Maintenance of Gene Expression in the Red Cell Lineage

描述(由申请人提供)：我们假设，在红细胞分化过程中发生的转录激活和沉默是基于表观遗传过程，如染色质结构、CpG甲基化和核定位。由于这些事件影响大的基因组区域，我们假设共调控基因不是随机定位在染色体上或间期核中。我们提出了三个目标，结合单基因座和基因组方法，从实验上解决这些假设。首先，我们将详细研究一个基因组整合位点，该位点显示的转录表型让人想起果蝇的“细胞记忆”。我们将绘制这个位置的染色体决定因素(染色质、CpG甲基化、核组织)，并定义记忆表型的特征。我们还建议确定可能的顺式元件，赋予细胞记忆表型，并通过靶向修饰剖析该元件的调节成分。在第二个目标中，我们将在单等位基因水平上研究特定基因组座位(β-珠蛋白)相对于其染色体区域的定位与建立/维持组织特异性转录状态之间的关系。我们还建议在不同的细胞类型中识别珠蛋白基因可能指向的不同的亚核区域，并研究顺式调控序列和转录在这些区域中基因位点定位中的作用。在第三个目标中，我们提出了一种生物信息学方法来确定在造血过程中差异表达的基因的表达状态和基因组分布之间的关系。我们还将通过使用微阵列方法和一种新的以免疫沉淀染色质为探针的荧光原位杂交(FISH)技术来测量全基因组染色质结构，从而确定红细胞生成过程中活跃和非活跃染色质区段的核组织。染色体分布、表达和染色质分析的结果将与红细胞生成过程中识别的共调控基因组的核定位分析相结合。我们相信，这些实验将揭示获得和维持红系特异性基因表达的表观遗传学机制，以及这些机制如何塑造红细胞系中基因表达协调调节所需的基因组组织。

DESCRIPTION (provided by applicant): We hypothesize that the transcriptional activation and silencing that occur during red cell differentiation are based in epigenetic processes such as chromatin structure, CpG methylation and nuclear positioning. Since these events affect large genomic regions, we hypothesize that co-regulated genes are not positioned randomly on the chromosome or in the interphase nucleus. We propose three aims, combining single locus and genomic approaches, to address these hypotheses experimentally. First, we will study in detail a genomic integration site that displays a transcriptional phenotype reminiscent of "cellular memory" in Drosophila. We will map the chromosomal determinants of this site (chromatin, CpG methylation, nuclear organization) and define the hallmarks of the memory phenotype. We also propose to identify the putative cis-element that confers the cellular memory phenotype and to dissect the regulatory components of this element by targeted modification. In the second aim, we will examine, at the single allele level, the relationship between the positioning of a specific genomic locus (beta-globin) relative to its chromosome territory and establishing/maintaining the tissue-specific transcription state. We also propose to identify the distinct subnuclear compartments to which the globin locus may be directed in different cell types and investigate the role of cis-regulatory sequences and transcription in the positioning of a gene locus in these compartments. In the third aim, we propose a bioinformatics approach to determine the relationship between expression status and genomic distribution of genes that are differentially expressed during hematopoiesis. We will also determine the nuclear organization of active and inactive chromatin compartments during erythropoiesis by measuring chromatin structure genome-wide using a microarray approach and a novel fluorescence in situ hybridization (FISH) technique with immunoprecipitated chromatin as probe. The results of the chromosome distribution, expression and chromatin analyses will be combined with an analysis of the nuclear positioning of identified groups of co-regulated genes during erythropoiesis. We believe these experiments will reveal the epigenetic mechanisms by which erythroid-specific gene expression is achieved and maintained and how these mechanisms have shaped the genomic organization required for the concerted regulation of gene expression in the red cell lineage.