Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency.
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.
AgResearch Ltd., Ruakura Research Centre, Reproductive Technologies, Hamilton, New Zealand.
Prasanna K
PK
Kallingappa
AgResearch Ltd., Ruakura Research Centre, Reproductive Technologies, Hamilton, New Zealand.
Pavla M
PM
Turner
AgResearch Ltd., Ruakura Research Centre, Reproductive Technologies, Hamilton, New Zealand.
Michael P
MP
Eichenlaub
AgResearch Ltd., Ruakura Research Centre, Reproductive Technologies, Hamilton, New Zealand.
Andria L
AL
Green
AgResearch Ltd., Ruakura Research Centre, Reproductive Technologies, Hamilton, New Zealand.
Fleur C
FC
Oback
AgResearch Ltd., Ruakura Research Centre, Reproductive Technologies, Hamilton, New Zealand.
Alice M
AM
Chibnall
AgResearch Ltd., Ruakura Research Centre, Reproductive Technologies, Hamilton, New Zealand.
David N
DN
Wells
AgResearch Ltd., Ruakura Research Centre, Reproductive Technologies, Hamilton, New Zealand bjorn.oback@agresearch.co.nz.
Björn
B
Oback
D000818:Animals; D002417:Cattle; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D065150:Cellular Reprogramming / Q000502:physiology*; D044127:Epigenesis, Genetic; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D008938:Mitosis / Q000502:physiology*; D053652:Nuclear Transfer Techniques
D018797:Cell Cycle Proteins; D006657:Histones
cell cycle; epigenetics; histone modifications; nuclear transfer; reprogramming