The long term goals of this proposal are to elucidate the molecular mechanisms involved in normal and abnormal expression of the human erythrocyte membrane protein ankyrin. Ankyrin-1, an important component of the red cell membrane skeleton, is expressed in erythroid, neural, and muscle cells.
The first aim of this proposal is the identification of ankyrin mutations in patients with ankyrin-linked hereditary spherocytosis (HS), a common inherited hemolytic anemia, and characterization of the effects of these abnormalities on ankyrin gene structure, expression, and regulation.
The second aim of this proposal is the identification and analysis of the key regulatory factors that control erythroid expression of the ankyrin gene, including the transcription factor EKLF. These results will be applied to the study of the genetic regulation of erythropoiesis and to the study of patients with hemolytic anemia with mutations in the ankyrin gene erythroid promoter.
The third aim of this proposal is to identify and characterize the isoforms of ankyrin-1 expressed in neural and muscle cells and to identify the c/s-acting sequences and trans-acting factors that regulate the tissue- and developmental stage-specific expression of these isoforms Reports of HS patients with co-segregating neural and muscular abnormalities provide the impetus for these studies. General methodology to be utilized in this research includes: study of genomic DNA from patients with ankyrin-linked HS by denaturing high performance liquid chromatography, followed by nucleotide sequencing;analysis of the cDNA and genomic fragments of the ankyrin gene relevant to its expression and regulation by the use of recombinant DNA technology;study of cis-acting sequences by gene manipulation and gene transfer/expression studies in tissue culture cells;studies of trans-acting factors by electrophoretic mobility shift assays, DNAse-l footprinting and hypersensitive site mapping, methylation interference techniques, guanine-adenine ligation-mediated PCR dimethyl sulfate in vivo footprinting;chromatin immunoprecipitation;developmental-, tissue-, and cell-specific studies of the regulatory sequences of ankyrin gene promoters and regulatory elements in transgenic mice using reporter gene and protein assays. These studies will provide important insights into the role of ankyrin in normal and disease states. This proposal seeks a clearer understanding of the red blood cell defects in many patients with hereditary spherocytosis, a common inherited anemia. These studies may provide important information to be used for diagnosis and treatment of patients with this disorder.
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