| Spermatogenesis is a complex process, including three important stages: spermatogonia mitosis, meiosis and sperm sperm cell deformability under the event of nuclear condensation, mitochondrial rearrangement, replacement of protein and transition protamine to histones, acrosome formation and sperm maturation. In high-throughput gene expression data in mice and rats showed that a lot of spermatogenesis testis-specific gene express in participation. It is estimated that about 4% of the specific gene expression occurs in all stages of spermatogenesis in the mouse genome.RNF138, namely RING (Really Interesting New Gene) Finger protein 138, located in the 18q12.1. Our previous work found that RNF138 had E3 ubiquitin ligase activity of the protein. Ubiquitination is an important way of post-translational modification of proteins. This reaction is processed by ubiquitin activating enzyme including El, ubiquitin conjugating enzyme E2 and E3 ubiquitin ligases, including a series of enzymatic reactions collaborative. Numerous studies confirmed that ubiquitylation involved in a series of cellular processes including protein degradation, activation of protein kinase, transport vesicles, DNA damage repair and chromatin remodeling. Some mammalian testis-specific of the E3 ubiquitin ligase perform a variety of different biological functions in different stages of spermatogenesis. Some E3 ubiquitin ligase complex play an important role in regulating early event of meiosis, DNA double strand breaks (DSB) and histone modifications.We examined testicular tissue of mice of different ages RNF138 protein expression levels showed that the expression of RNF138 increases with testicular developmental stage during the first wave of the seminiferous wave (5 weeks). Immunohistochemistry and immunofluorescence showed that RNF138 have different profile of expression in spermatogenic cells other than spermatogonial cells, especially in primary spermatocytes which is with highest expression. At this stage, primary spermatocytes began to enter the first division. Taken together, these results prompted RNF138 may be involved in early development of spermatogenic cells.In order to study the function of RNF138 in male reproduction, we used the Cre-loxP or Flp-FRT homologous recombination system constructed Vasa-Cre conditional knockout mouse models. Compared with control mice, testis weight and sperm count in various developmental stages reduced significantly. Based on the morphological evidence, the development of early stage of spermatogenesis is arrest in RNF138 gene knockout mice. Meanwhile, some early part of spermatogenic cell loss significantly, especially the spermatocytes TUNEL showed that apoptosis of spermatocytes increased.Transcriptome sequencing is widely used in studies of functional genomics. A large number of genetic data enriched, these data sources can help to carry out the biological mechanisms. To ravel the biological mechanisms, we performed transcriptome sequencing of testis of RNF138 conditional knockout mice. By analysis with control sample, a total of 152 differentially expressed genes were found, including 73 upregulated genes and 79 down-regulated genes. According to the analysis of differential gene expression RNAseq, COG display function classification involved in DNA replication, recombination and repair with the highest frequency, these results suggest RNF138 in response to DNA damage repair processes involved in important functions.Next, we identified Ring domain E3 ubiquitin ligases RNF138 as a DNA damage response protein that could be recruited to DNA damage site quickly through its Zinc finger domains. We found that RNF138 could be phosphorylated by ATM at Ser124. This phosphorylation was dispensable for recruitment to the DNA damage site. Our findings also indicated that depletion of RNF138 dramatically affected RAD51 assembly at DSB sites following irradiation. Hence, RNF138 might be involved in regulating homologous recombination repair pathway. To unravel the mechanism, we utilized modified tandem affinity purification technology to find that RAD51D directly interacted with RNF138 which was recruited to DNA lesion site. Depletion of RNF138 resulted in delayed and unstable recruitment of RAD51D to DNA damage site. RNF138-deficient cells were hypersensitive to DNA damage insults, such as IR and MMS. Furthermore, a comet assay confirmed that RNF138 directly participated in DNA damage repair.In addition, CCCTC-binding factor (CTCF) is a 11 zinc finger protein plays an essential role in regulating the structure of chromatin by binding DNA strands for defining the boundary between active and heterochromatic DNA. However, the role of CTCF in DNA damage response remains elusive. Here, we show that CTCF is quickly recruited to the sites of DNA damage site. The fast recruitment is mediated by the zinc finger domain and poly (ADP-ribose) (PAR). Further analyses show that only three zinc finger motifs are essential for PAR recognition. And lacking these three zinc finger motifs does not affect the DNA-binding. Moreover, CTCF-deficient cells are hypersensitive to genotoxic stress such as ionizing radiation (IR). Interestingly, the size control of IR-induced DNA damage foci is impaired in CTCF-deficient cells. Taken together, these results suggest that CTCF regulates the boundary of DNA damage repair region. |
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