Here we demonstrate that the rapsyn promoter has functional Kaiso binding sites and that rapsyn promoter activity is regulated by Kaiso and δ-catenin in mouse and chicken myotubes. Immunofluorescence analyses verified that Kaiso and δ-catenin are concentrated at the postsynaptic region of mouse and chicken skeletal muscles. Based on these observations, we hypothesize that there is a pathway involving Kaiso and δ-catenin that regulates synapse-specific transcription at the neuromuscular junction. Since Kaiso and δ-catenin specifically induced the rapsyn promoter in muscle cells, and not in nonmuscle 293T cells, we further hypothesize that myogenic factors are also involved in this pathway. Current experiments are directed at determining the exact interactions of these components both in vitro and in vivo. We have shown that Kaiso and δ-catenin induce rapsyn promoter activity, but we have yet to show that Kaiso and/or δ-catenin are required for rapsyn transcription in vivo. However, our results strongly support the existence of a pathway that would explain the synapse-specific transcription of rapsyn and other synapse-specific proteins at the neuromuscular junction and also at other synapses throughout the body.
The neuromuscular junction has long served as a model synapse (reviewed in reference 40
). The fact that we find antibody staining of Kaiso and δ-catenin at the neuromuscular junction is not surprising since many proteins found concentrated at synapses in the CNS are also found at the neuromuscular junction. Recently, another newly discovered member of the POZ-zinc finger family, myoneurin, has been reported to localize preferentially to subsynaptic nuclei at the neuromuscular junction (5
). Kaiso is ubiquitously expressed; however, Xenopus
Kaiso expression was detected predominantly in neural tissues (23
), providing the first clue that Kaiso may function in neural tissues. The Kaiso binding partner, p120 catenin, is also ubiquitously expressed (37
). However, the p120 family member δ-catenin has an expression pattern reported to be limited to the nervous system (18
). The observed colocalization between Kaiso and δ-catenin at the neuromuscular junction is consistent with our hypothesis that the Kaiso and δ-catenin interaction is integral in regulating synapse-specific transcription at the neuromuscular junction and raises the strong possibility that these molecules are involved with the regulation of synapse-specific transcription throughout the nervous system.
δ-Catenin was discovered in a two-hybrid assay as a bona fide interactor with presenilin-1 (46
), a protein which carries mutations that cause familial Alzheimer's disease. Presenilin-1 is expressed in the brain as well as at the postsynaptic domain of the neuromuscular junction (2
). δ-Catenin is a potent substrate and forms a stable complex with Abl kinase (27
). Abl kinase has been found in the postsynaptic domain of the neuromuscular junction and is a critical component of the tyrosine kinase signaling cascade downstream of agrin (15
). These results raise the possibility that δ-catenin can be phosphorylated by Abl kinase at the neuromuscular junction and this phosphorylation can modulate its activity. Experiments directed at testing this hypothesis are currently under way.
We found Kaiso and δ-catenin localized to nuclei of C2C12 myotubes. We additionally demonstrated for the first time that Kaiso either directly binds or forms an indirect complex with δ-catenin. Our data show that LMB treatment, which is a specific inhibitor of CRM-1-mediated nuclear export, leads to nuclear δ-catenin accumulation in C2C12 cells. Intriguingly, the rapsyn promoter is induced by LMB treatment in chicken myotubes. This induction is inhibited by mutations of the Kaiso binding site. Altogether, this supports the hypothesis that nuclearly localized δ-catenin is involved in transcriptional regulation in C2C12 myotubes and at the neuromuscular junction.
Mutations in the promoter regions of genes can often lead to disease. The mutation in the N box of the AChR
-subunit promoter causes AChR deficiency at the neuromuscular junction and, consequently, CMS (1
). This strongly implies a functional significance of the N box for transcription regulation of the AChR at the neuromuscular junction (41
). Similarly, two point mutations in the E box of the rapsyn promoter have been reported in a subset of patients with CMS (33
). One of these mutations (−38A to G) is located in the E box that partially overlaps the functional Kaiso binding site that we describe here. However, this mutation does not impact the core consensus Kaiso sequence as defined by Daniel et al. (8
). In contrast, it changes the nonconserved dinucleotide of the E-box consensus (CANNTG) and does not disrupt the binding of DNA with myogenic factors. Consistent with this view, Ohno et al. (33
) did not find the expected induction by MyoD of the simian virus 40 promoter when fused with several copies of this E box or the E box carrying the mutation −38A to G. In our study, however, this mutation reduces the activation of the rapsyn promoter by Kaiso and δ-catenin and implicates the influence of bases adjacent to the consensus Kaiso binding site. The possibility therefore exists that CMS associated with this mutation may result from disruption of the Kaiso binding site and not the adjacent E box.
Kaiso is a sequence-specific DNA-binding protein, and CTGCNA (where N is any nucleotide) was identified as the core consensus sequence (8
). Two copies of the optimal consensus were found in both the human and mouse matrilysin
promoters, and Kaiso was shown to demonstrate sequence-specific binding to matrilysin
-derived oligonucleotides (8
). These and other data implicate matrilysin
as a Kaiso target gene (C. M. Spring, K. F. Kelly, I. O'Kelly, M. Graham, H. C. Crawford, and J. M. Daniel, submitted for publication). While most POZ-zinc finger proteins are transcriptional repressors (10
), some are activators (24
), and some repress as well as activate transcription (4
). In our system, Kaiso behaves as a transcriptional activator, suggesting that the transcriptional properties of Kaiso may be context dependent. Consistent with this, Kaiso fails to activate the rapsyn promoter-reporter in nonmuscle human embryonic kidney 293T cells which do not express δ-catenin or myogenic factors. We hypothesize that the nuclear targeting of δ-catenin induces rapsyn transcription in muscle cells. We speculate that there is a balance between the inducing activity of the E-box-binding myogenic factors and Kaiso activity; this balance is likely regulated by δ-catenin nuclear trafficking. The mutation −38A to G decreases the induction by Kaiso and δ-catenin and apparently shifts the balance between myogenic factors and Kaiso and δ-catenin. This may cause the reduction or loss of rapsyn protein at the neuromuscular junction and the consequent endplate AChR deficiency and may explain the phenotype observed in CMS patients with a mutation in this region of the rapsyn promoter.
In conclusion, these results strongly suggest that Kaiso and δ-catenin are components of a mechanism that regulates synapse-specific transcription. We therefore propose the following novel model of synapse-specific transcription: activation of relevant target genes, including rapsyn, requires the binding of Kaiso and δ-catenin in addition to myogenic transcription factors. Promoter activation is modulated by the expression levels of Kaiso and δ-catenin as well as the translocation of δ-catenin from the subsynaptic cytoplasm to the subsynaptic nuclei. The proposed Kaiso/δ-catenin complex regulates the affinity of Kaiso-DNA binding and modulates the transcriptional activity of Kaiso target genes at synapses throughout the body.