Dynamic Change of R-Loop Implicates in the Regulation of Zygotic Genome Activation in Mouse

Abstract

In mice, zygotic genome activation (ZGA) occurs in two steps: minor ZGA at the one–cell stage and major ZGA at the two–cell stage. Regarding the regulation of gene transcription, minor ZGA is known to have unique features, including a transcriptionally permissive state of chromatin and insufficient splicing processes. The molecular characteristics may originate from extremely open chromatin states in the one–cell stage zygotes, yet the precise underlying mechanism has not been well studied. Recently, the R-loop, a triple–stranded nucleic acid structure of the DNA/RNA hybrid, has been implicated in gene transcription and DNA replication. Therefore, in the present study, we examined the changes in R-loop dynamics during mouse zygotic development, and its roles in zygotic transcription or DNA replication. Our analysis revealed that R-loops persist in the genome of metaphase II oocytes and preimplantation embryos from the zygote to the blastocyst stage. In particular, zygotic R-loop levels dynamically change as development proceeds, showing that R-loop levels decrease as pronucleus maturation occurs. Mechanistically, R-loop dynamics are likely linked to ZGA, as inhibition of either DNA replication or transcription at the time of minor ZGA decreases R-loop levels in the pronuclei of zygotes. However, the induction of DNA damage by treatment with anticancer agents, including cisplatin or doxorubicin, does not elicit genome-wide changes in zygotic R-loop levels. Therefore, our study suggests that R-loop formation is mechanistically associated with the regulation of mouse ZGA, especially minor ZGA, by modulating gene transcription and DNA replication.

Senataxin modulates resistance to cisplatin through an R-loop mediated mechanism in HPV-associated Head and Neck Squamous Cell Carcinoma

“…immunofluorescence, but no significant difference in R-loops levels as measured by S9.6 immunofluorescence following cisplatin treatment for 8 hours 61 . This contrasts with the findings here; however, it is likely that findings differ due to biological differences between mouse embryos and human cancer cells, alongside the different time points and methodology used in this study.…”

“…To the best of our knowledge, there are no papers in the literature that have shown an increase in R-loops following cisplatin treatment in human cells. There is a single study in mice that investigated the role of R-loops during mouse zygote genome activation 61 and identified changes in R-loop dynamics throughout this process, which were linked to transcription 61 . Interestingly, they treated mouse embryos with cisplatin and doxorubicin and noted an increase in gH2AX levels by immunofluorescence, but no significant difference in R-loops levels as measured by S9.6 immunofluorescence following cisplatin treatment for 8 hours 61 .…”

“…There is a single study in mice that investigated the role of R-loops during mouse zygote genome activation 61 and identified changes in R-loop dynamics throughout this process, which were linked to transcription 61 . Interestingly, they treated mouse embryos with cisplatin and doxorubicin and noted an increase in gH2AX levels by immunofluorescence, but no significant difference in R-loops levels as measured by S9.6 immunofluorescence following cisplatin treatment for 8 hours 61 . This contrasts with the findings here; however, it is likely that findings differ due to biological differences between mouse embryos and human cancer cells, alongside the different time points and methodology used in this study.…”

R-loop landscape in mature human sperm: Regulatory and evolutionary implications

“…Maternal and paternal pronuclei showed the highest intensities at pronuclear stage 1. The authors hypothesize that the R-loops detected in the early pronuclear stages may be related to epigenetic mechanisms such as histone modifications and chromatin structure and may contribute to the genomic reprogramming that occurs in separate pronuclei during this early zygotic development, and put forward that the dynamics of the R-loop landscape are likely mechanistically linked to zygotic genome activation (ZGA) ( Lee et al, 2022 ). This leaves open the question of whether R-loops occur in mature sperm cells and thus the search for their possible evolutionary consequences in the context of ‘non-genetic or epigenetic inheritance’ has not yet been addressed.…”