Section on Molecular Neuroscience, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892 USA
The
cis-acting elements of the VIP gene important for basal
and stimulated transcription have been studied by transfection
of VIP-reporter gene constructs into distinct human neuroblastoma
cell lines in which VIP transcription is constitutively high,
or can be induced to high levels by protein kinase stimulation.
The 5.2 kb flanking sequence of the VIP gene conferring correct
basal and inducible VIP gene expression onto a reporter gene
in these cell lines was systematically deleted to define its
minimal components. A 425-bp fragment (-4656 to -4231) fused
to the proximal 1.55 kb of the VIP promoterenhancer was absolutely
required for cell-specific basal and inducible transcription.
Four additional components of the VIP gene were required for
full cell-specific expression driven by the 425 bp TSE (region
A). Sequences from -1.55 to -1.37 (region B), -1.37 to -1.28
(region C), -1.28 to -.094 (region D), and the CRE-containing
proximal 94 bp (region E) were deleted in various combinations
to demonstrate the specific contributions of each region to
correct basal and inducible VIP gene expression. Deletion of
region B, or mutational inactivation of the CRE in region E,
resulted in constructs with low transcriptional activity in
VIP-expressing cell lines. Deletion of regions B and C together
resulted in a gain of transcriptional activity, but without
cell specificity. All five domains of the VIP gene were also
required for cell-specific induction of VIP gene expression
with phorbol ester. Gelshift analysis of putative regulatory
sequences in regions A-D suggests that both ubiquitous and neuron-specific
trans-acting proteins participate in VIP gene regulation.
