Determination of transcription start sites (TSS) for mouse CS-1 and CS-2
To further characterize the transcriptional control of the mouse CS-1 and CS-2 genes, we first determined their respective transcription start sites. RT-PCR analysis was performed using total RNA extracts of C2C12 cells and four different forward primers and one reverse primer for each gene. As shown in Table 1, the reverse primer is located in exon 1 and the different forward primers are located around the putative transcription start sites. The results of these analyses show that the first three primer pairs of CS-1, and first two for CS-2, successfully yielded PCR products. This indicates that the sequences contained within these oligonucleotides form part of the mRNA product of each gene (Fig. 1). We concluded that the TSS sites of mouse CS-1 and CS-2 are located at -191 to -176 and -82 to -74, respectively. The nucleotide immediately upstream of the translation initiation codon (ATG) is denoted as -1.
Transcriptional regulation of CS-1 and CS-2 during myogenic differentiation
Real-time PCR analysis was performed to determine the relative mRNA expression levels of both the mouse CS-1 and CS-2 genes during myoblast differentiation in C2C12 cells. Specific primers corresponding to the two genes were designed and the housekeeping gene β-actin was used as a control. We subsequently found that the CS-1 and CS-2 transcript levels were both low at the myoblast stage. CS-1 mRNA expression increases markedly, however, during the first two days of differentiation and is then maintained at relatively abundant levels throughout this process. In contrast, the CS-2 transcript levels remain low throughout myogenic differentiation (Fig. 2).
Promoter analysis of the mouse CS-1 and CS-2 gene
To test the minimal region required for promoter activity within the upstream CS-1 and CS-2 sequences, fragments corresponding to the regions -2554 to -144 of CS-1 and -2478 to -67 of CS-2 (relative to the ATG initiation codon), were inserted into the pGL3-basic vector and luciferase assays were performed. A significant increase in luciferase activity was observed in the C2C12 cell line (almost 20-fold and 16-fold increases following transfection with the promoter-luciferase vectors pCS1-2554/-144 and pCS2-2478/-67, respectively) compared with cells transfected with the empty vector (Fig. 3). Sequence analysis of these two promoter segments revealed that the flanking region harbours potential binding sites for multiple transcription factors including Sp1 and AP-1, in addition to the muscle specific transcription factors MEF-2 and MyoD. However, no TATA-boxes are present in these regions.
We next generated a series of deletion mutants for the CS-1 and CS-2 promoters via PCR-based approaches using the pCS1-2554/-144 and pCS2-2478/-67 constructs as templates. The amplified mutant fragments were then subcloned into the pGL3-basic vector and our results are shown in Figure 3. For the CS-1 promoter fragments, the deletion constructs pCS1-1954/-144 and pCS1-1206/-144 display high promoter activity. Further deletions at positions -926 (pCS1-926/-144) and -554 (pCS1-554/-144) resulted in a gradual decrease in promoter activity. However, the highest promoter activity levels were observed for the pCS1-427/-144 construct, suggesting that there are inhibitory elements within the region -554 to -427. Deletions at positions -337(pCS1-337/-144) and -244(pCS1-244/-144) also reduce promoter activity dramatically, whereas the pCS1-199/-144 construct shows almost no luciferase activity (Fig. 3A). Taken together, these reporter data indicate that the core region of the basal promoter of mouse CS-1 gene is located within the region -427 to -337. For the CS-2 promoter, the deletion constructs pCS2-1859/-67, pCS2-1166/-67 and pCS2-561/-67 display high promoter activity with only modest differences between them, suggesting that each harbours the core elements necessary for the basal promoter function of the mouse CS-2 gene. The promoter activity of pCS2-482/-67, PCS2-333/-67 and pCS2-185/-67 decreases gradually to relatively low levels and thus further defines the core region from -561 to -185 (Fig. 3B).
For our initial characterization of these two promoters, we searched for the presence of a consensus slow upstream regulatory element (SURE) and fast intronic regulatory element (FIRE), which have been shown to drive fibre-specific gene transcription [8]. However, neither of these sites was found to be present in the two respective promoter regions after careful analysis using TESS software. We then focused on the association of other transcription factors with the two gene promoters, such as the role of calcineurin signalling factors in activating the slow-fibre specific promoter of CS-1. It was noticeable that the pCS1-427/-144 construct exhibited maximum activity and that deletion of the region -427 to -337 of CS-1 5' flanking sequence reduced the promoter activity dramatically, indicating that a strongly positive element is located in this region. A putative binding site for the NF-κB transcription factor was detectable in this region using high-stringency analysis of the TESS database. However, a putative binding site for NF-κB was also assigned to the core region of CS-2 promoter in these searches. Since our reporter assay findings raised the possibility that transactivation of the mouse CS-1 and CS-2 gene may be achieved through NF-κB binding elements present in their core promoters, we attempted to determine which of these two potential NF-κB binding sites was authentic during this activation event.
Confirmation of NF-κB binding to the CS-1 promoter by EMSA
We synthesized specific oligonucleotides containing the NF-κB elements present in the CS-1 and CS-2 promoters and tested them with an NF-κB consensus sequence control in EMSA experiments with nuclear extracts from C2C12 myotubes. As shown in Figure 4A, incubation of C2C12 nuclear extracts with both the NF-κB consensus sequence and NF-κB-CS1 sequence produced a DNA-protein band shift. In contrast, the NF-κB-CS2 oligonucleotide probe failed to form such a complex in this experiment. These DNA-protein complexes were determined to be specific to the NF-κB sites by successful competition assays using excess unlabeled consensus and NF-κB-CS1 oligonucleotides (Fig. 4B). To confirm the binding of NF-κB family members to the NF-κB-CS1 sequence, these EMSA reactions were further incubated with antibodies raised against P50 of NF-κB. As shown in Figure 4B, the addition of this antibody resulted in a supershifted complex in addition to the DNA-protein band. These data confirm the presence of the NF-κB family member, P50, in the nuclear protein complex that binds the NF-κB binding site of the CS-1 promoter.
The inhibition of NF-κB downregulates CS-1 promoter activity but does not affect CS-2
To further illustrate the biological importance of NF-κB in the regulation of CS-1, we inhibited NF-κB transactivation by treatment with pyrrolidine dithiocarbamate (PDTC), a proven free radical scavenger that accelerates IκB dissociation with a resulting block in NF-κB transport to the nucleus and subsequent binding to DNA. As shown in Figure 5, when C2C12 cells were transfected with the 2.5-kb wild type CS-1 and CS-2 promoter luciferase reporter plasmids, a 20-fold and 16-fold increase in reporter activity was observed, respectively. The treatment of these cells with PDTC induces a modest decrease in luciferase activity for both wild-type reporter plasmids, and had no impact on the empty vector control (Fig. 5). Transfection of the shorter CS-1 and CS-2 promoter fragment reporters (pCS1-427/-144 and pCS2-561/-67), both containing their respective putative NF-κB elements, results in a 25-fold and 10-fold increase in luciferase activity, respectively. In this same experiment, PDTC treatment induced a 2–3-fold decrease in luciferase activity for CS-1 but no apparent changes were evident for the CS-2 reporter activities. However, when cells were transfected with the CS-1 and CS-2 reporter plasmids containing proximally shorter promoter fragments (pCS1-337/-144 and pCS2-482/-67), we again detected a large increase in luciferase activity compared with the empty vector, but PDTC has no effects. These data suggest that the NF-κB binding element located in the region -427 to -337 of CS-1 promoter plays an important role in the transcriptional activity of this gene.
The effects of NF-κB, NFAT and MEF2 overexpression upon the CS-1 and CS-2 promoters
Since putative NFAT and MEF2 binding elements are located within the CS-1 and CS-2 promoters, the sensitivities of the NFAT and MEF2 sites, as well as NF-κB site, to the overexpression of specific transcription factors were also determined. First, the 2.5-kb wild type CS-1 and CS-2 promoters were cotransfected with pReceiver-NF-κB1, which constitutively expresses the NF-κB subunit gene p50. The overexpression of p50 significantly induces CS-1 promoter activity and also the positive NF-κB reporter control (pNF-κB-Luc). A further 5' deletion of the NF-κB site totally abolishes the response to NF-κB overexpression, reconfirming the authenticity of this site. However, the activity of CS-2 promoter was found to be increased by 1.7-fold when cotransfected with NF-κB in these experiments, indicating that CS-2 is not independent of NF-κB. This partially contradicts the results of our gel shift and drug treatment experiments. Hence, other NF-κB elements may exist in the CS-2 promoter or it may be triggered by other factors induced by NF-κB.
As a preliminary test for the role of the calcineurin-NFAT-MEF2 signalling pathway [8] in the regulation of the calsarcin promoters, we next determined whether the CS-1 or CS-2 promoters are sensitive to the overexpression of NFAT or MEF2 in C2C12 cells. When cotransfected with the NFATc4 expression plasmid (pcDNA-NFATc4), both of the promoters show enhanced transcription activities, but to different extents (CS-1, 7.2 ± 2.3 fold; CS-2, 4.4 ± 1.8 fold; and pNFAT-Luc, 68.8 ± 13.6 fold). However, only a moderate decrease in promoter activity was observed after 5' deletion of the -1206 to -926 region of CS-1 promoter which harbours a NFAT consensus element, indicating that other putative binding motifs are present. Similarly, MEF2C overexpression was achieved by cotransfection of pReceiver-MEF2C with the luciferase reporter vector. This overexpression induces the activity of the CS-1 promoter by 3.5-fold but has only marginal effects upon the CS-2 promoter. This indicates that MEF2 also contributes to the differential transcription of the calsarcins in different fibres. However, it is noteworthy that both promoters contain several putative MEF2 binding elements (Fig. 6).