Chromosomal translocations represent frequent events in leukemia. In t(8;21)+ acute myeloid leukemia, RUNX1 is fused to nearly the entire ETO protein, which contains four conserved nervy homology regions, NHR1-4. Furthermore RUNX1/ETO interacts with ETO-homologous proteins via NHR2, thereby multiplying NHR domain contacts. As shown recently, RUNX1/ETO retains oncogenic activity upon either deletion of the NHR3 + 4 N-CoR/SMRT interaction domain or substitution of the NHR2 tetramer domain. Thus, we aimed to clarify the specificities of the NHR domains. A C-terminally NHR3 + 4 truncated RUNX1/ETO containing a heterologous, structurally highly related non-NHR2 tetramer interface translocated into the nucleus and bound to RUNX1 consensus motifs. However, it failed to interact with ETO-homologues, repress RUNX1 targets, and transform progenitors. Surprisingly, transforming capacity was fully restored by C-terminal fusion with ETO's NHR4 zinc-finger or the repressor domain 3 of N-CoR, while other repression domains failed. With an inducible protein assembly system, we further demonstrated that NHR4 domain activity is critically required early in the establishment of progenitor cultures expressing the NHR2 exchanged truncated RUNX1/ETO. Together, we can show that NHR2 and NHR4 domains can be replaced by heterologous protein domains conferring tetramerization and repressor functions, thus showing that the NHR2 and NHR4 domain structures do not have irreplaceable functions concerning RUNX1/ETO activity for the establishment of human CD34+ cell expansion. We could resemble the function of RUNX1/ETO through modular recomposition with protein domains from RUNX1, ETO, BCR and N-CoR without any NHR2 and NHR4 sequences. As most transcriptional repressor proteins do not comprise tetramerization domains, our results provide a possible explanation as to the reason that RUNX1 is recurrently found translocated to ETO family members, which all contain tetramer together with transcriptional repressor moieties.

中文翻译：

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驱动 CD34+ 人类祖细胞扩增的 RUNX1/ETO 融合蛋白模块的兼容性。

染色体易位代表了白血病中的常见事件。在 t(8;21)+ 急性髓系白血病中，RUNX1 几乎与整个 ETO 蛋白融合，其中包含四个保守的神经同源区域 NHR1-4。此外，RUNX1/ETO 通过 NHR2 与 ETO 同源蛋白相互作用，从而增加 NHR 域接触。如最近所示，RUNX1/ETO 在 NHR3 + 4 N-CoR/SMRT 相互作用域的缺失或 NHR2 四聚体域的替换后保留致癌活性。因此，我们旨在澄清 NHR 域的特殊性。C-末端 NHR3 + 4 截断的 RUNX1/ETO 含有异源的、结构高度相关的非 NHR2 四聚体界面，易位到细胞核中并与 RUNX1 共识基序结合。然而，它未能与 ETO 同源物相互作用、抑制 RUNX1 目标并转化祖细胞。令人惊讶的是，通过与 ETO 的 NHR4 锌指或 N-CoR 的抑制域 3 的 C 端融合，转化能力完全恢复，而其他抑制域失败。使用诱导型蛋白质组装系统，我们进一步证明，在建立表达 NHR2 交换的截短 RUNX1/ETO 的祖细胞培养物的早期，NHR4 域活性是至关重要的。总之，我们可以证明 NHR2 和 NHR4 结构域可以被赋予四聚化和阻遏功能的异源蛋白质结构域取代，从而表明 NHR2 和 NHR4 结构域结构在 RUNX1/ETO 活性方面​​没有不可替代的功能，用于建立人 CD34+细胞扩增. 我们可以通过模块化重组来自 RUNX1、ETO、没有任何 NHR2 和 NHR4 序列的 BCR 和 N-CoR。由于大多数转录抑制蛋白不包含四聚化结构域，我们的结果提供了一个可能的解释，说明 RUNX1 经常被发现易位到 ETO 家族成员的原因，这些成员都包含四聚体和转录抑制部分。