Introduction
Characterization of the mammalian acetylcholinesterase (AChE) gene, initially from isolated cDNAs (Rachinsky et al., 1990; Soreq and Zakut, 1990) and then from genomic clones (Li et al., 1991), reveals a compact gene confined to 7.4 kb from the primary transcription start site to the more distal of two polyadenylation signals. Three short introns are found in the invariantly spliced sequence between exons 1 and 4, and two alternative splice acceptors exist near the 3′-end of the gene, producing AChE with three distinct C-terminal sequences. Divergence in the very C-terminal sequences of AChE governs the oligomerization state of the enzyme and its membrane-tethering properties but does not directly affect the catalytic parameters (Massoulie, 2002; Radic and Taylor, 2006). In addition, there have been several reports describing alternative promoter usage and splicing at the 5′-end of the gene (Atanasova et al., 1999; Meshorer et al., 2004).
Despite the inherent complexity of gene expression arising from alternative splicing, the limited nucleotide span of the AChE gene with its short introns enables one to examine expression after transfection of an intact gene rather than monitoring expression of gene fragments attached to reporter genes.
AChE expression in excitable cells, such as skeletal muscle, is characterized by greatly enhanced expression after differentiation (Inestrosa et al., 1983). The enzyme is virtually absent in myoblasts or neuroblasts; differentiation into myotubes and neurons (Jones-Villeneuve et al., 1983) results in large increases in AChE mRNA and protein levels.
A series of studies using run-on transcription and reporter gene expression has demonstrated rather modest transcriptional control of mammalian AChE gene expression (Fuentes and Taylor, 1993; Mutero et al., 1995; Angus et al., 2001). Studies in cultured muscle cells reveal evidence for mRNA stabilization (Fuentes and Taylor, 1993; Deschenes-Furry et al., 2005), but stabilization of mRNA is not sufficient to explain the large increases in activity or mRNA levels associated with myoblast to myotube differentiation. Studies using inhibitors of RNA polymerase stabilize a labile RNA in myoblasts and provide evidence for superinduction of the mRNA (Fuentes and Taylor, 1993). Hence, labile proteins may control AChE mRNA stability, but here, again, differences in the rates of mRNA degradation do not appear sufficient to account for the marked increases in mRNA levels associated with myoblast to myotube conversion.
We have taken advantage of the compact nature of the AChE gene permitting us to transfect it in its entirety and compare the expression from transfected and endogenous genes in differentiating muscle cells. Through successive and reiterative gene deletions, we delineated a critical region in the first intron of the gene, between exons 1 and 2, that exerts dominant regulatory control of gene expression during the relatively rapid myoblast to myotube differentiation in culture. The identified region has multiple functional consensus sites for gene regulation (Angus et al., 2001) that are conserved in mammalian species. It functions only with its endogenous AChE promoter in concert with other areas of the gene, suggesting enhancesome-like behavior (Merika and Thanos, 2001; Arnosti and Kulkarni, 2005).
To examine whether the control of AChE gene expression is characteristic of particular tissues, and we deleted this intronic region by homologous recombination, allowing the altered gene to be expressed in the developing mouse. Remarkably, deletion of this intronic region in knock-out animals ablates AChE mRNA synthesis and protein expression in skeletal muscle, as indicated in cell culture expression, yet surprisingly, and in contrast to deletion of alternatively spliced exonic regions, brain and spinal cord expression patterns are not altered. Moreover, distinguishing differences in expression controlled by these intronic elements are found in two types of hematopoietic cell lineages; erythroid cells show normal AChE, whereas expression in the platelet arising from a megakarocyte lineage is ablated.
