The role of Ikaros in regulation of Notch target gene expression

描述（由申请人提供）：Ikaros是一种核蛋白，是T淋巴细胞生成的关键调节因子。Ikaros缺陷导致小鼠T细胞发育异常，并且在100%转化率的情况下导致T细胞白血病发生。然而，Ikaros在这些事件中的机械作用尚不清楚。T细胞发育中的另一个重要蛋白是Notch。Notch信号的失调，就像Ikaros功能的失调一样，不仅导致T细胞发育缺陷，还导致白血病发生。由于小鼠和人白血病细胞中Ikaros的缺失和Notch的组成性激活之间的相关性，Ikaros和Notch的失调被假设在白血病发生中协同作用。因此，这两条途径可能在T细胞发育过程中也协同作用。我们假设，Notch信号通路的失调，这是在Ikaros的情况下观察到的，是一个主要的促成因素，不仅白血病，而且在Ikaros null小鼠中观察到的T细胞发育缺陷。Notch是一种跨膜蛋白，其在识别其配体时被切割，释放“细胞内”结构域（ICN），其行进到细胞核。在这里，ICN通过结合并激活CSL转录因子来调节Notch靶基因的转录。CSL被认为在不存在ICN的情况下充当Notch靶基因表达的活性阻遏物。我们已经证明，在没有Ikaros的情况下，Notch靶基因被去阻遏，这表明Ikaros是这些基因的专性阻遏物。但是，相对于CSL，Ikaros在基因抑制中是如何发挥作用的呢？Notch靶基因表达的去抑制在多大程度上有助于在Ikaros敲除小鼠中观察到的T细胞缺陷？本文描述的研究将开始使用离体和体内方法来解决这些问题。在第一个具体目标中，我们建议利用shRNA敲除和生物化学方法的组合来定义Ikaros在Notch靶基因Hes 1的调节中的作用，最终目标是理解CSL/Ikaros介导的T细胞中Notch靶基因的抑制机制。在第二个具体目标中，我们建议利用条件性CSL敲除小鼠来确定Notch靶基因的去抑制在Ikaros敲除小鼠中观察到的T细胞发育缺陷的程度。总之，这些研究将提供CSL和Ikaros之间的相互作用在基因抑制，这反过来，将阐明在T细胞发育的一个重要途径的功能解剖。公共卫生相关性：在免疫细胞发育过程中发生的遗传异常可能导致灾难性疾病，如白血病。识别调控发育的基因是了解疾病过程的一种方法，这是设计更有效疗法的第一步。在这里提出的研究中，我们将研究两个重要的发育基因Ikaros和Notch之间的相互作用，这两个基因在人类白血病中都发生了突变。

DESCRIPTION (provided by applicant): Ikaros is a nuclear protein and key regulator of T lymphopoiesis. Ikaros deficiency in mice results in abnormal T cell development and, with 100% penetrance, T cell leukemogenesis. However, the mechanistic role of Ikaros in these events is not yet known. Another important protein in T cell development is Notch. Deregulation of Notch signaling, like deregulation of Ikaros function, leads not only to defects in T cell development but to leukemogenesis as well. Deregulation of Ikaros and Notch are hypothesized to cooperate in leukemogenesis due to a correlation between loss of Ikaros and constitutive activation of Notch in leukemia cells in mouse and man. Therefore, it is likely that these two pathways also function cooperatively during T cell development. We hypothesize that deregulation of Notch signaling pathways, which is observed in the absence of Ikaros, is a major contributing factor, not only to the leukemogenesis, but also to the T cell developmental defects observed in Ikaros null mice. Notch is a transmembrane protein that is cleaved upon recognition of its ligand, freeing the "intracellular" domain (ICN), which travels to the nucleus. Here, ICN regulates transcription of Notch target genes through binding to and activation of the CSL transcription factor. CSL is believed to function as an active repressor of Notch target gene expression in the absence of ICN. We have demonstrated that in the absence of Ikaros, Notch target genes are de-repressed, suggesting that Ikaros is an obligate repressor of these genes. But how does Ikaros function in gene repression relative to CSL? And to what degree does de-repression of Notch target gene expression contribute to the T cell defects observed in Ikaros null mice? The studies described here will begin to address these questions using both ex vivo and in vivo approaches. In the first specific aim, we propose to utilize a combination of shRNA knockdown and biochemical approaches to define the role of Ikaros in regulation of the Notch target gene Hes1, with the ultimate goal of understanding the mechanism of CSL/Ikaros-mediated repression of Notch target genes in T cells. In the second specific aim, we propose to utilize conditional CSL knockout mice to determine the extent to which de-repression of Notch target genes underlies T cell developmental defects observed in Ikaros null mice. Taken together, these studies will provide a functional dissection of the interplay between CSL and Ikaros in gene repression, which, in turn, will illuminate a centrally important pathway in T cell development. PUBLIC HEALTH RELEVANCE: Genetic abnormalities occurring within an immune cell during its development can lead to catastrophic disease, such as leukemia. Identifying genes that regulate development is one approach to understanding the disease process, which is the first step to designing more effective therapies. In the studies proposed here, we will investigate the interaction between two important developmental genes, Ikaros and Notch, both of which are mutated in human leukemia.