Diabetes Volume 68, April 2019 761

Bromodomain and Extra Terminal Protein Inhibitors Promote Pancreatic Endocrine Fate

Lukas Huijbregts,1 Maja Borup Kjær Petersen,2 Claire Berthault,1 Mattias Hansson,3 Virginie Aiello,1 Latif Rachdi,1 Anne Grapin-Botton,2 Christian Honore,4 and Raphael Scharfmann1

Diabetes 2019;68:761–773 | https://doi.org/10.2337/db18-0224

Bromodomain and extraterminal (BET) proteins are epi- derived from surrounding mesodermal cells and next genetic readers that interact with acetylated lysines of differentiate into cells with exocrine and endocrine prop- histone tails. Recent studies have demonstrated their erties, including b-cells (1,2). Chronic failure to reduce role in cancer progression because they recruit key high blood glucose levels results in diabetes, which in most components of the transcriptional machinery to modu- patients is due to impaired functional b-cell mass. Recent late gene expression. However, their role during em- efforts, therefore, have concentrated on developing in bryonic development of the has never been vitro protocols to produce functional b-cells from induced studied. Using mouse embryonic pancreatic explants pluripotent stem cells (iPSCs) or embryonic stem cells to SE STUDIES ISLET and human induced pluripotent stem cells (hiPSCs), replenish the decreasing b-cell mass (3). These protocols we show that BET protein inhibition with I-BET151 or are based on our knowledge of the molecular mechanisms JQ1 enhances the number of neurogenin3 (NEUROG3) underlying in vivo b-cell development (4,5). Despite recent endocrine progenitors. In mouse explants, BET protein b considerable progress, generating a homogeneous popula- inhibition further led to increased expression of -cell b markers but in the meantime, strongly downregulated tion of fully mature, glucose-responsive -cells remains Ins1 expression. Similarly, although acinar markers, such a challenge (6). as Cpa1 and CelA, were upregulated, Amy expression The mouse embryonic pancreas starts developing at was repressed. In hiPSCs, BET inhibitors strongly re- approximately embryonic day 9.0 (E9.0), with the dorsal pressed C-peptide and glucagon during endocrine dif- and ventral budding of the foregut endoderm under the ferentiation. Explants and hiPSCs were then pulsed with influence of surrounding mesodermal structures. At this BET inhibitors to increase NEUROG3 expression and stage, multipotent proliferating epithelial pancreatic pro- further chased without inhibitors. Endocrine develop- genitors express a specific set of transcription factors, such ment was enhanced in explants with higher expression as PDX1 and NKX6.1 (7). They can undergo exocrine and of and maturation markers, such as UCN3 and endocrine cell fate until approximately E11.5 and then pro- MAFA. In hiPSCs, the outcome was different because gressively segregate into acinar progenitors or bipotent C-peptide expression remained lower than in controls, endocrine/duct progenitors (8,9). Endocrine progenitors but expression was increased. Altogether, by using then transiently express the basic helix-loop-helix tran- two independent models of pancreatic development, we scription factor neurogenin3 (NEUROG3) to give rise to all show that BET proteins regulate multiple aspects of pan- pancreatic endocrine cells (10). creatic development. Major progress has been made on growth factors, such as FGF7 and FGF10, that signal through FGFR2b (11–15) and activate the proliferation of pancreatic progenitors During pancreatic development, multipotent endodermal as well as on small molecules acting through yet-to-be- pancreatic progenitors proliferate in response to signals discovered pathways (16) or upon coculture on a layer of

1INSERM U1016, Institut Cochin, Université Paris Descartes, Paris, France This article contains Supplementary Data online at http://diabetes 2Novo Nordisk Foundation Center for Stem (DanStem), University of .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0224/-/DC1. Copenhagen, Copenhagen, Denmark © 2019 by the American Diabetes Association. Readers may use this article as 3 Stem Cell Research, Novo Nordisk A/S, Måløv, Denmark long as the work is properly cited, the use is educational and not for profit, and the 4 Department of Stem Cell Biology, Novo Nordisk A/S, Måløv, Denmark work is not altered. More information is available at http://www.diabetesjournals Corresponding author: Raphael Scharfmann, [email protected] .org/content/license. Received 23 February 2018 and accepted 7 January 2019 762 I-BET151, JQ1, and Pancreatic Endocrine Cells Diabetes Volume 68, April 2019

3T3-J2 feeder cells (17). On the other hand, information proliferation assay, 10 mmol/L BrdU (Sigma-Aldrich) was regarding signals that modulate the differentiation of added during the last hour of culture. pancreatic progenitors into functional b-cells remains scarce, and the objective of the current work was to Culturing and Differentiation of hiPSCs increase our knowledge on this topic. Wild-type SBAD03-01 and SBAD03-04 hiPSCs were ob- The bromodomain and extraterminal (BET) family of tained through the Innovative Medicines Initiative/European proteins comprises four members: BRD2, BRD3, BRD4, Union–sponsored StemBANCC consortium through the and BRDT. The latter is specifically expressed in the testis, Human Biomaterials Resource Centre, Birmingham Univer- whereas the other three are more ubiquitously expressed sity (www.birmingham.ac.uk/facilities/hbrc). Cells were (18–20). The BET proteins recently emerged as a major cultured and differentiated as previously described (29); class of epigenetic readers and modulators of gene expres- BETi or DMSO control was added daily to culture medium sion by their ability to recognize and bind through their during stage 4 (pancreatic endoderm) or 5 (endocrine two bromodomains N-acetylated-lysine residues of histone progenitors) of the differentiation. Data presented in this tails (21). They subsequently induce an opened chromatin article are from the SBAD03-01 line; similar data were structure and can tether various transcription factors at obtained with SBAD03-04 hiPSCs (Supplementary Fig. 1). target promoters and enhancer regions to promote tran- scription (22). The BET family of proteins largely has been RNA Extraction and Real-time PCR associated with cancer progression, and recent efforts have Total RNA was extracted as previously described (29,30). concentrated on developing potent and specific inhibitors Real-time PCR was performed with a QuantStudio 3 or of BET proteins (BETis), such as (+)-JQ1 and I-BET151 OneStep Plus Real-Time PCR system (Applied Biosystems) (21,23,24). BET proteins play a crucial role throughout or an Mx3005P quantitative PCR system (Stratagene). development, as Brd2- and 4-null mice are embryonic Each reaction consisted of either a mix of Power SYBR lethal (25,26). Recent work has shown that BRD4 partic- Green PCR Master Mix (Applied Biosystems) with a specific ipates in adipogenesis and myogenesis by modulating gene pair of designed primers or a mix of Taqman Universal PCR transcription at specific enhancer regions (27), yet the role Master Mix with a specific labeled probe (Applied Biosys- of BET proteins in organogenesis and cell fate remains tems). Data are presented relative to cyclophylin A (for poorly characterized. rodent samples) or HPRT1 and ACTB (for hiPSC samples). In this study, we have evaluated the effect of BET protein inhibition during pancreatic development. We Immunohistochemistry and Quantification used two different in vitro models: 1) a culture of rodent Mouse fetal pancreata were processed for immunohisto- fetal pancreas under conditions that replicate the major chemistry, as previously described (30). All primary anti- steps of in vivo pancreatic development (13) and 2) in vitro bodies and dilutions are described in the Supplementary differentiation of human iPSCs (hiPSCs) into insulin- Data. The fluorescent secondary antibodies were purchased producing cells (4,28). We report that BET inhibition from Jackson ImmunoResearch (1/400). The biotin-labeled using either I-BET151 or JQ1 increases the pool of secondary antibodies were purchased from Vector Labo- NEUROG3+ endocrine progenitors in mouse embryonic ratories (1/200). NEUROG3 and MAFA were detected pancreatic explants and in pancreatic progenitors derived using the VECTASTAIN Elite ABC Kit (Vector Laborato- from hiPSCs. This increased number of endocrine pro- ries). The nuclei were stained using the Hoechst 33342 fluo- genitors resulted in enhanced endocrine differentiation rescent stain (0.3 mg/mL) (Invitrogen). Surface area from pancreatic explants, whereas GHRL expression was quantifications were performed on one out of three con- increased in hiPSCs. The current data demonstrate the secutive sections (i.e., sections separated by 12 mm) to avoid importance of the BET protein family during pancreatic counting the same cell twice. The signal was quantified using endocrine lineage differentiation. ImageJ software (National Institutes of Health) and summed to obtain the surface area per explant (expressed RESEARCH DESIGN AND METHODS in mm2). NEUROG3+ and MAFA+ nuclei were manually Animals and Dorsal Pancreatic Bud Dissection counted on one out of three consecutive sections with Pregnant C57BL/6J mice were purchased from the Janvier ImageJ and then summed to obtain the number of positive Breeding Center and killed by CO2 asphyxiation according nuclei per explant. to French Animal Care Committee guidelines. Dorsal pan- creatic buds were dissected as previously described (13). Immunocytochemistry hiPSCs were fixed with 4% paraformaldehyde at room Pancreatic Bud Culture, Treatment With BETis, and temperature for 30 min and then processed for immuno- BrdU Incorporation cytochemistry as previously described (31). E11.5 pancreatic buds were cultured as previously de- scribed (13). The medium was changed daily and supple- Flow Cytometry and Cell Sorting mented with either 0.1% DMSO, 500 nmol/L I-BET151 Cells were sorted as previously described (32,33). hiPSCs (Sigma-Aldrich), or 100 nmol/L JQ1 (Abcam). For the cell were harvested to a single-cell solution using TrypLE Select diabetes.diabetesjournals.org Huijbregts and Associates 763 and subsequently fixed and stained as previously described (Pierce). Secreted insulin and content were measured in (28). Additional information on antibodies is provided in duplicate by Mouse Ultra Sensitive ELISA Kit (Crystal the Supplementary Data. Chem) according to the manufacturer’s instructions.

Chromatin Immunoprecipitation Statistical Analysis Confluent Min6 cells were treated with either 0.1% DMSO All quantitative data are presented as the mean 6 SD. or 1 mmol/L JQ1 for 24 h at 37°C in a humidified 95% Statistical significancewassetat5%anddeterminedusing air/5% CO2 gas mixture. Chromatin immunoprecipitation ordinary one-way ANOVA with Dunett post hoc test except (ChIP) was performed as described by Cotney et al. (34) for ChIP data, which were determined using repeated- using 5 mg of BRD2, 3, and 4 rabbit polyclonal antibodies measures one-way ANOVA. (A302-583A; A302-368A; A301-985150; Bethyl Laboratories). Immunoprecipitated chromatin was analyzed by real-time RESULTS PCR. Primer sequences are detailed in the Supplementary Data. Brd2, 3, and 4 Are Expressed in the Developing Pancreas + Glucose-Stimulated Insulin Secretion Assay At E11.5, we separated the EpCAM fraction that contains 2 Pools of at least five pancreatic buds were incubated epithelial progenitors from EpCAM mesenchymal fraction overnight in culture medium containing 2.8 mmol/L glu- by FACS and performed real-time PCR. Brd2, 3,and4,but cose (Sigma-Aldrich) and 2% FCS (Eurobio), then incu- not Brdt, mRNAs were detected in both the epithelial and the mesenchymal fractions (Fig. 1A). At E16, EpCAM+ cells were bated in Krebs-Ringer buffer (125 mmol/L NaCl, 2 4.7 mmol/L KCl, 2.5 mmol/L CaCl2, 1.2 mmol/L MgSO4, separated from EpCAM cells by FACS and further divided 1.2 mmol/L KH2PO4, 25 mmol/L NaHCO3, pH 7.4) sup- into three fractions that contain exocrine cells, endocrine plemented with 0.2% BSA (Sigma-Aldrich) and 2.8 mmol/L progenitors, and hormone-producing cells (33). Brd2, 3,and glucose for 60 min. Insulin secretion was assessed by 4 were detected in the mesenchymal as well as in all three + sequential static incubations of 60 min in Krebs-Ringer EpCAM fractions, with the highest expression in the buffer containing 45 mmol/L 3-isobutyl-1-methylxanthine hormone-producing cells (Fig. 1B). We next cultured (Sigma-Aldrich) and increasing concentrations of glucose E11.5 pancreatic dorsal buds for 1, 3, 5, or 7 days under (2.8–22.4 mmol/L) and finally 30 mmol/L KCl without culture conditions that successfully replicated in vivo de- 3-isobutyl-1-methylxanthine. velopment of exocrine and endocrine lineages, with day 3 (d3) being comparable to E15.5 and d7 to E17.5–18.5 (35). Insulin Content We observed a sustained expression of Brd2, 3,and4 during Pancreatic explants were homogenized in radioimmuno- the 7-day culture period (Fig. 1B). These results hence show precipitation assay buffer, and protein concentration was that Brd2, 3,and4 are expressed as early as E11.5 and that assayed using a Bicinchoninic Acid Protein Assay Kit their expression is maintained during pancreas development.

Figure 1—BRD2, 3, and 4 are expressed in the developing pancreas. A: Real-time PCR analysis of Brd2, 3, 4, and Brdt expression in mesenchymal (EpCAM2) and epithelial (EpCAM+) fractions from E11.5 mouse pancreatic buds. B: Real-time PCR analysis of Brd2, 3, 4, and Brdt; Neurog3; Ins1; Cpa1; and Vim expression in E16.5 pancreata. EpCAM+ fractions were FACS sorted into three fractions: 1) CD49f high CD133+, enriched in exocrine cells; 2) CD49f low CD133+, enriched in endocrine progenitors; and 3) CD49f low CD1332, enriched in hormone-expressing cells. C: Real-time PCR analysis of Brd2, 3, and 4 in E11.5 mouse pancreatic buds cultured for 1, 3, 5, or 7 days. Data are mean 6 SD of at least three independent experiments. *P # 0.05, **P # 0.01, ***P # 0.001. nd, not detected; Rel., relative. 764 I-BET151, JQ1, and Pancreatic Endocrine Cells Diabetes Volume 68, April 2019

BET Bromodomain Inhibition Induces Neurog3 NEUROG3+ cells was not the result of an increased pro- Expression in Both Mouse and Human liferation of multipotent pancreatic progenitors during the We treated mouse pancreatic explants with I-BET151 and early stages of explant development: 1) There was no major analyzed by real-time PCR the expression of Neurog3, variations of multipotent progenitor markers Pdx1, Sox9, a specific marker of pancreatic endocrine progenitors and Ptf1a after 24 h of exposure to BETis (Supplementary (10). In control explants, Neurog3 mRNA level peaked Fig. 2), and 2) immunohistochemical analyses of BrdU after 3 days of culture when endocrine progenitors develop incorporation indicated that the proliferation of PDX1+ and decreased thereafter as they further differentiate pancreatic progenitors did not increase. In fact, BrdU into hormone-expressing cells (10,35). I-BET151 treat- incorporation by PDX1+ cells upon BETi treatment was ment enhanced Neurog3 expression after 3 days of culture decreased by 10% (Supplementary Fig. 3A and B). The with a 10-fold induction over control conditions at d5 and NEUROG3 surge did not result from an increased pro- d7 (Fig. 2A). Similar induction of Neurog3 levels was liferation of endocrine progenitors. Indeed, BrdU incor- obtained with another BETi, JQ1 (Fig. 2A). Immunohis- poration remained nearly undetectable in NEUROG3+ cells + tochemical analysis at d5 showed an increase of NEUROG3 from pancreatic buds cultured with I-BET151 or JQ1 cells upon treatment with either I-BET151 or JQ1 (Fig. (Supplementary Fig. 3C). TUNEL staining revealed no var- 2B and C for quantification). Finally, both inhibitors iations in apoptotic events (Supplementary Fig. 3D and E). enhanced the expression of NeuroD and Fev, two down- We next examined the effect of BETis on human stream targets of NEUROG3 (36,37) (Fig. 2D). The surge of endocrine progenitors derived from hiPSCs. They were first differentiated toward multipotent pancreatic progeni- tors and further cultured in the presence of I-BET151 and JQ1 (Fig. 3A). Both inhibitors increased NEUROG3 ex- pression (Fig. 3B). Immunohistochemistry and flow cytom- etry analysis further confirmed enhanced NEUROG3 expression (Fig. 3C and D), with an almost doubling of the NEUROG3+ population (Fig. 3E). The increase of NEUROG3 expression followed a dose-response curve, with no decline in cell viability (Supplementary Fig. 4). These results indicate that BET bromodomain inhibition increases the pool of endocrine progenitors both in mouse embryonic pancreatic explants and in a model of multi- potent pancreatic progenitors derived from hiPSCs.

Treatment With BETis Increases Endocrine Cell Development We next assessed whether the increased pool of NEUROG3+ cells that developed upon BETi treatment gives birth to more hormone-producing cells in mouse pancreatic buds. Somatostatin (Sst) and glucagon (Gcg), markers of d- and a-cells, respectively, were significantly increased by I-BET151 and JQ1 treatments (Fig. 4A). Expression of ghrelin (Ghrl), a marker of e-lineage expressed at low levels in control conditions, was markedly increased, peaking at d5 of culture with a 25- and a 47-fold increase with I-BET151 and JQ1, respectively (Fig. 4A). Immunohisto- logical analysis indicated a sharp increase in the number of GHRL+ cells at d7 with both inhibitors (Fig. 4B and C for Figure 2—BETis induce NEUROG3 expression in pancreatic fi + fi quanti cation). Most GHRL cells stained negative for explants. A: Real-time PCR quanti cation of Neurog3 expression + in mouse pancreatic buds after 1, 3, 5, or 7 days of culture in the GCG, and all GHRL cells stained negative for PDX1, presence of 0.1% DMSO, 0.5 mmol/L I-BET151, or 0.1 mmol/L JQ1. INS, and SST (Fig. 4B and Supplementary Fig. 5). Alto- B: Immunohistological analyses of NEUROG3 staining in pancreatic gether, these results show that BET bromodomain inhi- m explants cultured for 5 days with 0.1% DMSO, 0.5 mol/L I-BET151, bition induces an increase of a-, d-, and e-cell markers. The or 0.1 mmol/L JQ1. Scale bars = 100 mm. C: Quantification of the total number of NEUROG3+ nuclei per pancreatic bud cultured for 5 days expression of acinar markers also was evaluated, and we with DMSO, I-BET151, or JQ1. D: Real-time PCR quantification of observed a strong decrease in Amy expression, whereas NeuroD1 and Fev expression in mouse pancreatic buds after 1, 3, 5, Cpa1 and CelA were upregulated (Fig. 4D). This finding or 7 days of culture in the presence of 0.1% DMSO, 0.5 mmol/L I-BET151, or 0.1 mmol/L JQ1. Data are mean 6 SD of at least three suggests that BET inhibition enhances acinar development independent experiments. *P # 0.05, **P # 0.01, ***P # 0.001. Rel., but that in the meantime, BET activity is required for relative. amylase expression. diabetes.diabetesjournals.org Huijbregts and Associates 765

Figure 3—BETis increase NEUROG3 expression during pancreas endocrine differentiation of hiPSCs. hiPSC lines were differentiated into the pancreatic lineage with the effect of the BETis ( 400 nmol/L JQ1, 2 mmol/L I-BET151) tested during the endocrine progenitor stage of the protocol. A: Schematic overview of the differentiation protocol. B: Relative (Rel.) expression of NEUROG3 mRNA was evaluated by real-time PCR (n = 5 individual differentiations). C: Representative immunofluorescence images of NEUROG3 of iPSCs differentiated toward the pancreatic endocrine lineage in control conditions (DMSO) or in the presence of the BETis (JQ1 or I-BET151). Scale bars = 100 mm. D: Representative flow cytometric dot plots of iPSCs differentiated toward the pancreatic endocrine differentiation in control conditions (DMSO) or in the presence of the BETis (JQ1 or I-BET151) and analyzed for NEUROG3 expression. Gates were set on the basis of isotype controls and include NEUROG3+ cells. Numbers in dot plots indicate percentage of cells present in the gates. E: Quantification of NEUROG3+ cells evaluated as shown in C. Data are mean 6 SD of at least three independent experiments. **P # 0.01. inh, inhibition; SSC, side scatter; Vit.C, vitamin C. 766 I-BET151, JQ1, and Pancreatic Endocrine Cells Diabetes Volume 68, April 2019

800 250 A 25 Sst DMSO Gcg ** DMSO Ghrl DMSO ** *** *** I-BET151 I-BET151 20 I-BET151 200 JQ1 600 JQ1 JQ1 15 150 *** *** 400 10 100 *** *** *** 200 *** 50 Rel. Expression Rel. Expression Expression Rel.

Rel. Expression Expression Rel. 5 *** ** ** *** 0 0 0

d1 d3 d5 d7 d1 d3 d5 d7 d1 d3 d5 d7 B DMSO I-BET151 JQ1 C GHRL

) DMSO 2 0.20 *** I-BET151 JQ1 GHRL GHRL 0.15 **

0.10

0.05 GHRL GHRL /

PDX1 0.00 D Cumulated Surface per Rudiment (mm 2000 400 5000 *** DMSO Amy DMSO Cpa1 DMSO Cela1 I-BET151 I-BET151 *** 4000 I-BET151 *** 1500 JQ1 300 JQ1 ** JQ1 3000 1000 200 2000 *** *** 500 100 1000 Rel. Expression Rel. Expression Rel. Expression *** *** *** 0 *** *** *** 0 0 5 1 3 d1 d3 d5 d7 d1 d3 d d7 d d d5 d7

Figure 4—BETi increase endocrine a-, d-, and e-lineage differentiation in pancreatic explants. A: Real-time PCR quantification of Sst, Gcg, and Ghrl expression in mouse pancreatic buds after 1, 3, 5, or 7 days of culture in presence of 0.1% DMSO, 0.5 mmol/L I-BET151, or 0.1 mmol/L JQ1. B: Immunohistological analyses of GHRL expression in pancreatic explants cultured for 7 days with 0.1% DMSO, 0.5 mmol/L I-BET151, or 0.1 mmol/L JQ1. Scale bars = 100 mm. Highlighted sections were magnified and indicate the absence of GHRL/PDX1 costaining. Scale bars = 25 mm. C: Quantification of the absolute surface area occupied by GHRL+ cells after 7 days of culture with DMSO, I-BET151, or JQ1. D: Real-time PCR analysis of Amy, Cpa1, and Cela1 expression in mouse pancreatic buds cultured for 7 days in presence of 0.1% DMSO, 0.5 mmol/L I-BET151, or 0.1 mmol/L JQ1. Data are mean 6 SD of three independent experiments. **P # 0.01, ***P # 0.001. Rel., relative.

Chronic Treatment With BETis Activates the b-Cell Of note, however, we observed a major reduction of Ins1 Lineage but Inhibits Insulin Gene Expression expression upon BETi treatment. Ins1 mRNA levels were We next determined whether I-BET151 and JQ1 treatments reduced by 47- and 20-fold after 7 days of culture with influence b-cell development in mouse pancreatic buds. As I-BET151 and JQ1, respectively (Fig. 5D). This was further expected, Pcsk1/3, MafA,andIapp expression increased confirmed by immunohistochemistry (Fig. 5E and F for during the culture period in control conditions, whereas quantification) and by ELISA (Fig. 5G). On the other hand, Nkx6.1, which encodes a transcription factor first expressed Ins2 mRNA levels were reduced only mildly after I-BET151 in all early multipotent pancreatic progenitors and next and JQ1 treatment (Fig. 5H). This difference between restricted to b-cells, decreased (Fig. 5A, white bars). BETis Ins1 and Ins2 expression after BETi treatment was next sharply increased the expression of all four b-cell markers analyzed by immunohistochemistry. We used antibodies (Fig. 5A). The increase in MafA mRNA was further con- against either C-peptide 1 or C-peptide 2 as surrogate firmed by immunohistochemistry at d7 of culture. I-BET151 markers of INS1 and INS2 expression. We observed an and JQ1 treatments increased the number of MAFA+ cells almost complete loss of C-peptide (C-PEP) 1 signal with by 2.5 6 0.6- and 2.8 6 0.6-fold, respectively (Fig. 5B and C both inhibitors, whereas many cells remained positive for for quantification). These results demonstrate that b-cell C-PEP 2 (Supplementary Fig. 6). Such a differential effect development is induced upon BETi treatment. of BETis on Ins1 and Ins2 expression also was observed in diabetes.diabetesjournals.org Huijbregts and Associates 767

Figure 5—BETi effects on the expression of b-cells markers in pancreatic explants. A: Real-time PCR quantification of Pcsk1/3, MafA, Iapp, and Nkx6.1 expression in mouse pancreatic explants after 1, 3, 5, or 7 days of culture in the presence of 0.1% DMSO, 0.5 mmol/L I-BET151, or 0.1 mmol/L JQ1. B: Immunohistological analyses of MAFA expression in pancreatic explants cultured for 7 days with DMSO, I-BET151, or JQ1. Scale bars = 100 mm. C: Quantification of the total number of MAFA+ nuclei per pancreatic bud cultured for 5 days with DMSO, I-BET151, or JQ1. D: Real-time PCR quantification of Ins1 expression in mouse pancreatic buds after 1, 3, 5, or 7 days of culture in the presence of 0.1% DMSO, 0.5 mmol/L I-BET151, or 0.1 mmol/L JQ1. E: Immunohistological analyses of insulin staining in pancreatic explants cultured for 7 days with DMSO, I-BET151, or JQ1. Scale bars = 100 mm. F: Quantification of the absolute surface area occupied by INS+ cells after 7 days of culture with DMSO, I-BET151, or JQ1. G: Insulin content of pancreatic explants cultured for 7 days with DMSO, I-BET151, or JQ1. H: Expression of Ins1 and Ins2 by real-time PCR using Taqman probes in mouse pancreatic buds after 1, 3, 5, or 7 days of culture in the presence of 0.1% DMSO, 0.5 mmol/L I-BET151, or 0.1 mmol/L JQ1. Data are mean 6 SD of three independent experiments. *P # 0.05, **P # 0.01, ***P # 0.001. prot., protein; Rel. relative.

the mouse insulinoma cell line MIN6 (Supplementary Fig. to an increase of insulin-producing b-cells, we performed 7A) and in primary mouse islets (Supplementary Fig. 7B). To pulse-chase experiments. We cultured (pulse period) pan- determine whether such an effect was due to differential creatic explants with BETis for 5 days. At this stage, binding of BET proteins to the Ins1 and Ins2 promoters, Neurog3 expression is at its highest. Pancreatic explants ChIP of BRD2, 3, and 4 was performed in MIN6 cells. We were then cultured for 9 additional days (chase period) observed strong bindings of BRD2 and 4 to both promoters, without BETis and analyzed (Fig. 7A). Under this setting which strongly decreased upon JQ1 treatment. Of note, at d14, Ins1 and Ins2 mRNA levels increased three-to BRD2 and 4 were bound at the proximal region of the Ins1 fourfold (Fig. 7B) and insulin content was also strongly promoter, whereas they bound at a more distal position of increased (Fig. 7C). MafA, but not MafB, mRNA level also the Ins2 promoter (Fig. 6). This indicates that rather than was strongly upregulated, with a 13- and 16-fold increase reducing the ability of amplified NEUROG3+ endocrine after I-BET151 and JQ1 pulses/chases, respectively (Fig. progenitors to pursue b-cell differentiation, BETis promote 7D). It was also the case for Ucn3 mRNA levels (22- and b-cell development but in the meantime, downregulate 23-fold increases) (Fig. 7D). Gcg, Sst, and Ghrl expression Ins1 expression with little effect on Ins2. also were increased (Fig. 7D) as were other b-cell markers, such as Nkx6.1, Pcsk1/3, Pdx1, and Iapp (Supplementary Long-term Effect of Transient BETis on Endocrine Fig. 10A). Immunohistochemical labeling of MAFA (Fig. 7E Differentiation and F for quantification) and UCN3 (Fig. 7G) after BETi Our results demonstrate that exposure to either I-BET151 pulse/chase confirmed this massive increase observed by or JQ1 enhances pancreatic endocrine cell development. real-time PCR. There was no coexpression of INS with However, Ins1 levels are strongly downregulated upon either SST or GCG, showing that the resulting b-cells such chronic treatment. To determine whether BETi ex- are not polyhormonal (Supplementary Fig. 10B). ELISA posure during explant development could ultimately lead of insulin secretion did not show a response upon glucose 768 I-BET151, JQ1, and Pancreatic Endocrine Cells Diabetes Volume 68, April 2019

Figure 6—BRD2 and 4 are proximally bound to Ins1 promoter in MIN6 cells. ChIP of BRD2, 3, and 4 in MIN6 cells that were treated for 24 h with 0.1% DMSO or 1 mmol/L JQ1. The precipitated chromatin was analyzed by real-time PCR and is expressed as a percentage of the input chromatin signal. Data are mean 6 SD of at least three independent experiments. *P # 0.05, **P # 0.01. Ab, antibody; kb, kilobase.

stimulation (Fig. 7H), suggesting that the resulting b-cells remain not fully elucidated. Histone modifications are key yet remain not fully mature. Similar to what was observed epigenetic events that play major roles in cell proliferation in pancreatic buds, transient BETi treatment of hiPSCs and differentiation (38), and we previously showed that during stage 5 reduced C-PEP+ cells and INS expression inhibiting histone deacetylases modulates cell fate during (Fig. 8A and B). GCG+ and SST+ cells also were reduced (Fig. rat pancreas development (30). Here, we extended our 8A), whereas GHRL expression was upregulated (Fig. 8B). comprehension of epigenetic modulation of embryonic Cells were then further cultured without BETis during the pancreas development by studying histone code reader maturing endocrine cells stage (chase during stage 6). BET proteins. We provide evidence that BET inhibition C-PEP expression increased after removal of BETis (Fig. promotes the pool of NEUROG3+ endocrine progenitors, 8A and B compared with 8C and D), but remained lower which subsequently gives rise to an increased pool of than in control conditions (Fig. 8C and D). GCG and SST endocrine cells. expressions were similar to the control condition (Fig. 8C). To explore the role of BET proteins in embryonic On the other end, GHRL expression increased (Fig. 8C and pancreas development, we used an in vitro model of dorsal D). Of note, coimmunocytochemistry of C-PEP with either embryonic pancreatic buds that recapitulates the major SST and GHRL or GCG and GHRL showed that most steps of endocrine and exocrine development that occur endocrine cells at the end of stage 6 were monohormonal in vivo (13,35). We first observed that Brd2, 3, and 4 (Supplementary Fig. 11). expression at E11.5 was similar in the epithelial population Taken together, the data indicate that treatment with that is enriched in pancreatic progenitors and mesenchy- BETis induces an increased pool of Neurog3+ endocrine mal cells. This result was expected because Brd2, 3, and progenitors that will further differentiate into b-, a-, d-, or 4 have been described as ubiquitously expressed in the e-cells in mouse pancreatic buds or e-cells in hiPSCs. majority of tissues (22), with some exceptions such as the highly enriched expression of BRD2, 3, and 4 in crypts of DISCUSSION the small intestine compared with villi (39). Of note, Brd2, The molecular mechanisms underlying the transitions 3,and4 expression at E16.5 is higher in a fraction enriched from pancreatic progenitors toward mature endocrine cells in hormone-expressing cells than in fractions enriched in diabetes.diabetesjournals.org Huijbregts and Associates 769

Figure 7—Pulse-chase treatment with BETis promotes b-cell differentiation in pancreatic explants. A: Schematic representation of the timeline followed for the pulse-chase experiment. B: Real-time PCR quantification of Ins1 and Ins2 expression in mouse pancreatic buds cultured for 5 days with 0.1% DMSO, 0.5 mmol/L I-BET151, or 0.1 mmol/L JQ1 (pulse) followed by a 9-day chase period without BETis. C: ELISA quantification of insulin content after pulse-chase experiment with DMSO or 0.1 mmol/L JQ1. D: Real-time PCR quantification of MafA, Ucn3, MafB, Ghrl, Sst, and Gcg expression in mouse pancreatic buds after pulse-chase experiment. E: Immunohistological analysis of MAFA and insulin at the end of the pulse-chase period. Scale bars = 50 mm. F: Quantification of MAFA+ nuclei per bud after pulse-chase experiment. G: Immunohistological analysis of UCN3 staining at the end of the pulse-chase period with DMSO or JQ1. Scale bars = 50 mm. H: Analysis of glucose-stimulated insulin secretion in mouse pancreatic buds cultured for 5 days with 0.1% DMSO, 0.5 mmol/L I-BET151, or 0.1 mmol/L JQ1 followed by 9 additional days without DMSO or BETis. Data are mean 6 SD of three independent experiments. *P # 0.05, **P # 0.01, ***P # 0.001. prot., protein; Rel., relative.

acinar or pancreatic progenitor cells, suggesting an impor- mRNA is unstable. Finally, it is notable that the positive tant role of BET proteins in establishing or maintaining effect of BETis on NEUROG3 expression is context and endocrine cell fate. tissue dependent because BETi (CPI203 or I-BET151) In mouse pancreatic explants cultured with I-BET151 or treatment induces a loss of progenitor cell markers such JQ1, Neurog3 expression was amplified at both the mRNA as Neurog3 in differentiation in the and the protein level. NEUROG3 activity also was in- mouse adult small intestine (39). creased as demonstrated by the increased expression of In mouse explants, BETi treatment increased the ex- two of its targets, NeuroD1 (37) and Fev (36). This increase pressions of endocrine markers, with Ghrl being the most of NEUROG3+ cells was not the consequence of an up- strongly upregulated. Ghrelin is expressed by a rare pop- stream effect on proliferation of PDX1+ pancreatic pro- ulation of pancreatic endocrine cells named e-cells (41), genitors or NEUROG3+ endocrine progenitors themselves. and GHRL+ cells that developed with BETis resemble In mouse pancreatic buds, the expression pattern of e-cells. They do not express PDX1, INS, or SST, and Neurog3 was shifted because it peaked at d5 of culture only a few coexpress GCG (Supplementary Fig. 5). and remained 10 times more expressed than in controls at GHRL+ cell number was shown to be increased in the d7. A possibility is that BETis increase the half-life of pancreata of mice deficient for either nkx2.2, ,or NEUROG3. Indeed, NEUROG3 has been described as an (41,42). Our data suggest, however, that ghrelin unstable protein rapidly degraded by ubiquitin-mediated induction upon BETi treatment does not occur through proteolysis (40), and it can be suspected that Neurog3 Nkx2.2 or Pax6, whose expressions did not decrease after 770 I-BET151, JQ1, and Pancreatic Endocrine Cells Diabetes Volume 68, April 2019

Figure 8—Pulse-chase treatment with BETis during hiPSC pancreatic differentiation delays endocrine progenitor differentiation and promotes e-cell differentiation. hiPSCs differentiated toward the pancreatic lineage were treated with BETis (400 nmol/L JQ1, 2 mmol/L I-BET151) during the endocrine progenitor stage (stage 5) of the protocol. A: Flow cytometric analysis of C-PEP–, GCG-, SST-, and GHRL- expressing cells at the end of stage 5 (S5d3). B: Real-time PCR quantification of INS, GCG, and GHRL expression at S5d3. hiPSCs were further cultured in the absence of BETis during the next stage (stage 6) for 7 days. C: Flow cytometric analysis of C-PEP–, GCG-, SST-, and GHRL-expressing cells at the end of stage 6 (S6d7). D: Real-time PCR quantification of INS, GCG, and GHRL expression at S6d7. Data are fold change over control (DMSO) condition and mean 6 SD of three independent experiments. *P # 0.05, **P # 0.01, ***P # 0.001.

explant treatment (Supplementary Fig. 8A). The moderate BETis, yet other markers, such as Cela1 or Cpa1, were reduction of Pax4 expression (Supplementary Fig. 8B) upregulated by BETis (Fig. 4D and Supplementary Fig. 9), seems unlikely to explain the observed effect on ghrelin suggesting an incomplete development of the acinar 2 2 because supernumerary GHRL+ cells of Pax4 / mice compartment. coexpress GCG and low PDX1 levels (43), which was not Nkx6.1 and MafA are key transcription factors essential the case in our model. The mechanism involved in the for b-cell differentiation and maturation, respectively. effects of BETis on the development of e-cells hence Their upregulation should then reflect an increase of remains to be clarified. We also observed a strong decrease b-cell differentiation. We therefore were surprised to in amylase expression in pancreatic buds treated with observe that Ins1 expression was dramatically reduced diabetes.diabetesjournals.org Huijbregts and Associates 771 after treatment of pancreatic buds with BETis, whereas GCG+ cells. Such an effect was not observed when hiPSCs Ins2 did not vary with JQ1 and only by twofold with were cultured with BETis during the earlier pancreatic I-BET151. Our data indicate that this was also the case in endoderm stage (Supplementary Fig. 11). Further remov- two models of mature b-cells, adult mouse islets, and ing BETis from amplified NEUROG3+ progenitors replen- MIN6 cells, where Ins1, but not Ins2, was downregulated ished the C-PEP+ population that, however, did not quite by I-BET151 and JQ1. Brd4 has been the most studied BET reach control levels. Further testing of additional condi- member because it was shown to modulate RNA poly- tions may permit the reproduction of the increase in b-cell merase II activity either by interacting with members of population observed in mouse pancreatic buds. Indeed, the the transcription initiation complex, such as pTEFb and discrepancies between the two models may come from mediator (44–46), or by directly releasing proximally different susceptibility time windows to BET inhibition, paused RNA polymerase II (47). Of note, Brd4 was shown which should be tested. They also might be due to the to associate specifically with active promoters and absence, in hiPSCs, of mesenchymal tissue, which partic- enhancers of a given cell type and to tether various ipates in the control of b-cell differentiation (54) and could transcription factors through its extraterminal domain mediatesomeBETieffectsinexplants.Finally,weob- (48,49). The insulin promoter is hyperacetylated in served a rise of e-cells in hiPSCs that were pulsed with b-cells (50), and Ins1 is among the most-expressed genes BETis during the endocrine progenitor stage (Fig. 8), which in b-cells. ChIP of BRD2, 3, and 4 in BETi-treated MIN6 is consistent with what was observed in mouse pancreatic cells indicated that BRD4, and to a lesser extent, BRD2, buds. Therefore, this should be of particular interest to were strongly bound at the proximal promoter of Ins1 study e-cell differentiation, which remains poorly under- (285/+16). We also found them to bind Ins2 promoter, stood. Altogether, our results obtained across two very but at a more distal position (2683/2771). This could different models of pancreas development revealed novel explain the differences observed in BETi responses because roles of BET on pancreatic endocrine progenitors and their proximally bound BRD4 could be more likely to influence differentiation into endocrine cells. RNA polymerase II activity. Such an approach and high throughput ChIP sequencing analysis on purified b-cells or NEUROG3+ progenitors would be of great use for deci- Acknowledgments. The authors acknowledge the transcriptomic plat- phering the underlying mechanisms of BET on pancreas form from the Cochin Institute for performing array hybridizations and thank the fl development and insulin regulation. However, to date, ow cytometry platform at the Novo Nordisk Foundation Center for Stem Cell Biology. there are no available specific markers to efficiently purify Funding. This work was supported by the Agence Nationale de la Recherche such cells from mouse pancreatic buds. (BromoBeta) and the Fondation Francophone pour la Recherche sur le Diabéte Our data indicate that BETis can increase the propor- (2018). The R.S. laboratory is supported by Fondation Bettencourt Schueller and + tion of NEUROG3 endocrine progenitors both in a model belongs to the Laboratoire d’Excellence consortium Revive and to the Departement of rodent pancreatic development and in a model of b-cell Hospitalo-Universitaire Autoimmune and Hormonal disease. The research leading development from hiPSCs. However, the failure of the to these results has received support from the Innovative Medicines Initiative Joint generated b-cells to express insulin properly represents Undertaking under grant agreement number 115439, resources of which com- a limiting step. Our pulse-chase approach in rodent, how- prise a financial contribution from the European Union’s Seventh Framework ever, indicates that NEUROG3+ endocrine progenitors Programme (FP7/2007-2013) and an in-kind contribution from European Feder- amplified with BETis can develop into INS+ b-cells after ation of Pharmaceutical Industries and Associations companies. The Novo Nordisk BETi removal. Moreover, newly formed b-cells expressed Foundation Center for Stem Cell Biology is supported by Novo Nordisk Foundation grant number NNF17CC0027852. higher levels of MafA and Ucn3, both considered as fl ’ b This article re ects only the author s views, and neither the Innovative markers of -cell maturity (51,52). Their maturity, how- Medicines Initiative Joint Undertaking, the European Federation of Pharmaceutical ever, is not yet complete because their sensitivity to Industries and Associations, nor the European Commission is liable for any use that glucose in terms of insulin secretion was not activated, may be made of the information contained herein. suggesting that MafA or Ucn3 expression cannot be used as Duality of Interest. M.H. and C.H. are employees of Novo Nordisk A/S and sole markers of cell maturity. It is established that in may hold shares in this company. No other potential conflicts of interest relevant rodent, b-cells appear quite late during development com- to this article were reported. pared with, for example, a-cells (1). Early NEUROG3+ cells Author Contributions. L.H., C.B., V.A., and L.R. performed and analyzed also have been shown to differentiate into a-cells and experiments on mouse material. L.H. and R.S. designed the experiments, those appearing later on into b-cells (53). Thus, it could be interpreted the data, and wrote the manuscript. M.B.K.P. and C.H. designed, postulated that the timing of differentiation correlates performed, and analyzed experiments on hiPSCs. M.H. and A.G.-B. provided critical input and analyzed the data. R.S. supervised the study and obtained with maturity and that b-cells generated upon BETi treat- funding. R.S. is the guarantor of this work and, as such, had full access to all the ment are more mature because the NEUROG3 expression data in the study and takes responsibility for the integrity of the data and the peak was maintained for a longer period. The current accuracy of the data analysis. results in hiPSCs show that BET proteins are essential Data Availability. The data sets generated and/or analyzed during the for endocrine progenitor differentiation because BET in- current study are available from the corresponding author on reasonable hibition during the endocrine progenitor stage enhanced request. No applicable resources were generated or analyzed during the current NEUROG3+ cells and resulted in a total lack of C-PEP+ and study. 772 I-BET151, JQ1, and Pancreatic Endocrine Cells Diabetes Volume 68, April 2019

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