遺伝子制御、エピジェネティクス Gene Regulation and Epigenetics
P3-2-16 C6細胞における抗うつ薬によるHDAC4を介したGDNF遺伝子の制御機構 The mechanism of GDNF gene transcriptional regulation by antidepressants through HDAC4 in C6 cell ○大朏孝治1, 内田周作1, 山形弘隆1, 芳原輝之1, 渡邉義文1 ○Koji Otsuki1, Shusaku Uchida1, Hirotaka Yamagata1, Teruyuki Kobara1, Yoshifumi Watanabe1 山口大学大学院　医学系研究科　高次脳機能病態学分野1 Dept Neuroscience, Univ of Yamaguchi, Ube1 Antidepressants have been indicated to induce the expression of a variety of neurotrophic factors. We have previously developed an animal model of depression. Importantly, the reduction of GDNF mRNA in the striatum of stressed BALB/c mice was reversed by chronic treatment with an antidepressant imipramine. Therefore in this study, we aimed to clarify the molecular mechanism underlying the changes in the GDNF expression by antidepressants. First, we examined the effects of antidepressants, mood stabilizers, an anxiolytic drug and an antipsychotic drug on the expression of GDNF mRNA. The expression of GDNF mRNA was increased by all antidepressants used and valproate, whereas other drugs did not induce the GDNF expression. Next, since valproate has been known to act as a histone deacetylase inhibitor, we examined the effects of several histone deacetylase inhibitors on the expression of GDNF mRNA. The expression of GDNF mRNA was increased by all histone deacetylase inhibitors used. Therefore we hypothesized that the induction of GDNF mRNA by antidepressants could be mediated by the histone modification at the GDNF gene promoter. Actually, ChIP assay revealed that the levels of histone H3 and H4 acetylation at the GDNF promoter were enhanced and the recruitment of HDAC4 was decreased by antidepressants. In addition, HDAC4 significantly inhibited the induction of GDNF mRNA by antidepressants. Therefore we hypothesized that the induction of GDNF mRNA by antidepressants could be mediated by the histone modification at the GDNF gene promoter. Interestingly, the level of phosphorylation of HDAC4 was increased by antidepressants. Immunostaining showed that HDAC4 was translocated from nucleus to cytoplasm by antidepressants. Thus, our data provide novel insights that epigenetic mechanism of GDNF expression would be involved in the pathogenesis or pathophysiology of depression.	P3-2-17 BDNF誘導性Arc/Arg3.1遺伝子発現における転写因子SRFコファクターMKL1およびElk1の競合的関係 Competition between serum response factor (SRF) cofactors, MKL1 and Elk1, in the regulation of BDNF-induced Arc/Arg3.1 gene transcription ○石川充1, 福地守1, 菊池啓悦1, 石橋悠太1, 辻井惇也1, 津田正明1, 奥野浩行2, 尾藤晴彦2, 田渕明子1 ○Mitsuru Ishikawa1, Mamoru Fukuchi1, Keietsu Kikuchi1, Yuta Ishibashi1, Junya Tsujii1, Masaaki Tsuda1, Hiroyuki Okuno2, Haruhiko Bito2, Akiko Tabuchi1 富山大院・医薬・分子神経生物学1, 東京大学院・医・神経生化学2 Lab. of Mol. Neurobiol., Grad. Sch. of Med. & Pharm. Sci., Univ. of Toyama, Toyama, Japan1, Dep. of Neurochem., Grad. Sch. of Med., Univ. of Tokyo, Bunkyo-ku, Tokyo, Japan2 Regulation of gene expression is one of the major mechanisms influencing plasticity-associated alterations in neurons. Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), one of the immediate early genes (IEG), plays a key role in neuronal plasticity. Arc/Arg3.1 gene transcription induced by brain-derived neurotrophic factor (BDNF) is highly dependent upon the activation of synaptic-activity responsive element (SARE), located approximately at upstream 6.5 kbp of the Arc/Arg3.1 gene transcription start site. The SARE consists of the binding sites for transcription factors, cAMP response element-binding protein (CREB), myocyte enhancer factor 2 (MEF2), and serum response factor (SRF). However the mechanisms by which such transcription factors activate the SARE remain poorly understood. In this study, we have demonstrated the functional roles of SRF and its two distinct cofactors, megakaryoblastic leukemia 1 (MKL1) and a ternary complex factor (TCF) in Arc/Arg3.1 gene transcription in rat cortical neurons. A mutation in the binding site of SRF drastically reduces the SARE activity, whereas a mutation in the TCF-binding site increases. Additionally, the overexpression of MKL1 promotes the activation of the Arc gene promoter. Chromatin immunoprecipitation studies also show that following BDNF administration, endogenous MKL1 is recruited to chromatin including SARE, while Ets-like factor 1 (Elk1), a member of the TCF family, becomes dissociated. Moreover, overexpression of Elk1 decreases the activity of Arc/Arg3.1 gene transcription induced by MKL1. Therefore, we conclude that the SRF cofactors, MKL1 and Elk1, compete for binding to SRF on the SARE to activate Arc/Arg3.1 gene transcription.
P3-2-18 中枢神経系におけるSIRT1新規結合因子の同定 Identification of a novel binding partner of SIRT1 in neurons ○藤原慧1, 藤田幸1, 山下俊英1 ○Kei Fujiwara1, Yuki Fujita1, Toshihide Yamashita1 大阪大院　医　分子神経科学1 Dept Molecular neuroscience, Univ of Osaka, Osaka1 SIRT1 is a NAD+-dependent protein deacetylase that targets a wide variety of proteins to modulate those functions by deacetylation. Recent studies have revealed SIRT1 protects neurons from axonal degeneration. To identify novel binding partners for SIRT1 in the central nervous system, we performed yeast two-hybrid screening by using human fetal brain cDNA library. We found that histone demethylase JMJD2B is one of the possible binding partners of SIRT1. SIRT1 and JMJD2B were both expressed in developmental mouse cortical neurons. Transfected Myc-tagged SIRT1 and HA-tagged JMJD2B were predominantly localized in the nucleus in HEK293T cells. To confirm the interaction of SIRT1 with JMJD2B in mammalian cells, we performed co-immunoprecipitation assay and found that Myc-SIRT1 interacted with HA-JMJD2B in HEK 293T cells. It has been shown that SIRT1 regulates the histone methyl-transferase activity by deacetylation of lysine residue. In addition, we employed the catalytic domain of SIRT1 as the bait for the yeast two-hybrid screen. These observations prompted us to hypothesize that SIRT1 can deacetylate JMJD2B. Indeed, treatment of the HEK293T cells with SIRT1 inhibitor, Nicotinamide or EX-527, induced lysine acethylation of JMJD2B. These results suggest that SIRT1 can modulate deacetylation levels of JMJD2B in mammalian cells.	P3-2-19 ツニカマイシンによって誘導されるアポトーシスの抑制と神経突起伸長に寄与するHDAC阻害剤の作用機構 Prevention of tunicamycin-mediated apoptosis and induction of neurite by HDAC inhibitors in PC12 cells ○豊田雄資1, 丸岡弘規3, 藤木大地2, 上里新一1,2, 池内俊彦1,2, 下家浩二1,2 ○Yusuke Toyoda1, Hiroki Maruoka3, Daichi Fujiki2, Shinichi Uesato1,2, Toshihiko Ikeuchi1,2, Koji Shimoke1,2 関西大学　化学生命工学部　生命生物工学科1, 関西大学大学院　理工学研究科2, 倉敷紡績（株）　技術研究所3 Dept. Life Sci. Biotech., Fac. Chem, Mat and Bioengin., Kansai Univ., Osaka, Japan1, Grad. School. Sci. and Engin., Kansai Univ., Osaka, Japan2, Technol. Res. Lab., Kurabo Industries Ltd, Osaka, Japan3 Histone deacetylase (HDAC) is a key modulator of gene expression and the effect of HDAC inhibitors on neurodegenerative diseases has been elucidated. We have analyzed whether or not HDAC inhibitors, valploic acid (VPA), MS-275, and K-350 are effective for prevention of neurodegenerative diseases using PC12 cells. The inherent cellular toxicity of these agents can be avoided at low doses at which therapeutic benefits can be achieved. We have reported previously that HDAC inhibitors induce at least two genes that function in neurite outgrowth from PC12 cells. In our present study, we have found that tunicamycin-induced apoptosis reduces the cell viability which is marginally reversed by HDAC inhibitors. This suggests that HDAC inhibitors, including MS-275, VPA, and K-350, can prevent ER stress-mediated apoptosis leading to neurodegenerative diseases. Moreover, we have discovered the neurite outgrowth in PC12 cells exposed to HDAC inhibitors. Molecular analysis of these cells indicated that the acetylation of histone H3 and induction of Nur77 are important for the cell survival and the neurite outgrowth. The precise nature of the molecular mechanisms underlying the cell survival and the neurite outgrowth, including the epigenetic regulation of specific genes, is being investigated.
P3-2-20 F344ラット海馬における離乳後社会的隔離とDNAメチル化との関連性 Post-weaning social isolation alters expression of DNA methylation-related gene in the hippocampus of F344 rats ○岩田久彌1, 後藤直人1, 小倉祥一1, 寺本恭子1, 山室裕1,2 ○Hisaya Iwata1, Naoto Goto1, Shouichi Ogura1, Kyoko Teramoto1, Yutaka Yamamuro1,2 日本大院・生物資源・生産科学1, 日本大・生物資源・動物資源2 Dept Anim Sci, Coll Bioresource Sci, Nihon Univ, Kanagawa1, Dept Anim Sci, Coll Bioresource Sci, Nihon Univ, Kanagawa2 In rodent such as mice and rats, social isolation during adolescence is known to alter responses to a stressor, and change behavioral and neurochemical phenotypes in adulthood. These alterations by social isolation must be induced via epigenetic modifications, the mechanism, however, remains largely unclear. In the present study we investigated the effect of post-weaning social isolation (pwSI) on behavioral traits and genomic DNA methylation pattern using male F344 rats, highly stress-sensitive inbred strain. Rats 28 days of age were housed individually (pwSI group) or socially (3 rats; the control group). At 63-70 days of age in both groups, a novel-social exploratory behavior test was performed. Time of social interaction in pwSI rats increased from 1st to 2nd trial while that of group-reared rats decreased. MS-AP-PCR method was exerted to estimate global genomic DNA methylation patterns during the post-weaning period, and showed that the aspect of methylation pattern changed at 44 days in the hippocampus, whereas no changes observed at 34 days. Furthermore, to approach for the detail mechanism that changes DNA methylation, three hippocampal subregions, CA1, CA3, and dentate gyrus, were collected from both reared rats at 44 days of age using a laser-microdissection method, and the expression of DNA methylation-related genes was analyzed by real-time quantitative RT-PCR. The expression of DNA methyltransferase 3a gene in dentate gyrus of pwSI rats was dramatically higher than that of group-reared rats. In contrast, the expression of heterochromatin protein 1 gene in pwSI rats was significantly lower. In conclusion, these results suggest that pwSI alters several behavioral profiles including a cognitive behavior of rats and affects hippocampal global DNA methylation pattern. At least, these alterations were involved in changes of expression pattern of specific DNA methylation related-genes.	P3-2-21 Withdrawn
P3-2-22 末梢神経障害による脊髄内アストロサイトにおけるケモカイン遺伝子のエピジェネティクス修飾変動 Dynamic change in epigenetics modifications associated with transcriptional activation of chemokines in astrocytes by peripheral neuropathy ○池上大悟1, 今井哲司1, 伊達明利1, 成田道子1, 竹島秀幸2, 牛島俊和2, 成田年1 ○Daigo Ikegami1, Satoshi Imai1, Akitohi Date1, Michiko Narita1, Hideyuki Takeshima2, Toshikazu Ushijima2, Minoru Narita1 星薬科大学・薬・薬理1, 国立がん研究センター研究所・エピゲノム解析分野2 Dept. of Pharmacol., Hoshi Univ. Sch. of Pharm. and Pharmaceut. Sci., Tokyo, Japan1, Div. of Epigenomics, National Cancer Center Res. Institute, Tokyo, Japan.2 A multiplex analysis for profiling the expression of candidate genes along with epigenetic modification may lead to a better understanding of the complex machinery of neuropathic pain. In the present study, we found that partial sciatic nerve ligation most remarkably increased the expression of monocyte chemotactic protein-3 (MCP-3, known as CCL7) among a total of 33,541 genes in the spinal cord, which lasted for 4 weeks. Furthermore, intrathecal administration of MCP-3 recombinant protein caused a painful state and dramatic increased in signal intensity in pain-related brain regions, using a functional MRI analysis. A robust increase in MCP-3 protein, which lasted for up to 2 weeks after surgery, in the dorsal horn of the spinal cord of mice with partial sciatic nerve ligation was seen mostly in astrocytes, but not microglia or neurons. This increase in MCP-3 gene transcription was accompanied by the decreased trimethylation of histone H3 at lysine27 (H3K27me3) at the MCP-3 promoter. The increased MCP-3 expression associated with its epigenetic modification observed in the spinal cord was almost abolished in interleukin-6 (IL-6) knockout mice with partial sciatic nerve ligation. It has been reported that a Jumonji domein containing 3 (JMJD3) function as transcriptional activators that demethylate H3K27me3. Therefore we performed ChIP assays with antibodies against JMJD3 at the promoter region of MCP-3. As a result, sciatic nerve ligation significantly increased the induction of jmjd3 at MCP-3 promoter. These findings suggest that increased MCP-3 expression associated with IL-6-dependent epigenetic modification at the MCP-3 promoter after nerve injury, mostly in spinal astrocytes, may serve to facilitate astrocyte-microglia interaction in the spinal cord.	P3-2-23 C57BL/6マウスを用いたヒストン脱アセチル化酵素による抗うつ様作用の検討 Antidepressant-like effects on histone deacetylase expression in C57BL/6 mice ○大久保雅則1,2, 金井裕彦1, 青木治亮2, 山田尚登1 ○Masanori Ookubo1,2, Hirohiko Kanai1, Harusuke Aoki2, Naoto Yamada1 滋賀医科大学大学院　医学系研究科　精神医学講座1, 一般社団法人　水口病院2 Department of Psychiatry, Shiga University of Medical Science, Japan1, Department of Psychiatry, Minakuchi Hospital, Koka City, Japan2 Epigenetic modifications are considered to induce long-lasting but reversible alterations in neuronal function. Although histone deacetylase (HDAC) inhibitors have been reported to show various antidepressant actions, epigenetic changes in the brain underlying depressive state and antidepressant actions are still unclear.In this study, we used the following mood stabilizers and antidepressants; sodium valproate (VPA), lithium chloride (Li), carbamazepine (CBZ), lamotrigine (LTG), levetiracetam (LTM), olanzapine (OLZ) and clozapine (CLZ) for mood stabilizers, and clomipramine (CLM), (S)-citalopram (ECM), duloxetine (DLX), and mirtazapine (MIR) for antidepressants. We investigated the effects on tissue concentrations of monoamines, dopamine, serotonin, and norepinephrine, by HPLC, and protein expressions of HDACs (HDAC1, 2, 3, 4, 5, 7, 8 and 10) in the following regions of C57BL/6 mouse brain; striatum, nucleus accumbens, hippocampus, cingulate cortex, and amygdala. We then analyzed whether the treatment of these antidepressants / mood stabilizers have a similar region-specific change regarding tissue monoamine concentrations and protein expression of HDACs and acetylated histone H3 (AcH3).As a result, subchronic administration (14 days of i.p. injection) with OLZ, CLZ, ECM, DLX, or MIR commonly induced significant increase of dopamine content in the striatum. In contrast, we did not find any common profile in serotonin and norepinephrine contents. Furthermore, CBZ, LTM, OLZ, ECM, DLX, or MIR induced significant increase of HDAC3 and HDAC5 in the amygdala and in the striatum. Collectively, the changes of monoamine content were restricted, but increased expressions of HDAC3 and HDAC5 in the amygdala and cingulate cortex were often found, supporting the possibility that the effects of antidepressants / mood stabilizers involve epigenetic modifications associated with changes of HDAC expressions.
P3-2-24 プロトカドヘリンβ遺伝子改変マウスにおける発現制御メカニズムの解析 Analysis of the gene-regulatory mechanism in the deletion mice of Protocadherin-β cluster ○章瑠依1 ○Rui Sho1 大阪大学大学院　生命機能研究科　生命機能1 KOKORO-Biology Group, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan1 Neural development and the organization of complex neuronal circuits involve a number of processes that require cell-cell interaction. The clustered Protocadherin(Pcdh) are consist of Pcdh-α , Pcdh-β , and Pcdh-γ families. The Pcdh-β gene cluster encodes 22 variable exons, arranged in tandem between Pcdh-&alpha gene cluster and Pcdh-γ gene clusters on mouse chromosome 18. An isoform of Pcdh-β proteins is a single-pass transmembrane protein which consists of six extracellular cadherin domeins. Recentry we showed that Pcdh-β isoforms have differential and combinatorial expression patterns in individual neurons, and regulated monoallelically and combinatorially in single Purkinje cells, by using in situ hybridization and single-cell RT-PCR. Therefore, the Pcdh-βproteins would contribute for specifying neuronal identity and providing diversity of individual neurons. However function of Pcdh-βproteins have been unclear. To examine in vivo function of Pcdh-β proteins in the brain, we generated all Pcdh-β, and Pcdh-β3-β22 deletion mice. Now we are analyzing these mutant mice, by real-time PCR and in situ hybridization.

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