TRANSCRIPTIONAL REGULATORY PROTEINS 
Steven L. Mcknight, Ph.D., Investigator 
I. CCAAT/Enhancer Binding Protein. 
Two DNA sequence elements that occur quite fre- 
quently in association with promoters and en- 
hancers are the CCAAT homology and the enhancer 
core homology A heat-resistant DNA-binding activ- 
ity capable of specific interaction with both of these 
DNA sequence elements was identified in rat liver 
nuclear extracts. The polypeptide that specifies this 
activity, termed CCAAT/enhancer binding protein 
(C/EBP), was purified in the McKnight laboratory by 
Drs. Barbara Graves, Peter Johnson, and William 
Landschulz, allowing derivation of a partial amino 
acid sequence. The amino acid sequence was used 
to generate specific antibodies, which were in turn 
used to retrieve a molecular clone of the gene that 
encodes C/EBR 
A. Tissue distribution. The tissues and cell types 
that express C/EBP have been identified by a combi- 
nation of antibody staining and nucleic acid hybrid- 
ization assays. High levels of C/EBP and mRNA were 
observed in adipose, liver, placenta, lung, adrenal 
gland, and intestine. Each of these tissues metabo- 
lizes lipids at an exceptionally high rate, leading to 
the hypothesis that C/EBP might be a general regu- 
lator of proteins and enzymes involved in lipid syn- 
thesis. Evidence favoring this interpretation has 
begun to emerge from several different experimen- 
tal approaches that have been conducted in Dr. 
McKnight's laboratory and the laboratory of Dr. M. 
Daniel Lane (The Johns Hopkins University Medical 
School). Purified C/EBP binds to the promoters and 
enhancers of a number of genes that are expressed 
selectively in adipose and liver. These include the 
genes encoding serum albumin (the major lipid car- 
rier protein), stearoyl acyl-CoA desaturase (SCDl), 
and 422/aP2 protein (an intracellular lipid carrier 
protein). The significance of such binding is 
strongly substantiated by transient transfection as- 
says, in which a C/EBP expression vector has been 
found to be capable of trans- activating each of the 
aforementioned genes. 
B. Structural properties of the C/EBP DNA-binding 
domain. The amino acid sequence of C/EBP is sim- 
ilar to the sequences of several transforming pro- 
teins, including Fos, Jun, and Myc. This region of 
sequence relatedness, which is located within the 
DNA-binding domain of the C/EBP polypeptide, is 
free of amino acid residues that are incompatible 
with a-helical structure. It also contains a heptad 
repeat of leucine residues. On the basis of these 
properties, Drs. Landschulz, Johnson, and Mc- 
Knight speculated that this region of C/EBP might 
form an amphipathic a-helix and that the hydro- 
phobic surface of the helix would be used as a di- 
merization interface to bring two polypeptide 
chains together. This leucine zipper motif is charac- 
teristic of a newly discovered class of sequence-spe- 
cific DNA-binding proteins. Evidence supportive of 
the leucine zipper model has emerged from muta- 
tional and spectroscopic studies. 
11. Herpes Simplex Virus Protein 16. 
The lytic infectious cycle of herpes simplex virus 
(HSV) is characterized by a three-tiered cascade of 
viral gene expression. During the first 3 h postinfec- 
tion, five viral genes are transcribed. These five im- 
mediate early (IE) genes encode regulatory proteins 
that play a critical role in the expression of the sub- 
sequent class of viral genes, the delayed early (DE) 
genes. DE genes, which are expressed between 
3 and 9 h postinfection, encode enzymes and pro- 
teins necessary for the replication of viral DNA. The 
expression of the final class of viral genes, the late 
(L) genes, requires DNA replication and occurs be- 
tween 9 and 18 h postinfection. Most of the L genes 
encode structural proteins that form the mature vi- 
rion. However, the product of one L gene, HSV pro- 
tein 16 (VP 16), plays a regulatory role. When a vi- 
rion enters a newly infected cell, VP 16 moves to the 
nucleus and serves as a potent activator of IE gene 
expression. 
A. VP 16 attaches to the enhancers of IE genes indi- 
rectly. The DNA sequence elements of IE genes that 
are required for trans-activation by VP16 have been 
mapped and shown to represent binding sites for 
cellular DNA-binding proteins. Thus VP16 appears 
to "piggyback" its way onto IE genes via cellular 
transcription factors. One of the cellular proteins 
critical for VP16 function, octamer transcription fac- 
tor 1 (OTFl), has been studied extensively by Dr. 
Winship Herr and his colleagues at Cold Spring 
Harbor Laboratory. A second protein, immediate 
early facilitator GA (lEFga), has been purified by Dr. 
Karen LaMarco in the McKnight laboratory. Once at- 
tached to an IE enhancer via these cellular pro- 
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