LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation
Fat mass and obesity-associated protein (FTO) is α-ketoglutarate-dependent dioxygenase and responsible for demethylating N6-methyladenosine (m6A) in mRNA, 3-methylthymine (m3T) in single-stranded DNA (ssDNA) and 3-methyluracil (m3U) in single-stranded RNA (ssRNA). Its other function remains unknown...
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| creator | Zhu, Yuting Zhou, Guangyu Yu, Xuebin Xu, Qiang Wang, Kai Xie, Dan Yang, Qingkai Wang, Lina |
| description | Fat mass and obesity-associated protein (FTO) is α-ketoglutarate-dependent dioxygenase and responsible for demethylating N6-methyladenosine (m6A) in mRNA, 3-methylthymine (m3T) in single-stranded DNA (ssDNA) and 3-methyluracil (m3U) in single-stranded RNA (ssRNA). Its other function remains unknown but thousands of mammalian DNA show 5-methyl-2'-deoxycytidine (5mdC) modification and 5mdC demethylases are required for mammalian energy homeostasis and fertility. Here, we aimed to confirm whether FTO proteins can demethylate 5mdC in DNA. However, we found that FTO exhibits no potent demethylation activity against 5mdC in vitro and in vivo by using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The result showed FTO demethylase has the characteristics of high substrates specificity and selectivity. In addition, we also used immunofluorescence technique to demonstrate overexpression of wild type TET2, but not FTO and mutant TET2 in Hela cells results in higher levels of 5-hydroxymethyl-2'-deoxycytidine (5hmdC) generated from 5mdC. In conclusion, our results not only reveal the enzymatic activity of FTO, but also may facilitate the future discovery of proteins involved in epigenetic modification function. |
| doi_str_mv | 10.1371/journal.pone.0175849 |
| format | Article |
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Its other function remains unknown but thousands of mammalian DNA show 5-methyl-2'-deoxycytidine (5mdC) modification and 5mdC demethylases are required for mammalian energy homeostasis and fertility. Here, we aimed to confirm whether FTO proteins can demethylate 5mdC in DNA. However, we found that FTO exhibits no potent demethylation activity against 5mdC in vitro and in vivo by using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The result showed FTO demethylase has the characteristics of high substrates specificity and selectivity. In addition, we also used immunofluorescence technique to demonstrate overexpression of wild type TET2, but not FTO and mutant TET2 in Hela cells results in higher levels of 5-hydroxymethyl-2'-deoxycytidine (5hmdC) generated from 5mdC. In conclusion, our results not only reveal the enzymatic activity of FTO, but also may facilitate the future discovery of proteins involved in epigenetic modification function.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0175849</identifier><identifier>PMID: 28453518</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Alpha-Ketoglutarate-Dependent Dioxygenase FTO - metabolism ; Biocatalysis ; Biology and life sciences ; Body fat ; Body mass index ; Cancer ; Chromatography ; Chromatography, Liquid ; Demethylation ; Deoxycytidine - analogs & derivatives ; Deoxycytidine - metabolism ; Deoxyribonucleic acid ; Dioxygenase ; DNA ; DNA Methylation ; Energy balance ; Enzymatic activity ; Epigenesis, Genetic ; Epigenetics ; Fertility ; Gene expression ; Genomics ; Health aspects ; HEK293 Cells ; Homeostasis ; Hospitals ; Humans ; Immunofluorescence ; In vivo methods and tests ; Ketoglutaric acid ; Liquid chromatography ; Mammals ; Mass Spectrometry ; Mass spectroscopy ; Metabolism ; Methylation ; mRNA ; N6-methyladenosine ; Obesity ; Oncology ; Oxidation-Reduction ; Physical Sciences ; Proteins ; Quantitative analysis ; Research and Analysis Methods ; Selectivity ; Single-stranded DNA ; Stem cells ; Substrates ; Transfer RNA ; Trends</subject><ispartof>PloS one, 2017-04, Vol.12 (4), p.e0175849-e0175849</ispartof><rights>COPYRIGHT 2017 Public Library of Science</rights><rights>2017 Zhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2017 Zhu et al 2017 Zhu et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-dd235b5e5b1237c0303a02bd23d610d956f5e94030043656566f49da05c839863</citedby><cites>FETCH-LOGICAL-c692t-dd235b5e5b1237c0303a02bd23d610d956f5e94030043656566f49da05c839863</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409144/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409144/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2101,2927,23865,27923,27924,53790,53792,79471,79472</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28453518$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Wu, Qiang</contributor><creatorcontrib>Zhu, Yuting</creatorcontrib><creatorcontrib>Zhou, Guangyu</creatorcontrib><creatorcontrib>Yu, Xuebin</creatorcontrib><creatorcontrib>Xu, Qiang</creatorcontrib><creatorcontrib>Wang, Kai</creatorcontrib><creatorcontrib>Xie, Dan</creatorcontrib><creatorcontrib>Yang, Qingkai</creatorcontrib><creatorcontrib>Wang, Lina</creatorcontrib><title>LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Fat mass and obesity-associated protein (FTO) is α-ketoglutarate-dependent dioxygenase and responsible for demethylating N6-methyladenosine (m6A) in mRNA, 3-methylthymine (m3T) in single-stranded DNA (ssDNA) and 3-methyluracil (m3U) in single-stranded RNA (ssRNA). Its other function remains unknown but thousands of mammalian DNA show 5-methyl-2'-deoxycytidine (5mdC) modification and 5mdC demethylases are required for mammalian energy homeostasis and fertility. Here, we aimed to confirm whether FTO proteins can demethylate 5mdC in DNA. However, we found that FTO exhibits no potent demethylation activity against 5mdC in vitro and in vivo by using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The result showed FTO demethylase has the characteristics of high substrates specificity and selectivity. In addition, we also used immunofluorescence technique to demonstrate overexpression of wild type TET2, but not FTO and mutant TET2 in Hela cells results in higher levels of 5-hydroxymethyl-2'-deoxycytidine (5hmdC) generated from 5mdC. In conclusion, our results not only reveal the enzymatic activity of FTO, but also may facilitate the future discovery of proteins involved in epigenetic modification function.</description><subject>Alpha-Ketoglutarate-Dependent Dioxygenase FTO - metabolism</subject><subject>Biocatalysis</subject><subject>Biology and life sciences</subject><subject>Body fat</subject><subject>Body mass index</subject><subject>Cancer</subject><subject>Chromatography</subject><subject>Chromatography, Liquid</subject><subject>Demethylation</subject><subject>Deoxycytidine - analogs & derivatives</subject><subject>Deoxycytidine - metabolism</subject><subject>Deoxyribonucleic acid</subject><subject>Dioxygenase</subject><subject>DNA</subject><subject>DNA Methylation</subject><subject>Energy balance</subject><subject>Enzymatic activity</subject><subject>Epigenesis, Genetic</subject><subject>Epigenetics</subject><subject>Fertility</subject><subject>Gene expression</subject><subject>Genomics</subject><subject>Health aspects</subject><subject>HEK293 Cells</subject><subject>Homeostasis</subject><subject>Hospitals</subject><subject>Humans</subject><subject>Immunofluorescence</subject><subject>In vivo methods and tests</subject><subject>Ketoglutaric acid</subject><subject>Liquid chromatography</subject><subject>Mammals</subject><subject>Mass Spectrometry</subject><subject>Mass spectroscopy</subject><subject>Metabolism</subject><subject>Methylation</subject><subject>mRNA</subject><subject>N6-methyladenosine</subject><subject>Obesity</subject><subject>Oncology</subject><subject>Oxidation-Reduction</subject><subject>Physical Sciences</subject><subject>Proteins</subject><subject>Quantitative analysis</subject><subject>Research and Analysis Methods</subject><subject>Selectivity</subject><subject>Single-stranded DNA</subject><subject>Stem cells</subject><subject>Substrates</subject><subject>Transfer 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quantitative analysis reveals the association between FTO and DNA methylation</title><author>Zhu, Yuting ; Zhou, Guangyu ; Yu, Xuebin ; Xu, Qiang ; Wang, Kai ; Xie, Dan ; Yang, Qingkai ; Wang, Lina</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-dd235b5e5b1237c0303a02bd23d610d956f5e94030043656566f49da05c839863</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Alpha-Ketoglutarate-Dependent Dioxygenase FTO - metabolism</topic><topic>Biocatalysis</topic><topic>Biology and life sciences</topic><topic>Body fat</topic><topic>Body mass index</topic><topic>Cancer</topic><topic>Chromatography</topic><topic>Chromatography, Liquid</topic><topic>Demethylation</topic><topic>Deoxycytidine - analogs & derivatives</topic><topic>Deoxycytidine - metabolism</topic><topic>Deoxyribonucleic acid</topic><topic>Dioxygenase</topic><topic>DNA</topic><topic>DNA Methylation</topic><topic>Energy balance</topic><topic>Enzymatic activity</topic><topic>Epigenesis, Genetic</topic><topic>Epigenetics</topic><topic>Fertility</topic><topic>Gene expression</topic><topic>Genomics</topic><topic>Health aspects</topic><topic>HEK293 Cells</topic><topic>Homeostasis</topic><topic>Hospitals</topic><topic>Humans</topic><topic>Immunofluorescence</topic><topic>In vivo methods and tests</topic><topic>Ketoglutaric acid</topic><topic>Liquid chromatography</topic><topic>Mammals</topic><topic>Mass Spectrometry</topic><topic>Mass spectroscopy</topic><topic>Metabolism</topic><topic>Methylation</topic><topic>mRNA</topic><topic>N6-methyladenosine</topic><topic>Obesity</topic><topic>Oncology</topic><topic>Oxidation-Reduction</topic><topic>Physical Sciences</topic><topic>Proteins</topic><topic>Quantitative analysis</topic><topic>Research and Analysis Methods</topic><topic>Selectivity</topic><topic>Single-stranded DNA</topic><topic>Stem cells</topic><topic>Substrates</topic><topic>Transfer 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Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Zhu, Yuting</au><au>Zhou, Guangyu</au><au>Yu, Xuebin</au><au>Xu, Qiang</au><au>Wang, Kai</au><au>Xie, Dan</au><au>Yang, Qingkai</au><au>Wang, Lina</au><au>Wu, Qiang</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2017-04-28</date><risdate>2017</risdate><volume>12</volume><issue>4</issue><spage>e0175849</spage><epage>e0175849</epage><pages>e0175849-e0175849</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Fat mass and obesity-associated protein (FTO) is α-ketoglutarate-dependent dioxygenase and responsible for demethylating N6-methyladenosine (m6A) in mRNA, 3-methylthymine (m3T) in single-stranded DNA (ssDNA) and 3-methyluracil (m3U) in single-stranded RNA (ssRNA). Its other function remains unknown but thousands of mammalian DNA show 5-methyl-2'-deoxycytidine (5mdC) modification and 5mdC demethylases are required for mammalian energy homeostasis and fertility. Here, we aimed to confirm whether FTO proteins can demethylate 5mdC in DNA. However, we found that FTO exhibits no potent demethylation activity against 5mdC in vitro and in vivo by using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The result showed FTO demethylase has the characteristics of high substrates specificity and selectivity. In addition, we also used immunofluorescence technique to demonstrate overexpression of wild type TET2, but not FTO and mutant TET2 in Hela cells results in higher levels of 5-hydroxymethyl-2'-deoxycytidine (5hmdC) generated from 5mdC. In conclusion, our results not only reveal the enzymatic activity of FTO, but also may facilitate the future discovery of proteins involved in epigenetic modification function.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>28453518</pmid><doi>10.1371/journal.pone.0175849</doi><tpages>e0175849</tpages><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2017-04, Vol.12 (4), p.e0175849-e0175849 |
| issn | 1932-6203 1932-6203 |
| language | eng |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS); EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Alpha-Ketoglutarate-Dependent Dioxygenase FTO - metabolism Biocatalysis Biology and life sciences Body fat Body mass index Cancer Chromatography Chromatography, Liquid Demethylation Deoxycytidine - analogs & derivatives Deoxycytidine - metabolism Deoxyribonucleic acid Dioxygenase DNA DNA Methylation Energy balance Enzymatic activity Epigenesis, Genetic Epigenetics Fertility Gene expression Genomics Health aspects HEK293 Cells Homeostasis Hospitals Humans Immunofluorescence In vivo methods and tests Ketoglutaric acid Liquid chromatography Mammals Mass Spectrometry Mass spectroscopy Metabolism Methylation mRNA N6-methyladenosine Obesity Oncology Oxidation-Reduction Physical Sciences Proteins Quantitative analysis Research and Analysis Methods Selectivity Single-stranded DNA Stem cells Substrates Transfer RNA Trends |
| title | LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-08T10%3A36%3A54IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=LC-MS-MS%20quantitative%20analysis%20reveals%20the%20association%20between%20FTO%20and%20DNA%20methylation&rft.jtitle=PloS%20one&rft.au=Zhu,%20Yuting&rft.date=2017-04-28&rft.volume=12&rft.issue=4&rft.spage=e0175849&rft.epage=e0175849&rft.pages=e0175849-e0175849&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0175849&rft_dat=%3Cgale_plos_%3EA493765085%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1892971841&rft_id=info:pmid/28453518&rft_galeid=A493765085&rft_doaj_id=oai_doaj_org_article_4c36fbb7fdb244ca9823ae1e132adaf3&rfr_iscdi=true |