The focus of this thesis is the role of ribosomal proteins in hematopoiesis and development. Ribosomal proteins are mutated in patients with Diamond Blackfan anemia (DBA). These mutations primarily affect blood tissues, as DBA patients have a macrocytic anemia. We have identified hematopoietic defects in zebrafish with a mutation in ribosomal protein S29 (rps29). \(Rps29^{-/-}\) embryos have defects in hematopoietic stem cell formation, aorta specification, and hemoglobinization. Embryos also have increased numbers of apoptotic cells, and microarray analysis reveals up-regulation of a p53 gene signature. All of the hematopoietic phenotypes are rescued by p53 mutation, demonstrating that p53 activation induced by ribosomal protein knockdown is mediating the \(rps29^{-/-}\) mutant phenotype. In addition, polysome profiles of mutant embryos identify a decrease in 80s monosome and polysome fractions. Preliminary RNA sequencing analysis of the polysome fractions suggested a shift in genes being translated in the mutant. We performed a chemical screen on rps29 embryos. Using embryo morphology and vascular expression patterns as read-outs, 600 compounds of known bioactivity were screened. One compound, A-3, improves embryo morphology, and a structurally related compound, W-7, rescues the vasculature defect. These compounds are calmodulin inhibitors, and A-3 can also rescue the hemoglobin defect in \(rps29^{-/-}\) embryos. To elucidate the compounds’ mechanism of action, A549 and \(CD34^+\) cells with RPS19 knocked down by shRNA were treated with chemical hits. In these cells, calmodulin inhibitors cause a decrease of p21 even with p53 induction. These data support a model where calmodulin inhibition can inhibit the p53 pathway upon ribosomal protein knockdown. In parallel to our zebrafish studies, we generated induced pluripotent stem (iPS) cells from DBA patient fibroblasts as a part of a large-scale collaboration. Three iPS lines are validated, and a total of 27 lines will be generated from patients with mutations in RPS19, RPL5, and RPL11. Testing for defects in blood differentiation and determining the role of p53 in these lines will enable validation of this system as a model of DBA. The iPS lines can subsequently be used for chemical and genetic screening to identify novel DBA pathways and potential therapies.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/10288443 |
| Date | January 2012 |
| Creators | Taylor, Allison |
| Contributors | Zon, Leonard Ira |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
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