Ž . Molecular Brain Research 44 1997 309–322 Research report SEZ-6: promoter selectivity, genomic structure and localized expression in the brain 1 Ruth Herbst, Martin J.H. Nicklin ) Section of Molecular Medicine, Department of Medicine and Pharmacology, UniÕersity of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, UK Accepted 1 October 1996 Abstract Ž . AP-1-binding elements from promoter proximal DNA the small HpaII-digested fraction of mouse genomic DNA were affinity-selected with recombinant AP-1 complexes. One of the selected AP-1-binding elements originated from 1 kb 3 X of the transcription start site of SEZ-6. We show that the mouse SEZ-6 gene extends over 49 kbp and contains 17 exons. SEZ-6 has been reported as a mouse brain-specific transcript encoding an integral membrane protein with a short cytoplasmic tail which we note may have a signalling function. We show that SEZ-6 mRNA expression in rat brain is specific to neurons but shows sharp regional differences, unconnected with the localization of major neurotransmitters. Full-length and a 3 X truncated transcript are also abundant in testis. We define the origins of all reported sequence variants. The hypothetical domain structure of the protein is in excellent agreement with the exonic structure of Ž . the gene. The SEZ-6 promoter is a CpG island. In transient transfections, even the smallest promoter fragment tested 157 bp was extremely selective towards a mouse neuronal cell line, Neuro 2a, compared with NIH-3T3, a non-expressing line. Keywords: Neuron; Testis; Cerebral cortex; Gene expression; Promoter; Gene discovery; CpG island; Transmembrane protein 1. Introduction Identifying the target genes of sequence-specific tran- scriptional regulators by affinity selection has not been extensively pursued in large genomes. The complexity of the genome of mammals is such that apparent binding sites for transcription factors are expected to occur frequently in wx non-regulatory regions. We targeted the CpG island 3 fraction of DNA for affinity selection because it offers a concentrated source of promoter fragments. It has been argued that it contains 60% of all promoters within only 1–2% of the mass of the genome. Roughly half of these promoters are from ubiquitously expressed genes but the rest are tissue-specific. Among the former are examples of CpG island genes, such as fos and jun themselves, that are very highly inducible in all cells. To generate a CpG island-rich fraction, we used small DNA fragments gener- ) Ž . Corresponding author. Fax: q44 114 272-1104; E-mail: m.nick- lin@sheffield.ac.uk 1 The sequence described in this paper is available under database Accession number Y09922. ated by HpaII digestion as a starting material for the selection of AP-1-binding elements. We, thus, recovered a promoter which we have linked to the SEZ-6 gene, re- w x ported to be brain-specific 29 . The possible connection between the AP-1 element of the gene and its reported w x induction by convulsant drugs 22,29 led us to investigate the structure and expression of the gene in more detail. Because the reported level of induction of the gene is Ž small 1.9-fold by treatment with pentylenetetrazole in . cultured cortical neurones while its cell type specificity appears to be strong, we focused initially on investigating the stable, tissue-specific activity of the gene, rather than its potential induction by AP-1. The 4.0-kb cDNA encodes a putative integral membrane protein of 977 amino acids. The coding sequence of the cDNA shows no overall homology to other proteins but contains five cysteine-rich motifs that are homologous to the short consensus repeat Ž . of the complement C3brC4b receptor, CR1 SCR-CR1 w x 19 and two half repeats of a motif identified within the C1s and C1r complement proteins, known as the CUB w x domain 29,7 . Both protein motifs could be involved in signalling as well as structural associations. Beyond its likely cell surface location, the predicted primary structure 0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0169-328X 96 00274-4