DOT1L is essential for early hematopoiesis but the precise mechanisms remain largely unclear. The only known function of DOT1L is histone H3 lysine 79 (H3K79) methylation. We generated two mouse models; a Dot1L-knockout (Dot1L-KO), and another possessing a point mutation in its methyltransferase domain (Dot1L-MM) to determine the role of its catalytic activity during early hematopoiesis. We observed that Dot1L-KO embryos suffered from severe anemia, while Dot1L-MM embryos showed minimal to no anemia. However, ex vivo culture of Dot1L-MM hematopoietic progenitors (HPCs) exhibited defective development of myeloid and mixed progenitors. DOT1L is a well-recognized, cell-type specific epigenetic regulator of gene expression. To elucidate the mechanisms underlying such diverse hematopoietic properties of Dot1L-KO and Dot1L-MM HPCs, we examined their whole transcriptomes. Extensively self-renewing erythroblast (ESRE) cultures were established using yolk sac (YS) cells collected on embryonic day 10.5 (E10.5). Dot1l-KO and Dot1l-MM cells expanded significantly less than the wildtype cells and showed slower progression through the cell cycle. Total RNA extracted from the wildtype and Dot1l-mutant ESRE cells were subjected to RNA-seq analyses. We observed that the majority (~82%) of the differentially expressed genes (DEGs) were upregulated in both of the Dot1L-mutants, which suggests that DOT1L predominantly acts as a transcriptional repressor in HPCs. We also observed that about ~40% of the DEGs were unique to either of the mutant group, suggesting that DOT1L possesses both methyltransferase domain-dependent and -independent functions. We further analyzed Gene Ontology and signaling pathways relevant to the DEGs common to both mutant groups and those that were unique to either group. Among the common DEGs, we observed upregulation of CDK inhibitors, which explains the cell cycle arrest in both of the Dot1L-mutant progenitors.

中文翻译：

---

DOT1L主要充当造血祖细胞的转录阻遏物

DOT1L对于早期造血至关重要，但确切机制尚不清楚。DOT1L唯一已知的功能是组蛋白H3赖氨酸79（H3K79）甲基化。我们生成了两个鼠标模型。一个Dot1L基因敲除（Dot1L-KO），另一个在其甲基转移酶结构域（Dot1L-MM）中具有一个点突变，以确定其在早期造血过程中的催化活性。我们观察到，Dot1L-KO胚胎患有严重的贫血，而Dot1L-MM胚胎显示极少或没有贫血。但是，Dot1L-MM造血祖细胞（HPCs）的离体培养显示出髓样和混合祖细胞发育不良。DOT1L是公认的细胞类型基因表达的特定表观遗传调节剂。为了阐明潜在的Dot1L-KO和Dot1L-MM HPC多种造血特性的机制，我们检查了他们的整个转录组。使用在胚胎第10.5天（E10.5）收集的卵黄囊（YS）细胞建立广泛的自我更新型成红细胞（ESRE）培养物。Dot11-KO和Dot11-MM细胞的扩增明显小于野生型细胞，并且在整个细胞周期中显示出较慢的进程。从野生型和Dot11突变的ESRE细胞中提取的总RNA进行RNA序列分析。我们观察到大多数差异表达基因（DEG）（〜82％）在两个Dot1L突变体中均被上调，这表明DOT1L在HPC中主要充当转录抑制因子。我们还观察到，约40％的DEG对任一突变组而言都是唯一的，这表明DOT1L同时具有甲基转移酶结构域依赖性和非依赖性功能。我们进一步分析了与两个突变组和两个突变组均通用的DEG相关的基因本体论和信号通路。在常见的DEG中，我们观察到CDK抑制剂的上调，这解释了两个Dot1L突变祖细胞的细胞周期停滞。