Genetic Regulatory Mechanisms 
in Cellular Differentiation 
Gerald R. Crahtree, 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 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 in- 
teractions at the cell membrane. Building on con- 
cepts 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. 
Isolation of a Trans-acting Regulator 
of Homeodomain Protein Function 
Several years ago, we identified a tissue- 
specific transcription factor, HNF-la, that inter- 
acts with essential regions of the promoters of a 
large family of genes expressed in endodermally 
derived tissues. After purifying the protein and 
cloning its gene, we found that HNF-1 contains a 
homeodomain similar to that found in genes de- 
termining body form in insects. Curiously, the 
protein dimerizes through an amino acid se- 
quence unlike that found in other homeodomain- 
containing proteins. This led us to look for a pro- 
tein that might heterodimerize with it and hence 
diversify its regulatory capabilities. We found 
such a protein by screening a hepatocyte cDNA 
library at low stringency. This protein, HNF-1/8, 
which is expressed in a partially overlapping 
group of tissues with HNF-la, contains a dimer- 
ization and homeodomain similar to those of 
HNF-la, but a different transcriptional activation 
domain. 
Surprisingly, HNF-la was only able to activate 
transcription in certain tissues, suggesting the ex- 
istence of a second tissue-specific protein that 
regulated its function. Dirk Mendel began a 
search for such a protein by purifying HNF- 1 a by 
means that do not disturb hydrophobic interac- 
tions. HNF-la copurified with an 1 1-kDa protein 
that participated in the formation of a tetrameric 
complex. The 1 1 -kDa protein, which was cloned, 
expressed, and found to enhance the affinity of 
dimerization between HNF molecules, was 
named DCoH (dimerization cofactor for HNF- 1 ) . 
In both cases DCoH required the amino-terminal 
32 amino acids that constitute the dimerization 
domain for binding to the complex consisting of 
two molecules of HNF-la and two molecules of 
DCoH. Developmentally, the DCoH protein is ex- 
pressed early (about day 9 or 10 after fertiliza- 
tion) in mice and is expressed in a group of tis- 
sues that do not always express HNF-la or -1/3, 
suggesting that other tissues such as the brain 
contain proteins that can bind to DCoH. When 
co-expressed with HNF-la, DCoH enhances tran- 
scriptional activation by several hundredfold; we 
are presently investigating the mechanism by 
which this occurs. 
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 that coordinate the actions of 
other cells involved in the immune response by 
production of cytokines and cell-cell interac- 
tions. This differentiation pathway is initiated by 
a complex interaction between the T cell and an 
antigen-presenting cell. The essential require- 
ment for a commitment to specialized function is 
a highly specific interaction between histocom- 
patibility molecules, antigen, and the antigen re- 
ceptor. 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- 
presenting cell are accessory signals necessary to 
initiate 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- 
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