Molecular Biology of Human Erythrocyte alpha-Spectrin

Yale University, New Haven CT

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Abstract

[unreadable] DESCRIPTION (provided by applicant): The long-term goals of this proposal are to elucidate the molecular mechanisms involved in normal and abnormal expression of the erythrocyte membrane protein alpha-spectrin. Erythrocyte alpha-spectrin is a critical component of the erythrocyte membrane skeleton. The first aim of this proposal is to identify alpha- spectrin mutations in patients with recessively inherited hereditary spherocytosis (rHS) and hereditary pyropoikilocytosis (HPP), severe hemolytic anemias, and to characterize the effect of these mutations on spectrin structure, function, and/or gene regulation using genetic, biochemical, and molecular techniques, including a novel in vivo, lentivirus-based model of spectrin function. The second aim is to identify and characterize key regulatory factors that control expression of the erythrocyte alpha-spectrin gene. These results will be applied to the study of the role of alpha-spectrin gene transcription in erythropoiesis and membrane biogenesis and to the genetic study of patients with hemolytic anemia with mutations in these regulatory elements. The third aim is to analyze the regulation of erythrocyte membrane protein gene expression by the factor EKLF (Erythroid Krupple-Like Factor). The general methodology to be utilized includes: study of genomic DNA from patients with alpha-spectrin linked rHS and HPP using PCR-based DHPLC followed by nucleotide sequence analysis; cloning and structural analysis of the cDNA and genomic fragments of the alpha-spectrin gene relevant to its expression and regulation; study of cis-acting sequences by gene manipulation followed by 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 vitro and in vivo analyses, and guanine-adenine ligation-mediated PCR dimethyl sulfate in vivo footprinting; in vivo chromatin immunoprecipitation studies; tissue- and developmental-specific studies of the regulatory sequences of the alpha-spectrin gene in transgenic mice; in vitro transduction of alpha- spectrin deficient MEL cells with a lentivirus containing the alpha-spectrin cDNA; transduction of hematopoietic stem and progenitor cells from alpha-spectrin deficient sph/sph mice with the same lentivirus, followed by stem and progenitor cell-lentiviral gene transplant into W/Wv and sph/sph mice. These studies will provide important insights into the role of spectrin in normal and disease states. [unreadable] [unreadable]