A BSTR ACT mRNAs encoding acetylcholinesterase (AChE; EC 3.1.1.7) are highly concentrated within the postsynaptic sarcoplasm of adult skeletal muscle fibers, where their expression is markedly inf luenced by nerve-evoked electrical activity and trophic factors. To determine whether transcriptional regulatory mechanisms account for the synaptic accumulation of AChE transcripts at the mammalian neuromuscular synapse, we cloned a 5.3-kb DNA fragment that contained the 5 regulatory region of the rat AChE gene and generated several constructs in which AChE promoter fragments were placed upstream of the reporter gene lacZ and a nuclear localization signal (nls). Using a recently described transient expression assay system in intact skeletal muscle, we show that this AChE promoter fragment directs the synapsespecific expression of the reporter gene. Deletion analysis revealed that a 499-bp fragment located in the first intron of the AChE gene is essential for expression in muscle fibers. Further analysis showed that sequences contained within this intronic fragment were (i) functionally independent of position and orientation and (ii) inactive in hematopoietic cells. Disruption of an N-box motif located within this DNA fragment reduced by more than 80% the expression of the reporter gene in muscle fibers. In contrast, mutation of an adjacent CArG element had no effect on nlsLacZ expression. Taken together, these results indicate that a muscle-specific enhancer is present within the first intron of the AChE gene and that an intronic N-box is essential for the regulation of AChE along skeletal muscle fibers.

In proliferating non‐erythroid cells, the expression of transferrin receptors (TfR) is negatively regulated by the amount of intracellular iron. Fe‐dependent regulation of TfR occurs post‐transcriptionally and is mediated by iron‐responsive elements (IRE) located in the 3′ untranslated region of the TfR mRNA. IREs are recognized by a specific cytoplasmic binding protein (IRE‐BP) that, in the absence of Fe, binds with high affinity to TfR mRNA, preventing its degradation. While TfR numbers are positively correlated with proliferation in non‐erythroid cells, in hemoglobin‐synthesizing cells, their numbers increase during differentiation and are, therefore, negatively correlated with proliferation. This suggests a distinct regulation of erythroid TfR expression and evidence, as follows, for this was found in the present study. (a) With nuclear run‐on assays, our experiments show increased TfR mRNA transcription following induction of erythroid differentiation of murine erythroleukemia (MEL) with Me2SO. (b) Me2SO treatment of MEL cells does not increase IRE‐BP activity which is, however, increased in uninduced MEL cells by Fe chelators. (c) Following induction of MEL cells, there is an increase in the stability of TfR mRNA, whose level is only slightly affected by iron excess. (d) Heme‐synthesis inhibitors, such as succinylacetone and isonicotinic acid hydrazide, which inhibit numerous aspects of erythroid differentiation, also inhibit TfR mRNA expression in induced MEL cells. However, heme‐synthesis inhibition does not lead to a decrease in TfR mRNA levels in uninduced MEL cells. Thus, these studies indicate that TfR gene expression is regulated differently in hemoglobin synthesizing as compared to uninduced MEL cells.

In this study, we examined whether an intronic N-box motif is involved in the expression of acetylcholinesterase (AChE) during myogenesis. We determined that AChE transcripts are barely detectable in cultured myoblasts and that their levels increase dramatically in myotubes. Nuclear run-on assays revealed that this increase was accompanied by a parallel induction in the transcriptional activity of the AChE gene. These changes in transcription were also observed in transfection experiments using AChE promoter-reporter gene constructs. Mutation of the intronic N-box at position ؉755 base pairs (bp) reduced by more than 70% expression of the reporter gene in myotubes. Disruption of an adjacent E-box, at position ؉767 bp, also reduced expression of the reporter gene following myogenic differentiation. Co-transfection experiments using AChE promoter-reporter gene constructs and a myogenin expression vector showed that expression of this regulatory factor increased expression of the reporter gene in myotubes. Although the AChE promoter contains multiple E-boxes, mutation of this intronic one was sufficient to prevent the myogenin-induced increase in reporter gene expression. Together, these results indicate that changes in AChE gene transcription occur during myogenesis and highlight the contribution of the intronic Nand E-box motifs in the developmental regulation of the AChE gene in skeletal muscle.

Differentiation of hematopoietic cells is known to be accompanied by profound changes in acetylcholinesterase (AChE) enzyme activity, yet the basic mechanisms underlying this developmental regulation remain unknown. We initiated a series of experiments to examine the molecular mechanisms involved in regulating AChE expression during hematopoiesis. Differentiation of murine erythroleukemia (MEL) cells using dimethyl sulfoxide resulted in a 5-and 10-fold increase in intracellular and secreted AChE enzyme activity, respectively. Interestingly, these increases resulted from a preferential induction of the globular molecular form G 1 and a slight increase in G 4 instead of an increase in the levels of the G 2 membrane-bound form, a molecular form expressed in mature erythrocytes. Concomitantly, expression of the two predominant AChE transcripts (R and T, for read-through and tail, respectively) in MEL cells was induced to a similar extent with differentiation. Nuclear run-on assays performed with nuclei isolated from induced versus uninduced MEL cells revealed that in contrast to the large increases seen in the transcription of the ␤-globin gene, the transcriptional activity of the AChE gene remained largely unaffected after differentiation. Determination of the half-lives of the R and T transcripts demonstrated that they both exhibited an increase in stability in induced MEL cells. Taken together, results from these studies indicate that posttranscriptional regulatory mechanisms account for the increased expression of AChE in differentiated hematopoietic cells.

Abstract:The molecular mechanisms underlying the activity-linked plasticity of acetylcholinesterase (AChE) mRNA levels in mammalian skeletal muscle have yet to be established. Here, we demonstrate that denervation of adult muscle induces a dramatic (up to 90%) and rapid (within 24 h) decrease in the abundance of AChE mRNAs. By contrast, denervation of 14-day-old rats leads to a significantly less pronounced reduction (50% of control) in the expression of AChE mRNAs. Assessment of the transcriptional activity of the AChE gene reveals that it remains essentially unchanged in adult denervated muscles, whereas it displays an approximately two-to threefold increase ( p Ͻ 0.05) in denervated muscles from 2-to 14-day-old rats. In addition, we observed a higher rate of degradation of in vitro transcribed AChE mRNAs upon incubation with protein extracts from denervated muscles. Finally, UV-crosslinking experiments reveal that denervation increases the abundance of RNA-protein interactions in the 3Ј untranslated region of AChE transcripts. Taken together, these data suggest that the abundance of AChE transcripts in mature muscles is controlled primarily via posttranscriptional regulatory mechanisms, whereas in neo-and postnatal muscles, both transcriptional and posttranscriptional regulation appears critical in dictating AChE mRNA levels. Accordingly, the activitylinked transcriptional regulation of the AChE gene appears to demonstrate a high level of plasticity during muscle development when maturation of the neuromuscular junctions is still occurring.

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We determined levels of mRNA encoding acetylcholinesterase (AChE) in muscles of rats subjected to chronic enhancement of neuromuscular activation. After 8 wk of voluntary wheel running, extensor digitorum longus (EDL) muscles displayed a 72% increase in total AChE activity as a result of a selective threefold increase in the G4 content. Soleus muscles, on the other hand, exhibited a 30% decrease in A12 while displaying a small (33%) increase in total AChE activity. These enzymatic adaptations were paralleled by increases in the levels of AChE mRNAs in both EDL (32%; P < 0.03) and soleus (42%; P < 0.02) muscles. In addition, compensatory hypertrophy of the plantaris muscle increased total AChE activity by 75%. This change was reflected by an elevation in all AChE molecular forms with A12 (89%) and A8 (179%) showing the most prominent increases. Similar to exercise-trained muscles, hypertrophied plantaris muscles displayed an increase in AChE transcripts (25%; P < 0.04). These results indicate that increases in neuromuscular activity modulate expression of the AChE gene in vivo and suggest the involvement of pretranslational regulatory mechanisms in the adaptive response of AChE to enhanced neuromuscular activation.

Ferrochelatase, which catalyses the last step in haem biosynthesis, i.e. the insertion of Fe(II) into protophorphyrin IX, is present in all cells, but is particularly abundant in erythroid cells during haemoglobinization. Using mouse ferrochelatase cDNA as a probe two ferrochelatase transcripts, having lengths of 2.9 kb and 2.2 kb, were found in extracts of mouse liver, kidney, brain, muscle and spleen, the 2.9 kb transcript being more abundant in the non-erythroid tissues and the 2.2 kb transcript more predominant in spleen. In mouse erythroleukemia cells the 2.9 kb ferrochelatase transcript is also more abundant; however, following induction of erythroid differentiation by dimethyl sulphoxide there is a preferential increase in the 2.2 kb transcript, which eventually predominates. With mouse reticulocytes, the purest immature erythroid cell population available, over 90% of the total ferrochelatase mRNA is present as the 2.2 kb transcript. Since there is probably only one mouse ferrochelatase gene, the occurrence of two ferrochelatase transcripts could arise from the use of two putative polyadenylation signals in the 3' region of ferrochelatase DNA. This possibility was explored by using a 389 bp DNA fragment produced by PCR with synthetic oligoprimers having sequence similarity with a region between the polyadenylation sites. This fragment hybridized only to the 2.9 kb ferrochelatase transcript, indicating that the two transcripts differ at their 3' ends and suggesting that the 2.2 kb transcript results from the utilization of the upstream polyadenylation signal. The preferential utilization of the upstream polyadenylation signal may be an erythroid-specific characteristic of ferrochelatase gene expression.