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A novel pathway for MuSK to induce key genes in neuromuscular synapse formation.

Lacazette E, Le Calvez S, Gajendran N, Brenner HR - J. Cell Biol. (2003)

Bottom Line: Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes.The same pathways are used to regulate synaptic expression of AChR epsilon.We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChR epsilon. We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

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Regulatory sequences of the nsk2 gene. (A) Sequence comparison of the mouse (top) and the human (bottom) promoter regions with exon1 and part of the first intron of the nsk2/musk gene. The putative transcription start determined by 5′ RACE PCR is indicated as +1 for the mouse sequence. Boxes indicate E-boxes, the oval shows the N-box, black arrows show Sp1/Egr-1 putative binding sites, the large dotted line indicates a putative NF-κB binding site, the small dotted line indicates a putative TEF-1 binding site, and a putative AP-1 binding site is underlined. (B) Schematic representation of a larger region upstream and downstream the first exon. Potential binding sites for transcription factors are indicated. Prediction of the transcription factor binding site have been generated with the MatInspector software from Genomatix. (C) Schematic diagram of the different constructs used in vitro or in vivo experiments with either the luciferase gene or the lacZ gene as a reporter.
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fig1: Regulatory sequences of the nsk2 gene. (A) Sequence comparison of the mouse (top) and the human (bottom) promoter regions with exon1 and part of the first intron of the nsk2/musk gene. The putative transcription start determined by 5′ RACE PCR is indicated as +1 for the mouse sequence. Boxes indicate E-boxes, the oval shows the N-box, black arrows show Sp1/Egr-1 putative binding sites, the large dotted line indicates a putative NF-κB binding site, the small dotted line indicates a putative TEF-1 binding site, and a putative AP-1 binding site is underlined. (B) Schematic representation of a larger region upstream and downstream the first exon. Potential binding sites for transcription factors are indicated. Prediction of the transcription factor binding site have been generated with the MatInspector software from Genomatix. (C) Schematic diagram of the different constructs used in vitro or in vivo experiments with either the luciferase gene or the lacZ gene as a reporter.

Mentions: We screened a mouse genomic phage library with a 500-bp 5′ cDNA fragment of nsk2, the mouse ortholog of rat and human musk, and isolated 10 kb of mouse genomic sequence containing 5 kb each upstream and downstream of the first exon of nsk2 (Ganju et al., 1995). We denote this a nsk2/musk fragment. The transcription start site (+1) in nsk2/musk was determined by 5′ RACE (Fig. 1 A). The upstream region is TATA-less and lacks an obvious initiator element but contains several potential binding sites for transcription factors. Specifically, eight E-boxes are contained within the 1.6 kb upstream from the transcription start site (Fig. 1 B). The two proximal E-boxes (−40/−45 and –147/–152) are conserved between mouse nsk2 and the human musk ortholog (Fig. 1 A). E-boxes are thought to mediate muscle-specific and activity-dependent expression of AChR genes (Tang et al., 1994; Angus et al., 2001) by binding basic helix–loop–helix transcription factors of the myoD family (Schaeffer et al., 2001). Another putative E-box was found in the first intron at position +920 (Fig. 1, A and B). Importantly, the first intron of the nsk2 gene also contains at +871 an N-box, 5′-GTACCGGAAATA-3′ (Fig. 1, A and B). Its sequence is identical to the N-box of the AChRɛ subunit gene promoter where it is thought to mediate nerve- and NRG-induced transcription of the AChRɛ subunit gene (Duclert et al., 1993, 1996; Schaeffer et al., 1998). When compared with the human sequence, the overall identity of the first intron in mouse and human nsk2/musk is 74%, with the N-box located in a very highly conserved (91%) stretch of 80 bp which also contains putative-binding sites for TEF-1 and AP-1 (Fig. 1 A).


A novel pathway for MuSK to induce key genes in neuromuscular synapse formation.

Lacazette E, Le Calvez S, Gajendran N, Brenner HR - J. Cell Biol. (2003)

Regulatory sequences of the nsk2 gene. (A) Sequence comparison of the mouse (top) and the human (bottom) promoter regions with exon1 and part of the first intron of the nsk2/musk gene. The putative transcription start determined by 5′ RACE PCR is indicated as +1 for the mouse sequence. Boxes indicate E-boxes, the oval shows the N-box, black arrows show Sp1/Egr-1 putative binding sites, the large dotted line indicates a putative NF-κB binding site, the small dotted line indicates a putative TEF-1 binding site, and a putative AP-1 binding site is underlined. (B) Schematic representation of a larger region upstream and downstream the first exon. Potential binding sites for transcription factors are indicated. Prediction of the transcription factor binding site have been generated with the MatInspector software from Genomatix. (C) Schematic diagram of the different constructs used in vitro or in vivo experiments with either the luciferase gene or the lacZ gene as a reporter.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199368&req=5

fig1: Regulatory sequences of the nsk2 gene. (A) Sequence comparison of the mouse (top) and the human (bottom) promoter regions with exon1 and part of the first intron of the nsk2/musk gene. The putative transcription start determined by 5′ RACE PCR is indicated as +1 for the mouse sequence. Boxes indicate E-boxes, the oval shows the N-box, black arrows show Sp1/Egr-1 putative binding sites, the large dotted line indicates a putative NF-κB binding site, the small dotted line indicates a putative TEF-1 binding site, and a putative AP-1 binding site is underlined. (B) Schematic representation of a larger region upstream and downstream the first exon. Potential binding sites for transcription factors are indicated. Prediction of the transcription factor binding site have been generated with the MatInspector software from Genomatix. (C) Schematic diagram of the different constructs used in vitro or in vivo experiments with either the luciferase gene or the lacZ gene as a reporter.
Mentions: We screened a mouse genomic phage library with a 500-bp 5′ cDNA fragment of nsk2, the mouse ortholog of rat and human musk, and isolated 10 kb of mouse genomic sequence containing 5 kb each upstream and downstream of the first exon of nsk2 (Ganju et al., 1995). We denote this a nsk2/musk fragment. The transcription start site (+1) in nsk2/musk was determined by 5′ RACE (Fig. 1 A). The upstream region is TATA-less and lacks an obvious initiator element but contains several potential binding sites for transcription factors. Specifically, eight E-boxes are contained within the 1.6 kb upstream from the transcription start site (Fig. 1 B). The two proximal E-boxes (−40/−45 and –147/–152) are conserved between mouse nsk2 and the human musk ortholog (Fig. 1 A). E-boxes are thought to mediate muscle-specific and activity-dependent expression of AChR genes (Tang et al., 1994; Angus et al., 2001) by binding basic helix–loop–helix transcription factors of the myoD family (Schaeffer et al., 2001). Another putative E-box was found in the first intron at position +920 (Fig. 1, A and B). Importantly, the first intron of the nsk2 gene also contains at +871 an N-box, 5′-GTACCGGAAATA-3′ (Fig. 1, A and B). Its sequence is identical to the N-box of the AChRɛ subunit gene promoter where it is thought to mediate nerve- and NRG-induced transcription of the AChRɛ subunit gene (Duclert et al., 1993, 1996; Schaeffer et al., 1998). When compared with the human sequence, the overall identity of the first intron in mouse and human nsk2/musk is 74%, with the N-box located in a very highly conserved (91%) stretch of 80 bp which also contains putative-binding sites for TEF-1 and AP-1 (Fig. 1 A).

Bottom Line: Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes.The same pathways are used to regulate synaptic expression of AChR epsilon.We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChR epsilon. We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

Show MeSH
Related in: MedlinePlus