基本情報

所属

• 理学研究院　生物科学部門　生殖発生生物学分野

職名

• 教授

学位

• 博士（理学）（北海道大学）

ホームページURL

https://www.sci.hokudai.ac.jp/~akimura/Molecular/Welcome.APK.html

科研費研究者番号

• 90422005

J-Global ID

• 201301022226959232

研究キーワード

• プロテアーゼ   胎盤   精子形成   dual promoter‒enhancer   卵巣   精巣   ゲノム   転写   クロマチン   生殖   long noncoding RNA   卵巣顆粒膜細胞   エンハンサー   転写調節   プロモーター   エピジェネティクス   

研究分野

• ライフサイエンス / ゲノム生物学
• ライフサイエンス / 形態、構造

担当教育組織

•  学士課程　理学部
•  修士課程　生命科学院
•  博士課程　生命科学院

職歴

• 2022年04月 - 現在 北海道大学 大学院理学研究院 教授
• 2006年04月 - 2022年03月 北海道大学 大学院理学研究院 准教授

研究活動情報

論文

• Controlling the Rigidity of Kinesin-Propelled Microtubules in an In Vitro Gliding Assay Using the Deep-Sea Osmolyte Trimethylamine N-Oxide

  Arif Md. Rashedul Kabir, Tasrina Munmun, Tomohiko Hayashi, Satoshi Yasuda, Atsushi P. Kimura, Masahiro Kinoshita, Takeshi Murata, Kazuki Sada, Akira Kakugo

  ACS Omega 7 4 3796 - 3803 2022年02月01日
• Evidence for a functional role of Start, a long noncoding RNA, in mouse spermatocytes.

  Kai Otsuka, Hong Yang, Shin Matsubara, Akira Shiraishi, Misuzu Kurihara, Honoo Satake, Atsushi P Kimura

  PloS one 17 8 e0273279  2022年 

  A mouse testis-specific long noncoding RNA (lncRNA), Start, is localized in the cytosol of Leydig cells and in the nucleus of pachytene spermatocytes. We previously showed that Start regulates steroidogenesis through controlling the expression of Star and Hsd3b1 genes in Leydig cells, but its function in germ cells was not known. Here we verified that a spermatocyte-specific protease gene, Prss43/Tessp-3, was downregulated in Start-knockout testes. To investigate the transcriptional regulatory activity of Start in spermatocytes, we first performed a series of reporter gene assays using a thymidine kinase promoter in spermatocyte-derived GC-2spd(ts) cells. A 5.4-kb genome sequence encompassing Start exhibited enhancer activity for this promoter, and the activity was decreased by knockdown of Start. Deletion of the Start promoter and replacement of the Start sequence abolished the enhancer activity and, consistently, the activity was detected in further experiments only when Start was actively transcribed. We then examined whether the Prss43/Tessp-3 gene could be a target of Start. A reporter gene assay demonstrated that the 5.4-kb sequence exhibited enhancer activity for a Prss43/Tessp-3 promoter in GC-2spd(ts) cells and that the activity was significantly decreased by knockdown of Start. These results suggest that Start functions in transcriptional activation of the Prss43/Tessp-3 gene in spermatocytes. Given that Start is presumed to regulate steroidogenic genes at the posttranscriptional level in Leydig cells, the function in spermatocytes is a novel role of Start. These findings provide an insight into multifunctionality of lncRNAs in the testis.
• A Testis-Specific Long Noncoding RNA, Start, Is a Regulator of Steroidogenesis in Mouse Leydig Cells.

  Kai Otsuka, Shin Matsubara, Akira Shiraishi, Natsumi Takei, Yui Satoh, Miho Terao, Shuji Takada, Tomoya Kotani, Honoo Satake, Atsushi P Kimura

  Frontiers in endocrinology 12 665874 - 665874 2021年 [査読有り]
   

  The testis expresses many long noncoding RNAs (lncRNAs), but their functions and overview of lncRNA variety are not well understood. The mouse Prss/Tessp locus contains six serine protease genes and two lncRNAs that have been suggested to play important roles in spermatogenesis. Here, we found a novel testis-specific lncRNA, Start (Steroidogenesis activating lncRNA in testis), in this locus. Start is 1822 nucleotides in length and was found to be localized mostly in the cytosol of germ cells and Leydig cells, although nuclear localization was also observed. Start-knockout (KO) mice generated by the CRISPR/Cas9 system were fertile and showed no morphological abnormality in adults. However, in adult Start-KO testes, RNA-seq and qRT-PCR analyses revealed an increase in the expression of steroidogenic genes such as Star and Hsd3b1, while ELISA analysis revealed that the testosterone levels in serum and testis were significantly low. Interestingly, at 8 days postpartum, both steroidogenic gene expression and testosterone level were decreased in Start-KO mice. Since overexpression of Start in two Leydig-derived cell lines resulted in elevation of the expression of steroidogenic genes including Star and Hsd3b1, Start is likely to be involved in their upregulation. The increase in expression of steroidogenic genes in adult Start-KO testes might be caused by a secondary effect via the androgen receptor autocrine pathway or the hypothalamus-pituitary-gonadal axis. Additionally, we observed a reduced number of Leydig cells at 8 days postpartum. Collectively, our results strongly suggest that Start is a regulator of steroidogenesis in Leydig cells. The current study provides an insight into the overall picture of the function of testis lncRNAs.
• A dual enhancer-silencer element, DES-K16, in mouse spermatocyte-derived GC-2spd(ts) cells.

  Thusitha A M K Bandara, Kai Otsuka, Shin Matsubara, Akira Shiraishi, Honoo Satake, Atsushi P Kimura

  Biochemical and biophysical research communications 534 1007 - 1012 2020年10月26日 [査読有り]
   

  The multifunctionality of genome is suggested at some loci in different species but not well understood. Here we identified a DES-K16 region in an intron of the Kctd16 gene as the chromatin highly marked with epigenetic modifications of both enhancers (H3K4me1 and H3K27ac) and silencers (H3K27me3) in mouse spermatocytes. In vitro reporter gene assay demonstrated that DES-K16 exhibited significant enhancer activity in spermatocyte-derived GC-2spd(ts) and hepatic tumor-derived Hepa1-6 cells, and a deletion of this sequence in GC-2spd(ts) cells resulted in a decrease and increase of Yipf5 and Kctd16 expression, respectively. This was consistent with increased and decreased expression of Yipf5 and Kctd16, respectively, in primary spermatocytes during testis development. While known dual enhancer-silencers exert each activity in different tissues, our data suggest that DES-K16 functions as both enhancer and silencer in a single cell type, GC-2spd(ts) cells. This is the first report on a dual enhancer-silencer element which activates and suppresses gene expression in a single cell type.
• Transcriptional activation of the mouse Scd2 gene by interdependent enhancers and long noncoding RNAs in ovarian granulosa cells.

  Shota Mayama, Nobuhiko Hamazaki, Yuki Maruyama, Shin Matsubara, Atsushi P Kimura

  The Journal of reproduction and development 66 5 435 - 444 2020年10月13日 [査読有り]
   

  Specific gene expression in granulosa cells is key for the function of ovary, but the molecular mechanism of transcriptional activation is not well studied. Here we investigated the regulatory mechanism of the mouse stearoyl-CoA desaturase 2 (Scd2) gene encoding an enzyme for lipid metabolism. Northern blot and in situ hybridization indicated that the mouse Scd2 mRNA was highly expressed in ovarian granulosa cells. We found four conserved noncoding sequences (CNSs) and two long noncoding RNAs (lncRNAs) transcribed from regions upstream of the Scd2 gene as candidates of regulatory elements/factors. These lncRNAs were predominantly transcribed in the opposite direction to Scd2 and localized in nuclei and showed the correlation with Scd2 expression, raising the possibility of their transcriptional regulatory roles. Indeed, knockdown of both lncRNAs, lncRNA-sc1 and lncRNA-sc2, significantly decreased the Scd2 mRNA level in primary granulosa cells. Then, we investigated the histone modification pattern at this locus by a chromatin immunoprecipitation assay, and two CNSs, CNS1 and CNS2, were found to be marked with high levels of histone H3K9/K27 acetylation in primary granulosa cells. By a reporter gene assay, both CNS1 and CNS2 interdependently exhibited enhancer activity for the Scd2 promoter in primary granulosa cells. These data suggest that the mouse Scd2 gene is activated by two lncRNAs and interdependent enhancers in ovarian granulosa cells, which provides a new insight into transcriptional activation in granulosa cells.
• Knockdown of long noncoding RNA dreh facilitates cell surface GLUT4 expression and glucose uptake through the involvement of vimentin in 3T3-L1 adipocytes

  Nobuhiko Takahashi, Atsushi P. Kimura, Kazumasa Ohmura, Sumiyoshi Naito, Mika Yoshida, Masahiro Ieko

  Gene 735 144404  2020年04月 [査読有り][通常論文]
  
• Dreh, a long noncoding RNA repressed by metformin, regulates glucose transport in C2C12 skeletal muscle cells.

  Nobuhiko Takahashi, Atsushi P Kimura, Kai Otsuka, Kazumasa Ohmura, Sumiyoshi Naito, Mika Yoshida, Masahiro Ieko

  Life sciences 236 116906 - 116906 2019年11月01日 [査読有り][通常論文]
   

  AIMS: The anti-hyperglycemic action of metformin on skeletal muscles is presently unclear. Long noncoding RNAs (lncRNAs) are implicated in multiple cellular functions. This study aims to explore the role of lncRNAs in the glucometabolic action of metformin on skeletal muscle cells. MAIN METHODS: Metformin accumulation was assessed using [14C]-metformin. A lncRNA array was used to investigate metformin-regulated lncRNAs in C2C12 skeletal muscle cells. Knockdown studies were applied to evaluate the function of lncRNA Dreh. A colorimetric assay was used for the measurement of medium glucose concentration; glucose transport was assessed using [3H]-2-deoxyglucose; real-time PCR was used for RNA expression analysis, and western blotting was used to assess protein expression in myotubes. A Dreh overexpression plasmid was transfected into the cells. KEY FINDINGS: Metformin accumulated in C2C12 myotubes. Metformin reduced medium glucose concentration and repressed lncRNA Dreh expression in the myotubes. Knockdown of Dreh in the myotubes resulted in reduced glucose concentration in the culture medium, increased glucose transport, and increased levels of GLUT4 protein in the plasma membrane. Overexpression of Dreh attenuated the glucose-lowering effect of metformin in myotubes. SIGNIFICANCE: The glucoregulatory actions of metformin are mediated in part by a lncRNA, Dreh, in the skeletal muscle cells. Dreh is a novel regulator for glucose transport and could be a therapeutic target for diabetes.
• A molecular mechanism of mouse placental spongiotrophoblast differentiation regulated by prolyl oligopeptidase.

  Maruyama Y, Kimura AP

  Zygote (Cambridge, England) 27 1 49 - 53 2019年02月 [査読有り][通常論文]
  
• A novel testis-specific long noncoding RNA, Tesra, activates the Prss42/Tessp-2 gene during mouse spermatogenesis.

  Satoh Y, Takei N, Kawamura S, Takahashi N, Kotani T, Kimura AP

  Biology of reproduction 100 3 833 - 848 2018年10月 [査読有り][通常論文]
  
• Construction of artificial cilia from microtubules and kinesins through a well-designed bottom-up approach

  Ren Sasaki, Arif Md. Rashedul Kabir, Daisuke Inoue, Shizuka Anan, Atsushi P. Kimura, Akihiko Konagaya, Kazuki Sada, Akira Kakugo

  Nanoscale 10 14 6323 - 6332 2018年04月14日 [査読有り][通常論文]
   

  Self-organized structures of biomolecular motor systems, such as cilia and flagella, play key roles in the dynamic processes of living organisms, like locomotion or the transportation of materials. Although fabrication of such self-organized structures from reconstructed biomolecular motor systems has attracted much attention in recent years, a systematic construction methodology is still lacking. In this work, through a bottom-up approach, we fabricated artificial cilia from a reconstructed biomolecular motor system, microtubule/kinesin. The artificial cilia exhibited a beating motion upon the consumption, by the kinesins, of the chemical energy obtained from the hydrolysis of adenosine triphosphate (ATP). Several design parameters, such as the length of the microtubules, the density of the kinesins along the microtubules, the depletion force among the microtubules, etc., have been identified, which permit tuning of the beating frequency of the artificial cilia. The beating frequency of the artificial cilia increases upon increasing the length of the microtubules, but declines for the much longer microtubules. A high density of the kinesins along the microtubules is favorable for the beating motion of the cilia. The depletion force induced bundling of the microtubules accelerated the beating motion of the artificial cilia and increased the beating frequency. This work helps understand the role of self-assembled structures of the biomolecular motor systems in the dynamics of living organisms and is expected to expedite the development of artificial nanomachines, in which the biomolecular motors may serve as actuators.
• High-Sensitivity and High-Resolution in Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes

  Natsumi Takei, Takuma Nakamura, Shohei Kawamura, Yuki Takada, Yui Satoh, Atsushi P. Kimura, Tomoya Kotani

  Biological Procedures Online 20 1 6  2018年03月01日 [査読有り][通常論文]
   

  Background: Subcellular localization of coding and non-coding RNAs has emerged as major regulatory mechanisms of gene expression in various cell types and many organisms. However, techniques that enable detection of the subcellular distribution of these RNAs with high sensitivity and high resolution remain limited, particularly in vertebrate adult tissues and organs. In this study, we examined the expression and localization of mRNAs encoding Pou5f1/Oct4, Mos, Cyclin B1 and Deleted in Azoospermia-like (Dazl) in zebrafish and mouse ovaries by combining tyramide signal amplification (TSA)-based in situ hybridization with paraffin sections which can preserve cell morphology of tissues and organs at subcellular levels. In addition, the distribution of a long non-coding RNA (lncRNA), lncRNA-HSVIII, in mouse testes was examined by the same method. Results: The mRNAs encoding Mos, Cyclin B1 and Dazl were found to assemble into distinct granules that were distributed in different subcellular regions of zebrafish and mouse oocytes, suggesting conserved and specific regulations of these mRNAs. The lncRNA-HSVIII was first detected in the nucleus of spermatocytes at prophase I of the meiotic cell cycle and was then found in the cytoplasm of round spermatids, revealing expression patterns of lncRNA during germ cell development. Collectively, the in situ hybridization method demonstrated in this study achieved the detection and comparison of precise distribution patterns of coding and non-coding RNAs at subcellular levels in single cells of adult tissues and organs. Conclusions: This high-sensitivity and high-resolution in situ hybridization is applicable to many vertebrate species and to various tissues and organs and will be useful for studies on the subcellular regulation of gene expression at the level of RNA localization.
• A Testis-Specific Long Non-Coding RNA, lncRNA-Tcam1, Regulates Immune-Related Genes in Mouse Male Germ Cells

  Misuzu Kurihara, Kai Otsuka, Shin Matsubara, Akira Shiraishi, Honoo Satake, Atsushi P. Kimura

  FRONTIERS IN ENDOCRINOLOGY 8 299  2017年11月 [査読有り][通常論文]
   

  Spermatogenesis is precisely controlled by hormones from the hypothalamus-pituitary-gonadal axis and testis-specific genes, but the regulatory mechanism is not fully understood. Recently, a large number of long non-coding RNAs (lncRNAs) are found to be transcribed at each stage of meiosis of male germ cells, and their functions in spermatogenesis have yet to be fully investigated. lncRNA-testicular cell adhesion molecule 1 (lncRNA-Tcam1) is a nuclear lncRNA which is specifically expressed in mouse male germ cells and presumed to play a role in gene regulation during meiosis. Here, we present the identification of potential target genes of lncRNA-Tcam1 using spermatocyte-derived GC-2spd(ts) cells. Initially, 55 target gene candidates were detected by RNA-sequencing of two GC-2spd(ts) cell clones that were stably transfected with transgenes to express lncRNA-Tcam1 at different levels. Expression of 21 genes of the candidates was found to be correlated with lncRNA-Tcam1 at 7-14 postnatal days, when lncRNA-Tcam1 expression was elevated. Subsequently, we examined expression levels of the 21 genes in other two GC-2spd(ts) clones, and 11 genes exhibited the correlation with lncRNA-Tcam1. Induction of lncRNA-Tcam1 transcription using the Tet-off system verified that six genes, Trim30a, Ifit3, Tgtp2, Ifi47, Oas1g, and Gbp3, were upregulated in GC-2spd(ts) cells, indicating that lncRNA-Tcam1 is responsible for the regulation of gene expression of the six genes. In addition, five of the six genes, namely, Ifit3, Tgtp2, Ifi47, Oas1g, and Gbp3, are immune response genes, and Trim30a is a negative regulator of immune response. Altogether, the present study suggests that lncRNA-Tcam1 is responsible for gene regulation for the immune response during spermatogenesis.
• Sarcolipin expression is repressed by endoplasmic reticulum stress in C2C12 myotubes

  Nobuhiko Takahashi, Atsushi P. Kimura, Sumiyoshi Naito, Mika Yoshida, Osamu Kumano, Takeshi Suzuki, Satoshi Itaya, Mitsuru Moriya, Masahiro Tsuji, Masahiro Ieko

  JOURNAL OF PHYSIOLOGY AND BIOCHEMISTRY 73 4 531 - 538 2017年11月 [査読有り][通常論文]
   

  Sarcolipin is a transmembrane protein expressed in the sarco/endoplasmic reticulum of skeletal and atrial muscles in large animals. Sarcolipin plays crucial roles in heat production through modifying the function of sarco/endoplasmic reticulum Ca2+ ATPase, thereby being involved in thermogenesis and systemic metabolism. In skeletal muscle, endoplasmic reticulum (ER) stress has been implicated in several conditions, such as insulin resistance, muscle diseases, and hypo/hyper-contraction. Here, we investigated the effect of ER stress on sarcolipin expression in skeletal muscle cells, C2C12 myotubes. First, gene expression of sarcolipin was confirmed in the cells during myogenesis. Then, ER stress was induced in C2C12 myotubes by treatment with tunicamycin or thapsigargin. Sarcolipin messenger RNA (mRNA) and protein expression were significantly reduced by ER stress induction. The reduction was independent of inositol-requiring element 1 (IRE1), which is activated by ER stress and has potent endonuclease activity, when evaluated by treatment with an IRE1 inhibitor, 4 mu 8C. On the other hand, sarcolipin mRNA stability was reduced under the ER stress when evaluated by treatment with actinomycin D. In conclusion, these results show that ER stress represses sarcolipin expression due to changes in mRNA stability in C2C12 myotubes.
• A Long Noncoding RNA, lncRNA-Amhr2, Plays a Role in Amhr2 Gene Activation in Mouse Ovarian Granulosa Cells

  Atsushi P. Kimura, Ryoma Yoneda, Misuzu Kurihara, Shota Mayama, Shin Matsubara

  ENDOCRINOLOGY 158 11 4105 - 4121 2017年11月 [査読有り][通常論文]
   

  Anti-Mullerian hormone (AMH) is critical to the regression of Mullerian ducts during mammalian male differentiation and targets ovarian granulosa cells and testicular Sertoli and Leydig cells of adults. Specific effects of AMH are exerted via its receptor, AMH type II receptor (Amhr2), but the mechanism by which the Amhr2 gene is specifically activated is not fully understood. To see whether a proximal promoter was sufficient for Amhr2 gene activation, we generated transgenic mice that bore the enhanced green fluorescent protein (EGFP) gene driven by a 500-bp mouse Amhr2 gene promoter. None of the established 10 lines, however, showed appropriate EGFP expression, indicating that the 500-bp promoter was insufficient for Amhr2 gene activation. As a regulatory element, we found a long noncoding RNA, lncRNA-Amhr2, transcribed from upstream of the Amhr2 gene in ovarian granulosa cells and testicular Sertoli cells. In primary granulosa cells, knockdown of lncRNA-Amhr2 resulted in a decrease of Amhr2 messnger RNA level, and a transient reporter gene assay showed that lncRNA-Amhr2 activation increased Amhr2 promoter activity. The activity was correlated with lncRNA-Amhr2 transcription in stably transfected OV3121 cells derived from mouse granulosa cells. Moreover, by the Tet-on system, the induction of lncRNA-Amhr2 transcription dramatically increased Amhr2 promoter activity in OV3121 cells. These results indicate that lncRNA-Amhr2 plays a role in Amhr2 gene activation in ovarian granulosa cells by enhancing promoter activity, providing insight into Amhr2 gene regulation underlying the AMH signaling in the female reproductive system.
• Mouse prolyl oligopeptidase plays a role in trophoblast stem cell differentiation into trophoblast giant cell and spongiotrophoblast

  Yuki Maruyama, Shin Matsubara, Atsushi P. Kimura

  PLACENTA 53 8 - 15 2017年05月 [査読有り][通常論文]
   

  Introduction: Prolyl oligopeptidase (prolyl endopeptidase, Prep), a multifunctional protease hydrolyzing -Pro-X-peptide bonds, is highly expressed in the mouse placenta, but the function during development is not known. We explored the possibility of Prep's involvement in placental differentiation. Methods: We cultured trophoblast stem cells (TSCs) derived from the E6.5 mouse embryo and investigated the detailed expression pattern of Prep during their differentiation. Prep-specific inhibitors were added to the TSC culture, and the effect on the differentiation was assessed by microscopic observation and the expression of marker gene for each placental cell. Results: During TSC differentiation for 6 days, Prep was constantly detected at mRNA, protein, and activity levels, and the proteinwas found mainly in the cytoplasm. The addition of 30 mu M and 10 mu M SUAM-14746, a Prep-specific inhibitor, effectively inhibited the differentiation into spongiotrophoblasts (SpTs) and trophoblast giant cells (TGCs), while the TSC viability was not affected. 5 mu M SUAM-14746 impaired the differentiation into SpTs, and 1 mu M SUAM-14746 exhibited no effects. Another Prep-specific inhibitor, KYP-2047, did not affect the differentiation. We confirmed efficient inhibition of Prep enzymatic activity in TSCs by both inhibitors. Conclusion: The dose-dependent effect of SUAM-14746 on TSCs suggests that Prep plays an important role in the differentiation into SpTs and TGCs in the mouse placenta. (C) 2017 Elsevier Ltd. All rights reserved.
• A Genomic Region Transcribed Into a Long Noncoding RNA Interacts With the Prss42/Tessp-2 Promoter in Spermatocytes During Mouse Spermatogenesis, and Its Flanking Sequences Can Function as Enhancers

  Ryoma Yoneda, Yui Satoh, Ikuya Yoshida, Shohei Kawamura, Tomoya Kotani, Atsushi P. Kimura

  MOLECULAR REPRODUCTION AND DEVELOPMENT 83 6 541 - 557 2016年06月 [査読有り][通常論文]
   

  Spermatogenesis is regulated by many meiotic stage-specific genes, but how they coordinate the many individual processes is not fully understood. The Prss/Tessp gene cluster is located on mouse chromosome 9F2-F3, and the three genes at this site (Prss42/Tessp-2, Prss43/Tessp-3, and Prss44/Tessp-4) are specifically activated during meiosis in pachytene spermatocytes. We searched for DNase I hypersensitive sites (HSs) and long noncoding RNAs (lncRNAs) at the Prss/Tessp locus to elucidate how they are activated. We found eight DNase I HSs, three of which were testis germ cell-specific at or close to the Prss42/Tessp-2 promoter, and a testisspecific lncRNA, lncRNA-HSVIII, that was transcribed from a region adjacent to the Prss42/Tessp-2 gene. lncRNA-HSVIII transcripts localized to nuclei of most pachytene spermatocytes and the cytosol of stage-X pachytene spermatocytes and spermatids. Chromosome conformation capture revealed that the lncRNA-HSVIII locus specifically interacted with the Prss42/Tessp-2 promoter in primary and secondary spermatocytes. A 5.8-kb genome sequence, encompassing the entire lncRNA-HSVIII sequence and its flanking regions, significantly increased Prss42/Tessp-2 promoter activity using a reporter-gene assay, yet this construct did not change lncRNA-HSVIII expression, indicating that the elevated promoter activity was likely through enhancer activity. Indeed, both upstreamand downstream regions of the lncRNA-HSVIII sequence significantly increased Prss42/Tessp-2 promoter activity. Our data therefore identified the direct interaction of a genomic region in the lncRNA-HSVIII locus with the Prss42/Tessp-2 promoter in spermatocytes, and suggested that sequences adjacent to the lncRNA function as enhancers for the Prss42/Tessp-2 gene. (C) 2016 Wiley Periodicals, Inc.
• Characterization of the human TCAM1P pseudogene and its activation by a potential dual promoter-enhancer: Comparison with a protein-coding mouse orthologue

  Misuzu Kurihara, Atsushi P. Kimura

  FEBS LETTERS 589 4 540 - 547 2015年02月 [査読有り][通常論文]
   

  TCAM1P is a unitary pseudogene, which was disabled since the human-mouse divergence. Here we found that TCAM1P was specifically expressed in the human testis, with different cell type-specificity from mouse Tcam1, and characterized its transcripts. At the mouse locus, a multifunctional dual promoter-enhancer (DPE) controls the expression of Tcam1 and Smarcd2 genes. The corresponding human sequence was found to potentially function as a DPE, although the molecular mechanism was different from mouse. Interestingly, the change in DPE activity occurred before pseudogenization of TCAM1P. These data suggest the presence of a DPE in the human genome for the first time, and provide an important model of evolutionary changes in the regulatory mechanism of a pseudogene. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
• A Conserved Noncoding Sequence Can Function as a Spermatocyte-Specific Enhancer and a Bidirectional Promoter for a Ubiquitously Expressed Gene and a Testis-Specific Long Noncoding RNA

  Misuzu Kurihara, Akira Shiraishi, Honoo Satake, Atsushi P. Kimura

  JOURNAL OF MOLECULAR BIOLOGY 426 17 3069 - 3093 2014年08月 [査読有り][通常論文]
   

  Tissue-specific gene expression is tightly regulated by various elements such as promoters, enhancers, and long noncoding RNAs (IncRNAs). In the present study, we identified a conserved noncoding sequence (CNS1) as a novel enhancer for the spermatocyte-specific mouse testicular cell adhesion molecule 1 (Tcam1) gene. CNS1 was located 3.4 kb upstream of the Tcam1 gene and associated with histone H3K4 mono-methylation in testicular germ cells. By the in vitro reporter gene assay, CNS1 could enhance Tcam1 promoter activity only in GC-2spd(ts) cells, which were derived from mouse spermatocytes. When we integrated the 6.9-kb 5'-flanking sequence of Tcam1 with or without a deletion of CNS1 linked to the enhanced green fluorescent protein gene into the chromatin of GC-2spd(ts) cells, CNS1 significantly enhanced Tcam1 promoter activity. These results indicate that CNS1 could function as a spermatocyte-specific enhancer. Interestingly, CNS1 also showed high bidirectional promoter activity in the reporter assay, and consistent with this, the Smarcd2 gene and IncRNA, designated IncRNA-Tcam1, were transcribed from adjacent regions of CNS1. While Smarcd2 was ubiquitously expressed, IncRNA-Tcam1 expression was restricted to testicular germ cells, although this IncRNA did not participate in Tcam1 activation. Ubiquitous Smarcd2 expression was correlated to CpG hypo-methylation of CNS1 and partially controlled by Sp1. However, for IncRNA-Tcam1 transcription, the strong association with histone acetylation and histone H3K4 tri-methylation also appeared to be required. The present data suggest that CNS1 is a spermatocyte-specific enhancer for the Tcam1 gene and a bidirectional promoter of Smarcd2 and IncRNA-Tcam1. (C) 2014 Elsevier Ltd. All rights reserved.
• A long non-coding RNA transcribed from conserved non-coding sequences contributes to the mouse prolyl oligopeptidase gene activation

  Shin Matsubara, Misuzu Kurihara, Atsushi P. Kimura

  JOURNAL OF BIOCHEMISTRY 155 4 243 - 256 2014年04月 [査読有り][通常論文]
   

  Prolyl oligopeptidase (POP) is a multifunctional protease which is involved in many physiological events, but its gene regulatory mechanism is poorly understood. To identify novel regulatory elements of the POP gene, we compared the genomic sequences at the mouse and human POP loci and found six conserved non-coding sequences (CNSs) at adjacent intergenic regions. From these CNSs, four long non-coding RNAs (lncRNAs) were transcribed and the expression pattern of one (lncPrep+96kb) was correlated with that of POP. lncPrep+96kb was transcribed as two forms due to the different transcriptional start sites and was localized at the nucleus and cytoplasm, although more was present at the nucleus. When we knocked down lncPrep+96kb in the primary ovarian granulosa cell and a hepatic cell line, the POP expression was decreased in both cells. In contrast, overexpression of lncPrep+96kb increased the POP expression only in the granulosa cell. Because lncPrep+96kb was upregulated with the same timing as POP in the hormone-treated ovary, this lncRNA could play a role in the POP gene activation in the granulosa cell. Moreover, a downstream region of the human POP gene was also transcribed. We propose a novel mechanism for the POP gene activation.
• A testis-specific serine protease, Prss41/Tessp-1, is necessary for the progression of meiosis during murine in vitro spermatogenesis

  Ryoma Yoneda, Atsushi P. Kimura

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 441 1 120 - 125 2013年11月 [査読有り][通常論文]
   

  The function of protease during male meiosis has not been well studied. We previously cloned and characterized four testis-specific serine proteases in the mouse testis. One of the proteases, Prss41/Tessp-1, was expressed in the germ and Sertoli cell. This time, to examine the involvement of Prss41/Tessp-1 in spermatogenesis, we conducted the organ culture of testis fragments in the presence of the anti-Prss41/Tessp-1 antibody. Because in the Sertoli cell, the Prss41/Tessp-1 protein was mostly associated with the membrane of intracellular organelles by glycosylphosphatidylinositol, the antibody was expected to affect Prss41/Tessp-1 at the plasma membrane of spermatogonia. By adding the antibody, the number of germ cells was decreased in some seminiferous tubules. The marker genes expression strongly suggested that meiosis was arrested at spermatogonia, and the number of apoptotic germ cells increased by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. These data indicated that Prss41/Tessp-1 was necessary for the progression of meiosis at the stage of spermatogonia during in vitro spermatogenesis. Together with our previous study, the current results suggest that the Prss/Tessp proteases are important for the progression of meiosis at each stage. (C) 2013 Elsevier Inc. All rights reserved.
• A 914-bp promoter is sufficient to reproduce the endogenous prolyl oligopeptidase gene localization in the mouse placenta if not subject to position effect

  Shin Matsubara, Yuki Maruyama, Atsushi P. Kimura

  GENE 524 2 114 - 123 2013年07月 [査読有り][通常論文]
   

  Prolyl oligopeptidase (POP) is a widely distributed multifunctional protein which has an endopeptidase activity to cleave a -Pro-X- peptide bond. In spite of numerous studies about POP, the mechanism by which its transcription is controlled has not been well investigated. Here we generated transgenic mice bearing a transgene which contained a 914-bp POP gene promoter linked to the enhanced green fluorescent protein (EGFP) gene to assess the in vivo promoter activity. We established six transgenic lines with different copy numbers, but no EGFP signal was observed in four lines due to a high level of DNA methylation, which suggested that the transgene was subject to position effect. However, in the other two lines, we detected the EGFP expression in many tissues, and its placental localization showed a similar change to POP. A strong EGFP signal was observed in the junctional and labyrinthine zones of E10.5-E12.5 placentas and in the junctional zone and the maternal decidua after that. This placental gene activation might be attributed to AP-2 gamma because we detected its binding to the POP promoter. In contrast, we did not obtain any evidence that EGFP was expressed in a similar pattern compared with POP in the ovary. The current data demonstrated that the 914-bp promoter had sufficient activity to reproduce the POP localization in the placenta if it was not subject to position effect and suggest that the regulatory mechanism of the POP gene expression differs between tissues.(C) 2013 Elsevier B.V. All rights reserved.
• Three Testis-Specific Paralogous Serine Proteases Play Different Roles in Murine Spermatogenesis and Are Involved in Germ Cell Survival During Meiosis

  Ryoma Yoneda, Takayuki Takahashi, Hitoshi Matsui, Naoharu Takano, Yuko Hasebe, Katsueki Ogiwara, Atsushi P. Kimura

  BIOLOGY OF REPRODUCTION 88 5 118  2013年05月 [査読有り][通常論文]
   

  Spermatogenesis is a complex process that generates spermatozoa; its molecular mechanisms are not completely understood. Here we focused on the functions of three testis-specific serine proteases: Prss42/Tessp-2, Prss43/Tessp-3, and Prss44/Tessp-4. These protease genes, which constitute a gene cluster on chromosome 9F2-F3, were presumed to be paralogs and were expressed only in the testis. By investigating their mRNA distribution, we found that all three genes were expressed in primary and secondary spermatocytes. However, interestingly, the translated proteins were produced at different locations. Prss42/Tessp-2 was found in the membranes and cytoplasm of secondary spermatocytes and spermatids, whereas Prss43/Tessp-3 was present only in the membranes of spermatocytes and spermatids. Prss44/Tessp-4 was detected in the cytoplasm of spermatocytes and spermatids. To assess the roles of these proteases in spermatogenesis, we used organ culture of mouse testis fragments. Adding antibodies against Prss42/Tessp-2 and Prss43/Tessp-3 resulted in meiotic arrest at the stage when each protease was beginning to be translated. Furthermore, the number of apoptotic cells dramatically increased after the addition of these antibodies. These results strongly suggest that the three paralogous Prss/Tessp proteases play different roles in spermatogenesis and that Prss42/Tessp-2 and Prss43/Tessp-3 are required for germ cell survival during meiosis.
• Localization and subcellular distribution of prolyl oligopeptidase in the mouse placenta

  Shin Matsubara, Takayuki Takahashi, Atsushi P. Kimura

  JOURNAL OF MOLECULAR HISTOLOGY 42 3 251 - 264 2011年06月 [査読有り][通常論文]
   

  Prolyl oligopeptidase (POP) is a serine endopeptidase which selectively digests a -Pro-X-peptide bond. Our previous study showed that POP mRNA was strongly expressed in the spongiotrophoblast of the mouse placenta at E17.5, suggesting its importance in development. To gain more insight into POP's role during gestation, we investigated its expression using different developmental stages of placenta. As a result of in situ hybridization, we found that localization of POP mRNA changed at E12.5. POP mRNA was strongly expressed in the spongiotrophoblast and labyrinth at E10.5 and E11.5 but thereafter only in the spongiotrophoblast. Immunohistochemistry revealed that POP was present in the parietal trophoblast giant cell, the spongiotrophoblast cell, and the labyrinth at E11.5 but the strong expression in the labyrinth was maintained only in the canal-associated and sinusoidal trophoblast giant cells at E16.5 and E18.5. To determine subcellular distribution of the POP protein, we fractionated the placental extract into cytoplasmic, membrane, and nuclear subfractions. By Western blot analysis, POP was detected in the cytoplasmic and membrane fractions but not in the nuclear fraction at E11.5 and E16.5. Interestingly, the cytoplasmic POP exhibited higher enzymatic activity than the membrane-associated type. These data suggest that the cytoplasmic and membrane-associated POP have distinct roles in different types of placental cells.
• Expression of cyclooxygenase-2 and prostaglandin receptor EP4b mRNA in the ovary of the medaka fish, Oryzias latipes: Possible involvement in ovulation

  Chika Fujimori, Katsueki Ogiwara, Akane Hagiwara, Sanath Rajapakse, Atsushi Kimura, Takayuki Takahashi

  MOLECULAR AND CELLULAR ENDOCRINOLOGY 332 1-2 67 - 77 2011年01月 [査読有り][通常論文]
   

  In vitro ovulation of mature medaka ovarian follicles was inhibited by inhibitors of cyclooxygenase (COX) or by an antagonist of the prostaglandin E-2 receptor (EP). Of the three medaka COX genes, ptgs2 was most dominantly expressed in the fish ovary. The ptgs2 transcript was detected in all sizes of growing follicles. In a 24-h spawning cycle, large-sized follicles contained ptgs2 mRNA at a fairly constant level. The levels of COX enzyme activity and prostaglandin E-2 were also constant in the large-sized follicles during the spawning cycle. The expression of prostaglandin E-2 receptor EP4b (ptger419) mRNA was drastically upregulated in the large-sized follicles as the ovulation time approached. The current results indicate that prostaglandin E-2. which might be produced by COX-2, is involved in the ovulation of medaka, and that EP4b is likely the receptor responsible for exerting the action of prostaglandin E-2 in the process. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
• Epigenetic patterns at the mouse prolyl oligopeptidase gene locus suggest the CpG island in the gene body to be a novel regulator for gene expression

  Shin Matsubara, Takayuki Takahashi, Atsushi P. Kimura

  GENE 465 1-2 17 - 29 2010年10月 [査読有り][通常論文]
   

  Prolyl oligopeptidase (POP) is a widely distributed serine peptidase which hydrolyzes small peptides on the carboxyl side of an internal proline residue. While its physiological role has been intensely studied, the regulatory mechanism of the gene expression is poorly understood. This time we assessed the POP mRNA expression in mouse embryos and tissues related to reproduction and development and found that POP mRNA was highly expressed in the ovarian granulosa cell, placental spongiotrophoblast, and blastocyst embryo. To elucidate the mechanism by which POP expression is regulated, we investigated DNA methylation and histone modification patterns of the two CpG islands (CGIs) found at the, mouse POP locus. Whereas the CGI including the POP promoter (CGI-1) was completely hypomethylated in all the tissues examined, DNA methylation level of the CGI in the gene body (CGI-2) was lower in the granulosa cell, placenta, and blastocyst than in the liver. Some specific CpGs in CGI-2 were significantly demethylated in the three tissues. An in vitro reporter analysis indicated that CGI-2 enhanced POP promoter activity and its effect was significantly reduced by DNA methylation. Moreover, histone H3 acetylation and H3K4 methylation levels of CGI-2 were higher in the granulosa cell than liver. The results suggest that the CGI-2 region is a cis-element for the POP gene expression. (C) 2010 Elsevier B.V. All rights reserved.
• Expression and localization of collagen type IV alpha 1 chain in medaka ovary

  Yumiko Kato, Katsueki Ogiwara, Chika Fujimori, Atsushi Kimura, Takayuki Takahashi

  CELL AND TISSUE RESEARCH 340 3 595 - 605 2010年06月 [査読有り][通常論文]
   

  A cDNA clone coding for the collagen type IV alpha 1 chain was obtained from the ovary of the medaka, Oryzias latipes. The clone encoded a protein of 1639 amino acids including a putative 21-residue signal peptide, and the deduced amino acid sequence of the alpha 1 chain was homologous to those of the proteins from other species. The mRNA of the collagen type IV alpha 1 chain was expressed in various tissues of the adult fish. In situ hybridization analysis revealed that the alpha 1 chain mRNA was localized in the follicle layer of all growing follicles. In the post-ovulatory follicle that had released its oocyte during ovulation, the alpha 1 chain transcript was detected in a winding line surrounding the tissue. This localization pattern was different from that of gelatinase B, a marker gene for granulosa cells. A specific antibody was prepared for the medaka collagen type IV alpha 1 chain. Immunohistochemical analysis with this antibody yielded results consistent with those obtained by in situ hybridization. These data indicate that, in the medaka ovary, collagen type IV is synthesized by theca cells and is localized in the basement membrane.
• Two distinct localization patterns of testis-specific serine protease 1 (TESSP1) in the seminiferous tubules of the mouse testis.

  Takano N, Kimura A, Takahashi T

  Zoological science 26 4 294 - 300 Zoological Society of Japan 2009年04月 [査読有り][通常論文]
  
• Epigenetic activation of the human growth hormone gene cluster during placental cytotrophoblast differentiation

  Atsushi P. Kimura, Daria Sizova, Stuart Handwerger, Nancy E. Cooke, Stephen A. Liebhaber

  MOLECULAR AND CELLULAR BIOLOGY 27 18 6555 - 6568 2007年09月 [査読有り][通常論文]
   

  The hGH cluster contains a single human pituitary growth hormone gene (hGH-N) and four placenta-specific paralogs. Activation of the cluster in both tissues depends on 5' remote regulatory elements. The pituitary-specific locus control elements DNase I-hypersensitive site I (HSI) and HSII, located 14.5 kb 5' of the cluster (position -14.5), establish a continuous domain of histone acetylation that extends to and activates hGH-N in the pituitary gland. In contrast, histone modifications in placental chromatin are restricted to the more 5'-remote HSV-HSIII region (kb -28 to -32) and to the placentally expressed genes in the cluster, with minimal modification between these two regions. These data predict distinct modes of hGH cluster gene activation in the pituitary and placenta. Here we used cell culture models to track structural changes at the hGH locus through placental-gene activation. The data revealed that this process was initiated in primary cytotrophoblasts by histone H3K4 di- and trimethylation and H4 acetylation restricted to HSV and to the individual placental-gene repeat (PGR) units within the cluster. Later stages of transcriptional induction were accompanied by enhancement and extension of these modifications and by robust H3 acetylation at HSV, at HSIII, and throughout the placental-gene regions. These data suggested that elements restricted to HSIII-HSV regions and each individual PGR might be sufficient for activation of the hCS genes. This model was tested by comparing hCS transgene expression in the placentas of mouse embryos carrying a full hGH cluster to that in placentas in which the HSIII-HSV region was directly linked to the individual hCS-A PGR unit. The findings indicate that the HSIII-HSV region and the PGR units, although targeted for initial chromatin structural modifications, are insufficient to activate gene expression and that this process is dependent on additional, as-yet-unidentified chromatin determinants.
• Characterization of mouse tissue kallikrein 5

  Sanath Rajapakse, Katsueki Ogiwara, Noriko Yamano, Atsushi Kimura, Kensaku Hirata, Sumio Takahashi, Takayuki Takahashi

  ZOOLOGICAL SCIENCE 23 11 963 - 968 2006年11月 [査読有り][通常論文]
   

  Mouse tissue kallikreins (Klks) are members of a large, multigene family consisting of 37 genes, 26 of which can code for functional proteins. Mouse tissue kallikrein 5 (KIk5) has long been thought to be one of these functional genes, but the gene product, mK5, has not been isolated and characterized. In the present study, we prepared active recombinant mK5 using an Escherichia coli expression system, followed by column chromatography. We then determined the biochemical and enzymatic properties of purified mK5. mK5 had trypsin-like activity for Arg at the P1 position, and its activity was inhibited by typical serine protease inhibitors. mK5 degraded gelatin, fibronectin, collagen type IV, high-molecular-weight kininogen, and insulin-like growth factor binding protein-3. Our data suggest that mK5 may be implicated in the process of extracellular matrix remodeling.
• Tissue-specific chromatin modifications at a multigene locus generate asymmetric transcriptional interactions

  Eung Jae Yoo, Isabela Cajiao, Jeong-Seon Kim, Atsushi P. Kimura, Aiwen Zhang, Nancy E. Cooke, Stephen A. Liebhaber

  MOLECULAR AND CELLULAR BIOLOGY 26 15 5569 - 5579 2006年08月 [査読有り][通常論文]
   

  Random assortment within mammalian genomes juxtaposes genes with distinct expression profiles. This organization, along with the prevalence of long-range regulatory controls, generates a potential for aberrant transcriptional interactions. The human CD79b/GH locus contains six tightly linked genes with three mutually exclusive tissue specificities and interdigitated control elements. One consequence of this compact organization is that the pituitary cell-specific transcriptional events that activate hGH-N also trigger ectopic activation of CD79b. However, the B-cell-specific events that activate CD79b do not trigger reciprocal activation of hGH-N. Here we utilized DNase I hypersensitive site mapping, chromatin immunoprecipitation, and transgenic models to explore the basis for this asymmetric relationship. The results reveal tissue-specific patterns of chromatin structures and transcriptional controls at the CD79b/GH locus in B cells distinct from those in the pituitary gland and placenta. These three unique transcriptional environments suggest a set of corresponding gene expression pathways and transcriptional interactions that are likely to be found juxtaposed at multiple sites within the eukaryotic genome.
• Epigenetic modifications at the human growth hormone locus predict distinct roles for histone acetylation and methylation in placental gene activation

  AP Kimura, SA Liebhaber, NE Cooke

  MOLECULAR ENDOCRINOLOGY 18 4 1018 - 1032 2004年04月 [査読有り][通常論文]
   

  Developmental control of eukaryotic gene expression is tightly linked to alterations in chromatin structure. Studies of the hGH multigene cluster suggest that the four placental genes are activated by a pathway of histone modification distinct from the pathway leading to activation of the single pituitary hGH-N gene. The relationship between histone acetylation and hGH-N activation in the pituitary has been previously defined using a combination of epigenetic mapping and transgenic analyses. The repeated gene structures within the hGH cluster had been an impediment to comparable analysis of placental gene activation. In the present report we defined patterns of core histone acetylation and methylation within and flanking the hGH cluster in human placental chromatin. These data highlight differences between placental and pituitary pathways of transcriptional control at the hGH cluster and suggest that selective activation of the placental genes reflects distinct roles for histone acetyltransferase and histone methyltransferase coactivator complexes.

その他活動・業績

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  木村 敦, 佐藤 優衣, 丸山 優樹 比較内分泌学 44 (164) 58 -62 2018年06月03日 [査読無し][通常論文]
  
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  木村 敦 比較内分泌学 44 (164) 65 -68 2018年06月03日 [査読無し][通常論文]
  
• ヒト成長ホルモン遺伝子クラスターのLCRによる発現制御

  木村 敦, COOKE Nancy E, LIEBHABER Stephen A 日本比較内分泌学会ニュース = Newsletter of Japan Society for Comparative Endocrinology 0 (122) 23 -31 2006年08月01日 [査読無し][通常論文]
  

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