The goal of this application is to understand how trans-acting factors and coregulators function through the beta-globin locus control region (LCR) to confer strong transcription to the beta-globin genes over a long distance on a chromosome. We discovered that RNA polymerase II (pol II) resides at the LCR in erythroid cells and formulated a new model of LCR function called long-range pol II transfer (LPT). This model proposes that LCR-bound pol II relocalizes to beta-globin promoters in a regulated fashion, thereby constituting a critical step in activation. The following Specific Aims will address how pol II is recruited to the LCR and the resulting functional consequences.
Aim 1. To define the role of GATA-1 and Friend of GATA-1 (FOG-l) in pol II recruitment to the LCR. Pol II associates with the LCR in erythroid cells, and GATA-1 induces pol II recruitment. We hypothesize that GATA-1 functions with other factors to recruit pol II. GATA-I-null cells will be used to define how GATA-1 recruits pol II. Cells lacking the GATA-1 coregulator FOG-1 will be used to define if FOG-1 is required. Experiments will address whether single hypersensitive sites autonomously recruit pol II.
Aim 2. To determine if pol II recruitment to the LCR is dynamic during erythropoiesis. As the concentrations and activities of erythroid-specific factors change during erythropoiesis, we hypothesize that pol II loading on the LCR is developmentally dynamic. Experiments will assess if pol II recruitment to the LCR differs between embryonic and adult erythroid cells.
Aim 3. To discriminate among models for understanding the function of pol II at the LCR. Besides LPT, LCR-bound pol II might engage in transcription-dependent chromatin remodeling or the generation of regulatory transcripts. Experiments will discriminate among models to explain the function of pol II at the LCR. These studies will increase our understanding of how the LCR regulates the beta-globin genes, which should have broad relevance to mechanisms controlling diverse cellular and organismal processes. The long-term objective is to therapeutically control beta-globin genes in humans with hemoglobinopathies by regulating specific steps of the LCR mechanism.
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