Identification of Atat1 As a Major Alpha-Tubulin Acetyltransferase

Home , Acetyltransferase

Identification of Atat1 as a major alpha-tubulin

acetyltransferase dispensable for mouse development

Go Woon Kim

Department of Anatomy and Cell Biology

Faculty of Medicine

McGill University

Montreal, Quebec, Canada

December 2012

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of

Master of Science

ABSTRACT

Post-translational modification is one crucial cellular mechanism through which proteins are altered in structure and function. A variety of proteins different in function and cellular localization is affected by such modification. With a wide range of influence within a cell, lysine acetylation has emerged as one major post- translational modification that rivals to phosphorylation. Indeed, recent proteomic studies have revealed that lysine acetylation affects 5-10 % of mammalian and bacterial proteins. This modification is catalyzed by the opposing actions of lysine acetyltransferases and deacetylases. One of the first acetylated proteins identified in the mid-1980s is α-tubulin, a subunit of microtubules that comprise the cytoskeletal network. Although deacetylation of α-tubulin is well known to be catalyzed by HDAC6, the responsible acetyltransferase remained elusive until recently. The recent discovery of Atat1 (α-tubulin acetyltransferase 1) has yielded important insights into the role of acetylated α-tubulin in lower organisms such as

C. elegans, but it remains unclear whether this is also the case in vivo in higher organisms such as mammals. Here, using mice devoid of Atat1, I demonstrate that

Atat1 is a major acetyltransferase of α-tubulin dispensable for mouse development. I also show that Atat1 is highly expressed in testis, renal pelvis, gastrointestinal tract, and hippocampus, but development of these tissues appears normal in the absence of Atat1. Moreover, in a set of molecular and cellular studies, I have identified ATAT1 as a potential interaction partner of several proteins. Together, these findings provide direct support of Atat1 as an authentic

α-tubulin acetyltransferase, thereby setting up a solid foundation for additional studies of this enzyme and tubulin acetylation in mice and humans.

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RÉ SUMÉ

La modification post-traductionnelles des protéines est un mécanisme cellulaire essentiel par laquel la structure et la fonction des protéines peuvent-être affectées. Les modifications sont présentes dans la grande majorité de voie cellulaire et les protéines cibles possèdent une grande diversité de fonction et de localisation cellulaire. L’acétylation des lysines a émergé comme une modification de grande importance, ayant un impact majeur sur les cellules, à l’instar de la phosphorylation. Effet, des études récentes de protéomique ont révélées que de 5 à 10 % des protéines bactériennes et animales étaient affectées par l’acétylation des lysines. Cette mondification est catalysé par l’effet inverse de deux classes de protéines, les lysines acétyltransférase et les déactylases. L'une des premières protéines identifiées dans acétylés milieu des années 1980 est l’α- tubuline, une sous-unité des microtubules qui composent le réseau du cytosquelette. Malgré que l’enzyme responsable de la déacétylation de l’α- tubuline est connue sous le nom HDAC6, l’enzyme responsable de son acétylation est restée inconnue jusqu’à récemment. La découverte d’Atat1 (α- tubuline acétyltransférase1) a donné des informations importantes sur le rôle de l’ aétylation sur l’α-tubuline dans les organismses inférieurs comme C. elegans, mais il reste difficile de savoir si c’est aussi le cas in vivo dans les organismes supérieurs commes les mammifères. En utilisant des modèles murin de Atat1, je démontre que Atat1 est une acétyltransférase majeur de l’α-tubuline dispensable pour le développement normal. De plus, je démontre que le niveau d’expression d’Atat1 est particulièrement élevé dans les testicules, le bassinet du rein, le tractus

IV gastro-intestinal, et l’hippocampe, pourtant, le développement de ces tissus semble normal en l'absence de Atat1. En outre, dans une série d'études moléculaires et cellulaires, j'ai identifié ATAT1 comme un partenaire potentiel d'interaction de plusieurs autres protéines. Ces résultats non seulement apporter un soutien supplémentaire de la Atat1 comme une acétyltransférase authentique de l’α-tubuline, mais aussi mettre en place une bonne base pour des études supplémentaires de cette protéine chez les souris et les humains.

ACKNOWLEDGEMENTS

This thesis work presented herein has been completed with the help of many people. Firstly, I would like to thank my supervisor, Dr. Xiang-Jiao Yang for guiding me during the course of the studies and providing valuable suggestions and advice. His vast amount of experience and passion for science also helped me gain insight into the scientific research in depth. No matter whether it is short or long, science-related or not, discussion with him always made me learn and stimulated me to do better.

Secondly, I would like to thank my colleagues, former and current, including Dr. H. Taniguchi, Dr. D. Walkinshaw, Dr. S. Tahmasebi, Dr. J. Nie, G.

Gocevski, R. Weist, Y. Lu, C.J. Wu, K. Yan, L. You, H. Salem, M. Ghorbani, T.

Sabri, H.I. Kim, L. Chen, H.Y. Ham, and many others for sharing scientific knowledge and laughs during the course of my research. I am particularly grateful to Dr. Taniguchi for teaching me very patiently about various techniques, especially those related to mice, in the first six months or so after I joined the lab.

The experience that I have had with him and others in the lab is one memory so valuable that it can never be forgotten.

Thirdly, special thanks should be given to M. Flamand in the neighbor lab for French translation of the abstract. I am also thankful to Dr. Craig Mandato, Dr.

Stefano Stefani, and Dr. René St-Arnaud for serving as my mentor/committee members. Their kind and intellectual suggestions helped improve the research to be described herein. I would like to thank Dr. Jaeok Park for carefully reading and editing a close-to-final version of this thesis.

Finally, I must thank all my friends and family, who supported me with never-failing love in every situation I have been. Their constant support equipped me with courage and strength to step forward to this achievement.

The research presented here was supported by funding from CIHR,

NSERC, and MDEIE (to Dr. X.J. Yang).

VII

PUBLICATIONS

1. Kim, G.W., et al., Dietary, metabolic, and potentially environmental

modulation of the lysine acetylation machinery. Int J Cell Biol, 2010. 2010:

p. 632739.

2. Kim, G.W. and X.J. Yang, Comprehensive lysine acetylomes emerging from

bacteria to humans. Trends Biochem Sci, 2011. 36(4): p. 211-20.

3. Aka, J.A., G.W. Kim, and X.J. Yang, K-acetylation and its enzymes: overview

and new developments. Handb Exp Pharmacol, 2011. 206: p. 1-12.

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CONTRIBUTIONS OF AUTHORS

CHAPTER II:

Identification of Atat1 as a major α-tubulin acetyltransferase dispensable for mouse development

I performed most of the experiments presented in this chapter, with several expression constructs given by others (see below). I also prepared all the figures and wrote the manuscript. In collaboration with Dr. Yang, I designed the experiments, edited, and finalized the manuscript.

Dr. Jianyun Nie prepared the HA-AKAP8, HA-AKAP8L, HA-TUBB4, HA-

TIF1β, and HA-MAGED2 expression constructs. Dr. Ed Seto and his colleagues at University of South Florida engineered the HA-RAC1 and FLAG-cortactin expression constructs. The FLAG-HSP90 plasmid construct was provided by Dr.

Jason Young at McGill University.

The mass spectrometry shown in Table 1 was carried out in collaboration with Dr.

Jin Jing in Dr. Tony Pawson’s laboratory at University of Toronto.

TABLE OF CONTENTS

ABSTRACT...... II RÉSUMÉ ...... IV ACKNOWLEDGEMENTS...... VI PUBLICATIONS ...... VIII CONTRIBUTIONS OF AUTHORS ...... IX TABLE OF CONTENTS ...... X LIST OF ABBREVIATIONS...... XII

CHAPTER I

Literature Review

1. Post-translational modifications...... 2 1.1 Cellular function of post-translational modifications ...... 3 1.2 Lysine acetylation ...... 3 2. Lysine acetyltransferases and deacetylases ...... 4 2.1 Families of lysine acetyltransferases...... 5 2.2 Histone deacetylase superfamily ...... 5 2.3 Lysine acetylation in different cellular processes ...... 6 2.3.1 Lysine acetylation in chromatid cohesion complex ...... 6 2.3.2 Lysine acetylation in metabolism ...... 7 2.3.3 Lysine acetylation in other cytoplasmic events ...... 8 3. Microtubule ...... 9 3.1 Microtubule regulation ...... 9 3.2 α-Tubulin modifications ...... 11 3.2.1 Poly-glutamylation and poly-glycylation ...... 11 3.2.2 Tyrosination and detyrosination ...... 12 3.2.3 Acetylation ...... 12 3.3 Lysine acetyltransferases and deacetylases of α-tubulin ...... 12 X

4. Physiological roles of acetylated lysine 40 of α-tubulin ...... 14 4.1 Acetylated α-tubulin in primary cilia ...... 14 4.2 Acetylated α-tubulin in intracellular trafficking...... 15 4.3 Acetylated α-tubulin in the central nervous system (CNS)...... 16 4.4 Acetylated α-tubulin in cell cycle control and cancer ...... 16 5. Rationale for the thesis research project ...... 18 6. Figures and Legends ...... 19 7. References ...... 25

CHAPTER II

Identification of Atat1 as a major α-tubulin acetyltransferase dispensable for mouse development

1. Abstract ...... 34 2. Introduction ...... 35 3. Materials and Methods ...... 38 4. Results ...... 46 5. Discussion and Conclusion ...... 55 6. Illustrations ...... 60 7. References ...... 80

LIST OF ABBREVIATIONS

AKAP8 A-kinase anchor protein 8 AKAP8L AKAP8-like ARD1 ADP-ribosylation factor domain protein 1 ATAT1 α-Tubulin acetyltransferase ATP Adenosine-5’-triphosphate AurA Aurora A BBS4 Bardet-Biedl syndrome 4 protein BDNF Brain-derived neurotrophic factor BMAL1 Brain and muscle Arnt-like protein 1 B. subtilis Bacillus subtilis CBP CREB-binding protein C. difficile Clostridium difficile cDNA Complementary DNA C. elegans Caenorhabditis elegans Chk1 Checkpoint kinase 1 CMV Cytomegalovirus CNS Central nervous system CoA Coenzyme A Co-IP Co-immunoprecipitation CYLD Cylindromatosis DAPI 4’,6-Diamidino-2-phenylindole DEPC Diethylpyrocarbonate DMEM Dulbecco’s modified eagle medium DNA Deoxyribonucleic acid DTT Dithiothreitol E2F E2 transcription factor Eco1 Establishment of cohesion 1 E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid EF1 Elongation factor 1 EGTA Ethylene glycol tetraacetic acid ELP Elongator protein F-actin Filamentous actin FBS Fetal bovine serum FOXO Forkhead box, class O Gapdh Glyceraldehyde 3-phosphate dehydrogenase GCN5 General control of amino acid synthesis 5 GFP Green fluorescent protein GI Gastrointestinal GNAT Gcn5-related N-acetyltransfearse GTP Guanosine-5’-triphosphate HA Human influenza hemagglutinin HAT Histone acetyltransferase Hda3 Histone deacetylase 1 XII

HDAC Histone deacetylase H&E Hematoxyline and eosin HEK 293 Human embryonic kidney 293 cell line HeLa Human epithelial carcinoma cell line from a fatal cervical carcinoma HIV Human immunodeficiency virus hnRNPA1 heterogeneous nuclear ribonucleoprotein A1 HRP Horseradish peroxidase HSP Heat Shock Protein IgG Immunoglobulin G KAT Lysine acetyltransferase KD Knockdown KO Knockout MAGED2 Melanoma-associated antigen D2 MAP Microtubule-associated protein Mec17 C. elegans protein mechanosensory abnormality 17 MOZ Monocytic leukemia zinc finger protein mRNA Messenger RNA MS Mass spectrometry MTOC Microtubule-organizing center MYST MOZ, Ybf2/Sas3, SAS2, and Tip60 NAD Nicotinamide adenine dinucleotide NAT1 N-acetyltransferase 1 NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells NP-40 Nonyl phenoxypolyethoxylethanol-40 ORF Open reading frame p300 E1A-associated protein of 300 kDa p(A) Polyadenylation site Pat Protein acetyltransferase PBS Phosphate buffered saline PCR Polymerase chain reaction PFA Paraformaldehyde PIPES Piperazine-N,N’-bis(2-ethanesulfonic acid) PKA Protein kinase A PMSF Phenylmethanesulfonylfluoride pRB Retinoblastoma protein PTM Post-translational modification RhoGDI Rho GDP-dissociation inhibitor RNA Ribonucleic acid Rpd3 Reduced potassium dependency 3 RT-PCR Reverse transcription-PCR Rtt109 Fungal-specific regulator of ty1 transposition 109 Sas Something about silencing Scc Sister chromatid cohesion SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis S. enterica Salmonella enterica XIII

SIRT2 Sirtuin 2 Smc Structural maintenance of chromosomes SrtN Sirtuin STAT Signal transducer and activator of transcription TAF1 Transcription initiation factor TFIID subunit 1 Tat Transactivator of transcription TCA Tricarboxylic acid cycle TFIID Transcription factor II D T. thermophila Tetrahymena thermophila TIF1β Transcription intermediary factor 1β TIP Microtubule plus-end tracking protein TTL Tubulin tyrosine ligase TTLL TTL-like TUBB4 Tubulin β4 WT Wild-type

XIV

Chapter I

Literature Review

1. Post-translational modifications

The genetic blueprint of life, DNA in the nucleus of eukaryotic cells, is packaged into nucleosomes with histones, which together constitute a higher structure called chromatin. Orchestrating the organization and the function of chromatin is post-translational modifications (PTMs), which in turn regulate differential gene expression. The impact of such PTMs is not only restricted in chromatin-templated nuclear events but also is extended to manipulation of cytoplasmic proteins. In cytoplasm, PTMs serve as one of the key mechanisms that mediate intracellular signaling by shaping protein structure and regulating protein function. A wide range of PTMs exists to meet distinct cellular demands including phosphorylation, ubiquitination, sumoylation, glutamylation, tyrosination, and acetylation, which together or alone form a sophisticated network of protein dialogues. One emerging notion based on accumulating data is that multisite modifications within a protein cooperate or compete with each other in order to create a specific PTM code exerting certain cellular effects [1-4]. A representative example is differential PTMs that occur on p53. It has been observed that ubiquitination at lysine 370, 372, 373, 381, and 382 induces nuclear export and subsequent proteasomal degradation of p53 [5]. Lysine 370, 372, and

382 are also subject to methylation, suggesting that these sites are competed by different PTMs [4]. With such differential and multifaceted impacts on protein, cells are required to retain tight regulation of PTMs in order to maintain cellular homeostasis. Indeed, deregulated PTMs are often observed in many diseases, with one exemplified by histone H4 hypo-acetylation at lysine 16 that serves as a

2 hallmark of cancer cells [6].

1.1 Cellular function of post-translational modifications

PTMs act by adding chemical moieties or peptides to one or more certain amino acids of a protein, resulting in different protein function depending on the type of PTMs. For example, initially identified in histones, acetylation results in activation of gene expression by neutralizing the positive charge of a lysine residue on core histone tails, thus disrupting the interaction of histones with DNA and enabling transcription factors to have access to DNA. A number of cytosolic proteins unambiguously have been witnessed to be regulated by phosphorylation, which can turn on and off their activation. Proteins are targeted for degradation upon getting tagged with poly-ubiquitin chains.

In addition, the net cellular results induced by a certain PTM also depend on the amino acid site(s) on which it occurs. For example, mono-acetylation of p53 at lysine 382 increases its affinity to the bromodomain of CREB-binding protein (CBP) [4, 7], while di-acetylation of lysine 373 and 382 induces p53 association with the tandem bromodomains of TAF1, a TFIID subunit [4, 8].

Furthermore, certain protein domains recognize specific PTMs for the sequential protein-protein interaction, thereby using PTMs as a docking site.

1.2 Lysine acetylation

Lysine acetylation refers to the transfer of an acetyl moiety from acetyl-

CoA to the ε-amino group of a lysine residue (Fig. 1). Because lysine acetylation

3 has been first discovered in histones in the 1960s [9], its role has long been recognized to be associated with chromatin structure and transcriptional regulation and thus extensively investigated in this regard. Along with the sequential discovery of acetylated non-histone proteins, the recent proteomic studies have widened the scope of lysine acetylation through the identification of numerous acetylated proteins involved in a diverse array of cellular processes including transcription, DNA repair, protein folding, and metabolism (Fig. 2) [10-

16]. Furthermore, a large number of acetylated proteins were found in different bacterial species, indicating lysine acetylation as an evolutionarily conserved mechanism. As such, lysine acetylation has begun to gain much attention as a major PTM comparable to other major PTMs like phosphorylation.

2. Lysine acetyltransferases and deacetylases

Highly dynamic and reversible, lysine acetylation is governed by two families of counteracting enzymes called lysine acetyltransferases (KATs) and histone deacetylases (HDACs). Due to a historical reason, KATs and HDACs were initially referred to histone acetyltransferases (HATs) and HDACs. Since the nomenclature of these names is no longer precise based on the fact that these enzymes affect acetylation of other non-histone proteins, HATs have been renamed as KATs but the nomenclature of HDAC remained because it has already become coherent [17]. Therefore, the acronyms, KAT and HDAC, will be used herein.

2.1 Families of lysine acetyltransferases

Since the first discovery in the mid-1990s, the list of KATs has been continuously increasing. KATs are mainly divided into three groups: GCN5- related N-acetyltransferases (GNATs), MOZ, Ybf2/Sas3, Sas2, and Tip60 (MYST) proteins, and the E1A-associated protein of 300 kDa (p300)/CBP. The recent emergence of new KATs includes the fungal-specific regulator of ty1 transposition 109 (Rtt109), which exhibits structural similarity to p300 [18], and

Mec17 in C. elegans, which is related to Gcn5 [19]. Many of the KATs are found as a catalytic subunit of multiprotein complexes and often conserved from yeast to humans [20]. Compared to the large size of KATs found in eukaryotes, KATs in bacteria are sparse in spite of the high number of acetylated proteins. Therefore, potential KATs remain to be identified [21-22].

2.2 Histone deacetylase superfamily

There are two families of HDACs, which can be further divided into four classes based on sequence homology and phylogenetic analysis [23]. The

Rpd3/Hda1 family is comprised of class I (HDAC1, 2, 3, and 8), IIa (HDAC4, 5,

7, and 9), IIb (HDAC6 and 10), and IV (HDAC11), while the sirtuin family contains seven sirtuin members, comprising class III [24-26]. Class I HDACs are homologous to yeast Rpd3 with class II HDACs related to Hda1. Class IV shows similarities to both Rpd3 and Hda1. Whereas the mechanistic action of enzymes belonging to the Rpd3/Hdac1 family requires Zn2+, that of sirtuins is NAD+- dependent (Fig. 1B). Like KATs, many HDACs are evolutionarily conserved from

5 yeast to humans.

2.3 Lysine acetylation in different cellular processes

As the diversity of proteins subject to undergo lysine acetylation expands along with the number of acetylation sites, the scope of functional influence of lysine acetylation in a cell has become enlarged accordingly (Fig. 2).

2.3.1 Lysine acetylation in chromatid cohesion complex

Coupled to DNA replication, adherence of the sister chromatids ensures the genome integrity and proper chromosome segregation during cell division.

The cohesion establishment of the sister chromatids requires the cohesion complex containing Smc1, Smc3 (two homologous ATPases), and Scc1 that together form a ring-like structure [27]. Among the regulatory proteins that associate with these proteins is Eco1, a GNAT-related KAT [27-29]. Via targeting different substrates under certain cellular conditions, Eco1 results in distinct functional consequences such as stabilization of the cohesion complex [28-30], inhibition of the interaction of the cohesion complex with negative regulators [27-

28, 31-32], and maintenance of replication fork velocity [33]. For example, during

S-phase, Eco1 acetylates of Smc3, which leads to the stabilization of the cohesion establishment. There are three evolutionarily conserved lysine residues in Smc3

(lysine 112, 113, and 931), two of which (lysine 112 and 113) cause cell lethality due to severe defects in cohesion establishment of the sister chromatids when mutated [28, 30]. Eco1 also targets Scc1 for acetylation upon DNA damage

6 during G2/M phase [34]. In response to DNA damage, Chk1 is activated and subsequently phosphorylates Scc1 at serine 83. This phosphorylation triggers

Eco1 to acetylate Scc1 at lysine 84 and 210.

2.3.2 Lysine acetylation in metabolism

The recent investigation of the role of lysine acetylation beyond transcriptional regulation led to identify a large number of proteins implicated in metabolic processes. However, it was initially noticed in studies of acetyl-CoA synthetase in S. enterica that acetylation might control metabolism. In S. enterica, acetylation functions as a switch whereby acetylation of lysine 609 blocks the catalytic activity of acetyl-CoA synthetase [35]. The responsible KAT and HDAC were subsequently discovered to be Pat and CobB, which pushes and pulls to maintain the catalytically inactive and active status of the enzyme, respectively.

Similar results were obtained from B. subtilis with AcuA identified as a KAT and

AcuC and SrtN as HDACs. Lysine 609 of acetyl-CoA synthetase is evolutionarily conserved from bacteria to mammals, suggesting that this enzyme is regulated in a similar manner in the mammalian system [36].

The proteome-wide analysis revealed the presence of lysine acetylation in more than 50% of metabolic enzymes in E.coli [37-39]. Moreover, many enzymes involved in glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid metabolism, and glycogen metabolism in human liver tissues were preferentially acetylated with acetylation having a direct impact on the enzymatic activity or stability [16]. It was found that extracellular nutrient

7 availability changed the acetylation level of some metabolic enzymes, suggesting that nutritional information in the environment is decoded in part by acetylation of metabolic enzymes in order to produce a proper cellular response [39]. The participation of lysine acetylation in metabolism is further witnessed by the presence of acetylation in more than 20% of mitochondrial proteins [15, 40].

Although the functional importance of each individual acetylation event needs to be elucidated, the extensive number of acetylated metabolic enzymes found in both prokaryotes and humans indicates that lysine acetylation is evolutionarily conserved mechanism from bacteria to humans in regulating metabolism.

2.3.3 Lysine acetylation in other cytoplasmic events

One of the prominent features of HDAC6 distinct from other HDACs is the predominant cytoplasmic localization where HDAC6 acts on different cytosolic proteins including cortactin, HSP90, and α-tubulin. It has been demonstrated that acetylation of cortactin causes disruption of its interaction with

F-actin, affecting cell motility [41]. Similarly, HSP90 has been found to be involved in HDAC6-dependent actin remodeling [42]. In addition, acetylation status of HSP90 has an impact on its chaperone function [43]. Considering that

HSP90 chaperons proteins involved in nuclear receptors, protein kinases, and small/micro RNA-containing silencing complexes, the influence of HSP90 acetylation may be more widespread [36].

3. Microtubules

Comprised of actin filaments, intermediate filaments, and microtubules, the cytoskeleton forms a complex intracellular network that controls a multitude of cellular function including biophysical integrity, morphology, motility, and life cycle of the cell. Microtubules are found in a majority of eukaryotic cells and are highly dynamic, thus exist heterogeneously in length. This dynamic nature of microtubules is conferred by rapid cycling of polymerization and depolymerization, allowing microtubules to expedite various cellular requests.

The basic building block of microtubules is a heteromer containing α- and

β-tubulins. These monomeric proteins associate with each other in a head-to-tail manner to form a heteromer, which assembles laterally to further form a hollow cylindrical tube. In cilia, tubulins assemble as heterodimers whereas in centrioles and basal bodies, they form into heterotrimers. The microtubule polymers are polarized stemming from the microtubule-organizing center (MTOC) with plus- ends exploring throughout the cytoplasm. Microtubule minus-ends can be also polarized in vitro, but they mostly reside in a stable or depolarizing state.

3.1 Microtubule regulation

Microtubule-associated proteins (MAPs) interact with microtubules to regulate microtubule dynamics and organization. The most heavily studied microtubule-stabilizing MAPs are tau, MAP2, and MAP4. There are also microtubule-destabilizing MAPs, which function either by severing or by depolarizing the microtubule lattice, such as spastin and katanin. Another group of

MAPs is the microtubule plus-end tracking proteins (TIPs). They track and stabilize the plus-ends of microtubules that extend towards the leading edge of a cell and control the interaction of microtubules with other organelles and subcellular domains [44]. In addition, microtubule motor proteins constitute an important frame for the regulation of microtubule function, particularly in intracellular trafficking by generating force while associated with microtubules.

The two major groups of microtubule motor proteins are kinesins and dyneins.

Apart from MAPs and microtubule motor proteins, another emerging regulator of microtubule function are PTMs that act in a sole or combinatorial manner to generate a specific molecular signature on microtubules. Different types of PTMs are known to occur on tubulins, including de/tyrosination, glutamylation, glycylation, and acetylation. These tubulin PTMs occur not only post-translationally, but also post-polymerized. In spite of the vigorous research, there is no coherent evidence for tubulin modifications directly linking to microtubule dynamics or stability. Although PTMs themselves may directly mediate microtubule function by modulating microtubule structure, it was also suggested that certain PTMs mark for the recruitment of MAPs and affect the interaction with the motor proteins. For example, tau, MAP1B, and MAP2 have higher affinity towards moderately poly-glutamylated tubulins, whereas MAP1A optimally binds to heavily poly-glutamylated tubulins [45]. Likewise, lysine 40 acetylation of α-tubulin was observed to affect the binding to p58, a MAP involved in microtubule interaction with the Golgi membrane [45-46].

3.2 α-Tubulin modifications

The tubulin PTMs preferentially occur on tubulin subunits that have already been incorporated into the polymers, with the exception of β-tubulin phosphoryation at serine 172 on unpolymerized tubulins in mitotic cells [45]. An accumulating number of reports point to a notion that PTMs establish a ‘tubulin code’. In a manner highly similar to the postulated histone code, where specific recognition of histone modification recruits other proteins [2, 4], the tubulin code hypothesizes that MAPs or motor proteins decode and interpret differential PTMs of tubulins to dictate microtubule-based function [45]. Moreover, certain PTMs are evolutionarily conserved and mark subpopulations of microtubules [45].

3.2.1 Poly-glutamylation and poly-glycylation

Compared to other PTMs that add only a single moiety, glutamylation and glycylation add chains of glutamate and glycine residues varied in length to the C-terminus of tubulins. The enzyme responsible for poly-glutamylation, the poly-glutamylase, has been initially identified in brain and found to exist as a catalytic subunit in a multiprotein complex with TTL-like 1 (TTLL1) [47].

Sequential investigations have found that all tubulin glutamylases [48-50] and glycylases [51-53] comprise the TTLL family. The functional impact of polyglutamylation and poly-glycylation has been associated with microtubule stability

[54] and regulation of motor proteins [55-56].

3.2.2 Tyrosination and detyrosination

The C-terminus of α-tubulin contains tyrosines, which can be removed and re-attached to α-tubulin [57-58]. Tyrosination of α-tubulin is usually found in dynamic microtubules [59]. Although the enzyme that tyrosynates α-tubulin, tubulin tyrosine ligase (TTL), has been identified, the counteracting enzyme that detyrosynates α-tubulin remains elusive [60]. Tyrosination of α-tubulin has been found to play a role in recruiting proteins that regulate microtubule dynamics [60].

3.2.3 Acetylation

The acetylation of lysine 40 on α-tubulin was the second tubulin PTM discovered after α-tubulin tyrosination [61-62] and initially found in the flagella of Chlamydomonas [62-63]. Whereas most of the α-tubulin PTMs decorate the C- terminus of α-tubulin located on the outer surface of the microtubule shaft, acetylation is predicted to occur on the luminal side of microtubules [64].

Acetylated lysine 40 of α-tubulin is known to be distributed in microtubules of specific cellular domains, including the cytoplasmic network, centrioles, and axonemes of cilia and flagella [45, 65-67].

3.3 Lysine acetyltransferases and deacetylases of α-tubulin

HDAC6 has been discovered as the first deacetylase of α-tubulin. Both in vitro and in vivo analysis firmly established that HDAC6 deacetylates lysine 40 of

α-tubulin [68-69] and Hdac6 deficiency in mice leads to α-tubulin hyper- acetylation in many tissues [70]. Nonetheless, the role of HDAC6 and acetylation

12 of α-tubulin in the microtubule stability and dynamics remain unclear and is hotly debated. Although several research groups suggested that microtubule stability is not influenced by α-tubulin acetylation [71-72], the initial hypothesis was that acetylation of α-tubulin confers microtubules more stable [73]. This was supported by the observation that over-expression of HDAC6 increased chemotactic cell motility and that neuronal growth cones and the leading edges of fibroblast are devoid of acetylated microtubules [69]. Irrespective of whether or not HDAC6 directs microtubule stability, it is still surprising that Hdac6 KO mice are viable and grow normally without any obvious phenotype except mild developmental defects [70], given that acetylated α-tubulin is present in several important organelles and that HDAC6 affects acetylation levels of other cytosolic proteins, as mentioned previously.

The second enzyme suggested to deacetylate α-tubulin was SIRT2 [74].

Interestingly, co-immunoprecipitation suggested that SIRT2 interacts with

HDAC6, suggesting their interdependency [74]. However, it was shown that the acetylation of α-tubulin is not affected in mouse embryonic fibroblasts [70] and in brain tissues [75] of Sirt2 KO mice, indicating HDAC6 as a major deacetylase of

α-tubulin.

Despite of the discovery of HDAC6 as the α-tubulin deacetylase about a decade ago [69, 74, 76], the identity of the corresponding acetyltransferase had long remained elusive until recently. During this blackout period, several proteins were shown to affect acetylation of α-tubulin including, ARD1-NAT1 [77], ELP3 complex [78], and GCN5 [79]. However, recent investigations of the proteins

13 required for the maintenance of C. elegans touch receptor neurons led to the identification of Mec17 (C. elegans homolog of Atat1, α-tubulin acetyltransferase). Through a series of in vitro and in vivo analysis using

Tetrahymena, C. elegans, zebrafish, and HeLa cells, Mec17 was shown to be the bona-fide α-tubulin acetyltransferase (Fig. 3) [19]. Related to the Gcn5 family proteins, Mec17 homologs exist in most eukaryotes excluding fungi and plants

[19]. Interestingly, several recent reports in the past few months suggested that by acetylating α-tubulin at lysine 40, Mec17 increases cohesion between tubulin subunits and therefore promotes microtubule stabilization [80-81].

4. Physiological roles of acetylated lysine 40 of α-tubulin

It has been established that long-lived microtubules found in ciliary axonemes, neurons, and migrating cells contain acetylated α-tubulin at lysine 40.

Although acetylation of α-tubulin is not significantly required for survival of lower organisms [82-83], it was suggested that vertebrates are more sensitive to the loss of acetylated lysine 40 of α-tubulin [19, 84].

4.1 Acetylated α-tubulin in primary cilia

The role of α-tubulin acetylation in ciliary assembly and resorption was initially overlooked based on the observation that a non-acetylable form of α- tubulin (lysine 40 mutated to arginine) did not affect cilia in T. thermophila [83].

However, an increasing number of data suggest that acetylation of α-tubulin at lysine 40 may play an important role in mammalian primary cilia. AurA, which is

14 required for cilia resorption in Chlamydomonas flagella, also promoted resorption of the primary cilia in the human retinal pigmental epithelial cell line [85-87]. In vitro studies have shown that HDAC6 interacts with and is regulated by AurA and

HDAC6 inhibition leads to reduced resorption of primary cilia [87]. Interestingly,

ATAT1 was found to be required for normal kinetics of cilium assembly in mammalian cells [84], consistent with HDAC6 implicated in cilium disassembly.

Although acetylated lysine 40 of α-tubulin was abundant in zebrafish cilia [88],

Atat1 KD still exhibited lysine 40 acetylation to some degree, suggesting the presence of another putative α-tubulin acetyltransferase in zebrafish cilia [19]

4.2 Acetylated α-tubulin in intracellular trafficking

It has been well documented that α-tubulin acetylation is important for intracellular trafficking. The first evidence arose from the co-localization of

HDAC6 with the dynein-dynactin microtubule motor complex, p150glued, suggesting acetylation as a potential means to regulate motor-based cargo transport [69]. In line with this, α-tubulin acetylation elevated the velocity of kinesin-1 and enhanced the affinity to microtubules in the presence of other PTMs.

Pharmacological inhibition of HDAC6 produced a higher rate of kinesin-1 cargo protein transport [89] and facilitated anterograde and retrograde transport of vesicles containing brain-derived neurotrophic factor (BDNF) in neurons [90].

Furthermore, HDAC6 has been shown to play a role in the recruitment of misfolded proteins onto dynein motors for their transport to aggresomes [91] and in autophagic degradation of misfolded α-syn aggregates [92]. Together, it is

15 suggested that acetylation of α-tubulin is crucial for neurological processes, possibly leading to neuropathogenesis such as Parkinson’s disease when deregulated. Indeed, HDAC6 was identified as an essential factor for the degradation of aggregated huntington [93].

4.3 Acetylated α-tubulin in the central nervous system (CNS)

A recent study revealed that HDAC6 is expressed specifically at the tip of an axon, regulating the development of the axonal initial segment and axonal growth rate by modulating microtubule behavior [94]. In fact, acetylated α-tubulin is non-uniformly distributed and enriched in the proximal site of the axon and present less in the cell body and growth cone [95-96].

Interestingly, HDAC6 is implicated in emotional behavior of mice [97]. A variety of behavioral tests applied to Hdac6 KO mice revealed that these mice are hyper-active and less depressed. These phenotypes concur with abundant HDAC6 expression in serotonergic neurons in the dorsal and median raphe nuclei, which regulate emotional behaviors. In addition, deletion of Mec17 in C. elegans and zebrafish exhibited reduced touch responsiveness along with neuromuscular defects, supporting the importance of α-tubulin acetylation in the CNS function

[19].

4.4 HDAC6 in cell cycle control and cancer

In the effort to generate the expression profiling of estrogen-responsive genes using cDNA microarray, HDAC6 was found to be implicated in

16 tumorigenesis, cell proliferation, and cell cycle [98]. A tumor-suppressor gene,

CYLD constitutively was co-localized with acetylated microtubules in the perinuclear region, where it inhibits HDAC6 resulting in reduced rate of cytokinesis and a delay in the G1/S phase transition [99]. In addition, HDAC6 over-expression has been observed in many cancer cell lines and mouse tumor models. For example, HDAC6 is proposed to induce anchorage-independent proliferation and cell motility and plays an important role in maintaining transformed phenotypes [100-101]. Since microtubules are highly implicated in obtaining cellular motility, deregulated acetylation of α-tubulin may serve as one of the intracellular mechanisms that lead to acquisition of invasive nature of cancer cells.

5. Rationale for the thesis research project

Since it was first discovered in the mid-1980s, the in-depth investigation of acetylated α-tubulin at lysine 40 has been delayed partially due to the undefined status of the responsible enzymes. With HDAC6 identified as the α- tubulin deacetylase in the early 2000s, several reports have been recently made on the identification of responsible acetyltransferases, including ARD1-NAT1 [77],

ELP3 [78], and GCN5 [79]. This raises the question whether there are multiple α- tubulin acetyltransferases. Related to this, Mec17 was shown to be the bona-fide acetyltransferase of α-tubulin in C. elegans [19, 84]. Although this recent discovery determined the physiological impact of Atat1 in lower organisms including C. elegans [19], the in vivo function of this enzyme has not been examined in mammals such as mice and humans. In my thesis project, I have employed in vitro approaches to unmask other potential substrates of ATAT1, which may elucidate the intracellular influence of ATAT1 beyond acetylating α- tubulin. In addition, by using mice that lack Atat1, I have also demonstrated that

Atat1 is an authentic acetyltransferase of α-tubulin dispensable for mouse development. These findings will provide an important foundation for interpreting the impact of α-tubulin PTMs in mammals.

6. Figures and Legends

Figure 1. (A) The opposing actions of a KAT and an HDAC. A KAT catalyzes the transfer of an acetyl group (boxed in light green) from acetyl-CoA onto the ε- amino group of a lysine residue. Conversely, an HDAC hydrolyzes the acetylated lysine residue to remove the acetyl group from a substrate, releasing acetate. (B)

Deacetylation by sirtuins. Different from other HDACs which are Zn2+-dependent, sirtuins mediate deacetylation from an acetylated lysine residue using NAD+. This figure is derived from Kim et al [36].

Figure 2. Substrate diversity of lysine acetylation. Although initially discovered on histones, lysine acetylation has been identified in a variety of proteins with different function. Not only found in eukaryotes, lysine acetylation is known to occur in many prokaryotes, suggesting it is an evolutionarily conserved mechanism across various species. Recent proteomic studies have shown that lysine acetylation is highly implicated in metabolism, indicating lysine acetylation as a potential mechanism of metabolic regulation [15, 36]. Ac in a circle denotes acetylation.

Figure 3. ATAT1 is a bona-fide acetyltransferase of α-tubulin. (A to C)

Immunofluorescent images of Mec17 WT (two entities on the left side) and KO

(arrowed) Tetrahymena show that the Mec17 KO animal is devoid of acetylated α- tubulin at lysine 40. (D) In vitro acetylation assay using recombinant ATAT1.

ATAT1 efficiently acetylates α-tubulin shown by Western blotting (top) and by phosphor-imaging (middle). (E to H) Acetylated lysine 40 (acetyl-K40) of α- tubulin is significantly decreased in zebrafish embryos injected with ATG-MEF17 morpholinos (F and H) compared to the control embryos (E and G). The boxed area shown in E and F are enlarged in G and H at higher magnifications. All of these figures are adopted from Akella et al [19], except D, which is derived from

Shida et al [84].

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Chapter II

Identification of Atat1 as a major alpha- tubulin acetyltransferase dispensable for

mouse development

1. ABSTRACT

Proteomic studies in the past few years have suggested lysine acetylation as one major post-translational modification influencing thousands of proteins involved in diverse cellular function inside various organisms ranging from bacteria to humans. Among such proteins that are affected by lysine acetylation is

α-tubulin, a subunit of microtubules that serve as a cytoskeletal component along with actin filaments and intermediate filaments. Lysine acetylation of α-tubulin occurs at lysine 40 as a major site and is unique among other α-tubulin post- translational modifications for two reasons: 1) α-tubulin is one of the first acetylated proteins identified in the mid-1980s and 2) this acetylation occurs on the luminal side of microtubules. With the responsible deacetylase discovered to be HDAC6 about a decade ago, the counteracting acetyltransferase had long awaited discovery until recently. Although several acetyltransferases have been shown to affect the acetylation of α-tubulin, including ARD1-NAT1, ELP3, and

GCN5, Atat1 was demonstrated to be the bona-fide acetyltransferase of α-tubulin in C. elegans. An important question is whether this is also the case in vivo in mammalian systems. By using mice devoid of Atat1, I set out to determine the physiological importance of Atat1 and α-tubulin acetylation in mice. Here, I show that Atat1 is an authentic acetyltransferase of α-tubulin at lysine 40 in most of the tissues examined in mice but the loss of Atat1 is not detrimental to mouse development. In addition, through a set of proteomic and molecular studies, I have identified ATAT1 as a potential interaction partner of other proteins in addition to α-tubulin.

2. INTRODUCTION

Post-translational modifications are an important cellular mechanism that affects protein structure and function in addition to their own intrinsic properties.

Among various other post-translational modifications, lysine acetylation has emerged as a major modification comparable to phosphorylation [1-3]. The importance of lysine acetylation initially arose from its relation with chromatin organization and its consecutive impact on gene expression. However, it is now clear that the spectrum of lysine acetylation is broader than initially anticipated, comprising not only nuclear events but also a diversity of other cytoplasmic processes. Lysine acetylation is catalyzed by two groups of counteracting enzymes called lysine (K) acetyltransferases (KATs) and histone deacetylases

(HDACs). Although the nomenclature HDAC is longer accurate due to the substrate diversity of the enzymes, it will be used herein for a historical reason [4].

According to the sequence homology and structural similarity, KATs are mainly grouped into three families [5] and HDACs are divided into four classes (class I to

IV). Whereas HDACs in class I, II, and IV are Zn2+-dependent, sirtuins in class III require NAD+ for reaction.

One of the acetylated proteins discovered about three decades ago is α- tubulin, an important subunit of microtubules that comprise the cytoskeleton along with actin filaments and intermediate filaments. By structural analysis, acetylation of α-tubulin is predicted to occur on the luminal side of microtubules at lysine 40 which serves as a major site. A body of evidences has linked α-tubulin acetylation to microtubules in specific organelles, including the cytoplasmic

35 network, centrioles, and axonemes of cilia and flagella [6-9]. In 2002, HDAC6 was discovered to be the α-tubulin deacetylase [10] and the subsequent study confirmed that Hdac6 KO mice show global hyper-acetylation of α-tubulin in various tissues [11]. Another HDAC that had been suggested to deacetylate α- tubulin was SIRT2 [12]. However, genetic deletion of Sirt2 in mice did not affect acetylation of α-tubulin [11, 13], indicating HDAC6 as the major deacetylase of

α-tubulin. Interestingly, in spite of unique and crucial function, mice devoid of

Hdac6 were viable and fertile [11] and did not produce any obvious phenotype except anti-depressant like characteristics [14].

Due to veiled discovery of the α-tubulin acetyltransferase, ablation of α- tubulin acetylation was simulated by mutation of the lysine 40 residue (changed to arginine). However, in lower organisms such as Tetrahymena and

Chlamydomonas, lysine 40 mutation had no impact on survival [15-16]. In the past few years, several acetyltransferases have been shown to affect the acetylation of α-tubulin, including ARD1-NAT1 [17], ELP3 [18-19], and GCN5

[20]. Creppe et al. showed that ELP3, a multisubunit KAT, regulates the migration of cortical neurons in the developing mouse brain through acetylating α-tubulin

[19]. In the consecutive year, another group suggested that Gcn5 is recruited by

Myc-nick, a cytoplasmic form of Myc, and acetylates α-tubulin [20]. However, by an array of biochemical and cell-based approaches, it was demonstrated that

Mec17, the C. elegans homolog of α-tubulin acetyltransferase 1 (Atat1), is the bona-fide α-tubulin acetyltransferase that acts on lysine 40. The following lines of evidence support this finding. Firstly, ATAT1 exclusively acetylates lysine 40 of

α-tubulin in vitro and is necessary and sufficient for acetylation of α-tubulin at lysine 40 in mammalian cells [21]. Secondly, genetic disruption of Atat1 phenocopies the α-tubulin mutation at lysine 40 in Tetrahymena [22]. Thirdly, the induction of ATAT1 efficiently leads to acetylation of α-tubulin in cells devoid of acetylated α-tubulin, while ELP3 fails to do so [21].

Though the recent discovery of ATAT1 provided important insights into the function of α-tubulin acetylation in several organisms [21-22], the analysis of

Atat1 in mammals has not been examined yet. Here, by using mice deficient of

Atat1 by genetic deletion, we demonstrate that Atat1 is a major α-tubulin acetyltransferase dispensable for mouse development. We also show that Atat1 is robustly expressed in testis, renal pelvis, gastrointestinal (GI) tract, and hippocampus in mice, yet the development of these tissues appears normal in the absence of Atat1. Furthermore, through proteomic and molecular analyses, we suggest that Atat1 may have other substrates in addition to α-tubulin.

3. MATERIALS AND METHODS

Cell culture

Human embryonic kidney 293 (HEK 293) cells were cultured in

Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin/streptomycin (Invitrogen).

Cell transfection

Cells were seeded in a 12-well plate at a density of 0.04 X 106 cells/well, or in a 6 cm-dish at 0.15 X 106 cells/dish. Transfection was performed using 1.5

µL of lipofectamin (Invitrogen) for 12-well plates with 1.5 µg of DNA or 6 µL for

6 cm-dishes with 5 µg of DNA, according to the manufacturer’s instructions. The

GFP-HDAC6 plasmid construct, whose usage has been published in Bertos et al

[23], was used here.

Co-Immunoprecipitation (Co-IP)

HEK 293 cells were seeded and transfected in 6 cm-dishes as described above. The plasmids bearing a FLAG epitope were used as baits and either HA- or GFP-tagged plasmids were expressed as preys in the same cells. 48 hours of post-transfection, cells were washed with 3 mL of 1X PBS and lysed with 600 µL of buffer K (20 mM sodium phosphate pH 7.0, 150 mM potassium chloride, 30 mM sodium pyrophosphate, 0.1 % NP-40, 50 mM EDTA, 10 mM sodium fluoride,

0.1 mM sodium vanadate, and protease inhibitor cocktail including pepstatin, aprotinin,, leupeptin, and PMSF) on ice. Cell lysates were harvested in microtubes

38 and sonicated for 5 sec at setting 5 with subsequent rotation for 30 min at 4℃.

Meanwhile, 250 µL of M2 agarose beads (for seven 6 cm-dishes) were washed twice with 300 µL of buffer K. The beads were added with 500 µL of buffer K and aliquoted 100 µL to each microtube placed on ice. After 30 min of rotation, the cell lysates were centrifuged at 14,000 rpm for 6 min. 50 µL of cell lysates was kept for input and the rest was poured to the washed beads. Cell lysate/bead mixture was left rotated in the microtubes for 4 hours and processed for four times of washing. Proteins were eluted in 35 µL of buffer K containing 3.5 µL of FLAG peptide and further rotated for 45 min at 4℃. The eluants were collected and kept at -80℃. The input and purified eluants were analyzed by Western blotting.

Luciferase-based reporter gene assay

The ORF of ATAT1 was subcloned to a vector containing a

DNA/promoter binding domain (Gal4) which controls the expression of the luciferase reporter. For normalization control among different wells, β- galactosidase (CMV-β-Gal) was co-transfected in HEK 293 cells in a 12-well plate. 48 hours post-transfection, the subsequent procedures were performed.

Wash the cells twice with cold 1X PBS. Add 100 µL of lysis buffer (17 mM

KH2PO4, 18.3 mM K2HPO4, 1 % Triton X-100, 0.2 mM DTT, and 0.1 % glycerol).

Shake the plate for 10 min and collect the cell lysate in microtubes at 4℃. Spin at

14,000 rpm for 10 min at 4℃ and transfer the supernatant to new microtubes.

Transfer 20 µL of lysates to a flat-bottom 96-well white plate for luciferase reading and add 20 µL of lysis buffer to the adjacent well as a control. 70 µL of luciferin was added for luciferase reading. Transfer 5 µL of cell lysates for β- galactosidase reading and add 5 µL of lysis buffer to the adjacent well as a control.

Mix 990 µL of Galacto Reaction Buffer Diluent and 10 µL of Galacto-plus

(Applied Biosystems). Add this reaction mix to each well for β-galactosidase reading.

Animals

All the experimental procedures performed on mice were carried out according to standard operating procedures created by the Veterinary Care

Subcommittee and approved by the University Animal Care Committee.

The mice used for this thesis project are on C57BL/6NTac background and were housed in groups of one to five animals. Food and water were provided ad libitum (Animal Care Services at Goodman Cancer Center Animal Facility).

All the mice that have been used for the experiments were 5-7 weeks old males, unless indicated.

Mouse genotyping

Mice were genotyped by PCR with DNA extracted from ear punch. The protocol for DNA extraction is as follows. After punching mouse ears, ear pieces are immersed in 200 µL of 50 mM NaOH and incubated at 95℃ for 10 min. Add

20 µL of 1M Tris-HCl pH 6.8. Vortex samples for 7 sec. Centrifuge at 14,000

40 rpm for 6 min. Use 2 µL for PCR. Primer sequences for Atat1 and LacZ detection are,

Atat-F1 (5’-ACTGAAGGACACCTCAGCCCGA-3’),

Atat-R1 (5’-TACCTCATTGTGAGCCTCCCGG-3’),

LacInF (5’-GGTAAACTGGCTCGGATTAGGG-3’) and,

LacInR (5’-TTGACTGTAGCGGCTGATGTTG-3’).

The PCR mixture was made with 5 µL of 2X GoTaq Green Master Mix

(Promega), 2 µL of DNA, 0.25 µL of 10 pM primers, and 2.5 µL of nuclease-free water. Cycling conditions are as follows: 95℃ for 3 min, (95℃ for 30 sec, 50℃ for 30 sec, and 72℃ for 30 sec) X 30, followed by 2 min at 72℃. The PCR product was kept at 4℃ afterwards.

RT-PCR

Tissues collected from the mice were rinsed in ice-cold PBS containing

0.1 % of DEPC and put in microtubes. Tissues were added to 200 µL of PBS containing DEPC and crushed. 1 mL of Trizol (Invitrogen) was applied followed by vortexing. Crushed tissues were then left at the room temperature for 5 min.

Samples were centrifuged at 13000 rpm for 10 min at 4℃ and the supernatant was taken to new microtubes. 200 µL of chloroform was added to the supernatant and mixed by shaking. Samples were centrifuged for 15 min at 13000 rpm and the upper phase was taken and mixed well with 500 µL of isopropanol. Samples were again centrifuged for 15 min at 13000 rpm. Isopropanol was removed carefully

41 and pellet was washed with 750 µL of 75 % ethanol in DEPC water. Following the washing step with ethanol, pellet was air-dried and dissolved in an appropriate amount of DEPC water. After quantification of RNA concentration, cDNA was synthesized using QuantiTect Rev. Transcription kit (Qiagen) according to the manufacturer’s instructions. PCR was then performed using the synthesized cDNA. The PCR cycling condition is identical to that for genotyping (the same condition for Gapdh, too), but the reaction volume was adjusted to 25 µL according to the manufacturer’s instructions. Primers for Atat1 and LacZ are indicated in the genotyping method section. Primer sequences for Gapdh are, mGAPDH-RT01 (5’-GCA CAG TCA AGG CCG AGA AT-3’) and mGAPDH-RT02 (5’- GCC TTC TCC ATG GTG GTG AA-3’).

Tissue preparation for SDS-PAGE and Western blotting

Tissues were weighted and homogenized with a pestle in 3 volumes of

RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 % NP-40, 1 % sodium deoxycholate, 0.1 % SDS, and a cocktail of protease inhibitors described in the co-IP method section). Further homogenization was done by sonication at setting

5. After the centrifugation at 13000 rpm for 10 min, the supernatant was kept and stored at -80℃. All these procedures were done on ice. Subsequently, protein concentration was measured by Bradford assay.

SDS-PAGE and Western blotting

Samples were diluted in 3X RSB SDS-PAGE loading buffer and

42 denatured in boiling water for 5 min. Proteins were loaded and ran in a 10 % SDS polyacrylamide gel at 120 voltage (V) for 15 min and at 150 V until the blue dyes are off the gel. Proteins were then transferred onto nitrocellulose membranes at

200 mA for 3 hours at 4℃, using transfer apparatuses (Biorad). Membranes were blocked for 1 hour with 5 % non-fat dried milk in PBS containing 0.15 % Tween-

20 (Sigma). Primary antibodies [anti-FLAG antibodies (Sigma) and anti-HA antibodies (Covance) in 1:1000 dilution, anti-α-tubulin antibodies (Sigma) in

1:2500 dilution, and anti-acetylated α-tubulin antibodies (Sigma) in 1:10,000 dilution] were applied in the blocking solution overnight at 4℃. Blots were washed five times for 8 min each in PBS containing 0.15 % Tween-20 and incubated with HRP-conjugated anti-mouse IgG diluted in 1:5000 (GE healthcare

Life Sciences). For signal detection, chemi-luminescence solution (Pierce) and hyperfilms (Denville) were used.

Whole-mount β-galactosidase staining of tissues or embryos

Mice were anesthetized and perfused transcardially first with PBS and then with 2 % PFA in PBS. After perfusion is completed, tissues or embryos were collected and fixed at 4℃ for 2-3 hours (up to 1 hour for embryos) in a fixative

containing 2 % PFA, 0.2 % glutaldehyde, 0.02 % NP-40, and 0.2 mM MgCl2 in

PBS. The fixative was then changed to detergent rinse (0.1 M phosphate buffer pH 7.3, 2 mM MgCl2, 0.01 % sodium deoxycholate, and 0.02 % NP-40). After 2 hours to overnight incubation in the detergent rinse (changed 3 to 5 times), the

43 whole-mount tissues or embryos were incubated in X-gal staining solution at 37℃

{detergent rinse containing 5 mM of K3[Fe(CN)6] and K4[Fe(CN)6], and 1 mg/ml of X-gal} until the desired color is developed.

Preparation of frozen mouse tissue sections

Mice were anesthetized and sacrificed by cervical dislocation and subsequent procedures were followed. Fix the tissues overnight in 4 % PFA in

PBS at 4℃. The next morning, equilibrate in 15 % sucrose in PBS at 4℃ for about 8 hours. Equilibrate in 30 % sucrose in PBS at 4℃ overnight. Equilibrate further in OCT (Tissue-Tek) for 30 min at 4℃. Embed in OCT with careful consideration of orientation and quick-freeze on dry-ice. Store blocks at -80℃. 30 min prior to sectioning, place blocks in a styrofoam box containing dry-ice at a

-20℃ freezer. Section cryostat blocks 10-30 µm onto slides. Air-dry and store the slides at -80℃.

β-galactosidase staining of frozen sections

Sections were post-fixed in a fixative (0.2 % PFA in 0.1 M PIPES buffer pH. 6.9, 2 mM MgCl2, and 5 mM EGTA) on ice for 10 min and rinsed in PBS with 2 mM MgCl2, followed by washing in the same solution for 10 min. Sections were then permeabilized in detergent rinse on ice for 10 min. After staining at 37℃

44 in the dark (up to overnight), the slides were washed three times in PBS with 2 mM MgCl2 at room temperature for 5 min, followed by washing in distilled water.

Counterstaining was carried out in nuclear fast red solution (Sigma), with dehydration through a series of methanol (5 min each in 50 %, 70 %, and 100 %).

Sections were cleared in Histoclear (Diamed) for 5 min and mounted using a mounting medium (American Master Tech).

Immunofluorescence

Sections were fixed in ice-cold acetone at -20℃ for 10 min and the subsequent procedures were followed. Draw a circle with a PAP pen (Invitrogen) around the tissue sections. Rinse with PBS three times for 5 min. Permeabilize with IF buffer [PBS containing 0.2 % Triton X-100 (Biorad) and 0.05 % Tween-

20] for 30 min. Incubate in blocking solution [IF buffer with 2% bovine serum albumin (Sigma)] for 1 hour. Incubate with the primary antibodies (anti-α-tubulin antibodies in 1:500 dilution and anti-acetylated α-tubulin antibodies in 1:1000 dilution) for 3 hours. Wash three times with IF buffer. From the next step on, all the procedures were carried out in the dark. Incubate with the secondary antibodies [Alexa fluor-labeled anti-mouse IgG (Invitrogen)] for 1 hour. Wash three times with IF buffer. Incubate in PBS containing DAPI (0.5 ng/mL) for 5 min with subsequent washing in distilled water. Mount using a mounting medium

(Thermo Scientific).

4. RESULTS

Perhaps due to the recent identification, little has been defined about other roles of ATAT1 besides acetylation of α-tubulin. So, here we have employed a set of in vitro experiments to explore other potential function (if any) of ATAT1.

The putative substrates of ATAT1 suggested by mass spectrometry (MS)

Although it was well established by previous in vitro and in vivo data that

α-tubulin is a faithful substrate of ATAT1 [21-22], it is possible that ATAT1 has impact on the acetylation status of other proteins, given that the counteracting enzyme, HDAC6 acts on other cytosolic proteins in addition to α-tubulin. To survey the range of ATAT1 substrate diversity, mass spectrometry (MS) was executed to find other potential targets of ATAT1 (Table 1). As expected, high protein abundance of α-tubulin was observed.

We then explored to validate if the proteins listed in the MS data physically associate with ATAT1. For this, co-immunoprecipitation (co-IP) was performed, followed by Western blotting. We tested some of the proteins listed in the MS data (Fig. 1) using either ATAT1 or the candidate proteins as a FLAG- tagged bait. p53 and 14-3-3 did not co-immunoprecipitate with ATAT1 (Fig. 1A), suggesting that ATAT1 does not interact with these proteins. AKAP8 and

AKAP8L, the proteins that associate with regulatory subunit of PKA, were well expressed but only AKAP8L was shown to bind to ATAT1 with much high affinity, consistent with its MS output (Fig. 1A). Likewise, tubulin-β4 (TUBB4),

MAGED2 which is known as a negative regulator of p53 [24], and the

46 transcription intermediary factor 1β (TIF1B) were well associated with ATAT1 in accordance with MS result (Fig. 1A and 1B). To our curiosity, we tested if there is physical interaction between ATAT1 and HDAC6 and found that they do not bind to each other (Fig. 1C). In summary, the verification of the MS data by co-IP showed that ATAT1 may have other potential targets in addition to α-tubulin.

ATAT1 does not bind to the known HDAC6 substrates and their effector

Although not listed in the MS data, we set out to determine if the known substrates of HDAC6 (cortactin and HSP90) and their effector (RAC1) bind to

ATAT1. Similar biochemical approaches using co-IP followed by Western blotting were executed. As a result, ATAT1 did not co-immunoprecipitate with any of these proteins (Fig. 2). This suggests that acetylation of cortactin, HSP90, and

RAC1 may be modulated by a KAT other than ATAT1 and therefore, ATAT1 may not counteract with HDAC6 for these proteins.

ATAT1 is co-localized with BBS4, a cilium marker

Previous studies have demonstrated that acetylated α-tubulin is enriched in ciliary microtubules [6, 9]. To investigate if ATAT1 is involved in cilia, we examined whether ATAT1 is co-localized with a cilium marker, BBS4. GFP- tagged ATAT1 was co-transfected with mCherry-tagged BBS4 in HEK 293 cells and 48 hours of post-transfection, cells were observed under the fluorescence microscope. As shown in Fig. 3Aa and 3Ab, ATAT1 was mostly localized to cytoplasm and co-localized with BBS4 (Fig. 3Aa to Cb), suggesting a possible

47 role of ATAT1 in ciliary formation or maintenance. This is likely given that

ATAT1 is required for rapid ciliogenesis in mammalian cells [21] and that enrichment of acetylated α-tubulin is found in cilia. The mutant ATAT1 construct with only N-terminus, where the catalytic domain of ATAT1 is, produced similar results observed with the WT construct (Fig. 3D to F). In contrast, the mutant form of ATAT1 with only C-terminus, which does not exhibit homology to any known domain, was mostly pan-cellular (Fig. 3G to I). Together, these results show that ATAT1 is co-localized with BBS4 in HEK 293 cells and that this co- localization is no longer observed when the catalytic domain is removed, suggesting that the catalytic activity may play an important role in the subcellular localization of ATAT1.

Transcriptional activity of ATAT1

In spite of its predominant localization in cytoplasm, HDAC6 affects expression of several genes [25]. Thus, we set out to determine if ATAT1 possesses any transcriptional activity by the luciferase-based reporter gene assay.

For this, the luciferase reporter (Gal4-TK-Luc) and β-galactosidase reporter

(CMV-β-Gal) were co-transfected without ATAT1 or with an increasing amount of an ATAT1 construct bearing a Gal4 DNA-binding domain. The luciferase activity was measured and normalized by internal β-galactosidase activity to rule out any possibility that the calculated reporter activity is due to differential transfection efficiency. Upon the Gal4-Gal4 association, the luciferase is either transactivated or inhibited depending on the transcriptional activity of ATAT1. As shown in Fig.

4, ATAT1 reduced the expression of the reporter gene and this effect was not dose- dependent. Direct inhibitory action of ATAT1 is less likely in that it is predominantly localized to the cytoplasm (shown in Fig. 3Aa and Ab). On the other hand, ATAT1 may indirectly inhibit gene expression through acetylating factors required for transcription, conferring the factor repressive to transcription.

In vivo analysis of mouse Atat1

KO animals are an absolute requirement to investigate in vivo function of the gene of interest. We have obtained Atat1 KO mice from U.C. Davis

(KnockOut Mouse Project Repository) and maintained them.

Targeted deletion of Atat1 in mice

To determine the necessity of Atat1 in mice and to observe what roles

Atat1 plays at the physiological level, we used and examined mice in which Atat1 is genetically deleted in C57BL/6NTac background. These mice were generated by gene trapping and tactical description is as follows (Fig. 5A). The targeting vector (middle) with a gene trapping cassette containing a promoterless lacZ and a selection marker flanked by a splice acceptor (SA) and a polyadenylation site

[p(A)] was targeted to the intron between exon 6 and exon 7 of the Atat1 (top) gene by homologous recombination. The lacZ is expressed under the endogenous promoter of Atat1 with the resulting gene product expressed as a fusion protein with truncated and non-functional Atat1.

Heterozygous mice were mated to produce litters containing WT

(Atat1+/+), heterozygous (Atat1+/-), or homozygous KO (Atat1-/-) mice. The genotypes of mice were determined by PCR using DNA extracted from ear punch.

Mating between Atat1+/- mice yielded mice with different genotypes at the expected Mandelian ratio.

Overall, the Atat1-/- mice were viable and did not produce any obvious phenotype compared to their Atat1+/+ and Atat1+/- littermates. Both male and female Atat1-/- mice were indistinguishable from their Atat1+/+ or Atat1+/- littermates by gross examination. Genetic deletion of Atat1 did not affect the size and the overall growth (Fig. 5B to F) and both Atat1+/- and Atat1-/- mice were fertile. The viability and fertility of Atat1-/- mice were somewhat expected in that the abrogation of α-tubulin acetylation by mutating lysine 40 did not affect the survival of Tetrahymena and Chlamydomonas. Also, Hdac6 KO mice were viable and developed normally, suggesting that tight regulation of α-tubulin acetylation is not an absolute requirement for normal growth.

To validate that these Atat1-/- mice do not express Atat1, RNA from different tissues in Atat1+/+ and Atat1-/- mice was extracted and compared for the mRNA levels of endogenous Atat1 and inserted lacZ reporter gene by RT-PCR.

As shown in Fig. 6A, the mRNA transcript of Atat1 was completely absent in the

Atat1-/- mouse, while the lacZ reporter gene was expressed only in the Atat1-/- mouse, confirming the accuracy of the genetic strategy.

Atat1 is a major acetyltransferase of α-tubulin in mice

To substantiate the genetic ablation of Atat1 at the functional level, we

50 extracted proteins from various tissues in Atat1+/+ and Atat1-/- mice and performed Western blotting using antibodies against α-tubulin or against acetylated lysine 40 of α-tubulin. As shown in Fig. 6B, α-tubulin acetylation was totally ablated in many tissues such as testis, kidney, liver, and brain in the Atat1-/- mouse, while acetylation of histone H3 was not affected (data not shown).

Although acetylated α-tubulin was still observed in lung, spleen, and fat, it is dramatically decreased in these tissues except fat. Taken together, these results demonstrate that Atat1-/- mice exhibit significant loss of acetylated α-tubulin, indicating Atat1 as the major α-tubulin acetyltransferase. The presence of acetylated α-tubulin in lung, spleen, and fat proposes the existence of another putative acetyltransferase of α-tubulin. However, the significant decrease of α- tubulin acetylation in these tissues (except fat) also reflects the minor role of the putative acetyltransferase in acetylating α-tubulin. Whether it is Ard1-Nat1, Elp3, or Gcn5 that is responsible for the retained acetylated α-tubulin in these tissues remains to be determined.

ATAT1 is expressed ubiquitously in embryos

Taking advantage of the inserted lacZ reporter geneby the gene trapping strategy (Fig. 5A) that reports the expression of Atat1, we next sought the expression pattern of Atat1 during mouse embryonic development (Fig. 7). For this, embryos at E10.75 and E13.5 were examined by whole-mount β- galactosidase staining. Embryonic days were calculated according to the convention that E0.5 corresponds to the morning of finding the copulation plug,

51 assuming mating occurred at midnight.

Gross examination of the Atat1 WT and KO embryos did not reveal any developmental and morphological defect during this embryonic period. At both

E10.75 and E13.5, Atat1 is expressed ubiquitously throughout the embryos (Fig.

7A to D), with the spinal cord more intensely stained in the first place at E13.5

(data not shown). In summary, Atat1 deletion is not detrimental to mouse embryonic development and Atat1 does not exhibit a specific expression pattern but is rather expressed ubiquitously, suggesting that Atat1 may not play a vital role during mouse embryonic development.

ATAT1 expression in adult mouse tissues

We then explored to obtain the global expression pattern of Atat1 in adult mice. For this, we collected a variety of tissues from Atat1+/+ and Atat1-/- mice and performed whole-mount β-galactosidase staining. No sign of pathogenecity in any tissue, such as tumor formation, was found in the Atat1-/- mice and the size of the tissues was similar between Atat1+/+ and Atat1-/- mice by gross examination.

As shown in Fig. 8, the expression of Atat1 varied among different tissues, with the testis, renal pelvis, GI tract (stomach, small intestine, and rectum), and brain, having the most intense expression (Fig. 8A, B, H to J, and L to M respectively).

No expression of Atat1 was observed in spleen and heart (Fig. 8E and F, respectively). In fact, the robust levels of Atat1 in the brain and the testis are consistent with the previous finding that acetylated α-tubulin is associated with stable, long-lived microtubules found in axons of neurons [26-29] and basal

52 bodies and axonemes of flagella [30-31]. Also, it has been reported that in pathogenesis induced by C. difficile toxin A, the levels of acetylated α-tubulin are decreased, leading to acute inflammation in intestine [32]. This suggests that α- tubulin acetylation is required for normal function of the intestine and disrupted acetylation of α-tubulin is implicated in intestinal pathogenesis, implying a possible role of Atat1 in this tissue. Conversely, the high expression of Atat1 in renal pelvis was surprising, but it is feasible that acetylation of α-tubulin serves for primary cilia in kidney. Together, the robust expression of Atat1 in the testis, renal pelvis, GI tract, and brain, and yet no obvious defects of these tissues upon

Atat1 deletion, suggests that Atat1 may play essential roles in these tissues under certain conditions.

Atat1 expression in mouse brain and testis sections

We then explored to gain further insight into the Atat1 expression in these tissues by histological methods. For this, brain and testis tissues were prepared in frozen blocks and sectioned. β-galactosidase staining was performed to examine where exactly Atat1 is expressed within these tissues.

Overall, tissue histology of the brain from the Atat1-/- mouse appeared normal (by H&E staining, data not shown). Expression pattern of Atat1 in the brain seemed ubiquitous throughout all brain regions, as exemplified in the cerebellum and the cerebral cortex (Fig. 9A to B, respectively). Interestingly, specific and strong expression of Atat1 was detected in hippocampal regions (Fig.

9C), suggesting a potential role of Atat1 in the hippocampus.

In the testis, predominant expression of Atat1 was observed in a region between the lumen and the basal lamina of the seminiferous tubule, where spermatids and spematocytes reside (Fig. 10A to B). By immunofluorescence, we observed that α-tubulin is present in almost every cell in the seminiferous tubules localizing exclusively to the cytoplasm (Fig. 10C to E and I to K, in Atat1+/+ and

Atat1-/-, respectively). However, acetylated α-tubulin seemed to be specific for cells in the central region of the seminiferous tubules (Fig. 10F to H), consistent with the expression pattern of Atat1 shown in Fig. 10B. This observation was absent in the testis section of the Atat1-/- mouse (Fig. 10L to N). The fluorescence emanating from interstitium (areas between the seminiferous tubules) was found as non-specific auto-fluorescence since the negative control (immunofluorescent staining without the primary antibody incubation) also exhibits signal in these regions (data not shown). Together, these results suggest that Atat1 and acetylated

α-tubulin are presumably present in spermatids and spermatocytes located in the central region of the seminiferous tubules of adult mouse testis. However, further validation using specific markers of spermatids and spermatocytes will complement these findings and the identification of the specific role of Atat1 in these cells requires other functional assays.

5. DISCUSSION AND CONCLUSION

Other potential targets of Atat1 in addition to α-tubulin – Although previous studies firmly established that Atat1 faithfully acetylates α-tubulin, we took a further step to find other potential substrates of ATAT1. Through MS analysis and validation by co-IP, we determined that α-tubulin may not be the only substrate of ATAT1. Indeed, we identified AKAP8L, TUBB4, MAGED2, and

TIF1B as the potential interaction partners of ATAT1. Because we selected some of the proteins listed in the MS result, it is possible that more interaction partners of ATAT1 exist. With these ATAT1 binding proteins at hand, there are two questions to be addressed: 1) does acetylation actually occur on these proteins? and 2) if it does, how does it affect the function of these proteins? Elucidation of these two issues will reveal how ATAT1 exerts its effect in the cell in addition to acetylating α-tubulin. We also showed that ATAT1 does not interact with the known substrates of HDAC6 and their effector, suggesting that another KAT counteracts with HDAC6 over the acetylation status of these proteins.

Possible roles of ATAT1 in cilia - Among the organelles associated with acetylated α-tubulin, we tested if ATAT1 is implicated in cilia by looking at BBS4 and its co-localization with ATAT1. We observed that ATAT1 is mostly expressed in the cytoplasm and co-localized with BBS4. Mammalian cells require and produce primary cilia under certain extracellular conditions in order to sense the environmental changes through intracellular signaling. Though our results are only preliminary, based on the fact that cilia are formed of acetylated microtubule bundles and that acetylation of α-tubulin is implicated in kinetics of ciliogenesis

[21], the possibility of ATAT1 involvement in dynamics of ciliary microtubules is highly feasible.

Transcriptional activity of ATAT1 - BLAST search to find functional domain(s) of ATAT1 beside the GCN5-like acetyltransferase domain in the N- terminus did not yield any known functional domain, suggesting that acetylation is a primary function of ATAT1. However, as a method to predict any transcriptional activity of ATAT1, we took advantage of the luciferase-based reporter gene assay and showed that ATAT1 down-regulates luciferase expression.

Although this could be due to a direct effect of ATAT1, it is more likely that

ATAT1 indirectly mediates down-regulation of transcription by acetylating other putative proteins which are capable of binding to Gal4 DNA-binding domain for two reasons. First, ATAT1 is predominantly localized in cytoplasm. Second, one of the candidate substrates of ATAT1 found in the MS data and co-IP is TIF1B, a protein involved in transcription. In both cases, it is interesting to observe the inhibitory effect of ATAT1 on transcription since HDAC6 also has impact on transcription of several genes [25].

Viability of Atat1-/- mice – We showed that Atat1-/- mice exhibit significant loss of acetylated α-tubulin at lysine 40 in a majority of tissues. However, Atat1-/- mice did not produce any obvious phenotypes or defects. These mice were viable, fertile, and healthy like their WT littermates. These results were somewhat anticipated based on the viability and normal development of Hdac6 KO mice

[11]. To date, our oldest Atat1-/- mice (around 5 months-old by 2012 August) have not exhibited any obvious physical or behavioral abnormalities compared to their

WT littermates. Nonetheless, we cannot preclude the possibility that Atat1 plays important function in later stages of life and/or under certain conditions. For example, a critical part of the treatment for spinal cord injury depends on axon regeneration and appropriate synapse formation. Indeed, there have been several reports, which implicated microtubule stabilization in facilitating axon regeneration [33-34]. Because our results showed that Atat1 is the only α-tubulin acetyltransferase in brain (Fig. 6B) and is highly expressed in the CNS (Fig. 8K to

M and Fig. 9) and it was previously reported that Atat1, as well as acetylated α- tubulin, is involved in microtubule stabilization [35-38], Atat1 may be a key target to treat spinal cord injuries. HDAC6 over-expression observed in many cancer cell lines also implicates deregulated acetylation of α-tubulin in cancer.

Furthermore, decreased levels of acetylated α-tubulin in response to intestinal inflammation induced by C. difficile toxin A support the idea that acetylation of α- tubulin plays a critical role under pathogenic conditions [32].

Atat1 in microtubule stabilization – Recent studies showed that Atat1 functions to maintain microtubule architecture [35-36]. In touch receptor neurons of C. elegans that lack Mec17, fewer and shorter microtubules were present and more strikingly, these microtubules were varied in protofilament number both between and within microtubules [35-36]. Mec17 increased the cohesion between tubulin subunits in the microtubule lattice and constrained the protofilament number by acetylating lysine 40 of α-tubulin. The absence of Mec17 resulted in reduced cohesion and destabilization of the microtubules, suggesting that lysine

40 acetylation of α-tubulin by Mec17 provides mechanical assistance to

57 microtubule integrity. Whether similar observations are acquired in cells derived from Atat1-/- mice remains to be determined and will provide an important insight into the precise role of α-tubulin acetylation in mammals.

Histological examination of Atat1 - To detect any subtle change in tissues where Atat1 is highly expressed, histological examination was employed and showed that Atat1 is highly and specifically expressed in the mouse hippocampus

(Fig. 8M and 9C). The hippocampus is well known as a region of the brain responsible for processing spatial memory and navigation, and therefore, the robust expression of Atat1 suggests that Atat1 may be involved in these neurological processes and subsequent brain function. Although not performed here, applying various behavioral tests to the Atat1-/- mice such as the Morris water maze, which tests spatial learning and memory, will help determine if deficiency of Atat1 leads to any defect in hippocampal function. In the testis,

Atat1 was expressed presumably in the spermatids and spermatocytes located in the central region of the seminiferous tubules (Fig. 10B). This expression pattern was consistent with the immunofluorescent image obtained with antibodies against acetylated lysine 40 of α-tubulin, suggesting that Atat1 may regulate the acetylation of α-tubulin at lysine 40 in these cells (Fig. 10H). Indeed, our Western blotting result (Fig. 6B), showed Atat1 as the sole α-tubulin acetyltransferase in the testis. Although possible roles of acetylated α-tubulin in positioning and orientation of spermatogonia in the porcine testis have been reported, the exact function of Atat1 in spermatids and spermatocytes of the mouse testis remains to be discovered. Histological analysis of other tissues such as renal pelvis and GI

58 tract will also shed light on of finding physiological implication of Atat1 and of acetylated α-tubulin.

Conclusion – We have demonstrated that Atat1 is an authentic acetyltransferase of α-tubulin in a majority of tissues in mice with a potential to acetylate other substrates in addition to α-tubulin. This is the first demonstration that Atat1 is a bona-fide α-tubulin acetyltransferase in mammals. Viability of the

Atat1-/- mice suggests that the loss of Atat1 is not detrimental to mouse development. Though Atat1 is highly expressed in the testis, renal pelvis, GI tract, and the hippocampus, normal development of these tissues is achieved in the absence of Atat1 and therefore, further functional and behavioral analysis should follow to elucidate whether Atat1 plays an important role in these tissues under certain conditions. Given the importance of microtubule regulation in a diverse range of physiological processes, the results found herein will be an important framework for documenting the physiological impact of α-tubulin PTMs.

6. ILLUSTRATIONS

Table 1. Mass spectral counts of proteins associated with ATAT1. MS was done using recombinant FLAG-tagged ATAT1 as a bait and shows that α-tubulin is a faithful substrate of ATAT1 as previously reported. Other listed proteins were validated for their physical association with ATAT1 by co-IP in the following figures.

Figure 1. Validation of MS analysis by co-IP. Proteins listed in the MS result were subject to co-IP to confirm their physical interaction with ATAT1. Stronger bands of FLAG-tagged proteins in the immunoprecipitated samples indicate concentrated amount of the proteins. (A) As accordance with the MS data,

AKAP8L, TUBB4, and MAGED2, well associated with ATAT1. However, p53 and 14-3-3 did not associate with ATAT1. Bars along the right side of the figure indicate the size of the proteins. (B) TIFIB is also validated to interact with, and therefore, suggested to be a potential target of ATAT1. (C) HDAC6 does not physically interact with ATAT1. The light band (on the right side of the figure) shown at the bottom is a heavy chain contamination of FLAG-tagged antibodies that are attached to M2 beads.

Figure 2. The known substrates of HDAC6 (cortactin and Hsp90) and their effector protein (Rac1) do not co-immunoprecipitate with ATAT1, suggesting absence of their interaction with ATAT1. This result also suggests that acetylation of these proteins is not modulated by ATAT1 and therefore, HDAC6 counteracts with another acetyltransferase over these proteins. N-ATAT1 is the ATAT1 mutant containing only N-terminal region. The asterisks denote heavy chain contamination of FLAG-tagged antibodies that are attached to M2 beads.

Figure 3. BBS4 is co-localized with ATAT1. GFP-tagged WT or mutant constructs of ATAT1 were co-transfected in HEK 293 cells with mCherry-tagged

BBS4, a cilium marker. In most of the cases, only WT (Aa to Cb) and the mutant containing only N-terminal part of ATAT1 (D to F), and not the mutant containing only C-terminal part of ATAT1 (G to I), were co-localized with BBS4.

Figure 4. Transcriptional activity of ATAT1. ATAT1 (Gal4-ATAT1) has an inhibitory effect on the luciferase expression (Gal4-SV40-Luc) in a dose- independent manner. In all cases, CMV-β-galactosidase was co-expressed as an internal control.

Figure 5. Targeted deletion of Atat1 in mice. (A) Genetic strategy of generating

Atat1-/- mice. The targeting vector (in the middle) was inserted into the intron between exon 6 and 7 by homologous recombination. The gene trapping cassette in the targeting vector contains a promoterless lacZ and a selection marker (h-

UBC promoter-neor) flanked by a splice acceptor (SA) and polyadenylation sites

[p(A)]. Due to the p(A) site just downstream of the LacZ gene, the final gene product, which is truncated and non-functional, is expressed as a fusion protein with β-gal. (B to E) Atat1-/- mice appear normal and are indistinguishable from their WT littermates by gross examination. (F) Atat1+/+ (WT), Atat1+/-

(heterozygous), and Atat1-/- (KO) littermate male mice were weighted weekly and showed no obvious difference in gaining weight and overall growth. Each graph figure represents an individual litter, with averaged weight of mice having the same genotype.

Figure 6. Atat1 is the major acetyltransferase of α-tubulin at lysine 40 in mice. (A)

The genetic strategy of generating Atat1-/- mice was confirmed by RT-PCR. No mRNA of Atat1 was detected in the Atat1-/- mouse, whereas the mRNA expression of the inserted LacZ gene was expressed only in the Atat1-/- mouse. (B) Protein extracted from various tissues of Atat1+/+ and Atat1-/- mice was subject to Western blotting using antibodies against α-tubulin (left) or antibodies specifically against acetylated lysine 40 of α-tubulin (right). In a majority of tissues, Atat1 deficiency led to absence or significant decrease of acetylated lysine 40 of α-tubulin, indicating that Atat1 is the major acetyltransferase of α-tubulin.

Figure 7. Expression of Atat1 in mouse embryos. At both E10.75 (A to B) and

E13.5 (C to D), Atat1 is expressed ubiquitously throughout the embryos. Atat1-/- embryos do not show any morphological defect, suggesting that Atat1 is not an absolute requirement for normal embryonic development.

Figure 8. Expression of Atat1 in adult mouse tissues. Atat1 in (A) testis, (B) kidney, (C) liver, (D) lung, (E) spleen, (F) heart, (G) pancreas, (H) stomach, (I) small intestine, (J) rectum, (K) spinal cord, (L) sagitally cut brain, and (M) coronally cut brain are shown following β-galactosidase staining. Strong expression of Atat1 is observed in the testis, GI tract, and brain, as well as renal pelvis.

Figure 9. β-galactosidase staining on frozen sections of Atat1+/+ and Atat1-/- mouse brain. Though Atat1 was expressed uniformly throughout all brain regions including (A) cerebellum and (B) cerebral cortex, specifically high expression of

Atat1 was detected in hippocampus (C).

Figure 10. Frozen sections of Atat1+/+ and Atat1-/- mouse testis. (A to B) β- galactosidase staining shows that Atat1 is mostly expressed in a central layer of seminiferous tubule. (C to E, and I to K, respectively) Immunofluorescence using anti-α-tubulin antibodies on Atat1+/+ and Atat1-/- testis sections shows α-tubulin present in almost every cell in the seminiferous tubules, localizing exclusively in cytoplasm. Immunofluorescent staining with antibodies against acetylated lysine

40 of α-tubulin (F to H) shows specific localization of acetylated α-tubulin in the central region of the seminiferous tubules, consistent with the Atat1 expression shown in (B). This is no longer observed on the testis section of the Atat1-/- mouse

(L to N).

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