A restrictive element 1 (RE‐1) in the VIP gene modulates transcription in neuronal and non‐neuronal cells in collaboration with an upstream tissue specifier element

Abstract

The vasoactive intestinal peptide (VIP) gene has been studied extensively as a prototype neuronal gene containing multiple cis-active elements that confer responsiveness to cell lineage, neurotrophic, and activity-dependent intrinsic and extrinsic cues. However, reporter genes containing the presumptive complete regulatory region 5¢ to the start of transcription do not confer tissue-specific gene expression in vivo. We therefore sought cis-regulatory elements downstream of the transcriptional start that might confer additional tissue-specific and tissue-restrictive properties to the VIP transcriptional unit. We report here a repressor element, similar to the canonical restrictive element-1 (RE-1), located within the first non-coding exon of the human VIP gene. The ability of this element to regulate VIP reporter gene expression in neuroblastoma and fibroblastic cells was examined. Endogenous VIP expression is high in SH-EP neuroblastoma cells, low but inducible in SH-SY5Y cells, and absent in HeLa cells. Endogenous RE-1 silencer factor (REST) expression was highest in SH-EP and HeLa cells, and significantly lower in SH-SY5Y cells. Transient transfection of a VIP reporter gene containing a mutated RE-1 sequence revealed an RE-1-dependent regulation of VIP gene expression in all three cell types, with regulation greatest in cells (SH-EP, HeLa) with highest levels of REST expression. Serial truncation of the VIP reporter gene further revealed a specific interaction between the RE-1 and a tissuespecifier element located 5 kb upstream in the VIP gene. Thus, REST can regulate VIP gene expression in both neuroblastic and non-neuronal cells, but requires coupling to the upstream tissue specifier element. Vasoactive intestinal peptide (VIP) is a neuropeptide uniquely distributed within the brain and peripheral nervous system with pleiotropic functions in the immune, endocrine and nervous systems. VIP plays an important role in neurodevelopment with mitogenic and trophic effects on embryonic neurons in the CNS and PNS (Brenneman and Eiden 1986;Gressens et al. 1993). It is transiently expressed at high levels in rat stellate ganglion in utero, possibly regulating neuroblast and glial cell proliferation and survival (Tyrrell and Landis 1994;Waschek 1995). Control and regulation of the VIP gene is likely, therefore, to have far reaching effects on the organism. Although multiple regulatory regions have been mapped on the VIP gene, complete recapitulation of VIP gene expression has not yet been achieved using VIP reporter genes in transgenic animal models, suggesting the presence of other as yet unidentified regulatory regions (Agoston et al. 1990;Tsuruda et al. 1996;Waschek et al. 1999).The human VIP gene sequence was searched using the web-based transcription factor analysis software package Transfac (Wingender et al. 2000), and a restrictive element-1 Received July 15, 2003; revised manuscript received October 21, 2003; accepted October 22, 2003. Address correspondence and reprint requests to Lee Eiden, Building...

Developmental expression of the high affinity choline transporter in cholinergic sympathetic neurons

“…The VIP gene has been the most extensively studied, and a number of cis-regulatory elements have been identified (Hahm et al, 2003;Hamelink et al, 2004;Waschek et al, 1988), including a 180 base pair cytokine response element that confers responsiveness to LIF and related cytokines (Pitts et al, 2001;Symes et al, 1994). Our data indicate that expression of CHT is induced together with ChAT, VAChT, and VIP in the developing sweat gland innervation.…”

Novel Small Molecule Positive Allosteric Modulator SPAM1 Triggers the Nuclear Translocation of PAC1-R to Exert Neuroprotective Effects through Neuron-Restrictive Silencer Factor

“…Both hydrazide and doxycycline (DOX) have been reported to bind to the PAC1-R-EC1 domain, but in different ways [8,9]. DOX, with clinical neuroprotective activity, binds PAC1-R-EC1 similarly to PACAP (30)(31)(32)(33)(34)(35)(36)(37), while hydrazide, without clinical neuroprotective activity, binds similarly to PACAP (20)(21)(22)(23)(24)(25)(26)(27) [9], indicating that PACAP (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) (GKRYKQRVKNK) plays an important role in the regulation of the activity of PACAP/PAC1-R, a finding which has also been recently recognized by other groups [10,11]. Moreover, some hydrazide and hydrazone analogs have been found to have significant neuroprotective effects, including phenyl hydrazide J147 [12], L-carnosine hydrazide [13], dantrolene-like hydrazide, and hydrazone analogues [14], etc., which are predicted to bind to the site at PAC1-R-EC1 overlapped by PACAP (28)(29)(30)(31)(32)(33)(34)(35)…”

Identification of Repressor Element 1 in Secretin/PACAP/VIP Genes

“…In the brain, majority of these peptides have already been identified as neurotransmitters, neuromodulators, neurotrophic factors, and/or neurohormones 15,16 . Recently, functional RE‐1 sites have been located in VIP and PACAP genes 17,18 . These findings and the neuronal specificity of this peptide family prompted us to investigate the presence of potential functional RE‐1 sites in this peptide gene family.…”

“…15,16 Recently, functional RE-1 sites have been located in VIP and PACAP genes. 17,18 These findings and the neuronal specificity of this peptide family prompted us to investigate the presence of potential functional RE-1 sites in this peptide gene family. By searching the RE-1 database, RE1db (http:// bioinformatics.leeds.ac.uk/group/online/RE1db/re1db home.htm), 5 we could only locate the RE-1 sites in VIP and glucagon genes (TABLE 1).…”