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

急性淋巴细胞性白血病是儿童最常见的癌症。其潜在的发病机制尚不完全清楚，包括成熟停滞是急性白血病的一个普遍特征。在指导B淋巴细胞生成的转录因子中已经描述了各种结构异常，这些转录因子导致祖细胞分化停滞，如在PAX5、IKZF1和EBF-1中。我们最近发现锌指因子423？(ZNF423？)的一种干扰的表观遗传和转录调控是阻碍ALL中B细胞分化的一种新机制。ZNF423增强子序列的低甲基化与BMP2信号的结合导致ZNF423的反式激活，ZNF423？亚型编码核小体重塑和组蛋白去乙酰基酶复合体相互作用的结构域。ZNF423及其小鼠同系物Zfp423在嗅神经发生和小脑发育过程中分别在胚胎干细胞和中枢神经系统中生理性表达，但在正常造血中缺失。ZFN423在淋巴生成过程中的异常表达导致依赖EBF-1的转录程序中断，至少部分是通过ZNF423和EBF-1之间的物理相互作用。为了增加另一层调控复杂性，ZNF423已被证明在三元Smad1-Smad4转录因子复合体中直接骨形态发生蛋白2介导的信号活性，而该复合体又被EBF-1抑制。因此，ZNF423似乎以相互排斥的方式在BMP和EBF-1介导的独立反应途径中发挥作用。ETV6-RUNX1重排均显示出显著高的ZNF423和SMAD活性，这提出了ZNF423是通过截留EBF-1、通过转录调控SMAD复合体还是通过未知的机制来抑制分化的问题。此外，在ETV6-RUNX1的背景下，评估异常的ZNF423是否构成诱发直接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ZNF423和EBF-1的全基因组DNA结合研究和转录组测序，破译ZNF423在B-前体中的作用模式，所有这些都整合了ZNF423与EBF-1和Smad1-Smad4复合体的蛋白质-蛋白质相互作用。此外，将利用条件敲入/移植小鼠模型，在体内ETV6-RUNX1/ETV6-RUNX1阳性造血干细胞中研究ZNF423ü异常对白血病发生的影响。在相互实验中，我们将评估ZNF423缺失/缺失对所有细胞在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.