Direct interaction of PU.1 with oncogenic transcription factors reduces its serine phosphorylation and promoter binding

The homeostasis of hematopoiesis in the bone marrow is governed by a small number of key transcription factors, including PU.1, GATA-1 and c/EBP alpha. PU.1, a member of the E-twenty-six family of transcription factors, is indispensable for normal hematopoiesis. Inactivation of PU.1 induces acute leukemia in mice. Recent data suggest that the leukemia-associated fusion protein pro-myelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha) inhibits PU.1, but the mechanism mediating this inhibition is unclear. Here, we investigated the mechanisms by which the fusion proteins PML/RAR alpha and pro-myelocytic leukemia zinc finger/RAR alpha (PLZF/RAR alpha) (X-RAR alpha) interfere with the function of PU.1. We found that X-RAR alpha proteins functionally inactivate PU.1 by reducing its promoter-binding capacity, resulting in a reduction in PU.1-dependent transcriptional transactivation. In fact, X-RAR alpha proteins directly interact with PU.1, leading to both the sequestration of PU.1 from its target promoters and a reduction in its serine phosphorylation, which is crucial for its promoter binding and transcriptional activity. We found that the functional inactivation of PU.1 could be overcome by the forced overexpression of PU.1 in PML/RAR alpha- or PLZF/RAR alpha-positive murine hematopoietic progenitor cells; evidently, this overexpression rescued the leukemic differentiation block induced by X-RAR alpha proteins. Our data thus provide strong evidence that X-RARa proteins functionally inhibit PU.1, shedding light on the mechanism by which X-RAR alpha proteins induce leukemogenesis.