Specific Silencing of the REST Target Genes in Insulin- Secreting Cells Uncovers Their Participation in Beta Cell Survival David Martin1., Florent Allagnat1., Emilie Gesina2, Dorothee Caille3, Asllan Gjinovci3, Gerard Waeber1, Paolo Meda3, Jacques-Antoine Haefliger1* 1 Service of Internal Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, 2 Ecole Polytechnique Fe´de´rale de Lausanne, Faculte´ des Sciences de la Vie, Lausanne, Switzerland, 3 Department of Cell Physiology and Metabolism, University Medical Center, Geneva, Switzerland Abstract The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP- REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock- down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival. Citation: Martin D, Allagnat F, Gesina E, Caille D, Gjinovci A, et al. (2012) Specific Silencing of the REST Target Genes in Insulin-Secreting Cells Uncovers Their Participation in Beta Cell Survival. PLoS ONE 7(9): e45844. doi:10.1371/journal.pone.0045844 Editor: Jianping Ye, Pennington Biomedical Research Center, United States of America Received March 19, 2012; Accepted August 24, 2012; Published September 20, 2012 Copyright: 2012 Martin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported by grants from The Swiss National Science Foundation [31003A_138528/1; 310000-109402], the Juvenile Diabetes Research Foundation [1- 2007-158], the European Union [BETAIMAGE 222980; IMIDIA, C2008-T7], the National Institutes of Health HL64232, the Novartis and the Octav and the Marcella Botnar Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Jacques-Antoine.Haefliger@chuv.ch . These authors contributed equally to this work. Introduction Type 1 (T1D) and type 2 (T2D) diabetes are characterized by an absolute or relative insulin deficiency, respectively. In both diseases, loss of functional beta cell mass occurs through beta cell apoptosis [1,2]. While the triggering events and the nature of the molecular effectors leading to diabetes-associated apoptosis are still disputed, several critical regulators of beta cell survival have been identified (Reviewed in [3]). Importantly, the search for intrinsic pro-survival factors has identified several key proteins, including insulin receptor substrate 2 (IRS2) [4,5], the anti-apoptotic members of the BCL2 family: BCL2 [6,7] and BCL2L1 (also called BCLXL) [8], MAPK8IP1 (also called islet brain 1) [9], protein tyrosine phosphatase, receptor type, N (PTPRN, also called islet cell antigen 512) [10], AKT1 (also called AKT/PKB) [11]. Our incomplete knowledge of the mechanisms responsible for the unusual susceptibility of beta cells to metabolic stress and inflammation, imposes that specific positive regulators of beta cell mass are identified. To better understand what is a beta cell, and to attempt improving it under pathological situations [12], we initiated a search for new beta cell-specific genes. Generating transgenic mice expressing the transcriptional repressor REST specifically in beta cells (RIP-REST mice) allowed us to identify the function of a wide group of uncharacterized genes that contains the REST binding motif, called Repressor Element 1 (RE-1) [13]. REST is a zinc finger transcription factor which blocks the expression of neuroendocrine traits in all cell types, but neurons and beta cells. Indeed, REST is commonly absent in mature insulin-producing cells and neurons [14–16], but sup- presses elsewhere the expression of a large set of RE-1-containing genes, thereby ensuring that their expression is specific to neuroendocrine cell types. Upon ectopic REST expression in the RIP-REST transgenic mice, REST target genes were specifically silenced in beta cells. The resulting phenotype showed that, in vivo, RE-1-containing genes are crucial for proper beta cell function [13]. The identification that several bona fide REST target genes code for proteins that are key to exocytosis further substantiated our observations [13]. Here, we report the characterization of a novel line of RIP-REST founder mice which demonstrates that RE-1-containing genes are also essential to beta cell survival. These mice featured diabetes as a consequence of a gradual but extensive loss of beta cells through apoptosis. In vitro experiments PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45844