CHROMATIN STRUCTURE IN REGULATION OF MAMMALIAN DEVELOPMENTAL GENE EXPRESSION

了解基因表达是如何在一个时间和空间上被控制的， 组织特异性方式是发育生物学的基本问题。 人类珠蛋白家族的个体成员在时间上是 调节，以实现连续生产 个体发育过程中的胚胎、胎儿和成人血红蛋白。 我们 对这些基因在染色质结构上的变化感兴趣， 在发育过程中被激活。 我们已经使用 电泳迁移率变动分析检测相互作用 存在于细胞中的假定的反式作用调节因子之间 细胞核积极转录珠蛋白基因和顺式作用 位于γ-珠蛋白和γ-珠蛋白基因侧翼的序列。 我们检测到 K562核蛋白与 人β珠蛋白启动子的片段。 dna酶I 足迹和核酸外切酶保护试验表明， 这些相互作用发生在许多受调控的 真核基因，即，在CCAAT和ATA序列上， 主要转录起始位点。 一个强结合位点， 真核生物转录因子Sp1在细胞内表达， ε-珠蛋白启动子。 参与其中的蛋白质 在DNA-琼脂糖柱上可以部分解析相互作用。 然而，没有一个是红系特异性。 强结合位点， K562细胞核提取物中存在的红系特异性蛋白质， 位于β-、γ-和 β-珠蛋白基因。 γ-和β-珠蛋白位点 已经显示具有增强子活性的区域， 各自的基因。 这些位点位于DNA的 其显示组织和发育阶段特异性DNA酶1 基因表达时的超敏感位点。 但 因子存在于不同发育阶段的红系细胞中， 阶段 我们将继续进行旨在了解 这种结合的结构和功能意义 DNA因素。

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.