组蛋白去甲基化酶KDM5B影响猪SCNT重编程的分子机制研究

The low clone efficiency caused by little understanding of porcine somatic cell reprogramming, impeded it’s use in establishing transgenetic models. Accumulating evidence suggests that mammalian SCNT reprogramming could be improved via regulating epigenetic modifications of donor cells or SCNT embryos. Our preliminary data suggested that the in vitro development capacity of porcine SCNT embryos could be improved by knockout histone demethylase KDM5B in the donor cell. Research in mouse also identified Kdm5b as a critical barrier to somatic reprogramming, the efficiency of induced pluripotent stem cells (iPSCs) could be improved by knockdown Kdm5b. . So in this research, CRISPR/Cas9 was used to generate KDM5B-/- cell line, the obtained cell line was used as somatic cell donor to build porcine SCNT recombined embryos, their developmental ability will be evaluated. It’s mechanism of KDM5B to porcine SCNT reprogramming will also be discussed in this research. . Differences in cell proliferation, cell cycle and chromatin accessibility between wild-type and KDM5B-/- cell lines will be compared, and the signal pathway regulated by KDM5B through cell proliferation and chromatin to epigenetic reprogramming will be addressed by combining researches in iPSCs. The status of epigenetic modification such as histone methylation, histone acetylation, DNA methylation and RNA methylation in wild-type and KDM5B-/- cell lines and their obtained SCNT 4-cell stage embryos will be described, and the epigenetic modifications regulated directly or indirectly via KDM5B to epigenetic reprogramming will be explored. Furthermore, we want to identify and analyze the genome wide transcriptomes of pig in SCNT 4-cell embryos using single cell mRNA deep sequencing, and the signal pathway regulated by KDM5B through zygotic gene activation to epigenetic reprogramming will be found by combining their results of STAR-ChIP-seq against H3K4me3. The candidate genes might be regulated by KDM5B will be screened, and their spatial and temporal expression changes during the porcine embryonic development will be stated, including their function to porcine SCNT reprogramming. The molecular mechanism of KDM5B to porcine SCNT reprogramming will be finally deeply explained to offer proof that mammalian clone efficiency could be improved by rescuing aberrant epigenetic modifications.

体细胞重编程机制不明，导致克隆效率低下，阻碍了猪基因编辑模型的制备。前期研究结果表明：调控供体细胞的表观修饰，可以有效的改善体细胞重编程。初步研究发现：在供体细胞中敲除KDM5B，能够显著提高猪克隆胚胎的体外发育能力。因此，本研究将进一步检测KDM5B功能缺失对克隆胚胎发育能力的影响，并全面分析其作用机制。对比野生型和KDM5B-/-细胞增殖、周期和染色质结构区别，结合iPSCs的研究，解析KDM5B通过调控细胞染色质结构等，改善重编程的信号网络；分析野生型和KDM5B-/-供体细胞，以及构建的SCNT 4-细胞胚胎的表观修饰模式，探讨KDM5B通过调控表观修饰，改善重编程的分子机制；利用单细胞转录组测序获得4-细胞胚胎的转录组数据，结合4-细胞胚胎中H3K4me3的STAR-ChIP-seq结果，阐明KDM5B通过调控ZGA，改善重编程的机制。为提高体细胞重编程提供理论依据。

大量的研究证明了表观重编程的不完全很可能就是导致体细胞核移植效率低下以及克隆动物异常的主要原因。而改善供核体细胞或者重构胚胎在合子基因组激活之前的表观修饰，可能是提高克隆效率的有效手段。我们的研究发现，在供体细胞中敲除KDM5B能够显著改善猪SCNT胚胎发育潜能。细胞水平的研究发现野生型细胞系和KDM5B-/-细胞，在细胞增殖、细胞周期和染色质结构方面存在显著区别，尤其是KDM5B-/-细胞系中染色体的松散程度显著高于野生型细胞系。单细胞转录组研究发现，以KDM5B-/-细胞系作为供体早期4-细胞胚胎较之于野生型细胞构建4-细胞胚胎的转录组与体外受精来源的4-细胞胚胎转录组更为接近，均表现出较高的与基因表达及调控、RNA生物合成/代谢及调控、细胞代谢等相关的信号基因表达模式，即在供体细胞中敲除KDM5B，有利于弥补猪体细胞核移植胚胎在合子基因组激活早期生命过程不足的现象；供体细胞KDM5B缺失，还降低了体细胞核移植来源胚胎在4-细胞早期-即合子基因组激活早期HOX家族基因的表达，可能是其促进猪体细胞核移植胚胎发育潜能的重要机制。利用KDM5B抑制剂PBIT和促染色质松散小分子化合物氯喹（CC）处理供体细胞，能够极显著提高克隆胚胎的发育潜能，进一步验证了供体来源KDM5B是猪体细胞核移植重编程的重要障碍，在供体细胞中敲除KDM5B有利于染色体重塑，从而提高猪体细胞核移植胚胎的发育能力。本项目研究结果不仅从理论上丰富我们对哺乳动物体细胞核移植重编程机制的认识；在实践上，提高猪克隆效率可为创制人类疾病模型猪和基因编辑育种猪提供技术保障。