binding to its target sequence? A cotransfection assay 
utilizing an Egr- 1 expression vector and a reporter with 
three Egr- 1 -binding sites placed upstream of a minimal 
promoter and the chloramphenicol acetyltransferase 
(CAT) gene showed that Egr- 7 is a transcriptional acti- 
vator. This system is being used to delineate activation 
domain (s), nuclear localization signals, and DNA-bind- 
ing requirements. Not surprisingly, the three zinc 
fingers are sufficient for DNA binding. Nuclear localiza- 
tion depends on a bipartite sequence, one similar to the 
corresponding signal in the simian virus 40 (SV40) T 
antigen, the other part of the zinc finger motif. Two 
activation domains in the amino-terminal region of the 
protein have been mapped. An unexpected finding is a 
region of 50 amino acids 5' of the three fingers, which 
when deleted from the wild-type protein results in a 
superactivator mutant. Moreover, this region functions 
as a modular repressor, as demonstrated by studies with 
chimeric constructs with the yeast GAL4 (1-147) frag- 
ment. This fragment is one of the smallest mammalian 
repressor "proteins" defined to date. This region, as 
well as the nuclear localization signals and the DNA- 
binding domain, is exquisitely conserved in the zebra- 
fish, the earliest evolutionary Egr-1 homologue found. 
Transcription Factors 
in Early Kidney Development 
The molecular events that characterize the devel- 
opment of the kidney are the second focus of Dr. 
Sukhatme's laboratory. It is well known that meta- 
nephric mesenchymal cells convert into epithelial 
cells over a 4- to 5-day period in response to inva- 
sion of the ureteric bud. Little is known at the molec- 
ular and cellular level of the events that transpire 
during this process. Dr. Sukhatme's approach is 
based on the premise that characterization of a hier- 
archy of transcriptional regulators that occur during 
this period will be important in understanding this 
process. Toward this end, recent studies have fo- 
cused on the Wilms' tumor gene WTl. 
The WTl protein in three of its four zinc fingers 
shows a 60-70% similarity to the amino acid se- 
quence of the Egr-1 finger domain. Antibodies 
raised against the Wilms' tumor antibody cross-react 
with Egr-1, and both proteins can bind to the 
same target sequence (GCGGGGGCG) (Dr. Frank 
Rauscher, Wistar Institute). However, WTl is a 
negative regulator of transcription, whereas Egr-1 
is a positive regulator (in collaboration with Dr. 
Rauscher) . 
What then are the physiologic targets for WTl ac- 
tion in the kidney? Several binding sites exist for the 
Egr-1 /Wilms' tumor proteins in the promoter se- 
quence of insulin-like growth factor II (IGF-II). It 
has been known that IGF-II levels are high in Wilms' 
tumor and that during development IGF-II levels 
fall. Dr. Sukhatme's laboratory, in collaboration 
with Dr. Rauscher, used cotransfection studies to 
investigate the possibility that IGF-II is a direct tar- 
get for the repressive action of the Wilms' tumor 
protein, and found this to be the case. Two critical 
target sites (one upstream and one downstream of 
the transcription site) were identified by deletion 
analysis and mutagenesis. Gel shift and footprint 
analysis provided evidence for a direct interaction, 
and these sequences were sufficient to confer re- 
pression when ligated to a heterologous promoter. 
These studies constitute the identification of the 
first target gene for WTl and suggest a mechanism 
for the genesis of some Wilms' tumors. Recently it 
has been noted that a major alternatively spliced 
variant of WTl also represses the IGF-II promoter. 
This variant, which contains a three-amino acid in- 
sertion between the third and fourth zinc fingers, 
does not bind to the Egr-1 target consensus. 
In preliminary studies in collaboration with Dr. 
Tucker Collins (Harvard Medical School), Dr. Suk- 
hatme and his colleagues have shown that a 900-bp 
promoter region of the platelet-derived growth fac- 
tor (PDGF) human A chain is strikingly repressed by 
WTl. This region of the promoter contains several 
putative sites for WTl /Egr-1 binding. Studies are 
currently under way utilizing gel shift analysis and 
DNase I footprinting. Previous studies have shown 
that the PDGF A chain is transcribed in human fetal 
kidney, and overexpression of this gene has been 
described in several Wilms' tumors. Thus these stud- 
ies suggest that lack of WTl repressor activity may 
lead to the deregulation of A chain expression, fur- 
ther contributing to the tumorigenic pathway in 
Wilms' tumors. 
Dr. Sukhatme is also Associate Professor of Med- 
icine and of Molecular Genetics and Cell Biology 
at the University of Chicago. 
Articles 
Cao, X.M., Guy, G R., Sukhatme, V.P,, and Tan, 
Y.H. 1992. Regulation of the Egr- 1 gene by tumor 
necrosis factor and interferons in primary human 
fibroblasts./ 5/0/ Chem 267:1345-1349. 
Darland, T., Samuels, M., Edwards, S.A., Sukhatme, 
V.P., and Adamson, E.D. 1991. Regulation of Egr- 
1 (Zfp-6) and c-fos expression in differentiating 
embryonal carcinoma cells. Oncogene 6:1367- 
1376. 
Drummond, I.A., Madden, S.L., Rohwer-Nutter, 
P., Bell, G.I., Sukhatme, V.P., and Rauscher, F.J., 
GENETICS 269 
