Structure and function of the human beta-globin locus control region

 描述（由申请人提供）：人型珠蛋白基因仅在红系细胞中表达，并由位于基因远上游的基因座控制区（LCR）调节。β-珠蛋白基因座的突变是人群中最常见的致病突变之一。这些突变导致地中海贫血，其特征是成人珠蛋白基因表达减少或缺失，或镰状细胞性贫血。这些血红蛋白病的一种可能的治疗方法是重新激活成人红系细胞中通常被抑制的β-珠蛋白基因。我们建议继续我们的努力，以了解如何LCR调节珠蛋白基因的表达在发展和分化，并将开发新的方法来改变珠蛋白基因座的表达模式。我们建立了一个新的程序，利用小的合成DNA结合域分析和中和特定的顺式调节DNA元件的珠蛋白基因座。我们将针对这些合成的DNA结合域已知的阻遏物结合位点的-珠蛋白基因启动子和激活剂结合位点的-珠蛋白阻遏物Bcl11A的基因编码。用合成的DNA结合蛋白靶向这些位点，预期会增加成人红系细胞中β-珠蛋白的表达。我们和其他人以前已经表明，LCR招募产生增强子RNA（eRNA）的转录复合物。转录因子NF-E2和USF与转录复合物向LCR的募集有关。我们将利用合成的DNA结合域来分析LCR中与转录因子NF-E2和USF相互作用的顺式调节元件的贡献。此外，我们将检查是否LCR相关的转录本或转录过程有助于LCR介导的珠蛋白基因表达的激活。本提案的四个目标将集中于优化合成DNA结合蛋白的DNA结合特异性和递送方法（目标1），使用合成DNA结合蛋白分析β-珠蛋白基因座中的顺式调节元件并重新激活成人红系细胞中的β-珠蛋白表达（目标2），鉴定转基因小鼠的红系细胞中与人LCR相关的所有蛋白（目标3），并分析LCR和LCR相关的非编码转录激活珠蛋白基因的机制（目的4）。

 DESCRIPTION (provided by applicant):The human -type globin genes are expressed exclusively in erythroid cells and regulated by a locus control region (LCR) that is located far upstream of the genes. Mutations in the -globin locus are among the most common disease-causing mutations in the human population. These mutations lead to thalassemias, characterized by reduced or absent adult -globin gene expression, or sickle cell anemia. A possible therapy for these hemoglobinopathies is the reactivation of the normally repressed -globin genes in adult erythroid cells. We propose to continue our efforts to understand how the LCR regulates globin gene expression during development and differentiation and will develop new methodology for changing expression patterns in the - globin gene locus. We established a novel procedure utilizing small synthetic DNA binding domains to analyze and neutralize specific cis-regulatory DNA elements in the -globin gene locus. We will target these synthetic DNA binding domains to known repressor binding sites in the -globin gene promoters and to activator binding sites in the gene encoding for the -globin repressor Bcl11A. Targeting these sites with synthetic DNA binding proteins is expected to increase -globin expression in adult erythroid cells. We and others have shown previously that the LCR recruits transcription complexes which produce enhancer RNAs (eRNAs). Transcription factors NF-E2 and USF have been implicated in transcription complex recruitment to the LCR. We will utilize the synthetic DNA binding domains to analyze the contribution of cis-regulatory elements in the LCR that interact with transcription factors NF-E2 and USF. Furthermore, we will examine if LCR associated transcripts or the process of transcription contributes to LCR mediated activation of globin gene expression. The four aims of this proposal will focus on optimizing the DNA binding specificity and delivery methods for synthetic DNA binding proteins (aim 1), to use synthetic DNA binding proteins to analyze cis-regulatory elements in the -globin locus and to reactivate -globin expression in adult erythroid cells (aim 2), to identify all proteins associated with the human LCR in erythroid cells of transgenic mice (aim 3), and to analyze the mechanism(s) by which the LCR and LCR associated non-coding transcription activates the globin genes (aim 4).