The role of Ikaros in regulation of Notch target gene expression

描述（申请人提供）：Ikaros是一种核蛋白，是T淋巴生成的关键调节因子。小鼠的Ikaros缺乏导致T细胞发育异常，并且100%外显率导致T细胞白血病的发生。然而，伊卡洛斯在这些事件中的机制作用尚不清楚。T细胞发育过程中另一个重要的蛋白是Notch。Notch信号的失调，就像Ikaros功能的失调一样，不仅会导致T细胞发育的缺陷，还会导致白血病的发生。由于小鼠和人白血病细胞中Ikaros的缺失和Notch的组成性激活之间存在相关性，Ikaros和Notch的失调被假设在白血病发生中合作。因此，这两种途径很可能在T细胞发育过程中协同起作用。我们假设，在Ikaros缺失时观察到的Notch信号通路的失调，不仅是白血病发生的主要因素，也是Ikaros缺失小鼠中观察到的T细胞发育缺陷的主要因素。Notch是一种跨膜蛋白，在识别其配体时被切割，释放“细胞内”结构域（ICN），并将其转移到细胞核。在这里，ICN通过结合和激活CSL转录因子来调节Notch靶基因的转录。在ICN缺失的情况下，CSL被认为是Notch靶基因表达的活性抑制因子。我们已经证明，在缺乏Ikaros的情况下，Notch靶基因被去抑制，这表明Ikaros是这些基因的专性抑制因子。但是Ikaros在与CSL相关的基因抑制中是如何起作用的呢？Notch靶基因表达的去抑制在多大程度上导致了Ikaros缺失小鼠中观察到的T细胞缺陷？这里描述的研究将开始使用体外和体内的方法来解决这些问题。在第一个具体目标中，我们建议结合shRNA敲低和生化方法来确定Ikaros在Notch靶基因Hes1调控中的作用，最终目的是了解CSL/Ikaros介导的T细胞中Notch靶基因抑制的机制。在第二个特定目标中，我们建议利用条件CSL敲除小鼠来确定Notch靶基因的去抑制在Ikaros null小鼠中观察到的T细胞发育缺陷的程度。综上所述，这些研究将提供CSL和Ikaros在基因抑制中的相互作用的功能解剖，这反过来将阐明T细胞发育的一个重要途径。公共卫生相关性：免疫细胞在发育过程中发生的遗传异常可导致灾难性疾病，如白血病。识别调节发育的基因是了解疾病过程的一种方法，也是设计更有效治疗方法的第一步。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，这两个基因都在人类白血病中发生突变。

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.