application): During red cell differentiation the genome is progressively packaged into heterochromatin and gene expression silenced. To define the mechanisms by which LCRs and enhancers prevent silencing, the ability of LCR 5'HS2 to suppress silencing of transgenes was tested at ectopic genomic sites in K562 cells. Active transgenes containing an intact enhancer localize away from centromeric heterochromatin while silenced transgenes are in closer proximity to centromeric heterochromatin. Mutations that impair the ability of the enhancer to suppress silencing cause the transgene, even when in an active state, to remain in proximity to centromeres. The molecular basis for activation and maintenance of gene expression in erythroid cells will be defined by: 1) isolation of the transgene integration sites to examine interactions between the transgene and flanking region. 2) determining the cis sequences and trans factors associated with activation and silencing of defined genomic loci, focusing on nuclear factors associated with active and inactive nuclear compartments. 3) deriving new K562 integration sites that can be targeted without selection for expression to address fundamental questions regarding the nature of LCRs and chromatin insulators, and 4) developing a mouse model to study enhancer function during red cell maturation in vivo. This will allow investigation of the mechanism by which erythroid enhancers suppress silencing during erythroid maturation in mice and determine if suppression of silencing in vivo involves relocalization of the transgene within the nucleus.
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