Regulation of the DNA Damage Response and Gene Expression by the Dot1L Histone Methyltransferase and the 53Bp1 Tumour Suppressor

BackgroundDot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined.Methodology/Principal FindingsUsing the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L−/− and wild type cells are equally resistant to ionising radiation, whereas 53Bp1−/−/Dot1L−/− cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of over 1200 genes involved in diverse biological functions. These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression. In 53Bp1−/− cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed. To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line.Conclusions/SignificanceTaken together, our results uncover a negative role for Dot1L and H3K79 methylation in the DNA damage response in the absence of 53Bp1. They also enlighten the roles of Dot1L and 53Bp1 in gene expression and the control of DNA double-strand repair pathways in the context of chromatin.

研究背景
Dot1L是一类靶向组蛋白H3赖氨酸79（H3K79）的组蛋白甲基转移酶，尽管其在基因调控与DNA损伤应答过程中的具体功能尚未得到清晰阐释，但已有研究提示其参与上述两类生物学过程。

材料与方法/研究结果
本研究采用鸡DT40模型系统，构建了Dot1L基因敲除细胞系，以探究DNA损伤应答蛋白53Bp1的功能及其局灶募集机制。详细的动力学与剂量响应实验结果显示，尽管质谱（mass spectrometry）检测证实细胞中完全缺失H3K79甲基化修饰，但53Bp1的局灶募集过程并未受到影响。本研究还首次报道了同时缺失Dot1L与53Bp1的细胞系的表型特征。
Dot1L敲除（Dot1L−/−）细胞与野生型细胞对电离辐射的耐受性无显著差异，而同时缺失53Bp1与Dot1L的细胞则表现出显著的DNA损伤耐受表型。
Dot1L与53Bp1同样可影响大量基因的表达。Dot1L活性缺失会显著改变超过1200个参与多种生物学功能的基因的mRNA水平。上述结果结合此前已报道的Dot1L敲低小鼠ES细胞中差异表达基因的列表，证实了Dot1L依赖的基因表达调控在细胞类型与物种间存在出乎意料的保守性。在53Bp1敲除（53Bp1−/−）细胞中，超过300个基因（其中多数参与免疫应答与细胞凋亡过程）呈现差异表达。截至目前，本研究是首次对53Bp1缺陷细胞系进行全基因组基因表达分析。

结论与意义
综上，本研究结果揭示了在缺失53Bp1的情况下，Dot1L与H3K79甲基化在DNA损伤应答中发挥负调控作用；同时也阐明了Dot1L与53Bp1在染色质背景下参与基因表达调控以及DNA双链断裂修复通路的调控机制。