Quiescence induces epigenetic changes in bovine fibroblasts and improves their reprogramming into cloned embryos

Cloning by somatic cell nuclear transfer (SCNT) forces cells to lose their lineage-specific epigenetic marks and become totipotent again. This reprogramming process often results in epigenetic and transcriptional aberrations that compromise development. Development rates after SCNT can thus serve as a functional assay for genome-wide epigenetic reprogramming. Dolly the sheep, the first mammalian SCNT clone, was derived from a donor cell that was induced into quiescence by serum starvation. We hypothesized that quiescence alters the epigenetic status of donor cells and elevates their reprogrammability. In order to test this idea, we compared chromatin composition and cloning efficiency of serum-starved, quiescent (G₀), bovine fibroblasts vs non-starved, diploid G₁ controls. Mechanically synchronized G₁ cells were generated by mitotic shake-off and harvested within 3 h post-mitosis. Based on morphological assessment and EdU incorporation during continuous labeling, >93% of cells were captured in G₁. Using quantitative confocal immunofluorescence microscopy and fluorometric ELISA, we show that G₀ fibroblasts were significantly hypomethylated at lysines (K) of histone 3 (H3), specifically H3K4me3, H3K9me2, H3K9me3 and H3K27me3, but not H3K9me1. Histone acetylation was reduced at H3K9 and H4K5, increased at H3K12 and remained unchanged at H3K16. G₀ cells also significantly reduced DNAme. In addition, they significantly down-regulated the nuclear abundance of RNA polymerase II, histone variant H2A.Z, as well as Polycomb group (PcG) proteins EED, SUZ12, PHC1 and RING2. Histone variant H3.3, PcG proteins EZH2 and histone deacetylase HDAC1 did not change compared to the G₁ controls. Following NT into metaphase-arrested oocytes, G₀ DNA condensed slower than that of G₁ cells, indicating a more relaxed chromatin configuration. After seven days of in vitro culture, H3K9me3, but not H3K4me3, H3K27me3, SUZ12 and RING2, remained hypomethylated in G₀- vs G₁-derived NT blastocysts, both in the inner cell mass and trophectoderm. Furthermore, G₀ donors significantly improved development into cloned blastocysts. In conclusion, quiescence induced long-term epigenetic changes, specifically H3K9me3 hypomethylation, that correlated with increased donor cell reprogrammability

Effect of ageing and single nucleotide polymorphisms associated with the risk of aggressive prostate cancer in a New Zealand population

Prostate cancer is one of the most significant male health concerns worldwide, and various researchers carrying out molecular diagnostics have indicated that genetic interactions with biological and behavioral factors play an important role in the overall risk and prognosis of this disease. Single nucleotide polymorphisms are increasingly becoming strong biomarker candidates to identify the susceptibility of individuals to prostate cancer. We carried out risk association of different stages of prostate cancer to a number of single nucleotide polymorphisms to identify the susceptible alleles in a New Zealand population and checked the interaction with environmental factors as well. We identified a number of single nucleotide polymorphisms to have associations specifically to the risk of prostate cancer and aggressiveness of the disease, and also certain single nucleotide polymorphisms to be vulnerable to the reported behavioral factors. We have addressed “special” environmental conditions prevalent in New Zealand, which can be used as a model for a bigger worldwide study

A virus-free poly-promoter vector induces pluripotency in quiescent bovine cells under chemically defined conditions of dual kinase inhibition.

Authentic induced pluripotent stem cells (iPSCs), capable of giving rise to all cell types of an adult animal, are currently only available in mouse. Here, we report the first generation of bovine iPSC-like cells following transfection with a novel virus-free poly-promoter vector. This vector contains the bovine cDNAs for OCT4, SOX2, KLF4 and c-MYC, each controlled by its own independent promoter. Bovine fibroblasts were cultured without feeders in a chemically defined medium containing leukaemia inhibitory factor (LIF) and inhibitors of MEK1/2 and glycogen synthase kinase-3 signaling ('2i'). Non-invasive real-time kinetic profiling revealed a different response of bovine vs human and mouse cells to culture in 2i/LIF. In bovine, 2i was necessary and sufficient to induce the appearance of tightly packed alkaline phosphatase-positive iPSC-like colonies. These colonies formed in the absence of DNA synthesis and did not expand after passaging. Following transfection, non-proliferative primary colonies expressed discriminatory markers of pluripotency, including endogenous iPSC factors, CDH1, DPPA3, NANOG, SOCS3, ZFP42, telomerase activity, Tra-1-60/81 and SSEA-3/4, but not SSEA-1. This indicates that they had initiated a self-sustaining pluripotency programme. Bovine iPSC-like cells maintained a normal karyotype and differentiated into derivatives of all three germ layers in vitro and in teratomas. Our study demonstrates that conversion into induced pluripotency can occur in quiescent cells, following a previously undescribed route of direct cell reprogramming. This identifies a major species-specific barrier for generating iPSCs and provides a chemically defined screening platform for factors that induce proliferation and maintain pluripotency of embryo-derived pluripotent stem cells in livestock