Overexpression of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) is linked to a number of autoimmune diseases and cancer. MIF production has been correlated to the number of CATT repeats in a microsatellite region upstream of the MIF gene. We have characterized the interaction of pituitary-specific positive transcription factor 1 (Pit-1) with a portion of the MIF promoter region flanking a microsatellite polymorphism (−794 CATT_5–8). Using fluorescence anisotropy, we quantified tight complex formation between Pit-1 and an oligonucleotide consisting of eight consecutive CATT repeats (8xCATT) with an apparent K_d of 35 nM. Using competition experiments we found a 23 base pair oligonucleotide with 4xCATT repeats to be the minimum DNA sequence necessary for high affinity interaction with Pit-1. The stoichiometry of the Pit-1 DNA interaction was determined to be 2:1 and binding is cooperative in nature. We subsequently structurally characterized the complex and discovered a completely novel binding mode for Pit-1 in contrast to previously described Pit-1 complex structures. The affinity of Pit-1 for the CATT target sequence was found to be highly dependent on cooperativity. This work lays the groundwork for understanding transcriptional regulation of MIF and pursuing Pit-1 as a therapeutic target to treat MIF-mediated inflammatory disorders.

中文翻译：

---

Pit-1与巨噬细胞迁移抑制因子启动子中CATT重复的新型合作结合模式的生化和结构表征

促炎性细胞因子巨噬细胞迁移抑制因子（MIF）的过度表达与多种自身免疫性疾病和癌症有关。MIF的产生与MIF基因上游的微卫星区域中CATT重复的数量有关。我们已经表征了垂体特异性正转录因子1（Pit-1）与MIF启动子区域的侧翼微卫星多态性（−794 CATT _5-8）的相互作用。使用荧光各向异性，我们定量了Pit-1和寡核苷酸之间紧密紧密的复合物形成，该寡核苷酸由八个连续的CATT重复序列（8xCATT）和一个明显的K _d组成为35 nM。通过竞争实验，我们发现具有4xCATT重复序列的23个碱基对的寡核苷酸是与Pit-1进行高亲和力相互作用所需的最小DNA序列。Pit-1 DNA相互作用的化学计量确定为2：1，结合本质上是协作的。我们随后在结构上对复合物进行了表征，并发现了与先前描述的Pit-1复合物结构相反的Pit-1全新结合模式。发现Pit-1对CATT靶序列的亲和力高度依赖于协同作用。这项工作为理解MIF的转录调控和追求Pit-1作为治疗MIF介导的炎性疾病的治疗靶标奠定了基础。