pathophysiology and management of inherited and 
acquired disorders of hematopoiesis. 
Erythroid Cell Development 
During red blood cell development, globin genes 
are activated and precisely regulated, both with re- 
spect to cell lineage and temporal expression of in- 
dividual members. These genes are expressed in a 
developmental sequence from embryonic through 
adult life. To address molecular mechanisms of red 
cell development, Dr. Orkin and his colleagues have 
focused on a DNA-binding protein, GATA- 1 , that par- 
ticipates in transcriptional control of all known 
erythroid-expressed genes. 
This protein, which recognizes a consensus GATA 
motif in its DNA targets, is part of a larger family of 
proteins that are related by virtue of highly similar 
"finger" DNA-binding domains. GATA-1 is ex- 
pressed at the very earliest stages of blood cell devel- 
opment and in multipotential progenitor hemato- 
poietic cells that have not yet chosen a unique 
development path. 
Previously, gene-targeting experiments in mouse 
embryo-derived stem (ES) cells established an es- 
sential role for GATA-1 in erythroid development. 
More recently, a stringent assay for GATA- 1 function 
in vivo has been devised that employs introduction 
of the GATA-1 gene into GATA-1 ES cells to rescue 
erythroid development. This assay permits delinea- 
tion of the specific domains of GATA- 1 required for 
promoting erythroid gene expression within devel- 
oping cells. 
In complementary studies, the binding of pro- 
teins to the critical regulatory elements of the hu- 
man globin gene clusters, the locus control regions, 
has been evaluated by an improved in vivo foot- 
printing method. These studies define GATA-1 and a 
second erythroid protein, NF-E2, as the critical com- 
ponents for establishing erythroid-specific gene ex- 
pression. This latter protein, recently purified, and 
its cDNA cloned, will be the focus of future studies. 
White Blood Cell Development 
Phagocytic cells (neutrophils and macrophages) 
produce superoxide via an NADPH-oxidase com- 
plex as part of a major bactericidal host-defense sys- 
tem. Deficiency of this oxidase leads to an immune 
condition, chronic granulomatous disease (CGD). 
Previously the gene encoding a major component of 
the oxidase (gp91-phox) was identified through 
positional cloning in the laboratory. Subsequent 
studies have shown that mutations in the gp9 1 -phox 
gene lead to one form of CGD. The gp9 1 -phox gene 
is expressed only in developing phagocytic cells. To 
identify important regulators of white cell gene ex- 
pression and development, the gp9 1 -phox gene and 
its controlling elements are under study. 
In transgenic mice the promoter region alone di- 
rects limited cell specificity and can target malig- 
nancy to a subset of macrophages when linked to the 
simian virus 40 (SV40) T antigen. Protein-binding 
and functional studies revealed that the gp9 1 -phox 
promoter is under negative regulation through a pu- 
tative repressor protein CCAAT displacement factor 
(CDP). Molecular cloning revealed that CDP is the 
counterpart of a Drosophila homeodomain protein, 
encoded by the cut gene, which is involved in cell- 
fate decisions in several tissues in the fly. 
Experiments under way are aimed at determining 
how CDP recognizes DNA and functions to repress 
gene expression in undifferentiated white cells. 
Other experiments are aimed at defining the posi- 
tive regulatory elements specifying gp9 1 -phox ex- 
pression in white cells. 
Dr. Orkin is also Leland Fikes Professor of Pedi- 
atric Medicine at Harvard Medical School and the 
Children's Hospital, Boston. 
Articles 
Dinauer, M.C., Pierce, E.A., Erickson, R.W., Muhle- 
bach, T.J., Messner, H., Orkin, S.H., Seger, R.A., 
and Curnutte, J.T. 1991. Point mutation in the 
cytoplasmic domain of the neutrophil p22-phox 
cytochrome b subunit is associated with a non- 
functional NADPH oxidase and chronic gran- 
ulomatous disease. Proc Natl Acad Sci USA 
88:11231-11235. 
Dorfman, D.M., Wilson, D.B., Bruns, G AP., and 
Orkin, S.H. 1992. Human transcription factor 
GATA-2: evidence for regulation of preproen- 
dotheIin-1 gene expression in endothelial cells./ 
Biol Chem 267:1279-1285. 
Neufeld, E.J., Skalnik, D.G., Elevens, P.M. -J., and 
Orkin, S.H. 1992. Human CCAAT displacement 
protein is homologous to the Drosophila homeo- 
protein, cut. Nature Genet 1:50-55. 
Orkin, S.H. 1992. GATA-binding transcription fac- 
tors in hematopoietic cells. Blood 80:575-581. 
Simon, M.C., Pevny, L., Wiles, M.V., Keller, G., Cos- 
tantini, F., and Orkin, S.H. 1992. Rescue of ery- 
throid development in gene targeted GATA-1" 
mouse embryonic stem cells. Nature Genet 1: 
92-98. 
Skalnik, D.G., Dorfman, D.M., Perkins, A.S., Jenkins, 
N.A., Copeland, N.G., and Orkin, S.H. 1991 . Tar- 
geting of the transgene expression to monocyte/ 
macrophages by the gp91-phox promoter and 
consequent histiocytic malignancies. Proc Natl 
Acad Sci USA 88:8505-8509. 
GENETICS 243 
