Determinants of Developmental Programs 
of Gene Activation 
Michael G. Rosenfeld, M.D. — Investigator 
Dr. Rosenfeld is also Professor of Medicine in the Eukaryotic Regulatory Biology Program at the School of 
Medicine of the University of California, San Diego. He received his undergraduate degree from the Johns 
Hopkins University and his medical degree from the University of Rochester. His internship and medical 
residency ivere completed at Barnes Hospital, St. Louis. Before accepting his current position, he received 
postdoctoral training at the NIH. Dr. Rosenfeld also holds an adjunct position at the Salk Institute. 
OVER the past year the central research focus 
in this laboratory has been to define the mo- 
lecular mechanisms that dictate the developmen- 
tal and regulated expression of neuroendocrine 
genes and to begin to apply emerging principles 
to analysis of neuronal gene expression. 
The neuroendocrine system coordinates the 
complex pattern of regulation necessary to 
achieve the precise temporal, spatial, and homeo- 
static patterns of gene expression required by 
complex eukaryotic organisms. Development of 
the central nervous system and endocrine organs 
involves precise patterns of responses to morpho- 
gens and other regulatory signals that ultimately 
establish the intricate patterns of neural and en- 
docrine phenotypes. The cloning and analysis of 
specific genes encoding regulatory genes, recep- 
tors, and cell-specific transcription factors have 
permitted an initial definition of developmental 
and regulatory transcriptional and post-transcrip- 
tional strategies in the neuroendocrine system. 
We are using the anterior pituitary gland as a 
model to investigate the molecular basis for gen- 
eration of specific cell phenotypes in an organ. 
The rat genes for growth hormone and prolactin 
(a pituitary hormone that stimulates and sustains 
lactation) exhibit precisely restricted expression 
in somatotrophs and lactotrophs, respectively. 
Prolactin gene expression is dictated by two sepa- 
rate regions, a distal enhancer and a proximal re- 
gion, each containing at least four critical ele- 
ments. The two regions, each capable of targeting 
tissue-specific gene expression, act synergisti- 
cally to generate high levels of prolactin gene ex- 
pression in transgenic mice. 
The consensus binding site for cell-specific 
transcription-regulating protein (s), referred to as 
Pit-1 , was characterized; the 33-kDa Pit- 1 protein 
was purified; and, using in vitro transcription as- 
says, it was shown to exert functional effects. 
Pit-1 cDNAs were cloned from rat pituitary ex- 
pression libraries. Bacterially expressed Pit-1 
bound, specifically and with high affinity, to pro- 
lactin and growth hormone promoters. Addi- 
tional elements and factors are required to 
achieve the full physiological levels and re- 
stricted patterns of expression of the prolactin 
and growth hormone genes. 
The carboxyl terminus of Pit- 1 contains a 60- 
amino acid region similar to the homeodomain of 
several Drosophila and vertebrate regulatory 
genes. Pit-1 contains a second, 76-amino acid 
sequence with striking homology to other tran- 
scription regulators. These two regions, together 
with a nonconserved region between them, are 
referred to as the POU domain. We have found 
that the 76-amino acid POU-specific domain 
functions in high-affinity DNA binding, in confer- 
ring site specificity, and in protein-protein inter- 
actions. The major transcription-activating do- 
main of Pit-1 is a 70-amino acid amino-terminal 
region rich in serine and threonine and distinct 
from recognized motifs. A detailed structure- 
function analysis has suggested that the determi- 
nants of DNA binding by POU-domain proteins 
are distinct from those of the classical homeodo- 
main proteins. A genetic approach was utilized to 
determine the functional role of Pit- 1 during or- 
ganogenesis. Based on the demonstration of 
disrupted patterns of the Pit l gene in genetic 
dwarf mice, it appears that the POU-domain pro- 
tein acts as a developmental regulator to deter- 
mine patterns of commitment, progression, and 
proliferation of specific cell types in the anterior 
pituitary gland. 
We devised a strategy to isolate new members 
of the POU-domain gene family. Four were iden- 
tified from cDNA in brain and testes. Transcripts 
from the Brn-1 and Brn-2 genes exhibited vir- 
tually identical patterns of expression in the cen- 
tral nervous system, although Brn-1 was clearly 
expressed in the medullary zone of the kidney, 
and Brn-2 was not. Brn-3 mRNA was present pre- 
dominantly in the peripheral nervous system; 
Tst- 1 transcripts were present in testes and brain. 
Subsequently, other POU domains have been 
identified expressed in early development or 
during organogenesis. 
Most POU-domain genes were widely ex- 
pressed in all levels of the neural tube (including 
the retina) during early development, and hybrid- 
ization in the ventricular (proliferative) zone of 
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