HDAC-dependent regulation and functional relevance of WT1 during replicative stress

该项目旨在分析控制转录因子WT 1的分子机制以及WT 1在经历复制应激和DNA损伤的原发性和永久性白血病细胞中的生物学功能。我们专注于属于I类组蛋白脱乙酰酶（HDAC）组的表观遗传修饰剂的WT 1的调节。这些酶的抑制剂诱导WT 1的蛋白酶体降解（涉及E2泛素缀合酶UBCH 8）和WT 1 mRNA表达的损失。我们想要定义哪种I类HDAC控制WT 1基因和编码UBCH 8的UBE 2L 6基因的表达。此外，我们希望确定WT 1的目标，影响DNA复制干扰后的细胞凋亡。此外，我们将测试WT 1是否直接控制复制应激信号，DNA复制检查点和DNA损伤。此外，我们将分析检查点激酶ATM、ATR和CHK 1/CHK 2在具有失活的I类HDAC的细胞中与复制应激的相关性。我们还旨在了解HDAC依赖性调节和促凋亡因子NOXA的生物学相关性白血病细胞的生存经历复制停滞和WT 1的损失。我们有一个生化和分子的方法。我们用羟基脲耗尽细胞dNTP池，我们使用HDACi MS-275，其特异性阻断I类HDAC HDAC 1、HDAC-2和HDAC-3，并且我们从遗传上消除感兴趣的蛋白质。我们采用的方法有Western blot检测蛋白质表达和磷酸化，定量实时PCR，共聚焦显微镜，定量蛋白质组学，i-POND，DNA纤维测定，流式细胞术评估细胞活力，CRSPR-Cas9和RNAi。

This project aims to analyze molecular mechanisms controlling the transcription factor WT1 as well as the biological functions of WT1 in primary and permanent leukemic cells undergoing replicative stress and DNA damage. We focus on the regulation of WT1 by epigenetic modifiers belonging to the group of class I histone deacetylases (HDACs). Inhibitors of these enzymes induce the proteasomal degradation of WT1 (involving the E2 ubiquitin conjugase UBCH8) and a loss of WT1 mRNA expression. We want to define, which class I HDACs control the expression of the WT1 gene and of the UBE2L6 gene encoding UBCH8. Furthermore, we want to identify targets of WT1 that affect apoptosis upon disturbed DNA replication. Additionally we will test whether WT1 directly controls replicative stress signaling, the DNA replication checkpoint, and DNA damage. Moreover, we will analyze the relevance of the checkpoint kinases ATM, ATR, and CHK1/CHK2 in cells with inactivated class I HDACs combined with replicative stress. We also aim to understand the HDAC-dependent regulation and the biological relevance of the pro-apoptotic factor NOXA for the survival of leukemic cells undergoing replication arrest and a loss of WT1. We have a biochemical and molecular approach. We deplete the cellular dNTP pool with hydroxyurea, we use the HDACi MS-275 that specifically block the class I HDACs HDAC1, -2, and -3, and we eliminate proteins of interest genetically. The methods we employ are Western blot to detect protein expression and phosphorylation, quantitative real-time PCR, confocal microscopy, quantitative proteomics, i-POND, DNA fiber assay, flow cytometry assessing cell vitality, CRSPR-Cas9 and RNAi.