Transcriptional Modulation by the Zinc Finger Factor 423ß in Leukemogenesis at Childhood

急性淋巴细胞白血病是儿童最常见的癌症。疾病的潜在机制尚未完全理解，包括作为急性白血病的一般标志的成熟停滞。在引导B淋巴细胞生成的转录因子中已经描述了多种结构畸变，其导致祖细胞中的分化停滞，例如PAX 5、IKZF 1和EBF-1。我们最近确定了一个扰动的表观遗传和转录调控的锌指因子423 β（ZNF 423 β）作为一种新的机制，阻碍B细胞分化的ALL。ZNF 423增强子序列的低甲基化和BMP 2信号传导以组合方式导致ZNF 423的反式激活，ZNF 423的β-同种型编码核小体重塑和组蛋白脱乙酰酶复合物相互作用结构域。ZNF 423及其鼠同源物Zfp 423分别在嗅觉神经发生和小脑发育期间的胚胎干细胞和中枢神经系统中生理性表达，但它们在正常造血中不存在。淋巴细胞生成过程中ZFN 423的异常表达导致EBF-1依赖性转录程序的破坏，至少部分是通过ZNF 423和EBF-1之间的物理相互作用。为了增加另一层调控复杂性，ZNF 423已被证明在三元SMAD 1-SMAD 4转录因子复合物中指导骨形态发生蛋白2介导的信号传导活性，而这反过来又被EBF-1抑制。因此，ZNF 423似乎在独立的BMP-和EBF-1介导的反应途径中以相互排斥的方式起作用。ETV 6-RUNX 1重排的ALL显示出显著高的ZNF 423和SMAD活性，这提出了ZNF 423是否通过隔离EBF-1、通过SMAD复合物的转录调节或通过尚不清楚的机制来发挥其对分化的抑制作用的问题。此外，评估异常ZNF 423是否构成在ETV 6-RUNX 1背景下诱导明显白血病的决定性动力将是非常有意义的，本文提出的研究旨在通过基因组分析，结合ZNF 423与EBF-1和SMAD 1-SMAD 4复合物的蛋白质-蛋白质相互作用，破译ZNF 423在B前体ALL中的作用模式。ZNF 423和EBF-1的广泛DNA结合研究，并辅以转录组测序。此外，将利用条件性敲入/移植小鼠模型在体内ETV 6-RUNX 1/Etv 6-RUNX 1阳性造血干细胞中研究异常ZNF 423 β对白血病发生的影响。在一个交互实验中，我们将评估ZNF 423缺失/缺失对ALL细胞体外和体内分化的影响。

The acute lymphoblastic leukemia is the most common cancer of childhood. The underlying mechanisms of disease are incompletely understood including the maturation arrest as a general hallmark of acute leukemia. A variety of structural aberrations have been described in transcription factors directing B-lymphopoiesis that cause a differentiation arrest in progenitor cells such as in PAX5, IKZF1, and EBF-1. We have recently identified a perturbed epigenetic and transcriptional regulation of the zinc finger factor 423ß (ZNF423ß) as a novel mechanism hampering B-cell differentiation in ALL. In a combinatorial manner hypomethylation of ZNF423 enhancer sequences and BMP2 signaling lead to transactivation of ZNF423 whose ß-isoform encodes a nucleosome remodeling and histone deacetylase complex-interacting domain. ZNF423 and its murine homologue Zfp423 are physiologically expressed in embryonic stem cells and in the central nervous system during olfactory neurogenesis and cerebellar development, respectively, but they are absent in normal hematopoiesis. Aberrant expression of ZFN423 during lymphopoiesis results in disruption of the EBF-1 dependent transcriptional program at least in part through physical interaction between ZNF423 and EBF-1. To add another layer of regulatory complexity ZNF423 has been shown to direct bone morphogenetic protein 2-mediated signaling activity in a ternary SMAD1-SMAD4 transcription factor complex, which in turn is inhibited by EBF-1. Hence ZNF423 appears to operate in a mutually exclusive manner in independent BMP- and EBF-1 mediated reaction pathways. ETV6-RUNX1 rearranged ALL revealed a remarkably high ZNF423 and SMAD activity, which raises the question whether ZNF423 exerts its inhibitory effect on differentiation by sequestration of EBF-1, by transcriptional modulation of SMAD complexes or by yet unknown mechanisms. Furthermore, it will be of great interest to evaluate if aberrant ZNF423 constitutes a decisive momentum to induce frank leukemia in the context of ETV6-RUNX1, which is not leukemogenic in its own right.The research proposed herein aims at deciphering the mode of action of ZNF423 in B-precursor ALL integrating its protein-protein interaction with EBF-1 and SMAD1-SMAD4 complexes by genome-wide DNA binding studies of ZNF423 and EBF-1 complemented with transcriptome sequencing. Moreover, the impact of aberrant ZNF423ß on leukemogenesis will be studied in ETV6-RUNX1/Etv6-RUNX1 positive hematopoietic stem cells in vivo utilizing conditional knock-in/transplantation mouse models. In a reciprocal experiment we will assess the effect of ZNF423 depletion/deletion on the differentiation of ALL cells in vitro and in vivo.