MOLECULAR MECHANISMS IN EVI1-INDUCED MYELOID LEUKEMIA

描述：(改编自研究人员的摘要)急性髓系 白血病是一种致命性疾病，其分子病因尚不清楚， 但这很可能涉及髓系细胞 控制生长，执行正常成熟。申请者的长期身份 目的是了解这种疾病的分子机制。 通过对锌指基因Evi1的深入研究。Evi1已经被 在小鼠和人类中都与急性髓系白血病有关，但 其致病机制尚不清楚。一种可能性是 它会导致对促进分化的细胞因子的异常反应， 如粒细胞集落刺激因子。已知与两个序列具有高特异性结合 (GACAAGATA和GAAGATGAG)通过锌指的两个独立结构域，尽管 它作用的目标基因还没有被描述。申请人 假设EVI1蛋白导致白血病的机制是 通过改变某些靶基因的转录调控。这个 申请人声称，通过识别和表征 通过这些基因，他可以开始了解EVI1 会导致白血病。这条调查路线也将导致建立 他可以用来进行结构功能研究的可行的生物检测方法。 他在初步数据中显示，他已经建立了两个不同的 识别EVI1靶基因的方法，并使用这些技术 隔离几个候选人。在本申请中，他建议进一步 确定这些基因的特征，并研究EVI1在其 转录调控。他打算在他的选择上有所改进。 战略，以确定更多可能相关的目标 来探讨S在EVI1在造血和白血病中的作用。他还提议 研究EVI1在转录调控中的作用，使用 靶基因在手中作为底物。 在第二个具体目标中，申请人希望解决假设 EVI1导致转录抑制，至少部分通过 蛋白质之间的相互作用。已知EVI1的结合位点 和GATA-1重叠，EVI1可以抑制转录激活 GATA1的属性，导致假设EVI1可能有助于 白血病通过阻断GATA-1功能发挥作用。但是，EVI1需要额外的 GATA基序之外的碱基用于DNA结合，因此不能与GATA结合 到目前为止描述的网站。尽管如此，他观察到EVI1可以抑制 含有EVI1失败的GATA基序的记者的GATA-1激活 在体内结合。这些发现表明，EVI1介导的抑制 转录可能通过蛋白质-蛋白质相互作用来调节，而不是 DNA-蛋白质相互作用。为了进一步研究EVI1对哪些蛋白质的作用 为了在细胞内结合，他提议在酵母中进行双杂交筛选。 这将导致识别与EVI1结合的蛋白质，并将 可能对EVI1效应的机制有重要的洞察力 转录抑制，并由此导致疾病。

DESCRIPTION: (adapted from the investigator's abstract) Acute myeloid leukemia is a lethal illness whose molecular etiology is poorly understood, but which likely involves alterations in the ability of myeloid cells to control growth and execute normal maturation. The applicant's long-term goal is to understand the molecular mechanisms involved in this disease through the in-depth study of the zinc finger gene, Evi1. Evi1 has been implicated in acute myeloid leukemia both in mouse and human, but the mechanism by which it causes disease is not known. One possibility is that it causes abnormal responsiveness to differentiation-promoting cytokines, such as G-CSF. It is known to bind with high specificity to two sequences (GACAAGATA and GAAGATGAG) via two separate domains of zinc fingers, although the target genes on which it acts have not been described. The applicant hypothesizes that the mechanism by which the EVI1 protein causes leukemia is by altering the transcriptional regulation of certain target genes. The applicant asserts that through the identification and characterization of these genes he can begin to understand the exact mechanism by which EVI1 causes leukemia. This line of inquiry will also result in the establishment of viable bioassays with which he can perform structure-function studies. He shows in his Preliminary data that he has established two different approaches to identify EVI1 target genes, and has used these techniques to isolate several candidates. In this application, he proposes to further characterize these genes, and to study the role of EVI1 in their transcriptional regulation. He intends to improve upon his selection strategies, to identify greater numbers of targets that may be of relevance to EVI1 s role in hematopoiesis and leukemia. He also proposes to investigate the effect of EVI1 on transcriptional regulation, using the target genes in hand as substrates. In the second Specific Aim, the applicant wishes to address the hypothesis that EVI1 causes transcriptional repression, at least in part, through protein-protein interactions. It is known that the binding sites of EVI1 and GATA-1 overlap, and that EVI1 can repress the transcriptional activation properties of GATA1, leading to the hypothesis that EVI1 may contribute to leukemia by blocking GATA-1 function. However, EVI1 requires additional bases beyond the GATA motif for DNA binding, and thus can not bind to GATA sites described to date. Nonetheless, he has observed that EVI1 can repress GATA-1 activation of reporters that contain GATA motifs to which EVI1 fails to bind in vivo. These findings suggest that EVI1-mediated repression of transcription may be mediated by protein-protein interactions, rather than DNA-protein interaction. To further investigate the proteins to which EVI1 binds within the cell, he proposes to perform a two-hybrid screen in yeast. This will lead to the identification of proteins that bind to EVI1, and will likely yield significant insight into the mechanism by which EVI1 effects transcriptional repression, and by which it causes disease.