Exonic Sp1 sites are required for neural-specific expression of the glycine receptor beta subunit gene.
The gene encoding the beta subunit of the inhibitory glycine receptor (GlyR) is widely expressed throughout the mammalian central nervous system. To unravel the elements regulating its transcription, we isolated its 5' non-coding and upstream flanking regions from mouse. Sequence analysis revealed significant differences between the 5' region of the beta subunit gene and the corresponding regions of the homologous GlyR alpha subunit genes; it also identified a novel exon (exon 0) that encodes most of the 5'-untranslated portion of the GlyR beta mRNA. Primer extension experiments disclosed multiple transcriptional start sites. Transfection experiments with luciferase reporter gene constructs showed that sequences encompassing 1.58 kb of upstream flanking region and 180 bp of exon 0 displayed high promoter activity in two neuroblastoma cell lines but not in non-neural cells. Analysis of various deletion constructs showed that the 5' flanking region preceding the transcriptional start sites silences expression in non-neural cells but is not essential for general promoter activity. In contrast, the deletion of sequences within exon 0 drastically decreased or abolished transcription; the removal of sequences harbouring Sp1 consensus sequences within exon 0 decreased expression specifically in a neuroblastoma cell line. Band-shift assays confirmed the binding of Sp1 to sites within the deleted sequence. Our results indicate that neural-specific expression of the GlyR beta subunit gene might depend on a direct interaction of Sp1 transcription factors with cis elements located downstream from transcription initiation sites.
Tintrup
H
H
Department of Neurochemistry, Max Planck Institute for Brain Research, Deutschordenstrasse 46, D-60528 Frankfurt, Germany.
Fischer
M
M
Betz
H
H
Kuhse
J
J
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D005091:Exons; D017403:In Situ Hybridization; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D018009:Receptors, Glycine / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism*
C491949:Glrb protein, mouse; D012333:RNA, Messenger; D018009:Receptors, Glycine; D016329:Sp1 Transcription Factor