VARIATION IN THE METHYLATION PROFILE AND STRUCTURE OF PAX3 AND PAX7 AMONG DIFFERENT MOUSE STRAINS AND DURING EXPRESSION

Structural alterations within the myogenic and neurogenic developmenta l gene Pax7 which involve TaqI recognition sequences have previously b een reported. These alterations are associated with differences in the efficiency of regrowth of damaged skeletal muscle. To identify other structural features of Pas genes which may influence skeletal muscle r egrowth, variation in the structure and methylation status of Pax7 and the closely related gene Pax3 has been sought among different mouse s trains and during gene expression using the restriction endonucleases MspI and HpaII. Following MspI digestion, RFLPs within Pax7 have been found which most likely reflect intron size variability within the pai red box. Differences in the size of MspI and HpaII fragments hybridisi ng with Pax-7 and Pax3 region specific sub-probes indicate that the pa ired boxes are hypomethylated, whereas the region encoding the homeodo main of each gene is highly methylated in the spleen and other tissues from adult mice. In the skeletal muscle precursor cell line C2C12, wh ich expresses Pax7 but not Pax3, the homeodomain encoding region of Pa x7 is hypomethylated. In spleen cells, the Pax7 paired box is transcri bed but the homeodomain encoding region is not. By contrast, both the paired box and the homeobox of Pax3 are hypermethylated in C2C12 cells indicating that generation of alternate transcripts from Pas genes ma y be controlled by DNA methylation. In contrast to Pax3, reference to the size of fragments hybridising with a Pax7 homeobox specific probe provides evidence for C(p)N(p)G methylation within and immediately dow nstream from the region encoding the homeodomain. Interestingly, C(p)N (p)G methylation remains when the Pax7 homeobox is expressed. Structur al variation recognised by MspI digestion and differences in the methy lation profile of Pax7 are not associated with the ability to regrow d amaged skeletal muscle.