A Comparative Approach To Globin Regulation/Thalassemia

描述（由申请人提供）：血红蛋白产生是一种进化上保守的过程，对红细胞谱系具有特异性。我们已经利用斑马鱼作为研究这一过程的模型。我们对低色素小红细胞性贫血突变鱼的分析为调节血红蛋白合成的因素提供了关键的见解。这些因素分为四类：血红素的生产，铁的利用，珠蛋白的表达，和铁硫簇的生产。我们已经发现了几个新的基因参与血红蛋白的生产，包括ferroportin，mitoferrin和谷氧还蛋白5。这些因子中的前两个在人类疾病患者中被发现发生突变，从而将斑马鱼系统建立为人类疾病的模型。到目前为止，我们已经发现了超过八个互补组的低色素性贫血。在这个提议中，我们计划通过表征斑马鱼的珠蛋白位点，包括其相关的染色质结构来扩展我们的观察。还将审查监管要素。斑马鱼基因座控制区（LCR）是一个代表？LCR。在转基因中，使斑马鱼LCR与珠蛋白基因并置驱动红系细胞内的高水平表达。转基因鱼正在与人类珠蛋白BAC创建，以评估监管元件的保护。突变的仙粉黛缺乏胚胎珠蛋白的生产，但恢复并具有正常的成人血红蛋白形成。该基因定位于3号染色体上的珠蛋白基因座，并且没有珠蛋白编码区突变。一个高分辨率的定位克隆项目正在进行中，以确定这种新的胚胎特异性珠蛋白调控突变。我们还开发了一种方法，用于遗传分析调节血红蛋白产生的染色质因子。将针对特定基因的反义寡核苷酸注射到单细胞胚胎中的吗啉代技术可以敲低基因功能，并将揭示红细胞生成的变化。已经创建了超过300种作为人类基因直系同源物的染色质重塑因子的纲要，并且将通过在斑马鱼胚胎中的基因敲低来研究每种因子。将评价基因敲减对红系基因表达（包括LCR活化和珠蛋白基因表达）的影响。这些因子将在斑马鱼中进行遗传学研究，并通过双敲低和过表达研究与特定转录因子的相互作用。使用人类红系细胞，我们计划生化纯化通过这种遗传筛选发现的染色质因子直向同源物的复合物。对影响血红蛋白生成的染色质因素的了解将对我们对血红蛋白生成的理解以及对地中海贫血、镰状细胞性贫血和珠蛋白突变患者的治疗产生巨大影响。

DESCRIPTION (provided by applicant): Hemoglobin production is an evolutionarily conserved process that is specific to the red blood cell lineage. We have utilized the zebrafish as a model for studying this process. Our analysis of mutant fish with a hypochromic microcytic anemia has provided key insights into factors that regulate hemoglobin synthesis. These factors fall into four categories: the production of heme, iron utilization, globin expression, and iron sulfur cluster production. We have found several novel genes involved in hemoglobin production, including ferroportin, mitoferrin, and glutaredoxin 5. The first two of these factors were found to be mutated in patients with human disorders, establishing the zebrafish system as a model for human disease. To date, we have uncovered over eight complementation groups of hypochromic anemia. In this proposal, we plan to extend our observations by characterizing the globin locus of the zebrafish, including its associated chromatin structure. Regulatory elements will also be examined. The zebrafish locus control region (LCR) is representative of an ?LCR. In transgenics, bringing the zebrafish LCR into apposition with globin genes drives high level of expression within erythroid cells. Transgenic fish are being created with human globin BACs to evaluate conservation of regulatory elements. The mutant zinfandel lacks embryonic globin production, but recovers and has normal adult hemoglobin formation. This gene was mapped to the globin locus on chromosome 3 and no globin coding regions are mutated. A high-resolution positional cloning project is underway to define this novel embryonic-specific globin regulatory mutation. We also developed a method for genetically analyzing chromatin factors that regulate hemoglobin production. The technology of morpholinos in which antisense oligonucleotides against specific genes are injected into the one cell embryo can knock-down gene function and will reveal changes in erythropoiesis. A compendium of over 300 chromatin remodeling factors that are orthologs of human genes has been created, and each will be studied by gene knock-down in zebrafish embryos. The effect of gene knock-down on erythroid gene expression (including LCR activation and globin gene expression) will be evaluated. These factors will be studied genetically in the zebrafish and interactions with specific transcription factors will be investigated by double knock-down and overexpression studies. Using human erythroid cells, we plan to biochemically purify a complex of chromatin factor orthologs found through this genetic screen. The understanding of chromatin factors that affect hemoglobin production will have a tremendous impact on our understanding of hemoglobin production and for the treatment of patients with thalassemia, sickle cell anemia, and globin mutations.