Regulation of Gene Expression 
in Developing Lymphocytes 
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, 
Massachusetts Institute of Technology. 
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HEMATOPOIESIS refers to a complex develop- 
mental 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 the host from 
infection by a variety of means. When defects 
arise in the hematopoietic 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 must explain the regulation of hema- 
topoiesis at the molecular level. 
The approach used in our laboratory to study 
the regulation of the hematopoietic pathway is to 
identify proteins that directly activate or inacti- 
vate genes expressed at specific stages of B and T 
lymphocyte development. Lymphocytes play a 
central role in the immune response by mediat- 
ing the recognition of foreign and infectious mat- 
ter. As B and T cells mature, a wide variety of 
genes are turned on and off in a specific temporal 
pattern. An analysis of the mechanisms by which 
these genes are precisely controlled is a good 
starting point for studying the regulation of 
hematopoiesis. 
Our laboratory currently focuses on the regula- 
tion of the gene encoding terminal deoxynucleo- 
tidyltransferase (TdT) . This gene is turned on for 
only a short time during both B and T cell devel- 
opment. The TdT protein is a template-indepen- 
dent DNA polymerase that appears to play a role 
in generating diversity within the antibody and T 
cell receptor molecules. 
We chose to analyze the TdT gene because of 
its expression patterns in normal and leukemic 
cells. In normal cells TdT expression is unusual 
because it is found in both early B and T cells, 
suggesting that it may be regulated by transcrip- 
tion factors common to these two related but dis- 
tinct lineages. Moreover, TdT is expressed at high 
levels in acute lymphocytic leukemias (ALLs) and 
at lower levels in many acute myeloid leukemias 
(AMLs). TdT expression in AMLs is particularly 
intriguing because it does not occur in normal 
myeloid cells or myeloid precursors. Therefore 
the abnormal expression of TdT in AMLs suggests 
that the protein or proteins that deregulate TdT 
may also play a role in leukemogenesis. 
To study the mechanisms of regulating TdT ex- 
pression, we have analyzed the promoter, the 
transcriptional control region surrounding the 
start site for TdT RNA synthesis. Our analysis fo- 
cuses on two aspects of promoter function: 1) the 
DNA sequence elements and proteins responsible 
for directing TdT expression specifically in lym- 
phoid cells and 2) the unusual architecture of the 
TdT promoter region, which is fundamentally 
different from that of most other mammalian pro- 
moters that have been studied in detail. Our pro- 
gress in these two areas is described below. 
Regulated Expression of the TdT Gene 
To understand the specific activation of the 
TdT gene in B and T lymphocytes, we are 
currently focusing on a DNA sequence element 
located 60 nucleotides upstream from the TdT 
transcription start site. We have shown that this 
element of approximately 15 base pairs, called 
D', is essential for efficient promoter activity in 
lymphocytes. We have identified two different 
classes of proteins that interact with the D' re- 
gion, one or both of which may be important for 
TdT transcription. We identified the first protein, 
called LyF- 1 , by looking for protein-DNA interac- 
tions at the D' site in protein extracts derived 
from lymphocytes. We have characterized this 
novel protein after purifying it from a murine 
thymoma cell line. LyF-1 is a 50-kDa protein and 
was found to be highly enriched in lymphoid 
cells relative to a variety of nonlymphoid cells. 
Moreover, LyF- 1 was found to bind to two differ- 
ent sites in the TdT promoter as well as to the 
promoters for four other lymphocyte-specific 
genes. 
The second class of proteins that bind to the 
TdT D' site are those related to the ets family of 
DNA-binding proteins. Mammalian ets-1 was first 
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