- 學位論文
雄鼠減數分裂生殖細胞表現之 新型類3號DNA甲基酶異構體研究
Novel DNA Methyltransferase 3-Like Isoforms in Meiotic Male Germ Cells: Beyond Epigenomic Modulation
摘要
類3號DNA甲基化轉移酶（DNA methyltransferase 3-like, DNMT3L）為表觀遺傳調節分子，協助胚胎發育中新生DNA甲基化之建立。在DNMT3L 缺失的小鼠中，轉座子表現失調並且導致生精作用中斷，造成雄性不育。本研究室發現新穎的DNMT3L蛋白異構物，由於此異構物之RNA最初於成年小鼠睪丸被證實，同樣地此蛋白異構物大量被表現於成年小鼠睪丸中，故命名為DNMT3L_AT (adult testis)。DNMT3L_AT 主要座落在精母細胞 (spermatocytes) 與精細胞 (spermatids) 之細胞質中，不同於典型DNMT3L_S (stem cell) 多存在於前精原細胞 (prospermatogonia) 與精原細胞 (spermatogonia) 之細胞核當中。鑑於其胞內位置與表現時期的差異，我們推論此DNMT3L_AT具有不同於典型構型DNMT3L_S的生物功能。本研究試圖利用in vivo以及in vitro 的策略來驗證此蛋白並且探索其交互作用因子以及潛在功能探討。研究結果顯示在5%-40%蔗糖密度梯度離心分離中，發現DNMT3L_AT 座落於germ granule的成員如VASA 與PIWI 聚集的分層當中。免疫染色也得到DNMT3L_AT與在intermitochondrial cement (IMC) 中表現的 MILI以及chromatoid body (CB)之標記 VASA具有高度訊號重疊，而與MIWI的交互作用經由免疫沈澱實驗佐證。因此我推測其參與germ granule的功能如調節piRNA生成與作用、信使RNA儲存以及其他後轉錄修飾功能。
並列摘要
DNA methyltransferase 3-like (DNMT3L) is an important epigenetic modulator which facilitates de novo DNA methylation by cooperating with other DNA methyltransferases (DNMTs). In Dnmt3l knockout mice, males are infertile because of the spermatogenic arrest in early meiosis and derepression of transposons elements (TEs). Strikingly, our lab identifies a novel protein isoform of DNMT3L, DNMT3L adult testis form (DNMT3L_AT), in mouse testes. It is a much smaller protein compared with canonical DNMT3L stem cell form (DNMT3L_S). I confirmed the transcriptional variants and protein expression in postnatal testes of different developmental stages. Spermatocytes and spermatids are the major developmental stages with high DNMT3L_AT expression level. Besides, the subcellular pattern of DNMT3L_AT displays granule-like structures in the cytoplasm, which is distinct from DNMT3L_S that mainly resides in the nucleus. Based on the characteristic protein size, subcellular localization, and the expression period, we deduced that DNMT3L_AT has functions beyond epigenomic modulation during spermatogenesis. I analyzed male germ cell lysates separated in the 5%-40% sucrose density gradient. DNMT3L_AT coexists in the fractions enriched with germ granule’s proteins. Co-immunoprecipitation assay shows potential interaction of DNMT3L_AT with MIWI, the only PIWI protein expressed at later spermatogenesis. In spermatocytes and spermatids, there are substantial overlaps between the immunofluorescent signal of DNMT3L_AT with that of MILI or VASA, marking germ granules, i.e., intermitochondrial cement (IMC) and chromatoid body (CB). IMC or CB has been suggested as the subcellular membrane-less compartment accommodating mRNA regulation and small RNA mediated processes. With potential interaction between DNMT3L_AT and germ granules, we propose that DNMT3L_AT may play roles in post-transcriptional gene regulation for later male germ cell development.
參考文獻
延伸閱讀
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