Elevated levels of fetal hemoglobin lead to a significant amelioration of symptoms in patients with sickle cell disease and beta-thalassemia. This observation suggests that treatment strategies capable of re-activating fetal hemoglobin expression after birth should be explored. To achieve this goal we must first understand the mechanisms underlying the developmental regulation of the beta-globin locus, particularly the factors that can modulate gamma- globin expression. We have previously identified a candidate factor for this aim, the stage selector protein (SSP). The SSP is an erythroid-specific protein complex consisting of the ubiquitously expressed transcription factor CP2, and a tissue-restricted partner, p22 NF-E4. We have recently identified a second CP2-like gene, LBP-1a that also can form an SSP complex with NF-E4. We have also identified a 14 kD isoform of NF-E4 which acts in direct contrast to p22 NF-E4 and represses gamma-gene expression in a fetal/erythroid cell line. We have coupled these recent observations to our pre-existing knowledge of the SSP to develop three aims that will enhance our understanding of the mechanisms of action of this complex. The first specific aim focuses on the mechanistic roles of LBP-1a and p14 NF-E4 in regulating gamma-globin gene expression in the fetal erythroid environment and in hemoglobin switching models. These experiments will employ gene-targeting and transgenic strategies to elucidate the role of LBP-1a and p14 NF-E4 in vitro and in vivo.
Specific aim 2 will expand on our observation that p14 NF-E4 fails to bind CP2/LBP-1a, full length NF-E4 or DNA and thus appears to exert is dominant negative effect through sequestration of a protein associating with the SSP. We will define the protein partners of both isoforms of NF-E4 using molecular and biochemical approaches. The work described in specific aim 3 will focus on the identification and characterisation of the core regions of the NF-E4 promoter using transcription assays in cell lines and transgenic mice. Taken together, these aims address many of the issues raised in the RFA. In particular, they seek to validate an existing trans-activator by direct function analysis and by investigation of mechanism of action. They also examine induction of the structural gene of such an activator with the emphasis on developing an assay system for high throughput drug screening. Finally, through protein interaction studies they may identify additional novel factors important in the activation (or repression) of gamma-globin gene expression.
