Divergent Members of the SRY Family 
of Transcriptional Regulators Bind an 
Insulin-Responsive Element, IRE-A 
Maria C. Alexander-Bridges, M.D., Ph.D. — Assistant Investigator 
Dr. Alexander- Bridges is also Assistant Professor of Medicine at Harvard Medical School and Assistant in 
Medicine at Massachusetts General Hospital, Boston. She received her M.D. and Ph.D. degrees from 
Harvard University Medical School, where she was a member of the Harvard-MIT Health Sciences and 
Technology program, which is geared toward students interested in academic medicine. She developed an 
/ abiding interest in hormonal regulation of cellular metabolism and, as a graduate student in physiology, 
L investigated the mechanism of insulin- stimulated phosphorylation of cellular proteins. Dr. Alexander- 
Bridges then served as an intern and resident at the Johns Hopkins University. After subspecialty training 
in endocrinology at Massachusetts General Hospital, she was a postdoctoral fellow with Howard Goodman. 
THE main function of insulin is to allow a 
starved animal to adapt to a glucose load. In- 
sulin does this by activating enzymes that pro- 
mote energy storage and inactivating enzymes 
that break down energy stores. Over the long 
term, insulin alters the expression of these en- 
zymes by regulating the transcription of their 
genes. The work in our laboratory is directed to- 
ward understanding the mechanism of insulin ac- 
tion on expression of metabolically active genes 
in tissues that modulate glucose utilization. We 
use the glyceraldehyde- 3 -phosphate dehydroge- 
nase (GAPDH) gene as a model gene for the ana- 
bolic effects of insulin, because it is highly regu- 
lated by insulin 1) in cultured 3T3 adipocytes, 2) 
during nutritional manipulations such as fasting 
and refeeding, and 3) during the induction and 
treatment of diabetes. 
Transgenic animals that express GAPDH- 
growth hormone fusion genes have been made in 
collaboration with Jeung Yun and Tom Wagner 
(Ohio State University). Using these animals, we 
have confirmed that GAPDH gene transcription is 
regulated in vivo by nutritional manipulations 
that lead to hyperinsulinemia. For example, fast- 
ing a rat and refeeding it a high-carbohydrate, 
low-fat diet increases circulating glucose and in- 
sulin levels, resulting in the induction of glyco- 
lytic and lipogenic enzymes and the repression of 
gluconeogenic and lipolytic enzymes. 
We have identified a cis-acting sequence, 
insulin-responsive element A (IRE-A), in the up- 
stream region of the GAPDH gene that interacts 
with an insulin-responsive DNA-binding protein 
(IRP-A) . Activation of GAPDH gene expression in 
insulin-responsive tissues correlates with the 
presence of IRP-A. Within one hour of exposure 
of 3T3 adipocytes or H35 hepatoma cells to insu- 
lin, the activity of this protein is increased two- to 
fourfold. The activity of IRP-A is induced four- to 
eightfold in liver and fat during the process of 
refeeding a fasted rat a high-carbohydrate, low-fat 
diet. These observations support the importance 
of GAPDH gene regulation. In muscle, where 
GAPDH activity is not rate limiting and is not reg- 
ulated by insulin, IRP-A binding is not detectable. 
The wild-type IRE-A motif was used to isolate a 
clone from a rat adipocyte library, using the 
Singh-Sharp Southwestern screening approach. 
The cloned cDNA encodes a protein (IRE-ABP) 
that binds IRE-A DNA with sequence specificity 
that overlaps that of the adipocyte IRP-A nuclear 
extract protein. IRE-ABP is expressed in liver and 
fat but not in muscle, which provides an explana- 
tion for the tissue-specific regulation of GAPDH 
gene expression. Expression of IRP-A mRNA is in- 
duced during the process of fasting and refeed- 
ing. In contrast, one hour of insulin exposure of 
cells does not appear to alter expression of the 
IRP-A mRNA. IRE-ABP footprints the upstream re- 
gion of genes that are inhibited and genes that are 
stimulated by insulin. We presume that IRE-ABP 
will regulate the transcription of metabolic genes 
with diverse functions to change the phenotype 
of the fat and liver cell during the switch from the 
fasted to the refed state that is initiated by glucose 
and insulin. Studies are in progress to determine 
whether IRE-ABP mediates the effect of insulin 
alone or in association with another protein. 
IRE-A DNA-binding Protein Shares Binding 
Specificity with the Testis-determining 
Factor 
Surprisingly, the HMG (high-mobility group) 
box domain of IRE-ABP is 68 percent identical to 
SRY, the testis-determining factor, and is 98 per- 
cent identical to an autosomal gene that was iso- 
lated during the process of screening a whole 
mouse embryo cDNA library for 5i?F-related se- 
quences. Furthermore, IRE-ABP and SRY share 
DNA-binding specificity for IRE-A. Although IRE- 
ABP shows markedly higher affinity for the IRE-A 
motif, the nucleotides protected by these two di- 
vergent family members are essentially identical. 
The sequence in IRE-A that is contacted by IRE- 
ABP and SRY is highly conserved: the sequence 
5'-Py-ctttg(a/t)-3', previously defined by Kather- 
ine Jones and her colleagues as a consensus motif, 
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