isolation and characterization of a protein fraction … journal of biological chemistry vol. 264,...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264, No. 36, Issue of December 25, pp. 21915-21922,1989 Printed in U.S.A.

Isolation and Characterization of a Protein Fraction That Binds to Enhancer Core Sequences in Intracisternal A-particle Long Terminal Repeats*

(Received for publication, June 9, 1989)

M. Falzon and E. L. Kuff From the Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

The U3 region of mouse intracisternal A-particle (IAP) long terminal repeats (LTRs) contains several nuclear protein-binding domains. Two of these contain sequences with homology to the SV40 enhancer core. We refer to these two domains as Enhl and Enh2. The Enh2 domain is an important determinant of promoter activity in vivo. We report here the isolation of nuclear fractions from human 293 and mouse MOPC-3 15 cells which interact with Enhl and Enh2. Purification was achieved via DNA-affinity chromatography on a multimerized oligonucleotide representing the Enh2 region from the LTR of the mouse genomic IAP element, MIA14. Glycerol gradient sedimentation suggested a native M, of -80-100 for the binding component(s) in both crude and affinity-purified fractions. UV cross- linking showed that the binding activity involved two polypeptides within this size range. The affinity-isolated fraction from each cell line was highly purified, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in vitro binding analysis. Ex- onuclease I11 footprinting showed that the two polypeptides interacted preferentially with the Enhl and Enh2 domains within a 139-base pair segment from the MIA14 LTR. The polypeptides interacted in a sequence-specific manner with oligonucleotides representing these domains within the IAP LTR and with oligonucleotides containing the enhancer core sequence from SV40 and polyoma virus. Equilibrium binding studies indicated that the apparent dissociation constants for the polypeptides binding to the enhancer core sequence from MIA14, SV40, and polyoma virus were similar. Therefore, this affinity-purified fraction may represent a novel enhancer core-binding component which is distinct from the previously characterized rat CCAATlenhancer-binding protein, C/EBP.

Intracisternal A-particles (IAPs)’ are encoded by endogenous genetic elements that have the overall structural orga- nization of integrated retroviral proviruses, with long terminal repeats (LTRs) flanking the protein-coding sequences (re- viewed by Kuff and Lueders (1)). There are about 1000 IAP elements per haploid genome in Mus musculus. Certain (un- determined) members of this multicopy gene family are tran-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: IAP, intracisternal A-particle; LTR, long terminal repeats; hp, base pair(s); Hepes, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid; DTT, dithiothreitol; PMSF, phenyl- methylsulfonyl fluoride; SDS, sodium dodecyl sulfate; CAT, chloramphenicol acetyltransferase; EBP, enhancer-binding protein.

scriptionally active during early mouse development, in some normal mouse tissues, and in many mouse tumor cells. IAP elements can transpose and act as endogenous mutagens by inserting at new locations in the genome. For example, IAP transpositions have been shown to activate the c-mos gene in a transplantable BALB/c plasmacytoma (2) and to induce the constitutive production of interleukin-3 in a murine myelo- monocytic leukemia (3).

Cloned LTRs from a number of IAP elements have effec- tively promoted transcription of linked reporter genes when transfected into both homologous and heterologous mamma- lian cells (4-6). Analysis of deletion mutants has shown that the U3 region is essential for promoter activity of IAP LTRs (4). Different segments of the U3 region fulfill different func- tions in promoting IAP LTR activity. Sequences downstream of the CCAAT box determine the basal transcriptional activity, while sequences upstream of the CCAAT box modulate these basal transcription levels (4).

We have previously demonstrated (7) by DNase I and exonuclease 111 footprinting that the LTR from a mouse genomic IAP element, designated MIA14 (8, 9) contains five binding domains for nuclear proteins in a 139-bp region upstream of the CCAAT and TATA motifs (Fig. la). Two of these domains include sequences homologous with the SV40 enhancer “core” (12). We refer to these domains as Enhl and Enh2 (Fig. lb ) .

In this study, we provide evidence that the Enh2 domain is an important determinant of IAP LTR promoter activity in. uiuo. To address in finer detail the role of the Enh2 domain in transcription regulation, we have sought to isolate and characterize the DNA binding protein(s) that interact with this region. Nuclear extracts from 293 and MOPC-315 cells, which were previously found to be rich in enhancer-binding activity (7), were fractionated by chromatography on heparin- Sepharose and DNA-affinity columns. A protein fraction was isolated that binds in a sequence-specific manner to the Enhl and Enh2 domains within the IAP LTR, as well as to oligonucleotides containing the SV40 and polyoma virus enhancer core sequences.

MATERIALS AND METHODS

Cell Lines and Nuclear Extracts-Nuclear extracts were prepared from tissue culture cells by the method of Parker and Topol (13) as modified by Ohlsson and Edlund (14). The following cell lines were used 293, an adenovirus type 5-transformed human kidney line (15) and MOPC-315, a mouse myeloma line. The cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum.

Plasmids and Oligonucleotides-The expression plasmids pMIAcat- 5’L and pMIAcat-3’L have been described (5). The plasmidpMIAcat- 3’L(del) was derived from pMIAcat-3’L by deleting the LTR region upstream of the PuuII restriction site at nucleotide position 48 (Fig. la) . All of these constructs contain the CAT gene downstream of the

21915

21916 Isolation of Enhancer Core Binding Protein IAP sequence and were prepared by cloning the respective 5’- or 3’- ends of the mouse IAP gene MIA14 into the plasmid pSVOcat (16). In this vector, the entire SV40 promoter region has been deleted, and a unique Hind111 site has been substituted for the insertion of other promoter sequences. A 173-bp HaeIII-HaeIII fragment extending from a position 33 bp upstream of the LTR to position 139 within the LTR was used as probe for exonuclease I11 footprinting. This fragment was cloned into pUC13 and has been described (7).

Transfection Procedures and ChloramphenicolAcetyltransferaseAs- say-293 cell transfections and measurement of the chloramphenicol acetyltransferase activity from the transfected cells were carried out as previously described (17).

In Vitro Methylation of Plasmids-The plasmids pMIAcat-5’L, pMIAcat-3’L, and pMIAcat-3’L(del) were methylated with HhaI methylase (New England Biolabs) as previously described (17).

Protein Purification-Nuclear extract protein (250-300 mg) in a volume of 30 ml was loaded onto a 20-ml heparin-Sepharose column equilibrated in 50 mM Hepes, pH 7.8, 100 mM KC1, 12.5 mM MgC12, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 20% glycerol (buffer A). The column was washed with buffer A, and the protein was subsequently eluted with st,epwise additions of buffer A containing KC1 of 0.1 M increments. Protein was monitored by absorbance a t 280 nm. The peak fractions from each elution step were pooled and dialyzed against buffer A. Activity was monitored by the gel retardation assay and by exonuclease I11 footprinting.

The sequence-specific DNA-affinity column was prepared by cou- pling of cyanogen bromide-activated Sepharose-CL4B with the double-stranded oligonucleotide (5’CTGCGCATATGCCGAGGGTGG- TTCTCTACT3’) that had been multimerized (average length = 15 monomers) by self-ligation via a Sau3AI 4-bp overhang at the 5’-end of each strand (18). We refer to the monomeric oligonucleotide as Enh2 (Fig. lb ) . The column (2.5 ml) was equilibrated with 50 mM Hepes, pH 7.8, 100 mM KC1, 12.5 mM MgC12, 1 mM DTT, 1 mM PMSF, 20% glycerol, and 0.1% Nonidet P-40 (buffer B). The active fraction from the heparin-Sepharose column (0.3 M KC1 eluate, 60- 70 mg of protein) was preincubated on ice for 10 min with 100 pg of sheared calf thymus DNA, as nonspecific competitor, and loaded onto the oligonucleotide affinity column. The column was washed with buffer B and eluted with 0.2 M, 0.5 M, and 1.0 M KC1 in buffer B. Aliquots (25 pl) of each fraction (0.35 ml) were individually dialyzed and assayed for binding activity by the gel retardation assay. The binding activity eluted with 0.5 M KC1 in buffer B. Further purification of the active fraction from 293 extracts was achieved by a second passage through the affinity column. Fractions were diluted to 0.1 M KC1 with buffer B, mixed with 20 Fg of sheared calf thymus DNA, and reapplied to the column. Active fractions were pooled, dialyzed against buffer B, and stored in 50-p1 aliquots a t -80 “C after quick freezing in liquid nitrogen. The fractions were stable for at least 6 months under these conditions and could be thawed and refrozen up to four times without loss of activity.

Gel Retardation and Exonuclease 111 Footprinting Assays-The binding reaction for the gel retardation assay was carried out in a final volume of 25 p1 as previously described (7), using poly(d1-dC). poly(d1-dC) as nonspecific competitor in the quantities indicated under “Results” and in the figure legends. Oligonucleotide competition experiments were carried out under the same conditions. The unlabeled competitor fragments (Fig. l b and “Results”) were added prior to the labeled probe. The binding mixture was fractionated by electrophoresis through a native 2% polyacrylamide, 2% agarose gel (19). The exonuclease I11 assay was performed as described (7, 20), using the amounts of exonuclease I11 and protein indicated in the figure legend.

For quantitation of protein enrichment, binding activity was determined by the gel retardation assay, in the presence of 0.2 ng (8 fmol) of Enh2 oligonucleotide as probe and 100 ng of poly(dI-dC). poly(d1-dC) as nonspecific competitor. One unit of activity is defined as the amount of protein required to shift 50% of the probe under these standard conditions. Protein from the unfractionated and heparin-Sepharose fractions was determined by the method of Lowry et al. (21). The protein concentration for the affinity-isolated fraction was estimated from silver-stained gels by comparison with known amounts of protein markers (phosphorylase B and bovine serum albumin).

Equilibrium Binding Studies-Binding curves were obtained by the gel retardation assay in the presence of 5 ng of affinity-isolated protein and increasing amounts of DNA probe (over a 60-fold range). The binding data were quantified with a Zieneh Soft Laser Scanning Densitometer (Biomed Instruments, Inc.). Scatchard plot analysis of

binding data was conducted, and a best fit line was obtained by linear regression.

The Kd for an oligonucleotide of random sequence was determined by the method described by Prywes and Roeder (22). Binding reac- tions contained 8 fmol of Enh2(MIA14) probe, 5 ng of affinity-isolated fraction, and different concentrations of the nonspecific DNA. The data were quantified as above, and the nonspecific DNA concentration that produced 50% inhibition of binding was determined.

35% glycerol in 50 mM KC1, 50 mM Hepes, pH 7.8, 0.1 mM EDTA, 1 Glycerol Gradient Sedimentation-Linear gradients of 4.8 ml (10-

mM DTT, 1 mM PMSF, and 0.1% Nonidet P-40) were prepared. Protein from the unfractionated (500 pg), heparin-Sepharose (200 pg), and affinity-isolated (approximately 50 ng) fractions was loaded onto the gradient. The gradients were cent,rifuged at 48,000 rpm in a Beckman sW50.1 rotor for 22 h at 4 “C. Protein standards (20 pg each of phosphorylase A, bovine serum albumin, and ovalbumin) were prepared in the same buffer and run in parallel. Fractions (200 pl) were collected from the bottom of the tube and assayed for DNA binding activity by gel retardation. For the unfractionated and heparin Sepharose extracts, 2 pl of fraction and 100 ng of poly(dI-dC). poly(d1-dC), as nonspecific competitor, were used. For the affinity- isolated material, 15 pl of fraction in the absence of nonspecific competitor was used. The position of the protein standards was determined by electrophoresis in SDS-polyacrylamide gels.

UV Cross-linking-For UV cross-linking experiments, the double- stranded Enh2 oligonucleotide was cloned into the BamHI site of the polylinker in pUC13. The plasmid was digested with EcoRI and primer-extended using the M13 reverse primer, [w3’P]dATP, [a-”P] dCTP (800 Ci/mmol), dGTP, and TTP/BrdU (1:l). It was then cut with XbaI; the fragment was isolated on a 12% acrylamide gel and electroeluted.

UV cross-linking was carried out essentially as described by Wu et al. (23). Labeled probe was incubated with protein samples from the DNA-affinity column. An amount of protein which gave an 80% shift in the gel retardation assay was used, in the presence of 20 ng of poly(d1-dC) .poly(dI-dC) as nonspecific competitor. Binding was carried out in a final volume of 25 pl of 10 mM Tris hydrochloride, pH 7.5, 50 mM NaCl, 1 mM EDTA, and 5% glycerol for 30 min a t 25 “C. The open 1.5-ml Eppendorf centrifuge tube was placed under an inverted 302 nm UV transilluminator (UV Products Model TM 40) (about 5-cm sample to source separation) and irradiated at 25 “C for 20 min. The mixture was brought to 10 mM CaCI’, and the DNA was digested with 2.5 units of DNase I a t 30 “C for 10 min. The reaction was terminated with 1 p1 of 500 mM EDTA; Laemmli loading dye was added, and the samples were electrophoresed in a 10% SDS- polyacrylamide gel. The gel was dried and autoradiographed.

Oligonucleotide (Fig. l b and “Results”) competition studies were carried out under the same conditions. The competitors (50-fold molar excess) were added prior to the labeled DNA.

Other Procedures-Protein analysis by SDS-PAGE was performed by the method of Laemmli (24). G + A ladders used to calibrate exonuclease I11 footprints were generated by the method of Maxam and Gilbert (25).

RESULTS

DNase I and exonuclease I11 footprinting have shown that nuclear extracts from a variety of mouse and primate cell lines contain protein(s) that interact with the Enh2 region (sequence shown in Fig. lb) of the MIA14 LTR (7). TWO cell lines, 293 (human) and MOPC-315 (mouse), are particularly rich in components with EnhZ affinity. The cell line 293 was used to evaluate the functional role of the Enh2 domain.

Role of the Enh2 Domain in the in Vivo Promoter Activity of the IAP LTR-The promoter activity of the 5‘- and 3’- LTRs from the mouse genomic IAP element, MIA14, was measured following transfection into 293 cells, using the CAT gene as a reporter. The two LTRs had similar promoter activities in this cell line (Fig. 2). Previous work has shown that this is also the case following transfection into COS7 cells (5). The 5’- and 3‘-LTRs from this particular IAP element share the same sequence. I n vitro methylation of three HhaI sites within the 5’- and 3’-LTRs (Fig. la) caused a major decrease in promoter activity following transfection into 293 cells (Fig. 2, compare lanes I and 3 with lanes 2 and

Isolation of Enhancer Core Binding Protein

-250 -216 -170 - 78 +1 1 I ) - 6 I

I I CAAT TAiA k A P Poly A

I I I I I I I 1 50 100 150 200 250 300

I B1 I I ”SP1“ I

TGTTGGGAG~CGCGCCCAC~TTCGCCGTT~CAAGATGGC~CTGACAGC- 48

I Enhl I I

0 .. 0 EnhZ TQTGTTCTAAG~GGTAAACAA~TAATCTGCG~ATATGCCGA~GGTGGTTC 98

1 1 82 I T~TACTCCATG~GCTCTGCCT~CCCCGTGAC~TCAACTCGG

0

0 0 0 0 0 COO

030 139

I “ATF“ I FIG. 1. a, schematic representation of the 3’-LTR and immediate

upstream region of the mouse IAP clone, MIA14. The numbers at the top represent the nucleotide position with respect to the RNA start site. The numbers at the bottom represent the nucleotide position in the LTR. The heavy line shows the position of a polypurine stretch. Restriction sites used in this study are also shown. The 173-bp HaeIII- HaeIII fragment used as probe is shown as a horizontal line. b, sequence of the first 139 bp from the LTR of the mouse IAP element, MIA14. Oligonucleotides used as probes and competitors are indicated. The sequences indicated by bold lettering in the B1 and B2 regions bear homology to the consensus sequences for the transcription factors SP1 (10) and ATF (11) (we have previously referred to the ATF binding site as an AP-1 binding site (7)). A 5-bp sequence common to the Enhl and Enh2 domains and forming part of the enhancer core sequence (as first described for SV40) is shown in bold lettering. The exonuclease 111 stop sites (over the Enhl and Enh2 sequences) in the presence of the affinity-purified fraction are also shown (the exonuclease I11 footprints are presented in Fig. 5b). Symbols: 0 = negative strand; 0 = positive strand.

4 ) . A decrease in promoter activity following HhaI methylation of the 5’-IAP LTR and transfection into COS7 cells has previously been described (17).

A PuuII cleavage site at position 48 of the LTR separates the two more 5‘-HhaI sites from the third site at position 77 (Fig. 1, a and b). This PuuII site also separates the first two protein binding domains from the other more downstream domains (7). The promoter activity of a construct lacking the two upstream HhaI sites was studied. In this construct, the vector (pSVOcat) sequence was linked directly to the PuuII site. The promoter activity of this truncated construct, pMIAcat-3’L(del), was compared to the activities of pMIAcat- 5’L and pMIAcat-3’L. Deletion of the LTR region upstream of PuuII reduced chloramphenicol acetyltransferase activity in 293 cells to 40% of that of the whole LTR (Fig. 2, compare lanes 3 and 5 ) . I n vitro methylation of the single HhaI site in pMIAcat-3’L(del) resulted in a 75% reduction in promoter activity compared to the unmethylated construct, following transfection into 293 cells (Fig. 2, compare lanes 5 and 6). Since the HhaI site lies within the Enh2 binding domain (Fig. lb), this observation suggests that this domain is an important determinant of IAP LTR promoter activity in uiuo.

Purification of Enh2 Binding Actiuity-The cell lines 293 and MOPC-315 were used for isolation of Enh2 binding proteins.

Nuclear extracts were passed over a heparin-Sepharose column and binding activity in the eluted fractions was fol-

FIG. 2. Promoter activity of the MIA14 5’- and 3’-LTRs, and effect of in vitro methylation of the 5’-GCGC-3’ (HhaI) sequence(s) on the promoter activity. The conversion of [“C] chloramphenicol (0) to its acetylated forms ( I and 2) reflects the extent of expression of the CAT gene and, therefore, the promoter activity. pMIAcat-5’L = 5’-LTR pMIAcat-3’L = 3’-LTR, pMIAcat- B’L(de1) = 3’-LTR lacking the 48-bp upstream of the PvuII site. Extracts of 293 cells transfected with HhaI-methylated (meth) or unmethylated plasmids were assayed for chloramphenicol acetyltransferase activity as described under “Materials and Methods.” Similar results were obtained in three additional experiments using different preparations of the plasmids.

lowed by the gel retardation assay. Binding activity for the Enh2 oligonucleotide eluted mainly with 0.3 M KCl. As shown in Fig. 3a, the eluate from 293 cells was more active in terms of binding activity per pg of protein than that from MOPC- 315 cells.

Active fractions from the heparin-Sepharose 0.3 M KC1 eluate were applied to a DNA-affinity column containing the multimerized Enh2 oligonucleotide. The active protein fraction eluted with 0.5 M KC1 (Fig. 3b). A summary of the purification of Enh2 binding activity from 293 cell nuclear extracts is shown in Table I. The heparin-Sepharose column resulted in a %fold increase in activity compared to the crude nuclear extract. The major purification step occurred with the DNA-affinity column, such that there was a 4000-fold purification of the final product compared to the crude fraction. The percent yield and -fold purification observed here are within the range reported for other purified DNA-binding proteins; for example, comparable data were reported for the enhancer core-binding protein from rat liver (26), the eryth- roid cell proteins that bind to the a-globin gene promoter (27), the human immunodeficiency virus type 1 enhancer and TAR binding proteins (EBP-1 and UBP-1) (28), and the human lymphoid-specific octamer-binding protein (29).

Samples of the active fractions from each step of the purification procedure were analyzed by SDS-polyacrylamide gel electrophoresis. Both the unfractionated and heparin- Sepharose fractions contained a large number of polypeptide species, revealed by Coomassie Blue staining (Fig. 4). The predominant species in the affinity-isolated fractions from both 293 and MOPC-315 cells were polypeptides of about 85 kDa and 75 kDa (Fig. 4). These polypeptides were not detect- able in inactive fractions from the affinity column (not

21918 Isolation of Enhancer Core Binding Protein

B-

F-

’ 293 I

-” .I

MOPC-315 I

B -

F - l 1 2 4 6 1 2 4 6 8 1 2 1 6

pg total protein

293 Fraction number

7 8 9 10 11 12 13 14 15 5 6 7 8 9 10 11

MOPC-315

FIG. 3. Gel retardation assay of chromatography fractions from the heparin-Sepharose and sequence-specific DNA-affinity columns. The Enh2 oligonucleotide (Fig. l b ) was used as probe. a, 0.3 M KC1 eluate from the heparin-Sepharose column. The binding reaction was carried out in the presence of 1 pg of nonspecific competitor. The amount of protein per reaction, in pg, is indicated. b, 0.5 M KC1 eluate from the affinity column. 20 ng of nonspecific competitor were used in the binding reaction. The fraction number is indicated above each lane. The position of migration of the DNA- protein complex ( B ) and free probe DNA (F) are indicated.

shown). Polypeptides from the two cell lines were close in size, with only minor differences becoming apparent when the preparations were run side by side. The heterodisperse polypeptides of about 66 and 60 kDa which appear in the fraction from MOPC-315 are probably degradation products of the 85- kDa and 75-kDa species, since they were the predominant electrophoretic components in the affinity-purified fractions that had lost binding activity.

Exonuclease 111 Footprint Analysis-Exonuclease I11 footprinting was carried out at the various stages of purification using the 173-bp HaeIII-HaeIII fragment (Fig. la) as probe.

We have previously shown footprinting results for the unfractionated nuclear extract from both 293 and MOPC-315 cells (7). The footprint for the heparin-Sepharose eluates showed that binding activity for all regions of the LTR co-eluted mainly with 0.3 M KCl. Thus, no separation of the various DNA-binding proteins was apparent at this step (Fig. 5a). In contrast, the affinity-purified fraction interacted almost ex- clusively over the Enhl and Enh2 domains (Fig. 5b). The interaction appeared to be stronger with the negative strand than with the positive strand. A summary of the exonuclease I11 stop sites in the presence of affinity-isolated protein is shown in Fig. lb. The footprinting data presented here were obtained with protein from 293 cells. Fractions from MOPC- 315 cells gave similar footprints (data not shown).

Sequence Specificity of the Affinity-isolated Fractions-The nucleotide sequence specificity of active fractions from the DNA-affinity column was further examined by competition binding studies with synthetic oligonucleotides representing the various protein-binding domains in the IAP LTR, as determined in a previous study (Fig. l b and Ref. 7). Oligonu- cleotides B1 and B2 contained putative binding sites on the IAP LTR for the SP1 (10) and ATF (11) transcription factors, respectively, while oligonucleotide Enhl contained the more 5’-enhancer core sequence. In the experiment shown in Fig. 6a, the affinity-isolated protein fraction from 293 cells was preincubated with 50-fold molar excess of unlabeled competitor before addition of the labeled Enh2 probe. Only the Enhl and Enh2 oligonucleotides competed significantly. An oligonucleotide lacking 7 bp from the 3’-end of the original Enhl oligonucleotide (Fig. l b ) competed to the same extent as Enhl. This finding suggests that the 7-bp overlap between Enhl and Enh2 is not responsible for the cross-competition between the Enhl and Enh2 sequence elements.

Oligonucleotides containing the enhancer core sequence from the SV40 (30) and polyoma virus (31) were effective competitors (Fig. 6b), as was an oligonucleotide containing the core motif from the murine sarcoma virus enhancer (31) (data not shown). The profiles presented in Fig. 6, a and b, were also obtained with the protein fraction from MOPC-315 cells. It therefore appears that the protein fractions from the DNA-affinity column interacted specifically with sequences showing homology to the enhancer core motif.

Equilibrium Binding Studies-We determined the relative affinity of the purified protein fraction for oligonucleotides containing the IAP, SV40, and polyoma virus enhancer core motifs. Aliquots containing approximately 5 ng of affinity- isolated protein were mixed in a volume of 25 p1 at 100 mM KC1 with amounts of labeled oligonucleotide covering a 60- fold concentration range. Free and bound oligonucleotides were separated by gel electrophoresis and quantified by au- toradiography followed by densitometric scanning. Scatchard plots of the binding data are shown in Fig. 7. These plots are linear and do not show any deviation at the top or bottom. This indicates no crowding effect a t high probe levels and no

TABLE I Purification of enhancer core-binding activity

One unit of activity is the amount of protein required to shift 50% of the oligonucleotide probe in a gel retardation assay, under conditions described under “Materials and Methods.”

Fraction Total Activity Specific protein activity

Purification

Total Per steD %

yield

mg units unitslmg -fold units Nuclear extract 300 140,000 470 Heparin-Sepharose 64 80,000 1,250 2.7 2.7 57 DNA-affinity 0.002 3,700 1,850,000 3,940 1,480 2.6

Isolation of Enhancer Core Binding Protein 21919

' 293 '

.3 1

-2 1

FIG. 4. SDS-polyacrylamide gel analysis of extract from 293 and MOPC-315 cells at different stages of purification. Abbreviations: NUC. Ext., unfractionated nuclear extract; hep. Seph., heparin-Sepharose; Aff. Col., oligonucleotide affinity column. 1 and 2 refer to the 1st and 2nd pass through the affinity column, respectively. Samples (unfractionated and heparin-Sepharose, 30 fig of protein; affinity column, approximately 0.1 pg of protein) were analyzed on a 10% gel and visualized by Coomassie Blue staining (unfractionated extracts and heparin-Sepharose fractions) or silver staining (affinity column fractions). M = size markers in kilodaltons.

cooperative effect at low probe levels. In these plots, the slope is proportional to the equilibrium dissociation constant and is equal to I/-&. The Kd value for Enh2 under these conditions is 6 X lo-" M, and maximal probe bound is 2 X lo-' M. The Enh2(MIA14) and SV40 oligonucleotides had essentially the same apparent affinity for the protein fraction (Kd of 6 and 8 X lo-" M, respectively), whereas the Kd of the polyoma sequence was slightly different (16 X lo-" M). The plots for the three oligonucleotides had intercepts on the abscissa between 20 and 26 X 10"' M. Assuming a single binding site per protein molecule and 5 ng of active binding protein per binding mixture, these values yield a range of estimated molecular weights for the binding component of 77,000- 100,000.

We also determined the equilibrium dissociation constant for the binding of the affinity-isolated material to an oligonucleotide of random sequence but of the same size as Enh2. In this experiment, the concentrations of protein and DNA probe were kept constant while the concentration of the nonspecific DNA was varied. A 100,000-fold molar excess of nonspecific oligonucleotide was required to reduce binding of the protein to Enh2(MIA14) by 50%. This indicates an apparent equilibrium dissociation constant in the order of 10"j

Molecular Weight Determination of the Enh2 Binding Ac- tiuity-The native molecular weight of the Enh2 binding activity from 293 cells was determined at various stages of purification by glycerol gradient centrifugation. Marker proteins were run in parallel gradients. Gradient fractions were assayed by the gel retardation assay, using the Enh2 oligonucleotide as probe. The results from such an analysis are shown in Fig. 8. Binding activity for the unfractionated ex-

M.

tract sedimented as a fairly broad band at a rate corresponding to 78-93 kDa, with the peak fraction at 86 kDa. The peak binding activity from heparin-Sepharose and affinity-isolated fractions corresponded to 84 and 90 kDa, respectively. There was some binding activity in the lower fractions from the gradient containing affinity-isolated protein. This may reflect some aggregation of the binding proteins in their purified state.

To examine the polypeptide components responsible for DNA-binding activity in the affinity-purified preparations, we carried out UV cross-linking experiments using a bromo- deoxyuridine-substituted probe prepared from a pUC13 clone containing the Enh2 sequence. Label was associated with two polypeptides (Fig. 9). In lanes 2 to 6, the 293 fraction used was derived from the first pass through the DNA-affinity column. This fraction contained several polypeptides, in addition to the predominant M , = 85,000 and 75,000 species (Fig. 4); none of these other polypeptides was cross-linked to DNA. The labeled polypeptides had an Mr of 100,000 and 83,000. The molecular weights of proteins determined by UV cross-linking to DNA are always higher than the molecular weights of the free proteins because of DNA sequences which remain attached to the protein after nuclease treatment (23, 28). For the top band, the shift in M, corresponds roughly to 22 bp of DNA being attached to the protein. This shift is larger than that observed with other DNA-binding proteins (23, 28). It therefore appears that UV cross-linking of the protein has rendered a large portion of the Enh2 oligonucleotide resistant to DNase I digestion. The DNA was cross- linked more strongly to the larger protein from 293 cells. The two proteins from MOPC-315 cells interacted with the DNA to the same extent (Fig. 9, compare lanes 1 and 6). Fractions from the affinity column which were not active in the gel retardation assay gave no cross-linked bands.

The sequence specificity of cross-linking was established by competition experiments with synthetic olignucleotides (sequence shown in Fig. lb). Only oligonucleotides representing the Enhl and Enh2 domains were effective competitors (Fig. 9, data shown for fraction from 293 cells). However, oligonucleotides representing two binding sites elsewhere in the LTR (SP1 and ATF) had an effect on enhancer-binding activity not exerted by the nonspecific competitor poly(d1- dC) -poly(dI-dC) (Fig. 9, compare lane 6 with lanes 3 and 4 ) . This effect indicates some interaction of the SP1 and ATF oligonucleotides with one or both of the protein components of the affinity-isolated fractions. However, the nature of this interaction is a t present not clear.

DISCUSSION

We have used sequence-specific DNA-affinity chromatography to isolate from 293 cells a protein fraction that interacts preferentially with nucleotide sequences homologous to the SV40 enhancer core. The fraction contains two polypeptides with apparent sizes of 85 kDa and 75 kDa as assessed by SDS- polyacrylamide gel electrophoresis. UV cross-linking and glycerol gradient centrifugation experiments confirm the associ- ation of binding activity with polypeptides in this size range. The sedimentation experiments have also shown that proteins of similar size are responsible for binding activity to the Enh2 domain in both the crude and purified fractions. Finally, exonuclease I11 footprinting has shown that the heparin- Sepharose and affinity-isolated fractions interact at the same positions over the Enhl and Enh2 regions. Therefore, we believe that the affinity-purified material represents the major enhancer-binding activity present in the crude extracts. For purposes of reference, we refer to this purified protein fraction


a b Negative Strand Positive Strand Negative Strand Positive Strand

FIG. 5. Exonuclease I11 footprint analysis. a, heparin-Sepharose fractions eluted at the different stages of the KC1 gradient. The binding reaction was carried out in the presence of 10 pg of protein from 293 cells. b, affinity isolated fractions. Lanes I and 7 are blanks (absence of added protein). In lanes 2, 4,8, and 9, the reaction was carried out in the presence of 5-10 ng of protein. In lanes 3 and 5, the protein was incubated with proteinase K (60 pg) prior to addition to the binding reaction. In lane 6, a fraction which has no binding activity, as determined by the gel retardation assay, was used. Lanes G + A represent Maxam- Gilbert (25) sequence ladders. Enhl, Enh2, B1, and B2 refer to the position of the respective oligonucleotides within the sequence (see Fig. lb). In a and b, the probe was the 173-bp HaeIII-Hue111 fragment (Fig. la), and 500 units of exonuclease I11 were used. A summary of the exonuclease I11 stop sites in the presence of affinity-isolated protein is shown in Fig. lb.

as EBP-80 (enhancer-binding protein, M, = 75,000-85,000). Fractions isolated from 293 and MOPC-315 behave identi-

cally in gel retardation competition and exonuclease I11 footprinting experiments. We suggest that they represent homologous proteins from the two cell lines and that the small variations in apparent molecular weight and in reaction with the Enh2 probe in UV cross-linking experiments reflect differences in the species and/or cell type from which the fractions were made. Both polypeptides have DNA-binding activity and can bind to the same oligonucleotide probes. Further- more, both species can be simultaneously displaced by a single competing oligonucleotide. At present, we do not yet know whether they represent different proteins or modifications of a single molecular species.

The K d for these polypeptides and the Enh2 oligonucleotide is approximately 6 X lo-” M, a value comparable with Kd

values for other high affinity DNA binding proteins (23, 33). This value represents an apparent equilibrium dissociation constant (which we refer to as &*) because there may be perturbations in the protein-DNA complex during migration through the gel and because the fraction used in the analysis contains more than one polypeptide which binds to DNA. However, UV cross-linking data (in the absence of competitor DNA) for the protein fraction from 293 cells, which was used in the binding experiments, have shown that Enh2 interacts predominantly with only one of the species present. In addition, the similarity of the dissociation constants for three oligonucleotides which only share homology over the enhancer core sequence (Enh2(MIA14), SV, and Py) suggests that this motif is a major determinant of the binding reaction. Other purified transcription factors have been recovered as multiple species (e.g. EBP-1 and UBP-1 (28), SP1, (10)). It is possible that the polypeptides compete with each other for the same binding site, with one polypeptide having an overall greater affinity.

Johnson et al. (26) have purified from rat liver a nuclear

N

protein, termed C/EBP, that binds selectively to DNA containing the SV40 enhancer core sequence. This protein has been cloned and expressed in bacteria, and has a molecular size of about 42 kDa (34). The liver-derived protein differs from the present enhancer-binding proteins not only in molecular size but in heat stability: while C/EBP is stable to treatment at 80 “C for 5 min, the fractions from 293 and MOPC-315 cells lost all binding activity under the same conditions.2 Furthermore, while C/EBP interacts equally well with the enhancer core sequence and the CCAAT sequence, the enhancer-binding protein characterized in this study does not interact with an oligonucleotide containing the CCAAT sequence from MIA14.2 We therefore feel that our affinity- isolated fraction contains a novel enhancer core-binding protein. However, the present data do not establish that the core motif itself is sufficient for binding or eliminate the possibility that the protein(s) can interact with other types of sequence elements.

The 293 and MOPC-315 cells were chosen in the present study because they are relatively rich in factors that bind to the Enhl and Enh2 sites (7). This property is shared by the COS7 monkey cell line. All three cell lines contain active nuclear oncogenes: the adenovirus Ela and Elb genes in 293 cells, the SV40 large T-antigen in COS7, and a transposed c- myc in MOPC-315. Luria and Horowitz (6) reported increased in vivo transcription from the rc-mos IAP LTR when it was transfected into cells expressing these same oncogenes. They suggested that the effect could be due to direct transactivation of the LTR by the oncogene products themselves, or, more likely, to interaction of the LTR with oncogene-activated cellular proteins. Recent work has shown that some viral gene and nuclear oncogene products can combine with cellular factors to form sequence-specific DNA-binding complexes (35-39). We are currently testing for this type of interaction between nuclear oncogene products and the 85- and 75-kDa

M. Falzon, unpublished observations.

Isolation of Enhancer Core Binding Protein 21921

- B

- B

- F

FIG. 6 . Sequence specificity of the affinity-purified protein fraction from 293 cells. The binding reaction was carried out in the presence of 20 ng of poly(d1-dC).poly(dI-dC). a, competitor oligonucleotides representing different regions of the LTR from MIA14 (sequence and position shown in Fig. l b ) , in 50-fold molar excess, are indicated above each lune. b, competition between sequences within the SV40 and polyoma virus enhancers and Enh2(MIA14) (used as probe). Oligonucleotides from SV40 (30) and polyoma (31) are aligned such that the enhancer core homologies are in register. B = protein-DNA complex and F = free probe.

BOUND ( ~ 1 0 - l ~ MI

FIG. 7. Equilibrium binding analysis with affinity-purified protein. Scatchard plots are shown. The oligonucleotides used as probes are described above (Enh2(MIA14) (Fig. l b ) , SV40 and Py (Fig. 66)) . Kd* = apparent dissociation constant.

I PH

I BSA OV

I I

4 1 Aff. Col. n

BOTTOM ,4 TOP

FRACTION NUMBER

FIG. 8. Glycerol gradient sedimentation of Enh2 binding activity of extracts from 293 cells. Fractions were collected and analyzed for DNA-binding activity by gel retardation as described under “Materials and Methods,” with the Enh2 oligonucleotide as probe. The relative binding activity is shown. Individual fraction numbers are indicated below the lunes. PH, phosphorylase A; BSA, bovine serum albumin; and OV, ovalbumin.

200-

97 -

66 -

43-

M 1 2 3 4 5 6

FIG. 9. UV cross-linking of Enh2 to the affinity-isolated protein fractions from 293 and MOPC-315 cells. An amount of protein which gave an 80% shift in the gel retardation assay was used, in the presence of 20 ng of nonspecific competitor. Lune 1, fraction from MOPC-315; lunes 2 to 6, fraction from 293 cells. In lunes 2 to 5, the binding reaction was carried out in the presence of a 50-fold molar excess of oligonucleotide competitors, as follows: lune 2, Enhl; lune 3, SP1; lune 4 , ATF; and lune 5, Enh2. M = molecular weight markers; the numbers refer to the sizes in kilodaltons.

enhancer-binding proteins. We also entertain the possibility that an elevated level of enhancer-binding proteins is char- acteristically associated with heightened expression of certain nuclear oncogenes and proto-oncogenes and is in turn re- flected in the IAP expression observed in early mouse development and many transformed mouse cells (cf. Ref. 6).

The promoter activities of a number of cloned IAP LTRs transfected into cultured mouse fibroblasts (3T3 and Ltk- cells) varied greatly in effectiveness (4). Measurement of the


chloramphenicol acetyltransferase activities produced by constructs containing various deleted and chimeric LTRs led to the conclusion that the U3 region upstream of position 48 was required for optimal promoter activity in these cells. Sequences between positions 48 and 110, including what we refer to as the Enhl and Enh2 domains, were important primarily in determining the relative promoter efficiency of the LTR in the conventional and reverse directions. We do not believe that the results of our transfection experiments, which suggest that the enhancer domain is an important determinant of LTR promoter activity, are in conflict with the above observations. In an earlier study, the MIA14 LTR was shown to be a relatively poor promoter of chloramphenicol acetyltransferase activity when transfected into mouse 3T3 and Ltk- cells (6); subsequently, we found that nuclear extracts from these cells contain low levels of enhancer binding proteins (7). If, as seems likely, overall promoter activity is determined by interactions with and among multiple proteins, then the relative contributions of different protein- binding domains may vary in accord with the levels of their cognate factors in any given cell type. I t would not be sur- prising then if the enhancer-binding domains assumed a different functional significance when transfected into 3T3 and Ltk- cells on the one hand and into the factor-rich 293 cells on the other. We are presently carrying out mutational analysis to define the role of the Enh2 domain further.

Acknowledgments-We thank C. Klee, K. K. Lueders, and S. H. Wilson for a critical review of the manuscript; M. Brownstein for oligonucleotide synthesis; and M. Liu for preparing the manuscript.

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