Molecular Genetic Studies of 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 NIH under the 
supervision of Philip Leder. Upon returning to Harvard, Dr. Orkin received specialty training in pediatric 
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 a member of the National Academy of Sciences. 
ALL mature blood cells are derived from plur- 
ipotem hematopoietic stem cells, which 
constitute a rare population in the bone marrow. 
The decision of stem cells to differentiate leads to 
the production of a heterogeneous array of cells 
with varying developmental potentials and with 
commitment to expression of lineage-specific 
protein products. A major goal of this laboratory 
is an improved understanding of hematopoietic 
cell development and the expression and func- 
tion of specific genes that relate to the normal 
biology of hematopoietic cells. 
Efforts are directed to analyses of both red and 
white blood cells. These cell types are important 
in severe, clinically significant genetic disorders 
in which the capacity to produce specific pro- 
teins is impaired by mutation. In these studies we 
seek to describe the molecular basis of inherited 
disorders, understand the normal regulation of 
the affected genes, and utilize the findings from 
this basic work to formulate novel treatments 
based on molecular biologic considerations. 
One of the major, classical disorders of red 
cells is ;8-thalassemia (also known as Cooley's ane- 
mia) , in which the synthesis of hemoglobin is de- 
fective. Through molecular cloning and gene ex- 
pression, the molecular basis of the disease was 
determined in this laboratory several years ago. 
Now the unsolved problems are related to how 
globin genes are normally regulated in develop- 
ing erythroid precursor cells. Specifically, how 
are the globin genes activated only in red cells? 
How are different globin genes regulated in devel- 
opment? How do erythroid precursor cells arise 
during development from progenitor cells that 
have the potential to yield either red or white 
cells? 
To approach these general problems, 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 first discovered that rec- 
ognizes a small DNA motif (GATA) found in 
the promoters or enhancers of all erythroid- 
expressed genes. Through molecular cloning, 
mammalian, avian, and amphibian homologues 
were characterized. 
The protein is modular, consisting of a novel 
two-finger structure required for DNA-binding 
and other domains that serve as potent positive 
activators of gene transcription. Expression of the 
protein in two other hematopoietic cell types, 
megakaryocytes and mast cells, suggests that it is 
first expressed in a multipotential progenitor cell 
and may regulate genes in those cell types as 
well. Recent data have supported these conclu- 
sions. Attention has been directed to how this 
transcription factor is itself regulated in hemato- 
poietic cells. An improved understanding may 
provide important insights into the initial events 
involved in erythroid decision-making and 
maturation. 
Studies of the gene revealed an element in the 
promoter region that serves as a site for positive 
autoregulation. In this manner, expression of the 
factor tends to maintain its own expression and 
stabilize the differentiated state. Furthermore, 
the promoter for the receptor for the erythroid- 
specific growth factor, erythropoietin, is under 
control by this transcription factor. By such cir- 
cuitry, expression of the factor tends to guarantee 
subsequent erythroid development and viability. 
Site-specific gene disruption in mouse embryo- 
derived stem cells and generation of chimeric an- 
imals has also revealed that the protein is essen- 
tial for normal erythroid development and that 
related proteins that bind the GATA motif cannot 
compensate for its absence. Using in vitro differ- 
entiation of embryo-derived stem cells into hema- 
topoietic cell types, we have developed an exper- 
imental system that permits assessment of the 
role of GATA-transcription factor in erythroid de- 
velopment and systematic testing of various 
aspects of the function and/or regulation of the 
protein. 
In separate but conceptually related studies, a 
gene that encodes an essential component of the 
white blood cell (phagocytic) system responsi- 
ble for killing ingested microorganisms is being 
examined in an effort to understand how this 
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