Super-resolution structured illumination (SR-SIM) confirms the localization of LSH to the inner centromere in both male and female germ cells at the pachytene stage (scale bar = 1 μm). Unpaired t-test, two tailed and resulted in P < 0.0001. Box plots depict the median value as well as upper and lower quartiles with whiskers representing the minimum and maximum values observed in n = 15 male and n = 15 female pachytene cells examined over three independent experiments.
Note the absence of LSH staining in Lsh −/− pachytene oocytes. In pachytene oocytes, LSH (red) exhibits a diffuse nucleoplasmic localization, resulting in a significant increase in fluorescence intensity, but is also enriched at the inner centromere (arrow inset). In pachytene spermatocytes, LSH (red) is present at the inner centromere (arrow) in close apposition with the kinetochores (arrowhead) detected by CREST (green inset). Our results indicate that loss of LSH affects the levels and chromosomal localization of H3T3ph and provide evidence that, by maintaining transcriptionally repressive heterochromatin, LSH may be essential to prevent deleterious meiotic recombination events at repetitive centromeric sequences.Ī Sexual dimorphism in the patterns of LSH nuclear localization in mouse male and female germ cells. Moreover, knockout oocytes exhibit centromere fusions, ectopic kinetochore formation and abnormal exchange of chromatin fibers between paired bivalents and asynapsed chromosomes. Notably, mutant oocytes show a striking increase in histone H3 phosphorylation at threonine 3 (H3T3ph) and accumulation of major satellite transcripts in both prophase-I and metaphase-I chromosomes.
LSH knockout pachytene oocytes exhibit reduced HDAC2 and DNMT-1. Superresolution chromatin analysis resolves LSH at the inner centromere and kinetochores during oocyte meiosis. Here, we demonstrate that LSH is enriched at meiotic kinetochores and its targeted deletion induces centromere instability and abnormal chromosome segregation. However, the mechanisms regulating centromere and kinetochore chromatin modifications are not known. Centromeres are epigenetically determined nuclear domains strictly required for chromosome segregation and genome stability.
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