Article
Reference
Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes
YANG, Beatrice T, et al.
Abstract
Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic β-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal β-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P
= 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, [...]
YANG, Beatrice T,
et al. Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes.
Molecular Endocrinology, 2012, vol. 26, no. 7, p. 1203-12
DOI : 10.1210/me.2012-1004 PMID : 22570331
Available at:
http://archive-ouverte.unige.ch/unige:33356
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1 Supplemental information
Supplemental Table 1
Primer sequences and positions in relation to the transcription start site for the assays used to analyze DNA methylation of the human and rat PDX-1 gene by Pyrosequencing and EpiTYPER. Lower case denotes sequence tags necessary for the EpiTyper protocol.
Position Direction Sequence (5' to 3') Human
Proximal
promoter -120 -27 Reverse Biotin-AATCTCAAAAAAAACCCACAACCAA
Forward AGTTGAGAGAGAAAATTGGAATAAAAGTA Sequencing AGTGAGGTTAGTTAGGTTAAT
Distal
promoter -908 -472 Reverse cagtaatacgactcactatagggagaaggctTTTACAAACCCAACTCTCTAAATCAA Forward aggaagagagTGTAAGTAGAAGAGAGTGAGTGTTTTTTG
Enhancer -3767 -3292 Reverse cagtaatacgactcactatagggagaaggctCCCAACACTTTAAAAAACTAAAACAAA Forward aggaagagagTTTAGGGATTATTGAATTATTTGGAA
Rat
Promoter -235 -38 Reverse cagtaatacgactcactatagggagaaggctAAAAAACTACAAACCAAACCTTAAA Forward aggaagagagTTAAGGTAGGTTAGGTTAATGGTGG
2 Supplemental Table 2
Degree of DNA methylation (%) of the analyzed CpG sites of the distal promoter and enhancer regions of PDX-1 in pancreatic islets from non-diabetic donors and patients with T2D.
CpG site Non-diabetic T2D Delta (%) Fold change P-value Q-value Distal promoter
-567 40.3 ± 7.6 47.8 ± 5.5 7.5 1.19 0.0072 0.031
-633 41.9 ±15.4 42.4 ± 12.2 0.6 1.01 0.75 0.75
-746, -741 42.6 ± 8.3 48.8 ± 7.3 6.2 1.15 0.030 0.052
-799 42.1 ± 7.4 48.3 ± 5.8 6.1 1.15 0.021 0.044
-822 34.1 ± 7.7 40.7 ± 9.4 6.6 1.19 0.050 0.064
-842 33.6 ± 10.5 43.2 ± 10.3 9.6 1.29 0.096 0.10
-857, -852 51.2 ± 8.8 57.0 ± 7.5 5.8 1.11 0.055 0.064
-868 38.0 ± 8.1 45.7 ± 6.2 7.7 1.20 0.010 0.031
-878 35.6 ± 8.6 42.1 ± 7.4 6.5 1.18 0.036 0.057
Enhancer
-3321 49.9 ± 9.5 56.9 ± 8.4 6.9 1.14 0.053 0.064
-3342 51.9 ± 13.7 66.4 ± 10.5 14.5 1.28 0.0015 0.019
-3373 37.2 ± 12.8 48.7 ± 11.1 11.5 1.31 0.012 0.031
-3408, -3404 50.6 ± 12.6 66.0 ± 10.7 15.4 1.30 0.0019 0.019
-3420, -3416 57.2 ± 11.4 66.7 ± 8.4 9.5 1.17 0.0095 0.031
-3479 66.6 ± 11.1 75.4 ± 9.6 8.8 1.13 0.050 0.064
-3496 31.1 ± 13.1 45.7 ± 11.7 14.6 1.47 0.021 0.044
-3504, -3502 41.3 ± 13.2 55.4 ± 9.4 14.1 1.34 0.0054 0.031
-3534 48.2 ± 12.4 66.2 ± 9.9 18.0 1.37 0.00008 0.0053
Data are expressed as mean ± SD. False discovery rate (FDR) was used to correct for multiple testing and to generate Q-values. Q 0.05 is considered to be significant.
3 Supplemental Figure 1
The three PDX-1 sequences used the luciferase reporter assays to test the effect of DNA methylation on PDX-1 promoter activity. The three sequences contain either 908 bp of the human PDX-1 promoter, 606 bp of the human PDX-1 enhancer region or 3800 bp of a sequence containing both the PDX-1 promoter and the enhancer region.
Promoter region 908bp
5’-
TGCAAGCAGAAGAGAGTGAGTGTTCTCTGCCGGCAAACTCCGCCAGGGTCCCGG CCCGTAGAGAGTCGTCAAGGGTCTGGAACCCCCGTGCCAACACCTGCCCCTGCTT CGCAGCCCCAAGAGGAAGGCCGCGTCTTTCCCCCTCGCTGTATTGGGAAGCTACG TTCCGGGCTGGCCAAATGGGCCCCAATTTTCCAAAACCCAAATTTGTAATACCCT TCAATTTTTTAAAAAAAAGAATTTAAAAAAGTCTCTGTGAATGCTTCAGAAGTTA CCGTTTACACCCCAGAAGTACTTGCAGCACATCCACAAGTAAAAACACACAACG AATGCCAGAGTTTCGTGTGTTTTTTAACCGACATCTTTGTGGCTGTGAACAAACTT CATAAATAAAATAGAATCAAATGCTTCTGACCTAGAGAGCTGGGTCTGCAAA]CT TTTTTTTTATCGTATTCCGCAACAGTTAAATAAAAAATTAAAAACTCAACATGTCT CCTTGTAAACTACATCAATTAACAAACACACTATGTCCATTATCAAATATAATAG AAAAAATATAGGAAAATAGAAAATAGAAAAATATAGGAAAATAGAAACTTTTAA GCCACGGTGAAAATGTTTCTATAAATGAGTGGTTCTAATGTTTTCGTGAGCGCCC ATTTTGGGGAGCACCGCCAGCTGCCCGTTCAGGAGTGTGCAGCAAACTCAGCTGA GAGAGAAAATTGGAACAAAAGCAGGTGCTCGCGGGTACCTGGGCCTAGCCTCTT AGTGCGGCCAGCCAGGCCAATCACGGCCCCCGGCTGAACCACGTGGGGCCCCGC GGAGCCTATGGTGCGGCGGCCGGCCCGCCGGTCCGCGCTGGCTGTGGGTTCCCTC TGAGATCAGTGCGGAGCTGTCAAAGCGAGCAG – 3’TSS-
Enhancer and promoter region 3800bp
5’ –
AATTCGGTTTTTAAGCTGCTTTTATCTTTACAG[TTCAGGGATCACTGAATCATCTG GAAAATAATTTCAGCTCTCTGCTTATTTTCACTAGATTGCTTAGCCCTAGGAATAT TGGAAAACCCTTCATTCAAAGTCTGAGGTCCTTTCCTCTCTCCAATCCCCACAACC CAAGCTGGGTTTCCCTGGCCCCTCTCAGACCTGGATACCAAATTCTGGGTATTTAT TTATATATATATATATATTTTTAATTTTTTGAGACGGAGCCTCGCTCTGTCTCCCA GGCTGGAGGGCAGTGGCGCGATCTCGGATCACTGCAAGCTCCGCCTCCTGGGTTC ACGCCATTCTCCTTCCTCAGCCTCCCAAGTAGCTGGAACTACAGGCGCCCGCCAC CACGCCCGGCTAAATTTTTTTGTAGGTTTAGTAGAGACGGGGTTTCATCATGTTAG CCAGGATGGTCTCGATCTCCTGACCTCATGATCCGCCTGTCTCAGCCTCCCAAAGT GCTGGG]ATTACAGGCGTGAGCCACCGCCCTCGGCCAATTCTGGGTATTCATAAA CCAGCAGGAGGGAAACATTGATTCGACTTAGGAAAATGAAAACCTGCCTGCATT AGTGCTCATTGAAGGAAATGCTAATGAACTAAAGTGCAATCAATTATAGAAGTAT TCTCATTCCACACATCACATCGTTTGCTTTACAGAATTTTCCAACTCAAGAAACAA TTTTTATTTTCAACAGAGATTTAATTATAAAATGAATATAAATTCCACGTGCATTC ATGATGTATACCTTGGAGTTAAGTGGGATTCATCTGTTTTTTTCATTTATAATGGG ATGCAGTGGCTTAAAAATCCTATCACCAAGCCAAATTCAGCCCTTAGCATGCCTC TGAAACCACCGAGGAAGACTTAGCCACTTACCAAGAACAAGGTAACCTGTGTTCT
4 CCCGGGTGTAAACCGCGTCTCTGTGAAGGGAAAGGGGGAAGAGGGCCGGCAAGG ACCTCCAGTATCAGCGAGGACTATCAGGACGTCCAGCTAATAAACGACTTTTTCA CTGTCCACACTTTAATTGGTTTACAGCCTTTTTTGTTTATTTATCCATAAGAGCTGC TGTTAAATGGCTCGGGAAGGTGCTCGCCTAATGGCTCGGTATCGCTGCCGGGCCT GTTGTGGGCGAGGCGGCTTGGAGGGGGCGCAGGCGTCAGCGGGGATTCAGAGCG GAAACGCATTTCGCCTGTAATGGACTCGGCGATTTTACGATACCAGGAGCTAGAT CCACGGGGAAGTACAACACACGACCTTACTAAGCGGAGGGTTCCGCTTGATGCGT TTTTTATACGGCTGTACGTGATAAAATTCCAGATAACTTTGTTTCATCTAATAATA ATAATAACAATAATAAATGGTTGTTTGACCTTTGCTCCCTGGGTCCCTTCTATAGG TCGCAGGCAGCAGAAAATGCTAATTGTGAAAGTGAGAACGCGCCTCCATGGAAT ACCCCCGGCGATCCCCTCCTCCTCCCCCACAGCCGCGGGTACCCGGGTTTTTGCCG GCGACCGCAACCGGCGGATTTGCTTTCTGCTGAGAGCCTCTTTTCTTTCTGCAGGG CCGAGCAAAAATATTAAATGGAAGCAAATGAAGCATCGAAATGGAGACCAATTT ACAGAAATGTGCAATTAGTTGAGAAGTGCAAAGTAAACACTCCGGGGGTGGCAG GCGAGGCAGGGAGCAGGGGAGGGAGAGTGGGGCGCAGACTGAGTCCAGAGTGG GTGTAGGGGTGCCCAGGGAGGAGGGGAAGGGGGAGGAAAAGGAGGGGGAGGGG AAAAGTGGCCGATCTTTGTCCTGAGCGGAGCTTGCCGTCCGCTCATAACCGCGTC GCCAAGTCAACCCTCGGCGACTCGGCAGATTATCTCCAAGGGTCAACACGCCGCG GCCAGGCAACCCCCTCCAGCCTGCCCTCCTGATCGATGTGGTCACTCGCGGCCAG GGTGATAGGGTAGAGTTCTCCGCGGCCGCAGACAATGGACTCATTGACGCGGGC GCACAGAGATTAGGTGCCCGGGCCCCACGGCCTTTCACTCTCTGCCCCCACCAAA ACTGCAGGTGGCACTTTTCCCTGAAGAAATCACCGAAACTCTCCCCGCCCGCCGC CGTGCTTCCCTTGGCCTGTCTTCGGTGACAGCAAGGGCATCTACAAGCCAGGGAG CCACACATCAGAGCCACACACATGAGGCCACAGGCCAGGAAGCCACACACATGA GTTCTCACACATCGAAGCAAATGCTGAGACTATACCGGCCTCCCGCAAGAGAAG GAATCTGCAGAGAAGTCCACATTCCCGACTGGCTTTTTTGTCTTTTAATAAAAATA AAGAGTCCTCAGAATAGAGCACTCAATCCGCACTTAAAACACAAATATTTCCAGT AGGAAGAAAGAAATACATTAAGATTGACTCTCAAAGGTCTGGATGTTGGTGCGTT GTTTAGGAAAGGCGGAGCAGTGATTTTTCTCTGGCTTGGTTCTTTGTGGTTCAAGC TCTCTCTTGCTGAGACGTTTCTGCAAAGCTGTCTAGTTTTTCTAAAACCCACGGTG GCGTTTCGAGAAACGTCCTCATTTCGGGAGTATCCAGTCAGAGGCTGGTCAGGCC GCCAGGCCTAGCGGATCATGCCCAGGCAGGGGGTTGGACGTCACCGCCACCCGG AGGTCATCCATCCGCGTCAGTGGGTGCAGAAAAAGTGGCCCTGTTTAAGTCAGGA CCCCAGAATGCCCAGAAGTTACTGAATGGTTTGAAGCCAAGCACAGATGTTATCA TGGAAAATGCAGCGTTTTTATTTCTTTTTCTAAATATGTAACTCTTCCTCCACTTCC CCCTCTCCTGCTTGCCTTATTTCAAT[TGCAAGCAGAAGAGAGTGAGTGTTCTCTG CCGGCAAACTCCGCCAGGGTCCCGGCCCGTAGAGAGTCGTCAAGGGTCTGGAAC CCCCGTGCCAACACCTGCCCCTGCTTCGCAGCCCCAAGAGGAAGGCCGCGTCTTT CCCCCTCGCTGTATTGGGAAGCTACGTTCCGGGCTGGCCAAATGGGCCCCAATTT TCCAAAACCCAAATTTGTAATACCCTTCAATTTTTTAAAAAAAAGAATTTAAAAA AGTCTCTGTGAATGCTTCAGAAGTTACCGTTTACACCCCAGAAGTACTTGCAGCA CATCCACAAGTAAAAACACACAACGAATGCCAGAGTTTCGTGTGTTTTTTAACCG ACATCTTTGTGGCTGTGAACAAACTTCATAAATAAAATAGAATCAAATGCTTCTG ACCTAGAGAGCTGGGTCTGCAAA]CTTTTTTTTTATCGTATTCCGCAACAGTTAAA TAAAAAATTAAAAACTCAACATGTCTCCTTGTAAACTACATCAATTAACAAACAC ACTATGTCCATTATCAAATATAATAGAAAAAATATAGGAAAATAGAAAATAGAA AAATATAGGAAAATAGAAACTTTTAAGCCACGGTGAAAATGTTTCTATAAATGAG TGGTTCTAATGTTTTCGTGAGCGCCCATTTTGGGGAGCACCGCCAGCTGCCCGTTC AGGAGTGTGCAGCAAACTCAGCTGAGAGAGAAAATTGGAACAAAAGCAGGTGCT CGCGGGTACCTGGGCCTAGCCTCTTAGTGCGGCCAGCCAGGCCAATCACGGCCCC
5 CGGCTGAACCACGTGGGGCCCCGCGGAGCCTATGGTGCGGCGGCCGGCCCGCCG GTCCGCGCTGGCTGTGGGTTCCCTCTGAGATCAGTGCGGAGCTGTCAAAGCGAGC AG– 3’TSS-
Enhancer_region 606bp
5’-
AATTCGGTTTTTAAGCTGCTTTTATCTTTACAG[TTCAGGGATCACTGAATCATCTG GAAAATAATTTCAGCTCTCTGCTTATTTTCACTAGATTGCTTAGCCCTAGGAATAT TGGAAAACCCTTCATTCAAAGTCTGAGGTCCTTTCCTCTCTCCAATCCCCACAACC CAAGCTGGGTTTCCCTGGCCCCTCTCAGACCTGGATACCAAATTCTGGGTATTTAT TTATATATATATATATATTTTTAATTTTTTGAGACGGAGCCTCGCTCTGTCTCCCA GGCTGGAGGGCAGTGGCGCGATCTCGGATCACTGCAAGCTCCGCCTCCTGGGTTC ACGCCATTCTCCTTCCTCAGCCTCCCAAGTAGCTGGAACTACAGGCGCCCGCCAC CACGCCCGGCTAAATTTTTTTGTAGGTTTAGTAGAGACGGGGTTTCATCATGTTAG CCAGGATGGTCTCGATCTCCTGACCTCATGATCCGCCTGTCTCAGCCTCCCAAAGT GCTGGG]ATTACAGGCGTGAGCCACCGCCCTCGGCCAATTCTGGGTATTCATAAA CCAGCAGGAGGGAAACATTGATTCGACTTAGGAAAATGAAAACCTGCCTG– 3’-
