Screening for inhibitors of an essential chromatin remodeler in mouse embryonic stem cells by monitoring transcriptional regulation.
Screening for inhibitors of an essential chromatin remodeler in mouse embryonic stem cells by monitoring transcriptional regulation.
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DOI：
10.1177/1087057112455060
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发表时间：
2012-10
影响因子：
--
通讯作者：
Palmer MA
中科院分区：
文献类型：
--
作者：
Dykhuizen EC;Carmody LC;Tolliday N;Crabtree GR;Palmer MA
SWI/SNF样三磷酸腺苷（ATP）依赖性染色质重塑复合物，esBAF，是必要的，在某些情况下，足以诱导多能状态。此外，各种BAF亚基的突变与癌症相关。关于这种复合物发挥其活动的精确机制知之甚少。因此，目前尚不清楚哪些蛋白质相互作用对破坏以分离相关机制读数是重要的。为了解决这个问题，我们开发了一种基于基因表达的测定来鉴定天然esBAF复合物的抑制剂。具体而言，在小鼠胚胎干（ES）细胞中开发了定量逆转录酶聚合酶链反应（qRT-PCR）试验，以监测Bmi 1（一种受esBAF复合物抑制的发育重要基因）的表达。该测定被小型化为384孔格式，并用于筛选各种化合物，包括多样性导向合成（DOS）的新产品。确认的命中使用基因敲入ES细胞报告细胞系进行验证，其中荧光素酶插入Bmi 1基因座。几个经验证的命中调节一组靶基因的方式类似于BAF染色质重塑复合物。总之，这些数据表明，使用qRT-PCR的基于表达的筛选是鉴定靶向ES细胞中关键发育基因调控的化合物的成功方法。
The SWI/SNF-like adenosine triphosphate (ATP)–dependent chromatin remodeling complex, esBAF, is both necessary and, in some contexts, sufficient to induce the pluripotent state. Furthermore, mutations in various BAF subunits are associated with cancer. Little is known regarding the precise mechanism(s) by which this complex exerts its activities. Thus, it is unclear which protein interactions would be important to disrupt to isolate a relevant readout of mechanism. To address this, we developed a gene expression–based assay to identify inhibitors of the native esBAF complex. Specifically, a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay was developed in mouse embryonic stem (ES) cells to monitor expression of Bmi1, a developmentally important gene repressed by the esBAF complex. The assay was miniaturized to a 384-well format and used to screen a diverse collection of compounds, including novel products of diversity-oriented synthesis (DOS). Confirmed hits were validated using a knock-in ES cell reporter line in which luciferase is inserted into the Bmi1 locus. Several of the validated hits regulate a panel of target genes in a manner similar to the BAF chromatin-remodeling complex. Together these data indicate that expression-based screening using qRT-PCR is a successful approach to identify compounds targeting the regulation of key developmental genes in ES cells.
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影响因子：
64.8
作者：
Varela, Ignacio;Tarpey, Patrick;Raine, Keiran;Huang, Dachuan;Ong, Choon Kiat;Stephens, Philip;Davies, Helen;Jones, David;Lin, Meng-Lay;Teague, Jon;Bignell, Graham;Butler, Adam;Cho, Juok;Dalgliesh, Gillian L.;Galappaththige, Danushka;Greenman, Chris;Hardy, Claire;Jia, Mingming;Latimer, Calli;Lau, King Wai;Marshall, John;McLaren, Stuart;Menzies, Andrew;Mudie, Laura;Stebbings, Lucy;Largaespada, David A.;Wessels, L. F. A.;Richard, Stephane;Kahnoski, Richard J.;Anema, John;Tuveson, David A.;Perez-Mancera, Pedro A.;Mustonen, Ville;Fischer, Andrej;Adams, David J.;Rust, Alistair;Chan-on, Waraporn;Subimerb, Chutima;Dykema, Karl;Furge, Kyle;Campbell, Peter J.;Teh, Bin Tean;Stratton, Michael R.;Futreal, P. Andrew
通讯作者：
Futreal, P. Andrew
影响因子：
64.5
作者：
Singhal, Nishant;Graumann, Johannes;Schoeler, Hans R.
通讯作者：
Schoeler, Hans R.
DOI：
10.1126/science.1198056
发表时间：
2011-01-28
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Parsons DW;Li M;Zhang X;Jones S;Leary RJ;Lin JC;Boca SM;Carter H;Samayoa J;Bettegowda C;Gallia GL;Jallo GI;Binder ZA;Nikolsky Y;Hartigan J;Smith DR;Gerhard DS;Fults DW;VandenBerg S;Berger MS;Marie SK;Shinjo SM;Clara C;Phillips PC;Minturn JE;Biegel JA;Judkins AR;Resnick AC;Storm PB;Curran T;He Y;Rasheed BA;Friedman HS;Keir ST;McLendon R;Northcott PA;Taylor MD;Burger PC;Riggins GJ;Karchin R;Parmigiani G;Bigner DD;Yan H;Papadopoulos N;Vogelstein B;Kinzler KW;Velculescu VE
通讯作者：
Velculescu VE
影响因子：
10.5
作者：
Bultman, SJ;Gebuhr, TC;Magnuson, T
通讯作者：
Magnuson, T
影响因子：
30.8
作者：
Wang, Kai;Kan, Junsuo;Leung, Suet Yi
通讯作者：
Leung, Suet Yi