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。脊椎动物GATA因子有两个锌指，包括DNA结合域。c端指是主要的DNA结合指，GATA-1与DNA结合的c端指的三维结构已经通过核磁共振得到了解析。该结构揭示了一个手指和螺旋结合DNA的主要凹槽和一个相邻的基本臂结合在次要凹槽。然而，GATA-1 n端指在DNA结合和蛋白质功能中也起着重要作用。干扰n指与DNA结合能力的突变与人类和转基因小鼠的贫血有关。GATA-1的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具有结合位点特异性的不同构象，这些变化可以通过电泳迁移率转移试验中的迁移改变或对蛋白酶的不同抗性来检测。这些变化不是由于DNA弯曲，因为我们已经确定GATA-1以一种与结合位点无关的方式弯曲DNA。我们还证明GATA-1在与某些DNA位点结合时不能刺激转录，这表明它具有变构调节作用。已经确定了与GATA-1的锌指相互作用的几个关键辅因子，并且它们与GATA-1结合的能力可能受到GATA-1响应DNA时采用的构象的影响。因此，我们正试图通过x射线晶体学来解决一些DNA结合位点上连接的GATA-1锌指的结构。通过一个结合位点，我们得到了衍射5.5埃的共晶体，我们正致力于改进这些结果。同时，我们采用了生化方法来证明n端手指与DNA的相互作用方式类似于c指。c指螺旋中的三个氨基酸与DNA进行碱基特异性接触，我们已经证明，n指与DNA结合需要两个类似位置的n指氨基酸。这有力地表明，两根手指的DNA识别模式是相似的。此外，N端和c端手指的结合特异性可能有所不同。采用GATA-1型c指、GATA-1型n指与c指基本臂融合或GATA-2型n指进行的结合位点选择实验表明，GATA-2型的n指更倾向于含有GATC的位点，而GATA-1型的手指更倾向于含有GATA的位点。然而，将GATA-1 c指的基本臂与GATA-2 n指融合后，GATA-1的特异性从GATC转向GATA，表明GATA-1的基本臂控制了核心结合位点最后一个碱基的特异性。由于一些生物学上重要的GATA结合位点包含GATA序列，因此这些位点的DNA识别模式是重要的。GATA蛋白的n指可能在这些位点上特别重要。GATA-3的n指识别DNA对某些基因的调控也很重要。IL13基因启动子中的三个GATA识别序列形成两个GATA-3分子的高亲和力结合位点。在限制GATA-3浓度的情况下，这三个位点都是该启动子充分发挥活性所必需的，并且n指参与了与这些位点的结合。IL - 5基因还含有一些对基因表达重要的回文GATA结合位点，这些位点需要GATA-3的n指。

The interaction of the erythroid transcription factor, GATA-1 with DNA is a major focus of our research. Vertebrate GATA factors have two zinc fingers that comprise the DNA binding domain. The C-terminal finger is the main DNA binding finger and the three dimensional structure of this finger of GATA-1 bound to DNA has been solved by NMR. The structure reveals a finger and helix that bind to the major groove of DNA and an adjacent basic arm that binds in the minor groove. However, the GATA-1 N-terminal finger also plays an important role in DNA binding and in the function of the protein. Mutations in the N-finger that interfere with its ability to bind to DNA are associated with anemia in humans and transgenic mice. The two zinc fingers of GATA-1 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, and these variations can be detected by altered migration in electrophoretic mobility shift assays or by differential resistance to proteases. These variations are not due to DNA bending since we have established that GATA-1 bends DNA in a binding site independent manner. We have also demonstrated 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 influenced by the conformation that GATA-1 adopts in response to DNA. Consequently, we are attempting to solve the structure of the linked GATA-1 zinc fingers on a number of DNA binding sites by Xray crystallography. With one binding site, we have co-crystals that diffract at 5.5 angstroms and we are focusing on improving these results. Meanwhile we have taken a biochemical approach to show that the N-terminal finger interacts with DNA in a manner similar to the C-finger. Three amino acids in the C-finger helix make base specific contacts with DNA, and we have shown that two analogously positioned N-finger amino acids are required for N-finger binding to DNA. This strongly suggests that the mode of DNA recognition is similar for both fingers. In addition, the binding specificity of the N- and C-terminal fingers may be somewhat different. Binding site selection experiments using the GATA-1 C-finger, the GATA-1 N-finger fused to the basic arm of the C-finger, or the GATA-2 N-finger, show that the N-finger of GATA-2 prefers sites containing GATC while both of the GATA-1 fingers tested prefer GATA containing sites. However, fusing the basic arm of the GATA-1 C-finger to the GATA-2 N-finger changes the preference from GATC to GATA, suggesting that the GATA-1 basic arm controls the specificity at the last base of the core binding site. Because some biologically important GATA binding sites contain the GATC sequence, the mode of DNA recognition at these sites is significant. The N-finger of the GATA proteins may be particularly important at these sites. DNA recognition by the N-finger of GATA-3 is also important for the regulation of some genes. Three GATA recognition sequences in the IL13 gene promoter form a high affinity binding site for two molecules of GATA-3. All three sites are necessary for full activity of this promoter at limiting GATA-3 concentrations and the N-finger is involved in binding to these sites. The IL 5 gene also contains some palindromic GATA binding sites that are important for gene expression and require the N-finger of GATA-3.