Osada Tomoharu, Nozaki Tadashige, Masutani Mitsuko
Drug Development Service Segment LSI Medience Corporation, 13-4, Uchikanda 1-chome, Chiyodaku, Tokyo 101-8517, Japan.
Curr Protein Pept Sci. 2016;17(7):693-704. doi: 10.2174/1389203717666160419144502.
Accumulating evidence suggests that cloned mice production by the injection of a somatic cell nucleus into an enucleated oocyte is inefficient. DNA damage and chromatin remodeling failures that occur during embryogenesis following nuclear transfer (NT) might explain the poor development of cloned embryos. To avoid these problems, it is important to elucidate somatic chromatin remodeling after NT. Because polyADP-ribosylation, which is catalyzed mainly by poly(ADP-ribose) polymerase 1 (Parp1), is a major post-translational modification that facilitates DNA repair and chromatin remodeling, we examined the effects of Parp1 deficiency in developing NT embryos. Parp1 was located within the pseudo-pronuclei (PPN) of NT eggs. We observed that NT eggs, after activation by Sr2+, formed PPN with significantly more efficiency in Parp1-null embryos than in wild-type NT embryos. However, most the Parp1-null embryos stopped developing by the four-cell stage. Immunostaining for γH2AX foci, a marker of DNA double strand breaks, showed longer retention in the PPN of Parp1-/- donor NT embryos than in wild-type NT embryos, suggesting that, in the absence of Parp1, DNA breaks are slowly repaired and consequently, entry into the S phase might be delayed. Furthermore, decreases in histone H3 acetylation, H3 monomethylation at lysine 4, and H3 trimethylation at lysine 27 after the Sr2+ activation step were observed in the PPN of Parp1-/- donor embryos. Taken together, our data suggest that Parp1 is involved in the plastic remodeling of chromatin structure after NT by supporting DNA repair and specific histone code modifications.
越来越多的证据表明，通过将体细胞核注入去核卵母细胞来生产克隆小鼠的效率很低。核移植（NT）后胚胎发育过程中发生的DNA损伤和染色质重塑失败可能解释了克隆胚胎发育不良的原因。为了避免这些问题，阐明NT后的体染色质重塑很重要。由于主要由聚（ADP-核糖）聚合酶1（Parp1）催化的聚ADP-核糖基化是一种促进DNA修复和染色质重塑的主要翻译后修饰，我们研究了Parp1缺陷对发育中的NT胚胎的影响。Parp1位于NT卵的假原核（PPN）内。我们观察到，在用Sr2+激活后，NT卵在Parp1基因敲除胚胎中形成PPN的效率明显高于野生型NT胚胎。然而，大多数Parp1基因敲除胚胎在四细胞阶段停止发育。对DNA双链断裂标记物γH2AX焦点的免疫染色显示，Parp1-/-供体NT胚胎的PPN中γH2AX焦点的保留时间比野生型NT胚胎更长，这表明在没有Parp1的情况下，DNA断裂修复缓慢，因此进入S期可能会延迟。此外，在Parp1-/-供体胚胎的PPN中，观察到Sr2+激活步骤后组蛋白H3乙酰化、赖氨酸4处的H3单甲基化和赖氨酸27处的H3三甲基化减少。综上所述，我们的数据表明，Parp1通过支持DNA修复和特定组蛋白密码修饰参与NT后染色质结构的可塑性重塑。
