Abstract
Vasoactive intestinal peptide (VIP) is a neuromodulator expressed with great anatomical specificity throughout the nervous system. Cell-specific expression of the VIP gene is mediated by a tissue specifier element (TSE) located within a 2.7-kilobase (kb) region between -5.2 and -2.5 kb upstream from the transcription start site, and requires an intact promoter proximal VIP-CRE (cyclic AMP-responsive element) (Hahm, S. H., and Eiden, L. E. (1997) J. Neurochem. 67, 1872-1881). We now report that the TSE comprises a 425-base pair domain located between -4.7 and -4.2 kb containing two AT-rich octamer-like sequences. The 425-base pair TSE is sufficient to provide full cell-specific regulation of the VIP gene, when fused to the 5' proximal 1.55 kb of the VIP gene. Mutational analysis and gel shift assays of these octamer-like sequences indicate that the binding of proteins related to the ubiquitously expressed POU-homeodomain proteins Oct-1 and/or Oct-2 to these octamer-like sequences plays a central role for the function of the TSE. The TSE interacts with three additional discrete domains besides the cAMP response element, which are located within the proximal 1.55 kb of the VIP gene, to provide cell-specific expression. An upstream domain from -1.55 to -1.37 kb contains E-boxes and MEF2-like motifs, and deletion of this domain results in complete abrogation of cell-specific transcriptional activity. The region from -1.37 to -1. 28 kb contains a STAT motif, and further removal of this domain allows the upstream TSE to act as an enhancer in both SH-EP and HeLa cells. The sequence from -1.28 to -0.9 kb containing a non-canonical AP-1 binding sequence (Symes, A., Gearan, T., Eby, J., and Fink, J. S. (1997) J. Biol. Chem. 272, 9648-9654), is absolutely required for TSE-dependent cellspecific expression of the VIP gene. Thus, five discrete domains of the VIP gene provide a combination of enhancer and repressor activities, each completely contingent on VIP gene context, that together result in cell-specific transcription of the VIP gene.
Highlights
Regulator, and neuroendocrine releasing factor [1, 2]
Using Vasoactive intestinal peptide (VIP)-luciferase reporter constructs in transient expression assays, we have previously shown that cell type-specific expression of the VIP gene in SH-EP cells requires at least two different cis-acting sequences within the VIP 5Ј-flanking region
We have previously shown from a deletional analysis that sequences within the region Ϫ5.2 to Ϫ2.5 kb upstream from the transcription start site of the human VIP gene are absolutely required for cell-specific expression of a VIP-luciferase reporter gene in SH-EP neuroblastoma cells [11]
Summary
Vol 273, No 27, Issue of July 3, pp. 17086 –17094, 1998 Printed in U.S.A. Five Discrete Cis-active Domains Direct Cell Type-specific Transcription of the Vasoactive Intestinal Peptide (VIP) Gene*. The TSE interacts with three additional discrete domains besides the cAMP response element, which are located within the proximal 1.55 kb of the VIP gene, to provide cell-specific expression. The anatomical and temporal specificity of VIP expression throughout the neuroendocrine axis is critical to neuronal function and endocrine homeostasis Understanding how this specificity is obtained, at the level of the cis-acting sequences of the VIP gene itself, should provide experimental access to the trans-acting factors that interact with these sequences, and to the signal transduction pathways that developmentally and physiologically link VIP gene expression within VIPergic cells with the extracellular environment. An upstream tissue-specifier element (TSE) located between Ϫ4.6 and Ϫ4.0 kb from the transcription start site was absolutely required for a cell type-specific expression of the VIP gene. This paper is available on line at http://www.jbc.org pression of the VIP gene requires combinatorial effects from multiple cis-acting sequences that both repress transcriptional activity of the TSE in HeLa cells, and enhance its activity in SH-EP cells
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