The KAP1 corepressor functions to coordinate the assembly of de novo HP1-demarcated microenvironments of heterochromatin required for KRAB zinc finger protein-mediated transcriptional repression
The KAP1 corepressor functions to coordinate the assembly of de novo HP1-demarcated microenvironments of heterochromatin required for KRAB zinc finger protein-mediated transcriptional repression
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DOI：
10.1128/mcb.00487-06
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发表时间：
2006-11-01
影响因子：
5.3
通讯作者：
Schultz, David C.
中科院分区：
文献类型：
--
作者：
Sripathy, Smitha P.;Stevens, Jessica;Schultz, David C.
KAP 1/TIF 1 β被认为是KRAB锌指蛋白（KRAB-zfp）超家族转录抑制子的通用辅抑制蛋白。为了表征KAP 1和KAP 1相互作用蛋白在转录抑制中的作用，我们研究了激素响应性KRAB和KAP 1抑制蛋白对稳定整合的报告转基因的调节。在这里，我们证明了内源性KAP 1水平的小干扰RNA（siRNA）的耗竭显着抑制KRAB介导的染色质模板的转录抑制。类似地，通过siRNA降低HP 1 α/β/γ和SETDB 1的细胞水平减弱了KRAB-KAP 1抑制。我们还发现，KAP 1与DNA的直接连接足以抑制整合的转基因的转录。这种活性是绝对依赖于KAP 1与HP 1的相互作用，并在一个完整的PHD手指和KAP 1的布罗莫结构域，这表明这些结构域的功能合作，在转录共抑制。通过野生型KAP 1实现抑制状态涉及RNA聚合酶11的募集减少，组蛋白H3 K9乙酰化和H3 K4甲基化水平降低，组蛋白占据增加，三甲基组蛋白H3 K9、H3 K36和组蛋白H4 K20富集，以及HP 1沉积在转基因的近端调控序列处。含有HP 1结合结构域突变的KAP 1蛋白未能诱导与转基因DNA序列相关的组蛋白修饰的任何变化，这意味着HP 1指导的核区室化是KRAB/KAP 1抑制复合物转录抑制所必需的。这些数据的组合表明，KAP 1的功能，以协调活动，动态调节组蛋白修饰和沉积的HP 1的变化，以建立一个从头异染色质的微环境，这是需要通过KRAB-zfps的基因转录的抑制。
KAP1/TIF1 beta is proposed to be a universal corepressor protein for the KRAB zinc finger protein (KRAB-zfp) superfamily of transcriptional repressors. To characterize the role of KAP1 and KAP1-interacting proteins in transcriptional repression, we investigated the regulation of stably integrated reporter transgenes by hormoneresponsive KRAB and KAP1 repressor proteins. Here, we demonstrate that depletion of endogenous KAP1 levels by small interfering RNA (siRNA) significantly inhibited KRAB-mediated transcriptional repression of a chromatin template. Similarly, reduction in cellular levels of HP1 alpha/beta/gamma and SETDB1 by siRNA attenuated KRAB-KAP1 repression. We also found that direct tethering of KAP1 to DNA was sufficient to repress transcription of an integrated transgene. This activity is absolutely dependent upon the interaction of KAP1 with HP1 and on an intact PHD finger and bromodomain of KAP1, suggesting that these domains function cooperatively in transcriptional corepression. The achievement of the repressed state by wild-type KAP1 involves decreased recruitment of RNA polymerase 11, reduced levels of histone H3 K9 acetylation and H3K4 methylation, an increase in histone occupancy, enrichment of trimethyl histone H3K9, H3K36, and histone H4K20, and HP1 deposition at proximal regulatory sequences of the transgene. A KAP1 protein containing a mutation of the HP1 binding domain failed to induce any change in the histone modifications associated with DNA sequences of the transgene, implying that HP1-directed nuclear compartmentalization is required for transcriptional repression by the KRAB/KAP1 repression complex. The combination of these data suggests that KAP1 functions to coordinate activities that dynamically regulate changes in histone modifications and deposition of HP1 to establish a de novo microenvironment of heterochromatin, which is required for repression of gene transcription by KRAB-zfps.