REGULATION OF ERYTHROID GENE EXPRESSION

红系转录因子加塔-1与DNA的相互作用是我们研究的一个主要焦点。用核磁共振方法解析了与DNA结合的加塔-1的C-末端锌指的三维结构。然而，加塔-1有两个锌指，我们已经表明，N-末端的手指也发挥了重要作用，在DNA结合。两个锌指相互作用的方式，可以导致增强或抑制DNA结合，这取决于结合位点的序列。我们已经表明，加塔-1采用不同的构象，结合位点特异性，这些变化可以检测到电泳迁移率变动测定或差异电阻蛋白酶的迁移。我们还发现，当与某些DNA位点结合时，加塔-1不能刺激转录，这表明了变构调节。已经鉴定了与加塔-1的锌指相互作用的几种关键辅因子，并且它们与加塔-1结合的能力可能受加塔-1响应DNA而采取的构象控制。因此，我们正试图通过X射线晶体学来解决DNA结合位点上的两个加塔-1锌指的结构。两个紧密间隔的加塔位点在许多物种的加塔-1启动子中是保守的，并且对于小鼠中加塔-1的正确表达是必需的。我们已经表明，这种特殊的结合位点的安排是非常有效的支持反式激活的加塔-1。加塔-2在红系发育期间先于加塔-1表达，显示出与该位点相互作用的独特模式，这取决于加塔-2的N-末端指的独特结合特性。我们正在表征这两种蛋白质与该结合位点的相互作用，试图理解该位点的效率的基础以及这两种加塔因子与它的差异相互作用。在染色质边界的相对侧的珠蛋白位点，鸡HS 4绝缘子元件。无论是FR还是？珠蛋白基因受加塔因子调控，尽管它们在发育过程中的不同时间表达。fr启动子内的加塔位点对于红系前体细胞系中fr的拷贝数依赖性表达至关重要，并且正在研究这种依赖性的基础。它可能是发育阶段特异性染色质开放活动的一部分。

The interaction of the erythroid transcription factor, GATA-1 with DNA is a major focus of our research. The three dimensional structure of the C-terminal zinc finger of GATA-1 bound to DNA has been solved by NMR. However, GATA-1 has two zinc fingers and we have shown that the N-terminal finger also plays an important role in DNA binding. The two zinc fingers interact with each other in ways that can lead either to enhancement or to inhibition of DNA binding, depending on the sequence of the binding site. We have shown that GATA-1 adopts different conformations that are binding site specific; these variations can be detected by altered migration in electrophoretic mobility shift assays or by differential resistance to proteases. We have also shown that GATA-1 is unable to stimulate transcription when bound to some DNA sites, suggesting allosteric regulation. Several crucial cofactors that interact with the zinc fingers of GATA-1 have been identified, and their ability to bind to GATA-1 may be controlled by the conformation that GATA-1 adopts in response to DNA. Consequently, we are attempting to solve the structure of the two GATA-1 zinc fingers on a number of DNA binding sites by Xray crystallography. Two closely spaced GATA sites are conserved in the GATA-1 promoters of many species and are essential for correct expression of GATA-1 in mice. We have shown that this particular arrangement of binding sites is extremely efficient at supporting transactivation by GATA-1. GATA-2, which is expressed before GATA-1 during erythroid development, shows a distinct mode of interaction with this site, which is dependent on the unique binding properties of N-terminal finger of GATA-2. We are characterizing the interactions of these two proteins with this binding site, in an attempt to understand the basis for the efficiency of this site and the differential interaction of these two GATA factors with it. The chicken folate receptor gene (fr) lies upstream of the ?-globin locus on the opposite side of a chromatin boundary, the chicken HS4 insulator element. Both fr and the ?-globin genes are regulated by GATA factors, although they are expressed at different times during development. A GATA site within the fr promoter is critical to the copy number dependent expression of fr in an erythroid precursor cell line and the basis of this dependence is being investigated. It could be a part of a developmental stage- specific chromatin opening activity.