Hereditary gingival fibromatosis (HGF) is a rare genetic disorder featured by nonsyndromic pathological overgrowth of gingiva. The excessive gingival tissues can cause dental, masticatory, and phonetic problems, which impose severe functional and esthetic burdens on affected individuals. Due to its high recurrent rate, patients with HGF have to undergo repeated surgical procedures of gingival resection, from childhood to adulthood, which significantly compromises their quality of life. Unraveling the genetic etiology and molecular pathogenesis of HGF not only gains insight into gingival physiology and homeostasis but also opens avenues for developing potential therapeutic strategies for this disorder. Recently, mutations in REST (OMIM *600571), encoding a transcription repressor, were reported to cause HGF (GINGF5; OMIM #617626) in 3 Turkish families. However, the functions of REST in gingival homeostasis and pathogenesis of REST-associated HGF remain largely unknown. In this study, we characterized 2 HGF families and identified 2 novel REST mutations, c.2449C>T (p.Arg817*) and c.2771_2793dup (p.Glu932Lysfs*3). All 5 mutations reported to date are nonsenses or frameshifts in the last exon of REST and would presumably truncate the protein. In vitro reporter gene assays demonstrated a partial or complete loss of repressor activity for these truncated RESTs. When coexpressed with the full-length protein, the truncated RESTs impaired the repressive ability of wild-type REST, suggesting a dominant negative effect. Immunofluorescent studies showed nuclear localization of overexpressed wild-type and truncated RESTs in vitro, indicating preservation of the nuclear localization signal in shortened proteins. Immunohistochemistry demonstrated a comparable pattern of ubiquitous REST expression in both epithelium and lamina propria of normal and HGF gingival tissues despite a reduced reactivity in HGF gingiva. Results of this study confirm the pathogenicity of REST truncation mutations occurring in the last exon causing HGF and suggest the pathosis is caused by an antimorphic (dominant negative) disease mechanism.

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...