利用生物資訊工具探討類第三型 DNA 甲基化酶 DNMT3L 於piRNA 生成過程扮演之角色

Abstract

生殖細胞在基因延續與物種演化中扮演重要的角色。因此，性腺與生殖幹細胞能適當且正確地維持與分化極為重要。跳躍子 (transposon) 能在基因間移動的特性，可能影響生殖幹細胞的基因穩定度而造成缺失。為了調控跳躍子的表現，生殖細胞發展出轉錄與後轉錄機制，分別於不同層面調節跳躍子。然而部分報導指出，兩個機制可能合作並協同調控跳躍子的表現。類第三型 DNA 甲基化酶 (DNMT3L) 是轉錄調控機制中參與 DNA 甲基化的重要蛋白，而研究顯示 DNMT3L 可能會參與後轉錄調控機制 PIWI-interacting RNA pathway，於成熟 piRNA 進入細胞核調控表觀基因體時，協助建立DNA 甲基化以調節跳躍子的表現。近期，我們實驗室的初步研究顯示 DNMT3L 極可能不只於 piRNA pathway 下游間接透過轉錄機制調控跳躍子，還可能參與 piRNA pathway 其他層面。於不同發育階段的小鼠生殖細胞研究結果顯示，剔除 DNMT3L 會造成 8 天大之小鼠睪丸中 piRNA pathway 相關蛋白的 RNA 表現量改變。其中一個相關蛋白 MAEL 於細胞內的表現量及位置，會因生殖細胞發育階段之不同而有不同程度之變異。此外，DNMT3L 的缺失也會影響 piRNA 的族群結構。儘管種種證據指出 DNMT3L 不只在 piRNA pathway 的下游作用，目前仍不清楚 DNMT3L 是如何對 piRNA pathway 造成影響。本研究利用生物資訊工具進行正常小鼠及 Dnmt3l 基因剔除小鼠 piRNA 序列分析，探討在 0 天小鼠生殖細胞與 8 天小鼠精原母細胞 (spermatogonial stem cell THY1+ enriched population) 中 DNMT3L 可能參與 piRNA pathway 的層面為何。結果顯示，microRNA 及 piRNA 的相對比例並沒有因為 DNMT3L 剔除出現明顯變化。然而，在 DNMT3L 缺失的 8 天小鼠精原母細胞中，piRNA 生成的位置與組成有極大的變化，且位於基因上的位置相較於野生型明顯不同。進一步研究 piRNA前驅物 (precursor) 發現，DNMT3L 缺失基因型生成的 piRNA 前驅物有極高比例帶有 ZBTB12、STAT3 或 FOXP1 蛋白的結合位。本研究顯示 DNMT3L 除了 piRNA pathway下游外，還會影響 piRNA 前驅物的生成位置並導致 piRNA 組成改變。然而 DNMT3L 是否會參與更多層面並影響 piRNA pathway 仍須更深入的研究。

It is crucial to preserve appropriate germline development, differentiation and maintenance, as germ cells are responsible for genetic inheritance, reproduction and evolution. Transposons may cause genome instability and lead to germ cell defects and infertility due to their dysregulation and derepression. To combat with transposons, transcriptional as well as post-transcriptional regulations have been developed in germline, and these two pathways have been indicated to cooperate. In mice, DNA methyltransferase 3-like protein (DNMT3L), an important factor participating in de novo DNA methylation for transcriptional regulation. It is also suggested that DNMT3L may act downstream of PIWI-interacting RNA (piRNA) pathway, which is mainly responsible for post-transcriptional regulation. Mature piRNA-PIWI complex may also guide sequence specific histone modification and subsequent de novo DNA methylation potentially facilitated partly by DNMT3L. However, according to our following preliminary data, DNMT3L seems to act not only downstream but also participate in other level of piRNA pathway. As the result of studying mice spermatogonial stem cells (SSCs) from different developmental stage, in 8 dpp Dnmt3l-deficiency samples, RNA expression of several piRNA pathway components are down regulated; MAEL, one of the piRNA pathway components, is mislocalized in E18.5 dpc germ cells from Dnmt3l KO embryos, and its expression level varies in the mutant spertmatogonia cells of different developmental stages compared to those from wild type littermates; besides, Dnmt3l-deficiency results in distinct piRNA composition. Though evidence from different developmental stages above suggests the noncanonical role of DNMT3L, it is still unclear how DNMT3L participates in and makes influence on piRNA pathway. In this study, we apply bioinformatics tools to analyze small RNA sequencing datasets to investigate the potential role of DNMT3L in 0 dpp germ cells and 8 dpp spermatogonial stem cell (SSC) enriched THY1+ population. Results indicated that relative proportion of microRNA and piRNA were similar between WT and Dnmt3l KO mice. However, piRNA origin and composition significantly differed in 8 dpp Dnmt3l-deficiency samples, and genomic location of predicted precursors remarkably shifted as well. Further analysis revealed that the binding sites of ZBTB12, STAT3 and FOXP1 highly enriched among predicted precursors with high piRNA production in 8 dpp Dnmt3l KO samples. This report suggests that DNMT3L should act not only downstream of piRNA pathway, especially affecting piRNA precursor production and thus resulting in distinct piRNA composition. However, whether DNMT3L participates in other levels of piRNA pathway needs more investigation.