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: We have created a mouse model of Diamond Blackfan Anemia expressing the RPS19 R62W mutation. The model has all of the features of DBA. We will perform transcriptional and proteomic analyses of these animals to determine how erythropoiesis is inhibited. We will also generate other mutant RPS19 mice as well as haploinsufficient mice with other Ribosomal protein genes. ? 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. We have performed ChIP seq of HA-Tagged EKLF on chromatin from erythroid progenitors. We will compare these data to a transcriptional profile of mRNA expressed in the same cells to determine direct targets of EKLF. We will extend this combined approach into terminally differentiation erythroid cells to determine the role EKLF plays in terminal differentiation. 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 this we are preparing to knock out this Hb binding region to determine the physiological consequences of disrupting this association. Analysis of red cell membranes will be performed to determine whether this association alters red cell stability. 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 |
