FETAL GLOBIN GENES--EVOLUTION OF ONTOGENETIC PROGRAMS

这个项目的目标是找出调节 基因编程改变了类β珠蛋白基因的活性， 发生在人类发育过程中。对这些控制因素的阐明可能 建议通过治疗性的方法来控制 Epsilon或Gamma基因以有益于患有 镰状细胞性贫血和β地中海贫血。因为珠蛋白的控制 切换是复杂的，对监管机构的准确识别已经证明 很难。该项目采用了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，胎盘哺乳动物的贝塔样珠蛋白簇是 来自相同的五个基因簇和相似的发育 基因活性的开关存在于所有哺乳动物的谱系中。自关键字 因此，开关机械的部件必须是保守的、对齐的 来自灵长类和其他哺乳动物的聚类序列被用来识别 中性DNA中较短的、古代保守的顺式元件。TRANS 与这些元素结合的因子则以凝胶移动为特征 化验。这一策略在epsilon和Gamma珠蛋白基因中的应用 揭示了几个潜在的重要交互作用，包括 阻遏蛋白和推测的多个结合位点 阶段选择器蛋白。这些蛋白在发育过程中的作用 在本提案中将直接测试epsilon和Gamma的表达 通过体内足迹和转基因小鼠的表达研究。 还将分析珠蛋白星系团的其他重要区域 通过系统发育足迹(LCR，β启动子β3‘增强子)。一个 第二种方法，差异系统发育足迹，可能揭示 猿猴(类人猿)招募中的顺式和反式因子 伽马基因到胎儿的表达模式。通过比较来自 猿猴(胎儿伽马)和非猿猴(胚胎伽马)灵长类，基础 与这种伽马表达变化相关的差异是 使用识别和功能测试(结合和表达研究 来测试这种联系。最近发现了一组配置项更改 改变反式因子模式的伽马基因的CCAAT盒 绑定(到SIMIAN与非SIMAN探针)，并更改相对 猿猴与非猿猴启动子在红系细胞中的表达水平。 要确定这些配置项更改是否也会修改 他们将在转基因小鼠身上进行测试。 此外，还将扩大对类人猿特有成分的搜索 到珠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 在类人灵长类动物中，基因沿着不同的路线进行： 在鸭嘴兽中，Gamma2是胎儿表达的主要基因，而在 卡特拉犀类，以Gamma1为主。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 属于几种鸭嘴兽。它们之间的序列差异 高伽马-2的白颈鹿和卡他鼻可能会表现出额外的 调节伽马基因表达的因素；这些因素的阐明 对操控伽马基因活性的最终目标很重要 在成年珠蛋白基因有缺陷的个体中。

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.