Polycomb protein Ezh2 regulates pancreatic β-cell Ink4a/Arf expression and regeneration in streptozotocin-induced diabetes mellitus

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1 Chen et al. 1 Polycomb protein Ezh2 regulates pancreatic β-cell Ink4a/Arf expression and regeneration in streptozotocin-induced diabetes mellitus Hainan Chen 1, Xueying Gu 1, I-hsin Su 2, Rita Bottino 3, Juan L. Contreras 4, Alexander Tarakhovsky 2, Seung K. Kim 1,5 1 Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 2 Laboratory of Lymphocyte Signaling, The Rockefeller University, New York, NY 3 Department of Pediatrics, Division of Immunogenetics, Diabetes Institute, University of Pittsburgh, School of Medicine, Pittsburgh, PA 4 Department of Surgery, Division of Transplantation, University of Alabama School of Medicine, Birmingham, AL 5 Department of Medicine, Oncology Division, Stanford University School of Medicine, Stanford, CA Current address: Dept. of Genomics and Genetics, Nanyang Technological University, Singapore Running title: β-cell Ink4a/Arf regulation by Ezh2 Keywords: pancreas; islet of Langerhans; histone; epigenetics; diabetes; cell cycle Supplementary Information Supplementary Tables 1 and 2 Supplementary Figures 1, 2 and 3, and Figure legends

2 Chen et al. 2 Supplementary Tables Table 1. qrt-pcr s used for mouse islet mrna expression assays Gene Forward Probe Reverse Ezh2 5 -GACAAATACATGTGCAGCTTTCTGT 5 -CAACTTGAACAATGATTTTGTGGTG 5 -GCCCTTTCGGGTTGCAT Ezh1 5 -GGATGCTACCCGGAAAGGA 5 -CGCTTTGCAAACCATTCAGTGAACCC 5 -ACCATAACCACTTTGGCATAACAG Suz12 5 -GAAGCTGTGGAACCTCCATGTC 5 -TGAAGCATGGATTTATTGCTGACAA 5 -ACAGCATACAGGCATGATTCATTT Eed 5 -GCGATGGTTAGGCGATTTGAT 5 -TCCAAGTCTTGTGAAAATGCCATT 5 -TTTTGCCAGGTTTCCAGCAT Bmi-1 5 -CAGATTGGATCGGAAAGTAAATAAAGAG 5 -AGCCTAAGGAAGAGGTGAATGATAAAAGGT 5 -TTGCTGCTGGGCATCGTA Phc1 5 -CCCAGCAGAGCAGTTTCGA 5 -TCTAAGAGGTTCTGCTCCATGACGT 5 -CTGCAGCTCACATTGTACCTCTTT Cbx4 5 -TGCTGATCGCCTTCCAGAA 5 -AGGGAAAGGCAGGAGCAGCTGATG 5 -GGGCCCTCTCTTGCGATATC Ink4a 5 -GTACCCCGATTCAGGTGATGA 5 -CGTTCACGTAGCAGCTCTTCTGC 5 -CAGTTCGAATCTGCACCGTAGT Arf 5 -TGAGGCTAGAGAGGATCTTGAGAAG 5 -CCGGAATCCTGGACCAGGTGA 5 -CGTGAACGTTGCCCATCAT Ink4b 5 -GCAGGCCTTCCAAAACTTGA 5 -CCTACCCAGTAAGACAAAGCCAATCAGAAATA 5 -AGCTGCAGAAAATGCGTAGGA Ink4c 5 -AAATAATGTAAACGTCAACGCTCAAA 5 -TGGATTTGGGAGAACTGCGCTGC 5 -CCGGATTTCCAAGTTTCATAACC Ink4d 5 -AGACGGCCTTGCAGGTCAT 5 -CCTGAAGCAAGGTGCCAGCCC 5 -CCGGAGGCATCTTGGACAT p21 Cip1 5 -CCACAGCGATATCCAGACATTC 5 -CCACAGGCACCATGTCCAATCC 5 -CGGAACAGGTCGGACATCA p27 Kip1 5 -CAGACAATCCGGCTGGGTTA 5 -AGGGATGAGGAAGCGACCTGCTG 5 -GCCCTTTTGTTTTGCGAAGA Trp53 5 -CAAAAGAAAAAACCACTTGATGGA 5 -TTCACCCTCAAGATCCGCGGGC 5 -CGGAACATCTCGAAGCGTTTA PPIA 5 -TGCTGGACCAAACACAAACG 5 -TTCCCAGTTTTTTATCTGCACTG 5 -TGCTTGCCATCCAGCCATTCA

3 Chen et al. 3 Table 2. qpcr s used for ChIP analysis on mouse Ink4b and Ink4a/Arf Loci Location Forward Probe Reverse set 1 set 2 set 3 set 4 set 5 Ink4b promoter Arf promoter Ink4a promoter End of exon 1α Between exon 2 and 3 5 -CGACGGGAGGCAGGTTTT 5 -CAAGAGCAAAAATCCTGAAAAACAAAAACGATC 5 -CAATCTAGTGCCGAGGGATGTT 5 -GAGTACAGCAGCGGGAGCAT 5 -TGAGGATTCAGCGCGCGGG 5 -GAACTTCACCAAGAAAACCCTCTCT 5 -GTCCGATCCTTTAGCGCTGTT 5 -ACGCCCAGCTCTCCTCCTGAA 5 -AGCCCGGACTACAGAAGAGATG 5 -CCGGAGCCACCCATTAAACTA 5 -TAGTGAAACCATTTTGGCATCGAG 5 -CAAGACTTCTCAAAAATAAGACACTGAAA 5 -CCCAACACCCACTTGAGGAA 5 -TATATCAGCCTGGAATTTCAGCTCC 5 -CAGAGGTCACAGGCATCGAA

4 Chen et al. 4 Supplementary figure legends Supplementary Fig. 1. Expression of genes encoding Polycomb group factors in pancreatic islets from C57BL/6 mice during aging. Relative mrna levels of Eed, Suz12, Cbx4 and Phc1 in islets from the mice of the indicated ages were determined by real-time RT- PCR. n=3 to 10 mice per time point. Unlike Ezh2, no significant change in the levels of mrna expression of these genes in pancreatic islets was detected during aging. Supplementary Fig. 2. Expression of Ezh2 and genes encoding CDK inhibitors or p53 and the association of H3K9/14Ac and H3K9me3 with the Ink4a/Arf locus in βezh2ko islets. (A) Representative Western blot analysis of the indicated proteins in islets from one-month-old Ezh2 f/f and βezh2ko mice. (B) Relative mrna levels of the indicated CDK inhibitors in isolated islets from one-month-old Ezh2 f/f and βezh2ko mice. n=4 to 10 mice. (C-E) ChIP analysis of the Ink4b and Ink4a/Arf loci using the indicated antibodies in islets isolated from one-month-old control Ezh2 f/f and βezh2ko mice. Three to five independent ChIP experiments were performed for each antibody using separate islet preparations. Supplementary Fig. 3. βezh2ko mice have normal insulin sensitivity and islet insulin secretion function. (A) Mass of βezh2ko and littermate Ezh2 f/f mice during aging. n=11 to 29 for each group at each time point. (B) Glucose tolerance testing and (C) Area under curve (AUC) from (B) performed in 2-month-old mice with glucose injection (2 g/kg body weight). n=3 to 5 mice for each group. Glucose regulation in Ezh2 f/+ ; RipCre mice was indistinguishable from Ezh2 f/f control mice, indicating that the RIP-Cre transgene alone did not impair glucose regulation in vivo. (D) Box-whisker plot showing fasting blood glucose and (E) Glucose tolerance testing performed in 8-month-old mice (3 g/kg body weight glucose). n=5 for each group. (F) Insulin tolerance test performed in one-month-old mice. n=4 mice for each group. Pancreatic islet insulin content (G) and in vitro insulin secretion by glucose (H) or arginine (I)

5 Chen et al. 5 stimulation in one-month-old βezh2ko and Ezh2 f/f mouse islets. No significant difference in islet insulin content and insulin secretion between βezh2ko and control was detected (P>0.05). n=8-30 independent measurements from 4 independent islet preparations per group.

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