Hyeonji Lee, Dong Wook Han, Seonho Yoo, Ohbeom Kwon, Hyeonwoo La, Chanhyeok Park, Heeji Lee, Kiye Kang, Sang Jun Uhm, Hyuk Song, Jeong Tae Do, Youngsok Choi, Kwonho Hong
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引用次数: 0
Abstract
Objective: R-loops are DNA:RNA triplex hybrids, and their metabolism is tightly regulated by transcriptional regulation, DNA damage response, and chromatin structure dynamics. R-loop homeostasis is dynamically regulated and closely associated with gene transcription in mouse zygotes. However, the factors responsible for regulating these dynamic changes in the R-loops of fertilized mouse eggs have not yet been investigated. This study examined the functions of candidate factors that interact with R-loops during zygotic gene activation.
Methods: In this study, we used publicly available next-generation sequencing datasets, including low-input ribosome profiling analysis and polymerase II chromatin immunoprecipitation-sequencing (ChIP-seq), to identify potential regulators of R-loop dynamics in zygotes. These datasets were downloaded, reanalyzed, and compared with mass spectrometry data to identify candidate factors involved in regulating R-loop dynamics. To validate the functions of these candidate factors, we treated mouse zygotes with chemical inhibitors using in vitro fertilization. Immunofluorescence with an anti-R-loop antibody was then performed to quantify changes in R-loop metabolism.
Results: We identified DEAD-box-5 (DDX5) and histone deacetylase-2 (HDAC2) as candidates that potentially regulate R-loop metabolism in oocytes, zygotes and two-cell embryos based on change of their gene translation. Our analysis revealed that the DDX5 inhibition of activity led to decreased R-loop accumulation in pronuclei, indicating its involvement in regulating R-loop dynamics. However, the inhibition of histone deacetylase-2 activity did not significantly affect R-loop levels in pronuclei.
Conclusion: These findings suggest that dynamic changes in R-loops during mouse zygote development are likely regulated by RNA helicases, particularly DDX5, in conjunction with transcriptional processes. Our study provides compelling evidence for the involvement of these factors in regulating R-loop dynamics during early embryonic development.
目的R环是DNA:RNA三重杂交体,其新陈代谢受到转录调控、DNA损伤反应和染色质结构动态的严格调控。R 环的平衡是动态调节的,与小鼠子代的基因转录密切相关。然而,调节小鼠受精卵R环动态变化的因子尚未得到研究。本研究考察了在子代基因激活过程中与R环相互作用的候选因子的功能:在这项研究中,我们使用了公开的新一代测序数据集,包括低输入核糖体图谱分析和聚合酶 II 染色质免疫沉淀测序(ChIP-sequq),以确定子代中 R 环动态的潜在调控因子。对这些数据集进行下载、重新分析,并与质谱数据进行比较,以确定参与调节 R 环动态的候选因子。为了验证这些候选因子的功能,我们利用体外受精技术用化学抑制剂处理了小鼠的子代。然后用抗R环抗体进行免疫荧光,以量化R环代谢的变化:结果:根据DEAD-box-5(DDX5)和组蛋白去乙酰化酶-2(HDAC2)基因翻译的变化,我们发现它们可能调控卵母细胞、合子和两细胞胚胎中的R环代谢。我们的分析发现,抑制 DDX5 的活性会导致 R 环在原核中的积累减少,这表明它参与了 R 环动态的调控。然而,抑制组蛋白去乙酰化酶-2的活性并不会显著影响原核中的R环水平:这些研究结果表明,在小鼠胚胎发育过程中,R环的动态变化很可能是由RNA螺旋酶(尤其是DDX5)与转录过程共同调控的。我们的研究为这些因子参与调节早期胚胎发育过程中的 R 环动态提供了有力证据。