Characterization of GAR22 function in the regulation of red blood cell differentiation and cell migration
- Charakterisierung der Funktion von GAR22 in der Regulierung der erythroiden Differenzierung und Zellmigration
Gamper, Ivonne; Zenke, Martin (Thesis advisor)
Aachen : Publikationsserver der RWTH Aachen University (2009)
Dissertation / PhD Thesis
Aachen, Techn. Hochsch., Diss., 2009
Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that have a major impact on erythroid cell development. In this study, I have investigated the influence of TR activity on red blood cell gene expression and identified TR target genes by employing a genome-wide approach with DNA microarray. Ligand-activated TR potently accelerated differentiation of SCF/Epo-dependent progenitors in vitro, concomitantly with inducing growth arrest. By comparing the gene expression profile of untreated and T3 treated SCF/Epo progenitor cells, I demonstrated that T3 potently regulated a subset of genes involved in erythroid differentiation, such as GATA-2, c-kit and Band 3. Furthermore, T3 regulated genes previously not implicated in differentiation of erythroid cells, including BTEB1 (basic transcription binding protein 1/ Krüppel-like factor 9) and the novel TR target gene GAR22 (growth arrest specific gene 2 on chromosome 22). The upregulation of GAR22 in terminally differentiated cells suggests that GAR22 might be involved in regulation of growth control and cell cycle. Accordingly, ectopic expression of GAR22 in SCF/Epo progenitor cells lengthened the cell cycle, but did not affect red cell gene expression. This finding is consistent with the proposed role of GAR22 as a tumor suppressor. GAR22 binds to microtubules and microfilaments, and this might contribute to the intense cytoskeletal remodeling that takes place during red blood cell differentiation. To investigate GAR22 binding to microfilaments and microtubules, I generated a panel of green fluorescent protein (GFP)-tagged GAR22 fusion proteins. For studying GAR22 dynamics, B16F1 mouse melanoma cells and NIH3T3 fibroblasts were chosen, since they have a flat morphology and are fairly motile, which makes these cells particularly well suitable for such study. GAR22 genes encodes for two splice variants, GAR22alpha and GAR22beta. Both GAR22 variants perfectly co-localized with actin in B16F1 and NIH3T3 cells. It was found that the dynamics of GAR22 proteins resembled that of actin suggesting that GAR22 is involved in the regulation of actin cytoskeleton remodeling and cell migration. Consistent with this hypothesis I found that overexpression of GAR22beta greatly impaired directional cell migration. To further characterize GAR22 function, I searched for GAR22 interacting proteins employing proteomics and mass spectrometry, and identified EB1 (end binding protein 1). GAR22beta interacts with EB1 through its carboxy-terminal domain. EB1 is a microtubule plus end binding protein that regulates stability and dynamics of microtubules. Thus, the GAR22-EB1 interaction might be involved in the regulation of microtubule dynamics and/ or function. This possibility is supported by the observation that overexpression of GAR22beta displaced EB1 from the plus tips of microtubules, causing the collapse of microtubule cytoskeleton. Moreover, overexpression of GAR22beta also impaired the reassembly of microtubules in nocoazole treated cells. Finally, the deletion of either the actin binding domain or the microtubule-binding domain within GAR22beta reduced the inhibitory phenotype of GAR22beta on cell migration. These findings uncovered a novel function of GAR22beta in regulating directional cell migration via the control of EB1 localization and microtubule dynamics. In this context through its dual binding to microfilaments and microtubules GAR22beta is proposed to support the functional interplay between actin and microtubule cytoskeletons. Thus, this GAR22beta activity is of fundamental importance for cell motility, and extends beyond its function in red cell differentiation.