京都大学0048新制・課程博士博士(薬科学)甲第18923号薬科博第37号新制||薬||5(附属図書館)31874京都大学大学院薬学研究科医薬創成情報科学専攻(主査)教授 岡村 均, 教授 中山 和久, 教授 竹島 浩学位規則第4条第1項該当Doctor of Pharmaceutical SciencesKyoto UniversityDFA

Atobe, Yuta

Kyoto University Research Information Repository

Title Mechanism of circadian oscillation of the mammalian coreclock gene Per2( Abstract_要旨 )Author(s)Atobe, YutaCitationKyoto University (京都大学)Issue Date2015-03-23URL http://dx.doi.org/10.14989/doctor.k18923Right学位規則第9条第2項により要約公開; 許諾条件により要約は2016/03/22に公開; 許諾条件により要旨は2015/05/23に公開Type Thesis or DissertationTextversionnoneKyoto University （ 続紙 １ ） 京都大学 博士（薬科学） 氏名 跡部 祐太 論文題目 Mechanism of circadian oscillation of the mammalian core clock gene Per2 （時計遺伝子Per2の発現制御機構） （論文内容の要旨）  Daily rhythms in behavior and physiology are controlled by an internal self-sustained molecular oscillator, referred to as the circadian clock. Circadian rhythms are generated in a cell-autonomous manner through transcription/translation-based autoregulatory feedback loops, wherein protein products of three Period genes (Per1, Per2 and Per3) periodically suppress their own transcription. Genetic studies suggest that, of the three Period genes, Per2 has a dominant role in both humans and mice. However, little is known about the regulatory mechanism of this gene. In this thesis, I investigate the molecular mechanism driving the circadian transcription of the mouse Per2 gene. I find that Per2 promoter contains a circadian cis-element, termed Rhythm-box (R-box) (Chapter 1). Furthermore, I generated novel mutant mice carrying a point mutation on the sequence of the Per2 promoter R-box (Chapter 2). Analysis of the mutant mice reveals a previously unknown in vivo function of this element in regulation of circadian transcription of the Per2 gene (Chapter 3).  Chapter 1: Autoregulatory feedback model of Per2 transcription through R-box  Firstly, I determined the circadian oscillation profiles of Per2 mRNA and protein in the mouse livers. Mice were sacrificed at 4 h intervals over 24-h cycle. Quantitative RT-PCR and Western blot analyses revealed that the levels of Per2 mRNA and protein exhibit an overt circadian oscillation with a typical phase-relationship. PER2 protein began to increase 8 hours behind mRNA expression and reached its peak when the mRNA levels were decreased. These observations corroborate the current negative feedback model in which PER2 represses its own transcription. In an attempt to further test this model, I next explored the regulatory cis-element on the Per2 promoter. The mouse embryonic fibroblasts were transfected with a luciferase reporter vector containing the 1.7 kb fragment of the 5’-flanking region of Per2, and its activities were monitored using a real-time luminometer system. I observed a robust circadian oscillation of the Per2 promoter activity over three cycles. Furthermore, I demonstrated that a single point mutation that was introduced into the R-box sequence located in the vicinity of the transcription start site of Per2 resulted in a complete abrogation of this rhythmicity. These in vitro data strongly suggest that this single R-box element is indispensable for the circadian activation and repression of Per2 gene.      Chapter 2: Generation of mice carrying a point mutation of the Per2 R-box  To investigate the role of R-box in regulation of Per2 oscillation in vivo, I generated Per2 R-box mutant (P2Rmut) mice. To introduce a point mutation into this element, I employed piggyBac (PB) transposon system. PB is a moth-derived DNA transposon whose functionality retains in mammals. Unlike the well-established methods relying on the Cre/loxP and Flp/FRT-based recombination systems, PB transposase (PBase)/PB-based system achieves a ‘seamless’ excision of a PB-flanked target sequence without any residual ‘footprint’. This allows, for example, ‘complete’ removal of a neomycin resistant gene-coding cassette from a mutant mouse genome without leaving any unwanted residual sequence. I therefore constructed the targeting vector, carrying a mutant R-box sequence and a neomycin resistant cassette flanked by PB terminal repeats. Targeted mouse embryonic stem cell clones were injected into blastocysts to generate chimeric mice. To remove the PB-flanked neomycin cassette from the genome, F1 heterozygotes were intercrossed with ROSA26-PBase mice that carry a ubiquitously expressed PBase gene at the ROSA26 locus. I performed Southern blot analysis and confirmed that the neomycin cassette was deleted without reintegration into the host genome. Finally, a seamless excision of the cassette was confirmed by DNA sequencing. The sequence analysis also verified that a point mutation was indeed introduced into the R-box. Moreover, the established mutant mice were backcrossed to the C57BL/6J background over ten generations, and at the end of backcrossing, I confirmed that 64 microsatellite markers covering all individual mouse chromosomes were replaced with those of the C57BL/6J mouse strain.  Chapter 3: Characterization of in vivo role of the Per2 R-box  To evaluate whether the R-box mutation in vivo abrogates rhythmicity of Per2 expression, I measured circadian profiles of Per2 mRNA in the mouse liver samples collected every 4 hours in constant darkness. Quantitative RT-PCR analysis revealed that the point mutation of the Per2 R-box caused alteration of the circadian rhythm of Per2. This mutation impairs circadian repression of Per2 expression and thereby elevates the levels of the baseline. Similar results were also observed for the suprachiasmatic nucleus (SCN) in the brain, which serves as the mammalian circadian center. Thus, this R-box appears to be important for the repression of Per2 transcription in vivo. I therefore hypothesized that this R-box might play a role in recruitment of the negative circadian transcriptional regulators. To test this hypothesis, I performed chromatin immunoprecipitation (ChIP) assays. In the wildtype mouse liver, the negative regulators, PER1, CRY1 and CRY2 were all recruited to the R-box at the time of Per2 repression. However, in the mutant mice, no recruitment was observed throughout the day, demonstrating that this R-box is required for binding of the negative regulators. Based on these results, I conclude that the R-box is responsible for circadian repression, rather than activation, of Per2 transcription in vivo.    （続紙 ２ ）                             （論文審査の結果の要旨）  生体リズムを司る生体時計の振動は、時計遺伝子の転写調節を介した自己制御型フィードバック機構によって生み出される。すなわち、転写促進因子であるCLOCK:BMAL1 複合体が Period (Per) 遺伝子の転写を促進し、翻訳された PER 蛋白質が転写抑制因子 CRY と複合体を形成して核内へ移り、CLOCK:BMAL1 複合体の転写活性を抑制する。これにより PER 蛋白質が減少すれば、転写抑制の効果が弱まり、再び CLOCK:BMAL1 複合体による転写が開始されることになる。申請者は、本論文の第一章において、哺乳類の時計機能において中心的な役割を担うと考えられているPer2 遺伝子に着目し、本遺伝子の mRNA 並びに蛋白質レベルの発現変動をマウス肝において追跡し、上述のフィードバック理論が成立することを明らかにした。さらに、申請者は、PiggyBac トランスポゾン系による遺伝子改変によって目的の点変異マウスを作製することに成功した。さらに、第三章においてクロマチン免疫沈降法による解析から、点変異によって転写抑制因子群が Per2 プロモーターへリクルートされなくなっていることを明らかにした。これは、遺伝子発現制御部位の変異で初めてリズム変動が起こることを明らかにした研究で有り、リズム形成機構に新たな発見がもたらされたといえる。よって、本論文は博士（薬科学）の学位論文として価値あるものと認める。また、平成 27 年 2 月 23 日、論文内容とそれに関連した事項について試問を行った結果、合格と認めた。 なお、本論文は、京都大学学位規程第１４条第２項に該当するものと判断し、公表に際しては、（当分の間）当該論文の全文に代えてその内容を要約したものとすることを認める。  要旨公表可能日：  平成 27  年 5  月  23 日以降     

時計遺伝子Per2の発現制御機構

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時計遺伝子Per2の発現制御機構

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