Tet2调节骨髓间充质干细胞功能
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摘要
Tet2(Tet家族成员2)在DNA去甲基化修饰、表观遗传调控及骨髓造血中起着重要作用。笔者课题组前期研究发现,随着年龄增长,Tet2敲除小鼠逐步发展为淋系白血病和髓系白血病。但Tet2在骨髓微环境中的作用仍不清楚。进一步研究发现,Tet2敲除的骨髓间充质干细胞(Mesenchymal stem cells,MSC)更多处于G2/M分裂期,其细胞分裂时间缩短,生长速度加快。长周期培养-起始细胞实验表明,Tet2敲除的MSC支持造血干细胞扩增和髓系分化的能力增强。通过点杂交实验发现,Tet2敲除后,骨髓细胞DNA总甲基化水平升高。对Tet2缺失的骨髓细胞进行甲基化测序,结果表明:基因组转录调控区域等多个功能性结构域的甲基化水平明显升高。同时,敲除Tet2的MSC分泌IL-8、IL-18等炎性细胞因子的能力下降;敲除Tet2的MSC更多分泌促进造血干细胞髓系分化的GM-CSF和CCL-3等细胞因子。Tet2可以影响间充质干细胞造血支持作用,进而调节造血。
Tet2(member 2 of the Tet family) plays an important role in DNA demethylation modification,epigenetic regulation,and hematopoiesis.In our previous study,we found that Tet2 knockout mice progressively developed lymphocytic leukemia and myeloid leukemia with aging.However,the role of Tet2 in bone marrow microenvironment is unclear.Here in this study,we found that more Tet2~(-/-)mesenchymal stem cells(MSCs) from bone marrow were in the G2/M cell cycle stages.The division time of Tet2~(-/-)MSCs was shorter than that of the control cells.The growth rate of Tet2~(-/-)MSCs was accelerated.The cobblestone area-forming cells assay(CAFC) showed that Tet2 knockout MSCs supported the expansion of hematopoieticstem cells(HSCs) and the differentiation of HSCs was skewed towards myeloid cells.Through the dot blotting experiment,we found that the total methylation level was increased in Tet2~(-/-)bone marrow cells(BM).We used the methylation-chip to analyze the methylation level of Tet2~(-/-)bone marrow cells and found that the level of methylation was increased in the transcriptional starting area(TSS),exons(EXONS) and 3untranslated region(3UTR).Moreover,we found that the cytokines secreted by Tet2~(-/-)MSCs,such as IL-8 and IL-18,were decreased.While the expressions of GM-CSF and CCL-3,which supported hematopoietic stem cells to differentiate to myeloid cells,were increased in Tet2~(-/-)MSCs.Our results demonstrated that Tet2 regulates MSCs to support hematopoiesis.
Tet2(member 2 of the Tet family) plays an important role in DNA demethylation modification,epigenetic regulation,and hematopoiesis.In our previous study,we found that Tet2 knockout mice progressively developed lymphocytic leukemia and myeloid leukemia with aging.However,the role of Tet2 in bone marrow microenvironment is unclear.Here in this study,we found that more Tet2~(-/-)mesenchymal stem cells(MSCs) from bone marrow were in the G2/M cell cycle stages.The division time of Tet2~(-/-)MSCs was shorter than that of the control cells.The growth rate of Tet2~(-/-)MSCs was accelerated.The cobblestone area-forming cells assay(CAFC) showed that Tet2 knockout MSCs supported the expansion of hematopoieticstem cells(HSCs) and the differentiation of HSCs was skewed towards myeloid cells.Through the dot blotting experiment,we found that the total methylation level was increased in Tet2~(-/-)bone marrow cells(BM).We used the methylation-chip to analyze the methylation level of Tet2~(-/-)bone marrow cells and found that the level of methylation was increased in the transcriptional starting area(TSS),exons(EXONS) and 3untranslated region(3UTR).Moreover,we found that the cytokines secreted by Tet2~(-/-)MSCs,such as IL-8 and IL-18,were decreased.While the expressions of GM-CSF and CCL-3,which supported hematopoietic stem cells to differentiate to myeloid cells,were increased in Tet2~(-/-)MSCs.Our results demonstrated that Tet2 regulates MSCs to support hematopoiesis.
引文
[1]Ziller MJ,Gu HC,Müller F,et al.Charting a dynamic DNA methylation landscape of the human genome.Nature,2013,500(7463):477-481.
[2]Robertson KD.DNA methylation and human disease.Nat Rev Genet,2005,6(8):597-610.
[3]Tahiliani M,Koh KP,Shen YH,et al.Conversion of5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science,2009,324(5929):930-935.
[4]Ito S,D’Alessio AC,Taranova OV,et al.Role of Tet proteins in 5mC to 5hmC conversion,ES-cell self-renewal and inner cell mass specification.Nature,2010,466(7310):1129-1133.
[5]Guo JU,Su YJ,Zhong C,et al.Hydroxylation of5-methylcytosine by TET1 promotes active DNAdemethylation in the adult brain.Cell,2011,145(3):423-434.
[6]Jankowska AM,Szpurka H,Tiu RV,et al.Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms.Blood,2009,113(25):6403-6410.
[7]Ko M,Huang Y,Jankowska AM,et al.Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.Nature,2010,468(7325):839-843.
[8]Delhommeau F,Dupont S,Della Valle V,et al.Mutation in TET2 in myeloid cancers.N Engl J Med,2009,360(22):2289-2301.
[9]Fouse SD,Shen Y,Pellegrini M,et al.Promoter CpGmethylation contributes to ES cell gene regulation in parallel with Oct4/Nanog,PcG complex,and histone H3 K4/K27 trimethylation.Cell Stem Cell,2008,2(2):160-169.
[10]Itzykson R,Kosmider O,Renneville A,et al.Clonal architecture of chronic myelomonocytic leukemias.Blood,2013,121(12):2186-2198.
[11]Smith AE,Mohamedali AM,Kulasekararaj A,et al.Next-generation sequencing of the TET2 gene in 355MDS and CMML patients reveals low-abundance mutant clones with early origins,but indicates no definite prognostic value.Blood,2010,116(19):3923-3932.
[12]Song ZQ,Zhang QS.Design,synthesis,and incorporation of fluorous 5-methylcytosines into oligonucleotides.J Org Chem,2011,76(24):10263-10268.
[13]Song ZJ,Zhou RB,Li D,et al.Detection of p16INK4a promoter methylation status in non-small cell lung cancer by a fluorescence polarization assay.Diagn Mol Pathol,2011,20(3):138-142.
[14]Szulwach KE,Li XK,Li YJ,et al.5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging.Nat Neurosci,2011,14(12):1607-1616.
[15]Szulwach KE,Li XK,Li YJ,et al.Integrating5-hydroxymethylcytosine into the epigenomic landscape of human embryonic stem cells.PLoSGenet,2011,7(6):e1002154.
[16]Yao LS,Li YY,Du FX,et al.Histone H4 Lys 20methyltransferase SET8 promotes androgen receptor-mediated transcription activation in prostate cancer.Biochem Biophys Res Commun,2014,450(1):692-696.
[17]Yao B,Lin L,Street RC,et al.Genome-wide alteration of 5-hydroxymethylcytosine in a mouse model of fragile X-associated tremor/ataxia syndrome.Hum Mol Genet,2014,23(4):1095-1107.
[18]Mendelson A,Frenette PS.Hematopoietic stem cell niche maintenance during homeostasis and regeneration.Nat Med,2014,20(8):833-846.
[19]Krause DS,Scadden DT,Preffer FI.The hematopoietic stem cell niche-home for friend and foe?Cytometry B Clin Cytom,2013,84(1):7-20.
[20]Pan F,Wingo TS,Zhao ZG,et al.Tet2 loss leads to hypermutagenicity in haematopoietic stem/progenitor cells.Nat Commun,2017,8:15102.
[21]Li Z,Cai XQ,Cai CL,et al.Deletion of Tet2 in mice leads to dysregulated hematopoietic stem cells and subsequent development of myeloid malignancies.Blood,2011,118(17):4509-4518.
[22]Zhao ZQ,Chen L,Dawlaty MM,et al.Combined loss of Tet1 and Tet2 promotes B cell,but not myeloid malignancies,in mice.Cell Rep,2015,13(8):1692-1704.
[23]Zhao Z,Chen S,Zhu X,et al.The catalytic activity of TET2 is essential for its myeloid malignancysuppressive function in hematopoietic stem/progenitor cells.Leukemia,2016,30(8):1784-1788.
[24]Zhang P,Xing CH,Rhodes SD,et al.Loss of Asxl1alters self-renewal and cell fate of bone marrow stromal cells,leading to bohring-opitz-like syndrome in mice.Stem Cell Rep,2016,6(6):914-925.
[25]Zhang Q,Zhao K,Shen QC,et al.Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically repress IL-6.Nature,2015,525(7569):389-393.
[26]Greenbaum A,Hsu YMS,Day RB,et al.CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance.Nature,2013,495(7440):227-230.
[2]Robertson KD.DNA methylation and human disease.Nat Rev Genet,2005,6(8):597-610.
[3]Tahiliani M,Koh KP,Shen YH,et al.Conversion of5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science,2009,324(5929):930-935.
[4]Ito S,D’Alessio AC,Taranova OV,et al.Role of Tet proteins in 5mC to 5hmC conversion,ES-cell self-renewal and inner cell mass specification.Nature,2010,466(7310):1129-1133.
[5]Guo JU,Su YJ,Zhong C,et al.Hydroxylation of5-methylcytosine by TET1 promotes active DNAdemethylation in the adult brain.Cell,2011,145(3):423-434.
[6]Jankowska AM,Szpurka H,Tiu RV,et al.Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms.Blood,2009,113(25):6403-6410.
[7]Ko M,Huang Y,Jankowska AM,et al.Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.Nature,2010,468(7325):839-843.
[8]Delhommeau F,Dupont S,Della Valle V,et al.Mutation in TET2 in myeloid cancers.N Engl J Med,2009,360(22):2289-2301.
[9]Fouse SD,Shen Y,Pellegrini M,et al.Promoter CpGmethylation contributes to ES cell gene regulation in parallel with Oct4/Nanog,PcG complex,and histone H3 K4/K27 trimethylation.Cell Stem Cell,2008,2(2):160-169.
[10]Itzykson R,Kosmider O,Renneville A,et al.Clonal architecture of chronic myelomonocytic leukemias.Blood,2013,121(12):2186-2198.
[11]Smith AE,Mohamedali AM,Kulasekararaj A,et al.Next-generation sequencing of the TET2 gene in 355MDS and CMML patients reveals low-abundance mutant clones with early origins,but indicates no definite prognostic value.Blood,2010,116(19):3923-3932.
[12]Song ZQ,Zhang QS.Design,synthesis,and incorporation of fluorous 5-methylcytosines into oligonucleotides.J Org Chem,2011,76(24):10263-10268.
[13]Song ZJ,Zhou RB,Li D,et al.Detection of p16INK4a promoter methylation status in non-small cell lung cancer by a fluorescence polarization assay.Diagn Mol Pathol,2011,20(3):138-142.
[14]Szulwach KE,Li XK,Li YJ,et al.5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging.Nat Neurosci,2011,14(12):1607-1616.
[15]Szulwach KE,Li XK,Li YJ,et al.Integrating5-hydroxymethylcytosine into the epigenomic landscape of human embryonic stem cells.PLoSGenet,2011,7(6):e1002154.
[16]Yao LS,Li YY,Du FX,et al.Histone H4 Lys 20methyltransferase SET8 promotes androgen receptor-mediated transcription activation in prostate cancer.Biochem Biophys Res Commun,2014,450(1):692-696.
[17]Yao B,Lin L,Street RC,et al.Genome-wide alteration of 5-hydroxymethylcytosine in a mouse model of fragile X-associated tremor/ataxia syndrome.Hum Mol Genet,2014,23(4):1095-1107.
[18]Mendelson A,Frenette PS.Hematopoietic stem cell niche maintenance during homeostasis and regeneration.Nat Med,2014,20(8):833-846.
[19]Krause DS,Scadden DT,Preffer FI.The hematopoietic stem cell niche-home for friend and foe?Cytometry B Clin Cytom,2013,84(1):7-20.
[20]Pan F,Wingo TS,Zhao ZG,et al.Tet2 loss leads to hypermutagenicity in haematopoietic stem/progenitor cells.Nat Commun,2017,8:15102.
[21]Li Z,Cai XQ,Cai CL,et al.Deletion of Tet2 in mice leads to dysregulated hematopoietic stem cells and subsequent development of myeloid malignancies.Blood,2011,118(17):4509-4518.
[22]Zhao ZQ,Chen L,Dawlaty MM,et al.Combined loss of Tet1 and Tet2 promotes B cell,but not myeloid malignancies,in mice.Cell Rep,2015,13(8):1692-1704.
[23]Zhao Z,Chen S,Zhu X,et al.The catalytic activity of TET2 is essential for its myeloid malignancysuppressive function in hematopoietic stem/progenitor cells.Leukemia,2016,30(8):1784-1788.
[24]Zhang P,Xing CH,Rhodes SD,et al.Loss of Asxl1alters self-renewal and cell fate of bone marrow stromal cells,leading to bohring-opitz-like syndrome in mice.Stem Cell Rep,2016,6(6):914-925.
[25]Zhang Q,Zhao K,Shen QC,et al.Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically repress IL-6.Nature,2015,525(7569):389-393.
[26]Greenbaum A,Hsu YMS,Day RB,et al.CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance.Nature,2013,495(7440):227-230.
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