類3號DNA甲基化酶對於骨髓間葉幹細胞之長期影響

Abstract

類3號DNA甲基化酶（DNA methyltransferase 3-lke, DNMT3L）為主要表現於胚胎幹細胞和生殖細胞的表觀遺傳因子，其雖不具酵素功能，但可透過與DNA甲基轉移酶3A和3B作用以催化DNA甲基化之建立。先前實驗室觀察到一歲以上缺少Dnmt3l的小鼠和野生型相比，體型明顯瘦小，且擁有較少量的白色脂肪組織，以及高於3的骨骼結構模型指數（Structure model index, SMI）; 此指數數值是在較年長者中可觀察到的趨勢，並且暗示缺少Dnmt3l的小鼠可能有提早衰老現象。除此之外，我們也證實可分化成脂肪以及硬骨的前驅細胞-間葉幹細胞（Mesenchymal stem/stromal cells, MSCs），從缺乏Dnmt3l的小鼠骨髓中分離進行體外培養後，成骨能力明顯下降。 雖然使用通用的蛋白質表現量偵測技術，例如西方墨點法，DNMT3L在一般體細胞譜系中檢測不到，包括間葉幹細胞也未偵測到DNMT3L的表現。然而本實驗室過去針對完全沒有DNMT3L表現之小鼠胚胎纖維母細胞的研究顯示，相較於野生型同胎胚胎取得之纖維母細胞，來自Dnmt3l缺失小鼠胚胎的胚胎纖維母細胞，體外培養後觀察到提早衰老和相對鬆散的染色質結構，說明在胚胎幹細胞（Embryonic stem cells, ESCs）及某些前驅細胞中短暫表達的DNMT3L，不只影響胚胎幹細胞當下的表觀遺傳標記與基因表現，經由部分表觀基因體標記之保留性，於幹細胞分化後，DNMT3L不再表現時，仍得以影響體細胞的染色質結構以及細胞功能。因此，本論文著重於探討在胚胎幹細胞時DNMT3L存在與否所影響的表觀基因體標示，其效應是否會延續至間葉幹細胞，而對間葉幹細胞及其衍生細胞系的基因表達造成影響，使得從缺乏Dnmt3l的小鼠骨髓中分離之間葉幹細胞，分化成骨骼系細胞的潛能下降。 於本論文中，我們首先利用細胞表面抗原表現概觀從野生型以及缺乏Dnmt3l小鼠所分離的間葉幹細胞細胞族群組成相似，另外，除了間葉幹細胞硬骨分化能力受到基因缺乏Dnmt3l影響外，根據細胞在低密度環境下形成群落的效率測試中發現，來自缺乏Dnmt3l的間葉幹細胞形成聚落的效率較低。然而細胞生長速率在較前期（P4）的細胞代數中並無明顯差異，但過去實驗室結果顯示從骨骺分離的間葉幹細胞族群中，缺乏Dnmt3l小鼠的間葉幹細胞在相對後期的細胞代數（P6）有較低的生長速率。綜合自我更新以及生長速率測試結果暗示缺乏Dnmt3l可能造成小鼠的間葉幹細胞提早衰老，造成幹細胞耗竭。 另外，我們藉由RNA定序結果中剖析來自野生型和缺乏Dnmt3l小鼠的間葉幹細胞在誘導硬骨分化前以及分化第3天的基因表達。結果顯示在硬骨分化前，野生型和缺乏Dnmt3l小鼠來源的間葉幹細胞之間差異表達基因（Differentially expressed genes, DEGs）與骨頭型態的相關途徑相關 ; 而誘導硬骨分化三天的差異表達基因則顯示和微管（microtubule）的動態活性有關。這些基因表現分析暗示缺乏Dnmt3l小鼠來源的間葉幹細胞不正常的基因表達可能是失去幹細胞特性和硬骨分化能力有關。 接著我們試圖了解DNMT3L在胚胎幹細胞時期大量表現對於表觀基因組和轉錄組的影響，且探究此影響是否持續至間葉幹細胞並和維持幹細胞特性以及硬骨分化潛能有關。我們下載並重新分析三個已發表的資料集，包含DNMT3L直接結合位點（使用 ChIP-seq）、Dnmt3l knockdown的胚胎幹細胞以及控制組的基因表達 (Microarray) 和DNA甲基化程度 (MeDIP-seq)。並將上述的資料集個別和野生型以及缺失Dnmt3l間葉幹細胞之間，於硬骨分化前、以及誘導硬骨分化第三天，差異表現的基因進行聯集，以找出DNMT3L於胚胎幹細胞之存在與否所造成的基因表現以及DNA甲基化程度不同，和兩種基因型之間，間葉幹細胞的轉錄體組成有無直接的關聯性。我們發現在Dnmt3l knockdown的胚胎幹細胞中，總體啟動子和基因本體上的甲基化水平較高。此外，我們發現於兩種基因型間葉幹細胞中，差異表現之基因，有較高比例亦屬於胚胎幹細胞中，因DNMT3L存在與否而造成基因本體上甲基化的差異所導致。 總論，我們發現胚胎幹細胞中DNMT3L的表現，其效應可持續至間葉幹細胞並影響幹細胞特性以及硬骨分化。

DNA methyltransferase 3-like (DNMT3L) is an epigenetic modulating factor, mainly expressed in embryonic stem cells and germ cells. It’s a well-known factor for transcriptional silencing and facilitating de novo DNA methylation with DNMT3A and DNMT3B. Previously, we observed that Dnmt3l knockout (KO) mice over one year old display lower body weight and reduced white adipose tissue associated with a higher structural model index, which is a trend observed in older animals and human, insinuating the pre-mature aging phenomenon. Moreover, we demonstrated that Dnmt3l KO mice derived mesenchymal stem/stromal cells (MSCs), the progenitor cells of adipocytes and osteoblasts, have seriously impaired osteogenesis ability in vitro. Although we cannot exclude the lower DNMT3L expression in some subpopulation of MSCs, DNMT3L is certainly not expressed in mouse embryonic fibroblasts (MEFs). However, premature senescence and relatively relaxed chromatin structure are observed in cultured mouse embryonic fibroblast derived from Dnmt3l KO embryos compared to counterparts from wild type littermates. These results suggested that transiently expressed DNMT3L mediated epigenetic marks established in embryonic stem cells (ESCs) may have long-lasting effect on the chromatin structure and phenotype of differentiated cells, long after DNMT3L is silenced. Therefore, this thesis focuses on studying whether DNMT3L mediated epigenetic legacy in ESCs may affect their differentiation downstream gene expression and osteogenic potential in MSCs in vitro. First, we investigated the expressions of typical cell surface markers on mesenchymal stem cell populations isolated from WT and Dnmt3l-deficient mice, and observed that these cells exhibit similar cell surface markers expression. However, besides the osteogenic potential that was affected by the lack of DNMT3L, the lower colony forming efficiency in Dnmt3l KO mice-derived MSCs was observed. Although there was no significant difference in growth rate between WT and Dnmt3l KO mice-derived MSCs at the early passage (P4), our results showed that a decreased rate of cell growth at late-passage (P6) in the epiphysis-derived MSCs population from Dnmt3l KO mice. Furthermore, the results of colony forming efficiency assay and growth curve test implicated that MSCs from Dnmt3l KO mice have the lower self-renewal capacity and premature senescence phenotype. In the second part of the thesis, I investigated the gene expression pattern using RNA-sequencing analysis of MSCs derived from WT and Dnmt3l KO mice. I compared the transcriptome from the two genotypes for both undifferentiated MSCs and differentiating derivatives on day 3 of osteogenic induction, and found that differentially expressed genes (DEGs) between WT and Dnmt3l KO mice-derived MSCs are highlighted in various pathways related to bone morphology in undifferentiated group and associated with microtubule activity in 3 days of osteogenic induction. These gene ontology analyses implied that distinct gene expression on proliferation and differentiation may serve as the cue of compromised stemness in Dnmt3l KO mice-derived MSCs. In order to investigate the mechanisms of how DNMT3L in ESCs trigger a cascade of epigenomic and transcriptome changes that lead to long-term effect on the MSCs and modulate the differentiation potential of MSCs for osteogenesis. We downloaded and reanalyzed the published dataset including DNMT3L binding region (by ChIP-seq), gene expression profile (by Microarray) and DNA methylation level (by MeDIP-seq) in Dnmt3l knockdown and control ESCs. We compared these DNMT3L associated changes in ESCs with Dnmt3l genotype dependent gene expression profile of undifferentiated and differentiating MSCs. The global methylation level of promoter and gene body regions are both higher in Dnmt3l knockdown ESCs. In addition, we found that the obvious differentially expressed genes between WT and Dnmt3l KO MSCs are also genes associated with DNMT3L-dependent methylation on gene bodies in ESCs. Collectively, this thesis reveals that transient DNMT3L expression in ESCs has lasting effect in MSCs, including their self-renew and osteogenic differentiation.