Analysis of the Nuclear Distribution of the Translocation t(8;21)-Derived Fusion Protein AML1/ETO by Confocal Laser Scanning Microscopy.

摘要

The AML1/ETO protein derived from the t(8;21) translocation retains the DNA binding domain of AML1, the runt homology domain (RHD), and nearly the complete ETO protein with its four nervy homology regions (NHR1-4). To analyze which domains of AML1/ETO are responsible for its intranuclear transport and its subnuclear distribution, AML1/ETO deletion constructs tagged with green fluorescence protein were expressed transiently in 293 cells. The subcellular distribution was analyzed by confocal laser scanning microscopy. The nuclear localization signal (NLS) of AML1/ETO was mapped to a region encoded by the carboxy-terminal part of NHR1 and the sequences following up to NHR2 corresponding to the amino acids 304-489 of the AML1/ETO protein. A speckled subnuclear distribution was found with those constructs containing the NHR2 and/or the NHR3 and NHR4 domains. Co-localization with AML1/ETO was complete with constructs containing the NHR2 domain, indicating that NHR2 has a crucial role in the subnuclear distribution of AML1/ETO. Co-localization with AML1 seems to be supported by RHD, whereas the NHR3 and NHR4 regions possibly counterbalance this effect. Finally, AML1/ETO could not be co-localized with PML and SUMO-1, indicating that AML1/ETO is not part of the nuclear bodies and probably not SUMOylated.