REGULATION OF GENE EXPRESSION DURING CELL DIFFERENTIATION AND ACTIVATION 
Jeffrey M. Leiden, M.D., Ph.D., Assistant Investigator 
Dr. Leiden's objective is to understand better the 
molecular mechanisms that regulate gene expres- 
sion during the processes of cellular activation and 
differentiation. Toward this end, Dr. Leiden and his 
colleagues have been studying transcriptional regu- 
lation in two different experimental systems, the 
human T lymphocyte and the mouse myocyte. The 
studies of human T cells have focused on the regu- 
lation of the T cell receptor (TCR) a and P genes 
during T cell differentiation and the regulation of 
the 4F2 heavy-chain (4F2HC) gene during T cell ac- 
tivation. The studies of mouse myocyte differentia- 
tion have utilized the skeletal muscle troponin C 
(sTnC) and cardiac troponin C (cTnC) genes as 
models of developmentally regulated, lineage-spe- 
cific myocyte gene expression. 
L Regulation of Human TCR a Gene Expression 
During T Cell Ontogeny. 
A. Identification and characterization of a T cell- 
specific transcriptional enhancer 3' of the Ca 
gene in the human TCR a locus. Human T lympho- 
cytes can be divided into two distinct subsets, 
based on their cell surface expression of antigen re- 
ceptor molecules. The majority of peripheral blood 
T cells, including most cells of the helper and 
cytotoxic phenotypes, express a CD3-associated a/p 
TCR. A second smaller subset of T cells of unknown 
function express a CD3-associated 7/8 TCR. Al- 
though a great deal has been learned about the 
structure of the four TCR genes, little is understood 
about the molecular mechanisms that regulate the 
rearrangement and expression of these genes dur- 
ing T cell ontogeny. 
Dr. Leiden's laboratory has identified a transcrip- 
tional enhancer element located 4.5 kilobases (kb) 
3' of Ca in the human TCR a-chain locus. This en- 
hancer is necessary for transcription from a TCR Va 
promoter and is also active upon the minimal SV40 
promoter in TCR a/p^ T cells. However, it is inac- 
tive in human TCR 7/8''" T cells, B cells, and fibro- 
blasts. The enhancer has been localized to a 1 16 bp 
BstXl/Dral restriction enzyme fragment that lacks 
immunoglobulin octamer and kB enhancer motifs 
but does contain a consensus cAMP-responsive ele- 
ment (CRE). DNase I footprint and electrophoretic 
mobility shift analyses demonstrated that the mini- 
mal enhancer contains two binding sites for Jurkat 
nuclear proteins. One of these sites corresponds to 
the CRE, while the other does not correspond to a 
known transcriptional enhancer motif These data 
support a model in which TCR a gene transcription 
is regulated by a unique set of cis-acting sequences 
and trans-acting factors that are differentially active 
in cells of the TCR a/P"*" lineage. Activation of the 
TCR a enhancer may be a key step in determining 
the differentiation of TCR a/p"*" T cells. In addition, 
the TCR a enhancer may play a role in activating 
oncogene expression in T lymphoblastoid tumors, 
which have previously been shown to display chro- 
mosomal translocations into the human TCR a 
locus. Ongoing studies are aimed at determining 
the importance of each of the nuclear protein-bind- 
ing sites in enhancer function and elucidating the 
molecular mechanisms responsible for the prefer- 
ential activity of the enhancer in TCR a/p^, as com- 
pared with TCR 7/5^, T cells. Dr. Leiden's labora- 
tory is also in the process of cloning the 
DNA-binding proteins that regulate the activity of 
this enhancer. 
B. Molecular cloning of CREB-2, a novel CRE-bind- 
ing protein that interacts with the human TCR a 
transcriptional enhancer. The TCR a enhancer 
contains a nuclear protein-binding site, Tal, which 
includes the palindromic sequence TGACGTCA 
that is identical to the sequence motif that confers 
cAMP responsiveness on multiple eukaryotic pro- 
moters (CRE). Using electrophoretic mobility shift 
analyses. Dr. Leiden's laboratory has shown that a 
CRE-binding protein (CREB) present in T cell nu- 
clear extracts binds specifically to the Tal site of 
the TCR a enhancer. By using a radiolabeled TCR a 
CRE probe to screen a Jurkat Xgtll cDNA expres- 
sion library, they have identified a human cDNA 
clone that encodes a novel protein that binds spe- 
cifically to the TCR a enhancer CRE (CREB-2). The 
351-amino acid CREB-2 protein contains a car- 
boxyl-terminal leucine zipper motif and an adjacent 
basic domain that are structurally related to similar 
domains in the c-jun and c-fos 12-O-tetradecanoyl- 
phorbol-13-acetate (TPA) response element (TRE)- 
binding proteins, as well as to the previously de- 
scribed CRE-binding proteins, CREB and CRE-BPl. 
Comparison of the basic domains of the known 
TRE- and CRE-binding proteins has allowed the 
identification of three amino acid residues that may 
account for the different binding specificities of 
these two related families of transcription factors. 
Continued 
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