Interaction of EVI1 with cAMP-responsive element-binding protein-binding protein (CBP) and p300/CBP-associated factor (P/CAF) results in reversible acetylation of EVI1 and in co-localization in nuclear speckles
Interaction of EVI1 with cAMP-responsive element-binding protein-binding protein (CBP) and p300/CBP-associated factor (P/CAF) results in reversible acetylation of EVI1 and in co-localization in nuclear speckles
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DOI：
10.1074/jbc.m106733200
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发表时间：
2001-11-30
影响因子：
4.8
通讯作者：
Nucifora, G
中科院分区：
文献类型：
--
作者：
Chakraborty, S;Senyuk, V;Nucifora, G
EVIL是一种非常复杂的蛋白质，具有两个锌指结构域，并且在许多类型的人髓性白血病中不适当地表达。使用报告基因测定，一些研究人员表明，EVI 1是一个转录阻遏物，最近它被证明，EVI 1与辅阻遏羧基末端结合蛋白1（CtBP 1）相互作用。早些时候，我们发现，在小鼠造血前体细胞中不适当的EVI 1表达导致其异常分化和增殖增加。使用生物化学测定，我们已经确定了两组转录辅助调节因子，与EVIL可能调节基因表达。一组共调节因子包括CtBP 1和组蛋白脱乙酰酶。第二组包括两种共激活剂cAMP反应元件结合蛋白结合蛋白（CBP）和p300/CBP相关因子（P/CAF），两者都具有组蛋白乙酰转移酶（HAT）活性。所有这些蛋白质需要EVI 1的单独区域进行有效的相互作用，并且它们在报告基因测定中不同地影响EVI 1调节基因转录的能力。共聚焦显微镜分析表明，在大多数细胞中，EVI 1是核和扩散，而在约10%的细胞中，EVI 1定位于核斑点。然而，在添加外源性辅抑制剂组蛋白脱乙酰酶或CtBP 1的情况下，所有细胞核都具有弥漫性EVI 1染色，并且蛋白质似乎并不一起存在于明显的细胞核结构中。相反，当加入CBP或P/CAF时，细胞核中出现明确的斑点状小体。染色模式的分析表明，EVI 1和CBP或EVIL和P/CAF包含在这些结构中。当CBP被点突变的HAT失活CBP取代时，这些核结构未观察到，EVI 1也与点突变的HAT失活CBP发生物理相互作用。最后，我们发现EVI 1与CBP或P/CAF的相互作用导致EVI 1的乙酰化。这些结果表明，在核斑点的EVIL的组装需要完整的HAT活性的共激活剂。
EVIL is a very complex protein with two domains of zinc fingers and is inappropriately expressed in many types of human myeloid leukemias. Using reporter gene assays, several investigators showed that EVI1 is a transcription repressor, and recently it was shown that EVI1 interacts with the co-repressor carboxyl-terminal binding protein 1 (CtBP1). Earlier, we showed that the inappropriate expression of EVI1 in murine hematopoietic precursor cells leads to their abnormal differentiation and to increased proliferation. Using biochemical assays, we have identified two groups of transcription co-regulators that associate with EVIL presumably to regulate gene expression. One group of co-regulators includes the CtBP1 and histone deacetylase. The second group includes the two co-activators cAMP-responsive element-binding protein-binding protein (CBP) and p300/CBP-associated factor (P/CAF), both of which have histone acetyltransferase (HAT) activity. All of these proteins require separate regions of EVI1 for efficient interaction, and they divergently affect the ability of EVI1 to regulate gene transcription in reporter gene assays. Confocal microscopy analysis shows that in the majority of the cells, EVI1 is nuclear and diffused, whereas in about 10%, of the cells EVI1 localizes in nuclear speckles. However, in the presence of the added exogenous co-repressors histone deacetylase or CtBP1, all of the nuclei have a diffuse EVI1 staining, and the proteins do not appear to reside together in obvious nuclear structures. In contrast, when CBP or P/CAF are added, defined speckled bodies appear in the nucleus. Analysis of the staining pattern indicates that EVI1 and CBP or EVIL and P/CAF are contained within these structures. These nuclear structures are not observed when CBP is substituted with a point mutant HAT-inactive CBP with which EVI1 also physically interacts. Finally, we show that the interaction of EVI1 with either CBP or P/CAF leads to acetylation of EVI1. These results suggest that the assembly of EVIL in nuclear speckles requires the intact HAT activity of the co-activators.