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

 描述(申请人提供)：人类类型的珠蛋白基因仅在红系细胞中表达，并受位于基因上游的一个基因座控制区(LCR)调控。珠蛋白基因突变是人类群体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。这些血红蛋白疾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珠蛋白基因。我们建议继续努力了解LCR如何在发育和分化过程中调节珠蛋白基因的表达，并将开发新的方法学来改变珠蛋白基因位点的表达模式。我们建立了一种新的方法，利用合成的小DNA结合域来分析和中和珠蛋白基因座位上特定的顺式调控DNA元件。我们将这些合成的DNA结合域定位于-珠蛋白基因启动子中已知的抑制物结合部位，以及编码-珠蛋白抑制物BCL11A的基因中的激活物结合部位。用合成的DNA结合蛋白靶向这些位置有望增加成人红系细胞中珠蛋白的表达。我们和其他人以前已经证明，LCR招募转录复合体，产生增强子RNA(ERNAs)。转录因子NF-E2和USF参与了LCR的转录复合体募集。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作用。此外，我们将检查LCR相关转录本或转录过程是否有助于LCR介导的珠蛋白基因表达的激活。这项建议的四个目标将集中于优化合成DNA结合蛋白的DNA结合特异性和传递方法(目标1)，利用合成的DNA结合蛋白分析珠蛋白基因座上的顺式调节元件并重新激活成人红系细胞中的珠蛋白表达(目标2)，在转基因小鼠的红系细胞中鉴定所有与人LCR相关的蛋白质(目标3)，以及分析LCR和LCR相关的非编码转录激活珠蛋白基因的机制(S)。

 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).