An intronic enhancer containing an N-box motif is required for synapse- and tissue-specific expression of the acetylcholinesterase gene in skeletal muscle fibers

Abstract

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 influenced 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 synapse-specific 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.

Figure 1

5′ regulatory region of the rat AChE gene. (Upper) Nucleotide sequence of the rat AChE promoter aligned with corresponding regions from the mouse and human genes. The first nucleotide in the initiator element (Inr; underlined) is designated as +1. Note that the sequence in the rectangular box (+5 to +75) exhibits more than 97% identity with exon 1 in the mouse and human AChE genes. Black arrows indicate EGR-1/Sp1 clusters; oval shows an AP2 site; and white arrows indicate E-boxes. (Lower) Schematic representation of the Inr and other consensus sites for DNA-binding proteins present in a larger promoter fragment as well as in the first intron of the gene.

Figure 2

Expression of AChE promoter–reporter gene constructs in synaptic compartments of mouse TA muscle fibers. (A and B) Cryostat sections stained histochemically for the simultaneous demonstration of β-gal (blue staining) and AChE (brown staining) activity. Note that the presence of blue nuclei coincides with the occurrence of neuromuscular junctions, reflecting AChE promoter activity within junctional myonuclei. (Bar = 75 μm.)

Figure 3

(A) Schematic representation of several AChE promoter–reporter gene constructs used for the in vivo studies. Note that the four N-boxes (N) found in GRAP are retained in NRAP, whereas only the first intronic N-box at position +755 bp is still present in FRAP (R indicates those that are in reverse orientation). Arrow points to the first nucleotide of the Inr. (B and C) Total number of events (B) and percentage of synaptic events (C) seen in TA muscles after injection of the different constructs. Note that the pattern of expression of the reporter gene is unchanged despite large deletions of the 5′ and 3′ regions in the original 5.3-kb fragment (compare GRAP with NRAP and FRAP). However, note that deletion of an additional 499-bp intronic region abolished AChE promoter activity (compare FRAP with RAP). Star indicates no expression. Mean ± SE is shown; a minimum of 10 muscles were analyzed per construct.

Figure 4

Expression of AChE promoter–reporter gene constructs in hematopoietic and myogenic cells grown in culture. MEL and C2 cells were transfected with plasmids containing the reporter gene nlsLacZ and the AChE promoter fragment FRAP or RAP. Note that in contrast to myogenic cells (A; see also Fig. 3), both constructs were equally active in hematopoietic cells (B). In these assays (three independent experiments performed in triplicate), transfection efficiency was monitored by determining the expression of a constitutively expressed CAT plasmid. Star indicates no expression. Mean ± SE is shown.

Figure 5

Expression of β-gal in TA muscle fibers after injection of a plasmid containing the reporter gene lacZ with and without the intronic fragment placed in either orientation (r = reverse) upstream of the thymidine kinase (TK) promoter. Note the significant (P < 0.001; ANOVA) ≈6-fold induction in β-gal activity with the presence of the intronic DNA fragment in either orientation. Expression of β-gal was normalized to the activity of a coinjected CAT plasmid used to monitor transduction efficiency. Mean ± SE is shown; a minimum of 12 muscles were analyzed per construct.

Figure 6

Disruption of the CArG element does not affect expression of AChE promoter–reporter gene constructs in TA muscle. (A) EMSA using radiolabeled oligonucleotides containing the CArG-box. Note the presence of two major DNA–protein complexes (arrows) in muscle nuclear extracts whose formation was specifically blocked by competition with a 250-fold molar excess of the wild-type (WT) but not the mutant oligonucleotide. (B) Schematic representation of the nucleotides that were mutated (underlined) in the core region of the CArG element in NRAP to generate the mutant CArG-NRAP promoter fragment (mC-NRAP). As shown in A, this mutation resulted in a failure to compete for formation of specific DNA–protein complexes. (C) Expression of β-gal in TA muscles injected with reporter plasmids containing either NRAP or mC-NRAP. Note that disruption of this DNA regulatory element did not affect significantly (P > 0.05; Student’s t test) expression of the reporter gene. Expression of β-gal was normalized to the activity of a coinjected CAT plasmid used to monitor transduction efficiency. Mean ± SE is shown; a minimum of 10 muscles were analyzed per construct.

Figure 7

Disruption of the N-box motif reduces drastically expression of AChE promoter–reporter gene constructs in TA muscle. (A) EMSA using radiolabeled oligonucleotides containing the N-box motif. Note that one specific DNA–protein complex (arrow) was formed when a 24-bp oligonucleotide encompassing the first intronic N-box at position +755 (N int-1) was used. Formation of this protein complex was blocked by competition with a 250-fold molar excess of unlabeled wild-type oligonucleotides (WT oligo). Mutation of the core sequence as shown in C, abolished its protein-binding capacity, as indicated by the inability of the mutant oligonucleotides to compete in formation of specific protein complex. Note also that oligonucleotides containing the two palindromic N-box motifs located in the promoter region (N prom) displayed a weaker affinity for specific protein complexes. Arrowhead indicates the amount of unbound radioactive oligonucleotides present in each sample. (B) The protein complex was supershifted (white arrow) by an additional incubation with antibodies against either GABP α or GABP β. (C) Schematic representation of the nucleotides that were mutated (underlined) in the core region of the first intronic N-box motif at position +755 in NRAP to generate the mutant N-box-NRAP promoter fragment (mN-NRAP). As shown in A, this mutation resulted in a failure to compete in formation of specific DNA–protein complexes. (D) Expression of β-gal in TA muscles injected with reporter plasmids containing either NRAP or mN-NRAP. Note that disruption of this DNA regulatory element essentially abolished (P < 0.001; Student’s t test) expression of the reporter gene, indicating that the N-box is involved in enhancing expression of AChE in muscle. Expression of β-gal was normalized to the activity of a coinjected CAT plasmid used to monitor transduction efficiency. (E) The percentage of synaptic events was also significantly reduced (P < 0.005; Student’s t test) in muscles injected with reporter plasmids containing mN-NRAP. Mean ± SE is shown; a minimum of 10 muscles were analyzed per construct.