POLYPEPTIDE HORMONE GENE REGULATION 
Joel F. Habener, M.D., Investigator 
Major emphasis in Dr. Habener's laboratory is 
presently in two areas: 1) identification of DNA- 
binding proteins responsible for regulated and tis- 
sue-specific expression of polypeptide hormone 
genes and 2) determination of the bioactivities of 
peptides identified through nucleotide sequencing 
of precursors encoding peptide hormones. 
Nuclear proteins bind to specific DNA sequences 
in or around the polypeptide hormone genes either 
to up- or downregulate the transcription of genes. 
A major goal is to isolate and characterize the struc- 
tures and functions of the DNA-binding proteins. 
Dr. Habener is investigating the cell-specific expres- 
sion of the glucagon, somatostatin, and an- 
giotensinogen genes, using islet cell lines with dis- 
tinct hormone-expressing phenotypes. Similar 
studies of expression of the gonadotropin subunit 
and angiotensinogen genes are being analyzed in 
placental and liver cell lines, respectively. 
Dr. Habener has focused on analyses of the ex- 
pression of the human glycoprotein hormone a- 
subunit gene, utilizing the JEG-3 placental cell line, 
in which the transcription of the a gene is greatly 
stimulated by cAMP. The transcription of the a and 
CG-P genes is stimulated by analogues of cAMP, and 
synthetic cAMP/enhancer-like cassettes linked to 
the a-promoter in bacterial chloramphenicol acetyl 
transferase (CAT) reporters confer a 40- to 50-fold 
induction of gene transcription. Dr. Habener is in- 
vestigating the molecular workings and interactions 
of the multiple cooperating cis elements and DNA- 
binding proteins involved in cellular cAMP re- 
sponsivity at the level of the genome. 
One cis element of the a gene consists of two di- 
rect 18 bp repeats, each of which contains the palin- 
dromic cAMP-responsive octamer moti^ TGACGTCA 
(CRE). This sequence element confers cAMP re- 
sponsiveness and enhancer-like properties to the 
gonadotropin a-subunit, glucagon, and somato- 
statin genes. 
The structural and functional properties of the 
CRE were investigated. Dr. Habener found that the 
CRE only functions as a transcriptional response 
element when present in the proper context of 
adjacent bases surrounding the elements. The 
cAMP responsiveness of the CRE depends on the 
catalytic subunit of cAMP-dependent protein kinase 
A, because all responsiveness was either stimulated 
or attenuated by coexpression of minigenes en- 
coding either the catalytic subunit of protein ki- 
nase A or the protein kinase A inhibitor peptide, re- 
spectively 
Dr. Habener determined the amino acid se- 
quence of CREB from a cloned cDNA that he iso- 
lated from a placental Agtll expression library 
using a radioactive CRE probe. CREB is a 327- 
amino acid protein that belongs to a newly recog- 
nized class of transcriptional proteins, the "leucine 
zipper" proteins. Other members of this class of 
proteins include myc, fos, c-jun, C/EBP, and GCN4. 
The zipper, located at the carboxyl terminus of 
CREB, consists of a heptad repeat of leucine resi- 
dues interspersed with charged residues that form 
an amphipathic a-helix with a hydrophobic face, al- 
lowing two CREB proteins to interact to form a par- 
allel coiled-coil CREB homodimer. A positively 
charged sequence with —40% amino acid identities 
with jun and fos (basic region) lies adjacent to the 
leucine zipper region. Dr. Habener proposes that 
the carboxyl-terminal basic region juxtaposed to 
the zipper region forms a helix-turn-helix with two 
amphipathic a-helices with basic faces, which con- 
stitutes the DNA-binding domain of CREB that 
binds to the symmetrical CRE palindromes as a 
homodimer. A synthetic peptide consisting of the 
carboxyl-terminal 66 residues of CREB readily 
forms dimers and binds the CRE, but a peptide of 
50 residues, missing one helix of the basic region, 
forms dimers but does not bind the CRE. Further- 
more, cell-free cotranslation of CREB, jun, and fos 
in different combinations shows the formation of 
CREB homodimers and jun/fos heterodimers but 
no CREB/jun or CREB/fos heterodimers, indicating 
that the CREB/CREB dimer is a highly favored con- 
figuration. The amino-terminal region of CREB is 
negatively charged and exists in the conformation 
of a random coil— characteristic of a "negative noo- 
dle" believed to be involved in transcriptional acti- 
vation. In addition, this random coil region of CREB 
contains a sequence of 50 residues containing 
phosphorylation sites for protein kinases A and C, 
casein kinase II, and glycogen synthase kinase III. 
This phosphorylation box (P-box) sequence has the 
potential for forming an amphipathic a-helix with 
an acid face when the serines are converted to 
phosphoserines. Dr. Habener has shown that the P 
box is responsible for transcriptional activation in 
response to cAMP, because fusion genes consisting 
of the CREB sequence 1-260 (negative noodle), 
both with and without the P box, linked to the 
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