Five Discrete Cis-active Domains Direct Cell Type-specific Transcription of the Vasoactive Intestinal Peptide (VIP) Gene

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. 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. VIP 1The abbreviations used are: VIP, vasoactive intestinal peptide; TSE, tissue-specifier element; bp, base pair(s); kb, kilobase(s); GnRH, gonadotropin releasing hormone; MEF2, myocyte enhancer factor-2; CyRE, cytokine response element; CRE, cAMP response element. 1The abbreviations used are: VIP, vasoactive intestinal peptide; TSE, tissue-specifier element; bp, base pair(s); kb, kilobase(s); GnRH, gonadotropin releasing hormone; MEF2, myocyte enhancer factor-2; CyRE, cytokine response element; CRE, cAMP response element. is distributed throughout the central and peripheral nervous systems and functions as a neuromodulator, growth regulator, and neuroendocrine releasing factor (1Brenneman D.E. Nicol T. Warren D. Bowers L.M. J. Neurosci. Res. 1990; 25: 386-394Crossref PubMed Scopus (175) Google Scholar, 2Gozes I. Brenneman D.E. Mol. Neurobiol. 1989; 3: 1-36Crossref PubMed Scopus (229) Google Scholar). It is a 28-amino acid peptide that is processed from a larger precursor peptide that also encodes a second VIP-like peptide called PHI in rodents and PHM in humans (3Mutt V. Said S.I. Mutt V. Vasoactive Intestinal Polypeptide and Related Peptides. The New York Academy of Sciences, New York1988: 1-19Google Scholar). VIP is found in high concentrations in specific regions of the brain including cerebral cortex, hypothalamus, and hippocampus (4Emson P.C. Gilbert R.F.T. Loren I. Fahrenkrug J. Sundler F. Muckadell O.B.S.D. Brain Res. 1979; 177: 437-444Crossref PubMed Scopus (97) Google Scholar), and in the peripheral nervous system in cholinergic and sensory nerves (5Fahrenkrug J. Digestion. 1979; 19: 149-169Crossref PubMed Scopus (266) Google Scholar). VIP is a co-transmitter mediating vasodilatation in the brain, gut, and other organs. VIP is also expressed in the anterior pituitary and functions in the regulation of endocrine homeostasis (2Gozes I. Brenneman D.E. Mol. Neurobiol. 1989; 3: 1-36Crossref PubMed Scopus (229) Google Scholar, 6Rosténe W.H. Prog. Neurobiol. 1984; 22: 103-129Crossref PubMed Scopus (169) Google Scholar). An important role for VIP in development is suggested by striking growth regulatory effects of exogenously administered VIP during mouse embryogenesis (7Gressens P. Hill J.M. Gozes I. Fridkin M. Brenneman D.E. Nature. 1993; 362: 155-158Crossref PubMed Scopus (257) Google Scholar). The anatomical and temporal specificity of VIP expression throughout the neuroendocrine axis is therefore 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 ultimately to the signal transduction pathways that developmentally and physiologically link VIP gene expression within VIPergic cells with the extracellular environment. We have relied on neuroblastoma cell lines, subclones of which recapitulate various aspects of VIP gene regulation in vivo(8Cooper M.J. Hutchins G.M. Cohen P.S. Helman L.J. Mennie R.J. Israel M.A. Cell Growth Differ. 1990; 1: 149-159PubMed Google Scholar, 9Waschek J.A. Pruss R.M. Siegel R.E. Eiden L.E. Bader M.-F. Aunis D. Ann. N. Y. Acad. Sci. 1987; 493: 308-323Crossref PubMed Scopus (47) Google Scholar, 10Waschek J.A. Hsu C.-M. Eiden L.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9547-9551Crossref PubMed Scopus (39) Google Scholar), to study the mechanism of cell-specific regulation of the VIP gene underlying its anatomically precise expression in vivo. Subclones from the SK-N-SH neuroblastoma cell line have been characterized with respect to cell type-specific modes of basal and inducible regulation of the VIP gene. In particular, the SH-EP subline constitutively expresses high levels of VIP mRNA (10Waschek J.A. Hsu C.-M. Eiden L.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9547-9551Crossref PubMed Scopus (39) Google Scholar, 11Hahm S.H. Eiden L.E. J. Neurochem. 1996; 67: 1872-1881Crossref PubMed Scopus (20) Google Scholar). Using 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 5′-flanking region. An upstream tissue-specifier element (TSE) located between −4.6 and −4.0 kb from the transcription start site was absolutely required for a cell type-specific expression of the VIP gene. A deletion in this region abolished high level expression of the reporter gene in SH-EP cells (11Hahm S.H. Eiden L.E. J. Neurochem. 1996; 67: 1872-1881Crossref PubMed Scopus (20) Google Scholar). Inactivation of a 17-bp promoter proximal element (the VIP-CRE), originally defined by its ability to mediate cAMP-dependent induction of the VIP gene (12Fink J.S. Verhave M. Kasper S. Tsukada T. Mandel G. Goodman R.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6662-6666Crossref PubMed Scopus (151) Google Scholar), caused an approximately 60% decrease in the level of expression of the reporter gene in SH-EP cells (11Hahm S.H. Eiden L.E. J. Neurochem. 1996; 67: 1872-1881Crossref PubMed Scopus (20) Google Scholar), indicating that the upstream TSE cannot fully direct cell-specific expression of the VIP gene without the participation of the promoter-proximal CRE. We hypothesized that additional elements in the VIP gene might be required for full tissue-specific gene expression directed by the TSE. Stepwise deletion of domains of the VIP gene between the TSE and the promoter proximal CRE, and assay of cell-specific transcription in SH-EP versus HeLa cells, now show this hypothesis to be correct. Cell type-specific expression 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. Cell culture reagents, LipofectAMINE, and synthetic oligonucleotides were obtained from Life Technologies, Inc. (Gaithersburg, MD), fetal bovine serum from BioWhittaker (Walkersville, MD), and culture plates from Costar Corp. (Cambridge, MA). pGL3 vectors, pGEM vectors, luciferase assay reagents, reporter lysis buffers, and core footprinting kits were purchased from Promega Corp. (Madison, WI). DNA ligation kit was from Boehringer Mannheim Corp. (Indianapolis, IN). Sequencing reagents were purchased from U. S. Biochemical Corp. and Sequagel-6 ready to use 6% sequencing gel solutions were from National Diagnostics (Atlanta, GA). Oct-1 and Oct-2 supershift antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). SH-EP neuroblastoma cells and HeLa cells were cultured in Dulbecco's modified Eagle's medium with 4.5 g of glucose per liter, containing 10% fetal bovine serum (heat-inactivated) supplemented with glutamine (0.03%), penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells were maintained in a humidified 95% air, 5% carbon dioxide atmosphere. Culture medium was changed every 3–4 days to a fresh medium and SH-EP cells were subcultured before the cells reach approximately 70% confluency. The methods used to construct VIP-luciferase reporter plasmids were essentially as described previously (11Hahm S.H. Eiden L.E. J. Neurochem. 1996; 67: 1872-1881Crossref PubMed Scopus (20) Google Scholar). All VIP-luciferase reporter constructs contained the first exon, the first intron, and the second exon extending to the first codon of the human VIP gene fused to the luciferase coding sequence of the pGL3-basic vector, in addition to various regions of 5′ flank of the VIP gene. The promoter proximal 251 nucleotides upstream of the VIP transcription initiation site, which includes the minimal VIP promoter and the previously to be required for full cell-specific as as inducible expression in SH-EP and SK-N-SH cells is domain this minimal VIP gene was fused various of the VIP gene between the TSE in and the proximal promoter constructs were for transcriptional activity as described both with and without the TSE fused to the of the transcriptional The minimal TSE to was defined by both 5′ and of the −5.2 to −2.5 previously shown to be required for cell-specific expression of VIP (10Waschek J.A. Hsu C.-M. Eiden L.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9547-9551Crossref PubMed Scopus (39) Google fused to the VIP minimal and SH-EP cells. SH-EP and HeLa cells were as described previously (11Hahm S.H. Eiden L.E. J. Neurochem. 1996; 67: 1872-1881Crossref PubMed Scopus (20) Google with Cells were in Costar tissue culture plates at of and for SH-EP and HeLa cells, in of culture Cells were to to approximately and with of DNA and of for in of medium was and with complete Cells were for an additional before were in of the reporter lysis the luciferase of cell was with of the luciferase and were for a SH-EP and HeLa cell were to R.E. J.A. in New Scholar). of DNA used in the footprinting analysis different of the TSE sequence each of the two AT-rich sequences to and to DNA used as a was first and in the of and from a gel the gel kit CA). was to the of and T. PubMed Scopus Google Scholar), with of DNA was of was used in each and with with of SH-EP of HeLa was at for WI). sequencing for A was as described in J. T. A was the in system DNA was from in which the VIP TSE to was DNA was the and by J. T. A Scholar). The base within the AT-rich upstream AT-rich sequence AT-rich sequence The AT-rich are with in SH-EP and HeLa cell were to E. P. Res. 1989; PubMed Scopus Google with Cells were in approximately confluency. Cells in two were and in of A and Cells were to on for and of 10% was Cell was in a for and the was in of and proteins were on by on a for The was at g for at and the which contains the proteins was to a fresh concentrations were by to the and were by in and at in oligonucleotides were and by of was with of SH-EP HeLa in a of containing and were at for and a gel in were at for and at for approximately at were and supershift assays, of Oct-1 Oct-2 was to the binding used for the VIP gene upstream and AT-rich sequences and 5′ used as a was and its 5′ We have previously shown from a analysis that sequences within the region −5.2 to −2.5 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 (11Hahm S.H. Eiden L.E. J. Neurochem. 1996; 67: 1872-1881Crossref PubMed Scopus (20) Google Scholar). A sequence to as the VIP tissue-specifier element, two located at and from the start of transcription completely cell-specific transcription by the kb of upstream sequence between −5.2 and −2.5 kb, when fused to kb of the VIP gene 5′ flank Thus, of the TSE to the which is a in both SH-EP and HeLa cells, in an in transcription in SH-EP cells, with a in expression in HeLa cells. the TSE as a cell-specific when in of the of fully expression of the reporter gene in cells. The TSE contains two AT-rich sequences to the site found within the enhancer domain of the human gonadotropin releasing gene Mol. Biol. PubMed Scopus Google Scholar). the sequences of the VIP TSE with the site of the gene, the and binding found in other The AT-rich sequence of the human VIP gene from the Oct-1 binding sequence of the gene by versus and the of L.M. D. M. G. 1988; PubMed Scopus Google Scholar). in of the AT-rich sequences of the VIP gene reporter gene that both of these AT-rich sequences are required for cell-specific expression of the VIP gene. The results in show that the TSE fully recapitulate cell-specific expression when fused 5′ to 1.55 kb of the VIP gene 5′ flank nucleotides within the upstream AT-rich sequence of the TSE caused an approximately decrease in the of the TSE. in the AT-rich sequence the level of reporter gene expression by the TSE by approximately in SH-EP cells. a deletion of from the region of the TSE that the AT-rich sequence caused a decrease in results indicate that both of the AT-rich sequences an important role for the function of the TSE. shift assays were synthetic oligonucleotides each of the AT-rich of the VIP gene, to proteins that interact with these sequences. The the AT-rich sequence of the VIP gene, multiple with both SH-EP and HeLa proteins of these was specific as a of an to with the of an Oct-1 an Oct-2 in the binding caused a supershift of the as Oct-1 and the upstream AT-rich with for binding in with an Oct-1 with binding in by an Oct-2 an Oct-1 for the of the with that is with Oct-1 and the by the Oct-2 Oct-1 and Oct-2 were in both SH-EP and HeLa The within the AT-rich to for the of these indicating that Oct-1 and Oct-2 cannot to this AT-rich In the shown in the within the AT-rich sequence caused an decrease in cell-specific VIP gene transcription mediated by the TSE, indicating that binding of Oct-1 and Oct-2 to the AT-rich sequence is critical for the function of the TSE. an the upstream AT-rich sequence was used as a for the gel shift three were in both SH-EP and HeLa All were specific on with and the containing to for of these of Oct-1 specific caused a supershift of the as Oct-1 in the other two was by an addition of Oct-1 was in this Thus, the AT-rich sequence which both Oct-1 and Oct-2 the upstream AT-rich sequence the core sequences of and are sequences the AT-rich be critical for the specificity of these two In footprinting analysis was to regions within the TSE. The result in show that the AT-rich sequence was by SH-EP HeLa In an approximately region the 5′ flank of the AT-rich element was by SH-EP that a be at this region of DNA to cell-specific enhancer function of the TSE, including POU-homeodomain proteins Oct-1 and Oct-2 and other of which are expressed in SH-EP cells. In addition to within the TSE itself, the of TSE function on intact cAMP response sequences within the proximal promoter of the VIP gene (11Hahm S.H. Eiden L.E. J. Neurochem. 1996; 67: 1872-1881Crossref PubMed Scopus (20) Google suggested that other regions of the VIP gene between the TSE and VIP core promoter be in mediating TSE function as as a minimal VIP-luciferase construct containing the TSE fused to the construct of the VIP gene 5′-flanking sequence including the to fully recapitulate cell-specific expression of the reporter gene in SH-EP cells The construct was expressed at a level the construct in SH-EP cells. In five the was expressed on at approximately of the level of the indicating that both the TSE and VIP-CRE are sufficient for a cell-specific expression of the VIP gene. additional cis-acting sequences required for full cell-specific expression of the VIP gene, transient assays were reporter constructs in which the TSE was fused to VIP gene 5′-flanking sequences with from −2.5 kb to cell-specific expression of the reporter gene was in SH-EP cells when the TSE was fused upstream of at least 1.55 kb of promoter proximal VIP gene 5′ constructs transcriptional activity in HeLa cells. of from −1.55 to −1.28 kb from the VIP gene 5′ flank caused a decrease in reporter gene transcription in SH-EP cells. deletion of the VIP gene 5′ flank −1.55 kb caused a in expression of the reporter gene in HeLa cells, that these constructs cell-specific transcriptional activity. the role of the domain from −1.55 to −1.28 kb, a construct was by the upstream of the domain from the The construct a complete of cell-specific expression in SH-EP cells indicating that sequences located between −1.55 and −1.37 kb enhance the of TSE in SH-EP cells in HeLa cells. deletion of the 5′ flank to −1.28 kb caused an in the level of reporter gene expression in both SH-EP and HeLa cells, indicating that the region between −1.37 and −1.28 kb repressor activity in cells. In when the of the domain between −1.55 and −1.37 kb was in the of the domain between −1.37 and −1.28 kb domain from the the construct was expressed at a level of the construct in SH-EP cells that sequences between −1.37 and −1.28 kb are also required for the function of the TSE The region −1.55 to −1.28 kb and of the human VIP gene is to the region of the mouse VIP gene M. J.A. DNA PubMed Scopus Google Scholar), and contains two two enhancer sequences, a STAT site, and a pair of element The STAT site previously been as a critical in VIP gene of neuroblastoma cells to factor A. S. L. P. J.S. Mol. PubMed Google Scholar). The domain between −1.55 and −1.28 kb when fused of the TSE upstream of 251 of the VIP gene promoter proximal sequence to recapitulate a full cell-specific expression of the reporter gene the construct was expressed at an level both in SH-EP and HeLa cells, the construct the that additional sequences, located within domain the domain and the promoter proximal VIP-CRE), are required for full cell-specific expression. to the an additional construct was by approximately and of from the an upstream region of the containing the non-canonical site A. T. J. J.S. J. Biol. PubMed Scopus Google Scholar). The deletion of from the domain cell-specific expression of the reporter gene a full cell-specific expression of the VIP gene requires the upstream TSE promoter proximal and the region between −1.55 kb and of approximately We have shown that at least five regions of the human VIP gene 5′ flank in to mediate cell-specific expression of the reporter gene in SH-EP neuroblastoma cells. analysis is in A TSE located at approximately kb fully recapitulate cell-specific transcription when fused to at least 1.55 kb of the VIP gene promoter proximal in cells The VIP TSE contains two AT-rich sequences, as and of proteins of the POU-homeodomain for and Oct-1 and Oct-2 for related proteins to the TSE is to be required for transcriptional of the VIP gene. is on the that is in of SH-EP cells, and that in the TSE that Oct-1 binding also the cell-specific enhancer activity of this element. have that in a Oct-1 binding in the gene reporter gene expression by 95% in the neuronal cell line binding of Oct-1 a related to the AT-rich sequences plays a role in cell-specific transcription of at least two for VIP and Oct-1 is to be ubiquitously expressed in been shown to interact with other factors that are expressed in a to mediate cell-specific transcriptional transcription of requires binding of the Oct-1 also requires a also called L.M. D. M. G. 1988; PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar, M. L. Nature. PubMed Scopus Google Scholar, M. P. PubMed Scopus Google Scholar). Oct-1 interact with factors expressed in cells to mediate a expression of the VIP gene in this neuronal cell footprinting of the VIP TSE that expressed in SH-EP in HeLa cells in a for the specificity of VIP gene expression in neuroblastoma cells. −1.55 and −1.37 plays a critical role in cell-specific enhancer activity to the TSE. domain contains an and two MEF2-like sequences. that between proteins and cell type-specific proteins are important for tissue-specific transcription in both and neuronal cell J. Biol. 1996; PubMed Scopus (39) Google Scholar, Y. J. Biol. 1996; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). 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Res. 1990; PubMed Scopus Google Scholar). between −1.37 and of the VIP gene function to transcription of the VIP gene in VIP cells. from the −1.28 to domain is required for both expression in SH-EP cells and full of reporter constructs in HeLa cells. The region from −1.37 to −1.28 kb with the of the cytokine element which induction of the VIP gene by factor A. S. L. P. J.S. Mol. PubMed Google Scholar, J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). region contains a binding site for and which important in of transcription in systems A. S. L. P. J.S. Mol. PubMed Google Scholar, PubMed Scopus Google Scholar). The role of this STAT site and other regions of the cytokine element in cell-specific second expression of the VIP gene is these that the participation of five domains within the VIP are required for full cell-specific expression of the VIP gene in neuroblastoma cells. domain is to multiple including ubiquitously expressed proteins as and cAMP response and as cell-specific transcription factors that mediate both transcriptional in cells and transcriptional in VIP cell The of cell-specific regulation of VIP transcription at the level of the gene the for of multiple and extracellular to the and expression of the VIP gene in vivo. We G. for in