Molecular Genetics Studies on Hematopoietic Cells 
Stuart H. Orkin, M.D. — Investigator 
Dr. Orkin is also Leland Pikes Professor of Pediatric Medicine at Harvard Medical School. He received his 
B.S. degree in biology from the Massachusetts Institute of Technology and his M.D. degree from Harvard 
Medical School. His postdoctoral research was in the Laboratory of Molecular Genetics at the NIH under 
the supervision of Philip Leder. Upon returning to Harvard, Dr. Orkin received specialty training in pedi- 
atric hematology at the Children's Hospital, where he later joined the faculty. His many honors include 
the Clinical Investigator Award from the American Federation for Clinical Research and the Dameshek 
prize of the American Society of Hematology. He is Past President of the American Society of Clinical 
Investigation. Dr. Orkin was recently elected to the National Academy of Sciences. 
ALL blood cells derive from pluripotent stem 
cells in the bone marrow. The decision of 
stem cells to differentiate leads to the production 
of a heterogeneous population of cells with vary- 
ing developmental potentials and commitment to 
expression of lineage-specific protein products. 
A goal of this laboratory is an improved under- 
standing of hematopoietic development and the 
expression and function of specific genes that re- 
late to the normal biology of hematopoietic cells. 
Our efforts are concentrated on the analysis of 
both red and white blood cells. These cell types 
are important in severe, clinically significant hu- 
man genetic disorders in which the capacity to 
produce specific proteins is impaired by muta- 
tion. We seek to understand the molecular basis 
of these inherited disorders, delineate the normal 
regulation of affected genes, and utilize what is 
learned to formulate novel treatments based on 
molecular biologic considerations. 
One of the major disorders of red blood cells is 
/3-thalassemia (Cooley's anemia), in which the 
synthesis of hemoglobin is defective. Through 
molecular cloning and gene expression the mo- 
lecular basis of the disease was determined in this 
laboratory several years ago. The major, unsolved 
problems now relate to how globin genes are nor- 
mally regulated in the developing erythroid pre- 
cursor cells. Specifically, how are globin genes 
activated only in red cells but not in other tissues? 
How are different globin genes regulated in devel- 
opment? To examine these general issues we have 
concentrated on identifying and characterizing 
unique DNA-binding proteins that appear to be 
major transcriptional regulators in erythroid 
cells. 
A prominent, apparently erythroid-specific 
DNA-binding protein was discovered that recog- 
nizes a small DNA motif found in the promoters 
or enhancers of virtually all erythroid-expressed 
genes, and the human, mouse, and frog homo- 
logues were cloned. The protein is modular, con- 
sisting of a novel two-finger structure required 
for DNA binding and other domains that serve as 
potent activators of gene transcription. The ex- 
pression of this protein in two other hematopoi- 
etic cell types, megakaryocytes and mast cells, 
suggests that it is first expressed in a multipoten- 
tial progenitor cell and may regulate genes in 
those cell types as well. 
An understanding of how this transcription fac- 
tor is itself regulated in erythroid cells may pro- 
vide important insights into the initial events in 
erythroid decision making and maturation. Re- 
cent findings indicate that the gene is activated 
early in hematopoietic development and is later 
subject to positive autoregulation of its pro- 
moter. Site-specific gene disruption in mouse 
embryo stem cells and generation of chimeric an- 
imals has also revealed that the protein is essen- 
tial for normal erythroid differentiation and that 
other proteins binding to the GATA motif cannot 
compensate for its absence. The focus of our stud- 
ies is to understand in detail how this transcrip- 
tion factor functions in normal erythroid develop- 
ment and how its expression is first turned on in 
early progenitor differentiation. Ultimately these 
studies may provide new clues to differential reg- 
ulation of globin genes and the prospects for di- 
rected manipulation of their expression for the 
treatment of hemoglobinopathies. 
In a separate but conceptually related group of 
studies a gene that encodes an essential compo- 
nent of the white blood cell (phagocytic) system 
responsible for killing ingested microorganisms 
is being examined. We wish to understand how 
this clinically important host defense system is 
regulated and, more generally, how cell-specific 
gene expression is achieved in this lineage, also 
descendent from the pluripotent stem cell. The 
gene under study encodes a subunit of a unique 
cytochrome that is defective in chronic granulo- 
matous disease, an X-linked condition. By reverse 
genetics (positional cloning) we previously iso- 
lated the relevant gene, determined its structure, 
and demonstrated the presence of protein prod- 
uct in the cytochrome complex of phagocytic 
cells. In addition, because interferon-7 stimu- 
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