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. ? ? ?
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