Opposing and complementary roles of KLF1 and KLF2 in erythropoiesis

这项建议的目的是研究Kr样因子1(EKLf或EKLf)的相反和互补作用 KLF1)和KLF2在胚胎和胎儿红细胞生成中的作用。这在生物学上很重要，因为它可以促进 针对血红蛋白疾病的有效治疗策略。KLF1和KLF2是密切相关的转录 激活小鼠胚胎样珠蛋白基因的因素。目标1是定义KLF1和KLF2在 ？-珠蛋白基因表达。KLF1结合体外分化的CD34+人脐血中的珠蛋白启动子 细胞，但与其在胚胎珠蛋白基因表达中的作用不同，它负调控 胎儿细胞。KLF2与胎儿细胞中的珠蛋白启动子紧密结合，符合CACCC共识 地点。我们将使用KLF2基因敲除(KD)来确定KLF2是否正向调节珠蛋白的表达。 在体外分化的人脐血CD34+细胞中高表达。假设KLF1有一个 排除KLF2结合对珠蛋白基因表达的净负面影响将通过击倒 KLF1在体外分化为CD34+细胞。目标2是定义KLF1和KLF2在 在胚胎和胎儿红系细胞中组织珠蛋白基因座的整体染色质结构。Kd In In 将使用体外分化的CD34+人脐血细胞和基因敲除(KO)小鼠模型来确定 如果需要KLF1和KLF2来将BRG1和CBP/p300染色质重塑复合体招募到 ？-珠蛋白LCR和启动子。我们将测试是否需要这些因素来形成5‘HS2和 5‘HS3脱氧核糖核酸酶I敏感部位。我们还将确定是否需要KLF1和KLF2 RNA聚合酶II在LCR和？-珠蛋白基因之间的募集和染色质环路。在《目标3》中， 我们将研究KLF1和KLF2对小鼠胚胎红细胞生成的影响。在E10.5，KLF1-/-KLF2- /-(双KO)胚胎面色苍白，贫血，但KLF1-/-和KLF2-/-大体正常。KLF1--KLF2-/- 胚胎的外周血细胞较少，形成原始的红系祖细胞集落，成熟程度较高 速度较慢，且比正常对照组的血红素化程度较低。KLF1和KLF2似乎协调控制着 小鼠原始红系祖细胞的形成和成熟。为了测试这一点，KLF1和KLF2在 确定红系祖细胞集落形成的数量和性质，细胞增殖，成熟和 将使用单KO小鼠胚胎和双KO小鼠胚胎来测试细胞凋亡。根据大体表型 胚胎，我们期望从KLF1-/-KLF2+/-的祖细胞分析中获得更多和更成熟的克隆。 而不是KLF1+/-KLF2-/-。初步数据表明，KLF1和KLF2调节与 扩散。我们将确定KLF1和KLF2靶基因的信使核糖核酸的量，如FOXM1，Myc， KLF1+/-KLF2-/-、KLF1-/-中CD24a抗原、鞘氨醇激酶1、PURA和甲状旁腺激素1受体 KLF2+/-、KLF2-/-、KLF1-/-和KLF1-/-KLF2-/-胚胎，以及来源于红系祖细胞的集落。

The goal of this proposal is to study the opposing and complementary roles of Kr¿ppel-like factor 1 (EKLF or KLF1) and KLF2 in embryonic and fetal erythropoiesis. This is biologically important because it could facilitate effective therapeutic strategies for the hemoglobinopathies. KLF1 and KLF2 are closely related transcription factors that activate the mouse embryonic ¿-like globin genes. Aim 1 is to define the roles of KLF1 and KLF2 in ¿-globin gene expression. KLF1 binds the ¿-globin promoter in in vitro differentiated CD34+ human cord blood cells, but in contrast to its role in embryonic globin gene expression, it negatively regulates the ¿-globin gene in fetal cells. KLF2 binds robustly to the ¿-globin promoter in fetal cells, at the same ¿-globin CACCC consensus site. We will determine whether KLF2 positively regulates ¿-globin expression, using KLF2 knockdown (KD) and overexpression in in vitro differentiated CD34+ human cord blood cells. The hypothesis that KLF1 has a net negative effect on ¿-globin gene expression by precluding KLF2 binding will be tested, by knocking down KLF1 in in vitro differentiated CD34+ cells. Aim 2 is to define the mechanistic roles of KLF1 and KLF2 in organizing the global chromatin structure of the ¿-globin locus in embryonic and fetal erythroid cells. KD in in vitro differentiated CD34+ human cord blood cells and knockout (KO) mouse models will be used to determine if KLF1 and KLF2 are required for recruitment of BRG1 and CBP/p300 chromatin remodeling complexes to the ¿-globin LCR and promoters. We will test whether the factors are required for formation of the 5'HS2 and 5'HS3 DNase I hypersensitive sites. We will also determine whether KLF1 and KLF2 are required for recruitment of RNA polymerase II to, and chromatin looping between, the LCR and ¿-globin genes. In Aim 3, the effects of KLF1 and KLF2 on mouse embryonic erythropoiesis will be investigated. At E10.5, KLF1-/-KLF2- /- (double KO) embryos are pale and anemic, but KLF1-/- and KLF2-/- are grossly normal. KLF1-/-KLF2-/- embryos have less peripheral blood cells, and form primitive erythroid progenitor colonies that mature more slowly and are less hemoglobinized than normal controls. KLF1 and KLF2 appear to coordinately control the formation and maturation of mouse primitive erythroid progenitors. To test this, the roles of KLF1 and KLF2 in determining the number and nature of erythroid progenitor colonies formed, cell proliferation, maturation and apoptosis will be tested using single and double KO mouse embryos. Based on the gross phenotypes of embryos, we expect a larger number and more mature colonies from progenitor assays with KLF1-/-KLF2+/- than with KLF1+/-KLF2-/-. Preliminary data suggests that KLF1 and KLF2 regulate genes involved in proliferation. We will determine the amounts of mRNA for KLF1 and KLF2 target genes, such as FoxM1, Myc, CD24a antigen, sphingosine kinase 1, Pura and parathyroid hormone 1 receptor, in KLF1+/-KLF2-/-, KLF1-/- KLF2+/-, KLF2-/-, KLF1-/- and KLF1-/-KLF2-/- embryos, and in colonies derived from erythroid progenitor cells.