Fusion of NPM to RARA in t(5;17) Disrupts Its Ability To Interact with YY1 Transcription Factor.

The transcription factor Ying-Yang 1 (YY1) can function either as a transcriptional activator or transcriptional repressor, depending on context. It has been reported that YY1 is a myeloid transforming gene interfering with neutrophilic differentiation. Further, it has been shown that the nucleolar phosphoprotein Nucleophosmin (NPM) directly interacts with YY1 in cells. As a consequence of NPM overexpression, YY1 repression is diminished. We previously reported the expression of an NPM fusion protein in the t(5;17)(q35;q21) variant of acute promyelocytic leukemia. The fusion protein encodes the N-terminal 118 amino acids of NPM fused to the retinoic acid receptor alpha (RARA). We previously showed that NPM-RAR inhibits myeloid differentiation in both U937 and transgenic model systems. We hypothesized that NPM-RAR might contribute to abnormal myeloid differentiation by altering YY1-mediated transcription pathways. To test this hypothesis, we sought to determine whether the NPM-RAR protein interacted with YY1 in the same manner as wild-type NPM. We generated a eukaryotic expression vector encoding FLAG-tagged YY1, and used it to transform COS cells. Full-length NPM, or NPM-RAR, were subcloned into a vector encoding the maltose binding protein (MBP), in the appropriate reading frame to generate MBP-fusions. The MBP proteins were expressed in bacteria, and purified by affinity chromatography. MBP, MPB-NPM, or MBP-NPM-RAR were incubated with YY1-COS lysates, and complexes were captured on amylose resin. The resin-complexes were washed, and the presence of YY1 was detected by immunoblotting using an anti-FLAG antibody. As expected, MBP-NPM pulled down FLAG-YY1 under these conditions. However, we were unable to detect association between MBP-NPM-RAR and FLAG-YY1. To determine whether this finding might be attributable to lack of post-translational modification of NPM-RAR in the bacterial system, we co-transfected expression vectors for FLAG-YY1 and NPM-RAR into COS cells. Lysates were subjected to immunoprecipitation using anti-RARA polyclonal antisera, and the presence of co-precipitating YY1 was detected by immunoblotting using an anti-FLAG antibody. Again, we were unable to detect direct interaction between the proteins. We conclude that NPM-RAR does not directly interact with YY1. This data does not exclude the possibility that YY1 activity could be altered in t(5;17) by decreased expression of wild-type NPM.