ThePDX1HomeodomainTranscriptionFactorNegatively ... ·...

The PDX1 Homeodomain Transcription Factor NegativelyRegulates the Pancreatic Ductal Cell-specificKeratin 19 Promoter*

Received for publication, June 20, 2006, and in revised form, October 12, 2006 Published, JBC Papers in Press, October 20, 2006, DOI 10.1074/jbc.M605891200

Therese B. Deramaudt‡, Mira M. Sachdeva§, Melanie P. Wescott‡, Yuting Chen‡, Doris A. Stoffers§,and Anil K. Rustgi‡1

From the ‡Division of Gastroenterology, Departments of Medicine and Genetics, Abramson Cancer Center and §Division ofEndocrinology, Diabetes and Metabolism, the Institute of Diabetes, Obesity and Metabolism, University of Pennsylvania,Philadelphia, Pennsylvania 19104

Keratin 19 is a member of the cytokeratin family that is crit-ical for maintenance of cellular architecture and organiza-tion, especially of epithelia. The pancreas has three distinctcell types, ductal, acinar, and islet, each with differentfunctions. Embryologically, the pancreatic and duodenalhomeobox 1 (PDX1) homeodomain protein is critical for theinitiation of all pancreatic lineages; however, the later differ-entiation of the endocrine pancreas is uniquely dependentupon high PDX1 expression, whereas PDX1 is down-regu-lated in the ductal and acinar cell lineages. We find that thisdown-regulation may be required for normal ductal expres-sion of cytokeratin K19. The K19 promoter-reporter geneassay demonstrates that ectopic PDX1 inhibits K19 reportergene activity in primary pancreatic ductal cells. This is rein-forced by our findings that retrovirally mediated stable trans-duction of PDX1 in primary pancreatic ductal cells sup-presses K19 expression, and short interfering RNA to PDX1in Min6 insulinoma cells results in the induction of normallyundetectable K19. Complementary functional and biochem-ical approaches led to the unexpected finding that a multim-eric complex of PDX1 and two members of the TALE home-odomain factor family, MEIS1a and PBX1b, regulates K19gene transcription through a specific cis-regulatory element(�341 to �325) upstream of the K19 transcription start site.These data suggest a unifying mechanism whereby PDX1,myeloid ecotropic viral insertion site (MEIS), and pre-B-cellleukemia transcription factor 1 (PBX) may regulate ductaland acinar lineage specification during pancreatic develop-ment. Specifically, concomitant PDX1 suppression andMEISisoform expression result in proper ductal and acinar lineage

specification. Furthermore, PDX1 may inhibit the ductal dif-ferentiation program in the pancreatic endocrine compart-ment, particularly beta cells.

Cytokeratins are members of the intermediate filament fam-ily that are critical to themaintenance of cell and tissue integrity(1). In addition, they influencemembrane and subcellular local-ization of proteins. The cytokeratin family consists of at least 20members that are categorized as acidic type I, comprising kera-tins 9–20, or basic type II, comprising keratins 1–8. Typically,cytokeratins form heterodimers between one type I memberand one type II member. Keratin 19 (K19)2 is expressed in epi-thelia and substitutes for keratin 18 in heterodimerization withkeratin 8. Among the pancreatic cell types, K19 is specificallyexpressed in pancreatic ducts in vivo and in primary pancreaticductal cells in vitro that our laboratory has successfully isolatedand characterized (2). We have previously demonstrated thatK19 expression is modulated by the KLF4 and Sp1 zinc-fingertranscription factors, contributing to its tissue specificity in thepancreas (3). This activity is mediated by a short cis-regulatoryregion containing an overlapping binding site for KLF4 and Sp1within the K19 promoter. KLF4 has a higher binding affinityand is the predominant binding factor in pancreatic ductal cellswith low Sp1 protein levels (3).PDX1 (pancreatic and duodenal homeobox 1) is a Hox type

homeodomain transcription factor that is critical for the tran-scriptional regulation of beta cell development (4–9). PDX1expression and function are noteworthy in the emergence ofpancreatic buds from the endoderm and the maintenance ofputative pancreatic progenitor cells during development, and itis, thereafter, highly expressed in the endocrine beta cell line-age. Within the pancreas PDX1 expression is minimallydetected in ductal and acinar cells. Mice deficient for Pdx1exhibit pancreatic agenesis (10–12). Humans with germ linePDX1 mutations develop early (designated maturity onset dia-

* This work was supported by NIDDK, National Institutes of Health Grants R01DK50306 (to A. K. R., T. B. D., Y. C., and D. A. S.), R01 DK068157 (to D. A. S.),and P01 DK49210 (to D. A. S. and M. M. S.), and by the National PancreasFoundation (to T. B. D.), Department of Genetics Training Grant 5-T32-GM-08216-19 (to M. M. S.), NIDDK, National Institutes of Health Center forMolecular Studies in Digestive and Liver Diseases Grant P30 DK50306 andits Morphology, Molecular Biology, Mouse, and Cell Culture Core Facilities,and the Penn Diabetes and Endocrinology Research Center (Grant P30DK19525) of the Institute of Diabetes, Obesity, and Metabolism. The costsof publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: GI division, 600 CRB, Uni-versity of Pennsylvania, 415 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-0154; Fax: 215-573-5412; E-mail:: [email protected].

2 The abbreviations used are: K19, cytokeratin 19; WT, wild type; PDC, pancre-atic ductal cells; PDX1, pancreatic and duodenal homeobox 1; MEIS1, mye-loid ecotropic viral insertion site 1; PBX1, pre-B-cell leukemia transcriptionfactor 1; TALE, three-amino acid loop extension; siRNA, short interferingRNA; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immuno-precipitation; CMV, cytomegalovirus; GST, glutathione S-transferase; Ct,threshold cycle.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 50, pp. 38385–38395, December 15, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

DECEMBER 15, 2006 • VOLUME 281 • NUMBER 50 JOURNAL OF BIOLOGICAL CHEMISTRY 38385

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betes of the young, or MODY4) and late-onset forms of type 2diabetes (13–19). Use of a tet-regulatory system formodulationof PDX1 expression in utero has shown that, in addition to itsnecessity for early pancreatic development, PDX1 is alsorequired later for the formation of acinar cell compartments. Inthe absence of PDX1, acini do not form, and in addition theprecursor epithelium develops a truncated ductal tree consist-ing of immature duct-like cells. Thus, the temporal and spatialregulation of PDX1 expression appears to be critical for cell fatedetermination during development and has implications forcell autonomous and non-autonomous behavior during adultdifferentiation and regeneration (20–24).It is precisely the potential interplay between PDX1 and duc-

tal cell morphogenesis that motivated us to investigate theimpact of PDX1 upon ductal epithelial cell-specific geneexpression and, in this particular context, K19 gene transcrip-tion. We report herein that the transcription factor PDX1 neg-atively regulates K19 expression in pancreatic cells. K19 expres-sion is reduced in pancreatic ductal cells that stably expressPDX1 mediated by retroviral transduction, and conversely,Min6 insulinoma cells that normally express high levels ofPDX1 exhibit increased K19 expression after short interferingRNA (siRNA) knockdown of PDX1.We identified a cis-regula-tory region of 16 nucleotides in the K19 promoter that is nega-tively regulated by PDX1 and is distinct from the region regu-lated byKLF4 and Sp1. Surprisingly, consensus binding sites fortheMEIS (myeloid ecotropic viral insertion site) and PBX (pre-B-cell leukemia transcription factor) three-amino acid loopextension (TALE) homeodomain transcription factors werealso found in this region. This 16-bp regulatory region is mod-ulated byMEIS1a acting as a positive regulator, whereas PBX1bfunctions as a repressor of K19 reporter gene activity. Complexformation of PDX1 with MEIS1a and PBX1b leads to PDX1-mediated repression of K19. We conclude that low level PDX1expression, as observed in ductal cells, permits higher levels ofK19 expression as compared with islet cells that express highlevels of PDX1 and undetectable levels of K19. This dynamicinterplay between PDX1 and K19 has important implicationsfor cell fate decisions during development and regeneration.

EXPERIMENTAL PROCEDURES

Cell Lines—Themouseprimarypancreatic ductal cell line,WT-PDC, was isolated and characterized as previously described andmaintained in a serum-free Dulbecco’s modified Eagle’s medium/F-12 medium (2). The mouse insulinoma �-cell line, Min6, wasmaintained in Dulbecco’s modified Eagle’s medium (high glucoseand supplementedwith pyroxidinehydrochloride)with 10%heat-inactivated fetal calf serum.HeLaandPANC-1cellsweregrown inDulbecco’s modified Eagle’s medium (Invitrogen) supplementedwith 10% fetal bovine serum. Cell lines were kept as subconfluentmonolayers andweremaintained in a 5%CO2 humidified incuba-tor at 37 °C.K19 Promoter-Luciferase Reporter and Serial Deletion

Constructs—The 5�-flanking region of the mouse K19 genefrom�1970 to�46 bpwas inserted into pGL3-Basic (Promega,Madison, WI) (3). Similarly, plasmids pK19-654 and pK19-288were generated by a PCR-based technique (3). pK19-408, pK19-365, pK19-341, and pK19-325 were generated using the

QuikChange site-directedmutagenesis kit (Stratagene, La Jolla,CA). The primers used to construct the deletion mutations ofthe K19 promoter were as follows 5�-ctatcgataggtaccATTAT-TCCAGAGGGG-3� (K19–408), 5�-ctatcgataggtaccGGGCTC-AGAGGG-3� (K19–365), 5�-ctatcgataggtaccAGGGTGTCAA-ATTCC-3� (K19–341), and 5�-ctatcgataggtaccGGAGGTTTT-AAAGGG-3� (K19–325). Bases in lowercase correspond topGL3-Basic, whereas bases in uppercase correspond to the 5�-flanking region of K19. The antisense primers matched thecomplementary sequences of each sense primer. Deletion orsite-directed mutations of the pK19-1970 plasmid between�352 and �325 bp were generated using the QuikChangesite-directed mutagenesis kit (Stratagene). Primer sequencesused to generate pK19-1970� (oligonucleotide K19-352�),pK19-1970m (oligonucleotide K19-352m), pK19-1970m2 (oli-gonucleotide K19-352m2), and pK19-1970m4 (oligonucleotideK19-352m4) are described in the electrophoretic mobility shiftassay (EMSA) section below. The sequences of the plasmidswere verified by the DNA sequencing facility at the Universityof Pennsylvania.Expression Vectors—The pCMX-PDX1 vector expressing

mouse PDX1 was described previously (25). pCS2-MEIS1a,pCS2-MEIS1b, and pCS2-PBX1a were gifts from Dr. MarkFeatherstone (26). pCDNA1.1-MEIS2b and pcDNA1.1-PBX1bwere gifts from Dr. Galvin Swift (27). The pGEX-PDX1 vectorexpressing a glutathione S-transferase (GST) fused to the intactrat PDX1 (residues 1–283) was described elsewhere (7). ThepGEX-4T-1-PDX1 (144–283) and the pGEX-4T-1-PDX1(206–283) vectors were described previously (25) andexpressed the GST fused to the mouse PDX1 homeodomain/Cterminus regions, GST-PDX (144–283), and PDX1 C terminusdomain, GST-PDX (206–283), respectively.Transient Transfections and Luciferase Activity Assays—

Cells were plated 24 h before transfection in 24-well plates andtransiently transfected with 0.15 �g of pGL3-Basic or pGL3-K19 plasmid, 1 ng of pRL-CMV plasmid expressing the renillaluciferase reporter gene with or without 50 ng to 0.3 �g ofpCMX-PDX1, 0.3 �g of pCS2-MEIS1a, 0.3 �g of pCS2-PBX1a,0.3 �g of pcDNA1.1-PBX1b expression plasmids using Lipo-fectamine 2000 (Invitrogen) as directed by the manufacturer.The total amount of DNA in each transfection was adjustedwith the corresponding empty expression vector. pGL3-Basicwas used as a standard control. Twenty-four hours after trans-fection, the cells were harvested, assayed for firefly luciferaseactivity and normalized to renilla luciferase activity using theDual-luciferase Reporter assay system (Promega). Luciferaseactivities were detected by the Orion microplate luminometer(Berthold detection system).Antibodies—Rabbit anti-cyclophilin B was obtained from

Affinity Bioreagents (Golden, CO). Mouse anti-MEIS1/2/3antibody was obtained from Upstate (now Millipore, Bil-lerica, MA). Antibodies against mouse anti-PBX1/2/3/4,rabbit anti-PBX1/2/3, and goat anti-PDX1 (clone A-17) wereobtained from Santa Cruz Biotechnology Inc. (Santa Cruz,CA). Mouse monoclonal anti-�-actin antibodies wereobtained from Sigma. Horseradish peroxidase-conjugatedsheep anti-mouse or donkey anti-rabbit IgG were obtainedfrom Amersham Biosciences.

PDX1 and Keratin 19 Gene Regulation

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In Vitro Transcription-Translation—MEIS1a, MEIS1b, andPBX1a were in vitro transcribed and translated with the SP6TNT rabbit reticulocyte lysate coupled transcription-transla-tion system (Promega) according to the manufacturer’s proto-col. MEIS2b, PBX1b, and PDX1 were translated using the T7polymerase from a similar coupled reaction kit. Controls of thetranslation efficiency were performed using [35S]methionine(Redivue, Amersham Biosciences) in the reaction mixes, andsamples were resolved by SDS-PAGE (4–12%).EMSAs—For EMSAs, nuclear extracts were prepared from

WT-PDC cells and Min6 cells as described previously (28). Syn-thetic oligonucleotides and their respective complementary oligo-nucleotides for K19-352 probe (�352 to �323, 5�-GGTG-TGATTTCTAAGGGTGTCAAATTCCTGG-3�), TSE2-PDX1(5�-GATCTCAGTAATTAATCATGCA-3�) (29), mutant K19-352� (5�-GAGGGGTGTGATTTCT-�-GGAGGTTTTAAAG-GGCC-3�), mutant K19-352m (5�GGTGTGATTTCTAAGatca-TCAAATTCCTGGAGG-3�), mutant K19-352m2 (5�-CTAAG-GGTGTCAAtcagCTGGAGGTTTTAAAGG-3�), and mutant352m4 (5�-GTGATTTCTAAGGGTGatcaATTCCTGGAGG-3�) were diluted to a final concentration of 5�M (lowercase lettersrepresent mutated nucleotides, and bold letters represent regionof interest in the K19 promoter). The sense oligonucleotides wereend-labeled using T4 polynucleotide kinase (New England Bio-labs, Beverly, MA) in the presence of 50 �Ci of [�-32P]ATP andthen purified using Microspin G-25 columns (Amersham Bio-sciences) following the manufacturer’s instructions. The comple-mentary antisense oligonucleotides were then added to the radio-labeled oligonucleotides and annealed by heating at 100 °C for 5min followed by slow cooling to room temperature. EMSA exper-iments were carried out by mixing the following components tothe reactionmixture: 5 �g of nuclear extract,�0.5 pmol of radio-labeled probe, 25mMHEPES, pH7.9, 150mMKCl, 10% glycerol, 5mMdithiothreitol, and 0.5�g of poly(dI-dC). The various compo-nents were incubated at room temperature for 30 min. Nuclearextracts fromPDCandMin6werepreincubated in thepresenceorabsence of 100-fold excess of competitor DNAs at room temper-ature for 20 min before the addition to the reaction mixture. Forsupershift analysis, nuclear extractswerepreincubatedwith3�l ofgoat polyclonal antibody to PDX1, mouse anti-PBX, mouse anti-MEIS, or purified immunoglobulin G at room temperature for 20min before the addition of the labeled probes. Free and boundDNAwere separated on a 4% non-denaturing polyacrylamide gel,whichwas runat a constant voltageof 120V inTris-glycinebuffer.After drying the gel, the results were visualized by phosphorimag-ing or exposed to BioMaxMR film (Eastman Kodak Co.).Western Blot Analysis—For immunoblot analysis, cells were

washedwith phosphate-buffered saline and lysedwith radioim-mune precipitation assay buffer (150 mM NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl,pH 7.5, and a mixture of protease inhibitors (Complete mini,Roche Applied Science)). Protein concentration was deter-mined with the Bradford reagent (Bio-Rad). A total of 10 �g oftotal protein was resolved by SDS-PAGE (4–12%) and trans-ferred to polyvinylidene difluoride membrane (Immobilon,Millipore Corp., Bedford, MA). To check for equal proteintransfer, the membranes were stained briefly with Ponceau Ssolution (Sigma). Blocking was performed in 5% milk, 10 mM

Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20 for 1 hbefore incubation with primary antibodies. Horseradish perox-idase-conjugated secondary antibodies were used according tothe manufacturer’s protocol. Immunoreactivity was visualizedusing the ECL� system (Amersham Biosciences) and exposedto BioMax MR film.DNA Affinity Precipitation Assay—5�-Biotinylated oligonu-

cleotides and their respective complementary oligonucleotideswere synthesized, gel-purified by Integrated DNA Technologies(Coralville, IA), and annealed in Tris-EDTA buffer with 150 mM

NaCl by heating at 100 °C for 5 min followed by a slow cooling toroom temperature. The positive control oligonucleotide (CMV2)was 5�-biotin-TAATCAATTACGGGGTCATTA-3�. The nega-tive control oligonucleotide (scramble) was 5�-biotin-GCCGCC-GCCGCCGCCGCCGC-3�. K19 oligonucleotides were 5�-biotinK19-352 (as described above).Thebiotinylatedprobes (2�g)wereincubated on icewith 20�g ofMin6 or 60�g ofWT-PDCnuclearextract in 400 �l of binding buffer (20 mM HEPES, pH 7.9, 10%glycerol, 0.2 mM EDTA, 1.5 mMMgCl2, 50 mM KCl, 1 mM dithio-threitol, 0.25% Triton X-100). Increasing amounts of excesscompetitor (0–50-fold excess) were included in the binding reac-tion for competition assays. The negative control was done byomitting the biotinylatedprobes.After 30minof incubation, 20�lof streptavidin-agarose beads prewashed 3 times with the bindingbuffer were added to each reaction mixture, and the reaction wasconducted for an additional hour on ice with gentle shaking. Thestreptavidin-agarose beads were washed 4 times with 1 ml ofbinding buffer before adding 30 �l of protein sample buffer (with1% 2-� mercaptoethanol). All the samples were denatured byheating at 85 °C for 5min and resolved by SDS-PAGE (4–12%).Quantitative Chromatin Immunoprecipitation (ChIP)—

ChIP assays were performed as described previously (30, 31)with a few modifications. Briefly, for each antiserum, one con-fluent 10-cm plate of pancreatic ductal cells (�1 � 107 cells)was cross-linked with 1% formaldehyde in phosphate-bufferedsaline for 10 min at room temperature and quenched with gly-cine to a final concentration of 0.125 M. Chromatin was soni-cated to create �500-bp fragments in size and precleared withnormal goat ormouse IgG (Santa Cruz) overnight at 4 °C. Afterremoval of an aliquot for analysis as input, precleared chroma-tin was divided equally for immunoprecipitation with eithergoat polyclonal anti-PDX1 antibody, mouse monoclonal anti-MEIS, or normal IgG for 3 h at 4 °C. Data were analyzed quan-titatively in duplicate by real-time PCR.GST Pulldown Assays—The GST pull-down assays were car-

ried out using the ProFoundTM pulldown GST protein-proteininteraction kit (Pierce) according to the manufacturer’s proto-col. Briefly, GST fusion proteins orGST alonewere produced inEscherichia coliBL21-Gold(DE3)pLysS cells inducedwith 1mM

isopropyl-�-D-thiogalactopyranoside for 3 h at 37 °C. The puri-fied GST proteins were then incubated with immobilized glu-tathione for 1 h at 4 °C. After 3 washes (ProFound lysis buffer:Tris-buffered saline, 1:1), the immobilized baits were incubatedovernight at 4 °C with 5 �l of in vitro translated 35S-labeledPDX1, MEIS1a, or PBX1b diluted in washing buffer. The beadswere then washed 4 times, the captured proteins were eluted in100 �l of sample buffer and heated at 85 °C for 5 min, and 20 �l




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of proteins were fractionated by SDS-PAGE (4–12%). The gelswere dried under vacuum at 80 °C and autoradiographed.Viral Infection and RNA Isolation—Min6 cells were infected

with recombinant adenovirus containing a siRNA sequencedesigned to target either PDX1 or luciferase as described (32).Briefly, 5 � 105 cells were infected at a multiplicity of infectionof 2500 plaque-forming units/cell for 6.5 h. Cells were har-vested 72 h post-infection and processed for RNA isolationusing Trizol (Invitrogen). RNA samples were treated withDNase I (Ambion), analyzed for integrity with an Agilent 2100Bioanalyzer, and reverse-transcribed with Superscript II(Invitrogen) using oligo(dT) for priming.Full-length mouse PDX1 cDNA was obtained by digesting

pCMX-PDX1 expression vector with the restriction enzymesSalI and BamHI. The extremities of the insert were then filledblunt with T4 DNA polymerase, and the PDX1 cDNA wasinserted into the SnaBI-digested pBABE/puro retroviral vector(33). Retrovirus preparation andWT-PDC infection and selec-tion were conducted as previously described (34).Reverse Transcription-PCR and Real-time PCR Analysis—

Total RNA was isolated from monolayer cultures using Trizolreagent, and cDNAwas synthesized by oligo(dT) priming from1 �g of total RNA using a Superscript first-strand synthesissystem (Invitrogen) according to themanufacturer’s directions.The semiquantitative analysis of transcripts encoding variousisoforms of MEIS and PBX was carried out with a mixture ofcDNAderived fromWT-PDCorMin6, 0.2�Meachof the senseand antisense primers, 0.2�MdNTP, and 2.5 units of PfuTurboDNA polymerase (Stratagene) in a final reaction volume of 50�l. The specific primers to detectMEIS1, -2, and -3 and PBX1a,-1b, -2, and -3were described elsewhere (35). ThePCRprogramwas 90 °C for 2min and 30 cycles of 94 °C for 30 s, 60 °C for 30 s,and 68 °C for 1 min with a final extension at 68 °C for 7 min.Data shown were obtained with 30 PCR cycles. Analysis ofdigested and undigested PCR products was done by electro-phoresis on a 1% agarose gel.For quantification of duplicate or triplicate samples, real-time

PCR was performed with SYBR green using a Bio-Rad iCycler(Apply Biosystems) by cycling 40 times using the conditions 95 °Cfor10 s, 60 °C for45 s (PDX1and�-actinprimers), or 55 °C for45 s(K19 primers). PCR product signals were referenced to a dilutionseries of the relevant input to account for different efficiencies ofprimer sets: K19 promoter, sense, 5�-TGTCAAATTCCTGGAG-GTTTTAAAG-3�, and antisense, 5�-GCCCCTTACTACACAG-GCTTAGAC-3�; albumin promoter, sense, 5�-TGGGAAAACT-GGGAAAACCATC-3�, and antisense, 5�-CACTCTCACACAT-ACACTCCTGCTG-3� (30). The following forward and reverseprimers were used to amplify PDX1 mRNA (forward, 5�-GAAC-CCGAGGAAAACAAGAGG-3�, and reverse, 5�-GTTCAACAT-CACTGCCAGCTC-3�), K19 mRNA (forward, 5�-TCCCAGCT-CAGCATGAAAGCT-3�, and reverse, 5�-AAAACCGCTGATC-ACGCTCTG-3�), Pbx1 mRNA (forward, 5�- AACCTCCTTCG-AGAGCAAAGC-3�, and reverse, 5�- GCATCTGGATGGAGC-TGAACT-3�), Meis2mRNA (forward, 5�-CCCGTCCATGTGT-CCTTTAGT-3�, and reverse, 5�-TGAAGAAGCCTTCGCT-CTGTC-3�), hypoxanthine-guanine phosphoribosyltransferase(forward, 5�-GGCCAGACTTTGTTGGATTTG-3�, and reverse,5�-TGCGCTCATCTTAGGCTTTGT-3�), or �-actin (forward,

5�-GAAGTGTGACGTTGACATCCG-3�, and reverse 5�-GTC-AGCAATGCCTGGGTACAT-3�).Densitometry Measurements and Statistical Analysis—Re-

sults are expressed as mean � S.E. Densitometry measure-ments were performed using Scion Image Beta 4.02 software(Frederick,MD) and calibratedwith the�-actin signal. Analysisof variance with a Tukey post hoc test was used for statisticalanalysis. A p � 0.05 was considered statistically significant.

RESULTS

Expression of PDX1 in Primary Pancreatic Ductal Cells—Todetermine the level of expression of PDX1 in our primary pan-creatic cell lines, designated as wild-type pancreatic ductal cells(WT-PDC), we performedWestern blots on whole cell lysates.The results show that PDX1 is expressed at low levels in thepancreatic ductal cells, and its expression is significantly higherin the insulinoma cell line Min6 (PDX1 is 22 � 3.5-fold higherin Min6 cells compared with WT-PDC, p � 0.01). PDX1expressionwas undetectable in PANC-1 andHeLa cells (Fig. 1).PDX1 Negatively Regulates K19 Expression—The K19-1970

plasmid containing a luciferase reporter gene under the controlof the 5� regulatory region of the mouse K19 gene was con-structed as previously described (3). A set of 5� deletion con-structs were generated using a PCR-based technique, and the

FIGURE 1. Low PDX1 expression detected in primary pancreatic ductalcells. 10 �g of whole-cell lysates prepared from WT-PDC, PANC-1, Min6, andHeLa cell lines were fractionated by SDS-PAGE, and Western blot analysis forPDX1 was performed. �-Actin was used as a loading control. Three independ-ent experiments were carried out, and densitometry was performed usingScion Image Beta 4.02 software (Frederick, MD). Signals were calibrated withthat of �-actin, and the signals from WT-PDC were set arbitrarily at 1. Error barsrepresent the means � S.E.




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reporter plasmids were transiently transfected into WT-PDC(Fig. 2A). PDX1 had no effect on the basal luciferase activity ofthe empty pGL3-Basic vector (Fig. 2B). Data from the luciferasereporter assays revealed that co-expression of PDX1 in WT-PDC negatively regulated the K19 promoter. PDX1 partiallysuppressed K19 expression by about 50% when using the K19promoter constructs from �1970/�46 to �341/�46. How-ever, PDX1 had no effect on K19 expression when the K19promoter was reduced to �325/�46 and �288/�46. Thisresult suggests that the negative regulation of K19 by PDX1 isalleviated when the region between �341 and �325 of the K19promoter is deleted. Interestingly, this region does not containan obvious AT-rich consensus DNA binding site for PDX1.PDX1Occupies theK19Promoter inVivo—Toprovide in vivo

evidence for PDX1 binding to the K19 promoter, we carried outChIP in WT-PDC and analyzed the results using quantitativereal-time PCR. The compiled data from 5 independent ChIPexperiments demonstrate that PDX1 specifically occupies theK19 promoter in WT-PDC, resulting in an �2.5-fold higherenrichment than observed with the control IgG immunopre-

cipitation (Fig. 3A). In these assays the lack of PDX1 occupancyat the albumin promoter serves as a negative control. Thesedata suggest that PDX1 associates with the K19 promoterdespite the absence of a TAA(T/T)TAT consensus sequence,perhaps via interaction with another/other DNA bindingfactor(s).To further confirm the interaction of PDX1 with the K19

promoter, the DNA affinity precipitation assay was carried outusing both Min6 and WT-PDC nuclear extracts and a double-stranded K19 oligonucleotide. As a positive control for PDX1binding, we used a well established element taken from thehuman cytomegalovirus immediate early (CMV IE) promoter(36). Our results showed that the K19-352 and the CMV oligo-nucleotides were able to pull down PDX1 protein from both

FIGURE 2. Effect of exogenous PDX1 on K19 promoter in WT-PDC. A, sche-matic representation of serial deletions of K19 promoter regulating the fireflyluciferase reporter gene. The 5� regulatory region from �1970 to �46 corre-sponding to the full-length K19 promoter (�2 kilobases) was inserted in thefirefly luciferase reporter plasmid pGL3-Basic. Serial deletions of K19 pro-moter were generated by a PCR based technique. B, mutation analysis of theK19 promoter. WT-PDC cells were transiently transfected with pK19-1970 ordeletion constructs in presence (black columns) or absence (white columns) ofpCMX-PDX1 expression vector. After 24 h luciferase activities were measured,and firefly luciferase activity was normalized to renilla luciferase activity. Therelative luciferase activities of WT-PDC transfected with pGL3-Basic and/orpCMX-PDX1 were adjusted to 1.0. The values are expressed as the average �S.D. of three independent experiments done in triplicate.

FIGURE 3. PDX1 occupancy of the K19 promoter in PDC cells and DNAaffinity precipitation assay. A, after formaldehyde cross-linking and sonica-tion, chromatin from pancreatic ductal cells was immunoprecipitated witheither anti-PDX1 antiserum or normal IgG. The data were analyzed quantita-tively using real-time PCR with primers designed to amplify the putativePDX1 binding region of the K19 promoter (�337 to �253) or a distal TAAT-containing element of the albumin promoter. For each primer pair, ChIP sig-nals were compared with a dilution series of input chromatin to account fordiffering amplification efficiencies and are expressed here as such. These datarepresent the means � S.E. of five independent experiments. The asterisksignifies a p value of 0.01. B and C, 20 �g of nuclear extracts (NE) from Min6 (B)or WT-PDC (C) were incubated with 2 �g of biotinylated K19-352, CMV2, orscramble oligonucleotides (containing GCC repeats) for 30 min. The DNA-protein complexes were then precipitated by streptavidin-agarose beads fol-lowed by extensive washes, and the reaction mixtures were resolved by SDS-PAGE. PDX1 expression was detected by Western blot. D, increasing amounts(2–50-fold excess) of unlabeled K19-352 oligonucleotides were used as spe-cific competitors.




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Min6 andWT-PDC nuclear extracts, whereas a scrambled oli-gonucleotide with GC repeats did not (Fig. 3, B and C). Thebinding specificity was further confirmed by competitionassays with cold K19 oligonucleotide incubated withWT-PDCnuclear extract (Fig. 3D) and Min6 nuclear extracts (data notshown). The cold K19 oligonucleotide competed for binding ina dose-dependent manner that almost completely abrogatedbinding at 10-fold or greater concentration of the biotinylatedoligonucleotide.PDX1Negatively Regulates K19 Expression inMin6 andWT-

PDC Cell Lines—We infected WT-PDC with a retrovirusexpressing PDX1 or the control empty pBABE vector.We con-firmed higher expression of PDX1 byWestern blot and showedby real-time PCR that induction of PDX1 results in a 54%decrease of K19 expression (Fig. 4A). Conversely, to comple-

ment the PDX1 overexpression studies, we employed a siRNA-mediated approach to knockdown endogenous PDX1 in Min6cells, whichnormally express high levels of PDX1 andundetect-able levels of K19. Min6 cells were infected with an adenovirusencoding siRNAdesigned to target either PDX1 (AdsiPDX1) orluciferase (AdsiLUC), and we confirmed specific reduction ofPDX1 at both the protein and mRNA levels (Figs. 4, B and C,respectively) (32). Furthermore, we observed a 2.5-fold increasein K19 transcript levels as assessed by quantitative real-timePCR in the AdsiPDX1-infected cells relative to both untreatedcells and those infected with AdsiLUC (Fig. 4C). These datasupport a role for PDX1 in the negative transcriptional regula-tion of K19 and suggest that the high PDX1 levels in beta cellsmight contribute to the repression of K19 expression in thesecells.Analysis of the Upstream K19 Promoter Region by EMSA—

The cis-regulatory region of 16 nucleotides between �341 and�325 delineated by the promoter-luciferase reporter geneassays to be regulated by PDX1 was then analyzed by EMSAs.Nuclear extracts from WT-PDC were used. The specificity ofthe retarded band observed was verified by competition with100-fold excess of unlabeled K19-352 probe, whereas no com-petition was apparent with the nonspecific oligonucleotide(Fig. 5A). Supershift assays were performed by preincubatingtheWT-PDC nuclear extracts with increasing amounts of anti-PDX1 antibody before the addition of the radiolabeled K19-352probe. These results suggest that PDX1 may interact with theK19-352 probe (Fig. 5B).Mutagenesis of the Region Regulated by PDX1 in the K19 Pro-

moter Reveals Interaction with MEIS and PBX—Systematicanalysis of the 5� upstream region of K19 that is regulated byPDX1 revealed the presence of consensus binding sites forMEIS (TGTCA) and PBX (TGATT), twomembers of theTALEfamily of homeodomain proteins (37). Previous work has dem-onstrated that PDX1 is able to form a trimeric complex withPBX1b and MEIS2b to activate the elastase ELA1 mini-en-hancer in HeLa cells (27, 38).We used reverse transcription-PCR to determine MEIS and

PBX expression levels in WT-PDC and Min6 cells. Total RNAwas purified, and the cDNAwas synthesized by oligo(dT) prim-ing. Using specific primers that were described previously forMEIS1, -2, and -3 and PBX1a, -1b, -2, and -3 (35), the PCRresults demonstrate thatMEIS 2 and 3 are present in bothWT-PDC and Min6 cells, whereas MEIS1 is detected only in WT-PDC. Both cell lines contain several isoforms of PBX, namelyPBX1, -2, and -3 (Fig. 6A). To further quantify expression levels,real-time PCR was performed for PBX1 and MEIS2 (Fig. 6B).These results reveal significantly greater levels of both PBX1andMEIS2 inWT-PDC compared with Min6 cells. The differ-ences in MEIS and PBX isoform expression may contribute tothe differential regulation of K19 expression in distinct pancre-atic cell types.MEIS1a Cannot Relieve Repression of K19 Expression by

PDX1 or PBX1b—The K19 promoter was analyzed further bymutating different subregions within the 16-bp region of inter-est regulated by PDX1. Four K19 mutant reporter constructswere generated, designated as pK19-1970�, pK19-1970m,pK19-1970m2, and pK19-1970m4, using a PCR-based strategy

FIGURE 4. siRNA-mediated knockdown of PDX1 in Min6 cells and PDX1-overexpressing WT-PDC. A, WT-PDC cells retrovirally infected with pBABE orpBABE-PDX1 were verified by Western blot for PDX1 expression. Real-timePCR results for K19 showed that K19 mRNA levels are significantly decreasedin WT-PDC overexpressing PDX1 compared with cells infected with theempty pBABE vector (value set at 1.0). Data are presented as the means � S.E.of two independent experiments done in triplicate. The asterisk indicates a pvalue of �0.0003. B, approximately 5 � 105 Min6 cells were either untreatedor treated with an adenovirus expressing siRNA against PDX1 (AdsiPDX1) orluciferase (AdsiLUC) at a multiplicity of infection of 2500 plaque-forming unit/cell for 6.5 h. Cells were harvested 72 h later. Expression of PDX1 protein wasassessed by Western blot. C, PDX1 and K19 mRNA levels were assessed usingquantitative real-time PCR, and Ct values were normalized to �-actin. -Foldchanges are expressed relative to untreated cells (value set at 1.0). Error barsrepresent the means � S.E. for three independent experiments done in eitherduplicate or triplicate. The reduction in PDX1 levels and the increase in K19levels in the AdsiPDX1-infected cells relative to untreated cells are statisticallysignificant (*, p � 0.01; **, p � 0.001) as is PDX1 or K19 expression inAdsiPDX1-infected samples as compared with AdsiLUC-infected cells (#, p 0.05; ##, p � 0.05).




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and the specific oligonucleotides K19-352�, K19-352m, K19-352m2, and K19-352m4, respectively (Fig. 7A). In addition,Western blotswere performed to verify the efficiency of expres-sion of PDX1, MEIS1a, MEIS1b, PBX1a, and PBX1b proteins(data not shown).Deletion of the 16-bp region abrogated PDX1-mediated

repression of K19 and demonstrated a decrease in promoteractivity that suggests a loss of positive regulatory elements (Fig.7B). Mutations 352m and 352m2 had little effect upon PDX1-mediated repression (Fig. 7B and data not shown), whereasmutation 352m4, which mutates part of the consensus bindingsite for MEIS, relieved the PDX1 repression of K19 transcrip-tion (Fig. 7B). We then co-expressed the MEIS1a and MEIS1bexpression vectors along with the K19 reporter-luciferase genein WT-PDC. The results of these transfection assays demon-strate that MEIS1a up-regulates K19 expression, whereasMEIS2b does not (Fig. 7C). PBX1a co-expressed with the K19reporter-luciferase gene does not appear to directly regulate

K19 transcription (Fig. 7D). Noticeably, expression of PBX1b inWT-PDC appears to negatively regulate K19 in a mannercomparable with PDX1 (Fig. 7E). Moreover, co-expression ofMEIS1a and/or PBX1a in WT-PDC that express high levelsof ectopic PDX1 is not able to rescue the negative regulation ofK19 by PDX1 (Fig. 7E and data not shown). Similar results areobserved for PBX1b expressed in WT-PDC, with MEIS1aunable to rescue the down-regulation of K19 by PBX1b (Fig.7E). These results indicate that a high level of PDX1 or PBX1binWT-PDC is sufficient to down-regulate K19 expression evenin the presence of the transactivator MEIS1a or MEIS1b.MEIS1a Interacts with the K19 Promoter—EMSA was per-

formed to determine whether the MEIS and PBX homeodo-main proteins are involved in the direct binding to the 16-bpregion of the K19 promoter.Mousemonoclonal anti-MEIS andanti-PBX antibodies were added to the reaction mixtures con-taining WT-PDC nuclear extracts before the addition of theradiolabeled probe. The antibody against PDX1 specificallyeliminates the retarded-mobility complex as demonstratedpreviously. The antibody against MEIS supershifted the samecomplex, although the level of intensity was different from thePDX1 EMSA. Of note, the MEIS supershift revealed two bandsor a doublet, perhaps consistent with two MEIS isoforms,MEIS1a and MEIS1b. However, the antibody against PBX did

FIGURE 5. EMSA with PDX1 and K19 promoter radiolabeled cis elementprobes. A, the radiolabeled K19-352 probe located between �352 and �325of the K19 promoter was incubated in the presence of 5 �g of WT-PDC nuclearextract for 30 min at room temperature in presence or absence of a 100�excess-fold nonspecific (NS, TFIID double-strand oligonucleotide obtainedfrom Santa Cruz) or specific (S, cold K19-352 oligonucleotide) competitor. Thearrow indicates bound probe, and the star indicates free probe. B, radiola-beled K19-352 probe was incubated in the presence of 5 �g of WT-PDCnuclear extract (NE) for 20 min at room temperature. For supershift assay theWT-PDC nuclear extracts were preincubated as indicated with increasingamounts (0.5–3 �l) of control goat IgG or goat anti-PDX1 for 20 min on icebefore incubation with radiolabeled probe. PDX1 antibody was able to shiftthe retarded band as indicated by an arrow.

FIGURE 6. MEIS and PBX gene expression in WT-PDC and Min6 cells.A, MEIS1, -2, and -3 and PBX1, -2, and -3 gene expression in WT-PDC and Min6cells. Reverse transcription-PCR was performed using reverse transcriptionproducts and primers specific for each of the MEIS and PBX isoforms. ForPBX1, the lower band corresponds to PBX1b, whereas the upper band corre-sponds to PBX1a. B, relative PBX1 and MEIS2 transcript levels in WT-PDC (des-ignated as PDC) and Min6 cells by real-time PCR. PBX1 and MEIS2 mRNA levelswere assessed using quantitative real-time PCR from either PDC or Min6lysates (n 3 of each done independently). PBX1 and MEIS2 primers weredesigned and optimized for linear amplification of cDNA to ensure accu-rate quantification, and the Ct values were normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT). Error bars represent themeans � S.E. *, p � 0.01; **, is p � 0.001.




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not appear to interfere with the same complex formation (Fig.8A), although we cannot rule out the lack of efficiency of thisantibody in EMSA assays. These results suggest that in WT-PDC, MEIS binds to the K19-352 probe.To confirm in vivo occupancy of MEIS protein to the K19

promoter, the ChIP assay was performed inWT-PDCusing themouse monoclonal anti-MEIS1/2/3 antibody for immunopre-cipitation. Real-time PCR results indicate a 5-fold enrichmentusingmouse anti-MEIS as comparedwith control IgG (Fig. 8B).The albumin promoter served as a negative control, showing noMEIS occupancy. These results were further confirmed by theDNA affinity precipitation assay (Fig. 8C). The same mem-branes used previously to demonstrate the interaction of PDX1with the K19 promoter were probed with anti-MEIS antibody.The results demonstrated that MEIS proteins indeed interactwith the �352/�323 fragment of the K19 promoter and thatthis interaction is specific since cold K19 oligonucleotide abro-gated the signal in a dose-dependent manner. In addition, weshowed an interaction of PBX1 to the same region of K19 pro-moter, most likely to the consensus binding site located at�345/�350 of the K19 promoter (Fig. 8C).MEIS1a Interacts with PDX1 Independently of PBX1—Next,

we performed GST pulldown assays to determine whetherPDX1 was involved in a trimeric complex with MEIS1a andPBX1b. GST-PDX1 fusion protein was incubated with in vitrotranslated 35S-labeled MEIS1a, and the result was visualized byautoradiography. The results show that GST-PDX1 interactswith MEIS1a (Fig. 9). Interestingly, GST-PDX1 also interactswith in vitro translated MEIS1b (data not shown). Of note, theaddition of PBX1b in the pulldown reactions does not appear toincrease MEIS1a interaction with PDX1, suggesting thatPBX1b has little function in stabilizing the PDX1/MEIS1a com-plex. Furthermore, GST-PDX1 (144–283), which contains thehomeodomain and C terminus domain of PDX1, and GST-PDX1 (206–283), which contains the C terminus domain ofPDX1, are still able to interact with in vitro translated MEIS1a(data not shown and Fig. 9), thereby indicating that the C ter-minus of PDX1mediates the interaction with MEIS1a and thatthis interaction is PBX1-independent. The N terminus domainof PDX1 has been shown to interact with PBX1, whereas ourdata suggest a new interaction between theC terminus domainsof PDX1 with MEIS1a.

DISCUSSION

The objective of this study was to understand the molecularbasis underlying the regulation of pancreatic ductal epithelial

FIGURE 7. Deletion of the 16-bp region of K19 promoter leads to loss ofPDX1 repression. A, analysis of the 5� regulatory region of K19 repressed byPDX1 (boldface (boldface, deleted in pK19 –1970 � construct)) revealed twoconsensus binding sites for MEIS and PBX. The 4-nucleotide region targetedby each K19 mutant oligonucleotide (K19 –352m, K19 –352m2, and K19 –352m4) is indicated. B, mutant 5� regulatory region of K19 was co-transfectedwith PDX1 expression vector in WT-PDC, and luciferase activities were meas-ured 24 h after transfection. Firefly luciferase activity was relative to that of therenilla luciferase activity obtained from the control plasmid pRL-CMV.

pK19-1970� and pK19-1970m4 lost the region regulated by PDX1. M4mutation corresponds to mutation of the consensus binding site for MEIS.C, MEIS1a up-regulated the transcription of the K19 reporter gene. Dele-tion of the 16-bp (pK19-1970�) or mutation of the MEIS binding site(pK19-1970m4) abolished the regulation by MEIS1a. Interestingly, MEIS2bis not involved in the regulation of K19. D, similarly, PBX1a does not reg-ulate K19 transcription, whereas PBX1b seems to have a negative regula-tory effect on K19 transcription. This negative regulation by PBX1b is abol-ished in pK19-1970�. E, in co-transfection assays with PBX1b and MEIS1awith or without PDX1 expression vectors, PBX1b abolished the positiveregulatory effect of MEIS1a on K19 expression. MEIS1a cannot relieve therepression by PDX1 in the presence or absence of PBX1a. Similar results toMEIS1a were obtained when K19 promoter was co-transfected withMEIS1b (not shown). The values are expressed as the average � S.D. ofthree independent experiments done in triplicate.




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cell-specific K19 promoter gene activity by the PDX1 home-odomain protein. We found that PDX1 negatively regulatesK19 expression inWT-PDC andMin6 cells as demonstrated bya reduction in K19 expression in WT-PDC expressing ectopicPDX1, whereas the introduction of a siRNA to PDX1 in Min6leads to increasedK19 expression.More specifically, PDX1 reg-ulates a 16-bp region located between �341 and �325upstream of the transcription start site of the K19 gene. Thisregion does not contain any consensus DNA binding sites forPDX1. Instead, the consensus binding sites for two homeodo-main transcription factors MEIS1 (located at position �332 to�337) and PBX1 (located at position �345 to �350) arepresent.

MEIS1 and PBX1 are members of the TALE superclass ofatypical homeodomain-containing proteins (39). Additionally,MEIS andPBXhave several isoforms. In our primary pancreaticductal cells, MEIS1, -2, and -3 are present, whereas in Min6cells, MEIS2 and -3 are present, but MEIS1 is absent. Similaranalysis reveals that PBX1b, -,2 and -3 are present in primarypancreatic ductal cells, but PBX1a is absent. However, in Min6cells, PBX1a, -1b, -2, and -3 are present. HOX proteins, such asPDX1, can form complexes with either PBX or MEIS isoformsto result in a heterotrimeric complex. To that end it has beendemonstrated that PDX1 forms a multimeric complex withPBX1b and MEIS2b to regulate the 10-bp B element of thetranscriptional enhancer of the pancreatic elastase I gene pro-moter (27). The complex binds to overlapping half-sites forPDX1 and PBX in the promoter. Whereas in pancreatic acinarcells the B element requires other elements of the ELA1enhancer for promoter activity, in beta cells the B element canactivate a promoter in the absence of other enhancer elements.Our results indicate that a multimeric complex of PDX1,

MEIS1a, andPBX1bmodulatesK19 transcriptional activity andthat the in vitro complex between PDX1 and MEIS1a is mostcritical. MEIS1a alone can induce K19 gene transcriptionthrough direct binding with its cognate DNA consensusbinding site (also evident with MEIS1b). Mutation of theMEIS consensus binding site (located �332 to �337) abol-ished MEIS1a positive regulation of K19 gene transcriptionand, interestingly, abolished also PDX1-mediated repres-sion. Noticeably, the recruitment of PDX1 or PBX1b negatestheMEIS1a-mediated effect, thereby leading to negative reg-ulation of K19 gene transcription. Our data suggest thatPDX1 regulates K19 gene transcription indirectly by inter-action of its C terminus domain with MEIS1a and, thus,potentially modulating MEIS1a binding to DNA. However,we have no direct evidence for DNA binding of PBX1b to theK19 promoter, suggesting that PDX1 alone or possiblythrough the known PDX1/PBX1b complex interacts withMEIS1a, and this heterocomplex of PDX1/MEIS1a and/orPDX1/PBX1b/MEIS1a modulates K19 gene transcription ina manner that may suppress the effects of direct DNA bind-ing by MEIS1a. Thus, the nature of the association betweenPDX1 and the TALE proteins, MEIS, and PBX controls genetranscription in pancreatic acinar cells (27, 37) and ductalcells. Furthermore, we would propose that the particular

FIGURE 8. PDX1 and MEIS antibodies supershift in EMSA assays. A, radio-labeled 352 probe is incubated in presence of 5 �g of WT-PDC nuclear extractfor 20 min at room temperature. For supershift assay, WT-PDC nuclearextract (NE) is preincubated with nonspecific IgG, anti-PDX1 (3 �l), anti-MEIS (3 and 6 �l), or anti-PBX (3 �l) antibodies for 20 min on ice beforeincubation with the radiolabeled 352 probe. Arrows indicate bands shiftedand supershifted. B, after formaldehyde cross-linking and sonication,chromatin from WT-PDC was immunoprecipitated with either anti-MEISantiserum or normal IgG. The data were analyzed quantitatively usingreal-time PCR with primers designed to amplify the putative MEIS bindingregion of the K19 promoter (�337 to �253) or a distal TAAT-containingelement of the albumin promoter. For each primer pair ChIP signals werecompared with a dilution series of input chromatin to account for differingamplification efficiencies and are expressed here as such. These data rep-resent the means � S.E. of two independent experiments done in dupli-cates. The asterisk signifies a p value�0.02. C, 20 �g of nuclear extractsfrom WT-PDC were incubated with 2 �g of biotinylated K19-352 oligonu-cleotide, CMV2 oligonucleotide, scramble oligonucleotide, or increasingamounts (2–50-fold excess) of unlabeled K19-352 oligonucleotide (forcompetition assay) for 30 min. The DNA-protein complexes were thenprecipitated by streptavidin-agarose beads and washed, and the reactionmixtures were resolved by SDS-PAGE. MEIS and PBX interactions weredetected by Western blot.

FIGURE 9. The C terminus domain of PDX1 interacts with MEIS1a. GSTpulldown assays are shown. A purified fusion protein consisting of GSTfused to the intact PDX1 or the C terminus domain of PDX1 (206 –283) wasincubated with in vitro transcribed-translated 35S-labeled MEIS1a alone orin combination with 35S-labeled PBX1b. GST incubated with 35S-labeledMEIS1a served as negative control. After washing, proteins bound to glu-tathione beads were resolved by SDS-PAGE, dried and exposed to film.Western blot staining for PDX1 was used as a control for equal proteinloading.




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MEIS isoform is critical in the regulation of gene transcrip-tion in acinar (MEIS2b) versus ductal cells (MEIS1a/1b),whereas PBX1b expression is likely constant. We would alsosuggest that whereas in acinar cells PDX1/MEIS2b/PBX1b(domain B) and p48/PTF1 (domain A) are necessary for theacinar specific elastase transcriptional activity, PDX1/MEIS1a/PBX1b (domain B “equivalent”) and KLF4 (domainA equivalent) are necessary for the ductal K19 transcrip-tional activity (3, 27, 37). This does not preclude the possi-bility of recruitment of coactivators (e.g. p300, CBP (cAMP-response element-binding protein (CREB)-binding protein))and/or co-repressors by PDX1 (30, 40–46). Our results arefurther highlighted by the functional characterization ofthese transcription factors in primary pancreatic ductal cellsthat provide biological relevance. The potential role of PDX1in ductal cells either as a marker of putative progenitor cellsor during states of pancreatic regeneration has beenreported previously (47–49). Additionally, phosphorylatedPDX1 has been noted in ductal cells and in islet cells; how-ever, how this form of PDX1 modulates PDX1-mediatedgene expression is not known (50).A model that emerges from our studies would complement

developmental studies that indicate PDX1 is necessary for earlypancreatic development and proper specification of the endo-crine lineage (20). PDX1may be necessary for proper formationof the acinar cellular compartment. In the absence of PDX1,achieved by a regulatable system, acini do not form in theappropriate fashion, and yet, immature ductal cells do form(20). This might mean that PDX1 needs to be degraded orsequestered fromductal cells during development, thereby per-mitting MEIS1a and/or MEIS1b as well as KLF4 to exert theirpositive regulatory effects upon the ductal lineage, such as inK19. Future investigations are geared to understand how thistranscriptional regulatorymachinery exerts its effects inmousemodels.

Acknowledgments—We thankmembers of the Rustgi and Stoffers lab-oratories for discussions.

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Stoffers and Anil K. RustgiTherese B. Deramaudt, Mira M. Sachdeva, Melanie P. Wescott, Yuting Chen, Doris A.

Pancreatic Ductal Cell-specific Keratin 19 PromoterThe PDX1 Homeodomain Transcription Factor Negatively Regulates the

doi: 10.1074/jbc.M605891200 originally published online October 20, 20062006, 281:38385-38395.J. Biol. Chem.

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