The long-term goals of this proposal are to elucidate the molecular mechanisms involved in normal and abnormal expression of the protein 1-spectrin, a critical component of the erythrocyte membrane skeleton. Abnormalities of ?-spectrin are associated with inherited hemolytic anemia, which is sometimes severe.
The first aim of this proposal is to identify and characterize the cis-sequences, trans-factors, and epigenetic state, including chromatin architecture, across the ?-spectrin gene locus that regulate its expression in erythroid and nonerythroid cells. These studies address the hypothesis that common regulatory signatures in ?-spectrin and other erythrocyte membrane protein genes control their tissue-specific expression. Integration of mRNA transcript composition, genomic organization, RNA polymerase II binding, transcription factor and regulatory protein binding, and histone architecture will provide detailed knowledge of erythrocyte membrane gene structure, function, and regulation and allow us to identify a common regulatory signature that controls expression in erythroid cells. The studies in this aim combine high throughput genomic technologies with functional studies of gene expression. Techniques to be utilized include chromatin immunoprecipitation experiments followed by array hybridization (ChIP-chip) or whole genome sequencing (ChIP-seq), high throughput DNase I hypersensitive site mapping, and functional studies of gene expression. Results obtained from these studies will allow study of the role of 1-spectrin in erythropoiesis, membrane biogenesis, and inherited erythrocyte disorders.
The second aim of this proposal is the identification of mutations that perturb ?- spectrin spectrin gene regulation and/or expression in patients with spectrin-linked inherited hemolytic anemias and characterization of the effect of these mutations on ?-spectrin gene structure, function, and expression. These studies address the hypothesis that defects of ?-spectrin occur in regions of functional importance and their elucidation will provide important information about the structure, function, and regulation of the ?-spectrin gene in normal and mutant erythrocytes. Nucleotide sequence analysis of amplified patient genomic DNA will be performed to identify genetic defects in cases of inherited hemolytic anemia associated with qualitative and quantitative defects of ?-spectrin. Previously known and newly identified cis-regulatory elements in the ?- spectrin gene will be interrogated and characterized in functional studies of gene regulation. Together, results from these studies will provide important information on the structure, function, and regulation of spectrin in erythroid and nonerythroid cells and shed additional insight into the pathogenesis of spectrin-linked disorders of the erythrocyte.
Many people suffer from anemia, or low blood count, due to abnormalities in the membranes, or lining, of their red blood cells. Some people require surgery or blood transfusions to treat the anemia. This application studies the what, why, and how of abnormalities of the lining of the red blood cell that cause the anemia.
|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|
|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|
|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|
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