AgResearch Ruakura Research Centre, Hamilton, New Zealand.
Institute of Nutrition, University Jena, Jena, Germany.
Biol Reprod. 2020 Jun 23;103(1):114-125. doi: 10.1093/biolre/ioaa053.
Correct reprogramming of epigenetic marks in the donor nucleus is a prerequisite for successful cloning by somatic cell transfer (SCT). In several mammalian species, repressive histone (H) lysine (K) trimethylation (me3) marks, in particular H3K9me3, form a major barrier to somatic cell reprogramming into pluripotency and totipotency. We engineered bovine embryonic fibroblasts (BEFs) for the doxycycline-inducible expression of a biologically active, truncated form of murine Kdm4b, a demethylase that removes H3K9me3 and H3K36me3 marks. Upon inducing Kdm4b, H3K9me3 and H3K36me3 levels were reduced about 3-fold and 5-fold, respectively, compared with noninduced controls. Donor cell quiescence has been previously associated with reduced somatic trimethylation levels and increased cloning efficiency in cattle. Simultaneously inducing Kdm4b expression (via doxycycline) and quiescence (via serum starvation) further reduced global H3K9me3 and H3K36me3 levels by a total of 18-fold and 35-fold, respectively, compared with noninduced, nonstarved control fibroblasts. Following SCT, Kdm4b-BEFs reprogrammed significantly better into cloned blastocysts than noninduced donor cells. However, detrimethylated donors and sustained Kdm4b-induction during embryo culture did not increase the rates of postblastocyst development from implantation to survival into adulthood. In summary, overexpressing Kdm4b in donor cells only improved their reprogramming into early preimplantation stages, highlighting the need for alternative experimental approaches to reliably improve somatic cloning efficiency in cattle.
正确重编程供体核中的表观遗传标记是体细胞转移（SCT）成功克隆的前提条件。在几种哺乳动物中，抑制性组蛋白（H）赖氨酸（K）三甲基化（me3）标记，特别是 H3K9me3，是体细胞重编程为多能性和全能性的主要障碍。我们设计了牛胚胎成纤维细胞（BEFs），使其能够诱导表达生物活性的、截断的鼠 Kdm4b 形式，该脱甲基酶可以去除 H3K9me3 和 H3K36me3 标记。与非诱导对照相比，诱导 Kdm4b 后，H3K9me3 和 H3K36me3 水平分别降低了约 3 倍和 5 倍。先前已经发现，供体细胞静止与牛中体细胞甲基化水平降低和克隆效率提高有关。同时诱导 Kdm4b 表达（通过强力霉素）和静止（通过血清饥饿），与非诱导、非饥饿对照成纤维细胞相比，总共有 18 倍和 35 倍分别进一步降低了全局 H3K9me3 和 H3K36me3 水平。SCT 后，Kdm4b-BEFs 比非诱导供体细胞更有效地重编程为克隆囊胚。然而，去甲基化供体和胚胎培养期间持续的 Kdm4b 诱导并没有增加囊胚从着床到成年存活的后期发育率。总之，在供体细胞中过表达 Kdm4b 仅改善了它们向早期植入前阶段的重编程，突出了需要替代实验方法来可靠地提高牛体细胞克隆效率的必要性。
