Developmental regulation of human beta-like globin gene switching is controlled by several parameters, primarily the trans-acting transcriptional milieu and cis-acting DNA elements. These cis motifs include, but are not limited to, individual gene associated sequences involved in activation, silencing and competition for interaction with the locus control region (LCR), gene order, and distance from the LCR. Other potential points of cis-regulation may comprise intergenic sequences such as domain boundaries or barriers and chromatin architecture. The goals of this grant proposal are to delineate the cis-control of human beta-globin gene switching and the structure/function relationships of the beta-globin LCR. The proposed study will utilize transgenic mice produced with beta-globin locus yeast artificial chromosomes (beta-YACs) so that the effect of mutations introduced into cis-linked sequences may be analyzed in the context of the entire locus throughout development.
Specific Aim 1 will examine the structure/function relationship of the LCR and globin gene switching by: a) determining the function of the individual DNAseI-hypersensitive sites (HSs), and b) testing whether formation of an LCR complex is required for LCR function.
Specific Aim 2 will identify Agamma-globin gene proximal sequences responsible for autonomous silencing of gamma-globin gene expression during development.
Specific Aim 3 will employ constructs to: a) determine whether a putative delta-beta boundary element demarcates embryonic and fetal chromatin domains from an adult chromatin domain and b) test the role of this delta-beta boundary element in regulating embryonic, fetal and adult globin gene expression during development.
Specific Aim 4 will investigate the role of the human beta-globin LCR in globin gene switching when the human locus is integrated in place of the murine beta-globin locus by: a) deleting one copy of the endogenous locus to provide a globin locus insertion site, b) testing whether the locus is open or closed in the absence of the beta-LCR after passage through the mouse germ line, and c) testing whether the human beta-globin LCR functions exclusively as an enhancer or has additional roles in the temporal regulation of downstream genes by replacing it with alternate enhancers.
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