Role of zfp148 (ZBP-89) in Erythroid Development

贝塔血球疾病，如镰状细胞性贫血和贝塔地中海贫血，会导致相当大的发病率和 全球范围内的死亡率。血液学领域的一个长期目标是找到重新激活的方法 这些患者的胎儿球蛋白产生情况。合理设计的方法首先需要有坚定的理解 控制正常发育珠蛋白基因表达的分子机制。之前的工作已经完成 描述了人类β-珠蛋白基因位点控制区(LCR)以及伽马和β-珠蛋白基因启动子中的关键顺式作用DNA调控元件。聚集在这些区域内的是GATA、NF-E2的结合位点 含有核心“CACCC”基序的转录因子和序列。这些类似于CACCC的子集 序列在珠蛋白基因切换中起着重要作用。然而，哪些因素结合了这些序列呢？ 生理条件尚未完全确立。在初步研究中，我们纯化了含有GATA-1的 诱导小鼠红白血病(MEL)细胞的多蛋白复合体及鉴定Kruppe型锌 手指转录因子zfp148作为一种新的结合蛋白。这一因素承认了共识 序列CC(A/T)CCCCC，作为转录激活因子或抑制因子。最近，格罗丁 和他的同事们独立地发现zfp148是一种富含NF-E2/mafK复合体的因子 梅尔细胞。我们使用zfp148基因诱捕ES细胞(zfp148 GT/GT)的嵌合小鼠研究表明，它们 对成熟的成人红细胞的贡献很小。这项提案的主要目标是进一步审查 Zfp148在体内正常红系发育中的作用，并验证zfp148在红系发育中的作用假说。 人类珠蛋白基因转换。具体目标包括(1)产生和分析红系特异性 条件性小鼠zfp148基因敲除小鼠(zfp148+/-嵌合小鼠不育)(2)体内检测 染色质免疫沉淀(CHIP)对先前鉴定的CACCC序列的zfp148占有率 化验。这将在携带人β-珠蛋白的转基因小鼠的红系祖细胞上进行。 定位酵母人工染色体(YAC)；(3)通过慢病毒siRNA或逆转录病毒过度表达控制表达水平，确定zfp148是否是人从伽马到β珠蛋白基因转换所必需的 然后测量人β-珠蛋白YAC转基因中人-β-珠蛋白与β-珠蛋白的表达比率 小鼠(与Stuart Orkin博士(项目2)；和(4)通过芯片识别额外的zfp148直接靶基因 然后是小鼠启动子阵列杂交(芯片上芯片)，并与GATA-1，FOG-1，STAT5， 和FOXO3a目标基因(与哈维·洛迪什博士合作(项目1)。这些研究的结果应该提供 关于一种新发现的红系转录因子的重要信息，它可能是一种有价值的 治疗β-血红蛋白病和β-地中海贫血的药物操作靶点。

Beta-hemogloblinopathies, such as sickle cell anemia, and beta-thalassemia cause considerable morbidity and mortality worldwide. A long-standing goal in the field of hematology has been to find means to re-activate fetal globin production in these patients. Rationally designed approaches first require a firm understanding of the molecular mechanisms controlling normal developmental globin gene expression. Prior work has delineated key cis-acting DNA regulatory elements in the human beta-globin locus control region (LCR) and gamma and beta-globin gene promoters. Clustered within these regions are binding sites for GATA, NF-E2 transcription factors and sequences containing a core "CACCC" motif. A subset of these "CACCC"-like sequences play important roles in globin gene switching. Yet, which factors bind these sequences under physiologic conditions has not been fully established. In preliminary studies, we purified GATA-1 containing multiprotein complexes from induced mouse erythroleukemia (MEL) cells and identified the Kruppel-type zinc finger transcription factor zfp148 as a novel associated protein. This factor recognizes the consensus sequence CC(A/T)CCCCC and acts as either a transcriptional activator or represser. Recently, Groudine and his colleagues independently identified zfp148 as a factor enriched in NF-E2/mafK complexes in induced MEL cells. Our chimeric mouse studies using zfp148 genetrap ES cells (zfp148 gt/gt) show that they contribute poorly to mature adult erythrocytes. The main objectives of this proposal are to further examine the role of zfp148 in normal erythroid development in vivo, and test the hypothesis that zfp148 plays a role in human globin gene switching. Specific aims include (1) generation and analysis of erythroid-specific conditional murine zfp148 knock-out mice (zfp148+/- chimeric mice are infertile) (2) Examination of in vivo zfp148 occupancy of the previously identified CACCC sequences by chromatin immunoprecipitation (ChIP) assays. This will be performed on erythroid progenitor cells of transgenic mice harboring a human beta-globin locus yeast artificial chromosome (YAC); (3) determination of whether zfp148 is required for human gamma- to beta-globin gene switching by manipulation of expression levels by lentiviral SiRNA or retroviral overexpression followed by measurement of human gamma- to beta-globin expression ratios in the human beta-globin YAC transgenic mice (with Dr. Stuart Orkin (Project 2); and (4) Identifcation of additional zfp148 direct target genes by ChiP followed by mouse promoter array hybridization ("ChIP on Chip"), and comparison to GATA-1, FOG-1, stat5, and FoxO3a target genes (with Dr. Harvey Lodish (Project 1). The results of these studies should provide important information about a newly recognized erythroid transcription factor that may serve as a valuable target for pharmacologic manipulation in the treatment of beta-hemoglobinopathies and beta-thalassemia.