Role of zfp148 (ZBP-89) in Erythroid Development

β-血红蛋白病，如镰状细胞性贫血和β-地中海贫血引起相当大的发病率， 全世界的死亡率。血液学领域的一个长期目标是找到重新激活 胎儿珠蛋白的产生。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。以前的工作有 描述了人β-珠蛋白基因座控制区（LCR）和γ-珠蛋白和β-珠蛋白基因启动子中的关键顺式作用DNA调控元件。在这些区域内存在加塔、NF-E2 转录因子和含有核心“CACCC”基序的序列。这些“CACCC”类的子集 序列在珠蛋白基因转换中起重要作用。然而，哪些因素结合这些序列下， 生理条件尚未完全建立。在初步研究中，我们纯化了含有加塔-1的 诱导小鼠红白血病（MEL）细胞的多蛋白复合物，并确定了Kruppel型锌 指状转录因子ZFP 148作为一种新的相关蛋白。这一因素承认共识 序列CC（A/T）CCCCC，并且充当转录激活因子或阻遏因子。最近，Groudine 和他的同事们独立地鉴定出zfp 148是一种在诱导的神经细胞凋亡中富集NF-E2/mafK复合物的因子。 MEL细胞。我们使用zfp 148 genetrap ES细胞（zfp 148 gt/gt）的嵌合小鼠研究表明， 对成熟的成人红细胞的贡献很小。这项建议的主要目的是进一步研究 ZFP 148在体内正常红系细胞发育中的作用，并检验ZFP 148在 人类珠蛋白基因转换具体目标包括：（1）红系特异性抗体的产生和分析。 条件性鼠ZFP 148敲除小鼠（ZFP 148 +/-嵌合小鼠是不育的）（2）体内 通过染色质免疫沉淀（ChIP）对先前鉴定的CACCC序列的zfp 148占有率 测定。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 基因座酵母人工染色体（YAC）;（3）通过慢病毒SiRNA或逆转录病毒过表达操纵表达水平，确定zfp 148是否是人γ-至β-珠蛋白基因转换所需的 随后测量人β-珠蛋白YAC转基因小鼠中人γ-珠蛋白与β-珠蛋白的表达比率， 小鼠（与StuartOrkin博士（项目2））;和（4）通过芯片鉴定额外的zfp 148直接靶基因 随后进行小鼠启动子阵列杂交（“ChIP on Chip”），并与加塔-1，FOG-1，stat 5， 和FoxO 3a靶基因（与Harvey Lodish博士（项目1）。这些研究的结果应该提供 关于一个新认识的红细胞转录因子的重要信息，可以作为一个有价值的 用于治疗β-血红蛋白病和β-地中海贫血的药理学操作的靶点。

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.