Transcriptional Regulation by Activation and Repression Elements Located at the 5′-Noncoding Region of the Human α9 Nicotinic Receptor Subunit Gene

Luis M Valor,Mar Castillo,José A Ortiz,Manuel Criado

doi:10.1074/jbc.m307043200

Abstract

The alpha9 subunit is a component of the neuronal nicotinic acetylcholine receptor gene superfamily that is expressed in very restricted locations. The promoter of the human gene has been analyzed in the human neuroblastoma SH-SY5Y, where alpha9 subunit expression was detected, and in C2C12 cells that do not express alpha9. A proximal promoter region (from -322 to +113) showed maximal transcriptional activity in SH-SY5Y cells, whereas its activity in C1C12 cells was much lower. Two elements unusually located at the 5'-noncoding region exhibited opposite roles. A negative element located between +15 and +48 appears to be cell-specific because it was effective in C2C12 but not in SH-SY5Y cells, where it was counterbalanced by the presence of the promoter region 5' to the initiation site. An activating element located between +66 and +79 and formed by two adjacent Sox boxes increased the activity of the alpha9 promoter about 4-fold and was even able to activate other promoters. This element interacts with Sox proteins, probably through a cooperative mechanism in which the two Sox boxes are necessary. We propose that the Sox complex provides an initial scaffold that facilitates the recruiting of the transcriptional machinery responsible for alpha9 subunit expression.

Highlights

Neuronal nicotinic acetylcholine receptors1 are members of a supergene family of ion channels gated by neurotransmitters [1]
The ␣9 subunit is a component of the neuronal nicotinic acetylcholine receptor gene superfamily that is expressed in very restricted locations
A negative element located between ؉15 and ؉48 appears to be cell-specific because it was effective in C2C12 but not in SH-SY5Y cells, where it was counterbalanced by the presence of the promoter region 5؅ to the initiation site

Summary

EXPERIMENTAL PROCEDURES

Isolation and Analysis of the 5Ј-Flanking Sequence of the ␣9 Subunit—The human ␣9 coding sequence was obtained by PCR from a human pituitary cDNA library (Clontech, Heidelberg, Germany) by using the information contained in the GenBankTM sequence AJ243342. 5Ј-RACE Analysis of 5Ј mRNA Ends—The 5Ј-end of ␣9 mRNA was mapped by 5Ј-RACE, as primer extension and RNase protection methods did not yield satisfactory results For this purpose the Marathon Ready cDNA system from Clontech was applied to the previously mentioned cDNA library from human pituitary as indicated by the manufacturer. Deletion analysis of the most promoter-proximal region was performed by generating either appropriate restriction enzyme fragments or PCR fragments with suitable sense oligonucleotides and an antisense primer

Regulatory Elements of Nicotinic Receptor Gene Expression

RESULTS

DISCUSSION