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

描述（由申请人提供）：红细胞克虏伯样因子（EKLF）是唯一已知的红细胞特异性转录因子，这种锌指蛋白对于正确的γ -到β -珠蛋白基因转换至关重要。尽管EKLF在几年前就被发现，并且对其功能已经有了很多了解，但我们对这种关键转录因子在体内调节珠蛋白基因表达的方式的理解仍然存在很大差距。对EKLF作用机制的更详细的了解将提供对珠蛋白基因调控的见解，并将为维持或重新激活人类胎儿珠蛋白基因表达的血红蛋白病（如-地中海贫血和镰状细胞病）患者提供新的方法。与本提案中描述的EKLF结合谱一致的珠蛋白基因转换新模型如下。EKLF在原始红细胞和终末红细胞中形成不同的复合物，这些复合物直接影响珠蛋白基因的转换。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。在最终细胞中，形成新的EKLF复合物（可能是由于EKLF蛋白水平增加3倍），该复合物特异性地与成人基因CACCC盒结合。在这种情况下，更远的成年基因优先与LCR相互作用，因此在高水平上表达。该建议的中心假设是原始和最终红细胞中的EKLF复合物是不同的，这些差异对于正确的珠蛋白基因转换是必不可少的。为验证这一假设而设计的具体目标是：用串联亲和纯化（Tandem affinity Purification， TAP）标签对内源性EKLF转录因子基因进行亲和标记，并产生适于该修饰的纯合子小鼠。2. 目的：从TAP标记小鼠的原始和最终红系组织中纯化蛋白质复合物，并用质谱法确定这些复合物的成分。3. 确定EKLF复合物在珠蛋白基因转换中的作用。这些实验将为球蛋白基因转换提供新的机制见解，并为开发镰状细胞病等血红蛋白病的新疗法提供基础。

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.