Garriga-Canut, Mireia; (2001) Control of neurone-specific gene expression: Transcriptional regulation of the M1 muscarinic acetylcholine receptor gene. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The establishment of differentiated neuronal phenotype remains an outstanding problem of molecular neurobiology. One of the clearest manifestations of this molecular diversity is provided by the G-protein coupled receptor (GPCR) family. Each of the more than 1,000 members of this gene family has a unique expression profile and thereby offer an ideal model to examine the transcriptional mechanisms that underwrite this molecular diversity. Muscarinic acetylcholine receptors (M1- M5) make up one of the subfamilies of GPCR genes. The M1 gene is the most abundant of the muscarinic receptor genes and is mainly expressed in telencephalic regions and autonomic ganglia. I have now investigated the regions of this gene that are capable of driving expression of a reporter gene in an M1 specific manner. One of these regions is a polypyrimidine/polypurine (PPY/PPU) sequence capable of forming single stranded DNA, and the other (found downstream of the PPY/PPU tract) is conserved across species, has no recognisable motifs and is not able to form single stranded DNA by itself although it shows sensitivity to specific single stranded nucleases when next to the PPY/PPU tract. Both the PPY/PPU tract and the conserved region are bound by nucleolin, a multifunctional phosphoprotein, and act as cis-enhancing elements. A second region important for expression of the M1 gene has been identified to be bound by SHARP-1, a basic helix loop helix protein of unknown function expressed in the adult nervous system. Gal4 fusion experiments have shown that SHARP-1 functions as a repressor of both basal and activated transcription driven either by a TATA-containing or a TATA-less promoter in a position independent manner. Furthermore, SHARP-1 contains two independent repression domains, one at the C- terminus, which acts by a mechanism sensitive to the histone deacetylase inhibitor Trichostatin A (TSA), and the other at the bHLH domain, whichworks through a TSA insensitive mechanism. Co-transfection assays showed that SHARP-1 downregulates expression of the M1 gene in M1 expressing cells. Data presented here shows that the trancriptional mechanisms that control expression of the M1 gene are different to those that control expression of the other members of the same family. These results provideinsight into the molecules and mechanisms employed in the stablishment of aspects of differentiated neuronal phenotype.

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