Nuclear quiescence and histone hyper-acetylation jointly improve protamine-mediated nuclear remodeling in sheep fibroblasts

Abstract

Recently we have demonstrated the possibility to replace histones with protamine, through the heterologous expression of human protamine 1 (hPrm1) gene in sheep fibroblasts. Here we have optimized protaminization of somatic nucleus by adjusting the best concentration and exposure time to trichostatin A (TSA) in serum-starved fibroblasts (nuclear quiescence), before expressing Prm1 gene. To stop cell proliferation, we starved cells in 0.5% FBS in MEM (“starved”—ST group), whereas in the Control group (CTR) the cells were cultured in 10% FBS in MEM. To find the most effective TSA concentration, we treated the cells with increasing concentrations of TSA in MEM + 10% FBS. Our results show that combination of cell culture conditions in 50 nM TSA, is more effective in terminating cell proliferation than ST and CTR groups (respectively 8%, 17.8% and 90.2% p<0.0001). Moreover, nuclear quiescence marker genes expression (Dicer1, Smarca 2, Ezh1 and Ddx39) confirmed that our culture conditions kept the cells in a nuclear quiescent state. Finally, ST and 50 nM TSA jointly increased the number of spermatid-like cell (39.4%) at higher rate compared to 25 nM TSA (20.4%, p<0.05) and 100 nM TSA (13.7%, p<0.05). To conclude, we have demonstrated that nuclear quiescence in ST cells and the open nuclear structure conferred by TSA resulted in an improved Prm1-mediated conversion of somatic nuclei into spermatid-like structures. This finding might improve nuclear reprogramming of somatic cells following nuclear transfer.

Karyotype structure and NOR activity in Brazilian Smilax Linnaeus, 1753 species (Smilacaceae)

“…For instance, in Atropa belladonna Linnaeus, 1753 derived from a tetraploid and a diploid ancestor species, only four out of six rDNA sites are transcriptionally active, as revealed by silver-staining (Volkov et al 2017). In Quercus robur Linnaeus, 1753, two rDNA loci were observed, NOR-1 and NOR-2 (Bockor et al 2014). Only NOR-1 showed decondensed chromatin in FISH preparations and positive silver signals.…”

Impacts of Epigenetic Processes on the Health and Productivity of Livestock

“…Wool is an economic product of high regard with increasing value in the goat industry, but the limited yield of cashmere wool (or cashmere) was recently speculated to be potentially regulated by epigenetic modifications ( Wang et al, 2017f , 2020d ). DNA methylation and histone acetylation were found to actively contribute to the regulation of goat fetal fibroblast cells, which is critical for cashmere production ( Wang et al, 2017f ; Palazzese et al, 2018 ). Recently, DNA methylation was associated to the genetic stability of cashmere traits between generations of cashmere goats ( Dai et al, 2019 ).…”

The expression, function, and utilization of Protamine1: a literature review

“…Moreover, biofunction of PRM1 on cell growth is thought to be intricate. It has been reported that the increased rate of tumorigenesis was observed by subcutaneous injection of protamines (subtracted from salmon sperm), as well as the impaired proliferation of Hela cells and E. coli by repeated transfection of PcDNA-PRM1 (19)(20)(21)(22)(23)(24). Additionally, due to the high affinity to DNA, PRM1 has been applied in various biomedicine fields such as transgenic animal research, development of drug delivery system, and gene therapy (20,25).…”

“…It has been reported that the increased rate of tumorigenesis was observed by subcutaneous injection of protamines (subtracted from salmon sperm), as well as the impaired proliferation of Hela cells and E. coli by repeated transfection of PcDNA-PRM1 (19)(20)(21)(22)(23)(24). Additionally, due to the high affinity to DNA, PRM1 has been applied in various biomedicine fields such as transgenic animal research, development of drug delivery system, and gene therapy (20,25). Spermatidlike structures of somatic cell nucleus have been observed after heterologous expression of PRM1, which facilitates somatic cell nucleus transfer (SCNT) (19)(20)(21).…”

“…Additionally, due to the high affinity to DNA, PRM1 has been applied in various biomedicine fields such as transgenic animal research, development of drug delivery system, and gene therapy (20,25). Spermatidlike structures of somatic cell nucleus have been observed after heterologous expression of PRM1, which facilitates somatic cell nucleus transfer (SCNT) (19)(20)(21). Moreover, researchers applied peptide-derived approaches using PRM1 as a nucleic acid vector to protect and facilitate the delivery of siRNA or mRNA to specific cell populations (25)(26)(27)(28).…”