The p300 and CBP Transcriptional Coactivators Are Required for β-Cell and α-Cell Proliferation

The expression of specific transcription factors both determines and maintains the identities of pancreatic endocrine cells through activation of endocrine genes. For example, Pdx1, MafA, and NeuroD1 form a transcriptional complex at the insulin promoters and enhancers to activate insulin expression synergistically in β-cells (1). These transcription factors also recruit coregulators to fine-tune gene expression. p300 and CBP (p300/CBP) are transcription coactivators that share >60% protein sequence identity and exhibit highly similar functions. These coactivators acetylate lysine residues on histones to modulate chromatin structure or function, and lysine residues on nonhistone proteins to modulate their activities (2). Although p300/CBP can acetylate most histone proteins, they are absolutely essential for acetylating histone H3 lysine 27 (3). The H3K27Ac mark tags tissue-specific promoters and enhancers and signals transcription of the tissue-specific target genes (3,4).

p300/CBP appear to regulate important β-cell functions in vitro. For instance, p300/CBP coactivate insulin gene expression in vitro by binding synergistically to Pdx1 and NeuroD1/E47 (5). Small interfering RNA knockdown of p300/CBP in INS1 cells reduced glucose-stimulated insulin gene expression (6). In contrast, CRISPR-Cas9–mediated deletion of p300 in INS1 832/13 cells induced a subtle increase in glucose-stimulated insulin secretion and reduced high glucose-mediated apoptosis (7). Mice with the S436A variant in both copies of CBP, a mutation that renders CBP unresponsive to insulin-triggered phosphorylation, had increased islet mass but relatively normal β-cell function (8). These data left unresolved whether p300/CBP expression in pancreatic islets is necessary for establishing glucose homeostasis in vivo. We hypothesized that the removal of p300/CBP from pancreatic endocrine progenitors would lead to postnatal glucose intolerance due to defects in islet mass and function. In this study, we generated and phenotyped Neurog3-Cre–driven pancreatic islet-specific p300 and CBP knockout (KO) mice to study the roles of these coactivators in pancreatic islets in vivo.

All procedures were approved by the University of British Columbia Animal Care Committee. Mice housed at BC Children’s Hospital Research Institute were under a 12-h light/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’s Hospital Research Institute, Vancouver, British Columbia, Canada (9). Ep300^fl/fl mice were obtained from Dr. Paul Brindle, St. Jude Children’s Research Hospital, Memphis, TN (10). Crebbp^fl/fl mice were purchased from The Jackson Laboratory (Bar Harbor, ME) (10). All mice were kept on C57BL/6J background. Cre-negative littermates from each breeding setup were used as wild-type (WT) controls. Timed matings were used to study embryonic day 15.5 (E15.5), E18.5, postnatal day 0 (P0), and P7 mice; the morning when a vaginal plug was found on the dams was designated as E0.5.

For glucose tolerance test, mice were fasted for 5 h and then injected intraperitoneally with 2 g/kg glucose. For insulin tolerance test, mice were injected with 0.7 units/kg Humulin R (Eli Lily, Indianapolis, IN). Blood was sampled from mouse tails, and blood glucose levels were assessed using a OneTouch Ultra Mini Glucometer (Johnson & Johnson, New Brunswick, NJ). For plasma insulin measurement, mice were fasted for 5 h, and blood was sampled from the saphenous veins using heparinized capillary tubes before and after glucose injection. Heparinized blood was centrifuged at 2,000g for 15 min at 4°C to separate plasma. Body composition analysis and metabolic cage experiments were performed as previously described (11).

Insulin was quantified using STELLUX Chemiluminescent Immunoassays (ALPCO, Salem, NH). Glucagon was quantified using Glucagon ELISA (Mercodia, Uppsala, Sweden). Somatostatin was quantified using a Somatostatin EIA (enzyme immunosorbent assay) Kit (Phoenix Pharmaceuticals, Burlingame, CA). Total glucagon-like peptide 1 (GLP-1) was quantified using Multi Species GLP-1 Total ELISA (Merck Millipore, Burlington, MA).

Mouse pancreatic islets were isolated as described previously (12). For glucose-stimulated insulin secretion assay, overnight recovered islets were incubated in Krebs-Ringer buffer containing 2.8 mmol/L glucose for 1 h at 37°C. After the preincubation, 30 islets were incubated in Krebs-Ringer buffer containing either 2.8, 16.7, or 2.8 mmol/L glucose with 30 mmol/L KCl for 1 h at 37°C. Supernatants were collected for insulin measurement. Fura-2 calcium imaging was performed as described previously (13). Perifusion assays were performed on a Biorep Perifusion System per manufacturer instructions using 100 islets per chamber (Biorep Technologies, Miami Lakes, FL).

Adult pancreata were fixed in 10% formalin for 24 h at 4°C, dehydrated, and embedded in paraffin, and 5-µm serial sections were made. A total of four to five sections, each separated by 150 µm, were obtained per adult pancreas. For E15.5 and E18.5, sections were obtained from the entire pancreas and sections separated by 30 µm were stained. For P0 and P7, sections separated by 60 µm were obtained. These sections were stained for insulin, glucagon, somatostatin, ghrelin, chromogranin A, and/or Ki67. Other proteins were stained using randomly chosen sections. After blocking, the sections were incubated with primary antibodies overnight at 4°C followed by incubation with secondary antibodies. Primary antibodies used include rabbit anti-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; Santa Cruz Biotechnology), rabbit anti-Ki67 (1/200; CST America), rabbit anti-chromogranin A (1/200; Abcam), goat anti-chromogranin A (1/200; Santa Cruz Biotechnology), mouse anti-Ngn3 (1/50; Developmental Studies Hybridoma Bank 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 performed with the In Situ Cell Death Detection Kit (Sigma-Aldrich, St. Louis, MO). Representative images of individual islets were taken on a SP5 Confocal Microscope (Leica, Wetzlar, Germany). Images of whole pancreas sections were tiled on a BX61 Fluorescence Microscope (Olympus, Tokyo, Japan) and quantified using Fiji (14). Islet endocrine area were calculated by dividing the corresponding stained area by the total pancreas area. For E15.5 studies, the total number of cells per population were counted and normalized to total DAPI count per section.

Total RNA was extracted from islets using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA) and the RNeasy Micro Kit (Qiagen, Hilden, Germany). For RNA sequencing (RNA-seq), six WT, three p300^IsletKO, three CBP^IsletKO, and three CBP^Het;p300^KO samples were sequenced in two batches at the University of British Columbia Biomedical Research Centre Sequencing Core. RNA samples with an RNA integrity number >8.5 as measured on a model 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) were prepared into sequencing libraries on NeoPrep using TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA). Each library was sequenced to a depth of at least 20 million paired end reads on a NextSEq 500 Sequencing System (Illumina). Quality filtered reads were aligned to reference mouse genome mm10 with TopHat2 (15). Count tables for the aligned reads were generated, and batch effects were corrected using SVA (Surrogate Variable Analysis) (16), followed by calling of differentially expressed genes using DESeq2 with default settings in R (17). Genes were defined as differentially expressed based on an adjusted P value of <0.05. The Venn diagram for overlapping downregulated genes was generated using BioVenn (18). Downregulated gene lists were uploaded to Webgestalt for gene ontology (GO) terms analysis and transcription factor target prediction (19), using a reference list of 15,999 islet genes from the WT islet transcriptome for accurate overrepresentation analyses. The lowest false discovery rate from Webgestalt was capped at 10⁻¹⁵. Manual gene set enrichment analyses were performed by applying hypergeometric test with Bonferroni correction to the overlapped genes between different gene sets. All RNA-seq data were deposited in the Gene Expression Omnibus database (GSE101537).

For quantitative PCR (qPCR), RNAs were reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) and quantified by SYBR green–based reaction using GoTaq qPCR Master Mix (Promega, Madison, WI) and primers (Supplementary Table 1) on an ABI 7500 Real-Time PCR System (Thermo Fisher Scientific). Data were normalized to 18s rRNA, and fold changes were calculated with the ∆∆CT method (20).

Islet nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific). One microgram of nuclear lysate was subject to Western 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 binding protein (1/5,000; Abcam), as described previously (21).

The low-input native chromatin immunoprecipitation (N-ChIP) protocol was carried out as previously described (22). One hundred islets were dispersed in 0.05% trypsin and flash frozen prior to lysis. Buffers were supplemented with 10 mmol/L sodium butyrate to retain acetylation signals. Cells were lysed and digested with MNase for 5 min, as validated using WT islet cells. Chromatin equivalent to 10 islets per sample was subjected to ChIP using rabbit anti-H3K27Ac or rabbit anti-H3K27me3 (both 2 µL/ChIP; CST America). ChIP’d DNA was quantified by qPCR and expressed as the percentage of input.

Data were shown as mean ± SD. Statistical significance was tested using the Student t test, one-way ANOVA, or two-way ANOVA, as appropriate, with P < 0.05 considered to be statistically significant.

We first characterized Neurog3-Cre; Ep300^fl/WT mice that did not develop glucose intolerance up to 24 weeks of age. They also appeared phenotypically identical to Cre-negative Ep300^fl/fl mice and to mice bearing the Neurog3-Cre transgene alone (data not shown). We then made Neurog3-Cre; Ep300^fl/fl mice (p300^IsletKO mice). At E15.5, p300 was effectively removed using Neurog3-Cre in up to 95% of chromogranin A–positive cells, while Ngn3-positive progenitors remained p300 positive (Supplementary Fig. 1). This is likely due to a time lag between the onset of Cre expression and the onset of recombination. Among the chromogranin A–positive cells, recombination occurred in newly formed β-cells, α-cells, δ-cells, and ε-cells (Supplementary Fig. 1, δ-cells; other cell types are not shown). In p300^IsletKO mouse pancreata, p300 was absent in islet nuclei but was expressed normally in the exocrine tissues (Fig. 1A and B). The protein levels of CBP were similar between WT and p300-null islets, indicating the absence of compensatory overexpression of a CBP paralog.

p300^IsletKO mice were glucose intolerant at 8 weeks of age but displayed normal insulin tolerance (Fig. 1C and D). Plasma insulin levels of p300^IsletKO mice were 60% lower than those of WT mice both before and after glucose injection (Fig. 1E). Theoretically, defective glucose metabolism in p300^IsletKO mice could be in part due to the Neurog3-Cre–mediated recombination outside of islets such as in the ventromedial hypothalamus and enteroendocrine cells (9,23). We found that WT and p300^IsletKO mice had similar body composition, energy expenditure, locomotor activity, and food intake (Supplementary Fig. 2A–D). Also, plasma total GLP-1 levels were normal in p300^IsletKO mice (Supplementary Fig. 2E). In the absence of observable extrapancreatic phenotypes, the glucose intolerance and hypoinsulinemia can be attributed to the recombination within pancreatic islets rather than other Neurog3-expressing tissues.

To examine how p300^IsletKO mice developed glucose intolerance and hypoinsulinemia, we first quantified islet cell area in adult mice. Although the weights of p300^IsletKO mouse pancreata were comparable to WT mice (Supplementary Fig. 3A), they showed a 25% reduction in α-cell and β-cell area (Fig. 2A). The δ-cell area was unaffected. The reduced α-cell and β-cell areas were attributable to a reduced number of islets rather than islet size (Fig. 2B and Supplementary Fig. 3B). The β-cell-to-α cell ratios were similar between WT and p300^IsletKO mouse pancreata (Supplementary Fig. 3C). Insulin content of p300-null islets was reduced by 19%, whereas glucagon content was unchanged (Fig. 2C). The somatostatin content was elevated by nearly twofold. Fasting plasma glucagon levels of p300^IsletKO mice were unaffected (Supplementary Fig. 3D). To assess the function of p300-null islet explants, we stimulated the islets with low glucose, high glucose, or KCl and measured their insulin release. Although both WT and p300-null islets responded similarly to low or high glucose, p300-null islets had increased insulin secretion and calcium response upon KCl stimulation (Fig. 2D and E). Glucagon secretion from p300-null islets under low-glucose conditions was not significantly different from that of the WT islets (Supplementary Fig. 3E). Thus, the combined defects in islet mass and islet insulin content result in deficient glucose-stimulated insulin secretion in p300^IsletKO mice.

To understand whether p300 and CBP function similarly in pancreatic islets, we generated Neurog3-Cre; Crebbp^fl/fl (CBP^IsletKO) mice. The deletion of CBP in islets was not compensated for by the overexpression of p300 (Fig. 3A). CBP^IsletKO mice developed glucose phenotypes similar to those of p300^IsletKO mice, including glucose intolerance at 8 weeks of age without insulin resistance and defective insulin release upon glucose injection (Fig. 3B–D). Unlike p300^IsletKO mice, fasting plasma insulin levels of CBP^IsletKO mice did not differ from those of controls (Fig. 3D). The pancreata of CBP^IsletKO mice had 40% less α-cell area and 30% less β-cell area, with δ-cell area unaffected (Fig. 3E). In contrast to p300-null islets, CBP-null islets had reduced glucagon content but normal somatostatin content compared with controls (Fig. 3F). Similar to p300-null islets, CBP-null islets had reduced insulin content, but their β-cell secretory function appeared to be normal (Fig. 3F and G). Overall, both p300^IsletKO and CBP^IsletKO mice exhibited reduced β-cell area and islet insulin contents, features that explain their glucose intolerance.

Since mice lacking p300 or CBP had similar phenotypes, we hypothesized that p300^IsletKO or CBP^IsletKO mice lacking an additional copy of p300 or CBP would develop more severe glucose phenotypes. To test this, we generated Neurog3-Cre; Crebbp^fl/WT; Ep300^fl/fl mice and Neurog3-Cre; Crebbp^fl/fl; Ep300^fl/WT mice (CBP^Het;p300^KO and CBP^KO;p300^Het mice, respectively). These triallelic mice developed severe glucose intolerance by 8 weeks of age with no defects in insulin tolerance (Fig. 4A and B and Supplementary Fig. 4, CBP^KO;p300^Het mice data). Unlike the biallelic mice, triallelic p300/CBP mice of either genotype failed to mount an insulin response to glucose challenge (Fig. 4C). In addition, CBP^Het;p300^KO mice had 58% less α-cell area and 45% less β-cell area (Fig. 4D). Their islets contained 72% less insulin than WT islets (Fig. 4E). These phenotypes recapitulated those seen in p300^IsletKO or CBP^IsletKO mice despite being more severe.

Since reduced α-cell and β-cell areas could be caused by defects in differentiation, proliferation, and/or apoptosis, we examined these processes throughout pancreas development in WT and p300^IsletKO mice. We first excluded apoptosis as a possible cause of islet cell loss by performing TUNEL assays on E18.5 p300^IsletKO mouse pancreata and adult p300^IsletKO mouse pancreata; apoptotic events in p300-null islets were as rare as in WT islets (data not shown). Next, the number of newly differentiated endocrine cells and Ngn3-positive endocrine progenitors were unaffected in E15.5 p300^IsletKO mouse pancreata (Supplementary Fig. 5A). At E18.5, α-cell, β-cell, and pan-endocrine cell areas were normal in p300^IsletKO mouse pancreata (Supplementary Fig. 5B). At P7, the α-cell and β-cell areas were reduced in p300^IsletKO mouse pancreata (Fig. 5A). The percentages of Ki67⁺ α-cell and β-cells in these pancreata were lower than that of WT; however, the overall percentage of Ki67⁺ cells in the pancreata was unchanged (Fig. 5B and C). This indicated that the proliferation of neonatal α-cells and β-cells was reduced in p300-null islets. Hence, the reduced α-cell and β-cell mass in the adult p300^IsletKO mouse pancreata originated after E18.5 and is attributable to impaired proliferation.

We attempted to breed for Neurog3-Cre; Crebbp^fl/fl; Ep300^fl/fl mice (p300/CBP double-KO mice), but we did not observe 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 (Supplementary Table 2). We speculated that these p300/CBP double-KO mice might die shortly after birth because of a failure to establish sufficient β-cell mass. At P0, some double-KO pups survived, but their pancreata lacked α-cells and β-cells completely (Fig. 5E and F). Surprisingly, their δ-cell and ε-cell populations were unaffected (Fig. 5E). Immunostaining showed that a few ε-cells, which are normally absent in adult mouse pancreata, persist in the biallelic and triallelic mouse pancreata (Supplementary Fig. 6A). Thus, at least one allele of p300 or CBP is necessary for normal development of α-cells and β-cells, but not for δ-cells or ε-cells.

Because p300/CBP are transcriptional coactivators, the loss of p300/CBP may reduce the expression of genes important for islet function or development. We examined gene expression by performing RNA-seq on islet mRNAs from WT, p300^IsletKO, CBP^IsletKO, and CBP^Het;p300^KO mice. We identified 761 (477 down, 284 up), 923 (513 down, 410 up), and 5,589 (2,411 down, 3,178 up) differentially expressed genes in p300-null, CBP-null, and triallelic islets relative to WT islets, respectively (Supplementary Table 3).

We focused our analyses on the downregulated genes. The aggregation of the downregulated gene sets revealed 230 downregulated genes overlapped between p300-null islets (48.2%) and CBP-null islets (44.8%) (Fig. 6A). The genes downregulated in CBP^Het;p300^KO islets overlapped with 436 (91.4%) and 437 (85.2%) of the downregulated genes from p300-null and CBP-null islets, respectively. Enrichment analyses of the Biological Process GO terms on all three sets suggested three common themes of genes downregulated by the loss of p300/CBP: lipid metabolic process, regulation of hormone levels, and ion transport (Fig. 6B and Supplementary Table 4). Transcription factor target prediction from Webgestalt showed that all three gene sets were significantly enriched for the predicted transcription factor Hnf1α (Fig. 6C and Supplementary Table 5). We also performed gene set enrichment analysis by comparing our gene sets to published downregulated genes in mouse islets lacking factors important for β-cell development and function, including Pdx1, NeuroD1, Hnf1α, Pax6, MafA, Nkx6.1, and Nkx2.2 (24–30). Our gene sets overlapped more significantly with the gene sets of Hnf1α and Nkx2.2, followed by MafA, Nkx6.1, Pdx1, and NeuroD1 (Fig. 6D and Supplementary Tables 6 and 7).

Because Hnf1α could recruit p300/CBP for coactivation (31), we further examined the genes that overlap between the Hnf1α gene set and the gene sets we had defined as downregulated genes in p300-null islets, CBP-null islets, and CBP^Het;p300^KO islets. Tmem27, a known Hnf1α-mediated regulator of β-cell proliferation (32), was reduced in all three models. Other loci downregulated in Hnf1α-null islets, including Pklr, Slc2a2, and G6pc2, were also downregulated in triallelic islets as validated by qPCR (Fig. 6E) (24). β-Cell transcription factors were not specifically downregulated in either p300-null or CBP-null islets, whereas Hnf4a, Hnf1b, and NeuroD1 were downregulated in triallelic islets (Supplementary Table 8). Insulin-processing genes were not altered in the biallelic mouse islets. Ins1 and Ins2 mRNAs were normally expressed in the biallelic mouse islets, although both were downregulated by >50% in the triallelic islets.

Because p300/CBP coactivate transcription factors in part by acetylating H3K27 at target promoters and enhancers, we hypothesized that the loss of p300/CBP would reduce H3K27 acetylation at the loci downregulated in Hnf1α-null islets. We assessed the acetylation and methylation statuses of H3K27 at various loci using low-input N-ChIP, and found that there was significantly less H3K27Ac at the promoters of G6pc2, Hnf4a, Pklr, and Tmem27 in the triallelic islets (Fig. 6F and Supplementary Fig. 6B, negative loci). Pdx1-associated genes also showed reduced H3K27Ac at their promoters and enhancers in the triallelic islets (Fig. 6G). The H3K27Ac levels at these loci were reduced in CBP-null islets, although the reduction did not reach statistical significance. These loci-specific H3K27Ac levels clearly correlated with the total dosages of p300/CBP in the cells. We confirmed an ∼60% reduction of H3K27Ac globally in the triallelic islet nuclei (Fig. 6H). Total and loci-specific H3K27me3 levels were unaffected in triallelic islets (Fig. 6H and Supplementary Fig. 6B). Overall, the reduced dosages of p300/CBP impaired coactivation of downregulated genes in Hnf1α-null islets, which we attribute to reductions in global and loci-specific H3K27Ac levels.

In this study, the loss of either p300 or CBP alone in the pancreatic islets was sufficient to perturb whole-body glucose homeostasis. Mice lacking p300 or CBP in islets developed similar β-cell phenotypes, including reduced β-cell area and insulin content. Mechanistically, p300 and CBP are known to coactivate Pdx1, NeuroD1, Hnf4α, and Hnf1α/β in vitro (33–35). Our RNA-seq data suggested that genes downregulated in Hnf1α-null islets became downregulated once the dosages of either p300 or CBP were reduced in the islets. Hnf1α/β are homeobox transcription factors that are critical for pancreas and β-cell development (36,37). In particular, impaired Hnf1α coactivation in our mouse models could attenuate β-cell proliferation through genes such as Tmem27 (32). The role of Hnf1α in α-cells remains unclear, although high levels of HNF1α were found in FACS-sorted human α-cells, thereby implying that p300/CBP might also regulate aspects of α-cell biology, such as proliferation, through HNF1α (38). p300/CBP bind to Hnf1α/β through their transactivation domains, and coactivate their downstream targets by acetylating the histones bound to regulatory elements affiliated with these targets (39). The observed loss of H3K27Ac in triallelic islets at loci downregulated in Hnf1α-null islets appears to be in line with such a mechanism.

Although Hnf1α might be one of the targets in p300-null/CBP-null islets, the coactivation of other transcription factors could also account for the phenotypes of p300/CBP-null islets. Our data suggest that p300/CBP do not appear to have major importance in the development of δ-cells or ε-cells. The lack of effect on δ-cells and ε-cells in the double-KO mice shows striking similarity to the phenotypes of Nkx2.2-null mice (40,41). Although Nkx2.2 is not known to interact with p300/CBP, the significant overlapping between the gene set of Nkx2.2 and our p300/CBP gene sets suggested that Nkx2.2 might mediate some of the phenotypes seen in the p300/CBP mutant mice. Intriguingly, Nkx2.2 is mainly known for its repressor function, so p300/CBP might be recruited by Nkx2.2 to initiate its activator function instead (30,42). In the future, it will be interesting to explore whether p300/CBP interact physically with Nkx2.2 and acetylate the H3K27 residues at Nkx2.2-associated loci, and whether the genomic occupancy of Nkx2.2 or Hnf1α in islets is affected by p300 deletion.

Both Ins1 and Ins2 mRNAs were reduced in the triallelic p300/CBP islets but not in p300-null islets or CBP-null islets. Reduced transcriptional activities of MafA and Nkx6.1, which are not known to recruit p300/CBP previously, might contribute indirectly to the reduced insulin gene expression seen in triallelic mice. Alternatively, p300/CBP might regulate insulin gene expression by binding to Pdx1 and NeuroD1 (6,35). The acetylation of H3K27 at the Ins1 promoter correlated with the dosages of p300/CBP present in the islets. The reduced insulin gene expression in triallelic islets could be a consequence of less p300/CBP available to β-cell transcription factors, which in turn impairs the acetylation of H3K27 at insulin promoters. Taken together, p300/CBP may coordinate transcriptional networks in β-cells by coactivating various β-cell transcription factors, perhaps through Hnf1α/β and/or Nkx2.2. Mutations in many of these transcription factors are known to cause monogenic diabetes, including HNF1A, HNF1B, PDX1, and NEUROD1 (43), suggesting that p300/CBP could have relevancy to the underlying pathophysiology.

Overall, mice lacking p300 or CBP alone in islets developed glucose intolerance and hypoinsulinemia associated with reduced islet area and insulin content. Mice lacking three copies of p300/CBP in islets developed similar yet exacerbated phenotypes. Mice lacking all copies of p300/CBP died postnatally due to their failure to establish β-cell mass. Islet genes mediated by p300/CBP overlapped significantly with genes downregulated in islets lacking transcription factors such as Hnf1α and Nkx2.2. p300/CBP expression was required to acetylate H3K27 at the loci downregulated in Hnf1α-null islets including Slc2a2, Pklr, Hnf4a, and particularly Tmem27, which could regulate β-cell proliferation. Thus, the expression of p300/CBP family of coactivators in islets is critical to drive β-cell genesis and to maintain β-cell proliferation and insulin content. In the pancreatic endocrine lineage, p300 and CBP serve as functionally similar yet limiting cofactors to coordinate various islet transcription factors and maintain whole-body glucose homeostasis.

Acknowledgments. The authors thank The Canucks for Kids Childhood Diabetes Laboratories at BC Children’s Hospital Research Institute (BCCHR) for institutional support and Dr. Jingsong Wang (BCCHR) for technical assistance at the Imaging Core. The authors also thank Ryan Vander Werff and the University of British Columbia (UBC) Biomedical Research Centre Sequencing Core for support on RNA-seq experiments, Dr. Julie Brind’Amour and Dr. Matthew Lorincz (UBC) for advice on low-input ChIP, and Dr. Lawryn Kasper (St. Jude Children’s Research Hospital) for advice on p300 Western blotting.

Funding. The salary for C.K.W. is supported by a BCCHR Graduate Studentship, and the investigator salary for W.T.G. is supported by BCCHR Intramural IGAP Award. This study was supported by grants to W.T.G. from the Natural Sciences and Engineering Research Council of Canada (RGPIN 402576-11) and the Canadian Institutes of Health Research Institute of Nutrition, Metabolism and Diabetes (MOP-119595 and PJT-148695).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. C.K.W. and W.T.G. conceived the study and designed the experiments, generated and analyzed the data, wrote the manuscript, revised the article’s intellectual content, revised the manuscript, and approved the final version of the manuscript. A.K.W.-V. generated and analyzed the data, contributed to the study design, revised the article’s intellectual content, revised the manuscript, and approved the final version of the manuscript. D.S.L. helped with the calcium imaging experiment, contributed to the study design, revised the article’s intellectual content, revised the manuscript, and approved the final version of the manuscript. P.K.B. and F.C.L. contributed to the study design, revised the article’s intellectual content, revised the manuscript, and approved the final version of the manuscript. C.K.W. and W.T.G. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.