of the hormone's action to maintain steroidogenic 
competence. The objective of these studies is to de- 
fine the factors that regulate the adrenal steroido- 
genic enzymes; in particular, the studies have fo- 
cused on defining shared regulatory proteins that 
determine the coordinate expression of this gene 
network. 
Mouse Homologue of the Nuclear Receptor 
fushi tarazu Factor I Regulates 
Steroidogenic Enzyme Gene Expression 
Dr. Parker's laboratory has characterized the pro- 
moter regions of mouse genes encoding SCC, 21- 
OHase, and 1 1/3-OHase. Initial studies showed that 
the 5'-flanking regions of these genes are sufficient 
for regulated expression and define key regulatory 
elements. Of particular interest, several promoter 
elements contain variations of an AGGTCA motif, 
suggesting they interact with the same protein. This 
protein, which was designated steroidogenic factor 
1 (SF- 1 ) , interacts with promoter elements from all 
of the mouse steroidogenic enzymes and is selec- 
tively expressed in steroidogenic cell lines, estab- 
lishing it as a likely determinant of coordinate, cell- 
selective expression of these essential genes. 
The AGGTCA recognition sequence of SF-1 
closely resembles that of several members of the nu- 
clear receptor family, a superfamily of structurally 
related, regulatory proteins that mediate transcrip- 
tional induction by such diverse agents as steroid 
and thyroid hormones, vitamin D, and retinoic acid. 
On the assumption that the similar recognition mo- 
tifs reflect structural similarities between nuclear 
receptors and SF-1, a Yl adrenocortical cell cDNA 
library was screened with a probe comprising the 
DNA-binding domain of H-2R11BP, one family 
member. One clone hybridized specifically to tran- 
scripts that are only expressed in steroidogenic tis- 
sues, the same pattern seen with SF-1. Moreover, 
expression of this cDNA as a glutathione 5-transfer- 
ase fusion protein yielded a protein that interacted 
with the steroidogenic regulatory elements in a 
manner indistinguishable from SF- 1 . 
When the nucleotide sequence of the SF- 1 cDNA 
was determined and compared with other se- 
quences, a surprising finding emerged. This se- 
quence is highly related to the nuclear receptor fam- 
ily member FTZ-Fl, which regulates the fushi 
tarazu (ftz) homeobox gene in Drosophila. Two 
distinct forms of FTZ-Fl had been identified: an 
early form, which correlated with ftz expression 
during development, and a late form, which was ex- 
pressed after /te expression was extinguished. Both 
forms had identical DNA-binding specificities but 
apparently differed slightly in their primary struc- 
tures. These results suggest that the FTZ-Fl gene in 
Drosophila encodes developmentally specific tran- 
scripts that provide alternative functions: a form 
that is essential for early embryonic development 
and a form that is expressed later. 
A similar situation is found in the mouse. The 
mouse SF- 1 transcript strikingly resembles a cDNA, 
termed embryonal long terminal repeat-binding 
protein (ELF) , that silenced retroviral gene expres- 
sion in embryonal carcinoma cells. The two tran- 
scripts are essentially identical for 1,100 bp, in- 
cluding the zinc finger DNA-binding domain, but 
diverge at the 5' and 3' ends. These results indicated 
that they either derive from distinct but highly re- 
lated genes or result from alternative splicing of the 
same gene. Consistent with the latter hypothesis, 
genomic Southern analyses showed that both the 
ELF and SF-1 transcripts are encoded by the same 
gene. This finding suggests that a critical regulator 
of steroidogenic enzyme gene expression in adult 
animals is either identical or closely related to a pro- 
tein that is expressed at high levels in mouse em- 
bryonal carcinoma cells. The ELF cDNA presumably 
corresponds to the early FTZ-Fl protein, whereas 
the SF-1 cDNA corresponds to late FTZ-Fl. This 
close conservation from Drosophila to mouse of de- 
velopmentally regulated transcripts derived from 
the FTZ-Fl gene leads to the intriguing speculation 
that the late form of FTZ-Fl regulates the enzymes 
that make ecdysteroids in Drosophila. 
NGFI-B, Another Nuclear Receptor Protein, 
May Mediate ACTH Induction of 
Steroid 21 -Hydroxylase 
The sequence of the 21-OHase —65 regulatory el- 
ement closely resembled the canonical response ele- 
ment for nerve growth factor-induced B (NGFI-B), 
an immediate-early nuclear receptor that is rapidly 
induced by a variety of growth factors and trophic 
hormones. The possible role of NGFI-B in regulating 
steroidogenic enzyme expression was therefore ad- 
dressed. In situ hybridization studies demonstrated 
high levels of NGFI-B expression in the adrenal cor- 
tex, and treatment of mouse Yl adrenocortical cells 
with ACTH or cAMF rapidly increased levels of 
NGFI-B transcripts and protein. Gel mobility shift 
and DNase I footprinting experiments showed that 
recombinantly expressed NGFI-B interacts specifi- 
cally with the 2 1 -OHase —65 element and identified 
one complex formed by Yl nuclear extracts that 
contains NGFI-B. Expression of NGFl-B significantly 
augmented the activity of the intact 2 1 -OHase pro- 
moter and reporter constructs containing the 21- 
CELL BIOLOGY AND REGULATION 
101 
