Genetic Regulatory Mechanisms in Cellular 
Differentiation 
Gerald R. Crabtree, M.D. — Associate Investigator 
Dr. Crabtree is also Associate Professor of Pathology at Stanford University School of Medicine. He received 
his B.S. degree from West Liberty State College, West Virginia, and his M.D. degree from Temple University 
School of Medicine, Philadelphia. He was a Senior Investigator at the NIH before coming to Stanford 
University. 
CELLS acquire their final differentiated func- 
tion by a complex interplay between primary 
genetic regulatory events in the nucleus and 
interactions at the cell membrane. Building on 
concepts largely provided from studies on lower 
animals, our laboratory has been exploring regu- 
latory mechanisms that help determine how 
mammalian cells differentiate to assume their 
normal functions. 
T Lymphocyte Activation and 
Differentiation 
T lymphocytes undergo two biologically and 
medically important types of differentiation. The 
first occurs in the thymus and generates cells ca- 
pable of directing an immune response to nearly 
any antigen. However, the cells produced by the 
thymus that circulate in our blood are immuno- 
logically nearly inert. They acquire immunologic 
function as a result of a second process of cellular 
differentiation that takes about 10-14 days and 
produces T cells able to coordinate the actions of 
other cells involved in the immune response and 
carry out a variety of immune functions. This 
pathway of differentiation is initiated by a com- 
plex interaction between the T cell and an anti- 
gen-presenting cell. The essential requirement 
for a commitment to specialized function is a 
highly specific interaction between histocompati- 
bility molecules, antigen, and the antigen recep- 
tor. This critical interaction is only effective 
when stabilized by transient nonspecific interac- 
tions based on intracellular adhesive molecules. 
Finally, lymphokines such as interleukin- 1 and -6 
that are the secreted products of the antigen-pre- 
senting cell are necessary accessory signals to ini- 
tiate differentiation. These three requirements 
for the initiation of differentiation — a highly spe- 
cific cell-cell interaction, a nonspecific adhesive 
interaction, and cytokines — are similar to the re- 
quirements for the cellular commitment to dif- 
ferentiate in other systems. 
Because the interaction between the antigen- 
presenting cell and a T lymphocyte is transient 
(lasting only about 30 minutes), all of the molec- 
ular events required for the decision to proceed 
down this 1 0- to 1 4 -day process of cellular differ- 
entiation must occur during this short period. 
Our laboratory is seeking an understanding of the 
molecular basis of this cellular decision. 
By analyzing the regulatory regions of genes 
that are essential for T cell activation, such as in- 
terleukin- 2, we have identified several molecules 
that appear critical to initiating T cell activation. 
These molecules interact on the promoters of 
genes necessary for T cell activation in such a way 
that all must be present for the gene to be acti- 
vated. Thus the all-or-none decision of a T lym- 
phocyte to differentiate appears related to coop- 
erative interactions between molecules binding 
to DNA and activating the genes, again in an on- 
or-off manner, essential for progression toward 
differentiated function. 
Recently, our studies of the immunosuppres- 
sive drug cyclosporin A have given us insight into 
the relative importance of the events involved in 
the initiation of T cell activation. Although the 
mechanism of action of this drug is largely un- 
known, it appears to work early during the com- 
mitment period for T cells. By doing this, it 
blocks the late functions of T cells and also many 
of the functions of B lymphocytes and other hema- 
topoietic cells that are directed by T cells. One of 
the proteins we identified in our early studies, 
nuclear factor of activated T cells (NFAT) , is ex- 
quisitely sensitive to the effects of cyclosporin A, 
while nearly all other proteins are unaffected. 
Other groups had found that cyclosporin A binds 
and inhibits the function of a cis-trans prolyl iso- 
merase. These enzymes accelerate the folding of 
newly synthesized proteins. In studies with Stuart 
Schreiber (Harvard University), we have shown 
that the isomerase activity is not involved in the 
action of cyclosporin A and FK-506 (another im- 
munosuppressant) but rather that an inhibitory 
complex formed between the isomerase and the 
drug blocks signal transduction. In other studies 
we found that the specific transcriptional activity 
of NFAT, but not its DNA-binding activity, is af- 
fected by cyclosporin A and FK-506, suggesting 
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