GENE REGULATION IN ANIMAL CELLS 
Joseph R. Nevins, Ph.D., Investigator 
The determination of cellular phenotype is a 
function of the regulated expression of a set of 
unique genes under a particular set of circum- 
stances. Therefore an understanding of the molecu- 
lar mechanisms that control gene expression is cen- 
tral to an understanding of the complex cellular 
events that take place during such events as em- 
bryogenesis and oncogenesis. Dr. Nevins's labora- 
tory is focused on the elucidation of the molecular 
events that control gene expression, beginning 
with the identification and isolation of factors that 
directly interact with the gene or primary tran- 
scripts of the gene. 
L Trans-Activation of Transcription. 
Complex cellular events are often best studied 
through the use of simple model systems. The con- 
trol of transcription mediated by the adenovirus 
gene product has served as a useful means to un- 
derstand the workings and control of transcription 
factor activity in eukaryotic cells. Dr. Nevins is at- 
tempting to elucidate the underlying mechanisms 
of trans-activation brought about by viral regulatory 
genes such as ElA. The early adenovirus E2 gene, a 
viral transcription unit, has been used to define the 
mechanism of ElA action. Previous experiments 
demonstrated that adenovirus infection of human 
cells leads to an ElA-dependent activation of the 
DNA-binding capacity of a cellular transcription fac- 
tor, E2F, that binds to two sites in the viral E2 early 
promoter. These E2F recognition sequences have 
been shown to be critical for ElA-dependent E2 
transcription, and the E2F-binding sites can confer 
ElA-induced transcription to a heterologous pro- 
moter. In addition, under a variety of circumstances 
the increase in E2F-binding activity coincides with 
the activation of E2 transcription. Recent experi- 
ments have demonstrated that in addition to the 
ElA gene, the early viral E4 gene is also necessary 
for the activation of E2F-binding activity. Measure- 
ments of E2 RNA production in cells infected with 
an E4 mutant, as well as various transfection assays, 
demonstrate that the E4 gene can trans-activate the 
E2 promoter and that the E4 gene is essential for 
full E2 transcription in addition to ElA. A further 
analysis of the activation of the E2F factor has 
yielded information concerning the respective roles 
of the ElA and E4 gene products. 
These experiments demonstrate that the en- 
hanced binding of E2F to the E2 promoter is the re- 
sult of two separate events. First, there is a stimula- 
tion of the DNA-binding activity of the E2F factor, 
and this stimulation is ElA-dependent but indepen- 
dent of the E4 gene. Second, there is an induction 
of a stabilized interaction between two E2F mole- 
cules bound to adjacent promoter sites. An E2F fac- 
tor bound to a single site rapidly dissociates from 
the DNA, whereas the dissociation from a double- 
site interaction is much slower. This is only true, 
however, for the E2F factor isolated from infected 
cells. The low level of E2F from mock-infected cells 
shows no evidence of this stable binding, even at 
high concentrations, where two E2F factors can be 
driven onto the promoter. The induction of this sta- 
ble binding capacity requires the E4 gene. Thus the 
activation of E2F during an adenovirus infection is a 
two-step process involving a change in both the 
DNA-binding activity of the factor and the capacity 
to stabilize the interaction through protein-protein 
contacts. This latter function is of particular interest 
because of the potential importance of combinato- 
rial interactions of transcription factors with pro- 
moter elements. 
Cell-free extracts have been used to develop an 
assay for the in vitro activation of the DNA-binding 
activity of E2F. E2F activity is undetectable in ex- 
tracts of HeLa cells, but upon incubation with a 
fractionated extract from adenovirus-infected cells 
there is an ATP-dependent increase in E2F DNA- 
binding activity. This increase does not occur when 
an equivalent fraction from ElA mutant-infected 
cells or E4 mutant-infected cells is used. Incubation 
of E2F with phosphatase inactivates the E2F DNA- 
binding activity. Incubation of the phosphatase- 
inactivated E2F with an infected cell fraction re- 
stores E2F activity, as does incubation with a known 
protein kinase. In contrast, incubation with an ex- 
tract from mock-infected cells does not restore ac- 
tivity, thus indicating that at least part of the activa- 
tion of the DNA-binding activity of E2F is regulated 
by phosphorylation. 
Finally, it has been found that the normal cellular 
role of E2F may involve participation in a signal 
transduction pathway relating to cell proliferation. 
E2F binds to two sequence elements within the P2 
promoter of the human c-myc proto-oncogene that 
are within a region that is critical for myc promoter 
activity. Furthermore, analysis of the gene sequence 
library for potential E2F-binding sites identified a 
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