Diabetes Volume 69, March 2020 355

PRMT1 Is Required for the Maintenance of Mature b-Cell Identity

Hyunki Kim,1 Byoung-Ha Yoon,2,3 Chang-Myung Oh,4 Joonyub Lee,1 Kanghoon Lee,1 Heein Song,1 Eunha Kim,5 Kijong Yi,1 Mi-Young Kim,6,7 Hyeongseok Kim,1 Yong Kyung Kim,8 Eun-Hye Seo,2,3 Haejeong Heo,2,3 Hee-Jin Kim,2 Junguee Lee,9 Jae Myoung Suh,1 Seung-Hoi Koo,10 Je Kyung Seong,6,11 Seyun Kim,5 Young Seok Ju,1 Minho Shong,8 Mirang Kim,2,3 and Hail Kim1,12

Diabetes 2020;69:355–368 | https://doi.org/10.2337/db19-0685

Loss of functional b-cell mass is an essential feature of our results indicate that PRMT1 plays an essential role type 2 diabetes, and maintaining mature b-cell identity is in maintaining b-cell identity by regulating chromatin important for preserving a functional b-cell mass. How- accessibility. ever, it is unclear how b-cells achieve and maintain their mature identity. Here we demonstrate a novel function of Maintaining the functional b-cell mass is crucial for pre- arginine methyltransferase 1 (PRMT1) in main- b

venting diabetes, which develops when -cells fail to meet STUDIES ISLET taining mature b-cell identity. Prmt1 knockout in fetal and the demand (1,2). Although b-cell death is thought adult b-cells induced diabetes, which was aggravated by to be the major mechanism of b-cell failure (3), recent – Prmt1 high-fat diet induced metabolic stress. Deletion of studies indicate that b-cell dedifferentiation can decrease b in adult -cells resulted in the immediate loss of histone the functional b-cell mass and thereby deteriorate sys- H4 arginine 3 asymmetric dimethylation (H4R3me2a) temic glucose homeostasis (4,5). Maintaining mature and the subsequent loss of b-cell identity. The expression b-cell identity is also important for maintaining b-cell levels of involved in mature b-cell function and function (6,7). A hierarchy of transcription factor (TF) identity were robustly downregulated as soon as Prmt1 deletion was induced in adult b-cells. Chromatin immu- cascades directs b-cell differentiation, and b-cells require noprecipitation sequencing and assay for transposase- continuous activation of these TFs to maintain their – accessible chromatin sequencing analyses revealed that function and identity (8 10). The genetic identity of PRMT1-dependent H4R3me2a increases chromatin ac- a differentiated cell is generally controlled by the chroma- cessibility at the binding sites for CCCTC-binding fac- tin state, which is overall stable and has limited epigenomic tor (CTCF) and b-cell transcription factors. In addition, flexibility (11,12). Likewise, epigenetic regulation plays an PRMT1-dependent open chromatin regions may show an essential role in the postnatal maturation of b-cells and association with the risk of diabetes in humans. Together, the maintenance of mature b-cell identity (13–16).

1Graduate School of Medical Science and Engineering, Korea Advanced Institute of 10Division of Life Sciences, Korea University, Seoul, Republic of Korea Science and Technology, Daejeon, Republic of Korea 11Interdisciplinary Program for Bioinformatics, Program for Cancer Biology and 2Personalized Genomic Medicine Research Center, Korea Research Institute of BIO-MAX/N-Bio Institute, Seoul National University, Seoul, Republic of Korea Bioscience and Biotechnology, Daejeon, Republic of Korea 12KAIST Institute for the BioCentury, Korea Advanced Institute of Science and 3Department of Functional Genomics, University of Science and Technology, Technology, Daejeon, Republic of Korea Daejeon, Republic of Korea Corresponding authors: Hail Kim, [email protected], and Mirang Kim, mirang@ 4 Department of Biomedical Science and Engineering, Gwangju Institute of Science kribb.re.kr and Technology, Gwangju, Republic of Korea Received 16 July 2019 and accepted 12 December 2019 5Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea This article contains Supplementary Data online at https://diabetes 6Laboratory of Developmental Biology and Genomics, Research Institute for .diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0685/-/DC1. Veterinary Science, BK21 PLUS Program for Creative Veterinary Science Research, Hyu. Kim, B.-H.Y., C.-M.O., and Jo. Lee contributed equally to this work. College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea © 2019 by the American Diabetes Association. Readers may use this article as 7Korea Mouse Phenotyping Center, Seoul, Republic of Korea long as the work is properly cited, the use is educational and not for profit, and the 8Research Center for Endocrine and Metabolic Diseases, Chungnam National work is not altered. More information is available at https://www.diabetesjournals University School of Medicine, Daejeon, Republic of Korea .org/content/license. 9Department of Pathology, College of Medicine, Seoul St. Mary’s Hospital, The Catholic University of Korea, Daejeon, Republic of Korea 356 Essential Role of PRMT1 in b-Cell Identity Diabetes Volume 69, March 2020

Histone arginine methylation, which is regulated by Pancreatic Insulin Content protein arginine methyltransferase (PRMT), can affect Pancreatic tissues were dissected, placed in acid-ethanol chromatin structures to facilitate the recruitment of (1.5% HCl in 70% ethanol), homogenized, and incubated at protein complexes that regulate transcription 4°C for 16 h. The aqueous phase of pancreatic insulin (17,18). PRMT4-dependent histone H3 arginine 17 asym- extract was neutralized with an equal amount of 1 mol/L metric dimethylation (H3R17me2a) in b-cells has been Tris-Cl buffer (pH 7.5). The pancreatic insulin content was reported to regulate glucose-stimulated insulin secretion calculated by dividing the total pancreatic insulin by the (GSIS) (19). However, the role of PRMT-induced histone weight of the . arginine methylation in regulating b-cell identity has not GSIS yet been elucidated. Among the nine members of the For the in vivo GSIS assay, mice were fasted for 16 h and PRMT family, PRMT1 predominates in mammalian cells then given an intraperitoneal injection of D-glucose in PBS (20). It appears to be associated with diabetes, as its (2 g/kg). For the ex vivo islet GSIS assay, pancreatic islets catalytic activity is decreased in the liver and pan- were isolated from mice as described previously (25), and creas of diabetic Goto-Kakizaki rats (21). PRMT1 has the assay was performed as described in Supplementary also been shown to specifically induce the active histone Data. code, histone H4 arginine 3 asymmetric dimethylation (H4R3me2a), which potentiates subsequent histone acety- Oxygen Consumption Rate lation and contributes to establishing euchromatin struc- Pancreatic islets were isolated from mice as described ture (22,23). Based on these previous findings, we herein previously (25), and the oxygen consumption rate (OCR) explored the role of PRMT1-dependent H4R3me2a in ma- assay was performed as described in Supplementary ture b-cells. Data.

RESEARCH DESIGN AND METHODS Quantitative RT-PCR Animals Total RNA was extracted from mouse tissues, and quan- fl/fl Prmt1-floxed (Prmt1 )(MouseGenomeInformatics titative RT-PCR (qRT-PCR) was performed as described in [MGI]: 4432476) mice were crossed with Rip2-Cre (MGI: Supplementary Data. The sequences of the primers used ERT2 are listed in Supplementary Table 1. 2387567) and Pdx1-Cre (MGI: 2684321) mice to gen- Prmt1 – erate b-cell knockout (bKO) and inducible b-cell Chromatin Immunoprecipitation Sequencing, RNA fi Prmt1 Prmt1 speci c KO ( biKO) mice, respectively. Sequencing, and Assay for Transposase-Accessible R26-eYFP (MGI: 2449038) mice were crossed for lineage- Chromatin Sequencing Analyses tracing experiments and b-cell sorting. All mice were Chromatin immunoprecipitation (ChIP) experiments were backcrossed and maintained on a C57BL/6J background. performed in MIN6 cells as previously described (26) with Cre recombination for CreERT2 was induced by a total of modifications. RNA sequencing (RNA-seq) experiments were five intraperitoneal injections of corn oil–dissolved ta- performed using wild-type (WT) and Prmt1-null islets. Assay moxifen (75 mg/kg) over 2 weeks. Mice were housed in for transposase-accessible chromatin (ATAC) experiments climate-controlled, specific pathogen-free barrier facili- were performed as previously described (27), using MIN6 ties under a 12-h light/dark cycle, and chow and water cells and FACS WT and Prmt1-null b-cells. ChIP sequencing were provided ad libitum. Mice were fed either a standard (ChIP-seq), RNA-seq, and ATAC sequencing (ATAC-seq) chow diet or high-fat diet (HFD) (60% kcal fat). The animal analyses were performed as described in Supplementary experiment protocols for this study were approved by the Data. Institutional Animal Care and Use Committee at the Korea Conformation Capture PCR Advanced Institute of Science and Technology. All experi- Chromosome conformation capture (3C) experiments ments were performed in accordance with the relevant were performed in MIN6 cells as previously described guidelines and regulations. (28) with modifications. The data were normalized with respect to those obtained using internal primers that rec- Metabolic Assays ognized sequences within the Gapdh gene. At least three Body weight and random blood glucose levels were mea- independent biological replicates were included for each 3C- sured in the afternoon hours each day. The glucose toler- PCR assay. The sequences of the primers used are listed in ance test and insulin tolerance test were performed as Supplementary Table 1. previously described (24). Statistics Histological Analyses All values are expressed as mean 6 SEM. The two-tailed For histological analyses, formalin-fixed paraffin-embedded Student t test or one-way ANOVA followed by post hoc pancreatic slides were prepared, stained, and analyzed as Tukey test was used to compare groups. P values ,0.05 described in Supplementary Data. were considered statistically significant. diabetes.diabetesjournals.org Kim and Associates 357

Figure 1—Prmt1 bKO mice develop a progressive diabetes phenotype. A: Intraperitoneal glucose tolerance test (IPGTT) of 6-week-old male control and Prmt1 bKO mice after a 16-h fast; n 5 4/group. B–F: Male control and Prmt1 bKO mice aged 12–13 weeks were fed a standard chow diet and used for experiments. B: IPGTT after a 16-h fasting; n 5 5/group. C: Ex vivo islet GSIS assay; n 5 4/group. D: In vivo GSIS assay after a 16-h fast; n 5 3/group. E: OCR analysis of isolated islets; n 5 6/group. F: Representative b-cell images obtained by transmission electron microscopy. Arrows indicate immature insulin granules (blue), dilated endoplasmic reticulum (green), and dysmorphic mitochondria (red); n 5 3/group. G–O: Male control and Prmt1 bKO mice (8 weeks old) were fed HFD for 18 weeks and used for experiments. G: IPGTT after a 16-h fast; n 5 5/group. H: Representative islet images obtained by immunofluorescence (IF) staining of INS (green), GCG (red), and DAPI 358 Essential Role of PRMT1 in b-Cell Identity Diabetes Volume 69, March 2020

Data and Resource Availability PRMT1 and H4R3me2a became enriched in pancreatic The ChIP-seq, RNA-seq, and ATAC-seq data that support islets after weaning at 3 weeks of age, when the b-cells the findings of this study have been deposited in the become mature. In Prmt1 bKO mice, PRMT1 was nearly National Center for Biotechnology Information Gene Ex- undetectable at postnatal day 7, whereas H4R3me2a pression Omnibus (GEO) under accession code GSE117100. remained detectable in a substantial number of b-cells All data that support the findings of this study are available until 3 weeks of age, suggesting the more important role from the authors on reasonable request. No applicable resour- of PRMT1-dependent H4R3me2a in mature b-cells. In- ces were generated or analyzed during the current study. deed, the pancreatic islets of Prmt1 bKO mice developed normally and did not show any abnormality in the RESULTS markers of b-cell development (Supplementary Fig. 3A Prmt1 bKO Mice Develop Progressive Glucose and B). Prmt1 bKO mice grew normally and showed Intolerance normal glucose tolerance until they developed glucose We first checked the of the Prmt family intolerance at 12 weeks of age (Fig. 1A and B and genes in pancreatic islets and confirmed that Prmt1 Supplementary Fig. 3C). Despite this glucose intoler- exhibited the highest expression level among them (Sup- ance, Prmt1 bKO mice showed no defects in insulin plementary Fig. 1A and B). The expression level of Prmt1 sensitivity and insulin production (Supplementary Fig. washigherinpancreaticisletsthaninliverorbrain,and 4A–C). Instead, GSIS was impaired in Prmt1 bKO islets PRMT1 and H4R3me2a were enriched in pancreatic islets (Fig. 1C). Basal insulin secretion was increased when of both mice and humans (Supplementary Fig. 1C and Prmt1 bKO islets were treated with 2.8 mmol/L glu- D).TotestthepossibleroleofH4R3me2ainb-cells, we cose, and those treated with 20 mmol/L glucose failed performed ChIP-seq for H4R3me2a and ATAC-seq in to show any further increase of insulin secretion. The MIN6 cells. Motif analysis of the H4R3me2a ChIP- impairment of GSIS was further confirmed by analyses seq data suggested that H4R3me2a was significantly of plasma insulin levels and mitochondrial OCRs in the associated with the b-cell TFs: MAFA, NEUROD1, and isolated islets (Fig. 1D and E). To investigate whether FOXA1 (Supplementary Fig. 1E). Intriguingly, the most a transcriptional change was responsible for the defect significantly associated TF was CCCTC-binding factor of GSIS in Prmt1 bKO mice, we performed RNA-seq (CTCF), which is known to play a crucial role in regu- analysis in the islets of Prmt1 bKO mice at 12 weeks of lating the chromatin architecture (29,30). In order to age (Supplementary Fig. 5A). Although there was no assess the association of H4R3me2a with CTCF and b-cell robust change in global gene expression, Prmt1 bKO TF, we performed ChIP-seq for CTCF in MIN6 cells. islets exhibited downregulation of mature b-cell genes Analyses of ChIP-seq data obtained for H4R3me2a and that are involved in GSIS and misexpression of genes CTCF, combined with publicly available ChIP-seq data that are disallowed to be expressed in mature b-cells for MAFA, indicated that CTCF and MAFA bind near (31–33) (Supplementary Fig. 5B). These gene expres- H4R3me2a-occupied chromatin regions (Supplementary sion changes were further confirmed by qRT-PCR anal- Fig. 1F). The association of H4R3me2a with CTCF ysis (Supplementary Fig. 5C–F). Pathway analysis showed and MAFA, together with the enrichment of PRMT1 and that most of the genes downregulated in Prmt1 bKO H4R3me2a in adult b-cells, suggests that PRMT1 and islets were involved in pancreas development and maturity- H4R3me2a may play roles in b-cells. onset diabetes of the young (Supplementary Fig. 5G). This To further investigate the physiological role of PRMT1 notion was further supported by electron microscopic and H4R3me2a in b-cells, we generated b-cell–specific analysis (Fig. 1F), which showed that Prmt1-null b-cells fl/fl Prmt1 KO (Rip2-Cre; Prmt1 , herein called Prmt1 bKO) exhibited ultrastructural changes resembling those mice. Immunofluorescence staining confirmed the dele- found in the b-cells of patients with type 2 diabetes tion of PRMT1 at postnatal day 7 and the subsequent (34,35). The volume and density of insulin granules were removal of H4R3me2a in the b-cells of these mice reduced, the endoplasmic reticulum was dilated, and the around weaning at 3 weeks of age (Supplementary Fig. mitochondria appeared round and swollen. Condensed 2).InWTcontrolmice,thefluorescence signals of both chromatin was observed in the nuclei of Prmt1-null b-cells

(blue) from HFD-fed 26-week-old Prmt1 bKO mice; n 5 3/group. I: Representative islet images obtained by IF staining of eYFP (green), INS (blue), and GCG, SST, or PPY (red) from HFD-fed 26-week-old R26-eYFP; Rip2-Cre (control) and R26-eYFP; Prmt1 bKO mice. White arrows indicate GCG/SST/PPY-expressing eYFP1 cells. Quantification analysis of eYFP-copositive cells expressing INS (J), GCG (K), SST (L), and PPY (M) in the islets of HFD-fed 26-week-old R26-eYFP; Rip2-Cre (WT) and R26-eYFP; Prmt1 bKO (KO) mice; n 5 3/group. N: Representative islet images obtained by IF staining of INS (green) and PDX1, NKX6.1, MAFA, or SLC2A2 (red); n 5 3/group. O: Representative islet images obtained by IF staining of INS (green), FOXO1 (red), and DAPI (blue); n 5 3/group. Scale bars, 50 mm(H, I, N, and O); 2.5 mm(F). Prmt1fl/fl or Rip2-Cre mice were used as controls (A–G, N, and O). Data are expressed as mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 by Student t test (A, B, E, G, and J–M) or one-way ANOVA with post hoc Tukey test (C and D). CCCP, carbonyl cyanide m-chlorophenylhydrazone; Glc, glucose; Olig., oligomycin, Rot., rotenone. diabetes.diabetesjournals.org Kim and Associates 359

Figure 2—PRMT1 is required for the maintenance of mature b-cell identity. A: Representative islet images obtained by immunofluorescence staining of INS (green) and PRMT1, H4R3me2a, PDX1, NKX6.1, MAFA, or SLC2A2 (red) in 8- and 12-week-old control and Prmt1 biKO mice; n 5 3/group. B: Representative islet images obtained by immunofluorescence staining of eYFP (green), INS (blue), and GCG, SST, PPY, and UCN3 (red) from 8- and 12-week-old R26-eYFP; Pdx1-CreERT2 (control) and R26-eYFP; Prmt1 biKO mice; n 5 3/group. White arrows indicate GCG/SST/PPY-expressing or UCN32 eYFP1 cells. Quantification analysis of eYFP-copositive cells expressing GCG (C), SST (D), PPY (E), INS (F), and UCN3 (G) in the islets of 8- and 12-week-old R26-eYFP; Pdx1-CreERT2 (WT) and R26-eYFP; Prmt1 biKO (KO) mice; n 5 3/group. Scale bars, 50 mm. Tamoxifen-injected Prmt1fl/fl or Pdx1-CreERT2 mice were used as controls. Data are expressed as mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 by Student t test (C–G). 360 Essential Role of PRMT1 in b-Cell Identity Diabetes Volume 69, March 2020

Figure 3—HFD exacerbates mature b-cell identity in Prmt1 biKO mice. Male control and Prmt1 biKO mice (12 weeks) were fed HFD for 14 weeks and used for experiments. A: Representative islet images obtained by immunofluorescence (IF) staining of eYFP (green), INS (blue), and GCG, SST, and PPY (red) from HFD-fed 26-week-old R26-eYFP; Pdx1-CreERT2 (control) and R26-eYFP; Prmt1 biKO mice; n 5 3/group. White arrows indicate GCG/SST/PPY-expressing eYFP1 cells. Quantification analysis of eYFP-copositive cells expressing INS (B), GCG (C), SST (D), and PPY (E) in the islets of HFD-fed 26-week-old R26-eYFP; Pdx1-CreERT2 (WT) and R26-eYFP; Prmt1 biKO (KO) mice; n 5 3/group. F: Representative islet images obtained by IF staining of INS (green) and PDX1, NKX6.1, or MAFA (red); n 5 3/group. G: Representative islet images obtained by IF staining of INS (green), FOXO1 (red), and DAPI (blue); n 5 3/group. Scale bars, 50 mm. Tamoxifen-injected Prmt1fl/fl or Pdx1-CreERT2 mice were used as controls. Data are expressed as mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 by Student t test (B–E).

(Fig. 1F), but apoptosis was not observed in the islets of indicate that PRMT1 is neededtomaintainthefunction, Prmt1 bKO mice (Supplementary Fig. 5H). In addition, not the survival, of b-cells. islet hormones and b-cell TFs were normally expressed in As the phenotypes of Prmt1 bKO mice resembled the these mice (Supplementary Fig. 5I and J). These data early features of type 2 diabetes (34–36), we fed Prmt1 diabetes.diabetesjournals.org Kim and Associates 361

Figure 4—Deletion of Prmt1 from mature b-cells induces robust transcriptomic changes. RNA-seq analysis of islets from Prmt1 biKO mice at the early (8 weeks) and late (12 weeks) stages of loss of mature b-cell identity. Age- and sex (male)-matched, tamoxifen (TAM)-injected littermates (Prmt1fl/fl) were used as controls at the two different stages; n 5 2/group. Differentially expressed genes (DEGs) were identified using the following parameters: log2(fold change of counts per million mapped reads) #21.5 or $1.5; false discovery rate ,0.05. A: Schematic representation of time points at which RNA-seq sampling was performed for islets of control and Prmt1 biKO mice. B: Top ranked of commonly downregulated (n 5 260) and upregulated (n 5 310) DEGs. C: Expression heat maps of gene subsets relative to the functional categories identified in our islet RNA-seq analysis; n 5 2/group. Gene lists for the heat maps are presented in Supplementary Table 2. D–I: RNA-seq and qRT-PCR analyses of islets from Prmt1 biKO mice at the early (8 weeks) and late (12 weeks) stages of loss of mature b-cell identity; n 5 2/group for RNA-seq and n 5 4/group for qRT-PCR. Line (RNA-seq) and bar (qRT-PCR) graphs showing relative 362 Essential Role of PRMT1 in b-Cell Identity Diabetes Volume 69, March 2020 bKO mice with an HFD from 8 weeks of age to test how 2A). At 8 weeks of age, insulin expression was robustly these mice respond to metabolic stress (Supplementary Fig. reduced in the b-cells of Prmt1 biKO mice, and sub- 2 1 2 1 6A). HFD exacerbated the glucose intolerance in Prmt1 stantial numbers of INS /PDX1 ,INS /NKX6.1 ,and 2 1 bKO mice without perturbing compensatory b-cell expan- INS /MAFA cells were observed in the islets. At 12 weeks sion (Fig. 1G and Supplementary Fig. 6B). Interestingly, of age, the insulin signals were slightly recovered in Prmt1 the islets of HFD-fed Prmt1 bKO mice contained poly- biKO mice, but their intensities were still low, and most of hormonal cells that coexpressed insulin and glucagon the b-cells had lost MAFA and SLC2A2. A lineage-tracing (GCG) (Fig. 1H). A lineage-tracing analysis revealed that analysis showed that a number of b-cells lost their identity 2 2 these polyhormonal cells were originated from insulin- and became INS cells (;30%), urocortin 3 (UCN3) cells producing b-cells (GCG, ;0.4%; [SST], (;33%), polyhormonal cells, or other endocrine cells ;2.4%; and pancreatic polypeptide [PPY], ;0.19%), (GCG, ;1.5%; SST, ;3.5%; and PPY, ;0.24%) after in- suggesting that the b-cells of HFD-fed Prmt1 bKO mice duction of Prmt1 KO (Fig. 2B–G and Supplementary Fig. had undergone some changes in their differentiated 8A and B). Electron microscopic analyses showed similar states (Fig. 1I–M). Immunofluorescence staining of ma- ultrastructural changes in Prmt1 biKO and Prmt1 bKO ture b-cell markers further confirmed the loss of mature mice (Fig. 1F and Supplementary Fig. 8C). b-cell identity in HFD-fed Prmt1 bKO mice; such cells Furthermore, HFD aggravated the metabolic pheno- showed loss of MAFA and SLC2A2, cytoplasmic locali- types of Prmt1 biKO mice (Supplementary Fig. 9A–D). zation of NKX6.1, and blockage of HFD-induced FOXO1 Random blood glucose levels were continuously elevated, nuclear translocation (4,37,38) (Fig. 1N and O). These and glucose intolerance became more severe in HFD-fed phenotypes of HFD-fed Prmt1 bKO mice suggest that Prmt1 biKO mice, but the insulin sensitivity and b-cell PRMT1 plays an essential role in maintaining the ma- mass were comparable to those of the WT mice (Supple- ture b-cell identity. mentary Fig. 9C–F). Consistent with these findings, b-cells lost their identity in HFD-fed Prmt1 biKO mice: loss of PRMT1 Is Required for the Maintenance of b-Cell insulin (;30%) and expression of other hormones (GCG, 2 Identity ;1.7%; SST, ;3.8%; and PPY, ;0.37%), presence of INS / Prmt1 1 Although bKO mice presented the features of PDX1 cells, cytoplasmic localization of NKX6.1, and loss of loss of b-cell identity, these phenotypes were weak. MAFA (Fig. 3A–F). In addition, the HFD-induced nuclear fl This could re ect the presence of metabolic compensa- translocation of FOXO1 was blocked in Prmt1 biKO mice tion, which often comes into playingeneticKOmodels. (Fig. 3G). These data indicate that b-cells require PRMT1 to To minimize the involvement of any compensatory maintain their mature identity and that the loss of PRMT1 fi mechanism and further con rm the role of PRMT1 leads to the aberrant reprogramming of b-cells to express in mature b-cells, we generated an inducible b-cell– fl/fl other hormones. specific Prmt1 KO mouse model by crossing Prmt1 ERT2 mice with Pdx1 promoter-driven CreERT2 (Pdx1-Cre ) PRMT1 Regulates the Transcriptomic Program of mice (hereafter called Prmt1 biKO). Prmt1 KO was in- Mature b-Cells duced in adult b-cells by intraperitoneally injecting the In Prmt1 biKO mice, b-cells lost their identity soon after mice with tamoxifen (75 mg/kg) five times over 2 weeks, Prmt1 was ablated and before the glucose homeostasis beginning at 6 weeks of age (Supplementary Fig. 7A). At deteriorated. To examine the molecular mechanism un- 8 weeks of age, these mice exhibited deletion of PRMT1 derlying the observed loss of mature b-cell identity, we in b-cells and subsequent removal of H4R3me2a, but explored the global gene expression pattern in islets of maintained normoglycemia (Fig. 2A and Supplementary Prmt1 biKO mice at two different stages: the early stage at Fig. 7B and C). At 12 weeks of age, Prmt1 biKO mice 8 weeks of age and the late stage at 12 weeks of age (Fig. developed glucose intolerance and exhibited elevated 4A). RNA-seq analysis revealed robust changes in gene random glucose levels due to impaired GSIS (Supple- expression at both stages (Supplementary Fig. 10A and B). mentary Fig. 7C–E). Thus, acute loss of PRMT1 in adult In particular, genes involved in cellular energy production b-cells is sufficient to induce the loss of mature b-cell processes, such as oxidation reduction and the electron function. transport chain, were commonly downregulated at both Further immunofluorescence staining revealed more stages (Fig. 4B). Our stage-specific gene expression anal- severe defects in the b-cells of Prmt1 biKO mice (Fig. ysis showed that the gene expression patterns differed

expressions of the representative mature b-cell genes Ins1 (D), Ins2 (E), Ucn3 (F), Pdx1 (G), Mafa (H), and Slc2a2 (I) at the two different stages of loss of mature b-cell identity. Fold changes of counts per million are plotted, and false discovery rate values are indicated in line graphs for each stage. Expression levels of genes were normalized to Actb in each sample in qRT-PCR analysis. Heat maps of gene ontology (GO) (J) and Kyoto Encyclopedia of Genes and Genomes pathway (K) analyses of the stage-specific downregulated and upregulated DEGs. Data are expressed as mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 by one-way ANOVA with post hoc Tukey test (D–I). 8w, 8 weeks; 12w, 12 weeks; MAPK, mitogen-activated protein kinase; NOD, nucleotide-binding oligomerization domain; TCA, tricarboxylic acid. diabetes.diabetesjournals.org Kim and Associates 363

Figure 5—PRMT1-dependent H4R3me2a regulates the chromatin accessibility of mature b-cells. A–L, N, and P: ATAC-seq analysis performed on b-cells purified from 8-week-old R26-eYFP; Pdx1-CreERT2 (WT) and R26-eYFP; Prmt1 biKO (KO) mice; n 5 12/group were used for one ATAC-seq replicate. ATAC-seq peaks of WT and KO b-cells; n 5 2 were used for replicates. A: Schematic representation of the ATAC- seq analysis. B: Volcano plot showing differential ATAC-seq peaks in WT and KO b-cells. Differentially changed ATAC-seq peaks were identified using the following parameters: log2(fold change; WT/KO) #21or$1. P , 0.01. Average ChIP-seq peak intensities for H4R3me2a (C), H3K27ac (D), and H3K4me1 (E) in PRMT1-independent (indep.) and -dependent (dep.) open chromatin regions. F: Heat maps of normalized ATAC-seq and ChIP-seq signals at PRMT1-dependent open chromatin regions. Each row represents a peak. Normalized ChIP- seq signals of histone marks, CTCF, and b-cell TFs are shown. G: Known TF-binding motifs returned by Hypergeometric Optimization of Motif EnRichment analysis for PRMT1-dependent open chromatin regions. Gene ontology (GO) and pathway enrichment analyses of the genes nearby promoters (H) and enhancers (I) of PRMT1-dependent open chromatin regions. Scatter plots showing correlation between gene expression and open chromatin changes in WT and KO b-cells for b-cell genes (J) and OXPHOS genes (K). The x-axis represents log2(fold change; KO/WT) of RNA-seq data from 8-week-old Prmt1 biKO islets. The y-axis is a change of the highest ATAC-seq peak from the same gene promoters (TSS 6 2 kb). Integrative maps of ChIP-seq (histone marks and b-cell TFs), ATAC-seq (MIN6, WT, and KO b-cells), and RNA-seq (WT and KO islets) data obtained for the Ins1 (L), Ucn3 (N), and Pdx1 (P) genes. Red boxes indicate the regions in which the ATAC- seq signals of KO b-cells are decreased. Asterisks (red) indicate PRMT1-dependent open chromatin regions. 3C-PCR assays were 364 Essential Role of PRMT1 in b-Cell Identity Diabetes Volume 69, March 2020 between the early and late stages (Fig. 4C and Supplemen- (Fig. 5B). The average ChIP-seq peak intensities of tary Table 2). The mature b-cell genes showed various H4R3me2a and H3K27ac (active enhancer marks) were changes at the early stage, whereas most of these genes higher in PRMT1-dependent open chromatin regions were downregulated at the late stage. In particular, the compared with PRMT1-independent open chromatin expression levels of Ins1, Ins2, Ucn3,andPdx1 were de- regions (Fig. 5C and D). However, the average ChIP-seq creased in Prmt1 biKOmiceatbothstages,whereasthoseof peak intensity of H3K4me1 (a poised enhancer mark) Mafa and Slc2a2 were downregulated only at the late stage. was similar in PRMT1-dependent and -independent These results were further confirmed by qRT-PCR analysis open chromatin regions (Fig. 5E). These data indi- (Fig. 4D–I). The notable novel features in the b-cells of cate that H4R3me2a is responsible for the PRMT1- Prmt1 biKO mice included the robust downregulation of dependent chromatin openings in mature b-cells. The Ins1 at the early stage and its recovery at the late stage and PRMT1-dependent open chromatin regions were also corre- the downregulations of Ins2 and Ucn3 at the early stage (Fig. lated with the binding sites of CTCF and b-cell TFs, in- 4D–F). These findings correlated with our immunofluores- cluding NKX6.1, NKX2.2, MAFA, NEUROD1, and PDX1 cence staining observations in Prmt1 biKO mice (Fig. 2B). (Fig. 5F). Further motif analysis showed that the DNA- Meanwhile, RNA-seq and qRT-PCR analyses revealed binding motifs of CTCF and b-cell TFs were highly associ- that most of the genes related to oxidative phosphor- ated with PRMT1-dependent open chromatin regions (Fig. ylation (OXPHOS) were downregulated in Prmt1 biKO 5G and Supplementary Fig. 11). Intriguingly, genes near mice at both stages, suggesting that mitochondrial dys- PRMT1-dependent open chromatin regions were significantly function could be a feature of the loss of mature b-cell associated with mature b-cell function and identity (Fig. identity (Fig. 4C and Supplementary Fig. 10C). Stage- 5H and I). These data indicate that PRMT1-dependent specific gene ontology and pathway analyses revealed H4R3me2a is needed to maintain the unique chromatin that most of the genes downregulated at the early stage architecture of mature b-cells and that the loss of were linked with the electron transport chain and mature b-cell identity in Prmt1 biKO mice is associated maturity-onset diabetes of the young, whereas genes re- with widespread alterations of the chromatin landscape. lated to GSIS function (e.g., those related to vesicle To delineate how PRMT1-dependent chromatin open- transport and intracellular protein trafficking) were ings relate to the transcriptional changes observed in downregulated at the late stage (Fig. 4J and K). Taken Prmt1-null b-cells at the early stage, we closely examined together, these data indicate that PRMT1 is required the regulatory regions of genes downregulated in the to maintain the transcriptional program of mature islets of Prmt1 biKO mice at 8 weeks of age in parallel b-cells. with RNA-seq and ChIP-seq data of b-cell TFs. Indeed, the PRMT1-dependent open chromatin regions included PRMT1-Dependent H4R3me2a Regulates Chromatin multiple promoter or enhancer regions of b-cell and Accessibility in Mature b-Cells OXPHOS genes that were downregulated in the islets of Cell type–specific chromatin state is essential for main- Prmt1 biKO mice. A comparative analysis of data from taining the transcriptional program and identity of a dif- RNA-seq and ATAC-seq showed a correlation between ferentiated cell (39). Given the extensive gene expression thegeneexpressionchangesofb-cell genes and OXPHOS changes in Prmt1 biKOisletsandtheassociationof genes and the chromatin accessibility in the promoters of H4R3me2a with CTCF and b-cell TFs in MIN6 cells, we these genes (Fig. 5J and K and Supplementary Table 3). speculated that PRMT1-dependent H4R3me2a could Moreover, the average ATAC-seq peak intensities of regulate gene transcription through the actions on the Prmt1-null b-cells were decreased in the promoters of chromatin status of mature b-cells. To test our hy- b-cell genes and OXPHOS genes, indicating that the pothesis, we performed ATAC-seq with b-cells purified decreased expression of these genes at the early stage from Prmt1 biKO and WT mice at 8 weeks of age (Fig. could be attributed to the loss of PRMT1-dependent 5A). Unlike MIN6 cells that showed clear and distinc- H4R3me2a (Supplementary Fig. 12A and B). tive ATAC-seq peaks, ATAC-seq with purified b-cells We also identified PRMT1-dependent open chromatin showed some background noise in the peaks. There- regions at 12 kb upstream of the transcription start site fore, we analyzed ATAC-seq data from WT and Prmt1- (TSS) for the Ins1 gene and at 40 kb (E1) and 60 kb (E2) null b-cells along with ATAC-seq data from MIN6 cells upstream of the TSS for the Ucn3 gene (Fig. 5L and N). and identified 5,044 peaks corresponding to the PRMT1- These regions contained binding sites for b-cell TFs, in- dependent open chromatin regions in mature b-cells cluding PDX1, MAFA, NKX2.2, and NKX6.1. Although the

performed in MIN6 cells for Ins1 (M) and Ucn3 (O) genes. Arrows indicate 3C-PCR primers. L, N, and P: ATAC-seq peaks of MIN6 cells; n 5 3 were used for replicates. C, F, L, N, and P: H4R3me2a ChIP-seq peaks of MIN6 cells; n 5 3 were used for replicates. F: CTCF ChIP-seq peaks of MIN6 cells; n 5 2 were used for replicates. bp, ; E, enhancer; gDNA, genomic DNA; HIF, hypoxia-inducible factor; MAPK, mitogen-activated protein kinase; P, promoter; TGF-b, transforming growth factor-b. diabetes.diabetesjournals.org Kim and Associates 365

Figure 6—PRMT1-dependent open chromatin regions are conserved in the . Human genome alignment of PRMT1- dependent open chromatin regions in mouse b-cells for the Ucn3 (A), Pdx1 (B), and Slc30a8 (C) genes. Asterisks (red) indicate PRMT1- dependent open chromatin regions. Red bars and numbers indicate genomic locations and percentages of sequence identity, respectively. Type 2 diabetes susceptibility loci are indicated in green. Multiple alignments (Multiz Align) were performed for the genomes of 100 vertebrate species, which were captured from the University of California Santa Cruz Genome Browser (https://genome.ucsc.edu/). D: Cumulative plot of human conserved ATAC-seq peaks with the distances to the closest diabetes susceptibility locus. Human conserved PRMT1-dependent peaks tended to be closer to the diabetes susceptibility loci compared with the PRMT1-independent peaks; P 5 0.0003739 by paired Student t test, with matched sampling. E: Schematic representation describing the physiological role of PRMT1-dependent H4R3me2a in mature b-cells. Vert. Cons, vertebrate conservation. expression levels of Ins1 and Ucn3 were robustly and conserved regulatory elements in these regions. The E2 rapidly downregulated in Prmt1 biKO mice at the early element of the mouse Ucn3 gene and area IV of the mouse stage, the ATAC-seq peaks at the promoters of both genes Pdx1 gene were highly conserved in the human UCN3 and were not significantly reduced at this point. Instead, 3C- PDX1 genes, which are found at similar genomic locations PCR experiments showed the long-range interactions (Fig. 6A and B). A type 2 diabetes–associated locus was between the upstream enhancer elements and the pro- found near area IV of PDX1, and mice lacking endogenous moters of the Ins1 and Ucn3 genes, indicating that the area IV showed the impairment of b-cell maturation (40,45). loss of chromatin accessibility for the b-cell TFs at the We also found a highly conserved PRMT1-dependent open upstream enhancer elements had reduced the promoter chromatinregioninthehumanSLC30A8 gene, which is activities of these genes (Fig. 5M and O). We also iden- strongly associated with type 2 diabetes (45–48) (Fig. 6C). tified a PRMT1-dependent open chromatin region at 5 kb These findings prompted us to examine the association of upstream of the TSS for the Pdx1 gene (Fig. 5P); this human orthologous sequences of PRMT1-dependent open region, which is called area IV, was recently reported to chromatin regions with diabetes genome-wide association play an essential role in b-cell maturation during the study single nucleotide polymorphisms. Interestingly, the weaning period (40). PDX1 has also been shown to di- human diabetes-associated loci were more closely local- rectly regulate the gene expression of numerous mito- ized with PRMT1-dependent open chromatin regions chondrial genes that were downregulated in Prmt1-null than with PRMT1-independent open chromatin regions b-cells (41–44). These data suggest that PRMT1-dependent (Fig. 6D). This suggests that there may be a link between H4R3me2a plays a critical role in maintaining the unique PRMT1-dependent H4R3me2a and the susceptibility to chromatin architecture of mature b-cells and that the type 2 diabetes in humans. alteration of this chromatin architecture can result in the loss of mature b-cell identity. DISCUSSION In an effort to test the possible implication of PRMT1- Epigenetic regulation is crucial for b-cell maturation and dependent H4R3me2a in human diabetes, we performed the maintenance of mature b-cell identity (13–16). As one sequence-alignment analysis of PRMT1-dependent open of the major mechanisms of epigenetic regulation, histone chromatin regions in the human genome and searched for arginine methylation plays important roles in transcriptional 366 Essential Role of PRMT1 in b-Cell Identity Diabetes Volume 69, March 2020 regulation (17,18). However, its role in b-cells has not b-cells to lose their cell-specific chromatin architecture and yet been explored. Here, we demonstrate a novel func- gene expression program and thereby lose their identity. tion of PRMT1-dependent H4R3me2a in maintaining These b-cells that lose their identity then undergo differ- mature b-cell identity. Both Prmt1 bKO and Prmt1 ent changes based on their genetic heterogeneity in re- biKO mice developed diabetes, which was aggravated by sponse to metabolic stress. However, further study will be HFD-induced metabolic stress (Supplementary Fig. 13). needed to elucidate the precise mechanisms underlying Deletion of Prmt1 in adult b-cells resulted in the imme- the severe b-cell phenotypes of HFD-fed Prmt1 bKO and diate loss of H4R3me2a, which induced robust changes in Prmt1 biKO mice. The chromatin changes driven by HFD the transcriptions of genes necessary for the maintenance of feeding together with the losses of H4R3me2a and argi- mature b-cell function and identity. PRMT1-dependent nine methylation in nonhistone substrates of PRMT1 may H4R3me2a worked as an active histone code that in- have affected these phenotypes. FOXO1 and HNF4a are creased chromatin accessibility at the binding sites for PRMT1’s nonhistone targets that are also known to play CTCF and b-cell TFs, including NKX6.1, MAFA, PDX1, roles in b-cell function and identity (4,52–54). However, E and NEUROD1 (Fig. 6 ). Furthermore, PRMT1-dependent the phenotypes of b-cell–specific Foxo1 or Hnf4a KO mice open chromatin regions appear to be associated with genes differed from those of Prmt1 bKO mice (4,54). In this that have been associated with diabetes susceptibility in regard, we think that the phenotypes of Prmt1 bKO and humans. Prmt1 biKO mice may be largely attributed to the loss of Genome-wide association studies have indicated that H4R3me2a in b-cells. most diabetes-susceptibility genes are related to b-cells, Here, we provide novel insight into the importance and thus, b-cell failure is thought to be an essential feature of epigenetic control of PRMT1-dependent H4R3me2a b of diabetes (49,50). The dysfunction of -cells occurs long in maintaining mature b-cell identity. Taken together before hyperglycemia develops in humans (51), suggesting with the associations seen among CTCF, b-cell TFs, and that b-cell dysfunction is an early feature of diabetic b-cell H4R3me2a, our work reveals previously unknown func- failure. However, due to the lack of an appropriate animal tions of PRMT1-dependent open chromatin regions that model, researchers have been limited in their ability to govern mature b-cell identity. Thus, our phenotypic, tran- study how b-cell dysfunction begins in response to met- scriptomic, and epigenomic analyses of stage-specific Prmt1 abolic stress and how b-cell failure progresses before the KO in b-cells provide a new mechanistic insight into the apoptosis or dedifferentiation of b-cells is observed. In this regulation of mature b-cell identity. regard, Prmt1 biKO mice provide the following useful features: initial events of loss of b-cell identity, which has not previously been described in an animal model; loss of Acknowledgments. The authors thank Hee-Saeng Jung, Jueun Kim, and INS and UCN3 prior to the loss of other mature b-cell Hanna Jung (Korea Advanced Institute of Science and Technology) for technical TFs; decreased expression of b-cell genes; decreased support, Dr. Dahee Choi (Korea University) and Hyun Jung Hong (Chungnam National expression of mitochondrial genes; and subsequent mi- University) for technical advice and support, Drs. Yong-Ho Ahn and Joo-Man Park tochondrial dysfunction. Despite these extensive changes (Yonsei University) for help with electron microscopic analysis, Jeong-Hwan Kim of gene expression in the b-cells of Prmt1 biKO mice, (Korea Research Institute of Bioscience and Biotechnology) for technical assistance their metabolic phenotype was unexpectedly mild and with next-generation sequencing library preparation, Drs. Kyong Soo Park (Seoul became severe upon HFD feeding. This discrepancy National University), Kun-Ho Yoon (Catholic University of Korea), Soo Heon Kwak prompted us to propose the following explanations: 1) (Seoul National University), and Kyoung-Jae Won (University of Copenhagen) since b-cells are highly dedicated to insulin production for helpful discussions, and Drs. Mark O. Huising (University of California, Davis) and Paul E. Sawchenko (Salk Institute) for the gift of the UCN3 antibody. and secretion, even though the b-cells of Prmt1 biKO Funding. This work was supported by grants from the National Research mice are not fully functional, they can maintain glycemic Foundation funded by the Ministry of Science, ICT and Future Planning, Republic of 2 control as long as mice are insulin sensitive; and )there Korea (grants NRF-2013M3A9D5072550 to J.K.S., NRF-2017M3C9A5028693 to may be compensatory mechanisms to maintain glycemic M.K., and NRF-2014M3A9D5A01073546, NRF-2018R1A2A3074646, and NRF- control in Prmt1 biKO mice. The restoration of Ins1 2015M3A9B3028218 to Hai. Kim), the Korea Research Institute of Bioscience and expression in the late stage of Prmt1 biKO mice (12 weeks Biotechnology Research Initiative (to M.K.), and the Korea Advanced Institute of of age) supports the existence of these compensatory Science and Technology Institute for the BioCentury (grant N10180027 to Hai. Kim). mechanisms. Duality of Interest. No potential conflicts of interest relevant to this article Since the phenotype of Prmt1 biKO mice resembles the were reported. natural history of type 2 diabetes, this mouse model may Author Contributions. Hyu. Kim, B.-H.Y., C.-M.O., Jo. Lee, M.K., and Hai. be useful for studying how b-cells behave in response to Kim generated the hypothesis, designed the experiments, and analyzed the results. Hyu. Kim, C.-M.O., Jo. Lee, K.L., H.S., M.-Y.K., Hye. Kim, Y.K.K., and metabolic stress during the development of type 2 diabetes. Prmt1 J.K.S. performed the animal experiments. Hyu. Kim, C.-M.O., E.K., E.-H.S., H.H., biKO mice showed the following features in the H.-J.K., Ju. Lee, J.M.S., S.-H.K., S.K., and M.S. performed the cell and in vitro progression of b-cell failure: loss of b-cell identity, aber- experiments. Hyu. Kim, B.-H.Y., C.-M.O., K.Y., Y.S.J., M.K., and Hai. Kim analyzed rant expression of b-cell TFs, and cell type change of b-cells the next-generation sequencing data. Hyu. Kim, B.-H.Y., C.-M.O., Jo. Lee, M.K., to other endocrine cells. We speculate that Prmt1 deletion and Hai. Kim wrote the manuscript. M.K. and Hai. Kim supervised the research. resulted in the loss of H4R3me2a and that this causes M.K. and Hai. Kim are the guarantors of this work and, as such, had full access to diabetes.diabetesjournals.org Kim and Associates 367

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