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M. Antony, A. V. Das, K. Mallya, I. Ahmad; Chromatin Remodeling ATPase, Brg1 Promotes Maintenance of Retinal Stem Cells/Progenitors by Facilitating the Canonical Wnt Signaling. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4428.
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We have recently demonstrated that chromatin remodeling ATPase, Brm, is developmentally regulated and facilitates the differentiation of retinal stem cells/progenitors (Das et al., 2007, J. Biol. Chem. 282: 3517). Transcripts corresponding to Brm homologue, Brg1 is detected in the developing retina with constitutive patterns of expression. Here, we have tested the hypothesis that Brg1, in contrast to the role of Brm, promotes proliferation of retinal stem cells/progenitors by facilitating Wnt signaling.
E14 rat retinal stem cells/ progenitors, enriched as neurospheres, were cultured in differentiation conditions and RT-PCR analysis was carried on every 24 hours to obtain a temporal trend in the expression of Brg1, Brm, Lef1 and CyclinD1. E14 Neurospheres, transduced with Brg1, dominant negative (DN) Brg1, and empty retrovirus, were cultured in the presence of Wnt2b to examine the effects of perturbation of Brg1 expression/function on Wnt2b-driven Wnt signaling. Promoter-reporter (Lef1-Lacz) assay was carried out in 293T cells transduced with recombinant retrovirus to perturb Brg1 expression to test whether Brg1 has a direct effect on Lef1 promoter. Co-immunoprecipitation was carried out on nuclear extracts from E14 neurospheres that were exposed to FGF2/Wnt2b to examine complex formation between beta catenin and Wnt2b.
We observed that as E14 retinal stem cells/progenitor differentiate, the level of transcripts corresponding to Lef1 and CyclinD1 decreased and that of Brm increased. Levels of Brg1 transcript appeared to be unchanged. Cells in Wnt-exposed neurospheres, transduced with Brg1 retrovirus, expressed higher levels Lef1 and CyclinD1 transcripts compared to controls. In contrast, levels of these transcripts decreased in neurospheres, transduced with DN Brg1. A significant increase in Lacz activities was observed in 293T cells transduced with Brg1 retrovirus, compared to those infected with empty or DN Brg1 retrovirus. A complex, immunoreactive to Brg1 and beta-catenin antibodies was observed whose levels were higher in E14 cells cultured in the presence of Wnt2b, compared to controls.
Our observations suggest that the two homologous chromatin remodeling ATPases serve different functions in the regulation of retinal stem cells/progenitors. While Brm facilitates their differentiation, Brg1 maintains them in proliferating and undifferentiated states by facilitating Wnt signaling.
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