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。脊椎动物加塔因子具有两个锌指，其包含DNA结合结构域。C-末端指是主要的DNA结合指，并且已经通过NMR解析了加塔-1与DNA结合的该指的三维结构。该结构揭示了一个与DNA大沟结合的指状和螺旋，以及一个与小沟结合的相邻基本臂。然而，加塔-1 N-末端指在DNA结合和蛋白质功能中也起重要作用。干扰其与DNA结合能力的N指突变与人类和转基因小鼠的贫血有关。加塔-1的两个锌指以可导致DNA结合增强或抑制的方式相互作用，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，是结合位点特异性，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。这些变化不是由于DNA弯曲，因为我们已经确定加塔-1以不依赖于结合位点的方式弯曲DNA。我们还证明了加塔-1不能刺激转录时，结合到一些DNA位点，这表明变构调节。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结合的能力可能受到加塔-1响应DNA而采取的构象的影响。因此，我们试图通过X射线晶体学来解决连接的加塔-1锌指在许多DNA结合位点上的结构。有了一个结合位点，我们就有了在5.5埃处起作用的共晶体，我们正致力于改善这些结果。同时，我们已经采取了一种生物化学的方法来显示，N-末端的手指与DNA相互作用的方式类似的C-手指。C指螺旋中的三个氨基酸与DNA进行碱基特异性接触，并且我们已经表明，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，两个手指的DNA识别模式是相似的。此外，N-和C-末端指的结合特异性可能有些不同。使用加塔-1 C-指、与C-指的基本臂融合的加塔-1 N-指或加塔-2 N-指的结合位点选择实验表明，加塔-2的N-指优选含有GATC的位点，而测试的两个加塔-1指优选含有加塔的位点。然而，将加塔-1 C-指的基本臂融合到加塔-2 N-指改变了从GATC到加塔的偏好，表明加塔-1基本臂控制核心结合位点的最后一个碱基处的特异性。因为一些生物学上重要的加塔结合位点含有GATC序列，所以在这些位点的DNA识别模式是重要的。加塔蛋白的N-指在这些位点可能特别重要。加塔-3的N-指对DNA的识别对于某些基因的调节也很重要。IL 13基因启动子中的三个加塔识别序列形成两个加塔-3分子的高亲和力结合位点。所有这三个位点对于该启动子在限制性加塔-3浓度下的完全活性是必需的，并且N-指参与与这些位点的结合。IL 5基因还含有一些回文加塔结合位点，这些位点对基因表达很重要，并且需要加塔-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.