Role of GATA-1 in p21 Gene Transcription

GATA-1 在 p21 基因转录中的作用

• 批准号: 6739973
• 负责人: MICHAEL J PAPETTI
• 金额: $ 4.73万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6739973
• 关键词: HeLa cells; cell cycle; cell differentiation; cell growth regulation; cell proliferation; chromatin immunoprecipitation; erythrocytes; gel mobility shift assay; gene expression; genetic promoter element; genetic transcription; leukemia; neoplastic growth; nucleic acid sequence; postdoctoral investigator; small interfering RNA; transcription factor

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.

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子如何维持细胞处于正常与致瘤状态。 我们的方法是分析细胞周期调控因子p21和红系特异性转录因子加塔-1在小鼠红白血病（MEL）细胞之间的关系。 MEL细胞由于PU.1（一种由病毒整合激活的髓样转录因子）对加塔-1的直接结合和抑制而被阻断分化。 p21表达在未分化的MEL细胞中较低，但在分化期间随着PU.1下降而显著上调。 此外，外源性加塔-1和p21可以克服这种阻断并触发终末分化。 初步数据表明，加塔-1可以诱导p21基因转录。 我们将通过以下三个具体目标来检验p21基因是加塔-1的直接转录靶点的假设：1）使用报告分析和电泳迁移率变化来鉴定p21启动子中加塔- 1刺激所需的DNA序列2）通过使用染色质免疫沉淀测试内源性p21启动子的加塔- 1占有率，确定p21基因是否是分化MEL细胞中的加塔-1靶标;和3）确定p21基因是否是未分化MEL细胞中PU. 1介导的加塔-1抑制的靶。 我们将研究是否减少PU.1水平与短干扰RNA（siRNA）去抑制p21基因转录和测试占用的p21启动子PU.1和转录辅阻遏物，如Rb。 这些研究将阐明红系细胞中增殖和分化如何协调，以及这种协调在肿瘤发生期间如何被破坏。