Page 1 of 72 Diabetes 1 Full title 2 PRMT1 is required for the maintenance of mature β cell identity 3 4 Short running title 5 Essential role of PRMT1 in β cell identity 6 7 Authors 8 Hyunki Kim1, #, young-Ha Yoon2, 3, #, Chang-Myung Oh4, #, Joonyub Lee1, #, Kanghoon Lee1, Heein 9 Song1, Eunha Kim5, Kijong Yi1, Mi-Young Kim6, Hyeongseok Kim1, Yong Kyung Kim7, Eun-Hye 10 Seo2, 3, Haejeong Heo2, 3, Hee-Jin Kim2, Junguee Lee8, Jae Myoung Suh1, Seung-Hoi Koo9, Je Kyung 11 Seong6,10, Seyun Kim5, Young Seok Ju1, Minho Shong7, Mirang Kim2, 3, * and Hail Kim1, 11, * 12 13 Affiliations 14 1Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and 15 Technology, Daejeon 34141, Republic of Korea 16 2Personalized Genomic Medicine Research Center, Korea Research Institute of Bioscience and 17 Biotechnology, Daejeon 34141, Republic of Korea 18 3Department of Functional Genomics, University of Science and Technology, Daejeon 34113, 19 Republic of Korea 20 4Department of Biomedical Science and Engineering, Gwangju Institute of Science and Technology, 21 Gwangju 61005, Republic of Korea 22 5Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 23 34141, Republic of Korea 24 6Laboratory of Developmental Biology and Genomics, Research Institute for Veterinary Science, 25 BK21 PLUS Program for Creative Veterinary Science Research, College of Veterinary Medicine, 1 Diabetes Publish Ahead of Print, published online December 17, 2019 Diabetes Page 2 of 72 26 Seoul National University, and Korea Mouse Phenotyping Center (KMPC), Seoul 08826, Republic 27 of Korea 28 7Research Center for Endocrine and Metabolic Diseases, Chungnam National University School of 29 Medicine, 282 Munhwaro, Daejeon 35015, Republic of Korea 30 8Department of Pathology, Daejeon St. Mary's Hospital, College of Medicine, The Catholic 31 University of Korea, 64 Daeheung-ro, Jung-gu, Daejeon 34943, Republic of Korea 32 9Division of Life Sciences, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul 02841, Republic 33 of Korea 34 10 Interdisciplinary Program for Bioinformatics, Program for Cancer Biology and BIO-MAX/N-Bio 35 Institute, Seoul National University, Seoul 08826, Republic of Korea 36 11KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, 37 Daejeon 34141, Republic of Korea 38 #These authors contributed equally to this work 39 40 * Corresponding authors 41 Hail Kim, M.D., Ph.D. 42 Graduate School of Medical Science and Engineering 43 Korea Advanced Institute of Science and Technology 44 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea 45 Phone: +82-42-350-4243 46 e-mail: [email protected] 47 48 Mirang Kim, Ph.D. 49 Personalized Genomic Medicine Research Center 50 Korea Research Institute of Bioscience and Biotechnology 2 Page 3 of 72 Diabetes 51 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea 52 Phone: +82-42-879-8113 53 e-mail: [email protected] 54 55 Word count: 4,444 56 57 Number of figures: 6 3 Diabetes Page 4 of 72 58 Abstract 59 Loss of functional β cell mass is an essential feature of type 2 diabetes, and maintaining mature 60 cell identity is important for preserving a functional β cell mass. However, it is unclear how β cells 61 achieve and maintain their mature identity. Here, we demonstrate a novel function of PRMT1 in 62 maintaining mature β cell identity. Prmt1 knockout in fetal and adult β cells induced diabetes, which 63 was aggravated by high fat diet-induced metabolic stress. Deletion of Prmt1 in adult β cells resulted 64 in the immediate loss of histone H4 arginine 3 asymmetric di-methylation (H4R3me2a) and the 65 subsequent loss of β cell identity. The expression levels of genes involved in mature β cell function 66 and identity were robustly downregulated as soon as Prmt1 deletion was induced in adult β cells. ChIP- 67 seq and ATAC-seq analyses revealed that PRMT1-dependent H4R3me2a increases chromatin 68 accessibility at the binding sites for CTCF and β cell transcription factors. In addition, PRMT1- 69 dependent open chromatin regions may show an association with the risk of diabetes in humans. 70 Together, our results indicate that PRMT1 plays an essential role in maintaining β cell identity by 71 regulating chromatin accessibility. 4 Page 5 of 72 Diabetes 72 Introduction 73 Maintaining the functional β cell mass is crucial for preventing diabetes, which develops when 74 β cells fail to meet the insulin demand (1,2). Although β cell death is thought to be the major 75 mechanism of β cell failure (3), recent studies indicate that β cell dedifferentiation can decrease the 76 functional β cell mass and thereby deteriorate systemic glucose homeostasis (4,5). Maintaining mature 77 cell identity is also important for maintaining cell function (6,7). A hierarchy of transcription factor 78 (TF) cascades directs β cell differentiation, and β cells require continuous activation of these TFs to 79 maintain their function and identity (8–10). The genetic identity of a differentiated cell is generally 80 controlled by the chromatin state, which is overall stable and has a limited epigenomic flexibility 81 (11,12). Likewise, epigenetic regulation plays an essential role in the postnatal maturation of β cells 82 and the maintenance of mature β cell identity (13–16). 83 Histone arginine methylation, which is regulated by protein arginine methyltransferase 84 (PRMT), can affect chromatin structures to facilitate the recruitment of protein complexes that regulate 85 gene transcription (17,18). PRMT4-dependent histone H3 arginine 17 asymmetric di-methylation 86 (H3R17me2a) in β cells has been reported to regulate glucose-stimulated insulin secretion (GSIS) (19). 87 However, the role of PRMT-induced histone arginine methylation in regulating β cell identity has not 88 yet been elucidated. Among the nine members of the PRMT family, PRMT1 predominates in 89 mammalian cells (20). It appears to be associated with diabetes, as its catalytic activity is decreased in 90 the liver and pancreas of diabetic Goto-Kakizaki rats (21). PRMT1 has also been shown to specifically 91 induce the active histone code, histone H4 arginine 3 asymmetric di-methylation (H4R3me2a), which 92 potentiates subsequent histone acetylation and contributes to establishing euchromatin structure 93 (22,23). Based on these previous findings, we herein explored the role of PRMT1-dependent 94 H4R3me2a in mature β cells. 5 Diabetes Page 6 of 72 95 Research Design and Methods 96 Animals 97 Prmt1 floxed (Prmt1fl/fl) [MGI: 4432476] mice were crossed with Rip2-Cre [MGI: 2387567] and 98 Pdx1-CreERT2 [MGI: 2684321] mice to generate Prmt1 βKO and Prmt1 βiKO mice, respectively. 99 R26-eYFP [MGI: 2449038] mice were crossed for lineage-tracing experiments and β cell sorting. All 100 mice were backcrossed and maintained on a C57BL/6J background. Cre recombination for CreERT2 101 was induced by a total of five intraperitoneal injections of corn oil-dissolved tamoxifen (75 mg/kg) 102 over 2 weeks. Mice were housed in climate-controlled, specific pathogen-free barrier facilities under 103 a 12-hour light/dark cycle, and chow and water were provided ad libitum. Mice were fed either a 104 standard chow diet or high-fat diet (HFD; 60% kcal fat). The animal experiment protocols for this 105 study were approved by the Institutional Animal Care and Use Committee at the Korea Advanced 106 Institute of Science and Technology. All experiments were performed in accordance with the 107 relevant guidelines and regulations. 108 109 Metabolic assays 110 Body weight and random blood glucose levels were measured in the afternoon of the daytime. The 111 glucose tolerance test and the insulin tolerance test were performed as previously described (24). 112 113 Histological analyses 114 For histological analyses, formalin-fixed paraffin-embedded pancreatic slides were prepared, stained 115 and analyzed as described in Supplementary Materials. 116 117 Pancreatic insulin content 118 Pancreatic tissues were dissected, placed in acid-ethanol (1.5% HCl in 70% ethanol), homogenized 119 and incubated at 4°C for 16 hours. The aqueous phase of pancreatic insulin extract was neutralized 6 Page 7 of 72 Diabetes 120 with an equal amount of 1 M Tris-Cl buffer (pH 7.5). The pancreatic insulin content was calculated by 121 dividing the total pancreatic insulin by the weight of the pancreas. 122 123 Glucose-stimulated insulin secretion (GSIS) 124 For the in vivo GSIS assay, mice were fasted for 16 hours and then given an intraperitoneal injection 125 of D-glucose in PBS (2 g/kg). For the ex vivo islet GSIS assay, pancreatic islets were isolated from 126 mice as described previously (25), and the assay was performed as described in the Supplementary 127 Materials. 128 129 Oxygen consumption rate (OCR) 130 Pancreatic islets were isolated from mice as described previously (25), and the OCR assay was 131 performed as described in the Supplementary Materials. 132 133 Quantitative reverse transcription PCR (qRT-PCR) 134 Total RNA was extracted from mouse tissues and qRT-PCR was performed as described in the 135 Supplementary Materials. The sequences of the utilized primers are listed in Supplementary Table 1. 136 137 ChIP-seq, RNA-seq and ATAC-seq analyses 138 ChIP experiments were performed in MIN6 cells as previously described (26) with modifications. 139 RNA-seq experiments were performed using WT and Prmt1-null islets. ATAC experiments were 140 performed as previously described (27), using MIN6 cells and FACS-sorted WT and Prmt1-null β 141 cells. ChIP-seq, RNA-seq and ATAC-seq analyses were performed as described in the Supplementary 142 Materials. 143 144 Chromatin conformation capture PCR (3C-PCR) 7 Diabetes Page 8 of 72 145 3C experiments were performed in MIN6 cells as previously described (28) with modifications.