Alm-Kristiansen A H, Saether T, Matre V, Gilfillan S, Dahle O, Gabrielsen O S
Department of Molecular Biosciences, University of Oslo, Oslo, Norway.
Oncogene. 2008 Aug 7;27(34):4644-56. doi: 10.1038/onc.2008.105. Epub 2008 Apr 14.
The c-Myb oncoprotein is a DNA-binding transcription factor with a key role in early stages of hematopoiesis. To expand our knowledge of partners cooperating with c-Myb, we performed a yeast two-hybrid screening with full-length c-Myb as bait. Here, we report FLICE-associated huge protein (FLASH)/CASP8AP2 as a novel Myb-interacting protein. We show that FLASH interacts with the DNA-binding domain of c-Myb and enhances c-Myb-dependent reporter activity and expression of endogenous c-Myb target genes. Chromatin immunoprecipitation assays revealed that FLASH and c-Myb both associate with the MYC promoter region as well as with the intronic enhancer of the c-Myb target gene ADA. Furthermore, siRNA knock-down of FLASH or c-Myb both result in a reduction of MYC and ADA expression. The co-activator effect is mediated through the C-terminal part of FLASH, which binds c-Myb. The FLASH-induced enhancement is comparable with the increase seen with the c-Myb co-activator p300. We find FLASH localized in discrete nuclear speckles in several cell lines, co-localized with c-Myb in active RNA polymerase II foci. These results imply a novel molecular mechanism of regulation of c-Myb activity. We propose that c-Myb cooperates with FLASH in foci associated with active RNA polymerase II, leading to enhancement of Myb-dependent gene activation.
c-Myb癌蛋白是一种DNA结合转录因子，在造血早期阶段起关键作用。为了拓展我们对与c-Myb协同作用的伙伴的认识，我们以全长c-Myb作为诱饵进行了酵母双杂交筛选。在此，我们报告FLICE相关巨蛋白（FLASH）/半胱天冬酶8相关蛋白2（CASP8AP2）是一种新型的与Myb相互作用的蛋白。我们发现FLASH与c-Myb的DNA结合结构域相互作用，并增强c-Myb依赖的报告基因活性以及内源性c-Myb靶基因的表达。染色质免疫沉淀分析显示，FLASH和c-Myb均与MYC启动子区域以及c-Myb靶基因ADA的内含子增强子相关联。此外，对FLASH或c-Myb进行小干扰RNA敲低均导致MYC和ADA表达降低。共激活效应是通过FLASH的C末端介导的，该末端与c-Myb结合。FLASH诱导的增强作用与c-Myb共激活因子p300所导致的增加相当。我们发现FLASH定位于几种细胞系中离散的核斑点中，与c-Myb在活跃的RNA聚合酶II位点共定位。这些结果暗示了一种调节c-Myb活性的新分子机制。我们提出c-Myb在与活跃的RNA聚合酶II相关的位点与FLASH协同作用，导致Myb依赖的基因激活增强。
