Chromatin loops at the beta globin locus

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DESCRIPTION (provided by applicant): We propose to manipulate cellular gene expression using a novel strategy involving the generation of chromatin loops, focusing on the ¿-globin locus in developing erythrocytes. The locus control region (LCR) of the ¿-globin locus is required for the expression of all ¿-like globin genes and functions over great distances. Throughout development, the LCR interacts dynamically with the active globin gene promoters via the formation of chromatin loops. Here we will examine the cause-effect relationships between loop formation and gene transcription by establishing chromatin loops at endogenous cellular genes in vivo. Specifically, we will examine 1) whether forced loop formation between the LCR and a designated ?-globin gene leads to transcriptional activation in immature erythroid cells, 2) whether inducing the LCR to loop to a developmentally silenced globin gene can lead to its reactivation, 3) whether a silent non-globin gene can be activated by bringing the LCR into close proximity by forced loop formation. Our approach makes use of artificial zinc finger proteins designed to bind to unique, predetermined sequences at the endogenous LCR and the target gene promoters. This will be accomplished by an already established collaboration with Sangamo Biosciences. This company has in its possession a large archive of zinc finger proteins and the required bioinformatics tools to assemble polydactyl zinc finger proteins to bind to the desired target sequences with high selectivity and affinity. These zinc finger proteins will be fused to protein moieties that allow drug-induced heterodimerization such as FKBP and FRB from the Argent(tm) system (ARIAD Pharmaceuticals). This system, which we have acquired, is well suited to generate high affinity interactions between FKBP and FRB bearing proteins upon exposure to the cell permeable, non-toxic drug AP21967. Alternative approaches to manipulate cellular gene expression typically involve the introduction of transcriptional activators, repressors or molecules that interfere with such transcription factors. A potential advantage of the system proposed here is that it does not rely on the effects of a single factor but harnesses the regulatory potential of an entire enhancer or LCR. The long-term goal of this study is to explore whether this strategy is suitable to activate the ?-globin gene in human primary adult erythroid cells. If successful, this might provide a novel tool for the treatment of sickle cell disease and the ¿-thalassemias.