TRANSCRIPTIONAL REGULATION OF HEMOGLOBIN SWITCHING

血红蛋白病(如镰状细胞病)和地中海贫血是 表现为中度至中度的红细胞功能缺陷 危及生命的贫血。胎儿珠蛋白的重新激活提供了一个 代偿缺勤对这些成年患者的治疗益处 β-珠蛋白链(在β-地中海贫血中)或通过干扰 聚合或突变的血红蛋白(镰状细胞病)。这样的课程 是在胎儿遗传性持续性的条件下自然发现的 或通过药物干预，从而使胎儿 在审计中，血红蛋白的表达水平异常高。 控制类β珠蛋白合成的研究取得成功 专注于破译基因中的每个基因的机制 簇是最大限度地表达的。其中一个极其重要的因素 转录激活物是红系Kruppel因子(EKLF)。EKLF是一种 与成人β-珠蛋白CACCC元件结合的锌指蛋白和 是优先建立高水平的β-珠蛋白所必需的 表情。EKLF的遗传消融导致了深刻的β- 基因转换为成人时的地中海贫血和胚胎死亡 珠蛋白合成。分子数据表明EKLF不能很好地结合 胎儿伽马珠蛋白CAC部位与 β珠蛋白CAC位点)位于序列的预测部分 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(S)的同一性。 除了它与阐明机械细节的内在关联之外 珠蛋白的发育调控、相关蛋白的鉴定 增加了它可以被用来转录重新激活的可能性 成人红系细胞内的胚胎和/或胎儿珠蛋白基因。 改善镰状细胞病和贝塔病毒对生命的威胁 地中海贫血为寻求NOT提供了相当大的临床理由 就是这个目标，但也是为了研究一种利用已有的方法 试剂也能达到同样的目的。本提案通过以下方式解决这些问题 具体目标如下：1.设计一种修饰的EKLF蛋白 这样它将以高亲和力识别伽马珠蛋白CAC位点 通过用EKLF Finger-1对残基进行突变，其变化将正确 改变靶标的特异性；2)γ-珠蛋白CAC结合 蛋白质将通过分离EKLF样指蛋白来鉴定 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份转录的 将对其重新激活胎儿珠蛋白的能力进行测试的试剂 成人红系环境中的基因。这些研究将得到资助 极大地依靠造血核心设施和 与中心其他合作调查员的合作。

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.