m6A Methylated Long Noncoding RNA LOC339803 Regulates Intestinal Inflammatory Response.

Cytokine mediated sustained inflammation increases the risk to develop different complex chronic inflammatory diseases, but the implicated mechanisms remain unclear. Increasing evidence shows that long noncoding RNAs (lncRNAs) play key roles in the pathogenesis of inflammatory disorders, while inflammation associated variants are described to affect their function or essential RNA modifications as N6-methyladenosine (m6A) methylation, increasing predisposition to inflammatory diseases. Here, the functional implication of the intestinal inflammation associated lncRNA LOC339803 in the production of cytokines by intestinal epithelial cells is described. Allele-specific m6A methylation is found to affect YTHDC1 mediated protein binding affinity. LOC339803-YTHDC1 interaction dictates chromatin localization of LOC339803 ultimately inducing the expression of NFκB mediated proinflammatory cytokines and contributing to the development of intestinal inflammation. These findings are confirmed using human intestinal biopsy samples from different intestinal inflammatory conditions and controls. Additionally, it is demonstrated that LOC339803 targeting can be a useful strategy for the amelioration of intestinal inflammation in vitro and ex vivo. Overall, the results support the importance of the methylated LOC339803 lncRNA as a mediator of intestinal inflammation, explaining genetic susceptibility and presenting this lncRNA as a potential novel therapeutic target for the treatment of inflammatory intestinal disorders.

Preparation of pepsin trypsin digested gliadin for stimulation experiments.

Celiac disease (CD) is a complex immune disorder of the intestine that developes in genetically susceptible individuals. CD develops as an intolerance to ingested gluten proteins (gliadins, secalins, hordeins and avenins), being gliadin one of the most immunogenic. Here we present a protocol for the preparation of digested gliadin for laboratory use, a fundamental axis for in vitro and in vivo stimulation studies related to celiac disease research. The importance of a scrupulous handling of materials, products and laboratory instruments to achieve a lipopolysaccharide free gliadin is explained and emphasized. Therefore, in the present chapter, a step-by-step set-up of the protocol for pepsin trypsin gliadin digestion is explained.

LncRNA ARGI Contributes to Virus-Induced Pancreatic ß Cell Inflammation Through Transcriptional Activation of IFN-Stimulated Genes.

Type 1 diabetes (T1D) is a complex autoimmune disease that develops in genetically susceptible individuals. Most T1D-associated single nucleotide polymorphisms (SNPs) are located in non-coding regions of the human genome. Interestingly, SNPs in long non-coding RNAs (lncRNAs) may result in the disruption of their secondary structure, affecting their function, and in turn, the expression of potentially pathogenic pathways. In the present work, the function of a virus-induced T1D-associated lncRNA named ARGI (Antiviral Response Gene Inducer) is characterized. Upon a viral insult, ARGI is upregulated in the nuclei of pancreatic ß cells and binds to CTCF to interact with the promoter and enhancer regions of IFNß and interferon-stimulated genes, promoting their transcriptional activation in an allele-specific manner. The presence of the T1D risk allele in ARGI induces a change in its secondary structure. Interestingly, the T1D risk genotype induces hyperactivation of type I IFN response in pancreatic ß cells, an expression signature that is present in the pancreas of T1D patients. These data shed light on the molecular mechanisms by which T1D-related SNPs in lncRNAs influence pathogenesis at the pancreatic ß cell level and opens the door for the development of therapeutic strategies based on lncRNA modulation to delay or avoid pancreatic ß cell inflammation in T1D.

A long non-coding RNA that harbors a SNP associated with type 2 diabetes regulates the expression of TGM2 gene in pancreatic beta cells.

Introduction: Most of the disease-associated single nucleotide polymorphisms (SNPs) lie in non- coding regions of the human genome. Many of these variants have been predicted to impact the expression and function of long non-coding RNAs (lncRNA), but the contribution of these molecules to the development of complex diseases remains to be clarified. Methods: Here, we performed a genetic association study between a SNP located in a lncRNA known as LncTGM2 and the risk of developing type 2 diabetes (T2D), and analyzed its implication in disease pathogenesis at pancreatic beta cell level. Genetic association study was performed on human samples linking the rs2076380 polymorphism with T2D and glycemic traits. The pancreatic beta cell line EndoC-bH1 was employed for functional studies based on LncTGM2 silencing and overexpression experiments. Human pancreatic islets were used for eQTL analysis. Results: We have identified a genetic association between LncTGM2 and T2D risk. Functional characterization of the LncTGM2 revealed its implication in the transcriptional regulation of TGM2, coding for a transglutaminase. The T2Dassociated risk allele in LncTGM2 disrupts the secondary structure of this lncRNA, affecting its stability and the expression of TGM2 in pancreatic beta cells. Diminished LncTGM2 in human beta cells impairs glucose-stimulated insulin release. Conclusions: These findings provide novel information on the molecular mechanisms by which T2D-associated SNPs in lncRNAs may contribute to disease, paving the way for the development of new therapies based on the modulation of lncRNAs.

Boosting Cholesterol Efflux from Foam Cells by Sequential Administration of rHDL to Deliver MicroRNA and to Remove Cholesterol in a Triple-Cell 2D Atherosclerosis Model.

Cardiovascular disease, the leading cause of mortality worldwide, is primarily caused by atherosclerosis, which is characterized by lipid and inflammatory cell accumulation in blood vessels and carotid intima thickening. Although disease management has improved significantly, new therapeutic strategies focused on accelerating atherosclerosis regression must be developed. Atherosclerosis models mimicking in vivo-like conditions provide essential information for research and new advances toward clinical application. New nanotechnology-based therapeutic opportunities have emerged with apoA-I nanoparticles (recombinant/reconstituted high-density lipoproteins, rHDL) as ideal carriers to deliver molecules and the discovery that microRNAs participate in atherosclerosis establishment and progression. Here, a therapeutic strategy to improve cholesterol efflux is developed based on a two-step administration of rHDL consisting of a first dose of antagomiR-33a-loaded rHDLs to induce adenosine triphosphate-binding cassette transporters A1 overexpression, followed by a second dose of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine rHDLs, which efficiently remove cholesterol from foam cells. A triple-cell 2D-atheroma plaque model reflecting the cellular complexity of atherosclerosis is used to improve efficiency of the nanoparticles in promoting cholesterol efflux. The results show that sequential administration of rHDL potentiates cholesterol efflux indicating that this approach may be used in vivo to more efficiently target atherosclerotic lesions and improve prognosis of the disease.

Long non-coding RNA-regulated pathways in pancreatic ß cells: Their role in diabetes.

Long non-coding RNAs (lncRNAs) are transcripts of more than 200 nucleotides that have not coding potential, but act as gene expression regulators through several molecular mechanisms. Several studies have identified tons of lncRNAs that are expressed in pancreatic ß cells and many of them have been shown to have ß cell-specific expression, suggesting a potential role in the regulation of basal ß cell functions. Indeed, accumulating evidence based on numerous studies, has highlighted the implication of lncRNAs in the regulation of pancreatic ß cell differentiation and proliferation, insulin synthesis and secretion, and apoptosis. In addition, several lncRNAs have shown to be implicated in pancreatic ß cell dysfunction linked to different types of diabetes, including type 1 and type 2 diabetes, and monogenic forms of the disease. Pathogenic conditions linked to diabetes (inflammation or lipoglucotoxicity, for example) dysregulate the expression of several lncRNAs, suggesting that changes in lncRNA may alter potentially important pathways for ß cell function, and eventually leading to ß cell dysfunction and diabetes development. In this sense, functional characterization of some lncRNAs has demonstrated that these non-coding molecules participate in the regulation of several crucial pathways at the pancreatic ß cell level, and dysregulation of these pathways leads to pathogenic phenotypes. In this review, we provide an overview of the action mechanisms of functionally characterized lncRNAs in healthy ß cells and describe the contribution of some diabetes-associated lncRNAs to pancreatic ß cell failure.

The Role of lncRNAs in Gene Expression Regulation through mRNA Stabilization.

mRNA stability influences gene expression and translation in almost all living organisms, and the levels of mRNA molecules in the cell are determined by a balance between production and decay. Maintaining an accurate balance is crucial for the correct function of a wide variety of biological processes and to maintain an appropriate cellular homeostasis. Long non-coding RNAs (lncRNAs) have been shown to participate in the regulation of gene expression through different molecular mechanisms, including mRNA stabilization. In this review we provide an overview on the molecular mechanisms by which lncRNAs modulate mRNA stability and decay. We focus on how lncRNAs interact with RNA binding proteins and microRNAs to avoid mRNA degradation, and also on how lncRNAs modulate epitranscriptomic marks that directly impact on mRNA stability.

The T1D-associated lncRNA Lnc13 modulates human pancreatic ß cell inflammation by allele-specific stabilization of STAT1 mRNA.

The vast majority of type 1 diabetes (T1D) genetic association signals lie in noncoding regions of the human genome. Many have been predicted to affect the expression and secondary structure of long noncoding RNAs (lncRNAs), but the contribution of these lncRNAs to the pathogenesis of T1D remains to be clarified. Here, we performed a complete functional characterization of a lncRNA that harbors a single nucleotide polymorphism (SNP) associated with T1D, namely, Lnc13 Human pancreatic islets harboring the T1D-associated SNP risk genotype in Lnc13 (rs917997*CC) showed higher STAT1 expression than islets harboring the heterozygous genotype (rs917997*CT). Up-regulation of Lnc13 in pancreatic ß-cells increased activation of the proinflammatory STAT1 pathway, which correlated with increased production of chemokines in an allele-specific manner. In a mirror image, Lnc13 gene disruption in ß-cells partially counteracts polyinosinic-polycytidylic acid (PIC)-induced STAT1 and proinflammatory chemokine expression. Furthermore, we observed that PIC, a viral mimetic, induces Lnc13 translocation from the nucleus to the cytoplasm promoting the interaction of STAT1 mRNA with (poly[rC] binding protein 2) (PCBP2). Interestingly, Lnc13-PCBP2 interaction regulates the stability of the STAT1 mRNA, sustaining inflammation in ß-cells in an allele-specific manner. Our results show that the T1D-associated Lnc13 may contribute to the pathogenesis of T1D by increasing pancreatic ß-cell inflammation. These findings provide information on the molecular mechanisms by which disease-associated SNPs in lncRNAs influence disease pathogenesis and open the door to the development of diagnostic and therapeutic approaches based on lncRNA targeting.

Implication of m6A mRNA Methylation in Susceptibility to Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract that develops due to the interaction between genetic and environmental factors. More than 160 loci have been associated with IBD, but the functional implication of many of the associated genes remains unclear. N6-Methyladenosine (m6A) is the most abundant internal modification in mRNA. m6A methylation regulates many aspects of mRNA metabolism, playing important roles in the development of several pathologies. Interestingly, SNPs located near or within m6A motifs have been proposed as possible contributors to disease pathogenesis. We hypothesized that certain IBD-associated SNPs could regulate the function of genes involved in IBD development via m6A-dependent mechanisms. We used online available GWAS, m6A and transcriptome data to find differentially expressed genes that harbored m6A-SNPs associated with IBD. Our analysis resulted in five candidate genes corresponding to two of the major IBD subtypes: UBE2L3 and SLC22A4 for Crohn's Disease and TCF19, C6orf47 and SNAPC4 for Ulcerative Colitis. Further analysis using in silico predictions and co-expression analyses in combination with in vitro functional studies showed that our candidate genes seem to be regulated by m6A-dependent mechanisms. These findings provide the first indication of the implication of RNA methylation events in IBD pathogenesis.

A novel RT-QPCR-based assay for the relative quantification of residue specific m6A RNA methylation.

N6-methyladenosine (m6A) is the most common and abundant RNA modification. Recent studies have shown its importance in the regulation of several biological processes, including the immune response, and different approaches have been developed in order to map and quantify m6A marks. However, site specific detection of m6A methylation has been technically challenging, and existing protocols are long and tedious and often involve next-generation sequencing. Here, we describe a simple RT-QPCR based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues. Using this technique, we have been able to confirm the recently described m6A methylation in the 3'UTR of SOCS1 and SOCS3 transcripts. Moreover, using the method presented here, we have also observed alterations in the relative levels of m6A in specific motifs of SOCS genes in celiac disease patients and in pancreatic ß-cells exposed to inflammatory stimuli.

MAGI2 Gene Region and Celiac Disease.

Celiac disease (CD) patients present a loss of intestinal barrier function due to structural alterations in the tight junction (TJ) network, the most apical unions between epithelial cells. The association of TJ-related gene variants points to an implication of this network in disease susceptibility. This work aims to characterize the functional implication of TJ-related, disease-associated loci in CD pathogenesis. We performed an association study of 8 TJ-related gene variants in a cohort of 270 CD and 91 non-CD controls. The expression level of transcripts located in the associated SNP region was analyzed by RT-PCR in several human tissues and in duodenal biopsies of celiac patients and non-CD controls. (si)RNA-driven silencing combined with gliadin in the Caco2 intestinal cell line was used to analyze the implication of transcripts from the associated region in the regulation of TJ genes. We replicated the association of rs6962966*A variant [p = 0.0029; OR = 1.88 (95%1.24-2.87)], located in an intron of TJ-related MAGI2 coding gene and upstream of RP4-587D13.2 transcript, bioinformatically classified as a long non-coding RNA (lncRNA). The expression of both genes is correlated and constitutively downregulated in CD intestine. Silencing of lncRNA decreases the levels of MAGI2 protein. At the same time, silencing of MAGI2 affects the expression of several TJ-related genes. The associated region is functionally altered in disease, probably affecting CD-related TJ genes.

DEXI, a candidate gene for type 1 diabetes, modulates rat and human pancreatic beta cell inflammation via regulation of the type I IFN/STAT signalling pathway.

AIMS/HYPOTHESIS: The initial stages of type 1 diabetes are characterised by an aberrant islet inflammation that is in part regulated by the interaction between type 1 diabetes susceptibility genes and environmental factors. Chromosome 16p13 is associated with type 1 diabetes and CLEC16A is thought to be the aetiological gene in the region. Recent gene expression analysis has, however, indicated that SNPs in CLEC16A modulate the expression of a neighbouring gene with unknown function named DEXI, encoding dexamethasone-induced protein (DEXI). We therefore evaluated the role of DEXI in beta cell responses to 'danger signals' and determined the mechanisms involved. METHODS: Functional studies based on silencing or overexpression of DEXI were performed in rat and human pancreatic beta cells. Beta cell inflammation and apoptosis, driven by a synthetic viral double-stranded RNA, were evaluated by real-time PCR, western blotting and luciferase assays. RESULTS: DEXI-silenced beta cells exposed to a synthetic double-stranded RNA (polyinosinic:polycytidylic acid [PIC], a by-product of viral replication) showed reduced activation of signal transducer and activator of transcription (STAT) 1 and lower production of proinflammatory chemokines that was preceded by a reduction in IFNß levels. Exposure to PIC increased chromatin-bound DEXI and IFNß promoter activity. This effect on IFNß promoter was inhibited in DEXI-silenced beta cells, suggesting that DEXI is implicated in the regulation of IFNß transcription. In a mirror image of knockdown experiments, DEXI overexpression led to increased levels of STAT1 and proinflammatory chemokines. CONCLUSIONS/INTERPRETATION: These observations support DEXI as the aetiological gene in the type 1 diabetes-associated 16p13 genomic region, and provide the first indication of a link between this candidate gene and the regulation of local antiviral immune responses in beta cells. Moreover, our results provide initial information on the function of DEXI.

Functional implication of celiac disease associated lncRNAs in disease pathogenesis.

Celiac disease (CD) is a chronic immune-mediated disorder triggered by the consumption of dietary gluten that develops in genetically susceptible individuals. Genome-wide association studies (GWAS) and RNA sequencing technology (RNAseq) have helped in the detection of genes and genetic mechanisms involved in CD pathogenesis. However, the majority of the CD-associated variants reside in non-coding regions, which are mainly functionally uncharacterized. New evidences indicate that long non-coding RNAs (lncRNAs) play crucial roles in various biological processes and they have emerged as key regulatory molecules involved in the development of a wide range of diseases, including intestinal inflammatory disorders. This paper reviews the work performed by our group in the identification and characterization of lncRNAs associated with CD, highlighting the validity of some of the available bioinformatic resources to decipher the function of disease related lncRNAs.

Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens.

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the localization analysis of specific proteins or protein complexes in different tissue states (i.e., healthy vs. disease). This method is useful for the fractionation of both fresh and frozen intestinal tissue samples; it is easily accessible for all laboratories and not time consuming.

Transcription Factor Binding Site Enrichment Analysis in Co-Expression Modules in Celiac Disease.

The aim of this study was to construct celiac co-expression patterns at a whole genome level and to identify transcription factors (TFs) that could drive the gliadin-related changes in coordination of gene expression observed in celiac disease (CD). Differential co-expression modules were identified in the acute and chronic responses to gliadin using expression data from a previous microarray study in duodenal biopsies. Transcription factor binding site (TFBS) and Gene Ontology (GO) annotation enrichment analyses were performed in differentially co-expressed genes (DCGs) and selection of candidate regulators was performed. Expression of candidates was measured in clinical samples and the activation of the TFs was further characterized in C2BBe1 cells upon gliadin challenge. Enrichment analyses of the DCGs identified 10 TFs and five were selected for further investigation. Expression changes related to active CD were detected in four TFs, as well as in several of their in silico predicted targets. The activation of TFs was further characterized in C2BBe1 cells upon gliadin challenge, and an increase in nuclear translocation of CAMP Responsive Element Binding Protein 1 (CREB1) and IFN regulatory factor-1 (IRF1) in response to gliadin was observed. Using transcriptome-wide co-expression analyses we are able to propose novel genes involved in CD pathogenesis that respond upon gliadin stimulation, also in non-celiac models.

Celiac Diasease-associated lncRNA Named HCG14 Regulates NOD1 Expression in Intestinal Cells.

OBJECTIVE: The aim of the study is to identify additional celiac disease associated loci in the major histocompatibility complex (MHC) independent from classical HLA risk alleles (HLA-DR3-DQ2) and to characterize their potential functional impact in celiac disease pathogenesis at the intestinal level. METHODS: We performed a high-resolution single-nucleotide polymorphism (SNP) genotyping of the MHC region, comparing HLA-DR3 homozygous celiac patients and non-celiac controls carrying a single copy of the B8-DR3-DQ2 conserved extended haplotype. Expression level of potential novel risk genes was determined by RT-PCR in intestinal biopsies and in intestinal and immune cells isolated from control and celiac individuals. Small interfering RNA-driven silencing of selected genes was performed in the intestinal cell line T84. RESULTS: MHC genotyping revealed 2 associated SNPs, one located in TRIM27 gene and another in the non-coding gene HCG14. After stratification analysis, only HCG14 showed significant association independent from HLA-DR-DQ loci. Expression of HCG14 was slightly downregulated in epithelial cells isolated from duodenal biopsies of celiac patients, and eQTL analysis revealed that polymorphisms in HCG14 region were associated with decreased NOD1 expression in duodenal intestinal cells. CONCLUSIONS: We have successfully employed a conserved extended haplotype-matching strategy and identified a novel additional celiac disease risk variant in the lncRNA HCG14. This lncRNA seems to regulate the expression of NOD1 in an allele-specific manner. Further functional studies are needed to clarify the role of HCG14 in the regulation of gene expression and to determine the molecular mechanisms by which the risk variant in HCG14 contributes to celiac disease pathogenesis.

An Activating Mutation in STAT3 Results in Neonatal Diabetes Through Reduced Insulin Synthesis.

Neonatal diabetes mellitus (NDM) is a rare form of diabetes diagnosed within the first 6 months of life. Genetic studies have allowed the identification of several genes linked to the development of NDM; however, genetic causes for ∼20% of the cases remain to be clarified. Most cases of NDM involve isolated diabetes, but sometimes NDM appears in association with other pathological conditions, including autoimmune diseases. Recent reports have linked activating mutations in STAT3 with early-onset autoimmune disorders that include diabetes of autoimmune origin, but the functional impact of STAT3-activating mutations have not been characterized at the pancreatic ß-cell level. By using whole-exome sequencing, we identified a novel missense mutation in the binding domain of the STAT3 protein in a patient with NDM. The functional analyses showed that the mutation results in an aberrant activation of STAT3, leading to deleterious downstream effects in pancreatic ß-cells. The identified mutation leads to hyperinhibition of the transcription factor Isl-1 and, consequently, to a decrease in insulin expression. These findings represent the first functional indication of a direct link between an NDM-linked activating mutation in STAT3 and pancreatic ß-cell dysfunction.

Pancreatic Beta Cell Survival and Signaling Pathways: Effects of Type 1 Diabetes-Associated Genetic Variants.

Type 1 diabetes (T1D) is a complex autoimmune disease in which pancreatic beta cells are specifically destroyed by the immune system. The disease has an important genetic component and more than 50 loci across the genome have been associated with risk of developing T1D. The molecular mechanisms by which these putative T1D candidate genes modulate disease risk, however, remain poorly characterized and little is known about their effects in pancreatic beta cells. Functional studies in in vitro models of pancreatic beta cells, based on techniques to inhibit or overexpress T1D candidate genes, allow the functional characterization of several T1D candidate genes. This requires a multistage procedure comprising two major steps, namely accurate selection of genes of potential interest and then in vitro and/or in vivo mechanistic approaches to characterize their role in pancreatic beta cell dysfunction and death in T1D. This chapter details the methods and settings used by our groups to characterize the role of T1D candidate genes on pancreatic beta cell survival and signaling pathways, with particular focus on potentially relevant pathways in the pathogenesis of T1D, i.e., inflammation and innate immune responses, apoptosis, beta cell metabolism and function.

TYK2, a Candidate Gene for Type 1 Diabetes, Modulates Apoptosis and the Innate Immune Response in Human Pancreatic ß-Cells.

Pancreatic ß-cells are destroyed by an autoimmune attack in type 1 diabetes. Linkage and genome-wide association studies point to >50 loci that are associated with the disease in the human genome. Pathway analysis of candidate genes expressed in human islets identified a central role for interferon (IFN)-regulated pathways and tyrosine kinase 2 (TYK2). Polymorphisms in the TYK2 gene predicted to decrease function are associated with a decreased risk of developing type 1 diabetes. We presently evaluated whether TYK2 plays a role in human pancreatic ß-cell apoptosis and production of proinflammatory mediators. TYK2-silenced human ß-cells exposed to polyinosinic-polycitidilic acid (PIC) (a mimick of double-stranded RNA produced during viral infection) showed less type I IFN pathway activation and lower production of IFNα and CXCL10. These cells also had decreased expression of major histocompatibility complex (MHC) class I proteins, a hallmark of early ß-cell inflammation in type 1 diabetes. Importantly, TYK2 inhibition prevented PIC-induced ß-cell apoptosis via the mitochondrial pathway of cell death. The present findings suggest that TYK2 regulates apoptotic and proinflammatory pathways in pancreatic ß-cells via modulation of IFNα signaling, subsequent increase in MHC class I protein, and modulation of chemokines such as CXCL10 that are important for recruitment of T cells to the islets.

BACH2, a candidate risk gene for type 1 diabetes, regulates apoptosis in pancreatic ß-cells via JNK1 modulation and crosstalk with the candidate gene PTPN2.

Type 1 diabetes is a chronic autoimmune disease characterized by specific destruction of pancreatic ß-cells by the immune system. Linkage and genome-wide association studies have identified more than 50 loci across the human genome associated with risk of type 1 diabetes. Recently, basic leucine zipper transcription factor 2 (BACH2) has been associated with genetic risk to develop type 1 diabetes, in an effect ascribed to the immune system. We evaluated whether BACH2 may also play a role in immune-mediated pancreatic ß-cell apoptosis. BACH2 inhibition exacerbated cytokine-induced ß-cell apoptosis in human and rodent ß-cells by the mitochondrial pathway of cell death, whereas BACH2 overexpression had protective effects. BACH2 silencing and exposure to proinflammatory cytokines increased phosphorylation of the proapoptotic protein JNK1 by upregulation of mitogen-activated protein kinase kinase 7 (MKK7) and downregulation of PTPN2. JNK1 increased phosphorylation of the proapoptotic protein BIM, and both JNK1 and BIM knockdown protected ß-cells against cytokine-induced apoptosis in BACH2-silenced cells. The present findings suggest that the type 1 diabetes candidate gene BACH2 regulates proinflammatory cytokine-induced apoptotic pathways in pancreatic ß-cells by crosstalk with another candidate gene, PTPN2, and activation of JNK1 and BIM. This clarifies an unexpected and relevant mechanism by which BACH2 may contribute to diabetes.