TRANSCRIPTIONAL REGULATION OF HEMOGLOBIN SWITCHING

血红蛋白病（如镰状细胞病）和地中海贫血是 红细胞功能缺陷，表现为中度至 危及生命的贫血胎儿珠蛋白的重新激活提供了一种 这些成年患者的治疗益处， β-珠蛋白链（β-地中海贫血）或通过干扰 聚合或突变血红蛋白（镰状细胞病）。这样的课程 在胎儿遗传性持续存在的条件下自然发现 血红蛋白（HPFH）或通过药物干预， 血红蛋白在审计中以异常高的水平表达。 控制β-样珠蛋白合成的研究已经成功地 专注于破译每个基因的机制， 最大限度地表达了集群。一个至关重要的 转录激活因子是红细胞Kruppel因子（EKLF）。EKLF是一个 与成人β-珠蛋白CACCC元件结合的锌指蛋白， 是优先建立高水平β-珠蛋白所必需的 表情EKLF的基因切除导致了深刻的β- 地中海贫血和胚胎死亡时的遗传开关到成人 珠蛋白合成分子数据表明，EKLF不能很好地结合到 胎儿γ-珠蛋白CAC位点，这是由于与 β珠蛋白CAC位点）的序列的一部分，该部分被预测为 与EKLF的finger-1相互作用。因此，问题仍然是， 占据胎儿CACCC元件的蛋白质的身份。 除了其内在的相关性，以照亮机械细节， 珠蛋白发育调控，相关蛋白的鉴定 增加了它可以用来转录激活 成人红系细胞内的胚胎和/或胎儿珠蛋白基因。 改善镰状细胞病和β- 地中海贫血提供了一个相当大的临床理由， 只是这个目标，但也为了研究一种方法，利用现有的 以达到同样的目的。本提案通过以下方式解决这些问题： 具体目标如下：1.将设计一种修饰的EKLF蛋白， 这样它将以高亲和力识别γ-珠蛋白CAC位点 通过用EKLF指-1突变残基，其变化将正确地 改变靶特异性; 2）γ-珠蛋白CAC-位点结合 蛋白质将通过分离EKLF样指状蛋白来鉴定， 表达胚胎/胎儿珠蛋白基因的来源。 这些目标的最终结果将是提供一个转录 将测试其重新激活胎儿珠蛋白的能力的试剂 在成人红细胞环境中的基因。这些研究将有助于 通过造血核心设施的服务， 与该中心的其他合作研究者合作。

Hemoglobinopathies (such as sickle cell disease) and thalassemias are defects of red blood cell function that are manifested as moderate to life-threatening anemias. Reactivation of fetal globin provides a therapeutic benefit to these adult patients by compensating for absent beta-globin chains (in beta-thalassemia) or by interfering with polymerization or mutant hemoglobin(in sickle cell disease). Such a course is naturally found under conditions of hereditary persistence of fetal hemoglobin (HPFH) or by pharmacological intervention, whereby fetal hemoglobins are expressed at abnormally high levels in the audit. Studies on the control of beta-like globin synthesis have successfully focused on deciphering the mechanisms by which each gene within the cluster is maximally expressed. One of the critically important transcriptional activators is Erythroid Kruppel Factor (EKLF). EKLF is a zinc finger protein that binds to the adult beta-globin CACCC element and is required for preferentially establishing high levels of beta-globin expression. Genetic ablation of EKLF results in a profound beta- thalassemia and embryonic death at the time of the genetic switch to adult globin synthesis. Molecular data indicates that EKLF does not bind well to the fetal gamma-globin CAC site due to a subtle difference compared to the beta globin CAC site) in the portion of the sequence that is predicted to interact with finger-1 of EKLF. As a result, the question remains as to the identity of the protein(s) that occupies the fetal CACCC element. Apart from its intrinsic relevance to illuminating mechanistic details of globin developmental regulation, identification of the relevant protein raises the possibility that it can be used to transcriptionally reactivate the embryonic and/or fetal globin gene within the adult erythroid cell. Ameliorating the life-threatening effects of sickle cell disease and beta- thalassemias provides a considerable clinical rationale for pursuing not just this goal, but also for examining a way to utilize already available reagents to achieve the same end. This proposal addresses these issues by the following specific aims: 1. A modified EKLF protein will be designed such that it will recognize, with high affinity, the gamma-globin CAC site by mutagenesis of residues with EKLF finger-1 whose changes will correctly changes the target specificity; 2) The gamma-globin CAC-site binding protein will be identified by isolation of EKLF-like finger proteins from sources that are expressing the embryonic/fetal globin genes. The end results of these aims will be to make available a transcriptional reagent that will be tested for its ability to reactivate the fetal globin gene within the adult erythroid environment. These studies will be aided immensely by the services of the Hematopoiesis Core Facility and collaborations with the other co-investigators in the Center.