つくばリポジトリ SR 8 1 425

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Mi ce def i ci ent i n t he Shmt 2 gene have mi t ochondr i al r espi r at i on def ect s and ar e embr yoni c l et hal 著者 j our nal or publ i cat i on t i t l e vol ume page r ange year 権利 URL Tani Har una, Ohni shi Saki ko, Shi t ar a Hi r oshi ,Mi t o Takayuki ,Yamaguchi Mi dor i ,Yonekawa Hi r omi chi ,Hashi zume Osamu, I shi kawa Kaor i ,Nakada Kazut o, Hayashi J un- I chi Sci ent i f i c r epor t s 8 425 2018- 01 (C) The Aut hor (s) 2017 Thi s ar t i cl e i s l i censed under a Cr eat i ve Commons At t r i but i on 4. 0 I nt er nat i onal Li cense, whi ch per mi t s use, shar i ng, adapt at i on, di st r i but i on and r epr oduct i on i n any medi um or f or mat ,as l ong as you gi ve appr opr i at e cr edi t t o t he or i gi nal aut hor (s) and t he sour ce, pr ovi de a l i nk t o t he Cr eat i ve Commons l i cense, and i ndi cat e i f changes wer e made. The i mages or ot her t hi r d par t y mat er i al i n t hi s ar t i cl e ar e i ncl uded i n t he ar t i cl e’ s Cr eat i ve Commons l i cense, unl ess i ndi cat ed ot her wi se i n a cr edi t l i ne t o t he mat er i al .I f mat er i al i s not i ncl uded i n t he ar t i cl e’ s Cr eat i ve Commons l i cense and your i nt ended use i s not per -mi t t ed by st at ut or y r egul at i on or exceeds t he per mi t t ed use, you wi l l need t o obt ai n per mi ssi on di r ect l y f r om t he copyr i ght hol der .To vi ew a copy of t hi s .ht t p: hdl .handl e. net /2241/ 00150985 doi: 10.1038/s41598-017-18828-3 Cr eat i ve Commons :表示 ht t p: cr eat i vecommons. or g/ l i censes/ by/ 3. 0/ deed. j a www.nature.com/scientificreports OPEN Received: 15 August 2017 Accepted: 12 December 2017 Published: xx xx xxxx Mice deicient in the Shmt2 gene have mitochondrial respiration defects and are embryonic lethal Haruna Tani1, Sakiko Ohnishi1, Hiroshi Shitara1,2, Takayuki Mito3, Midori Yamaguchi2, Hiromichi Yonekawa2, Osamu Hashizume3, Kaori Ishikawa1,3, Kazuto Nakada1,3 &Jun-Ichi Hayashi4 Accumulation of somatic mutations in mitochondrial DNA (mtDNA) has been proposed to be responsible for human aging and age-associated mitochondrial respiration defects. However, our previous indings suggested an alternative hypothesis of human aging—that epigenetic changes but not mutations regulate age-associated mitochondrial respiration defects, and that epigenetic downregulation of nuclear-coded genes responsible for mitochondrial translation [e.g.,glycine C-acetyltransferase (GCAT),serine hydroxymethyltransferase 2 (SHMT2)]is related to age-associated respiration defects. To examine our hypothesis, here we generated mice deicient in Gcat or Shmt2 and investigated whether they have respiration defects and premature aging phenotypes. Gcat-deicient mice showed no macroscopic abnormalities including premature aging phenotypes for up to 9 months after birth. In contrast, Shmt2-deicient mice showed embryonic lethality after 13.5 days post coitum (dpc),and ibroblasts obtained from 12.5-dpc Shmt2-deicient embryos had respiration defects and retardation of cell growth. Because Shmt2 substantially controls production of N-formylmethioninetRNA (fMet-tRNA) in mitochondria, its suppression would reduce mitochondrial translation, resulting in expression of the respiration defects in ibroblasts from Shmt2-deicient embryos. These indings support our hypothesis that age-associated respiration defects in ibroblasts of elderly humans are caused not by mtDNA mutations but by epigenetic regulation of nuclear genes including SHMT2. Because mitochondria produce reactive oxygen species endogenously and preferentially accumulate exogenous chemical carcinogens, mitochondrial DNA (mtDNA) is exposed to these mutagens, resulting in accumulation of somatic mutations with age1–5. Some of these somatic mutations in human mtDNA are pathogenic, because the same mutations are found in patients with mitochondrial diseases caused by mitochondrial respiration defects. herefore, the mitochondrial theory of aging1–5 proposes that accumulation of somatic mutations in mtDNA is responsible for human aging and age-associated mitochondrial respiration defects. However, it is also possible that abnormalities in nuclear DNA but not in mtDNA induce age-associated mitochondrial respiration defects, because both nuclear DNA and mtDNA encode proteins required for mitochondrial respiratory function1. To determine which genome, nuclear or mitochondrial, is responsible for the respiration defects in the ibroblasts of elderly humans, we previously carried out intercellular transfer of mtDNA6 or nuclear DNA7 by using mtDNA-less HeLa cells8; the results led us to propose that nuclear recessive mutations induce the age-associated respiration defects. In contrast, the mitochondrial theory of aging has been supported by studies of mtDNA mutator mice9,10, which were generated by introducing a proofreading-deicient mtDNA polymerase gene. hese mice showed accelerated accumulation of somatic mutations in mtDNA, resulting in accelerated expression of respiration defects and premature aging phenotypes9,10. herefore, it has been controversial whether human aging and age-associated respiration defects are controlled by the accumulation of somatic mutations in mtDNA9,10 or by nuclear recessive mutations7. More recently, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki, Japan. Laboratory for Transgenic Technology, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku, Tokyo, Japan. Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki, Japan. University of Tsukuba, Tennodai, Tsukuba, Ibaraki, Japan. Haruna Tani, Sakiko Ohnishi and Hiroshi Shitara contributed equally to this work. Correspondence and requests for materials should be addressed to J.-I.H. email: jih @biol.tsukuba.ac.jp) SCIENTIFIC REPORTS |2018) 8:425 |DOI: s 1 www.nature.com/scientificreports/ Figure 1. Genotyping of F1 pups obtained by mating heterozygous females and males. a) Mutations in Gcat. PCR products of 266 bp and 234 bp correspond to Gcat without mutations (and with mutations (m/m; see Supplementary Fig. S1 for sequences),respectively. he presence of both fragments indicates heterozygosity (m/+Arrowhead shows an additional fragment that may represent heteroduplex molecules. b) Mutation in Shmt2. Because an XcmI site was eliminated by an insertion of T (Supplementary Fig. S1),XcmI digestion of PCR products without the mutation produces two fragments (318 bp and 280 bp),whereas digestion of PCR products with the T insertion produces a single fragment (599 bp including the T insertion).he presence of all three fragments indicates heterozygosity (m/+No mice with homozygous mutations (m/m) were found, indicating their embryonic lethality. epigenetic regulation of cellular senescence has been proposed in human fibroblasts11. Our recent study12 addressed these issues by deep sequencing analysis of mtDNA and showed that mtDNA in ibroblasts from elderly humans does not accumulate somatic mutations. Moreover, reprogramming of these ibroblasts by generating induced pluripotent stem cells (iPSCs) restores normal respiratory function12. his led us to hypothesise that age-associated respiration defects are controlled not by mutations in either nuclear or mtDNA, but by epigenetic regulation of nuclear genes. Our microarray screening results suggest that epigenetic downregulation of the nuclear genes glycine C-acetyltransferase (GCAT) and serine hydroxymethyltransferase 2 (SHMT2) is involved in age-associated respiration defects of the ibroblasts of elderly humans12. Because the products of both genes are localized in mitochondria and regulate glycine production in mitochondria13,14, their downregulation would induce defects in mitochondrial translation and respiratory function, resulting in the age-associated respiration defects found in the ibroblasts of elderly humans6,7. To examine this possibility, we generated mice deicient in Gcat or Shmt2, and investigated whether these mice would have mitochondrial respiration defects and premature aging phenotypes. Results Generation of mice deicient in the Gcat or Shmt2 genes. We generated knockout mouse strains deicient in the Gcat gene or the Shmt2 gene by using the CRISPR/Cas9 system. Target sequences were designed according to the mouse Gcat and Shmt2 sequences (Supplementary Fig. S1).Cas9 mRNA and single-guide RNAs (sgRNAs) were synthesized as reported previously15, and were microinjected into fertilized eggs (pronuclear stage) from C57BL/6J (hereater referred to as B6J) mice. he microinjected eggs were transferred to the oviducts of pseudo-pregnant females. In the case of Gcat knockout mice, 41 of 70 mice were mutation-positive in the Surveyor assay (see Methods).We analysed the sequence around the target region in the mice with mutations and selected one male mouse with an insertion and a deletion that would disrupt Gcat gene function (Supplementary Fig. S1);we used this mouse as a founder for further breeding to obtain heterozygous (Gcat m/+females and males. By mating heterozygous females with heterozygous males, we obtained 34 pups. Genotyping showed that 11 pups had no mutation, 19 were heterozygous, and 4 were homozygous (Gcat m/m) Fig. 1a).We then obtained ofspring (Gcat+/m/+m/m) by in vitro fertilization using heterozygous females and a heterozygous male. In the case of Shmt2 knockout mice, 20 of 25 mice were mutation-positive in the Surveyor assay. We selected one female mouse with a single-nucleotide insertion (T) resulting in a frame shit that would disrupt Shmt2 gene function (Supplementary Fig. S1) and used this mouse as a founder for further breeding to obtain heterozygous (Shmt2 m/+females and males. By mating heterozygous females with heterozygous males, we obtained 45 pups. Genotyping by Xcm I digestion of the PCR products showed that 14 mice had no mutation and 31 were heterozygous, but no mice had a homozygous mutation (Fig. 1b),indicating the lethality of embryos with a homozygous mutation in Shmt2 (Shmt2 m/m).Characterization of mice deicient in the Gcat gene. Because the Gcat m/m mice did not show embryonic lethality, we performed Western blot analysis to conirm the suppression of the Gcat gene in these mice. he GCAT protein was not detectable in livers of 5-month-old Gcat m/m mice, but was detectable in wild-type (or heterozygous (m/+mice (Fig. 2).hese observations conirm complete absence of the GCAT protein in Gcat m/m mice. However, no growth retardation or obvious macroscopic abnormalities including premature aging phenotypes, such as hair greying, alopecia, or kyphosis, were observed in Gcat m/m mice for at least 9 months ater SCIENTIFIC REPORTS |2018) 8:425 |DOI: s 2 www.nature.com/scientificreports/ Figure 2. Suppression of GCAT protein production in 5-month-old Gcat m/m mice. a) Western blot analysis of the GCAT protein in the livers of mice of the indicated genotypes. b) Quantiication of Western blot data. Figure 3. Lethality of 13.5-dpc F1 embryos with a homozygous mutation in Shmt2 obtained by mating heterozygous females and males. a) Macroscopic abnormalities. b) Genotyping of the Shmt2 mutation. c) Examination of the presence of the SHMT2 protein by Western blot analysis. Experiments were performed in triplicate. Data are means ±s.e.m. P

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