MOLECULAR MECHANISMS IN EVI1-INDUCED MYELOID LEUKEMIA

描述：（改编自研究者摘要）急性髓系 白血病是一种致命疾病，其分子病因学知之甚少， 但这可能涉及骨髓细胞的能力改变， 控制生长并执行正常成熟。 申请人长期 目的是了解这种疾病的分子机制 通过对锌指基因Evi 1. Evi 1已经 与小鼠和人类的急性髓系白血病有关，但 它引起疾病的机制尚不清楚。 一种可能是 它引起对促进分化的细胞因子的异常反应， 例如G-CSF。 已知其以高特异性结合两个序列 （GACAAGATA和GAAGATGAG）通过两个独立的锌指结构域，尽管 其作用的靶基因尚未被描述。 申请人 假设EVI 1蛋白引起白血病的机制是 通过改变某些靶基因的转录调控。 的 申请人声称，通过鉴定和表征 这些基因，他可以开始了解EVI 1 会导致白血病 这一调查路线也将导致建立 可行的生物测定，他可以进行结构功能研究。 他在他的初步数据中表明，他已经建立了两个不同的 方法，以确定EVI 1靶基因，并已使用这些技术， 隔离几个候选人 在本申请中，他建议进一步 描述这些基因，并研究EVI 1在其 转录调控 他打算改进他的选择 战略，以确定更多可能相关的目标 EVI 1在造血和白血病中的作用 他还建议， 研究EVI 1对转录调控的影响，使用 目标基因作为底物。 在第二个具体目标中，申请人希望解决假设 EVI 1导致转录抑制，至少部分是通过 蛋白质相互作用 已知EVI 1的结合位点 EVI 1与加塔-1的转录激活存在重叠，EVI 1可抑制转录激活 GATA 1的特性，导致假设EVI 1可能有助于 通过阻断加塔-1功能来治疗白血病。 然而，EVI 1需要额外的 DNA结合的加塔基序以外的碱基，因此不能结合加塔 网站描述到目前为止。 尽管如此，他观察到EVI 1可以抑制 加塔-1激活包含加塔基序的报告基因，EVI 1不能激活该基序 to bind结合in vivo体内. 这些发现表明，EVI 1介导的抑制， 转录可能是由蛋白质-蛋白质相互作用介导的，而不是 DNA-蛋白质相互作用 为了进一步研究EVI 1与之结合的蛋白质， 结合在细胞内，他建议在酵母中进行双杂交筛选。 这将导致识别与EVI 1结合的蛋白质， 这可能会对EVI 1的作用机制产生重大影响， 转录抑制，并通过它引起疾病。

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.