Nucleosome Binding by the Constitutive Transcription Factor Spl*

THEJOURNAL OF BIOLWICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 10, Issue of March 11, pp. 7756-7763, 1994 Printed in U.S.A.

Nucleosome Bindingby the Constitutive Transcription Factor Spl* (Received for publication, September 27, 1993, and in revised form, November 17, 1993)

Baiyong Li, Christopher C. Adam&, and Jerry L. Workman§ From the Department of Biochemistry and Molecular Biology and the Center for Gene Regulation, Pennsylvania State University, University Park, Pennsyliania 16802-4500

The constitutive transcription factorSpl plays a role in the transcription of numerousviralandcellular genes including constitutive “housekeeping” genes and inducible genes. Spl has also been implicatedin the formation of a nucleosome-free region in Simian virus40 (SV40)minichromosomes. To investigate the potential functions of Spl in remodeling chromatin structures, Spl was analyzed forthe ability to bind its recognition sites (GC boxes) in DNA fragments reconstituted into nucleosome cores.Spl was foundto bind to the GC boxes of the SV40 early promoter when this element was reconstituted into nucleosome cores. The affinity of Spl for the nucleosomal SV40 early promoter DNA was reduced approximately 10-20-foldrelative to naked DNA. A peptide containing only the zinc fingers of Spl was also capable of binding nucleosomal DNA, indicating that the glutamine-rich and serinelthreonine-rich domains of Spl are not required for nucleosome binding. The binding of Spl to nucleosome coresresulted in the formation of a ternary Spl-nucleosome complex.

yeast pH05 promoter are disruptedupon induction of the promoter by the PH04activator independent of DNA replication (Schmid et al.,1992, and references therein). Similarly, disruption of a nucleosome containing glucocorticoid response elements occurs on the mouse mammary tumor virus (MMTV)l promoter and the rat tyrosine aminotransferase promoter upon hormone induction (Richard-Foy and Hager, 1987; Carr and Richard-Foy, 1990; Bresnick et al., 1992). Insight into the mechanism of nucleosome disruption at the MMTV promoter resulted from the observation that purified glucocorticoid receptor is able to bind glucocorticoid response elements contained in a nucleosome core reconstituted over the promoter (Perlmann and Wrange, 1988; Pina et al., 1990; Archer et al., 1991; Perlmann, 1992; Li and Wrange, 1993). Thus, binding of the receptor apparently initiates disruptionof the nucleosome which allows the subsequent bindingof additional factors. The involvement of the glucocorticoid receptor and PH04 in the inducible disruption of nucleosomes on the MMTV and PH05 promoters raises the question as to whether nucleasehypersensitive sites on other promoters might arise by similar replication-independent mechanismsvia the action of constituRemodeling of chromatin structureoccurs prior to or concur- tive factors.For example, the six Spl-binding sites (in the three rent with the transcriptional activation of eukaryotic genes. 21-bp repeats) in the SV40 early promoter have been impliThis has been evidenced by the formation of regions lacking cated in the formation of the nuclease-hypersensitive site on archetypal nucleosomes (nuclease hypersensitive sites)a t pro- SV40 minichromosomes (Jongstra et al., 1984, and references therein). Thefraction of SV40 minichromosomes bound by S p l moter and enhancer elements of cellular and viral genes (Elgin, 1988,1990;Gross and Garrard, 1988). Two general mecha- in vivo (10-20%) correlates with the fraction containing the nisms have been envisaged for the formation of nuclease-hy- nuclease-hypersensitive site (Buchanan and Gralla, 1987) and fraction of minichromosomes whichis persensitive sites. The binding of trans-acting factors to pro- appears to represent the competent for transcription initiation in vitro (Batson et al., moters and enhancers might occur immediately following DNA replication prior to the reassembly or maturation of nucleo- 1992, 1993). In uiuo, pulse-labeling experiments indicate that forms de novo subsequent to viralDNA somes. Bound factors would then exclude nucleosome forma- this hypersensitive site tion over these crucial transcription elementsallowing the sub- replication (Solomon and Varshavsky, 1987). To participate in the de nouo formation of nucleosome-free sequent association of additionalregulatoryandgeneral regions, S p l would have to access the GC boxes (Spl binding transcription factors (Svaren and Chalkley, 1990; Wolffe, 1991). Alternatively, nucleosome-free regions may result from the dis- elements) subsequent to theirincorporation into nucleosomes. placement of pre-existing nucleosomes as a result of the bind- To investigatethis possibility, we haveinitiallyexamined bind to GC boxes ing of trans-acting factors. By the latter mechanism, nucleo- whether S p l contains any inherent ability to when reconstituted into nucleosome cores. some disruption would occur independent of DNA replication (Workman and Buchman, 1993; Svaren and Horz, 1993). Evidence for replication-independent nucleosome disruption has come from the analysisof inducible genes inboth yeast and mammals. For example, four positioned nucleosomes over the

* This work was supported part in by Grant GM47867 fromthe Public Health Service (to J. L. W.) and a research initiation grant from Pennsylvania State University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be herebymarked “aduertisement” inaccordancewith18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by Public Health ServicePostdoctoralFellowship GM15861. 9 LeukemiaSocietyScholar. Towhom correspondenceand reprint requests should be addressed: Dept. of Biochemistry and Molecular Biology, 301 Althouse Laboratory, PennsylvaniaState University, University Park, PA 16802-4500. Tel.: 814-863-8256; Fax: 814-863-7024.

EXPERIMENTALPROCEDURES DNA Probes-The 198-bp SV40 early probe was generatedby digestingplasmidpSp72SV40 (Promega)with the restriction enzymes HindIII and NsiI. The fragment was end-labeled with Klenow at the HindIII site (the end farthest from the GC boxes). In this probe, the center of the first GC box is 30 bp away fromthe NsiI end. The plasmid pBendSpl-2 was generated by inserting two copies of an oligo containing a GC box (5’-CTAGATCGGGGCGGGGCGm-3’)into theXbaI site of pTK401(Kerppolaand Curran, 1991).The155-bp2Sp1(155/51,71) probe was generated by digesting pBendSpl-2 with the restriction enzymes XhoI and NheIand labelingthe NheI end with Klenow(the end farthest from the GC boxes). The centers of the two GC boxes were 51 and 71 bp away fromtheXhoI end of the probe, respectively. The 160-bp The abbreviations used are: MMW, mouse mammary tumor virus; SV40, Simian virus 40; bp, base pairs.

7756

S p l Binding toCores Nucleosome

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dures"). Fig. IA shows a mobility shift gel of the three DNA fragments used inthis study before andafter nucleosome core reconstitution. For each of the three DNA fragments shown (SV40 early, 2Sp1(155/51, 71) a n d 2Sp1(160/33, 53)), following the reconstitution procedure (lanes 2 , 4 , a n d 6 )over 80% of t h e fragment was containedin the nucleosome-reconstituted band (Nucl.)while the remainder was free DNA probe (DNA).This result demonstratesthat the probe DNAsused were efficiently reconstituted into nucleosome cores. The SV40 early fragment contains six distinctGCboxes (21-bp repeats) located from 2882tobp from the upstream end of the fragment. Nucleosome reconstitution on this DNA is further evidenced by the fact that this 198-bp fragment takes up a specific rotational frame (orientationof t h e DNA helix on the surface of the histone octamer) upon reconstitution into nucleosome cores (Fig.1 B ) as previously shown for overlapping et al., 1985). DNase I digesSV40 promoter fragments (Clarke tion of the nucleosome-reconstituted SV40 early fragment illustrates a ladder of DNase I cleavage approximating 10 bp (lanes 3-5) which is not apparent upon digestionof the naked DNA (lanes 1 and 2). Micrococcal nuclease trimming of the reconstituted SV40 early promoter nucleosome cores resulted in the generation of an approximate 145-bp digestion product indicating that the histone octamer was fully complexed with 145 bp of the DNA fragment (data not shown). However, we have not observed a specific translational position of the nuThis is consistent with earlier cleosome cores on this fragment. studies suggesting random or multiple translational positions of nucleosomes over the early promoter in vivo a n d in uitro (Ambrose et al., 1989, 1990; Stein, 1987). Moreover, while the 72-bp repeats (not containedin the SV40 early fragment used here) apparently have some translational positioning properties in uitro, the 21-bp repeats do not (Clarke et al., 1985). S p l Binding to Nucleosome Cores Reconstituted on the SV40 Early Promoter-The ability of S p l t obind to the nucleosomereconstituted SV40 early fragment was initially analyzed by DNase I footprinting (Fig. 2). Protection of all six GC boxes from DNase I was observed onthe naked DNA fragmentat 0.5 units of a d d e d S p l (lane 2 ) , a n d a complete footprint was achieved with 1 unit of factor (lane 3 ) . An S p l footprint was also observed onthe nucleosome-reconstituted probe (compare lanes 9 a n d 10).However, Spl binding to the nucleosome-reconstituted probe required higher concentrations of S p l . While a footprint was apparent at 0.5 units of S p l o n n a k e d DNA (lane 21, protection of the GC boxes from DNase I on the nucleosome probes required 6 units of Spl (compare lanes9 a n d 10).Thus, the affinity of S p l for the GC boxes on the nucleosome cores was reduced approximately 10-fold relative to naked DNA. Importantly, the Spl bound to GCboxes within the lower 145 bp of the probe where the DNase I ladder is most regular (the lower two-thirds of the bracketed region). Since the SV40 early probe DNA (198 bp) was greater than nucleosome core length (146 bp) part of the DNA(52 bp) extends beyond the limits of each core particle. Therefore, one or two GC boxes may extend beyond the surface of the core particlein the fraction of the probe where the nucleosome cores were positioned at the opposite end.Above 145 bp,the initial digestion pattern on the reconstituted nucleosome (lane 6 ) also includes digestion of these naked DNA ends and therefore represents overlapping RESULTS digestion patterns of naked DNA and nucleosome cores (lane To analyze the binding of S p l t o nucleosome cores, we have 1).At 1unit of S p l (lane 7), the naked DNA fractionat this end utilized a purified system to reconstitute nucleosome cores onto was protected (bound by Spl), and the 10 bp ladder from the DNA fragments containing Spl-binding sites. Labeled DNA nucleosomes occupying that end of the probe became more apat high parent. Protection of the remaining 10-bp digestion pattern fragments were mixed with purified HeLa core histones salt, reconstitutedinto nucleosomes by dialysis to low salt, and occurred at the higher concentrationsof S p l . T h u s , S p clearly l purified by gradient sedimentation (see "Experimental Proce- bound to those GC boxes within the core particle, protecting

2Sp1(160/33,53) probe was generated by digesting pBendSp1-2 with the restriction enzymesNheI and SalI followed byend-labelingthe SalI site with Klenow (the end farthest from the GC boxes). In this probe, the centers of the two GC boxes are 33 and 53 bp away from the NheI end, respectively. The probes were purified by elution from 8%native acrylamide gels. Nucleosome Reconstitution-For nucleosome reconstitution, approximately 200 ng of end-labeled probe, 75 pgof calf thymus DNA, and 75 pg of purified HeLa corehistones were mixedin a totalvolume of 50 pl with final concentrations of 2 M NaCl, 10 m~ HEPES, pH 8.0, 1 m~ EDTA, 1 mg/ml bovine serum albumin, and 1 m~ 2-mercaptoethanol. Nucleosome cores were reconstituted by salt gradient dialysis as described (Workman and Kingston, 1992) and purified on sucrose gradients. Briefly,following dialysis, the reconstituted nucleosomecores were loaded on a 5-25% sucrose density gradient containing 10 m~ HEPES, pH 8.0, 1 m~ EGTA, 0.1 m~ phenylmethylsulfonyl fluoride, 0.1% Nonidet P-40 and centrifuged for 18 h at 34,500 revolutiondmin at 4 "C in a Beckman SW55 rotor. Fractions were countedand analyzed on a native 4% acrylamide, 0.5 x TridboratelEDTA(TBE) electrophoresis buffer. The gel was dried and exposed to Kodak X-OmatAR film. Peak fractions containing the nucleosome core-reconstituted probeswere pooled and divided into small aliquots, frozen on dry ice, and stored at -80 "C. Binding Reactions-Naked DNA probes used as controls forall binding reactions were derived from the purified nucleosome cores by extracting the histone proteins with phenol and chloroform followed by precipitation and resuspension. For the binding reactions with full-length Spl, 5 p1 of purified nucleosome cores,containing less than 25 fmol of nucleosome cores with Spl sites, or the equivalent amount of naked DNA probe (diluted in the appropriate mixture of the sucrose gradient solutions) was mixed with the amounts of Spl indicated in the figures in a final volume of 12 pl containing 25 m~ Tris-HC1, pH 8.0,6.25 mM MgC12,0.5 m~ EDTA, 10% glycerol, and 0.5 m~ dithiothreitol. For the binding reactions with SplZn92, nucleosome coresor DNAand the amounts of Spl-Zn92 indicated in the figures were mixed in a final volume of 12 pl containing 10 m~ Tris-HC1, pH8.0,50 m~ NaCI, 1001.1~ZnSO,, and 10% glycerol. Binding reactions proceeded for 30min at 30 "C. Full-length Spl was purchased from Promega or purified from HeLa cells according to Jackson and Tjian (1989). The experiments shown utilized Promega Spl, and the amounts added to binding reactions are indicated in theirunits. Spl-Zn92 was generously provided by Kriwacki and Caradonna (see Kriwacki et al., 1992). Mobility Shifts and DNase I Footprinting-For the mobility shift experiments, samples were loaded directly on 4 or 6%, 0.5 x TBE acrylamide gels immediately after the30 min binding incubation. Following electrophoresis, the gels were dried and subjected t o autoradiography at -80 "C. DNase I footprinting was performed following binding reactions in a final volume of 24 pl. 3 pl of DNase I dilutions containing the amounts of DNase I (Boehringer) indicated in the figures, 5 m~ MgCl,, and 10 m~ Tris-HCI, pH 8.0, were added to the 24-pl binding reaction and incubated 1min at 30 "C. DNase I digestions were terminated by the addition of 25 pl of stop mixture which contained 0.5% Sarkosyl, 10 mM EDTA, 50 pg/ml protease K, and 0.1mg/ml yeast tRNA. This mixture was incubated at 37 "C for 30 min and then phenol and chloroform extracted, ethanol precipitated, and then resuspended in 5 pl of sequencing loading buffer containing deionized formamide and dyes. Samples were heated to 100 "C for 5 min and placed on ice before being loaded onan 8%acrylamide (acrylamidehis = 29:1), 7 M urea, 1 x TBE gel. The gel was dried and exposed to film at -80 "C. Oligonucleotide and Plasmid CompetitionExperiments-For the competition experiments shown in Fig. 4, competitor Spl site plasmid DNA or the double-stranded oligonucleotides wereadded after the 30min binding reactions. Competition proceeded for 30 min at 30 "C before the samples were loadedon mobility shift gels. The Spl site-containing plasmid used was plasmid pSp72SV40 (Promega). The sequence of the double-stranded USF oligonucleotide was 5'-CAGGCCACGTGACCGGCATG-3'. The sequence of the double-stranded Spl oligonucleotide was 5'-AGCITGGGGCGGGGAGGGGCGGGGCA-3'.

S p l Binding to Nucleosome Cores

7758

A Fragment

DNA

Nucleosome Core

SV40 2Spl 2Spl " Early (155/51,71) (160/33,53) 0 0.5 1 3 M 0 1 3 6 0 Spl (units) "

1

z n 1

3

4 5

2

3

4

-

5

6

7

8

9

1

_"

0

6

Nucl. -

DNA -

4

B

DNA Nucleosome

'

12'3

4

5 '

- 124 - .~ "-11011

" "

"89

-

" "

-a

-80

-"

-64

- 57 " "

-51

r,

FIG.1. In vitro nucleosome reconstitution of DNA fragments containing Spl sites. A, mobility shift analysis of reconstituted nucleosome cores. The extent of nucleosome core reconstitution on the DNA fragments is illustrated by the reduced mobility of nucleosomereconstituted probes (Nucl.) relative to nakedDNA probes (DNAj on a native 4% acrylamide gel. The fragments shown as naked DNA and reconstituted nucleosomes, respectively, include the 198-bp SV40 early fragment (lanes 1 and 2), 2Sp1(155/51,71) (lanes 3 and 4 ) , and 2Sp1(160/33,53)(lanes 5 and 6 ) . Naked DNAcontrols were derived by extracting the histonesfrom the reconstituted nucleosome samples. B ,

FIG.2. Spl binding to the nucleosome-reconstituted SV40 early fragment. Binding of S p l to the SV40 early fragment as naked DNA(lanes 1 4 )or a s reconstituted nucleosome cores (lanes 6-10). The amount of Spl (in units)included in each binding reaction is indicated at the top above the lanes. The vertical bar indicates the region of the six GC boxes. Note that protection by S p l occurs over regions which previously demonstrated an approximate 10 bp periodicity of DNase I cleavage (compare lanes 9 and IO).Lane 5 contains labeled DNA molecular weight markers, the size (inof bp) which are 80,89,104,124 and 184 from the bottom to the top, respectively.

sequences that previously demonstrated a IO-bp digestion ladder. This is more dramatically illustrated in Fig. 3A. The Zinc Fingers of S p l Alone Bound to the Reconstituted Nucleosome Cores-To investigate the domainsof the S p l protein which were required for nucleosome binding, we analyzed the ability of peptides containing only the three zinc fingers to bindnucleosomecores. The peptide used, Spl-Zn92 (kindly provided by R. W. Kriwacki and J. P. Caradonna), contains DNase I digestion analysis of nucleosome reconstitution on the SV40 early fragment. Purifiednucleosome cores (as inlane 2 of A ) and DNA extracted from the nucleosome (as in lane 1 of A ) were digested with increasing amounts of DNase I. DNA was extracted from the digested samples and analyzed on a 8% denaturing acrylamide gel. The DNase I digestion pattern of the naked DNA probe is shown in lanes 1 and 2, while the DNase I digestion pattern of the reconstituted nucleosome cores is shown in lanes 3-5. Note the repeated pattern of DNase I cutting for the nucleosome core samples (lanes 3 4 ) which is not apparent on naked DNA (lanes 1 and 2 ) . The position of molecular weight markers also includedon the gel are indicated on the right of the figure. The amounts of DNase I in each digestion were: for naked DNA: 0.01 unit (lane 1), 0.1 unit (lane 2; for nucleosome cores: 2 units (lane 3 ) , 5 units (lane 4 ) . and 10 units (lane 5).

S p l Binding to Nucleosome Cores

A

Nucl. Core

DNA I

1 0 0.1 0.2 0.4

1

2

3

4

I 0

2

5

6

4 7

8

Spl-Zn92(ng)

8

"

B Nucleosome DNA I

I

Core I

SpllNu.

6SplIDNA

Nucl.

-

SSplIDNA 4SplIDNA

-

2SplIDNA -

3SplIDNA

1SplIDNADNA-

Flo. 3. Spl-Zn92bindingtothe

nucleosome-reconstituted

SV40 early fragment. A . 1)Nasr I footprinting titration of Spl-Zn92 hinding to SV.10 wrly DNA and nuclrosomrs. Rinding o f Spl-Zn92 to

7759

amino acids 533-623 of t h e S p l p r o t r i n . w h i c h i n c l u d r s t h r three zinc fingers, and hinds to GC hoxes with similar affinity and specificityas t h e n a t i v r S p l p r o t r i n ( K r i w a crkt in l . . 1992). The hinding of Spl-Zn92 to the SV4O early fragment rithrr as naked DNA or after reconstitutionintonuclrosomecnrrs is illustrated by t h e D N a s e I footprinting data prrsrntrd in Fig. 3A. While 0.4 n g of Spl-7,1192 produced a complrtr fontprint o v e r t h e GC boxes on the nakrd DNA templatrs ilnnr 4 I , 8 n g a complrtr footprint on thr nuclrowas required to achieve some-reconstituted prohe ( l n n r HI. Hrncr. thr affinity of t h r zinc finger peptide for the rrconstitutrd nuclrosome corrsw a s reduced approximately 2O-fold. s o m r w h a t s i m i l a r t o t h r i n h i 2 1 .This bition observed with the full-length Spl protrin (Fig. result suggests that the observed rrduction in Spl affinity was largely due to an inherently lowcr affinity of thc. zinc fingrrs for DNAonthenucleosomesurface,ratherthandurtostrric clashes of t h e 105-kDa full-lrngthprotrinwiththrhistonr octamer. Note again in Fig. 3A that the rrgion protrctrd from wrll into the IO-hp DNase I digestion hy Spl-Zn92 extended digestion ladder tlanrs 6 - 8 ) indicating that thr zinc fingrrs bound to the GC hoxrs which wrrr containrd within nuclrosome cores. Binding of the Spl-Zn92 pcptide to nuclrosomr corrs was X I ). Incubation of a l s o a p p a r e n tby mohility shift analysis (Fig. a srrirs o f the naked DNA prohe with Spl-Zn92 resultrd in shifted complexes reprrsenting incrrasing numhrrs of hound peptide ( f n n r s I-?; also see Kriwacki r t a/..19921. Thrsrs e n r as m a r k e r s for the nucleosome grl shift in l o n r s 4-9. Upon Spl-Zn92 binding to the nucleosome cores, thcrr was a clear shift of the nucleosome complex to slowrr migrating complrxrs (compare lnnrs 4 with 9 1. However, hrcausr of t h r s m a l ls i z r of the peptide, nucleosome complexes with diffrrrnt numhrrs o f bound peptide werr not as clrarly resolvrd as t h r y w r r r o n naked DNA. Importantly, the complexrs formrd upon Spl-Zn92 binding to the nucleosome cores wrrr suprrshiftrdrrl a t'I V P to the nucleosome core and to thr Spl-Zn92-1)NAcomplrxrs Icompare lanes .?, 4. a n d 9).This ohsrrvation indicatrs that thr binding of thezincfingersdidnotdisplacethrundrrlying nucleosome core (alsosee h e l o w ) . T h r s e d a t a i l l u s t r a t c t h a t t h r zinc fingers of Spl alone were ahle to hind to nuclrosomal DNA (Fig. 3, A a n d R ) as they are able to hind naked DNA (Kriwacki et a f . , 1992). Therefore, no additional domains of t h r S p l p r o tein beyond those required for DNA hinding, for rxample the i Pascal and glutamine-rich or the serine/threonine-rich regions Tjian, 1991,and rrferences therrin). wrre rrquirrd for nuclrosome binding. S p l Rinding Forms n T r r n n p Cnmplrx w i t h thr Ntrrlro.somr ~~~

-~

the SV40 early f r a p r n t as nakrd DNA ~ l n n e s1-1 1 or following r w o n stitution into nuclrosomr corrs ilnnrs .5& I w a s nnnlyzvd hy 1)Nnsr I footprinting. Binding reactions contninrd thr amount of Spl-ZnW shown above rach lane in the figure. Thr r r ~ o nhound hy S p t 1 1 . r . t h r CC hoxrsl is indicatrd hy the hnr at t h r rcght of t h r fipurr. Notr t h n t t h v protected rrkion extends well within thr rrkion of t h r ohsc*n.rd IO-hp cleavage ladder Ilnnrs 6 4 I. R . analysis o f Spl-Zn92 hindinc t o nurlcwsome-reconstltuted SV.10 early DNA hy a 6 ' ; ncrylamidr prl m n h ~ l ~ t y shift assay. Nucleosome cnrrs containing thv S\.'N) r n r l y frapmrnt ( N u r l . . fnnr 4 ) werr incuhntrd with inrrrnslng ronrrntr:ttlnns of t h r Spl-Zn92 peptide as indicntrd in thr fi~nlrr~ l n n c s5-91, At t n r r v n s i n p concentrations of Spl-ZnW. thr mikTatton of t h r nucleosomr-r(.constttuted fragment was rrduccd rrsulting In a suprrshiftrd complrx ( S p l i N u . )Apadual rrduction in mohllity w a s ohsrn.rd dur tn thr drpnwtton of incrrasing numhcrs o f hound prptidr. Individual S p l - Z n W Nucleosome complrxrs ww(- n o t rcsolvahlv dur to t h c prpttdrs small size relativr to thr nuclrosomr corr. For sin- m:lrkrrs. nnkrd SV40 DNA prohe i l n n ~I 1 hound hy a n incrrnsing numhrr of p'ptidrs Ilrrn~as2 :tnd 3 ) is shown. Notr thnt n 1 1 of thr complrxrs of t h r prptldr wlth DNA ifrom onr to six hound p r p t i d r s l m l g m t r d f:tstrr t h a n thv nuclrnsomr core. The amounts of S p l tndicntcd corrrspnnd t o 2 4 . ~ 1rvnrttons l t h r samr as thr fontprint rrartinns shown in A I. whilc- t h r nrtunl volumv of thr hinding rractions londvd on thr mohtlity shtft grl wns 12 111

S p l Binding to Nucleosome Cores

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Ba

A

E s1

E

;-

Nucleosome +Spl

1

2

Nucleosome +Spl

3

4

5

6

7

8

Complex

- Nucl.

- Nucl.

core

core

- DNA

b

-DNA

FIG.4. The nucleosome core remains in a complex with bound Spl. A. S p l sitr plasmid comprtition of Spl/SV40 mrly nuclrnsnme complexes. liinding rrnctions includrd nuclrnsome-rrconstitutrd SV40 early probes and fi.2 units of S p l a n d r w u l t r d in thv formatinn of S p l containingcomplrxes CSpl cornplrxl which appeared at t h e vrry top ofthe.Ir: acrylamidr gel fInnr.2I. Following S p l hlnding, thrsr complrxt*sw w suhjrcted to comprtition with increasing amounts of a plasmid DNA containing six S p l sites rlonrs % 1 0 ) . I'pnn compt-titlnn nf S p l frnm t h r complrx. the nucleosome core reappearrd tNucI. C o w ) w h i l r a t t h e h i g h r s t a m o u n ot f comprtitor. thr small fraction of l r r r I ) S A In t h r r r x t ~ n n also rmergrd ( D N A , Innr IO). T h r a m o u n t of plasmid usrd was: 0.1 nhl in lnnr 4 , 0 . 3 nsc in lnnr 4 . 1 nsl in lnnc 5 . 3 n\c in Irrnv 6. 1 0 n\c in I r t w i . 30 nM in Innr 8,1 0 0 nM in lnnr 9 , and 300 m e in lnnr 10. The hand ahovr the nuclrosomr cow inlnncs 4-c) rcymwmts nnr S p l mnnnmor hnund w t h the nucleosome cow.Imnc I containrd marker nucleosomes indicating thr mohility of frrr DNAand nuclrosomv cnrrs and cnnt:llns mnrr prolw 1 h:ln lnncs 2-10, R . oligo competition o f Spl from complrxes with nuclrosome cores. Spl hinding to nuclrosomr cnrrs was t h r s n m r a s In A : hnwrvvr. comprtition of S p l employrd a S p l s l t r oligo ( S J or a USF site oligo ( ~ JatJ the concentrations indicatrd.Imnr I shnws markrrs fnr thv nuclrmnmt~ corr and the DNA f r a q n r n t . Imnr 2 shows thr complrx formrd upon S p l binding to nuclrosnmrs In t h r a h s r n c r of rnmpt-titor nllg')nllcll.r,tlrlc. Incrrasing amounts o f douhlc-strandrd oligonucleotidrs containing Spl-binding sites f l n n r s 5 . and 7 I or I'SF-hindinc s ~ t r sI/nnt*s4 . ti. and 89I werr added after the initial hindingof S p l to nuclrosnmrs. Oncr again, thr hand ahovr thc nuclrosomr cnrr in lnnrs 3 , 5. and 7 rvprcwmts onv SpI monomrr hound with thr nuclrosomr core.

3.

Core-The supershifted complex shown in Fig. 3B illustrates competitors as illustratcd inFig. 4B. Incubation of t h r S p l the co-occupancy of t h e Spl-7,1192 peptide and the histone oc- hound nuclrosome cows ( S p l conrplrx. Innr 2 I with an S p l sitr tamer when bound to the SV40 early promoter fragment. In oligonucleotide resulted in the renppcarancc of the nuclrnsomr contrast, bindingof full-length S p l to the multiple GCboxes on cores (lancs .?, 5 . and 7 ).whilc incuhation of the Spl cnmpltw-s t h e SV40 early fragment as nakedDNA or after reconstitution with identical concentrations of a n nligonuclrotidr cnnt:lining :I into nucleosome cores resulted in the shift of the labeled probe USF site did not result in thr rrappcarancc of thc nuclcwsomr to the well of mobility shift gels (Fig.4A, lnnr 2 ). This is due to core (Iancs 4 , 6, and 8 ). theself-aggregationproperties of S p l which is mediatedTheseohservationsindicatethatinspite of t h r fact t h a t through the glutamine-rich activation domains (Courey et af., full-length Spl binding aggrcgatcd the nuclensnmr c n r r - r c w m 1989; Pascal and Tjian, 1991; Hoey r f nf., 1993). Thus, we were stituted SV40 probe, t h r histone octamrr rcmainrd assoriatctl unable to observe supershifted complexes of full-length Spl with the Spl-hound complex. Thus. Spl hinding to thf. SY.20 bound to nucleosome cores on the SV40 early prohe. early nucleosome resulted in the formation of a n S p l / To determine whether the nucleosome core remained in the nucleosome core ternary cnmplrx fcnntaining S p l . histonc.s, S p l aggregate ( S p l complex),weutilized an assay similar to and DNA). S p l Binding t o f l c w s i t r Nuclcoson~r C