CHROMATIN STRUCTURE IN REGULATION OF MAMMALIAN DEVELOPMENTAL GENE EXPRESSION

哺乳动物发育基因表达调节中的染色质结构

• 批准号: 3917369
• 负责人: A DEAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3917369
• 关键词: DNA binding protein; DNA footprinting; chemical binding; chromatin; developmental genetics; endonuclease; gene expression; gene interaction; genetic promoter element; genetic regulation; globin; nucleic acid sequence; nucleoproteins

Understanding how gene expression is controlled in a temporal and tissue specific manner is a basic problem in developmental biology. The individual members of the human globin family are temporally regulated so as to bring about the sequential production of embryonic, fetal, and adult hemoglobins during ontogeny. We are interested in the changes in chromatin structure that these genes undergo when they are activated during development. We have used the electrophoretic mobility shift assay to detect interactions between putative trans-acting regulatory factors present in the nuclei of cells actively transcribing globin genes and cis-acting sequences flanking the epsilon and gamma globin genes. We detected the formation of several complexes between K562 nuclear protein and a fragment of the human epsilon globin promoter. DNAse I footprinting and exonuclease protection assays suggested some of these interactions occurred at elements common to many regulated eukaryotic genes, i.e., at the CCAAT and ATA sequences and over the major transcription initiation site. One strong binding site for the general eukaryotic transcription factor Spl was observed in the epsilon-globin promoter. The proteins participating in these interactions could be partially resolved on DNA-agarose columns. however, none was erythroid specific. Strong binding sites for an erythroid specific protein present in K562 nuclear extracts were located in the 3' flanking regions of the epsilon-, gamma-, and beta-globin genes. The gamma- and beta-globin sites corresponded to regions which have been shown to possess enhancer activity for their respective genes. These sites are situated in regions of DNA which display tissue and developmental stage specific DNAse 1 hypersensitive sites when the genes are expressed. However, the factor is present in erythroid cells of different developmental stages. We will pursue studies aimed at understanding the structural and functional significance of the binding of this factor to DNA.

了解基因表达如何在时间和时间上受到控制 组织特异性方式是发育生物学中的一个基本问题。 人类珠蛋白家族的个体成员在时间上 进行调整以实现……的连续生产 个体发育过程中的胚胎、胎儿和成人的血红蛋白。我们是 感兴趣的是这些基因染色质结构的变化 当它们在发育过程中被激活时会经历。我们已经使用了 检测相互作用的凝胶迁移率改变分析 在假定的反式作用调节因子之间存在于 主动转录珠蛋白基因和顺式作用的细胞核 位于epsilon和Gamma珠蛋白基因两侧的序列。我们检测到 K562核蛋白与K562核蛋白形成的几种复合体 人类epsilon珠蛋白启动子的片段。DNA酶I 足迹和核酸外切酶保护分析表明 这些相互作用发生在许多受调控的元素中 真核基因，即在CCAAT和ATA序列上以及在 主要转录起始点。一个强结合位点 在小鼠中观察到了普遍的真核转录因子Sp1 Epsilon-珠蛋白启动子。参与这些活动的蛋白质 相互作用可以在DNA-琼脂糖柱上部分分离。 然而，没有一种是红系特异性的。An的强结合位点 K562核提取液中存在红系特异性蛋白 位于epsilon-、Gamma-和 β-珠蛋白基因。伽马和贝塔球蛋白的位置对应 到已被证明具有增强基因活性的区域 它们各自的基因。这些位点位于DNA区域 显示组织和发育阶段特异的DNase1 基因表达时的过敏性部位。然而， 因子存在于不同发育阶段的红系细胞中 各阶段。我们会继续进行研究，以了解 这一约束的结构和功能意义 与DNA有关的因素。