Methylation of chick UbI and UbII polyubiquitin genes and their differential expression during spermatogenesis

Northern analysis demonstrated that levels of ubiquitin transcript increased during the chicken testis maturation process, in agreement with the previously published increase of ubiquitin during this differentiation process. Specific probes for four different ubiquitin genes (two polyubiquitins, UbI and UbII, and two ubiquitin-fusion genes, UbCep52 and UbCep80) allowed us to analyse the expression of each individual gene. UbI polyubiquitin gene was expressed in all the tissues tested, and its transcript was the most abundant ubiquitin RNA in all of them. Unspliced UbI transcript, already detected in stressed chicken-embryo fibroblast, was also present in immature testis and reticulocytes. UbII, a chicken polyubiquitin gene not previously found expressed and not heat-shock-inducible, was specifically stimulated during the testis maturation process. Two minor ubiquitin fusion transcripts of 0.6 and 0.7 kb, corresponding to UbCep52 and UbCep80 respectively, were also found in chicken testis. Although differentially expressed, it was found that UbI and UbII chicken polyubiquitin genes had an HTF ('HpaII tiny fragments') island (CpG-rich and constitutively unmethylated region) in their 5' proximal non-coding region. In addition, we demonstrated the coexistence of 3' and/or 5' relatively distal methylated sites together with these 5' proximal HTF islands in both chicken polyubiquitin genes. 3' and 5' distal UbI CCGG sites were specifically hypermethylated in mature testis, whereas a 3' distal UbII CCGG site was found to be about 50% methylated in all DNAs tested.