Research report Relaxation of glycine receptor and onconeural gene transcription control in NRSF deficient small cell lung cancer cell lines Sabine B. Neumann a , Randolf Seitz a , Anke Gorzella a , Angelien Heister a , Magnus von Knebel Doeberitz b , Cord-Michael Becker a, * a Institut fu ¨r Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander Universita ¨t Erlangen-Nu ¨rnberg, Fahrstrasse 17, 91054 Erlangen, Germany b Abteilung Molekulare Pathologie, Pathologisches Institut der Universita ¨t Heidelberg, Im Neuenheimer Feld 220/221, 69120 Heidelberg, Germany Accepted 31 October 2003 Abstract Negative regulation of many neuronal genes is mediated by the neuron-restrictive silencer factor (NRSF/repressor element-1 binding transcription factor, REST), which binds to the neuron-restrictive silencer element (NRSE/repressor element-1, RE-1) and thereby represses transcription of neuronal genes in non-neuronal cells. Sequence analysis of 5V -flanking regions of glycine receptor (GlyR) subunit genes revealed a consensus motif for NRSE in the GLRA1 and GLRA3, but not in GLRB, genes. In this study, we examined tumor cell lines for the expression of NRSF, GlyR subunits and onconeural genes. We identified two small cell lung cancer (SCLC) cell lines lacking full-length NRSF/REST as well as its neuronal splice variants. Presence or absence of NRSF as well as its functionality in different SCLC cell lines was additionally shown in reporter gene assays. As GlyR a1 is selectively transcribed in NRSF/REST free cells, GlyR a1 transcripts might serve as positive signals for NRSF deficient cells. In contrast, GlyR h is nearly ubiquitously transcribed in the cell lines analyzed and, therefore, should represent a useful marker for neoplastic cells. Sequence analysis of GlyR h transcripts led to the identification of a new splice variant lacking exon 8, GlyR h D8. This suggests that the lack of NRSF in SCLC cells, resulting in the relaxation of neuronal gene suppression, is an important mechanism underlying paraneoplastic expression. D 2003 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Neurotransmitter receptors: glycine Keywords: Glycine receptor; NRSE; NRSF; Small cell lung cancer; Paraneoplastic expression 1. Introduction Multiple DNA elements and sequence motifs contribute to transcriptional regulation in development and tissue-specific gene expression in the mammalian central nervous system (CNS). Negative transcriptional regulation has emerged as an important determinant of neuron specific gene expression. The neuron-specific silencer element (NRSE/repressor ele- ment-1, RE-1) is a 21bp genomic element present in many mammalian genes that are specifically expressed in neurons [36]. NRSE consensus motifs have been identified in the genes for type II sodium channel [25], neuronal nicotinic acetylcholine receptor h2 [5], synapsin I [34] and GABA A R g2 subunit [26]. Consistent with general properties of silencer elements, NRSE function in non-neuronal cells has been found to be independent of its localization, orientation [37], or position in intronic or coding regions [36]. Genomic NRSE sequences represent binding sites for the neuron-restrictive silencer factor (NRSF/repressor element-1 binding transcrip- tion factor, REST/repressor binding to the X2 box, XBR), which is selectively expressed in non-neuronal cells [35]. Functional studies show that binding of the NRSF/REST protein to NRSE restricts gene expression to neurally differ- entiated cells [36]. Inhibitory glycine receptors (GlyRs) represent a family of ligand gated chloride channels that mediate inhibitory neurotransmission in the central nervous system. Develop- mentally regulated GlyR isoforms are oligomeric assemblies of variants of the ligand binding a subunit and a h polypeptide, all of which are encoded by distinct genes 0169-328X/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2003.10.021 * Corresponding author. Tel.: +49-9131-852-4190; fax: +49-9131-852- 2485. E-mail address: cmb@biochem.uni-erlangen.de (C.-M. Becker). www.elsevier.com/locate/molbrainres Molecular Brain Research 120 (2004) 173 – 181