Role of GATA-1 in p21 Gene Transcription

描述（由申请人提供）：本提案的总体目标是了解介导分化和增殖之间平衡的因素如何维持细胞处于正常与致瘤状态。我们的方法是分析小鼠红细胞白血病（MEL）细胞中细胞周期调节因子p21与红细胞特异性转录因子GATA-1之间的关系。由于PU.1直接结合和抑制GATA-1， MEL细胞无法分化，PU.1是一种由病毒整合激活的髓系转录因子。p21在未分化的MEL细胞中表达较低，但在分化过程中随着PU.1的下降而显著上调。此外，外源性GATA-1和p21可以克服这一障碍并触发终端分化。初步数据表明GATA-1可以诱导p21基因转录。我们将通过三个具体目标来验证p21基因是GATA-1的直接转录靶点的假设：1)通过报告基因分析和电泳迁移转移（EMS ' s）鉴定GATA-1刺激所需的p21启动子中的DNA序列；2)利用染色质免疫沉淀法检测内源性p21启动子中GATA-1的占用情况，确定p21基因是否为GATA-1在MEL细胞分化中的靶点；3)确定p21基因是否是pu .1介导的未分化MEL细胞中GATA-1抑制的靶标。我们将研究用短干扰RNA （siRNA）降低PU.1水平是否会抑制p21基因的转录，并测试PU.1和转录共阻子（如Rb）是否占据p21启动子。这些研究将阐明红系细胞的增殖和分化是如何协调的，以及这种协调在肿瘤发生过程中是如何被破坏的。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand how factors that mediate the balance between differentiation and proliferation maintain cells in a normal versus tumorigenic state. Our approach is to analyze the relationship between the cell cycle regulator p21 and the erythroid-specific transcription factor GATA-1 in murine erythroleukemia (MEL) cells. MEL cells are blocked from differentiating due to direct binding and repression of GATA-1 by PU.1, a myeloid transcription factor activated by viral integration. p21 expression is low in undifferentiated MEL cells but is significantly upregulated as PU.1 declines during differentiation. Furthermore, exogenous GATA-1 and p21 can overcome this block and trigger terminal differentiation. Preliminary data indicates that GATA-1 can induce p21 gene transcription. We will test the hypothesis that the p21 gene is a direct transcriptional target of GATA-1 by pursuing three specific aims: 1) To identify DNA sequences in the p21 promoter required for GATA- 1 stimulation using reporter assays and electrophoretic mobility shifts (EMS A' s); 2) To determine if the p21 gene is a GATA-1 target in differentiating MEL cells by testing for GATA- 1 occupancy of the endogenous p21 promoter using chromatin immunoprecipitation; and 3) To determine whether the p21 gene is a target for PU.1-mediated repression of GATA-1 in undifferentiated MEL cells. We will examine whether reducing PU.1 levels with short interfering RNA (siRNA) derepresses p21 gene transcription and test for occupancy of the p21 promoter by PU.1 and transcriptional corepressors such as Rb. These studies will elucidate how proliferation and differentiation are coordinated in erythroid cells and how this coordination is disrupted during oncogenesis.