Role of Wdr13 Gene in Beta Cell Proliferation Figure 5. Effect of WRD13 levels on pancreatic beta cell proliferation and cell cycle regulators. A) Transfection with AdWdr13 and AdGFP viruses shows overexpression of WDR13 protein in MIN6 cells as visualized by immunoblotting using anti WDR13 antibody. Lower panel shows beta actin as loading control. Overexpression of WDR13 protein results in retardation in cell proliferation after 48 h of transfection with 100 MOI. B) Overexpression of WDR13 protein results in accumulation of p21, whereas Cyclin D1, Cyclin D2, Cyclin E1 and p27 levels remain unaffected. C) siRNA knockdown of WDR13 in MIN6 cells. MIN6 cells were transfected with nonspecific scrambled (Scr) siRNAs and WDR13 specific siRNA. Immunoblot analysis shows Knockdown of WDR13 protein. Actin was used as loading control. Immunoblot, using p21 antibody shows reduction of p21 levels in WDR13 knockdown MIN6 cell. D) Cell cycle inhibitor p21 expression in purified pancreatic islets of Wdr13 knockout mice and that of wild type littermates by western blot analysis. Beta actin was used as loading control. p21 expression is less in the islets of knockout mice. E) Occupancy by WDR13 at p21 promoter revealed by chromatin immunoprecipitation using primers specific for p21 and GAPDH. doi:10.1371/journal.pone.0038685.g005 responsible for beta cell proliferation have been reported [12,16]. Many positive regulators of beta cells exist which include incretins [37], EGF [38], lactogens and growth hormones [39], HNF-4a [40], calcineurin/NFAT [41], Wnt3a [42] and integrins [43]. Various cell cycle inhibitors (p15, p16, p18, p19, p21, p27 and p57) have been identified, which target either various cyclins or cyclin dependent kinases to inhibit progression at various stages of the cell cycle [16]. In the present study, overexpression of WDR13 in pancreatic MIN6 cell line resulted in significantly higher p21 protein level, while cyclin D1, cyclin D2, cyclin E and p27 levels remained unaltered (Figure 5B). Further, through ChIP assay, we have identified occupancy of p21 promoter by WDR13 in pancreatic MIN6 cells. Consistent with these results, knockdown of WDR13 using siRNA in MIN6 cells and Wdr13 knockout pancreatic islets showed downregulation of p21 protein (Figure 5C, D). Cozar-Castellano et al., [44] observed an inhibitory role of p21 on beta cell proliferation in mice transgenic for hepatocyte growth factor and placental lactogen. However, in another study this group showed that knockout of p21 did not affect beta cell proliferation [45]. It might be possible that other protein(s) compensated the complete loss of p21 in beta cells in the latter study. WD-repeat proteins provide a platform for protein-protein interactions [1]. WDR13 protein contains five putative WD repeats and a LxxLL motif at c-terminal. It is possible that WDR13 mediates its effect (s) by interacting with other proteins. The effect of WDR13 on p21 levels observed by us in the present study may be the result of either direct or indirect interactions of this protein with p21 promoter. However, no DNA binding domain has been predicted in WDR13 [8]. Therefore, it is probable that WDR13 may either be activating p21 promoter by interacting with other proteins or repressing a repressor of p21. Our results will be useful in further unraveling the mechanisms of beta cell cycle regulation. It may be recalled that Wdr13 gene is expressed in most of the tissues and some of the interactions of this protein with its partners may even turn out to be tissue-specific; consequently, the precise mechanism(s) of action may differ in different tissues and/or at different target genes. Extensive CHIP experiments will be necessary to unravel different target genes of WDR13 and to understand the mode of action of this protein at these loci in various cell types. In conclusion, we provide evidence that WDR13 deficiency in mice leads to increased pancreatic islet mass, hyperinsulinemia, better glucose clearance and mild obesity. Our present study reveals a role of WDR13 in regulation of beta cell proliferation and these results provide a basis for further investigations aimed at delineating the mechanisms of action of WDR13 in cell cycle regulation through p21 and other as yet unidentified targets of this protein. We propose that given the higher random insulin levels and better glucose clearance in Wdr13 knockout mice, this protein may be explored as a potential candidate drug target for ameliorating impaired glucose metabolism in diabetes. PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e38685