The Tetrameric Structure of NF-pNR Provides a Mechanism for

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 1, Issue of January 5, $: 624-634,1991 rrnted rn (I. S. A.

The Tetrameric Structure of NF-pNR Provides a Mechanism for Cooperative Binding to the Immunoglobulin Heavy Chain p Enhancer*

(Received for publication, May 30, 1991)

Richard H. ScheuermannS From the Basel Institute for Immunology, Grenzacherstrmse 487, CH-4005 Basel, Switzerland

We have analyzed the structure and DNA-binding characteristics of the IgH enhancer-binding regulatory protein NF-pNR. We estimate that there are at least 5000 molecules of NF-pNR protein/nucleus of non-B cells, based on the yield of active protein following purification. Purified NF-pNR exists as a tetramer of 40-kDa subunits, even in the absence of its DNA-binding substrate. The protein complex binds to four binding sites flanking the immunoglobulin heavy chain p enhancer core. Separately, individual sites bind to the tetrameric complex with affinities varying over a 100- fold range. However, when a low affinity site and a high affinity site are present on the same DNA molecule, both are occupied at the same NF-pNR concentration; thus, there is a strong cooperative interaction between binding sites. The tetrameric structure provides a mechanism for binding cooperativity in which initial binding is mediated through a high affinity site on the DNA molecule followed by the engagement of the low affinity site juxtaposed to adjacent protein subunits. The presence of multiple binding sites flanking the p enhancer core may reflect the influence of NF-pNR binding on enhancer three-dimensional structure, transcription factor binding, and/or nuclear matrix interactions for cell type-specific enhancer function.

The differential control of gene expression provides the basis for defining discrete cell types and organs in a multicel- lular organism. While expression can be regulated at any step between the gene and the final functional protein, one of the most important regulatory points is the control of transcription initiation. Several types of cis-acting regulatory sequences are important for initiating transcription, and all of them are potential sites for differential regulation: promoter elements located relatively close and 5’ to the site of transcription initiation, enhancers which stimulate transcription from a defined promoter and can act at a relatively large distance from the transcription initiation site, and dominant control regions which probably affect transcription regulation through a higher order change in chromatin structure.

The expression of the immunoglobulin heavy chain gene in higher eukaryotes is a model for tissue-specific transcription regulation. Each V-region contains a promoter element located 5’ of the transcription initiation site, which is regulated

* This work was supported by the F. Hoffman-La Roche Ltd. Co., Basel, Switzerland. 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.

$ Present address: Dept. of Pathology, Southwestern Medical Cen- ter, University of Texas, Dallas, TX 75235-9072; Tel.: 214-688-7255; Fax: 214-688-7578.

in a tissue-specific manner (Grosschedl and Baltimore, 1985; Mason et al., 1985). There are at least two transcriptional enhancers associated with the IgH locus, one located between the JH elements and Cp (Banerji et aL, 1983; Gillies et al., 1983; Mercola et al., 1983; Neuberger, 1983), and the other located 3‘ of the Ca region (Pettersson et al., 1990) I will refer to these as the p and the a enhancers, respectively. Both of these enhancers are regulated in a tissue-specific manner; that is, they function to stimulate transcription in B cells but not in other kinds of cells. Thus, these cis-acting transcriptional elements are at least partly responsible for the restriction of antibody synthesis to cells of the B lymphocyte lineage.

It has become apparent that these cis-acting regulatory elements function as the binding sites for nuclear proteins, which in turn affect the transcription machinery (reviewed in Maniatis et al., 1987; Mitchell and Tjian, 1989). Indeed, much of the initial work defining the role of these DNA-binding proteins analyzed their binding to the immunoglobulin gene enhancers and promoters (Augereau and Chambon, 1986; Landofli et al., 1986; Peterson et al., 1986; Schlokat et aL, 1986; Sen and Baltimore 1986; Singh et al., 1986; Staudt et al., 1986; Weinberger et al., 1986). While the exact mechanism by which most of these DNA-binding proteins function is still unclear, a subset has been shown to function as transcription factors in uitro (Fletcher et al., 1987; Scheidereit et al., 1987). One important question is how these DNA-binding proteins are involved in tissue-specific regulation. While a large number of distinct binding activities can associate with the p enhancer, most of these were found to be present in every cell type examined. Some of these ubiquitous proteins bound to sites (E boxes) that show a tissue-specific protection pattern i n vivo (Church et al., 1985; Ephrussi et al., 1985). On the other hand, one of these binding activities, OTF2 or Oct2, was specific for lymphoid cells. OTF2 recognizes a specific octamer sequence present not only in the p enhancer but also in every VH and VL promoter (Parslow et al., 1984; Falkner and Zachau, 1984; Bergman et al., 1984; Mason et al., 1985). Interestingly, another nuclear protein, OTF1, also recognizes the octamer site but is present in all cell types. Both of these octamer-binding proteins can function as transcription factors in uitro. In addition, the octamer site is found in the promoters of several housekeeping genes, e.g. histone H2b (Harvey et al., 1982; Perry et al., 1985; LaBella et al., 1988) and some UsnRNA genes (e.g. Zamrod and Stumph, 1990), and it is important for the transcription of these genes in all cell types. Another recently identified protein, pB, shows a B-cell-specific distribution (Libermann et al., 1990; Nelsen et al., 1990); however, its relevance for the tissue specificity of the p enhancer remains to be demonstrated. Taken together these results raise several questions: if the octamer site within the enhancer is responsible for its tissue specificity due to the action of OTF2, then why is OTFl nonfunctional with the 1.1 enhancer i n uiuo; if OTFl does not function in vivo then why

624

Cooperative Binding of Tetrameric NF-pNR 625

are the housekeeping genes like histone H2b not expressed in a B-cell-specific manner, reflecting OTF2 activity; and if the E box-binding proteins are present in the nuclei from all cells, why do their binding sites show a B-cell-specific protection pattern in uiuo?

We recently described another nuclear protein, NF-pNR,' that binds to several sites flanking the core enhancer (Scheuermann and Chen, 1989). NF-pNR is present in nuclear extracts from non-B cells but absent from mature B-cell extracts, the tissue distribution one would expect for an enhancer suppressor. Indeed, when NF-pNR binding regions are deleted from the p enhancer, it functions in non-B cells; i.e. it loses its tight tissue specificity (Imler et al., 1987; Scheuermann and Chen, 1989). This finding supports the notion that NF-pNR functions as an enhancer suppressor. The fact that the NF-pNR-binding sites surround the binding sites of enhancer-stimulating transcription factors suggested that the mechanism of enhancer suppression was distinct from the simple binding site overlap seen with several prokaryotic repressors (Reznikoff et al., 1985). In a previous paper, we proposed several models for the molecular mechanism of enhancer suppression. In order to elucidate this mechanism we have begun by examining some of the biochem- ical properties of NF-pNR and its interaction with its different recognition sites. Here we report that the NF-pNR binding activity resides within a 40-kDa protein which is part of a tetrameric complex in nuclear extracts. While NF-pNR was found to bind to the individual recognition sites with varying affinities when assayed separately, there was a strong cooperative interaction when more than one recognition site was present on the same DNA fragment. The multimeric structure of NF-pNR and its cooperativity in binding strongly support models for enhancer suppression where the p enhancer is rendered inaccessible by NF-pNR binding. Thus, the tissue specificity of the p enhancer may be dominantly regulated by suppression over activation.

MATERIALS AND METHODS

NF-pNR Purification-The PD31 preB cell line was grown in 3- liter batches of Iscove's modified Dulbecco's medium supplemented with 3% fetal calf serum, 1.43 mM @-mercaptoethanol, and penicillin/ steptomycin (GIBCO) to a cell density of -5 X lo6 cells/ml. Nuclei were prepared from each 3-liter batch by lysis of the cell pellet in 10 ml of Buffer A (0.1% (v/v) Nonidet P-40,5.4% (w/v) sucrose, 15 mM

mM spermidine, 2 mM DTT) facilitated with Dounce homogenization (-5 strokes with a type B pestle), and sedimentation through a 30- ml sucrose cushion (30% (w/v) sucrose in Buffer A without Nonidet P-40) at 2100 X g, 4 "C for 30 min. The resulting nuclear pellet was resuspended with 5 ml of Solution E (50% (v/v) glycerol, 20 mM Tris/ HCl, pH 8.0, 75 mM NaCl, 0.5 mM EDTA, 0.15 mM PMSF). The nuclear extracts were prepared by adding an equal volume of 0.6 M NaCl (0.34 M final concentration). Following a 30-min incubation on ice, the nuclear debris was removed by centrifugation at 2100 X g, 4 "C for 30 min, and the extracts were frozen in liquid N2 and stored a t -70 'C. When nuclear extracts had been prepared from -100 liters of tissue culture cells (-5 X 10" cells), the extracts were pooled and dialyzed extensively against cold column Buffer A (CBA, 10% (v/v) glycerol, 50 mM Tris/HCl, pH 7.5, 10 mM KCl, 10 mM NaC1, 0.5 mM EDTA, 0.1 mM PMSF). The dialyzed extract was clarified by centrifugation and the supernatant referred to as fraction FI.

Fraction FI was applied to a 500-ml DEAE-Sephacel column in CBA. NF-pNR activity was eluted with CBA containing 100 mM NaCl. Column fractions were assayed for NF-pNR binding activity

' The abbreviations used are: NF-pNR, nuclear factor-p enhancer negative regulator; DTT, dithiothreitol; MSA, mobility shift assay; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate, kb, kilo- base(s); HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; CHAPS, 3[(3-cholamidopropyl)dimethylammonio]-l-propane- sulfonic acid.

HEPES/KOH, pH 7.9,60 mM KCI, 15 mM NaCl, 2 mM EDTA, 0.4

using mobility shift assays with the 5'-En restriction fragment (see below) as probe. Fractions with 250% peak activity were pooled and referred to as fraction Fir. The FIX fraction was loaded on a 35-ml heparin-agarose column in CBA. Protein was eluted with a 400-ml 0.2-0.7 M NaCl gradient, with NF-pNR binding activity eluting at -0.35 M NaCl. Fractions with 250% peak activity were pooled, giving fraction FII~.

NF-pNR was further purified using an oligonucleotide affinity column (Kadonaga and Tjian, 1986) with DNA corresponding to the binding site P2 identified by DNase protection experiments with FIII (see Fig. IC). Complementary oligonucleotides used for the affinity column correspond to sequences from position 288 to 334 numbered from the first nucleotide of the J H proximal XbaI site of the p enhancer. The two complementary oligonucleotides (1.5 mg each) were annealed in 0.2 ml of 50 mM Tris/HCl, pH 7.5, 10 mM MgC12, 5 mM DTT, 1 mM spermidine, 1 mM EDTA by first heating the mixture to 90 "C for 5 min, then 10 min at 65 "C, 10 min at 37 "C, and then 5 min at 25 "C. The annealed oligonucleotides, which contain 4 base complementary overhangs, were kinased, and then multimerized with T4 DNA ligase to an average length of -250 base pairs. The multimerized oligonucleotides were attached to 5 g of CNBr-activated Sepharose by incubation for 4 h in 10 mM KPO4, pH 8.0, at 4 "C. Unreacted groups were blocked with ethanolamine and the affinity column matrix washed extensively first with 1.0 M KPO4, pH 8.0, then with 1.0 M KCl, then with deionized, distilled HzO, and finally with Buffer Z (25 mM HEPES/KOH, pH 7.0, 12.5 mM MgC12, 20% (v/v) glycerol, 0.1% (v/v) Nonidet P-40) to remove any nonattached oligonucleotides. FI11 (10 ml) dialyzed against Buffer Z with 100 mM KC1 was incubated for 15 min on ice with 0.6 mg of poly(d1-dC) and loaded onto the oligonucleotide affinity column (-30 ml of packed volume). NF-pNR was eluted with 300 mM KC1 in Buffer Z. Fractions containing ~ 5 0 % peak activity were pooled giving FIV. The analysis of the purification fractions are described under "Results," Table I, and Fig. 1.

Mobility Shift Assays-DNA-binding assays (Fried and Crothers, 1981; Garner and Revzin, 1981) were performed essentially as described previously (Scheuermann and Chen, 1989; Scheuermann, 1990). Briefly, protein fractions (added last) are incubated with a 32P- labeled DNA probe in (20 pl) MSA Buffer (10% (v/v) glycerol, 20 mM Tris/HCl, pH 7.5,lO mM NaC1,0.5 mM EDTA) with 5 pg of BSA (fraction V, Miles Laboratory), and 1 pg of poly(d1-dC) as a nonspecific competitor. Following a 20-min incubation at room temperature, the samples are loaded directly onto a 4% polyacrylamide (30:1, acrylamidebis) gel in TBE Buffer (22 mM Tris base, 22 mM boric acid, 0.5 mM EDTA). Following electrophoresis at 10 V/cm for 1.5- 2.5 h, the gels are fixed for 30 min in 10% methanol, 10% acetic acid, vacuum dried, and exposed for autoradiography or for phosphorimaging. Quantitative binding was measured with the Molecular Dynamics Phosphorimager using Imagequant software.

For the binding reaction where,

DNA + NF + NF.DNA complex (c)

the equilibrium binding constant K is given by

K = [c]/[DNA][NF] (1)

where [c], [DNA], and [NF] are the concentrations of DNA-protein complex, free DNA, and free nuclear factor, respectively. Since [DNA] is equal to [DNA]; - [c], where [DNA]; is the input DNA concentration, then

K = [cl/([DNAli - [C])[NFI (2)

Rearrangement gives

[cl/[NF] = K[DNAli - K[c] (3)

In a graph of [c] versus [c]/[NF], the slope of the curve would correspond to -K and the y intercept to K[DNA],.

Likewise, since [NF] is equal to [NF], - [c], where [NF], is the initial nuclear factor concentration, Equation 1 becomes

[cl/[DNA] = KtNFli - Ktc] (4) For weak binding constant determinations (for binding site P3), when [NFI, >> [cl

[cl/[DNAl - K[NFli (5)

The concentration of active NF-pNR protein in fraction Flv ([NF];) was determined using a Scatchard analysis in which the

626 Cooperative Binding of Tetrameric NF-pNR

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FIG. 1. Analysis of NF-pNR purification fractions. NF-pNR protein was purified from preB cell nuclei as described under "Materials and Methods." Panel A , SDS-gel fractionation of purification fractions. Lane I contains 2.0 pg of the nuclear extract fraction; lane 2 contains 2.0 pg of fraction F1; lane 3 contains 1.0 pg of fraction FII; lane 4 contains 1.0 pg of fraction FIII; lane 5 contains 0.1 pg of fraction FN. The molecular mass markers indicated in kDa to the left side of the gel include myosin, phosphorylase b, BSA, ovalbumin, carbonic anhydrase, and trysin inhibitor. Samples were run on a 4-15% acrylamide gradient gel and proteins identified with silver staining. Panel B, mobility shift assay of NF-pNR purification fractions. Samples were assayed for specific DNA binding using the labeled 3'-En fragment as a probe (-20 ng/reaction). Reaction mixes included lane 1, no extract; lane 2, 1.26 pg of nuclear extract; lane 3, 0.70 pg of Fl; lane 4, 0.20 pg of Fll; lane 5, 0.27 pg of Frll; and lane 6, 0.01 pg of FIV. The arrow indicates the characteristic NF-pNR/DNA complex. Panel C, quantification of NF-pNR purification fraction binding activity. Mobility shift assays from reaction mixes containing 2 ng of 32P-labeled 3"En fragment, and varying amounts of purification fractions as indicated were quantified by phosphorimaging. Panel D, DNase footprint analysis of NF-pNR purification fractions. The specific binding sites on the 5"En and 3"En fragments of the final two purification fractions, Fill and FIv, were determined by protection from DNase digestion. In the left panel (lanes 1-8) the 5"En fragment was 32P-labeled at the PouII site, and in the right panel (lanes 9-16) the 3'-En fragment was 32P-labeled at the EcoRI site with polynucleotide kinase. Reaction mixes contained 2 ng of labeled DNA and either 0.32 pg (lanes 2 and 10) or 0.96 pg (lanes 3 and 11) of fraction F11l or 0.036 pg (lanes 6 and 14) or 0.11 pg (lanes 7 and 15) of fraction Flv. Regions protected from DNase digestion are indicated with solid bars as Prot. 1-4. (reprinted with permission from Scheuermann and Chen, 1989). Panel E, mobility shift assays of purification fraction FIV in the presence of various DNA competitors. Reaction mixes contained 0.063 ng of 32P- labeled binding site P3, either 0 (lane 1 ) or 0.009 pg (lanes 2-12) of NF-pNR fraction F ~ v , and a 5-fold (lanes 3 ,5 ,


TABLE I Analysis of NF-pNR purification fractions

Fraction Purification step“ Vol. Proteinb Binding activity’ Specific activityd Purification factor Yield ml mg units X lo-‘ units fpg %

Nuclear extract 0.3 M NaCl extract 600 3780 4.98 1.32 (1.0) (100) FI Clarified dialysate 610 2140 1.16 0.54 0.41 23.3 FII DEAE pool 185 185 1.76 9.51 7.20 35.3 FIII Heparin pool 102 27.5 0.57 20.7 15.7 11.4 Flv Oligo affinity 20.v 0.18 0.11 611 463 2.21

Details of the purification procedure are described under “Results.” * Protein concentration was determined in a dye-binding assay (Bradford, 1976) with BSA as a standard. e 1 unit of NF-pNR binding activity corresponds to the amount binding half of the DNA probe in a standard

mobility shift assay mix containing 2 ng of labeled 3”En DNA. NF-pNR specific activity was determined from the data presented in Fig. 1C.

‘Since only half of fraction FIII was applied to the oligonucleotide affinity column the actual quantities for fraction FIv (10 ml) were doubled for the purpose of this table.

concentration of DNA was varied in a mobility shift assay (similar to the analysis indicated in Fig. 5). In a graph of [c] versus [c]/[DNA], they intercept will be K[NF],. Under these conditions the concentration of active NF-pNR in fraction F ~ v is 4.0 X M (average of six experiments, using binding sites P1, P2, and P4, and the restriction fragments 5”En and 3’-En as binding substrates; standard deviation is * 4.0 X 10”’).

The velocity (v) of molecules during electrophoresis in the mobility shift assay gel can be determined by

v = qE/f (6)

where q is the total charge of the molecule and E is the electric field. Since the frictional coefficient (f) is related to the mass and shape of the molecule, and if we assume that the shapes are similar, then

v a charge/mass (7)

DNA Fragments-The p enhancer is contained within a 1.0-kb XbaI restriction fragment which can be divided into three parts by digestion with PvuII and EcoRI. The internal PvuII-EcoRI fragment (the enhancer core) contains the octamer and pB motifs, and most of the E boxes described in the text. DNA probes used for binding and competition assays are as follows: 5”En is the 381-base pair XbaI- PuuII restriction fragment derived from the p enhancer; 3’-En is the 309-base pair EcoRI-XbaI fragment derived from the p enhancer; binding site P1 corresponds to sequences from 171 to 200 numbered from the first nucleotide of the JH proximal XbaI site of the p enhancer; site P2 from 281 to 334; site P3 from 732 to 753; site P4 from 773 to 827; octa from 540 to 549; pB from 412 to 427; E5 from 371 to 380. (A schematic representation of the NF-pNR-binding sites is given in Fig. 6A.) Complementary oligonucleotides corresponding to each of the sites P1-P4 were synthesized with BglII 3’ overhangs, or octa and pB with XbaI overhangs, annealed, and cloned into the BamHI site or the XbaI site of pUC19, respectively. Substrates for mobility shift assays were generated by polymerase chain reaction (Saiki et al., 1988) using polylinker sequences as primers.

DNA substrates used for some experiments were derived from a mutant version of the 1.0-kb XbaI fragment of the p enhancer (mutagenized enhancer). The p enhancer was modified using a uracil- mutagenesis system (Bio-Rad) to insert specific restriction enzymes sites at the limits of the binding sites Pl-P4. SpeI restrictions sites replaced sequences starting at positions 162 and 203 flanking P1; BglII sites at 277 and 341 flanking P2; XhoI sites a t 697 and 759 flanking P3; and EagI sites at 782 and 825 flanking P4. Binding site deletion mutants were generated by digestion with the appropriate restriction enzyme, followed by recircularization with T4 DNA ligase, and the subclones were analyzed by restriction enzyme digestion.

UV Cross-linking of DNA-Protein Cornple~es-~~P-Labeled DNA fragments were cross-linked to bound protein using UV irradiation essentially as described (Biswas and Kornberg 1984; Peterson and Calame 1989). DNA probes used were internally labeled with 32P and BrdU, and were generated in standard polymerase chain reactions

(100 pl) which contained in addition 100 pCi of [(u-~’P]~TTP and 1 mM BrdUTP. The internally labeled DNA was incubated with protein fractions under standard mobility shift assay conditions. Following electrophoresis the wet gel was irradiated for 15 min with 254-nm UV light. Radioactive bands corresponding to complexed and free DNA identified by autoradiography were excised, placed in 0.4 ml of Buffer Z, and the labeled DNA eluted by diffusion overnight at 4 ‘C. Following the addition of CaClz to 5 mM, excess DNA was removed from the protein by digestion with 10 pg of DNase1 (Bethesda Research Laboratories) for 10 min at 37 “C. The protein was then run on a standard SDS-gel (as described below) and the protein directly involved in the DNA interaction identified following autoradiography.

DNase Footprinting-DNase footprinting experiments (Kadonaga and Tjian, 1986) were performed essentially as described (Scheuer- mann and Chen, 1989; Scheuermann, 1990). Following isopropyl alcohol precipitation samples were electrophoresed on 8% polyacrylamide (382, acrylamide/bis) denaturing gels in a Tris-borate-EDTA buffer. Chemical sequencing reactions (Maxam and Gilbert, 1980) of the labeled fragments were included as position markers.

Denaturing Gel Electrophoresis-Standard discontinuous SDS-gel electrophoresis was used (Laemmli, 1970). For the UV cross-linking experiments, a 15% polyacrylamide gel was used. For the analysis of the NF-pNR purification fractions 4-15% polyacrylamide gradient minigels (Bio-Rad) were used, and protein was detected with silver staining (Oakley et al., 1980).

Glycerol Gradient Sedimentation-Proteins were sedimented through a 5.0-ml 10-30% glycerol gradient in 20 mM Tris/HCl, pH 7.5, 10 mM NaCl, 0.5 mM EDTA. Sedimentation was performed in a TST 55.5 rotor at 52,000 rpm (342,000 RCF,,), 4 “C for 8 h. One- drop fractions (-60 pl) were collected from the bottom through a 21- gauge needle. As sedimentation standards BSA (250 pg; Miles Sci- entific, Fraction Fv) and catalase (500 pg; Sigma C3155) were included with 50 pl of NF-pNR FW in a total volume of 100 pl.

From the sedimentation coefficient (s) the molecular weight (M) of a protein can be estimated (Young 1989) as

s = 0.00242 (8)

or

M = (423.2~)’.~’~ (9)

In an isokinetic gradient, the migration of a molecule will be directly proportional to its sedimentation coefficient. Therefore the sedimentation coefficient of a species with unknown molecular weight, in this case NF-pNR, can be determined by comparison with the sedimentation of two standard molecules, catalase and BSA, as

sn = s b + [(sc - sb)(ln - lb)/(C - lb)] (10)

where s,, s,, and s b are the sedimentation coefficients and l,,, l,, and lb the migrations of NF-pNR, catalase, and BSA, respectively.

7,9, and 11 ) or 25-fold (lanes 4,6,8,10, and 12) molar excess of unlabeled competitor DNA. P3 and P4 correspond to NF-pNR-binding sites identified by DNase footprinting; sites octa, p B , and E5 correspond to enhancer sequences bound by other nuclear proteins.

628 Cooperative Binding of Tetrameric NF-pNR

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1 2 3 4 5 6 7 8

FIG. 2. Mobility shift assay showing the effects of divalent cations on NF-pNR binding activity. NF-pNR fraction F ~ v , at the indicated amounts, was incubated with the 5’-En fragment under standard conditions. In addition, the sample in lane 5 contained 2.5 mM ZnC12; lane 6 contained 5.0 mM MgClz; lanes 7 and 8 contained 0.75 mM ( 1 P ) and 3.0 mM (4P) 1,lO-phenanthroline, respectively. The migration positions of the free DNA fragment and the DNA- protein complex are indicated.

RESULTS

Purification of NF-pNR Indicates That There Are at Least 5000 Molecules/ Nucleus in an Early preB Cell-The nuclear protein NF-pNR binds to two DNA fragments flanking the core of the p enhancer (Scheuermann and Chen, 1989). The protein is generally present in nuclear extracts of non-B cells and early preB cells but absent from extracts of mature B cells and preB cells representing late stages of B lineage differentiation. The effect of binding site deletion mutations on p enhancer activity has suggested that NF-pNR functions as an enhancer suppressor, keeping the p enhancer inactive in non-B cells. NF-pNR was purified from an early preB cell line, PD31, by a combination of classical and affinity chro- matographic techniques. NF-pNR in nuclear extracts binds to DNA segments flanking the enhancer thereby generating a large DNA/protein complex that migrates to a position close to the sample wells in a standard mobility shift assay (Fig. 1B). This characteristic MSA pattern was used as the assay system to follow NF-pNR binding activity during purification.

In the first column step the nuclear extract was separated on a DEAE-Sephacel column. This column is useful for the removal of nucleic acids and other cellular components present in the crude nuclear extract preparation. A modest purification was obtained following heparin-agarose chromatography (Table I), and the heparin column fraction, FIII , was used to define the NF-pNR-binding sites more precisely on the two flanking enhancer fragments by DNase footprint analysis (Fig. 1D; Scheuermann and Chen, 1989). Two binding sites on each of the flanking fragments were identified and designated Pl-P4 from the 5’ end to the 3‘ end of the p enhancer. Oligonucleotides corresponding to binding site P2 were used to generate an affinity column, which gave by far the greatest purification.

The protein which bound to this affinity column in the presence of an excess of nonspecific poly(d1-dC) competitor and eluted with 0.3 M KC1 (fraction FIV) shows strong NF- pNR binding activity assayed with the MSA (Fig. 1, B and C). In addition to the 30-fold purification, an analysis of the protein complexity by SDS-polyacrylamide gel electrophoresis shows that this final step has substantially reduced the number of visible protein bands (Fig. lA). The final purification fraction, FIv, contains only a few discernable protein bands, one of which presumably corresponds to NF-pNR.

Fraction FIv was examined for binding activity by DNase protection. Even though this fraction was purified based on its ability to bind to site P2, it still demonstrates protection of all four sites Pl-P4 (Fig. 1D). Indeed, the specific activity in FIV for binding to these sites appears to be the same. At the lower concentration (lanes 6 and 14) partial protection is seen, while at the higher concentration (lanes 7 and 15) there is more complete protection; all four sites are occupied at the same concentrations of Flv. This result not only suggests that the same activity is binding to these sites but also that under these conditions the binding affinities for the four sites are similar. The DNA binding activity in fraction FIV is indeed specific for these sites since in mobility shift assays sites P3 and P4 are both able to compete for NF-pNR binding to a P3 probe, whereas the octamer, pB, or E5 sites do not compete for P3 binding even at a 25-fold excess (Fig. 1E).

In summary, a 463-fold purification of NF-pNR activity was obtained from the initial nuclear extract, with a yield of 180 pg of protein in 20 ml. Fraction FIv represents a yield of 2.2% of the binding activity in the initial nuclear extract isolated from -5 x 10” cells. As described under “Materials and Methods,’’ the active NF-pNR concentration in Frv was determined to be 4.0 X lo-’ M. Therefore there would be at least 5000 molecules of NF-pNR in the nucleus of a single preB cell. This final fraction, FIv, was used for the subsequent analysis of NF-pNR structure and binding characteristics.

NF-pNR May Be a Zn Finger Protein-Several classes of DNA-binding proteins have been identified containing homeo domains, helix-loop-helix motifs, or zinc finger domains. We found that NF-pNR activity did not require added components like M e , ATP, or Zn2+, and would function in a relatively simple mixture (Fig. 2).2 However, we wanted to determine whether divalent cations already present in the purified fraction were important for binding activity. Indeed, the addition of the Zn2+ chelator 1,lO-phenanthroline was inhibitory to binding activity (Fig. 2). This is most easily seen as an increase in the level of free DNA in lane 8. Interestingly, the addition of excess Zn2+ was also inhibitory to NF-pNR binding (lane 5). Both of these effects strongly suggest that NF-pNR contains a zinc finger(s) necessary for specific binding. The inhibitory effect of phenanthroline is presumably due to the chelation of Zn2+ bound within the protein. The inhibitory effect of excess Zn2+ may be due to the disruption of the presumptive Zn finger structure in which each Zn2+ ion would only coordinate two amino acids instead of four in the standard finger structure (Freedman et al., 1988; Thiesen and Bach, 1991). Alternatively, the effect of excess Zn2+ or Zn2+ chelators may reflect the disruption of quartenary structure rather than the presence of a Zn finger, as suggested for their inhibitory effect on NF-KB binding (Zabel et al., 1991).

NF-pNR Exists As a Tetramer of 40-kDa Subunits- Throughout our analysis of NF-pNR/DNA interactions, specific retarded bands often appeared in the MSA at a position intermediate between the characteristic complex and the free DNA, even with our most pure fraction (see Fig. 1E). Using the 5”En fragment, we see a band about halfway between the free fragment and the normal complex (Fig. 3A); this intermediate band is only present at low NF-pNR or high DNA concentrations. With isolated binding sites the pattern is often more complex; as many as three extra bands can be seen at low NF-pNR concentrations (Fig. 3B, e.g. panel P3, bands b-d). In order to determine the molecular weight of the NF-pNR binding activity and to test whether these intermediate bands are a result of binding to the same protein in the purification fraction, we cross-linked the radioactively labeled

R. H. Scheuermann, unpublished results.


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46kD F

30kD

1 2 3 4 5

DNA to the bound protein in a mobility shift gel with UV irradiation. This effectively transfers the label from the DNA to the bound protein. Following digestion with DNase to remove most of the DNA, the protein was separated by SDS- gel electrophoresis to determine the molecular weight of the binding protein in the NF-pNR fraction. We found that the protein responsible for the characteristic NF-pNR band ( A ) and the intermediate band ( B ) was the same -40-kDa protein (Fig. 3C). This low molecular weight was somewhat surprising in view of the large shift in mobility of the protein-DNA complex. Indeed, NF-pNR gives larger mobility shifts than the 90-kDa OTFl protein (Fletcher et al., 1987) interacting with the octamer motif under similar conditions? Under nonreducing conditions we failed to detect a larger protein band (Fig. 3C), indicating that the MSA complex does not result from 40-kDa subunits that are disulfide linked. In fact, the protein migrates slightly faster under nonreducing conditions suggesting the presence of intramolecular disulfide bonds. This same 40-kDa protein is detected from either retarded band A or B with either the 5'-En or 3"En fragment as a binding probe (Fig. 3D), again indicating that the same protein interacts with both enhancer-flanking fragments. In Fig. 3 0 an additional faint band is observed at -100 kDa from each of the retarded bands. This band might represent the linkage of two 40-kDa subunits by a stretch of DNA between the two binding sites which was not completely digested with DNase I.

The relatively low molecular weight of the binding protein combined with the large mobility shift suggested that NF- pNR binds to DNA as a multimeric complex. In order to determine whether this large protein complex is only formed in the presence of DNA or whether it preexists in the purification fraction before the addition of DNA, we sedimented fraction Flv in a glycerol gradient with BSA and catalase as sedimentation standards. All of the binding activity migrated between the BSA and catalase peak (Fig. 4A). The sedimentation constants for catalase (232,000 kDa), s,, and BSA (68,750 kDa), sb, can be calculated according to Equation 8 under "Materials and Methods" as 9.52 and 4.21, respectively. The migrations of the three molecules in the gradient, 1, for catalase, lb for BSA, and 1, for NF-pNR were 3.075,1.700, and 2.525 ml, respectively. Hence, the sedimentation coefficient for NF-pNR, s,, can be calculated from Equation 10 to be

FIG. 3. Binding and UV cross-linking analysis of NF-aNR- DNA complexes. Panel A , mobility shift assay with the labeled 5'- En fragment as a binding probe for NF-pNR fraction FIV binding. DNA probe was added as follows: lanes 1 and 2,50 ng; lanes 3 and 6- 11, 100 ng; lane 4 , 200 ng; lane 5, 400 ng. NF-pNR fraction F ~ v was added as follows: lanes I and 6 none added; lanes 2-5, 20 ng; lane 7, 5 ng; lane 8, 10 ng; lane 9, 20 ng; lane 10,40 ng; lane I I , 8 0 ng. The position of the free DNA fragment (F) and the two retarded bands ( A and B ) are indicated. Panel B, mobility shift assay with independent binding sites as probes. The binding site probes used are indicated above each panel. Lanes 1-3 contained 0, 5, and 20 ng of NF-pNR F ~ V , respectively. Panel C, SDS-polyacrylamide gel of samples following UV cross-linking. An internally labeled 5"En fragment was used as a probe for fraction Frv binding in an MSA; the gel was irradiated with UV and the protein-DNA complexes in retarded bands A (lanes 3 ,4 , and 6 ) and B (lanes I, 2, and 5) were purified. Following DNase digestion, SDS sample buffer with (lanes 1-4) or without (lanes 5 and 6 ) DTT was added prior to electrophoresis on a 15% SDS-polyacrylamide gel. Following electrophoresis the gel was fixed, dried, and the proteins examined by autoradiography. Panel D, UV cross-linking with two different DNA fragments. Samples were prepared as above with sample buffer containing DTT except that for lanes 4 and 5 the 3'-En fragment was used as the binding probe. Lane I contains a sample derived from the position of the free DNA fragment in the MSA gel. Open arrows indicate the migration of a major and minor protein species detected by UV cross-linking.

630 Cooperative Binding of Tetrameric NF-PNR

4 - + + + +

1 2 3 4 6

FIG. 4. Native molecular weight of NF-pNR determined by glycerol gradient sedimentation. Panel A, glycerol gradient sedimentation of NF-pNR fraction FIv under nondenaturing conditions. NF-pNR binding activity (closed circles) was measured in a standard mobility shift assay, and protein concentrations (open circles) determined in a dye binding assay (Bradford 1976). Panel B, mobility shift assay of NF-pNR-DNA complexes in the presence of detergent. R2P- Labeled 3”En fragment was incubated with ( lanes 2-5) or without ( l a n e I ) 20 ng of NF-pNR FIV. In addition, lanes 3-5 contained 0.0025% (w/v), 0.005%, and 0.0075% SDS, respectively. The solid arrow indicates the position of the characteristic NF-pNR-retarded band, and the open arrow indicates a novel band seen in the presence of SDS.

7.40, and the molecular mass calculated from Equation 9 to be 160,000 kDa. Assuming this complex to be composed of a single type of subunit, it would correspond to a tetramer of 40-kDa proteins in the absence of its DNA-binding substrate.

Additional support for the multimeric structure of NF-pNR activity comes from experiments where’low concentrations of detergent are used to disrupt the complex into single subunits. While detergents like Nonidet P-40 and CHAPS have no effect on binding activity, detergents like SDS and deoxycholate can destroy binding? However, a t low concentrations of these detergents a novel band intermediate between the characteristic NF-pNR band and the free DNA band can be seen (Fig. 4B). We presume that this represents the disruption of the multimeric complex into its individual binding subunits. This relatively small mobility shift is consistant with the binding of a relatively small (40 kDa) protein.

If fraction Frv were composed entirely of the 160-kDa NF- pNR complex, it’s concentration would be 5.6 X M based on the protein determination. However, we have measured the “active” NF-pNR concentration in FIV to be 4 X lo-’ M

D

x I

d

1

I

4

a-pR cone. (pMl

a-vm cone. 1pMl

FIG. 5. Measurements of NF-pNR/DNA binding affinities. Panel A, binding of NF-pNR to different DNA substrates. Binding of NF-pNR fraction Frv was measured with standard mobility shift assays. The concentration of NF-pNR-binding protein was varied while the concentration of DNA substrates was held constant at 50 p ~ . DNA substrates included 5”En (closed circles), 3”En (closed squares), binding sites P1 (open circles), P2 (open triangles), P3 (open squares), and P4 (closed triangles). Panel B, Scatchard analysis of binding site data to determine equilibrium binding constant, K. The binding data for binding sites Pl-P4 given in panel A were rearranged in order to give values of [c]/[NF-pNR] versus [c] in order to determine the equilibrium binding constant, K (see “Results” for descrip- tion of the analysis; for the weak binding site P3, K was determined instead according to Equation 5). The equation of the “best fit” line is indicated within each panel. Panel C, effects of binding site deletions on binding to the 5’-En fragment. DNA-binding substrates used were the wild type 5”En fragment (open circles), and mutant versions containing deletions of site P1 ( d l , closed circles), site P2 (d2, closed sqwres), or both P1 and P2 (d ld2 , closed triangle). Panel D, effects of binding site deletions on binding to the 3’-En fragment. DNA-

Cooperative Binding of Tetrameric NF-pNR 63 1

TABLE I1 Equilibrium binding constants of protein DNA interactions

The equilibrium binding constants, K, for sites P1, P2, and P4 were determined by the Scatchard analysis described in the text. Values represent the average of three or more determinations. In the case of the weak binding site, P3, K was determined by Equation 5 from individual binding reactions. Standard deviations for Pl-P4 binding constants were 13, 28,7.5, and 29%, respectively. The binding constants for other proteins including nuclear factor I (NFI), adenovirus major late transcription factor (MLTF), heat shock transcription factor (HSTF), nuclear factor-Kappa B (NF-kB), and the E. coli lactose operon repressor (lacR) were determined elsewhere. DNA fragment

protein Binding constant K Refs.

"' P1 4.9 X lo9 This study P2 1.6 X 10" P3 1.2 x lo8 This study P4 1.1 x 1O'O This study

This study

NF-pNR SP1 NFI MLTF HSTF

lacR 5.7 x 1O'O Fried and Crothers 1981

1.2 X 10' - 1.6 X 10" This study 1.9 X 109 - 2.4 X lo9 Letovsky and Dynan 1989

2 x 10' - 2 x 1O'O Meisterernst et al., 1988; Rosenfeld and Kelly, 1986 1 X 10" - 2.5 X 10" Chodosh et aL, 1986

2.5 X 10" Wu et al., 1987 NF-kB 3.7 X 10l1 - 2.5 X 10'' Zabel et al., 1991

based on the DNA binding activity ("Materials and Meth- ods"). This would seem to imply that the vast majority of NF- pNR in Flv is inactive, however, there are several alternatives to explain this discrepancy. While the major protein band in an SDS-gel of fraction FIv is -40 kDa there are also other minor bands in this molecular mass range (Fig. L4). It is possible that NF-pNR is actually one of these minor bands. A second possibility is that the active complex is composed of two different subunits and that one of them is limiting in the final purification fraction. While we have no direct evidence to rule out this last possibility, all of our results are consistent with the structure being a tetramer of identical subunits.

The NF-pNR Tetramer Binds Cooperatively to Adjacent Binding Sites-The binding protein affinity of the NF-pNR for its different DNA substrates was measured with the standard MSA. At constant DNA concentrations, increasing concentrations of NF-pNR results in increasing complex forma- tion with saturation binding curves (Fig. 5A). From these curves we can estimate that NF-pNR binds with highest affinity to the restriction fragments containing two binding sites, also with high affinity to sites P2 and P4, intermediate affinity to site PI, and low affinity to site P3. This low affinity interaction with site P3 is still specific; the assays are done in the presence of a large excess of poly(dI-dC), DNA-binding sites for other nuclear proteins are not able to effectively compete for P3 binding (Fig. lE), and we have not been able to detect direct binding to the octamer sequence under similar conditions (data not shown).

The equilibrium binding constant ( K ) for this type of protein-DNA interaction is given by Equation 1. Re- arrangement of Equation 1 to the form of Equation 3 allows graphing of the binding data as a Scatchard plot of [c] versm [c]/[NF-pNR] in which the slope is equal to -K. When the binding data for sites Pl-P4 were so plotted (Fig. 5B), a straight line could be fitted (Cricket Graph version 1.3.2). The equations for the lines are indicated in each panel and the binding constant determined by the negative slope (e.g. for P1, K = 4.19 X lo9). From this analysis binding to sites P2 and P4 is strong, binding to P1 intermediate, and binding

binding substrates used were the wild type 3'-En fragment (open circles), and mutant versions containing deletions of site P3 (d3, closed circles), site P4 (d4, closed squares), or both P3 and P4 (d3d4, closed triangles).

to P3 weak. The binding affinities for the four binding sites are similar to the binding affinities of other prokaryotic and eukaryotic transcription regulators (Table 11). Binding to either the 5'-En or 3"En fragment is somewhat stronger (Fig. 5A). In the case of fragment 5'-En, deletion of either site P1 or site P2 results in a significant reduction in binding affinity (Fig. 5C). Deletion of both sites results in even weaker binding. A similar result is seen with the 3'-En fragment except that deletion of the weaker site (P3) has less effect than deletion of the stronger site (P4) (Fig. 50) . Again, deletion of both results in a substantial reduction in binding affinity (Fig. 50). Interestingly, even when both DNase protected regions are deleted from a restriction fragment, the binding affinity is still higher than to site P3 by itself. It is possible that deletion of the normal binding sites uncovers cryptic binding sites located elsewhere within these fragments.

The equilibrium binding constants for sites P3 and P4 differ by about two orders of magnitude, yet in the DNase protection experiment in Fig. 1D both of these sites are filled at the same NF-pNR concentration when they are present on the same DNA fragment. A similar phenomenon is also seen with sites P1 and P2. This suggested cooperative interaction between binding sites. In order to test this hypothesis, we examined the effects of downstream enhancer sequences on binding to site P1 using DNase protection (Fig. 6A). With the entire 1.0-kb p enhancer fragment, we observe strong binding to site P1 ( l a n e 3). Deletion of the 3'-En fragment containing binding sites P3 and P4 has little effect on binding to P1 ( l a n e 6). Likewise deletion of the enhancer core also has little effect ( l a n e 9). However, when site P2 is deleted, strong binding to site P1 is lost ( l a n e 12). Thus, binding to site P2 increases the apparent affinity of binding to site P1, indicative of a cooperative interaction. On the other hand binding to site P2 does not appear to be affected by the presence or absence of site P1 (Fig. 6B, compare P2 footprints in lanes 4 and 5 with lanes 10 and 11 ). Thus, the stronger site affects binding to the weaker site but not vice versa. A similar effect is seen in the interaction between sites P3 and P4. Deletion of site P4 results in a lower affinity for binding to site P3 (Fig. 6C), but deletion of P3 has little effect on binding to P4 (Fig. 60) .

DISCUSSION

The Structure of NF-pNR and Its Complexes with DNA- The results presented in this paper show that the enhancer

Cooperative Binding of Tetrameric NF-pNR

C

P4

P3

0 . 3 1 3 8 0 "1 NF.uNR Prv

" - " " - -

-"* - "

"

FIG. 6. DNase footprint analysis of NF-pNR binding cooperativity. DNA fragments were incubated with NF-pNR fraction F ~ v as indicated, before DNase digestion. Following extraction and purification, the digested DNA was electrophoresed on an 8% polyacrylamide sequencing gel. Panel A, effect of 3' enhancer segments on binding to site P1. The mutagenized 1.0-kb p enhancer fragment (see "Materials and Methods") was '*P-labeled near the 5' XbaI site and fragments isolated following mock digestion ( a ) , digestion with EcoRI ( b ) , digestion with P o d 1 (c), and digestion with BglII ( d ) . Panel B, effect of site P1 on binding to site P2. The mutagenized enhancer was labeled at the PuuII site and isolated either mock- digested (lanes 1-6) or digested with SpeI to remove site P1 (lanes 7- 12). Panel C, effect of site P4 on binding to site P3. The mutagenized enhancer was labeled at the EcoRI site and isolated either mock- digested (lanes 1-6) or digested with EagI to remove site P4 (lanes 7-12). Panel D, effect of site P3 on binding to site P4. The mutagen-

suppressor protein NF-pNR binds cooperatively to its multiple binding sites in and around the p enhancer and that the activity exists as a large multimeric protein complex. Several lines of evidence support the notion that this is a specific complex and not the result of a nonspecific aggregation formed during the isolation procedure. We were unable to disrupt the complex with high concentrations of CHAPS detergent which has been used to disrupt nonspecific aggre- gates. Detergents such as SDS and deoxycholate apparently did dissociate the protein subunits but only a t concentrations just below those which totally destroyed binding activity (see Fig. 4B).' In addition, a single sharp peak is seen in the glycerol gradient profile rather than a broad smear one might expect if the high molecular weight were due to nonspecific aggregation. This large complex is not merely a result of protein purification since the characteristic retarded band is also seen in mobility shift assays with simple nuclear extracts (Fig. 1B; see also Scheuermann and Chen, 1989). These results all argue that the large NF-pNR complex is the normal form of this DNA binding activity. Indeed, this multimeric form would seem to be appropriate for an activity that binds tightly to several independent sites in and around the p enhancer.

Occasionally novel bands are seen during the mobility shift analysis, even with our most purified NF-pNR preparation. The results of the UV cross-linking experiments indicate that a t least one of these novel bands (band B in Fig. 3A) is due to the binding of the same protein that gives rise to the characteristic retarded band near the sample well (band A in Fig. 3A) . Several possibilities for the origin of these various DNA-protein complexes come to mind. The characteristic band might result from the binding of the tetrameric NF- pNR structure to a single DNA molecule, and the intermediate band might result from the binding of a dimeric form of NF- pNR. However, using sizing chromatography or glycerol gradient sedimentation we were never able to demonstrate the presence of smaller protein complexes in quantities large enough to account for the amount of the intermediate bands seen. Another possibility is that the intermediate band results from the binding of one tetramer with one DNA molecule, and the characteristic band due to the binding of two tetra- mers to one DNA molecule, since there are two binding sites in the large restriction fragment. This possibility is unlikely for two reasons. Since the binding to the two sites within the restriction fragments show strong cooperativity one would expect the one tetramer-one DNA complex (the intermediate band) to predominate, which is not the case. Moreover, this does not explain the existence of several intermediate bands when single binding sites are used as substrates (Fig. 3B) . While there are other possible explanations, we favor the hypothesis that the retarded bands are a result of the binding of different numbers of DNA molecules to the NF-pNR tetramer. We assume that the characteristic retarded band, which predominates under most assay conditions, is the result of the interaction of one tetramer with one DNA molecule. The relative slow mobility of this complex during electrophoresis would be due to a relatively large protein complex bound to a DNA fragment with a certain charge. The migration of these complexes during gel electrophoresis is given by Equation 6 ("Materials and Methods"), which means that the distance migrated would be proportional to the charge to mass ratio (Equation 7). Therefore, the migration of these complexes in the MSA gel can be increased by increasing the charge or decreasing the mass. Band B in Fig. 3A would be due to the

ized enhancer was labeled near the 3' XbaI site and isolated either mock-digested (lanes 1-6) or digested with XhoI to remove site P3 (lanes 7-12).


binding of two DNA molecules to one tetramer thereby increasing the charge to mass ratio. This type of molecule would be expected to increase with respect to the one-to-one complex with increasing DNA or decreasing protein. The intermediate bands seen with individual binding site probes would also correspond to multiple DNA molecules bound to a single tetramer. For example, in Fig. 3B, panel P3, bands a-d would correspond to a single tetramer of NF-pNR bound by one, two, three, and four DNA molecules, respectively.

A variety of proteins regulating eukaryotic transcription have now been found to bind to DNA in a cooperative fashion (Topol et al., 1985; Davidson et al., 1988; Martinez and Wahli, 1989; Poellinger et al., 1989; Schmid et al., 1989; Tsai et al., 1989; Weintraub et al., 1990; this paper). The cooperative binding of the positive transcription factors, however, differs in one important aspect from the type described here; the proteins are initially monomeric and interact only after DNA binding. This type of cooperative binding would allow transcription to react to very small changes in transcription factor concentration (Poellinger et al., 1989). In the case of NF-pNR and enhancer suppression, cooperative interaction could simply allow a switch from the off state (transcription suppression) to the on state (responsive to transcription activators) during B lineage development.

The tetrameric structure of NF-pNR provides a simple mechanism to achieve binding cooperativity. A DNA fragment containing two binding sites for NF-pNR, one with high affinity and one with relatively low affinity, would be initially bound through the high affinity site. This initial binding would bring the low affinity site into close proximity to another protein subunit in the complex increasing the effec- tive concentration of the weak site, which would then be bound. This would result in the binding of both weak and strong sites at the same NF-pNR concentrations. In this system strong binding sites would exhibit cooperative effects on weak sites but not vice versa, as observed experimentally for NF-pNR. An alternative model where binding of one site would change the conformation of NF-pNR in such a way as to increase its affinity for subsequent binding is probably not correct, since cooperativity does not work in trans.* Thus, in the case of NF-pNR, cooperativity in DNA binding is simply a result of its multimeric structure.

Relationship between Multimeric Structure and Functwn- The fact that NF-pNR exists as a large protein complex is probably related to its function. It is interesting that a wide variety of DNA-binding proteins are found as homomeric or heteromeric complexes. In the case of transcription factors, specific domains containing amphipathic helices with leucine repeats or helix-loop-helix motifs have been identified which are involved in dimerization. It is possible that the dimerization of these transcription factors might be important for mediating interactions between promoter-proximal and promoter-distal (enhancer) elements, as seen with proteins binding to the SV40 promoter and enhancer (Takahashi et al., 1986), and with the Escherichia coli glnA promoter (Su et al., 1990), by an as yet ill-defined “looping-out” mechanism (Dy- nan and Tjian, 1985; Robbins and Botchan, 1985).

In the case of NF-pNR, the tetrameric structure and multiple sites flanking the p enhancer core probably reflect the molecular mechanism of enhancer suppression, which may be quite distinct from the mechanism of transcription activation. The binding of repressor proteins to multiple sites separated by intervening DNA has been demonstrated in several prokaryotic systems (Irani et al., 1983; Dunn et al., 1984; Majumar and Adhya, 1984). Indeed, demonstration that the interaction between protein subunits bound at distal site is required for

repression has also been demonstrated (Mandal et al., 1990). NF-pNR might be suppressing IgH enhancer function by a mechanism similar to the effects of these prokaryotic repressors on their target promoters.

Several mechanisms can be imagined to explain suppression by binding to sequences flanking transcription activator sites. By binding to all four binding sites with a single protein complex the intervening DNA might be made inaccessible for binding by the various transcription factors which recognize sequences in this region through steric hindrance. This mechanism of enhancer suppression would provide an explanation for the observation that proteins which bind to the E boxes can be isolated from any cell, but their binding sites are only occupied in a tissue-specific manner in uiuo. It would also explain why OTFl does not stimulate p enhancer activity in non-B cells. An alternative mechanism to “steric hindrance” proposes that the bend produced in the intervening DNA (in this case the p enhancer core) by the large protein-DNA complex would alter the three-dimensional structure of the transcription factor binding sites in such a way as to prevent binding. Since the agents used for chemical modification in uiuo (e.g. dimethyl sulfoxide) are probably less sensitive to this kind of structural alteration, this mechanism for enhancer suppression could also produce a cell type-specific chemical protection pattern for ubiquitous nuclear proteins.

If the function of NF-pNR is simply to prevent binding of positive transcription factors, why are four binding sites required rather than just one on either side of the p enhancer core? Indeed, all four binding sites are necessary for optimal suppression since deletion of a single site allows partial enhancer function in inappropriate cells (Scheuermann and Chen, 1989). Many regions regulating DNA transactions are composed of multiple binding sites for the same or different proteins. The requirement for multiple binding sites is probably, in part, due to the difficulty in achieving high precision in a large genome (Echols, 1986). It is advantageous to require multiple binding sites for function rather than to build a huge protein which would recognize only one site within the entire eukaryotic genome; a genome of lo1’ base pairs would require direct recognition of 17 nucleotides in order for that site to be represented only once. Since this specific recognition would probably occur on one face of the DNA helix it would correspond to at least four helical turns and require a relatively large protein if encoded by a single polypeptide chain. Pre- sumably, the complex of four 40-kDa subunits in NF-pNR has achieved the possibility of high precision interactions without the expense of using an excessive amount of DNA to encode the protein.

The requirement for so many binding sites, however, may reflect a different mechanism for enhancer suppression. Pre- viously, we proposed a model for enhancer suppression in which NF-pNR inhibited enhancer function by preventing attachment of the enhancer to the nuclear matrix (or nuclear scaffold) (Scheuermann and Chen, 1989). This was based on the observation that the two restriction fragments bound by NF-pNR (5’-En and 3‘-En, here) would also specifically attach to the nuclear matrix (Cockerill et al., 1987) and that short consensus sequences proposed for nuclear matrix attachment (Cockerill and Garrard, 1986) overlapped NF-pNR- binding sites. However, more recently it has become apparent that strong binding to the nuclear matrix probably requires A-T-rich regions that may be several hundred nucleotides long (Amati et al., 1990). Thus, the requirement for multiple NF-pNR-binding sites might reflect the need to occupy a large stretch of enhancer DNA in order to prevent attachment to the nuclear matrix.

634 Cooperative Binding of Tetrameric NF-wNR

Clearly the immunoglobulin heavy chain p enhancer has evolved an elaborate mechanism for achieving enhancer function and specificity. The elucidation of the molecular mechanisms underlying cell type specificity will require the further analysis of the effects of NF-pNR binding on enhancer core three-dimensional structure and transcription factor binding and a better understanding of the role nuclear matrix attachment plays in the regulation of gene transcription.

Acknowledgment.+” thank J.-M. Buerstedde, J. F. Kaufman, and C. M. Steinberg for critical reading of the manuscript, H. Echols for advice on velocity sedimentation, and all of the members of our institute for helpful discussion. I would also especially like to thank D. Lenig for excellent technical assistance throughout the course of this work.

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