MECHANISM OF THE HUMAN BETA-GLOBIN LOCUS CONTROL REGION

University Of Wisconsin Madison, Madison WI

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Abstract

Description: (Investigator's abstract) We propose to study how the beta-globin locus control region (LCR) confers high-level transcriptional activity to the beta-globin genes over a long distance on a chromosome. Mechanisms of long-range transactivation are poorly understood. In Specific Aim 1, we will define protein components of the LCR in living cells and will determine if they differ during erythroid differentiation. Despite extensive in vitro studies, little is known about the composition of native LCR complexes. We hypothesize that the components of the LCR vary during erythropoiesis to accommodate differential requirements for transactivation during development. A chromatin immunoprecipitation (ChIP) assay will be used to measure the binding of candidate proteins to the LCR. In Specific Aim 2, we will define the histone acetylation and phosphorylation patterns of the beta-globin locus and will determine if the patterns change during erythroid differentiation. We hypothesize that the LCR recruits coactivators that establish a specific pattern of histone modifications throughout the beta-globin locus, which is necessary for long-range transactivation. We will define the pattern of histone modifications, will assess whether it changes during erythropoiesis, and will determine whether pharmacological inducers of fetal hemoglobin specifically modulate the pattern. We hypothesize that the histone modification pattern is established via primary and modulatory determinants. In Specific Aim 3, we will identify primary determinants of the acetylation pattern. We have shown that the histone acetylase CBP/p300 is critical for LCR-mediated transactivation. We will test whether CBP/p300 is a primary determinant and will identify CBP/p300-interacting coactivators in erythroid cells. We will also assess whether specific histone deacetylases are primary determinants. The native structure of enhancer and LCR complexes and the histone modification pattern of a domain have not been defined in any system. Since long-range mechanisms control the transcription of multiple crucial genes that regulate cell proliferation and differentiation, our studies will yield principles of broad physiological and pathophysiological relevance. The long-term objective is to therapeutically modulate beta-globin gene expression in humans with hemoglobinopathies by perturbing specific steps of the mechanism by which the LCR regulate the beta-globin genes.