In man, there is a switch from hemoglobin (Hb) F to A during ontogeny. Patients with hemoglobinopathies would be clinically improved if Hb F persisted or were reactivated. We plan to use a mouse model for studies of Hb regulation. Our hypothesis is that globin gene expression in mice is influenced by the same factors which affect Hb regulation in humans. We will study mice in which the % beta mu iota (of beta mu alpha+beta mu iota) serves as a model for % Eta Beta F (of Eta Beta F + Eta Beta F + Eta Beta Alpha). We will also investigate mice in which the % alpha4 (of alpha4+alpha1) undergoes changes, as another example of globin modulation. There are 4 aims: 1) We will examine conditions which increase Beta Mu Iota when added in vivo. Mice will be manipulated with physiologic hemopoietins as well as cell cycle specific drugs, and we will follow blood counts, globin chain composition, and globin chain synthesis using gel electrophoresis. 2. We will use cultures of erythroid progenitor cells to evaluate agents which may increase Beta Mu Iota when added in vitro, and to assess changes in progenitor cell pools in animals treated with such agents. Bone marrow cells will be cultured in methylcellulose, colonies counted, and globin chain synthesis examined in colonies. 3) Murine erythroleukemia cell lines (ELC) will be used to examine agents which might influence beta mu iota. These will be added at various times, and globin synthesis patterns examined. Chromatin structure of uninduced and induced ELC will be examined for differences in DNase I hypersensitivity or methylation, and line 9mu compared with line 25-66. 4. Mice with alpha1 and alpha4 globin genes will be evaluated to determine the physiologic significance of these hemoglobins, and the molecular explanation for the spontaneous appearance of mice with an apparent hybrid alpha4/1 gene. The mice will be inbred, and blood counts and sigma globin phenotype monitored. DNA will be examined by enzyme mapping, cloning, and sequencing, and RNA by studies of transcription, accumulation, and translation. The molecular explanation for the apparent higher expression of the 5' compared to the 3' gene will be sought. The expression of these genes will be examined in transient systems and in transgenic mice. Studies of these mouse models should increase our understanding of globin gene regulation, and be applicable to humans with hemoglobin disorders.
