REGULATION OF ERYTHROID GENE EXPRESSION

红系转录因子GATA-1与DNA的相互作用是我们研究的重点。用核磁共振技术研究了GATA-1与DNA结合的C-末端锌指的三维结构。然而，GATA-1有两个锌指，我们已经证明N末端的手指在DNA结合中也起着重要的作用。这两个锌指相互作用的方式可以导致DNA结合的增强或抑制，这取决于结合位点的序列。我们已经证明，GATA-1采用不同的结合位点特异的构象；这些变化可以通过电泳迁移率改变分析中的迁移或对蛋白酶的不同抗性来检测。我们还表明，当GATA-1与一些DNA位点结合时，不能刺激转录，这表明变构调节。已经确定了几个与GATA-1的锌指相互作用的关键辅因子，它们与GATA-1结合的能力可能受GATA-1对DNA的反应所采用的构象控制。因此，我们试图用X射线结晶学来解决两个GATA-1锌指在一些DNA结合位点上的结构。在许多物种的GATA-1启动子中，两个紧密排列的GATA位点是保守的，对于GATA-1在小鼠中的正确表达是必不可少的。我们已经证明，这种特殊的结合位点排列在支持GATA-1的反式激活方面是非常有效的。在红系发育过程中，GATA-2在GATA-1之前表达，它与这个位点的相互作用方式是独特的，这取决于GATA-2的N-末端手指的独特结合性质。我们正在鉴定这两个蛋白质与这个结合位点的相互作用，试图了解这个结合位点效率的基础，以及这两个GATA因子与它的差异相互作用。鸡叶酸受体基因(Fr)位于β-珠蛋白基因的上游，位于染色质边界的另一侧，即鸡HS4绝缘体元件。Fr和β-珠蛋白基因都受到GATA因子的调控，尽管它们在发育过程中表达的时间不同。Fr启动子中的GATA位点对于fr在红系前体细胞系中拷贝数依赖的表达至关重要，这种依赖的基础正在研究中。它可能是发育阶段特异的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.