Regulation of Globin Gene Switching in Human ES Cells

描述（由申请人提供）：类人珠蛋白基因表达的发育调控受几种因素控制，主要是反式作用转录环境和顺式作用DNA元件。鉴定这些DNA序列和理解珠蛋白基因转换的顺式调节机制对于开发用于治疗血红蛋白病如镰状细胞病和B-地中海贫血的治疗策略至关重要。该项目的母基金R 01 HL 67336“珠蛋白基因转换的基因座相关调控基序”一直在使用转基因小鼠或常规细胞系研究这些DNA调控区的功能。然而，在人类细胞系中珠蛋白基因表达的研究可能会提供新的见解珠蛋白基因调控没有揭示在小鼠系统。因此，本补充申请的目标是实现使用人胚胎干细胞（hESC）进行B样珠蛋白基因转换的广泛研究，最初的重点是检查人B-珠蛋白LCR在其内源性基因组位置的作用。具体目标1将建立hESC系H1培养条件，并确定从该系获得红系细胞所需的参数。在特定目标2中，将构建hESC系H1基因组文库，用于当前和未来的基因靶向应用。特异性目标3的目标是在LCR内产生突变，以测试其在天然人珠蛋白基因座内的功能。这将通过删除LCR或用CMV增强子或B-珠蛋白HS-40控制区替换LCR来实现。这些修饰的ES细胞将用于测试在B-珠蛋白LCR不存在的情况下，基因座染色质是开放的还是闭合的。用替代增强子替换LCR将测试人B-珠蛋白LCR是否仅作为增强子起作用或在下游基因的时间调节中具有额外作用。具体目标4将分析小鼠B样珠蛋白基因在人类细胞中的表达，以更好地了解这两个物种之间的基因调控和表达模式的进化关系和保守性。

DESCRIPTION (provided by applicant): Developmental regulation of human-like globin gene expression is controlled by several factors, primarily the trans-acting transcriptional milieu and cis-acting DNA elements. Identifying these DNA sequences and understanding the cis regulatory mechanisms underlying globin gene switching are essential to the development of therapeutic strategies for treatment of hemoglobinopathies such as sickle cell disease and B-thalassemias. The parent grant to this project, R01 HL67336, "Locus-linked Regulatory Motifs of Globin Gene Switching," has been investigating the function of these DNA regulatory regions using transgenic mice or routine cell lines. However, studies of globin gene expression in human cell lines may provide novel insights into globin gene regulation not revealed in mouse systems. Thus, the goal of this supplement application is to implement the use of human embryonic stem cells (hESCs) for broad-based studies of B-like globin gene switching, with an initial emphasis on examining the role of the human B-globin LCR at its endogenous genomic location. Specific Aim 1 will establish hESC line H1 culture conditions and define the parameters necessary to derive erythroid cells from this line. In Specific Aim 2, a hESC line H1 genomic library will be constructed for use in current and future gene targeting applications. The goal of Specific Aim 3 will be to produce mutations within the LCR to test its function within the native human globin locus. This will be accomplished by deleting the LCR or replacing it with the CMV enhancer or the B-globin HS -40 control region. These modified ES cells will be used to test whether locus chromatin is open or closed in the absence of the B-globin LCR. Replacement of the LCR with alternate enhancers will test whether the human B-globin LCR functions exclusively as an enhancer or has additional roles in the temporal regulation of downstream genes. Specific Aim 4 will analyze the expression of the murine B-like globin genes in human cells to better understand the evolutionary relationships and conservation of gene regulation and expression patterns between these two species.