Pax proteins are the key factors in the development of tissues and organs during embryogenesis. These proteins are characterised by the presence of a paired domain (PD), which specifically binds to DNA sequences where it acts as a transcription repressor or activator, and is generally located in the N-terminal part. Pax genes may also have an octapeptide (OP) motif and a homeobox DNA-binding domain (homeodomain; HD). Pax genes can be categorised into four subfamilies according to their structural characteristics. Within these subfamilies there is some overlap in expression during the development of tissues or organs in determining the choice of cell fate (1)
Pax7 is expressed specifically during the development of the nervous and muscular system. A study by Merrick et al. (2009) showed that Pax7 can be detected as early as E11.5 in wild-type embryos and its level increases throughout the gestational period (2). It plays a role in the regulation of muscle precursor cell proliferation by recruiting the histone-3-lysine-4 methyltransferase (H3K4 HMT) complex via Pax3/7BP and the Wdr5 adaptor. This complex then target genes, i.e. Cdc20 and Id3, which are involved in cell-division cycle activation and the transcription inhibition process, respectively (3). The down-regulation of pax7 protein levels in an embryo correlates with an increased level of apoptosis in myoblasts isolated from mdx and cav3−/− mice (2).
Further observations have shown the attrition of a Pax7-positive cell population in mdx and cav3−/− embryos until late in gestation. Pax7-positive cells fragments were found in dystrophic muscle (E15.5: cav3−/− and mdx; E17.5: mdx) and was much more severe in mdx/cav3+/−, suggesting that these cells may undergo apoptosis (2). From the results, the severity of the double mutant showed that dystrophin and cav-3 play an important role for the survival of Pax7-positive cells during myogenesis. However, in vitro analysis of apoptosis in dystrophin-deficient myoblasts is lacking, which would demonstrate the frequency of apoptosis, as well as the significance of the event.
Recently, it has been reported that Pax7 is regulated/modified by SUMOylation. Pax7 interacts with the SUMO conjugating enzyme known as UBC9, which SUMOylates Pax7 at lysine85 (K85) within the DNA binding domain (4). SUMOylated-Pax7 is essential during neural crest development, C2C12 myogenic differentiation, and transcriptional transactivation. In adult satellite cells, the arginine methyltransferase Carm1, specifically methylates an arginine present in the N-terminus of Pax7, and thus is recruited to the WDR5-ASH2L-MLL2 complex for up-regulating Pax7 target genes (5). Pax3/7 have also been shown to interact with other proteins involved in chromatin binding, including HIRA, DAXX and PAX3/7BP (6). This binding is important for the up-regulation of Pax7 target genes, such as Id3 and Cdc20. Id3 is a helix-loop-helix (HLH) protein that can also bind with other HLH proteins as a heterodimer. The lack of a basic DNA-binding domain inhibits other HLH proteins from binding to DNA, thus acting as a transcription inhibitor. Binding of Pax7 to the Id3 promoter is also thought to up-regulate and increase its expression during differentiation. Cdc20 is a regulatory protein that interacts with other proteins during cell division and is highly expressed in proliferating cells. It is responsible for mediating the association of the anaphase protein complex with the mitotic spindle checkpoint protein, MAD1L1 (7).
Different from prokaryotic cells, the nucleus and cytoplasm are separated by a double lipid bilayer nuclear envelope (NE) in eukaryotic cells. The nuclear pore complex (NPC), in the NE, tightly mediates the bidirectional traffic of molecules, including proteins, nucleic acids and small molecules, via aqueous channel either passively or actively (8,9). The nuclear-cytoplasmic machinery involves the NPC, cellular apoptosis susceptibility (CAS), RanGTP, karyopherins (also known as importins/exportins) as well as the specific proteins/RNA complex to be transported. Alterations to any of these factors can cause the failure of protein regulation within a cell, thus affecting the whole process, i.e. up-regulation or down-regulation of genes.
The regulation of translated proteins in the cytoplasm is control by various signalling mechanisms, either for secretion or to remain within a cell. For those proteins that play a role as an activator, adaptor or gene regulator, specific transcription factors are need to already be present in the nucleus for the up-regulation, as well as down-regulation of genes, thus they need to be transported into the nucleus. As most of these proteins are larger than 40 kDa, protein adaptors (β-karyopherins) are needed to shuttle them through the nuclear membrane.
Transport receptors, also known as β-karyopherins (importins/exportins), only recognise proteins containing a nuclear localisation signal (NLS). This is a sequence highly enriched with the basic amino acids lysine (K), arginine (R) and histidine (H). It has been reported that phosphorylation of the NLS enhances the binding affinity of importin-α to become part of a complex (10), which then binds to importin-β and is translocated into the nucleus via the NPC as the karyopherin-β has a binding affinity for nucleoporins within the NPC. This translocation process is also regulated by the gradient of RanGTP, which is asymmetrically distributed and predominantly found in the nucleus. Dissociation of the NLS-protein complex by RanGTP occurs within the nucleus, where the concentration of RanGTP is controlled by RanGEF which converts RanGDP to RanGTP. Importin-β is carried out of the nucleus by RanGTP, while importin-α needs a protein receptor (CAS) to form a complex with in order for RanGTP to be recycled back into the cytoplasm (8).
Earlier, our group has reported that Pax7 cell is attenuated in the mdx embryo during gestation. Pax7 cell fragments have also been found in dystrophic muscle, indicating that the cells are undergoing apoptosis. Immunoblot analysis of whole embryos showed that Pax7 expression was also substantially reduced with age (2). This finding has led to the suggestion that dysregulation of Pax7 takes place in MD as early as during the embryonic stages. Therefore, in this study the Pax7 expression pattern has been further examined during the postnatal/juvenile stage in Pax7 myoblasts isolated from a 5-week old mdx mouse.
As a transcription factor, Pax7 is mainly found in the nucleus of myoblasts, but surprisingly, it has been found not only in the nucleus, but also found in cytoplasmic region (i.e. organelles) of dystrophin-deficient myoblasts. However, there are a lack of studies to show what actually happens to Pax7 in dystrophin-deficient myoblasts. There may be a problem in protein trafficking in or out of the nucleus which involves the importin and exportin mediated pathway, or there could be an alteration in the NLS which causes the failure of Pax7 to bind to importins. This could be the cause of the failure of these cells to be differentiated into mature myotubes prior to the development of muscle tissue. Initially, the expression of Pax7 upon differentiation was determined. Furthermore, the cytoplasmic-Pax7 subcellular localisation, i.e. endoplasmic reticulum, golgi, recycling endosome and lysosome, for protein synthesis and degradation was examined using subcellular markers. Finally, protein prediction tools were utilised which suggested that a Pax7-karyopherin protein interaction is responsible for nuclear localisation.