Pax6 maintains pancreatic beta-cell identity by repressing alternative islet cell genes Legends to Supplemental Figures and Tables Supplementary Figure S1: Expression of β cell transcription factors in islets of db/db mice. Expression level of Nkx6.1, MafA and Pdx1 mRNA as a function of blood glucose levels in db/db and control mice. The mice used for this analysis are the same as in figure 1E. Note the early and deeper drop in MafA expression suggesting greater sensitivity to high blood glucose levels as compared with the other factors. Supplementary Figure S2: Pax6 deletion in adult β cells. A. Representative images of islets from Pax6lox/lox (lox/lox) and MIP-CreER; Pax6lox/lox (βPAX6) mice stained for PAX6 (green) and insulin (blue). Mice are 3 months old, 1 week after injection of tamoxifen or vehicle. Original magnification 400x. B. Number of Pax6 positive β cells in Pax6lox/lox mice (no Cre), MIP-CreER; Pax6lox/lox; Rosa26-LSL-YFP mice without tamoxifen (no TM), and MIP- CreER; Pax6lox/lox; Rosa26-LSL-YFP mice 1 week after tamoxifen injection (βPAX6). n=3 for each group of mice. C. Pax6 mRNA in sorted YFP+ β cells from MIP-CreER; Rosa26-LSL-YFP (YFP, n=5) and MIP-CreER; Pax6lox/lox; Rosa26-LSL-YFP (βPAX6, n=3) mice. Mice were 6 weeks old, 1 week after tamoxifen injection. D. Measurements of blood glucose following tamoxifen (TM) injection in an additional cohort of βPAX6 mice showing development of severe hyperglycemia. n=24 lox/lox and 23 βPAX6 mice. ***P < 0.001 as determined by 2-tailed Student’s t-test. Supplemental Figure S3: Correlation between blood ketones and insulin or glucagon in βPAX6 mice. Blood ketones in βPAX6 mice are plotted against plasma insulin (A) and plasma glucagon (B). Note that the highest levels of ketone bodies were observed in hyperglucagonemic mice. Supplementary Figure S4: TUNEL staining for detection of cell death in βPAX6 islets. Pancreatic sections from lox/lox and βPAX6 mice at different time points after tamoxifen injection as indicated, stained for insulin (red), TUNEL (green) and DNA (blue). Original magnification 400x. Supplemental Figure S5: Immunostaining of islet hormones following Pax6 deletion. Immunohistochemical staining of pancreatic sections from control and βPAX6 mice for chromogranin A, glucagon, somatostatin, pancreatic polypeptide and ghrelin. βPAX6 mice were 1 week or 1 month after tamoxifen. Ghrelin cells appeared shortly after Pax6 deletion, while expansion of glucagon and somatostatin cells was seen only 1 month after deletion. Original magnification 400x. Supplemental Figure S6: Ultrastructural resemblance of granules in βPAX6 islet cells and in gastric ghrelin cells. Left panel, EM images of cells from a βPAX6 islet, 2 months after tamoxifen administration. Original magnification 1000x or 3000x (indicated). Right panel, EM images of gastric ghrelin cells, from Kojima et al. (with permission). Image C is presented in main figure 3E and is shown here again for comparison with the gastric ghrelin cells. Supplemental Figure S7: Origins of somatostatin and pancreatic polypeptide-expressing cells in βPAX6 mice. Pax6-deleted β cells do not express detectable somatostatin or pancreatic polypeptide. Staining for somatostatin or pancreatic polypeptide (red), YFP (green) and insulin (blue). Original magnification 400x. Supplemental Figure S8: Changes in protein level of key β cell factors following Pax6 ablation. A. Representative islets stained for insulin and for β cell specific markers that were differentially expressed in βPAX6 as observed by RNA-seq. Maf- A and Glut2 were most dramatically reduced in βPAX6 islets. Original magnification 400x. B. Elevation of Ngn3 protein in Pax6-deficient β cells. Immunohistochemistry of Ngn3 in lox/lox and βPAX6 islets, 1 week after tamoxifen. The pancreas of an embryonic day 14.5 mouse serves as a positive control. Original magnification 400x. Supplemental Table S1 Expression levels of β cell genes following Pax6 deletion, divided into functional categories. Upregulated genes are highlighted in red and downregulated are in green. FC=fold change. We considered FC of 1.5 and FDR<0.05 as significant change in expression. Supplemental Table S2 Expression levels of specific and general transcription factors in β cells, obtained from RNA-seq analysis of sorted beta cells from βPAX6 mice as described in Figure 5. We considered FC of 1.5 and FDR<0.05 as significant change in expression. Supplemental Table S3 List of genes significantly changed during maturation of mouse beta cells between post-natal day 18 and p35. RNA was extracted from sorted beta cells. Supplemental Table S4 A combined list of all Pax6 bound sites obtained from ChIP-seq analysis on min6 cells, and their association with histone marks (H3K27ac, H3K4me1, H3K27me3, H3K9ac) and TF maps (Nkx6-1, Foxa2, Pdx1, MafA, NeuroD1) as well as expression data from RNAseq analysis on βPAX6 mice (1-3). Pax6 ChIP-seq was done in three replicates and all peaks were combined for our analyses. For full description of integrated analysis see second and third tab in the S4 file. Supplemental Table S5 A short list obtained from table S4, showing central islet genes that are directly regulated by Pax6 including details of nearby Pax6 binding sites, additional previously published TFs binding sites and histone marks as well as differential expression data from βPAX6 RNA-seq. Supplemental Table S6 A combined list of all PAX6 bound sites obtained from ChIP-seq analysis on EndoC-betaH2 cells, and their association with histone marks (H3K27ac, H3K4me1, H3K4me3, H3K27me3) and TF maps (PDX1, NKX2-2, NKX6-1, FOXA2, MAFB) performed on human islets or sorted beta cells (4, 5). Pax6 ChIP-seq was done in two replicates and all peaks were combined for our analyses. For full description of integrated analysis see 2nd and 3rd tab in the S6 file. For a list of common genes bound by Pax6 in Min6 and EndoC cells, see 4th tab. Supplemental Table S7 Primary antibodies used for immunohistochemistry, including their dilutions and commercial details, as well as sequence of primers used for qPCR in this study and for amplifying mouse genomic regions cloned into the luciferase reporter vector. Supplemental References 1. Avrahami D, Li C, Zhang J, Schug J, Avrahami R, Rao S, Stadler MB, Burger L, Schubeler D, Glaser B, et al. Aging-Dependent Demethylation of Regulatory Elements Correlates with Chromatin State and Improved beta Cell Function. Cell Metab. 2015;22(4):619-32. 2. Taylor BL, Liu FF, and Sander M. Nkx6.1 is essential for maintaining the functional state of pancreatic beta cells. Cell Rep. 2013;4(6):1262-75. 3. Tennant BR, Robertson AG, Kramer M, Li L, Zhang X, Beach M, Thiessen N, Chiu R, Mungall K, Whiting CJ, et al. Identification and analysis of murine pancreatic islet enhancers. Diabetologia. 2013;56(3):542-52. 4. Bramswig NC, Everett LJ, Schug J, Dorrell C, Liu C, Luo Y, Streeter PR, Naji A, Grompe M, and Kaestner KH. Epigenomic plasticity enables human pancreatic alpha to beta cell reprogramming. J Clin Invest. 2013;123(3):1275-84. 5. Pasquali L, Gaulton KJ, Rodriguez-Segui SA, Mularoni L, Miguel-Escalada I, Akerman I, Tena JJ, Moran I, Gomez-Marin C, van de Bunt M, et al. Pancreatic islet enhancer clusters enriched in type 2 diabetes risk- associated variants. Nature genetics. 2014;46(2):136-43. 1.8 1.6 1.4 1.2 WT 1 0.8 db/db-4W 0.6 db/db-5-7W 0.4 db/db-3M Nkx6.1 relave expression 0.2 0 0 100 200 300 400 500 600 700 Blood glucose (mg/dL) 1.6 1.4 1.2 1 WT 0.8 db/db-4W 0.6 db/db-5-7W 0.4 MafA relave expression db/db-3M 0.2 0 0 100 200 300 400 500 600 700 Blood glucose (mg/dL) 2 1.8 1.6 1.4 1.2 WT 1 db/db-4W 0.8 db/db-5-7W 0.6 Pdx1 relave expression 0.4 db/db-3M 0.2 0 0 100 200 300 400 500 600 700 Blood glucose (mg/dL) Supplemental Figure S1 A lox/lox No TM βPAX6 INSULIN PAX6 PAX6 50 μm B C D 100 1.2 700 lox/lox βPAX6 ) *** 600 *** 80 1 dL *** 500 *** 0.8 *** 60 400 0.6 40 300 0.4 *** 200 20 Pax6 HprtRNA / 0.2 Blood glucose (mg/ Pax6+ / Ins+ cells (%) 100 0 0 0 0 50 100 150 200 Days aer TM injecFon Supplemental Figure S2 A B 7 8 6 7 ) 6 5 mM 5 4 4 3 3 2 3-OH-butyrate ( 3-OH-butyrate (mM) 2 1 1 0 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0 20 40 60 80 100 120 140 160 180 Plasma insulin (ng/ml) Plasma glucagon (pg) Supplemental Figure S3 lox/lox βPAX6 – 12 days βPAX6 – 7 months lox/lox βPAX6 – 6 months βPAX6 – 6 months 20 μm INSULIN TUNEL DAPI Supplemental Figure S4 Control βPAX6-1 week βPAX6-1 month Chromogranin 20 μm Glucagon Somatostatin Pancreaticpolypeptide Ghrelin Supplemental Figure S5 βPAX6 islets Oxyntic gland in the stomach A D 2 μm E μm x1000 2 B C 2 μm x1000 1 μm x3000 0.5 μm Supplemental Figure S6 YFP INSULIN SOMATOSTATIN YFP INSULIN PANCREATIC POLYPEPTIDE 1 week 50 μm 6 months Supplemental Figure S7 A PDX1 MafA Nkx-6.1 Nkx-2.2 GLUT2 lox/lox INSULIN PAX6 β 50 μm B lox/lox E14.5 βPAX6 βPAX6 NGN3 20 μm Supplemental Figure S8 Supplementary table S1 Insulin Secretion Gene Full name FC FDR Control βPAX6 Acox2 acyl-Coenzyme A oxidase 2, branched chain 2.43 0.839 1.00 1.90 Acss1 acyl-CoA synthetase short-chain family member 1 3.31 0.612 1.21 2.13 Acss3 acyl-CoA synthetase short-chain family member 3 2.73 0.710 1.21 2.42 Aldoa aldolase A, fructose-bisphosphate 1.16 0.822 1944.54 2064.87 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C3 (subunit Atp5G3 9) 1.39 0.312 778.06 1031.06 Cox4I1 cytochrome c oxidase subunit IV isoform 1 1.46 0.167 611.77 823.55 Gpi1 glucose phosphate isomerase 1 1.37 0.450 412.57 496.70 Nnt nicotinamide nucleotide transhydrogenase -1.93 0.009 952.05 456.20 Pcsk1 proprotein