In this study, we performed the first comprehensive DNA methylation profiling of human T2D pancreatic islets identifying 276 differentially methylated CpG sites that are affiliated to 254 genes. These generated data illustrate, for the first time, the epigenetic dimension of T2D in islets and how this is associated with transcriptional alterations. The pathophysiological significance of eight genes found to be differentially methylated in their promoters was validated by RNAi experiments.
It has been suggested that progressively occurring DNA methylation errors lead to diminished gene responsiveness to external stimuli and might thus contribute to the development of T2D (Gallou-Kabani and Junien, 2005
). Our findings of prevalent promoter hypomethylation in T2D islets are indicative of active biological processes involved in adaptation to the diabetic environment as well as biological pathways associated with β-cell dysfunction and apoptosis ( and ). The functional relevance of some of the differentially methylated genes in β-cells was documented by screening for β-cell survival/death following RNAi and subsequent exposure to stresses relevant to T2D (). Given the increased evidence that ER stress-induced apoptosis is one of the mechanisms of β-cell loss in T2D (Eizirik et al, 2008
), it was of interest to further assess the biological functions of two putative ER stress-related genes that we found to be hypomethylated in T2D islets, namely NIBAN
. We observed that these two genes are upregulated by synthetic ER stressors and by the more physiologically relevant saturated fatty acid palmitate in human islets, while knockdown of their expression by specific RNAi demonstrated their modulatory role in apoptosis (cf
. ). While NIBAN
protects against ER stress-induced apoptosis, CHAC1
seems to contribute to cell death. The hypomethylation observed at both genes could be explained by competing proapoptotic and antiapoptotic processes during ER stress response in diabetic islets. NIBAN
is a negative regulator of translation initiation factor eIF2α (Sun et al, 2007
). Therefore, its hypomethylation may indicate an attempt to re-establish ER homeostasis by reduction of protein synthesis (Eizirik et al, 2008
). Pending the outcome of these attempts, ER stress-induced apoptosis may be triggered by CHAC1
and other proapoptotic genes.
An important question with regard to epigenetic changes is: are the observed DNA methylation changes reflected in gene activity? By comparing the obtained DNA methylation profiles with microarray gene expression data, we were able to determine that a high proportion of genes in whose promoter T2D-related differential DNA methylation occurs are actively transcribed in pancreatic islets. A comparison with expression data of islet cell types (Dorrell et al, 2011
) showed that most of the differentially methylated genes are expressed in β-cells. This allowed us to conclude that T2D-related aberrant DNA methylation partially happens in the promoters of active genes. One has to keep in mind though that the expression studies in islets as well as in the β-cells analysed non-diabetic material. We observed mostly DNA hypomethylation in diabetic islets, not infrequently accompanied by elevated gene expression. Therefore, it can be assumed that the T2D-related hypomethylation leads, in part, to the induction of formerly silent genes.
Regarding differential gene expression in T2D islets, we observed an inverse correlation between differential promoter methylation and differential gene expression for a subset of genes. It is worth mentioning that for a significant proportion of differentially methylated genes, no statistically significant differential expression was observed (compare Supplementary Tables S4
). This may partially be due to the incompatibilities between methylation assays and the design of expression microarrays mentioned above that hamper in-depth comparisons between methylation and expression. However, for many genes the link between differential methylation and gene activity may be quite complex. Methylation of the cytosine base changes the topology of the major DNA groove, which may affect the binding of many transcription factors and DNA-binding proteins. Supporting this possibility, binding of two transcription factors whose target genes are differentially methylated in T2D islets has been described as methylation sensitive, namely CTCF (which binds to IGF2
, Bell and Felsenfeld, 2000
; Filippova et al, 2005
) and YY1 (which binds to ZIM2
, Kim et al, 2003
). Additionally, we found several genes encoding chromatin-associated proteins and transcription factor genes differentially methylated in T2D islets (cf
. and Supplementary Table S2
). Should the expression of these genes and/or their binding motifs be influenced by differential DNA methylation in T2D islets, it might further add to the complexity of the methylation–expression relationship. This could potentially explain the observations made in this study as well as others (Eckhardt et al, 2006
; Illingworth et al, 2008
; Suzuki and Bird, 2008
) that the relationship between DNA methylation and gene expression is rather complex.
In terms of genomic features, we detected T2D-associated differential DNA methylation mainly in LCP and ICP, while HCPs are underrepresented. Analysing LCP and a subset of ICP genes (CpG ratio <0.5), we discovered GATA
family transcription factors that are predicted to regulate a significant subset of these genes. Interestingly, the GATA
transcription factor family members are critical regulators in endocrine development, function and pathologies (Viger et al, 2008
). The physiological roles of many differentially methylated loci in T2D can be described as genes responding to (external) stimuli and to stress. Of note, Saxonov et al (2006)
found that a disproportionately high percentage of genes affiliated to these biological functions possess promoters with a low CpG density. This might indicate a general principle with regard to the promoter class of the differentially methylated gene loci: while in chronic diseases such as T2D and lupus (Javierre et al, 2010
) LCP genes are overrepresented, in diseases associated with cellular overgrowth (such as cancer) there is increased prevalence of HCP and relatively few LCP genes (Richter et al, 2009
; Martin-Subero et al, 2009a
). Further studies are required to test this intriguing possibility.
A key issue is whether the methylation changes we report play a causal role in T2D or are secondary to the diabetic condition. Indeed, the hypomethylation observed in oxidative stress, ER stress and apoptotic pathways may result from chronic exposure to the stressful metabolic environment of T2D, for example, high-glucose concentrations (Cnop et al, 2005
). An interesting example in this respect is CASP10
: we found significant hypomethylation in its promoter () and since caspase 10 is inducible by advanced glycation end products (Lecomte et al, 2004
; Obrenovich and Monnier, 2005
), this hypomethylation could be indicative of gene activation caused by chronically elevated blood glucose levels and consequently heightened non-enzymatic glycosylation events. Interestingly, experimental exposure of islets from non-diabetic donors to high-glucose concentrations (28 mM) for 72 h did not induce differential DNA methylation in any of the genes that display methylation changes in T2D islets. Even though these findings do not exclude an impact of chronic exposure to stressors like hyperglycaemia on the islet epigenome, they do make it unlikely that the observed alterations in DNA methylation are merely a consequence of relatively short metabolic insults. By inferring from the functions of the differentially methylated genes, it is possible that some of the identified epigenetic changes play a role in the progressive islet dysfunction in T2D, that is, they have potentially been acquired at different time points during pathological decline. Thus, the hypomethylation observed at some genes, like CASP10
, may be a consequence of T2D and severe and long-lasting hyperglycaemia. On the other hand, some genes, for example, those related to insulin secretion, may have obtained alterations in promoter methylation much earlier. For instance, defects in acute insulin response to glucose (AIRg) are among the earliest impairments and even precede the onset of pre-diabetic IGT (Bogardus and Tataranni, 2002
; Fukushima et al, 2004
; Leahy, 2005
; Bunt et al, 2007
). If aberrant AIRg arises from epigenetic aberrations at genes involved in insulin secretion (which is an established function for many genes in our study), these defects should manifest ahead of clinical T2D development. Whether such changes can be designated ultimately causal for the decline into T2D will remain to be proven. Overall, from the data at hand the changed methylation in the promoters of some genes identified in our study might thus be consequential and represent reactions to the diabetic environment. At other genes, the methylation aberrations could be interpreted to play a causal role, driving the islet dysfunction and T2D pathogenesis. Future, large-scale studies involving multiple stages of T2D development will be needed to elucidate the role of the epigenetic changes in the various stages of T2D pathogenesis. Due to medical ethics, it is impossible to obtain repeated pancreatic biopsies. Therefore, these studies will need to rely on surrogate tissues that remain to be validated. The availability of the presently described human islet methylation profiling will allow future search and validation of surrogate tissues.
However, identification and validation of tissues whose T2D-related DNA methylation profiles can serve as a proxy for pancreatic islets might prove difficult. The apparent absence of significant T2D-related differential DNA methylation in blood raises the possibility that T2D-related epigenetic aberrations are tissue-specific although more tissues will have to be screened to substantiate this. The finding of almost no differential DNA methylation in blood cells of T2D patients versus the significant changes in pancreatic islets implies the question whether the observed blood–islet difference is attributable to the different lifespan of the cells, for blood cells being days to months while β-cells have a lifespan of many decades (Cnop et al, 2010
). The validity of blood for epigenetic analysis has, however, been established by previous studies that uncovered differential methylation in DNA isolated from whole blood of individuals that were prenatally exposed to famine (Heijmans et al, 2008
; Tobi et al, 2009
). Further investigations into T2D-related epigenetic changes in surrogate tissues for pancreatic islets might elucidate their causative role or expose them as consequences of the disease.
A possible confounding factor for the identification of T2D-related epigenetic profiles is the medication that T2D patients receive and that may influence gene regulation. Histone deacetylase inhibitors (HDACi), for example, have been demonstrated to increase insulin sensitivity in muscle and liver and partially thwart diabetic nephropathy and retinopathy (Christensen et al, 2011
). It is possible that diabetes drugs like rosiglitazone, a PPAR-γ agonist, or metformin will alter gene activity patterns and confound profiling approaches. Adequately powered epigenetic profiling studies of surrogate tissues that consider the patients' medication may yield new insight of relevance for drug-based T2D therapy.
As acknowledged by McCarthy and Zeggini (2009)
, the >40 gene variants of T2D susceptibility genes known to date cannot fully explain T2D predisposition. Our study points to the involvement of epigenetic alterations in T2D thus underscoring the previously established contribution of lifestyle habits to its development. Combining the advantages of genome-scanning techniques and epigenome analyses might pave the way to better comprehend the pathogenesis of T2D. It will be of great interest to examine SNPs in the differentially methylated genes in T2D described in this study since the interplay between SNPs and differential (allele-specific) DNA methylation has recently been described (Shoemaker et al, 2010
). Linked to the topic of allele-specific DNA methylation, it is noteworthy that a number of the genes found to display differential methylation are also reported to be imprinted (Supplementary data
). It could hence be speculated that at least a partial loss of imprinting occurs in T2D islets.
In conclusion, we report the first comprehensive and detailed analysis of epigenetic changes in T2D, specifically an altered DNA methylation profile in the pancreatic islets of T2D patients with a major preponderance of hypomethylation in sequences outside CGIs. These aberrant methylation events affect over 250 genes, a subset of which is also differentially expressed. The dysregulation of these genes in T2D may notably be linked to β-cell functionality, cell death and adaptation to metabolic stress. Examination of two genes identified by methylation profiling, NIBAN and CHAC1, revealed their biological functions in distinct processes of the ER stress response. Furthermore, our data highlight genes belonging to biological processes whose involvement in T2D is not yet fully understood, such as inflammation and ion transporters/channels/sensors. Importantly, it can be envisaged that the uncovered DNA methylation changes might be, on one part, indicative of reactions of the islet cells to the diabetic condition and on another part, might be causal of T2D. A challenge in the future is to provide further evidence for the primary effects of methylation changes in the diabetic condition. Taken together, our DNA methylation study on human islets thus lays the ground to further unravel the biological complexity of T2D and outlines an unexpected level of epigenetic regulation in islets, which must be taken into account in future studies aiming to understand the pathogenesis of T2D.