Maintenance of Gene Expression in the Red Cell Lineage

描述（申请者提供）：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，我们假设共调节基因不是随机定位在染色体上或间期核中。我们提出三个目标，结合单位点和基因组的方法，以解决这些假设实验。首先，我们将详细研究一个基因组整合位点，该位点显示了一种与果蝇“细胞记忆”相似的转录表型。我们将绘制该位点的染色体决定因素（染色质，CpG甲基化，核组织）并定义记忆表型的标志。我们还建议鉴定假定的赋予细胞记忆表型的顺式元件，并通过靶向修饰剖析该元件的调控成分。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位点（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。我们还建议确定不同细胞类型中珠蛋白位点可能指向的不同亚核区室，并研究顺式调控序列和转录在这些区室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造血过程中差异表达基因的表达状态和基因组分布之间的关系。我们还将利用微阵列方法和一种新的荧光原位杂交（FISH）技术，以免疫沉淀的染色质为探针，在全基因组范围内测量染色质结构，从而确定红细胞生成过程中活性和非活性染色质室的核组织。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确定的共调节基因组的核定位分析相结合。我们相信这些实验将揭示红细胞特异性基因表达实现和维持的表观遗传机制，以及这些机制如何塑造了红细胞谱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.