多发性骨髓瘤U266细胞株DNA甲基转移酶和甲基结合蛋白的表达

引用本文

许闪闪, 李智, 何丽梅, 翁文浩, 于慧. 多发性骨髓瘤U266细胞株DNA甲基转移酶和甲基结合蛋白的表达. 25(8): 632-636
XU Shanshan, LI Zhi, HE Limei, WENG Wenhao, YU Hui. The expression of DNA methyltransferases and methyl-DNA binding proteins in multiple myeloma U266 cell line. Labratory Medicine, 25(8): 632-636 复制到剪切板

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《检验医学》编辑部

多发性骨髓瘤U266细胞株DNA甲基转移酶和甲基结合蛋白的表达

许闪闪, 李智, 何丽梅, 翁文浩, 于慧

同济大学附属第十人民医院检验科,上海 200072

通讯作者：李智,联系电话：021-66306909。

作者简介：许闪闪,女,1984年生,硕士,主要从事临床常见病的发病机制及其诊断方法学研究。

摘要

目的

检测多发性骨髓瘤(MM)细胞DNA甲基转移酶(DNMTs)及甲基结合结构域(MBD)蛋白[包括甲基CpG结合结构域蛋白(MeCP2)和MBD2]的表达状况,探索其在基因甲基化过程的作用。并观察5-氮杂-2'-脱氧胞苷(5-Aza-CdR)和/或曲古霉素A(TSA)对DNMTs及MeCP2和MBD2的作用。

方法

利用荧光定量聚合酶链反应(PCR)分析MM U266细胞株DNMTs(DNMT1、DNMT3a、DNMT3b)及MeCP2和MBD2蛋白的mRNA水平,与10名正常人单个核细胞(PBMC)mRNA比较。4 μmol/L 5-Aza-CdR和/或0.1 μmol/L TSA与U266细胞共培养,分析MBD2和MeCP2蛋白mRNA的变化情况。Western Blot 检测这2种药物对U266细胞MBD2蛋白的影响。

结果

各甲基化调控蛋白mRNA与PBMC mRNA相比,差异有统计学意义( P均<0.05)。其中DNMTs和MBD2蛋白在U266细胞中表达升高,而MeCP2蛋白表达下降。经5-Aza-CdR和/或TSA作用后,MBD2表达下降,MeCP2升高。5-Aza-CdR在蛋白水平对MBD2的作用亦不显著;TSA可明显降低MBD2的表达,且具有时间依赖性。

结论

DNMTs和MBD2、MeCP2蛋白在MM U266细胞中表达异常,可能与U266细胞多个抑癌基因启动子高甲基化相关。5-Aza-CdR和TSA可以逆转U266细胞MBD2和MeCP2的表达。

关键词: 多发性骨髓瘤; 表观遗传学; DNA甲基转移酶; 甲基结合结构域蛋白

中图分类号:Q503 文献标志码:A 文章编号:1673-8640(2010)08-0632-05

The expression of DNA methyltransferases and methyl-DNA binding proteins in multiple myeloma U266 cell line

XU Shanshan, LI Zhi, HE Limei, WENG Wenhao, YU Hui

Department of Clinical Laboratory, the Tenth People's Hospital of Tongji University, Shanghai 200072, China

Abstract

Objective

To investigate the expression of DNA methyltransferases (DNMTs) and methyl-CpG-binding domain (MBD) proteins including MeCP2 and MBD2 in multiple myeloma (MM) cell and the influence on methylation, and observe the effect of 5-aza-2-deoxycitydine (5-Aza-CdR) and/or trichostatin A (TSA) on DNMTs, MeCP2 and MBD2.

Methods

The mRNA levels of DNMTs (DNMT1, DNMT3a and DNMT3b), MeCP2 and MBD2 proteins in MM U266 cell line and the mRNA level of peripheral blood mononuclear cell(PBMC)from 10 healthy controls were analyzed and compared by fluorescence quantitative polymerase chain reaction (PCR). U266 with 4 μmol/L 5-Aza-CdR and/or 0.1μmol/L TSA was cultured, and the mRNA of MBD2 and MeCP2 proteins was analyzed. In addition,the influence on MBD2 protein in U266 cell was evaluated by Western blot.

Results

There were statistical significance between the mRNA levels of methylation regulating proteins and PBMC ( P<0.05). The expressions of DNMTs and MBD2 increased in U266,while the expression of MeCP2 decreased. After treated with 5-Aza-CdR and/or TSA, the expression of MBD2 decreased, and the expression of MeCP2 increased. The effect of 5-Aza-CdR to MBD2 was not obvious. TSA with time dependence reduced the expression of MBD2.

Conclusions

DNMTs and MBD2, MeCP2 proteins are aberrant in MM U266 cell. It may be related with promoter hypermethylation in multiple tumor suppressor genes. 5-Aza-CdR and TSA can reverse the expression of MBD2 and MeCP2 in U266 cell.

Keyword: Multiple myeloma; Epigenesis; DNA methyltransferase; Methyl-CpG-binding domain protein

Show Figures

多发性骨髓瘤(multiple myeloma, MM)是以大量浆细胞异常增殖、单克隆免疫球蛋白过表达为特征的第二大恶性血液病。1997年以来, 国内外就不断有文献报导MM细胞存在抑癌基因p15^[1] 、p16^[1]、MGMT^[2]、DAPK^{[2, 3]}及干扰素相关因子家族19等^[4]启动子区异常高甲基化, 导致这些蛋白表达减少。甲基化改变主要受DNA甲基化转移酶(DNA methyl transferases, DNMTs, 包括DNMT1、DNMT3a、DNMT3b)和甲基结合结构域家族(methyl-binding domain, MBD)蛋白[包括MBD2和甲基CpG结合结构域蛋白(MeCp2)]的调节。我们以分离的健康成人外周血单个核细胞(peripheral blood mononuclear cell, PBMC)为对照, 分析MM U266细胞系DNA甲基调控蛋白的表达情况, 探索MM基因异常高甲基化的机制, 观察去甲基化药物和组蛋白乙酰化酶抑制剂对这些蛋白表达的影响。

材料和方法

一、实验材料

1.MM细胞 U266细胞株由上海市第十人民医院血液科施菊妹教授惠赠。细胞悬浮生长于RPMI1640培养液(内含15%胎牛血清)(美国Gibco公司)、青-链霉素100 U/mL、1%HEPES、1%丙酮酸钠和1%葡萄糖。置37 ℃、5%CO₂、100%饱和湿度孵箱中培养。维持细胞密度和培养液营养, 以使细胞处于对数生长期。实验前台盼蓝染色计数, 保持活细胞数95%100%。

2.引物设计合成根据GENEBANK各基因的RNA序列设计特异性引物。以β -肌动蛋白(β -actin)作为内源性参照, 引物序列和产物信息见表1。

表1 甲基化调控蛋白引物序列

二、实验方法

1.外周血单个核细胞分离 10名健康体检者, 男5名, 女5名, 年龄分布在1961岁。各采集枸橼酸钠抗凝血2 mL, 经血常规检查排除外周血异常后, 用Ficoll400淋巴细胞分离液(华昌药业)分离PBMC。

2.氯仿-异丙醇法抽提标本总mRNA Trizol (Invitrogen公司)裂解U266细胞及PBMCs, 氯仿-异丙醇法抽提总RNA, 紫外分光光度计检测其浓度和纯度。PrimeScript TM逆转录聚合酶链反应(RT-PCR)试剂盒(TaKaRa公司)20 μ L体系逆转录2 000 ng总RNA, 获得各标本cDNA。

3.荧光定量分析各基因mRNA转录水平以上述获得的2 μ L cDNA为模板, 加入18 μ L PCR反应液[4 μ mol/L基因引物1 μ L, 2× 高灵敏DNA荧光染料(SYBR) Premix Ex Taq 10 μ L, 红色荧光染料ROX Dye(阳性参比信号)(50× )0.4 μ L, 双蒸水(ddH₂O) 6.6 μ L]。在ABI 7900实时荧光定量PCR仪上, 95℃预变性1 min, 95℃ 15 s, 56℃ 30 s, 72℃ 45 s, 共40个循环, 72℃ 5 min。分析各扩增曲线, 读取各标本荧光阈值(cycle threshold value, Ct)值。

4. 5-氮杂-2'-脱氧胞苷(5-Aza-CdR)和曲右霉素(TSA)作用以68× 10⁵/mL浓度U266细胞接种6孔板。根据前期MTT试验结果选择药物浓度和作用时间。药物作用分为4组, 第1组(PBS对照组):加入与药物等量的PBS; 第2组(5-Aza-CdR组):加入终浓度为4 μ mol/L 5-Aza-CdR(Sigma公司); 第3组(TSA组):加入终浓度为0.1 μ mol/L TSA(sigma公司); 第4组(5-Aza-CdR和TSA共同作用组):同时加入4 μ mol/L 5-Aza-CdR和0.1 μ mol/L TSA。37℃、5%CO₂共培养48 h。药物作用重复3次, 在相应时间点收集细胞并抽提总RNA。

5.Western blot检测药物作用前后MBD2蛋白表达的改变 4 μ mol/L 5-Aza-CdR和/或0.1 μ mol/L TSA与U266细胞共培养48 h。收集细胞, 用核蛋白和浆蛋白抽提试剂盒(凯基公司)抽提核蛋白, BCA法定量, 100 ℃煮沸变性5 min。实验结果发现TSA可以降低MBD2蛋白的表达, 为观察该作用是否具有时间依赖性, 将U266细胞在0.1 μ mol/L TSA作用下分别培养24、72 h, 并检测MBD2蛋白表达情况。配制分离胶浓度为10%的十二烷基磺酸钠-聚丙烯酰胺凝胶(SDS-PAG)。核蛋白30 μ g上样, 80 V 15 min, 150 V 45 min电泳后, 18 V 15 min转移至聚偏-氟乙烯膜(PVDF)膜上。5%脱脂奶粉室温封闭1 h。与羊抗人MBD2抗体(Santa Cruz公司)4 ℃孵育过夜。Tris-硼酸吐温缓冲液(TBST)洗涤10 min× 3次, 驴抗羊IgG-F(ab')2-辣根过氧化物酶(HRP) 37 ℃孵育30 min, TBST洗涤10 min× 3次。Pro-light HRP(天根公司)化学发光检测试剂A液和B液等比例混合, 滴加至PVDF膜上, X光胶片曝光显像。

三、统计学方法

应用SPSS17.0软件包对实验数据进行统计分析, 实验结果以 $\begin{matrix} \bar{x} \end{matrix}$ ± s表示, 采用独立样本t检验, 比较U266细胞和PBMC之间各指标2^{-Δ Δ Ct}的差异。用单因素方差分析作两两比较。P< 0.05表示差异有统计学意义。

结果

一、DNMTs、MBD2和MeCP2蛋白mRNA的表达情况

经实时荧光定量PCR扩增得到各目的基因的Ct值, 以2^{-Δ Δ Ct}方法计算各基因与β -actin拷贝数的比值, 比较实验组和对照组之间的差异, 见表2。与对照组PBMC相比, DNMTs在U266细胞中表达均增高, 而MBD家族中MBD2蛋白表达增高, MeCP2表达降低。

表2 实时荧光定量PCR扩增各基因

二、5-Aza-CdR和TSA对MBD2和MeCP2蛋白mRNA的影响

4 μ mol/L 5-Aza-CdR对MBD2的转录无影响, 见图1(a); 但可以加强MeCP2的转录, 见图1(b)。在0.1 μ mol/L TSA作用下, 2种蛋白mRNA均出现逆转, MBD2 mRNA减少而MeCP2增加, 对MeCP2而言, 5-Aza-CdR和TSA共同作用比单一药物作用mRNA增加更显著。

三、5-Aza-CdR和TSA作用后MBD2蛋白表达的改变

4 μ mol/L 5-Aza-CdR作用48 h后, U266细胞MBD2蛋白表达无差异。0.1 μ mol/L TSA作用48 h, 可明显抑制U266细胞MBD2蛋白的表达。增加或减少TSA对U266细胞的作用时间发现, TSA对MBD2蛋白表达的影响具有时间依赖性, 即随着作用时间的延长蛋白表达降低。见图2。

	Figure Option View Download New Window
	图1 5-Aza-CdR和TSA作用48 h后U266细胞MBD2、MeCP2 mRNA的改变

	Figure Option View Download New Window
	图2 5-Aza-CdR和TSA对U266细胞MBD2蛋白的影响

讨论

自Ng等^[1]发表了首篇关于MM完全由5'CpG岛高甲基化引起p16和p15基因异常, 并且这可能是MM发生的特殊机制之一后, 国内外关于MM甲基化的研究逐渐增多, 人们发现抑癌基因尤其是p16^[5]和p15基因启动子区CpG岛高甲基化导致基因表达沉默, 是MM发生的重要表观遗传因素。近期Yuregir等^[3]对MM细胞甲基化谱进行筛选, 除p16基因外, MGMT、DAPK^[6]及ECAD等至少还存在1个基因启动子高甲基化。

筛选MM细胞DNA甲基化谱固然重要, 然而关于引起这些基因高甲基化改变机制的研究甚少。本研究旨在检测甲基化调控蛋白mRNA转录水平探索其在MM甲基化调控中的作用。与PBMC相比, U266细胞DNMTs和MBD2转录水平升高, 前者与傅海英等^[7]的研究结果相符合。DNMTs即胞嘧啶5-甲基转移酶, 其功能就是将甲基转移至胞嘧啶5位碳原子上。DNA甲基化的发生和甲基化程度的高低主要取决于DNMTs。U266细胞DNMTs比PBMC增高, 这可能是MM细胞多个抑癌基因沉默的主要原因。首先, DNMTs催化甲基转移, 直接使基因启动子发生甲基化, 阻碍转录因子与甲基化CpG结合从而直接抑制基因表达^[8]; 其次, DNMTs招募DNA甲基结合蛋白等, 与特定DNA序列结合, 形成复合物, 间接抑制基因表达^[9]。本研究结果MBD2蛋白明显增高可能也是解释这一机制的依据之一。若想进一步证实该机制, 可用染色质免疫沉淀技术芯片(ChIP-ChIP)分析DNMTs和MBD2的结合位点。Fu等^[10]的报导已经证实甲基转移酶抑制剂5-Aza-CdR可以通过抑制DNMTs mRNA提高抑癌基因p16的表达。本研究结果说明5-Aza-CdR不通过MBD2发挥作用。

MeCP2是MBD蛋白家族的另一重要成员, 是唯一能识别单个甲基化CpG位点的结合蛋白, 与MBD2能够募集组蛋白去乙酰化酶(histone-deacetylase, HDAC), 与转录抑制相关^[11]。然而MeCP2多定位于异染色质, 不但可以结合甲基化DNA, 而且与非甲基化DNA序列也有较弱的亲和力。采用ChIP-ChIP研究表明59%MeCP2结合位点位于基因间, 仅6%位于CpG位点^[12]。这说明MeCP2可能不是直接与甲基化抑癌基因结合, 而是通过其他途径间接调控这些基因的表达。或许MM细胞同时存在癌基因启动子低甲基化, MeCP2调节该过程。5-Aza-CdR和TSA可以协同作用促进MeCP2转录。

TSA是HDAC抑制剂, 抑制HDAC、促进基因表达的高效试剂。TSA作用后, DNA甲基化过程中MBD2与HDAC复合物减少。因此用于结合MBD2的HDAC减少, 反馈下调MBD2转录, 一并抑制下游相关反应。因此, TSA可能成为MM患者治疗的有效药物。

综上所述, DNMTs和MBD2、MeCP2蛋白在MM U266细胞中表达异常, 可能与U266细胞多个抑癌基因启动子高甲基化相关。通过RNA干扰或过表达, 观察这些基因沉默后抑癌基因启动子的甲基化状态及基因的转录水平来验证。5-Aza-CdR和TSA可以逆转U266细胞MBD2和MeCP2蛋白的表达。

The authors have declared that no competing interests exist.

参考文献

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1997

9.06

0.0

... 1997年以来,国内外就不断有文献报导MM细胞存在抑癌基因p15^[1] 、p16^[1]、MGMT^[2]、DAPK^[2,3]及干扰素相关因子家族19等^[4]启动子区异常高甲基化,导致这些蛋白表达减少 ...

... 讨论自Ng等^[1]发表了首篇关于MM完全由5'CpG岛高甲基化引起p16和p15基因异常,并且这可能是MM发生的特殊机制之一后,国内外关于MM甲基化的研究逐渐增多,人们发现抑癌基因尤其是p16^[5]和p15基因启动子区CpG岛高甲基化导致基因表达沉默,是MM发生的重要表观遗传因素 ...

2008

2.128

0.0

. 2008, 84(3):256-261 DOI:10.1016/j.yexmp.2008.02.003

Aberrant gene promoter methylation in plasma cell dyscrasias

a Laboratory of Molecular Pathology, Department of Pathology, Hospital Universitario Puerta de Hierro, Madrid, Spain b Laboratory of Molecular Pathology, Department of Pathology, Hospital Ramon y Cajal, Madrid, Spain

The aberrant methylation of promoter CpG island is known to be a major inactivation mechanism of tumour-related genes. To determine the clinicopathological significance of gene promoter methylation in monoclonal gammopathies, we analysed the methylation status of 6 tumour suppressor genes and their association with loss of gene function. Methylation status of the genes p14, p15, p16, hMLH1, MGMT, and DAPK was determined by methylation-specific PCR in 52 cases: 30 MM, 13 MGUS, and 9 plasmacytomas, comparing them with their protein expression by immunohistochemistry, and association between methylation status, protein expression, and clinical characteristics was assessed. The methylation frequencies were 50% for p16, 17% for p15, 10% for hMLH1, 23% for MGMT and 30% for DAPK in MM samples, and 38%, 15%, 8%, and 15% for p16, p15, MGMT and DAPK respectively in MGUS samples. In plasmacytomas samples we found methylation of p16 in 55%, p15 in 22%, MGMT in 67% and DAPK in 44%. hMLH1 was unmethylated in all cases of MGUS and plasmacytomas. Immunohistochemistry showed that gene methylation was closely associated with a loss of protein expression. Our study demonstrates that methylation-mediated silencing is a frequent event in monoclonal gammopathies: 83% of MM, 46% of MGUS and 77% of plasmacytomas have at least one gene methylated, affecting different molecular pathways involved in cell cycle, DNA repair and apoptosis. This high prevalence of aberrant promoter hypermethylation suggests that monoclonal gammopathies carry a CpG island methylator phenotype, therefore the development of new DNA demethylation agents may be a potential therapeutic use in this disease.

2010

1.293

0.0

. 2010, 32(2):142-149

Detecting methylation patterns of p16, MGMT, DAPK and E-cadherin genes in multiple myeloma patients

O. OZALP YUREGIR 1 ,E. YURTCU 2 ,E. KIZILKILIC 3 ,N. E. KOCER 4 ,H. OZDOGU 3 andF. I. SAHIN 1

1 Department of Medical Genetics, Baskent University Faculty of Medicine, Ankara, Turkey 2 Department of Medical Biology and Genetics, Baskent University Faculty of Medicine, Ankara, Turkey 3 Department of Hematology, Baskent University Faculty of Medicine, Ankara, Turkey 4 Department of Pathology, Baskent University Faculty of Medicine, Ankara, Turkey * Prof. Dr Feride Iffet Sahin, Department of Medical Genetics, Baskent University Faculty of Medicine, Kubilay Sokak No: 36 Maltepe, 06570 Ankara, Turkey. Tel.: 90 312 2324400/138; Fax: 90 312 2319134; E-mail: feridesahin@hotmail.com

> Multiple myeloma (MM) is a B-cell neoplasia characterized by the clonal proliferation of plasma cells. Besides known genetic abnormalities, epigenetic changes are also known to effect MM pathogenesis. DNA methylation is an epigenetic mechanism that silences genes by adding methyl groups to cytosine-guanine dinucleotides at the promoter regions. In this study, the methylation status of four genes; p16 , O6-methyl guanine DNA methyl transferase ( MGMT ), death-associated protein kinase ( DAPK ) and E-cadherin ( ECAD ); at the time of diagnosis was investigated using methylation-specific polymerase chain reaction (MS-PCR). In the 20 cases studied; methylation of the promoter regions of p16 , MGMT , DAPK and ECAD genes was detected in 10%, 40%, 10% and 45% of the cases, respectively. In 65% (13/20) of cases, at least one of the genes studied had promoter methylation; while 35% of cases (7/20) had methylated promoters of more than one gene. There was a significant correlation between promoter hypermethylation of MGMT and the presence of extramedullary involvement; but for the other genes no correlation was found regarding disease properties like age, disease stage, clinical course and the presence of lytic bone lesions. Determining the methylation profiles of genes in MM, could lead to a new understanding of the disease pathogenesis and guide the assessment of treatment options.

... 近期Yuregir等^[3]对MM细胞甲基化谱进行筛选,除p16基因外,MGMT、DAPK^[6]及ECAD等至少还存在1个基因启动子高甲基化 ...

2008

2.907

0.0

. 2008, 36(12):1673-1681 DOI:10.1016/j.exphem.2008.08.001

Epigenetic silencing of the interferon regulatory factor ICSBP/IRF8 in human multiple myeloma

Objective Multiple myeloma (MM) is presently an incurable malignant plasma cell tumor. The objective of this study was to investigate expression of the interferon regulatory factor family (IRF1-9) and the potential role of DNA methylation in silencing IRF genes in MM cell lines and purified MM cells from patients. Materials and Methods Using a panel of 13 human MM cell lines and purified CD138 + cells from nine MM patients, expression of IRF genes was investigated by quantitative reverse transcriptase polymerase chain reaction and Western blot. DNA methylation of the interferon consensus sequence–binding protein (ICSBP/IRF8) gene was measured using pyrosequencing, and the effect of promoter methylation on expression was analyzed by in vitro methylation of a cloned ICSBP/IRF8 promoter, and treatment of MM cells with 5-aza-2′-deoxycytidine (DAC). Results Eight of thirteen of the MM cell lines were found to lack ICSBP/IRF8 expression, associated with hypermethylation of the CpG island in the ICSBP/IRF8 promoter. We also found that ICSBP/IRF8 was significantly underexpressed in primary MM cells, whereas the ICSBP/IRF8 promoter was methylated in only one of nine of primary purified CD138 + MM samples. DAC-mediated demethylation restored endogenous ICSBP/IRF8 expression, whereas in vitro methylation silenced the promoter. Conclusion Expression of the ICSBP/IRF8 gene is silenced in a majority of MM cell lines and primary CD138 + MM cells. DNA methylation of the ICSBP/IRF8 gene is a frequent event in MM cell lines, but silencing is also observed in the absence of methylation. These results suggest that silencing of ICSBP/IRF8 expression, by DNA methylation or other epigenetic mechanisms, may be associated with the malignant phenotype of MM.

2000

10.164

0.0

. 2000, 14(1):-

De novo methylation of tumor suppressor gene p16/INK4a is a frequent finding in multiple myeloma patients at diagnosis

Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.

2008

1.13

0.0

. 2008, 8(6):null-null

Gene hypermethyla-tion in multiple myeloma: lessons from a cancer pathway approach

... 近期Yuregir等^[3]对MM细胞甲基化谱进行筛选,除p16基因外,MGMT、DAPK^[6]及ECAD等至少还存在1个基因启动子高甲基化 ...

2007

0.0

... 与PBMC相比,U266细胞DNMTs和MBD2转录水平升高,前者与傅海英等^[7]的研究结果相符合 ...

1988

12.444

0.0

... 首先,DNMTs催化甲基转移,直接使基因启动子发生甲基化,阻碍转录因子与甲基化CpG结合从而直接抑制基因表达^[8] ...

2002

12.444

0.0

... 其次,DNMTs招募DNA甲基结合蛋白等,与特定DNA序列结合,形成复合物,间接抑制基因表达^[9] ...

2007

0.0

... Fu等^[10]的报导已经证实甲基转移酶抑制剂5-Aza-CdR可以通过抑制DNMTs mRNA提高抑癌基因p16的表达 ...

1999

9.737

0.0

PNAS. 1999, 96(7):3969-3974

MED1, a novel human methyl-CpG-binding endonuclease, interacts with DNA mismatch repair protein MLH1

Alfonso Bellacosa*†‡, Lucia Cicchillitti*, Filippo Schepis*, Antonio Riccio*, Anthony T. Yeung§, Yoshihiro Matsumoto¶, Erica A. Golemis§, Maurizio Genuardi†, and Giovanni Neri†

* Divisions of Population Science, § Basic Science, and ¶ Medical Science, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111; and † Department of Medical Genetics, Catholic University Medical School, Largo F. Vito 1, 00168, Rome, Italy

The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenanceof genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Usingthe MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases,and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associatedwith microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involvedin MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these resultssuggest that cytosine methylation may play a role in human DNA repair.

... MeCP2是MBD蛋白家族的另一重要成员,是唯一能识别单个甲基化CpG位点的结合蛋白,与MBD2能够募集组蛋白去乙酰化酶(histone-deacetylase,HDAC),与转录抑制相关^[11] ...

2007

9.737

0.0

PNAS. 2007, 104(49):19416-19421

Integrated epigenomic analyses of neuronal MeCP2 reveal a role for long-range interaction with active genes

Dag H. Yasui* , † , Sailaja Peddada* , † , Mark C. Bieda ‡ , § , Roxanne O. Vallero* , † , Amber Hogart* , † , Raman P. Nagarajan* , † , Karen N. Thatcher* , † , Peggy J. Farnham ‡ , § ,and Janine M. LaSalle* , † , ¶

Departments of *Medical Microbiology and Immunology and ‡ Pharmacology, † Rowe Program in Human Genetics, and § Genome Center, School of Medicine, University of California, 1 Shields Avenue, Davis, CA 95616

Mutations in MECP2 cause the autism-spectrum disorder Rett syndrome. MeCP2 is predicted to bind to methylated promoters and silence transcription.However, the first large-scale mapping of neuronal MeCP2-binding sites on 26.3 Mb of imprinted and nonimprinted loci revealedthat 59% of MeCP2-binding sites are outside of genes and that only 6% are in CpG islands. Integrated genome-wide promoteranalysis of MeCP2 binding, CpG methylation, and gene expression revealed that 63% of MeCP2-bound promoters are actively expressedand that only 6% are highly methylated. These results indicate that the primary function of MeCP2 is not the silencing ofmethylated promoters.

... 采用ChIP-ChIP研究表明59%MeCP2结合位点位于基因间,仅6%位于CpG位点^[12] ...