The p300 and CBP Transcriptional Coactivators AreRequired for b-Cell and a-Cell ProliferationChi Kin Wong,1,2 Adam K. Wade-Vallance,2 Dan S. Luciani,2,3 Paul K. Brindle,4 Francis C. Lynn,2,3,5 andWilliam T. Gibson1,2

Diabetes 2018;67:412–422 | https://doi.org/10.2337/db17-0237

p300 (EP300) and CBP (CREBBP) are transcriptional co-activators with histone acetyltransferase activity. Variousb-cell transcription factors can recruit p300/CBP, andthus the coactivators could be important for b-cell func-tion and health in vivo. We hypothesized that p300/CBPcontribute to the development and proper function of pan-creatic islets. To test this, we bred and studied mice lack-ing p300/CBP in their islets. Mice lacking either p300or CBP in islets developed glucose intolerance attribut-able to impaired insulin secretion, together with reduceda- and b-cell area and islet insulin content. These pheno-types were exacerbated in mice with only a single copyof p300 or CBP expressed in islets. Removing p300 inpancreatic endocrine progenitors impaired proliferationof neonatal a- and b-cells. Mice lacking all four copiesof p300/CBP in pancreatic endocrine progenitors failedto establisha- and b-cell mass postnatally. Transcriptomicanalyses revealed significant overlaps between p300/CBP-downregulated genes and genes downregulated inHnf1a-null islets and Nkx2.2-null islets, among others.Furthermore, p300/CBP are important for the acetylationof H3K27 at loci downregulated in Hnf1a-null islets. Weconclude that p300 and CBP are limiting cofactors for isletdevelopment, and hence for postnatal glucose homeosta-sis, with some functional redundancy.

The expression of specific transcription factors both deter-mines and maintains the identities of pancreatic endocrinecells through activation of endocrine genes. For example,Pdx1, MafA, and NeuroD1 form a transcriptional complexat the insulin promoters and enhancers to activate insulin

expression synergistically in b-cells (1). These transcriptionfactors also recruit coregulators to fine-tune gene expres-sion. p300 and CBP (p300/CBP) are transcription coac-tivators that share .60% protein sequence identity andexhibit highly similar functions. These coactivators acet-ylate lysine residues on histones to modulate chromatinstructure or function, and lysine residues on nonhistoneproteins to modulate their activities (2). Although p300/CBPcan acetylate most histone proteins, they are absolutely es-sential for acetylating histone H3 lysine 27 (3). The H3K27Acmark tags tissue-specific promoters and enhancers and sig-nals transcription of the tissue-specific target genes (3,4).

p300/CBP appear to regulate important b-cell functionsin vitro. For instance, p300/CBP coactivate insulin geneexpression in vitro by binding synergistically to Pdx1 andNeuroD1/E47 (5). Small interfering RNA knockdown ofp300/CBP in INS1 cells reduced glucose-stimulated insulingene expression (6). In contrast, CRISPR-Cas9–mediateddeletion of p300 in INS1 832/13 cells induced a subtle in-crease in glucose-stimulated insulin secretion and reducedhigh glucose-mediated apoptosis (7). Mice with the S436Avariant in both copies of CBP, a mutation that renders CBPunresponsive to insulin-triggered phosphorylation, had in-creased islet mass but relatively normal b-cell function (8).These data left unresolved whether p300/CBP expression inpancreatic islets is necessary for establishing glucose ho-meostasis in vivo. We hypothesized that the removal ofp300/CBP from pancreatic endocrine progenitors wouldlead to postnatal glucose intolerance due to defects in isletmass and function. In this study, we generated andphenotyped Neurog3-Cre–driven pancreatic islet-specific

1Department of Medical Genetics, University of British Columbia, Vancouver,British Columbia, Canada2BC Children’s Hospital Research Institute, Vancouver, British Columbia, Canada3Department of Surgery, University of British Columbia, Vancouver, British Co-lumbia, Canada4St. Jude Children’s Research Hospital, Memphis, TN5Department of Cellular & Physiological Sciences, University of British Columbia,Vancouver, British Columbia, Canada

Corresponding author: Chi Kin Wong, ckwong@bcchr.ca.

Received 21 February 2017 and accepted 21 November 2017.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0237/-/DC1.

© 2017 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

412 Diabetes Volume 67, March 2018

ISLETSTUDIES

p300 and CBP knockout (KO) mice to study the roles ofthese coactivators in pancreatic islets in vivo.

RESEARCH DESIGN AND METHODS

AnimalsAll procedures were approved by the University of BritishColumbia Animal Care Committee. Mice housed at BCChildren’s Hospital Research Institute were under a 12-hlight/dark cycle with ad libitum access to standard chow(Teklad 2918; Envigo, Huntingdon, U.K.) and water. Neurog3-Cre mice were obtained from Dr. Francis Lynn, BC Children’sHospital Research Institute, Vancouver, British Columbia,Canada (9). Ep300fl/fl mice were obtained from Dr. PaulBrindle, St. Jude Children’s Research Hospital, Memphis,TN (10). Crebbpfl/fl mice were purchased from The JacksonLaboratory (Bar Harbor, ME) (10). All mice were kept onC57BL/6J background. Cre-negative littermates from eachbreeding setup were used as wild-type (WT) controls. Timedmatings were used to study embryonic day 15.5 (E15.5), E18.5,postnatal day 0 (P0), and P7 mice; the morning when a vaginalplug was found on the dams was designated as E0.5.

Metabolic PhenotypingFor glucose tolerance test, mice were fasted for 5 h and theninjected intraperitoneally with 2 g/kg glucose. For insulintolerance test, mice were injected with 0.7 units/kg HumulinR (Eli Lily, Indianapolis, IN). Blood was sampled from mousetails, and blood glucose levels were assessed using a OneTouchUltra Mini Glucometer (Johnson & Johnson, New Bruns-wick, NJ). For plasma insulin measurement, mice werefasted for 5 h, and blood was sampled from the saphenousveins using heparinized capillary tubes before and afterglucose injection. Heparinized blood was centrifuged at2,000g for 15 min at 4°C to separate plasma. Body compo-sition analysis and metabolic cage experiments were per-formed as previously described (11).

Analyte MeasurementInsulin was quantified using STELLUX ChemiluminescentImmunoassays (ALPCO, Salem, NH). Glucagon was quanti-fied using Glucagon ELISA (Mercodia, Uppsala, Sweden).Somatostatin was quantified using a Somatostatin EIA(enzyme immunosorbent assay) Kit (Phoenix Pharmaceut-icals, Burlingame, CA). Total glucagon-like peptide 1 (GLP-1)was quantified using Multi Species GLP-1 Total ELISA(Merck Millipore, Burlington, MA).

Ex Vivo Islet AssaysMouse pancreatic islets were isolated as described pre-viously (12). For glucose-stimulated insulin secretion assay,overnight recovered islets were incubated in Krebs-Ringerbuffer containing 2.8 mmol/L glucose for 1 h at 37°C. Afterthe preincubation, 30 islets were incubated in Krebs-Ringerbuffer containing either 2.8, 16.7, or 2.8 mmol/L glucosewith 30 mmol/L KCl for 1 h at 37°C. Supernatants werecollected for insulin measurement. Fura-2 calcium imagingwas performed as described previously (13). Perifusion

assays were performed on a Biorep Perifusion System permanufacturer instructions using 100 islets per chamber(Biorep Technologies, Miami Lakes, FL).

Immunofluorescence StainingAdult pancreata were fixed in 10% formalin for 24 h at 4°C,dehydrated, and embedded in paraffin, and 5-mm serialsections were made. A total of four to five sections, eachseparated by 150 mm, were obtained per adult pancreas. ForE15.5 and E18.5, sections were obtained from the entirepancreas and sections separated by 30 mm were stained.For P0 and P7, sections separated by 60 mm were obtained.These sections were stained for insulin, glucagon, somato-statin, ghrelin, chromogranin A, and/or Ki67. Other pro-teins were stained using randomly chosen sections. Afterblocking, the sections were incubated with primary anti-bodies overnight at 4°C followed by incubation with second-ary antibodies. Primary antibodies used include rabbitanti-p300 (N-15 + C-20 1:1, 1/50; Santa Cruz Biotechnology,Dallas, TX), rabbit anti-CBP (1/200; CST America, Framingham,MA), guinea pig anti-insulin (1/200; Abcam, Cambridge,U.K.), mouse anti-glucagon (1/1,000; Abcam), rabbit anti-somatostatin (1/400; Abcam), goat anti-somatostatin (1/200;Santa Cruz Biotechnology), goat anti-ghrelin (1/100; SantaCruz Biotechnology), rabbit anti-Ki67 (1/200; CST Amer-ica), rabbit anti-chromogranin A (1/200; Abcam), goat anti-chromogranin A (1/200; Santa Cruz Biotechnology),mouse anti-Ngn3 (1/50; Developmental Studies HybridomaBank at the University of Iowa, Iowa City, IA), rabbit anti-H3K27Ac (1/200; CST America), and rabbit anti-H3K27me3(1/200; CST America). The TUNEL assays were performedwith the In Situ Cell Death Detection Kit (Sigma-Aldrich,St. Louis, MO). Representative images of individual isletswere taken on a SP5 Confocal Microscope (Leica, Wetzlar,Germany). Images of whole pancreas sections were tiled ona BX61 Fluorescence Microscope (Olympus, Tokyo, Japan)and quantified using Fiji (14). Islet endocrine area werecalculated by dividing the corresponding stained area bythe total pancreas area. For E15.5 studies, the total numberof cells per population were counted and normalized tototal DAPI count per section.

Transcriptomic AnalysesTotal RNA was extracted from islets using TRIzol Reagent(Thermo Fisher Scientific, Waltham, MA) and the RNeasyMicro Kit (Qiagen, Hilden, Germany). For RNA sequencing(RNA-seq), six WT, three p300IsletKO, three CBPIsletKO, andthree CBPHet;p300KO samples were sequenced in twobatches at the University of British Columbia Biomedical Re-search Centre Sequencing Core. RNA samples with an RNAintegrity number .8.5 as measured on a model 2100 Bio-analyzer (Agilent Technologies, Santa Clara, CA) were pre-pared into sequencing libraries on NeoPrep using TruSeqStranded mRNA Library Prep Kit (Illumina, San Diego, CA).Each library was sequenced to a depth of at least 20 millionpaired end reads on a NextSEq 500 Sequencing System(Illumina). Quality filtered reads were aligned to referencemouse genome mm10 with TopHat2 (15). Count tables for

diabetes.diabetesjournals.org Wong and Associates 413

the aligned reads were generated, and batch effects werecorrected using SVA (Surrogate Variable Analysis) (16),followed by calling of differentially expressed genes usingDESeq2 with default settings in R (17). Genes were definedas differentially expressed based on an adjusted P valueof ,0.05. The Venn diagram for overlapping downregu-lated genes was generated using BioVenn (18). Downregu-lated gene lists were uploaded to Webgestalt for geneontology (GO) terms analysis and transcription factor tar-get prediction (19), using a reference list of 15,999 isletgenes from the WT islet transcriptome for accurate over-representation analyses. The lowest false discovery rate fromWebgestalt was capped at 10215. Manual gene set enrich-ment analyses were performed by applying hypergeomet-ric test with Bonferroni correction to the overlapped genesbetween different gene sets. All RNA-seq data were depositedin the Gene Expression Omnibus database (GSE101537).

For quantitative PCR (qPCR), RNAs were reversetranscribed using the iScript cDNA Synthesis Kit (Bio-Rad,Hercules, CA) and quantified by SYBR green–based reactionusing GoTaq qPCR Master Mix (Promega, Madison, WI) andprimers (Supplementary Table 1) on an ABI 7500 Real-TimePCR System (Thermo Fisher Scientific). Data were normal-ized to 18s rRNA, and fold changes were calculated with theΔΔCT method (20).

Western BlottingIslet nuclear extracts were prepared using the NE-PERNuclear and Cytoplasmic Extraction Kit (Thermo FisherScientific). One microgram of nuclear lysate was subject toWestern blotting using rabbit anti-p300 (N-15 + C-20 1:1,1/1,000; Santa Cruz Biotechnology), rabbit anti-CBP (1/1,000;Santa Cruz Biotechnology), and mouse anti–TATA bindingprotein (1/5,000; Abcam), as described previously (21).

Low-Input Native Chromatin ImmunoprecipitationThe low-input native chromatin immunoprecipitation (N-ChIP)protocol was carried out as previously described (22). One hun-dred islets were dispersed in 0.05% trypsin and flash frozenprior to lysis. Buffers were supplemented with 10 mmol/Lsodium butyrate to retain acetylation signals. Cells were lysedand digested with MNase for 5 min, as validated using WTislet cells. Chromatin equivalent to 10 islets per sample wassubjected to ChIP using rabbit anti-H3K27Ac or rabbit anti-H3K27me3 (both 2mL/ChIP; CST America). ChIP’d DNA wasquantified by qPCR and expressed as the percentage of input.

StatisticsData were shown as mean6 SD. Statistical significance wastested using the Student t test, one-way ANOVA, or two-wayANOVA, as appropriate, with P , 0.05 considered to bestatistically significant.

RESULTS

Mice Lacking p300 in Pancreatic Islets Develop GlucoseIntolerance Due to HypoinsulinemiaWe first characterized Neurog3-Cre; Ep300fl/WT mice thatdid not develop glucose intolerance up to 24 weeks of age.

They also appeared phenotypically identical to Cre-negativeEp300fl/fl mice and to mice bearing the Neurog3-Cre trans-gene alone (data not shown). We then made Neurog3-Cre;Ep300fl/fl mice (p300IsletKO mice). At E15.5, p300 was effec-tively removed using Neurog3-Cre in up to 95% of chromog-ranin A–positive cells, while Ngn3-positive progenitorsremained p300 positive (Supplementary Fig. 1). This is likelydue to a time lag between the onset of Cre expression andthe onset of recombination. Among the chromograninA–positive cells, recombination occurred in newly formedb-cells, a-cells, d-cells, and e-cells (Supplementary Fig. 1,d-cells; other cell types are not shown). In p300IsletKO mousepancreata, p300 was absent in islet nuclei but was expressednormally in the exocrine tissues (Fig. 1A and B). The proteinlevels of CBP were similar between WT and p300-null islets,indicating the absence of compensatory overexpression ofa CBP paralog.

p300IsletKO mice were glucose intolerant at 8 weeks ofage but displayed normal insulin tolerance (Fig. 1C and D).Plasma insulin levels of p300IsletKO mice were 60% lowerthan those of WT mice both before and after glucose in-jection (Fig. 1E). Theoretically, defective glucose metabolismin p300IsletKO mice could be in part due to the Neurog3-Cre–mediated recombination outside of islets such as in the ven-tromedial hypothalamus and enteroendocrine cells (9,23).We found that WT and p300IsletKO mice had similar bodycomposition, energy expenditure, locomotor activity, andfood intake (Supplementary Fig. 2A–D). Also, plasma totalGLP-1 levels were normal in p300IsletKO mice (Supplemen-tary Fig. 2E). In the absence of observable extrapancreaticphenotypes, the glucose intolerance and hypoinsulinemiacan be attributed to the recombination within pancreaticislets rather than other Neurog3-expressing tissues.

p300IsletKO Mice Have Reduced Islet Area and IsletInsulin ContentTo examine how p300IsletKO mice developed glucose intol-erance and hypoinsulinemia, we first quantified islet cellarea in adult mice. Although the weights of p300IsletKO

mouse pancreata were comparable to WT mice (Supplemen-tary Fig. 3A), they showed a 25% reduction in a-cell andb-cell area (Fig. 2A). The d-cell area was unaffected. Thereduced a-cell and b-cell areas were attributable to a reducednumber of islets rather than islet size (Fig. 2B and Supple-mentary Fig. 3B). The b-cell-to-a cell ratios were similarbetween WT and p300IsletKO mouse pancreata (Supplemen-tary Fig. 3C). Insulin content of p300-null islets was re-duced by 19%, whereas glucagon content was unchanged(Fig. 2C). The somatostatin content was elevated by nearlytwofold. Fasting plasma glucagon levels of p300IsletKO micewere unaffected (Supplementary Fig. 3D). To assess thefunction of p300-null islet explants, we stimulated the isletswith low glucose, high glucose, or KCl and measured theirinsulin release. Although both WT and p300-null isletsresponded similarly to low or high glucose, p300-null isletshad increased insulin secretion and calcium response uponKCl stimulation (Fig. 2D and E). Glucagon secretion from

414 Roles of p300/CBP in Pancreatic Islets Diabetes Volume 67, March 2018

p300-null islets under low-glucose conditions was not sig-nificantly different from that of the WT islets (Supplemen-tary Fig. 3E). Thus, the combined defects in islet mass andislet insulin content result in deficient glucose-stimulatedinsulin secretion in p300IsletKO mice.

CBPIsletKO Mice Share Similar b-Cell Phenotypes Withp300IsletKO MiceTo understand whether p300 and CBP function similarly inpancreatic islets, we generated Neurog3-Cre; Crebbpfl/fl

(CBPIsletKO) mice. The deletion of CBP in islets was not com-pensated for by the overexpression of p300 (Fig. 3A).CBPIsletKO mice developed glucose phenotypes similar tothose of p300IsletKO mice, including glucose intolerance at8 weeks of age without insulin resistance and defective in-sulin release upon glucose injection (Fig. 3B–D). Unlikep300IsletKO mice, fasting plasma insulin levels of CBPIsletKO

mice did not differ from those of controls (Fig. 3D). Thepancreata of CBPIsletKO mice had 40% less a-cell area and30% less b-cell area, with d-cell area unaffected (Fig. 3E). Incontrast to p300-null islets, CBP-null islets had reduced glu-cagon content but normal somatostatin content comparedwith controls (Fig. 3F). Similar to p300-null islets, CBP-nullislets had reduced insulin content, but their b-cell secretoryfunction appeared to be normal (Fig. 3F and G). Overall,both p300IsletKO and CBPIsletKO mice exhibited reducedb-cell area and islet insulin contents, features that explaintheir glucose intolerance.

Mice With Only a Single Copy of p300 or CBP in IsletsDevelop More Severe Glucose and Islet PhenotypesThan Mice Lacking p300 or CBP Alone in IsletsSince mice lacking p300 or CBP had similar phenotypes, wehypothesized that p300IsletKO or CBPIsletKO mice lacking anadditional copy of p300 or CBP would develop more severeglucose phenotypes. To test this, we generated Neurog3-Cre;Crebbpfl/WT; Ep300fl/fl mice and Neurog3-Cre; Crebbpfl/fl;Ep300fl/WT mice (CBPHet;p300KO and CBPKO;p300Het mice,respectively). These triallelic mice developed severe glucoseintolerance by 8 weeks of age with no defects in insulintolerance (Fig. 4A and B and Supplementary Fig. 4,CBPKO;p300Het mice data). Unlike the biallelic mice, triallelicp300/CBP mice of either genotype failed to mount an in-sulin response to glucose challenge (Fig. 4C). In addition,CBPHet;p300KO mice had 58% less a-cell area and 45% lessb-cell area (Fig. 4D). Their islets contained 72% less insulinthan WT islets (Fig. 4E). These phenotypes recapitulatedthose seen in p300IsletKO or CBPIsletKO mice despite beingmore severe.

Expression of p300/CBP Is Necessary for Neonatalb-Cell and a-Cell ProliferationSince reduced a-cell and b-cell areas could be caused bydefects in differentiation, proliferation, and/or apoptosis,we examined these processes throughout pancreas develop-ment in WT and p300IsletKO mice. We first excluded apo-ptosis as a possible cause of islet cell loss by performing

Figure 1—Mice lacking p300 in pancreatic islets develop glucose intolerance due to hypoinsulinemia. A: Western blotting for p300 and CBPin isolated WT or p300-null islet nuclear extracts. TBP (TATA binding protein) was used as a loading control. The experiment was replicatedonce. B: Representative immunofluorescence images of p300 and CBP in the pancreatic islets of WT and p300IsletKO mice. n = 3. Scale bar =50 mm. Insulin, Ins; glucagon, Gcg. C: Glucose tolerance test of 8-week-old WT and p300IsletKO mice. n = 8–9. D: Insulin tolerance test of9-week-old WT and p300IsletKO mice. n = 5–7. E: Plasma insulin measurement before and 15 min after glucose injection. n = 4–5. Two-wayANOVA for C–E. *P , 0.05; **P , 0.01; ***P , 0.001.

diabetes.diabetesjournals.org Wong and Associates 415

TUNEL assays on E18.5 p300IsletKO mouse pancreata andadult p300IsletKO mouse pancreata; apoptotic events inp300-null islets were as rare as in WT islets (data notshown). Next, the number of newly differentiated endo-crine cells and Ngn3-positive endocrine progenitors wereunaffected in E15.5 p300IsletKO mouse pancreata (Supple-mentary Fig. 5A). At E18.5, a-cell, b-cell, and pan-endocrinecell areas were normal in p300IsletKO mouse pancreata(Supplementary Fig. 5B). At P7, the a-cell and b-cell areaswere reduced in p300IsletKO mouse pancreata (Fig. 5A). Thepercentages of Ki67+ a-cell and b-cells in these pancreatawere lower than that of WT; however, the overall percent-age of Ki67+ cells in the pancreata was unchanged (Fig. 5Band C). This indicated that the proliferation of neonatala-cells and b-cells was reduced in p300-null islets. Hence,the reduced a-cell and b-cell mass in the adult p300IsletKO

mouse pancreata originated after E18.5 and is attributableto impaired proliferation.

We attempted to breed for Neurog3-Cre; Crebbpfl/fl;Ep300fl/fl mice (p300/CBP double-KO mice), but we did notobserve any of the double-KO mice in a cohort of 59 pups at

the weaning age, in contrast to the triallelic p300/CBP mice,which were observed at the expected Mendelian ratio (Sup-plementary Table 2). We speculated that these p300/CBPdouble-KO mice might die shortly after birth because ofa failure to establish sufficient b-cell mass. At P0, somedouble-KO pups survived, but their pancreata lackeda-cells and b-cells completely (Fig. 5E and F). Surprisingly,their d-cell and e-cell populations were unaffected (Fig. 5E).Immunostaining showed that a few e-cells, which are nor-mally absent in adult mouse pancreata, persist in the bial-lelic and triallelic mouse pancreata (Supplementary Fig. 6A).Thus, at least one allele of p300 or CBP is necessary fornormal development of a-cells and b-cells, but not ford-cells or e-cells.

Loss of p300/CBP Impairs Genes Associated WithMultiple Islet/b-Cell Transcription Factors and Impairsthe Coactivation of Hnf1a-Associated Genes In VivoBecause p300/CBP are transcriptional coactivators, the lossof p300/CBP may reduce the expression of genes importantfor islet function or development. We examined gene

Figure 2—p300IsletKO mice display defects in islet mass and islet insulin content. A: Quantification of b-cell, a-cell, and d-cell areas of adult WTand p300IsletKO mice pancreata. n = 8 for b-cells; n = 4–5 for a-cells and d-cells. B: Islet density of adult WT and p300IsletKO mice pancreata. n = 5.C: Insulin (Ins), glucagon (Gcg), and somatostatin (Sst) content of WT and p300-null islets. n = 5–6. D: Glucose-stimulated insulin secretionassays on isolated WT and p300-null islets. n = 5–7. E: Fura-2 calcium imaging on isolated WT and p300-null islets. n = 3. Student t test for A–D.Two-way ANOVA for E. *P , 0.05; **P , 0.01.

416 Roles of p300/CBP in Pancreatic Islets Diabetes Volume 67, March 2018

expression by performing RNA-seq on islet mRNAs fromWT, p300IsletKO, CBPIsletKO, and CBPHet;p300KO mice. Weidentified 761 (477 down, 284 up), 923 (513 down,410 up), and 5,589 (2,411 down, 3,178 up) differentiallyexpressed genes in p300-null, CBP-null, and triallelic isletsrelative to WT islets, respectively (Supplementary Table 3).

We focused our analyses on the downregulated genes.The aggregation of the downregulated gene sets revealed230 downregulated genes overlapped between p300-nullislets (48.2%) and CBP-null islets (44.8%) (Fig. 6A). Thegenes downregulated in CBPHet;p300KO islets overlappedwith 436 (91.4%) and 437 (85.2%) of the downregulatedgenes from p300-null and CBP-null islets, respectively. En-richment analyses of the Biological Process GO terms on allthree sets suggested three common themes of genes down-regulated by the loss of p300/CBP: lipid metabolic process,regulation of hormone levels, and ion transport (Fig. 6B andSupplementary Table 4). Transcription factor target predic-tion from Webgestalt showed that all three gene sets weresignificantly enriched for the predicted transcription factor

Hnf1a (Fig. 6C and Supplementary Table 5). We also per-formed gene set enrichment analysis by comparing our genesets to published downregulated genes in mouse islets lack-ing factors important for b-cell development and function,including Pdx1, NeuroD1, Hnf1a, Pax6, MafA, Nkx6.1, andNkx2.2 (24–30). Our gene sets overlapped more signifi-cantly with the gene sets of Hnf1a and Nkx2.2, followedby MafA, Nkx6.1, Pdx1, and NeuroD1 (Fig. 6D and Supple-mentary Tables 6 and 7).

Because Hnf1a could recruit p300/CBP for coactivation(31), we further examined the genes that overlap betweenthe Hnf1a gene set and the gene sets we had defined asdownregulated genes in p300-null islets, CBP-null islets, andCBPHet;p300KO islets. Tmem27, a known Hnf1a-mediatedregulator of b-cell proliferation (32), was reduced in all threemodels. Other loci downregulated in Hnf1a-null islets, in-cluding Pklr, Slc2a2, and G6pc2, were also downregulatedin triallelic islets as validated by qPCR (Fig. 6E) (24). b-Celltranscription factors were not specifically downregulatedin either p300-null or CBP-null islets, whereas Hnf4a,

Figure 3—CBPIsletKO mice share similar phenotypes with p300IsletKO mice. A: Representative immunofluorescence images of p300 and CBP inthe pancreatic islets of WT and CBPIsletKO mice. Scale bar = 50 mm. B: Glucose tolerance test of 8-week-old WT and CBPIsletKO mice. n = 4–11.C: Insulin tolerance test of 9-week-old WT and CBPIsletKO mice. n = 5–8. D: Plasma insulin measurement of WT and CBPIsletKO mice before and15 min after glucose injection. n = 6–7. E: Quantification of b-cell, a-cell, and d-cell areas of adult WT and CBPIsletKO mice pancreata as thepercentage of total pancreas area. n = 4–5 for a-cells and b-cells; n = 5–6 for d-cells. F: Islet insulin (Ins), glucagon (Gcg), and somatostatin (Sst)content of WT and CBP-null islets as quantified by ELISA. n = 4. G: Perifusion assay for insulin secretion of WT and CBP-null islets. n = 3. Two-way ANOVA for B–D. Student t test for E and F. *P , 0.05; **P , 0.01; ***P , 0.001.

diabetes.diabetesjournals.org Wong and Associates 417

Hnf1b, and NeuroD1 were downregulated in triallelic islets(Supplementary Table 8). Insulin-processing genes were notaltered in the biallelic mouse islets. Ins1 and Ins2 mRNAswere normally expressed in the biallelic mouse islets, althoughboth were downregulated by .50% in the triallelic islets.

Because p300/CBP coactivate transcription factors inpart by acetylating H3K27 at target promoters andenhancers, we hypothesized that the loss of p300/CBPwould reduce H3K27 acetylation at the loci downregulatedin Hnf1a-null islets. We assessed the acetylation andmethylation statuses of H3K27 at various loci using low-input N-ChIP, and found that there was significantly lessH3K27Ac at the promoters of G6pc2, Hnf4a, Pklr, andTmem27 in the triallelic islets (Fig. 6F and SupplementaryFig. 6B, negative loci). Pdx1-associated genes also showedreduced H3K27Ac at their promoters and enhancers in thetriallelic islets (Fig. 6G). The H3K27Ac levels at these lociwere reduced in CBP-null islets, although the reductiondid not reach statistical significance. These loci-specificH3K27Ac levels clearly correlated with the total dosagesof p300/CBP in the cells. We confirmed an;60% reductionof H3K27Ac globally in the triallelic islet nuclei (Fig. 6H).Total and loci-specific H3K27me3 levels were unaffected intriallelic islets (Fig. 6H and Supplementary Fig. 6B). Overall,

the reduced dosages of p300/CBP impaired coactivation ofdownregulated genes in Hnf1a-null islets, which we attri-bute to reductions in global and loci-specific H3K27Ac levels.

DISCUSSION

In this study, the loss of either p300 or CBP alone in thepancreatic islets was sufficient to perturb whole-body glu-cose homeostasis. Mice lacking p300 or CBP in islets devel-oped similar b-cell phenotypes, including reduced b-cellarea and insulin content. Mechanistically, p300 and CBPare known to coactivate Pdx1, NeuroD1, Hnf4a, and Hnf1a/b in vitro (33–35). Our RNA-seq data suggested that genesdownregulated in Hnf1a-null islets became downregulatedonce the dosages of either p300 or CBP were reduced in theislets. Hnf1a/b are homeobox transcription factors that arecritical for pancreas and b-cell development (36,37). Inparticular, impaired Hnf1a coactivation in our mouse mod-els could attenuate b-cell proliferation through genes suchas Tmem27 (32). The role of Hnf1a in a-cells remains un-clear, although high levels of HNF1a were found in FACS-sorted human a-cells, thereby implying that p300/CBPmight also regulate aspects of a-cell biology, such as prolif-eration, through HNF1a (38). p300/CBP bind to Hnf1a/bthrough their transactivation domains, and coactivate their

Figure 4—Triallelic deletion of p300/CBP in islets leads to severe glucose intolerance. A: Intraperitoneal glucose tolerance test of 8-week-old WTand CBPHet;p300KO mice. n = 6. B: Insulin tolerance test of adult WT and CBPHet;p300KO mice. n = 6–7. C: Plasma insulin measurement of WTand CBPHet;p300KO mice before and 15 min after glucose injection. n = 7. D: Quantification of b-cell, a-cell, and d-cell areas of adult WT andCBPHet;p300KO mouse pancreata as percent of total pancreas area. n = 4–6 for a-cells and b-cells; n = 4 for d-cells. E: Islet insulin content of WTand CBPHet;p300KO islets as quantified by ELISA. n = 4. Two-way ANOVA for A–C. Student t test for D and E. **P , 0.01; ***P , 0.001.

418 Roles of p300/CBP in Pancreatic Islets Diabetes Volume 67, March 2018

downstream targets by acetylating the histones bound toregulatory elements affiliated with these targets (39). Theobserved loss of H3K27Ac in triallelic islets at loci down-regulated in Hnf1a-null islets appears to be in line withsuch a mechanism.

Although Hnf1a might be one of the targets in p300-null/CBP-null islets, the coactivation of other transcriptionfactors could also account for the phenotypes of p300/CBP-null islets. Our data suggest that p300/CBP do not appearto have major importance in the development of d-cellsor e-cells. The lack of effect on d-cells and e-cells in thedouble-KO mice shows striking similarity to the phenotypesof Nkx2.2-null mice (40,41). Although Nkx2.2 is not knownto interact with p300/CBP, the significant overlapping be-tween the gene set of Nkx2.2 and our p300/CBP gene setssuggested that Nkx2.2 might mediate some of the pheno-types seen in the p300/CBP mutant mice. Intriguingly,Nkx2.2 is mainly known for its repressor function, sop300/CBP might be recruited by Nkx2.2 to initiate its

activator function instead (30,42). In the future, it will beinteresting to explore whether p300/CBP interact physicallywith Nkx2.2 and acetylate the H3K27 residues at Nkx2.2-associated loci, and whether the genomic occupancy ofNkx2.2 or Hnf1a in islets is affected by p300 deletion.

Both Ins1 and Ins2 mRNAs were reduced in the triallelicp300/CBP islets but not in p300-null islets or CBP-nullislets. Reduced transcriptional activities of MafA andNkx6.1, which are not known to recruit p300/CBP previ-ously, might contribute indirectly to the reduced insulingene expression seen in triallelic mice. Alternatively, p300/CBP might regulate insulin gene expression by binding toPdx1 and NeuroD1 (6,35). The acetylation of H3K27 at theIns1 promoter correlated with the dosages of p300/CBPpresent in the islets. The reduced insulin gene expressionin triallelic islets could be a consequence of less p300/CBPavailable to b-cell transcription factors, which in turn im-pairs the acetylation of H3K27 at insulin promoters. Takentogether, p300/CBP may coordinate transcriptional networks

Figure 5—Expression of p300/CBP is necessary for b-cell and a-cell development. A: Quantification of b-cell, a-cell, and d-cell areas of P7 WTand p300IsletKO mouse pancreata as the percentage of total pancreas area. n = 4–6. B: Representative immunofluorescence images of insulin,glucagon, and Ki67 in P7 WT and p300IsletKO mouse pancreata. Scale bar = 50 mm. C: Quantification of Ki67+ b-cells, a-cells, and all pancreaticcells in P7 WT and p300IsletKO mouse pancreata as the percentage of total b-cells, a-cells, and total pancreatic cells. n = 8. D: Quantification ofb-cell, a-cell, d-cell, e-cell, and chromogranin A–positive pan-endocrine cell areas of P0 WT and p300/CBP double-KO (dKO) mouse pancreataas the percentage of total pancreas area. n = 3. E: Representative immunofluorescence images of insulin (Ins), glucagon (Gcg), somatostatin(Sst), ghrelin (Ghrl), chromogranin A (ChrA), and DAPI in P0 WT and p300/CBP double-KO mouse pancreata. Scale bar = 50 mm. Student t testfor A, C, and D. *P , 0.05; **P , 0.01.

diabetes.diabetesjournals.org Wong and Associates 419

Figure 6—Loss of p300/CBP impairs coactivation of Hnf1a through reduced H3K27 acetylation. A: Venn diagram of overlapping downregulatedgenes of p300IsletKO, CBPIsletKO, and CBPHet;p300KO mouse islets compared with WT islets. B: The three Biological Process GO terms commonlyoverrepresented in the downregulated genes of p300IsletKO, CBPIsletKO, and CBPHet;p300KO mouse islets. All significantly overrepresented GOterms and their associated genes can be found in Supplementary Table 4. C: Transcription factor target analysis by Webgestalt on thedownregulated genes of p300IsletKO, CBPIsletKO, and CBPHet;p300KO mouse islets. Hnf1a was commonly overrepresented in all three genesets. D: Gene set enrichment analysis on downregulated gene sets derived from microarray or RNA-seq data of mice lacking b-cell transcriptionfactors in islets or b-cells. Random 1 and 2 were control gene lists generated randomly from the 15,999 genes in the WT reference list. E: qPCRof islet Hnf1a-associated genes in WT and CBPHet;p300KO mouse islets. n = 5–6. F: Low-input N-ChIP for H3K27Ac at Hnf1a-associated genesin WT, CBPIsletKO, and CBPHet;p300KO mouse islets. n = 3–5.G: Low-input N-ChIP for H3K27Ac at Pdx1-associated loci in in WT, CBPIsletKO, andCBPHet;p300KO mouse islets. n = 3–5.H: Representative immunofluorescence images of insulin, H3K27Ac, andH3K27me3 inWT andCBPHet;p300KO

mouse islets. Scale bar = 50mm. FDR, false discovery rate. Student t test for E. One-way ANOVA for F andG. *P, 0.05; **P, 0.01; ***P, 0.001.

420 Roles of p300/CBP in Pancreatic Islets Diabetes Volume 67, March 2018

in b-cells by coactivating various b-cell transcription fac-tors, perhaps through Hnf1a/b and/or Nkx2.2. Mutationsin many of these transcription factors are known to causemonogenic diabetes, including HNF1A, HNF1B, PDX1, andNEUROD1 (43), suggesting that p300/CBP could have rel-evancy to the underlying pathophysiology.

Overall, mice lacking p300 or CBP alone in isletsdeveloped glucose intolerance and hypoinsulinemia associ-ated with reduced islet area and insulin content. Micelacking three copies of p300/CBP in islets developed similaryet exacerbated phenotypes. Mice lacking all copies ofp300/CBP died postnatally due to their failure to establishb-cell mass. Islet genes mediated by p300/CBP overlappedsignificantly with genes downregulated in islets lacking tran-scription factors such as Hnf1a and Nkx2.2. p300/CBPexpression was required to acetylate H3K27 at the locidownregulated in Hnf1a-null islets including Slc2a2, Pklr,Hnf4a, and particularly Tmem27, which could regulate b-cellproliferation. Thus, the expression of p300/CBP family ofcoactivators in islets is critical to drive b-cell genesis and tomaintain b-cell proliferation and insulin content. In thepancreatic endocrine lineage, p300 and CBP serve as func-tionally similar yet limiting cofactors to coordinate variousislet transcription factors and maintain whole-body glucosehomeostasis.

Acknowledgments. The authors thank The Canucks for Kids ChildhoodDiabetes Laboratories at BC Children’s Hospital Research Institute (BCCHR) forinstitutional support and Dr. Jingsong Wang (BCCHR) for technical assistanceat the Imaging Core. The authors also thank Ryan Vander Werff and the Uni-versity of British Columbia (UBC) Biomedical Research Centre Sequencing Corefor support on RNA-seq experiments, Dr. Julie Brind’Amour and Dr. MatthewLorincz (UBC) for advice on low-input ChIP, and Dr. Lawryn Kasper (St. Jude Child-ren’s Research Hospital) for advice on p300 Western blotting.Funding. The salary for C.K.W. is supported by a BCCHR Graduate Student-ship, and the investigator salary for W.T.G. is supported by BCCHR IntramuralIGAP Award. This study was supported by grants to W.T.G. from the NaturalSciences and Engineering Research Council of Canada (RGPIN 402576-11) andthe Canadian Institutes of Health Research Institute of Nutrition, Metabolismand Diabetes (MOP-119595 and PJT-148695).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. C.K.W. and W.T.G. conceived the study anddesigned the experiments, generated and analyzed the data, wrote the manuscript,revised the article’s intellectual content, revised the manuscript, and approved thefinal version of the manuscript. A.K.W.-V. generated and analyzed the data, contrib-uted to the study design, revised the article’s intellectual content, revised the man-uscript, and approved the final version of the manuscript. D.S.L. helped with thecalcium imaging experiment, contributed to the study design, revised the article’sintellectual content, revised the manuscript, and approved the final version of themanuscript. P.K.B. and F.C.L. contributed to the study design, revised the article’sintellectual content, revised the manuscript, and approved the final version of themanuscript. C.K.W. and W.T.G. are the guarantors of this work and, as such, had fullaccess to all the data in the study and take responsibility for the integrity of the dataand the accuracy of the data analysis.

References1. Melloul D, Marshak S, Cerasi E. Regulation of insulin gene transcription. Dia-betologia 2002;45:309–326

2. Bedford DC, Brindle PK. Is histone acetylation the most important physiologicalfunction for CBP and p300? Aging (Albany NY) 2012;4:247–2553. Heinz S, Romanoski CE, Benner C, Glass CK. The selection and function of celltype-specific enhancers. Nat Rev Mol Cell Biol 2015;16:144–1544. Bose DA, Donahue G, Reinberg D, Shiekhattar R, Bonasio R, Berger SL. RNAbinding to CBP stimulates histone acetylation and transcription. Cell 2017;168:135–149.e225. Qiu Y, Guo M, Huang S, Stein R. Acetylation of the BETA2 transcription factor byp300-associated factor is important in insulin gene expression. J Biol Chem 2004;279:9796–98026. Sampley ML, Ozcan S. Regulation of insulin gene transcription by multiplehistone acetyltransferases. DNA Cell Biol 2012;31:8–147. Bompada P, Atac D, Luan C, et al. Histone acetylation of glucose-inducedthioredoxin-interacting protein gene expression in pancreatic islets. Int J BiochemCell Biol 2016;81(Pt A):82–918. Hussain MA, Porras DL, Rowe MH, et al. Increased pancreatic beta-cell pro-liferation mediated by CREB binding protein gene activation. Mol Cell Biol 2006;26:7747–77599. Schonhoff SE, Giel-Moloney M, Leiter AB. Neurogenin 3-expressing progenitorcells in the gastrointestinal tract differentiate into both endocrine and non-endocrinecell types. Dev Biol 2004;270:443–45410. Kasper LH, Fukuyama T, Biesen MA, et al. Conditional knockout mice revealdistinct functions for the global transcriptional coactivators CBP and p300 in T-celldevelopment. Mol Cell Biol 2006;26:789–80911. Wong CK, Botta A, Pither J, Dai C, Gibson WT, Ghosh S. A high-fat diet rich incorn oil reduces spontaneous locomotor activity and induces insulin resistancein mice. J Nutr Biochem 2015;26:319–32612. Li D-S, Yuan Y-H, Tu H-J, Liang Q-L, Dai L-J. A protocol for islet isolation frommouse pancreas. Nat Protoc 2009;4:1649–165213. Luciani DS, White SA, Widenmaier SB, et al. Bcl-2 and Bcl-xL suppress glucosesignaling in pancreatic b-cells. Diabetes 2013;62:170–18214. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform forbiological-image analysis. Nat Methods 2012;9:676–68215. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2:accurate alignment of transcriptomes in the presence of insertions, deletions andgene fusions. Genome Biol 2013;14:R3616. Leek JT, Johnson WE, Parker HS, Jaffe AE, Storey JD. The sva package forremoving batch effects and other unwanted variation in high-throughput experi-ments. Bioinformatics 2012;28:882–88317. Love MI, Huber W, Anders S. Moderated estimation of fold change and dis-persion for RNA-seq data with DESeq2. Genome Biol 2014;15:55018. Hulsen T, de Vlieg J, Alkema W. BioVenn—a web application for the com-parison and visualization of biological lists using area-proportional Venn diagrams.BMC Genomics 2008;9:48819. Wang J, Duncan D, Shi Z, Zhang B. WEB-based GEne SeT AnaLysis Toolkit(WebGestalt): update 2013. Nucleic Acids Res 2013;41:W77–W83.20. Livak KJ, Schmittgen TD. Analysis of relative gene expression data usingreal-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402–40821. Kasper LH, Boussouar F, Ney PA, et al. A transcription-factor-bindingsurface of coactivator p300 is required for haematopoiesis. Nature 2002;419:738–74322. Brind’Amour J, Liu S, Hudson M, Chen C, Karimi MM, Lorincz MC. An ultra-low-input native ChIP-seq protocol for genome-wide profiling of rare cell populations.Nat Commun 2015;6:603323. Song J, Xu Y, Hu X, Choi B, Tong Q. Brain expression of Cre recombinase drivenby pancreas-specific promoters. Genesis 2010;48:628–63424. Servitja J-M, Pignatelli M, Maestro MA, et al. Hnf1alpha (MODY3) controlstissue-specific transcriptional programs and exerts opposed effects on cell growth inpancreatic islets and liver. Mol Cell Biol 2009;29:2945–295925. Gu C, Stein GH, Pan N, et al. Pancreatic beta cells require NeuroD to achieveand maintain functional maturity. Cell Metab 2010;11:298–310

diabetes.diabetesjournals.org Wong and Associates 421

26. Taylor BL, Liu F-F, Sander M. Nkx6.1 is essential for maintaining the functionalstate of pancreatic beta cells. Cell Reports 2013;4:1262–127527. Gao T, McKenna B, Li C, et al. Pdx1 maintains b cell identity and function byrepressing an a cell program. Cell Metab 2014;19:259–27128. Hang Y, Yamamoto T, Benninger RKP, et al. The MafA transcription factorbecomes essential to islet b-cells soon after birth. Diabetes 2014;63:1994–200529. Swisa A, Avrahami D, Eden N, et al. PAX6 maintains b cell identity by re-pressing genes of alternative islet cell types. J Clin Invest 2017;127:230–24330. Gutiérrez GD, Bender AS, Cirulli V, et al. Pancreatic b cell identity requirescontinual repression of non-b cell programs. J Clin Invest 2017;127:244–25931. Soutoglou E, Papafotiou G, Katrakili N, Talianidis I. Transcriptional activation byhepatocyte nuclear factor-1 requires synergism between multiple coactivator pro-teins. J Biol Chem 2000;275:12515–1252032. Akpinar P, Kuwajima S, Krützfeldt J, Stoffel M. Tmem27: a cleaved and shedplasma membrane protein that stimulates pancreatic beta cell proliferation. CellMetab 2005;2:385–39733. Dell H, Hadzopoulou-Cladaras M. CREB-binding protein is a transcriptionalcoactivator for hepatocyte nuclear factor-4 and enhances apolipoprotein gene ex-pression. J Biol Chem 1999;274:9013–902134. Ban N, Yamada Y, Someya Y, et al. Hepatocyte nuclear factor-1alpha recruitsthe transcriptional co-activator p300 on the GLUT2 gene promoter. Diabetes 2002;51:1409–1418

35. Qiu Y, Guo M, Huang S, Stein R. Insulin gene transcription is mediated byinteractions between the p300 coactivator and PDX-1, BETA2, and E47. Mol Cell Biol2002;22:412–42036. Pontoglio M, Sreenan S, Roe M, et al. Defective insulin secretion in hepatocytenuclear factor 1alpha-deficient mice. J Clin Invest 1998;101:2215–222237. Haumaitre C, Barbacci E, Jenny M, Ott MO, Gradwohl G, Cereghini S. Lack of TCF2/vHNF1 in mice leads to pancreas agenesis. Proc Natl Acad Sci USA 2005;102:1490–149538. Bramswig NC, Everett LJ, Schug J, et al. Epigenomic plasticity enables humanpancreatic a to b cell reprogramming. J Clin Invest 2013;123:1275–128439. Barbacci E, Chalkiadaki A, Masdeu C, et al. HNF1beta/TCF2 mutations impairtransactivation potential through altered co-regulator recruitment. Hum Mol Genet2004;13:3139–314940. Prado CL, Pugh-Bernard AE, Elghazi L, Sosa-Pineda B, Sussel L. Ghrelin cellsreplace insulin-producing beta cells in two mouse models of pancreas development.Proc Natl Acad Sci USA 2004;101:2924–292941. Mastracci TL, Wilcox CL, Arnes L, et al. Nkx2.2 and Arx genetically interact toregulate pancreatic endocrine cell development and endocrine hormone expression.Dev Biol 2011;359:1–1142. Doyle MJ, Sussel L. Nkx2.2 regulates beta-cell function in the mature islet.Diabetes 2007;56:1999–200743. Fajans SS, Bell GI, Polonsky KS. Molecular mechanisms and clinical patho-physiology of maturity-onset diabetes of the young. N Engl J Med 2001;345:971–980

422 Roles of p300/CBP in Pancreatic Islets Diabetes Volume 67, March 2018