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The P300 and CBP Transcriptional Coactivators Are Required for Β 412 Diabetes Volume 67, March 2018 The p300 and CBP Transcriptional Coactivators Are Required for b-Cell and a-Cell Proliferation Chi Kin Wong,1,2 Adam K. Wade-Vallance,2 Dan S. Luciani,2,3 Paul K. Brindle,4 Francis C. Lynn,2,3,5 and William T. Gibson1,2 Diabetes 2018;67:412–422 | https://doi.org/10.2337/db17-0237 p300 (EP300)andCBP(CREBBP)aretranscriptionalco- expression synergistically in b-cells (1). These transcription activators with histone acetyltransferase activity. Various factors also recruit coregulators to fine-tunegeneexpres- b-cell transcription factors can recruit p300/CBP, and sion. p300 and CBP (p300/CBP) are transcription coac- thus the coactivators could be important for b-cell func- tivators that share .60% protein sequence identity and tion and health in vivo. We hypothesized that p300/CBP exhibit highly similar functions. These coactivators acet- contribute to the development and proper function of pan- ylate lysine residues on histones to modulate chromatin creatic islets. To test this, we bred and studied mice lack- structure or function, and lysine residues on nonhistone ing p300/CBP in their islets. Mice lacking either p300 proteins to modulate their activities (2). Although p300/CBP or CBP in islets developed glucose intolerance attribut- can acetylate most histone proteins, they are absolutely es- able to impaired insulin secretion, together with reduced sential for acetylating histone H3 lysine 27 (3). The H3K27Ac a-andb-cell area and islet insulin content. These pheno- mark tags tissue-specific promoters and enhancers and sig- types were exacerbated in mice with only a single copy nals transcription of the tissue-specifictargetgenes(3,4). of p300 or CBP expressed in islets. Removing p300 in b pancreatic endocrine progenitors impaired proliferation p300/CBP appear to regulate important -cell functions of neonatal a-andb-cells. Mice lacking all four copies in vitro. For instance, p300/CBP coactivate insulin gene ISLET STUDIES of p300/CBP in pancreatic endocrine progenitors failed expression in vitro by binding synergistically to Pdx1 and to establish a-andb-cell mass postnatally. Transcriptomic NeuroD1/E47 (5). Small interfering RNA knockdown of analyses revealed significant overlaps between p300/ p300/CBP in INS1 cells reduced glucose-stimulated insulin CBP-downregulated genes and genes downregulated in gene expression (6). In contrast, CRISPR-Cas9–mediated Hnf1a-null islets and Nkx2.2-null islets, among others. deletion of p300 in INS1 832/13 cells induced a subtle in- Furthermore, p300/CBP are important for the acetylation crease in glucose-stimulated insulin secretion and reduced of H3K27 at loci downregulated in Hnf1a-null islets. We high glucose-mediated apoptosis (7). Mice with the S436A conclude that p300 and CBP are limiting cofactors for islet variant in both copies of CBP, a mutation that renders CBP development, and hence for postnatal glucose homeosta- unresponsive to insulin-triggered phosphorylation, had in- sis, with some functional redundancy. creased islet mass but relatively normal b-cell function (8). These data left unresolved whether p300/CBP expression in pancreatic islets is necessary for establishing glucose ho- The expression of specific transcription factors both deter- meostasis in vivo. We hypothesized that the removal of mines and maintains the identities of pancreatic endocrine p300/CBP from pancreatic endocrine progenitors would cells through activation of endocrine genes. For example, lead to postnatal glucose intolerance due to defects in islet Pdx1, MafA, and NeuroD1 form a transcriptional complex mass and function. In this study, we generated and at the insulin promoters and enhancers to activate insulin phenotyped Neurog3-Cre–driven pancreatic islet-specific 1Department of Medical Genetics, University of British Columbia, Vancouver, Corresponding author: Chi Kin Wong, [email protected]. British Columbia, Canada Received 21 February 2017 and accepted 21 November 2017. 2BC Children’s Hospital Research Institute, Vancouver, British Columbia, Canada This article contains Supplementary Data online at http://diabetes 3Department of Surgery, University of British Columbia, Vancouver, British Co- .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0237/-/DC1. lumbia, Canada 4St. Jude Children’s Research Hospital, Memphis, TN © 2017 by the American Diabetes Association. Readers may use this article as 5Department of Cellular & Physiological Sciences, University of British Columbia, long as the work is properly cited, the use is educational and not for profit, and the Vancouver, British Columbia, Canada work is not altered. More information is available at http://www.diabetesjournals .org/content/license. diabetes.diabetesjournals.org Wong and Associates 413 p300 and CBP knockout (KO) mice to study the roles of assays were performed on a Biorep Perifusion System per these coactivators in pancreatic islets in vivo. manufacturer instructions using 100 islets per chamber (Biorep Technologies, Miami Lakes, FL). RESEARCH DESIGN AND METHODS Immunofluorescence Staining fi Animals Adult pancreata were xed in 10% formalin for 24 h at 4°C, fi m All procedures were approved by the University of British dehydrated, and embedded in paraf n, and 5- m serial fi Columbia Animal Care Committee. Mice housed at BC sections were made. A total of four to ve sections, each m Children’s Hospital Research Institute were under a 12-h separated by 150 m, were obtained per adult pancreas. For light/dark cycle with ad libitum access to standard chow E15.5 and E18.5, sections were obtained from the entire m (Teklad 2918; Envigo, Huntingdon, U.K.) and water. Neurog3- pancreas and sections separated by 30 m were stained. m Cre mice were obtained from Dr. Francis Lynn, BC Children’s For P0 and P7, sections separated by 60 m were obtained. Hospital Research Institute, Vancouver, British Columbia, These sections were stained for insulin, glucagon, somato- fl fl Canada (9). Ep300 / mice were obtained from Dr. Paul statin, ghrelin, chromogranin A, and/or Ki67. Other pro- Brindle, St. Jude Children’s Research Hospital, Memphis, teins were stained using randomly chosen sections. After fl fl TN (10). Crebbp / mice were purchased from The Jackson blocking, the sections were incubated with primary anti- Laboratory (Bar Harbor, ME) (10). All mice were kept on bodies overnight at 4°C followed by incubation with second- C57BL/6J background. Cre-negative littermates from each ary antibodies. Primary antibodies used include rabbit breeding setup were used as wild-type (WT) controls. Timed anti-p300 (N-15 + C-20 1:1, 1/50; Santa Cruz Biotechnology, matings were used to study embryonic day 15.5 (E15.5), E18.5, Dallas, TX), rabbit anti-CBP (1/200; CST America, Framingham, postnatal day 0 (P0), and P7 mice; the morning when a vaginal MA), guinea pig anti-insulin (1/200; Abcam, Cambridge, plug was found on the dams was designated as E0.5. U.K.), mouse anti-glucagon (1/1,000; Abcam), rabbit anti- somatostatin (1/400; Abcam), goat anti-somatostatin (1/200; Metabolic Phenotyping Santa Cruz Biotechnology), goat anti-ghrelin (1/100; Santa For glucose tolerance test, mice were fasted for 5 h and then Cruz Biotechnology), rabbit anti-Ki67 (1/200; CST Amer- injected intraperitoneally with 2 g/kg glucose. For insulin ica), rabbit anti-chromogranin A (1/200; Abcam), goat anti- tolerance test, mice were injected with 0.7 units/kg Humulin chromogranin A (1/200; Santa Cruz Biotechnology), R (Eli Lily, Indianapolis, IN). Blood was sampled from mouse mouse anti-Ngn3 (1/50; Developmental Studies Hybridoma tails, and blood glucose levels were assessed using a OneTouch Bank at the University of Iowa, IowaCity,IA),rabbitanti- Ultra Mini Glucometer (Johnson & Johnson, New Bruns- H3K27Ac (1/200; CST America), and rabbit anti-H3K27me3 wick, NJ). For plasma insulin measurement, mice were (1/200; CST America). The TUNEL assays were performed fasted for 5 h, and blood was sampled from the saphenous with the In Situ Cell Death Detection Kit (Sigma-Aldrich, veins using heparinized capillary tubes before and after St. Louis, MO). Representative images of individual islets glucose injection. Heparinized blood was centrifuged at were taken on a SP5 Confocal Microscope (Leica, Wetzlar, 2,000g for 15 min at 4°C to separate plasma. Body compo- Germany). Images of whole pancreas sections were tiled on sition analysis and metabolic cage experiments were per- a BX61 Fluorescence Microscope (Olympus, Tokyo, Japan) formed as previously described (11). and quantified using Fiji (14). Islet endocrine area were calculated by dividing the corresponding stained area by Analyte Measurement the total pancreas area. For E15.5 studies, the total number fi Insulin was quanti ed using STELLUX Chemiluminescent of cells per population were counted and normalized to Immunoassays (ALPCO, Salem, NH). Glucagon was quanti- total DAPI count per section. fied using Glucagon ELISA (Mercodia, Uppsala, Sweden). Somatostatin was quantified using a Somatostatin EIA Transcriptomic Analyses (enzyme immunosorbent assay) Kit (Phoenix Pharmaceut- Total RNA was extracted from islets using TRIzol Reagent fi icals, Burlingame, CA). Total glucagon-like peptide 1 (GLP-1) (Thermo Fisher Scienti c, Waltham, MA) and the RNeasy was quantified using Multi Species GLP-1 Total ELISA Micro Kit (Qiagen, Hilden, Germany). For RNA sequencing IsletKO IsletKO (Merck Millipore, Burlington, MA). (RNA-seq), six WT, three p300 ,threeCBP ,and three CBPHet;p300KO samples were sequenced in two Ex Vivo Islet Assays batches at the University of British Columbia Biomedical Re- Mouse pancreatic islets were isolated as described pre- search Centre Sequencing Core. RNA samples with an RNA viously (12). For glucose-stimulated insulin secretion assay, integrity number .8.5 as measured on a model 2100 Bio- overnight recovered islets were incubated in Krebs-Ringer analyzer (Agilent Technologies, Santa Clara, CA) were pre- buffer containing 2.8 mmol/L glucose for 1 h at 37°C. After pared into sequencing libraries on NeoPrep using TruSeq the preincubation, 30 islets were incubated in Krebs-Ringer Stranded mRNA Library Prep Kit (Illumina, San Diego, CA).
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