Role of KLF2 in erythropoiesis and globin expression

描述(申请人提供)：[KLF2和EKLF在原始红细胞生成中具有补偿和关键作用。]我们假设KLF2以红系细胞自主的方式发挥作用，直接与珠蛋白基因结合。第一个目的是进一步阐明KLF2在红细胞生成中的机制作用。[将用流式细胞仪测定KLF2/-胚胎红系细胞的成熟阶段，并评估这些细胞在体外与外界因素分化的能力。]带有KLF2基因条件性敲除(KO)的小鼠(KLF2flx等位基因)已经与带有红系特异性启动子的Cre重组酶小鼠(LCR-beta Pr-Cre)繁殖，并且90%的KLF2flx等位基因可以被删除。该系统将用于确定KLF2对原始红系细胞的细胞形态、珠蛋白基因表达、分化和凋亡作用是否如预期的那样是细胞自主的。使用这个系统，我们还将测试KLF2在最终(成人)红细胞生成中的作用，测量相同的参数。使用K562、HEL和MEL细胞株表达编码KLF2的可诱导、标记的构建体，将使用CHIP(染色质免疫沉淀)分析来确定KLF2是否在体内与β-珠蛋白基因中的CACCC元件相互作用。[如果可能，将对本地小鼠红系前体进行芯片检测]。第二个目的是利用我们建立的双KO小鼠模型，从机制上研究KLF2和EKLF在红细胞生成中的代偿作用。EKLF在原始红细胞生成中表达，在胚胎第10.5天，当单个KO看起来未受影响时，[EKLF-/-KLF2-/-胚胎]出现严重贫血。为了评估EKLF-/-KLF2-/-胚胎红系细胞的成熟和分化，将使用流式细胞术和祖细胞分析。]为了研究基因剂量对红细胞形态和珠蛋白基因表达的影响，将EKLF-/-KLF2和EKLF2-/-与野生型、EKLF-/-、KLF2-/-和EKLF-/-KLF2-/-胚胎进行比较。我们将使用具有人类β-珠蛋白基因的小鼠来确定EKLF-/-KLF2-/-胚胎与野生型相比是否降低了伽马珠蛋白mRNA。[EKLF-/-KLF2-/-卵黄囊内皮细胞异常，与单个KO卵黄囊不同。我们将只在红系细胞中产生缺失EKLF和KLF2的胚胎，以确定内皮缺陷是否由红系细胞引导。]我们的长期目标是将KLF2用于增加胚胎/胎儿β-珠蛋白的策略，以治疗患有β-血红蛋白疾病的患者。

DESCRIPTION (provided by applicant): [KLF2 and EKLF have compensatory and critical roles in primitive erythropoiesis.] We hypothesize that KLF2 acts in an erythroid cell autonomous manner, directly binding to globin genes. The first aim is to further elucidate the mechanistic role of KLF2 in erythropoiesis. [The stage of maturation of KLF2-/- embryonic erythroid cells will be determined with a flow cytometry assay, and the ability of these cells to differentiate in vitro with exogenous factors will be assessed.] Mice with a conditional knockout (KO) the KLF2 gene (KLF2flox allele) have been bred with Cre recombinase mice with an erythroid-specific promoter (LCR-beta pr-Cre), and >90% of the KLF2flox alleles can be deleted. This system will be used to determine whether the cell morphology, globin gene expression, differentiation and apoptotic effects of KLF2 on primitive erythroid cells are cell autonomous, as expected. Using this system, we will also test the role of KLF2 in definitive (adult) erythropoiesis, measuring the same parameters. ChIP (chromatin immunoprecipitation) assays will be used to determine if KLF2 interacts with CACCC elements in the beta-globin locus in vivo, using K562, HEL and MEL cell lines expressing an inducible, tagged construct encoding KLF2. [If possible, ChIP assays will be performed on native mouse erythroid precursors]. The second aim is to mechanistically study the compensatory roles of KLF2 and EKLF in erythropoiesis using [our established] double KO mouse model. EKLF is expressed in primitive erythropoiesis, and [EKLF-/-KLF2-/- embryos are severely anemic at embryonic day 10.5, when single KOs appear unaffected. To assess the maturation and differentiation of EKLF-/-KLF2-/- embryonic erythroid cells, flow cytometry and progenitor assays will be used.] To study the effects of gene dosage on red cell morphology and globin gene expression, EKLF-/-KLF2 and EKLF KLF2-/- will be compared to wildtype, EKLF-/-, KLF2-/- and EKLF-/-KLF2-/- embryos. We will determine whether EKLF-/-KLF2-/- embryos have reduced gamma-globin mRNA compared to wildtype, using mice with the human beta-globin locus. [EKLF-/-KLF2-/- yolk sacs have abnormal endothelial cells, which is not the case in single KO yolk sacs. We will generate embryos lacking EKLF and KLF2 in erythroid cells only, to determine whether the endothelial defects are directed by erythroid cells.] Our long-term goal is to use KLF2 in strategies to increase embryonic/fetal beta-globin, to treat patients with beta-hemoglobinopathies.