The E2 SUMO-conjugating enzyme UBE2I is essential for chromatin dynamics and transcriptional silencing in mouse oocytes
Ontology highlight
ABSTRACT: In mammals, meiotically competent oocytes develop cyclically during ovarian folliculogenesis. During their development, prophase I arrested oocytes are highly transcriptionally active in preparation for the resumption of meiosis and early stages of embryogenesis prior to the maternal to zygotic transition. Defective oocyte development during folliculogenesis leads to meiotic defects, aneuploidy, follicular atresia, or non-viable embryos. SUMOylation, a dynamic post-translational protein modification, is essential for oocyte development during folliculogenesis and to regulate meiotic maturation in mice. We generated a novel oocyte-specific knockout of Ube2i, the sole SUMO E2 ligase, to test its role growing ovarian follicles using Zp3-cre. Ube2i Zp3-cre+ female mice are sterile with oocytes with defects in meiotic progression but not meiotic resumption. Importantly, fully grown oocytes do not silence transcription and prematurely activate the zygotic transcriptional program. This work uncovers unknown functions of UBE2i as a key orchestrator of chromatin and transcriptional regulation in oocytes.
Project description:Persistence of unrepaired DNA damage in oocytes is detrimental and may cause genetic aberrations, miscarriage, and infertility. RPA, an ssDNA-binding complex, is essential for various DNA-related processes. Here we report that RPA plays a novel role in DNA damage repair during postnatal oocyte development after meiotic recombination. To investigate the role of RPA during oogenesis, we inactivated RPA1 (replication protein A1), the largest subunit of the heterotrimeric RPA complex, specifically in oocytes using two germline-specific Cre drivers (Ddx4-Cre and Zp3-Cre). We find that depletion of RPA1 leads to the disassembly of the RPA complex, as evidenced by the absence of RPA2 and RPA3 in RPA1-deficient oocytes. Strikingly, severe DNA damage occurs in RPA1-deficient germinal vesicle (GV)-stage oocytes. Loss of RPA in oocytes triggered the canonical DNA damage response mechanisms and pathways, such as activation of ATM, ATR, DNA-PK, and p53. In addition, the RPA deficiency causes chromosome misalignment at metaphase I and metaphase II stages of oocytes, which is consistent with altered transcript levels of genes involved in cytoskeleton organization in RPA1-deficient oocytes. Absence of the RPA complex in oocytes severely impairs folliculogenesis and leads to a significant reduction in oocyte number and female infertility. Our results demonstrate that RPA plays an unexpected role in DNA damage repair during mammalian folliculogenesis.
Project description:Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization, and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 down-regulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a novel meiotic and embryonic competence factor in meiosis and mitosis, safeguarding genome integrity at the onset of life. We performed expression profiling on pools of 16 denuded GV-oocytes isolated per mouse. We used oocytes from 4 Setdb1 f/+; Zp3-cre mice and 2 Setdb1 f/- mice as controls and oocytes from 4 Setdb1 f/-; Zp3-cre mice as mutant.
Project description:Oocytes are indispensable for mammalian life. Thus, it is important to understand how mature oocytes are generated. As a critical stage of oocytes development, meiosis has been extensively studied, yet how chromatin remodeling contributes to this process is largely unknown. Here, we demonstrate that the ATP-dependent chromatin remodeling factor Snf2h (also known as Smarca5) plays a critical role in regulating meiotic cell cycle progression. Females with oocyte-specific depletion of Snf2h are infertile and oocytes lacking Snf2h fail to undergo meiotic resumption. Mechanistically, depletion of Snf2h results in dysregulation of meiosis-related genes, which causes failure of maturation-promoting factor (MPF) activation. ATAC-seq analysis in oocytes revealed that Snf2h regulates transcription of a key meiotic gene, Prkar2b, by increasing its promoter chromatin accessibility. Our studies thus not only demonstrate the importance of Snf2h in oocyte meiotic resumption, but also reveal the mechanism underlying how a chromatin remodeling factor can regulate oocyte meiosis.
Project description:Maternal-to-zygotic transition (MZT) is the first and key step in the control of animal development and is intimately related to changes of chromatin structure and histone modifications. Mono-ubiquitination of histone H2A at lysine-119 (H2AK119ub1), an important epigenetic modification in regulating chromatin configuration and function, is primarily catalyzed by polycomb repressive complex 1 (PRC1) and contributes to resistance to transcriptional reprogramming in mouse embryos. In this study, the genome-wide dynamic distribution of H2AK119ub1 during MZT in mice was investigated by chromosome immunoprecipitation and sequencing. The results indicated that H2AK119ub1 accumulated in fully grown oocytes, enriched at the transcriptional start sequences (TSSs) of maternal genes, but was promptly removed after meiotic resumption, particularly at the TSSs of early zygotic genes, by a previously unidentified mechanism. Genetic evidences indicated that ubiquitin-specific peptidase 16 (USP16) is the major deubiquitinase of H2AK119ub1 in mouse oocytes. Conditional knockout of Usp16 in oocytes did not impair their survival, growth, andor meiotic maturation. However, oocytes lacking USP16 were unable to undergo zygotic genome activation or gain developmental competence after fertilization, due to high levels of H2AK119ub1 deposition on the zygotic genomes. Taken together, this study indicates that H2AK119ub1 is erased during oocyte maturation by an USP16-dependent mechanism, which ensures zygotic genome reprogramming and transcriptional activation of essential early zygotic genes.
Project description:The goal of this study is to identify the differentially expressed genes in Gdf9-Cre and Zp3-Cre mediated Mtor oocyte-specific knockout (CKO) GV-stage fully-grown oocytes (FGO) by comparing their transcriptomes with that of the wild-type (WT) via RNA-Seq Analysis.
Project description:The role of G-quadruplex (G4) structures and their effects on meiosis resumption and early embryo development remain unclear. We discovered that the G4 helicase DHX36 is essential for oocyte growth and the maternal-to-zygotic transition (MZT). Conditional knockout of DHX36 resulted in DNA G4 accumulation in mouse oocytes, reducing chromatin accessibility, and inhibiting RNA transcription, ultimately disrupting transcriptome homeostasis during oocyte growth and MZT.
Project description:Zar1 was the first mammalian maternal-effect gene to be identified. Embryos derived from Zar1-null female mice are blocked before zygotic genome activation; however, the underlying mechanism remains unclear. By knocking out Zar1 and its homolog Zar2 in mice, we revealed a novel function of these genes in oocyte meiotic maturation. Zar1/2-deleted oocytes displayed delayed meiotic resumption and polar body-1 emission and a higher incidence of abnormal meiotic spindle formation and chromosome aneuploidy. The grown oocytes of Zar1/2-null mice contained fewer total mRNAs and displayed a reduced level of protein synthesis. Key maturation-associated changes failed to occur in the Zar1/2-null oocytes, including the translational activation of maternal mRNAs encoding the cell cycle proteins cyclin B1 and WEE2, as well as maternal-to-zygotic transition (MZT) licensing factor BTG4. Consequently, maternal mRNA decay was impaired and MZT was abolished. ZAR1/2 bound mRNAs to regulate the translational activity of their 3ʹ-UTRs and interacted with other oocyte proteins, including mRNA-stabilizing protein MSY2 and cytoplasmic lattice components. These results countered the traditional view that ZAR1 only functions after fertilization and highlight a previously unrecognized role of ZAR1/2 in regulating the maternal transcriptome and translational activation in maturing oocytes.
Project description:In fully grown oocytes, the genome is considered to be globally transcriptionally silenced. However, this conclusion is primarily derived from the results obtained through immunofluorescence staining or inferred from the highly condensed state of chromosomes, lacking more direct evidence. Here, by using a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, we investigated the landscape of single-strand DNA (ssDNA) throughout the genome and provided a readout of the activity and dynamics of transcription during oocyte meiotic maturation. In non-surrounded nucleolus (NSN) oocytes, we observed a robust KAS-seq signal, indicating the high transcriptional activity. In surrounded nucleolus (SN) oocytes, the presence of ssDNA still persists although the KAS-seq signal was relatively weak, suggesting the presence of transcription. Accompanying with the meiotic resumption, the transcriptional activity gradually decreased, and global repression was detected in matured oocytes. Moreover, we preformed the integrative genomics analysis to dissect the transcriptional dynamics during mouse oocyte maturation. In sum, the present study delineates the detailed transcriptional activity during mammalian oocyte maturation.
Project description:The mechanisms of oocyte meiotic defects and low competence during ovarian aging remains elusive for decades. Using Hi-C (genome-wide chromatin conformation capture) and Smart RNA-seq of oocytes from 6- weeks or 10- months aged ovaries, the abnormal loose chromatin structures and disturbing expression of meiosis associated genes at metaphase I phase were disclosed. Furthermore, the transcriptomic landscape of granulosa cells (GCs) surrounding oocytes from young and aged ovaries reveled that oocyte meiotic maturation was accompanied with a robust increased expression of genes involved with the mevalonate (MVA) pathway in GCs from young ovaries but these genes expression was not upregulated to counterpart level in GCs from aged ovaries.The inhibitor of MVA pathway of GCs, Statins significantly decreased polar body extrusion rate and increased the rate of irregularly assembled spindles and misaligned chromosomes remarkably in oocytes of culumus-oocyte complex (COCs) from young ovaries . Correspondingly, the activitor of MVA pathway of GCs, Geranylgeraniol ameliorated ovarian reserve and reduced meiotic defects in oocytes of COCs from aged ovaries. Mechanistically, MVA pathway activation in GCs culminated oocyte meiotic maturation by upregulating EGF signaling via LH receptor on GCs surrounding oocytes. Together, MVA pathway is a promising therapeutic target for prompting quality of oocytes from aged ovaries.