the relevant stimulus (Sonnenberg et al. 1989a; Naranjo et al. 1991), 
demonstration that the proteins of interest bind specifically to the target 
gene with high affinity in vitro, and demonstration that the IEG activates 
(or represses) the target gene following cotransfection (Sonnenberg et al. 
1989a; Naranjo et al. 1991). Because of difficulties in culturing and transfecting 
many primary cell types within the nervous system, evidence that a particular 
IEG plays a role in the cell type of interest has been difficult to obtain to date. 
Additional experiments that could strengthen the interpretation of a causal 
interaction between IEG and target might include inhibition of expression of 
specific lEGs using antisense oligonucleotides or inhibition of transcription 
factor function by microinjection of specific antibodies into cells prior to 
stimulation. 
THE PROENKEPHALIN GENE IS A POTENTIAL TARGET FOR IEG- 
MEDIATED ACTIVATION 
The proenkephalin gene is widely investigated as a model of stimulus-induced 
gene expression in brain and as a potential target gene for regulation by lEGs. 
Hyman and colleagues (1988), Comb and colleagues (1988), and Sonnenberg 
and colleagues (1989a) have demonstrated that the AP-1 transcription factor 
(of which c -fos is a component; see Morgan and Curran 1991) binds the 
proenkephalin promoter and that AP-1 proteins can positively and negatively 
regulate proenkephalin gene expression (Sonnenberg et al. 1989a; Kobierski 
et al. 1991). 
The proenkephalin enhancer has been resolved into three closely spaced 
DNA regulatory elements (figure 1) by detailed mutational analysis and DNAse I 
footprinting with affinity purified protein (Hyman et al. 1988, 1989; Comb et al. 
1988). Within the enhancer, the element most distal from the TATA box is 
called ENKCRE-1 . It contains the sequence TGGCGTA and binds NF-1-like 
proteins (Chu et al. 1991). The element that is closest to the TATA box 
contains the sequence CCGCCGGC and binds nuclear factor AP-2 (Comb 
■110 -100 
I 
GCGGGGC 
CGCCCCG ACCGCAT CCCGG 
-70 
I 
GGCGATT 
CCGCTAA 
ENKCRE-1 ENKCRE-2 AP-2 
FIGURE 1 . The proenkephalin second messenger-inducible enhancer. The 
sequence of the enhancer is shown; the sequences of the boxed 
regulatory elements were determined by mutational analysis. 
28 
