Multiple promoters drive tissue-specific expression of the human M muscarinic acetylcholine receptor gene.
Despite the wealth of information on the functional and pharmacological properties of the M2 muscarinic receptor, we know relatively little of structure and regulation of the M2 receptor gene. Here, we describe the organisation of the human M2 gene and its promoters. Four exons are present in the 5' untranslated region of the human M2 mRNA distributed over 146 kb on chromosome 7 which produce eight different splice variants in the IMR-32 neuroblastoma cell line. The unexpectedly large size of this gene indicates that transcription initiates much further upstream of the coding region than earlier studies had indicated. We present evidence that there are three distinct human M2 promoters. Analysis of endogenous transcripts revealed that promoter 2 is preferentially used in neuroblastoma cells, whereas promoter 1 in cardiac cells. All promoters are highly conserved across human, mouse, rat and pig. They contain multiple start sites and none possess a TATA-box. In addition, we describe another M2 promoter that is specific for rat. We show that GATA-4 transcription factor binds to two sites within the regulatory regions of the M2 gene using reporter gene assays, electromobility shift assays and mutational analysis.
Krejci
Alena
A
Institute of Physiology, Prague, Czech Republic.
Bruce
Alexander W
AW
Dolezal
Vladimir
V
Tucek
Stanislav
S
Buckley
Noel J
NJ
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D005091:Exons; D050991:GATA4 Transcription Factor; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter / Q000502:physiology; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D032446:Myoblasts; D009447:Neuroblastoma; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D043585:Receptor, Muscarinic M2 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment / Q000379:methods; D035181:TATA-Box Binding Protein / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D002843:Chromatin; D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D012333:RNA, Messenger; D043585:Receptor, Muscarinic M2; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D008156:Luciferases