Role of KLF2 in erythropoiesis and globin expression

描述（由申请人提供）：[KLF 2和EKLF在原始红细胞生成中具有补偿和关键作用。]我们假设KLF 2以红系细胞自主方式发挥作用，直接与珠蛋白基因结合。第一个目的是进一步阐明KLF 2在红细胞生成中的机制作用。[The KLF 2-/-胚胎红系细胞的成熟阶段将用流式细胞术测定来确定，并且将评估这些细胞在体外用外源因子分化的能力。具有条件性敲除（KO）KLF 2基因（KLF 2flox等位基因）的小鼠已经与具有红系特异性启动子的Cre重组酶小鼠（LCR-β pr-Cre）一起繁殖，并且>90%的KLF 2flox等位基因可以缺失。该系统将用于确定KLF 2对原始红系细胞的细胞形态、球蛋白基因表达、分化和凋亡作用是否如预期的那样是细胞自主的。使用该系统，我们还将测试KLF 2在确定性（成人）红细胞生成中的作用，测量相同的参数。ChIP（染色质免疫沉淀）试验将用于确定KLF 2是否与β-珠蛋白基因座中的CACCC元件在体内相互作用，使用表达编码KLF 2的诱导型标记构建体的K562、HEL和MEL细胞系。[If可能的话，将对天然小鼠红细胞前体进行ChIP测定]。第二个目的是使用[我们建立的]双KO小鼠模型，从机制上研究KLF 2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，[EKLF-/-KLF 2-/-]胚胎在胚胎第10.5天严重贫血，此时单个科斯似乎未受影响。为了评估EKLF-/-KLF 2-/-胚胎红系细胞的成熟和分化，将使用流式细胞术和祖细胞测定。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-/-KLF 2和EKLF KLF 2-/-与野生型、EKLF-/-、KLF 2-/-和EKLF-/-KLF 2-/-胚胎进行比较。我们将使用具有人β-珠蛋白基因座的小鼠，确定与野生型相比，EKLF-/-KLF 2-/-胚胎是否具有减少的γ-珠蛋白mRNA。[EKLF-/-KLF 2-/-卵黄囊具有异常的内皮细胞，这在单个KO卵黄囊中不是这种情况。我们将仅在红系细胞中产生缺乏EKLF和KLF 2的胚胎，以确定内皮缺陷是否由红系细胞指导。我们的长期目标是使用KLF 2的策略，以增加胚胎/胎儿β-珠蛋白，治疗β-血红蛋白病患者。

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.