GENOMIC RESPONSE TO GROWTH FACTORS 
Daniel Nathans, M.D., Senior Investigator 
Extracellular signaling agents— hormones, growth 
and differentiation factors, neuromodulators, and 
depolarizing agents— act via second messenger sys- 
tems to induce genetic programs in target cells. Re- 
search in Dr. Nathans' laboratory is aimed at char- 
acterizing genes that are part of the program 
induced by growth factors and defining the func- 
tion of their encoded proteins. By screening mouse 
3T3 cell cDNA libraries for clones derived from 
mRNA present in cells stimulated by serum growth 
factors but not in unstimulated cells, Dr. Nathans 
and his colleagues have identified a set of genes 
that is activated coordinately with the proto- 
oncogene c-fos or c-myc during the first several 
minutes after addition of serum, platelet-derived 
growth factor, or fibroblast growth factor; other 
genes that are activated later have also been identi- 
fied. Several of the "immediate early" genes encode 
known or probable transcription factors that are 
thought to regulate the genetic program induced 
by growth factors. These genes and the proteins 
they encode have been the main focus of the 
laboratory's research during the past year. 
I. A New Member of the Steroid and Thyroid Hor- 
mone Receptor Superfamily. 
One of the cDNA clones (nur77) derived from an 
immediate early mRNA was found to encode a pro- 
tein of 601 amino acids homologous to steroid and 
thyroid hormone receptors. The Nur77 protein 
contains two regions of sequence similarity to the 
hormone receptors, corresponding to their DNA- 
binding and ligand -binding domains. In each case 
the degree of similarity resembles that found be- 
tween other members of the family. A homologous 
mRNA was detected by Dr. Jeffrey Milbrandt in rat 
pheochromocytoma cells after stimulation by nerve 
growth factor. Studies of Nur77 are continuing in 
the laboratory of Dr. Lester Lau. 
II. Characterization of the Zinc Finger Protein Zif268. 
As reported earlier, the zif268 gene encodes a 
protein of 533 amino acids with three tandemly re- 
peated, typical zinc finger sequences. (The cDNA 
has been isolated in three other laboratories and 
designated NGFI-A, erg-1, and Krox24.) Upstream 
of the gene are potential binding sites for known 
transcription factors: AP-2, AP-1, cAMP-binding pro- 
tein, and four serum response element (SRE) core 
sequences [CC(A or T)gGG], known to be part of a 
signal involved in the activation of the c-fos gene by 
serum growth factors. To determine whether the 
core SREs upstream of the zif268 gene mediate in- 
duction, various deletion constructs of the zif268 
promoter region and synthetic oligonucleotides 
corresponding to each of the four zif268 putative 
SREs were tested for responsiveness to serum, pla- 
telet-derived growth factor, and phorbol ester. Each 
of the SREs conferred inducibility to these agents, 
and multiple SREs resulted in greater inducibility 
than a single element. Each of the zif268 SREs also 
competed with the c-fos SRE for binding by serum 
response factor present in HeLa cell nuclear ex- 
tract, even though the sequences that flank the 
core SRE sequences are different. Thus the zif268 
SREs are functional and probably account for the 
coordinate induction of the zif268 and c-fos genes. 
Based on its three zinc finger sequences, Zif268 
is thought to be a sequence-specific DNA-binding 
protein that regulates transcription of genes that 
are part of the growth factor-induced genetic pro- 
gram. To begin a search for genes that are regulated 
by Zif268, DNA sequences to which Zif268 binds 
have been identified. On the assumption that the 
zif268 gene itself has a binding site in its promoter 
region, fragments of DNA derived from the up- 
stream region of the gene were incubated with 
Zif268 produced in Escherichia coli, and binding 
was assessed by electrophoretic separation of 
bound and unbound fragments. Two binding sites 
were found and confirmed by DNase I footprinting. 
Similar sequences are present in the promoter re- 
gions of a number of other genes, including other 
immediate early genes. Several were shown to be 
Zif268-binding sites. The consensus binding site se- 
quence, inferred from natural and synthetic sites, is 
GCG ^GGGCG. 
III. Interaction of Jun and Fos. 
Two immediate early genes encode proteins re- 
lated to the oncoprotein v-Jun and the transcrip- 
tion factor AP-1. One of these is the proto-oncogene 
product c-Jun, and the other is Jun-B. A third mem- 
ber of the jun gene family (jun-D) was isolated by 
screening murine genomic and cDNA libraries. It is 
expressed in nonproliferating cells and is only 
slightly induced by serum growth factors. The three 
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