Our overall goal is to develope mouse models to determine sdpecific aspects of red blood cell production, terminal red cell differentiation, erythroid specific gene expression and the role of the band 3 trans membrane protein in red cell physiology. Our basic approiach is to use transgenic mouse technology to create null animals by gene replacement strategies (knock out) or to introduce specific mutations into red cell genes by knock in strategies. Our work is divided into 4 specific aims.? Specific Aim 1: To create a mouse model of Diamond Blackfan Anemia. We have chosen to focus on Diamond Blackfan Anemia, which is thought to affect early erythroid progenitors. Over 25% of patients with DBA have mutations in the Ribosomal Protein S19 gene (RPS19). RPS19 is a component of the large ribosomal subunit, but how mutant RPS19 mutations affects differentiating erythroid cells is unknown. Because the DBA mutations are dominant, ectopic expression of mutant RPS19 in transgenic mice produced a lethal phenotype. We are using gene targeting to place the mutant RPS19 gene downstream of the normal allele. Upon Cre mediated excision of the wild type allele, we will induce mutant RPS19 expression . We will compare wild type and mutant transgenic mice for the relative number of HSC, CMP, BFU-E and CFU-E. The terminal stages of erythroid maturation cells at each stage of erythroid maturation in mutant and WT mice will be compared using the markers CD71 and TER11936. The cell cycle status at each stage will be analyzed by PI staining and apoptosis will be analyzed with Annexin V. We anticipate that these studies will identify the stage and the nature of the defect caused by mutant RPS19 expression. ? Specific Aim 2; Characterization of defective erythropoiesis in EKLF deficient mice. EKLF deficient mice die at day 14.5 of gestation of a severe anemia. FACS analysis of EKLF-- fetal liver cells stained with antibodies against CD71 and Ter119 demonstrated that the terminal stages of erythroid maturation (Ter119BRIGHT) were absent. To determine which genes are regulated by EKLF in red cells, we performed microarray analysis and RT-PCR and RNase protection validation to document more than 70 genes that are misregulated in EKLF-- fetal liver cells. Among the genes down regulated in EKLF deficient fetal liver cells are those involved in the cell cycle and apoptosis pathways as well as structural proteins of the red cell membrane, and channel proteins that regulate the hydration or red blood cells. We will use FACS to sort the primitive cells and compare the number of colony forming cells, their cell cycle status and the degree of apoptosis in mutant and wild type cells. We will perform osmotic fragility assays tp compare the stability of the red cell membrane and hydration of primitive and definitive cells, using hemoglobin markers for each stage. ? Specific Aim 3: To generate a comprehensive profile of chromatin changes associated with the activation of specific Ankyrin promoters.? Identification and Characterization of Ank-1 DNaseI Hypersensitive Sites: Regulatory elements such as promoters, barriers and enhancers often co-localize with DNsae I hypersensitive sites (HSs). We have developed a high throughput quantitative PCR assay to detect DNase I HS across a 119 kb region of the Ank-1 promoter region, which includes an neuromuscular, an erythroid and a ubiquitous promoter. We have identified 6 discrete HSs within the region, which flank the three promoters. We have shown that those surrounding the erythroid promotr are barrier elements, but do not have either enhancer or enhancer blocking activity. We will use the Chip Chromatin Conformation Capture assay (4C) to determine how these HSs interact with each other. We have already shown that the sites surrounding the erythroid promoter are in contact in erythroid cells, but separated in non-erythroid cells. We will identify the proteins associated with the HSs by Chromatin Immune Preciptiation (ChIP). We hypothesize that the deletion of the erythroid promoter may allow one of the other promoters to become active in erythroid cells. To test this we have developed a targeted deletion of this region in ES cells for evaluation. ? Specific Aim 4: StructureFunction analysis of band 3 protein in red cells. Band 3 (B3) is an integral membrane protein that serves as the major anion channel for red blood cells and binds ankyrin, tethering the actinspectrin network to the red cell membrane. In addition to binding ankyrin, Band 3 is the major anion exchange protein of the red cell membrane and plays a critical role in maintaining red cell hydration, which is important to prevent the concentration dependent polymerization of deoxy HbS. We have shown that the N-terminus of band 3 lies in the cytoplasm and is a high affinity binding site for deoxy hemoglobin, and that this binding serves as a catalyst for HbS polymerization. Because of these varied functions, all of which may modify the severity of SCD, we have embarked on a genetic analysis of the structure and function of band 3. Molecular models using peptide fragments suggested that a b-hairpin loop in band 3 between amino acids 175 and 185 is responsible for ankyrin binding. We have used homologous recombination in ES cells to delete the nucleotides encoding the 11 amino acid hairpin loop in exon 7 of the mouse band 3 gene and replace them with a di-glycine bridge sequence. Analysis of red cell membranes will be performed to determine whether the mutant band 3 can bind ankyrin and the consequence of disrupting the band 3ankyrin bridge. We will use a similar gene targeting approach to generate a Band 3 N-terminus deleted mouse model to test the effects of mutations in the deoxy hemoglobin binding domain of band 3. In addition to analyzing the binding of Hb to band 3, we will breed these animals to mouse models of SCD to determine whether preventing deoxy Hb binding to band 3 can modulate the severity of SCD.
|Stefanovic, Marko; Markham, Nicholas O; Parry, Erin M et al. (2007) An 11-amino acid beta-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes. Proc Natl Acad Sci U S A 104:13972-7|
|Nilson, Douglas G; Sabatino, Denise E; Bodine, David M et al. (2006) Major erythrocyte membrane protein genes in EKLF-deficient mice. Exp Hematol 34:705-12|
|Pilon, Andre M; Nilson, Douglas G; Zhou, Dewang et al. (2006) Alterations in expression and chromatin configuration of the alpha hemoglobin-stabilizing protein gene in erythroid Kruppel-like factor-deficient mice. Mol Cell Biol 26:4368-77|