Adenovirus as a Model for Control 
of Gene Expression 
Thomas E. Shenk, Ph.D. — Investigator 
Dr. Shenk is also Elkins Professor of Molecular Biology at Princeton University and Adjunct Professor of 
Biochemistry at the Robert Wood Johnson Medical School, University of Medicine and Dentistry of New 
Jersey. He received his Ph.D. degree in microbiology from Rutgers University for studies with Victor Stollar, 
and his postdoctoral training with Paul Berg at Stanford University. Before coming to Princeton, he was 
Assistant Professor of Microbiology at the University of Connecticut Health Center and then Professor of 
Microbiology at the State University of New York School of Medicine at Stony Brook. Dr. Shenk counts 
among his honors the Eli Lilly Award in Microbiology from the American Society for Microbiology and an 
American Cancer Society Professorship. 
ADENOVIRUSES are widespread, and humans 
are first infected when quite young. Gener- 
ally the infection results in cold-like symptoms 
and resolves without complication. Some human 
adenoviruses, however, induce a variety of be- 
nign and malignant tumors if injected into a rat or 
hamster. Since these viruses contain DNAand are 
tumorigenic under certain conditions, they are 
classified as DNA tumor viruses. 
Adenoviruses can be propagated easily in cul- 
tured cells. When human cells are infected, the 
approximately 30 viral genes are expressed, the 
viral chromosome is replicated, and individual 
DNA molecules are packaged into protein shells 
to produce virus progeny. Since viral genes are 
expressed at high levels compared with most cel- 
lular genes, and since this expression is tightly 
regulated, the adenovirus is a useful probe for 
studying the control of gene expression. 
During the past year, much of our effort has 
focused on transcriptional control of viral gene 
expression. The first viral gene to be expressed 
after infection of a cell is the ElA gene, which 
encodes a protein that activates expression of ad- 
ditional viral genes. The ElA protein appears to 
activate transcription (copying of genetic infor- 
mation) through several physiologically distinct 
mechanisms. One of these involves a cellular 
transcription factor that we have termed YY- 1 
We first identified the binding site for YY- 1 in 
the P5 transcriptional control region of adeno- 
associated virus, a defective virus that depends on 
a variety of adenovirus gene products for its repli- 
cation. The ElA protein activates expression of 
the P5 control region, and the critical sequence 
element required for activation is a DNA segment 
constituting the binding site for YY-1 . To investi- 
gate its function, this site was inserted upstream 
of several heterologous promoters, and it re- 
pressed their activity. The repression was re- 
lieved in the presence of ElA protein. In fact, the 
protein also activated transcription through the 
YY-1 binding site. The combination of these two 
effects, relief of repression and activation, in- 
duced transcription by a factor of 1,000 in some 
test genes. Thus the combination of the YY-1 
binding site and ElA protein formed a powerful 
biological on/off switch. 
In order to study the YY- 1 factor, we prepared 
some from cultured human cells and determined 
a short amino acid sequence from the purified 
protein. This sequence was used to design a short 
probe DNA, which enabled us to identify and iso- 
late a cDNA clone encoding the protein. Se- 
quence analysis of the clone revealed that YY- 1 is 
a 4l4-amino acid protein with a zinc finger 
DNA-binding motif. Protein was expressed from 
the clone and shown to bind specifically to the 
YY-1 recognition site. 
The binding activity of YY- 1 was altered by fus- 
ing it to the DNA-binding domain of the yeast 
GAL4 protein. This approach is widely used to 
study transcription factors, since it provides the 
opportunity to direct binding of the factor under 
study in a highly specific fashion to a test gene 
construct that contains the yeast GAL4 DNA- 
binding site. By redirecting the factor to bind to a 
novel site, it was possible to study the activity of 
the fusion protein in cells that contain high levels 
of endogenous YY-1 . As anticipated, the YY-1 fu- 
sion protein repressed transcription of the test 
gene in the absence of the ElA protein, and the 
repression was relieved by the ElA protein. Muta- 
tional analysis of the fusion protein has demon- 
strated that the YY- 1 zinc finger domain is respon- 
sible for its ability to repress transcription. 
Since the ElA protein would not be expected 
to cause the YY-1 fusion protein to detach from 
the GAL4 DNA-binding site, it appears likely that 
YY- 1 remains bound to the control region but is 
somehow altered in the presence of the ElA pro- 
tein so that transcription is not repressed. Several 
experiments indicate that the ElA protein can 
bind directly to YY-1. For example, if the two 
proteins are mixed, they sediment as a complex 
in a sucrose gradient. Furthermore, radioactively 
labeled ElA can bind to YY-1 that has been sepa- 
rated from a complex mixture of proteins by elec- 
trophoresis and bound to a membrane filter. 
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