Erythroid Krupple-Like Factor Complexes Defined in TAP-Tagged Knockin Mice

描述（由申请人提供）：红系Krupple样因子（EKLF）是唯一已知的红系特异性转录因子，这种锌指蛋白对于正确的γ-珠蛋白至β-珠蛋白基因转换是必需的。虽然EKLF被发现了数年前，已经了解了很多关于它的功能，仍然存在很大的差距，在我们的理解中，这一关键的转录因子调节珠蛋白基因的表达在体内。更详细地了解EKLF的作用机制将提供珠蛋白基因调控的见解，并将提出新的方法来维持或重新激活血红蛋白病患者，如β-地中海贫血和镰状细胞病的人胎儿珠蛋白基因的表达。一种新的珠蛋白基因转换模型，这是与EKLF结合在这个建议中描述的档案是一致的如下。EKLF在原始和定形红系细胞中形成不同的复合物，这些不同的复合物指导珠蛋白基因转换。在原始细胞中，EKLF复合物与？y2，？H1和？主要/次要珠蛋白基因CACCC盒等效;然而，？Y2和？h1基因优先表达，因为这些基因更接近LCR。在定形细胞中，形成新的EKLF复合物（可能是EKLF蛋白水平增加3倍的结果），该复合物特异性结合成人基因CACCC盒。在这种情况下，更远端的成体基因优先与LCR相互作用，因此以高水平表达。该建议的中心假设是原始和定形红系细胞中的EKLF复合物是不同的，并且这些差异对于正确的珠蛋白基因转换是必不可少的。测试这一假设的具体目的是：1。用几个串联亲和纯化（TAP）标签亲和标记内源性EKLF转录因子基因，并产生这种修饰的纯合小鼠。2.从TAP标记小鼠的原始和定形红系组织中纯化蛋白复合物，并通过质谱法确定这些复合物的组分。3.探讨EKLF复合物在珠蛋白基因转换中的作用。这些实验将为珠蛋白基因转换提供新的机制见解，并为血红蛋白病（如镰状细胞病）的新疗法的开发提供基础。

DESCRIPTION (provided by applicant): Erythroid Krupple Like Factor (EKLF) is the only known erythroid-specific transcription factor, and this zinc finger protein is essential for correct gamma to beta-globin gene switching. Although EKLF was discovered a number of years ago and much has been learned about its function, large gaps still exist in our understanding of the way in which this critical transcription factor regulates globin gene expression in vivo. A more detailed understanding of the mechanism of EKLF action will provide insights into globin gene regulation and will suggest novel methods for maintaining or reactivating human fetal globin gene expression in patients with hemoglobinopathies such as beta-thalassemia and sickle cell disease. A novel model for globin gene switching that is consistent with the EKLF binding profile described in this proposal is as follows. EKLF forms different complexes in primitive and definitive erythroid cells and these different complexes direct globin gene switching. In primitive cells, the EKLF complex binds to ?y2, ?h1 and ?-major/minor globin gene CACCC boxes equivalently; however, ?y2 and ?h1 genes are preferentially expressed because these genes are more proximal to the LCR. In definitive cells, a new EKLF complex is formed (perhaps as a result of the 3-fold increase in EKLF protein levels) and this complex binds specifically to the adult gene CACCC boxes. In this case, the more distal adult genes preferentially interact with the LCR and are consequently expressed at high levels. The central hypothesis of this proposal is that EKLF complexes in primitive and definitive erythroid cells are different and that these differences are essential for correct globin gene switching. The Specific Aims designed to test this hypothesis are: 1. To affinity tag the endogenous EKLF transcription factor gene with several Tandem Affinity Purification (TAP) tags and to produce mice that are homozygous for this modification. 2. To purify protein complexes from primitive and definitive erythroid tissues of the TAP tagged mice and to define the components of these complexes by mass spectrometry. 3. To determine the role of EKLF complexes in globin gene switching. These experiments will provide new mechanistic insights into globin gene switching and should provide a foundation for the development of new therapies for hemoglobinopathies such as sickle cell disease.