Mechanisms of cell type-specific gene transcription : Studies on the choline acetyltransferase gene

Sammanfattning: Choline acetyltransferase (ChAT) is the enzyme synthesizing the neurotransmitter acetylcholine. Previous studies from our laboratory on the regulation of the cholinergic phenotype have suggested the presence of regulatory elements in the ChAT gene controlling accurate time- and cell-specific expression. It has been shown that a 3862 base pair (bp) long, but not a 1520 bp-long, 5´ flanking segment from the rat ChAT gene upstream promoter directs cholinergic cell-specific expression of a reporter gene in cell culture, indicating that the intervening 2336 bp-long segment contains crucial regulatory elements. This upstream sequence was used to control the expression of a reporter gene in transgenic mice. Transgene expression targeted to several cholinergic regions of the central nervous system. The pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2336 bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in neurons. With the aim of increasing the expression levels and also making gene transcription easily regulatable in an on-off fashion, the tetracyclin responsive promoter system was adapted for cholinergic cell-specific expression. Directing the expression of a tetracyclin-dependent transcriptional activator (rtTA) to cholinergic neurons allowed for the dose-dependent activation of a responsive promoter by tetracyclin analogues in these cells, but not in other cell types. The 2336 bp regulatory region was analyzed for the presence of regulatory elements that direct cholinergic cell-specific gene expression. A neuron-restrictive silencer element (NRSE) was identified in the proximal part of this region, recognized by the neuron-restrictive silencer factor (NRSF). The ChAT NRSE was inactive in neuronal cells but repressed expression in non-neuronal cells. The distal part of the ChAT gene promoter showed cholinergic-cell-specific enhancing activity, but was inactive in non-cholinergic neuronal and non-neuronal cells. This enhancer region suppressed the activity of the ChAT NRSE in cholinergic cells, even after NRSF overexpression. We observed that the reported ChAT gene expression in spermatozoa is not driven by any of the known ChAT promoters, and isolated several testis-specific cDNAs from the ChAT gene. These cDNAs code for proteins with truncated N termini. When produced in cell- free extracts and in COS cells, the proteins do not show ChAT activity and do not compete with neuronal ChAT for either substrate in competition experiments. We suggest that a novel product of unknown function lacking ChAT activity is made in testis from the gene sequence that overlaps with the ChAT gene expressed in brain. Two further studies deal with gene regulation in the testis. Androgen binding protein (ABP) mRNA levels were found to be regulated through the cycle of spermatogenesis, with a maximum during stages VIII-XI. ABP mRNA expression was also elevated upon in vivo nerve growth factor (NGF) infusion of testes, possibly through a prolongation of stages VII-VIII induced by NGF. mRNAs for regulatory subunits of protein kinase A were also strictly and individually regulated with the stages of spermatogenesis, pointing to discrete functions for the different subunits.