Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, China.
Engineering Center for Animal Embryo Technology, Yangling, China.
FASEB J. 2023 Sep;37(9):e23111. doi: 10.1096/fj.202300131RRR.
The post-transfer developmental capacity of bovine somatic cell nuclear transfer (SCNT) blastocysts is reduced, implying that abnormalities in gene expression regulation are present at blastocyst stage. Chromatin accessibility, as an indicator for transcriptional regulatory elements mediating gene transcription activity, has heretofore been largely unexplored in SCNT embryos, especially at blastocyst stage. In the present study, single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) of in vivo and SCNT blastocysts were conducted to segregate lineages and demonstrate the aberrant chromatin accessibility of transcription factors (TFs) related to inner cell mass (ICM) development in SCNT blastocysts. Pseudotime analysis of lineage segregation further reflected dysregulated chromatin accessibility dynamics of TFs in the ICM of SCNT blastocysts compared to their in vivo counterparts. ATAC- and ChIP-seq results of SCNT donor cells revealed that the aberrant chromatin accessibility in the ICM of SCNT blastocysts was due to the persistence of chromatin accessibility memory at corresponding loci in the donor cells, with strong enrichment of trimethylation of histone H3 at lysine 4 (H3K4me3) at these loci. Correction of the aberrant chromatin accessibility through demethylation of H3K4me3 by KDM5B diminished the expression of related genes (e.g., BCL11B) and significantly improved the ICM proliferation in SCNT blastocysts. This effect was confirmed by knocking down BCL11B in SCNT embryos to down-regulate p21 and alleviate the inhibition of ICM proliferation. These findings expand our understanding of the chromatin accessibility abnormalities in SCNT blastocysts and BCL11B may be a potential target to improve SCNT efficiency.
牛体细胞核移植（SCNT）囊胚的转移后发育能力降低，这意味着在囊胚阶段存在基因表达调控异常。染色质可及性作为介导基因转录活性的转录调控元件的指标，迄今为止在 SCNT 胚胎中，尤其是在囊胚阶段，尚未得到广泛研究。在本研究中，对体内和 SCNT 囊胚进行了转座酶可及染色质（scATAC-seq）的单细胞测序分析，以分离谱系并证明 SCNT 囊胚中与内细胞团（ICM）发育相关的转录因子（TFs）的异常染色质可及性。谱系分离的拟时分析进一步反映了与体内对应物相比，SCNT 囊胚 ICM 中 TF 的染色质可及性动力学失调。SCNT 供体细胞的 ATAC 和 ChIP-seq 结果表明，SCNT 囊胚 ICM 中的异常染色质可及性是由于在供体细胞中相应基因座处的染色质可及性记忆持续存在，这些基因座处的组蛋白 H3 赖氨酸 4 三甲基化（H3K4me3）强烈富集。通过 KDM5B 去甲基化 H3K4me3 纠正异常染色质可及性，可减少相关基因（如 BCL11B）的表达，并显著改善 SCNT 囊胚的 ICM 增殖。通过在 SCNT 胚胎中敲低 BCL11B 来下调 p21 并减轻 ICM 增殖的抑制作用，证实了这一效应。这些发现扩展了我们对 SCNT 囊胚中染色质可及性异常的理解，BCL11B 可能是提高 SCNT 效率的潜在靶标。
