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(Received for publication, June 5, 1995; and in revised form, July 10, 1995) The combined factors that regulate the expression of cell
adhesion molecules (CAMs) during development of the nervous system are
largely unknown. To identify such factors for Ng-CAM, the neuron-glia
CAM, constructs containing portions of the 5` end of the Ng-CAM gene
were examined for activity after transfection into N2A neuroblastoma
and NIH3T3 cells. Positive regulatory elements active in both cell
types included an Ng-CAM proximal promoter with SP1 and cAMP response
element motifs extending 447 base pairs upstream of a single RNA start
site and a region within the first exon corresponding to
5`-untranslated sequences. Negative regulatory elements included five
neuron-restrictive silencer elements (NRSEs) and a binding site for Pax
gene products in a 305-base pair segment of the first intron.
Constructs containing the promoter together with the entire first
intron were active in N2A cells but were silenced in NIH3T3 cells. This
silencer activity was mapped to the NRSEs. In contrast, the Pax motif
inhibited activity of Ng-CAM constructs in both cell types. The DNA
elements defined in these transfection experiments were examined for
their ability to bind nuclear factors. The region within the first exon
formed a DNA-protein complex after exposure to nuclear extracts
prepared from both NIH3T3 and N2A cells. The NRSE region formed a more
prominent complex with proteins prepared from NIH3T3 cells than it did
with extracts from N2A cells. A member of the Pax protein family, Pax-3
bound to the Pax motif. Mutations introduced within the Pax motif in
its ATTA sequence eliminated this binding whereas mutations in its
GTTCC sequence did not, suggesting that paired homeodomain interactions
are important for the recognition of Pax-3 by this DNA target sequence.
The combined data suggest that negative regulation by NRSEs and Pax
proteins may play a key role in the place-dependent expression patterns
of Ng-CAM during development.
Volume 270,
Number 36,
Issue of September 08, pp. 21291-21298, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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