A comprehensive understanding of the molecular bases for the developmental stage-restricted expression of human gamma-globin is crucial to the generation of methods designed to reactivate its expression in individuals with congenital beta-chain defects. Although the fetal-to-adult globin switch is effected largely through mechanisms acting at the level of gene transcription, recent studies suggest that post-transcriptional events may also participate in this process. The current proposal is designed to investigate the possibility that physiological and pharmacological induction of gamma-globin expression may be linked to an increase in the stability of its mRNA. Pilot studies indicate that the stability of transgenic human gamma-globin mRNA increases more than six-fold in mice homozygous for an unlinked beta-thalassemic determinant, and is correspondingly reduced in otherwise identical mice that transcribe human beta-globin mRNA from an independent, randomly-integrated transgene. In vitro analyses indicate that this effect may be mediated by conserved elements within the gamma- and beta-globin 3' UTRs that co-regulate the stability of the two mRNAs. Three experimental aims have been designed to confirm and extend these preliminary results. First, the importance of the dynamic regulation of gamma-globin mRNA stability during fetal and adult development will be established in transgenic mice using a novel assay designed specifically for this purpose. The effect of increased stability on the structure and translational efficiency of the gamma-globin mRNA will also be studied. Second, the likelihood that elements within the 3'UTR participate in regulating gamma-globin mRNA stability will be investigated by a series of linked structure-function studies. The stabilities of structurally-informative chimeric gamma-globin mRNAs will be determined in transgenic mice, and the results used to direct fine-mapping of crucial cis elements using both conventional in vitro methods and a newly-developed in vitro mRNA decay assay. Third, whole animal experiments will be carried out to test cell-culture and transgenic mouse evidence indicating that the stability of gamma-globin mRNA is enhanced by hydroxyurea and alpha-amino butyric acid, two gamma-globin inducing agents that have been used in human trials. Results from the proposed experiments will provide a more comprehensive understanding of the regulation of human gamma globin in both normal and pathological settings, as well as identify new mechanisms that can be targeted for therapeutic dysregulation in individuals with beta-globin gene disorders.
| He, Zhenning; Russell, J Eric (2004) Antisickling effects of an endogenous human alpha-like globin. Nat Med 10:365-7 |
| He, Zhenning; Russell, J Eric (2004) Effect of zeta-globin substitution on the O2-transport properties of Hb S in vitro and in vivo. Biochem Biophys Res Commun 325:1376-82 |
