Regulation of Gene Expression in 
Developing Lymphocytes 
A ' U 
Stephen T. Smale, Ph.D. — Assistant Investigator 
Dr. Smale is also Assistant Professor of Microbiology and Immunology and a member of the Molecular 
Biology Institute at the University of California School of Medicine, los Angeles. He received his Ph.D. 
degree in biochemistry from the University of California, Berkeley, where he studied with Robert Tjian. 
Dr. Smale's postdoctoral research was done with David Baltimore at the Whitehead Institute, Massachu- 
setts Institute of Technology. 
TT w fcMATOPOIESIS refers to a complex develop- 
JL JLmental process through which pluripotent 
stem cells in mammalian fetal liver or adult bone 
marrow give rise to several types of terminally 
differentiated blood cells. The immune system in- 
corporates many of these cell types, which in- 
clude B and T lymphocytes, granulocytes, mono- 
cytes, and killer cells, to protect an organism 
from infection by a variety of means. Thus an ef- 
fective immune response depends on a properly 
functioning hematopoietic pathway. 
When defects arise in this pathway, the effects 
are often severe. In some cases, precursor cells 
cannot develop to maturity, resulting in immuno- 
deficiency. In other cases, uncontrolled prolifera- 
tion of developing cells results in leukemias and 
lymphomas. To understand the basis of these de- 
fects, we need to explain at the molecular level 
the regulation of the hematopoietic pathway. 
The primary objective of our research is to 
identify and characterize regulatory proteins for 
one stage of hematopoiesis: the maturation path- 
way for B lymphocytes. These cells produce anti- 
body molecules and therefore play a central role 
in an immune response. In numerous laboratories 
a variety of approaches have been employed to 
define molecules that regulate B cell maturation. 
One approach has been to identify components 
of the bone marrow microenvironment (which 
surrounds precursor B cells) essential for cell 
growth and differentiation. One such component 
is a soluble growth factor called interleukin-7, 
which is secreted by bone marrow-derived cells 
and promotes proliferation of specific pre-B cell 
types. Additional growth factors and stromal cell 
membrane proteins have been identified that may 
promote further stages of B cell development. 
A second approach has been to characterize 
genes that obstruct the differentiation process. 
These genes include several viral oncogenes and 
their cellular homologues. The Y-abl oncogene, 
for example, is leukemogenic in specific pre-B 
cell types, preventing them from further differ- 
entiation. The c-myc oncogene also facilitates 
pre-B cell transformation, and one study has 
shown that c-myc transformation was prevented 
by artificially altering the B cell differentiation 
pathway. Further studies of the structure and 
function of these oncogenes are providing insight 
into the control of differentiation. 
We are utilizing a third approach to define B 
cell regulatory molecules. We are identifying 
proteins that directly activate or inactivate genes 
expressed at specific stages of differentiation. 
The regulation of gene expression appears 
usually to be carried out by modulation of the 
rate of initiating transcription (RNA synthesis). 
Transcriptional analysis as a means of studying B 
cell differentiation has already been employed 
successfully and extensively with the genes that 
encode the antibody, or immunoglobulin (Ig), 
molecules. In fact, transcription of these Ig genes 
has been studied in more detail than that of al- 
most any other cellular gene. 
These studies have revealed at least two impor- 
tant regulators of B cell differentiation. Both regu- 
latory proteins, OCT-2 and NF-/cB, bind to DNA 
sequences in the control regions for Ig genes and 
play a role in specific activation of Ig RNA synthe- 
sis in developing B cells. Besides these two pro- 
teins, several other DNA-binding proteins, 
currently less well characterized, have been 
identified that also may play a role in regulating 
Ig transcription. 
The analysis of Ig transcription and the identifi- 
cation of OCT-2 and NF-/cB have contributed sig- 
nificantly to our understanding of B cell differen- 
tiation. Surprisingly, however, there has been 
little effort toward characterizing the transcrip- 
tional regulation of several other genes known to 
be expressed specifically in early B cells. We 
therefore have chosen to extend the analysis of B 
cell differentiation by focusing on the transcrip- 
tional regulation of a new gene that is regulated 
in a precise fashion during B cell ontogeny: 
the terminal deoxynucleotidyltransferase (TdT) 
gene. This gene encodes a DNA polymerase that 
appears to play a role in generating diversity 
within the antibody molecules. 
We chose to analyze TdT expression for four 
reasons. First, the gene is tightly regulated, being 
expressed exclusively in immature lymphocytes. 
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