LCR AND ITS COMPONENTS IN DEVELOPMENTAL REGULATION AND RESPONSE TO ANEMIC STRESS

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(Adapted from Applicant's Abstract) Regulation of expression of the Beta- globin gene cluster is under the control of the LCR. In this project the investigators will attempt to determine the role of the LCR during stress erythropoiesis and during development. In Aim 1 the investigators will crease a mouse line in which the beta globin LCR will be easy to modify. This will be achieved with the help of a powerful new technology that the investigators have termed Recombinase-Mediated Cassette Exchange (RMCE). RMCE is based on the CRE/LOX recombinase and allows single-copy integration of transgenes at pre-determined loci. In aim 2, the investigators will use this mouse line to study the role of the LCR in the developmental regulation of the Beta-globin genes. The investigators will replace the mouse LCR by its human counterpart, invert the mouse globin LCR, and study the role of mouse HS5. In aim 3, the investigators will study the role of the LCR in the up-regulation of the Betamin gene in the presence of hemin and in the presence of anemic stress using as an experimental model 5 mouse stains with targeted deletion of all the individual HS making up the LCR. This will be performed by inducing hemolytic or hemorragic anemia experimentally or genetically by breeding with thalassemic mice. The investigators have recently found out that HS2 but not HS3 was hemin inducible in single-copy in MEL cells; this lead the investigators to the hypothesis that HS2 might be important for the expression of the globin gene during erythroid stress. In aim 3C to 3F, the investigators will functionally and physically characterized the hemin response element within HS2. In Aim 4 the investigators will characterize the role of the human Beta-globin gene cluster polymorphisms in the level of gamma-globin gene expression in transgenic mice. The use of RMCE technology will allow the investigators to compare intact 80 kb fragments containing the Senegal an Benin Beta-globin gene clusters with integrated in single-copy at the same locus in transgenic mice. Understand the role of the LCR during stress erythropoiesis might help improve the treatment of sickle cell anemia by improving the ability to predict the course of the disease with the help of genetic markers and by improving of the understanding of the target drugs that induce an increase in the production of HbF in adults. Better understanding of the regulation of the Beta-globin gene cluster should also help devise new therapy strategies.