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The P300 and CBP Transcriptional Coactivators Are Required for Beta Cell and Alpha Cell

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The P300 and CBP Transcriptional Coactivators Are Required for Beta Cell and Alpha Cell Page 1 of 46 Diabetes The p300 and CBP transcriptional coactivators are required for beta cell and alpha cell proliferation Chi Kin Wong1,2, Adam K Wade-Vallance2, Dan S Luciani2,3, Paul K Brindle4, Francis C Lynn2,3,5, William T Gibson1,2 1. Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada 2. BC Children’s Hospital Research Institute, Vancouver, BC, Canada 3. Department of Surgery, University of British Columbia, Vancouver, BC, Canada 4. St. Jude Children’s Research Hospital, Memphis, TN, USA 5. Departments of Cellular & Physiological Sciences, University of British Columbia, Vancouver, BC, Canada *Corresponding author: Chi Kin Wong, [email protected], (604)-875-2000 ext 6783 Running title: roles of p300/CBP in pancreatic islets Word counts: 4000 Number of table: 0 Number of figure: 6 Diabetes Publish Ahead of Print, published online December 7, 2017 Diabetes Page 2 of 46 Abstract p300 (EP300) and CBP (CREBBP) are transcriptional coactivators with histone acetyltransferase activity. Various beta cell transcription factors can recruit p300/CBP, and thus the coactivators could be important for beta cell function and health in vivo. We hypothesized that p300/CBP contribute to the development and proper function of pancreatic islets. To test this, we bred and studied mice lacking p300/CBP in their islets. Mice lacking either p300 or CBP in islets developed glucose intolerance attributable to impaired insulin secretion, together with reduced alpha and beta cell area and islet insulin content. These phenotypes were exacerbated in mice with only a single copy of p300 or CBP expressed in islets. Removing p300 in pancreatic endocrine progenitors impaired proliferation of neonatal alpha and beta cells. Mice lacking all four copies of p300/CBP in pancreatic endocrine progenitors failed to establish alpha and beta cell mass postnatally. Transcriptomic analyses revealed significant overlaps between p300/CBP- downregulated genes and genes downregulated in Hnf1α-null islets and Nkx2.2-null islets, among others. Furthermore, p300/CBP are important for the acetylation of H3K27 at loci downregulated in Hnf1α-null islets. We conclude that p300 and CBP are limiting cofactors for islet development, and hence for postnatal glucose homeostasis, with some functional redundancy. Introduction 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 transcriptional complex at the insulin promoters and enhancers to activate insulin expression synergistically in beta cells (1). These transcription factors also recruit co-regulators Page 3 of 46 Diabetes to fine-tune gene expression. p300 and CBP (p300/CBP) are transcription coactivators that share more than 60% protein sequence identity and exhibit highly similar function. These coactivators acetylate lysine residues on histones to modulate chromatin structure or function, and lysine residues on non-histone proteins to modulate their activities (2). While p300/CBP can acetylate most histone proteins, they are absolutely essential for acetylating histone H3 lysine 27 (4). 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 beta cell functions in vitro. For instance, p300/CBP coactivate insulin gene expression in vitro by binding synergistically to Pdx1 and NeuroD1/E47 (5). siRNA knock down 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 which render CBP unresponsive to insulin-triggered phosphorylation, had increased islet mass but relatively normal beta 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 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 mice to study the roles of these coactivators in pancreatic islets in vivo. Research Design and Methods Diabetes Page 4 of 46 Animals All procedures were approved by the University of British Columbia Animal Care Committee. Mice housed at BC Children’s Hospital Research Institute were under 12-hour day-night cycle with ad libitum access to standard chow (Teklad 2918; Envigo, UK) and water. Neurog3-Cre mice were obtained from Dr. Francis Lynn (9). Ep300fl/fl mice were obtained from Dr. Paul Brindle (10). Crebbpfl/fl mice were purchased from The Jackson Laboratory (ME, USA) (13). All mice were kept on C57/BL6J background. Cre-negative littermates from each breeding setup were used as wildtype (WT) control. Timed matings were used to study embryonic day 15.5 (E15.5) and day 18.5 (E18.5), postnatal day 0 (P0) and day 7 (P7) mice; the morning when a vaginal plug was found on the dams was designated as E0.5. Metabolic phenotyping For glucose tolerance test, mice were fasted for five hours and then injected intraperitoneally with 2 g/kg glucose. For insulin tolerance test, mice were injected with 0.7 U/kg Humulin R (Eli Lily, IN, USA). Blood was sampled from mouse tails and blood glucose levels were assessed using a Onetouch UltraMini glucometer (Johnson & Johnson, NJ, USA). For plasma insulin measurement, mice were fasted for five hours and blood was sampled from the saphenous veins using heparinized capillary tubes before and after glucose injection. Heparinized blood was centrifuged at 2,000 g for 15 minutes at 4oC to separate plasma. Body composition analysis and metabolic cage experiments were performed as previously described (11). Analyte measurement Page 5 of 46 Diabetes Insulin was quantified using STELLUX Chemiluminescent Immunoassays (ALPCO, NH, USA). Glucagon was quantified using Glucagon ELISA (Mercodia, Sweden). Somatostatin was quantified using Somatostatin EIA Kit (Phoenix Pharmaceuticals, CA, USA). Total GLP1 was quantified using Multi Species GLP-1 Total ELISA (Merck Millipore, MA, USA). Ex vivo islet assays Mouse pancreatic islets were isolated as described previously (12). For glucose-stimulated insulin secretion assay, overnight recovered islets were incubated in Krebs-Ringer buffer (KRB) containing 2.8 mM glucose for 1 hour at 37°C. After the pre-incubation, 30 islets were incubated in KRB containing either 2.8 mM glucose, 16.7 mM glucose or 2.8 mM glucose with 30 mM KCl for 1 hour at 37°C. Supernatants were collected for insulin measurement. Fura-2 calcium imaging was performed as described before (13). Perifusion assays were performed on a Biorep Perifusion system per manufacturer’s instruction using 100 islets per chamber (Biorep, FL, USA). Immunofluorescence staining Adult pancreata were fixed in 10% formalin for 24 hours at 4oC, dehydrated, 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 4oC Diabetes Page 6 of 46 followed by incubation with secondary antibodies. Primary antibodies used include rabbit anti- p300 (N15 + C-20 1:1, 1/50; Santa Cruz, TX, USA), rabbit anti-CBP (1/200; CST, MA, USA), guinea pig anti-insulin (1/200; Abcam, UK), mouse anti-glucagon (1/1000; Abcam), rabbit anti- somatostatin (1/400; Abcam), goat anti-somatostatin (1/200; Santa Cruz), goat anti-ghrelin (1/100; Santa Cruz), rabbit anti-Ki67 (1/200; CST), rabbit anti-chromogranin A (1/200; Abcam), goat anti-chromogranin A (1/200; Santa Cruz), mouse anti-Ngn3 (1/50; DSHB, IA, USA), rabbit anti-H3K27Ac (1/200; CST) and rabbit anti-H3K27me3 (1/200; CST). The TUNEL assays were performed with the In Situ Cell Death Detection Kit (Sigma-Aldrich, MO, USA). Representative images of individual islets were taken on a SP5 confocal microscope (Leica, Germany). Images of whole pancreas sections were tiled on a BX61 fluorescence microscope (Olympus, 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, total number of cells per population were counted and normalized to total DAPI count per section. Transcriptomic analyses Total RNA was extracted from islets using TRIzol (ThemoFisher, MA, USA) and RNeasy Micro Kit (QIAgen, Germany). For RNA-seq, six WT, three p300IsletKO, three CBPIsletKO and three CBPHet; p300KO samples were sequenced in two batches at UBC Biomedical Research Centre Sequencing Core. RNA samples with RIN higher than 8.5 as measured on Bioanalyzer 2100 (Agilent, CA, USA) were prepared into sequencing libraries on NeoPrep using TruSeq Stranded mRNA Library Prep kit (Illumina, CA, USA). Each library was sequenced to a depth of at least 20 million
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