Identification and sequencing of the gene encoding DNA methyltransferase 3 ( DNMT3) from sea cucumber, Apostichopus japonicus Original Article First Online: 20 April 2019 Abstract
The sea cucumber
Apostichopus japonicus is well known as a traditional tonic food and as a commercially important cultured aquatic species. This species produces saponins, and has a high potential to cope with environmental stress, such as aestivation, organ regeneration, and wound healing. Recently, several studies have shown that cellular reprogramming and the physiological responses of the sea cucumber to environmental changes, including aestivation, are potentially mediated by epigenetic DNA methylation. The DNA methyltransferase ( DNMT) 1 and DNMT3 genes are independent participants in the maintenance and de novo methylation of specific sequences. Sea urchin ( Strongylocentrotus purpuratus) and starfish ( Asterina pectinifera), which belong to the same phylum as A. japonicus, have both DNMT1 and DNMT3 genes. However, it was previously reported that DNMT1 is present, but DNMT3 is absent, in A. japonicus. In the present study, we sequenced the full-length cDNA of the A. japonicus DNMT3 gene. The newly sequenced DNMT3 gene comprises three major conserved domains (Pro-Trp-Trp-Pro (PWWP), plant homeodomain (PHD), and S-adenosylmethionine-dependent methyltransferase (AdoMet-MTase)), indicating that the DNMT3 possibly has de novo DNA methylation catalytic activity. Gene structure and phylogenetic analysis showed that sea cucumber DNMT3 is evolutionarily conserved in the Echinodermata. Next, we demonstrated the conservation of DNMT3 gene expression in sea cucumber and starfish belong to same phylum, echinoderm. Using reverse transcription-polymerase chain reaction, sea cucumber DNMT3 mRNA was detected in testis tissue, but not in other tissues tested, including the respiratory tree, muscle, tentacle, intestine, and ovary. This is inconsistent with previous reports, which showed the expression of DNMT3 in ovary, but not in testis of the starfish A. pectinifera, indicating the tissue- and species-specific expression of DNMT3 gene. Although further studies are needed to clarify the epigenetic regulatory mechanisms of DNMT3 and its application to the aquaculture industry, our findings may provide insights into the sea cucumber biology. Keywords Sea cucumber Apostichopus japonicus DNA methylation DNMT3 RACE-PCR Phylogenetic analysis Electronic supplementary material
The online version of this article (
) contains supplementary material, which is available to authorized users. https://doi.org/10.1007/s11033-019-04821-8
Hyun-Hee Hong and Sung-Gwon Lee contributed equally to this work.
This research was supported by the Collaborative Genome Program (No. 20180430), and “Development of methods for controlling and managing marine ecological disturbance-causing and harmful organisms” (MEDHO), and “Research center for fishery resource management based on the information and communication technology (ICT)” of the Korea Institute of Marine Science and Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries of the Republic of Korea to C.P.
Compliance with ethical standards Conflict of interest
All authors declare that they have no conflict of interest.
This article does not contain any studies with human subject or animals performed by any of the authors.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Jaenisch R (1995) Developmental genetics—role for DNA methylation in genomic imprinting. Scientist 9:15
Tycko B (1997) DNA methylation in genomic imprinting. Rev Mutat Res 386:131–140.
https://doi.org/10.1016/S1383-5742(96)00049-X CrossRef Google Scholar
Beard C, Li E, Jaenisch R (1995) Loss of methylation activates Xist in somatic but not in embryonic-cells. Genes Dev 9:2325–2334.
https://doi.org/10.1101/gad.9.19.2325 CrossRef PubMed Google Scholar
Sharp AJ, Stathaki E, Migliavacca E, Brahmachary M, Montgomery SB, Dupre Y, Antonarakis SE (2011) DNA methylation profiles of human active and inactive X chromosomes. Genome Res 21:1592–1600.
https://doi.org/10.1101/gr.112680.110 CrossRef PubMed PubMedCentral Google Scholar
Borgel J et al (2010) Targets and dynamics of promoter DNA methylation during early mouse development. Nat Genet 42:1093.
https://doi.org/10.1038/ng.708 CrossRef PubMed Google Scholar
Franco R, Schoneveld O, Georgakilas AG, Panayiotidis MI (2008) Oxidative stress, DNA methylation and carcinogenesis. Cancer Lett 266:6–11.
https://doi.org/10.1016/j.canlet.2008.02.026 CrossRef PubMed Google Scholar
Curradi M, Izzo A, Badaracco G, Landsberger N (2002) Molecular mechanisms of gene silencing mediated by DNA methylation. Mol Cell Biol 22:3157–3173.
https://doi.org/10.1128/Mcb.22.9.3157-3173.2002 CrossRef PubMed PubMedCentral Google Scholar
Sanchez-Romero MA, Cota I, Casadesus J (2015) DNA methylation in bacteria: from the methyl group to the methylome. Curr Opin Microbiol 25:9–16.
https://doi.org/10.1016/j.mib.2015.03.004 CrossRef PubMed Google Scholar
Lyko F (2018) The DNA methyltransferase family: a versatile toolkit for epigenetic regulation. Nat Rev Genet 19:81–92.
https://doi.org/10.1038/nrg.2017.80 CrossRef PubMed Google Scholar
Bird AP, Wolffe AP (1999) Methylation-induced repression—belts, braces, and chromatin. Cell 99:451–454.
https://doi.org/10.1016/s0092-8674(00)81532-9 CrossRef PubMed Google Scholar
Smith ZD, Meissner A (2013) DNA methylation: roles in mammalian development. Nat Rev Genet 14:204–220.
https://doi.org/10.1038/nrg3354 CrossRef PubMed Google Scholar
Sasaki H, Matsui Y (2008) Epigenetic events in mammalian germ-cell development: reprogramming and beyond. Nat Rev Genet 9:129–140.
https://doi.org/10.1038/nrg2295 CrossRef PubMed Google Scholar
Rebollo R, Romanish MT, Mager DL (2012) Transposable elements: an abundant and natural source of regulatory sequences for host genes. Annu Rev Genet 46:21–42.
https://doi.org/10.1146/annurev-genet-110711-155621 CrossRef PubMed Google Scholar
Schultz MD et al (2015) Human body epigenome maps reveal noncanonical DNA methylation variation. Nature 523:212.
https://doi.org/10.1038/nature14465 CrossRef PubMed PubMedCentral Google Scholar
Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11:204–220.
https://doi.org/10.1038/nrg2719 CrossRef PubMed PubMedCentral Google Scholar
Lister R et al (2013) Global epigenomic reconfiguration during mammalian brain development. Science 341:629.
https://doi.org/10.1126/science.1237905 CrossRef Google Scholar
Xie W et al (2012) Base-resolution analyses of sequence and parent-of-origin dependent DNA methylation in the mouse genome. Cell 148:816–831.
https://doi.org/10.1016/j.cell.2011.12.035 CrossRef PubMed PubMedCentral Google Scholar
Colot V, Rossignol JL (1999) Eukaryotic DNA methylation as an evolutionary device. BioEssays 21:402
CrossRef PubMed Google Scholar
Suzuki MM, Bird A (2008) DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet 9:465–476.
https://doi.org/10.1038/nrg2341 CrossRef PubMed PubMedCentral Google Scholar
Drewell RA et al (2014) The dynamic DNA methylation cycle from egg to sperm in the honey bee Apis mellifera Development 141:2702–2711.
https://doi.org/10.1242/dev.110163 CrossRef PubMed Google Scholar
Riviere G, Wu GC, Fellous A, Goux D, Sourdaine P, Favrel P (2013) DNA methylation is crucial for the early development in the Oyster C gigas. Mar Biotechnol 15:739–753.
https://doi.org/10.1007/s10126-013-9523-2 CrossRef PubMed Google Scholar
Tweedie S, Charlton J, Clark V, Bird A (1997) Methylation of genomes and genes at the invertebrate-vertebrate boundary. Mol Cell Biol 17:1469–1475.
https://doi.org/10.1128/Mcb.17.3.1469 CrossRef PubMed PubMedCentral Google Scholar
Keller TE, Han P, Yi SV (2016) Evolutionary transition of promoter and gene body DNA methylation across invertebrate-vertebrate boundary. Mol Biol Evol 33:1019–1028.
https://doi.org/10.1093/molbev/msv345 CrossRef PubMed Google Scholar
Chen TP, Li E (2004) Structure and function of eukaryotic DNA methyltransferases. Curr Top Dev Biol 60:55–89
CrossRef PubMed Google Scholar
Lei H, Oh SP, Okano M, Juttermann R, Goss KA, Jaenisch R, Li E (1996) De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development 122:3195–3205
PubMed Google Scholar
Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69:915–926
CrossRef PubMed Google Scholar
Schermelleh L et al (2007) Dynamics of Dnmt1 interaction with the replication machinery and its role in postreplicative maintenance of DNA methylation. Nucleic Acids Res 35:4301–4312.
https://doi.org/10.1093/nar/gkm432 CrossRef PubMed PubMedCentral Google Scholar
Gao F et al (2012) Differential DNA methylation in discrete developmental stages of the parasitic nematode
. Genome Biol.
https://doi.org/10.1186/gb-2012-13-10-r100 CrossRef PubMed PubMedCentral Google Scholar
Adema CM et al (2017) Whole genome analysis of a schistosomiasis-transmitting freshwater snail. Nat Commun.
https://doi.org/10.1038/ncomms15451 CrossRef PubMed PubMedCentral Google Scholar
Geyer KK et al (2017) The
DNA methylation machinery displays spatial tissue expression, is differentially active in distinct snail populations and is modulated by interactions with
. PloS Neglect Trop D.
https://doi.org/10.1371/journal.pntd.0005246 CrossRef Google Scholar
Wang XT et al (2014) Genome-wide and single-base resolution DNA methylomes of the Pacific oyster Crassostrea gigas provide insight into the evolution of invertebrate CpG methylation. Bmc Genomics 15:4.
https://doi.org/10.1186/1471-2164-15-1119 CrossRef PubMed PubMedCentral Google Scholar
Choe S, Ohshima Y (1961) On the morphological and ecological differences between two commercial forms, green and red of the japanese common sea cucumber, Stichopus japonicus Selenka Nippon. Suisan Gakkaishi 27:97–106.
https://doi.org/10.2331/suisan.27.97 CrossRef Google Scholar
Kan-No M, Kijima A (2002) Quantitative and qualitative evaluation on the color variation of the japanese sea cucumber
aquaculture. Science 50:63–69.
https://doi.org/10.11233/aquaculturesci1953.50.63 CrossRef Google Scholar
Bahrami Y, Zhang W, Franco C (2014) Discovery of novel saponins from the viscera of the sea cucumber
. Mar Drugs 12:2633–2667.
https://doi.org/10.3390/md12052633 CrossRef PubMed PubMedCentral Google Scholar
Kim SK, Himaya SWA (2012) Chapter 20 - Triterpene glycosides from sea cucumbers and their biological activities. In: Advances in food and nutrition research, vol 65. Academic Press, pp 297–319.
Thimmappa R, Geisler K, Louveau T, O’Maille P, Osbourn A (2014) Triterpene biosynthesis in plants. Annu Rev Plant Biol 65:225–257.
https://doi.org/10.1146/annurev-arplant-050312-120229 CrossRef PubMed Google Scholar
Carnevali M, Burighel P (2010) Regeneration in echinoderms and ascidians. In: eLS.
Rychel AL, Swalla BJ (2009) Regeneration in hemichordates and echinoderms. Stem cells in marine organisms. Springer, Netherlands, pp 245–265.
https://doi.org/10.1007/978-90-481-2767-2_10 CrossRef Google Scholar
De Carvalho DD, You JS, Jones PA (2010) DNA methylation and cellular reprogramming. Trends Cell Biol 20:609–617.
https://doi.org/10.1016/j.tcb.2010.08.003 CrossRef PubMed PubMedCentral Google Scholar
Yakushiji N et al (2007) Correlation between Shh expression and DNA methylation status of the limb-specific Shh enhancer region during limb regeneration in amphibians. Dev Biol 312:171–182.
https://doi.org/10.1016/j.ydbio.2007.09.022 CrossRef PubMed Google Scholar
Zhao Y, Chen MY, Storey KB, Sun LN, Yang HS (2015) DNA methylation levels analysis in four tissues of sea cucumber
based on fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) during aestivation. Compar Biochem Physiol B 181:26–32.
https://doi.org/10.1016/j.cbpb.2014.11.001 CrossRef Google Scholar
Zhao Y, Chen MY, Su L, Wang TM, Liu SL, Yang HS (2013) Molecular cloning and expression-profile analysis of sea cucumber DNA (Cytosine-5)-methyltransferase 1 and methyl-CpG binding domain type 2/3 genes during aestivation. Compar Biochem Physiol B 165:26–35.
https://doi.org/10.1016/j.cbpb.2013.02.009 CrossRef Google Scholar
Cameron RA, Samanta M, Yuan A, He D, Davidson E (2009) SpBase: the sea urchin genome database and web site. Nucleic Acids Res 37:D750–D754.
https://doi.org/10.1093/nar/gkn887 CrossRef PubMed Google Scholar
Fujihara Y, Miyasako H, Kato K, Hayashi T, Toraya T (2012) Molecular cloning, expression, and characterization of starfish DNA (Cytosine-5)-methyltransferases. Biosci Biotechnol Biochem 76:1661–1671.
https://doi.org/10.1271/bbb.120161 CrossRef PubMed Google Scholar
Reich A, Dunn C, Akasaka K, Wessel G (2015) Phylogenomic analyses of Echinodermata support the sister groups of Asterozoa and Echinozoa. PLoS ONE 10:e0119627.
https://doi.org/10.1371/journal.pone.0119627 CrossRef PubMed PubMedCentral Google Scholar
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410.
https://doi.org/10.1016/S0022-2836(05)80360-2 CrossRef PubMed PubMedCentral Google Scholar
Finn RD et al (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44:D279–D285.
https://doi.org/10.1093/nar/gkv1344 CrossRef Google Scholar
Sievers F et al (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:5.
https://doi.org/10.1038/msb.2011.75 CrossRef Google Scholar
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797.
https://doi.org/10.1093/nar/gkh340 CrossRef PubMed PubMedCentral Google Scholar
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 60. Mol Biology and Evolution 30:2725–2729.
https://doi.org/10.1093/molbev/mst197 CrossRef Google Scholar
Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574.
https://doi.org/10.1093/bioinformatics/btg180 CrossRef PubMed PubMedCentral Google Scholar
Milne I, Wright F, Rowe G, Marshall DF, Husmeier D, McGuire G (2004) TOPALi: software for automatic identification of recombinant sequences within DNA multiple alignments. Bioinformatics 20:1806–1807.
https://doi.org/10.1093/bioinformatics/bth155 CrossRef PubMed Google Scholar
Yang A-F et al (2010) Stability comparison of cytb and β-actin gene expression in sea cucumber
. Journal of Agricultural Science and Technology 1:020
Wu H, Zhang Y (2014) Reversing DNA methylation: mechanisms, genomics, and biological functions. Cell 156:45–68.
https://doi.org/10.1016/j.cell.2013.12.019 CrossRef PubMed PubMedCentral Google Scholar
Ponger L, Li WH (2005) Evolutionary diversification of DNA methyltransferases in eukaryotic genomes. Mol Biol Evol 22:1119–1128.
https://doi.org/10.1093/molbev/msi098 CrossRef PubMed Google Scholar
Jo J et al (2016) Comparative transcriptome analysis of three color variants of the sea cucumber
. Mar Genom 28:21–24.
https://doi.org/10.1016/j.margen.2016.03.009 CrossRef Google Scholar
Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247–257.
https://doi.org/10.1016/s0092-8674(00)81656-6 CrossRef PubMed Google Scholar
Du HX et al (2012) Transcriptome sequencing and characterization for the sea cucumber
(Selenka, 1867). PLoS ONE 7:4.
https://doi.org/10.1371/journal.pone.0033311 CrossRef Google Scholar
Jo J et al (2017) Draft genome of the sea cucumber
and genetic polymorphism among color variants. Gigascience 6:5.
https://doi.org/10.1093/gigascience/giw006 CrossRef Google Scholar
Zhang XJ et al (2017) The sea cucumber genome provides insights into morphological evolution and visceral regeneration. PLoS Biol.
https://doi.org/10.1371/journal.pbio.2003790 CrossRef PubMed PubMedCentral Google Scholar
Golding MC, Westhusin ME (2003) Analysis of DNA (cytosine 5) methyltransferase mRNA sequence and expression in bovine preimplantation embryos, fetal and adult tissues. Gene Expr Patterns 3:551–558
CrossRef PubMed Google Scholar
Hirasawa R, Chiba H, Kaneda M, Tajima S, Li E, Jaenisch R, Sasaki H (2008) Maternal and zygotic Dnmt1 are necessary and sufficient for the maintenance of DNA methylation imprints during preimplantation development. Genes Dev 22:1607–1616.
https://doi.org/10.1101/gad.1667008 CrossRef PubMed PubMedCentral Google Scholar
Huntriss J et al (2004) Expression of mRNAs for DNA methyltransferases and methyl-CpG-binding proteins in the human female germ line, preimplantation embryos, and embryonic stem cells. Mol Reprod Dev 67:323–336.
https://doi.org/10.1002/mrd.20030 CrossRef PubMed Google Scholar
La Salle S, Mertineit C, Taketo T, Moens PB, Bestor TH, Trasler JM (2004) Windows for sex-specific methylation marked by DNA methyltransferase expression profiles in mouse germ cells. Dev Biol 268:403–415.
https://doi.org/10.1016/j.ydbio.2003.12.031 CrossRef PubMed Google Scholar
La Salle S, Trasler JM (2006) Dynamic expression of DNMT3a and DNMT3b isoforms during male germ cell development in the mouse. Dev Biol 296:71–82.
https://doi.org/10.1016/j.ydbio.2006.04.436 CrossRef PubMed Google Scholar
Marques CJ, Pinho MJ, Carvalho F, Bieche I, Barros A, Sousa M (2011) DNA methylation imprinting marks and DNA methyltransferase expression in human spermatogenic cell stages. Epigenetics 6:1354–1361.
https://doi.org/10.4161/epi.6.11.17993 CrossRef PubMed Google Scholar
Li Y et al (2019) Dynamics of DNA methylation and DNMT expression during gametogenesis and early development of scallop
. Mar Biotechnol 5:4.
https://doi.org/10.1007/s10126-018-09871-w CrossRef Google Scholar Copyright information
© Springer Nature B.V. 2019