Human pancreatic islets from deceased organ donors (n = 6, Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI66514DS1) were sorted into highly enriched α, β, and exocrine (duct and acinar) cell fractions using a recently developed cell-surface antibody panel (11) and the additional antibody 2D12 (Figure 1A). Sample purity of the sorted α and β cell populations was validated by quantitative RT-PCR (qRT-PCR) for relevant marker genes. We calculated the sample purity as percentage of contamination by the opposite cell type and found our α and β cell fractions to be on average 94% and 92% pure (Figure 1B, formula in Supplemental Methods). Next, we determined the transcriptomes and histone methylation profiles of the sorted cell fractions by RNA-Seq and ChIP/ultra high-throughput sequencing (ChIP-Seq) (Figure 1A). We analyzed the histone methylation profiles of each donor and cell type individually, pooled the H3K4me3 and H3K27me3 calls of each cell type to obtain cell-type–specific histone methylation profiles, and validated this approach by confirming the enrichment calls and their low interindividual variability in a heat map analysis (Figure 1C). As an example, the enrichment profiles for H3K4me3 and H3K27me3 for the diabetes gene PDX1 in α, β, and exocrine cells are shown in Figure 1D. PDX1 is expressed in mature β cells and at lower levels in exocrine cells, but not in α cells (15, 16), which is clearly reflected by the histone modifications, with H3K4me3 enrichment in all cell fractions, but an additional, repressive H3K27me3 mark present only in α cells. Thus, the PDX1 locus is marked monovalently by H3K4me3 in β and exocrine cells, but carries a bivalent mark (H3K4me3 and H3K27me3) in α cells.

Figure 1 Study design for determination of the transcriptome and differential histone marks in sorted human islet cells. (A) Human islets were dispersed and subjected to FACS to obtain cell populations highly enriched for α, β, and exocrine (duct and acinar) cells. Chromatin was prepared and precipitated with antibodies for H3K4me3 and H3K27me3 followed by high-throughput sequencing (ChIP-Seq) (H3K4me3: n = 4 α, n = 4 β, n = 2 exocrine, H3K27me3: n = 3 α, n = 3 β, n = 2 exocrine). RNA-Seq analysis was performed to determine mRNA and lncRNA levels (n = 3 α, n = 3 β, n = 2 exocrine). (B) Sample purity assessment. Normalized insulin and glucagon expression levels of the individual α and β cell populations were obtained by qRT-PCR to calculate the contamination by the opposite cell population, revealing high sample purity (2.5%–10.3% contamination in the α and 2-13.1% contamination in the β cell populations; details in Supplemental Methods). (C) Analysis pipeline for H3K4me3 and H3K27me3 ChIP-Seq data. Peak calling (H3K4me3: GLITR; H3K27me3: STAR) on individual replicates, followed by signal pooling, was employed to assess histone modification profiles of α, β, and exocrine cells. Heat map analysis confirmed reproducibility of replicates. (D) Genome browser image of the PDX1 locus showing H3K4me3 enrichment in α, β, and exocrine cells and H3K27me3 enrichment only in α cells (defined as monovalent H3K4me3 enrichment in β and exocrine cells, bivalent mark in α cells; CpG islands: red bars).

We performed RNA-Seq analysis to assess the genome-wide transcriptional landscape in our sorted cell populations and to analyze the purity of our cell populations on a genome-wide scale. Principal component analysis showed that our sorted cell populations were distinct and that the replicates (n = 3 α, n =3 β, n = 2 exocrine) clustered together tightly (Figure 2A). Next, we performed cluster analysis to identify groups of genes with distinct expression patterns across cell types, to focus on the cell-type–specific transcriptional differences, and to classify α, β, and exocrine cell–specific signature genes. We present our results in a heat map, in which the 3 cell populations are displayed in their respective columns, and we identify clusters of α, β, or exocrine cell–specific signature genes, which are marked as colored boxes next to the heat map (Figure 2B). Among the α cell–specific genes are, as expected, the α cell–specific transcription factor aristaless related homeobox (ARX) and the α cell hormone glucagon (GCG). In addition, the enzymes prohormone convertase 2 (PCSK2) and dipeptidyl peptidase-4 (DPP4) are expressed specifically in α cells, the latter an important target of the commonly used group of oral drugs for type 2 diabetes, the DPP4 inhibitors or gliptins. Not surprisingly, the exocrine cell–specific genes include many digestive enzymes, their inactive precursors, and their inhibitors, such as various amylase isoforms (AMY1A, AMY1B, AMY1C, AMY2A, and AMY2B), pancreatic trypsinogen III (PRSS3), chymotrypsinogen I and II (CTRB1 and CTRB2), and the trypsin inhibitor serine peptidase inhibitor, kazal type 1 (SPINK1). In addition, jagged 1 (JAG1), the transcription factor SOX9, and pancreas-specific transcription factor 1a (PTF1A) are also among the exocrine-specific genes. Our RNA-Seq based list of β cell–enriched genes includes the β cell–specific transcription factors MAFA, NKX6-1, and PDX1 as well as the β cell hormone insulin (INS) and one of the key enzymes for its synthesis, prohormone convertase 1 (PCSK1). The β cell–specific cluster also included histone deacetylase 9 (HDAC9), which has previously been shown to be enriched in murine β cells (4). Remarkably, we found many genes identified in genome-wide association studies for nonautoimmune forms of diabetes (17) among the α cell and β cell–specific genes, such as the hepatocyte nuclear transcription factor 1-α (HNF1A) and the protein-tyrosine phosphatase δ (PTPRD) in α cells, the potassium channels KCNQ2 and KCNJ11, and the zinc transporter SLC30A8 in β cells. The complete gene lists of α, β, and exocrine cell–specific clusters are provided in Supplemental Table 2. In addition, we provide the transcriptomes of our α, β, and exocrine cell populations, including normalized expression values of every gene (Supplemental Table 3).

Figure 2 Genome-wide transcriptome analysis using RNA-Seq confirms high purity of sorted cell populations and reveals cell-type–specific gene expression. (A) Principal component analysis displays distinct cell populations and clustering of replicates (n = 3 α, n = 3 β, n = 2 exocrine), which confirms the high purity of our sorted cell populations (dots, replicates; crosses, averages). (B) Heat map analysis shows groups of genes with distinct expression patterns across cell types (columns: cell types, rows: genes). The orange, blue, and yellow bars on the left side of the heat map indicate α, β, and exocrine cell–specific gene clusters, respectively. The darker portion of these bars indicates stronger cell-type specificity of the gene cluster. We highlight important genes, including genes found to be associated with diabetes in genome-wide association studies (marked with asterisks). The complete gene lists of α, β, and exocrine cell–specific genes are provided in Supplemental Table 2.

To extend the analysis of our human α and β cell–specific transcription atlas, we searched for novel, cell-type–specific long noncoding transcripts. Long noncoding RNA molecules (lncRNAs) have been implicated as important developmental regulators, cell lineage allocators, and contributors to disease development (18). Recently described human islet lncRNAs were regulated during development and dysregulated in type 2 diabetic islets (19). Therefore, discovery of novel lncRNAs and evaluation of their function can potentially provide insight into diabetes pathogenesis. We discovered 12 β cell–specific, and 5 α cell–specific noncoding transcripts, indicative of the valuable research resource represented by our transcriptome data (Supplemental Table 4).

Next, we focused on the genome-wide, monovalent histone modification landscapes of our sorted pancreatic cell populations. We identified monovalent H3K4me3-enriched regions in α, β, and exocrine cells and compared them among the 3 cell types (Figure 3A). Strikingly, the vast majority of monovalently H3K4me3-marked genes were shared among the 3 pancreatic cell lineages (83%–95%), reflecting both their related function in protein secretion and common embryonic descent (Figure 3A).

Figure 3 Human α, β, and exocrine cells exhibit convergent monovalent H3K4me3 and H3K27me3 profiles, which correlate highly with genome-wide expression data. (A) The majority of H3K4me3-marked genes are shared between α, β, and exocrine cells (their overlap is indicated in the purple portion of the bars, 83%–95%). (B) H3K27me3 modification patterns are similar among pancreatic cell types (73%–83%, dark blue portion of the bars). (C and D) Heat map analysis (columns, individual samples; rows, genes) confirms low interindividual variability for all H3K4me3 (C) and H3K27me3 (D) peaks identified from the pooled data (peaks called by algorithms are indicated by the solid bars on the left of the heat maps). All pairs of columns in every heat map are significantly correlated based on correlation t test assessed by R statistics software (P < 2.2 × 10–16). (E–G) Normalized expression values obtained by RNA-Seq for genes grouped by their histone modification status in each cell type are shifted significantly above or below baseline expression (Wilcoxon signed rank test, P < 2.2 × 10–16). A shift above 0 on this scale indicates highly expressed genes and was observed for gene groups marked solely by H3K4me3 in all cell types (pink boxes in E–G). A shift below 0 on this scale indicates low or nonexpressed genes and was observed in all bivalently marked gene groups (light blue boxes) and monovalently H3K27me3-marked genes (dark blue boxes) in all cell types. Therefore, the histone modification states are significantly correlated with gene expression levels.

To investigate the landscape of repressive histone modifications, we performed H3K27me3 ChIP-Seq analysis and detected monovalent H3K27me3 enrichment at 3,755 gene regions in α, 4,420 gene regions in β, and 5,628 gene regions in exocrine cells (Figure 3B). Similar to the H3K4me3 modification, we found a high degree of overlap of monovalently H3K27me3-marked genes among the 3 cell populations (73%–83%, Figure 3B). Our H3K4me3 and H3K27me3 enrichment calls were validated by heat map analysis of the biological α and β cell replicates showing low interindividual variability (Figure 3, C and D). The box-and-whisker plots display the gene expression levels of bivalently marked, H3K4me3 marked, H3K27me3 marked and “unmarked” genes in each cell population (Figure 3, E–G). We show that bivalent, monovalent H3K4me3, and monovalent H3K27me3 enrichment calls were correlated genome wide with their respective mRNA levels at high statistical significance.

Bernstein and colleagues observed bivalent marks to be common in undifferentiated cells, such as ES cells and pluripotent progenitor cells, and in most cases, one of the histone modification marks was lost during differentiation, accompanying lineage specification (12–14). Consequently, most genes in differentiated cells are marked by either H3K4me3 or H3K27me3, corresponding to an expressed or repressed state, respectively. Preserving the bivalent state in a subset of genes was suggested to maintain higher plasticity (12–14). Interestingly, α cells showed the highest incidence of bivalent marks (2915 gene regions), followed by β cells (1914) and exocrine cells (1368) (Figure 4). As an internally controlled data set, we determined the bivalent domains for all 3 cell types in 1 individual donor (CITH068) and confirmed the higher number of bivalent marks in α cells (Supplemental Figure 1).

Figure 4 Human α cells demonstrate a higher number of bivalently marked genes than β and exocrine cells. (A) Here, α cells display more bivalently marked loci than β and exocrine cells. Nearly half of the genes bivalently marked in α cells carry a monovalent mark in β cells (purple and dark blue portion of the far left bar corresponding to H3K4me3 and H3K27me3 marks in β cells, respectively). (B) 406 genes are marked bivalently in β cells, but monovalently by H3K4me3 in α cells, and gene ontology analysis for these genes shows 3 modestly enriched categories: regulation (reg.) of RNA metabolic process, regulation of transcription, and transcription. (C) Genes marked bivalently in α cells, but monovalently by H3K27me3 in β cells, are significantly enriched for developmental processes. For detailed GO analysis see Supplemental Table 6. (D) Comparison of transcriptional regulators marked bivalently in hESC (22) to the histone modification signatures of human α and β cells reveals a higher overlap between α cells and hESCs (44%, right pie chart) than between β cells and hESC (26%, left pie chart). Many of the genes marked bivalently both in α cells and hESCs carry the repressive mark in β cells (43%, dark blue portion of inset).

Analysis of genes carrying a bivalent mark in β cells showed that the majority of these genes were also marked bivalently in α cells (1,474 genes, 77%), while 26 genes carried a monovalent H3K27me3 mark and 406 genes carried a monovalent H3K4me3 mark in α cells. Gene ontology analysis (20, 21) of the 406 genes marked bivalently in β cells, but monovalently by H3K4me3 in α cells, revealed 3 significantly enriched categories (Figure 4B). However, no significantly enriched categories were identified in the 523 genes marked bivalently in exocrine, but monovalently by H3K4me3 in β cells, or the 467 genes marked bivalently in exocrine, but monovalently by H3K4me3 in α cells (data not shown). The histone methylation status (H3K4me3, H3K27me3, bivalent, none of the above) of every gene in α, β, and exocrine cells is provided in Supplemental Table 5.

Interestingly, nearly half of the genes that displayed a bivalent mark in α cells were marked only by H3K4me3 or H3K27me3 in β cells (48%, 1,406 genes) (Figure 4A) in contrast to 434 genes (22%) marked bivalently in β cells, but monovalently in α cells. Strikingly, gene ontology analysis of genes that were marked bivalently in α cells, but only by the repressive H3K27me3 mark in β cells, displayed highly significant enrichment for genes involved in developmental processes (Figure 4C and Supplemental Table 6), suggesting a more plastic epigenetic state of developmental genes in α cells and a more fixed epigenetic condition in β cells. To further strengthen these observations, we compared transcriptional regulators marked bivalently in human ES cells (hESCs) (22) to their histone profile in α and β cells. We found that only 26% of all transcriptional regulators marked bivalently in hESCs also showed a bivalent mark in β cells, while nearly half of them were marked bivalently in α cells (44%) (Figure 4D). Further analysis of genes marked bivalently in both hESCs and α cells showed that 43% of these genes were H3K27me3 modified in β cells (Figure 4D). These findings provide additional support for the enhanced epigenetic plasticity of human α cells.

Next, we integrated our histone modification ChIP-seq data sets with the specific transcriptional signatures of α, β, and exocrine cells identified from our RNA-seq analysis (Figure 2B). Quantitative analysis of H3K4me3 and H3K27me3 enrichment at α, β, and exocrine cell–specific signature genes in each of these cell types showed increased H3K4me3 levels in their respective signature gene group, as expected (Supplemental Figure 2A). Interestingly, H3K27me3 levels of α cell–specific genes were comparable between α and β cells, whereas H3K27me3 levels of β cell–specific genes were increased in α cells and decreased in β cells, supporting higher prevalence of the H3K27me3 mark repressing β-signature genes in α cells rather than vice versa.

Next, we wanted to assess whether the increased H3K27me3 mark in α cells was indicative of higher bivalency in functionally relevant β cell–specific genes, such as the transcriptional regulators that control cell-type–specific gene expression. We analyzed functional gene categories within the strongly α and β cell–specific genes (Figure 2B). Analysis of β cell–specific genes implicated in “ion transport” (34 genes) showed that 29% of the genes were marked bivalently in α, 15% in β, and 6% in exocrine cells. Interestingly, analysis of β cell–specific genes implicated in “regulation of transcription” (31 genes) displayed a much higher percentage of bivalently marked genes in α cells (42%) than in β or exocrine cells in reverse (16% and 13%), which was not observed in any of these functional categories in α cell–specific signature genes (Supplemental Figure 2B). In summary, a large fraction of β cell–specific transcriptional regulatory genes are in a bivalent state in α cells.

Finally, we focused our cell-type–specific analysis of the histone modification landscape on α and β cell–specific genes known to be important for pancreatic development and endocrine cell function. Analysis of the histone marks of α cell–specific genes in β cells identified many as being marked monovalently by H3K4me3 (HNF1A, PCSK2) or by H3K27me3 (Iroquois related homeobox 2 [IRX2], GCG, IRX1, ARX), whereas only 2 genes showed a bivalent histone modification profile (DPP4, PTPRD) (Figure 5B). As expected, most α cell–specific genes were marked only by H3K4me3 in α cells. However, the genes PTPRD, IRX1, and the locus encoding the α cell–specific transcription factor ARX were marked bivalently (Figure 5B). Strikingly, most β cell–specific genes important for β cell function displayed monovalent H3K4me3 enrichment (with the exception of HDAC9) in β cells (Figure 5C), while 7 of 12 β cell–specific loci were marked bivalently in α cells, including the functionally relevant genes PCSK1 and GLP1R and the genes encoding the crucial β cell–specific transcription factors MAFA and PDX1. We extended our findings by utilizing previously published H3K4me3 and H3K27me3 data of CD4-positive T cells (23) and compared our pancreatic histone modification profiles to the histone modification landscape of this extrapancreatic cell type. The histone modification profiles of all strongly α, β, and exocrine cell–specific genes in α, β, exocrine, and CD4+ T cells can be found in Supplemental Table 7. As expected, we found significant monovalent enrichment for the activating H3K4me3 mark in only 4 of the 34 selected α, β, and exocrine cell–specific genes (Supplemental Table 8), as these pancreatic genes are not active in lymphoid cells. In summary, α cells preserve high bivalency in many genes known to be crucial for endocrine cell development and function.

Figure 5 Human α cells display higher bivalency in genes encoding β cell transcriptional regulatory proteins. (A) The epigenetic status of β cell signature genes (Figure 2B) functioning in ion transport or regulation of transcription was analyzed separately for α, β, and exocrine cells. Of the β cell–enriched ion transport genes, only 6% and 15% were marked bivalently in exocrine and β cells, respectively, while 29% carried this mark in α cells. For β cell signature genes involved in transcriptional regulation, 42% were marked as bivalent in α cells, but only 16% and 13% in β cells and exocrine cells, respectively. Thus α cells display a higher degree of bivalency for genes important in transcriptional regulation than for genes implicated in ion transport. (B and C) Schematic representation of the histone modification status of a relevant subset of human α and β cell signature genes (Figure 2B). The histone modification status of β cells is shown below each gene of interest. (B) As expected, most α cell signature genes are marked monovalently by H3K4me3 in α cells, and many of them carry a monovalent H3K27me3 mark in β cells. Interestingly, IRX1 and ARX are marked bivalently in α cells. (C) Within this subgroup of genes, β cell–expressed genes are marked monovalently by H3K4me3 in β cells, with the exception of HDAC9, which is marked bivalently. Remarkably, many β cell–expressed genes are marked bivalently in α cells, including the crucial insulin-synthesis enzyme PCSK1, the GLP1-receptor (GLP1R), and 2 essential β cell–specific transcription factors, MAFA and PDX1.

The high incidence of bivalent marks in α cells, the interesting bivalent pattern of β and α cell–specific transcription factors in α cells, and the large overlap with bivalently marked transcriptional regulators in hESCs raised the possibility that epigenomic manipulations could be exploited to reprogram human α cells toward the β cell phenotype. Several drugs, such as adenosine dialdehyde (Adox) and 3-deazaneplanocin A (DZNep), interfere with histone methylation (24). We employed the general histone methyltransferase inhibitor Adox, which among others decreases H3K27me3 levels (24), to test whether modulation of the histone methylation status of human pancreatic islets could promote reprogramming. To validate the effectiveness of the histone methyltransferase inhibitor Adox in human islet tissue, we investigated the H3K27me3 modification landscape after Adox treatment. The small numbers of cells recovered after Adox-treatment and FACS analysis did not allow us to perform cell-type–specific analysis of the H3K27me3 profiles, so we compared the H3K27me3 profiles of whole human islets cultured in the absence or presence of Adox. This experiment allowed us to assess whether H3K27me3 levels of repressed genes are decreased after Adox treatment. Indeed, analysis of genes that carry a bivalent mark or a monovalent H3K27me3 mark in all pancreatic cell populations displayed a strong decrease in H3K27me3 enrichment after Adox treatment (Supplemental Figure 3A). In addition, this experiment approximated a cell-type–specific analysis of the H3K27me3 profiles of a subset of β cell–specific pancreatic transcription factors, namely those which are marked bivalently in α cells, but monovalently by H3K4me3 in β cells (MAFA and PDX1). Since α and β cells compose the vast majority (approximately 90%) of the human islet (25) and exocrine cells do not survive in culture (26), any change in H3K27me3 enrichment levels at these loci is thus likely indicative of changes in α cells at the MAFA and PDX1 loci, as there is no H3K27me3 present at these genes in β cells to begin with. In addition, we investigated the H3K27me3 profile of the α cell–specific transcription factor ARX, which is marked bivalently in α cells, but monovalently by H3K27me3 in β cells. Our H3K27me3 ChIP-Seq analysis confirmed the expected decrease of H3K27me3 enrichment at these 3 gene loci after Adox treatment (Figure 6A).

Figure 6 Inhibition of histone methyltransferases leads to partial endocrine cell-fate conversion. (A) H3K27me3 ChIP-Seq analysis of human islets shows decreased H3K27me3 levels at the ARX, MAFA, and PDX1 loci following treatment of human islets with the histone methyltransferase inhibitor Adox. (B) Adox-treatment of human islets results in colocalization of glucagon (red) and insulin (green) granules within the same cell (yellow arrows), suggesting partial endocrine cell fate conversion, which was not seen in vehicle-treated islets (control). Original magnification, ×63. For Z-stack confocal images see Supplemental Videos 1 and 2. (C) Treatment of human islets with Adox results in colocalization of the β cell–specific transcription factor Pdx1 (white) and glucagon (red), further indicating endocrine reprogramming (white arrows: glucagon-positive, Pdx1-negative cells; yellow arrows: glucagon-positive, Pdx1-positive cells). The images on the right correspond to the area within yellow box. Original magnification, ×63. (D) Quantification of glucagon-positive, Pdx1-positive cells in untreated and Adox-treated human islets reveals many double-positive cells after Adox treatment, indicating initiation of reprogramming events in α cells. (E) Adox treatment of human islets leads to a decrease in NKX6-1 and MAFA levels in β cells (n = 3 α, n = 3 β, n = 2 treated α, n = 2 treated β), an increase in PDX1-levels, and no change in INS and GCG levels. (F) In Adox-treated α cells, we observe no change in INS and GCG expression, a slight decrease in NKX6-1 and MAFA levels, and an increase of ARX and PDX1 expression.

Strikingly, we found that treatment of human islets with Adox resulted in the occasional cooccurrence of glucagon and insulin granules within the same islet cell, which was not observed in untreated islets (Figure 6B, Supplemental Videos 1 and 2, and Supplemental Figure 3B). A priori, the colocalization of glucagon with insulin could be due to α to β cell fate or β to α cell fate conversion. As lineage tracing is not possible in human samples, we next employed a murine genetic lineage tracing model to assess the origin of the dual hormone–positive cells. For this purpose, we treated islets from GlucagonCre;Rosa26EYFP mice, in which α cells are permanently marked by yellow fluorescence protein (YFP) expression (27, 28) with Adox in vitro. Adox-treated islets showed insulin granules in YFP+ cells, which were not observed in untreated control islets (Supplemental Figure 3, C and D), supporting partial conversion of α cells to the β cell fate. Unfortunately, at the present time, no inducible Glucagon-Cre ER line exists, and the possibility that Adox-treated β cells activated the Glucagon-Cre promoter cannot be excluded. Since the colocalization of glucagon and insulin granules was observed in a small number of cells, we wanted to investigate whether the important β cell marker PDX1 was present in glucagon-positive cells, indicating the initiation of partial reprogramming events. Strikingly, we found that Adox treatment caused nuclear PDX1 expression in many glucagon-positive cells (Figure 6, C and D).

To investigate the transcriptional changes in Adox-treated human islets and confirm increased PDX1 expression, we performed FACS analysis after Adox treatment using the same antibody panel as described above, followed by RNA-Seq analysis (n = 2 treated α, n = 2 treated β, technical replicates) and focused on cell-type–specific hormones and transcription factors. To assess whether Adox-treated β cells become more α cell like or vice versa, we first compared the ratio of expression levels in untreated β cells and untreated α cells (β/α) to the ratio in Adox-treated β cells and untreated α cells (Adox-β/α) (Figure 6E). We found a decrease in NKX6-1 and MAFA levels, an increase in PDX1 levels, and no change in insulin expression, but observed no change in the expression levels of the α cell–specific genes glucagon or ARX, giving no indication for a gain of α cell identity. Second, we compared the ratio of expression values in untreated α cells and untreated β cells (α/β) to the ratio in Adox-treated α cells and untreated β cells (Adox-α/β) to elucidate whether Adox-treated α cells become more “β cell like” (Figure 6F). Our analysis revealed no change in insulin and glucagon and an increase in ARX levels. Interestingly, we detected a slight decrease in NKX6-1 and MAFA levels, but an increase in PDX1 expression. Taken together, these results confirm our observations of PDX1 expression in glucagon-positive cells in the immunofluorescent staining and favor partial α to β cell fate conversion over the alternative.