REGULATION OF HUMAN B GLOBIN GENE IN ERYTHROID CELLS

地中海贫血综合征和镰状细胞性贫血的基因治疗需要 引入的珠蛋白基因模仿了原位正常的功能， 珠蛋白基因的组织特异性和转录效率。 DNA介导的基因转移实验表明， 人β-珠蛋白转基因的转录需要存在 一个远上游的增强子 在基因组中，这种红细胞特异性和 一个发育阶段独立的增强子位于一个DNA酶1超级 胚胎红细胞珠蛋白基因5'端-11Kb超敏位点（HS 2） 因此位于成人β-珠蛋白基因的~ 54 Kb 5 ′。 HS 2增强子 指导红细胞特异性长增强子转录物的合成 其似乎在增强子功能中起关键作用， 下游顺式连接基因的转录激活。 拟议工作的长期目标是研究分子 这种远上游增强子激活54 Kb DNA的机制 红系细胞中的胚胎、胎儿和成人珠蛋白基因， 各自的发展阶段。 具体目的是检验假设 长增强子转录本的合成是通过追踪和 转录机制是必需的，并构成了先决条件， β-样珠蛋白基因的转录激活， 红细胞生成 以下提出的实验旨在测试 这一假设的某些方面。 (1)评估跟踪与成环 在远上游HS 2增强子的功能中的机制。 长 增强子转录本，从而跟踪机制，将被阻断， 转录终止子。 这种阻塞对表达的影响 将研究顺式连接的CAT基因。 (2)增强剂分析 转染入红系细胞的含HS 2的CAT质粒中的转录 不同发育阶段的细胞。 (3)检测内源性长 未转染的K562细胞和原代人BFU-1中的增强子转录物 E和CFU-E。 (4)在增强子转录中的作用 26 bp HS 2增强子核心和与该增强子核心结合的转录因子。 核心 (5)两个相邻的协同效应的研究 HS 2驱动增强子上的DNase 1超敏位点HS 3和HS 4 转录。 通过寡核苷酸合成，PCR， 重组CAT质粒构建、转染实验、CAT测定 和RNA保护试验，在增强子的生物学意义 启动上游长转录本，因此跟踪机制将 被评价。

Gene therapy for the thalassemia syndromes and sickle cell anemia requires that the introduced globin gene mimic the function of the in situ normal globin gene both in tissue specificity and efficiency of transcription. DNA-mediated gene transfer experiments have shown that efficient transcription of the human beta-globin transgene requires the presence of a far upstream enhancer. In the genome, this erythroid specific and development-stage independent enhancer is located in a DNase 1 super hypersensitive site (HS2) at -11Kb 5' of the embryonic epsilon globin gene and thus at -54 Kb 5' of the adult beta-globin gene. The HS2 enhancer directs the synthesis of erythroid specific, long enhancer transcripts which appear to play a pivotal role in enhancer function and transcriptional activation of the downstream cis-linked gene. The long-term objective of the proposed work is to study the molecular mechanism by which this far upstream enhancer activates across 54 Kb of DNA the embryonic, fetal and adult globin genes in erythroid cells at the respective developmental stage. The specific aim is to test the hypothesis that the synthesis of long enhancer transcripts by a tracking and transcription mechanism is required, and constitutes a precondition, for the transcriptional activation of the beta-like globin genes during erythropoiesis. The following proposed experiments are designed to test selected aspects of this hypothesis. (1) Assessment of the relative importance of the tracking v.s. the looping mechanisms in the function of the far upstream HS2 enhancer. The long enhancer transcripts, and thus the tracking mechanism, will be blocked by transcriptional terminators. The effect of this blockage on the expression of the cis-linked CAT gene will be studied. (2) Analysis of enhancer transcription in HS2-containing CAT plasmids transfected into erythroid cells at different developmental stages. (3) Detection of endogenous long enhancer transcripts in non-transfected K562 cells and primary human BFU- E's and CFU-E's. (4) Examination of the role in enhancer transcription of a 26 bp HS2 enhancer core and of the transcription factors that bind to the core. (5) Investigation of the synergistic effects of two neighboring DNase 1 hypersensitive sites HS3 and HS4 on the HS2-driven enhancer transcription. By a combination of oligonucleotide synthesis, PCR, recombinant CAT plasmids construction, transfection experiments, CAT assays and RNA protection assays, in the biological significance of the enhancer initiated upstream long transcript and thus of the tracking mechanism will be evaluated.