Epo, Integrins and the Control of Erythrpoiesis

促红细胞生成素(EPO)激活多种细胞内信号转导途径，包括STATS、PI-3 KK/Akt和Shc/Ras/Mark，与关键的红系转录因子相互作用，包括GATA1和 FOG可阻止CFU-E祖细胞的凋亡，触发3-5个末端细胞分裂，并诱导多个 红细胞形成所必需的基因。STATS、GATA1和FOG诱导的大多数红系特异基因 都是未知的。在这里，我们将使用染色质免疫沉淀和小鼠启动子的组合 用于识别红系祖细胞中Stat5b、GATA1和FOG结合启动子的微阵列(ChlP-on-Chip) 以及从小鼠胎肝中提纯的红系分化的后续阶段。同时，我们将 红系末梢血不同时期祖细胞基因表达谱的测定 分化并确定由Stat5和Akt信号通路直接和间接调控的基因。 为此，我们将获得从Stat5a-/-b-/-胚胎中分离的红系细胞的转录图谱； Stat5b的异位再表达可以证实某些基因是Stat5的直接靶点。同时，我们将 检测EPOR介导的Akt激活或缺失的原代红系细胞的转录图谱 过表达一种具有内在活性的Akt激酶。最近我们证明了红系祖细胞必须 通过α4beta1或alpha5beta1整合素粘着纤维连接蛋白，以经历正常的末端增殖和 差异化。红系细胞中由整合素激活的信号蛋白未知，其特性也是未知的。 上调或下调的基因。因此，我们将获得纯化的CFU-E的转录图谱 在整合素与纤维连接蛋白黏附的条件下培养的祖细胞，并确定信号 转导通路被特别激活。最后，我们将创建一个转录的框架 通过结合表达分析和启动子结合数据在红细胞生成中活跃的调控网络 确定Stat5和GATA1结合基因的哪个子集在红系中受到活跃调控 分化，以及哪些依赖GATA1的基因子集需要FOG。我们将结合使用 确定由这些因素激活的基因(S)的生物信息学和实验技术，以及如何 多个转录因子可能相互作用，调节红系特异基因。这些信息将形成一个 建立红细胞终末分化转录调控网络的基础 并确定对决定红细胞表型至关重要的关键基因。

Erythropoietin (Epo) activates multiple intracellular signal transduction pathways, including StatS, PI -3 kinase/Akt, and Shc/Ras/ MARK, that interact with key erythroid transcription factors including GATA1 and FOG to prevent apoptosis of CFU-E progenitors, trigger 3-5 terminal cell divisions, and induce multiple genes essential for erythrocyte formation. Most erythroid- specific genes induced by StatS, GATA1, and FOG are unknown. Here we will use a combination of chromatin immunoprecipitation with mouse promoter microarrays (ChlP-on-chip) to identify promoters bound by Stat5b, GATA1, and FOG in erythroid progenitors and subsequent stages of erythroid differentiation purified from mouse fetal livers. In parallel we will determine the mRNA expression profiles of erythroid progenitors at different stages of terminal erythroid differentiation and determine genes directly and indirectly regulated by the Stat5 and Akt signaling pathways. To this end we will obtain transcriptional profiles of erythroid cells isolated from Stat5a-/-b-/- embryos; ectopic reexpression of Stat5b can confirm certain genes as direct targets of Stat5. In parallel we will examine the transcriptional profiles in primary erythroid cells deficient in EpoR- mediated Akt activation or overexpressing a constitutively active Akt kinase. Recently we showed that erythroid progenitors must adhere to fibronectin via alpha4beta1, or alpha5beta1 integrin in order to undergo normal terminal proliferation and differentiation. Signaling proteins activated by integrins in erythroid cells are unknown as are the identities of genes that are up- or down- regulated. Thus we will obtain transcriptional profiles of purified CFU-E progenitors cultured under conditions where integrins are adherent to fibronectin, and determine the signal transduction pathways specifically activated. Finally we will create a framework for the transcriptional regulatory networks active in erythropoiesis by combining expression analysis with promoter binding data to determine which subset of Stat5 and GATA1- bound genes are actively regulated during erythroid differentiation, and which subset of GATA1- dependent genes require FOG. We will use a combination of bioinformatic and experimental techniques to determine the gene(s) activated by these factors, and how multiple transcription factors might interact to regulate erythroid- specific genes. This information will form a basis on which to establish a transcriptional regulatory network for the terminal differentiation of erythrocytes and identify key genes crucial to determining the phenotype of erythrocytes.