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

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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.