The ultimate goal of this work is to understand the molecular mechanisms for the switch from human fetal (gamma) to adult (beta) beta-like globin gene expression. This will provide novel approaches to treatment of the hemoglobinopathies by the augmentation of transcription of the fetal gene. The system is also a paradigm for developmental gene expression.
The first aim i s to continue to identify elements in the gamma-globin promoter and in 5'HS2 from the locus control region (LCR) which are required to suppress the beta-globin gene in early erythroid cells. The competition model states that protein interactions between the LCR and gamma-globin promoter preclude expression of the beta-globin gene. Preliminary studies in transgenic mice and stable transfection assays suggest that sequences downstream of base position -161 in the gamma-globin promoter, including the TATA and CACCC but not the SSE elements, are necessary for this effect. 5'HS2 but not 5'HS3 from the LCR participates in competitive inhibition of beta-globin gene expression. Other gamma-globin promoter sequences and known transcription factor binding sites in 5'HS2 which are involved in the effect will be identified by mutagenesis of LCR-gamma-beta constructs. The constructs will be stably transfected into embryonic/fetal cultured cells, and mutations in elements that are important for competition should lead to higher than normal levels beta-globin mRNA. As an alternative, a chicken primary erythrocyte transfection system may be used for these initial analyses.
The second aim i s to confirm the effects of selected elements on suppression of beta-globin expression in vivo in embryonic transgenic mice using a genuine developmental system. The final goal is to determine if the most influential beta-globin promoter and LCR elements can suppress the beta-globin gene in the intact human beta-globin locus in mouse embryos. Mutations will be made prior to ligating together two cosmids, to generate a 70 kb locus containing all of the 5'HS sites and beta-like globin genes. The linked cosmids will be microinjected to generate transgenic mice, or as an alternative recombination mediated cassette exchange in ES cells may be used. These results will predict the proteins involved in early beta-globin gene suppression.
