Genome-wide analysis of binding sites and direct target genes of the orphan nuclear receptor NR2F1/COUP-TFI.
Genome-wide analysis of binding sites and direct target genes of the orphan nuclear receptor NR2F1/COUP-TFI.
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DOI：
10.1371/journal.pone.0008910
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发表时间：
2010-01-27
期刊：
影响因子：
3.7
通讯作者：
Pereira FA
中科院分区：
文献类型：
--
作者：
Montemayor C;Montemayor OA;Ridgeway A;Lin F;Wheeler DA;Pletcher SD;Pereira FA
鉴定真正的直接核受体基因靶点一直具有挑战性，但对于理解生物体生理过程的调节至关重要。我们描述了一种通过交叉微阵列数据、计算结合位点查询和进化守恒来鉴定体内转录因子结合位点和靶基因的方法。我们提供了每个步骤的详细实验验证，并且作为原理证明，利用该方法确定孤儿核受体NR2F1 （COUP-TFI）的新直接靶点。第一步涉及验证从野生型和COUP-TFI - / -内耳组织中获得的微阵列基因表达谱。其次，我们开发了一个生物信息学工具，使用分类型隐马尔科夫模型训练49个已发表的COUP-TF响应元素，在基因组中搜索COUP-TFI DNA结合位点。接下来，我们根据结合位点进化保守程度和微阵列统计显著性，通过整合芯片和生物信息学分析，获得候选体内直接COUP-TFI靶点的排名。最后，作为概念验证，5个特定基因被验证为直接调控。例如，脂肪酸结合蛋白7 （Fabp7）基因是体内直接的COUP-TFI靶点，因为：i)我们在Fabp7启动子中发现了2个保守的COUP-TFI结合位点；ii)在COUP-TFI−/−组织和mef中，Fapb7转录物和蛋白水平显著降低；iii)染色质免疫沉淀表明，在体外和体内，COUP-TFI被募集到Fabp7启动子上；iv)它与新生儿大脑中H3K9乙酰化增加和CBP和SRC-1结合富集的活性染色质相关。我们已经开发并验证了一种方法来鉴定体内直接核受体靶基因。这个生物信息学工具可以修改扫描转录因子，顺式调控模块，或任何灵活的DNA模式的响应元件。
Identification of bona fide direct nuclear receptor gene targets has been challenging but essential for understanding regulation of organismal physiological processes. We describe a methodology to identify transcription factor binding sites and target genes in vivo by intersecting microarray data, computational binding site queries, and evolutionary conservation. We provide detailed experimental validation of each step and, as a proof of principle, utilize the methodology to identify novel direct targets of the orphan nuclear receptor NR2F1 (COUP-TFI). The first step involved validation of microarray gene expression profiles obtained from wild-type and COUP-TFI−/− inner ear tissues. Secondly, we developed a bioinformatic tool to search for COUP-TFI DNA binding sites in genomes, using a classification-type Hidden Markov Model trained with 49 published COUP-TF response elements. We next obtained a ranked list of candidate in vivo direct COUP-TFI targets by integrating the microarray and bioinformatics analyses according to the degree of binding site evolutionary conservation and microarray statistical significance. Lastly, as proof-of-concept, 5 specific genes were validated for direct regulation. For example, the fatty acid binding protein 7 (Fabp7) gene is a direct COUP-TFI target in vivo because: i) we identified 2 conserved COUP-TFI binding sites in the Fabp7 promoter; ii) Fapb7 transcript and protein levels are significantly reduced in COUP-TFI−/− tissues and in MEFs; iii) chromatin immunoprecipitation demonstrates that COUP-TFI is recruited to the Fabp7 promoter in vitro and in vivo and iv) it is associated with active chromatin having increased H3K9 acetylation and enrichment for CBP and SRC-1 binding in the newborn brain. We have developed and validated a methodology to identify in vivo direct nuclear receptor target genes. This bioinformatics tool can be modified to scan for response elements of transcription factors, cis-regulatory modules, or any flexible DNA pattern.
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影响因子：
4.5
作者：
Lin CY;Vega VB;Thomsen JS;Zhang T;Kong SL;Xie M;Chiu KP;Lipovich L;Barnett DH;Stossi F;Yeo A;George J;Kuznetsov VA;Lee YK;Charn TH;Palanisamy N;Miller LD;Cheung E;Katzenellenbogen BS;Ruan Y;Bourque G;Wei CL;Liu ET
通讯作者：
Liu ET
影响因子：
30.8
作者：
Carroll, Jason S.;Meyer, Clifford A.;Brown, Myles
通讯作者：
Brown, Myles
影响因子：
3.6
作者：
Cabrero, A;Jové, M;Vázquez-Carrera, M
通讯作者：
Vázquez-Carrera, M
DOI：
10.1007/s10162-002-2058-2
发表时间：
2003-03-01
影响因子：
2.4
作者：
Klockars, T;Perheentupa, T;Dahl, HHM
通讯作者：
Dahl, HHM
影响因子：
2
作者：
Brown, Kerry K.;Alkuraya, Fowzan S.;Matos, Michael;Robertson, Richard L.;Kimonis, Virginia E.;Morton, Cynthia C.
通讯作者：
Morton, Cynthia C.