and CREB-like products arising from alternative 
splicing of exons, creating tissue-type-specific 
mRNAs with deleted, inserted, and substituted 
exons. Dr. Habener has discovered a particularly 
high level of CREB gene expression in the Sertoli 
and germ cells of the testis (in work supported in 
part by the National Institutes of Health). This ex- 
pression is regulated cyclically in the Sertoli cells, 
waxing and waning every 1 2 days in the seminifer- 
ous tubules. The rise and fall coincides with the 
splicing out or in, respectively, of an exon con- 
taining multiple stop codons, resulting in the 
translation of a truncated CREB lacking a nuclear 
translocation signal and the DNA-binding domain. 
Examination of the promoter region of the CREB 
gene revealed numerous control elements, includ- 
ing three CREs, indicating that the transcriptional 
expression of this gene is likely under autopositive 
feedback control. Dr. Habener postulates that the 
increase in expression of the CREB gene in the Ser- 
toli cells results from the increased intracellular 
cAMP levels stimulated by the cyclical rise in the 
pituitary gonadotropin (follicle-stimulating hor- 
mone) that interacts with receptors known to exist 
on the Sertoli cells. The fall in expression is a conse- 
quence of the splicing in of the exon, resulting in a 
CREB that can no longer enter the nucleus and au- 
toactivate the CREB gene. 
In addition. Dr. Habener has discovered a second 
gene that encodes the protein CREM, which also 
comprises many exons, several of which are nearly 
identical to those of CREB. Pre-mRNAs transcribed 
from both the CREB and CREM genes undergo com- 
plex patterns of alternative splicing of exons in a 
highly tissue-specific manner. The goal is to under- 
stand the functional consequences of these alterna- 
tively spliced mRNAs and how they alter the func- 
tions of the CREB and CREM proteins. 
The transcriptional activation of the angiotensin- 
ogen gene in liver cells in response to acute-phase 
reactants is mediated by a complex interplay of 
DNA-binding proteins, including glucocorticoid 
receptors, nuclear factor kB (NF-kB), and C/EBP 
(CCAAT/enhancer-binding protein) or C/EBP- 
related protein (s). Studies indicate that in basal 
(unstimulated) conditions in hepatocytes, constitu- 
tive ly expressed C/EBP binds the acute-phase re- 
sponse element (APRE), flanked on both sides by 
glucocorticoid response elements (GREs), provid- 
ing a modest glucocorticoid-inducible transcrip- 
tion. However, upon the activation of the acute- 
phase response (for example, by tissue injury, 
foreign antigens, or pyrogenic toxins), the resultant 
cytokines that are produced (such as interleukin- 1 
[IL- 1 ] and tumor necrosis factor [TNF]) activate he- 
patic receptors coupled to both cAMP/protein ki- 
nase A and phospholipid/protein kinase C path- 
ways. These pathways lead to the translocation of 
NF-kB from the cytoplasm to the nucleus, where it 
competitively displaces C/EBP for binding to the 
APRE. Inasmuch as NF-/cB is a more potent transacti- 
vator than C/EBP, the rate of angiotensinogen gene 
transcription is markedly elevated. 
Dr. Habener has identified a novel nuclear protein 
that serves as a dominant negative inhibitor of tran- 
scription factors C/EBP and LAP (liver-activating 
protein) . A '^P-labeled LAP DNA-binding and dimer- 
ization domain zipper probe was used to isolate a 
clone that encodes CHOP 10, a new C/EBP homolo- 
gous protein. CHOP 10 has strong sequence similar- 
ity to C/EBP-like proteins within the bZIP region 
corresponding to the DNA-binding domain consist- 
ing of a leucine zipper and a basic region. Notably, 
however, CHOP 10 contains two prolines substitut- 
ing for two residues in the basic region, critical for 
binding to DNA. Thus heterodimers of CHOP 10 and 
C/EBP-like proteins are unable to bind their cognate 
DNA enhancer element. CHOP 10 mRNA is ex- 
pressed in many different rat tissues. Antisera raised 
against CHOP 10 recognize a nuclear protein with 
an apparent molecular mass of 29 kDa. CHOP 10 is 
induced upon differentiation of 3T3-L1 fibroblasts 
to adipocytes, and cytokine-induced dedifferentia- 
tion of adipocytes is preceded by the loss of nuclear 
CHOPIO. 
Co-immunoprecipitation of CHOPIO and LAP 
from transfected COS- 1 cells demonstrated a direct 
interaction between the two proteins in vivo. Con- 
sistent with the structure of its defective basic re- 
gion, bacterially expressed CHOPIO inhibits the 
DNA-binding activity of C/EBP and LAP by forming 
heterodimers that cannot bind DNA. In transfected 
HepG2 cells, expression of CHOPIO attenuates ac- 
tivation of C/EBP- and LAP-driven promoters. Dr. 
Habener proposes that CHOPIO is a negative modu- 
lator of the activity of C/EBP-like proteins in certain 
terminally differentiated cells; this is similar to the 
regulatory function of Id on the activity of MyoD and 
MyoD-related proteins important in the develop- 
ment of muscle cells. 
During the next year research will be focused on 
more-detailed investigations of the functional do- 
mains of the cAMP-responsive DNA-binding pro- 
teins. Studies will be aimed at defining the DNA- 
binding properties and the molecular processes of 
transcriptional activation. Particular emphasis will 
be on investigations of the roles of phosphorylations 
in nuclear transport, DNA binding, dimerization for- 
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