Recently we demonstrated the possibility of the direct histone-protamine exchange in somatic cells by the exogenous induction of protamine 1 (Prm1) gene expression. Here we have further advanced our protocol, by mimicking the nuclear remodelling taking place in spermatogenesis. The spermiogenesis starts by cell quiescence and the open of nuclear chromatin (by histone-hyperacetylation) of post-meiotic round spermatids. Our aim was to test if protaminization of somatic nucleus increases through the correct union of the induction of G0 stage and exposure to Trichostatin A (TSA). To stop the cell proliferation, the cells were cultured in 0.5% FBS in MEM (G0 group) whereas in the control group (CTR) the cells were culture in 10% FBS in MEM. To check the proper TSA concentration, we treated the cells with 25 nM (25 TSA), 50 nM (50 TSA) and 100 nM (100 TSA) in MEM + 10% FBS. Our results showed that combination of the cell culture condition in G0 and 50 TSA stopped the cell proliferation vs CTR (respectively 17.8%, 8% and 90.2%, p<0.0001). Moreover, quiescent markers gene expression analysis (Dicer1, Smarca 2, Ezh1, Ddx39, H2afz and Pink1) demonstrated that our cell culture condition drive the cell in a quiescent state. Finally, the union of G0 and 50 TSA produced a higher number of spermatid-like cell (39.4%) than the 25 TSA (20.4%, p<0.05) and 100 TSA (13.7%, p<0.05). To conclude, we have demonstrated that the open chromatin structure conferred by G0 stage and TSA, resulted in a more efficient Prm1-mediated conversion of somatic nuclei into spermatid-like structures.
|Titolo:||Nuclear quiescence and histone hyper-acethylation jointly improve protamine-mediated nuclear remodeling in sheep fibroblasts.|
|Data di pubblicazione:||2017|
|Appare nelle tipologie:||4.3 Poster|