8_氯腺苷诱导的组蛋白H3K79双甲基化促进NBS1和p21的基因转录激活

ISSN 1007 7626CN 11 3870 Q

中国生物化学与分子生物学报

Chinese Journal of Biochemistry and Molecular Biology

2009年11月

25(11):1035~1040

收稿日期:2009 08 27;接受日期:2009 09 14

国家自然科学基金委项目(No.30471975,30671062)及北京市教委人才强教计划项目资助

*

联系人 Tel:010 ********;E mail:jiahongti@bj https://www.360docs.net/doc/ed3978033.html,

Received:Augus t 27,2009;Accepted:Septe mber 24,2009

Supported by National Natural Science Foundation of China (No.30471975,No.30671062)and Education Co mmittee of Beijing Teac hing Reinforcing Plan.

*

Corresponding author Tel:010 ********;E mail:jiahongti@bj https://www.360docs.net/doc/ed3978033.html,

8 氯腺苷诱导的组蛋白H3K79双甲基化促进NBS1和p21的基因转录激活

刘 畅1), 李文娟1), 陈 瑜2), 丁 卫1), 安国顺3)

,

李淑艳3), 倪菊华3), 贾弘

1),3)*

(1)首都医科大学基础医学院生物化学与分子生物学系,北京 100069;

2)福建医科大学生物化学与分子生物学系,福州 350004;3)

北京大学医学部生物化学与分子生物学系,北京 100191)

摘要 组蛋白H3第79位赖氨酸甲基化(H3K79me)修饰有单甲基、双甲基及三甲基3种形式,是常染色质的标志.然而,对于组蛋白H3K79三种甲基化各自在基因转录、DNA 损伤修复中所起的作用尚不十分清楚.本研究以8 氯腺苷(8 Cl Ado )为DNA 双链断裂(DNA double stranded breaks,DSB)诱导剂,采用Western 印迹,在人肺癌细胞H1299检测出了DNA 修复分子NBS1、细胞周期检验点相关分子p21,并发现H3K79me1、H3K79me2和H3K79me3三种甲基化修饰的组蛋白明显增加;染色质免疫共沉淀结合实时定量PCR 实验显示,只有H3K79me2与DNA 损伤检验点分子p21、DNA 修复分子NBS1的启动子区域相结合,说明H3K79双甲基化修饰与这些基因的转录激活有关.结果提示,在8 氯腺苷引起DSB 时,是H3K79me2、而不是H3K79me1和H3K79me3参与NBS1和p21基因转录激活时的染色质重塑.8 氯腺苷诱导H3K79双甲基化增强、促进H3K79me2所在染色质区域的NB S1和p21基因转录激活可能是8 Cl Ado 抑制肿瘤细胞生长作用机制之一.关键词 8 氯腺苷;DNA 双链断裂;H3K79甲基化;NBS1;p21;表观遗传调控中图分类号 Q78;Q26

8 Chloro adenosine induced H3K79me2Promotes Transcriptional

Activation of NBS1and p21Genes in Response to DNA

Double stranded Breaks

LIU C hang 1),LI Wen Juan 1),C HEN Yu 2),DING Wei 1),AN Guo Shun 3)

,

LI Shu Yan 3),NI Ju Hua 3),JI A Hong Ti

1),3)*

(1)De partment o f Biochemist ry and Mole cular Biology ,Capital Me dical U ni versity ,Be ijing 100069,China ;2)

De partment o f Bioche mistry and Molec ular Biolo gy ,Fujian Me dical U nive rsity ,Fuzhou 350004,China ;

3)

De partme nt o f Bioc he mistry and Molec ular Biology ,Peking U nive rsity Health Science Cente r ,Be ijing 100191,China )

Abstract Methylation of H3K79,including mono,di and trimethyled H3K79(H3K79me1,H3K79me2and H3K79me3),is one of the landmark modifications associated with euchromatin.However,the roles of methylated H3K79in gene regulation,DNA damage and repair are unclear. In this study,we showed that the levels of NB S1(a DNA damage response protein),p21(a DNA dama ge checkpoint protein),and three methylated H3K79were markedly up regulated in 8 Cl Ado exposed H1299cells,evidenced by Western

blotting.The c ombination of chromatin immunoprecipita tion(ChIP)with real time quantitative PC R(RT qPC R)revealed that only H3K79me2was associated with the NB S1and p21promoters,indicating the contribution of H3K79me2,to transcriptional activa tion of these genes.These results suggest that H3K79me2, but not H3K79me1and H3K79me3,is involved in the chromatin remodeling associated with the transcription activation of the NBS1and p21genes.Induction of H3K79me2by8 Cl Ado in the chromatin surrounding the NBS1and p21genes may be a way by that8 Cl Ado inhibits tumor cell proliferation.

Key words 8 Cl Ado;DNA double stranded breaks;H3K79me;NBS1;p21;epigenetic control

几种组蛋白N端结构域的各种共价修饰(甲基化、乙酰化、磷酸化、泛素化等)共同构成了 组蛋白密码(!histone code?)[1],在基因转录调控、DNA复制、DNA修复和染色体分离中发挥重要作用.过去一直认为,与其它化学修饰不同,甲基化和乙酰化修饰被认为是一类 持续或 不变的修饰;但近来认为,这类修饰是可逆的、动力学调节的[2].组蛋白甲基化多发生在组蛋白H3、H4的赖氨酸(K)和精氨酸(R)残基上[3].组蛋白H3中有5个赖氨酸(K4、K9、K27、K36、K79)残基,组蛋白H4中有1个赖氨酸残基(K20)可被特异的组蛋白赖氨酸甲基转移酶甲基化.不同位点的甲基化对基因转录激活的效应不同.一般认为,H3K9、H3K27、H4K20甲基化具有阻遏效应,H3K4、H3K36、H3K79甲基化具有激活效应[4].甲基化H3K79有H3K79me1、H3K79me2和H3K79me3三种形式.目前,H3K79me1研究较多,偶见H3K79me2、H3K79me3检测,至于H3K79me2 3的功能尚未见报告.在真核细胞中,组蛋白H3K79甲基化(H3K79me)可调节发育期的基因表达[5],并在常染色质(euchromatin)活化、重塑及相应转录区的基因激活中发挥作用[6].然而,近年也有研究显示其与基因阻遏相关.

此外,最近研究发现,H3K79me1和H3K79me2可与DNA修复蛋白53BP1的Tudor结构域结合,使53BP1定位在DNA损害部位而发挥修复作用[7]. DNA双链断裂(DNA double stranded breaks,DSB)发生可改变高度有序的染色质结构, 松弛局部高度压缩的核小体排列,从而使甲基化的H3K79暴露,变得有利于修复分子如NB S1,53B P1等的接近[7].在酵母中也存在着类似的机制来激活DNA损伤检验点[8,9].

8 氯腺苷(8 chloro adenosine,8 Cl Ado)[10~13]是一种腺苷衍生物,对多种肿瘤和肿瘤细胞系具有生长抑制的作用.我们以往的研究[14,15]证明,8 Cl Ado 可诱导细胞周期阻滞、有丝分裂灾难和凋亡,抑制肿瘤细胞生长;最近我们发现,8 Cl Ado可抑制#型拓扑异构酶(Topo#)活性,引起DNA双链断裂[16,17].DNA损伤激活ATM C HK1 CDC25C CDC2及BRC A1 C HK1信号,引起G2 M阻滞[17];同时,8 Cl Ado刺激了转录因子E2F1上调,后者激活p14ARF[18], p14ARF引起细胞晚期凋亡[19].此外,我们还发现其它一些DNA损伤反应性蛋白表达上调(未发表资料),这说明靶细胞启动了DNA损伤修复机制.无论是基因转录激活还是DNA损伤修复,必然涉及染色质重塑(chromatin remodeling).其中,组蛋白H3K79甲基化修饰具有重要意义[20,21].本研究以人肺癌H1299细胞系为对象,初步探讨了8 Cl Ado处理对组蛋白H3第79位赖氨酸甲基化状态的影响及其生物学意义.

1 材料与方法

1.1 细胞培养及药物处理

人肺癌细胞H1299购自ATCC(American type culture collection).细胞采用含10%小牛血清(Hyclone公司)的RPMI1640培养基(Gibco公司)加100I U ml青霉素、100 g ml链霉素,在CO2孵箱(37?)中孵育.8 Cl Ado由北京大学药学院天然药物及仿生药物国家重点实验室张礼和院士馈赠,用时用生理盐水溶解.8 Cl Ado终浓度10 mol L.细胞于接种后24h用8 Cl Ado处理.

1.2 抗体

抗组蛋白H3单甲基、三甲基化抗体及抗NB S1、p21抗体均购自美国Abcam公司;抗组蛋白H3双甲基化及抗 H2AX抗体购自美国Cell Signaling Technology公司;抗肌动蛋白多克隆抗体购自美国Santa Cruz生物技术公司.

1.3 蛋白质免疫印迹实验

H1299细胞经8 Cl Ado(10 mol L)分别处理0、12、24、48h后,用B CA蛋白定量试剂盒测定细胞上清裂解液中蛋白含量.取等量蛋白(50 g),经10% ~15%SDS PAGE分离后,用湿转法将分离胶上蛋白电转至硝酸纤维素膜上(恒流180m A,4?7h).用含5%脱脂奶粉的TBST(20m mol L Tris HCl,pH 7 6,135mmol L NaCl,011%Tween10%)封闭膜1

1036中国生物化学与分子生物学报25卷

h,用一抗(1%1000)4?孵育过夜后,含5%脱脂奶粉的TBST漂洗3次,每次10min;二抗室温孵育1h后,TB ST漂洗3次,每次10min.最后经ECL试剂显色成像.

1.4 染色质免疫共沉淀(chromatin immunopreci pitation,C hIP)

8 C L Ado以10 mol L浓度处理细胞48h;对照组以0 85%生理盐水处理.甲醛处理10min.收细胞加SDS裂解缓冲液(1%SDS、10mmol L EDTA、50 mmol L Tris HCl)(加入蛋白酶抑制剂cocktail)冰上10min.超声25s,4?,pulse on:5s,pulse off:5s,强度(ampl):20%,至样品清亮.13000r min,10 min,4?,收上清.加Chip稀释缓冲液(0 01%SDS、1 1%Triton X100、1 2mol L EDTA、16.7m mol L Tris HCl、16 7mmol L NaCl)稀释10倍(加蛋白酶抑制剂cocktail)各取1%作为阳性对照(input).加入鲑精DNA protein A agarose 50%slurry,4?,2h.12000 r min,3min,4?.分别取H3K79me1、H3K79me2、H3K79me3抗体1%100加入蛋白中,4?过夜.对照组加同等鲑精DNA protein A agarose 50%slurry.加鲑精DNA protein A agarose 50%slurry,4?,旋转8 h.弃上清.洗脱液洗脱弃上清.新鲜配制洗脱缓冲液250 l加入样品混匀,旋转室温15min,1000r min, 1min,收上清.加20 l,5mol L NaCl,65?,4h,解交联(包括阳性对照).提纯DNA:用Wiard Pcr preps DNA purification试剂盒(Promega公司).常规或实时定量PC R引物:p21:(sense)5& CACC ACTGAGCC TT CC TCAC 3&,(antisense)5& CTGACTCCC AGCACACAC TC 3&(449bp);NBS1:(sense)5& ATTGGC AAAGAT CTATGTAGAG 3&,(antisense)5& TGCCCTACCAGAT GGCAAAAT 3&(440bp).

2 结果

2.1 8 氯腺苷处理引起靶细胞DNA损伤???双链断裂

当细胞暴露于8 氯 腺苷(8 Cl Ado)时,组蛋白H2A的异构体H2AX第139位丝氨酸会发生磷酸化,即 H2AX. H2AX出现在DNA断裂点,提示DSBs的发生,用以招募细胞周期检验点分子和损伤修复因子.采用 H2AX特异性抗体进行的Western 印迹显示,8 Cl Ado处理的肺癌细胞H1299中的 H2AX蛋白水平随8 Cl Ado处理时间延长(6 48h)而增高.结果提示,8 Cl Ado可引起DNA损伤,发生

DSBs(Fig.1).

Fig.1 8 C l Ado up regulates the levels of H2AX

H1299cells were exposed to8 Cl Ado at the concentration of10

mol L for0,1,3,6,12,24,48hours,respectively.The levels of H2AX were detected by Western blotting. Actin was used as a loading control.Con,unexposed(0hour)

Fig.2 8 Cl Ado up regulates the levels of methylated

H3K79 H1299cells were exposed to8 Cl Ado at the concentration of10 mol L for0,12,24and48hours, respectively.The levels of H3K79me1,H3K79me2,

H3K79me3and total H3were detected by Western blotting with specific anti bodies. Acti n was used as a loading control

2.2 8 氯腺苷处理增强细胞组蛋白H3K79的单、双、三甲基化修饰

DNA发生损伤后即启动染色质重塑,以利于损伤修复过程的顺利进行,同时有利于修复分子及某些细胞周期检验点调控分子编码基因的转录激活.我们采用Western印迹检测了H3K79me1、H3K79me2、H3K79me3的表达水平.结果显示,与对照相比,在8 Cl Ado处理的H1299细胞中H3K79的3种甲基化形式在处理12h后即出现升高,且随时间延长(24~48h)而增加(Fig.2).3种甲基化H3K79在肿瘤细胞A549和HeLa中也呈明显升高趋势(资料未显示).结果提示,8 Cl Ado引起的DNA损伤启动了染色质重塑.

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第11期刘 畅等:8 氯腺苷诱导的组蛋白H3K79双甲基化促进NB S1和p21的基因转录激活

Fig.4 ChIP ana lyses of increased association of methylated H3K79with activated Nbs1and p21promoters H1299cells were unexposed (ctrl)or exposed to 8 Cl Ado (10 mol L)for 48hours.Chromatin was precipi tated with antibodies specific to H3K79me1,H3K79me2,H3K79me3,and H4K20me2.Regular PCR (A and C panels)and real time quantitative PCR (B and D)were performed using specific primers for p21(A,B)and NBS1(C,D)promoter sequences.In B and D,relative promoter binding in unexp osed cells was normalized to 1;n =3

2.3 8 氯腺苷上调D NA 损伤反应蛋白NBS1和细胞周期调控蛋白p21的表达

DNA 损伤后常引起DNA 损伤修复相关蛋白及细胞周期调控蛋白表达上调.为此,我们采用Western 印迹检测了DNA 损伤反应性蛋白NBS1以及一种细胞周期素依赖的蛋白激酶抑制物(inhibitor of cyclin dependent kinase,CKI)之一???p21的蛋白质表达及细胞核内水平.结果显示,总NBS1、p21的蛋白质表达水平、核内水平均随8 Cl Ado 处理时间的延长而增加(Fig.3).结果提示,8 Cl Ado 处理诱导了靶细胞中NBS1、p21基因的转录激活及二者的核转位.这与大多文献报告一致.

2.4 组蛋白H3K79双甲基化特异发生在NBS1和p21基因所在染色质区域

已有报告,组蛋白H3第79位赖氨酸甲基化是一种染色质活化分子

[22]

.但是,H3K79甲基化是否

与基因转录调控相关,3种甲基化形式哪种起主要

作用目前尚未见报告.为探索8 Cl Ado 诱导H3K79甲基化发生的生物学意义,分别采用抗H3K79me1、H3K79me2、H3K79me3、H4K20me2抗体进行染色质免疫共沉淀(ChIP)结合PC R 技术检测、鉴定了3种H3K79me 与DNA 损伤反应修复及细胞周期检验点调控分子NBS1、p21基因激活的关系.采用启动子序列特异引物经反转录PCR,在免疫沉淀中回收的

Fig.3 8 C l Ado up regulates the levels of NBS1and p21H1299cells were exposed to 8 Cl Ado at the concentration of 10mol L for 0,12,24,48hours,respectively.The nuclear protein and total protein levels of NBS1(A )and p21(B)were detected by Western blotting. A ctin was used as a loading control

DNA 中扩增NBS1、p21基因启动子特异序列.结果显示,只有H3K79me2,而非H3K79me1、H3K79me3或

H4K20me2发生在NBS1、p21基因所在的染色质区域内,即H3K79me2与相关基因的转录激活有关

1038中国生物化学与分子生物学报25卷

(Fig.4A、C).

为进一步定量地分析8 Cl Ado处理后H1299细胞中组蛋白H3第79位赖氨酸甲基化发生与NBS1、p21基因启动子激活的关系,采用实时定量PCR (real time quantitative PC R,RT qPC R)重复了ChIP实验.结果显示,与未处理细胞相比,在经8 Cl Ado处理的H1299细胞中,与H3K79me2所在染色质结合的2个基因启动子活性提高了约20~50倍(Fig.4B、D).结果进一步证明,H3K79me2与相关基因的转录激活有关.

3 讨论

我们曾经证明,8 Cl Ado可促进细胞凋亡、抑制细胞生长[14,15,19,23,24].最近发现,8 Cl Ado可在细胞内通过转化为8 Cl ATP,后者与ATP竞争、结合DNA 拓扑异构酶#(Topo#)而抑制Tpop#活性,从而引起DNA双链断裂[16,17].染色质重塑在基因转录、DNA复制和损伤修复中发挥重要作用.组蛋白甲基化可影响染色质高级结构,对基因转录、DNA修复具有重要影响.尽管有综述描述组蛋白H3K79甲基化通常处于常染色质活跃基因区[4],但关于H3K79me2与特异基因转录激活相关尚未见报告.本文证明,8 Cl Ado可引起肿瘤细胞DNA双链断裂损伤并诱导H3K79发生3种形式的甲基化修饰增强,使染色质发生重塑;H3K79双甲基化与DNA损伤修复分子NBS1和细胞周期检验点分子p21基因转录激活相关.这可能是H3K79双甲基化修饰在细胞周期检验点调节和DNA损伤修复功能调节的部分作用.

H3K79甲基化标志着染色质处于开放的常染色质状态[3].DOT1介导的H3K79甲基化可能阻止Sir2、Sir3蛋白与染色质结合,而解除Sir2 3导致的基因沉默[22,25].甲基化的H3K79抑制Sir2 3结合染色质,从而阻止了染色质凝缩,这又进一步促使DOT1催化邻近的H3K79发生甲基化;反之,足够量的Sir蛋白沉默复合物结合到未发生H3K79甲基化的染色质区域,将导致染色质发生凝缩,这会阻止DOT1催化该区域及其周围的组蛋白H3K79甲基化.这样一种动态平衡过程与建立、维持常染色质和异染色质两种状态密切相关.本研究用染色质免疫共沉淀技术证明H3K79me2主要发生在DNA损伤修复分子、细胞周期检验点分子NB S1、p21基因周围的染色质区域(Fig.4),与这些基因的转录激活相关.业已证明,p21可引起细胞周期停滞、促进细胞凋亡;NBS1参与细胞周期停滞和DNA损伤修复.这是8 Cl Ado抗肿瘤细胞生长的重要机制之一,此前未见报告.

DNA发生双链损伤时可激活下游信号分子,组蛋白H2AX的139位丝氨酸磷酸化( H2AX)是DSB s 发生后最早期事件之一[26].DNA损伤后的反应分子包括磷脂酰肌醇激酶(PI3K)家族的ATM、ATR、DNA PK,以及53B P1、MRN复合物(MRE11、Rad50、NBS1)、B RC A1和很多细胞周期检验点分子.这些分子共同构成复杂、多样的信号通路,介导细胞周期停滞,使细胞获得充分时间修复损伤,或者促进凋亡. DNA损伤后细胞周期检验点的一个主要事件是p53蛋白磷酸化而积聚,促进与细胞周期阻滞和凋亡相关的基因转录,介导G1 S期、G2 M期阻滞[27].在DNA损伤应答中,活化的p53可激活下游基因p21的转录.p21是一种CDK抑制分子,其编码基因可通过p53依赖和非依赖两种方式被激活.上调的p21可与许多Cyclin Cdk复合物结合,抑制细胞进程.我们最近曾经描述过8 Cl Ado上调p21,后者的表达可被C HK1抑制剂G 6976增强[17].这里,我们证明p21基因的转录激活与8 Cl Ado诱导的染色质组蛋白H3K79me2上调有关.因为H1299细胞是p53缺陷型,其具体转录激活机制尚待进一步研究.

在DNA损伤位点,Dot1催化组蛋白H3K79发生甲基化修饰,使染色质发生重塑.这里我们仅初步证明了H3K79me2与染色质重塑、部分基因激活有关.但是,我们尚未揭示8 Cl Ado上调的H3K79me2与DNA损伤修复有何直接联系;目前,我们也没有直接的实验证据解释H3K79me1、H3k79me3上调在DNA损伤修复、维持基因组稳定性中的作用.我们相信,进一步研究8 Cl Ado引起3种形式H3K79me 上调的生物学意义将有助于对8 Cl Ado抗肿瘤机制和肿瘤发生机制的认识和理解.

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