm, endoderm and ectoderm just as well as E2f1-/-;E2f2-/-;E2f3fl/+ ES cells upon subcutaneous injection to athymic nude mice (Chong et al., 2009). Additionally, activating E2Fs were found not to be necessary for proliferation of cells in two mouse adult tissues, the intestine and lens (Chong et al., 2009).

22 3. The roles of activating E2Fs in apoptosis

Several experiments showed that E2F1 regulates both proliferation and apoptosis. The level of apoptosis, as well as proliferation, increased in cells overexpressing E2F1 (Qin et al.,

1994; Shan & Lee, 1994). E2F1 was shown to regulate apoptosis through both p53-dependent and independent pathways (Carnevale et al., 2012; Wu & Levine, 1994). Despite the existence of considerable data supporting a unique role for E2F1 in apoptosis, other activating E2Fs have also been linked to apoptosis (Degregori et al., 1997). For example, E2F3 loss in vivo rescues the apoptotic defects induced by loss of pRB (Ziebold et al., 2001). Also many pro-apoptotic genes p19ARF, Tp73, APAF1, Casp3, Casp7 and BH3-only family members are activated robustly by E2F1, but also partially by E2F2 and E2F3a (Irwin et al., 2000; Moroni et al., 2001; Muller et al., 2001; Nahle et al., 2002).

4. The roles of activating E2Fs in differentiation and development

Analyses of mice with a loss of one of the activating E2Fs revealed that, in addition to their regulatory role in cell cycle, they have context-dependent functions. E2f1-/- mice are viable at birth, mature to puberty and fertile. Wildtype and E2f1-/- fibroblasts show no difference in doubling time or cell cycle progression rate, suggesting a possible redundancy between the activating E2Fs (Field et al., 1996). Instead, E2f1 loss causes enlargement of the thymus and lymph nodes, due to defects in elimination of self-reactive immature T-cells during thymic negative selection and activation of induced death of mature T-cells (Field et al., 1996; Garcia et al., 2000). Additionally, E2F1 loss causes testicular atrophy, exocrine gland dysplasia and high rate of tumorigenesis in mice of certain genetic backgrounds (Yamasaki et al., 1996).

23 Interestingly, in Rb+/- mice, E2F1 loss reduces pituitary and thyroid tumorigenesis, in line with free E2F1’s critical role in proliferation (Tsai et al., 1998).

E2f2-/- mice are also viable and fertile, however, they die prematurely due to autoimmune disorders linked to defects in T-lymphocytes homeostasis (Murga et al., 2001). In addition, erythroid maturation defects and a higher incidence of tumors are observed in a subset of E2f2 null mice. Contrary to E2f1 and E2f2 null mice, E2f3-/- mice show embryonic lethality in a pure genetic background and reduced viability in a mixed background (Cloud et al., 2002; Humbert et al., 2000a). Surviving E2f3-/- mice die as a result of congestive heart failure. E2f3a-/- and E2f3b-/- single knockout mice were generated to determine whether one of the isoforms is responsible for these phenotypes. The observation that E2f3a-/- and E2f3b-/- animals are viable and develop normally suggested that these two isoforms are functionally redundant (Danielian et al., 2008;

Tsai et al., 2008). Studies of compound mutant mice for the activating E2Fs confirmed functional redundancy between E2F members in cell cycle and development. As an example,

E2f1 and E2f2 null mice show defects in hematopoietic cell lineages progression, suggesting their cooperation in the regulation of hematopoietic proliferation (Li et al., 2003). Additional defects in T-cell lineage commitments, pancreatic degeneration and non-autoimmune insulin dependent diabetes were observed (Li et al., 2003). ii. E2F4 and E2F5

1. Discovery and structure

The second subgroup of E2Fs, comprised of E2F4 and E2F5, were identified and cloned as binding partners of p107 and p130 (Beijersbergen et al., 1994; Ginsberg et al., 1994; Hijmans et al., 1995; Sardet et al., 1995). While E2F4 and E2F5 show 78% similarity to each other, they

24 have only 30-60% similarity to activating E2Fs. Like activating E2Fs, E2F4 and E2F5 contain domains for DNA binding, dimerization and transactivation/pocket protein binding (Figure 4).

Yet, E2F4 and E2F5 have much shorter amino-terminal domains and lack the nuclear localization signal and the cyclin A/CDK binding sites present within the corresponding region of the activating E2Fs (reviewed in Attwooll et al., 2004; Dimova & Dyson, 2005). E2F4 and

E2F5 contain two hydrophobic nuclear export signals (NES) that results in their cytoplasmic localization in cycling cells (Gaubatz et al., 2001). The localization of E2F4 and E2F5 to nucleus seems partially reliant on their interaction with the DP proteins and pocket protein family members (Lindeman et al., 1997; Magae et al., 1996; Verona et al., 1997). The nuclear export , chromosomal maintenance 1 (CRM1) is shown to mediate nuclear to cytoplasmic transport of free E2F4 or E2F5 (Gaubatz et al., 2001).

E2F protein family members show different preferences toward members of the pocket protein family in cell cycle. While activating E2Fs (E2F1-3) preferentially associate with pRB in G1 stage of cell cycle, E2F5 predominantly binds to p130 in G0 stage of cell cycle (Hijmans et al., 1995; Lees et al., 1993). On the other hand, E2F4 shows association with all three members under physiological conditions (Beijersbergen et al., 1994; Ginsberg et al., 1994;

Moberg et al., 1996). Since E2F4 is the most highly abundant E2F family member in MEFs and interacts with all pocket protein members, E2F4 comprises the majority of the E2F-pocket protein complexes in many cell types (Moberg et al., 1996). In G0 stage, where p130 is main pocket protein expressed, the E2F4-p130 complex is highly abundant, but as pRB and p107 expression get higher in G1, E2F4 partners with these more abundant pRB and p107 rather than p130. In vivo these interactions are much more complex and might change depending on the identity of the gene.

25 2. The roles of repressive E2Fs in cell cycle

E2F4 and E2F5 mainly function in G0/G1 stages of cell cycle to actively suppress the expression of E2F-responsive genes in a complex with pocket proteins. E2f4-/-, E2f5-/- and E2f4-/-

;E2f5-/- MEFs showed no defects in cell cycle progression, growth arrest after serum starvation or re-entry into cell cycle following re-stimulation of serum starved cells (Gaubatz et al., 2000).

In wildtype MEFs, overexpression of p16INK4A and dominant negative Ras (N17Ras) results in cell cycle arrest through accumulation of hypophosphorylated pRB. Loss of both E2F4 and

E2F5, but not one or the other of these proteins reverse the p16INK4A or N17Ras mediated cell cycle arrest (Gaubatz et al., 2000). Thus, E2F4 and E2F5 have redundant functions in cell cycle arrest only in response to specific growth arrest signals that function through pocket-protein family, such as p16INK4A and N17Ras. Additionally, forced expression of protein transporter,

CRM1 in G1 stage blocks p16INK4a-induced cell arrest due to nucleocytoplasmic translocation of

E2F4 and E2F5 and de-repression of E2F-responsive genes (Gaubatz et al., 2001).

Chromatin immunoprecipitation (ChIP) assays, performed to analyze in vivo promoter occupancy by E2Fs and pocket proteins, identified the E2F4/p130 complex as the predominant repressor complex bound to E2F-responsive genes in quiescence. E2F4/p130 is observed to recruit chromatin modifying factors, like HDAC1 and mSinB to their complex although mSinB recruitment can occur in E2F-independent manner (Rayman et al., 2002). In G1 stage as p107 and pRB levels increase, E2F4 is observed to associate with p107 and pRB. At the G1/S transition, pocket proteins become phosphorylated and release E2F4/5, which are exported from the nucleus due to absence of nuclear localization domain. Promoters of E2F-responsive genes become occupied by activating E2Fs and histone acetyltransferases (HAT) that are recruited by activating E2Fs (Dyson et al., 1989). In differentiated cells, where strong transcriptional

26 repression is necessary to maintain cell state, histone H3, lysine-specific demethylase 5A

(KDM5A) co-occupies the promoters of cell cycle genes with E2F4 (Beshiri et al., 2012).

Knockout models showed that KDM5A and E2F4 are recruited to the common promoters independent of each other without a physical interaction (Beshiri et al., 2012).

Studies in human keratinocyte cells identified a transcriptional repressive role for E2F4 in the transforming growth factor-β (TGF-β) induced growth arrest program. TGF-β treatment enhances the formation of E2F4/pRB and E2F4/p107 complexes, which in turn transcriptionally repress specific genes such as E2f1, b- and HsORC1, whose expression can potentially override TGF-β induced growth arrest (Li et al., 1997). Another study showed the presence of

E2F4/p130/HDAC1 transcriptionally suppressive complexes on the promoter of cdc25 gene following TGF-β induced cell arrest (Iavarone & Massagué, 1999). Recent studies showed cytoplasmic E2F4/p107 or E2F5/p107 complexes interact with Smad3, one of the signal transducers of TGF-β receptors, when pathway is inactive. TGF-β pathway activation leads to translocation of these complexes to nucleus, where they interact additionally with Smad4 and bind to inhibitor elements in promoter of c- (Chen et al., 2002). In a similar fashion, TGF-β induced repression of survivin gene requires the presence of the Smad2/Smad3/pRB/E2F4 complex at its promoter (Yang et al., 2008).

E2F4 transcriptional repressor complexes have the capacity to regulate cell cycle progression in response to distinct cellular stress factors to minimize the cellular damage.

E2F4/p130 complexes have a key role in maintaining a stable G2 arrest in order to prevent entry to mitosis with damaged DNA (Plesca et al., 2007). These E2F4/p130 complexes were detected in the nucleus shortly following irradiation, causing down-regulation of many mitotic genes and promoting a G0-like state. Irradiation of E2F4 deficient cells resulted in an increase in DNA

27 double strand break levels and apoptotic cell death relative to controls (DuPree et al., 2004;

Plesca et al., 2007). In hypoxia, a similar accumulation of nuclear E2F4/p130 complexes and suppression of homology-dependent DNA repair pathways is observed (Bindra & Glazer, 2007).

E2F4 is also a component of another repressor complex called the DREAM complex.

This large multiunit complex, generally consists of MuvB core proteins (LIN9, LIN37, LIN52,

LIN54 and RBBP4) as well as E2F4, DP1 and the pocket proteins p107 and p130 (Morkel et al.,

1997; Schmit et al., 2007). DREAM complexes are shown to bind to promoters at G0 and G1 phase of cell cycle to suppress transcription. When cells progress through the cell cycle,

E2F4/DP1 and p107/p130 dissociate from this multiunit complex and B-Myb incorporates to form the MMB (Myb-MuvB) complex, which transcriptionally activates genes in S phase.

3. The roles of repressive E2Fs in differentiation and development

In vivo and in vitro studies identified additional, context-dependent roles for E2F4 and

E2F5 such as establishment and maintenance of specific cell lineages. For example, E2F4 functions as a regulator of cell fate choice between osteogenic versus adipogenic lineages. Loss of E2F4 enhances the level of adipogenesis upon hormonal induction or confluence arrest in

MEFs and MSCs (Fajas et al., 2002; Landsberg et al., 2003). ChIP assays validated the presence of E2F4 in the promoter of PPARγ, a master regulator of adipogenic lineage (Fajas et al., 2002).

As another example, E2F4 functions at the molecular switch between muscle progenitor cells and brown adipocytes. Specifically, MyoD/Myf5, two muscle lineage-determining factors, repress Prmd6, a brown adipocyte lineage-determining factor, via E2F4/p130 or E2F4/p107 repressor complexes (An et al., 2017). These studies highlight E2F4’s capacity to suppress

28 developmental pathways by transcriptionally regulating well-established lineage-determining factors.

During differentiation, in certain contexts E2F4 functions as a transcriptional activator in contrast to its canonical role as a transcriptional repressor. In terminally differentiated L6 myotubes, E2F1, E2F3, E2F5 and to smaller extend E2F4 show cytoplasmic localization.

Overexpression of either E2F1 or E2F4 overcomes the cytoplasmic sequestering and allows post- mitotic cells to enter S phase (Gill & Hamel, 2000). Additionally, there is a correlation between the level of nuclear E2F4 and proliferation of the myocytes. Loss of E2F4 significantly decreases the mitotic cardiomyocyte number without altering the number of S-phase cardiomyocytes (van Amerongen et al., 2010).

E2f4-/- mice showed several subtle defects, including embryonic anemia, cell-autonomous defects in red blood cells, altered craniofacial morphology and reduced fertility (Humbert et al.,

2000b; Kinross et al., 2006; Rempel et al., 2000). However, E2f4-/- mice died postnatally as a result of chronic rhinitis and associated opportunistic bacterial infections (Humbert et al.,

2000b). Retrospective analysis of the nasal epithelia revealed the absence of multiciliated cells from the entire airway but no defects in the proliferation or apoptosis rates of these epithelial cells (Danielian et al., 2007). In a separate study, loss of E2F4 resulted in severe defects in early eye patterning including aberrant structure of the optic cup, coloboma and eye pigmentation defects (Ruzhynsky et al., 2009). Analyses of E2f4-/- embryos identified developmental defects in the ventral telencephalon region of the brain and in neuronal precursor cell self-renewal capacity, which arose due to reduced sonic hedgehog signaling (Ruzhynsky et al., 2007). This study suggests that E2F4’s ability to regulate sonic hedgehog signaling is important for proper neural development. Studies on the role of E2F4 in fetal liver progenitors

29 differentiation showed E2F4 regulates the lineage-specific factors during lymphoid versus myeloid lineage commitment and its deficiency leads to defects in lymphoid differentiation

(Enos et al., 2008). Transient depletion of E2F4 in the CD8+ T-cell priming lineage resulted in defects in memory T-cell formation due to its necessity in cell cycle exit (Bancos et al., 2009).

E2f5-/- mice die within first six weeks after birth due to . Detailed analysis showed no perturbation in neural/glial apoptosis or abnormal cranial or extra-cranial bone development (Lindeman et al., 1998). Instead, dilation of cerebral ventricles was caused by excessive production of cerebrospinal fluid (CSF) by epithelial cells of the choroid plexus, suggesting a unique role for E2F5 in CSF production (Lindeman et al., 1998).

In summary, E2F4 and E2F5 have been described as repressive E2Fs due to their role in

G1/S transition. Today we know they can function as both transcriptional activators as well as transcriptional repressors in a wide range of cellular processes, including cell cycle, cell fate determination and development. iii. E2F6, E2F7 and E2F8

E2F6, E2F7 and E2F8 form a distinct subgroup of repressive E2Fs because they do not have domains for transactivation or pocket protein binding and their suppressive function is independent of the pocket proteins (Morkel et al., 1997).

1. Discovery and structure

Early studies suggested a dominant negative inhibitory role for E2F6, as overexpressed

E2F6 binds to the E2F responsive promoters and blocks the access of other E2F members thus represses the classical E2F responsive genes (Cartwright et al., 1998; Gaubatz, Wood &

Livingston, 1998; Morkel et al., 1997; Trimarchi et al., 1998). Further studies determined that

30 E2F6’s repressive function requires its association with the members of Polycomb group (PcG) including polycomb ring finger (Bmi1), ring finger protein 1 (Ring1) and RING1 and YY1 binding protein (RYBP) (Attwooll et al., 2004; Ogawa et al., 2002; Trimarchi et al., 2001). The

PcG proteins are responsible for the repression of genes that regulate the anterior- posterior patterning of developing embryo.

E2F7 and E2F8 are designated as atypical E2Fs due to their unique features. They do not carry the dimerization domain present in other E2Fs and in its place have a duplicated DNA binding domain. As a result, they do not bind to DNA as a heterodimer with DP proteins, rather function as either homodimer or heterodimer with each other, thus functioning independent of the DP proteins (de Bruin et al., 2003; Christensen et al., 2005; Logan et al., 2004, 2005).

2. The roles of E2F6-8 in cell cycle and differentiation

E2F6-/- embryonic fibroblasts show no defects in proliferation or quiescence despite occupying the promoters of cell cycle genes in G0 cells together with chromatin modifiers

(Ogawa et al., 2002; Storre et al., 2002). However, in combination with E2F4 loss, de-repression of several cell cycle genes is observed during S-phase, suggesting that E2F6 plays a role in regulating the cell cycle (Giangrande et al., 2004). E2F6 null mice are viable and healthy yet they show defects in spermatogenesis and homeotic transformation of the axial skeleton (Courel et al., 2008; Storre et al., 2002). Similar, yet wider, transformation of the axial skeleton arises from de-repressed Hox upon the loss of polycomb proteins, such as Bmi1 that associates with E2F6 (Akasaka et al., 1996; van der Lugt et al., 1994). Accordingly, E2f6-/-;

Bmi1-/- mutant mice display enhanced axial skeletal defects compared to the single E2f6-/- and

Bmi1-/- single mutants (Courel et al., 2008). Interestingly, Bmi1-/- mice show additional

31 neurological and hematopoietic defects as a result of de-repression of p16INK4A-p19ARF (Molofsky et al., 2003; Park et al., 2003) but these defects are not observed in E2f6-/- single mutant or enhanced in E2f6-/-;Bmi1-/- compound mutants (Courel et al., 2008). Thus, E2F6 and Bmi1 act synergistically at only a subset of Bmi1 target genes.

E2F7 and E2F8 function as transcriptional repressors during cell cycle. In cycling cells, overexpression of E2F7 or E2F8 inhibits S-phase entry and its acute loss enhances cell-cycle progression. E2f7 and E2f8 expression is induced by E2F1 at late G1 and seems to act as a negative feedback loop necessary to turn off the transcription of E2F-responsive genes such as

E2f1, CDC6, MCM2 or miR-25 in S phase (Christensen et al., 2005; Westendorp et al., 2012).

While E2f7-/- or E2f8-/- mice are viable and lack any detectable developmental abnormalities, double knockout mice show vascular defects in yolk sac, multifocal hemorrhages, and massive apoptosis and fail to survive past E11.5, suggesting a high degree of functional redundancy between these two E2F family members (Li et al., 2008). De-repression of E2f1 following the loss of E2F7 and E2F8 clearly contributes to the apoptosis, as the apoptotic phenotype can be reversed by knockout of E2f1 or p53 (Li et al., 2008).

32 Part 2. Multiciliogenesis and the canonical suppressive E2Fs, E2F4 and E2F5 A. Primary cilia versus cilia in multiciliated cells a. Differences in structure and function

Cilia are evolutionarily conserved, -based that perform a variety of functions in cells. Primary cilia formation is initiated by the migration of the mother-daughter centrioles to the apical side of the plasma membrane during G0/G1 stage of cell cycle (review in

Goetz & Anderson, 2010; Ishikawa & Marshall, 2011; Tucker et al., 1979). Once the mother centriole docks to the plasma membrane, it forms the basal body, which acts as an anchor point for the growth of the (Figure 6). Basal bodies have unique barrel-shape structures and are made of nine-fold symmetrical microtubule triplets (each triplet composed of A-, B-, C- tubules) (Figure 6). The extension of doublets containing A- and B- tubules from the basal body generates nine pairs of microtubule doublets of axoneme structure (Figure 6) (review in Vertii et al., 2016). The associated with the primary cilia contains high number of receptors, ion channels and transporter proteins, and thus functions as a sensory antenna and a signaling hub that transmits signals into the cell (reviewed in Goetz & Anderson, 2010). The anterograde and retrograde of transport of signaling components is performed by intraflagellar transport proteins, IFT-B and IFT-A, coupled to kinesin and dynein motors, respectively. In this context, the basal body is responsible for regulating the entry and exit of proteins to .

Defects in cilia structure and function result in congenital conditions collectively referred to as ciliopathies, including polycystic kidney disorder, nephronophthisis, Senior-Loken syndrome,

Bardet-Biedl syndrome (BBS), Joubert syndrome (JBTS) and Meckel-Gruber syndrome (MKS)

(reviewed in Waters & Beales, 2011).

33

9 + 0 Axoneme 9 + 2 microtubules in axoneme of non-motile cilia in axoneme of motile cilia

Plasma membrane

Basal body

9+0 microtubule triplets 9+0 microtubule triplets STRUCTURE OF CILIA in developing centrioles in mature centrioles

Figure 6: Microtubule structure of basal body and axoneme of cilia. The central diagram shows the cilia generated by the basal body (in brown color) and axoneme (green and red color). Cross-sectional views of developing and mature centrioles with nine-fold symmetrical arrangement of microtubule triplets (colored in shades of brown) are shown in lower two diagrams. Cross-sectional views of ciliary from non-motile and motile cilia are shown in upper two diagrams. While both axonemes carry nine microtubule dimers, there is an extra central two microtubule dimers, dynein arms and radial spokes (colored in red) in motile cilia. (Derived from Shahid & Singh, 2018).

34 In multiciliated cells, cilia are motile structures with microtubule based skeletons referred to as axonemes that are surrounded by the ciliary membrane (reviewed in Shahid & Singh,

2018). The axoneme of motile cilia is composed of nine microtubule doublets that are arranged in a circle around two singlet microtubules (referred as 9 + 2 arrangement, shown in Figure 6)

(Afzelius, 1959). This is distinct from non-motile cilia that contain only nine outer microtubule doublets (referred as 9 + 0 arrangement, shown in Figure 6) (Sorokin, 1962). The systematic action of inner and outer axonemal dynein arms, which slide adjacent doublets relative to one another, is necessary for the ciliary motility (Gibbon, 1963; Gibbon & Rowe, 1965; Summers &

Gibbons, 1971). The constraint that is placed on the sliding action by the bridging proteins between the adjacent doublets and the basal body results in the bending of the cilium (reviewed in Satir et al., 2014). The effective motility of multicilia in the environments in which multiciliated cells are generally found depends on this bi-phasic nature of the ciliary stroke.

Multicilia are present in specific terminally differentiated epithelial cells of brain ventricles, trachea and reproductive organs (review in Brooks & Wallingford, 2014; Spassky &

Meunier, 2017). They are essential for driving fluid flow to allow: circulation of the cerebrospinal fluid and neuronal migration in human brain; movement of mucus in the airway epithelia to maintain the integrity and sterility of the airway; transport of ovum in the oviduct/fallopian tube; and stirring of the luminal fluid in the efferent ducts of the testis (Lyons et al., 2006; Sawamoto et al., 2006; Wanner et al., 1996). Hydrocephalus, chronic airway diseases and infertility are observed in humans in which the function of motile cilia is disrupted.

35 b. Multiciliated cell fate determination

Multiciliogenesis requires both a transcriptional program, to induce the expression of genes essential for the formation of multiciliated cells, and massive amplification of centrioles to generate hundreds of basal bodies to nucleate cilia formation (review in Brooks & Wallingford,

2014). The multiple signaling cascades functional in multiciliogenesis during airway epithelia formation are summarized in this section (Figure 7).

p63 and positive mouse tracheal progenitors of the basal lineage give rise to both multiciliated and secretory cells of the airway epithelia (Daniely et al., 2004; Que et al., 2009).

Notch signaling is one of the main determinants of cell fate choice for these progenitors as its inhibition favors multiciliogenesis and its activation suppresses multiciliogenesis, promoting differentiation towards the secretory lineage (Guseh et al., 2009; Mori et al., 2015; Tsao et al.,

2009). The microRNA, miR-499 promotes multiciliogenesis by inhibiting Notch1 and its ligand

Delta-like 1 as well as CP110, a centriolar protein that suppresses cilia assembly (Marcet et al.,

2011; Song et al., 2014). Inhibition of the bone morphogenic protein (BMP) pathway is also necessary for the initiation of multiciliated cell development in the airway epithelia whereas high

BMP levels allow goblet cell differentiation, suggesting a regulatory role for the BMP pathway in cell fate determination between multiciliated and goblets cells (Cibois et al., 2015).

Studies have shown Geminin family members, initially identified as regulators of DNA synthesis, are evolutionally conserved regulators of multiciliogenesis. Two related, coiled-coil proteins of this family, Geminin coiled-coil containing factor 1 (GemC1) and multicilin (also called McIdas), are the most upstream activators of multiciliogenesis program following Notch inhibition (Arbi et al., 2016; McGarry & Kirschner, 1998; Stubbs et al., 2012; Terré et al., 2016).

36

MiR- 449

Notch

GemC1 Multicilin +E2F4/5 +E2F4/5 +DP1 +DP1 Rfx2 Myb Rfx3 FoxJ1

Genes for Genes for cilia de novo centriole growth/motiliy amplification

Cell cycle Massive Apical Cilia exit Centriole docking Maturation Amplification Proliferating Multiciliated Cell Mother centrile dependent Cells

Deuterosome dependent

Figure 7. Schematic representation of the multiciliogenesis program that developing airway epithelial precursors undergo to generate multiciliated cells. The cellular processes that precursor cells undergo in order to differentiate into multiciliated cells with the corresponding major transcriptional factors essential for these steps of multiciliogenesis are illustrated. (Derived from Brooks & Wallingford, 2014).

37 Neither of these proteins have DNA binding domains and both were subsequently shown to complex with E2F4 or E2F5 and their dimerization partner DP1 via their homologous TERT domain, thereby functioning as transcriptional co-activators (Ma et al., 2014; Terré et al., 2016).

A single point mutation in multicilin which abolishes its interaction with E2F4/5 is associated with a rare mucociliary clearance human disorder named “Reduced Generation of Multiple

Motile Cilia” (Boon et al., 2014; Ma et al., 2014).

Geminin inhibits the transcriptional activity of GemC1 and multicilin in proliferating cells through direct binding (Ma et al., 2014). Thus, precursor cells can commit to the multiciliated cell fate only after a decrease in Geminin levels and subsequent increase in GemC1.

GemC1 upregulates multicilin and subsequently multicilin can autoregulate its own transcription locking cells into the multiciliogenesis pathway (Arbi et al., 2018).

Studies show a terniary complex composed of multicilin, E2F4 or E2F5 and DP1 promotes the multiciliogenesis program by activating the genes necessary for de novo centriole biogenesis including Deup1, Cep152, Plk4, Sas6, and Stil (Ma et al., 2014). Based on ChIP sequence analysis, expression of multicilin increases the levels of E2F4 present at the promoters of these centriole assembly genes and decreases its presence at the cell cycle gene promoters (Ma et al., 2014). GemC1 and multicilin complexes also enhance the expression of transcriptional factors required for motile , including p73, myeloblastosis proto- Myb (c-

Myb), regulatory factor x (Rfx) family proteins Rfx2/3, and forkhead box J1 (FoxJ1) (Arbi et al.,

2018; Ma et al., 2014; Terré et al., 2016). TAp73 enhances the expression of key transcription factors such as Rfx2, Rfx3, Myb, and FoxJ1 in addition to many structural and functional ciliary genes (Nemajerova et al., 2017; Marshall et al., 2016). Genes required for cilia assembly, cilia motility and polarized beating are targets of Rfx2, and FoxJ1 is a target of Rfx3 (Chung et al.,

38 2014; Didon et al., 2013). Myb transcriptionally activates genes necessary for de novo centriole biogenesis as well as the transcription factor, FoxJ1 (Tan et al., 2013). In FoxJ1-deficient mice, defects in centriole migration and/or docking to the apical membrane lead to the absence of multiciliated cells, underscoring FoxJ1’s role in basal body positioning and anchoring (Brody et al., 2000). In summary, multiciliogenesis depends on initiation of a transcriptional program that allows expression of genes necessary for deuterosome dependent centriole biogenesis as well as genes necessary for centriole/basal body docking and motile cilium assembly. The mouse phenotypes observed upon the homozygous loss of these transcription factors are listed in

Table1.

39 Genotype Lethality Major phenotype Reference p63-/- Perinatal At E18.5 an increase in multiciliated cells, (Daniely et al., Lethality neuroendocrine and mucin cells; reduction in basal 2004) club cells. p73-/- Increased Hippocampal dysgenesis; hydrocephalus; sterility; (Marshall et al., lethality. chronic infection and inflammation of lungs, 2016) middle ear and sinus; absence of cilia from the airway epithelia, sinuses, and reproductive organs. GemC1-/- Postnatal Growth defects; hydrocephalus; sterility; the (Terré et al., lethality. absence of multiciliated cells from the brain, 2016) trachea, and reproductive organs. Geminin-/- Embryonic Embryonic growth arrest; defect in inner cell mass (Gonzalez et al., lethality formation; premature endoduplication duplication; 2006) before whole embryo commits to trophoblast cell lineage implantation. and composed of trophoblast giant cells. E2f4-/- Postnatal Embryonic anemia; defects in red blood cells; (Danielian et lethality. altered craniofacial morphology; reduced fertility; al., 2007; Humbert et al., chronic rhinitis and associated bacterial infections 2000b; Rempel due to absence of multiciliated cells in the airway et al., 2000) epithelia. E2f5-/- Postnatal Hydrocephalus; reduced ciliogenesis in the (Danielian et lethality. efferent ducts and dilated rete testis. al., 2016; Lindeman et al., 1998) Rfx2-/- Viable. Infertility; defects in spermatid maturation at the (Wu et al., round spermatid phase; failure to assemble 2016) flagellum.

Rfx3-/- Embryonic Growth retardation; left-right asymmetry defects; (Bonnafe et al., lethality. hydrocephalus; abnormality in monocilia 2004) development in the node; abnormality in multiciliated cells of airway and ependymal cells of brain ventricles. FoxJ1-/- Postnatal Hydrocephalus; left-right assembly defects; cilia (Brody et al., lethality. are absent in the airway and oviduct, yet present at 2000; Chen et embryo nodes; basal bodies are present but al., 1998) disorganized near apical side of cells.

Table 1. A summary of the phenotypes observed in mice with loss of the listed multiciliogenesis associated transcription factors.

40 B. Centriole biogenesis and function a. Structure and distinct roles of centrioles

Centrioles are cylindrical organelles made up of triplet microtubules that are essential for the formation of many microtubule organizing structures, including , spindle poles, cilia, and flagella. Two centrioles positioned orthogonally to each other near the nucleus, together with PCM (), form the during interphase.

Centrosomes organize a microtubular cytoskeleton network that positions the nucleus and all cellular organelles. During mitosis, duplicated centrosomes move to the opposite poles of the cell and function as spindle poles to segregate duplicated (Figure 8). In some quiescent and terminally differentiated cell types, centrioles migrate to the apical side of the plasma membrane where they form basal bodies from which cilia are assembled via tubulin polymerization (reviewed in Vertii et al., 2016). Phylogenetic studies show centrioles/basal bodies were present in the last common ancestor of eukaryotes, yet are not conserved in certain branches like yeasts and vascular plants (Carvalho-Santos et al., 2011). A plethora of human diseases including ciliopathies, brain diseases and cancer arise as a result of defects in the development and functioning of centrioles/basal bodies and cilia, underlying the importance of studying this cellular (Bettencourt-Dias et al., 2011). b. Mother-centriole-dependent centriole biogenesis in proliferating cells

In proliferating cells, centriole duplication is tightly linked to cell cycle and thus a single centriole forms next to each of the parental centrioles in a process called canonical or mother- centriole-dependent centriole biogenesis (Figure 8). Aberrations in centriole number in cycling cells can lead to mistakes in segregation of chromosomes and aneuploidy.

41

(5) Spindle assembly

Monociliated cells

G0 M (1) Centriole Disengagement

G2 G1

(4) Centriole Maturation and Seperation S

(3) Procentriole (2) Procentriole Elongation Formation

Figure 8. Interplay between centriole duplication and cell cycle. Diagram shows the main steps of centriole duplication and their specific timing during cell cycle: (1) Centriole disengagement occurs as cells complete mitosis and enter G1 stage and results in displacement of the cohesion ring from centrosome. This step is regulated by several proteins including; Plk1, CDK1, PCM, and seperase. A new linker containing c-Nap1 forms between mother and daughter centrioles. (2) Initiation of centriole duplication where cartwheel structure of procentriole assembles. (3) Procentriole elongation. Daughter centriole microtubules elongate during S and G2 phases of cell cycle. (4) Centriole maturation is accompanied by the acquisition of PCM protein γ-TuRC and associated proteins and separation to generate two mature centrosomes. This step is regulated by Plk1, AurA, and Cdk1 kinases. (5) Mitotic spindle assembly. (Fujita et al., 2016; Kobayashi et al., 2011; Wang et al., 2014).

42 While hundreds of proteins have been identified as components of the centriole biogenesis complex in proteomic analysis, genetic and RNAi screens in C. elegans showed an evolutionary conserved small set of proteins essential for canonical centriole duplication

(Andersen et al., 2003; Jakobsen et al., 2011) (Figure 9). Centriole duplication starts with centrosomal protein 63 (Cep63) and centrosomal protein 152 (Cep152) associating to form a ring-like structure at the proximal end of the mother centrioles (Brown et al., 2013; Sir et al.,

2011). Subsequently, Cep63 and Cep152 recruit polo like kinase 4 (Plk4) to the complex. Plk4, which initially forms a ring around Cep152, later specifies the procentriole position by forming a dot-like structure on the Cep152 ring, marking the site for assembly of single procentriole

(Cizmecioglu et al., 2010; Hatch et al., 2010). SAS6 (spindle assembly 6 homologue), STIL

(SCL/TAL1 interrupting locus), CENP-J (centromere protein J, also known as CPAP) and

Centrin are colocalized with Plk4 in S phase to form a cartwheel-like structure that primes assembly of the daughter centriole (Arquint et al., 2012; LeDizet et al., 1998; Strnad et al., 2007;

Vulprecht et al., 2012). The duplication process is completed by G2, and during mitosis each cell receives a mother-daughter centriole pair (Nigg & Holland, 2018). Proliferating cells prevent reduplication of centrioles by tightly controlling the expression levels of STIL1 and

SAS6, and also the activity of Plk4. STIL1 and SAS6 undergo APC/Cdh1-dependent degradation near end of mitosis or early G1 thus their activity is absent until the G1/S transition of next cell cycle (Arquint et al., 2012; Strnad et al., 2007). Plk4 activity is controlled by auto- regulatory mechanisms including auto-phosphorylation and degradation (Guderian et al., 2010).

This tight control over centriole duplication is essential for the proper segregation of chromosomes during mitosis.

43

C. elegans D. melanogaster H. sapiens

SPD-2 Asl Cep192 Cep152

Zyg-1 Sak Plk4

SAS5 Ana2 Stil SAS6 SAS6 SAS6

SAS4 SAS4 CPAP

Figure 9. Early components of the centriole biogenesis complex in C. elegans, D. melanogaster and mammals. The early components of mother-centriole-dependent centriole biogenesis pathway are highly conserved. The functional homologues in Caenorhabditis elegans, Drosophila melanogaster and mammals are represented by the same colors.

44 c. De novo centriole biogenesis in multiciliated cells

Development of terminally differentiated multiciliated cells requires massive amplification of centrioles to produce hundreds of basal bodies that serve as the foundation to produce motile cilia. The mother-centriole-dependent pathway produces only a small fraction of centrioles necessary for the multiciliated cells. Instead, 95% of nascent centrioles are generated de novo on ring-shaped osmophilic cytoplasmic structures called deuterosomes (Sorokin, 1968).

Studies have shown that new centrioles are generated by the de novo pathway in response to centrosome loss by laser ablation in many different cell lines arrested at S phase (Khodjakov et al., 2002).

Despite identification of deuterosome by electron microscopy over 50 years ago, our understanding of the molecular mechanism of deuterosome-dependent de novo centriole biogenesis pathway is limited. The coiled-coil domain containing protein Ccdc78 was the first protein shown to localize specifically to the deuterosome through studies in multiciliated cells of

Xenopus and mouse tracheal epithelial cell cultures (MTECs) (Klos Dehring et al., 2013). A reduction in centriole number upon Ccdc78 knockdown illustrates its importance in the de novo centriole biogenesis pathway (Klos Dehring et al., 2013).

Another study identified a second protein, deuterosome assembly protein 1 (Deup1, also known as Ccdc67) that specifically localized to the deuterosome but not to the foci of the mother centriole during procentriole formation (Zhao et al., 2013). Phylogenetic analysis suggested that

Deup1 was generated by gene duplication and divergence from Cep63, a central regulator of mother-centriole dependent centriole pathway during vertebrate evolution (Brown et al., 2013;

Zhao et al., 2013). Deup1 overexpression is sufficient for centriole amplification and bacterially

45 expressed recombinant Deup1 can generate circular/spherical structures similar to deuterosome in multiciliated cells (Zhao et al., 2013).

Colocalization of deuterosome specific Ccdc78 and Deup1 with components of the canonical centriole biogenesis pathway like Cep152, Plk4 and SAS6, but not Cep63 suggests deuterosome driven centriole biogenesis process utilizes some of the proteins functioning in mother-centriole-dependent pathway (Figure 10A) (Klos Dehring et al., 2013; Zhao et al., 2013).

In the deuterosome-dependent pathway, Deup1 forms a complex with the Cep152 and, similar to the Cep63/Cep152 complex, recruits Plk4 (Brown et al., 2013; Zhao et al., 2013). Subsequent arrival of other early components of centriole biogenesis like SAS6, STIL1 and initiates the centriole amplification. The precise role of Ccdc78 in the deuterosome complex has not been determined but it is thought to act as a scaffold for recruitment of Cep152 and activation of Plk4

(Tang, 2013).

Deuterosome complexes are proposed to arise independently from the mother/daughter centriole (Figure 10B). Fibrous granules that are osmophilic structures are believed to determine the position, where deuterosome complexes form in non-centrosomal region of the cell (Kubo et al., 1999). In contrast to this, a recent study, proposed that deuterosomes develop from the proximal region of the daughter centriole and move to cytoplasm (Al Jord et al., 2014). Based on these data, the asymmetric structures of centrosomes (having a mother and daughter centrioles) initiate both the daughter centriole formation in proliferating cells and the deuterosome complex in differentiating precursor cells. This is in contrast to earlier studies where deuterosome are proposed to arise independent of mother/daughter centriole inside a fibrous granule structure. Further studies are necessary to determine whether these two synthesis mechanisms occur simultaneously or each specific to certain tissues.

46 A) Mother-Centriole-Dependent Deuterosome Dependent Centriole Biogenesis Centriole Biogenesis

STAGE 1

2 Centrin Deup1 Cep63 Cep152 3 Plk4

SAS6

4

5

6

B)

c c MC: Mother Centriole D D: Deuterosome MC c c C: Centriole c c c c

Figure 10. Comparison of the mother-centriole and deuterosome dependent centriole biogenesis complexes. (A) Stepwise procentriole biogenesis by the mother-centriole and deuterosome pathways. (B) Typical EM images. Compared to the mother-centriole (MC, arrow), deuterosome (D, arrow head) is much more osmophilic and lacks white MT triplets (the white dots seen in mother centriole). Centrioles emerging from both structures are designated as C. (Zhao et al., 2013).

47 d. Maturation of nascent centrioles

In order to form functional cilia, the nascent basal bodies arising from deuterosomes migrate to the apical side of the cell and undergo vesicle-mediated fusion with the apical surface as they acquire accessory structures for ciliogenesis. The apical migration and docking of basal- body depends on actin filament assembly, which is governed by FoxJ1 via actin regulator ezrin and the small GTPase Ras homolog family member A (RhoA) (Huang et al., 2003; Pan et al.,

2007). Members of planar cell polarity (PCP) signaling that regulate Rho activation such as

Cadherin EGF LAG seven-pass G-type receptor 2 and 3 (Celsr2 and Celsr3) are also part of this process (Tissir et al., 2010).

Another important step in maturation of nascent centrioles is the rotational polarization of the cilia (review in Wallingford, 2010). In order to effectively create fluid flow, all of the axonemes inside a cell are required to beat in a synchronized and polarized manner. Dishevelled segment polarity protein (Dvl), Celsr2 and Celsr3, active RhoA and actin assembly are necessary in setting the rotational polarization as well as the apical migration and docking processes.

Multiciliated cells are also required to acquire a unidirectional polarity relative to one another

(known as tissue-level polarization) in order to generate directional and productive fluid flow across the epithelium (review in Wallingford, 2010). The PCP proteins Vang-like protein 2

(Vangl2), Dvl1, Dvl2, Dvl3, Celsr2 and Celsr3 function in murine ependymal cells to provide tissue-level polarization. e. Regulation of multiciliogenesis by cyclin/CDK complexes

Cell cycle progression is highly controlled by a subgroup of cyclin dependent kinases

(CDKs) that form complex with cyclins that provide substrate specificity and promote kinase

48 activity. Recent studies discovered that multiciliogenesis is regulated by cyclin/CDKs, some of which are specific for the multiciliogenesis program, others of which also function during the cell cycle. Cyclin O (CCNO) is a multiciliated cell specific cyclin and its loss is associated with a failure to downregulate genes necessary for centriole biogenesis such as Deup1 and transcription factors like multicilin (Funk et al., 2015; Nunez-Olle et al., 2017). This leads to decrease in the deuterosome number per cell despite increase in their size, defects in centriole maturation and localization and ciliated cell formation (Funk et al., 2015). A subgroup of mitotic regulators including CDK1, CDK2 and APC/C are present at low levels in cells during multiciliogenesis. In cycling cells, cyclin E/CDK2 and /CDK2 complexes initiate G1/S transition and control progression through S phase ensuring that DNA and centriole replication takes place once per cell cycle (Knudson & Knudson, 2008). In multicilitiated cells CDK2 functions in a complex with downstream of Notch pathway to transcriptional regulate the multiciliated cell program (Vladar et al., 2018). Inhibition of Plk1 or CDK1 elongates the early stage of centriole biogenesis, and thus leads an increase in the deuterosomes number as well as number of centriole per cell (Al Jord et al., 2017). On the other hand, overexpression of

Plk1 or CDK1 shortens this step decreasing number of deuterosomes, and thus centriole number per cell (Al Jord et al., 2017). Inhibition of APC/C leads to a delay in the centriole disengagement step, resulting in presence of partially ciliated cells. Additionally, some cells were observed to undergo DNA condensation and kinetogenesis (Al Jord et al., 2017).

49 C. Role of suppressive E2Fs, E2F4 and E2F5 in multiciliogenesis

Analyses of mutant mouse models allowed identification of a partially overlapping developmental role for E2F4 and E2F5 in multiciliogenesis. E2f4-/- mice die postnatally as a result of chronic rhinitis and associated bacterial infections. These phenotypes arise due to the failure of multiciliated cell development in the entire airway epithelium and in the submucosal glands in the paranasal sinuses (Danielian et al., 2007; Humbert et al., 2000b; Rempel et al.,

2000). In the nasal epithelium of E2f4-/- mice, columnar secretory cells that are producing mucin-like substance replace the ciliated cells (Danielian et al., 2007). The loss of ciliated cells and enhanced presence of mucous secreting cells suggests an essential role for E2F4 in multiciliated cell fate determination in the airway epithelia and the submucosal glands. This ciliary defect was also observed in E2f4+/-;E2f5-/- double mutants, but not in E2f4+/- or E2f5-/- single mutants, underscoring partial redundancy between E2F4 and its closest homolog E2F5 in multiciliogenesis (Danielian et al., 2007). Further support for partially redundant roles for E2F4 and E2F5 in cilia formation came from the analysis of mice with tissue specific homozygous loss of E2f4 alone or with the deletion of one copy of E2f5 from reproductive tissues. The loss of

E2F4 alone does not alter the ciliated cells in the efferent ducts, but additional mutation of one copy of E2f5 abolishes multiciliated cell development in this tissue (Danielian et al., 2016).

Also, in these E2f4f/f;E2f5+/-;Vil-Cre mice non-ciliated absorptive cells are malfunctioning and have an increased secretory activity. This resembles the excessive secretory phenotype observed in the choroid plexus epithelium of E2f5-/- mice as well as the enhanced formation of secretary like cells in the airway epithelium of E2f4-/- (Danielian et al., 2007; Lindeman et al., 1998).

These studies suggest that E2F4 and E2F5 have partially overlapping functions first in establishment of ciliated cells and second in suppression of a secretory phenotype.

50 E2F transcription factors regulate the cellular differentiation programs by either directly regulating differentiation specific genes or, indirectly, through their role in cell cycle. During multiciliogenesis, loss of E2F4 or E2F5 does not affect the level of cell proliferation or apoptosis

(Danielian et al., 2007, 2016). Instead, E2F4 and E2F5 (along with DP) are directly involved in regulating multiciliogenesis in complexes with GemC1 and multicilin (Ma et al., 2014; Terré et al., 2016). GemC1 and multicilin binding redirects the E2F4/5 and DP complexes, away from the cell cycle genes, towards genes necessary for the centriole biogenesis. The interaction occurs between the dimerization domains of E2F4/5 and the TERT domains of GemC1 or multicilin

(Ma et al., 2014; Terré et al., 2016). The presence of separate domains in geminin family members that interact with E2Fs versus each other suggests that they may bind separately or together to E2F/DP1 proteins. Overexpression of multicilin alone or with E2F4 is not sufficient to induce multiciliogenesis in non-epithelial cells (Kim et al., 2018). Interestingly, when a chimeric version of E2F4 with a strong transcriptional activation domain is utilized, multiciliogenesis was observed (Kim et al., 2018).

Thus, there is accumulating data on the role of repressor E2Fs; E2F4 and E2F5 in transcriptional activation of multiciliogenesis. In this thesis, I describe my investigation of

E2F4’s role in multiciliogenesis program of mouse tracheal progenitors using an air liquid interface (ALI) in vitro differentiation system and transfected mammalian cells. This thesis confirms E2F4’s transcriptional role in multiciliogenesis and provides evidence for a novel, cytoplasmic function of E2F4 in the centriole biogenesis.

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70

Chapter two

Cytoplasmic E2F4 forms organizing centres for initiation of centriole amplification during multiciliogenesis.

Munemasa Mori, Renin Hazan, Paul S. Danielian, John E. Mahoney, Huijun Li, Jining Lu, Emily S. Miller, Jacqueline A. Lees and Wellington V. Cardoso

R.H. participated in the design of the study, and conducted the experiments establishing the association between E2F4 and Deup1 or Sas6. R.H. wrote the chapter two manuscript and P.S.D. and J.L. revised the chapter two manuscript.

Published: Nature Communication. 2017. 8:15857.

71 Abstract

Multiciliated cells are found in several mammalian tissues, including the trachea, brain ventricles and reproductive tissues where they play important roles in physiology. Multiciliogenesis depends primarily on the initiation of a unique transcriptional program, followed by massive centriole amplification. The vast majority of centrioles, necessary for cilia nucleation, are generated by the deuterosome complexes and only a small fraction of centrioles are formed through mother-centriole-dependent pathway. The analyses of mutant mouse models targeting members of the E2F transcription family established that E2F4 and E2F5 function redundantly in multiciliogenesis and that loss of E2F4 alone is sufficient to cause loss of multiciliated cells from the entire airway epithelium. In this study, we investigated the role of E2F4 in the differentiation of mouse tracheal epithelial precursor cells to multiciliated cells using an in vitro differentiation system. We found that E2F4 is required for the appropriate induction of centriole biogenesis genes during differentiation of mouse tracheal progenitors to multiciliated cells, consistent with the prior studies in Xenopus epithelial precursor. We further discovered that E2F4 undergoes a nucleocytoplasmic transition at subsequent stages of differentiation and the resulting cytoplasmic

E2F4 colocalizes with core components of the fibrous granules and deuterosome complex and binds to Deup1 and SAS6, two components of deuterosome complex. Moreover, reconstitution experiments established that the transcriptional and cytoplasmic functions of E2F4 are both required for multiciliogenesis. Thus, our data identified a novel cytoplasmic function for E2F4, as a scaffold protein for the assembly of early components of deuterosome complex, which enables the replication of centrioles required for multiciliogenesis.

72 Introduction

The E2F family of transcription factors regulates a variety of cellular processes, including cell cycle, apoptosis and cell fate determination (reviewed in Attwooll et al., 2004; Dimova &

Dyson, 2005). E2Fs utilize highly conserved DNA-binding domains to bind and transcriptionally regulate their responsive genes. E2Fs are subdivided as activators or repressors based on their structures and functions. The activator E2Fs; E2F1, E2F2 and E2F3 mainly transcriptionally activate genes that are responsible from proliferation and apoptosis (reviewed in

Bracken et al., 2004). They function in G1/S transition and proliferation by activating the transcription of E2F-responsive genes necessary for cell cycle progression and DNA synthesis

(Helin et al., 1993; Lees et al., 1993). On the other hand, the repressor E2Fs, E2F4 and E2F5, function mainly at G0 and G1 stages of cell cycle to actively suppress the genes necessary for cell cycle progression in complex with the pocket proteins and the recruited chromatin- remodeling complexes (Rayman et al., 2002; reviewed in Takahashi et al., 2000).

The transcriptional functions of E2F4 and E2F5 can be regulated through changes in its subcellular localization during cell cycle. Studies showed the localization of E2F4 and E2F5 to the nucleus depends on their interaction with the pocket proteins as a result of the absence of nuclear localization signals that are present in the activator E2Fs and the presence of nuclear export signals (Gaubatz et al., 2001). While E2F4 associates with all three members of the pocket proteins (Moberg et al., 1996), E2F5 shows more specific preference for p130 (Hijmans et al., 1995; Sardet et al., 1995).

Our lab has used E2F4 and/or E2F5 mutant mouse models to understand the role of these

E2Fs in cell cycle and development. While E2f4-/- mouse embryo fibroblasts (MEF) did not

73 exhibit any defects in regulation of cell cycle, combined loss of E2f4 and E2f5 disrupted cell cycle arrest, establishing redundancy between E2F4 and E2F5 in this context (Gaubatz et al.,

2001; Landsberg et al., 2003; Rempel et al., 2000). E2f4 null mice died postnatally as a result of chronic rhinitis and associated opportunistic bacterial infections (Humbert et al., 2000). Analysis of the nasal epithelium showed no defects in proliferation or any significant changes in rate of apoptosis (Danielian et al., 2007). Instead, E2F4 loss resulted with the absence of the multiciliated cells in the airway and nasal epithelium (Danielian et al., 2007). Additionally, the loss of E2F4 and heterozygosity of E2F5 (in a conditional E2F4f/f;E2F5+/-;VilCre mouse model), but not E2F4 deficiency alone, caused ciliary defects in the efferent ducts, indicating a partial redundancy between E2F4 and E2F5 in multiciliogenesis (Danielian et al., 2016).

Multiciliogenesis depends on the activation of a specific transcriptional program and a massive amplification of centrioles, which form the basal bodies on which cilia are assembled.

The mother-centriole-dependent centriole duplication pathway contributes to this process, but it is not sufficient to generate hundreds of centrioles required for multicilia formation (Al Jord et al., 2014; Klos Dehring et al., 2013). Instead, progenitors of multiciliated cells specifically assemble novel cytoplasmic structures, called deuterosomes, that are responsible for de novo centriole synthesis (Carvalho-Santos et al., 2010). Many proteins function in both the mother- centriole and deutersome complexes, including centrosomal protein 152 (Cep152), polo-like kinase 4 (Plk4), spindle assembly 6 homologue (SAS6) and Centrin (reviewed in Shahid &

Singh, 2018). In contrast, two paralogues, called centrosomal protein 63 (Cep63) and deuterosome assembly protein 1 (Deup1), play central roles in the formation and function of the mother-centriole and deuterosome, respectively (reviewed in Shahid & Singh, 2018).

74 To understand E2F4’s function in multiciliogenesis, in this study we used an in vitro differentiation system that allows mouse tracheal precursor cells to differentiate into a functionally active airway epithelium that includes multiciliated cells (Dvorak et al., 2011;

Kesimer et al., 2009; You et al., 2002). Our studies identified both temporally and spatially distinct functions for E2F4 in multiciliogenesis. Early in the differentiation process, E2F4 acts in the nucleus to transcriptionally activate genes necessary for multiciliogenesis including centriole biogenesis genes. Subsequently, E2F4 undergoes a nucleocytoplasmic shift and the resulting cytoplasmic E2F4 helps the assembly and development of deuterosome complexes that are responsible from massive centriole biogenesis.

75 Results

E2F4 undergoes a nuclear-cytoplasmic shift during multiciliogenesis

Previous in vivo studies from our lab have shown E2F4 is necessary for multiciliogenesis in the airway epithelia (Danielian et al., 2007). We used a well-established air-liquid interphase

(ALI) assay to investigate E2F4’s function during multiciliogenesis. In this assay, murine adult airway epithelial progenitors isolated from trachea are grown to confluence and exposed to ALI in order to trigger differentiation (Dvorak et al., 2011; Kesimer et al., 2009; You et al., 2002).

E2F4’s expression and localization pattern was determined at distinct stages of differentiation using immunofluorescence (IF) and confocal imaging. E2F4 showed nuclear-specific localization in confluent precursor cells prior to ALI induction (stage 1, day 0) (Figure 1A).

E2F4 underwent a striking nucleocytoplasmic shift following ALI induction (Figure 1A). While small apical cytoplasmic E2F4 granules (<1µm) were observed in monociliated cells in stage 2 of differentiation, larger apical E2F4 aggregates (>1 µm) formed in later stages as multicilia started to appear. A portion of E2F4 stayed in the apical side of cells as smaller granules and another portion moved back to the nucleus in differentiated cells bearing long mature multicilia marked by acetylated α-tubulin (stage 4, > day 4) (Figure 1A). A similar E2F4 localization pattern was observed by immunostaining of developing (E12.5-E18.5) and adult murine lungs

(data not shown). Additionally, in adult human sections, high levels of cytoplasmic E2F4 were detected in multiciliated cells in sharp contrast to higher levels of nuclear E2F4 in the airway progenitor basal cells (data not shown). This indicates that the nucleocytoplasmic shift of E2F4 during multiciliogenesis is evolutionary conserved between mouse and human airway epithelia.

76 Figure 1. E2F4 undergoes a nuclear to cytoplasmic translocation to generate apical granules with early components of centriole biogenesis. (A) Immunofluorescence (IF) and confocal imaging showing apical and basal views of mouse tracheal progenitors at different stages of multiciliogenesis in air-liquid interphase (ALI) cultures. (B) Confocal images depicting apical aggregates of E2F4 under primary cilium labeled with α- tubulin, γ-tubulin, glutamylated tubulin (Gt335) or in adjacent regions where E2F4 partially overlap with Plk4. (C) IF images and diagram showing cytoplasmic E2F4 at the centrosomal region (circled area in left panel), forming ring-like structures with Cep63 and associating with c- Nap1, centrin, and Cep152, and at non-centrosomal regions, forming structures with Cep152, Centrin, and Plk4 (right panels). Bars: a-c = 10, 2.5, 0.5, 2 μm, respectively.

77 Localization of cytoplasmic E2F4 to areas of procentriolar biogenesis

To understand the function of cytoplasmic E2F4 in multiciliogenesis, the distribution of

E2F4 protein in comparison to the early components of centriole biogenesis complexes was examined using IF followed by confocal and super-resolution three-dimensional-structural illumination microscopy (3D-SIM). Confocal analysis showed E2F4 apical aggregates in two apical positions at stage 2; below the primary cilia, marked by α-tubulin, γ-tubulin and glutamylated tubulin (Gt335), and in proximal, non-centrosomal regions colocalized with Plk4

(Figure 1B). 3D-SIM images revealed that E2F4 formed ring-like structures with Cep63 in proximity to Cep152, Centrin, and C-Nap1, which marks sites of procentriole nucleation at parental centrioles (Tsou and Stearns, 2006) at stage 2 (Figure 1C, in left panels). In the non- centrosomal regions, small E2F4 granules colocalized with Cep152, Plk4, centrin but not Cep63

(Figure 1C, in right panels). The colocalization of E2F4 with centriole biogenesis components suggests a cytoplasmic role as organizing centres for nucleation of centrioles.

Cytoplasmic E2F4 and PCM1 form organizing centres for the centriole biogenesis

The first indication that multiciliogenesis has initiated, is the appearance of 70-100 nm electron-dense granular organelles, called fibrous granules (Akiharu Kubo & Tsukita, 2003;

Sorokin, 1968). We realized that the timing of E2F4’s cytoplasmic appearance, as well as its cytoplasmic distribution, was highly similar to that of the fibrous granules in the in vitro differentiation system. Thus, we analyzed the location of cytoplasmic E2F4 with respect to that of a marker of fibrous granules, PCM1, using IF coupled to confocal imaging or 3D-SIM. At stage 2 of differentiation, when E2F4 moves from the nucleus to cytoplasm, PCM1 was observed as discrete punctate structures that closely associated with E2F4 (Figure 2A). Subsequently,

78

Figure 2. E2F4 colocalizes with PCM1, a marker of fibrous granules (FGs). (A) Immunoflourescence (IF) and confocal imaging of cells passing from stage 1 to 2. As E2F4 translocates from nucleus to cytoplasm, E2F4 and PCM1 signals are observed to be adjacent but not overlapping. (B) Confocal image of cells at stage 2 depicting PCM1 expressing granules proximal to primary cilia labeled with acetylated α-tubulin (left panel). Colocalization of PCM1 and E2F4 is shown by 3D-SIM (right panel). (C) Overlapping E2F4 and PCM1 signals are detected at day 4 and day 8 of ALI cultures. (D) IF and confocal imaging shows E2F4 at the core of PCM1 granules in stage 2-3 cells, with the intensity of signals across a line represented in the graph. (Graph: arbitrary units (AU); distance in micrometers; peaks depicted as * and ♯ in two cells outlined by F-actin and stained with DAPI.) Bars: a, b, d= 5, 5 (left panel), 0.5 (right panel), 5 μm, respectively.

79 E2F4 and PCM1 were found to form small apical aggregates around and below the monocilia, which is marked by acetylated α-tubulin (Figure 2B, in left panel). The localization of cytoplasmic E2F4 at the core of PCM1-containing fibrous granules was further confirmed using

3D-SIM imaging (Figure 2B, in right panel). The E2F4-PCM1 colocalization was still apparent at stage 4 (days 4 - 8) (Figure 2C). Additionally, quantification of the signal intensities in double-labeled sections demonstrated the colocalization of E2F4 and PCM1 in different sized granules (Figure 2D). The association of cytoplasmic E2F4 with fibrous granules suggests a novel, non-transcriptional role for E2F4 in centriole biogenesis.

Deuterosomes assemble in cytoplasmic E2F4-PCM1 granules

To further investigate the role of cytoplasmic E2F4 in centriole biogenesis, we next examined the assembly of deuterosome complexes in our in vitro differentiation system.

Deuterosomes are large non-membranous, electron-dense, spherical structures (75-400 nm) that form within or in proximity to fibrous granules (Kubo et al., 1999) and enable massive de novo centriole amplification. Deup1 governs deuterosome assembly by interacting with Cep152 and recruiting Plk4 during multiciliogenesis (Zhao et al., 2013). In cells entering stage 2, confocal and 3D-SIM images both showed that Deup1 was associated with cytoplasmic E2F4 while forming ring-like structures with Cep152 (Figure 3A). More extensive overlap of Deup1,

Cep152 and E2F4 was observed later in stage 2 as more granules of E2F4 accumulated (Figure

3B). Association of E2F4 and Deup1 was further validated using proximity ligation assay (PLA) in ALI cultures of E2F4f/f ;R26CreERT2/+ adult airway progenitors, with tamoxifen treatment being used to induce E2F4 loss as a negative control (Figure 3C). Strong E2F4-Deup1 proximal ligation assay signal was detected in cells wildtype, but not deficient, for E2F4 (Figure 3C).

80 A B E2f4 Deup1 Cep152 3D-SIM E2f4-aggregate (>1um) Deup1 E2f4 *

Deup1 Cep152

Deup1 Pcm1 Centrin DAPI C D Deup1 E2f4 DAPI E # deuterosomes Stage 1-2 Stage 1-2 Stage 2 per cell A)

Deup1 E2F4 DAPI E2f4 Deup1 Cep152 A B 3D-SIM E2f4-aggregate Deup1 E2F4 (>1um) Deup1 E2f4 * M I S -

Deup1 D 3 Cep152 Deup1 Cep152 Stage 2 3 4 Deup1 Pcm1 Centrin DAPI C D Deup1 E2f4 DAPI E # deuterosomes Stage 1-2 Stage 1-2 Stage 2 B) E2E2Ff44 Deup1Deup1p eCCep152r ceelpl 152 A B Deup1 Pcm1 Centrin DAPI 3D-SIM E2f4-aggregate (>1um) Deup1 F Stage 2-3 Stage 3 H E2f4 * 1 m n c o i P t - a i 1 c p o Deup1 Stage 2 3 4 u s e s D

Cep152 a

Deup1 Pcm1 Centrin D% API

C) E2f4F f/f;RSt2ag6e C2-r3eERT2 Stage 3 H Deup1 Pcm1 Centrin DAPI 1 m E 1-20 21n -40 41-60 61-80 Deup1 E2f4 DAPI c D o C i P t - Stage 2 # deuteroa somes per cell i # deuterosomes Stage 1-2 Stage 1-2 1 c p o per cell u s e s D

G Stage 4 a % I e m o

s 1-20 21-40 41-60 61-80

o # deuterosomes per cell r e Stage 4 t

G u I e e m d o / s

Deup1 Pcm1 o n r i e r

t t u e n

Figure 3. Assembly of the deuterosomes in cytoplasmic E2F4-PCM1 aggregates. d / e

Deup1 Pcm1 n c i

r t

Stage 2 3 4 # n

(A) Confocal microscopy analysis showing increasing colocalization of Deup1 and1 E2F4-20 2(upper1-40 4 1-6e 0 61-80 c

# deuterosomes # per cell two panels); IF and 3D-SIM of cells transiting from stage 1 to 2 showing the assembly of 1-20 21-40 41-60 61-80 # deuterosomes per cell cytoplasmicDeup Deup11 Pcm with1 C E2Fentr4i nor D Cep152API into ring-like structures (lower two panels). (B) E2F4, Deup1 and Cep152 colocalization confirmed by quantitative assessment of signal intensity F Stage 2-3 across a repreStagsene 3tative x-y plane in stage 2-3 cells. H(Graph: arbitrary units (AU), large granules 1 m highlighted by *, also shown in inset.) (C) Proximity ligationn assay (PLA): Deup1 and E2F4 c o

i f/f CreERT2/+ P t

proximity signal overlapping with PCM1 in ALI day 3 - from E2f4 ;R26 cultures in the a i 1 Suppl . Figure 3 Mori et al. c p

absence of 4-hydroxytamoxifen (Tm) administration (lefto and middle panels); E2f4 loss upon Tm u s e s

administration leads to a loss in signal (left panel). Bars:D a-c = 5 (upper panel), 1 (lower panel),

Suppl . Figua re 3 Mori et al.

5, 5 μm, respectively. %

1-20 21-40 41-60 61-80 # deuterosomes per cell 81 G Stage 4 I e m o s o r e t u e d Deup1 Pcm1 / n i r t n e c

# 1-20 21-40 41-60 61-80 # deuterosomes per cell

Suppl . Figure 3 Mori et al. Moreover, a fraction of this E2F4-Deup1 signal overlapped with PCM1 foci, showing that these three proteins reside in close proximity (Figure 3C). 3D-SIM images of E2F4 and Deup1 double labeled cells that are at stage 2 or transforming to stage 3 revealed that the Deup1 dots transformed into rings with small diameters inside E2F4 clouds (Figure 4A). Mature deuterosomes, harboring rings with larger diameters, were present in the periphery or outside

E2F4 granules (Figure 4B). These observations strongly suggest that Deup1 initiates formation of deuterosomes in PCM1-E2F4 rich granules and mature deuterosomes dissociate from these granules.

E2F4 binds to early components of centriole biogenesis

Analysis of E2F4’s cellular distribution during multiciliogenesis in ALI cultures revealed the presence of cytoplasmic apical E2F4 granules in proximity to early components of centriole biogenesis complex. To determine whether E2F4 bound any fibrous granules or the deuterosome associated proteins, we performed co-immunoprecipitation and western blot experiments on cell lysates from cells transiently transfected with E2F4 and/or Flag tagged version of candidate binding partners Deup1, PCM1, Cep152, Plk4, Centrin1 and SAS6. Although artificial, this system allowed us to express the test proteins at high levels and assay their binding capabilities in a rapid manner. In this overexpression system, we did not observe any significant interaction of E2F4 with PCM1, Cep152, Plk4 or Centrin1. We cannot know whether this reflects a lack of interaction in vivo, or absence of appropriate signals/post-translational modifications or bridging proteins in this overexpression system. In contrast to these negative results, we did detect clear interactions between E2F4 and both Deup1 and SAS6 (Figures 5A and 6). While Deup1 functions specifically in deuterosomes that mediates de novo centriole biogenesis,

82

Figure 4. Maturation of the deuterosomes in cytoplasmic E2F4 aggregates. (A) IF and 3D-SIM images of stage 2 cells and cells passing to stage 3 showing small Deup1 dots develop into rings inside E2F4 granules. Later, mature Deup1 with larger rings are observed at the periphery or outside the E2F4 aggregates. Arrows represent Deup1 rings from circled areas enlarged in lower panels from Deup1-E2F4 double labeling. (B) Quantitative assessment of deuterosome diameters. Deuterosome size determined by profile analysis of Deup1 signal intensity (distance between peaks in nanometers) across the largest and smallest diameters (depicted as a and b) and estimated as a+b/2. Assessment of the relationship between deuterosome size and position in the E2F4 granules. Graph shows the deuterosome diameters (nanometers) in regions of total (i), partial (ii) or no (iii) E2F4-Deup1 overlap counted in 57 deuterosomes from samples represented in Figure 4A lower panel by white arrows. Bars: 0.5 μl.

83

Figure 5. E2F4 interacts with Deup1. (A) Extracts of 293FT cells transiently transfected with E2F4 alone or with Flag tagged Deup1 were subjected to western blot analysis before (Input) or after immunoprecipitation (IP) with the indicated antibodies. α-E2F4 conjugated beads but not IgG control beads immunoprecipitated E2F4 and recovered Flag tagged Deup1 with E2F4. (B) Binding of endogenous E2F4 to Deup1 in extracts from ALI day 3 cells. α-E2F4 western blot shows immunoprecipitation of E2F4 only with α-E2F4 conjugated beads, but not IgG control beads. α-Deup1 western blot shows that Deup1 is recovered specifically by the α-E2F4 conjugated beads, but not the IgG control beads.

84

Figure 6. E2F4 interacts with Sas6. Co-immunoprecipitation was carried on extracts of 293FT cells transiently transfected with E2F4 and/or SAS6 using the indicated antibodies. α-SAS6 conjugated beads, but not IgG control, recovered SAS6 only from samples overexpressing E2F4 and SAS6.

85 SAS6 is a component of both deuterosomes as well as mother-centriole-dependent centriole biogenesis complex. Importantly, we were able to observe association between E2F4 and Deup1 in the in vivo setting by using lysates of day 3 ALI cultures to conduct co-immunoprecipitation experiments (Figure 5B).

The conserved role of E2F4 in transcriptional regulation of centriole biogenesis genes

Previous studies showed that E2F4 transcriptionally upregulates genes necessary for multiciliogenesis in Xenopus as a multiunit complex with multicilin and/or GemC1 (Ma et al.,

2014; Terré et al., 2016). E2f4-/- null mice are unable to form multicilia in the respiratory epithelium but the neonatal lethality of these animals limits their use to analyze E2F4’s role in multiciliogenesis. Thus, we generated E2f4f/f;R26CreERT2/+ conditional mice to allow inducible

E2F4 deletion in epithelial progenitors in vitro. We validated this system by showing that 4- hydroxytamoxifen-treatment of E2f4f/f;R26CreERT2/+ airway epithelial progenitors in vitro resulted in efficient deletion of E2f4 and the absence of multiciliated cells, without disrupting primary cilia formation (Figure 7A). To evaluate E2F4’s transcriptional role, we performed whole- genome expression profiling on control and E2F4-deficient airway epithelial progenitors prior to and during initiation of multiciliogenesis (ALI day 0, 2, and 4). Our analysis confirmed an enrichment of centriole biogenesis genes in the differentially expressed genes (Figure 7B) and we detected wide overlap with E2f4 targets formerly reported in Xenopus (Ma et al., 2014).

Based on this clustering analysis and subsequent qPCR, E2f4 deficiency resulted in significant reduction of Deup1, Ccno, Myb and others (Deup1 cluster) at ALI day 0, and Plk4, SAS6 and

STIL (Plk4 cluster) at ALI day 2 (Figures 7C-F). In E2F4-deficient E18.5 lungs and adult ALI cultures, the expression of Foxj1 and β-tubulin was lost and multiciliogenesis was disrupted.

86

Figure 7. Nuclear function of E2f4 in regulating transcription of centriole biogenesis genes in murine adult airway progenitors.

(A) Immunoflourescence and confocal imaging of Foxj1 and E2f4 (inset) in ALI day 4 control (Ctr) and 4-hydroxytamoxifen (Tm) treated adult E2f4f/f;R26CreERT2/LSLXGreen1 cells. qPCR shows significant reduction in E2F4 and Foxj1. (Bars, mean ±se; n=3 cultures per group; efficient Cre- mediated recombination as shown by LSLZsGreen1). (B) Heatmap, microarray analysis of E2f4;R26 of ALI days 0, 2, 4 cultures (Ctr and Tm, n=3 per group). Gene enrichment analysis and comparison with E2f4 targets reported in Xenopus identified Deup1 and Plk4 clusters. (C) Bars represent mean (+se) mRNA levels of Deup1, Plk4 and Cep63 in each group based on the raw microarray data. (D) Bars represent mean (+se) fold-change values (Ctr/Tm) for each gene and time point. (E-F) qPCR of Deup1 and Plk4 at ALI days 0, 2 and 4 (samples used for microarray) confirming the temporal expression seen in microarray analysis. Bars represent mean (+se) mRNA levels. Bar: 10μm.

87 E2F4’s both nuclear and cytoplasmic roles are necessary for multiciliogenesis

Our analyses of the diffentiation of mouse tracheal precursors to multiciliated cells in the

ALI cultures confirmed the reported transcriptional role of E2F4, and also revealed a secondary function for E2F4 in the cytoplasm. To determine whether the cytoplasmic role of E2F4 is essential for multiciliogenesis, we generated two E2F4 mutants that are either unable to bind to

DNA (E2F4ΔDBD) or lack the nuclear export signal (E2F4ΔNES). (Figure 8A). We confirmed the functions of wildtype and mutant proteins in transfection assays, showing that wildtype E2F4

(E2F4WT) and E2F4ΔDBD were present in both nucleus and cytoplasm but in contrast to E2F4WT,

E2F4ΔDBD was transcriptionally inactive (Figures 8A and B), and E2F4ΔNES was transcriptionally active by qPCR of classic E2F-responsive genes but its localization to cytoplasm was disrupted

(Figure 8C). We then tested the ability of these mutants to rescue the ciliary defect of cells deficient for E2F4. Specifically, E2F4WT, E2F4ΔDBD and/or E2F4ΔNES were transduced into adult mouse tracheal progenitors (E2F4f/f ;R26CreERT2/+) that were null for E2f4 as a result of 4- hydroxytamoxifen treatment and their capacity to undergo multiciliogenesis checked in ALI cultures. The expression of wildtype E2F4 rescued multiciliogenesis as Foxj1 and acetylated-α- tubulin labeled multicilia appeared by ALI day 6 (Figure 8A). Expression of either E2F4ΔNES or

E2F4ΔDBD alone was not sufficient to rescue the defect as no cilia were observed (Figures 8B and

C). However, following co-expression of these two mutants in E2f4 null precursor cells, the ciliary defect was rescued and multiciliated cells were detected with antibodies against acetylated-α-tubulin, thus recapitulating the E2F4 wildtype phenotype (Figure 9). These experiments clearly established that both nuclear and cytoplasmic roles of E2F4 are necessary for generation of multiciliated cells in ALI cultures derived from mouse airway epithelia.

88

Figure 8. Expression of E2F4ΔDBD or E2F4ΔNES alone is not sufficient to rescue multiciliogenesis in E2F4-deficient cells. E2F4WT, E2F4ΔDBD or E2F4ΔNES were transduced into E2f4f/f;R26CreERT2/+ airway epithelial progenitors treated with 4-hydroxytamoxifen to delete the endogenous E2F4 and examined in ALI assays by fluorescent-confocal imaging. (A) Multicilia formation is observed upon introduction of wildtype E2F4, as validated by staining with α-tubulin and Foxj1. (B) E2F4ΔDBD localizes to both the nucleus and cytoplasm but fail to produce cilia, confirmed by the absence of α-tubulin and Foxj1 staining. (C) E2F4ΔNES shows only nuclear localization and fails to produce cilia as shown by the loss of α-tubulin and Foxj1 staining. All bars = 5 μl.

89

Figure 9. E2F4ΔDBD and E2F4ΔNES are required to rescue multiciliogenesis in E2F4- deficient cells. E2F4ΔDBD and E2F4ΔNES were co-transduced into E2f4f/f;R26CreERT2/+ airway epithelial progenitors treated with 4-hydroxytamoxifen to delete the endogenous E2F4 and examined in ALI assays by flourescent-confocal imaging. E2F4ΔDBD and E2F4ΔNES expression restore multiciliogenesis, as verified by staining with α-tubulin. Apical and dorsal images of PCM1 and Deup1 show the rescue of their subcellular localization. Deup1 is seen to colocalize with cytoplasmic E2F4ΔDBD. All bars = 5 μl.

90 The rescue experiment additionally showed how both Deup1’s expression and localization depends on the function of E2F4. Co-transduction of E2F4ΔDBD and E2F4ΔNES restored the appearance of Deup1’s characteristic apical aggregates together with E2F4 and

PCM1 (Figure 9). Cells expressing E2F4ΔDBD lacked detectable PCM1 and Deup1 signals, due to the absence of transcriptional activation of PCM1 and Deup1, which are E2F4 responsive genes during multiciliogenesis. On the other hand, expression of E2F4ΔNES, allowed Deup1 expression but it accumulated in the nucleus as early as ALI day 2 and less frequently was present in cytoplasm as non-aggregates, thus unable to form the normal apical cytoplasmic aggregates (Figure 10A). This was in sharp contrast with E2F4WT, where Deup1 showed high level of cytoplasmic localization (Figure 10B). The mislocalization of Deup1 in the presence of

E2F4ΔNES was additionally confirmed by counting HA positive cells based on the subcellular localization of Deup1 (Figure 10C). Our data prove both nuclear and cytoplasmic functions of

E2F4 are required for the expression and appropriate assembly of central deuterosome components, and thereby multiciliogenesis.

91 .

Figure 10. Cytoplasmic E2F4 is required for proper subcellular localization of Deup1. (A-B) Immunoflourescence (IF) for HA, Deup1, DAPI and confocal imaging at the basal-lateral areas of 4-hydroxytamoxifen (Tm)-treated E2F4f/f;R26CreERT2 cells transduced with (A) E2F4ΔNES or (B) E2F4WT at day 2 and 6. (A) E2F4ΔNES rescues Deup1 expression but protein is mislocalized to the nucleus. Arrows; nuclei shown by DAPI in left panels and dotted areas in right panels. (B) E2F4WT rescues Deup1 expression and prevents its mislocalization to nucleus seen in E2F4ΔNES. (C) Quantitative analysis of the subcellular localization of Deup1 in Tm- treated E2F4f/f;R26CreERT2 cells transduced with distinct HA tagged E2F4 constructs. Bars mean ±se of the percentage of HA positive cells with Deup1 labeling in cytoplasm or nucleus counted in 5 fields in 3 independent experiments. Bars: 5μm.

92 Discussion

Previous studies from our lab uncovered developmental roles for the repressor E2Fs,

E2F4 and E2F5, in multiciliogenesis (Danielian et al., 2007, 2016; Humbert et al., 2000). E2f4 null mice suffered from chronic airway infections that contribute to their postnatal lethality as a result of the absence of the multiciliated cells in the airway epithelium (Danielian et al., 2007;

Humbert et al., 2000). Similar phenotypes were observed in E2f4+/-;E2f5-/- double mutant, but not E2f4+/- or E2f4-/- single mutants (Danielian et al., 2007). Additionally, multiciliated cells were absent and replaced by secretory like cells in the efferent ducts of E2f4f/f;E2f5+/-;Vil-cre double mutants, but not E2f4f/f;Vil-cre or E2f5-/- single mutants, strengthening partially redundant roles of E2F4 and E2F5 in multiciliogenesis (Danielian et al., 2016).

To study E2F4’s function in multiciliogenesis in the airway epithelia, we used an in vitro system (ALI culture) that enables differentiation of mouse tracheal precursor cells in well- described fashion to differentiated airway epithelia including the multiciliated cells (Dvorak et al., 2011; Kesimer et al., 2009; You et al., 2002). The ciliary defects that were initially observed in E2f4 null mice models were recapitulated in the ALI system. Moreover, our analyses of this model showed that E2f4-deficiency results in the down-regulation of many genes essential for centriole biogenesis, including Deup1, Ccno, Myb, Plk4, Sas6 and Stil, consistent with prior reports that E2F4, as well as E2F5, play critical roles in the transcriptional activation of multiciliogenesis genes in Xenopus skin progenitors (Ma et al., 2014). Importantly, our data also revealed an additional, novel role for E2F4, and specifically cytoplasmic E2F4, in the assembly of the centriole amplification machinery. First, after activating the multiciliogenesis gene program, E2F4 undergoes a nucleocytoplasmic transition and accumulates at the apical side of cells, where the assembly and maturation of centriole biogenesis complex takes place. Second,

93 E2F4 colocalizes with PCM1, a marker of fibrous granules that appears early in multiciliogenesis and determines the location of deuterosome complexes. Finally, E2F4 colocalizes with a range of early centrosomal proteins including: Cep63, specific for mother-centriole-dependent pathway; Deup1, its paralogue specific to de novo pathway; and many other components present in both pathways, such as Cep152, Plk4, Sas6 and Centrin.

Our co-immunoprecipitation and western blot experiments revealed an interaction between E2F4 and two centrosomal proteins, Deup1 and SAS6. While Deup1 is a specific component of the deuterosome and not the mother-centriole-dependent pathway, SAS6 participates in both complexes. This, together with our in vivo immunofluorescence analyses, suggests that E2F4 might function in both centriole pathways. However, in E2f4 null airway epithelial cells we observe no detectable defects in primary cilia formation, which is solely mediated by the mother-centriole-dependent pathway, despite the loss of multiciliated cells, which is predominantly dependent upon deuterosome action. Thus, at least in the context of this tissue, E2F4 is dispensable for the mother-centriole-dependent pathway but not for the de novo pathway.

Importantly, having established that E2F4 has two separate functions in multiciliated cell differentiation in mouse airway epithelia, our reconstitution experiments showed that both are required for multiciliogenesis. Additionally, our analysis of Deup1 and PCM1’s localization patterns in these experiments strongly suggests that E2F4 acts to enable formation of deuterosome structures. Specifically, while expression of a nuclear-specific form of E2F4

(E2F4ΔNES) in E2F4-deficient airway epithelia progenitors is sufficient to restore expression of the multiciliogenesis genes, including Deup1, this protein was mislocalized, and the apical deuterosome granules that are responsible for massive centriole amplification were not

94 assembled. This strongly suggests that E2F4 is required for Deup1 to achieve its appropriate subcellular localization and/or assembly into deuterosomes. More than a decade ago, it was discovered that E2F4 and E2F5 partitioned between nucleus and cytoplasm, in stark contrast to other E2F members that are predominantly nuclear (Verona et al., 1997). The role of this partitioning remained unknown, however, a favored idea was that cytoplasmic localization offered another layer of regulation on E2F4 and E2F5’s transcriptional functions. Our study now reveals a critical and novel function for the cytoplasmic E2F4 as a central hub for the assembly and maturation of deuterosome dependent centriole biogenesis machinery during multiciliogenesis.

95 Materials and Method

Air-liquid interphase cultures of airway epithelial progenitors

Airway epithelial progenitors that were isolated from adult mouse tracheas (E2f4+/+ or

E2F4f/f; R26CreERT2) were allowed to differentiate into multiciliated cells in air-liquid interphase

(ALI) cultures. Briefly, mouse tracheas were harvested, and treated with 0.5% pronase overnight. Next day, isolated airway epithelial progenitor cells were cultured on collagen1- coated Transwell dishes (Corning) under submerged conditions in media that allowed proliferation of airway progenitors to confluence (7 days). Multiciliogenesis was initiated by exposure of the apical side of culture to air (ALI Day 0) and culturing cells in differentiating media (mTEC/serum free, RA media) up to 8 days (ALI Day 8). To initiate E2F4 loss,

E2F4f/f;R26CreERT precursor cells were treated with 1μM 4-hydroxytamoxifen (Tm) from day -5 to day 0. Quantitative polymerase chain reaction (qPCR) and immunofluorescence (IF) were used to verify the loss of E2F4.

Immunofluorescence (IF) and Immunohistochemistry (IHC)

ALI cultures were fixed in 4% paraformaldehyde (PFA) for 10 minutes, room temperature or with 100% methanol for 20 minutes. Immunostaining protocol is described briefly below. Samples were incubated with primary antibodies for 2 hours at room temperature or overnight at 4°C, washed in PBS and incubated with secondary antibodies conjugated to

Alexa488, 567 or 647 (Life Technologies, anti-mouse: A21202, A10037, A31571, 1/300; anti- rabbit: A21206, A10042, A3157, 1:300) for another 2 hours. When necessary, antigen retrieval was performed using unmasking solution Tris-EDTA buffer (1mM EDTA/Tris-HCl, pH 8.3) for

15 minutes at 110°C in a pressure cooker. The antibodies used in IF are listed below: α-E2F4

96 (Millipore, LLF4.2, 1:100), α-glutamylated tubulin (Adipogene, GT335, 1:250), α-centrin

(Millipore, 04-1624, 1:500; Proteintech, 12794-AP-1, 1:500), α-PCM1 (Cell Signaling, 5213,

1:50; Santa Cruz, D-19, 1:50), α-Scgb3a2 (gift from Dr. S. Kimura, NIH, 1:1000), α-Foxj1

(eBioscience, 2A, 1:100), α-acetylated α-tubulin (Abcam, ab125356, 1:1000; Sigma, T7451,

1:2000), α-β-tubulin IV (Abcam, ab11315, 1:500), α-Cre (Millipore, MAB3120, 1:100; Cell

Signaling, 69050, 1:200), α-HA (Cell Signaling, 2367 and 3724, 1:100), α-C-Nap1

(Proteintech, 14498-1-AP, 1:100), α-CEP63 (Proteintech, 16268-1-AP, 1:100). α-Deup1

(1:500), α-CEP152 (1:500) and α-PLK4 (1:250) antibodies were produced in Dr. Xueliang

Zhu’s lab and previously published (Zhao et al., 2013). F-actin and nuclei were imaged using

Alexa Fluor 647 phalloidin (Life Technologies, A22287, 1:100) and NucBlue Fixed Cell Ready

Probes Reagent (DAPI) (Life Technologies, R37606), respectively.

Microscopy

Images were captured by Nikon Labophot 2 microscope equipped with a Nikon Digital

Sight DS-Ri1 charge-coupled device camera or on a Zeiss LSM 700 or LSM710 confocal laser-scanning microscope equipped with a Motorized Stage, an oil-immersion × 40 or × 63 objective lens and argon laser. For Z-stack analysis, scanning was performed at 0.25 μm per layer. Structured illumination microscopy (SIM) was performed with a Nikon N-SIM based on an Eclipse Ti inverted microscope using an SR Apo-TIRF × 100/1.49 oil-immersion objective and an Andor iXon 3 EMCCD camera. Images were acquired in 3D-SIM mode using excitation at 488 nm and 561 nm and standard filter sets for green and red emission. Image z-stacks were collected with a z interval of 125 nm. SIM image reconstruction, channel alignment and 3D reconstruction were performed using NIS-Elements AR software.

97 Proximity Ligation Assay (PLA)

Proximity ligation assay (PLA) was performed as described in manufacturer’s protocol

(Sigma, DUO92102). Briefly, ALI day 4 cultures from E2F4f/f;R26CreERT with or without 4- hydroxytamoxifen treatment were fixed with 4% PFA at room temperature for 10 minutes.

Samples were incubated with primary antibodies (α-E2F4, α-Deup1 or relevant mouse or rabbit

IgG control antibodies) using immunofluorescence protocol described previously, followed by incubation with PLA probe set (anti-rabbit plus strand/anti-mouse minus strand) at 37°C for 2 hours. Ligation step was performed at 37°C for 30 minutes followed by a 100 minute amplification step. Subsequently, immunofluorescence staining for α-PCM1 antibody was performed. ProLong Gold antifade reagent (Life Technologies, P36930) was used as mounting media and images were captured using a confocal microscopy system (Carl Zeiss, LSM 710).

Immunoprecipitation (IP)

293FT cells were transiently transfected with the following plasmids, pcDNA mouse

E2F4, human SAS6 (Addgene, 46382), Flag-Deup1, Flag-Cep152, Flag-Plk4, Centrin-1 using lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, 11668019) to determine interaction between overexpressed proteins. To assess endogenous interaction, mTECs at ALI day 3 were utilized. Proteins were extracted by incubating cells at 4°C for 30 minutes in an

NP40 based lysis buffer (20mM Hepes pH 7.8, 15% glycerol, 250 mM KCl, 0.2mM EDTA, 1%

NP40, 50mM L-, 50mM L-arginine, 1mM DTT, 50mM NaF, 1mM sodium orthovandate (NaVO3), 0.1mM phenylmethanesulfonyl fluoride (PMSF), leupeptine and aprotinin) followed by centrifugation at 1000 rpm for 10 minutes at 4°C.

98 In immunoprecipiation experiments, 0.8-2 mg protein lysate that was precleared by incubation with protein A/G conjugated agarose beads (Santa Cruz, sc-2003, 50μl) for 30 minutes at 4°C, was incubated for 1-2 hours at 4°C with one of the following antibody conjugated beads; α-E2F4 agarose beads (Santa Cruz, sc-866 AC, 40μl), α-Flag magnetic beads

(Sigma, M8823, 30μl), α-SAS6 agarose beads (Santa Cruz, sc-81431 AC, 40μl). To determine specificity of the antibodies, we used, as negative control, anti-mouse or anti-rabbit IgG agarose beads corresponding to the species of each primary antibody (Santa Cruz, sc-2343; Santa Cruz, sc-2345). Following incubation, depending on the type of beads, the samples were either centrifuge at 800 rpm for 3 minutes or placed in magnets for 3 minutes to separate beads from protein lysate. Next beads were washed five times with 1ml lysis buffer to remove non-specific interactions. To elute the bound proteins from beads, 90μl of loading dye with 20% DTT was added on beads prior to boiling the samples for 8 minutes at 95°C.

Western blot Analysis

Quantification of proteins concentration was done using BCA protein assay kit

(ThermoFisher Scientific, 23250). Samples were loaded in 2X sample buffer (25mM Tris HCl pH 6.8, 4% SDS, 20% (v/v) glycerol and 0.004% bromophenol blue), separated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane at 80 volts for 2 hours at 4°C, and blocked in 8% nonfat milk for 2 hour at room temperature or overnight at 4°C. The following primary antibodies were used in 8% nonfat milk for 2 hours at room temperature or overnight at

4°C: α-E2F4 (LLF4.2, 1:500), α-Deup1 (produced in Dr Xueliang Zhu’s lab, 1:3000), α-SAS6

(Santa Cruz, 91.390.21, 1:1000) α-Flag (Sigma, F1804, 1:1000), α-Centrin (Santa Cruz, 1:1000).

HRP-conjugated mouse or rabbit secondary antibodies generated against native, disulphide form of IgG (Rockland Mouse Trueblot Ultra, 18-8817-33 and Rockland Rabbit Trueblot Ultra, 18-

99 8816-33) were used at 1:1000 dilution in 8% nonfat milk for 1 hour at room temperature. Usage of native secondary antibodies lowered the appearance of non-specific, immunoglobulin bands

(55 kDa heavy and ~23kDa light chain) arising due to denaturation of antibodies used for immunoprecipitation experiments. 80 μg of ALI culture input samples, 1 - 8 μg of overexpressed protein total protein lysates were loaded.

E2F4 mutant construct and lentiviral-mediated gene transduction

For rescue experiments, the lentiviral vectors that express 2X Flag-HA tagged wildtype

E2F4, nuclear export signal mutant (ΔNES) or a DNA binding domain mutant (ΔDBD, carrying two amino acid replacement in the DNA recognition domain, R56E and R57F) were generated.

The lentiviral vectors were transduced into packaging cell line HEK293. After packaging the virus, supernatant was concentrated by ultracentrifugation to around 0.5-1x109 PFU ml-1 titre.

Lentiviral gene transduction was performed at the time of plating (Day -7 ALI) by infecting cells with lentivirus at ~30 MOI in MTEC proliferation media supplemented with 5 ul of the RHO kinase inhibitor Y-27632 (Sigma) and media was changed a few days after transduction. Overall transduction was efficiency was 30-60%, based on mCherry expression or indirect immunofluorescence using α-HA tag antibodies.

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103

Chapter three

Biochemical analysis of complexes composed of E2F4 and the early components of centriole biogenesis during multiciliogenesis.

Renin Hazan, Paul S. Danielian, Munemasa Mori, Wellington V. Cardoso and Jacqueline A. Lees

R.H. designed the study, and conducted all the experiments, expect the rescue experiments. P.S.D. contributed to the cloning and immunoflourescence experiments. M.M. conducted the rescue experiments and imaged immunostained cells by fluorescent microscopy. R.H. wrote the chapter 3 manuscript and P.S.D. and J.A.L. revised the chapter 3 manuscript.

104 Abstract

E2F4, a member of the E2F family of transcription factors, is required for multiciliogenesis in the airway epithelium. We recently identified a novel cytoplasmic function for E2F4 in the assembly and maturation of deuterosomes, which mediate the massive de novo amplification of centrioles required to produce basal bodies, from which cilia are assembled.

Here we used co-expression and immunoprecipitation experiments to assess the interactions between E2F4 and deuterosome components. This showed that E2F4 associates with both

Deup1 and SAS6, two core components of the centriole replication machinery. Our work uncovered important information about the nature of these interactions. Firstly, Deup1 and

SAS6 use distinct structural motifs to bind E2F4, a coiled coil domain in Deup1 and the pisa domain/motif II in SAS6. Secondly, in contrast to SAS6, Deup1 selectively binds and likely stabilizes a highly phosphorylated form of E2F4 and is itself subject to an E2F4-dependent C- terminal cleavage. Finally, the same region of E2F4, residues 1-197, is both necessary and sufficient to bind to Deup1 and SAS6. Importantly, reconstitution experiments showed that the minimal Deup1 and SAS6 binding domain of E2F4 (E2F41-197) was able to perform the cytoplasmic function of E2F4 in multiciliogenesis as efficiently as full-length E2F4. We analyzed other E2F family members, and showed that E2F4’s ability to bind Deup1 and SAS6 was conserved in its closest relative E2F5, but not the more distant E2F1. This enabled us to use

E2F4/E2F1 chimeras to narrow down the Deup1 binding interface to residues 1-89, and subsequently to identify residues 48-53 as being critical for binding to both Deup1 and SAS6.

Taken together, these data strongly suggest that E2F4 and E2F5 play essential roles in multiciliogenesis via their interaction with these two core components of the centriole biogenesis machinery.

105 Introduction

Multiciliogenesis occurs in specialized terminally differentiated cells in higher vertebrates to generate multiciliated cells in specific organs. Directional beating of multicilia is essential for the circulation of cerebrospinal fluid (CSF) in adult brain, ovum and sperm transport in the reproductive organs and mucus clearance in lungs. Thus, defects in multiciliogenesis or in cilia motility can give rise to multiple disorders, including hydrocephalus, infertility and chronic respiratory infections resulting poor outcomes for patients (reviewed in Brooks & Wallingford,

2014).

Multiciliogenesis depends on the initiation of a specific transcriptional program in progenitor cells, followed by the massive amplification of centrioles, which form the basal bodies of cilia (reviewed in Lewis and Stracker, 2020). In proliferating cells, centriole replication is tightly linked to the cell cycle and only two centrioles are generated per cell cycle through the mother-centriole-dependent centriole biogenesis (reviewed in Nigg & Raff, 2009).

In contrast, in differentiating multiciliated cell progenitors, this duplication control is bypassed and numerous centrioles are generated “de novo”, predominantly without involvement of the mother centriole (Sorokin, 1968). Instead, electron dense structures termed “deuterosomes” mediate this massive centriole replication process (Sorokin, 1968; Kubo et al., 1999). As these centrioles mature, they move to the apical face of the membrane and form a central component of the basal bodies upon which cilia are assembled.

Many of the proteins known to play central roles in the mother centriole, including centrosomal protein 152 (Cep152), polo like kinase 4 (Plk4) and centriolar assembly protein 6

(SAS6) also function in the deuterosomes (Zhao et al., 2013). There is limited number of

106 procentriolar proteins that are specific for either the mother centriole or deuterosome complex.

The coiled-coil domain containing 78 (Ccdc78) is the first procentriolar protein shown to localize specifically to the deuterosome through studies in multiciliated cells of Xenopus and mouse tracheal epithelial cell cultures (MTECs) (Klos Dehring et al., 2013). Deuterosome assembly protein 1 (Deup1, also known as Ccdc67) is another key deuterosome specific protein that enables the assembly and maturation of deuterosomes by recruiting other core components like Cep152, Plk4 and SAS6 (Zhao et al., 2013). Notably, a Deup1’s paralogue centrosomal protein 63 (Cep63) plays a similar, specific role in the mother centriole (Brown et al., 2013; Sir et al., 2011).

The first indication that E2F4 regulates multiciliogenesis came from the finding that

E2f4-/- mice die of respiratory failure due to an absence of multiciliated cells in the nasal and airway epithelia (Danielian et al., 2007; Humbert et al., 2000). This, and subsequent, studies established that E2F4 and E2F5 have functionally redundant and widespread roles in multiciliogenesis. Specifically, the cilia defects of the E2f4-/- airway epithelium is recapitulated in E2f4+/-;E2f5-/- compound mutant mice, but not in either E2f4+/- or E2f5-/- mice (Danielian et al., 2007). Moreover, loss of E2f4 and heterozygosity of E2f5 (in E2F4f/f;E2F5+/-;VilCre mice), but not E2f4 loss alone, impairs multiciliogenesis in the efferent ducts (Danielian et al., 2016).

Multicilin and GemC1, both critical transcriptional co-regulators of multiciliogenesis were subsequently shown to associate with E2F4 or E2F5 in a ternary complex with DP1 and regulate transcriptional program during multiciliogenesis in Xenopus (Ma et al., 2014; Terré et al., 2016). We have used an air-liquid interface (ALI) assay to study the in vitro differentiation of murine tracheal epithelial cells into the multiciliated lineage (Mori et al., 2017). Consistent with the Xenopus studies, we found that nuclear E2F4 acts early in the differentiation process to

107 induce the transcriptional activation of multiciliogenesis regulators. Additionally, our study revealed that E2F4 shifts from the nucleus to the cytoplasm at later differentiation stages, and colocalizes with the early components of deuterosome/centriole replication machinery.

Importantly, reconstitution experiments in E2F4-deficient cells showed that the nuclear and cytoplasmic forms of E2F4 are both necessary for multiciliogenesis and the deuterosome components are unable to assemble in the absence of cytoplasmic E2F4 (Mori et al., 2017). In support of these results, we established that E2F4 can associate with Deup1, and also with SAS6 in transiently transfected cells (Mori et al., 2017). The association between E2F4 and Deup1 was also validated using endogenous proteins isolated from mouse tracheal precursor cells at day 3

ALI cultures (Mori et al., 2017).

Here we investigate the nature of the complexes to better understand E2F4’s role in the deuterosome complex and whether binding to the components of deuterosome complex is a prerequisite for E2F4 to conduct its cytoplasmic functions in multiciliogenesis.

108 Results

E2F4 binds to the deuterosome specific protein, Deup1

Based upon our colocalization experiments and the fact that Deup1 was proposed to be uniquely essential for centriole amplification, we first tested whether E2F4 could bind to Deup1.

For this, we expressed E2F4 and/or Flag tagged Deup1 in a human cell line, 293FT. As these cells are not programmed for multiciliogenesis, they have no endogenous Deup1 but do express other deuterosome components that are shared with the mother centriole, such as Cep152, Plk4, and SAS6. In immunoprecipitation-western blot experiments, Flag-Deup1 was clearly detected in anti-E2F4, but not control mouse IgG, immunoprecipitates only when the two proteins were co-expressed (Figure 1A). Similarly, the reciprocal anti-Flag IP recovered Flag-Deup1 and the co-expressed E2F4 (Figure 1B). Thus, this overexpression system allows formation and detection of the E2F4-Deup1 complex.

This experiment also revealed an unexpected finding that E2F4 and Deup1 both displayed gel mobility shifts when co-expressed. The E2F4 higher molecular weight species and

Deup1 lower molecular weight species were highly enriched when purified via immunoprecipitation of the associated proteins (Figures 1A and B). Thus, these data suggest that

E2F4 and Deup1 undergo post-translational modifications that result from, or are highly stabilized by, the association of these proteins.

The Deup1 paralogue, Cep63 shows no interaction with E2F4

The deuterosome specific protein, Deup1, has a paralogue, called Cep63, that functions specifically in mother-centriole (Sir et al., 2011; Zhao et al., 2013). We previously found that cytoplasmic E2F4 colocalizes with both deuterosome and the mother-centriole structures in the

109

A) Input IP: E2F4 IP: IgG E2F4 + - + + - + + - + Flag-Deup1 - + + - + + - + + 75 α-E2F4 E2F4

50 75 α-Flag Flag-Deup1

B) Input IP: Flag Deup1 e t E2F4 + - + + - + a o s n

Flag-Deup1 - - y + + + + l 75 α-Flag Flag-Deup1

75 α-E2F4 E2F4

50

Figure 1. E2F4 associates with Deup1. (A-B) Western blots with the indicated α-E2F4 or α-Flag antibodies were conducted on either the input lysates from 293FT cells overexpressing E2F4 and/or Flag-Deup1 or immunoprecipitates resulting from (A) α-E2F4 or (B) α-Flag antibodies, compared to the mouse IgG negative control. These data verified the association of E2F4 with Deup1.

110 progenitors of multiciliated cells (Mori et al., 2017). Considering this dual localization and the presence of coiled-coil domains within both Deup1 and Cep63 (Figure 2A), we tested whether

E2F4 also binds to Cep63. When expressed in 293FT cells, full-length Flag-Cep63 accumulated at much higher levels than full-length Flag-Deup1 (Figure 2B). Despite its high expression, and in clear contrast to Flag-Deup1, Flag-Cep63 was unable to co-precipitate E2F4 (Figure 2B).

E2F4 also binds to the deuterosome complex component, SAS6

Our prior immunofluorescence studies showed that E2F4 colocalizes with many other early deuterosome components, beyond Deup1, during deuterosome assembly (Mori et al.,

2017). Thus, we expanded our co-transfection studies to ask whether these other deuterosome complex members could also bind to E2F4. In the context of this assay, we saw no detectable interaction between E2F4 and Cep152, Plk4, Stil or PCM1 (data not shown) but clearly identified SAS6 as interacting with E2F4 (Figures 3A and B). First, cells transfected with mCherry tagged E2F4 and/or SAS6 were subjected to co-immunoprecipitation with anti-E2F4 or mouse IgG control antibodies. Western blotting with an anti-SAS6 antibody detected both the overexpressed SAS6, and endogenous SAS6, in the total cell lysate (Figure 3A) and showed that the exogenous SAS6 was clearly recovered in anti-E2F4, but not control anti-IgG, immunoprecipitates (Figure 3A). Second, reciprocal immunoprecipitation experiments of the same cell lysates showed that the SAS6-E2F4 complexes can be immunoprecipitated with antibodies against SAS6, and detected by western blotting with anti-E2F4 antibodies (Figure

3B). In both of these experiments, the interaction between E2F4 and SAS6 appears to be limited to the exogenously expressed SAS6 and not the endogenous SAS6. The reason for this selectivity is unclear, but may reflect the fact that endogenous SAS6 is localized within the mother-centriole and thus not free to interact with the overexpressed E2F4 at non-centrosomal

111

A)

E2F4 14 59 86 196 226 277 490 521 Deup1 1 535 +

131 341 401 543 Cep63 1 596 -

Coiled coil domain

B) Input IP: Flag e E2F4 - - - - t

+ + + + + + a s

Flag-Deup1 y - + + - - - + + - - l

Flag-Cep63 o - - - + + - - - + + n

α-Flag Deup1/Cep63

50

α-E2F4 E2F4 50

Figure 2. E2F4 interacts with Deup1 and not Cep63. (A) Schematic of Deup1 and its paralogue, Cep63. (B) Cell lysates containing E2F4 and either Flag tagged Deup1 or Cep63 were subjected to western blotting with α-Flag (upper panel) or α- E2F4 (lower panel) antibodies before (Input) or after immunoprecipitation (IP) with α-Flag antibodies. Western blots showed the recovery of E2F4 with Deup1, but not its paralogue, Cep63.

112

Figure 3. E2F4 associates with SAS6. (A-B) Western blots with the indicated α-E2F4 or α-SAS6 antibodies were conducted on either the input lysates from 293FT cells overexpressing E2F4 and/or SAS6 or the immunoprecipitates resulting from (A) α-E2F4 or (B) α-SAS6 antibodies, compared with the mouse IgG negative control. E2F4 and SAS6 can immunoprecipitate each other indicating a robust interaction. IgG HC denotes IgG heavy chain that are present in the α-SAS immunoblot. * denotes non-specific bands that are present in the α-SAS6 immunoblot.

113 regions where co-localization was previously observed. Finally, we noted that SAS6 interaction did not induce a mobility shift in E2F4, in contrast to interaction with Deup1.

N-terminal regions of Deup1 and SAS6 are necessary for interaction with E2F4

Having identified Deup1 and SAS6 as binding partners of E2F4, we next assessed which regions of Deup1 and SAS6 mediate their interaction with E2F4. Coiled-coils, which often mediate oligomerization, are present in both Deup1 (four domains in the Deup1 isoform used in our study) and SAS6 (a single central motif) (Strnad et al., 2007; Zhao et al., 2013). Thus, we used a deletion mutant strategy that was based roughly around these motifs, starting with Deup1

(Figures 4A). The N-terminal region of Deup1 (Deup11-129, residues 1-129), which includes the first coiled-coil domain of Deup1, immunoprecipitated E2F4 (Figure 4B). Notably, the association with Deup11-129 caused E2F4 to undergo the same molecular weight increase that is triggered by its interaction with full-length Deup1 (Figure 4B). In contrast, all other Deup1 mutants, including one with deletion of only the N-terminal 59 residues that comprise the first coiled-coil (Deup160-535), failed to immunoprecipitate E2F4 or trigger its mobility shift (Figures

4B and C). Based on these observations, we conclude that Deup1’s amino-terminal region including first coiled-coil domain (Deup11-129) is able to complex with E2F4 and induce, or greatly stabilize, its mobility shift.

There is significant homology between the minimal E2F4 binding region of mouse

Deup1 (residues 1-129) and residues 67-192 of mouse Cep63 (Figures 5 and 6A). This raised the possibility that E2F4 binding sequences are preserved in Cep63 but occluded by structural differences elsewhere in Cep63 that prevent full-length Cep63 from binding to E2F4 (Figures 5 and 6A). To address this, we tested the deletion mutant Cep6367-192 in our interaction assay but

114 A) Deup1 E2F4 14 59 86 196 226 277 490 521 WT 1 535 + 1-129 1 129 + 309-535 309 535 - 401-535 401 535 - 130-309 130 309 - 60-535 60 535 - 86-535 86 535 - Coiled coil domain

B) Input IP: Flag-Deup1

5 5 9 5 5 9 3 3 0 3 3 0 9 -5 -5 -3 9 -5 -5 -3 e 2 9 1 0 2 9 1 0 t Flag-Deup1 -1 0 0 3 -1 0 0 3 a 1 3 4 1 1 3 4 1 ys l o E2F4 + + + + + + + + n 37 25 25 Flag-Deup1 α-Flag 20 mutants 15

α-E2F4 E2F4 50

C) Input IP: Flag-Deup1

5 5 5 5 3 3 3 3 e -5 -5 -5 -5 t Flag-Deup1 T 0 6 T 0 6 a - W 6 8 - W 6 8 s ly o E2F4 + + + + + + + + n 75 Flag-Deup1 α-Flag mutants 50 *

α-E2F4 E2F4

50

Figure 4. Identification of the E2F4 interaction domain within Deup1. (A) Schematic representation of Flag tagged Deup1 and deletion mutants. (B-C) Cell lysates containing E2F4 and Flag-Deup1 mutants were subjected to western blotting with α-Flag (upper panel) or α-E2F4 (lower panel) antibodies before (Input) or after immunoprecipitation (IP) with α-Flag antibodies. Western blotting with α-E2F4 antibody showed only the Deup11-129 mutant recovers E2F4. Numbers in the diagram indicate amino acid positions. * denotes IgG bands that are present in the α-Flag immunoblot.

115

Fig. 9

Figure 5. Sequence alignment of mouse Deup1 (KC211185) and Cep63 (KC211186). Sequence alignment shows the conserved amino acids between mouse Deup1 and Cep63. The coiled-coil domains are highlighted in yellow. Deup1 and Cep63 share a high level of conserved amino acids in region necessary for E2F4 binding to Deup1 despite the absence of shared coiled- coil domains.

116 A) E2F4 14 59 86 196 226 277 490 521 Deup1 1 535 +

Deup1 (1-129) 1 129 +

131 341 401 543 Cep63 1 596 -

Cep63 (67-192) 67 192 -

Coiled coil domain

B) Input IP: Flag e t

E2F4 + - + - + + - + - + a s

Flag-Deup1(1-129) y - + + - - - + + - - l

Flag-Cep63 (67-192) o

- - - + + - - - + + n 20 Deup1 1-129 α-Flag 15 Cep63 67-192

70 α-E2F4 E2F4 50 *

Figure 6. E2F4 interacts with amino terminal region of Deup1 and not Cep63. (A) Schematic of full-length and truncated versions of Deup1 and its paralogue, Cep63. (B) Cell lysates containing E2F4 and either truncated versions of Flag tagged Deup1 or Cep63 were subjected to western blotting with α-Flag (upper panel) or α-E2F4 (lower panel) antibodies before (Input) or after immunoprecipitation (IP) with α-Flag antibodies. Western blot with α- E2F4 showed that the N-terminal region of Cep63 (residues 67-192) does not co- immunoprecipitate E2F4 unlike N-terminal region of Deup1. * denotes the heavy chain of IgG.

117 again failed to detect any association with E2F4 (Figure 6B). Thus, we conclude that E2F4 interacts specifically with Deup1 and not Cep63.

We next examined a panel of SAS6 mutants (Figure 7A) for their ability to interact with

E2F4, and found that neither the fragment containing the sole coiled-coil domain (SAS6160-505) nor the C-terminal region (SAS6499-656) immunoprecipitated with E2F4 (Figure 7B). In contrast,

E2F4 interaction mapped to the amino-terminal region of SAS6 (SAS61-175; Figures 7A and B).

Interestingly, SAS61-175 encompasses both the structurally unique pisa motif (residues 39-91) and a second motif, (residues 123-140) named motif II (Figure 7A), which together constitute a continuous, highly conserved patch that is required for head to head dimerization of SAS6 dimers (Breugel et al., 2011). Interestingly, specific SAS6 point mutations have been identified within the pisa motif (I62T), in members of a consanguineous family afflicted with autosomal recessive primary microcephaly, a classic ciliopathy phenotype (Khan et al., 2014), and within motif II (F131D) as preventing multimerization of SAS6 dimers to generate the nine-fold cartwhell structure of SAS6 (van Breugel et al., 2011; Kitagawa et al., 2011). Thus, we generated these point mutants in SAS6 and asked whether they influenced the E2F4-SAS6 interaction. While SASI62T associated with E2F4 as well as wildtype E2F4, SAS6F131D failed to co-immunoprecipitate, arguing that multimerization of SAS6 is required for E2F4 interaction

(Figure 7C).

Partially overlapping domains of E2F4 mediate its association with Deup1 and SAS6

The E2F4 domains necessary for binding to DNA, its DP dimerization partners and other key regulators (e.g. the pocket protein family members) are well characterized. To further investigate the E2F4’s interaction with the deuterosome components, and thus its contribution to

118 A)

SAS6 E2F4 39 91 123 140 166 471 WT 1 656 + 1-175 1 175 + 160-505 160 505 - 499-656 499 656 - Coiled coil domain Pisa domain Motif II

B) Input IP: Flag-SAS6 5 6 5 6 0 5 0 5 5 5 6 5 5 6 te 7 - - 7 - - a 0 9 0 9 s Flag-SAS6 -1 6 9 -1 6 9 y - 1 1 4 - 1 1 4 l o E2F4 + + + + + + + + n 75 50 Flag-SAS6 α-Flag 37 mutants 25 20 *

15 α-E2F4 E2F4 50

C) Input IP: HA-E2F4 D D D D 1 1 1 1 T 3 T 3 T 3 T 3 T 2 1 T 2 1 T 2 1 T 2 1 Flag-SAS6 W I6 F W I6 F W I6 F W I6 F HA-E2F4 - - - + + + - - - + + + 75 α-HA HA-E2F4 50 100 75 Flag-SAS6 α-Flag point mutants

Figure 7. Identification of the E2F4 interaction domain within SAS6. (A) Schematic representation of Flag tagged SAS6 mutants. (B) Cell lysates containing E2F4 and Flag tagged SAS6 truncation mutants were subjected to western blotting with α-Flag or α- E2F4 antibodies before (Input) or after immunoprecipitation (IP) with α-Flag antibodies. Western blotting showed association of E2F4 with the SAS61-175 truncation mutant. (C) Cell lysates overexpressing E2F4 and SAS6 full-length or its point mutants (I62T or F131D) were subjected western blotting with α-HA or α-Flag antibodies before (Input) or after immunoprecipitation (IP) with HA antibodies. SAS6I62T, but not SAS6F131D mutant was recovered with E2F4. Single letters indicate amino acid identity. Numbers in the diagram indicate amino acid positions. * denotes IgG bands that are present in the anti-Flag immunoblots.

119 multiciliogenesis, we proceeded to identify the interaction domain(s). For this, we generated a set of plasmids encoding HA tagged truncation mutants of E2F4 whose boundaries were guided by the known E2F4 functional domains. We then expressed these proteins in 293FT cells either alone or with Flag-Deup1 or SAS6. Since differences in cellular localization could prevent the association, we first confirmed by immunofluorescence that these mutants all displayed some degree of cytoplasmic localization (Figures 8-10), which is the known site of interaction between endogenous E2F4 and the deuterosomes (Mori et al., 2017).

Having verified this subcellular localization, we then proceeded with the interaction mapping, beginning with Deup1 (Figure 11A). Initially, 293FT cells were transfected with HA tagged E2F4 mutants that retained either various N-terminal (HA-E2F41-100, HA-E2F41-130, HA-

E2F41-158 and HA-E2F411-197) or C-terminal (HA-E2F4198-410) regions in the absence or presence of Flag-Deup1. The resulting lysates were immunoprecipitated with an anti-Flag antibody and western blotted for E2F4 (Figure 11B). This showed that only HA tagged E2F41-197, which includes the DNA binding and dimerization/marked box domains of E2F4, could be recovered in the Flag-Deup1 immunoprecipitates (Figure 11B). To further map the minimal interaction domain, we generated three HA tagged internal E2F4 deletion mutants; HA-E2F4Δ101-197 (retains residues 1-100 and 198-410), HA-E2F4Δ137-197 (retains residues 1-136 and 198-410) and HA-

E2F4Δ101-137 (retains residues 1-100 and 138-410). Notably, we could screen for these mutants using both anti-HA and anti-E2F4 antibodies, which recognize sequences within the C-terminal half of E2F4. When co-expressed with Flag-Deup1, all three of these internal deletion mutants were detected in the anti-Flag-Deup1 immunoprecipitates with the anti-E2F4 antibody (Figure

11C), but not the anti-HA antibody (data not shown). This difference is not due to the loss of the

HA-tag, as the E2F4 internal mutants are all detected in the total lysate by the anti-HA antibody.

120 DAPI HA Merge 4 F 2 E - A H 4 ) F 0 2 0 1 E - - 1 A (

H 4 ) F 7 2 9 E 1 - - 1 A ( H ) 4 0 F 1 2 4 - E - 8 9 A 1 H ( ) 4 0 F 1 2 4 E - - 6 A 9 ( H 0 4 0 F 2 2 - E 0 - 0 A 1 H Δ

Figure 8. Subcellular localization of E2F4 mutants. 293FT cells expressing the indicated E2F4 mutants were immunostained with antibodies against the HA tag. Representative fields of cells are shown. Nuclear DAPI staining in blue, and HA tagged mutants, red signal.

121 DAPI DEUP1 HA Merge 4 F 2 E - A H 4 ) F 0 2 0 E 1 - - 1 A ( H 4 ) F 7 2 9 E 1 - - 1 A ( H ) 4 0 F 1 2 4 - E - 8 9 A 1 H ( ) 4 0 F 1 2 4 E - - 6 A 9 ( H

Figure 9. Subcellular localization of E2F4 mutants in the presence of Flag tagged Deup1. 293FT cells expressing Deup1 and the indicated E2F4 mutants were immunostained with antibodies against the HA tag of the E2F4 mutants and the Flag tag of Deup1. Representative fields of cells are shown. Nuclear DAPI staining in blue, Flag tagged Deup1, green signal and HA tagged mutants, red signal.

122 DAPI SAS6 HA Merge 4 F 2 E - A H 4 ) F 0 2 0 E 1 - - 1 A ( H 4 ) F 7 2 9 E 1 - - 1 A ( H ) 4 0 F 1 2 4 - E - 8 9 A 1 H ( ) 4 0 F 1 2 4 E - - 6 A 9 ( H 0 4 0 F 2 2 - E 0 - 0 A 1 H Δ

Figure 10. Subcellular localization of E2F4 mutants in the presence of Flag tagged SAS6. 293FT cells expressing SAS6 and the indicated E2F4 mutant were immunostained with antibodies against the HA tag of the E2F4 mutants or Flag tag of SAS6. Representative fields of cells are shown. Nuclear DAPI staining in blue, Flag tagged SAS6, green signal and HA tagged E2F4 mutants, red signal.

123 E2F4 Deup1 A) WT 1 DBD DD TA 410 + 1-100 1 DBD 100 - 1-130 1 DBD 130 - 1-158 1 DBD 158 - 1-197 1 DBD DD 197 + 198-410 198 TA 410 - Δ101-197 1 DBD 100 198 TA 410 + Δ137-197 1 DBD 136 198 TA 410 + Δ101-137 1 DBD TA 410 + 100 138

DNA binding Dimerization Transactivation Nuclear export signal

B) Input IP: Flag-Deup1 0 0 0 0 1 1 1 1 0 0 8 7 -4 0 0 8 7 -4 0 0 8 7 -4 0 0 8 7 -4 0 3 5 9 8 0 3 5 9 8 0 3 5 9 8 0 3 5 9 8 -1 -1 -1 -1 9 -1 -1 -1 -1 9 -1 -1 -1 -1 9 -1 -1 -1 -1 9 HA-E2F4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Flag-Deup1 - - - - - + + + + + - - - - - + + + + + 75 α-Flag Flag-Deup1

37 HA-E2F4 α-HA mutants 25 * 20

15

C) Input IP: Flag-Deup1 7 7 7 7 7 7 7 7 7 7 7 7 9 3 9 9 3 9 9 3 9 9 3 9 1 1 1 0 1 1 1 0 1 1 1 0 1 1 1 0 - - - 1 - - - 1 - - - 1 - - - 1 e 1 1 7 -4 1 1 7 -4 1 1 7 -4 1 1 7 -4 t 0 0 3 8 0 0 3 8 T 0 0 3 8 0 0 3 8 T a HA-E2F4 1 1 1 9 1 1 1 9 1 1 1 9 1 1 1 9 s

Δ Δ Δ 1 Δ Δ Δ 1 W Δ Δ Δ 1 Δ Δ Δ 1 W y l

Flag-Deup1 - - - - + + + + + - - - - + + + + + o n 75 α-Deup1 Flag-Deup1

HA-E2F4 α-E2F4 50 mutants

37

Figure 11. Identification of the domains in E2F4 necessary for interaction with Deup1. (A) Schematic of the HA tagged E2F4 mutants and their association with Deup1. (B) Cell lysates expressing the indicated HA tagged E2F4 mutants alone or with Flag-Deup1 were subjected to western blotting with the indicated α-Flag or α-HA, antibodies before (Input) or after immunoprecipitation (IP) with an antibody against the Flag tag of Deup1. Western blotting showed that Flag-Deup1 co-immunoprecipitates only the HA-E2F41-197 mutant. (C) For further mapping, lysates transfected with indicated HA tagged E2F4 internal deletion mutants alone or with Flag-Deup1 were subjected to immunoprecipitation using α-Flag antibody. Western blotting with an α-E2F4 antibody showed E2F4 mutants containing the DNA binding domain and C-terminal region also bind to Deup1. Numbers in the diagram indicate amino acid positions. * denotes IgG bands that are observed in the α-HA immunoblots.

124 Instead, we believe that reflects the fact that the anti-E2F4 antibody has a higher avidity than the anti-HA antibody. Our data do not rule out the possibility that E2F4’s carboxy-terminal sequences facilitate folding or provide some additional Deup1 interaction points. However, they show that E2F41-197 is the minimal region necessary and sufficient for Deup1 association in this co-expression assay, and argue that residues 1-100 appear to contain the major Deup1 interaction sites.

We next mapped the SAS6 binding domain of E2F4, to determine whether Deup1 and

SAS6 bind to the same or distinct regions. First, SAS6 was co-expressed with or without various

HA tagged E2F4 N- and C-terminal deletion mutants and then immunoprecipitated with an anti-

HA antibody (Figure 12A). Western blotting for SAS6 showed that it was recovered in immunoprecipitates of E2F41-197, but not any of the smaller N-terminal fragments E2F41-100,

E2F41-130 and E2F41-158 or the C-terminal fragment E2F4198-410 (Figure 12B). Thus, SAS6’s selectivity for these mutants mirrored that of Deup1 (Figure 11B). Given this finding, we also tested SAS6’s ability to interact with the E2F4 internal deletion mutants (Figure 12C). To allow direct comparison of E2F4’s Deup1 and SAS6 binding sites, we conducted this experiment using exactly the same antibodies and epitopes as for the E2F4-Deup1 experiment. Specifically, we expressed E2F4 internal deletion mutants with or without a Flag tagged version of SAS6, immunoprecipitated SAS6 with the anti-Flag antibody and then screened for E2F4 mutants using anti-E2F4 antibody. In contrast to Deup1, we did not detect any interaction between SAS6 and

E2F4Δ101-197, E2F4Δ137-197 and E2F4Δ101-137 (Figure 12C). Based on our data, we conclude that N- terminal region of E2F4 (E2F41-197), including the DNA binding and dimerization/marked box domains, is both necessary and sufficient to mediate efficient interaction with both Deup1 and

SAS6. However, our data argue that Deup1 and SAS6 do possess subtle differences in either

125 E2F4 SAS6 A) WT 1 DBD DD TA 410 + 1-100 1 DBD 100 - 1-130 1 DBD 130 - 1-158 1 DBD 158 - 1-197 1 DBD DD 197 + 198-410 198 TA 410 - Δ101-197 1 DBD 100 198 TA 410 - Δ137-197 1 DBD 136 198 TA 410 - Δ101-137 1 DBD TA 410 - 100 138

DNA binding Dimerization Transactivation Nuclear export signal

B) Input IP: α-HA 0 0 1 1 0 0 8 7 4 0 0 8 7 4 te - 0 3 5 9 - a 0 3 5 9 8 8 s HA-E2F4 -1 -1 -1 -1 9 -1 -1 -1 -1 9 y - 1 1 1 1 1 - 1 1 1 1 1 l o SAS6 + + + + + + + + + + + + n

37 HA-E2F4 α-HA 25 mutants

20

15

α-SAS6 75 SAS6

C) Input IP: Flag-SAS6 7 7 7 7 7 7 7 7 7 7 7 7 9 3 9 0 9 3 9 0 9 3 9 0 9 3 9 0 -1 -1 -1 1 -1 -1 -1 1 -1 -1 -1 1 -1 -1 -1 1 1 1 7 -4 1 1 7 -4 1 1 7 -4 1 1 7 -4 e T 0 0 3 8 T 0 0 3 8 T 0 0 3 8 T 0 0 3 8 t HA-E2F4 1 1 1 9 1 1 1 9 1 1 1 9 1 1 1 9 sa W Δ Δ Δ 1 W Δ Δ Δ 1 W Δ Δ Δ 1 W Δ Δ Δ 1 y l o Flag-SAS6 - - - - - + + + + + - - - - - + + + + + n

α-Flag 75 Flag-SAS6

75

HA-E2F4 α-E2F4 mutants 50

37

Figure 12. Identification of the domains in E2F4 necessary for interaction with SAS6. (A) Schematic representation of the HA tagged E2F4 mutants and their association with SAS6. (B) Cell lysates with SAS6 and the indicated HA tagged E2F4 mutants were subjected to western blotting with α-HA or α-SAS6 antibodies before (Input) or after immunoprecipitation (IP) with (B) α-HA or (C) α-Flag antibodies. Only the N-terminal region of E2F4 (1-197) is sufficient to bind to SAS6. Internal deletion of E2F4 sequences between 101-197 abrogates SAS6 binding implicating this region as being essential for efficient interaction, in contrast to Deup1. Numbers in the diagram indicate amino acid positions. Δ denotes the residues that are deleted from the full-length.

126 their specific binding site requirements and/or affinity for E2F4, at least in the context of our transfection assay.

E2F4 binding to Deup1 leads to post-translational modifications in both proteins.

Our experiments unexpectedly revealed gel mobility shifts for both E2F4 and Deup1 when co-expressed (Figure 1B). A similar mobility shift of E2F4 was observed when the amino- terminal region of Deup1 (Deup11-129) was used in place of full-length Deup1 (Figure 4B). This observation was specific for E2F4-Deup1 association, as similar changes were not detected following association with SAS6 (Figure 3B). We asked whether phosphorylation might account for, or contribute to, the observed mobility shifts by conducting in vitro phosphatase assays.

First, we recovered the Flag-Deup1/E2F4 complex through immunoprecipitation with anti-Flag antibodies and then incubated the immunoprecipitates with lambda protein phosphatase.

Western blotting with anti-E2F4 antibody showed that the phosphatase eliminated the higher molecular weight form of E2F4 and produced two distinct bands of intermediate and lower molecular weight (Figure 13A). Protein phosphatase treatment caused a similar downshift for full-length E2F4 that was immunoprecipitated with Flag tagged Deup11-129 (Figure 13B). We wondered whether this phosphorylation mapped to the amino-terminal 1-197 residues that constituted the Deup1 binding site and thus repeated this analysis on immunoprecipitated Flag-

Deup1/HA-E2F41-197 complexes. In contrast to full-length E2F4, we did not observe any mobility shift of HA tagged E2F41-197 and the lambda protein phosphatase had no detectable effect (Figure 13C). We therefore conclude that co-expression of E2F4 with Deup1 promotes or stabilizes phosphorylation, likely within the C-terminal domain, and that Deup1 preferentially binds to this phospho-E2F4 species.

127 A) IP: Flag E2F4 + + + + Flag-Deup1 - + - +

α-E2F4 E2F4

- - + + Phosphatase Treatment

B) Input IP: α-Flag Flag-Deup1 (1-129) - + + - - + + + + E2F4 + - + + + - - + + Flag-Deup1 α-Deup1 1-129

α-E2F4 E2F4

- + - + - + Phosphatase treatment

C) Input IP: α-Flag Flag-Deup1 - + + - + + - + + HA-E2F4 (1-197) + - + + - + + - + α-Deup1 Flag-Deup1

α-HA HA-E2F4 1-197 - - - + + + Phosphatase treatment

Figure 13. Phosphorylation of E2F4 contributes to the gel mobility shift detected upon E2F4-Deup1 association. (A) Cell lysates from transfected cells were subjected to western blotting with α-E2F4 antibodies after immunoprecipitation (IP) with α-Flag antibodies. These immunoprecipitates were subjected to lambda protein phosphatase treatment as indicated. (B-C) Cell lysates expressing the indicated (B) E2F4 and/or Flag tagged Deup11-129 or (C) HA tagged E2F41-197 and/or Flag tagged Deup1 were subjected to western blotting with the indicated α-Deup1, α-E2F4 and α-HA tag antibodies before (Input) or after immunoprecipitation (IP) with α-Flag antibodies and then treatment with or without lambda protein phosphatase as indicated. The shift in E2F4’s size following full-length or N-terminal (1-129) Deup1 interaction is reduced following phosphatase treatment and this shift is dependent upon the C-terminus of E2F4 as it is not observed in E2F41- 197.

128 We then examined the nature of the downward mobility shift of Deup1 that exist within

Flag-Deup1/E2F4 complex. In this case, we found that lower molecular weight Deup1 species seem to be largely unaffected from lambda protein phosphatase treatment (Figure 14A). Thus, we hypothesized that protein cleavage, rather than amino acid modification, might be responsible for the reduction in Deup1 molecular weight. To test this possibility, we generated a Deup1 construct with distinct tags at the N- (Flag) and C- (HA) termini and co-expressed the resulting

Flag-Deup1-HA protein with or without E2F4. When Flag-Deup1-HA protein was co-expressed with E2F4, we again observed the lower mobility Deup1 species, which was specifically detected by the anti-Flag, but not the anti-HA antibody (Figure 14B). In the same experiments, both antibodies efficiently detected the higher mobility Flag-Deup1 species (Figure 14B). This suggests that the lower molecular weight Deup1 species that are generated in the presence of

E2F4, and selectively associate with E2F4, result from the removal of C-terminal Deup1 sequences. Collectively, these data indicate that formation of the E2F4-Deup1 complex is associated with mobility shifts in both proteins that are partially caused by the phosphorylation of E2F4 and the removal of carboxy-terminal sequences from Deup1.

E2F4 1-197ΔDBD is sufficient for the cytoplasmic function of E2F4 in multiciliogenesis.

We have previously shown that co-expression of nuclear (E2F4ΔNES) and cytoplasmic

(E2F4ΔDBD) E2F4 was sufficient to rescue multiciliogenesis in an ALI assay using E2f4 knockout derived tracheal cells, confirming the essential contribution of both nuclear and cytoplasmic forms of E2F4 to multiciliogenesis (Mori et al., 2017). In this current study, we have shown that

E2F41-197 is sufficient to associate with both Deup1 and SAS6, two components of the early centriole biogenesis pathway. Thus, we wanted to determine whether this region of E2F4 is

129 A) Input Flag-Deup1 + + + + E2F4 - - + +

75

α-Flag Flag-Deup1

50 - + - + Phosphatase Treatment

B) Input IP: α-E2F4

E2F4 + - + + - + Flag-Deup1-HA - + + - + +

α-E2F4 E2F4

50

75 α-Flag Flag-Deup1-HA

50

75 α-HA Flag-Deup1-HA

50

Figure 14. Co-expression of Deup1 and E2F4 leads to the C-terminal cleavage of Deup1. (A) Cell lysates expressing Flag tagged Deup1 alone or with E2F4 were subjected to western blotting with Flag tag antibodies before and after treatment with lambda protein phosphatase. (B) Western blotting with the indicated α-E2F4, α-Flag or α-HA antibodies was performed using cell lysates expressing the indicated E2F4 and/or double tagged Deup1 (an amino terminal Flag tag and a C-terminal HA tag) before (input) or after immunoprecipitation (IP) with α-E2F4 antibodies. This showed that the C-terminal tag is lost following co-expression of E2F4, suggesting that the mobility change in Deup1 after co-expression with E2F4 includes cleavage of the carboxy-terminal domain.

130 sufficient to perform the cytoplasmic function of E2F4 in multiciliogenesis. To address this, we generated a non-DNA binding variant of this N-terminal fragment, E2F41-197ΔDBD, and transduced this into adult mouse tracheal progenitors derived from E2F4f/f;R26CreERT/+ mice, with or without HA-E2F4 ΔNES. The cells were then treated with 4-hydroxytamoxifen, to knockout the endogenous E2f4, and subjected to ALI assays. Consistent with our prior report (Mori et al.,

2017), co-expression of full-length HA-E2F4ΔDBD and HA-E2F4ΔNES were able to restore the formation of multicilia in the E2F4-deficient cells, as detected with antibodies against acetylated-

α-tubulin (Figure 15A, upper panels). As expected, expression of E2F41-197ΔDBD alone was insufficient to rescue the multicilia defect (Figure 15A, middle panels). However, co-expression of HA-E2F41-197ΔDBD and HA-E2F4ΔNES restored multicilia formation in E2f4 null precursor cells

(Figure 15A, lower panels). Indeed, quantification of the fraction of HA-positive cells with

1-197ΔDBD ΔDBD multicilia showed that HA-E2F4 was just as effective as the full-length HA-E2F4 in this assay (Figure 15B). Thus, we conclude that E2F41-197 is sufficient to perform E2F4’s cytoplasmic role in multiciliogenesis, presumably via interaction with Deup1 and/or SAS6.

Deup1 binds to specifically to E2F4 and E2F5, but not E2F1

The highest degree of homology between E2F family members maps to the DNA binding and dimerization/marked box domains that also serve as the Deup1 and SAS6 interacting region.

Thus, we used our 293FT co-transfection assay to test whether Deup1 and SAS6 interact with other members of the E2F family. For this analysis, we selected E2F5, which is closely related to E2F4 and plays an overlapping role in multiciliogenesis (Danielian et al., 2007, 2016), and

E2F1, which is the archetypal member of the activating E2F subgroup (Figure 16A). This analysis showed that Flag-Deup1 was able to co-immunoprecipitate HA tagged E2F5, but failed to bind to HA tagged E2F1 (Figure 16B). Moreover, SAS6 also co-precipitated E2F5,

131 A) F-actin HA α-tubulin Merge S D E B N D Δ

Δ

4 4 F F 2 2 E E - - A A H H +

D B D Δ

7 9 1 e - 1 n

o 4 l F a 2 E - A H

D S B E D N Δ

Δ

7 9 4 1 - F 1

2 4 E F - 2 A E H - + A H

B) n.s. * * * * * * d e t a i l i c i t l u m

%

S D S E B E N D N Δ Δ Δ 7 4 9 4 1 - F 1 F 2 2 E 4 E - F - A 2 A H E H - + A + D D B H B D D Δ Δ 7 4 9 1 - F 1 2 E 4 - F A 2 H E - A H

Figure 15. E2F41-197ΔDBD is sufficient for the cytoplasmic function of E2F4 in multiciliogenesis. E2F4f/f; R26CreERT2/+ airway epithelial progenitors were transduced with E2F4ΔDBD, E2F41-197ΔDBD and/or E2F4ΔNES as indicated, treated with 4-hydroxytamoxifen to delete the endogenous E2F4 and then subjected to the ALI differentiation assay and subsequent confocal imaging. Expression of the E2F4 constructs is detected by α-HA tag staining. (A) Multiciliated cells develop upon co-expression of E2F4ΔDBD and E2F4ΔNES as shown by staining with the cilia marker α-tubulin (upper panel). Expression of E2F41-197ΔDBD alone fails to produce cilia (middle panel). E2F41-197ΔDBD and E2F4ΔNES co-expression restores multiciliogenesis, as verified by staining with α-tubulin staining (lower panel). (B) Quantitation of the frequency of HA positive cells in ALI cultures that are multiciliated. (n = 2 experiments and a minimum of 200 cells counted per condition.) These data show E2F41-197ΔDBD rescued the multiciliogenesis defect of the E2f4-deleted cells as efficient as the full-length E2F4ΔDBD.

132 A) E2F family members Deup1 SAS6 E2F4 1 DBD DD TA 410 + + E2F5 1 DBD DD TA 346 + + E2F1 1 DBD DD TA 437 - -

DNA binding Dimerization Transactivation Nuclear export signal

Input IP: Flag-Deup1

B) Flag-Deup1 e + - + - + + - + - + t a

HA-E2F5 - + + - - - + + - - s y l

HA-E2F1 - - - + + - - - + + o n 75 α-Flag Flag-Deup1

E2F1 α-HA 50 E2F5

Input IP: HA

C) e SAS6 + - + - + + - + - + t a

HA-E2F5 ------s + + + + y l

HA-E2F1 - - - + + - - - + + o n

E2F1 α-HA 50 E2F5

75 α-SAS6 SAS6

Figure 16. Association of Deup1 and SAS6 with other E2F members. (A) Schematic representation of the full-length E2F4, E2F5 and E2F1. (B) Cell lysates containing the indicated HA tagged E2F species with or without Flag-Deup1 were subjected to western blotting with the indicted α-Flag and α-HA antibodies before (Input) or after immunoprecipitation (IP) to recover Flag-Deup1 and associated E2Fs. Immunoprecipitated Flag-Deup1 recovered HA tagged E2F5, but not E2F1. (C) Cell lysates overexpressing indicated HA tagged E2F species with or without SAS6 were subjected to western blotting with indicated α-HA or α- SAS6 antibodies before (Input) or after immunoprecipitation (IP) with α-HA antibodies. HA tagged E2F5, but not E2F1, was able to co-immunoprecipitate SAS6.

133 and not E2F1 (Figure 16C). The ability of Deup1 and SAS6 to associate with E2F4 and E2F5, but not E2F1, has the potential to explain the specific, and biological redundant roles of E2F4 and E2F5 in multiciliogenesis.

Residues 48-53 of E2F4 is necessary for its binding to Deup1 and SAS6

Since both E2F4 and E2F5 can interact with Deup1 and SAS6 but E2F1 cannot, we generated a series of E2F4-E2F1 chimeras with the goal of narrowing down the specific region(s) of E2F4 that are required for these interactions. This strategy was predicted on the notion that the 3D structure might be important in maintaining the integrity and/or presentation of the interacting motifs and thus the junctions in the chimeras were positioned in linker regions within the E2F4 structure (Figure 17A; Liban et al., 2017; Zhang et al., 1999). These mutants were tested in our 293FT co-transfection assay for their ability to co-immunoprecipitate with

Flag-Deup1 (Figure 17B). Chimera 1-4-4, which carries the DNA binding domain of E2F1

(residues 121-198) and the dimerization/marked box domains of E2F4 (residues 90-197), did not bind to Deup1, while chimeras 4-1-1 or 4-4-1 showed partial or strong binding, respectively

(Figure 17B). These data argue that E2F41-89 is the main interaction region of E2F4, in direct agreement with the internal E2F4 mutant analyses (Figure 11C).

To identify more specific interaction site candidates, we compared the sequence of residues 1-89 of E2F4 from various species with the analogous sequences from E2F1, E2F2 and

E2F3. Regions that were unique to E2F4 were then mapped in silico onto the E2F4 3D structure to assess whether they were potential interaction sites on the surface of the protein (Zhang et al.,

1999). Based on this analysis, three E2F4 mutants (M1, M2 and M1+2) were generated by swapping E2F448-53 (DTLAVR) with E2F1158-162 (EVLKV) and/or E2F484-92 (VGPGCNTRE) with E2F1193-198 (SHTTVG) in the context of either the N-terminal fragment, E2F41-197 or

134 B) Input IP: Flag-Deup1 A) M1 M2 e HA-E2F4/1 -4 -1 -4 -1 -1 -4 -1 -1 -4 -1 -4 -1 -1 -4 -1 -1 t 4 1 4 1 4 4 1 4 4 1 4 1 4 4 1 4 a 4-4-4 E2F4 1-197 ------s chimeras 4 1 1 4 4 1 4 4 4 1 1 4 4 1 4 4 y l Flag Deup1 ------o 1-1-1 E2F1 121-301 + + + + + + + + + + n

1-4-4 E2F1 121-198 E2F4 90-197 Deup1 75

4-1-1 E2F4 1-89 E2F1 199-301 37 4-4-1 E2F4 1-148 F1 255-301 E2F4/F1 chimeras (α-HA) 25

C) D) E) Input IP: HA M1 e 2 2 t + + a T 1 2 1 T 1 2 1 s HA-E2F4 1-197 y mE2F4 - W M M M - W M M M l o hE2F5 DP1 + + + + + + + + + + n hE2F1 E2F41-197 M2 mutants 25 (α-HA) 20 mE2F4 75 hE2F5 hE2F1 DP1 50

Input IP: FLAG-Deup1 F) 2 2 2 2 + + + + te T T T T a HA-E2F4 1-197 1 2 1 1 2 1 1 2 1 1 2 1 s W M M M W M M M W M M M W M M M y l o Flag-Deup1 - - - - + + + + - - - - + + + + n 75 α-Deup1 Flag-Deup1

α-E2F4 E2F41-197 mutants 25

G) Input IP: FLAG-Deup1 2 2 2 2 e T + T + T + T + t HA-E2F4 FL 1 2 1 1 2 1 1 2 1 1 2 1 sa W M M M W M M M W M M M W M M M y l o Flag-Deup1 - - - - + + + + - - - - + + + + n

75 α-Deup1 Flag-Deup1

75 α-E2F4 E2F4 mutants

H) Input IP: FLAG-SAS6

2 2 2 2 + + + + te HA-E2F4 T 1 2 1 T 1 2 1 T 1 2 1 T 1 2 1 a W M M M W M M M W M M M W M M M s ly o Flag-SAS6 - - - - + + + + - - - - + + + + n

α-Flag 75 Flag-SAS6

75

α-E2F4 E2F4 mutants

50

135 full-length E2F4 (Figures 17C and D). We first established that these small sequence swaps had no detectable effect on E2F4’s ability to dimerize with DP1 (Figure 17E), showing that these mutants retain their 3D structure. We then conducted immunoprecipitation experiments to determine their ability to bind to Deup1 and SAS6. The M2 mutation alone either showed a weak impact on the binding of E2F41-197 to Deup1 and full-length E2F4 to SAS6 (Figures 17F and H), or no effect on the ability of full-length E2F4 to co-immunoprecipitate with Flag-Deup1

(Figure G). In contrast, the M1 mutation, either alone or combination with M2, completely abolished association of E2F41-197 to Deup1 and full-length E2F4 to SAS6 (Figures 17F and H) and greatly impaired association of full-length E2F4 to Deup1 (Figure 17G). Thus, these experiments show that residues 48-53 of E2F4 are critical for efficient binding to Deup1 and

SAS6.

------Figure 17. A short region of E2F4 (aa 48-53) is necessary for efficient binding to Deup1 and SAS6. (A) Schematic representation of E2F41-197, E2F1121-301 and the E2F4/E2F1 chimeras. Red lines show the positions of the M1 and M2 mutations. (B) Cell lysates containing the HA tagged E2F species/chimeras, alone or with Flag tagged Deup1 were subjected to western blotting with the indicated α-Deup1 and α-HA antibodies before (Input) or after immunoprecipitation (IP) with α- Flag antibodies. These experiments show that the N-terminus of E2F4 (E2F41-89) is critical for efficient binding to Deup1. (C) Amino acid alignments of E2F4 (mouse), E2F5 (human) and E2F1 (human), with the red rectangle indicating the regions of E2F4 swapped with those from E2F1 in the M1 and M2 mutations. (D) The crystal structure of E2F4 DNA binding domain showing the M1 mutation site, in red. The M2 mutation site is an unstructured carboxy-terminal region that was not resolved in the crystal structure. (E) Western blots with the indicated α-HA and α-DP1 antibodies were conducted on either the input lysates from cells overexpressing DP1 alone or with HA tagged E2F41-197 or its mutant forms and the immunoprecipitates resulting from α-HA antibodies showing that all E2F4 mutants efficiently dimerize with DP1. (F, G, H) Cell lysates with the indicated HA tagged (G, H) full-length or (F) amino terminal mutants of E2F4 expressed alone or with (F, G) Flag tagged Deup1 or (H) Flag tagged SAS6 were subjected to western blotting with the indicated antibodies before (Input) or after immunoprecipitation (IP) with α-Flag antibodies. This shows that the M1 mutation greatly impairs or completely abolishes the ability of E2F4 to associate with Deup1 and SAS6.

136 Discussion

Using an air-liquid interface (ALI) in vitro differentiation system we previously established a novel role for cytoplasmic E2F4 in the assembly and maturation of centriole biogenesis complexes required for multiciliogenesis (Mori et al., 2017). Here, we utilized transfection into a non-multiciliated cell line to assess the interactions between E2F4 and the early components of deuterosome complex. This showed that E2F4 can physically associate with Deup1 and also SAS6, but not with other core deuterosome components that colocalize with

E2F4 in ALI cultures including Cep152, Plk4, Centrin1 or PCM1. These negative results do not rule out the participation of these proteins in higher order cytoplasmic E2F4 complexes. Indeed, based upon colocalization, we believe that such complexes do exist in multiciliogenesis.

However, our data suggest that the ability of Cep152, Plk4, Centrin1 and PCM1 to associate with

E2F4 requires bridging proteins and/or post-translational modifications that exist in multiciliated cell precursors, but not our transfection-based assay.

Our mapping studies also provide insight into the interaction mechanisms and potential multimeric nature of the E2F4-deutoresome complexes. We determined that Deup1 and SAS6 use structurally distinct amino terminal domains to bind E2F4. For Deup1, E2F4 binding maps to residues 1-129, which contains a coiled coil domain. In contrast, SAS6 possesses a coiled- domain but this is fully dispensable for E2F4 binding. Instead, SAS6 uses residues 1-175, which contain a structurally unique pisa motif (residues 39-91) and motif II (residues 123-140) that together constitute a continuous, highly conserved patch that is required for SAS6 homodimers to multimerize, via head-to-head contacts, to form the mature 9-fold SAS6 cartwheel structure

(van Breugel et al., 2011). To further probe the requirements for SAS6 to bind E2F4, we tested the effects of two previously reported SAS6 mutants, I62T and F131D. The I62T mutation was

137 found in members of a consanguineous family afflicted with autosomal recessive primary microcephaly, a classic ciliopathy syndrome (Khan et al., 2014) and it did not alter SAS6’s binding to E2F4. In contrast, the F131D mutation, a synthetic mutation within motif II, abolished SAS6’s binding to E2F4. Notably, F131D is known to prevent the multimerization of

SAS6 dimers (van Breugel et al., 2011; Kitagawa et al., 2011), while I62T maps outside of the head to head binding interface. The differential E2F4 binding ability of these two mutants suggests that E2F4 specifically targets the multimerization region of SAS6 and/or that SAS6 multimerization is required for its interaction with E2F4. In the first scenario, it seems possible that E2F4 might bind, and then facilitate multimerization, of the SAS6 dimers. At the other extreme, the second scenario raises the possibility that the E2F4 binding interface might only exist in the multimerized form of SAS6.

Our analyses of E2F4 deletion mutants showed that the smallest fragment that is necessary and sufficient for binding to Deup1, and also to SAS6, is the amino terminal sequences

1-197. We had previously used the ALI differentiation assay to show that the presence of both nuclear and cytoplasmic forms of E2F4 was required to rescue multiciliogenesis defect of E2f4- deficient tracheal cells (Mori et al., 2017). Using this same assay, we now show that E2F41-197 is able to mediate the cytoplasmic function of E2F4 in multiciliogenesis as efficiently as the full- length E2F4 protein. This established that the minimal region required to bind Deup1 and SAS6, is also sufficient to mediate E2F4’s cytoplasmic role in multiciliogenesis.

Our analyses also revealed that E2F4 and Deup1 both undergo significant size changes when co-expressed, and these new species are specifically enriched in the resulting E2F4-Deup1 complex. In the case of Deup1, we see a smaller molecular weight species that results from the loss of carboxy-terminal sequences, suggesting cleavage of Deup1 may enable or stabilize its

138 interaction with E2F4 or occur as a consequence of complex formation. For E2F4, we find that

Deup1 binds specifically to a higher molecular weight E2F4 species, and not other E2F4 forms.

Phosphatase treatment eliminates this upper E2F4 species and yields two distinct lower molecular weight E2F4 bands, establishing involvement of phosphorylation and arguing for two

(or more) distinct phosphorylation events. We note that the smallest phosphatase-produced

E2F4 form is still somewhat larger than the smallest E2F4 species in the whole cell lysate. It is unclear whether this reflects an accessibility issue for the phosphatase, or the existence of another (i.e. non-phosphorylation) post-translational modification. Notably, the phosphorylation of E2F4 is induced by either full-length Deup1 or Deup11-129, which contains the minimal E2F4 interaction site, but not triggered by SAS6. This strongly suggests E2F4 phosphorylation is triggered as a consequence of Deup1 binding, or is stabilized by E2F4/Deup1 complex formation. Interestingly, Deup1 binds to E2F41-197 but does not cause its phosphorylation, arguing that this modification requires the presence of E2F4’s C-terminal region and/or occurs within this region. Given this finding, we conclude that phosphorylation is not creating the

Deup1 binding interface directly, but it could facilitate a conformation change within the full- length E2F4 that exposes this domain.

Our finding that residues 1-197 mediated E2F4’s cytoplasmic multiciliogenesis role was somewhat unexpected, as this region also contains the DNA binding and dimerization/marked box domains, which are highly conserved between E2F family members. Despite this conservation, we found that Deup1 and SAS6 binding is limited to E2F4 and its closest homolog

E2F5, and not shared by the canonical activating E2F, E2F1. Thus, the amino-terminal domains of these two E2F subclasses have evolved to create differential roles in multiciliogenesis, without materially altering their DNA binding or dimerization activities.

139 We used the differential binding specificity of E2F4 and E2F1 to narrow down E2F4’s

Deup1 and SAS6 binding regions through the generation of chimeras. This showed that E2F4 residues 1-89 are particularly important for Deup1 interaction. Moreover, we found that swapping residues 48-53 of E2F4 with the corresponding, and non-homologous, region of E2F1 highly diminished binding to both Deup1 and SAS6. Importantly, this sequence swap did not alter E2F4’s ability to bind to DP1, arguing that this reflects a targeted event, not a more general perturbation of E2F4’s structure. Collectively, these data show that Deup1 and SAS6 bind to

E2F4 in highly overlapping ways with some subtle differences. These observations raise questions about the possibility of higher order complexes. In our prior in vivo studies, we observed co-localization of E2F4, SAS6 and Deup1. We cannot rule the possibility that E2F4 can bind simultaneously to Deup1 and SAS6. However, based on their shared requirement of residues 48-53, we favor the notion that E2F4 binds to Deup1 and SAS6 in a mutually exclusive manner. Indeed, we failed to detect a trimolecular complex between E2F4, Deup1 and SAS6 when we co-expressed these three proteins in our transfection assay (data not shown). This raises the possibility that E2F4 somehow facilitates an ordering of multiciliogenesis events through the sequential binding of Deup1 versus SAS6.

140 Materials and Method

Cell culture

Cells were grown at 37°C in 10% CO2-containing incubators. 293FT cells were maintained in Dulbecco modified Eagle medium (DMEM, sigma) supplemented with heat inactivated, 10% fetal bovine serum (FBS, HyClone) and 5% penicillin/streptomycin (P/S,

Corning, MT30002Cl). HEK293T and U2OS cells were maintained in DMEM supplemented with 10% FBS, 5% P/S and 5% L-glutamine (GE Healthcare, SH30034).

Cloning

HA tagged E2F4 truncation mutants; HA-E2F41-100, HA-E2F41-130, HA-E2F41-158, HA-

E2F41-197 and HA-E2F4198-410 and HA tagged internal E2F4 deletion mutants; HA-E2F4Δ101-197

(retains residues 1-100 plus 198-410), HA-E2F4Δ137-197 (retains residues 1-136 plus 198-410) and

E2F4Δ101-137 (retains residues 1-100 plus 138-410) were generated using PCR cloning techniques starting with pCMVSPORT2 murine E2F4 plasmid. DNA sequencing verified the sequences of plasmids. Plasmid expressing Flag tagged Deup1 full-length and its truncation mutants (Deup11-

129, Deup1309-535, Deup1401-535 and Deup1130-309), Flag tagged Cep62, Flag tagged Cep152, Flag tagged Plk4 were kind gifts of Dr. Xueliang Zhu’s lab. pEGFP-hPCM1 and hPCM1-EGFP plasmids are kind gifts of Dr. Song-Hai Shi. Flag tagged Deup60-535, Deup186-535 and Cep6367-

192 plasmids were cloned into pcDNA3.1 in our lab. Amplification of Deup1 using forward primer encoding Flag tag and reverse primer encoding HA tag allowed generation of double tagged Deup1 construct (pcDNA3.1-Flag-Deup1-HA). SAS6 plasmid (pENTR-age-Hs-

SAS6) was purchased from Addgene (46381). Amino-terminus Flag tagged SAS6 truncation mutants; Flag-SAS61-175, Flag-SAS6160-505 and Flag-SAS6499-656 were generated in our lab by

141 PCR amplification of human SAS6 gene from pENTR-age-Hs-SAS6 plasmid using primers including Flag tag sequences and cloning into pcDNA3.1 plasmid. Flag tagged SAS6 point mutants (SAS6I62T and SAS6F131D) were cloned in our lab using QuikChange II XL-site directed mutagenesis kit (Agilent Technologies, 200521). To generate pcDNA3.1-SAS6

I62T, following primer pair was used:

Forward 5’- CCATTTTTTTTATATAACCTTGTTACATCTGAGGAAGATTTTCAAAG

Reverse 5’- CTTTGAAAATCTTCCTCAGATGTAACAAGGTTATATAAAAAAAATGG

To generate pcDNA3.1-SAS6 F131D, following primer pair was used:

Forward 5’-GGTAGAGACAAATCCTGATAAGCATCTTACACACC

Reverse 5’- GGTGTGTAAGATGCTTATCAGGATTTGTCTCTACC

To express HA tagged DP1 and DP2, pCMV-Neo-Bam-HA-DP1 and pCMV-Neo-Bam-HA-

DP2 plasmids, previously generated in our lab, were used. To express HA tagged E2F5 and

E2F1, pCMV-Neo-Bam-HA-E2F5 and pCMV-Neo-Bam-HA-E2F1 plasmids that were present in our lab were used. Plasmids expressing HA tagged E2F4/E2F1 chimeras were generated using Gibson assembly cloning kit (New England BioLabs, E5510S). The details of the chimeras and primers that are used in Gibson cloning can be found in Table 1.

Immunoprecipitation (IP) In order to determine E2F4’s interaction deuterosome complex components co- immunoprecipitation experiments were carried on lysate of 293FT cells that were transiently transfected with plasmids expressing mCherry or HA tagged E2F4, Flag tagged Deup1, SAS6,

Flag tagged Cep63 as indicated using lipofectamine 2000 transfection reagent (Thermo Fisher

Scientific, 11668019). To map domain necessary for interaction, cell lysates of 293FT cells transfected with either HA tagged E2F truncation mutants, Flag tagged SAS6 truncation mutants

142 or Flag tagged Deup1 truncation mutants were utilized. To check the presence/absence of DP proteins in E2F4 complex, co-immunoprecipiation experiments were carried on lysate expressing

Flag tagged Deup1 or SAS6, together with either HA-DP1 or HA-DP2 in the absence or presence of E2F4. Cell lysates overexpressing E2F5 or E2F1 with or without Deup1 or SAS6 were used to in immunoprecipitation experiments to check the interaction of centriole proteins with other E2F family members.

Proteins were extracted by incubating collected 293FT cells at 4°C for 30 minutes in an freshly prepared NP40 based lysis buffer (20mM Hepes, pH 7.8, 15% glycerol, 250 mM potassium chloride (KCl), 0.2mM ethylenediaminetetraacetic acid (EDTA), 1% NP40, 50mM L- glutamic acid, 50mM L-arginine, 1mM dithiothreitol (DTT), 50mM sodium fluoride (NaF),

1mM sodium orthovandate (NaVO3), 0.1mM phenylmethanesulfonyl fluoride (PMSF), 1ug/ml leupeptine and 1ug/ml aprotinin) followed by centrifugation at 1000 rpm for 10 minutes at 4°C.

In immunoprecipiation experiments, 0.8-2 mg protein lysate that was pre-cleared by incubation with protein A/G agarose beads (Santa Cruz) for 30 minutes at 4°C, was incubated for one to two hours at 4°C with one of the following antibody conjugated beads; α-E2F4 agarose beads (Santa Cruz, sc-866 AC, 40μl), α-Flag magnetic beads (Sigma, M8823, 30ul), α-SAS6 agarose beads (Santa Cruz, 40μl). To determine specificity of the antibodies, we used, as negative control, anti-mouse or anti-rabbit IgG agarose beads corresponding to the species of each primary antibody (Santa Cruz, sc-2343; Santa Cruz, sc-2345). Following incubation, depending on the type of beads, the samples were either centrifuge at 800 rpm for 3 minutes or placed in magnets for 3 minutes to separate beads from protein lysate. Next beads were washed up to five times with 1ml lysis buffer to remove non-specific interactions. In order to elute the

143 bound proteins from IP beads, 90 μl of 2X loading buffer containing 20% DTT was added to samples prior to boiling for 8 minutes at 95°C.

Western blot Analysis

Quantification of proteins concentration was done using BCA protein assay kit

(ThermoFisher Scientific, 23250). Samples were loaded in 2X sample buffer (25mM TRIS, HCl pH 6.8, 4% SDS, 20% (v/v) glycerol and 0.004% bromophenol blue), separated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane at 80 volts for two hours at 4°C, and blocked in 8% nonfat milk for two hours at room temperature or overnight at 4°C. The following primary antibodies were used in 8% nonfat milk for two hours at room temperature or overnight at 4°C: α-E2F4 (LLF4.2, 1:500), α-Deup1 (produced in Dr Xueliang Zhu’s lab,

1:3000), α-SAS6 (Santa Cruz, sc-81431, 1:1000) α-Flag (Sigma, F1804, 1:1000), α-HA

(Roche12CA5, 1:2000). HRP-conjugated mouse or rabbit secondary antibodies generated against native, disulphide form of IgG (Rockland Mouse Trueblot Ultra, 18-8817-33 and

Rockland Rabbit Trueblot Ultra, 18-8816-33) were used at 1:1000-1:5000 dilutions in 8% nonfat milk for 1 hour at room temperature. Usage of native secondary antibodies lowered the appearance of non-specific, immunoglobulin bands (55 kDa heavy and ~23kDa light chain) arising due to denaturation of antibodies used for immunoprecipitation experiments. 1 - 8 μg of overexpressed protein total protein lysates were loaded.

Immunoflourescence

U2OS cells were plated at low density gelatin coated glass coverslips. Next day, cells were transiently transfected with HA tagged truncation mutants alone or with Flag tagged Deup1 or SAS6. Cells 48 hr following transfection were washed with PBS then fixed in 4%

144 paraformaldehyde (PFA) for 10 minutes. These cells were then incubated in PBS 50mM ammonium chloride for 10 minutes, washed then permeabilized with 0.25% Triton X-100 in

PBS. The cells were blocked in 5% goat serum in 0.2% Tween20/PBS for 30 minutes at 37°C.

The following primary antibodies were used 1/1000 dilution in goat serum dilution buffer for one hour; α-HA (Roche, 12CA5) α-Flag (Cell Signaling, 2368). Secondary antibodies (Thermo

Fisher, Alexafluor, FITC or Texas Red conjugated) were used in 1/1000 dilution in goat serum dilution buffer 1ug/ml DAPI for one hour in the dark and washed with 1x PBS 3 times.

Following the last wash step, coverslips were washed with water, one drop of mounting media was placed on the glass slide, the coverslip was placed on mounting media (Vector Labs,

Vectashield H1000) and sealed with nail polish. Zeiss axioplan II upright microscopy in Koch

Institute microscope facility was used to capture images.

Air-liquid interphase cultures of airway epithelial progenitors

Airway epithelial progenitors were isolated from E2F4f/f; R26CreERT2 adult mouse tracheas, transduced with virus, treated with 4-hydroxytamoxifen and allowed to differentiate into multiciliated cells in air-liquid interphase (ALI) cultures as described previously (Mori et al.,

2017). Briefly, mouse tracheas were harvested, and treated with 0.5% pronase overnight. Next day, isolated airway epithelial progenitor cells were cultured on collagen1-coated Transwell dishes (Corning) under submerged conditions in media that allowed proliferation of airway progenitors until confluence (7 days). Multiciliogenesis was initiated by exposure of the apical side of culture to air (ALI Day 0) and culturing cells in differentiating media (mTEC/serum free,

RA media) up to 8 days (ALI Day 8). To initiate E2F4 loss, E2F4f/f;R26CreERT precursor cells were treated with 1μM 4-hydroxytamoxifen (Tm) from day -5 to day 0.

145

Chimera 144: PCR reaction 1 AGGCCGGGCCACAGGCGCCGCCGTCCCCGGGGGAGAAGTCACG Forward Chimera 144: PCR reaction 1 AGCTTGTCAGCGATCTCCCGGGTGCCCACTGTGGTGTGGCTGC Reverse Chimera 144: PCR reaction 2 ACCCGGGAGATCGCTGACAAGC Forward Chimera 144: PCR reaction 2 CGGCGGCGCCTGTGGC Reverse Chimera 411: PCR reaction 1 AGGCCGGGCCACAGGCGCCGCCGTCCCCGGGGGAGAAGTCACGC Forward Chimera 411: PCR reaction 1 ATACTCTAGAGCGGCCGTCACTCAGGGCACAGGAAAACATCGATCG Reverse Chimera 411: PCR reaction 2 TGACGGCCGCTCTAGAGTATCCC Forward Chimera 411: PCR reaction 2 ATTGCAACCTGGCCCGACGC Reverse Chimera 441: PCR reaction 1 GAAGACATCTGCAGATGCGCAGACCCTGCAGAGCAGATGG Forward Chimera 441: PCR reaction 1 ATACTCTAGAGCGGCCGTCACTCAGGGCACAGGAAAACATCGATCG Reverse Chimera 441: PCR reaction 2 TGACGGCCGCTCTAGAGTATCCC Forward Chimera 441: PCR reaction 2 GCATCTGCAGATGTCTTCATGAGTCACG Reverse

Table 1. Designates the name and the sequences of PCR primers that are generated for Gibson cloning chimeras of E2F4/1.

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148

Chapter four:

DISCUSSION

149 The E2F family of transcription factors is highly conserved throughout evolution and

E2Fs have a wide range of regulatory functions in proliferation, cell cycle progression, apoptosis, development, and cell fate determination (reviewed in Dimova & Dyson, 2005; Kent and Leone,

2019). E2F4 and the closely related E2F5, initially classified as transcriptional repressors at cell cycle target genes, have been shown to play key roles in multiciliogenesis (Arbi et al., 2016;

Danielian et al., 2007, 2016; Ma et al., 2014). Initially, the molecular analysis of how E2F4 functions in this pathway was limited due to the postnatal lethality of E2F4-deficient mice and the consequent difficulty of isolating, manipulating and studying the molecular biology of airway epithelia in vivo. The use of air-liquid interface (ALI) cultures, which allow in vitro proliferation and differentiation of the airway epithelial precursor cells isolated from murine tracheas (You and Brody, 2013), allowed us to investigate the molecular basis of E2F4's role in multiciliogenesis in a rigorous manner.

The transcriptional role of E2F4 in the centriole biogenesis

Our analyses showed that E2F4 is nuclear early in the differentiation of airway epithelial precursor cells in ALI cultures. To understand the contribution of nuclear E2F4 to the transcriptional program during multiciliogenesis in mouse airway epithelia, we conducted whole- genome expression profiling of wildtype versus E2f4 deficient airway epithelial progenitors prior to and during, their differentiation into multiciliated cells (ALI day 0, 2 and 4). This analysis revealed that E2F4 is required for the appropriate transcriptional upregulation of genes involved in centriole biogenesis, including Deup1 (Ccdc67), Plk4, Stil, and Sas6. This set of genes has extensive overlap with the gene set identified as targets of E2F4 in RNA sequencing (RNA-Seq) analysis of skin progenitors isolated form Xenopus embryos expressing multicilin (a critical

150 transcriptional regulator of multiciliogenesis bound by E2F4) alone or with dominant negative form of E2F4 (Ma et al., 2014). In this study, ChIP-sequencing analysis of Xenopus skin cells expressing E2F4-GFP in the presence or absence of multicilin suggested that multicilin expression directs E2F4 away from the promoters of cell cycle genes, to a new set of promoters belonging to genes required for multiciliated cell differentiation including those encoding core centriole replication factors (Ma et al., 2014). Similar to multicilin, GemC1, a homolog of multicilin, transcriptionally regulates the multiciliated cell differentiation program in murine oviduct and trachea and also binds to human E2F4/DP or E2F5/DP heterodimers (Arbi et al.,

2016; Kyrousi et al., 2015; Lu et al., 2019; Terré et al., 2016, 2019; Zhou et at., 2015). These studies suggest that E2F4 or E2F5, together with DP, act in a complex with either multicilin and/or GemC1 to induce the transcriptional activation of genes required for multiciliogenesis.

To date, genome-wide ChIP analysis has not been performed to investigate the presence of complexes composed of E2F4/5 with multicilin and/or GemC1 on the promoters of E2F responsive genes during multiciliogenesis. In our whole-genome analysis, we observed down- regulation of both GemC1 and multicilin following the loss of E2F4 in the ALI differentiation assay, suggesting that E2F4 acts upstream of these regulators. Thus, further studies are needed to understand the components of the E2F4 complexes that regulate GemC1 and multicilin transcription versus the centriole biogenesis genes. Additionally, it is not known how the binding of multicilin and GemC1 to E2F4/5 alters the target gene preference of the E2F-DP complexes. E2F4 responsive sites in cell cycle genes and centriole component genes share similar sequences suggesting that E2F4 does not select promoters based purely on their E2F binding site sequence. We speculate that interaction of multicilin or GemC1 with centriole gene

151 specific regulators and subsequent changes in chromosomal accessibility could alter the availability of E2F binding sites.

The roles of E2F4 and E2F5 in centriole biogenesis and multiciliogenesis are highly conserved

The transcriptional roles of E2F4 and E2F5 in complex with DP proteins in centriole biogenesis were first observed in Xenopus and murine models. Expression of a dominant negative E2F4 or E2F5 in Xenopus embryos resulted in the failure to form multiciliated cells due to defects on transcriptional regulation of genes essential for centriole assembly (Ma et al.,

2014). Knockout of E2f4 in mice resulted in a loss of multiciliated cells from the airway epithelia and their replacement by secretory cells and, interestingly, this phenotype was also observed in E2f4+/-;E2f5-/- double mutants, but not in E2f4+/- or E2f5-/- single mutants (Danielian et al., 2007). Furthermore, multiciliated cells were absent from the efferent ducts of

E2f4f/f;E2f5+/-;Vil-Cre double mutant male mice, but not E2f4f/f;Vil-Cre single mutants, and present at reduced numbers in E2f5-/- mutants indicating functionally redundant roles for these two E2Fs in multiciliogenesis (Danielian et al., 2016).

Recent studies indicate the conservation of the transcriptional roles of E2F4 and E2F5 in centriole biogenesis in Caenorhabditis elegans and Danio rerio (Miller et al., 2016; Stracker,

2019). In C. elegans embryos, the E2F orthologue EFL-1 and its partner DPL-1 was shown to transcriptionally regulate centriole duplication (Miller et al., 2016). Many of the genes encoding core centriole duplication factors including spindle-defective protein 2 (spd-2), probable serine- threonine protein kinase (zyg-1), spindle assembly abnormal protein 5 (sas5) and spindle assembly abnormal protein 6 (sas6), contain putative EFL-1/DPL-1 binding sites in their

152 promoters and their expression is enhanced by EFL-1/DPL-1 (Miller et al., 2016). Interestingly,

EFL-1 or DPL-1 loss suppresses centriole defect of Plk4-deficient embryos by stabilizing SAS6, suggesting EFL-1 has indirect repressor role in centriole biogenesis (Miller et al., 2016).

In D. rerio embryos, E2f5 is required for multiciliated cell formation in the pronephric duct and E2F4 and E2F5 function redundantly in multiciliogenesis in the nasal placode (Chong et al., 2018, Xie et al., 2020). The lost expression of multicilin and foxj1b from the pronephric duct, or both foxj isoforms from the nasal placode, within E2f4+/-;E2f5-/- embryos validates these

E2F members’ role in the multiciliogenesis transcriptional program (Chong et al., 2018). The ability of zebrafish GemC1 to both increase expression of multicilin, foxj1a and fox1b and interact with zebrafish E2F4 and E2F5 strongly suggests that GemC1 and E2F4 or E2F5 also work as a complex to regulate multiciliated cell differentiation in D. rerio (Chong et al., 2018).

Overall, these results suggest the transcriptional regulatory function of E2Fs and DP complexes in centriole biogenesis is not limited to vertebrate organisms, such as D. rerio, X. leavis and mice, bearing multiciliated tissues, but is evolutionary conserved in lower organisms like C. elegans.

The roles of cytoplasmic E2F4 in multiciliogenesis

Our analysis of ALI cultures established that E2F4 undergoes a nucleocytoplasmic transition at the early stages of the multiciliogenesis (stage 2) and accumulates at the apical side of the cells. At the primary cilium, E2F4 was found in ring-like structures with Cep63, Cep152,

Plk4 and C-Nap1. Additionally, E2F4 was present in non-centrosomal regions, being part of ring-like structures with Cep152, centrin and the deuterosome-specific protein Deup1, but not its paralogue Cep63, which functions specifically in the mother-centriole-dependent centriole

153 duplication. The E2F4 staining pattern was distinct from the primary cilia staining pattern and resembled that of the fibrous granules; an electron-dense structure identified by electron microscopy, only present early in differentiation at regions where deuterosome complexes arise

(Kubo et al., 1999; Zhao et al., 2013). Thus, we analyzed the locations of fibrous granules and deuterosome complexes, with respect to the location of cytoplasmic E2F4. We observed that as

E2F4 begins to accumulate at the apical side of the cell, it co-localizes with PCM1, a marker of fibrous granules, and subsequently immature Deup1-containing deuterosomes arise in areas rich in PCM1 and E2F4. At later stages, mature deuterosomes with larger rings are observed to leave the E2F4-PCM1 cloud. These data suggest that E2F4, similar to fibrous granules, functions in the assembly and maturation of the deuterosome complexes.

Importantly, having established that E2F4 has two separate functions in multiciliogenesis in airway epithelia, reconstitution experiments were conducted by transducing wildtype, nuclear and/or cytoplasmic variants of E2F4 into mouse tracheal progenitors that are mutant for E2f4 and then tested in ALI differentiation assays. These experiments clearly showed that both the transcriptional and cytoplasmic roles of E2F4 are essential for multiciliogenesis in murine airway epithelia.

E2F4’s interactions with Deup1 and SAS6

We discovered that E2F4 associates with the deuterosome-specific protein, Deup1, in vivo by conducting co-immunoprecipitation experiments on lysates of day 3 ALI cultures. We showed that we could recapitulate the E2F4-Deup1 interaction by transiently expressing these proteins into 293-FT cells. Moreover, by screening other early components of centriole biogenesis pathway in this assay, we established that E2F4 also interacts with SAS6, a

154 centriolar protein which functions in both the mother-centriole and deuterosome-dependent centriole biogenesis pathways.

Our analysis of the E2F4 binding domains of Deup1 and SAS6 showed that these two early components of deuterosome complex use structurally distinct domains to bind E2F4. The murine Deup1 variant we used in our overexpression system is composed of four coiled-coil domains and E2F4 binding was mapped to Deup1’s amino-terminal region (Deup11-129) that includes the first coiled-coil domain. In contrast, the single coiled-coil domain of SAS6, which is involved in the formation of SAS6 homodimers, is fully dispensable for E2F4 binding and instead SAS6’s amino-terminal domain facilitates the interaction with E2F4. Interestingly, the formation of nine-fold symmetrical cartwheel structure of SAS6 homodimers is known to depend on the N-terminal regions of SAS6 (van Breugel et al., 2011; Kitagawa et al., 2011).

Mutagenesis analysis has identified specific SAS6 residues within the pisa motif (residues 39-91) and motif II (residues 123-140) that are located amino terminal binding interface and necessary for multimerization (van Breugel et al., 2011). We determined that the F131D mutation, that was previously shown to impair the head to head interaction required for multimerization (van

Breugel et al., 2011), abolishes SAS6’s binding to E2F4, while the I62T mutation, that was identified in patients with microcephaly and is located in pisa domain, does not alter SAS6’s binding to E2F4. Based on the SAS6 crystal structure, I62 seems to reside outside the head to head binding interface (van Breugel et al., 2011). Thus, the specific effect of F131D, but not

I62T, argues that the multimerization surface of SAS6 is important for its interaction with E2F4.

There are several possible explanations for this finding: E2F4 may bind directly at this site; E2F4 may specifically recognize the multimerized form of SAS6; or the mislocalization of the SAS6 dimers, which are known to result from the absence of multimerization (van Breugel et al.,

155 2011), may preclude E2F4 binding. Interestingly, the I62T mutation was discovered in members of a consanguineous family afflicted with autosomal recessive primary microcephaly, a classic ciliopathy syndrome (Khan et al., 2014). This is a classic ciliopathy syndrome that results from defects in primary ciliogenesis. In contrast, our data clearly show that primary ciliogenesis is not affected by loss of E2F4, either alone or in combination with E2F5 (unpublished data), even though these proteins are critical for multiciliogenesis. Collectively, these differential biological roles, and effects of the I62T mutation, suggest that SAS6 regulates primary ciliogenesis and

E2F4 binding, and thus potentially multiciliogenesis, through functionally separable mechanisms.

E2F41-197 is sufficient to bind to Deup1 and SAS6, and also enable multiciliogenesis

We mapped the domains of E2F4 necessary for its interaction with Deup1 and SAS6 by conducting co-immunoprecipitation experiments using lysates of cells transiently transfected with HA tagged E2F4 truncation mutants in the presence or absence of Flag tagged Deup1 or

SAS6. E2F41-197, which includes the DNA binding and dimerization/marked box domains, was identified as the smallest deletion that showed binding to both Deup1 and SAS6, as no association was detected with any of the smaller N-terminal or the C-terminal fragments. To determine whether the E2F4’s ability to promote multiciliogenesis also maps to this region, we conducted rescue experiments in which a DNA binding defective version of the amino terminal domains of E2F4 (E2F41-197ΔDBD) was co-expressed with full-length nuclear E2F4 (E2F4ΔNES) in

E2F4-deficient tracheal precursor cells. When subjected to the ALI assay, we found that these two proteins were sufficient to promote the development of multiciliated cells, indicating that

156 E2F41-197 is both necessary for Deup 1 and SAS6 binding, and sufficient to mediate E2F4’s cytoplasmic role in multiciliogenesis.

The residues 48-53 of E2F4 are necessary for binding to Deup1 and SAS6

We used two additional strategies to further map the E2F4 sequences required for Deup1 and SAS6 binding. First, we generated internal deletion mutants E2F4Δ101-197, E2F4Δ101-137 and

E2F4Δ137-197, which contain the DNA binding domain (residues 1-100) and the carboxy-terminal domain (residues 198-410) but lack all or part of the dimerization/marked box domains. When we analyzed the immunoprecipitated complexes with an anti-E2F4 monoclonal antibody (which has higher affinity than the anti-HA antibody), we detected binding to Deup1, but not SAS6.

Second, having established that Deup1 and SAS6 binding is a specific property of E2F4 and

E2F5, but not E2F1, we generated a series of E2F4-E2F1 chimeras in which we exchanged various regions of E2F41-197 with the corresponding regions of E2F1, based on the crystal structure. This strategy also identified E2F41-89 as the critical region for Deup1 binding. By swapping smaller fragments of E2F4 with E2F1, we identified residues 48-53 of E2F4 as being critical for binding to both Deup1 and SAS6 (the M1 mutation). Collectively, our data argue that

Deup1 and SAS6 possess significant similarities, but also some subtle differences, in their specific binding site requirements and/or affinity for E2F4, at least in the context of our transfection assay.

Currently we do not know whether E2F4’s cytoplasmic function depends fully on its interaction with Deup1 and SAS6. To further probe the necessity of the Deup1 and SAS6 interaction in E2F4’s role in multiciliogenesis, the DNA binding defective E2F4 bearing the M1 mutation could be co-expressed with nuclear E2F4 in E2F4-deficient tracheal cells and then tested in the ALI differentiation system. Absence of rescue of multiciliogenesis would prove the

157 necessity of E2F4 residues 48-53 in multiciliogenesis as well as binding to Deup1 and SAS6.

On the other hand, a rescue in multiciliogenesis would suggest that cytoplasmic E2F4 has other, as yet unidentified, binding partners that can enable its function in the absence of interaction with

Deup1 and SAS6.

Post-translational modification in both E2F4 and Deup1

Our mammalian transfection assays also revealed the existence of mobility shifts in both

E2F4 and Deup1 when co-immunoprecipitated, yet no such change occurring with E2F4 and

SAS6. The mobility shift of E2F4 was largely reversed when the co-immunoprecipitated Deup1 and E2F4 complexes were treated with lambda protein phosphatase, indicating that phosphorylated E2F4 is preferentially bound to Deup1. The absence of mobility shift in E2F41-

197 fragment indicated that this phosphorylation maps within, or is dependent upon the C- terminal domain, and it is not necessary for the specific association of E2F4 with Deup1.

Previous studies have shown that E2F4’s subcellular localization and function is highly influenced by the specific phosphorylation events. ERK kinase dependent phosphorylation of

E2F4 at serine 244 and serine 384 sites and p38MAP dependent phosphorylation at threonine 261 and threonine 262 sites enhances E2F4’s nuclear localization (Morillo et al., 2012; Paquin et al.,

2013), and phosphorylation of the E2F4/DP complexes by Cyclin D1/Cdk4 disrupts its DNA binding capacity as cells enter S phase (Scime et al., 2008). Given these observations, we speculate that E2F4’s phosphorylation might modulate its interaction and/or function within the deuterosome complex, for example by enhancing its cytoplasmic localization and/or modifying its interaction with other partners. Thus, we made some effort to identify the responsible kinases. Based on the literature at that time, Plk4, was an obvious candidate kinase, as it was a

158 component of the deuterosome complex that colocalizes with cytoplasmic E2F4 (Mori et al.,

2017), and E2F4’s carboxy-terminal domain contains multiple sequences that fit Plk4/1’s canonical target site (Yaffe M., unpublished data). Thus, we performed in vitro kinase assays in which E2F4 was either immunoprecipitated from mammalian cells or expressed and purified from E. coli and then incubated with active Plk4 or Plk1 in the presence of [-35P] ATP.

However, we detected no phosphorylation of E2F4, even though Plk4 activity was verified by autophosphorylation (data not shown) suggesting that other kinases are responsible for this modification. Notably, several recent studies showed that Plk4 is fully dispensable for both the development of deuterosome complexes and the massive centriole biogenesis required for multiciliogenesis (reviewed in Lewis and Stracker, 2020).

We also investigated the nature of lower molecular weight species of Flag-Deup1 that appears when it is co-expressed with E2F4, and is the only Deup1 species that is recovered in

E2F4 immunoprecipitates. Treatment with lambda protein phosphatase determined that Deup1 is a phosphoprotein, but this modification does not account for the mobility shift induced in the presence of E2F4. Instead, binding experiments using Deup1 with distinct amino- and carboxy- tags strongly suggested that the lower molecular weight species results from cleavage of Deup1’s

C-terminal domain. Protein cleavage is a major mechanism used to control proper cell cycle progression and amplification of centrioles; for example, SAS6 and pericentrin both contain caspase-cleavage sites (Seo & Rhee, 2018). Identification of the cleavage site of Deup1 may help to determine Deup1 regulation in presence of E2F4. Further experiments are necessary to determine whether this cleavage is necessary for endogenous interaction or is an artifact formed as a result of the cellular stress of overexpressing Deup1 in non-ciliated cells.

159 Interestingly, we did not observe either of these mobility shifts in experiments with E2F5 and Deup1 indicating that they are specific to E2F4. Since E2F4 and E2F5 are thought to function redundantly in multiciliogenesis the significance of these post-translational modifications to this process is not yet clear.

Direct versus indirect interaction of E2F4 with Deup1 and SAS6

One question that needs further investigation is whether the association between E2F4 and Deup1 or SAS6 is direct or occurs indirectly via other cellular proteins. As bacterially expressed Deup1 is capable of forming deuterosome-like granules or rings (Zhao et al., 2013), we performed binding experiments in E. coli to try to address this question. In these experiments, we detected no interaction between Deup1 and amino-terminal domain of E2F4 in the presence or absence of DP1, despite observing binding between E2F4 and DP1. It is unclear whether the lack of interaction reflects the absence of linker proteins, or post-translational modifications that allow the association of E2F4 and Deup1 in mammalian lysates.

Additionally, the kinetics of ring maturation and stability of interaction between E2F4 and

Deup1 may be different in E. coli versus mammalian cells, thus we might be missing the appropriate maturation stage for Deup1 and E2F4 association to occur. To further explore whether Deup1 and SAS6 can directly bind E2F4, it would be valuable to express E2F4, Deup1 and SAS6 in Sf9 insect cells, which would allow post-translational modifications to take place.

Another unanswered question is interplay between the E2F4/Deup1 and E2F4/SAS6 complexes. It is possible that a trimolecular complex between E2F4, Deup1 and SAS6 exists, although we were not able to detect such a complex when we co-expressed all three proteins in our transfection assay. Given that the binding surfaces in E2F4 for Deup1 and SAS6 are

160 overlapping, and that binding is affected by the same point mutations, it seems more likely that binding is mutually exclusive. E2F4 therefore may play sequential roles in multiciliogenesis by binding Deup1 and SAS6 at different times and/or creating separate pools of E2F4/Deup1 complexes and E2F4/SAS6 complexes.

E2F4 binds to Deup1 but not Cep63

Deup1’s paralogue, Cep63 was identified by a mass spectrometry-based proteomic screen of human centrosomes as one of the most abundant centrosomal proteins in the interface of cell cycle (Andersen et al., 2003). Further studies showed that Cep63 functions together with

Cep152 in mother-centriole-dependent centriole duplication and is a target of ATM and ATR kinases in mitosis (Brown et al., 2013; Smith et al., 2009). Human patients with homozygous

Cep63 mutations exhibit Seckel syndrome characterized by microcephaly and dwarfism and

Cep63-deficient mice recapitulate these pathological outcomes (Marjanović et al., 2015; Sir et al., 2011).

Based on phylogenetic analyses, Deup1 appeared to diverge from Cep63 in an ancestor of the lobe-finned fish during vertebrate evolution (Zhao et al., 2013). These two proteins have

25% identity and 34% similarity in amino acid level in humans and share common properties.

They both interact with numerous centrosomal proteins, including Cep152, Plk4, Stil1 and

SAS6, (Zhao et al., 2013) and are both categorized as tumor suppressors, as their loss is associated with tumorigenesis (Loffler et al, 2011; Yu et al., 2019). Deup1 is specifically expressed in tissues where multiciliated cells are present and its evolutionary emergence is thought to reflect the need to expand the centriole biogenesis to produce the basal bodies required for multiciliated cells during development.

161 Although Cep63 colocalizes with cytoplasmic E2F4 in centrosomal regions during ALI differentiation, we observed no association of Cep63 and E2F4 in our co-immunoprecipitation experiments. This suggests that the ability of Deup1 to interact with E2F4 was acquired after its evolutionary divergence from Cep63. This is in line with previous data demonstrating that

Deup-RFP (red fluorescent protein), but not Cep63-RFP, can rescue the defects in deuterosome complex formation and deuterosome-dependent centriole amplification of Deup1-depleted

MTECs (Zhao et al., 2013). The homology of the amino terminal region of Deup1 (Deup11-129) to the corresponding region is Cep63 (Cep67-192) is around 70%, much higher than the overall homology. To further identify the critical E2F binding sequences within Deup1, we could generate Deup1/Cep63 chimeras within this region, in a similar manner to our use of E2F4/E2F1 chimeras. Additionally, it has not been investigated whether Deup1 can compensate for the functions of Cep63 in cycling cells. It would be interesting to address this, and subsequently to use Deup1/Cep63 chimeras to determine the mechanism that would allow Cep63, and potentially

Deup1, to locate to mother centriole. Following this theme, the absence of Cep63 in neuronal cells causes neuronal defects (Marjanović et al., 2015). In this context, expression of Deup1 was not induced, and thus it is unclear whether, or not, it is capable of compensating for Cep63 loss.

Thus, it would be interesting to determine whether overexpression of Deup1 can substitute for

Cep63.

E2F4 and E2F5 function redundantly in multiciliogenesis

E2f4 and E2f5 conditional knockout murine models showed that there is redundancy between E2F4 and E2F5 in multiciliogenesis and that a critical level of E2F4/5 activity is required for this process to be successful. We were interested in further understanding the role

162 of E2F5 in multiciliogenesis as it had not been determined whether E2F5 functions solely at the transcriptional level, in a complex with GemC1 or multicilin, or whether it also has a secondary cytoplasmic role enabling deuterosome assembly. Our data shows that E2F5 is able to associate with Deup1 and SAS6, strongly suggesting that E2F5 shares E2F4’s cytoplasmic role.

Experiments in which full-length E2f5 is expressed in E2F4-deficient precursor cells, would allow us to investigate whether E2F4’s functions in multiciliogenesis in the airway epithelia can be performed by E2F5. If E2F5 can rescue the ciliary defect of E2F4-deficient precursor cells, as anticipated, we could then co-express nuclear E2F4 (E2F4ΔNES) with full-length or amino terminal cytoplasmic E2F5 (E2F5ΔDBD or E2F51-228 ΔDBD), or visa versa, to establish whether

E2F5 plays both nuclear and cytoplasmic roles in multiciliogenesis. Rescue of ciliary defect in this system would show that E2F4 and E2F5 are functionally redundant with respect to both their nuclear and cytoplasmic roles in multiciliogenesis. This would argue that insufficient expression of E2F5 in airway epithelia, not differences in its function, leads to the ciliary defects observed in the absence of E2F4.

E2F4 and pRB show partial redundancy in multiciliogenesis

Comparison of E2f4 and the pocket protein Rb single and double knockout mice have shown that E2F4 and pRB have independent functions in embryonic development, but have overlapping roles in placental development (Lee et al., 2009). Surprisingly, mice homozygous for a mutation of Rb and heterozygous for a mutation of E2f4 displayed a loss of multiciliated cells from the airway epithelium during embryogenesis indicating that pRB can contribute to multiciliogenesis (Danielian et al., 2007). In contrast no phenotype was observed in E2f4+/-

;p107-/- and E2f4+/-;p130-/- double mutants indicating that pRB is the only pocket protein

163 important for multiciliogenesis (Danielian et al., 2007). Further experiments are needed to determine whether pRB’s transcriptional versus cytoplasmic functions are required during multiciliogenesis and whether its function is dependent on E2F4 and E2F5. Preliminary data showed that pRB colocalizes with PCM1 in apical regions of trachea-derived precursor cells similar to E2F4 and E2F5, suggesting it might also have cytoplasmic roles in deuterosome assembly or function (data not shown). Based on these observations, we would expect mouse tracheal precursors isolated from E2f4+/f ;pRbf/f would be defective in multicilia formation in the

ALI differentiation assay. Rescue experiments using mutant variants of pRB would allow us determine the specific domains of pRB that are required for this activity, particularly whether or not it requires E2F binding functions.

An evolutionary analysis of the nuclear export of E2F4/5

Our study is pivotal as it is the first to assign a cytoplasmic function to a member of E2F transcription factors. Only two members of the E2F family of transcription factors, E2F4 and

E2F5 contain bipartite nuclear export signals (NES) and move between the nucleus and cytoplasm (Gaubatz et al., 2001). Since E2F4’s cytoplasmic function was necessary for multiciliogenesis in the airway epithelia, I hypothesized that there might be a link between the evolution of E2F4’s NES and the emergence of multiciliated cells. Sequence alignment shows that the first NES is remarkably evolutionarily conserved, existing from mouse down to C. elegans (Table 1). In contrast, the second NES first appears in vertebrates, being present in D. rerio and Tetraodon nigroviris, but absent from C. elegans and other invertebrates (Table 1).

From an evolutionary perspective, I speculate that gaining a second NES may increase the level of cytoplasmic E2F4/5, enhancing the time that these proteins spend in the cytoplasm and/or

164 2

S E N 1

S E N 2

S E N

Table 1. Sequence conservation of E2F4 and E2F5 in Caenorhabditis elegans (round worm), Drosophila melanogaster (fruit fly), Arabidopsis thaliana (thale cress plant), Ciona intestinalis (sea vase), Tetraodon nigroviridis (pufferfish), Danio rerio (zebrafish), Xenopus (frog) and mus musculus (mouse) with the conserved nuclear export signals indicated.

165 allowing them to have new cytoplasmic binding partners. Interestingly, the appearance of the second NES shortly preceded the duplication and divergence of Deup1 from Cep63 during vertebrate evolution in an ancestor of lobed-finned fish (Zhao et al., 2013).

Notably, the sequence of E2F4’s second NES (mouse E2F483-106) partially overlaps with one of the sites we found as contributing to binding to Deup1 and SAS6 (E2F4 M2, mutagenesis in residues 84-92). In our co-immunoprecipitation experiment, the M2 mutation alone did not abrogate the ability of full-length E2F4 to co-immunoprecipitate with Flag-Deup and showed a weak effect on the association of E2F41-197 with Flag-Deup1 and full-length E2F4 with Flag-

SAS6. Since mutations of the second NES alone is not sufficient to prevent export to the cytoplasm (Gaubatz et al., 2001) we propose that loss of the second NES would still allow

E2F4’s cytoplasmic function but perhaps at reduced efficiency. Using the aforementioned ALI differentiation assay with a mutant cytoplasmic E2F4 containing only the first NES we would assess whether loss of the second NES impairs centriole replication and E2F4’s cytoplasmic function during multiciliogenesis.

The origin and function of deuterosome complex in multiciliogenesis and the role of E2F4

Deuterosomes are ring-shaped osmophilic cytoplasmic structures that were proposed to contribute significantly to massive replication of centriole in multiciliated cells (Sorokin, 1968;

Zhao et al., 2013). However, there has been considerable debate about where deuterosomes form. An early study suggested that they form and mature de novo, independently of the mother/daughter centriole complexes, within fibrous granules (Dirksen, 1971; Kubo et al., 1999;

Sorokin, 1968; Zhao et al., 2013) but subsequent work argued they arise from the proximal region of the daughter centriole and later move to cytoplasm (Al Jord et al., 2014). Our data

166 showed that E2F4 is colocalized with PCM1, a component of fibrous granules and that immature

Deup1 - containing deuterosomes arise in the areas where PCM1 and E2F4 are colocalized, while mature deuterosomes move outside the E2F4-PCM1 cloud (Mori et al., 2017). Consistent with our findings, three recent studies addressed the consequences of deleting the parental centrioles from mouse tracheal or ependymal cells, and showed that deuterosome complexes can assemble and function independently (Mercey et al., 2019 #1; Nanjundappa et al., 2019; Zhao et al., 2019).

Previous studies of mother-centriole and deuterosome dependent centriole biogenesis showed that several proteins, including Cep152, Plk4, SAS6, function in both pathways, but

Deup1 is deuterosome-specific and Cep63 is centriole-specific. A recent paper, utilizing knockouts of Deup1 and Cep63, showed that multiciliogenesis can occur in the absence of deuterosomes, indicating that alternative pathways are present (Mercey et al., 2019 #2). Notably, they showed that in the absence of Deup1, and thus deuterosome formation, centriole amplification occurred within fibrous granules. Since E2F4 is essential for multiciliogenesis

(alone or in combination with E2F5), and it is located in fibrous granules, we propose that it might enable multiciliogenesis in the absence of Deup1, or parental centrioles, by binding to

SAS6 within fibrous granules. Our data suggest that E2F4, and by inference E2F5, are critical for the massive centriole replication required for multiciliogenesis and likely function via binding to Deup1 and/or SAS6, perhaps serving as scaffolding proteins or nucleation centres for the assembly of deuterosomes or other structures required for centriole replication.

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171 Biographical Note

Renin Hazan Massachusetts Institute of Technology 77 Massachusetts Avenue, Building 76-441 Cambridge, MA 02139

Education

2009-present Massachusetts Institute of Technology Cambridge, MA Ph.D. Candidate in Biology Department

2007-2009 Bogazici University Istanbul, Turkey M.Sc. Student in Department of Molecular Biology and Genetics

2002-2007 Bogazici University Istanbul, Turkey B.Sc. in Department of Molecular Biology and Genetics Double Major in Department of Chemistry

Research Experience

2011-present Koch Institute for Integrative Cancer Research Cambridge, MA Ph.D. Thesis Advisor: Prof. Jacqueline Lees Ph.D. Thesis Title: “Investigating the cytoplasmic role of E2F4 in multiciliogenesis”

2007-2009 Bogazici University Istanbul, Turkey Department of Molecular Biology and Genetics M.Sc. Thesis Advisor: Prof. Aslihan Tolun M.Sc. Thesis Title: “Locus and Gene Screen in Three Diseases”

2006 Harvard School of Public Health Boston, MA Department of Genetics and Complex Diseases Summer Intern (volunteer) at Prof. Dr. Gokhan Hotamisligil’s laboratory

2005-2007 Bogazici University Istanbul, Turkey Department of Molecular Biology and Genetics Undergraduate Researcher at Prof. Ahmet Koman’s laboratory

2005 Weizmann Institute of Technology Rehovot, Israel Department of Biological Chemistry Summer Intern (volunteer) at Prof. Anthony Futerman’s laborator

2004 Dokuz Eylul University Hospital Izmir, Turkey Department of Medical Biology and Genetics Summer Intern (volunteer) at Cytogenetics and Molecular Biology lab Advisor: Prof. Dr. Meral Sakizli

172 Awards

2013-2014 Graduate Student Fellowship awarded by Koch Institute at MIT

2007-2009 National M.Sc. Student Scholarship awarded by Scientific and Technological Research Council of Turkey

June 2007 Bogazici University award of the Rector

June 2007 High Honors Certificate awarded by Bogazici University

2002-2007 Outstanding Student Scholarship awarded by Bogazici University

Teaching experience

Summer 2016 MIT Kaufman Teaching Certificate Cambridge, MA

Spring 2013 Massachusetts Institute of Technology Cambridge, MA Teaching Assistant in 7.02: Intro to Experimental Biology and Communication.

Spring 2008 Bogazici University Istanbul, Turkey Lab Assistant in Bio 342:Physiology I lab course

Fall 2007 Bogazici University Istanbul, Turkey Lab Assistant in Bio 409: Molecular Biology Laboratory course.

Presentations

2017 Colrain Retreat Title of talk: Understanding the role of cytoplasmic E2F4 in multiciliogenesis

2017 Gordon Conference on Lung Development, Injury and Repair Title of poster: Investigating the role of cytoplasmic E2F4 during multiciliogenesis

2017 Ludwig Retreat Title of poster: Investigating how E2F4 functions in multiciliogenesis

2016 MIT Department of Biology Graduate Student Retreat Title of talk: Understanding the role of E2F4 in multiciliogenesis.

2008 EMBO Young Scientist Forum Title of poster: A search for an IDDM Locus

Publications

Mori M., Hazan R., Danielian P.S., Mahoney J., Li H., Lu J., Miller E. S., Zhu X, Lees J. and Cardoso W. (2017). Cytoplasmic E2f4 Forms Organizing Centres for Initiation of Centriole Amplification During Multiciliogenesis. Nature Communication, 8, 15857.

Ayhan O., Balkan A., Guven A., Hazan R., Atar M. and Tolun A. (2014). Truncating mutations in TAF4B and ZMYND15 causing recessive azoopsermia. J. Med Genet, 51, (4) 239-44.

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