Morphogenetic events during insect development are triggered by ecdysteroids. The major insect ecdysteroid, 20-hydroxyecdysone (20E), binds directly to a heterodimeric transcription factor comprising two nuclear receptors, the ecdysone receptor (EcR) and the ultraspiracle (USP), and regulates various cellular processes including cell proliferation, cell differentiation, and cell death. The EcR/USP heterodimer binds to ecdysone response elements in the promoter of various ecdysteroid-responsive genes, and activates a complex transcriptional cascade. The EcR gene was identified in several non-insect arthropods (crustaceans, arachnids, and scorpions) as well as insects, and the molt-regulating function of the ecdysteroid/EcR system is conserved in the major subgroups of Arthropoda.

The ecdysteroid/EcR system has been studied extensively in the selected holometabolous insects such as a fruit fly Drosophila melanogaster and a silkmoth Bombyx mori. In holometabolous insects, there are two or three EcR isoforms (A and B1 isoforms; Drosophila has an additional B2 isoform) that are produced from a single genetic locus by differential promoter usage and alternative splicing. EcR isoforms have a common C-terminal region that includes the DNA binding and ligand binding domains (the C-F domains), but also have isoform-specific regions in the N-terminal A/B domain (Fig. 1A). In holometabolous insects, different EcR isoforms govern distinct ecdysteroid-stimulated responses during metamorphosis. In Drosophila, the EcR-A isoform is predominantly expressed in the proliferative tissues during metamorphosis and is required for adultspecific developmental processes. In contrast, EcR-B isoforms (B1 and B2 isoforms) are expressed in larvaspecific tissues and are involved in 20E-triggered larval tissue remodeling during metamorphosis. Because only the A isoform has been identified in basal direct-developing (ametabolous and hemimetabolous) insects, it is not known whether direct-developing insects have multiple EcR isoforms with distinct physiologic functions.

The key to understand the mechanism of the isoform-specific responses to 20E is the isoform-specific transcription activation functions. Generally, the nuclear receptors have two transcriptional activation functions (AF-1 and AF-2), and both AF regions are involved in recruitment of co-regulatory proteins (Fig. 1B). The EcR isoforms have the isoform-specific region in the A/B domain, which contains the ligandindependent activation function (AF)-1 region. Several studies of the AF-1 region in Drosophila EcR isoforms have revealed that the A/B domain of Drosophila EcR-B isoforms have strong transactivation activity, whereas that of the A isoform has weaker transactivation activity. The AF-1 of Drosophila EcR-B1 mainly locates in the N-terminal region (amino acid residues 1-53), whose sequence is considerably conserved among higher holometabolous insects such as flies, mosquitoes, and moths. However, the structural basis and molecular mechanisms underlying the isoform-specific AF-1 functions remain obscure.

The ecdysteroid/EcR system is conserved among arthropods, and the EcR-A and -B1 isoforms were found in several non-insect arthropods as well as insects. Thus, I hypothesized that the mechanisms of the isoform-specific AF-1 region-mediated transcriptional regulation are essentially conserved across arthropod species. If so, even if the isoform-specific AF-1 region of each EcR isoform varies in length and sequence across species, the essential structural basis for transcriptional regulation might be conserved. In search for the essential structural basis of the isoform-specific AF-1 activation function of each EcR isoform, I performed a comprehensive structural comparison of the isoform-specific regions of insect EcR-A and -B1 isoforms. The EcR isoforms were newly identified from 51 species of insects and non-insect arthropods, including directdeveloping ametabolous and hemimetabolous insects. The comprehensive structural comparison revealed that the isoform-specific region of each EcR isoform contained evolutionally conserved microdomain structures and insect subgroup-specific structural modifications (Fig. 2): The A isoform-specific region generally contained four conserved microdomains, the SUMOylation motif, the nuclear localization signal, the (D/E)(D/E)W motif, and the A-box (Fig. 2A). On the other hand, the B1 isoform-specific region contained three conserved microdomains, the S-rich motif, the SP residues, and the DL-rich motif. In addition, the EcR-B1 isoform of holometabolous insects had a novel (K/R)RRW motif at the N-terminal end (Fig. 2B).

To evaluate the functional roles of the conserved microdomains in transcriptional regulation, I performed the luciferase reporter assay. The isoform-specific regions of the Drosophila EcR-A and -B1 isoforms were C-terminally fused to the DNA binding domain of the GAL4 transcription factor, and were expressed in the Drosophila S2 cells. The reporter assay revealed that the isoform-specific region of EcR-A and -B1 isoforms have a weak transactivation activity and a strong transactivation activity, respectively, in the S2 cells. In addition, the reporter assay using the microdomain-deletion mutants revealed that themicrodomain-mediated transcriptional regulations: In the A isoform-specific region, the SUMOylation motif and the A-box were involved in transcriptional regulation. In the B1 isoform-specific region, the (K/R)RRW motif and the DL-rich motif were involved in transcription activation activity (Fig. 3). Given that the nuclear receptor AF-1 is involved in cofactor recruitment, the microdomain structures identified in the isoform-specific region of each EcR isoform might function as signature motifs and/or as targets for cofactor proteins. To test this hypothesis, I examined the microdomain-protein(s) interaction in the isoform-specific region of the Drosophila EcR-B1 isoform by using the fluorescence correlation spectroscopy (FCS) and the pull-down assay. The FCS assay using the motif-deletion mutants of the EGFP-fused B1 isoform-specific region revealed that the (K/R)RRW motif is involved in protein-protein interaction in the nucleus of the Drosophila S2 cells. Moreover, I obtained a ~50 kDa (K/R)RRW-motif-interacting protein from the Drosophila S2 cells by pulldown assay. These data suggest that the holometabolous insect EcR-B1 isoform acquired additional coregulatory protein and transcriptional regulation mechanisms, which are mediated by the novel (K/R)RRW motif.

This is the first study to determine the structural basis of the nuclear receptor AF-1 activation function based on the comprehensive structural comparison and the molecular evolutionary analysis. This study provides crucial insights into the isoform-specific transcriptional regulation mechanism of the insect EcR isoforms, as well as the structure-function relationships within the EcR isoform-specific AF-1 regions. Moreover, this is the first report on the identification of the EcR-B1 isoform in direct-developing insects. Further comparative studies on the developmental functions of the each EcR isoform in the holometabolous and direct-developing insects will shed light on the molecular mechanisms underlying the evolution of the complete metamorphosis.

Fig. 1. A) Domain structure of insect EcR isoforms. EcR isoforms have the isoform-specific region in the N-terminal A/B domain, which contains the ligand-independent activation function (AF-1). EcR-B2 isoform was only found in Drosophila. B) Schematic representation of the functional EcR transcriptional complex. EcR/USP heterodimer bind to the EcRE and recruit co-regulatory proteins on the AF-1 and AF-2. The EcR isoform-specific AF-1 results in the differential recruitment of regulatory proteins.

Fig. 2. Structural diversities of the EcR-A isoform-specific AF-1 region in insects. A) Evolution model for the structural modification in the A isoform-specific AF-1 region in insects. B) Evolution model for the structural modification in the B1 isoform-specific AF-1 region in insects. Schematic representation shows the phylogenetic relationship of insect subgroups with the structural types of the A isoform-specific region. Conserved structures are indicated by colored boxes.

Fig. 3. Luciferase assay to evaluate the AF-1 functions of the Drosophila EcR-B1 isoform-specific regions. The isoformspecific region of EcR-A and B1 isoforms were C-terminally fused to the GAL4 DNA-binding domain (GAL4DBD). The isoform-specific region of the Drosophila EcR-B1 isoform showed strong transactivation activity in Drosophila S2 cells. The deletion of the (K/R)RRW motif and the DL-rich motif resulted in decreased transactivation activity.

昆虫の脱皮・変態時の形態形成は脱皮ホルモン、エクダイソンによって協調的に制御される。エクダイソンは核内受容体であるエクダイソン受容体に受容され、多数の標的遺伝子の遺伝子発現を調節する。昆虫は、N-末端領域にアイソフォーム固有なAF-1転写活性化領域をもつ複数のEcRアイソフォームをもつ。核内受容体のAF-1領域はリン酸化などの翻訳後修飾や転写コファクターなどとのタンパク質-タンパク質相互作用の場として機能することが知られており,EcRアイソフォーム固有なAF-1領域の構造・機能的特徴が、アイソフォーム固有の転写調節能力や標的遺伝子の選択などに寄与することが想定される。本研究で論文提出者は、EcRアイソフォーム固有のAF-1領域の構造・機能的特徴を理解することを目的として、比較構造解析によるEcR-AとEcR-B1アイソフォームに固有なAF-1領域の基本構造の特徴付け、及び昆虫培養細胞系をもちいたAF-1 領域の機能解析を行った。

本論文は2章立てで構成されている。第一章では、比較構造解析によるEcR-B1アイソフォームAF-1領域の特徴付けを行った。一般に核内受容体のAF-1領域は配列の保存性が低く、決まった立体構造をもたない天然変性タンパク質としての特徴をもつ。論文提出者はAF-1領域でも、その機能に重要な部分は進化の過程で保存されるのではないかとの作業仮説を立て、比較構造解析によって種間で保存性の高い配列を検索した。EcRの同定・機能解析はショウジョウバエやカイコなど、特定の完全変態昆虫で行われており、原始的な昆虫である不完全変態昆虫(バッタ・セミなど)や無変態昆虫(シミなど)からB1アイソフォームは同定されていなかった。本研究では不完全変態昆虫や無変態昆虫を含む51種の昆虫や節足動物より新たにB1アイソフォームを同定し、72種の昆虫や節足動物のB1アイソフォームAF-1領域のアライメント解析を行った。その結果、B1アイソフォーム固有なAF-1領域には全ての昆虫・節足動物で進化的に保存された2つのマイクロドメイン(S-richモチーフ、及びDL-richモチーフ)が存在すること,完全変態昆虫のEcR-B1アイソフォームがAF-1領域のN-末端部位に新規なマイクロドメイン、(K/R)RRWモチーフを獲得したことが明らかになった。またこれら3つの主要なマイクロドメインに加え、特定の系統・グループのみがもつマイクロドメインが存在することも明らかになった。

第二章では、ショウジョウバエの培養細胞を用いたショウジョウバエEcR-B1アイソフォームAF-1領域のマイクロドメインの機能解析を行った。ショウジョウバエEcR-B1アイソフォームのAF-1領域には(K/R)RRWモチーフ、S-richモチーフ、及びDL-richモチーフが存在する。これらのマイクロドメインがAF-1領域を介した転写調節機能に関与するか否かを検討するため、ルシフェラーゼアッセイによる転写活性化能の評価をおこなった。さらに、これらのマイクロドメインがタンパク質-タンパク質間相互作用に関与するか否かを検討するため、蛍光相関分光法(FCS)法を用いてタンパク質間相互作用を解析した。その結果、(K/R)RRWモチーフ、及びDL-richモチーフがAF-1領域を介した転写活性化能に寄与することが明らかになった。FCS 法によりS2 細胞細胞核で発現させたEcRの拡散速度を測定したところ、(K/R)RRWモチーフを欠いたコンストラクトで有為に拡散速度が上昇した。このことは、核内を浮遊しているEcR-B1アイソフォームAF-1領域-EGFP融合タンパク質が (K/R)RRWモチーフを介して何らかのタンパク質と相互作用していることを示している。EcR-Aアイソフォームについては、C-末端領域に存在するS/P-richなマイクロドメインやN-末端領域に存在するSUMOylation部位がAF-1 領域を介した転写調節機能に関与することが明らかになった。

本研究で見出されたEcRアイソフォーム固有なAF-1領域のマイクロドメインは、EcRの転写調節や昆虫の系統・グループ固有の転写調節機能に関与すると考えられる。今後、各々のマイクロドメインと相互作用するタンパク質の同定・機能解析や相互作用様式の進化を研究することで、EcRアイソフォーム固有なAF-1領域の機能的特徴や、その進化の理解につながると期待される。本研究は昆虫分生物学や内分泌学の発展に寄与するものである。

なお本論文の研究は竹内秀明、久保健雄(東京大学)との共同研究であるが、論文提出者が主体となって実験を計画し、遂行したもので、論文提出者の寄与が十分であると判断できる。従って、博士(理学)の学位を授与できると認める。