Opposing and complementary roles of KLF1 and KLF2 in erythropoiesis

这项建议的目的是研究Kr <$ppel样因子1（EKLF或EKLF）的对立和互补作用。 KLF 1）和KLF 2在胚胎和胎儿红细胞生成中的作用。这在生物学上很重要，因为它可以促进 血红蛋白病的有效治疗策略。KLF 1和KLF 2转录水平密切相关 激活小鼠胚胎类珠蛋白基因的因子。目的1是定义KLF 1和KLF 2在 - 珠蛋白基因表达。KLF 1在体外分化的CD 34+人脐带血中结合<$-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相反，它对胚胎珠蛋白基因的表达起负调节作用。 胎儿细胞KLF 2与胎儿细胞中的<$-珠蛋白启动子以相同的<$-珠蛋白CACCC共识牢固结合 绝佳的价钱我们将使用KLF 2敲低（KD）方法确定KLF 2是否正调控<$-珠蛋白表达。 和在体外分化的CD 34+人脐带血细胞中的过表达。假设KLF 1有一个 通过敲低KLF 2结合，将测试通过排除KLF 2结合对<$-珠蛋白基因表达的净负面影响。 体外分化的CD 34+细胞中的KLF 1。目的2是确定KLF 1和KLF 2在 在胚胎和胎儿红细胞中组织<$-珠蛋白位点的整体染色质结构。KD在 将使用体外分化的CD 34+人脐带血细胞和敲除（KO）小鼠模型来确定 如果KLF 1和KLF 2是募集BRG 1和CBP/p300染色质重塑复合物到 - 珠蛋白LCR和启动子。我们将测试这些因素是否是形成5 'HS 2所必需的， 5 'HS 3 DNA酶I超敏感位点。 我们还将确定是否需要KLF 1和KLF 2， RNA聚合酶II的募集，以及LCR和？-珠蛋白基因之间的染色质循环。在目标3中， 将研究KLF 1和KLF 2对小鼠胚胎红细胞生成的影响。在E10.5，KLF 1-/-KLF 2- /-（双KO）胚胎苍白且贫血，但KLF 1-/-和KLF 2-/-大体正常。KLF1-/-KLF2-/- 胚胎的外周血细胞较少，形成的原始红系祖细胞集落成熟得更快， 与正常对照相比，血红蛋白化更慢。KLF 1和KLF 2似乎协调控制 小鼠原始红系祖细胞的形成和成熟。为了验证这一点，KLF 1和KLF 2在 确定形成的红系祖细胞集落的数量和性质，细胞增殖，成熟和 使用单和双KO小鼠胚胎测试细胞凋亡。 根据总表型， 胚胎，我们预期从使用KLF 1-/-KLF 2 +/-的祖细胞测定中获得更大数量和更成熟的集落。 KLF1+/-KLF2-/-。 初步数据表明，KLF 1和KLF 2调节参与以下过程的基因： 增殖我们将确定KLF 1和KLF 2靶基因，如FoxM 1，Myc， CD 24 a抗原、鞘氨醇激酶1、Pura和甲状旁腺激素1受体，KLF 1 +/-KLF 2-/-、KLF 1-/- KLF 2 +/-、KLF 2-/-、KLF 1-/-和KLF 1-/-KLF 2-/-胚胎以及衍生自红系祖细胞的集落。

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.