Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
J Biol Chem. 2021 Dec;297(6):101367. doi: 10.1016/j.jbc.2021.101367. Epub 2021 Nov 1.
Rif1 regulates DNA replication timing and double-strand break repair, and its depletion induces transcriptional bursting of two-cell (2C) zygote-specific genes in mouse ES cells. However, how Rif1 regulates zygotic transcription is unclear. We show here that Rif1 depletion promotes the formation of a unique Zscan4 enhancer structure harboring both histone H3 lysine 27 acetylation (H3K27ac) and moderate levels of silencing chromatin mark H3K9me3. Curiously, another enhancer mark H3K4me1 is missing, whereas DNA methylation is still maintained in the structure, which spreads across gene bodies and neighboring regions within the Zscan4 gene cluster. We also found by function analyses of Rif1 domains in ES cells that ectopic expression of Rif1 lacking N-terminal domain results in upregulation of 2C transcripts. This appears to be caused by dominant negative inhibition of endogenous Rif1 protein localization at the nuclear periphery through formation of hetero-oligomers between the N-terminally truncated and endogenous forms. Strikingly, in murine 2C embryos, most of Rif1-derived polypeptides are expressed as truncated forms in soluble nuclear or cytosolic fraction and are likely nonfunctional. Toward the morula stage, the full-length form of Rif1 gradually increased. Our results suggest that the absence of the functional full-length Rif1 due to its instability or alternative splicing and potential inactivation of Rif1 through dominant inhibition by N-terminally truncated Rif1 polypeptides may be involved in 2C-specific transcription program.
Rif1 调节 DNA 复制时间和双链断裂修复，其缺失会诱导小鼠 ES 细胞中两细胞（2C）合子特异性基因的转录爆发。然而，Rif1 如何调节合子转录尚不清楚。我们在这里表明，Rif1 的缺失会促进一种独特的 Zscan4 增强子结构的形成，该结构同时含有组蛋白 H3 赖氨酸 27 乙酰化（H3K27ac）和中等水平的沉默染色质标记 H3K9me3。奇怪的是，另一个增强子标记 H3K4me1 缺失，而结构中仍然保持 DNA 甲基化，该结构跨越 Zscan4 基因簇的基因体和邻近区域。我们还通过 ES 细胞中 Rif1 结构域的功能分析发现，缺失 N 端结构域的 Rif1 的异位表达导致 2C 转录物的上调。这似乎是由于内源性 Rif1 蛋白通过与 N 端截断的和内源性形式形成异源寡聚体，在核周定位的显性负抑制所致。引人注目的是，在小鼠 2C 胚胎中，大多数 Rif1 衍生的多肽以可溶性核或细胞质部分中的截断形式表达，并且可能是非功能性的。随着胚胎向桑葚胚阶段的发展，全长形式的 Rif1 逐渐增加。我们的结果表明，由于其不稳定性或选择性剪接导致功能性全长 Rif1 的缺失，以及通过 N 端截断的 Rif1 多肽的显性抑制潜在失活，可能参与了 2C 特异性转录程序。
