REST Target Genes and Beta Cell Survival Figure 2. REST expression is associated with a major loss of beta cell mass. A. Immunostaining for insulin (Ins, red) and glucagon (Glu, green) reveals, when compared to wild type mice (WT, left panel), a major loss of insulin-positive cells, within a low number of disorganized islets in pancreas of adult diabetic RIP-REST mice (middle panel). REST nuclear staining (green, right panel) indicates that only a few surviving beta cells still express REST. Blue staining is the DAPI labeling of nuclei. Scale bar, 25 mm. B. Immunostaining for insulin (Ins, red) and glucagon (Glu, green) shows that, even if disorganized when compared to islets from wild type animals (WT, left panel), islets from diabetic RIP-REST mice at postnatal day 7 (P7) show a significant number of insulin-positive cells (middle panel). REST nuclear staining (green, right panel) indicates that a majority of beta cells express REST. Blue staining is the DAPI labeling of nuclei. Scale bar, 50 mm. C. Quantification of beta-(Ins+ cells) and alpha- (Gluc+ cells) cell mass in P2, P7 and adult diabetic RIP-REST (black bars, n = 3 each) and wild type mice (white bars, n = 3 each). A 30, 45 and 90% reduction in beta cell mass is observed in P2, P7, and adult diabetic RIP-REST mice, respectively, when compared with corresponding mass of controls. Results are mean 6 SD. *P,0.05, ***P,0.001 versus values of wild type mice. D. Confocal analysis of insulin (Ins, red) and glucagon (Glu, green) immunofluorescence shows scarce double insulin-and glucagon-expressing cells (arrows) in pancreatic sections of adult diabetic RIP-REST mice. Blue staining is the DAPI labeling of nuclei. Scale bar, 25 mm. doi:10.1371/journal.pone.0045844.g002 Figure 3. Beta cell loss in diabetic RIP-REST mice occurs through apoptosis. A. Nuclear PCNA (green) and insulin (red) staining show same level of proliferating beta cells (arrows) in both wild type (WT, left panel) and diabetic RIP-REST mice (right panel) at P2. Blue staining is the DAPI labeling of nuclei. Scale bar, 50 mm. B. Representative images of nuclear TUNEL (green) and insulin (red) staining showing apoptotic nuclei of beta cells in sections of P2 diabetic RIP-REST mice. No apoptotic beta cells were detected in wild type animals (not shown). Blue staining is the DAPI labeling of nuclei. Scale bar, 25 mm. C. Quantification of apoptotic nuclei in non-infected INS-1E cells (NI) and INS-1E cells transfected with a control (Ad-GFP) or REST- expressing adenovirus (Ad-REST) at different multiplicity of infection (MOI) as indicated, and treated 24 h with a mix of cytokines (black bars) or not (white bars). Results are mean 6 SD of six independent experiments. *P,0.05 versus respective controls in treated or untreated conditions. # P,0.05 versus MOI 2 of Ad-REST infection. doi:10.1371/journal.pone.0045844.g003 expression on the capacity of INS-1E cells to survive to a cytotoxic attack or palmitate incubation. To this end, 56 h after siRNA transfection, INS-1E cells were treated during 24 h with a mix of IL1-b, TNF-a and IFN-c. Cdk5r2 silencing per se had no effect on INS-1E cells viability, as compared to non-transfected cells (Fig. 6B). However, siCdk5r2#1 and siCdk5r2#2 transfection increased the cytokine-induced apoptosis by 60% and 50%, respectively, as compared to cells transfected with the control siRNA (Fig. 6B). Western blotting performed with total proteins from the same experiment confirmed that Cdk5r2 knock-down aggravated the cytokine-induced apoptosis, as quantified with increased cleavage of caspase-9 and 23, an early and late apoptosis marker, respectively (Fig. 6C). Similarly, siCdk5r2#1 and siCdk5r2#2 transfections increased apoptosis induced by a 24 h treatment with palmitate (0.5 mM) by 60% and 40%, respectively (Fig. 6D). These results indicate that Cdk5r2 contrib- utes to protect beta cell against both cytokines and free fatty acid- induced apoptosis. Discussion Recent bioinformatic studies on the distribution of RE-1 binding sites in the genome, combined with analysis of chromatin occupancy mediated by REST, have revealed the existence of several hundred putative REST target genes [19–24]. The caveat of these large output studies is the lack of functional assessment of the candidate genes. The generation of transgenic mice expressing REST in beta cells represents a reliable model to identify the importance of RE-1-containing genes in insulin-producing cells [13]. Specifically, analysis of the glucose-intolerant RIP-REST mice led to the identification of several REST target genes which are key to exocytosis of insulin-containing granules [13]. We now document a role for REST target genes in beta cell survival in a RIP-REST mouse line which displayed diabetes, as a result of a high level of REST expression, which was associated with a massive loss of adult beta cells. This massive loss is due to the down-regulation of multiple REST target genes, whose number is likely to increase with REST levels. Indeed, transcription factor abundance is an important parameter influencing the activity of many, though not necessarily all cognate DNA binding motifs [25]. Furthermore, sequence variations in the RE-1 binding motif establish a hierarchy of binding affinity for REST to its target genes, indicating that genes bearing a weakly conserved RE-1 motif have a suboptimal binding affinity for REST [26]. Thus, it is likely that more genes are down- regulated in the diabetic than in the glucose-intolerant RIP-REST transgenic mice, due to the higher expression of REST in the former animals. Using RE-1 database screenings and our in vitro model of REST gain of function, we further show that REST expression triggers 1) beta cell death under normal conditions 2) increased sensitivity to cytokines and palmitate 3) decreases the PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45844