NORMAL MYELOID DEVELOPMENT REQUIRES BOTH THE GLUTAMINE-RICH TRANSACTIVATION DOMAIN AND THE PEST REGION OF TRANSCRIPTION FACTOR PU.1 BUT NOTTHE POTENT ACIDIC TRANSACTIVATION DOMAIN

Gene targeting of transcription factor PU.1 results in an early block to fetal hematopoiesis, with no detectable lymphoid or myeloid cells p roduced in mouse embryos. Furthermore, PU.1(-/-) embryonic stem (ES) c ells fail to differentiate into Mac-1(+) and F4/80(+) macrophages in v itro. We have previously shown that a PU.1 transgene under the control of its own promoter restores the ability of PU.1(-/-) ES cells to dif ferentiate into macrophages. In this study, we take advantage of our P U.1(-/-) ES cell rescue system to genetically test which previously id entified PU.1 functional domains are necessary for the development of mature macrophages. PU.1 functional domains include multiple N-termina l acidic and glutamine-rich transactivation domains, a PEST domain, se veral serine phosphorylation sites, and a C-terminal Ets DNA binding d omain, all delineated and characterized by using standard biochemical and transactivational assays. By using the production of mature macrop hages as a functional readout in our assay system, we have established that the glutamine-rich transactivation domain, a portion of the PEST domain, and the DNA binding domain are required for myelopoiesis. Del etion of three acidic domains, which exhibit potent transactivation po tential in vitro, had no effect on the ability of PU.1 to promote macr ophage development. Furthermore, mutagenesis of four independent sites of serine phosphorylation also had no effect on myelopoiesis. Collect ively, our results indicate that PU.1 interacts with important regulat ory proteins during macrophage development via the glutamine-rich and PEST domains. The PU.1(-/-) ES cell rescue system represents a powerfu l, in vitro strategy to functionally map domains of PU.1 essential for normal hematopoiesis and the generation of mature macrophages.