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Novel Polymorphisms Influencing Transcription of the Human CHRM2 Gene in Airway Smooth Muscle

Department of Clinical Pharmacology and Therapeutics, and Department of Physiology and Biochemistry, University of Malta, Msida, Malta; and Division of Therapeutics, University of Nottingham, Nottingham, United Kingdom

Address correspondence to: Prof. Ian P. Hall, Head, Division of Therapeutics, C Floor, South Block, University Hospital, Nottingham NG7 2UH, UK. E-mail: ian.hall{at}nottingham.ac.uk

Muscarinic receptors are a functionally important family of G-protein–coupled receptors. Using a combination of rapid amplification of 5' cDNA ends and reporter gene assays, we characterized the 5' untranslated region of the CHRM2 gene as expressed in human airway smooth muscle (HASM) cells. A splice site is present 46 bp upstream from the ATG start codon. Five exons with alternative splicing patterns are present upstream of this splice site, separated by introns ranging from 87 bp to > 145 kb. There is evidence for the gene being under the control of a TATA-less promoter with Sp1, GATA, and activator protein-2 binding sites. Multiple transcription start sites (TSSs) were identified. We identified a novel 0.5-kb hypervariable region located 648 bp upstream of the most 5' TSS, a multiallelic (CA) tandem repeat 96 bp downstream of the most 5' TSS, and a common CA SNP located 136 bp upstream of the most 5' TSS. Functional studies in primary HASM cells and the BEAS-2B cell line demonstrated highest promoter activity to be upstream of the most 3' TSS, with potential repressor elements operating in a cell type–dependent manner, located upstream of the most 5' TSS. We present functional data to show that the CA repeat may influence the transcription of the gene in HASM and BEAS-2B cells.

Abbreviations: rapid amplification of 5' cDNA ends, 5'RACE • 5' untranslated region, 5'UTR • activating protein-2, AP-2 • airway smooth muscle, ASM • calf intestinal phosphatase, CIP • chronic obstructive pulmonary disease, COPD • cyclic AMP–responsive element binding protein, CREB • G-protein–coupled receptor, GPCR • human airway smooth muscle, HASM • polymerase chain reaction, PCR • pGL3 control plasmid, pGL3C • pGL3 enhancer plasmid, pGL3E • single nucleotide polymorphism, SNP • tobacco acid pyrophosphatase, TAP • transcription start sites, TSS

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