The long-term goals of this proposal are to elucidate the molecular mechanisms involved in normal and abnormal expression of the erythrocyte membrane protein a-spectrin. Erythrocyte a-spectrin is an important component of the erythrocyte membrane skeleton.
The first aim of this proposal is to identify a-spectrin mutations in patients with recessively inherited hereditary spherocytosis and hereditary pyropoikilocytosis, severe hemolytic anemias, and to analyze the structural and/or functional significance of these abnormalities.
The second aim of this proposal is to identify and characterize the promoter and other key regulatory factors that control expression of the erythrocyte a spectrin gene. These results will be applied to the study of the role of a-spectrin gene transcription in erythropoiesis and membrane biogenesis and to the genetic study of patients with hemolytic anemia who have been found to have mutations in the putative a-spectrin gene promoter.
The third aim of this proposal is to correct the defect in an a spectrin deficient murine erythroleukemia cell line and in the erythroid cells of sph/sph mice, an a-spectrin deficieny model of inherited hemolytic anemia, via retroviral transduction of the a-spectrin cDNA. The general methodology tc be utilized in this research includes: study of genomic DNA from patients with a-spectrin linked rHS and HPP using PCR-based single stranded conformational polymorphism (SSCP) analysis, followed by nucleotide sequence analysis cloning and structural analysis of the cDNA and genomic fragments of the a-spectrin gene relevant to its expression and regulation by the use of recombinant DNA technology; study of cis-acting sequences by gene manipulation followed b, gene transfer/expression studies in tissue culture cells; studies of trans-acting factors by electrophoretic mobility shift assays, DNAse-I footprinting, methylation interference techniques and site-directed mutagenesis followed by in vitrc and in vivo analyses, and guanine-adenine ligation-mediated PCR (GA-LMPCR) dimethyl sulfate in vivo footprinting developmental and tissue-specific studies of the regulatory sequences of the a-spectrin gene promoter in transgenic mice; In vitro transduction of a-spectrin deficient MEL cells with a retrovirus containing the a-spectrin cDNA transduction of hematopoietic stem cells (HSCs) from a-spectrin deficient sph/sph mice with an ecotropic retrovirus containing the a-spectrin cDNA, followed by HSC-retroviral gene transplant into the sph/sph mice. These studies will provide important insights into our studies of the role of a-spectrin in normal and disease states.
| Steiner, Laurie A; Schulz, Vincent; Makismova, Yelena et al. (2016) CTCF and CohesinSA-1 Mark Active Promoters and Boundaries of Repressive Chromatin Domains in Primary Human Erythroid Cells. PLoS One 11:e0155378 |
| Christensen, Robert D; Yaish, Hassan M; Gallagher, Patrick G (2015) A pediatrician's practical guide to diagnosing and treating hereditary spherocytosis in neonates. Pediatrics 135:1107-14 |
| An, Xiuli; Schulz, Vincent P; Mohandas, Narla et al. (2015) Human and murine erythropoiesis. Curr Opin Hematol 22:206-11 |
| An, Xiuli; Schulz, Vincent P; Li, Jie et al. (2014) Global transcriptome analyses of human and murine terminal erythroid differentiation. Blood 123:3466-77 |
| Weinstein, Jason S; Lezon-Geyda, Kimberly; Maksimova, Yelena et al. (2014) Global transcriptome analysis and enhancer landscape of human primary T follicular helper and T effector lymphocytes. Blood 124:3719-29 |
| Gallagher, Patrick G (2013) Abnormalities of the erythrocyte membrane. Pediatr Clin North Am 60:1349-62 |
| Gallagher, Patrick G (2013) Disorders of red cell volume regulation. Curr Opin Hematol 20:201-7 |
| Su, Mack Y; Steiner, Laurie A; Bogardus, Hannah et al. (2013) Identification of biologically relevant enhancers in human erythroid cells. J Biol Chem 288:8433-44 |
| Yocum, Ashley O; Steiner, Laurie A; Seidel, Nancy E et al. (2012) A tissue-specific chromatin loop activates the erythroid ankyrin-1 promoter. Blood 120:3586-93 |
| Bogardus, Hannah H; Maksimova, Yelena D; Forget, Bernard G et al. (2012) A de novo band 3 mutation in hereditary spherocytosis. Pediatr Blood Cancer 58:1004 |
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