MOLECULAR AND CELLULAR BIOLOGY, June 1989, p. 2464-24760270-7306/89/062464-13$02.00/0Copyright © 1989, American Society for Microbiology

Identification of a Cell-Specific DNA-Binding Activity ThatInteracts with a Transcriptional Activator of Genes

Expressed in the Acinar PancreasM. COCKELL, B. J. STEVENSON,+ M. STRUBIN, 0. HAGENBUCHLE, AND P. K. WELLAUER*

Sw iss Institiute for Exper-imental Canc er Research, 1066 Epalinges, Switzerland

Received 9 January 1989/Accepted 8 March 1989

Footprint analysis of the 5'-flanking regions of the oa-amylase 2, elastase 2, and trypsina genes, which are

expressed in the acinar pancreas, showed multiple sites of protein-DNA interaction for each gene. Competitionexperiments demonstrated that a region from each 5'-flanking region interacted with the same cell-specificDNA-binding activity. We show by in vitro binding assays that this DNA-binding activity also recognizes a

sequence within the 5'-flanking regions of elastase 1, chymotrypsinogen B, carboxypeptidase A, and trypsindgenes. Methylation interference and protection studies showed that the DNA-binding activity recognized a

bipartite motif, the subelements of which were separated by integral helical turns of DNA. The &-amylase 2cognate sequence was found to enhance in vivo transcription of its own promoter in a cell-specific manner,

which identified the DNA-binding activity as a transcription factor (PTF 1). The observation that PTF 1 boundto DNA sequences that have been defined as transcriptional enhancers by others suggests that this factor isinvolved in the coordinate expression of genes transcribed in the acinar pancreas.

The regulation of transcription in eucaryotes is mediatedby protein components that interact with specific DNAsequences and thereby modify the rate at which RNApolymerase initiates at adjacent promoter sites. A consider-able number of cis-acting DNA elements and trans-actingprotein factors have now been identified in viral and cellulargenes (for reviews, see references 10 and 14). The prevailingview is that regulation of transcription from a particularpromoter involves the concerted activity of multiple proteincomponents within the transcription complex. The details ofhow transcription factors interact with each other to form anactive transcription complex are still not understood. Theapparent complexity of these interactions may be dictated bythe need of the cell to modulate the promoter efficiency ofmany of its genes in a temporal manner during development,cell differentiation, and the cell cycle or in adaptation todifferent physiological stimuli.

Little is known about the regulatory factors involved inthe expression of genes encoding the specialized functions ofa cell, since elucidation of a general principle is hampered bythe fact that cell-specific expression of eucaryotic genes isregulated in a modular way. For instance, expression of thegenes encoding liver-specific functions depends on the con-certed activity of several factors that bind to differentpromoter elements (2, 8, 12). The activities of some of thesefactors are apparently restricted to hepatocytes (18).We have studied a set of genes that are expressed in acinar

pancreatic cells. Expression of the genes for o-amylase 2,elastase 2, and trypsina has been previously shown to beregulated at the transcriptional level by means of strongpromoters (6, 23). In this paper, we present evidence that thesame cell-specific transcription factor binds in vitro to con-

served DNA motifs that are located in the 5'-flanking regionsof these and other genes expressed in the acinar pancreas.

* Corresponding author.t Present address: Institute of Molecular Biology, University of

Oregon, Eugene, OR 97403.

MATERIALS AND METHODS

Tissue culture conditions. AR42J and AR4IP rat cells (9)were cultured at 37°C and 5% CO2 in a medium containingnutrient broth F12K (custom made by GIBCO Laboratories,Grand Island, N.Y.) supplemented with 15% fetal calf se-

rum, 3 mM thymidine, 330 mM hypoxanthine, 18 mMsodium bicarbonate, 100 mM dexamethasone, and penicillinand streptomycin (each at 100 UIml). Mouse L cells were

cultured in minimal essential medium (GIBCO) containing10% fetal calf serum. AR42J cells were maintained as

subconfluent cultures and were trypsinized every 8 to 9days. Subcultures were made at a subcultivation ratio of 1:5(106 cells per 10-cm-diameter petri dish). AR4IP cells were

grown to confluency and subcultivated at a ratio of 1:40 (5 x105 cells per 10-cm-diameter petri dish).

Preparation of nuclear extracts and affinity column purifi-cation. Crude nuclear extracts from AR42J, AR4IP, and Lcells were prepared essentially as described by Dignam et al.(4) except that cell homogenization and nuclear lysis bufferscontained a mixture of specific and nonspecific proteaseinhibitors. These were used at the following final concentra-tions: 0.1 mM phenylmethylsulfonyl fluoride, 10 p.M tolyl-sulfonyl phenylalanyl chloromethyl ketone, 10 p.m N-oa-p-tolyl-L-lysine chloromethyl ketone, and 8 p.l of Aprotininper ml (all from Sigma) and 0.5 p.g each of pepstatin,antipain, and leupeptin per ml (all from Peptide Institute).Extracts containing final concentrations of 7 to 10 mg ofprotein per ml were stored at -70°C.DNA-binding activity was partially purified by DNA af-

finity column chromatography. Oligonucleotides containingthe Amy2-IV cognate sequence were biotinylated and immo-bilized on a streptavidine-agarose matrix essentially as de-scribed by Chodosh et al. (3). Nuclear extract was applied toaffinity columns in 135 mM KCl-20 mM N-2-hydroxyeth-ylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.9)-0.5EDTA-5 mM MgCl2-12% glycerol-5 mM dithiothreitol-0.1% Triton X-100. The column was washed with 10 volumesof the same buffer containing 100 p.g of single-strandedsalmon sperm DNA per ml, followed by 10 volumes of the

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same buffer without competitor DNA. The binding activitywas eluted from the column in 450 mM KCl-20 mM HEPES(pH 7.9)-2 mM EDTA-5 mM MgCl,-12% glycerol-).1%Triton X-100-250 p.g of bovine serum albumin per ml.DNase I footprinting. Restriction fragments of cx-amylase 2

(nucleotides +15 to -224), elastase 2 (nucleotides +3 to-170), and trypsin' (nucleotides +3 to -189) were clonedinto the SiniaI site of an SP65 vector and end labeled at eitherthe HinzdIII (c-amylase 2) or EcoRI (elastase 2 and trypsin"')site of the vector, using kinase or Klenow enzyme. Frag-ments were excised from the vector by cleavage at the Sacl(ot-amylase 2) or BamtiHI (elastase and trypsin') site of thepolylinker. End-labeled fragment (1 ng: specific activity. 5 xIO4cpm/ng) was incubated with 25 Vig of crude nuclearextract (7 to 15 mg/ml) for 30 min at room temperature. Thebinding reactions (20 [l) were carried out in 12 mM HEPES(pH 7.9)-60 mM KCl-0.12 mM EDTA4.8 mM dithiothrei-tol-12% glycerol-1 mM MgCl,, with 1 ,ug of poly(dl-dC) as anonspecific competitor. For footprint competition experi-ments, appropriate concentrations of unlabeled syntheticoligonucleotides were added to nuclear extract in standardbinding conditions just before addition of the 32P-labeledDNA fragments. Immediately before DNase I digestion, theMgCl concentration was raised to 3 mM. DNase I wasadded to a final concentration of 5 pg/ml, and the mixturewas digested for 30 s to 3 min at room temperature. NakedDNA was digested under the same conditions (withoutnuclear extract) at approximately 10-fold-lower DNase Iconcentrations. Digestions were terminated by addition of100 mM Tris hydrochloride (pH 7.9)-100 mM NaCl-1%sodium dodecyl sulfate-10 mM EDTA-25 ,ug of Esclherichiacoli DNA per ml. Samples were treated with proteinase K(50 ,ug/ml) for 30 min at 37°C before nucleic acids wereextracted with phenol and precipitated with ethanol. TheDNase I-resistant material was analyzed on 6% sequencinggels.

Preparation of synthetic oligonucleotides. Sequences cor-responding to potential protein-binding sites were synthe-sized on an oligonucleotide synthesizer and purified on 20%sequencing gels. Oligonucleotides containing 5'-flankingsequences of rat genes were synthesized according to pub-lished sequences for carboxpeptidase A (20), chymotryp-sinogen B (1), and elastase 1 (7). The sequence of oligonu-cleotides was verified by sequence analysis, using thechemical method of Maxam and Gilbert (15). For competi-tion studies, complementary oligonucleotides were mixedand reannealed at a concentration of 50 [Lg/ml each in 10 mMTris hydrochloride (pH 7.9)-S5 mM MgCl, by heating to 60°Cfor 5 min, followed by slow cooling to room temperature.

Gel retardation assays. A 0.2-ng sample of 32P-labeledprobe and 10 [.g of crude nuclear extract were incubated for30 min at room temperature in 20 [l of binding buffer (4)containing, in addition to standard components, 5 mMdithiothreitol, 5 mM MgCl,, and 1 to 3 ,ug of single-strandedE. coli competitor DNA. In competition experiments, bind-ing reactions contained 10 ng of synthetic oligonucleotidecompetitor DNA, which was added just before the 32P-labeled probe. Formation of protein-DNA complexes wasassayed by electrophoresis of the binding mixtures on 2%agarose gels. Gels were run in 0.5x Tris-borate buffer (pH8.3) at 40 mA for 2 h in the cold. Binding assays using affinitycolumn-purified material were carried out under similarconditions in the absence of nonspecific competitor DNA.

G-methylation protection and interference. For interfer-ence studies, G residues were partially methylated withdimethyl sulfate (DMS) (19) and incubated with either crude

nuclear extract or affinity column-purified material as de-scribed above. For protection assays, binding reactions werecarried out under standard conditions for 30 min at roomtemperature. DMS was then added to a final concentration ofabout 0.5%, and incubation was continued for 5 min. Sam-ples were then applied immediately to the gel, withoutaddition of DMS stop buffer. Small portions of each bindingreaction were electrophoresed on analytical gels, whichwere dried down and exposed to ensure that gel retardationpatterns were not altered by DMS treatment. The bulk ofeach binding reaction was electrophoresed on preparative2% low-melting-point agarose gels, and lanes containing thereactions were cut into 2-mm slices. Peaks corresponding tobound and unbound DNA were identified by Cerenkovcounting of the slices. and their positions were verified bycomparison with positions on the analytical gels. DNA fromthe appropriate gel slices was electroeluted, phenol ex-tracted, and ethanol precipitated. DNA was cleaved withpiperidine and subjected to electrophoresis on 12% sequenc-ing gels as described by Maxam and Gilbert (15).

Construction of hybrid genes. 5' deletions of the cx-amylase5'-flanking region (21) containing a common 3' end (nucleo-tide position +15) were produced by restriction enzyme orBAL 31 digestion. HindIll linkers were added to the blunt-ended DNA fragments, which were then inserted into theunique Hindlll site of a pSVO-TK vector containing the1.477-base-pair (bp) BglII-NcoI fragment of the herpes sim-plex virus thymidine kinase (TK) gene (nucleotides +253 to+1730; 16). The BglII site of this fragment was blunt endedand converted into a unique HinzdIII site by linker addition.The pRSV-TK vector was generated by adding Hi,idIIIlinkers to the 545-bp NdeI-HphI restriction fragment ofpRSV-CAT, containing the Rous sarcoma virus (RSV) pro-moter (nucleotides +5 to -540; 5). This fragment was theninserted into the unique HindIll site of pSVO-TK. Thenewly generated boundaries of all of these constructs wereverified by Maxam and Gilbert (15) sequence analysis.

Transfection protocols. AR42J and AR4IP cells were trans-fected with DNA for transient expression 6 and 4 days,respectively, after trypsinization. cx-Amylase-TK DNA (10pLg) was cotransfected with 10 ,ug of RSV-TK DNA, whichwas used as an internal control to monitor transfectionefficiencies. DNA precipitates were formed at room temper-ature for 5 to 10 min by using a calcium phosphate precipi-tation method (13) and were then added to the culturemedium. Chloroquine was added 3 h later to a concentrationof 100 mM; after an additional 3h, cells were shocked for 2min with 20% glycerol in serum-free medium and washedrapidly three times with 5 ml of 30 mM Tris hydrochloride(pH 7.5-S150 mM NaCl. Fresh culture medium was thenadded.RNA analysis. At 24 h after transfection, cytoplasmic

poly(A)+ RNA prepared from cells transfected with TKhybrid genes was extracted from postnuclear supernatantswith chloropane as described by Schibler et al. (22). TKmRNAs containing correct 5' termini were identified byprimer-extended cDNA synthesis. The method of McKnightand Kingsbury (7) was used with the following modifica-tions: a 32P-end-labeled oligodeoxynucleotide primer spe-cific for TK mRNA (nucleotides +91 to +130 of the TKgene; 16) was hybridized to poly(A) RNA for 5 h at 60°C.cDNA synthesis was performed at 42°C, and the productsfrom this reaction were extracted by chloropane and precip-itated with ethanol. cDNA extension products were sub-jected to electrophoresis in 6% sequencing gels. Appropriate

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FIG. 2. Gel retardation analysis of a-amylase 2, elastase 2. and trypsina 5'-flanking sequences that are specifically protected by AR42Jnuclear extracts. Samples (0.2 ng) of 32P-end-labeled, double-stranded oligonucleotides corresponding to the a-amylase 2 sequence Amy2-IV(-122 to -158) (A), the elastase 2 sequence EL2-I (-65 to -92) (B), and the trypsin' sequence Trpa-II (-83 to -115) (C) were incubated withcrude nuclear extracts from AR42J, AR4IP, and L cells. The protein-DNA complexes were separated by electrophoresis on 2% agarose gels.Lanes: 1, no extract added; 2, binding reactions with AR42J nuclear extract: 3, binding reactions containing AR42J extract plus 10 ng of coldhomologous oligonucleotide; 4, binding reactions containing AR42J extract plus 50 ng of an equimolar mixture of heterologous oligonucle-otides (the sequences of these were derived from footprint regions Amy2-V, Amy2-111, Amy2-11, Amy2-I, and EL2-II); 5, binding reactionswith AR41P extract; 6, binding reactions with L-cell nuclear extract. Positions of protein-DNA complexes a., and y and of unbound DNAare indicated.

DNA molecular weight markers were migrated alongside forcomparison.

RESULTS

Footprint analysis of the 5'-flanking sequences of pancreas-

specific genes reveals cell-specific patterns of DNA-bindingproteins. In an attempt to identify nuclear factors that mightbe essential for expression of genes in the acinar pancreas ofrodents, we carried out in vitro DNase I footprinting of5'-flanking regions of the mouse ox-amylase 2, elastase 2, andtrypsina genes, using crude nuclear extracts from expressingand nonexpressing cell lines. The AR42J cell line, which wasderived from an acinar tumor of rats (9), transcribes oa-

amylase 2, elastase 2, and trypsin genes, although at a lowerrate than does mouse or rat pancreas. In contrast, neither theAR4IP rat cell line (which is derived from the same tumor as

are AR42J cells) nor mouse L cells express these pancreas-specific functions.The 5'-flanking regions of all three genes yielded complex

patterns of DNase I protection irrespective of the source ofcrude nuclear extract used (Fig. 1). The complexity of theseinteractions suggests that multiple factors may be involvedin the expression of each gene. The present analysis islimited to the search for factors that interact with DNAelements known to be essential for the control of pancreas-

specific transcription. In the (x-amylase 2 5'-flanking region

(+15 to -224), DNA domain Amy2-IV (-121 to -158) was

protected from nuclease digestion on the coding and non-

coding strands by nuclear protein of AR42J cells only (Fig.1A, lanes 2 and 5). The Amy2-IV domain lies within a DNAsequence that has been defined as the cell-specific transcrip-tional enhancer of the a-amylase gene (1). The noncodingDNA strand containing elastase 2 5'-flanking sequences (+3to -170) encompassed a DNA domain, EL2-I (-68 to -90),that showed more extensive protection with AR42J nuclearextract than with extract of nonexpressing cell lines (Fig.IA, lane 2). Likewise, the noncoding DNA strand containingthe 5'-flanking sequences (+3 to -189) of the trypsina gene

displayed a DNA domain, Trpa_II (-98 to -128), that was

protected from digestion in a distinct manner by extract ofAR42J cells (Fig. 1A, lane 1). The footprint analysis of theelastase 2- and trypsina_coding strands (not shown) did notaid in defining the boundaries of the different protectedregions because their free DNA patterns contained longtracts of poorly cut bases. Nucleotide sequence comparisonof Amy2-IV, EL2-I, and Trpa_II DNAs showed that theseelements shared many nucleotides (see Results). No signif-icant sequence homologies have been detected betweenother DNA domains that are specifically protected by AR42Jextract.The a-amylase, elastase, and trypsin genes contain a 5'-

flanking element that recognizes the same cell-specific DNA-

FIG. 1. DNase I footprint analysis of the ox-amylase 2, elastase 2. and trypsin;' 5'-flanking regions, using crude nuclear extracts fromexpressing (AR42J) and nonexpressing (AR41P and L) cells. End labeling of DNA fragments. binding reactions, and digestion conditions were

carried out as described in Materials and Methods. (A) Digestion products analyzed by electrophoresis on 6% sequencing gels. The a-amylase2 fragment (+15 to -224) was digested either without extract added (lanes 1 and 4) or in the presence of nuclear extract from AR42J (lanes2 and 5) and AR41P (lanes 3 and 6) cells: the elastase 2 fragment (+3 to -170) was digested either without extract added (lane 1) or in thepresence of nuclear extract from AR42J (lane 2). AR4IP (lane 3). and mouse L (lane 4) cells: the trypsin fragment (+3 to -188) was digestedeither without extract added (lane 1) or in the presence of extract from AR42J (lane 2). AR41P (lane 3). and mouse L (lane 4) cells. G+Achemical cleavage reactions were used as sequence markers. DNA domains protected by nuclear extracts from AR42J cells are indicated bvbrackets on the left side of each autoradiograph: those protected by nuclear extracts from AR41P and L cells are indicated on the right. Theposition of nucleotide + 1 is shown for the a-amylase 2 sequence. and the -40 position is shown for elastase 2 and and trypsin ' sequences.The dashed line extending from protected region 11 of the trypsin fragment indicates an area of sequence that demonstrated variable degreesof protection by AR42J extract in different experiments. (B) Summary of protein-DNA interactions in the a-amylase 2. elastase 2. and trypsin5'-flanking regions. Symbols: _. DNA domains protected by nuclear protein of AR42J cells: -. DNA domains protected by nuclearproteins of AR41P and L cells: *, end at which the DNA strand was labeled. Nucleotide positions of the boundaries of protected regions are

indicated. TAT motifs are boxed, and +1 positions are indicated.

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2468 COCKELL ET AL.

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FIG. 3. DNase I footprinting analysis of protein-DNA com-

plexes from gel retardation assays. An end-labeled 70-bp restriction

fragment (-86 to 155) of the oa-amylase 2 gene. containing Amy2-1IV sequences (-122 to -155), was incubated in the presence of

AR42J nuclear extract and then partially digested with DNase The

material from the binding reaction was subjected to electrophoresis

on 2% low-melting-point agarose gels, and bands corresponding to

complexes and to unbound DNA were eluted separately. DNA

extracted from the gel pieces was analyzed on a 14% sequencing gel.

DNAs from complex at (lane 2), complex (lane 3). and unbound

DNA (lane 1) were migrated alongside a DNase I digest of the naked

restriction fragment (lane 4). The position of theAmyi-IV sequence

within the 70-bp fragment is indicated.

binding activity. To determine whether the sequences of

individual footprint domains constitute recognition sites for

DNA-binding proteins when not in the context of their

natural flanking sequences, we carried out gel retardation

and competition assays. For this purpose, we synthesized

oligonucleotide probes bearing sequences that were pro-

tected from DNase I digestion in nuclear extracts of AR42J

cells (Fig. 1). 32P-labeled oligonucleotides were then assayed

separately for the ability to bind protein(s) in crude nuclear

extracts of expressing (AR42J) and nonexpressing (AR41P

and L) cells. Binding reactions with Amy2-IV (37-bp), EL2-I(26-bp), and Trp"'-II (34-bp) oligonucleotides and AR42Jextract yielded three bands (a, 3, and y), which definedprotein-DNA complexes of different electrophoretic mobili-ties (Fig. 2). Two of these complexes, a and ,B, were specificfor the AR42J extract, whereas a third. y, was common to allextracts. The y complex, which was the only reproducibleprotein-DNA interaction detected in nonexpressing cells,appeared not to be sequence specific and was not examinedin detail. The Amy2-IV, EL2-I, and Trp-II ax and 1 com-plexes were judged to be the results of sequence-specificprotein-DNA interactions, since their formation was inhib-ited by a 50-fold molar excess of unlabeled homologousoligonucleotide. They were not competed against by amixture of oligonucleotides derived from other footprints:Amy2-I, -II, and -III, EL2-II, and Trp'-III (Fig. 2, lanes 4).To confirm that the AR42J cell-specific DNA-binding

activities detected by the footprint and gel retardation as-says, respectively, resulted from the same protein-DNAinteractions, we carried out a gel retardation assay on DNaseI-treated material. A 70-bp restriction fragment of ac-amylase2 (-85 to -155), which encompassed the Amy2-IV se-quence, was incubated with crude nuclear AR42J extractand partially digested with DNase I. Complexes a and awere then separated from unbound Amy2-IV DNA byelectrophoresis. Amy2-IV DNA present in both complexeswas protected in the same manner from DNase I digestion(Fig. 3). The boundaries of the footprints were very similarto those previously determined for footprint Amy2-IV on thea-amylase 2 5'-flanking sequence (Fig. 1A). This resultindicates that the same protein-DNA interactions were visu-alized by the two methods.The gel retardation assays showed that Amy2-IV, EL2-I,

and Trp-II oligonucleotide sequences each formed specificcomplexes of similar electrophoretic mobilities with nuclearproteins of AR42J cells. To test whether a common DNA-binding activity recognizes all three sequences, we assayedthe ability of a number of oligonucleotides to inhibit forma-tion of the AR42J cell-specific footprint Amy2-IV (Fig. 4A).We observed that oligonucleotides, Amy2-IV, EL2-I, andTrp'-II efficiently competed for the DNA-binding activitythat generated footprint Amy2-IV (Fig. 4A). Titrations withall three oligonucleotides had an endpoint around 50-foldmolar excess. In contrast, a 1,000-fold molar excess ofoligonucleotides corresponding to a number of other foot-print domains did not compete against footprint Amy2-IV(Fig. 4A and data not shown). Similar results were obtainedin competition experiments directed against footprints of theelastase 2 (not shown) and the trypsinL' 5'-flanking regions(Fig. 4B). The Trp'-II footprint was peculiar in that only partof its sequence recovered the DNase I pattern of naked DNAupon competition. We interpret this to mean that additionalDNA-binding activities are responsible for the extensiveprotection of this region. Nevertheless, the results fromfootprint competition analysis are in favor of a commonDNA-binding activity that recognizes Amy2-IV, EL2-I, andTrp"'-II sequences. The minimal sequence requirements forbinding of this nuclear factor must be close to those specifiedby the oligonucleotides. This conclusion is based on theobservation that oligonucleotides Amy2-IV' and Amy2-IV",which contained deletions of terminal Amy2-IV sequences,competed against the Amy2-IV footprint in its entity at leastan order of magnitude less efficiently than did oligonucleo-tides with the full sequence (Fig. 4A).

Cell-specific DNA-binding activity recognizes elements inthe 5'-flanking regions of other genes coding for abundant

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FIG. 5. Analysis of complexes ffactor from AR42J cells and upstreare specifically expressed in theoligonucleotides used are the cogn;print and gel retardation analyses oatrypsina (Trpa_II), elastase 2 (EL2-sequence comparison in the mouse1 (ELI), carboxypeptidase A (C(ChyB) genes. Formation of comiments and factor from AR42J cells v

The AR42J factor was partially IAmy2-IV DNA affinity columns.incubated with equal amounts of(B) End-labeled Amy2-IV oligonulpurified binding activity in the abmolar excess of unlabeled homoloiotides. Positions of unbound DNA zand ,B are indicated.

products of the acinar pancreas. The nucleotide homologies-- _ of Amy2-IV, EL2-1, and Trpa-II DNA sequences were found

to be essentially clustered in two regions (see Fig. 7).>--< > Inspection of published sequences of the genes encoding

elastase 1 (7), chymotrypsinogen B (1), and carboxypepti-dase A (20) in the rat pancreas allowed us to identify, withintheir 5'-flanking regions, sequence elements bearing closeresemblance to the mouse gene motifs. On the basis of these

*u*Uii3 sequence comparisons, we synthesized oligonucleotides cor-responding to the three rat gene elements in order to testtheir ability to be recognized by the DNA-binding activitypresent in AR42J cells. The presence of nonspecific DNA-

'_IIIIhIhhIm' binding proteins in crude nuclear extracts precludes a pre-cise determination of the relative affinity of the specificfactor for the various elements. Therefore, the bindingpotential of candidate elements from the rat genes wasassayed in gel retardation experiments by using factor thatwas partially purified by chromatography on affinity columnscontaining Amy2-IV oligonucleotide. All three rat sequencesand an additional sequence found upstream of the mouse

-- - trypsind gene (23) formed two protein-DNA complexes (otand 3) that had mobilities similar to those formed by

K- A, -stv vAmy2-IV, EL2-I, and Trpa-II sequences (Fig. 5A). Further-s _ _ _ ' 'more, a 50-fold molar excess of each of these sequences

effectively inhibited formation of Amy2-IV complexes (Fig.SB). As with crude nuclear extracts, this competition wassequence specific, since a mixture of unrelated oligonucleo-tides did not compete (data not shown). Some variabilityexisted among individual sequence elements in both theefficiency of binding purified factor and the ability to inhibitAmy2-IV complex formation. Those differences in affinitywere comparatively minor and were resolved only with the

- partially purified binding activity. They were most conspic-uous for the elements found in front of the trypsin andelastase 2 genes and were not unexpected, since the variousDNA sequences are only partially homologous. The results

ormed between partially purified from gel retardation analysis with purified factor confirmedam DNA elements of genes that and extended the conclusion drawn from experiments withacinar pancreas. The synthetic crude nuclear extracts, that 5'-flanking elements of sevenate sequences identified by foot- different genes expressed in the pancreas of rodents recog-f mouse a-amylase 2 (Amy2-IV), nize a common DNA-binding activity.-1) genes and those identified by To define more precisely the sequence requirements fortrypsind (Trpd), the rat elastase binding of the factor to the different elements, we carried outpA), and chymotrypsinogen B comprehensive G-methylation interference and protectionwas analyzed on 2e agarose gels. footprinting. G residues that are protected from methylationpurified by chromatography on as a consequence of factor binding and those interfering withA) End-labeled oligonucleotides factor binding because of methylation are generally assumedAR42J-specific binding activity. to reflect regions of close protein-DNA contact. The probescleotide incubated with affinity- for these studies were prepared by cloning the oligonucleo-)sence or presence of a 50-fold tides of Fig. 5 into SP65. DNAs were end labeled at agous or heterologous oligonucle- restriction site of the polylinker and excised from the vectorand of protein-DNA complexes a by cleavage at a second site. The a and a complexes

obtained from such binding reactions were separated by gelelectrophoresis. The DNA patterns resulting from cleavage

FIG. 6. Interaction of partially purified factor from AR42J cells with its cognate sequences. The synthetic oligonucleotides used in thebinding reactions shown in Fig. 5 were cloned into plasmid vectors and excised by cleavage at restriction sites within the vector. DNAfragments containing 32P-end-labeled coding or noncoding strands were used in binding reactions with partially purified factor from AR42Jcells. The DNAs were methylated with DMS either before or after binding of the factor to generate methylation protection (P) or interference(I) patterns, respectively. Material from the various binding reactions was electrophoresed on 2% low-melting-point agarose gels. The DNAwas extracted from bands corresponding to protein-DNA complexes (a and 1) or to unbound DNA and then cleaved with piperidine. Thepatterns of methylated G residues were analyzed by electrophoresis on 12% sequencing gels. Lanes: 1, naked DNA; 2, unbound DNA frombinding reactions; 3, DNA from complex a: 4, DNA from complex P. Positions of factor cognate sequences within DNA fragments ([), of Gresidues that are strongly (4) and weakly (<) protected from methylation in the protein-DNA complexes, and of G residues for whichmethylation strongly (D) and weakly (O) interferes with binding of factor are indicated.

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at G residues were analyzed individually on sequencing gels.A few representative examples from binding reactions withpurified factor are shown in Fig. 6. Patterns derived frombinding reactions containing crude nuclear extract gavequalitatively the same results (data not shown). Methylationpatterns derived from ot and 1 complexes were found to beidentical for all DNA sequences (shown for Amy2-IV; Fig.6A).The data from this comparative study were compiled in

the maps presented in Fig. 7, which show that the protectionand interference patterns of all DNA sequences have certainfeatures in common. In general, the G residues that interferewith factor binding coincide with those protected by thefactor. In each DNA element, these G residues are clusteredin two distinct sequence domains, which are separated by aregion where no interaction with the protein can be detected.The topology of the two domains containing protected Gresidues is depicted in a helix diagram (Fig. 7B). The modelshows that the midpoints of the two domains are separatedfrom each other by approximately integral turns of B-formDNA. In six of seven sequences, the domains of interactingG residues are spaced about one turn apart. The exception tothis rule is the Amy2-IV element, in which these domains areseparated by approximately two helical turns. The DNAsequences that closely interact with the factor coincide ingeneral with those specifying two domains of high sequencehomology, termed the A box and the B box (Fig. 7A).Notable exceptions are the two trypsin elements, in whichonly one of the two domains of protected G residues mapswithin the A- or B-box homology. This finding is discussed indetail below (see Discussion). The maps of G-residue pro-tection and interference patterns are consistent with thenotion that the factor recognizes similar features within thevarious DNA elements.The Amy2-IV DNA sequence enhances transcription in vivo

in a cell-dependent manner. To determine the functional roleof the factor from AR42J cells identified by binding studies invitro, we assayed the activity of the factor indirectly bystudying the transcriptional effects of one of its cognatesequences in vivo. For this purpose, we constructed a seriesof hybrid plasmids containing coding sequences of the her-pes simplex virus TK gene under control of ox-amylase 25'-flanking sequences (Fig. 8A). These hybrid genes and aplasmid containing the TK gene under control of the RSVpromoter were cotransfected for transient expression intoAR42J and AR4IP cells. The RSV-TK hybrid gene served asa standard against which the transcriptional efficiency ofx-amylase 2-TK genes was measured.Cytoplasmic poly(A)+ RNAs of transfected cells were

assayed for content of TK RNA transcripts by primer-extended cDNA synthesis (Fig. 8B). The expected primerextension products from cx-amylase 2-TK and RSV-TKgenes measured 104 and 88 bases, respectively. The TKplasmid containing ox-amylase 2 sequences +15 to -224 was

efficiently transcribed in AR42J cells. as evidenced by astrong band at 104 bases (lane 3). The observed transcriptionwas cell specific, since this band was not detected in RNA ofAR4IP cells (lane 2). Deletion of sequences between -100and -224 reduced transcription in AR42J cells to barelydetectable levels (lane 4). Insertion of the Amy2-IV element(nucleotides -122 to -158) upstream of a hybrid genecontaining ox-amylase 2 sequences +15 to -100 restoredtranscription to a level comparable to that of the -224-TKgene (lane 5). These data show that the Amy2-IV elementhas properties of a transcriptional activator and identify thenuclear DNA-binding activity of AR42J cells as a cell-specific transcription factor, which we have termed PTF 1(pancreas-specific transcription factor 1).

DISCUSSION

PTF 1 cognate sequences act as positive regulatory elements.The 5'-flanking regions of genes encoding the abundantproducts of the acinar pancreas share a sequence elementthat is recognized in vitro by a DNA-binding activity from anexocrine pancreatic cell line. We have shown that theelement Amy2-IV is essential for high-level expression fromthe o-amylase 2 promoter in vivo, which indicates that theDNA-binding activity identified in vitro is a transcriptionfactor (PTF 1). Three of the seven PTF 1 binding sitesidentified by protein-binding studies in vitro lie within theboundaries of positive regulatory sequences as defined by invivo analyses. In addition to our studies, which show thatthe PTF 1 cognate sequence is required for expression of themouse a.-amylase 2 gene, others have shown that an analo-gous region of the rat x-amylase gene (-108 to -165) isrequired for cell-specific transcription (1). These authorsalso found chymotrypsinogen B 5'-flanking sequences withinthe region -192 to -225 to be necessary for efficientexpression in AR42J cells. Elastase 1 5'-flanking sequences-71 to -205 are sufficient to direct pancreas-specific expres-sion in transgenic mice (7). Furthermore, analysis of linker-scanning mutants has shown that elastase 1 sequences -90to -120 are essential for efficient expression in a cell-dependent manner (11).

All of the sequence elements delimited by functionalanalysis comply with properties that have been assigned totranscriptional enhancers. They have been shown to func-tion with heterologous promoters and to act at a distance andindependent of their orientation (1, 7). It is interesting tonote that some of the properties associated with enhancersare reflected by PTF 1 binding sites in situ. The PTF 1binding sites, including those for which a functional signifi-cance has been directly demonstrated, occur in both orien-tations and at a variable distance, although only within a250-bp limit, of the cap site. It is not excluded that therelative position. orientation, or both of a particular PTF 1binding site may influence its interaction with other pro-

FIG. 7. Comparison of G-methylation protection and interference patterns of complexes between oligonucleotides and nuclear factor fromAR42J cells. (A) Maps based on analyses of the gels shown in Fig. 6 and of data not shown. Positions of G residues that are protected strongly(V) and weakly (V) from methylation and of those that interfere strongly (D) and weakly (C]) with factor binding are indicated. Protected Gresidues within the DNA of each complex lie in two regions separated by a spacer. Because of the variable lengths of the spacers. sequencehomologies were derived in the following way. The A- and B-box homologies were determined by comparing nucleotides starting from theleft and the right end, respectively, of the DNA elements. The consensus sequence was derived by counting the number of nucleotides presentat each position. Nucleotides that are homologous to the consensus are boxed. Numbers below the consensus represent frequencies ofidentical nucleotides at each position. X. Positions having identical nucleotides in less than half of all DNA sequences. Arrows indicate in situorientations of the elements with respect to the cap site. (B) Helix model showing stereospecificity of protected G residues within each DNAelement. The midpoints of the two DNA domains defined by protected G residues are plotted for each element on B-form DNA. Thesequences have been aligned with respect to the midpoint of one of these domains.

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c; O. synthetic oligonucleotide used as a primer for analysis of RNA from transfected cells; -, size of the expected cDNA extensionproducts. (B) Gel analysis. The 5' ends of the RNAs produced from RSV-TK and cx-amylase-TK hybrid genes during transient expressionin AR42J (lanes 3 and 5) or AR41P (lane 2) cells were mapped by electrophoresis of primer-extended cDNAs on a 6% sequencing gel. RNAfrom mock-transfected AR42J cells is shown as a control (lane 1). The expected sizes (in bases) of cDNAs (-) were estimated by comparisonwith appropriate molecular weight markers.

moter elements, thereby affecting the transcriptional effi-ciency of the promoter as a whole. Indeed, we have ob-served some long-range-distance effects of the PTF 1 bindingsite on expression of the zx-amylase 2 promoter in vivo. If thePTF 1 binding site is placed at distances greater than 2kilobases away from the cap site, its potential as a transcrip-tional activator is greatly reduced (unpublished observa-tions).The DNA-binding activity of PTF 1 has also been detected

in crude nuclear extracts of pancreatic tissue but not in othertissues, such as liver, kidney, and spleen of mice (S.Petrucco et al., submitted for publication). These observa-tions support the conclusion that PTF 1 activity is pancreasspecific but do not rule out the possibility that an inactiveform(s) of PTF 1 exists in cells of other origin. PTF 1 maynot be the only factor involved in cell-specific transcription,since an oL-amylase 2 promoter from which the PTF 1 bindingsite has been deleted is still preferentially active in exocrinepancreatic cells, although at a much reduced level (Fig. 8B).PTF 1 cognate sequences constitute a single protein-binding

domain. The PTF 1 binding sites are relative large cis-actingelements that are reiterated in all of the genes encodingpancreas-specific functions that we have examined. Despitethis, we and others had failed to pinpoint their essentialcharacter by sequence comparison alone. The reasons forthis are the bipartite organization of the elements withrespect to their interactions with the protein as well as thedegenerate nature of the recognition motifs. The existence oftwo distinct domains of close protein-DNA interaction asinferred from G-methylation protection and interferencestudies suggests that the PTF 1 cognate sequence mayrepresent not one but rather two separable protein-bindingsites. However, such a model is contradicted by the exper-imental evidence from protein-binding studies. Truncatedversions of the PTF 1 cognate sequence containing only oneof the two protein contact domains compete against theAmy2-IV footprint less efficiently than does the completesequence, but at high excess of competitor the footprint iscompeted against in its entity. Furthermore, no cell-specificprotein-DNA complexes with contacts in either site alone

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COORDINATE EXPRESSION OF PANCREAS-SPECIFIC GENES 2475

have been detected in gel retardation assays. We take thesedata to indicate that both domains of DNA sequencesinteracting with PTF 1 protein are necessary to constitute ahigh-affinity binding site. In the light of these observations, itis unlikely that any previously documented transcriptionfactors are candidate PTF 1 binding activities per se. How-ever, we cannot exclude that a member of the GT-IIB-likefamily of binding proteins (which includes AP4) is a potentialcomponent of PTF 1 binding activity. The cognate sequenceand interactions with G residues of GT-IIB coincide pre-cisely with those of the Amy2-IV B-box homology (24).The observation that the PTF 1 cognate sequence consti-

tutes a single protein-binding unit is compatible with modelsinvolving the binding of either a single or multiple proteins.If multiple proteins are involved, they would have to bind ina mutually dependent manner. In each of these models.either the protein or the DNA must be able to assumealternative conformations, since domains of protein-DNAinteractions are spaced at variable distances.

Interaction of protein with PTF 1 cognate sequences issubject to steric constraints. Our methylation protection andinterference studies show that, in general, PTF 1 factorinteracts with G residues that are located within DNAdomains of high sequence homology (boxes A and B). In allPTF 1 cognate sequences, with the exception of trypsingenes, the centers of the two boxes A and B are separated byapproximately integral turns of B-form DNA (Fig. 7A).

In the case of the trypsin PTF 1 cognate sequences, thecenters of boxes A and B are separated by nonintegral turnsof the DNA helix. The actual choice of contacts made by thePTF 1 factor shows that steric considerations outweigh thoseof best sequence fit for both trypsin PTF 1 binding sites.Close inspection of the trypsind element shows that its B-boxsequence has a higher homology with the consensus se-quence than does its A-box sequence. In protecting Gresidues within box B, PTF 1 makes its second set ofcontacts about one helical turn of DNA away, in a regionthat lies downstream of box A. In the trypsina PTF 1 bindingsite, box A has a higher homology than box B. In this case,it is box A which dictates the position of the second domainof protein-DNA interaction, again about one helical turn ofDNA away. This latter region has no significant homology tothe B-box consensus sequence except for the spatial ar-rangement of some of the affected G residues. Evidently, theparticular arrangement of G residues within this regionproves sufficient for the protein to select it over randomsequence.The trypsin sequences have the lowest affinity for PTF 1

factor, at least in vitro, since they compete less efficiently forprotein-DNA complex formation than do the other bindingsites tested. This reduced protein-binding potential may beexplained by the inability of the protein to bind to thosesequences which are expected to constitute optimal proteincontact sites. The peculiar organization of the two differenttrypsin PTF 1 binding sites may be related to their commonevolutionary origin. As members of a multigene family.trypsin genes have presumably arisen by duplication from acommon ancestor sequence.

It is tempting to speculate that PTF 1 is involved in theexpression of most if not all genes encoding the abundantfunctions of the acinar pancreas. Consistent with such amodel is the observation that the DNA-binding activity ofPTF 1 can first be detected in embryonic mouse pancreas atthe stage when pancreas-specific promoters are activated(Petrucco et al.. submitted). The molecular basis for thecell-specific activity of PTF 1 is not known at present.

Purification of the protein(s) and the gene(s) will be neededto discriminate between transcriptional and posttranscrip-tional mechanisms.

ACKNOWLEDGNIENTS

We thank R. Bovey and P. Garessus for expert technical assis-tance. We are indebted to R. J. Hay, C. Gorman. and S. L.McKnight for generous gifts of materials. We thank L. Kuhn foroligonucleotide synthesis and B. Hirt for continuing support. O.H.was supported by a career development award from the CloettaFoundation. This work was supported by a grant from the SwissNational Science Foundation.

LITERATURE CITED

1. Boulet, A. M., C. R. Erwin, and W. J. Rutter. 1986. Cell-specificenhancers in the rat exocrine pancreas. Proc. Natl. Acad. Sci.USA 83:3599-3603.

2. Cereghini, S., M. Raymondjean, A. G. Carranca, P. Herbomel,and M. Yaniv. 1987. Factors involved in control of tissue-specific expression of albumin gene. Cell 50:627-638.

3. Chodosh, L. A., R. W. Carthew, and P. A. Sharp. 1986. A singlepolypeptide possesses the binding and transcription activities ofthe adenovirus major late transcription factor. Mol. Cell. Biol.6:4723-4733.

4. Dignam, J. D., R. M. Lebowitz, and R. G. Roeder. 1983.Accurate transcription initiation by RNA polymerase 11 in asoluble extract from isolated mammalian nuclei. Nucleic AcidsRes. 11:1475-1489.

5. Gorman, C. M., G. T. Merlino, R. C. Willingham, I. Pastan, andB. H. Howard. 1982. The Rous sarcoma virus long terminalrepeat is a strong promoter when introduced into a variety ofeukaryotic cells by DNA mediated transfection. Proc. Natl.Acad. Sci. USA 79:6777-6781.

6. Hagenbuchle, O., U. Schibler, S. Petrucco, G. C. Van Tuyle, andP. K. Wellauer. 1985. Expression of mouse amy-2a alpha-amylase genes is regulated by strong pancreas-specific promot-ers. J. Mol. Biol. 185:285-293.

7. Hammer, R. E., G. H. Swift, D. M. Ornitz, C. J. Quaife, R. D.Palmiter, R. L. Brinster, and R. J. MacDonald. 1987. The ratelastase I regulatory element is an enhancer that directs correctcell specificity and developmental onset of expression in trans-genic mice. Mol. Cell. Biol. 7:2956-2967.

8. Hardon, E. M., M. Frain, G. Paonessa, and R. Cortese. 1988.Two distinct factors interact with the promoter regions ofseveral liver specific genes. EMBO J. 7:1711-1719.

9. Jessop, N. W., and R. J. Hay. 1980. Characteristics of two ratpancreatic exocrine cell lines derived from transplantable tu-mors. In Vitro 16:212.

10. Jones, N. C., P. W. J. Rigby, and E. B. Ziff. 1988. Trans-actingprotein factors and the regulation of eukaryotic transcription:lessons from studies on DNA tumor viruses. Genes 2:267-281.

11. Kruse, F., C. Komro, C. Michnof, and R. MacDonald. 1988. Thecell-specific elastase I enhancer comprises two domains. Mol.Cell. Biol. 13:893-902.

12. Lichtsteiner, S., J. Wuarin, and U. Schibler. 1987. The interplayof DNA-binding proteins on the promoter of the mouse albumingene. Cell 51:963-973.

13. Luthmann, H., and G. Magnusson. 1983. High efficiency poly-oma DNA transfection of chloroquine treated cells. NucleicAcids Res. 11:1295-1308.

14. Maniatis, T., S. Goodburn, and J. A. Fischer. 1987. Regulationof inducible and tissue-specific gene expression. Science 236:1237-1244.

1-5. Maxam, A. M., and W. Gilbert. 1977. A new method forsequencing DNA. Proc. Natl. Acad. Sci. USA 74:560-564.

16. McKnight. S. L. 1980. The nucleotide sequence and transcriptmap of the herpes simplex virus thymidine kinase gene. NucleicAcids Res. 8:5949-5964.

17. McKnight, S. L., and R. C. Kingsbury. 1982. Transcriptionalcontrol signals of a eukaryotic protein-coding gene. Science217:316-324.

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18. Monaci, P., A. Nicosia, and R. Cortese. 1988. Two differentliver-specific factors stimulate in vitro transcription from thehuman al-antitrypsin promoter. EMBO J. 7:2075-2087.

19. Ogata, R., and W. Gilbert. 1979. DNA-binding site of lacrepressor probed by dimethylsulfate methylation of lac opera-

tor. J. Mol. Biol. 132:709-728.20. Quinto, C., M. Quiroga, W. F. Swain, W. C. Nikovits, D. N.

Standring, R. L. Pictet, P. Valenzuela, and W. J. Rutter. 1982.Rat preprocarboxypeptidase A: cDNA sequence and prelimi-nary characterization of the gene. Proc. NatI. Acad. Sci. USA79:31-35.

21. Schibler, U., A. C. Pittet, R. A. Young, 0. Hagenbuchle, M.Tosi, S. Gellman, and P. K. Wellauer. 1982. The mouse alpha-amylase multigene family. Sequence organization of members

expressed in the pancreas, salivary gland and liver. J. Mol. Biol.155:247-266.

22. Schibler, U., M. Tosi, A. C. Pittet, L. Fabiani, and P. K.Wellauer. 1980. Tissue-specific expression of mouse Qt-amylasegenes. J. Mol. Biol. 42:93-116.

23. Stevenson, B. J., 0. Hagenbuchle, and P. K. Wellauer. 1986.Sequence organisation and transcriptional regulation of themouse elastase 11 and trypsin genes. Nucleic Acids Res. 14:8307-8329.

24. Xiao, J. H., I. Davidson, D. Ferrandon, R. Rosales, M. Vigneron,M. Macchi, F. Ruffenach, and P. Chambon. 1987. One cell-specific and three ubiquitous nuclear proteins bind in vitro tooverlapping motifs in the domain Bi of the SV40 enhancer.EMBO J. 6:3005-3013.

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