binding of disparate transcriptional activators to nucleosomal dna
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MOLECULAR AND CELLULAR BIOLOGY, Mar. 1995, p. 14051421 Vol. 15, No. 30270-7306/95/$04.0010Copyright q 1995, American Society for Microbiology
Binding of Disparate Transcriptional Activators to NucleosomalDNA Is Inherently Cooperative
CHRISTOPHER C. ADAMS AND JERRY L. WORKMAN*
Department of Biochemistry and Molecular Biology and The Center for Gene Regulation,The Pennsylvania State University, University Park, Pennsylvania 16802-4500
Received 30 August 1994/Returned for modification 5 October 1994/Accepted 14 December 1994
To investigate mechanisms by which multiple transcription factors access complex promoters and enhancerswithin cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used apurified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kB(KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we showthat binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, thebinuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kB allparticipated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particularDNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors fornucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficientnucleosome binding by factors (USF and NF-kB) which independently possess little nucleosome-bindingability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-bindingand dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was respon-sible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of theorientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome bindingis a general mechanism that may play a significant role in loading complex enhancer and promoter elementswith multiple diverse factors in chromatin and contribute to the generation of threshold responses andtranscriptional synergy by multiple activator sites in vivo.
Mounting evidence suggests that controlling the dynamicequilibrium between competing functional and structural pro-teins (i.e., transcription factors versus nucleosome formation)plays a crucial role in regulating gene activity (20, 62, 75). It iswell established that nucleosomes are potent inhibitors of tran-scription in vitro and can directly interfere with transcriptionfactor binding (reviewed in references 34 and 77). However,increasing evidence from both yeast and mammalian systemsillustrates that nucleosomes are susceptible to replication-in-dependent disruption initiated by transcription factor bindingin vivo (reviewed in references 1 and 64). For example, nu-cleosomes located over the mouse mammary tumor virus andrat tyrosine aminotransferase promoters are disrupted by glu-cocorticoid receptor binding (3, 7, 38, 55, 57, 58, 81). A se-quence-positioned nucleosome within the integrated humanimmunodeficiency virus type 1 59 long terminal repeat is dis-rupted following tetradecanoyl phorbol acetate induction (71).In Saccharomyces cerevisiae, heat shock factor disrupts a rota-tionally phased nucleosome from the TATA box and initiationsite of the HSP82 heat shock gene upon transcriptional acti-vation (20). The DNA-binding domain of the GAL4 proteincan directly disrupt nucleosomes to which it binds (46), whileits activation domains lead to disruption of an adjacent nucleo-some on the GAL1 promoter (4). Similarly, binding of thePHO4 activator to the PHO5 promoter disrupts adjacent nu-cleosomes, in a process facilitated by the PHO4 activationdomain (60, 66). Thus, studies with S. cerevisiae suggest a role
of DNA-binding domains in binding or disruption of underly-ing nucleosomes and a function of activation domains which ismost apparent in disruption of adjacent nucleosomes (perhapsindirectly).Consistent with the in vivo studies of replication-indepen-
dent chromatin remodeling, complementary in vitro studieshave directly demonstrated the binding of transcription factorsto nucleosomal DNA and the disruption of underlying nucleo-somes. GAL4 derivatives (67, 72, 78), glucocorticoid receptor(2, 40, 50, 51, 53), Sp1 (39), TFIIIA (37), USF (12), andMax/Max and Myc/Max dimers (74) have all been shown to becapable of nucleosome binding under some circumstances.However, many variables govern the affinity of a given tran-scription factor for nucleosomal DNA (73). The inherent af-finity of a factor for its cognate site on a nucleosome appearsto be largely a property of its DNA-binding motif but is furtherinfluenced by dimerization domains and partners (67, 74). Nu-cleosome binding is affected by the location of binding siteswithin nucleosomes, the availability of accessory proteinswhich participate in nucleosome disruption, and the chemicalmodification of the histone amino-terminal tails (2, 12, 13, 37,39, 40, 53). In addition, the binding of GAL4 derivatives tonucleosomes is cooperative (67). The binding of multipleGAL4 dimers overcomes inhibition from the core histoneamino termini, alleviating nucleosome position effects (72).Chromatin assembly greatly enhances the ability of multiple
GAL4-VP16 activators to mount a synergistic transcriptionalresponse in vitro (11). The requirement for multiple GAL4-binding sites can provide for synergistic interactions of multi-ple activation domains with target proteins that form transcrip-tion initiation complexes at the core promoter (9, 42; reviewedin reference 24), a rate-limiting step on nucleosomal templates(32, 33, 44, 76, 79, 80). In addition, multiplicity of GAL4-
* Corresponding author. Mailing address: Department of Biochem-istry and Molecular Biology, 306 Althouse Laboratory, The Pennsyl-vania State University, University Park, PA 16804-4500. Phone: (814)863-8256. Fax: (814) 863-0099. Electronic mail address: JLW10@psuvm.psu.edu.
FIG. 1. Unrelated transcription factors USF and GAL4 bind cooperatively to nucleosome cores but not to naked DNA. (A) Diagram of nucleosome-length DNAfragments, containing GAL4 and USF binding sites, used as probes for binding studies. Probes GUB and UGB are identical except for the orientation of theoligonucleotide, containing both binding sites, that is shown. Probe GUBmUSF is identical to GUB except for the single base substitution within the 6-bp coreUSF-binding site. Nucleotides are numbered from the BamHI site (bp 1) used to excise all probe fragments from their respective plasmids (see Materials and Methods).Nucleotides used to denote the center of each binding site in all three fragments are indicated. M, MluI; G, BglII; X, XhoI; P, PvuII; S, SspI; B, BamHI. (B) EMSAanalysis of USF binding to probe GUB as mock-reconstituted naked DNA. Probe DNA was labeled at the BamHI site by Klenow polymerase. For mock reconstitution,labeled DNA probe was added to HeLa oligonucleosomes, after dilution to 0.1 M NaCl, such that the final concentrations of donor nucleosomes and template DNAare the same as in the reconstituted samples (see Materials and Methods). USF was titrated into binding reaction mixtures with (1; lanes 7 to 12) or without (2; lanes1 to 6) 40 nM GAL4-AH. The relative concentration of USF protein in each binding reaction mixture is shown. Protein/DNA complexes were resolved by 4% PAGEand visualized by autoradiography. Complexes representing free probe DNA and USF and/or GAL4 bound to DNA are labeled, DNA, USF/DNA, GAL4-AH/DNA,and USF/GAL4-AH/DNA, respectively. At high USF concentrations, nonspecific binding of additional USF proteins to the DNA is detected (USF2/DNA, USF3/DNA,and USF2/GAL4-AH/DNA; lanes 6 and 12). These complexes represent nonspecific binding of additional USF dimers and/or binding of USF tetramers. (C) EMSA(4% PAGE) analysis of USF binding to probe GUB reconstituted into nucleosome cores. Probe DNA was labeled at the BamHI site as described above. USF wastitrated into binding reaction mixtures containing radiolabeled probe DNA that had been mock reconstituted (DNA; lanes 1 to 5) or reconstituted into nucleosomecores (lanes 5 to 20), in the absence (2) or presence (1) of GAL4 derivatives GAL4-AH (lanes 2, 4, 5, and 12 to 17) and GAL4(1-94) (lanes 18 to 20). The concentrationof USF protein in each binding reaction is given. The concentrations of GAL4-AH used in binding reactions were 123 nM for naked DNA samples (lanes 2 and 4)and 2.1 mM for nucleosome samples; the concentration of GAL4(1-94) was 2.0 mM. Complexes representing proteins bound to free DNA (DNA) are labeled on theleft, while complexes resulting from proteins bound to nucleosome cores (Nuc) are labeled on the right. (D) Comparison of USF binding to DNA probes GUB (lanes1 to 7) and GUBmUSF (lanes 8 to 14) reconstituted into nucleosomes. USF titration, GAL4-AH concentration, and conditions of EMSA are the same as for panel C.
1406 ADAMS AND WORKMAN MOL. CELL. BIOL.
binding sites also provides for cooperative binding of GAL4derivatives to recognition elements in nucleosomes (67, 72).Together, these non-mutually exclusive mechanisms (cooper-ative binding and activation domain synergism) may lead tosubstantial synergistic effects and threshold responses fromcellular promoters and enhancers in vivo.A common feature of most enhancers and promoters is the
presence of multiple binding sites (enhansons), often for dis-parate regulatory factors (reviewed in reference 16). Cooper-ative binding of regulatory factors can result from direct pro-tein-protein interactions between the factors