Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency

Abstract

Reprogramming by nuclear transfer (NT) cloning forces cells to lose their lineage-specific epigenetic marks and reacquire totipotency. This process often produces molecular anomalies that compromise clone development. We hypothesized that quiescence alters the epigenetic status of somatic NT donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) fibroblasts versus nonstarved mitotically selected (G1) controls. We show that G0 chromatin contains reduced levels of Polycomb group proteins EED, SUZ12, PHC1, and RING2, as well as histone variant H2A.Z. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay, we further show that G0 induced DNA and histone hypomethylation, specifically at H3K4me3, H3K9me2/3 and H3K27me3, but not H3K9me1. Collectively, these changes resulted in a more relaxed G0 chromatin state. Following NT, G0 donors developed into blastocysts that retained H3K9me3 hypomethylation, both in the inner cell mass and trophectoderm. G0 blastocysts from different cell types and cell lines developed significantly better into adult offspring. In conclusion, serum starvation induced epigenetic changes, specifically hypotrimethylation, that provide a mechanistic correlate for increased somatic cell reprogrammability.

Vitamin C treatment of embryos, but not donor cells, improves the cloned embryonic development in sheep

“…Moreover, we found that starvation combined with vitamin C treatment induced not only 5hmC increase but also simultaneous 5mC decrease. This finding supports previous report that serum starvation facilitates epigenetic changes in cells (Kallingappa et al, ). Despite this, vitamin C treatment of the starved donor cells still failed to improve the blastocyst development of SCNT embryos, albeit the expression of some pluripotency genes ( KLF4 and SOX2 ) was significantly increased.…”

Targeted demethylation of H3K9me3 and H3K36me3 improves somatic cell reprogramming into cloned preimplantation but not postimplantation bovine concepti

“…2A, B). Serum starvation has been shown to globally reduce trimethylation levels in bovine fibroblasts and increase their cloning efficiency [24]. We therefore analysed the effect of Kdm4b overexpression in non-starved vs serum-starved BEFs separately.…”

“…We previously reported that serum starvation about halves the amount of H3K9me3 in non-transgenic primary bovine cell types, resulting in a more relaxed chromatin structure and providing a molecular correlate for the elevated reprogrammability of quiescent donor cells into totipotency. This halving may be due to starved cells acquiring non-methylated histones during S-phase but not re-instating trimethylation upon serum starvation, allowing completion of the last DNA replication and mitosis before entering a non-proliferative G 0 state [24].…”

“…For each comparison, induced and non-induced cells were processed and analyzed in parallel. Cells were fixed and permeabilized simultaneously in 3.6% (w/v) paraformaldehyde/1% (w/v) Triton X-100 in PBS for 10 min, blocked with 2.5% (w/v) bovine serum albumin in PBS, all at room temperature, and incubated overnight at 4-8°C with the primary antibody specific for H3K9me3 (for immunofluorescence: rabbit polyclonal, gift by T. Jenuwein and previously validated for bovine [24]; for western analysis: mouse monoclonal, #c15200146, Diagenode), H3K36me3 (rabbit polyclonal, #9763, Cell Signaling) or H3K27me3 (mouse monoclonal, # c15200181, Diagenode). The next day, cells were washed in PBS-0.05% Tween 20 and incubated for 1 h at room temperature with Alexa Fluor® 488 or 568 donkey anti-goat, -mouse, -rabbit or –sheep secondary IgG antibodies (Thermo Fisher).…”

Testes of DAZL null sheep lack spermatogonia and maintain normal somatic cells

“…The population with the highest proportion of HDR events was used for manual isolation of clonal cell strains. We modified our mitotic shake-off procedure for synchronising cells in the G1 cell cycle stage to obtain sufficient starting material for isolating genome-edited clones 66,67 . Briefly, 2.5x10 4 cells/cm 2 were seeded on 4-well plate and cultured for 18-20 h. Cells were washed once with PBS and cultured for another 2-3 h before mitotic shake-off by gently tapping the dish with moderate horizontal force.…”