Mechanism and relevance of active DNA methylation during monocyte differentiation

实验证据以及TET2和IDH基因在急性髓系白血病中的高频率突变表明DNA甲基化转换对于正常的骨髓生成是必不可少的。其他人之前的工作以及我们实验室的工作表明，转录因子在靶向DNA去甲基化到谱系特异性增强子的过程中发挥着重要作用。然而，在大多数情况下，实际上并不清楚所观察到的去甲基化是转录因子结合、染色质重塑和例如增强子激活的结果，还是后者所必需的。为了了解DNA去甲基化在正常骨髓细胞中的相关性和作用，以及在白血病发生过程中的作用，我们需要更好地了解和定义哪些序列特征和哪些因素需要去甲基化来建立稳定的转录因子-DNA相互作用并参与基因调控过程。在目前的应用中，我们建议研究有丝分裂后单核细胞的分化，以解决与活性DNA去甲基化机制相关的剩余关键问题。利用最近建立的高效、无毒/激活的siRNA转染方案，我们将下调关键基因(包括TET2和候选靶向转录因子)的表达，并研究其对转录、DNA甲基化、染色质可及性、组蛋白修饰和转录因子结合的影响。这将最终阐明DNA去甲基化的功能重要性，以及它是如何在髓系细胞中靶向的。这些发现不仅对单核细胞生物学的一般理解很重要，而且对我们理解DNA甲基化机制中的突变如何改变造血细胞以驱动白血病发生也是重要的。

Experimental evidence as well as the high frequency of mutations in TET2 and IDH genes in acute myeloid leukaemia suggests that DNA methylation turnover is essential for normal myelopoiesis. Previous work from others as well as work from our lab suggests a major role for transcription factors in targeting DNA demethylation to lineage-specific enhancers. However, in the large majority of cases it is not actually clear whether the observed demethylation is 'just' the consequence of transcription factor binding, chromatin remodelling and e.g. enhancer activation, or whether it is required for the latter. To understand the relevance and function of DNA demethylation both in normal myeloid cells as well during leukaemogenesis we need to understand better and define, which sequence features and which factors require demethylation to establish stable transcription factor -DNA interactions and to participate in gene regulatory processes. In the current application we propose to study the differentiation of post-mitotic monocytes to address remaining key questions related to the mechanisms of active DNA demethylation. Using a recently established, highly efficient and non-toxic/activating siRNA transfection protocol we will knock-down the expression of key genes (including TET2 as well as candidate targeting transcription factors) and study the effect on transcription, DNA methylation, chromatin accessibility, histone modifications and transcription factor binding. This will ultimately clarify the functional importance of DNA demethylation, and how it is targeted in myeloid cells. The findings will not only be important for the general understanding of monocyte biology but also for our understanding how mutations in the DNA methylation machinery might alter hematopoietic cells to drive leukaemogenesis.