Chakraborty S, Senyuk V, Sitailo S, Chi Y, Nucifora G
Department of Pathology and The Cancer Center, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
J Biol Chem. 2001 Nov 30;276(48):44936-43. doi: 10.1074/jbc.M106733200. Epub 2001 Sep 21.
EVI1 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 EVI1 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 EVI1 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 EVI1 in nuclear speckles requires the intact HAT activity of the co-activators.
EVI1是一种非常复杂的蛋白质，含有两个锌指结构域，在多种人类髓系白血病中异常表达。通过报告基因检测，几位研究人员表明EVI1是一种转录抑制因子，最近有研究表明EVI1与共抑制因子羧基末端结合蛋白1（CtBP1）相互作用。早些时候，我们发现EVI1在小鼠造血前体细胞中的异常表达会导致它们的异常分化和增殖增加。通过生化分析，我们鉴定出两组与EVI1相关的转录共调节因子，推测它们参与调节基因表达。一组共调节因子包括CtBP1和组蛋白去乙酰化酶。第二组包括两种共激活因子，即环磷酸腺苷反应元件结合蛋白结合蛋白（CBP）和p300/CBP相关因子（P/CAF），它们都具有组蛋白乙酰转移酶（HAT）活性。所有这些蛋白质都需要EVI1的不同区域才能有效相互作用，并且它们在报告基因检测中对EVI1调节基因转录的能力有不同影响。共聚焦显微镜分析表明，在大多数细胞中，EVI1位于细胞核且呈弥散分布，而在约10%的细胞中，EVI1定位于核斑。然而，在添加外源性共抑制因子组蛋白去乙酰化酶或CtBP1的情况下，所有细胞核中的EVI1染色均呈弥散状，且这些蛋白质似乎并不共同存在于明显的核结构中。相反，当添加CBP或P/CAF时，细胞核中会出现明确的斑点状结构。染色模式分析表明，EVI1与CBP或EVI1与P/CAF包含在这些结构中。当用与EVI1也有物理相互作用的点突变失活HAT的CBP替代CBP时，未观察到这些核结构。最后，我们表明EVI1与CBP或P/CAF的相互作用会导致EVI1的乙酰化。这些结果表明，EVI1在核斑中的组装需要共激活因子完整的HAT活性。
