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

描述(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伽马-β-珠蛋白基因转换是必不可少的。虽然EKLF早在几年前就被发现了，对其功能也有了很多了解，但对于这种关键转录因子在体内调节珠蛋白基因表达的方式，我们的理解仍然存在很大差距。更详细地了解EKLF的作用机制将为珠蛋白基因调控提供洞察力，并将为维持或重新激活患有贝塔-地中海贫血和镰状细胞病等血红蛋白疾病患者的胎儿珠蛋白基因表达提供新的方法。一种与本提案中描述的EKLF结合谱相一致的珠蛋白基因切换的新模型如下。EKLF在原始的和确定的红系细胞中形成不同的复合体，这些不同的复合体指导珠蛋白基因的切换。在原始细胞中，EKLF复合体与Y2、H1和主要/次要珠蛋白基因CACCC盒等效结合；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，形成了一个新的EKLF复合体(可能是由于EKLF蛋白水平增加了3倍)，该复合体与成体基因CACCC盒特异结合。在这种情况下，更远端的成体基因优先与LCR相互作用，因此在高水平表达。这一建议的中心假设是，原始和最终红系细胞中的EKLF复合体是不同的，这些差异对于正确的珠蛋白基因切换是必不可少的。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，并产生纯合子的小鼠。2.从TAP标记的小鼠原始红系组织和确定的红系组织中提纯蛋白质复合体，并用质谱仪确定其组成。3.确定EKLF复合体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洞察力，并应为开发治疗镰状细胞病等血红蛋白疾病的新疗法奠定基础。

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.