MECHANISM OF TRANSCRIPTIONAL REGULATION IN ANIMAL CELLS 
Robert Tjian, Ph.D., Investigator 
This past year, Dr. Tjian's efforts have been 
directed at the cloning and characterization of 
genes encoding eukaryotic DNA-binding tran- 
scription factors. These studies have defined novel 
protein structural domains responsible for DNA- 
binding specificity, protein dimerization, and tran- 
scriptional activation. In addition, Dr. Tjian's exper- 
iments have begun to address the important issue 
of mechanisms that operate to confer promoter- 
selective gene activation in animal cells. Finally, a 
number of unexpected findings have opened the 
way to defining new regulatory factors involved in 
Drosophila gene control during embryonic devel- 
opment and tissue-specific expression in the ner- 
vous system. 
I. Mechanism of Action of Transcription Factor Spl. 
The SV40 early promoter, which contains six 
Spl-binding sites (GC boxes), is induced up to 500- 
fold in cells expressing Spl, whereas promoters 
with fewer sites are activated less efficiently Analy- 
sis of Spl mutants reveals multiple distinct regions 
outside of the DNA-binding domain that are re- 
sponsible for mediating transcriptional activation. 
The two most active domains, which appear to be 
functionally redundant, consist of an unusual struc- 
ture with a very low charge density but a strikingly 
high glutamine content. A number of other se- 
quence-specific transcription factors, such as the 
Drosophila zeste protein and several homeo- 
domain proteins, contain glutamine-rich stretches. 
Dr. Tjian proposes that these glutamine-rich do- 
mains represent a novel structural motif for tran- 
scriptional activation. 
The regulation of gene expression depends, in 
part, on interactions between sequence-specific 
transcription factors. Dr. Tjian has been examining 
the role of protein-protein interactions in modulat- 
ing the activity of Spl, a human transcription factor 
that utilizes glutamine-rich activation domains. 
These glutamine-rich regions may represent a com- 
monly used structural motif, since a nonhomolo- 
gous glutamine-rich segment from the Drosophila 
Antennapedia protein is also a potent activator 
when fused to the Spl DNA-binding domain. Spl is 
generally considered a proximal promoter factor 
that can only stimulate transcription when bound 
close to the transcriptional start site. However, Dr. 
Tjian has obtained evidence that distally and proxi- 
mally bound molecules of Spl can stimulate tran- 
scription synergistically. In addition, a DNA-binding- 
deficient mutant of Spl that retains active gluta- 
mine-rich domains can interact productively with 
proximally bound Spl to superactivate transcrip- 
tion. Glutaraldehyde crosslinking provides direct 
evidence for a protein-protein interaction between 
Spl monomers. These findings provide evidence 
for an Spl: Spl interaction that may play an impor- 
tant role in Spl-mediated transcriptional activation. 
n. Structure and Function of the Jun/AP-1 Family of 
Enhancer Factors. 
The discovery by Dr. Tjian and his colleagues that 
the activator protein-1 (AP-1) family of enhancer 
binding factors includes a complex of the cellular 
Fos (c-Fos) and cellular Jun (c-Jun) proteins estab- 
lished a direct and important link between onco- 
genesis and transcriptional regulation. Recently Dr. 
Tjian showed that homodimeric c-Jun protein syn- 
thesized in vitro is capable of binding selectively to 
AP-1 recognition sites, whereas the c-Fos polypep- 
tide is not. When cotranslated, the c-Fos and c-Jun 
proteins can form a stable, heterodimeric complex 
with the DNA-binding properties of AP-l/c-Jun. The 
related proteins JunB and v-Jun are also able to 
form DNA-binding complexes with c-Fos. Directed 
mutagenesis of the c-Fos protein reveals that a leu- 
cine repeat structure is required for binding to 
c-Jun, in a manner consistent with the proposed 
function of the "leucine zipper." A novel domain 
adjacent to but distinct from the leucine repeat of 
c-Fos is required for DNA binding by c-Fos-c-Jun 
heterodimers. Thus experimental evidence is pre- 
sented that leucine repeats can mediate complex 
formation between heterologous proteins. This in- 
creases understanding of the molecular mecha- 
nisms underlying the function of two proto- 
oncogene products. 
The human proto-oncogene product c-Jun is a 
component of the AP-1 family of transcription fac- 
tors that mediates the regulation of gene expres- 
sion in response to extracellular signaling events. 
Dr. Tjian and his colleagues have carried out a bio- 
chemical study of the relationship between the 
structure and function of c-Jun and its viral coun- 
terpart, v-Jun. Most interestingly comparison of 
c-Jun and v-Jun by in vitro transcription assays re- 
vealed that the viral oncoprotein has significantly 
greater transcriptional activity than c-Jun. Analysis 
of a collection of Jun mutants expressed in bacteria 
Continued 
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