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.
|An, Xiuli; Schulz, Vincent P; Li, Jie et al. (2014) Global transcriptome analyses of human and murine terminal erythroid differentiation. Blood 123:3466-77|
|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|
|Stewart, Andrew K; Shmukler, Boris E; Vandorpe, David H et al. (2011) Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S. Am J Physiol Cell Physiol 301:C1325-43|
|Pilon, Andre M; Ajay, Subramanian S; Kumar, Swathi Ashok et al. (2011) Genome-wide ChIP-Seq reveals a dramatic shift in the binding of the transcription factor erythroid Kruppel-like factor during erythrocyte differentiation. Blood 118:e139-48|
|Isern, Joan; He, Zhiyong; Fraser, Stuart T et al. (2011) Single-lineage transcriptome analysis reveals key regulatory pathways in primitive erythroid progenitors in the mouse embryo. Blood 117:4924-34|
|Steiner, Laurie A; Schulz, Vincent P; Maksimova, Yelena et al. (2011) Patterns of histone H3 lysine 27 monomethylation and erythroid cell type-specific gene expression. J Biol Chem 286:39457-65|
|Li, Xingguo; Wang, Shaohua; Li, Ying et al. (2011) Chromatin boundaries require functional collaboration between the hSET1 and NURF complexes. Blood 118:1386-94|
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