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Mol Cell Biol, July 1998, p. 4347-4357, Vol. 18, No. 7
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Normal Myeloid Development Requires Both the
Glutamine-Rich Transactivation Domain and the PEST Region of
Transcription Factor PU.1 but Not the Potent Acidic
Transactivation Domain
Robert C.
Fisher,1
Marilyn C.
Olson,2
Jagan M. R.
Pongubala,3
Jeffrey M.
Perkel,3
Michael L.
Atchison,3
Edward W.
Scott,1 and
M. Celeste
Simon2 4
Institute for Human Gene
Therapy1 and
Department of Animal
Biology School of Veterinary Medicine,3
University of Pennsylvania, Philadelphia, Pennsylvania 19104, and
Howard Hughes Medical Institute2 and
Departments of Medicine and of Molecular Genetics and Cell
Biology,4 The University of Chicago, Chicago,
Illinois 60637
Received 15 January 1998/Returned for modification 28 February
1998/Accepted 23 March 1998
Gene targeting of transcription factor PU.1 results in an early
block to fetal hematopoiesis, with no detectable lymphoid or myeloid
cells produced in mouse embryos. Furthermore,
PU.1
/
embryonic stem (ES) cells fail to
differentiate into Mac-1+ and F4/80+
macrophages in vitro. 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 differentiate into
macrophages. In this study, we take advantage of our
PU.1
/
ES cell rescue system to genetically
test which previously identified PU.1 functional domains are necessary
for the development of mature macrophages. PU.1 functional domains
include multiple N-terminal acidic and glutamine-rich transactivation
domains, a PEST domain, several serine phosphorylation sites, and a
C-terminal Ets DNA binding domain, all delineated and characterized by
using standard biochemical and transactivational assays. By using the
production of mature macrophages 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. Deletion of three acidic domains, which
exhibit potent transactivation potential in vitro, had no effect on the ability of PU.1 to promote macrophage development. Furthermore, mutagenesis of four independent sites of serine phosphorylation also
had no effect on myelopoiesis. Collectively, our results indicate that
PU.1 interacts with important regulatory proteins during macrophage
development via the glutamine-rich and PEST domains. The
PU.1
/
ES cell rescue system represents a
powerful, in vitro strategy to functionally map domains of PU.1
essential for normal hematopoiesis and the generation of mature
macrophages.
Present address: Third Wave Technologies, Inc., Madison, Wis.
Mol Cell Biol, July 1998, p. 4347-4357, Vol. 18, No. 7
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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