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Unraveling the molecular physiology of the β-cell: genome wide analysis of binding sites for the transcription.. Beach, Michael 2009

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UNRAVELING THE MOLECULAR PHYSIOLOGY OF THE n-CELL:GENOME WIDE ANALYSIS OF BINDING SITES FORTHE TRANSCRIPTION FACTOR PDX1byMichael BeachHonours B.Sc. Trinity Western University, 2006A thesis submitted in partial fulfillment ofthe requirements for the degree ofMASTER OF SCIENCEinThe Faculty of Graduate Studies(Interdisciplinary Oncology)The University of British Columbia(Vancouver)July 2009© Michael Beach 2009ABSTRACTThe selected expression of the genome determines distinct cell types, properties,and conditions. In the pancreatic n-cell, our knowledge of how this is regulated andmaintained is incomplete. Deciphering the molecular physiology of the 13-cell is criticalto develop improvements for expanding pools of donor islets for transplantation,the mostpromising curative option for sufferers of diabetes.Genomic regulation is controlled primarily by transcription factors, ofwhichpancreatic duodenal homeobox 1 (Pdxl) plays a critical role in both thedeveloping andmature pancreas. As such, I begin to unlock the molecular physiologyof the f3-cell byidentifying the binding sites of Pdxl in pancreatic islets on a genome-widescale throughthe use of chromatin immunoprecipitation followedby sequencing (ChIP-Seq). Thisprovides the best picture of Pdxl binding that has ever been assembled.Moreover, Iidentifr a highly co-occurring relationshipbetween Pdxl and pre-B-cell leukemiahomeobox 1 (Pbxl) in adult islets.The coupling of this data with othergenome-wide analyses will prove invaluableto discovering novel transcriptional complexesand the genes they regulate. It will alsocontribute to the creation of an islet transcriptional network,thereby greatly enhancingour knowledge of 13-cell regulation.11TABLE OF CONTENTSABSTRACT iiTABLE OF CONTENTSiiiLIST OF TABLESvLIST OF FIGURESviLIST OF ABBREVIATIONSviiACKNOWLEDGEMENTSixCHAPTER 1- INTRODUCTION1.1 Pancreas Development, Structure, and Function 11.2 Islet Structure and Function21.3 Insulin Release and Glucose Regulation31.4 Diabetes Mellitus41.5 Expanding Islet Pools and Islet Transplant 61.6 Transcription Factor Biology71.7 Key Transcription Factors of the EndocrinePancreas 81.8 Pdxl and the Endocrine Pancreas101.9 Chromatin Immunoprecipitation and Platformsfor Sequencing 12Hypothesis, Aims, and Objectives 16CHAPTER 2- MATERIALS AND METHODS2.1 Tissue Culture 172.2 Mouse Colony 172.3 Western Blotting 172.4 Islet Isolations 192.5 Chromatin Immunoprecipitation 222.6 Phenol-Chloroform Extractions 242.7 Illumina Sequencing of DNA and Peak Building 252.8 Quantitative Real Time Polymerase Chain Reaction 291112.9 Islet siRNA Transfection.292.10 Fluorescence Activated Cell Sorting 302.11 RNA Isolation and RT 302.12 Tag-Seq-Lite 312.13 Tag-Seq-Lite Library Bioinformatics 342.14 Seeded Motif Discovery 34CHAPTER 3- RESULTS3.1 Pdxl ChIP-Seq Library Construction3.1.1 Identification of ChIP Quality Antibody and Targets 353.1.2 Collection of Islet Pdxl ChIP DNA 373.2 Pdxl ChIP-Seq Library Results and Validation3.2.1 Statistics and Visualizations of Pdxl ChIP-Seq Peaks 393.2.2 Validation of the Pdxl ChIP-Seq Library 433.2.3 Validation Through siPdxl Tag-Seq Library Construction 463.2.4 KEGG Pathways of Pdxl Genes 483.3 Pdxl ChIP-Seq Library Analysis3.3.1 Pdxl and Pbxl Binding Motif Identification 503.3.2 Validation and Analysis of Pbxl Containing Peaks 52CHAPTER 4— DISCUSSION 57CONCLUSION 64REFERENCES 65APPENDIX 71CERTIFICATES 74ivLIST OF TABLESTable 1 — Summary of MODY Genes.5Table 2— Significantly Over-Represented KEGG Pathways of allGenes with a Pdxl ChIP-Seq Peak49Table 3— Significantly Over-Represented KEGG Pathways of siPdxlTag-Seq Down regulated Genes with a ChIP-Seq Peak49Table 4 — Monomer and Heterodimer Gene Categories54VLIST OF FIGURESFigure 1 — Chromatin Immunoprecipitation13Figure 2 — Islet Isolations 21Figure 3 — Illumina Flow Cell Sequencing by Synthesis 26Figure 4 — Constructing Peaks from ChIP-Seq Data 28Figure 5 — Tag-Seq-Lite Library Construction 33Figure 6—Identification of a ChIP Quality Pdxl Antibody 36Figure 7— Validating the Islet Pdxl ChIP DNA 38Figure 8— UCSC Screenshots of Pdxl ChIP-Seq at Known Sites40Figure 9 — Distribution of Pdx 1 ChIP-Seq Peaks42Figure 10— ChIP-Seq Versus ChIP-Chip & Known Binding Sites44Figure 11 — ChIP-Seq Peaks are Validated Via ChIP-qPCR45Figure 12 — Down Regulated siPdxl Tag-Seq Genes areSignificantlyRepresented in ChIP-Seq Data and Include Expected Genes 47Figure 13 — Seeded Motif Discovery of Pdxl ChIP-Seq Data ReturnsPdxl-like and Pbxl-like Motifs 51Figure 14— Pbxl has no Greater Affect on Pdxl Bindingat HeterodimerSites Compared to Monomer Sites53Figure 15 — Analysis of Heterodimer and Monomer Containing Peaks 56Figure Al — UCSC Screenshots of Interest of Pdxl ChIP-Seq Binding Sites 71Figure A2 — FACSorted siCyclo Islets72Figure A3 — FACSorted siPdxl Islets 73viLIST OF ABBREVIATIONSATP Adenosine TriPhosphateChIP Chromatin ImmunoprecipitationEDTA Ethylenediaminetetraacetic AcidEM Enrichment MaximizationERa Estrogen Receptor AlphaES cell Embryonic Stem CellFACS Fluorescence Activated Cell SortingGADEM A Genetic Algorithm Guided Formation of Spaced Dyads Coupled with anEM Algorithm for Motif DiscoveryGCK GlucokinaseGLUT Glucose TransporterGMAT Genome-wide Mapping TechniqueHBSS Ranks Balanced Salt SolutionHNF Hepatocyte Nuclear FactorIAPP Islet Amyloid PolypeptideISL1 Islet-iKD KnockdownKEGG Kyoto Encyclopedia of Genes and GenomesMAFA V-maf Musculoaponeurotic Fibrosarcoma Oncogene Homolog AMTN6 Mouse Insulinoma 6MODY Mature Onset Diabetes of the YoungviiNEUROD 1 Neurogenic Differentiation 1NGN3 Neurogenin 3NKX NK HomeoboxPAX Paired BoxPBS Phosphate Buffered SalinePBX1 Pre-B-cell-Leukemia Homeobox 1PCR Polymerase Chain ReactionPDX1 Pancreatic Duodenal Homeobox IPET Paired End DiTagPWM Position Weight MatrixRT Reverse TranscriptionSABE Serial Analysis of Binding EnrichmentSACO Serial Analysis of Chromatin OccupancySAGE Serial Analysis of Gene ExpressionSTAGE Sequence Tag Analysis of Genomic EnrichmentTB ST Tris-Buffered Saline Tween-20TE Trypsin-EDTATSS Transcriptional Start SiteUCSC University of California, Santa CruzWHO World Health OrganizationviiiACKNOWLEDGEMENTSSpecial thanks to Dr. Brad Hoffman for his mentorship and training, as well as othermembers of the Helgason lab: Bo Zavaglia and Joy Witzsche, and my supervisorycommittee: Dr. Pamela Hoodless, Dr. Cheryl Helgason, Dr. Dixie Mager, and Dr. SylviaNg. Islet isolations at the Verchere lab were performed by Galina Soukhatcheva. ChIPsat the Genome Sciences Centre were performed by BalgitKamoh. Motif Discoveryanalysis courtesy Gordon Robertson and Leping Li.ixCHAPTER 1. INTRODUCTION1.1 Pancreas Development, Structure, and FunctionGerm layer formation at gastrulation establishes the endoderm, the germ layerfrom which the pancreas develops. Subsequently, distinct morphological eventsaccompanied by specific onsets of gene expression culminate in pancreas formation. Thepoint of pancreas determination is termed the primary transition, which occurs shortlyafter the onset of FoxA2 expression in the endoderm. As the embryo begins to rotate,FoxA2 induces Pdxl expression, driving cells towards the pancreatic fate’. Dorsal andventral pancreatic buds begin to form and Nkx6. 1 and NeuroD 1 become expressed in theepithelium. Expansion of the epithelium occurs before the secondary transition, whenterminal differentiation of islet and exocrine cells occurs. At this point, insulin orexocrine genes experience a 100-fold activation, with Pdxl, Nkx6.1, and Nkx2.2becoming13-cellrestricted’. Finally, at isletogenesis, endocrine cells group into the isletsand exocrine acinars form.The role of the pancreas is twofold, as its exocrine cells produceand secretedigestive enzymes into the intestine, while endocrine cells release hormones intothebloodstream that are crucial to maintain homeostatic body metabolism.Exocrine cellscompose the majority of the pancreas, and the enzymesthey produce enter the duodenumthrough the ampulla of Vater2 (also termedmajor duodenal papilla3)where the commonbile duct and main pancreatic duct join. With apyramidal shape and basal nuclei,exocrine cells possess an abundance of rough ER andmany secretory vesicles to releasetheir digestive enzymes4. Additionally, hydrogencarbonate is also produced by the1exocrine pancreas to neutralize the hydrochloric acid produced in the stomach5.Thus,the exocrine pancreas has a critical role in nutrient digestion in the small intestine. Whilethis exocrine function of the pancreas is of utmost importance, the endocrine role of thepancreas in metabolic homeostasis through the cells grouped in its islets has been a majorresearch focus.1.2 Islet Structure and FunctionIn the pancreas, endocrine cells comprise only about two percent of thetotalpancreas mass6. Nevertheless, this relatively small population ofcells is absolutelycritical for normal metabolic maintenance. Embedded withinthe exocrine tissue,endocrine cells are found in clusters termed Islets of Langerhans. Theislet is composedof several types of endocrine cells, and while their exact percentagecontribution to eachislet is variable, general proportions are agreed upon. The majorityof the cells in isletsare f3-cells. These account for 6O-80%’8, 9of the cell mass and release the hormoneinsulin. Typically, ce-cells are the next most abundant. These cellsrelease glucagon andcomprise anywhere from 10-28% of the islet7’8, 9The remaining cell types are typicallyless abundant and are as follows: somatostatin producings-cells 2-10%’8,9, 10pancreaticpolypeptide producing PP-cells3-19%’8, 9, 10and ghrelin producing s-cells 1%’“.Being released directly into the bloodstream,these hormones target primarily the liver,muscle, and fat cells5,as they play major roles in metabolichomeostasis. Consequently,islets receive a rich arterial blood supply viaa unique capillary system that allows themto receive ten times the amount of bloodper mass compared to exocrine cells’2. Isletcapillaries are also larger and containfenestrae that increase permeability13 and aid in2insulin uptake following its release from the islet’4.As the most dominant cell type in theislet, and the source of insulin, the f3-cell plays the most significant role in metabolicmaintenance through careful regulation of blood glucose levels.1.3 Insulin Release and Glucose RegulationInsulin release has been long understood to occur in two phases’5.The first phaseis a rapid response to increasing blood glucose levels and lasts approximately 2-4 minutesbefore decreasing to a plateau at 10-15 minutes. A more gradual process, the secondphase of insulin release lasts 2-3 hours during which a steady state of insulin levels isachieved. Sensing of glucose by the -cel1 does not occur via a glucose receptor, butrather through the metabolic products of glucose that trigger a molecular responseculminating in insulin secretion5.This process begins with glucose entering the 3-cellthrough the channel protein GLUT2’6. In the cytosol, glucokinase phosphorylatesglucose’7preventing it from exiting the cell though GLUT2. Highly efficientoxidativemetabolism breaks down glucose to CO2 and H20 resulting in an increase in ATPlevelsthrough oxidative phosphorylation via the mitochondrial electron transportchain. At thispoint, signal transduction moves from metabolic to electric, as membrane-boundpotassium channels close in response to the increased levels of cytosolic ATp’8.Closureof these channels causes depolarization of the plasmamembrane, eliciting the opening ofvoltage-gated calcium channels and allowing calcium ions to flood the cytosol’9.Thisrise in internal calcium levels stimulates thecortical actin network to disband, permittinginsulin containing granules to fuse with the cell membrane andrelease insulin20.Movingthrough the circulation, insulin stimulates glucose uptake, acting primarily atstriated3muscle tissue and adipose tissue. Upon binding of insulin to the insulin receptor, theglucose transporter GLUT4 moves to the cell surface and facilitates entry of glucose intothe cell21. Metabolism of glucose produces ATP to meet the energy demands of the cell,or results in production of high potential energy storage molecules such as glycogen.1.4 Diabetes MellitusIn the absence of proper insulin controlled regulation, blood glucose levelsbecome abnormally high, indicative of the disease diabetes mellitus. In the year 2000,the WHO reported 171 million cases of diabetes worldwide with the incidence continuingto rise particularly in developed countries22.The core symptoms of diabetes includefrequent urination, increased fluid uptake due to thirst, and increased appetite. If allowedto progress untreated, severe conditions can include diabetic coma, blindness,loss oflimbs, renal failure, and death. Both hereditary and environmental factorssignificantlycontribute to the progression of diabetes.In type I diabetes, the J3-cells of the pancreas are destroyed by T-cell mediatedautoimmune attack23. While individuals remain responsive to insulin, theseverereduction in f3-cell numbers results in insufficient production of thehormone for thedemands of the body. Conversely, type II diabetes stems fromdiminished insulinsensitivity leading to insulin resistance. Central obesity is a majorrisk factor fordevelopment of type II diabetes, and for thisreason exercise is often prescribed astreatment and can restore insulin sensitivity.While environmental factors play a significant role in thedevelopment ofdiabetes, there are also major contributing geneticfactors. This has been particularly4well characterized in a third form of diabetes, MODY (mature onset diabetes of theyoung). While not all contributing genes are known, several have been well established,most of which have been termed MODY factors. MODY genes typically have anautosomal dominant mode of inheritance, and their mutation disrupts insulinproduction.Depending on the gene mutation, MODY is categorized as MODY 1 through 8.Thegenes belonging to each category are shown below:Table 1 — Summary of MODY GenesMODY 1 Hnf4aMODY 2 GckMODY3 HnflaMODY4 PdxlMODY 5 HnflbMODY 6 NeuroDiMODY 7 Kruppel like factor 11MODY 8 Bile salt dependent lipaseCompared to type I and type II diabetes, the MODYforms are extremely rare. However,regardless of the type, no form of diabetes has a cure.While the disease can be wellmanaged through careful monitoring of blood glucoseand insulin administration, this isonly therapeutic in nature. The mostlikely curative option for individuals suffering fromdiabetes is islet transplantation. As such, there is asignificant amount of research beingfocused on how to maximize islettransplant success and how to expand islet pools invitro for transplant purposes.51.5 Expanding Islet Pools and Islet TransplantIslet transplantation accounts for only a small percentage of the total transplantprocedures being performed in British Columbia. In 2008, out of 266 transplants in BC,only 15 were pancreatic islets24.Despite this being the only curative option for personssuffering from diabetes, there are two main reasons why so few transplants are beingdone: 1) graft survival in islet transplants is not long lasting, with only 33% of recipientsclaiming insulin independence after 2 years25 and 2) there is a huge shortage of availabletissue for transplant. This has motivated the majority of islet research to focuson how toimprove islet graft survival, or how to increase islet survival and proliferation in cultureand/or differentiate stem cells into insulin producing cells suitable for transplant.Bothtypes of research are of critical importancefor islet transplantation to evolve into a truecurative therapy for diabetes.The latter of these research focuses, expansion of islet pools andstem celldifferentiation, is of vital importance because currently, islets from several deceaseddonors must be harvested to perform a single transplant. Moreover, once inculture,survival of islets is poor, and proliferation of the cells does notreadily occur26.To date,there has been some success in overcoming thisbarrier, but further refinements arerequired27.Consequently, advances in expanding isolated islet populationsare invaluableto provide more tissue for transplant. Similarly, stemcell research addresses this sameproblem through the generation of f3-cells from early lineageprecursors. This could alsoaddress the problem of graft rejection given that tissuecould be differentiated directlyfrom stem cells of the patient. While such endeavourshave yielded insulin-producingcells28,they are not true 13-cells and are not as of yetsuitable for transplant use29.6Whether the goal is to enhance existing islet survival and proliferation, or produce f3-cellsfrom a stem cell antecedent, it is clear that a better understanding of the molecularphysiology of the n-cell is needed to augment these efforts. This is because a cell’sproperties are determined by the information carried in its genome, the selectedexpression of which serves to define distinct cell types and conditions30.This controlledexpression of genomic information is regulated by transcription factors. Therefore, acomprehension of transcription factor binding and networks can aid in betterunderstanding how a given cell type employs its genome to arrive at and maintain itsfinal functions.1.6 Transcription Factor BiologyTranscription factors are proteins that possess DNA binding domains allowingthem to directly bind to DNA and regulate transcription through activation andJorrepression31.These factors are significant contributors to controlled expression of thegenome, along with microRNAs30.In addition to the DNA binding domain, transcriptionfactors can also contain trans-activating domains that serve as binding sites for otherproteins acting as coregulators. This allows multiple transcription factors toassociateand form complexes for highly controlled genomic regulation. Transcriptionfactors aregrouped into families based on the structure oftheir DNA binding domain. Pdxl, forexample, is grouped in the homeodomain protein family.71.7 Key Transcription Factors of the Endocrine PancreasMost transcription factors known to have essential roles in the pancreatic 13-cellhave been identified based on their roles developmentally, or from their direct influenceon insulin regulation. In pancreatic islets, critical transcription factors include but are notlimited to: FoxA2(Hnf313),Hnf4a, Hnflcx, Hnfl13, Nkx2.2, Nkx6.l, NeuroDi, Ngn3,Pax4, Pax6, Isli, Mafa, Pbxl, and Pdxl.The importance of FoxA2 rests primarily on its developmental role as an activatorof Pdx132,whose activity is often mediated by Pbx133’.Similar to FoxA2, Hnflct35 andHnf11336are also regulators of Pdxl, in addition to themselves being MODY genes. It hasbeen suggested that these transcription factors, as well as Hnf4a, act cooperativelywithPdxl in the adult13-cellto drive expression of essential 13-cell specific genes’.While the above Hnf family members serve to both regulate and act with Pdxl,the Nkx family members are suspected targets of Pdx 1 that are also crucialtranscriptionfactors in 13-cells”‘.Knockout studies of Nkx2.2 reveal that while endocrine cellsdifferentiate normally, the 13-cells are unable to activate the insulin gene andexpressionof Nkx6.1 is also lost38. Nkx6.1 is specific to the 13-cellsof the pancreas and is essentialfor 13-cell formation. Loss of Nkx6. 1 expressionresults in pancreases showing normaldevelopment of islet cells with the exception of the mature13-cell, which is completelyabsent39.This observation has led to speculation that Nkx6.1 serves to repress genes thatconfer u-cell fate, thereby stabilizingthe 13-cell phenotype.Before the stabilization of endocrine cell type can beconferred, the overallendocrine fate must first be selected; this is accomplishedthrough Ngn3. Forcedexpression of Ngn3 in pancreatic ductal cells has beenshown to activate an endocrine8program40.Similarly, transfection of endodermal ES-cells with Ngn3 induces insulingene transcription as well as expression of other endocrine type factors41.It is believedthat this occurs as a result of Ngn3 activation of another key pancreas transcription factor,NeuroD 1.NeuroD 1 is a basic helix-loop-helix factor that binds to E-box elements of the f3-cell insulin gene promoter in a complex with Pdxl and Mafa, although it is alsoexpressed in all other endocrine cell types of the pancreas”42 Despite its ubiquitous isletexpression, it does not appear to be necessary fOr endocrine differentiation as NeuroD 1knockout mice successfully produce all islet cell types. However, upon islet formation,these same mice develop diabetes due to 13-cell apoptosis and a reduction of islet cellnumbers43.Several other transcription factors have been identified as critical to pancreasdevelopment and/or function as their altered expression gives rise to definitivephenotypes. Isli was one of the first genes identified as having a role in pancreasdevelopment. The dorsal pancreatic bud fails to develop in Isli deficient mice, and in theventral bud glucagon expressing cells areabsentW.Distinct phenotypes are also observedin Pax4 and Pax6 inactivated mice. In both cases, micedie shortly after birth of similaryet opposite causes. In Pax4 knockout mice,both f3-cells and &cells are completelyabsent while cL-cells persist. Conversely, Pax6 knockout mice show theopposite trend inendocrine cell type presence. When both Pax4 and Pax6 are inactivated, no pancreasendocrine cell types are observed45.The factors that regulate Isli, Pax4,and Pax6expression in the pancreas are not well understood.9In almost every case, the abovementioned transcription factors have beenidentified as crucial to the pancreas as a result of clear presentation of phenotypes.Inaddition, a uniting thread of a relationship with the pancreatic master regulatorPdx 1 isapparent. Therefore due to its overarching role, Pdxl represents an idealstartingcandidate to decipher the molecular physiology of the 13-cell.1.8 Pdxl and the Endocrine PancreasIn addition to the suspected relationship of Pdxl to manyother pancreas criticaltranscription factors, Pdxl was also the first gene identified to be independentlyrequiredfor pancreas development in mice and humans46’47•Its expression begins at E8.5 in thedefinitive endoderm, where it drives pancreatic fate, andmore specifically 13-celldifferentiation’. Consequently, knockout of Pdx 1 results in embryoniclethality aspancreas formation does not progress past the initialbudding stages. In the absence ofPdxl, the undifferentiated cells of the pancreas fail to expandafter dorsal and ventralbudding occurs. Therefore, the onset of Pdxlexpression at this stage is typicallyregarded as the beginning of pancreagenesis. Pdxlprotein distribution remainshomogenous until the secondary transition, whenexocrine cells down regulate Pdxlwhile endocrine cells up regulate Pdxlresulting in a 100-fold difference in expression.In the mature islet, Pdxl is restricted to13-cells and a small set of ö-cells48.In addition to its essential role in development,Pdxl also maintains vitalimportance in the adult. The most recognizablegene that Pdxl regulates is insulin. Forthis reason, Pdxl is a MODY factor.Binding of Pdxl at the insulin promoter has beenreported to occur at two distinctE-box elements upstream of the transcriptional start10site49. Here, the protein is thought to form a transcriptional complex with NeuroD 1 andMafa42. While the insulin promoter possesses potential binding sites for a varietyoftranscription factors50,the binding of Pdx 1 to these elements is not only confirmed butindispensable, as the loss of even one of the elements results in insulin deficiency.Recently, a short-range DNA looping model of Pdxl regulation at the insulin gene hasbeen proposed that results in distal enhancer regions being brought intoclose proximityto the transcriptional start site42. In this model,only a single true Pdx 1 binding siteexists, with the second binding site indirectly linked through NeuroD 1.Insulin, though arguably the most important, is not the onlycritical f3-cell generegulated by Pdxl. It has also been shown to activateGck51,Glut252,IAPP53,Mafa54,and its own promoter35.From this, it seems that Pdxlfunctions not only as a masterregulator of pancreas development, but also amaster regulator of f3-cell function in theadult. Mouse models confirm the importance of Pdxl in matureislets. Since the Pdxlknockout is embryonic lethal, our best insight intohow the absence of Pdx 1 affects theadult13-cellcomes from conditional knockout studies. Thesemice show reduced insulinsecretion as well as reduced expression of Glut2,thereby substantiating in vivo theimportance of Pdxl in adulthood55.In both the embryo as well as the adult, the transcriptionalactivity of Pdxl ismoderated, at least in part, by Pbx 1. The formationof Pdx!Pbx heterodimers has beenshown to occur in vitro, and has been hypothesizedto play a role in refining Pdxl activityin exocrine versus endocrinecell types’. Developmentally, the importance of thePdxlPbx interaction has been demonstrated throughgeneration of Pdxl mice with amutated Pbx 1 interaction domain.In these mice, the quantity and organization of11endocrine cells is severely impaired, suggesting a critical role for Pdx/Pbx complexes inexpansion of precurser cell populations33.The significant nature of this interaction seemsto continue into the mature f3-cell, as mice heterozygous for Pdxl and Pbxl mutant allelesdevelop more severe diabetes and hypoinsulinemia than single mutants of either gene34.The importance of Pdxl to both pancreas development and adult function isunquestionable. However, only a handful of Pdxl target genes, though absolutelycritical, are known. Consequently, on a genome-wide scale there is still very littleknownabout how Pdxl is operating to maintain f3-cell function.1.9 Chromatin Immunoprecipitation and Platforms for SequencingThe chromatin immunoprecipitation (ChIP) procedure is a valuable toolforidentifying transcription factor binding at target sites.Transcription factors arecrosslinked to DNA from isolated cells and the membranes lysed torelease thechromatin. Sonication pulses are used to shear the DNAinto small fragments that aresubsequently incubated with an antibody directed againstthe transcription factor ofinterest. To isolate antibody bound DNA, protein Gbeads are added which bind theantibody-transcription factor-DNA complex, allowingfor isolation, elution, and retrievalof only those DNA fragments bound bythe transcription factor of interest. A schematicof the ChIP procedure is displayed in Figure 1.Classically, ChIP-DNA has been assessed throughpolymerase chain reaction(PCR) on a site-by-site basis, which requiresprior suspicion of a site of interest towarrant testing for binding enrichment. However,advancements of array and sequencingtechnologies have made identifying large numbers ofnovel binding sites more feasible12Cells are lysed and DNA sonicatedresulting in lOO-300bp fragments7/FIProteins are cross linked to DNA byfixing cells with formaldehydeIncubate DNA withantibody directed againstprotein of interestIsolate immunoprecipitated DNAwith protein G beadsReverse cross link protein DNA complex.Precipitate DNA and use for sequencingor PCR.Figure 1 - Chromatin Immunoprecipitation.The steps of the ChIP procedurearedepicted leading to the isolationof transcription factor boundDNA to be sequenced.I-,and increasingly cost effective. Over the last several years, there has been much variationin the precise technology employed to identify DNA fragments isolated by ChIP. Initialhybridization of ChIP DNA to promoter microarrays (ChIP-Chip) has proven extremelycost effective in identifying transcription factor binding regions56’57However, thesestudies are limited insofar as they bias their results solely to promoter regions,therebyfailing to account for the majority of genomic sequence. An attempt to addressthisshortcoming was first made through the use of Sanger sequencing in ChIP-SACO58,ChIP-SABE59,ChIP-STAGE60,and GMAT6’studies. Nevertheless, these methodsproved to be extremely cost limiting, and as such failed to presentas reasonable optionsfor identification of anything other than the most noteworthy ofbinding sites. Morerecently, ChIP-PET has made use of Roche 454 parallelpyrosequencing to identifybinding sites of p5362, Oct463,Nanog63,andERcM.While these studies markedsignificant improvement over previous techniques, ChIP-PET still cannotreach a cost-effective sequencing depth necessary to scrutinize an entire genome.It has only beenwith the emergence of flow cell sequencing technologiesthat our ability to confidentlyand cost-effectively identify binding sites at agenome wide level has truly emergedthrough the ChIP-Seq method65.The use of flow cell sequencing in ChIP-Seqallows for tens of millions of DNAfragments to be sequenced in a single run on parallel lanes.Currently, Roche 454 andIllumina represent the two most commonly usedflow cell sequencers. For the purposesof ChIP-Seq, the Illuminadevice is superior to that offered by Roche 454 due to itsability to generate ten times the numberof DNA sequences at approximately one tenththe cost. These flow celltechnologies require as little as 1 Ong of input DNA for14sequencing. Comparatively, ChIP-ChIP procedures require 4-5igof material65.Moreover, as a constantly advancing technology, the cost associated with flow cellsequencing is continually lessening. The improving cost-effectiveness of ChIP-Seq isnoteworthy, as the main competing methodology, despite its aforementioned bias andlimitations, continues to be ChIP-ChIP due to its low cost. In fact, a ChIP-ChIP study ofPdxl binding in an insulinoma NIT-i cell line has been published previously56.However, with this study being cell line based in addition to encompassing theinferiorities of ChIP-ChIP as compared to ChIP-Seq, our work sought to provide a farsuperior representation of genome-wide Pdxi binding through ChIP-Seq inprimarytissue, pancreatic islets.15Hypothesis, Aims, and ObjectivesCurative options for diabetes, an increasingly prevalent worldwide diseasecharacterized by an inability to regulate blood glucose levels, find the most substantialpromise in islet transplant. A major limitation to islet transplantation is the scarcity oftissue, a shortcoming that can be addressed if islet pools can be either expanded orderived from stem cell precursors. To manipulate these cells, a much clearerunderstanding of the molecular physiology of the f3-cell is required. A cell’s propertiesare defined by the selective expression of its genome, which is controlled largely bytranscription factors, and in the f3-cell the foremost of these is Pdxl. Therefore, to beginto develop a truly in depth knowledge of themolecular workings of the n-cell, thepurpose of this work is to attempt to characterize the genome-widenature of Pdxlbinding in the pancreatic islet through the use of ChIP-Seq. Ihypothesize that Pdxl playsa major role in the 13-cell transcriptional network, that asubstantial percentage of itsbinding occurs at DNA regions distal to transcriptional start sites, and thatmuch of itsbinding is facilitated by cooperative partners.16CHAPTER 2. MATERIALS AND METHODS2.1 Tissue CultureThe mouse insulinoma adherent cell line MIN6 was maintainedin 10cm tissueculture dishes (BD Biosystems) at a minimum 40% confluency and incubated at 37°Cand 5.2% CO2 in high glucose Dulbecco’s Modified Eagles Medium (DMEM) (StemCellTechnologies) containing 10% Fetal Bovine Serum (FBS) (Invitrogen)and 1% LGlutamine (Invitrogen). Cells were passaged once per week at a confluencyof 80-100%using Trypsin-EDTA (TE) (Invitrogen) and a centrifugation speedof 1200rpm for 5minutes.2.2 Mouse ColonyC57B1/6J and ICR mice were maintained in theAnimal Resource Centre at theBC Cancer Research Centre in Vancouver accordingto the guidelines of the CanadianCouncil on Animal Care and protocols approved by theAnimal Care Committee of UBC.2.3 Western BlottingA single well of a 24-well plate (BD Biosystems)of adherent MIN6 cells washarvested using TE, centrifuged at 1200rpm for 5minutes to pellet cells, washed withlrnL of ice-cold 1X Phosphate Buffered Saline(PBS) (StemCell Technologies), andcentrifuged again to obtain the clean cell pellet. 1 OOjiLof Radio Immuno PrecipitationAssay (RIPA) lysis buffer (75mMNaC1, 1mM ethylenediaminetetraacetic acid[EDTA],50mM Tris-HC1 pH 7.25, 0.5% Triton X-100, ProteaseInhibitor (P1)@1/100) wasadded and the tube incubated on icefor a minimum of 10 minutes. The lysate was heated17at 96°C for 5 minutes, and placed on ice. A pre-cast polyacrylamide gel (Invitrogen) wasloaded into the running dock and 3-(N-morpholino) propanesulfonic acid sodium dodecylsulfate (MOPS-SDS) running buffer added (Invitrogen). Precision Plus Protein Ladder(BioRad) and MIN6 lysate were added to independent wells and the gel run at150V for 1hour. A transfer membrane was submerged in methanol (Sigma Aldrich) for 30seconds,removed, and submerged in NuPAGE Transfer Buffer (Invitrogen) containing 10%methanol. The gel was removed from its casting tray and the transfer apparatusassembled using the soaked transfer membrane. Protein transfer to the membranewascanied out by running at 35V for 1 hour in NuPAGETransfer Buffer. Followingtransfer, the membrane was removed and blocked with 5mL Tris Buffered SalineTween20 (TBST) containing 5% milk powder for 1 hour at 4°C.Blocking solution wasremoved and 5mL of new blocking solution containing primary Pdxl antibody (UpstateChemicon) at 1 jiL/l000!ILwas added to the membrane and incubated overnight at 4°Con a rocking platform. The next day, the membrane waswashed 3X for 10 minutes withTBST after which 5mL blocking solution containingsecondary antibody atljiL/10,000jiL was added and the membrane incubated for 1hour on a rocking platformat room temperature. Washes withTBST were done 3X for 10 minutes each, after whicha 1:1 mix of Detection Reagent 1 andDetection Reagent 2 (Amersham) were added tothe membrane which was subsequentlytaken for exposure and film (KodakChemiluminescent BioMax Light)development in a dark room.182.4 Islet IsolationsTo isolate mouse pancreatic islets, C57B1/6J (Jackson labs) and ICR mice aged 6to 8 weeks were sacrificed via CO2 asphyxiation and a midline incision made to exposethe inner abdominal and thoracic cavities. Liver lobes were folded upwards to reveal thegall bladder and common bile duct running to the duodenum. A clamp was placed at themajor duodenal papilla, the point of connection between the common bile duct and theduodenum, preventing fluid flow to the intestine and limiting it exclusively to thepancreas. Using a 26-gauge needle, 3mL of chilled collagenase (Sigma Aldrich) at1000units/mL in 1X Hanks Balanced Salt Solution (HBSS) (Invitrogen) was injectedthroughthe common bile duct to perfuse the pancreas. The swelled pancreas was scrapedawayfrom the intestine and placed in a 5OmL Falcon tube. As multiple pancreaseswerecollected, they were distributed such that each 5OmLFalcon tube contained twopancreases and an additional 6mL of collagenase solutionwas added to each tube. Thetubes were immediately placed in a 37°Cwater bath for 15-20 minutes to facilitate tissuedigestion. Next, a transfer pipette was used to mechanically disruptthe contents of eachtube until the mixture became homogenous. To stopdigestive activity, 2OmL of ice-coldlx HBSS containing 0.25% Bovine Serum Albumin(BSA) (Roche) and 0. 1M CaC12wasadded and the tubes placed on ice. Thetubes were centrifuged for 1 minute at 1120rpm,the supernatant was poured off, theremaining pellet was washed with 2OmL of HBSS,and again centrifuged at 1120rpmfor 1 minute. This wash was repeated at least threetimes, or until the supernatant appeared clear.Pellets were resuspended in 2OmL HESSand exocrine tissue was removed byfiltering the solution through a pre-wetted 70iMnylon mesh filter (Fisher Scientific). The contentsof the filter were washed with HESS19into a 10cm petri dish (BD Biosystems) and placed under a stereomicroscope where isletswere handpicked into a microcentrifuge tube using a 20ji1 pipette. Once a clean prep ofislets was obtained, a single-cell suspension was created by adding 400uL of Enzymefree Cell Dissociation Buffer (Gibco) and incubating the tube at room temperature for 12-15 minutes. During this time, islets were gently pipetted up and down every 3 minutes tofacilitate dissociation. After a single-cell suspension was acquired, cells were centrifugedat 1200rpm for 1 minute, supernatant was removed, the cell pellet washed with lmL 1XPBS, and centrifuged again at 1200rpm. The resultant cell pellet was then ready to beused for subsequent experiments. Islet isolation images are shown in Figure 2.20Figure 2 - Islet Isolations. Panels A through F show images of pancreatic islet isolation.In panel A, the major duodenal papilla is labelled marking the site of clamp placement.The bile duct is labelled in panel B, marking the location of syringe insertion seen in panelC. The perfused pancreas is clearly seen in D and magnified in E. Following digestion,washes, and filtration a clean preparation of islets is obtained through pipette picking (F).Li212.5 Chromatin ImmunoprecipitationChIP experiments were carried out in a manner similar to published previously66.MIN6 cells or a single-cell suspension of islets were collected and washed in lmL1XPBS and centrifuged at 1200rpm for 2 minutes. The cell pellet was resuspended in1360iL of 1X PBS and 381iL of 37% formaldehyde (Fisher Scientific) was added tocrosslink the cells. This fixation was carried out for 10 minutes on a rotating platform atroom temperature, after which l75iiL of 1M glycine (Invitrogen) was added and thesuspension rotated for another 5 minutes to stop the fixation. Cellswere centrifuged at4000rpm for 2 minutes, washed in lmL 1X PBS, and again centrifugedat 4000rpm for 2minutes. To lyse the cellular membrane, 5001iL of cold ChIP cellularlysis buffer (10mMTris-Ci pH8.0, 10mM NaC1, 3mM MgC12,0.5% NP-40, PT@1/100) was added to thecell pellet and the solution dounce homogenized for 10 strokes.The resulting suspensionwas then incubated on ice for at least 5 minutes and centrifuged at13,200rpm for 3minutes. The nuclear membrane was lysed to releasethe chromatin by adding lOOj.tL ofcold ChIP nuclear lysis buffer (1% SDS, 5mMEDTA, 50mM Tris-Cl pH8.0, PT@1/100) to the cell pellet and resuspending the cells by passing themthrough a 26-gaugeneedle for 5 strokes. Shearing of the resultingchromatin was accomplished throughsonication (S3000 Ultrasonic Cell Disruptor Processor,Fisher) of the solution as follows:10 minutes total sonication time, 1 minute onfollowed by 30 seconds off, in an ice waterbath at 50% output power. Undissolved debris was pelleted andremoved by centrifugingat 13,200rpm for 10 minutes andmoving the supernatant to a new tube.1/20thof thissupernatant was removed to a new tube and ChIPnuclear lysis buffer added to a finalvolume of 200jiL. To this, 81iL of SM NaC1 was addedand the tube incubated at 65°C22overnight to reverse crosslink the sample as an input control. To the 95p1 of remainingsupernatant, 42.5 jiL of ChIP nuclear lysis buffer and 7.511L of ChIP spike buffer (lOXconcentrate of ChIP dilution buffer — 0.01% SDS, 1.1% Triton X-100, 167mM NaC1,16.7m1\4 Tris-Ci pH8.0, PT@1/100) were added making the total volume 150jiL. 20jiLof Protein G agarose beads (Pierce) were then added to pre-clear the solution by mixingon a rotating platform at 4°C for 1 hour. Beads were spun down at 13,200rpmfor 30seconds and the supernatant transferred to siliconized tubes. 3ig of Pdxl (Upstate—Chemicon) antibody was added to the supernatant and for each ChIP reaction aseparatetube of 20pL of protein G beads were added to lmL ofChIP dilution buffersupplemented with lmg/mL BSA, and 0.lmg/mL salmon spermDNA (Invitrogen) toblock the beads. Both the supernatant and the beadswere incubated overnight at 4°C ona rotating platform.The next day, the beads were centrifuged at 13,200rpm for 30 seconds andthesupernatant was removed. The antibody mixture was added toan aliquot of blockedbeads and placed back on the rotating platform at4°C for 3 hours. Beads werecentrifuged at 13,200rpm for 30seconds, supematant removed, and beads washed asfollows: 5 minutes in low salt buffer (0.1% SDS, 1%Triton X-100, 2mM EDTA, 20mMTris-Ci pH8.0, 150mM NaC1), 5 minutes in highsalt buffer (0.1% SDS, 1% Triton X100, 2m1v1 EDTA, 20mM Tris-Ci pH8.0, 500mMNaC1), 5 minutes in LiC1 buffer (0.25MLiC1, 1% NP-40, 1% Deoxycholate,1mM EDTA, 10mM Tris-Ci pH8.0), and 2 washesfor 5 minutes each in TE Buffer (10mMEDTA, 10mM Tris-C1 pH8.0). Following thesewashes, 1 5OjiL of elution buffer (1% SDS,0.1 M NaHCO3)was added to the beads, thesolution transferred to a fresh tube, and incubatedat 50°C on a rotating platform for 123hour. Beads were centrifuged at 13,200rpm for 30 seconds and the supernatantcontaining eluted chromatin was transferred to a new tube. An additional 50jiL of elutionbuffer was added to the beads and they were again centrifuged and the supernatantremoved and combined with the initial 1 50iL. To reverse crosslink the eluted chromatinin the ChIP sample, 81iL of 5M NaC1 was added and the tube incubated overnightat 65°Con a rotating platform. The DNA from the input sample reverse crosslinkedfrom theprevious day was extracted via phenol-chloroform extraction. Similarly,DNA from theChIP sample was also phenol-chloroform extracted the following day.2.6 Phenol-Chloroform ExtractionsTo extract the DNA from input and ChIP samples,Buffer Saturated Phenol(Invitrogen) was combined with chloroform (Fisher Scientific)in a 1:1 ratio. Anequivalent volume of this mixture was added to the sample to be extracted andthe tubeshaken vigorously to mix. After letting stand for 5 minutes toallow phase separation tobegin, the sample was centrifuged at 13,200rpm for10 minutes. The uppermost aqueousphase was removed and transferred to a new tubewhere a 3X volume of ice-cold 100%ethanol was added and the tube let stand for 30minutes to precipitate the DNA.Following precipitation, the tube wascentrifuged at 13,200rpm for 10 minutes and thesupernatant aspirated leaving the invisibleDNA pellet. This pellet was resuspended in201iL DNase RNase free water (Invitrogen).242.7 Illumina Sequencing of ChIP DNA and Peak BuildingChromatin of 100-300bp was selected by running the sample on a 12% PAGE gel,excising all material found in that size range, and purifying using a Spin-X filter column(Costar) and ethanol precipitation by Baljit Kamoh at the Genome Sciences Centre(GSC). Subsequently, the isolated DNA was sequenced using theIllumina genomeanalyzer67 located at the GSC. Briefly, PCR amplification of the DNA was performedusing ligated adapters to the size selected fragments for use asprimers. The resultantPCR products were affixed to a flow cell where “bridge” amplificationwas employed toproduce clonal clusters of identical DNA fragments. To sequence these fragments,aprimer homologous to the ligated adapters was annealed and sequence by synthesisperformed using reversibly terminated fluorescently labelled nucleotides.Following eachcycle of nucleotide addition, the flow cell imagewas captured using fluorescencemicroscopy. At the end of the sequencing run, the combined imageswere used to makebase calls providing sequence information for the affixedfragments. The Illuminasequencing method is displayed in Figure 3.25I II II II II II IIv$/liii/Sequence by synthesisAddition of DNA polymerase,primer, and fluorescentlylabelled nucleotidesRepeat denature, anneal, and synthesisto form clusters‘:1!:[ 1117/I I I I I I II I I I I I ILaser scanning of flow cell and image captureafter each round of nucleotide addition._________Adapters ligated to DNAPCR amplification___________DNA denaturedssDNA attached to flow cellAnnealing of free DNA ends tocomplementary primers on flow cellI I II’ ‘II I I I I IHfHPFigure 3 - Illumina Flow Cell Sequencingby Synthesis.26Peaks were constructed from sequenced DNA using the computational toolFindPeaks3.168.The FindPeaks algorithm is utilized to analyze short-read sequencingexperiments to identify areas of enrichment and produce a “wig” file that can be uploadedto the UCSC genome browser website. Sequence reads are aligned to the genome andregions of protein-DNA interaction have an enriched concentration of reads compared toan islet input control background model. Sites of enrichment between the protein ofinterest and the genomic DNA are defined as peaks. A representation of the peakbuilding process is depicted in Figure 4.27Reads from antibody ChIP sampleReads from control sampleAligned read density for ChIP sample1Aligned read density for control sample7c77c1Figure 4 - Constructing Peaks from ChIP-Seq Data. Sequenced reads fromChIPDNA are aligned to the genome. Read density is compared against a controlbackground sample to determine areas of read density enrichment. Where readdensityis greater than the background control employed, a “peak” is defined.Final “peak” detennined fromaligned reads enriched abovecontrol sample282.8 ciPCRReactions were set up with the following components: 4p.L SYBRFast(AppliedBiosystems), 0.51iL ChIP DNA, ijiL primer mix at lOiiM of both forward andreverse,and 4.5jiL dH2O. Reaction plates (Applied Biosystems) were run ona 7500 Fast RealTime PCR System (Applied Biosystems) with cycle conditions of 95°Cfor 20 seconds,followed by 40 cycles of 95°C for 3 seconds and 60°Cfor 30 seconds.2.9 Islet siRNA TransfectionIslets were extracted from C57B1/6J mice and asingle cell suspension created toplate islet cells to 24-well plates at an average confluencyof 100,000 cells per well.Cells were cultured overnight at 37°C, 5.2% CO2in Royal Park Memorial Institute(RPMI) (StemCell Technologies) media containing10% FBS and 1% L-Glutamine.Pdxl and control siRNAs (Dharmacon) were preparedto 21iM solutions in 1X siRNABuffer (Dharmacon). For each well, 20tL oftargeted siRNA was combined with 5jiLsiGLO indicator (Dharmacon) and 25tL OPTI-MEMserum free media (Invitrogen). In aseparate tube, 2jiL of DharmaFECT4 transfectionreagent was combined with 481iLOPTI-MEM and tubes incubated at room temperaturefor 5 minutes. The contents ofboth tubes were combined and incubatedat room temperature for an additional 20minutes and added to each well along with freshRPMI media. Transfected cells werecultured for 48 hours and harvested forFluorescence Activated Cell Sorting (FACS).292.10 FACSIslet cells were harvested into PBS and dead cells stained with 7-aminoactinomycinD (7AAD) at 1/100. Sorting was performed on the BD FACS Vantage SEDiVa in the Teny Fox Lab Flow Cytometry Unit at the BCCRC. Cells were gated toremove 7AAD positives and doublets, while cells positive for siGLO were sorted directlyinto Trizol (Invitrogen).2.11 RNA Isolation and RTCells from FACS were placed into Trizol and a 1/5 volume of chloroform wasadded and the tube shaken vigorously. Following a2-minute incubation at roomtemperature, samples were centrifuged at 13,200rpm for 10 minutes,supematantsremoved, and RNA extracted via manufacturer’s protocol using an RNEasyKit (Qiagen).RNA Pellets were suspended in 2OjiLDNase RNase free water and a small portion usedfor subsequent reverse transcription (RT), with theremainder being used for Tag-Seq-litelibrary construction (section 2.12).RT was performed as follows. 1 jiL of islet RNA was added to1 1iL lox DNase 1reaction buffer (Invitrogen), 1 jiL Amp grade DNase 1@1U/pL (Invitrogen), and DNaseRNase free water to a final volume of lOjiL. Tubeswere incubated for 15 minutes atroom temperature and DNase 1 inactivated byaddition of 1 jiL of 25mMethylenediaminetetraacetic acid (EDTA) (Invitrogen).Following a 10 minute incubationat 65°C, 250ng of random primers (Invitrogen) andl1iL of 10mM dNTP mix (Invitrogen)were added and the mixture heated for an additional 5minutes at 65°C. After letting thetube sit on ice for 1 minute, thefollowing were added: 4j.tL 5X First Strand Buffer30(Invitrogen), lp.L 0.1M dithiothreitol (DTT) (Invitrogen), liiL RNaseOUT RecombinantRNase Inhibitor (Invitrogen), and 1 jiL SuperScript III RT@200U/tL (Invitrogen).Contents were pipetted up and down and incubated at 25°C for 5 minutes. Incubationtemperature was increased to 50°C for an additional 60 minutes after which the reactionwas inactivated by again increasing the temperature to 70°C for another 15 minutes. Theresultant cDNA was subsequently used for qPCR analysis as outlined in 2.8.2.12 Tag-Seg-liteTag-Seq-lite library construction was performed by the Genome Sciences Centreas described previously69.First strand cDNA was synthesized from 4Ongof DNAse1treated islet RNA (control or siPdx 1 treated) with Superscript III Reverse Transcriptase(Invitrogen) and amplified by 20 cycles of PCR based on SMART (SwitchingMechanism At the 5’ end of RNA Transcripts) cDNA synthesis to generate full-lengthcDNA (Clontech). Subsequently, SOOng of cDNA was digested with theanchoringenzyme N1aIII and ligated to an Illumina specific adapter containing a recognitionsite forthe type ITS tagging enzyme Mmcl aswell as sequencing and PCR primers. Afterdigestion with Mmel and SAP (Shrimp AlkalinePhosphatase) treatment todephosphorylate the DNA, a second Illumina adapter containing a2bp 3’ overhang wasligated. The resultant “tags” flanked by adapters were amplifiedvia PCR using Phusionpolymerase with the following cycling conditions: 98°Cfor 30 seconds, followed by 13cycles of 98°C for 10 seconds, 60°C for 30 seconds, and72°C for 15 seconds, and then72°C for 5 minutes. PCR products were purified byrunning samples on a 12% PAGEgel, excising the 85bp band, and purified usinga Spin-X filter column and ethanol31precipitation. Quality assessment and DNA amount were determined using an AgilentDNA 1000 series II assay (Agilent) and DNA then diluted to lOnM. DNA wassequenced using the Illumina Genome Analyzer and 1 7bp Serial Analysis of GeneExpression (SAGE) tags extracted from the resulting reads. The process of Tag-Seq-liteis depicted in Figure 5.32Poly A+ RNASMART II A Oligo‘ polyA 3’CDS PrimerFirst strand synthesis by RTpolyA3’dC tailing by RT/\///_/_//\_/\..polyA3’cccTemplate switching and extension by RTGGG//‘‘/polyA 3’cccJcDNA amplified by LD PCRwith primer foreStrandedcDNANlallIDigestion with anchoring enzyme NlaIII‘Y•\ rY\ r’Arv-\ IAAAAGTACTTTT-beadsLigate adapter A containing Mme 1 site & primer sequencesSequencing Primer Digest with MmelPrimer jCATG’’\/’)(’\[)(’NNGTAC-”“ v”-”\.Ligation of adapter B containing PCR primer sequence_________CATGGTACFigure 5 - Tag-Seq Lite Library Construction. The process of Tag-Seqis depicted.Following the final step, 13-17 cycles of PCR are performed toamplify the DNAwhich is then purified on a PAGE gel and sequenced via INumina.332.13 Tag-Seq Library BioinformaticsTags were mapped to Refseq genes using Discovery Space4.070.Tags with acount greater than 5 were included in the analysis. To account for multiple tagsmappingto the same Refseq accession, the counts for all tagsmapping to the same Refseq werecombined, providing a Refseq and an associated count. Countswere normalized basedon library size and expressed as counts per million. Normalized counts of genesweresubsequently compared between the control and Pdxl siRNAlibrary to determine whichgenes were significantly down and up regulated in thePdxl siRNA library compared tothe control.2.14 Seeded Motif DiscoverySeeded motif discovery was performed byGordon Robertson and Leping Li.GADEM71 (A Genetic Algorithm Guided Formationof Spaced Dyads Coupled with anEM Algorithm for Motif Discovery) addresseslarge sequence sets and identifies highlyprevalent motifs based on a user specified threshold.A modified version of GADEMwas used that employed an initial “seed”position weight matrix (PWM) provided by theuser. A motif is deemed significantly present if its E-value,produced by both its p-valueand the number of all possible motif-length segmentsin the search space, falls below thisthreshold.Pdxl and Pbxl binding motifs were identified fromChIP-Seq data using the seedPWMs IPF1_Q4_01, TRANSFAC M101013for Pdxl, and PBX1_02, TRANSFACM00124 for Pbxl. Threshold was established by settingthe p-value limit to 5e4,and theGADEM run provided Pdxl-like and Pbx1-like motifs.34CHAPTER 3. RESULTS3.1 Pdxl ChIP-Seq Library Construction3.1 .1 Identification of a ChIP Quality Pdx 1 Antibody and Pdx 1 TargetsThe first step in a successful ChIP experiment is to identify asuitable antibody.Therefore, to identify the best Pdxl antibodycandidate for use in ChIP, antibodiesdirected against Pdxl were purchased from Developmental StudiesHybridoma Bank,Chemicon (Upstate), and SantaCruz. The initial comparativeChIP trials were performedusing MTN6 cells and enrichment at several ChIP-ChIP identifiedPdxl targets56 wastested using qPCR. Pdxl ChIPs were performed using afully confluent 10cm plate ofcells and enrichments established based on comparisonagainst a control IgG ChIP.Figure 6a shows that while all antibodies producedenrichment of Pdxl targets, in everycase the best performance was observedusing the Chemicon antibody, followed bySantaCruz and Developmental Studies. The mosthighly enriched target, Epb4.113, wasselected as a positive control to assessthe degree of success of future ChIPs performed inislets. To ensure Chemicon Pdxl antibodyfidelity, a Western Blot was performed.Figure 6b shows that a single clear band atthe expected size of roughly 35kDa,corresponding to Pdxl protein, was observed. Therefore,based on these results, theChemicon Pdxl antibody was selectedfor use in all future experiments.35AFigure 6 - Identification of a ChIP Quality Pdxl Antibody. (A) ChIP-qPCRfold enrichments compared to lgG of Pdxl targets following ChIP with thePdxl antibodies from Developmental Studies, Santa Cruz, and Chemicon.Thehigh levels of enrichment of targets over lgG controls indicate ChIPs wereallsuccessful. For all targets the best ChIP enrichments are clearly displayedusing the Pdxl antibody from Chemicon. Maximal enrichment at the Epb4.113target identify it as a good positive control for future ChIPs. (B) Western Blot forPdxl performed with the Chemicon Pdxl antibody using MIN6 cell lysate. Presenceof a strong and clean band at the expected size of -35kDa shows the highspecificity of the Chemicon antibody.360500 -400 -300 -200 -100 -0Pdxl Dev StudiesPdxl Santa CruzPdxl ChemiconL00c).0 CTargetBPdx135kDa3.1.2 Collection of Islet Pdxl ChIP DNATo obtain primary tissue for Pdxl ChIPs, seven islet isolations were performed byGalina Soukhatcheva at the Verchere lab at the Child and Family Research Institute, froma total of fifty-six C57B1/6J mice, over the course of three months. Each islet isolationyielded a minimum of one thousand islets, which were immediately taken asfar as thestop fixation step of the ChIP protocol. To maintain ChIPprocedural consistency withother ChIP libraries, the fixed cells were delivered to the Genome SciencesCentre (GSC)and ChIPs performed by the GSC gene expression pipeline by Balgit Kamoh.Islets fromthe first three isolations were used to optimize the ChIPprotocol for the creation of asingle-cell suspension as well as ideal chromatinshearing conditions. Following thisoptimization, islets from the remaining four isolations produced chromatin thatwas of thecorrect size range of 100-300bp and showed enrichment atthe Epb4.113 positive targetfor Pdxl. Figure 7a shows the agilent size rangeprofiles of the sheared DNA from eachof the four replicates, and Figure 7b displaysqPCR enrichment for the abovementionedEpb4.113 target. Taken together, these resultsindicate that islet chromatin has beensheared sufficiently, and that Pdxl ChIPs havebeen successful. Subsequently, thechromatin from these four successful ChIPs was pooled andused for Illumina sequencingand library creation.37ABI I100 150 200Figure 7 - Validating the Islet Pdxl ChIP DNA.(A) Agilents displaying the size range profiles of thesonicated islet DNA going into each ChIP are shown.The 1 OO-300bp size ranges are labeled betweenthe black bars, confirming presence of chromatin inthe desired size range.(B) ChIP-qPCR fold enrichments of the positiveEpb4. 113 target are shown for each of the foursuccessful ChIPs performed in islets. ChIP 1enrichment was calculated against an IgG control.ChIPs 2 to 4 were compared against an input DNAcontrol to maximize DNA amount going into thePdxl ChIPs.IChIP 1 (IIgG) -ChIP 2 (/input)ChIP 3 (/input)ChIP 4 (/input)I Q’ QIq::ChIP 1, ChIP2ChIP 350ZFold Enrichment--—fl.——— -IbP]ChIP43.2 Pdxl ChIP-Seq Library Results and Validation3.2.1 Statistics and Visualization ofPdxl ChIP-Seq PeaksIn total, 7 lanes of Pdxl ChIP material was sequenced using Illumina Flow Celltechnology at the GSC, resulting in 62.1 million reads and amapping efficiency of 24%to the mm8 mouse genome assembly. Following peak buildingusing FindPeaks3.l andthe establishment of a peak height threshold of 11, the number ofPdxl peaks was 13,448.To visually assess the data, the generated “wig” file was loadedinto the UCSC genomebrowser to scan for peaks at knownPdxl binding sites. Figure 8 shows the UCSCscreenshots of several previously identified Pdxl bindingsites: Insi, Ins2, Pdxl, Gck,IAPP, and Glut2. It clearly illustrates thatPdxl ChIP-Seq peaks are located at mostexpected sites. Additionally, scanning of ChIP-Seqdata in UCSC revealed Pdxl sites atsuspected, but previously unidentified, genes includingIsil, Nkx2.2, Nkx6.1, and Pax6(Appendix). Peak to gene associations were performedin the Galaxy Genome Browser(http://main.22bx.lsu.edu/) by mapping peaks to the closest Refseqtranscriptional start site eitherup or downstream. A peak wasdefined as being associated with a gene if the closesttranscriptional start site was within 50kbof the peak. Using this method, 5560 genespossessed a Pdxl peak.39Known SitesI IPeak Height48 —PDX1 ChIPIh8-IiNS 1Known SitesI28—PDXI ChIP6— INS2 -*--I:--*-:-I___Known Site20-PDX1 ChIP3—— PDX1Known Site39—PDX1 ChIP3- ..- GCK-Known SitesII32—PDXI ChIP4—IAPP-.—IKnown SitePDX1ChIPA AGJ_TJT2Figure 8 - UCSC Screenshots of Pdxl ChIP-Seq at Known Sites. The previously Pdxlidentified binding sites at the Insi, Ins2, Pdxl, Gck, IAPP, and Glut2 genes are shown. Theknown binding site(s) are labeled as black vertical dashes, ChIP-Seq peaks are shown in blue.ChIP-Seq peaks are present at all known sites with the exception of Glut2.40Because previous ChIP-Seq data had revealed an abundance of transcriptionfactor binding sites located distally from transcriptional start sites, I examined thedistribution of Pdxl peaks to determine if a similar trend was present in this data. Thefraction of Pdxl peaks was plotted against distance to the closest transcriptional start site(Figure 9a). Compared to a random distribution of sites, Pdxl peaks were highly centredat transcriptional start sites. Using Galaxy Genome Browser, peaks were overlapped withvarious genomic regions: promoters, enhancers, exons, introns, and regions>10kb fromthe TSS. This yielded a distribution of Pdxl peaks as follows: 11% promoter(0-1kbupstream of the TSS), 8% enhancer (1-10kb upstream of the TSS), 4% exons, 27%introns, and 49% >10kb (Figure 9b). This type of distribution is consistentwith previousChIP-Seq studies72,and illustrates that an abundance of sites wereoverlooked in ChIP-Chip experiments due to their bias towards promoter and enhancer regions only.41A-40000 -20000 0 20000 400001000.100.08Ca0)CCC0.040.020Distance to TSSB— Intronic80— ExonicCa— Enhancers60— Promoters40>10kb200Pdxl CIilP-Seq PeaksFigure 9 - Distribution of Pdxl ChIP-Seq Peaks. (A) Histogram of the fractionof sites occurring relative to the position of the TSS. Pdxl ChIP-Seq peaksarecentred around TSS. (B) Distribution of peaks into gene regions. Peaks are foundin each region as follows: >10kb away - 49%, promoters - 11%, enhancers- 8%,exonic - 4%, and intronic - 27%.423.2.2 Validation of the Pdxl ChIP-Seq LibraryTo validate the Pdxl ChIP-Seq data, Pdxl peaks were compared against knownPdxl binding sites and previously published genome-wide binding data from ChIP-Chipstudies performed in NIT-i insulinoma cells56.Figure 10 shows this comparison. A 35%overlap between Pdxl ChIP-Seq data and ChIP-Chip data was observed, and 75% ofknown Pdxl binding sites are accounted for in the ChIP-Seq dataset while previousChIP-ChIP data fails to identify these well-established binding sites. A table detailingand referencing the known sites is also displayed in Figure 10.Additional validation was carried out using ChIP-qPCR to assessenrichment of35 peaks identified from the ChIP-Seq data, aswell as four negative targets. For these,four replicate Pdxi ChIPs, as well as control IgG ChIPs, were performed on isletsisolated by me from ICR mice. Islets were isolated from ten mice yielding atleast onethousand islets for each of four replicate ChIPs, and enrichment of the positive Epb4.113target was confirmed. The four ChIPs werepooled and qPCR reactions setup inquadruplicate to determine the enrichments shown in Figure ha.All tested ChIP-Seqtarget sites were enriched over the negative controls.Importantly, a positive correlationwas observed between ChIP-Seq peak height andChIP-qPCR fold enrichment (Figurehib).43Pdxl ChIP-Seq Islets13,448Keller et al. Pdxl ChIP-ChIP NIT1 Cells—i81765.O%Literature Pdxl Binding SitesGene Reference ChIP PeakInsi Al Germanetal. 1995 YesInsi A3/4 German et al. 1995 YesIns2 Al Germanetal. 1995 YesIns2 A3/4 German et al. 1995 YesPdxl Gerrish et al. 2001 YesGck Shelton et al. 1992 YesGlut2 Waeberetal. 1996No*IAPP Al Carty et al. 1997 YesIAPP A2 Carty et al. 1997 YesSst TSE1 Leonard et al. 1993No**Sst TSE2 Leonard et al. 1993No**Mafa Raum et al. 2006 YesWe do identify Pdxl binding peaks at Glut2 but not at the exactlocation outlined by Waeber.The absence of Pdxl binding peaks at Sst in our data is notunexpected as Sst is expressed in delta cells which compose aminimal % of islet mass.Figure 10 - Comparison of ChIP-Seq Data with ChIP-ChIPand Known Binding Sites. Islet Pdxl ChIP-Seq datawas compared against Pdxl ChIP-ChIP data as well asa list of known binding sites. The Venn diagram shows that 35%of the binding regions identified in the ChIP-ChIP study arealso accounted for in the ChIP-Seq data. It also displays thatthe total number of sites identified was much greater in the ChIP-Seq study (13,448) versusthe ChIP-ChIP study (817).Of known Pdxl binding sites identified from a literature survey, 75%have ChIP-Seq peaks at the exact location.Conversely, the ChIP-ChIP data fails to directly identify any ofthese sites. A summary of the known sites is provided inthe expanded table, as well as explanations for the absence of PdxlChIP-Seq peaks at those sites not identified.APdxl ChIP-Seq Peak HeightFigure 11 - ChIP-Seq Peaks are Validated Via ChIP-qPCR. (A) Pdxl ChIP-qPCRresults for Pdxl targets identified in ChIP-Seq data. 35/35 peaks were validatedcompared to 0/4 negative control targets using isolated ICR islets. qPCR reactions wereperformed in quadruplicate on pooled material from 4 independent ChIPs. (B) The lineof best fit on the scatter plot shows the positive correlation observed between ChIP-Seqpeak height and ChIP-qPCR fold enrichment.ct4)I380- nI.,nIIiFII lflmflflnn.;;__(rBC.) C)—444)4<:i1E!4<4)F- —7Targetfl•400I)y = 2.708x - 37.81R=0.493050 100 150453.2.3 Validation Through siPdxl Tag-Seq Library ConstructionTo determine genes most highly impacted by direct Pdxl binding, geneexpression libraries were created from islets treated with either control or Pdxl siRNA.Following siRNA knockdown of Pdxl or cyclophilin control, islet cells wereharvestedand sorted via FACS. Cells positive for knockdown were labelledgreen due to thepresence of siGLO and collected directly into Trizol. FACS gating andresults from thecollection are shown in the appendix. A total of 30,00O cellswere collected for eachcondition. This approximately corresponded to a 10% transfection efficiencyfor bothcollections. RNA was extracted from the sorted cellsand a portion used for RT-PCR toconfirm Pdxl knockdown (Figure 12a), while the remainderwas sent to the GSC forTag-Seq expression library construction as outlined insection 2.12. Tag mapping andcomparison of the cyclophilin and Pdxl siRNA librarieswere performed using DiscoverySpace7°and up and down regulated genes determined.655 genes were up regulated,while 488 were down regulated, in thesiPdxl library as compared to the control.Relative expression levels of known Pdx 1 positivelyregulated genes such as Ins 1, Ins2,Pdxl, Glut2, IAPP, and Gck, displayedreduced expression in the knockdown library,corroborating Pdxl knockdown was having a quantifiableeffect on its targets (Figure1 2b). Hence, gene lists were comparedagainst the Pdx 1 ChIP-Seq data to determinewhat portion possessed a Pdxl peak.Figure 12c shows that 36% of unaltered genes, 39%of up regulated genes, and 45% ofdown regulated genes had a Pdxl ChIP-Seq peakassociation.46A B1.51.0siCyclo0.8sjPdxlI::flIEflIjjL[fsiCyclo siPdxl Insi Ins2 Glut2 Pdxl IAPP GckC4.16 e-5I IGenes without a Pdxl Peak100Genes with a Pdxl PeakFigure 12 - Down Regulated siPdxl Tag-Seq Genes Are SignificantlyRepresented in ChIP-Seq Data and Include Expected Genes. (A) RT-PCRfrom RNA collected from FACSorted siCyclo and siPdxl islets confirms thatPdxl expression is decreased. (B) The relative expression levels of knownPdxl positively regulated genes reveal the expected decreased expression inthe siPdxl Tag-Seq library. (C) Unaltered, down, and up regulated genes werecompared against ChIP-Seq genes. Using Fisher’s exact test, down regulatedgenes in the siPdxl Tag-Seq library are significantly more likely to possess aPdxl ChIP-Seq peak.473.2.4 KEGG Pathways of Pdxl GenesAll genes that were associated with a Pdx 1 ChIP-Seq peak were analyzed againstall Refseq genes using ‘WebGestalt (htt1:J/bioinfovanderbiJt.eduIwebestaJtJindex.php) to determinesignificantly represented KEGG pathways. The Kyoto Encyclopedia of Genes andGenomes (KEGG) is a knowledge base for analyzing gene functions in terms of genenetworks and molecules. Significant KEGG pathways are shown in Table 2, several ofwhich are expected and critical in the 13-cell including: Insulin Signalling Pathway,MODY, and Type II Diabetes Mellitus.To elucidate the gene pathways on which Pdxl had the most impact, KEGGpathways were determined for the down regulated genes of the siPdxl Tag-Seq librarythat possessed a ChIP-Seq peak. The obtained pathways are shown in Table 3 and againinclude expected pathways such as MODY and Type II Diabetes Mellitus.Based on the validative studies performed on the Pdxl ChIP-Seq data, itwas clearthat the library was a quality representation of Pdxl binding in islets.48Table 2 - Significantly Over-Represented KEGG Pathways of all Genes witha PdxlChIP-Seq PeakKEGGPäthway Observed Expecte R Value P ValueRegulation of actm cytoskeleton 65 53.5359 1.2141 0.0395MAPK Signalling Pathway 96 72.97781.3155 0.0011Focal adhesion 61 50.4364 1.2094 0.0483Wnt signalling pathway 54 39.7292 1.35920.00577Insulin Signalling Pathway 48 37.1933 1.29060.0246MODY 14 6.1989 2.2585 0.000558VEGF Signalling Pathway 32 19.442 1.6459 0.000959Apoptosis 32 21.6961 1.47490.00786Colorectal Cancer 32 22.8232 1.40210.018Pancreatic Cancer 30 20.2873 1.4788 0,00945Type II Diabetes Mellitus 20 12.3978 1.6132 0.0107Cell Cycle 38 29.8674 1.2723 0.0515Table 3 - Significantly Over-Represented KEGG Pathways of siPdxl Tag-Seq DownRegulated Genes that have a ChIP-Seq PeakKEGG Pathway Observe: Expected R Value P ValueRegulation of actin cytoskeleton 13 1.8372 7.076 7.47e -7Cytokine-cytokine receptor interaction5 0.5846 8.5529 0.00131MODY 4 0.501 7.984 0.00497Focal adhesion 7 1.9207 3.6445 0.00641Colorectal cancer 5 1.0021 4.9895 0.00744Casignallingpathway 5 1.1691 4.2768 0.0123mTORsignallingpathway 3 0.334 8.9820.0125Dorso-ventral axis formation 3 0.4175 7.18560.0189Pancreatic cancer 4 0.9186 4.3545 0.0238DRPLA 2 0.167 11.9760.0320Type II Diabetes Mellitus 3 0.5846 5.13 170.0361Glioma 3 0.6681 4.4903 0.0469Observed - Number of genes found in dataset of interestExpected - Number of genes expected to be found based on backgroundR Value - Ratio of observed to expectedP Value - Probability of result493.3 Pdxl ChIP-Seq Library Analysis3.3.1 Pdxl and Pbxl Binding Motif IdentificationBecause transcription factors frequently bind DNA in complexes, I wanted toexamine the sequences contained under Pdxl ChIP-Seq peaks for nucleotide sequencebinding motifs to deduce possible co-regulators acting with Pdxl. Pdxl ChIP-Seq peakswere scanned for binding motifs similar to the classic Transfac Pdxl DNA binding motifas well as the Transfac Pbxl DNA binding motif to see if peaks were enriched for thesenucleotide sequences. Pbxl was selected due to its well-documented embryonic coregulatory role with Pdxl.To perform this analysis, Gordon Robertson and Leping Li used theGADEMmotif discovery tool (outlined in section 2.14) on Pdxl ChIP-Seq sequences basedon1 lbp Pdxl and l5bp Pbxl sequences from Transfac. This returned a Pdxl-like motif thatoccurred in roughly 45% of peaks (Figure 13a), and a Pbxl-like motif that occurred inroughly 43% of peaks (Figure 13b). Taken together, at least one of the identified motifswas present in 63.8% of peaks. Interestingly, thePbxl-like motif appeared to be aheterodimer comprising core Pbxl and Pdxl binding sequences. Because Pdxland PbxlTransfac motifs contained similar core base pair sequences,some sequences wereidentified by both independent motif discoveryruns. To determine which wereduplicates, a histogram of the distance between sitetypes was created (Figure 13c). Sitesseparated by a distance of—6bp (Pbxl-like relative toPdxl-like) were identified by bothtypes of motif discovery runs. Consequently,Pdx 1-like sites that had Pbx 1-like siteslocated at a distance —6bp were removed fromthe Pbxl-like list.50T’’==010,-,—I—NNTAATGNNNN +3XXXNTGATTAATXXXNNTAATGNN1’fl’4XXXNTGATTAATXXXFigure 13 - Seeded Motif Discovery of Pdxl ChIP-Seq Data Returns Pdxl -likeand Pbxl-like Motifs. (A) Using a Pdxl seed, a Pdxl-like motif (monomer) isfound in 45% of peaks. (B) Using a Pbxl seed, a Pbxl-like motif (heterodimer) isfound in 43% of peaks. Taken together, 64% of peaks contain at least one of the twosite types. (C) Relative distance of heterodimers from monomers show primarydistributions of +3, -3, and -6 base pairs. Alignments of motifs reveals that at -6bp,the same sequences were called sites by both seeded motif runs.A PDX1-Iike MONOMERTB PBX:PDX HETERODIMERCC’,CIDCC3.NNTAATGNNNN6XXXNTGATTAATXXX -Distance from Monomer (bp)513.3.2 Validation and Analysis of Pbxl Containing PeaksIn order to explore the relationship of Pdxl and Pbxl, experimental confirmationof Pbxl binding at sites identified via motif discovery was needed. The terms monomerand heterodimer were used to describe site type. Monomer sites were thoseidentifiedfrom the Pdxl based motif discovery run, and were indicative of Pdxl binding alone toDNA. Heterodimer sites were those identified by Pbx 1 based motif discovery,and wereindicative of a Pdxl and Pbxl complex binding to DNA. To confirm Pbxl bindingatpeaks containing a heterodimer site, ChIP-qPCR was performed in MTN6 cellswith anantibody directed against Pbxl (SantaCruz), and heterodimer as well as monomertargetstested for enrichment. The results shown in Figure 1 4areveal that Pbx 1 is enriched atheterodimer containing peaks while at monomer containing peaks it isnot.To determine if Pbxl is necessary for Pdxl binding at heterodimer sites, IusedsiRNA to knockdown Pbxl expression in MIN6 cellsafter which Pdxl binding wastested by ChIP. As a control, siCyclo was transfectedalongside siPbxl and Pdxl ChIPsalso performed. The results of this experiment, shownin Figure 14b, revealed that whilePdxl binding at target sites was reducedthrough knockdown of Pbxl, the degree ofbinding reduction was not greater in peaks containingheterodimer sites than peakscontaining monomer sites. Taken with the Pbxl ChIPresult, this indicates that whilePbxl is binding at heterodimer sites, its impacton Pdxl binding is no greater atheterodimers as compared to monomers.52CSQCFigure 14 - Pbxl has no greater affect on Pdxl binding at heterodimer sitescompared to monomer sites. (A) ChIP-qPCR was performed in MIN6 cells withPbxl antibody. Primers for heterodimer sites and monomer sites were tested andenrichments confirm Pbxl binding at heterodimers but absence at monomerswith the exception of the S1c7a14 site. (B) ChIP-qPCR was performed using Pdxlantibody in MIN6 cells subjected to Pbxl knockdown or a control knockdown todetermine if Pbxl was necessary for Pdxl binding at heterodimer sites.ioHnrir-,HSCnABnfl20nfTrFJT]T •rin— - — — c . — —a —C —) )1)<zzCl)‘ Heterodimer Sites Monomer Sites200150100 • L6040—20— CycloKDPbxl KDC)C)enC r- C) )—ILHeterodimer SitesLMonomer Sites53We next investigated whether the presence of a monomer or heterodimer affectedgene expression andlor gene specificity of the nearest gene. To do so, genes were placedinto the following categories:Table 4 — Monomer and Heterodimer Gene CategoriesMonomer Gene has peak(s) with Pdxl-like motif onlyDimer Gene has peak(s) with Pbxl-like motif onlyMono + Di Gene has peaks with both motifs, but motifs never occur in same peakMono : Di Gene has at least one peak where motifs co-occurFor expression analysis, an existing Tag-Seq library of gene expression constructed usingwild-type islets was used as the basis of gene expression. A gene was defined asexpressed if its count in the Tag-Seq library was greater than five. The presenceofmonomer and heterodimer sites was not found to have a significant impact onwhether agene was expressed or not (Figure 15a).Moreover, the absolute expression level of thosegenes that were expressed was also not affected (Figure 15b). An examinationof thespecificity of the genes in each category was also performed usingSAGE librariesgenerated through the Mouse Atlas of Gene Expression project(www.mouseatlas.org). Geneswere assigned a score quantifying theirspecificity to islets based on the followingformula73:Specificity = Mr x3Log(Ac)3Log(Lc)54Where Mr is the ratio of the counts of the tag in the library of interest (islet) over themean of the counts of the tag in all other libraries, Ac is the absolute countof the tag inthe library of interest, and Lc is the number of libraries the tag is found in. The relativespecificity scores for the genes represented in each category is depicted in Figure 15c.Interestingly, when genes possess both a monomer as well as a dimer they are far morelikely to be islet specific, likely because a far greater proportion of high specificitygenesare found in these categories (Figure 15d), where highspecificity is defined as a scoregreater than 2, moderate a score between 0.2 and 2, and low a score less than0.2.55A B0,VC.,C.)C)VC.,C)C)C0Figure 15 - Analysis of Heterodimer and Monomer Containing Peaks. Expression andspecificity analysis was performed comparing genes with varying types of monomer andheterodimer site distributions. Site type was not seen to have an effect on whetheror not agene was expressed (A), or on its relative level of expression (B). However,genes thatcontained both a monomer and a heterodimer site were significantly more likely tobe isletspecific (C) due to a greater percentage of high specificity genes belonging to this group (D).100ExpressedUnexpressed10000001ooooTI T T T.100.VIC) —z -. .- .----0C.,10.E +0Z 0 0DNo Site— Monomer— Dimer— Mono+Di— Mono:Di*C*10000‘°°TTTTT0.10.01Cl) EE +0Z 0 056CHAPTER 4. DISCUSSIONThe aim of this study was to identify the genome-wide binding of Pdxl inpancreatic islets. This entire body of work was dependent on the first step of identifyinga ChIP grade Pdxl antibody. The reason for this was twofold; first, the ChIP-Seqprocedure requires isolation of sufficient amounts of DNA for sequencing to besuccessful, and second, all isolated DNA is used for sequencing. An antibody that is usedfor ChIP-Seq purposes must therefore bind its target protein with high affinity to providesufficient DNA, and must also be highly specific for only the target protein of interest sothat sequenced DNA is a reliable representation of regions bound by the transcriptionfactor. These criteria were fulfilled by the Pdxl antibody purchased from Chemicon(Figure 6), and was largely expected given that this antibody had also been used for56ChIP-ChIP experimentsOnce constructed, quality checks of the Pdxl ChIP-Seq library were conductedusing several approaches. The most basic tactic, but also the most reliable, was to scanthe generated data for peaks at binding sites that had been well documentedin theliterature. The UCSC visualizations in Figure 8, as well as the table shownin Figure 10,exemplify the reliability of the library based on this approach. Of the 12known sites thatwere surveyed, only 3 did not show Pdxl peaks in our data. However, 2 of thesesiteswere for somatostatin, a gene expressed in the delta cells of the islet which make up only2-10% of islet mass. Therefore, the DNA contribution from these cell typesinto theChIPs would have been extremely small, and would not have provided enough input forthe binding site to be enriched. The other sitenot identified was for the glucosetransporter Glut2. However, the UCSC gene depiction of Glut2shown in Figure 857clearly reveals that several Pdx 1 peaks are actually present for the gene. This suggeststhat Pdxl binding may actually be occurring elsewhere in the Glut2 gene regionandperhaps not at the previously reported site52. Hence, all well-knownPdxl binding siteswere either present in the ChIP-Seq data, or if not, readily explainable.In addition tothose binding sites shown in Figure 8, peak scanning in UCSC also revealed Pdxl peaksat several distal enhancer regions of genes suspected, but notyet confirmed, to beregulated directly by Pdxl’. Most notably, a novelbinding site at the Insi gene wasidentified, as well as distal sites upstream of the Nkx2.2 and Nkx6.1 promoters. Bindingsites at the Isll and Pax6 promoters were alsoobserved, which is significant as factorsregulating these f3-cell critical transcription factors remain largelyunknown (appendixfigure Al). These observations provided confidence thatwhile the mapping efficiencyof the Pdxl library (24%) was lower than has beenpreviously reported for ChIP-Seqlibraries (FoxA2 liver— 33%72),the data is a valid representation of Pdxl binding.Furthermore, the ChIP-qPCR (Figure 11) that was performed tovalidate ChIP-Seq peaksprovides additional strong evidence that the bindingsites are real. Nevertheless,improvements in the quality of our ChIP-Seq datawould likely be possible if a greateramount of starting DNA was contributed to eachChIP replicate. This is because DNAinput amount is a major limiting factor toChIP success. With islets contributing so fewcell numbers in comparison to other studied tissuessuch as liver, cell numbers are themost significant limitation facing islet ChIPstudies.As an additional measureof quality, our ChIP-Seq data was compared directlyagainst ChIP-ChIP data previously published forPdx156.While the 35% overlap that weobserve (Figure 10) is lower than previously reportedcomparisons of ChIP-Seq and58ChIP-Chip72,there are several reasons why it is not unexpected. The previous ChIP-Chipstudy had been performed in a NIT-i cell line whereas our data was generated usingprimary tissue. In addition, only putative promoter and enhancer elements were includedin the ChIP-Chip study. This is a major caveat as it ignores major portionsof thegenome, which as evidenced in the analysis of peak distribution in gene regions (Figure9), account for a significant portion of Pdxl binding and suggest enhancerelements mayin fact be functioning further upstream of transcriptional start sitesthan previouslythought. Moreover, a glaring concern exists with the ChIP-Chip data inthat it identifiesnone of the well-known Pdxl binding sites. This calls into question the reliability ofthisprevious work given that extremely significant targets such as Ins 1,Ins2, IAPP, and Gckfail to be recognized. From all of this, it is clear that we haveconstructed a morecomprehensive, accurate, and biologically relevant documentationof genome-wide Pdxlbinding than previously shown.The generated Tag-Seq libraries ofgene expression (control and Pdxlknockdown) were meant to serve as both a validativetool for the ChIP library as well asto begin to provide insight into those genes thatare most highly influenced by alteredPdxl expression. In the analysis of these libraries, multiple tag typeshad to be combinedthat mapped to the same gene. This was done becausealthough the libraries are cDNAbased, multiple tag types can result from alternativetranscripts and errors in enzymecutting during library construction. While the altered expressionlevels of known Pdxltargets such as insulin and Glut2 demonstratethe changes one would expect fromdiminished Pdxl expression (Figure 12),genes such as IAPP and Gck (also known Pdxltargets) show more moderate changes in expressionlevels. This coincides with what has59been shown in Pdxl conditional knockout studies where the most notable changesinclude severe impairment of insulin production as well as diminishedGlut2expression55.Therefore, significantly altered genes in the siPdxl library are likely highlyresponsive to changes in Pdxl expression. Based on this, one would expect a substantialportion of these genes to be represented in the Pdxl ChIP-Seq library. Though this isobserved for the down regulated gene set compared to unaltered genes, the same cannotbe said for those genes up regulated. Moreover, though statistically significantwhencompared to unaltered genes, the down regulated genes still only show a 45% correlationwith ChIP-Seq. This is lower than desired, and suggests severalchanges in islet cell geneexpression could be based on the stresses of culture and FACSortingrather thanknockdown of Pdxl, or that the changes stem from indirect effectsof Pdxl knockdown.Additionally, while substantial knockdown of Pdxl mRNA was observed at roughly 70%reduction, the possibility exists that even low levels of protein are sufficientto maintainregulation at several of its targets, or that at 48 hours postsiRNA transfection, originalprotein levels had not had sufficient time to drop. Consequently a true knockoutlibraryof Pdxl may be necessary to holisticallyaddress its effects. Nevertheless, despite thesecaveats, the statistical significance between down regulatedversus unaltered genes andtheir correlation to Pdxl ChIP-Seq does allowfor several insights. The most obvious ofthese is that Pdxl is clearly fhnctioning most oftenas an activator. Were it havingsignificant repressive effects, one would have expected tosee a greater portion of upregulated genes with ChIP-Seq peaks; this is notthe case. Moreover, to determine inwhich pathways Pdxl was having the largest activationalrole, KEGG pathway analysisof all ChIP-Seq genes, as well as those that weredown regulated and in possession of a60Pdxl ChIP-Seq peak, was performed. Tables 2 and 3 show KEGG pathways that aresignificantly over represented correspond to signalling pathways where Pdxl is expectedto have major influence, such as MODY and Type II diabetes. These are present in bothKEGG analyses as one would anticipate given that they are the most impactful subjectsof Pdx 1. Of most interest in addition to these expected pathways, we observe that severalpathways related to cell cycle are also represented, such as those pertaining to pancreaticor colorectal cancer, apoptosis, glioma, and cell cycle itself. While not all of thesearepresent in both KEGG analyses, their varied presence between both establishes a highlyprobable involvement of Pdx 1 in aspects of 13-cell cycle.A relationship between Pdxl and Pbxl is known to exist in both the embryo aswell as the adult33’ This association has a profound role in expansion and organizationof the developing pancreas, while in the adult a more precise control ofinsulin regulationhas been reported through the Pdxl/Pbxl affiliation. Giventhe role of theseheterodimers in proliferative function during development, coupledwith the observedover representation of KEGG pathways related to cell cycle from PdxlChIP-Seq genes,the Pdxl/Pbxl adult relationship may similarly drive cell cyclerelated processes. Thissuspicion arose as a result of the unexpectedly highpercentage of Pdxl ChIP-Seq peaksthat upon motif discovery analysis showed thepresence of a Pdxl/Pbxl heterodimerbinding site (Figure 13). To begin to address this, we first examinedPbxl binding atthese sites and the dependency of Pdxl on such binding. Sincethese heterodimers havebeen reported to bind DNA with up to ten times theaffinity of Pdxl alone33,it washypothesized that reduction of Pbxlwould significantly alter Pdxl binding atheterodimer type sites while singly bound Pdx1 sites would be relatively unaffected.61Despite the confirmation of Pbxl binding at heterodimer sites and not at monomer sites(Figure 14a), the dependency of Pdxl on Pbxl was not found to be any greater atheterodimer sites as compared to monomers (Figure 1 4b). This seems to contradict thenotion that Pdxl/Pbxl binds DNA with lOx affinity at target sites. In addition,expression and specificity analysis of genes with various distributions of site type didnotreveal any changes in expressivity, while a positive correlation between specificityandpossession of multiple sites was observed (Figure 15). The most likely explanationforthese results is that since heterodimer sites still contain the core TAAT motif requiredforPdx 1-DNA binding, Pdxl is still capable of binding theseregions without dimerizationwith Pbxl. However, this does not negate the possibilitythat Pdxl binding at these sitesmay not be able to fully drive gene expressionwithout Pbxl. The increased specificity ofgenes with multiple sites finds explanation in that the greaternumber of transcriptionfactor binding events occurring for a given gene within a giventissue, are indicative ofthat gene being highly specific for that tissue. Forexample, insulin, a highly specific13-cell gene, would have the most transcription factorbinding events in f3-cells than in anyother tissue. Hence, specificity and the numberof transcription factors binding aredirectly proportional.The work surrounding Pdxl/Pbxl performed in this studyfocused on confirmingthe presence and requirement for Pbxl at heterodimersites. While the former wasshown, this work revealed that Pbxl is notessential for Pdxl binding. As a next step,expression analysis could be performedon heterodimer versus monomer regulated genesfollowing Pbxl knockdown (siRNA KD-qPCR) todetermine if without Pbxl, Pdxl62cannot drive expression at heterodimer sites. If so, Pbxl, though not essential for Pdx 1binding, would be essential for Pdx 1 activation of genes at heterodimer sites.Coming full circle, this bears significance in that the majority of the cell cyclerelated genes identified from our KEGG analyses possess Pdxl ChIP-Seqpeakscontaining heterodimer as opposed to monomer sites. Genes with Pdxl peaks known tohave roles in f3-cell proliferation include: Ccndl, Ccnd2, p15, p21, E2F1, Menl, Rb, p13,Insulin, FoxO, NFAT, Stat5, and Pdx174. Of these 13 genes, 8 have heterodimersitesassociated with them. Therefore, an analysis of the necessity for Pbxl at heterodimersites for Pdxl mediated expression would be a logicaldirection in which to take thiswork in order to extract more insight into how Pdxl may befunctioning at cell cyclerelated genes.As a genome-wide dataset, there also exists much value in couplingthis PdxlChll-Seq information with future genome-wide studies forboth other transcriptionfactors and markers of DNA methylation. Since a transcriptionfactor complex involvingNeuroDi, Mafa, and Pdxl is already known to form at theinsulin gene, ChIP-Seq studiesof NeuroDi and Mafa would proveextremely beneficial to compile with this Pdxldataset to determine genome-wide sites of transcriptionalcomplex formation in islets.Additionally, considering this binding information inthe context of DNA methylationstatus would enable us to determine functionalityof sites. Furthermore, construction ofan embryonic Pdxl library at E8.5 (theonset of Pdxl expression) would allow forcomparison of Pdxl gene regulation developmentallyon a genome-wide scale.63CONCLUSIONThe purpose of this study was to characterize Pdx 1 binding inpancreatic islets ona genome-wide scale. This was clearly accomplished byutilizing the ChIP-Seq strategyto produce the most extensive dataset of Pdxlbinding generated to date. Novel bindingsites at genes of high interest includeIns 1, Nkx2.2, Nkx6. 1 and Isi 1. Additionally, ahighly occurring relationship with Pbxl isidentified and the binding of Pbxl confirmedat suspected sites. Given the reportedrole of PdxlPbx in cell expansion and organizationin islet precurser populations, as well as the factthat Pdxl ChIP-Seq and Tag-Seq genesshow significant representation in cellcycle pathways, future investigation into thePdxlPbx adult role in proliferation is warranted.The expansion of islet populations for usein transplant for diabetic patients willfind substantial improvement asthe molecular physiology of the f3-cell continues tobeexj,osed. 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Genome Research. 19(6).69OLj:ll3f°44a!’taJi11vUOfl1UJTjOJJIPDtI[OT11OJ3u1JJOSJ02Ifl13SUUWI900Zif‘H(frLozoiuivuauidolaa3J1ffuwdopAps3J3u1duunpsutojdnI/ownooouo!ssJdx800tPYH‘ulnuJJoH:woJpgipowt?InauoJ(ELt9c-6frcI7:9‘yawasatSj3J7’uiounbspj1dIOASSWUiSflA1jflnpSflOtUU!SPSrnpuiqyxO{OAIAUIJOSiSXUfO9oozia‘IT.’p(EL.6E-LTE:()9T-°i°rjvuopv,znduto3JoirnwwrXJ3A0Os!pJTow.iojmqTuoJy.jjui4IMpojdnoospCppndsJouotnuuojppinwquo1oIui:JAJJcJ\796001‘1‘vi(IL9’j:(j)g?OlO!qau10u30UO!BOJddsis(juiipA!PiJUTu:wdg1cJAODSiQL0OZi’N‘uosj.iqo>j(oLAPPENDIXachrl3: 1174200001 1174300001a13—IPc4x 1 0388_7L_rnrr3.94488 —•IIIj Conservat iOflI U I II II I II 11 I II II III II [ I 1 I II II III I•chr2:I 1468800001 1468900001I25—I°’16.89764_—INkx2—2--)Nkx2—2•Nkx2—2JConservat ioflII • I 11111 II I I I I •i I II II*4++-+-+-*-+I . . I. I .: .I I III IJchrS: 1019000001•118—Pdx 1 0388_7L_rnrrJ4.99213 —a•ConservationI• I I III III I II I IIJchr2:1055000001IPdx1_MM0388_7L_mw1-IIPax6:1I:’:’Pa::6 :‘ :‘IbI:’ :I 1:j : :III:1F.j:x:E.P.E<6EU’4IPax6osl ‘1I :. 1:1:.:.:.::1111:E1p4jConservat ionliii I III I I liii II liii I I •i ii I I I I I I II IFigure Al: Additional UCSC screenshots of interest of Pdxl ChIP-Seq bindingsites710-Ju_SirigletsII Iii(111111141150 100 150 200 250SSC-A(xl,000)siGLO PositiveFigure 42: FACSorted siCYCLO islets72ScknenOO1-Cydo Sr7ecimen OO1-Cvdo,<,“,‘7AADN?gativeI IIllI IIII I III Ill Fl50 100 150 200 250FSC-A(x 1,000)000C‘4)LL44)0.ci4’)Specimen OO1-CvcloSpecimen OCI-CycloU)0..4-- Ff[ITI1IIIIIII I I FIITIj I I Ililif I I111IIJ-320io210 I0 10FL1A‘4)LI)U)liii 111111I I111111I T I 111111332°10 10FLI-A‘510SnrimAn flfl1-ndSQecimen 001 -odxC)C,)Figure A3: FACSorted siPdxl islets73‘C00-(NSingletsIiliiiIjill IjI IllijI50 100 150 200 250SSC-A(xl 000)Specimen 001 -pdxlilijil III1111111111I50 100 150 200 250FSC-A(xl,000)• 7AAD Noative<0.Soecimen 001-pdxsiGLO Positive-JU-LUjjjI-26°10— I1IIHIlI I IIIIIIj I I 111111) I I IIlIIIj-28010210 10 l0FITC FLI-AVI I 111119 I LTHH9 1 1FFH1JI10 10 10FITC FL1-APage 1 of 1THE UNIVERSITY OF BRITISH COLUMBIAANIMAL CARE CERTIFICATEApplication Number: A05-1741Investigator or Course Director: Cheryl D. HelgasonDepartment: SurgeryAnimals:Mice icrTac:ICR 900Mice Pdxl-GFP Transgenic 150Mice NGN3GFP transgenic 150Start Date: January 1, 2006 Approval Date: April 1, 2009Funding Sources:Funding Agency: Genome CanadaFunding Title: Dissecting Gene Regulatory Networks in Mammalian OrganogenesisUnfunded title: N/AThe Animal Care Committee has examined and approved the use of animals for the above experimental project.This certificate is valid for one year from the above start or approval date (whichever is later) provided there is nochange in the experimental procedures. Annual review is required by the CCAC and some granting agencies.A copy of this certificate must be displayed in your animal facility.Office of Research Services and Administration102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3Phone: 604-827-5111 Fax: 604-822-5093https://rise.ubc.calrise/Doc/0/EDOAN5E9AIF4D6COIO4UQJLVB7/fromString.html 4/1/2009

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