Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer.
Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer.
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DOI：
10.1093/nar/gkt601
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发表时间：
2013-09
影响因子：
14.9
通讯作者：
Verma A
中科院分区：
文献类型：
--
作者：
Bhattacharyya S;Yu Y;Suzuki M;Campbell N;Mazdo J;Vasanthakumar A;Bhagat TD;Nischal S;Christopeit M;Parekh S;Steidl U;Godley L;Maitra A;Greally JM;Verma A
5-羟甲基胞嘧啶（5-hmC）是最近发现的在癌症中改变的表观遗传修饰。由于许多用于5-甲基胞嘧啶（5-mC）测定的技术（包括甲基敏感性限制性消化和亚硫酸氢盐测序）无法区分5-mC和5-hmC，因此需要进行全基因组测定。β-葡萄糖基转移酶（β-GT）对5-hmC残基的糖基化可以使CCGG残基对MspI的消化不敏感。在β-GT转化后通过HpaII、MspI或MspI进行限制性消化，然后进行接头连接、大规模平行测序和定制生物信息学分析，使我们能够在MspI限制性位点以单碱基对分辨率确定5-mC和5-hmC的分布。通过连接介导的PCR与β-GT（HELP-GT）测定法进行的HpaII微小片段富集鉴定了5-hmC位点，通过液相色谱-质谱（LC-MS）在全局水平上验证了5-hmC位点，并通过5-hmC下拉DNA的定量逆转录酶聚合酶链反应在位点特异性水平上验证了5-hmC位点。启动子和基因内位点的羟甲基化与基因表达呈正相关。胰腺癌样本的分析显示，在癌细胞中5-hmC位点的显著再分布，并证明了这种修饰在许多致癌启动子如GATA 6处的富集。HELP-GT检测允许从低量DNA和使用适度的测序资源中全面测定5-hmC和5-mC。在癌症中观察到的5-hmC的再分布突出了结合常规甲基化分析确定这种修饰的重要性。
5-hydroxymethylcytosine (5-hmC) is a recently discovered epigenetic modification that is altered in cancers. Genome-wide assays for 5-hmC determination are needed as many of the techniques for 5-methylcytosine (5-mC) determination, including methyl-sensitive restriction digestion and bisulfite sequencing cannot distinguish between 5-mC and 5-hmC. Glycosylation of 5-hmC residues by beta-glucosyl transferase (β-GT) can make CCGG residues insensitive to digestion by MspI. Restriction digestion by HpaII, MspI or MspI after β-GT conversion, followed by adapter ligation, massive parallel sequencing and custom bioinformatic analysis allowed us determine distribution of 5-mC and 5-hmC at single base pair resolution at MspI restriction sites. The resulting HpaII tiny fragment Enrichment by Ligation-mediated PCR with β-GT (HELP-GT) assay identified 5-hmC loci that were validated at global level by liquid chromatography-mass spectrometry (LC-MS) and the locus-specific level by quantitative reverse transcriptase polymerase chain reaction of 5-hmC pull-down DNA. Hydroxymethylation at both promoter and intragenic locations correlated positively with gene expression. Analysis of pancreatic cancer samples revealed striking redistribution of 5-hmC sites in cancer cells and demonstrated enrichment of this modification at many oncogenic promoters such as GATA6. The HELP-GT assay allowed global determination of 5-hmC and 5-mC from low amounts of DNA and with the use of modest sequencing resources. Redistribution of 5-hmC seen in cancer highlights the importance of determination of this modification in conjugation with conventional methylome analysis.
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影响因子：
50.3
作者：
Figueroa ME;Abdel-Wahab O;Lu C;Ward PS;Patel J;Shih A;Li Y;Bhagwat N;Vasanthakumar A;Fernandez HF;Tallman MS;Sun Z;Wolniak K;Peeters JK;Liu W;Choe SE;Fantin VR;Paietta E;Löwenberg B;Licht JD;Godley LA;Delwel R;Valk PJ;Thompson CB;Levine RL;Melnick A
通讯作者：
Melnick A
DOI：
10.1056/nejmoa1013343
发表时间：
2011-06-30
期刊：
The New England journal of medicine
影响因子：
--
作者：
Bejar R;Stevenson K;Abdel-Wahab O;Galili N;Nilsson B;Garcia-Manero G;Kantarjian H;Raza A;Levine RL;Neuberg D;Ebert BL
通讯作者：
Ebert BL
影响因子：
50.3
作者：
Moran-Crusio K;Reavie L;Shih A;Abdel-Wahab O;Ndiaye-Lobry D;Lobry C;Figueroa ME;Vasanthakumar A;Patel J;Zhao X;Perna F;Pandey S;Madzo J;Song C;Dai Q;He C;Ibrahim S;Beran M;Zavadil J;Nimer SD;Melnick A;Godley LA;Aifantis I;Levine RL
通讯作者：
Levine RL
DOI：
10.1158/1078-0432.ccr-10-3431
发表时间：
2011-07-01
期刊：
Clinical cancer research : an official journal of the American Association for Cancer Research
影响因子：
--
作者：
Vincent A;Omura N;Hong SM;Jaffe A;Eshleman J;Goggins M
通讯作者：
Goggins M
DOI：
10.1126/science.1170116
发表时间：
2009-05-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Tahiliani M;Koh KP;Shen Y;Pastor WA;Bandukwala H;Brudno Y;Agarwal S;Iyer LM;Liu DR;Aravind L;Rao A
通讯作者：
Rao A