Transcriptional regulation of the beta-globin locus is a historically important system for the study of tissue and developmentally regulated transcription in mammals. In addition to its importance for fundamental insights into mammalian transcription, understanding how this locus is regulated holds the potential for developing novel therapies for sickle cell anemia and beta-thalassemia, two very common human genetic disorders. Regulation of the beta-globin locus is analyzed predominantly in transgenic mice that carry transgenes from the human beta-globin locus. This system has been productive but suffers from problems that are inherent to studying human transgenes in mice, where the randomly generated integration site has powerful effects and the murine transcription factors have not coevolved with the cis regulatory elements of the transgene. Results from human locus transgenes have been complemented and clarified by analysis of the murine beta-like globin locus through mutation of the murine locus using homologous recombination in ES cells. Comparisons of the human transgene studies and the murine locus studies have formed a baseline of definitive studies that underlies general conclusions concerning the regulation of this locus regardless of species. ? This proposal continues with the mutational analysis of the routine beta-like globin locus and definitively tests the following hypotheses that have been suggested by studies of human beta-globin locus transgenes in transgenic mice:
Aim l tests the hypothesis that expression of one gene at the locus quantitatively suppresses expression of another gene at the locus which is expressed at the same developmental stage. This will clarify specific aspects of models of how the locus control region influences gene expression.
Aim 2 tests the hypothesis that expression of the embryonic genes suppresses expression of the fetal/adult genes in the embryo. This will prove or disprove the consensus hypothesis concerning the mechanism by which the genes expressed later in development are kept silent early in development.
Aim 3 tests the hypothesis that deletion of the core part of a hypersensitive site in the locus control region will have stronger suppressive effects than deletion of the entire site. Unexpected findings using human transgenes have suggested this hypothesis and the experiments proposed in aim 3 will demonstrate the generality of this hypothesis and develop a facile system to dissect the effect if it is also seen in the murine locus. ? ?
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