Abstract
NUP98-HOXA9 is the prototype of NUP98 fusion oncoproteins that cause acute myeloid leukemia. It consists of an N-terminal FG-rich portion of the nucleoporin NUP98 fused to the homeodomain region of the homeobox protein HOXA9, and acts as an aberrant transcription factor. To identify interacting partners of NUP98-HOXA9, we used a cytoplasmic yeast two-hybrid assay to avoid the nonspecific trans-activation that would occur with the traditional yeast two-hybrid assay due to the transactivating properties of NUP98-HOXA9. We identified amino-terminal enhancer of split (AES), a transcriptional regulator of the transducin-like enhancer/Groucho family as a novel interaction partner of NUP98-HOXA9. The interaction was confirmed by in vitro pulldown and co-immunoprecipitation assays and was shown to require the FG repeat region of NUP98-HOXA9. Immunofluorescence analysis showed that AES localizes primarily to the interior of the nucleus. AES also showed a strong interaction with wild-type NUP98. AES augmented the transcriptional activity of NUP98-HOXA9. In the presence of NUP98-HOXA9, AES caused an increase in long-term proliferation of primary human CD34+ cells with a marked increase in the numbers of primitive cells. These effects of AES were not observed in the absence of NUP98-HOXA9. AES knockdown diminished the transcriptional and proliferative effects of NUP98-HOXA9. AES caused a shift away from the erythroid lineage in cells expressing NUP98-HOXA9. These data establish AES as an interacting partner of NUP98-HOXA9 and show that it cooperates with NUP98-HOXA9 in transcriptional regulation and cell transformation.
Highlights
The oncoprotein NUP98-HOXA9 deregulates transcription and induces cell proliferation leading to acute myeloid leukemia (AML)
Cytotrap Two-hybrid Analysis Shows That NUP98-HOXA9 Interacts with amino-terminal enhancer of split (AES)—To identify possible interaction partners of the oncoprotein NUP98-HOXA9, we used a Cytotrap yeast two-hybrid assay in which the interaction between target and the bait proteins occurs in the cytoplasm
The traditional twohybrid assay relies on intranuclear interactions that result in transactivation and is likely to show many false positives when the bait contains a transactivation domain as is the case with NUP98-HOXA9 [24]
Summary
The oncoprotein NUP98-HOXA9 deregulates transcription and induces cell proliferation leading to acute myeloid leukemia (AML). NUP98-HOXA9 is the prototype of NUP98 fusion oncoproteins that cause acute myeloid leukemia It consists of an N-terminal FG-rich portion of the nucleoporin NUP98 fused to the homeodomain region of the homeobox protein HOXA9, and acts as an aberrant transcription factor. We identified amino-terminal enhancer of split (AES), a transcriptional regulator of the transducin-like enhancer/Groucho family as a novel interaction partner of NUP98-HOXA9. The transcriptional regulator amino-terminal enhancer of split (AES) was identified as a novel interaction partner of NUP98-HOXA9 by this method. This interaction was found to enhance the ability of NUP98-HOXA9 to transform primary human hematopoietic cells.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
