A Functional Insulator Screen Identifies NURF and dREAM Components to Be Required for Enhancer-Blocking
A Functional Insulator Screen Identifies NURF and dREAM Components to Be Required for Enhancer-Blocking
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DOI：
10.1371/journal.pone.0107765
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发表时间：
2014-09-23
期刊：
影响因子：
3.7
通讯作者：
Renkawitz, Rainer
中科院分区：
文献类型：
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作者：
Bohla, Dorte;Herold, Martin;Renkawitz, Rainer
高等真核生物的染色质绝缘体在功能上将基因组划分为活性区域和非活性区域。此外，绝缘子调节增强子/启动子的通信，这从果蝇双胸基因座中可以明显看出，在双胸基因座中，许多调控元件控制着片段特定基因的活性。中心体蛋白190 （CP190）被CTCF或其他绝缘子dna结合因子靶向于绝缘子。染色质分析显示，绝缘体的特征是开放和核小体耗尽区域。在这里，我们想确定增强子阻断功能所需的染色质修饰和重塑因子。我们使用已被充分研究的双胸基因座的Fab-8绝缘子，应用全基因组RNAi筛选CTCF和CP190增强子阻断功能的因子。在最佳的Fab-8介导的增强子阻断所需的78个基因中，NURF复合体的所有四个组分以及dREAM复合体的几个亚基最为明显。CTCF或CP190结合蛋白的质谱分析以及免疫沉淀证实了NURF和dREAM结合。两者都与基因组中大多数CP190结合位点共定位，染色质免疫沉淀显示CP190招募NURF和dREAM。核小体占用和组蛋白H3结合分析显示，CP190介导的NURF结合导致CP190结合位点的核小体缺失。因此，我们得出结论，CP190结合CTCF或其他DNA结合绝缘子因子介导NURF和dREAM的募集。此外，绝缘子的增强子阻断功能与核小体耗竭有关，需要NURF和dREAM。
Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.