FETAL GLOBIN GENES--EVOLUTION OF ONTOGENETIC PROGRAMS

该项目的目标是确定调节 基因编程开关在β-样珠蛋白基因的活动， 发生在人类发展过程中。阐明这些控制因素可能 提出了治疗性操纵表达的方法， 以有益于患有 镰状细胞性贫血和β地中海贫血。因为对球蛋白的控制 开关是复杂的，调节器的精确识别已被证明 难 该项目采用三种比较方法来确定 这些监管者。首先，系统发育足迹利用了 事实上，胎盘哺乳动物的β样珠蛋白簇是 来自相同的五个基因簇， 基因活性的转换发生在所有哺乳动物谱系中。由于键 因此，开关机械的部件必须被保存、对齐 来自灵长类和其他哺乳动物的簇序列用于鉴定 中性DNA中的短的、古代保守的顺式元件。反式 与这些元素结合的因子然后通过凝胶位移来表征 测定。将该策略应用于γ-珠蛋白和γ-珠蛋白基因 揭示了几种潜在的重要相互作用，包括 阻遏蛋白和推定的抑制蛋白的多个结合位点 阶段选择蛋白这些蛋白质在发育过程中的作用 在本建议书中，将直接测试λ和γ的表达式 通过体内足迹法和转基因小鼠中的表达研究。 球蛋白簇的其他重要区域也将被分析 通过系统发育足迹法（LCR，β启动子β 3'增强子）。一 第二种方法，差异系统发育足迹法，可以揭示 顺式和反式因子参与猿类的募集 伽马基因与胎儿表达模式的关系通过比较 类人猿（胎儿伽马）和非类人猿（胚胎伽马）灵长类，基础 与这种γ表达改变相关的差异是 使用鉴定和功能试验（结合和表达研究） 来测试这种关联。一组顺式变化已被确定为近 γ基因的CCAAT盒，改变了反式因子的模式 结合（对猿类与非猿类探针），并改变相对 类红细胞中猿猴启动子与非猿猴启动子的表达水平。 为了确定这些顺式变化是否也改变了 发育表达，它们将在转基因小鼠中进行测试。 此外，将扩展对特定于RAID的元素的搜索 到球蛋白簇的其他相关区域（LCR，β启动子）。 最后，初始序列数据表明，两个伽马的微调 基因在灵长类动物中沿着沿着不同的路线发展： 在阔鼻鱼中，γ 2是主要的胎儿表达基因，而在 卡他林，γ 1占主导地位。蛋白质和DNA测序研究将 用于测定胎儿和胚胎血液中的γ 1和γ 2水平 几种宽鼻鱼的化石之间的序列差异 高γ 2的阔鼻和卡他鼻可能揭示了额外的 调节γ基因表达的因子;这些因子的阐明 对于操纵伽马基因活性的最终目标是很重要的 成人珠蛋白基因有缺陷的个体。

The goal of this project is to identify key factors that regulate the genetically programmed switches in beta-like globin gene activity that occur during human development. Elucidation of these control factors may suggest approaches to therapeutically manipulate the expression of the epsilon or gamma genes in ways that are beneficial to individuals with sickle cell anemia and beta thalassemia. Because the control of globin switching is complex, precise identification of the regulators has proven difficult. This project employs three comparative approaches to pinpoint these regulators. First, phylogenetic footprinting takes advantage of the fact that the beta-like globin clusters of the placental mammals were derived from the same five gene cluster and that similar developmental switches in gene activity occur in all mammalian lineages. Since key components of the switching machinery must thus be conserved, aligned beta cluster sequences from primates and other mammals are used to identify short, anciently conserved cis elements within neutral DNA. The trans factors which bind to these elements are then characterized by gel shift assays. Application of this strategy to the epsilon and gamma globin genes has revealed several potentially important interactions, including multiple binding sites for both a repressor protein and for a putative stage-selector protein. The role of these proteins in the developmental expression of epsilon and gamma will be directly tested in this proposal by in vivo footprinting and by expression studies in transgenic mice. Additional important regions of the globin cluster will also be analyzed by phylogenetic footprinting (LCR, beta promoter beta 3' enhancer). A second approach, differential phylogenetic footprinting, may reveal the cis and trans factors involved in the recruitment of simian (anthropoid) gamma genes to a fetal expression pattern. By comparing sequences from simian (fetal gamma) and non-simian (embryonic gamma) primates, base differences associated with this alteration in gamma expression are identified and functional tests (binding and expression studies) are used to test this association. One set of cis changes has been identified near the CCAAT box of the gamma gene that alters the pattern of trans factor binding (to simian vs. non-simian probes) and changes the relative expression levels of simian vs. non-simian promoters in erythroid cells. To determine whether these cis changes also modify the pattern of developmental expression, they will be tested in transgenic mice. Additionally, the search for anthropoid-specific elements will be expanded to other relevant areas of the globin cluster (LCR, beta promoter). Finally, initial sequence data suggest that fine tuning of the two gamma genes has proceeded along different lines among the anthropoid primates: in platyrrhines, gamma2 is the major fetally expressed gene, while in catarrhines, gamma1 predominates. Protein and DNA sequencing studies will be used to determine gamma1 and gamma2 levels in fetal and embryonic blood of several platyrrhine species. The sequence differences between platyrrhines with high gamma2 and catarrhines may reveal additional elements which modulate gamma gene expression; elucidation of such factors is important to the ultimate goal of manipulation of gamma gene activity in individuals with defective adult globin genes.