RESUMEN
Objective: DOT1L is the only known histone H3K79 methyltransferase essential for the development of the embryonic cardiovascular system, including the heart, blood vessels, and lymphatic vessels, through transcriptional regulation. Our previous study demonstrated that Dot1l deletion results in aberrant lymphatic development and function. However, its precise function in the postnatal cardiovascular system remains unknown. Methods: Using conditional and inducible Dot1l knockout (KO) mice, along with a reporter strain carrying the Geo gene at the Dot1l locus, DOT1L expression and its function in the vascular system during postnatal life were investigated. To assess vessel morphology and vascular permeability, we administered Latex or Evans blue dye to KO mice. In addition, in vitro tube formation and cell migration assays were performed using DOT1L-depleted human umbilical vein endothelial cells (HUVECs). Changes in the expression of vascular genes in HUVECs were measured by quantitative polymerase chain reaction. Results: Our findings demonstrate that conditional Dot1l knockout in the Tg (Tie2-cre) strain results in abnormal blood vessel formation and lymphatic anomalies in the intestine. In a mouse model of Rosa26-creER-mediated inducible Dot1l knockout, we observed vascular phenotypes, including increased vascular permeability and brain hemorrhage, when DOT1L was deleted in adulthood. Additionally, DOT1L depletion in cultured HUVECs led to impaired cell migration and tube formation, likely due to altered gene transcription. These findings highlight the essential role of DOT1L in maintaining vascular integrity and function during embryonic development and postnatal life. Conclusion: Our study revealed that DOT1L is required for the maintenance of adult vascular function through the regulation of gene expression.
RESUMEN
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.
RESUMEN
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.
