Gene Regulation in Animal Cells 
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Joseph R. Nevins, Ph.D. — Investigator 
Dr. Nevins is also Professor of Microbiology and Immunology and of Genetics at Duke University Medical 
Center. He received his Ph.D. degree in virology at Duke University, where he studied with Wolfgang Joklik. 
His postdoctoral studies as a Jane Coffin Childs fellow focused on the mechanisms of mRNA biogenesis 
and were conducted with James Darnell at the Rockefeller University, where he later became a faculty 
member. 
THE regulation of gene expression is central to 
the complex cellular changes that take place 
during such events as embryogenesis and onco- 
genesis. An elucidation of the molecular mecha- 
nisms of gene control pathways is the focus of our 
laboratory, including the modification and regu- 
lation of factors that mediate the control of gene 
expression. 
Mechanisms of Viral-mediated 
Trans-activation of Transcription 
The primary event in the expression of a gene is 
the initiation of transcription leading to the for- 
mation of functional mRNA. The control of this 
event is a key step in the determination of cellular 
phenotype. Various studies have shown that tran- 
scription initiation is complex, resulting from 
the interaction of a large number of proteins with 
regulatory DNA sequences of the gene. 
The study of complex cellular events is often 
facilitated by the use of simple viral model sys- 
tems. An example is the trans-activation of tran- 
scription by viral regulatory proteins such as the 
adenovirus El A gene product. The ElA protein 
mediates the activation of transcription of a set of 
viral genes and several cellular chromosomal 
genes. Since the activation process involves the 
use of cellular transcription factors, this system 
has provided a means for the study of cellular 
transcription control. 
Work in our laboratory has led to the identifi- 
cation of several cellular transcription factors 
that the viral regulatory system utilizes and that 
contribute to the trans-activation event. In partic- 
ular, the activity of these factors is modified by 
the viral infection, which increases the levels and 
changes the nature of the DNA-binding activity of 
the proteins. The DNA-binding activity of several 
factors depends on the phosphorylation state of 
the protein, and phosphorylation appears to play 
a key role in the activation process. 
Recent studies have shown that the E2F tran- 
scription factor, as isolated from adenovirus- in- 
fected cells, binds to the E2 promoter with a high 
degree of cooperativity, resulting in the forma- 
tion of a very stable DNA-protein complex. This 
binding is important for full transcription activa- 
tion, requires the precise dyad arrangement of 
the E2F sites as found in the E2 promoter, and 
requires the interaction of a 19-kDa product of 
the early viral E4 gene with the E2F factor. The 
induction of cooperative E2F binding is striking 
in view of the specificity. Although E2F-binding 
sites have been identified in a variety of cellular 
promoters, no other instances have been found of 
this precise arrangement in the E2 promoter. It 
thus appears that the cellular E2F factor is con- 
verted into an E2 promoter-specific factor 
through the action of the E4 protein. 
The E2F factor has now been found to be com- 
plexed to cellular factors in extracts of a variety 
of cell lines. These complexes are significant 
with respect to a viral infection, since the E4 pro- 
tein cannot interact with E2F that is already com- 
plexed to a cellular factor, thus preventing the 
formation of the stable interaction on the E2 pro- 
moter. Strikingly, however, we have found that 
ElA proteins can dissociate these E2F-containing 
complexes, releasing free E2E that can associate 
with the E4 protein. Thus it would appear that a 
two-step mechanism has evolved to utilize the 
cellular E2F factor efficiently and redirect it for 
viral-specific purposes. 
Through the analysis of a large series of ElA 
mutants it has become clear that the E2F-disso- 
ciating activity is dependent on sequences within 
ElA that are important for transforming activity. 
Moreover, trans-activation assays have demon- 
strated that mutations that disrupt E2F dissocia- 
tion also disrupt cell-specific trans-activation 
function. These are also the amino acid se- 
quences that are shared with two other viral on- 
cogene products — SV40 T antigen and human 
papillomavirus E7. Previous experiments have 
shown that both possess trans-activation function 
dependent on the E2F factor. Recent experiments 
have shown that the E7 protein is capable of al- 
tering the E2F complexes in a manner similar to 
ElA. It therefore appears that these otherwise 
unrelated viruses have acquired a common regu- 
latory function through evolution, directed at a 
specific cellular transcription factor, that may be 
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