Epigenetic regulation of the DLK1-MEG3 microRNA cluster in human type 2 diabetic islets

Abstract

Type 2 diabetes mellitus (T2DM) is a complex disease characterized by the inability of the insulin-producing β cells in the endocrine pancreas to overcome insulin resistance in peripheral tissues. To determine if microRNAs are involved in the pathogenesis of human T2DM, we sequenced the small RNAs of human islets from diabetic and nondiabetic organ donors. We identified a cluster of microRNAs in an imprinted locus on human chromosome 14q32 that is highly and specifically expressed in human β cells and dramatically downregulated in islets from T2DM organ donors. The downregulation of this locus strongly correlates with hypermethylation of its promoter. Using HITS-CLIP for the essential RISC-component Argonaute, we identified disease-relevant targets of the chromosome 14q32 microRNAs, such as IAPP and TP53INP1, that cause increased β cell apoptosis upon overexpression in human islets. Our results support a role for microRNAs and their epigenetic control by DNA methylation in the pathogenesis of T2DM.

Fig 1. The imprinted chromosome 14q32 miRNA cluster is down-regulated in T2DM islets

Expression levels of the 15 most abundant miRNAs in (a) three non-diabetic and (b) four T2DM human islets as identified by small RNA sequencing. (c) Differentially expressed miRNAs between non-T2DM (n=3) and T2DM (n=4) islets, identified by a FDR of 20% and minimum fold change of 1.5×. MiRNAs up- and down-regulated in T2DM islets are highlighted, and miRNAs belonging to the Chr 14q32 cluster are underlined. (d) Distribution of expression levels of miRNAs identified as significantly differentially expressed by small RNA sequencing across clustered samples. (e) Relative expression of miRNAs in the Chr 14q32 cluster as determined by Taqman qPCR of 14 non-T2DM and 10 T2DM human islets. p-value calculated using two-tailed Student’s t-test. *p<0.05, **p-value<0.01, ***p-value<0.005. +/− SEM. See also Figure S1 and Table S2.

Fig 2. Chr 14q32 miRNAs are highly and specifically expressed in human β-cells

(a) Average expression of Chr 14q32 cluster miRNAs in human α- and β- cells. (b) Expression of Chr 14q32 miRNAs (minimal expression 50 reads per million (RPM)) in sorted human α- and β- cells. (c) Genome browser image of histone modification marks H3K4me3 (n=4) and H3K27me3 (n=3) at the MEG3 promoter (chromosomal location marked on top) of sorted α- and β- cells from healthy human donors. Grey bars represent sequence conservation. Positions of the miRNA cluster and other nearby transcripts are shown. See also Figure S2 and Table S3.

Fig 3. Increased methylation of the MEG3-Differentially methylated region (DMR) in T2DM islets

(a) Schematic representation of DLK1-MEG3 locus DMRs with allele specific gene expression depicted. Regions analyzed for Fig 3c and d are marked as green bars. (b) Methylation-specific PCR for MEG3 promoter methylation in two T2DM and two non-diabetic donors’ islets. The methylated band (Me) is 160 bp, the unmethylated band (Un) is 120 bp. Percent methylation was determined for multiple CpGs in the (c) IG-DMR and (d) MEG3-DMR in 5 non-diabetic and 9 T2DM donors’ islets by pyrosequencing of bisulfite converted DNA. Each bar represents an individual CpG, and the regions refer back to schema in part (a). p-value calculated using Student’s T-test. **p-value<0.005, ***p-value<0.001. +/− SEM. See also Figure S3.

Fig 4. Identification of miRNA targets in human islets by HITS-CLIP

(a) Schema of HITS-CLIP procedure and chimeric reads ligation. (b) Average read coverage of all HITS-CLIP target mRNA fragments over a standardized mRNA. (c) Argonaute footprint distribution across target library mRNAs in human islets. (d) Targeting of human IAPP mRNA by miR-432 and 376a was validated by luciferase reporter assays. Vectors with or without the 3’UTR of IAPP were co-transfected with either empty pCAG-eGFP vector or miR-432 and -376a. Error bars indicate mean +/− SEM. ***p-value calculated using Student’s t-test. p = 1.8 × 10⁻⁵. See Tables S4 and S5.

Fig 5. Determination of direct miRNA:mRNA targeting relationship from chimeric reads

Deep sequencing of our Argonaute HITS-CLIP library identified thousands of chimeric reads, consisting of a mature miRNA and a target mRNA fragment. (a) The fifteen most abundant mRNAs found in chimeric reads in human islets. (b) The fifteen most highly miRNA-regulated mRNAs in chimeric reads. The regulatory load ratio is the relative Ago-associated mRNA fraction of the chimeric reads, defined as the ratio of their sequence counts to their normalized abundance in human islets (c) The fifteen most abundant miRNAs found in chimeric reads in human islets. (d) Significantly enriched gene ontology biological processes in targets of human islet miRNAs. (e) Pie-chart representation of distribution of mRNA regions found in chimeras with miRNAs. (f) Average read coverage of chimeric mRNA fragments across an mRNA divided into 150 equal bins. See Figure S4 and Table S6.

Fig 6. Validation of the miR-495:TP53INP1 targeting relationship

A β-cell apoptotic factor, TP53INP1 is regulated by miR-495. (a) The sequence of the miR-495 (orange) and TP53INP1 3’UTR (green) chimera. Folded confirmation with base pairing between the miRNA and 3’UTR is indicated below. (b) Relative levels of TP53INP1 mRNA between T2DM and non-T2DM islet samples. Error bars indicate mean +/− SEM. (c) Anti-correlation between normalized TP53INP1 and miR-495 in nine T2DM islet donor samples. (d) Targeting of human TP53INP1 mRNA by miR-495 was validated by luciferase reporter assays. Vectors with or without the 3’UTR of TP53INP1 were co-transfected with either scramble or miR-495 mimics. Error bars indicate mean +/− SEM. p-value calculated using Student’s t-test. ***p-value = 1.94 × 10⁻⁵. (e) Relative mRNA levels of TP53INP1 and Onecut1 (normalized to the average of HPRT and beta actin transcript levels) in human islets transduced with lentivirus encoding tough decoy constructs for either scramble sequence (TuDctrl) or miRNA-495 (TuD495). Error bars indicate +/− SEM. **p-value = 0.0076, n=3.