Study of dynamics of histone H3 variants and H3 proteolysis during mouse spermatogenesis

Spermatogenesis is a complex differentiation process in which male gametes, known as spermatozoa, are produced from spermatogonial stem cells in the seminiferous tubules of the testis. The spermatogenesis process is typically divided into three phases: a mitotic phase, a meiotic phase and post-meiotic spermiogenesis. During mammalian spermiogenesis, haploid round spermatids undergo remarkable morphological changes and an extensive reorganization of chromatin to differentiate into mature spermatozoa. As part of the chromatin reorganization, most histones in round spermatids are replaced by transition proteins and subsequently by protamines. This histone-to-protamine exchange is required for efficient compaction of paternal genome into the sperm head and implicated in male fertility. Nonetheless, previous studies found that 1-10 % histones are still retained at specific loci, particularly at unmethylated CpG-rich promoters, in mouse and human sperm. How spermatid chromatin is reorganized genome-wide during spermiogenesis while some loci are exempted from histone eviction is still elusive. Our previous study has shown that the residual nucleosomes in mouse sperm largely contain the histone H3 variant, H3.3. The study also revealed differential histone turnover of canonical and variant H3 in round spermatids, which may underlie the final histone composition in mature sperm. In order to determine the dynamics of H3 variants during mouse spermatogenesis, I analyze protein expression of canonical and variant H3 proteins at different stages of male germ cells by triton-acetic acid-urea gel-Western blotting. Surprisingly, I find that mouse testis-specific H3 variant (H3t), not canonical H3, is the most abundant H3 protein from spermatogonia to spermatids and that most canonical H3 is replaced by H3.3 during meiosis. I further observe that a relatively large portion of H3t is removed from chromatin during the process of histone-to-protamine exchange compared with H3.3, which is consistent with that H3.3 is the predominant H3 in residual sperm nucleosomes. Taken together, the first part of my thesis reveals important findings on chromatin composition and dynamics of histone H3 variants during mouse spermatogenesis. In the second part of my thesis, I describe the discovery that histone H3 is cleaved at its N-terminal tail by a serine protease activity in nuclei of the late-stage mouse spermatids. Arginine 26 and lysine 27 on H3 are important to the H3 protease activity. This proteolytic cleavage of H3 tail may result in nucleosome destabilization and then contribute to nucleosome eviction during spermiogenesis. Interestingly, I find that the acetylation on H3 can prevent H3 from proteolytic cleavage in vitro and that the genome-wide distribution of H3 lysine 27 acetylation (H3K27Ac) is positively correlated to the occupancy of nucleosomes containing transcriptionally active mark in sperm, suggesting that the inhibition of H3 cleavage by acetylated lysine 27 in late-stage spermatids may lead to the nucleosome retention at specific loci in mature sperm. Overall, these findings provide novel insights into the mechanism of nucleosome eviction and retention during spermiogenesis through the regulation of H3 proteolytic cleavage

Retroperitoneal Metastatic Adenocarcinoma Complicated with Necrotizing Fasciitis of the Thigh in a Patient with Advanced Rectal Colon Cancer

Background: Necrotizing fasciitis of the thigh due to colon cancer has not been previously reported, especially during radiotherapy. Case Presentation: A 73-year-old woman admitted to our hospital was diagnosed with sigmoid colon cancer that had spread to the left psoas muscle; radiotherapy was performed. Three months after the initiation of radiotherapy, the patient developed gait disturbance, poor appetite and high fever and was therefore admitted to the emergency department of our hospital. Blood examination revealed generalized inflammation with a high white blood cell count and C-reactive protein level. Computed tomography of the abdomen revealed fluid and gas tracking from the retroperitoneum into the intramuscular plane of the grossly enlarged right thigh. Consequently, emergent debridement was not performed and conservative therapy was done. The patient died. Conclusion: Necrotizing fasciitis of the thigh due to the spread of rectal colon cancer is unusual, but this fatal complication should be considered during radiotherapy in patients with unresectable colorectal cancer

GCM1 Regulation of the Expression of Syncytin 2 and Its Cognate Receptor MFSD2A in Human Placenta

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Epigenetic Regulation of the Placental Fusogenic Protein, Syncytin2

Syncytin 2屬於人類內生性反轉錄病毒（human endogenous retrovirus）HERV-FRD之外鞘蛋白，其專一表現在胎盤滋養層細胞中，已知syncytin 2會促進細胞滋養層細胞進行細胞融合，而形成融合滋養層，故syncytin 2在胎盤發育上扮演重要的角色。而過去研究指出，人類胚胎發育過程中，胎盤組織裡的DNA甲基化程度明顯較胚胎本體組織來的低，且最近有報導發現，syncytin家族另一個成員syncytin 1，在胎盤細胞中，syncytin 1之5’-long terminal repeat（5’-LTR）啟動子上DNA甲基化程度非常低，使得syncytin 1能表現於胎盤細胞中，因此，本篇論文想探討是否細胞能透過改變5’LTR啟動子上DNA甲基化與組蛋白修飾等後基因修飾，而達到syncytin 2表現。實驗結果顯示，胎盤細胞或組織中的syncytin 2 5’LTR上甲基化程度顯著比非胎盤細胞來的低，且5’LTR上的組蛋白修飾皆為活化態的修飾，接著再利用報導基因方式發現syncytin 2 5’LTR若甲激化會影響到其轉錄活性，但有趣的是，含DNA甲基化syncytin 2 5’LTR的報導基因送胎盤細胞中，仍具有一定的轉錄活性，這表示胎盤細胞中具有特殊分子機制可對syncytin 2 5’LTR做去甲基化的動作；接著，發現胎盤組織特定轉錄因子GCMa可調控syncytin 2表現，且在非胎盤細胞MCF-7中表現GCMa後，會促進syncytin 2 5’LTR的DNA去甲基化，故GCMa轉錄因子除了調控基因表現外，同時在syncytin 2 5''LTR啟動子的去甲基化扮演重要角色；最後發現GCMa會與thymine DNA glycosylase（TDG）之間有結合能力，而TDG過去被指出可進行DNA去甲基化的酵素，因此GCMa可能透過吸引TDG至syncytin 2 5’LTR來進行DNA去甲基化的動作。綜合以上實驗結果，證實在胎盤細胞及非胎盤細胞中，syncytin 2 5’LTR上的後基因修飾有所不同，且胎盤特殊轉錄因子GCMa可促進syncytin 2 5’LTR DNA去甲基化以及啟動syncytin 2 mRNA表現。Syncytin 2, an envelope glycoprotein encoded by the human endogenous retrovirus FRD (HERV-FRD), is specifically expressed in placental trophoblasts. It has been reported that syncytin 2 is a fusogenic protein. Therefore, syncytin 2 may play an important role in placental development by regulation of trophoblastic fusion. Previous studies have shown that the placenta exhibits lower overall DNA methylation levels than the embryo. A recent study has shown that the 5’-long terminal repeat (5’LTR) promoter of syncytin 1, which encodes the envelope protein of HERV-W, is hypomehtylated and controls syncytin 1 expression in placenta. n this study, we further investigated whether DNA methylation and histone modification control syncytin 2 expression in placenta. We demonstrated that the 5’LTR of syncytin 2 is hypomethylated and harbors active histone modification in placental cells. In luciferase reporter assay, in vitro DNA methylation inhibited the promoter activity of syncytin 2 5’LTR. Interestingly, the promoter activity of in vitro methylated syncytin 2 5’LTR could be restored in placental cells. To study the mechanism that counteracts the suppressive effect of DNA methylation on syncytin 2 5’LTR promoter in placenta, we demonstrated that placental transcription factor, GCMa, not only regulates syncytin 2 expression but also promotes DNA demethylation on syncytin 2 5’LTR. We further demonstrated that GCMa associates with TDG, an enzyme that involves in DNA demethylation process. This implies that GCMa may recruit TDG to demethylate syncytin 2 5’LTR. Overall, our results reveal an epigenetic regulation of syncytin 2 gene expression and that GCMa is a key factor for DNA demethylation of syncytin 2 promoter and transcription activation of syncytin 2.目錄………………………………………………………………….…………………I文摘要…………………………………………………….………………..……...III文摘要…………………………………………………….……………………….IV一章 緒論. 胎盤.1 人類胎盤發育…………………………………………………………….1.2 胎盤功能………………………………………………………………….3. 後基因調控.1 簡介……………………………………………………………………….4.2去氧核糖酸甲基化………………………………………………….…….4.3 DNA methyltransferases………………………………………………..5.4 去氧核糖酸去甲基化……………………………………………………7.5 組蛋白修飾………………………………………………………………8. 人類內生性反轉錄病毒.1 簡介……………………………………………………………………….9.2 syncytin…………………………………………………………………10. gcm轉錄因子………………………………………………………………...12. 研究動機……………………………………………………………………14二章 材料與方法…………………………………………………………………15三章 結果. syncytin 2 5’LTR啟動子之DNA甲基化狀態...............................................25. syncytin 2 5’LTR啟動子上組蛋白修飾.........................................................26. 核酸甲基化對syncytin 2 5’LTR轉錄活性之影響………………………….26. GCMa調控syncytin2表現並促進syncytin 2 5’LTR區域的去甲基化…..28. GCMa在細胞中會與TDG作結合…………………………………………29四章 討論…………………………………………………………………………31五章 圖表…………………………………………………………………………35六章 參考文獻……………………………………………………………………4

Generation of endogenously tagged E-cadherin cells using gene editing via non-homologous end joining

Summary: We provide a protocol using non-homologous end joining to integrate an oligonucleotide sequence of a fluorescence protein at the CDH1 locus encoding for the epithelial glycoprotein E-cadherin. We describe steps for implementing the CRISPR-Cas9-mediated knock-in procedure by transfecting a cancer cell line with a pool of plasmids. The EGFP-tagged cells are traced by fluorescence-activated cell sorting and validated on DNA and protein levels. The protocol is flexible and can be applied in principle to any protein expressed in a cell line.For complete details on the use and execution of this protocol, please refer to Cumin et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics