抑制LSD1对hiPSCs向定型内胚层分化的调控作用
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摘要
目的:探讨抑制组蛋白赖氨酸特异性去甲基化酶1(LSD1)对人诱导性多能干细胞(hiPSCs)向定型内胚层(DE)分化的调控作用。方法:利用LSD1抑制剂或shRNA抑制LSD1表达,观察hiPSCs形态变化并检测LSD1活性水平,CCK-8方法检测细胞增殖活性,qPCR检测hiPSCs多能性基因及各胚层标志基因的表达,IP-WB方法检测LSD1调控靶基因的复合体模式,Ch IP-qPCR方法检测DE标志基因启动子区域组蛋白H3第4位赖氨酸二甲基化和三甲基化(H3K4me2/me3)及第9位赖氨酸乙酰化(H3K9ac)水平。结果:(1)抑制LSD1可显著下调hiPSCs多能性基因OCT4、Y染色体性别决定区域盒2(SOX2)及Nanog同源盒(NANOG)的表达水平(P <0. 05);显著上调外胚层标志基因β3-微管蛋白(TUBB3),DE标志基因Y染色体性别决定区域盒17(SOX17)和叉头盒A2(FOXA2),以及中胚层标志基因骨形态发生蛋白2(BMP2)的表达水平(P <0. 05);(2)当LSD1活性为正常水平的53. 4%时利于DE分化;(3) LSD1在hiPSCs核内与组蛋白去乙酰化酶1(HDAC1)及阻遏物元件1沉默转录因子辅阻遏物(CoREST)以复合体的形式调控靶基因;(4)抑制LSD1后,SOX17和FOXA2基因启动子区域LSD1与HDAC1结合水平均显著下降,同时H3K4me2/me3和H3K9ac富集水平显著提高(P <0. 01)。结论:LSD1通过调控DE分化关键基因启动子区域H3K4me和H3K9ac水平来影响hiPSCs向DE的分化能力。
AIM: To investigate the regulation of lysine specific demethylase 1( LSD1) inhibition on differentiation of human induced pluripotent stem cells( hiPSCs) into definitive endoderm( DE). METHODS: LSD1 expression was inhibited by LSD1 inhibitor or shRNA. The morphological changes of hiPSCs were traced by microscopy,and LSD1 activity was measured by corresponding kit. The cell viability was detected by CCK-8 assay. The expression of hiPSCs pluripotency genes and germ layer specific genes was analyzed by qPCR,and the complex pattern of LSD1 targeting genes was determined by immunoprecipitation of protein( IP-WB). Moreover,Ch IP-qPCR was used to evaluate the levels of H3K4 me2/me3 and H3K9 ac located within the promoter region of DE marker genes. RESULTS: Inhibition of LSD1 decreased the expression of hiPSCs pluripotency genes OCT4,sex determining region Y-box 2( SOX2),Nanog homeobox( NANOG)( P <0. 05),but up-regulated the levels of germ layer specific genes tubulin beta 3 class III( TUBB3),sex determining region Y-box 17( SOX17),forkhead box A2( FOXA2) and bone morphogenetic protein 2( BMP2)( P < 0. 05).The process of hiPSCs differentiating into DE was promoted when LSD1 activity reached 53. 4% of normal level. LSD1,together with histone deacetylase 1( HDAC1) and co-repressor for repressor element-1-silencing transcription factor( CoREST),regulated the target genes in the form of a complex in the nucleus of the hiPSCs. When the activity of LSD1 reduced,the binding of LSD1 and HDAC1 within the promoter regions of SOX17 and FOXA2 was limited,and enrichment levels of H3K4 me2/me3 and H3K9 ac were observed( P < 0. 01). CONCLUSION: LSD1 affects the differentiation ability of hiPSCs into DE by regulating the H3K4 methylation and H3K9 acetylation in the promoter region of key lineage specification genes for DE differentiation.
AIM: To investigate the regulation of lysine specific demethylase 1( LSD1) inhibition on differentiation of human induced pluripotent stem cells( hiPSCs) into definitive endoderm( DE). METHODS: LSD1 expression was inhibited by LSD1 inhibitor or shRNA. The morphological changes of hiPSCs were traced by microscopy,and LSD1 activity was measured by corresponding kit. The cell viability was detected by CCK-8 assay. The expression of hiPSCs pluripotency genes and germ layer specific genes was analyzed by qPCR,and the complex pattern of LSD1 targeting genes was determined by immunoprecipitation of protein( IP-WB). Moreover,Ch IP-qPCR was used to evaluate the levels of H3K4 me2/me3 and H3K9 ac located within the promoter region of DE marker genes. RESULTS: Inhibition of LSD1 decreased the expression of hiPSCs pluripotency genes OCT4,sex determining region Y-box 2( SOX2),Nanog homeobox( NANOG)( P <0. 05),but up-regulated the levels of germ layer specific genes tubulin beta 3 class III( TUBB3),sex determining region Y-box 17( SOX17),forkhead box A2( FOXA2) and bone morphogenetic protein 2( BMP2)( P < 0. 05).The process of hiPSCs differentiating into DE was promoted when LSD1 activity reached 53. 4% of normal level. LSD1,together with histone deacetylase 1( HDAC1) and co-repressor for repressor element-1-silencing transcription factor( CoREST),regulated the target genes in the form of a complex in the nucleus of the hiPSCs. When the activity of LSD1 reduced,the binding of LSD1 and HDAC1 within the promoter regions of SOX17 and FOXA2 was limited,and enrichment levels of H3K4 me2/me3 and H3K9 ac were observed( P < 0. 01). CONCLUSION: LSD1 affects the differentiation ability of hiPSCs into DE by regulating the H3K4 methylation and H3K9 acetylation in the promoter region of key lineage specification genes for DE differentiation.
引文
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[11]Wang Z,Oron E,Nelson B,et al.Distinct lineage specification roles for NANOG,OCT4,and SOX2 in human embryonic stem cells[J].Cell Stem Cell,2012,10(4):440-454.
[12]Thomson M,Liu SJ,Zou IN,et al.Pluripotency factors in embryonic stem cells regulate differentiation into germ layers[J].Cell,2011,145(6):875-889.
[13]Santos Rosa H,Schneider R,Bannister AJ,et al.Active genes are tri-methylated at K4 of histone H3[J].Nature,2002,419(6905):407-411.
[14]Wysocka J,Swigut T,Xiao H,et al.APHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodeling[J].Nature,2006,442(7098):86-90.
[15]Sonnemann J,Zimmermann M,Marx C,et al.LSD1(KDM1A)-independent effects of the LSD1 inhibitor SP2509 in cancer cells[J].Br J Haematol,2017,4(2):12-20.
[16]Cusan M,Cai SF,Mohammad HP,et al.LSD1 inhibition exerts its anti-leukemic effect by recommissioning PU1-and C/EBPα-dependent enhancers in AML[J].Blood,2018,16(3):123-133.
[17]Yang Y,Huang W,Qiu R,et al.LSD1 coordinates with the SIN3A/HDAC complex and maintains sensitivity to chemotherapy in breast cancer[J].Mol Cell Biol,2018,15(1):23-30.
[18]Tosic M,Allen A,Willmann D,et al.Lsd1 regulates skeletal muscle regeneration and directs the fate of satellite cells[J].Nat Commun,2018,9(1):36-42.
[19]Rudolph T,Beuch S,Reuter G.Lysine-specifice histone demethylase LSD1 and the dynamic control of chromation[J].Biol Chem,2013,394(8):1019-1028.
[20]Nair VD,Ge Y,Balasubramaniyan N,et al.Embryonic stem cells changes during cell cycle progression in shorttime-scale gene expression involvement of histone demethylase LSD1[J].Mol Cell Biol,2012,32(23):4861-4876.
[2]Yan HJ,Zhou SY,Li Y,et al.The effects of LSD1 inhibition on self-renewal and differentiation of human induced pluripotent stem cells[J].Exp Cell Res,2016,340(2):227-237.
[3]Sun GQ,Alzayady K,Stewart R,et al.Histone demethylase LSD1 regulates neural stem cell proliferation[J].Mol Cell Biol,2010,30(8):1997-2005.
[4]Hirano K,Namihira M.FAD influx enhances neuronal differentiation of human neural stem cells by facilitating nuclear localization of LSD1[J].FEBS Open Bio,2017,7(12):1932-1942.
[5]Adamo A,SeséB,Boue S,et al.LSD1 regulates the balance between self-renewal and differentiation in human embryonic stem cells[J].Nat Cell Biol,2011,13(6):652-659.
[6]Zhang XD,Han X,Chew JL,et al.Whole-genome mapping of histone H3 Lys4 and 27 trimethylations reveals distinct genomic compartments in human embryonic stem cells[J].Cell Stem Cell,2007,1(3):286-298.
[7]钟娃,夏忠胜,欧阳慧,等.激活Nodal信号通路促进小鼠胚胎干细胞向定型内胚层分化[J].中国病理生理杂志,2015,31(11):2070-2075.
[8]Yamanaka S.Induced pluripotent stem cells:past,present and future[J].Cell Stem Cell,2012,10(6):678-684.
[9]Shao GB,Ding HM,Gong AH,et al.Role of histone methylation in zygotic genome activation in the preimplantation mouse embryo[J].In Vitro Cell Dev Biol Anim,2008,44(3):115-120.
[10]Whyte WA,Bilodeau S,Orlando DA,et al.Enhancer decommissioning by LSD1 during embryonic stem cell differentiation[J].Nature,2012,482(7384):221-225.
[11]Wang Z,Oron E,Nelson B,et al.Distinct lineage specification roles for NANOG,OCT4,and SOX2 in human embryonic stem cells[J].Cell Stem Cell,2012,10(4):440-454.
[12]Thomson M,Liu SJ,Zou IN,et al.Pluripotency factors in embryonic stem cells regulate differentiation into germ layers[J].Cell,2011,145(6):875-889.
[13]Santos Rosa H,Schneider R,Bannister AJ,et al.Active genes are tri-methylated at K4 of histone H3[J].Nature,2002,419(6905):407-411.
[14]Wysocka J,Swigut T,Xiao H,et al.APHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodeling[J].Nature,2006,442(7098):86-90.
[15]Sonnemann J,Zimmermann M,Marx C,et al.LSD1(KDM1A)-independent effects of the LSD1 inhibitor SP2509 in cancer cells[J].Br J Haematol,2017,4(2):12-20.
[16]Cusan M,Cai SF,Mohammad HP,et al.LSD1 inhibition exerts its anti-leukemic effect by recommissioning PU1-and C/EBPα-dependent enhancers in AML[J].Blood,2018,16(3):123-133.
[17]Yang Y,Huang W,Qiu R,et al.LSD1 coordinates with the SIN3A/HDAC complex and maintains sensitivity to chemotherapy in breast cancer[J].Mol Cell Biol,2018,15(1):23-30.
[18]Tosic M,Allen A,Willmann D,et al.Lsd1 regulates skeletal muscle regeneration and directs the fate of satellite cells[J].Nat Commun,2018,9(1):36-42.
[19]Rudolph T,Beuch S,Reuter G.Lysine-specifice histone demethylase LSD1 and the dynamic control of chromation[J].Biol Chem,2013,394(8):1019-1028.
[20]Nair VD,Ge Y,Balasubramaniyan N,et al.Embryonic stem cells changes during cell cycle progression in shorttime-scale gene expression involvement of histone demethylase LSD1[J].Mol Cell Biol,2012,32(23):4861-4876.
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