Epigenetic memory in somatic cell nuclear transfer and induced pluripotency: Evidence and implications

2014 ◽  
Vol 88 (1) ◽  
pp. 29-32 ◽  
Author(s):  
Jaber Firas ◽  
Xiaodong Liu ◽  
Jose M Polo
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Epigenetic Memory ◽  
Induced Pluripotency

21 The contrasting role of histone methyltransferases during nuclear reprogramming: SUV39H knockdown improves bovine somatic cell nuclear transfer, while the absence of EHMT2 hampers it

2020 ◽  
Vol 32 (2) ◽  
pp. 136
Author(s):  
R. Sampaio ◽  
D. Ambrizi ◽  
R. Nociti ◽  
J. Pinzon ◽  
J. Sangalli ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Control Group ◽  
Nuclear Reprogramming ◽  
Control Vector ◽  
Epigenetic Memory ◽  
Histone Methyltransferases ◽  
Sirna Group

The persistence of somatic epigenetic memory is indicated as the main barrier for efficient nuclear reprogramming. The augmented levels of histone methylation on somatic nuclei have been shown as the major regulator of this aberrant remodelling. Although they occupy the same site, H3K9me2 and H3K9me3 are catalysed by different histone methyltransferases (HMTs), EHMT2 and SUV39H, respectively. However, the influence of these HMTs during nuclear reprogramming is unclear. Hence, the main goal of this project was to investigate the role of HMTs responsible for H3K9 methylation during nuclear reprogramming and its consequences on embryo development. For this, we employed a small interfering RNA (siRNA)-mediated knockdown approach targeting EHMT2, SUV39H1, and SUV39H2 in bovine fetal fibroblasts. The RT-qPCR analyses showed ~80% reduction in total RNA after siRNA treatments for the target genes when compared with the control vector. We then quantified the H3K9me2 and H3K9me3 levels by immunostaining. The analysis displayed that H3K9me2 levels were diminished ~50% compared with control, whereas the reduction of H3K9me3 levels was only ~25%. Cells transfected with siRNA targeting EHMT2, SUV39H1, and SUV39H2 (All-siRNA) or control vector (control) were used as a nuclear donor on somatic cell nuclear transfer (SCNT) in five biological replicates. The IVF embryos were used as a biological control for immunostaining analysis. Embryos at both 8- to 16-cell and blastocysts stage (n=10 from 5 replicates) were collected to evaluate the effect of HMT knockdown on H3K9me2 and H3K9me3 levels by immunostaining. We found a reduction of blastocyst rates in the treatment 28±6.3 (mean±s.e.m.) when compared with control 60±4.8 (P=0.004). The immunostaining analysis showed that the levels of H3K9me2 and H3K9me3 were higher in cloned (control) than IVF embryos (P<0.05). Moreover, the All-siRNA group displayed a reduction in H3K9me2 levels compared with the control group and IVF through developmental stages analysed (P<0.05). Differently, H3K9me3 levels were higher in the All-siRNA group at the 8- to 16-cell stage, but no difference was found between treated and control groups at the blastocyst stage. We, therefore, decided to test whether individual knockdown would display a different result. We then used cells transfected with siRNA targeting only EHMT2 (EHMT2-siRNA), targeting SUV39H1 + SUV39H2 (SUV-siRNA), or control vector (control) as a nuclear donor on SCNT in five biological replicates. Surprisingly, the SUV-siRNA group increased blastocyst production 38±4.4 when compared with the control group 29±4.4 (P=0.01), whereas the EHMT2-siRNA showed a reduction in blastocyst rates: 21±5.6 (P=0.04). Our results indicate that EHMT2 has a key role during SCNT, possibly by its crosstalk with other modifications. Even though the SUV39H knockdown induced a small reduction in H3K9me3 levels in the nuclear donor, it was enough to increase the blastocyst rates by 10%. These results will allow us to better understand the complex mechanisms involved in the persistent epigenetic memory during nuclear reprogramming.

scholarly journals Author Correction: Application of interspecific Somatic Cell Nuclear Transfer (iSCNT) in sturgeons and an unexpectedly produced gynogenetic sterlet with homozygous quadruple haploid

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Effrosyni Fatira ◽  
Miloš Havelka ◽  
Catherine Labbé ◽  
Alexandra Depincé ◽  
Viktoriia Iegorova ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer
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