Pancreatic β cell identity is maintained by DNA methylation-mediated repression of Arx

Abstract

Adult pancreatic β cells can replicate during growth and after injury to maintain glucose homeostasis. Here, we report that β cells deficient in Dnmt1, an enzyme that propagates DNA methylation patterns during cell division, were converted to α cells. We identified the lineage determination gene aristaless-related homeobox (Arx), as methylated and repressed in β cells, and hypomethylated and expressed in α cells and Dnmt1-deficient β cells. We show that the methylated region of the Arx locus in β cells was bound by methyl-binding protein MeCP2, which recruited PRMT6, an enzyme that methylates histone H3R2 resulting in repression of Arx. This suggests that propagation of DNA methylation during cell division also ensures recruitment of enzymatic machinery capable of modifying and transmitting histone marks. Our results reveal that propagation of DNA methylation during cell division is essential for repression of α cell lineage determination genes to maintain pancreatic β cell identity.

Figure 1. Loss of DNA methylation in beta cells results in an increase in the number of glucagon expressing cells

(A) Representative pancreatic sections from 3 months old Dnmt1^fl/fl (control) and RIP-Cre:Dnmt1^fl/fl (RC:Dnmt1^fl/fl) littermates were immunostained for glucagon (Glu; green) and Dnmt1 (red). (B) Immunostaining of representative pancreatic sections from 8 months old Dnmt1^fl/fl and RC:Dnmt1^fl/fl littermates showing insulin (green), glucagon (red) and overlay with DAPI (to counter-stain the nuclei; blue). (C) Quantification of glucagon and insulin area in 8 months old RIP-Cre:Dnmt1^fl/+ (control) and RIP-Cre:Dnmt1^fl/fl animals, shown as percentage of total islet area. N=4 animals. The error bars represent standard error (SEM) of the mean. See also Figure S1.

Figure 2. DNA methylation is required for maintenance of beta cell identity

(A) Glucose tolerance test (GTT) for RC: Dnmt1^fl/+ (control) and RC:Dnmt1^fl/fl animals at ages 3 months (left panel) and 6 months (right panel) (n=5 for each genotype). (B) Representative pancreatic section from an 8 months old RC:Dnmt1^fl/fl:R26RLacZ animal showing immunostaining for glucagon (green) in the left panel and an overlay of glucagon with beta galactosidase (b-gal; red), in the middle panel. The inset marks a representative area, magnified and shown in right panel. A number of beta-galactosidase and glucagon double labeled cells (white arrows) are evident in these RC:Dnmt1^fl/fl:R26RLacZ animals. Green arrows indicate normal alpha cells, which express glucagon and do not stain for beta galactosidase. (C) Quantification of endocrine cells co-staining for glucagon and RIP-Cre driven beta-galactosidase in RC: Dnmt1^fl/+:R26RLacZ (control) and RC:Dnmt1^fl/fl:R26RLacZ animals at 4 days and 8 months of age. N=3 animals per group. The error bars represent standard error (SEM) of the mean. See also Figure S2.

Figure 3. Dnmt1 methylates and leads to the repression of Arx locus in beta cells

(A) Bisulfite sequencing analysis and (B) quantification of the bisulfite sequencing data, shown as percentage DNA methylation (N=3) for the CpG rich UR2 region of Arx locus (−2103 to −1992 bp) in isolated beta and alpha cells. (C) Bisulfite sequencing analysis and (D) quantification of the bisulfite sequencing data, shown as percentage DNA methylation (N=3), for the UR2 region of Arx locus in cells marked with beta-galactosidase activity from RIP-Cre:Dnmt1^fl/fl:R26RLacZ (RC:Dnmt1^fl/fl:R26RLacZ) animals. Littermate RC:Dnmt1^fl/+:R26RLacZ mice were used as controls. Each horizontal line with dots is an independent clone and 20 clones are shown here. The UR2 region is almost fully DNA methylated (filled circles) in beta cells, but largely hypomethylated (open circles) in alpha cells. Cells isolated using beta-galactosidase activity from RC:Dnmt1^fl/fl:R26RLacZ animals showed a subset of clones that were hypomethylated, as marked by the bracket. (E) Fold changes in the levels of Dnmt1, Pax4, Pdx1, MafB and Arx transcripts determined by real-time RT-PCR in the cells marked with beta-galactosidase activity from RC:Dnmt1^fl/fl:R26RLacZ and control littermates, with the levels in control set as one arbitrary unit. The levels are depicted on a logarithmic scale. (F) Bisulfite sequencing analysis and (G) quantification of the bisulfite sequencing data, shown as percentage DNA methylation (N=3), for the UR2 region of Arx promoter from Min6 cells treated with control, scrambled (Scr) or Dnmt1 siRNAs. The error bars represent standard error (SEM) of the mean. See also Figure S3.

Figure 4. MeCP2 binds to the Arx locus in beta cells and recruits H3R2 methyltransferase PRMT6 to repress Arx expression

(A) Chromatin immunoprecipitation (ChIP) analysis showing the binding of MeCP2 to the two CpG rich regions, UR2 (−2111 to −1960) and UR1 (−184 to −254) of the Arx locus in beta and alpha cells. (B) Co-immunoprecipitation analysis examining the interaction of MeCP2 and PRMT6. Cell extracts from Min6 cells transfected with Flag-MeCP2 construct were used as input for immunoprecipitation (IP) with anti-Flag antibody and analyzed by immunoblotting with Flag and PRMT6 antibodies. (C) Co-immunoprecipitation analysis for the interaction of PRMT6 with MeCP2 using immunoprecipitation with myc-tag antibody on Min6 cells transfected with myc-PRMT6 construct, and Western blotting with myc-tag and MeCP2 antibodies. (D) ChIP analysis comparing the recruitment of PRMT6 to the UR2 region of Arx locus in beta and alpha cells. (E) ChIP analysis comparing the levels of asymmetric H3R2 di-methylation (H3R2me2a), H3K4 trimethylation (H3K4me3), H3K9 di- and tri-methylation (H3K9me2 and H3K9me3) and H3K9/14 acetylation (H3K9/14Ac) at the differentially methylated UR2 region of the Arx locus in beta and alpha cells purified from 5 months old wildtype mice. These analyses indicate enrichment of histone modification associated with repression of gene expression, namely, H3R2me2a, H3K9me2 and H3K9me3 at the Arx locus in pancreatic beta cells. The error bars represent standard error (SEM) of the mean. See also Figure S4.

Figure 5. Loss of DNA methylation affects the inheritance of the repressive histone modifications on the Arx locus in successive cell generations

Min6 cells transfected with scrambled or Dnmt1 siRNAs were grown in the exponential phase for 9 days in culture before cells were harvested. (A) Bisulfite sequencing of the UR2 region of Arx locus in Min6 cells transfected with Dnmt1 siRNA, harvested at day 0 and day 9. The UR2 region of the Arx locus that is almost fully DNA methylated (filled circles) at the start of the experiment and is hypomethylated (open circles) after 9 days of exponential growth. (B, C) ChIP analyses in Min6 cells transfected with scrambled or Dnmt1 siRNAs, 0 and 9 after transfection. The binding of MeCP2 to the UR2 region of Arx locus was reduced in Dnmt1 siRNAs transfected Min6 cells at D9 (B). The levels of H3R2me2a were reduced and H3K4me3 were increased in Dnmt1 siRNAs transfected Min6 cells at D9 (C). (D) Real-time RT-PCR analyses comparing the transcript levels of Dnmt1, Pdx1, Pax4, Insulin (Ins), Glucagon (Glu), MafB and Arx in Min6 cells after 0 and 9 days of transfection with Dnmt1 or control, scrambled (Scr) siRNAs. (E) Transcript levels of Dnmt1, Pdx1, Pax4, Insulin (Ins), Glucagon (Glu), MafB and Arx determined by real-time RT-PCRs in Min6 cells, after 9 days of transfection with scrambled (Scr), Dnmt1, Arx or Dnmt1+Arx siRNAs, showing a requirement for Arx in cell fate conversion upon loss of Dnmt1. n=3 for each experiment. The error bars represent standard error (SEM) of the mean. See also Figure S5.

Figure 6. Effect of loss of MeCP2 and PRMT6 mimics on Arx expression the loss of DNA methylation

(A) Transcript levels of MeCP2, Pdx1, Pax4, Insulin (Ins), Glucagon (Glu), MafB and Arx determined by real-time RT-PCRs, (B) binding of MeCP2 and PRMT6 to the UR2 region of Arx locus and (C) levels of H3R2me2a and H3K4me3 at the UR2 region of the Arx locus in Min6 cells, after 9 days of transfection with scrambled (Scr) and MeCP2 siRNAs. (D) Transcript levels of Prmt6, Pdx1, Pax4, Insulin (Ins), Glucagon (Glu), MafB and Arx determined by real-time RT-PCRs, (E) binding of MeCP2 and PRMT6 to the UR2 region of Arx locus and (F) levels of H3R2me2a and H3K4me3 at the UR2 region of the Arx locus in Min6 cells, after 9 days of transfection with scrambled (Scr) and Prmt6 siRNAs. See also Figure S6.