Genome stabilization by RAD51 stimulatory compound-1 enhances efficiency of somatic cell nuclear transfer-mediated reprogramming and full-term development of cloned mouse embryos
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ABSTRACT:
PROVIDER: PRJNA634550 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11832800_1.fastq.gz | Fastqsanger.gz | |||
| SRR11832800_2.fastq.gz | Fastqsanger.gz | |||
| SRR11832801_1.fastq.gz | Fastqsanger.gz | |||
| SRR11832801_2.fastq.gz | Fastqsanger.gz | |||
| SRR11832802_1.fastq.gz | Fastqsanger.gz |
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Project description:Here, we observed that the activity of Rad 51, a key modulator of homologous recombination repair (HRR) is lower in somatic cell nuclear transfer (SCNT)-derived eggs than in conventional IVF, and found a significantly lower level of DNA double-strand breaks (DSBs) in SCNT eggs during reprogramming. To address this difference, supplementation with RS-1, an activator of Rad51, during the activation of SCNT eggs can increase RAD51 expression and DSB foci and thereby overcome the developmental block. This protocol finally increased the efficiency of SCNT reprogramming. Through subsequent single-cell RNA-seq analysis, we observed the reactivation of a large number of genes that were not expressed in SCNT-2-cell embryos by the upregulation of HRR, which may be related to overcoming the developmental block. Additionally, there may be an independent pathway involving histone demethylase that can reduce reprograming resistance regions. This technology can contribute to the production of comparable cell sources for regenerative medicine.
2020-05-23 | GSE151074 | GEO
Project description:ObjectivesThe genetic instability and DNA damage arise during transcription factor-mediated reprogramming of somatic cells, and its efficiency may be reduced due to abnormal chromatin remodelling. The efficiency in somatic cell nuclear transfer (SCNT)-mediated reprogramming is also very low, and it is caused by development arrest of most reconstituted embryos.Materials and methodsWhether the repair of genetic instability or double-strand breaks (DSBs) during SCNT reprogramming may play an important role in embryonic development, we observed and analysed the effect of Rad 51, a key modulator of DNA damage response (DDR) in SCNT-derived embryos.ResultsHere, we observed that the activity of Rad 51 is lower in SCNT eggs than in conventional IVF and found a significantly lower level of DSBs in SCNT embryos during reprogramming. To address this difference, supplementation with RS-1, an activator of Rad51, during the activation of SCNT embryos can increase RAD51 expression and DSB foci and thereby increased the efficiency of SCNT reprogramming. Through subsequent single-cell RNA-seq analysis, we observed the reactivation of a large number of genes that were not expressed in SCNT-2-cell embryos by the upregulation of DDR, which may be related to overcoming the developmental block. Additionally, there may be an independent pathway involving histone demethylase that can reduce reprograming-resistance regions.ConclusionsThis technology can contribute to the production of comparable cell sources for regenerative medicine.
| S-EPMC8249786 | biostudies-literature
Project description:RAD51 is the central protein that catalyzes DNA repair via homologous recombination, a process that ensures genomic stability. RAD51 protein is commonly expressed at high levels in cancer cells relative to their noncancerous precursors. High levels of RAD51 expression can lead to the formation of genotoxic RAD51 protein complexes on undamaged chromatin. We developed a therapeutic approach that exploits this potentially toxic feature of malignancy, using compounds that stimulate the DNA-binding activity of RAD51 to promote cancer cell death. A panel of immortalized cell lines was challenged with the RAD51-stimulatory compound RS-1. Resistance to RS-1 tended to occur in cells with higher levels of RAD54L and RAD54B, which are Swi2/Snf2-related translocases known to dissociate RAD51 filaments from dsDNA. In PC3 prostate cancer cells, RS-1-induced lethality was accompanied by the formation of microscopically visible RAD51 nuclear protein foci occurring in the absence of any DNA-damaging treatment. Treatment with RS-1 promoted significant antitumor responses in a mouse model, providing proof-of-principle for this novel therapeutic strategy.
| S-EPMC4079730 | biostudies-literature
Project description:Long non-coding RNAs (lncRNA) have been shown to play crucial roles in tumorigenesis. Little is known about lncRNA RAD51-AS1 in diseases. Here, we investigated the role of RAD51-AS1 in Epithelial ovarian cancer. Silencing RAD51-AS1 inhibited proliferation through cell cycle arrest and promotion in apoptosis, both in vitro and in vivo. But the mechanism of RAD51-AS1 downstream regulation remains unclear. In order to identify the downstream regulation of RAD51-AS1 in epithelial ovarian cancer, we used antisene oligotides targeting RAD51-AS1 / negative control to transfect Skov3.ip cells. Then, we used microarrays to identified differential expression genes and to look for possible target genes by knockdown RAD51-AS1 expression.
2017-08-17 | GSE89374 | GEO
Project description:RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity.
2017-02-18 | GSE95050 | GEO
Project description:To investigate the role of RAD51 in HCC, we established Huh7 cell lines in which RAD51 has been knocked down(KD) by sgRNA,we performed RNA-seq for RAD51-KD Huh7 cells and parental Huh7 cells(control group,sgcon).
2023-05-17 | GSE216728 | GEO
Project description:Early relapse after platinum chemotherapy in epithelial ovarian cancer (EOC) portends poor survival. A-priori identification of platinum-resistance is therefore crucial to improve on standard first-line carboplatin-paclitaxel treatment. The DNA repair pathway Homologous Recombination (HR) repairs platinum-induced damage, and the HR recombinase RAD51 is overexpressed in cancer. We therefore designed a REMARK-compliant study of pre-treatment RAD51 expression in EOC, using fluorescent quantitative immunohistochemistry (qIHC) to overcome challenges in quantitation of protein-expression in-situ. In a discovery cohort (n=284), RAD51-High tumours had shorter progression-free and overall survival compared to RAD51-Low cases in univariate and multivariate analyses. The association of RAD51 with relapse/survival was validated in a carboplatin-monotherapy SCOTROC4 clinical trial cohort (n=264), and was predominantly noted in HR-proficient cancers (Myriad HRDscore<42). Interestingly, overexpression of RAD51 modified expression of immune-regulatory pathways in-vitro, while high-RAD51 tumours showed exclusion of cytotoxic-T-cells in-situ. Our findings highlight RAD51 expression as a determinant of platinum resistance, and suggest possible roles for therapy to overcome immune exclusion in high-RAD51 EOC. The qIHC approach is generalizable to other proteins with a continuum instead of discrete/bimodal expression.
2021-02-11 | GSE166539 | GEO
Project description:The formation of RAD51/DMC1 filaments on single-stranded (ss)DNAs, which is essential for homology search and strand exchange in DNA double-strand break (DSB) repair and for the protection of stalled DNA replication forks, is tightly regulated in time and space. FIGNL1 AAA+++ ATPase plays positive and negative roles in the RAD51-mediated recombination in human cells. However, the role of FIGNL1 in gametogenesis remains unsolved. Here, we characterized a germ-line-specific conditional knockout (cKO) mouse of FIGNL1. The Fignl1 cKO male mice showed defective chromosome synapsis and impaired meiotic DSB repair with the accumulation of RAD51/DMC1 on chromosomes in mid-meiotic prophase I, supporting a role of FIGNL1 in a post-assembly stage of RAD51/DMC1 filaments. Fignl1 cKO spermatocytes accumulate RAD51 and DMC1 ensembles on chromosomes not only in early meiotic prophase I but also in meiotic S-phase. These RAD51/DMC1 assemblies are independent of meiotic DSB formation. Finally, we showed that purified FIGNL1 dismantles RAD51 filament on double-stranded (ds)DNA as well as ssDNA. These results suggest a critical role of FIGNL1 to limit the uncontrolled assembly of RAD51 and DMC1 on native dsDNAs during the meiotic S-phase and meiotic prophase I.
2023-03-31 | GSE227944 | GEO
Project description:RAD51 protein is an evolutionarily conserved recombinase that plays a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. RAD51 inactivation by small molecules has been proposed as a strategy to impair the BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim to make cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully assayed in vitro by using myr-BRC4, a peptide derived from the fourth BRC repeat of BRCA2, being the strongest reported natural RAD51 binder. The present study applies a method to obtain a proteomic fingerprint for RAD51 inhibition by the myr-BRC4 peptide (designed for a more efficient cell entry) using a mass spectroscopy (MS) proteomic approach. We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteomic hits, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3) and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of RAD51 inhibition, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment.
2023-08-15 | PXD042696 | Pride