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

本项目旨在分析转录因子WT1的分子调控机制以及WT1在原代和永久性白血病细胞复制应激和DNA损伤中的生物学功能。我们主要关注属于I类组蛋白脱乙酰基酶(HDACs)的表观遗传修饰物对WT1的调控。这些酶的抑制剂诱导WT1的蛋白酶体降解(包括E2泛素结合酶UBCH8)和WT1mRNA表达的丧失。我们想要定义，哪种I类HDAC控制WT1基因和编码UBCH8的UBE2L6基因的表达。此外，我们希望确定WT1在DNA复制受扰时影响细胞凋亡的靶点。此外，我们还将测试WT1是否直接控制复制应激信号、DNA复制检查点和DNA损伤。此外，我们将分析失活的I类HDAC结合复制应激的细胞中检查点激酶ATM、ATR和CHK1/CHK2的相关性。我们还旨在了解HDAC依赖的调节以及促凋亡因子NOXA与经历复制停止和WT1丢失的白血病细胞生存的生物学相关性。我们有一种生化和分子方法。我们用羟基脲耗尽细胞dNTP池，我们使用HDACi MS-275专门阻断I类HDACs HDAC1、-2和-3，我们从基因上消除感兴趣的蛋白质。我们使用的方法有蛋白质印迹检测蛋白表达和磷酸化、实时定量聚合酶链式反应、共聚焦显微镜、定量蛋白质组学、I-POND、DNA纤维分析、流式细胞仪检测细胞活力、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.