Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6203-6207, July 1993 Biochemustry Histone octamer dissociation is not required for transcript elongation through arrays of nucleosome cores by phage T7 RNA polymerase in vitro (RNA synthesis/SS RNA-encoding DNA/chromatin/dlmethyl suberimidate) TIMOTHY E. O'NEILL*t, JANET G. SMITH*t, AND E. MORTON BRADBURY*§ *Department of Biological Chemistry, School of Medicine, University of California, Davis, CA 95616; and §Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545 Communicated by Robert G. Roeder, April 6, 1993 ABSTRACT We have examined whether dissociation of ing of the nucleosome into halves, each containing a hetero- the histone octamer is required for elongation of RNA tran- typic tetramer (8); the formation of an unfolded lexosome scripts through arrays of nucleosome cores in vitro. Control or structure containing nonhistone proteins (9); the splitting of dimethyl suberimidate-crosslinked histone octamers were re- nucleosomes by transcription-induced positive supercoiling constituted onto supercoiled, closed circular pT207-18 DNA, (10); the loss of an (H2A-H2B) dimer, resulting in a depleted which contains tandemly repeated 207-base-pair (bp) 5S rDNA nucleosome that more easily complexes with RNA polymer- nucleosome positioning sequences inserted between the T7 and ase (11); the progressive transient displacement of histone SP6 transcription promoters of pGEM-3Z. Double label tran- H2A-H2B dimers as RNA polymerase passes through the scription experiments showed that there was little or no effect nucleosome (12); and the transfer ofhistone octamers to sites of extensive crosslinking of the histone octamers on transcrip- behind the transcribing RNA polymerase (13, 14). The phys- tion initiation and elongation by T7 RNA polymerase in vitro. iological validity of these mechanisms remains speculative Continuous regularly spaced linear arrays ofeither crosslinked for lack of definitive experimental evidence. or control nucleosome cores were obtained by digesting recon- In a previous study (4) we made a 6.6-kilobase-pair (kbp) stituted nucleosomal pT207-18 templates with Dra I, a site that DNA construct, pT207-18, which contains an insert of 18 is protected from digestion by the presence of positioned tandem repeats of the 207-bp nucleosome positioning se- nucleosome cores in the 207-bp sequence. After in vitro tran- quence from the Lytechinus variegatus 5S RNA gene adja- scription with T7 RNA polymerase, an RNA ladder with cent to a bacteriophage T7 transcription promoter. Because 207-nucleotide spacing was obtained from templates reconsti- nucleosome tuted both with crosslinked and with control histone octamers, cores are assembled into regularly spaced posi- demonstrating clearly that neither partial nor complete disso- tioned arrays on these tandem repeat sequences (15-18), the ciation of the histone octamer is essential for transcription pT207-18 construct can be used to assay the efficiency of elongation through arrays of nucleosome cores in vitro. transcript elongation through homogeneous arrays of regu- larly spaced nucleosome cores in a well-defined in vitro The mechanism of passage of RNA polymerase through system. This construct was used to show that bacteriophage nucleosomes has been the subject ofintense interest over the T7 RNA polymerase can elongate transcripts through arrays past few years. It has been shown clearly in in vitro exper- ofup to at least 10 nucleosome cores, but that with increasing iments that bacteriophage SP6 polymerase can transcribe numbers of nucleosome cores transcription elongation is through one nucleosome (1, 2) or short stretches of a few partially inhibited. In the current study, we use the pT207-18 nucleosomes (3). Recent work from this laboratory has construct to assay the effects ofextensive chemical crosslink- shown that T7 RNA polymerase can transcribe through ing of histone octamers on elongation of transcripts through regularly spaced arrays of at least 10 positioned nucleosome reconstituted arrays of nucleosome cores by T7 RNA poly- cores, but the efficiency of transcription elongation is signif- merase in vitro. These experiments directly test models for icantly decreased compared to free DNA templates (4). transcription elongation through nucleosome cores that in- Similar results have been obtained in studies of T7 RNA volve splitting or dissociation of the octamer prior to or polymerase transcription through linear DNA fragments con- during transcription. We demonstrate that dissociation of taining 4-7 nucleosome cores (5). Greater inhibition of tran- histone octamers is not necessary for transcription elongation scription elongation is observed in transcription of nucleo- through nucleosome cores in vitro. It is shown that nucleo- somal templates by RNA polymerase 11 (6). somal arrays reconstituted from histone octamers that have Hence, abundant evidence indicates that although RNA been extensively crosslinked with dimethyl suberimidate polymerases can transcribe through individual nucleosome behave similarly to arrays reconstituted from control histone cores, arrays of nucleosome cores do present a significant octamers in transcription assays with T7 RNA polymerase. obstacle to transcription elongation. At this time how RNA Transcription occurs through arrays of both control and polymerase gains access to DNA wrapped into nucleosomes during transcription elongation remains unclear. A number of crosslinked nucleosome cores, albeit at a lower efficiency models have been proposed for this process that involve the than for free DNA templates. Thus, dissociation or splitting transient disruption of nucleosome structure during the pro- of the histone octamer is not essential for transcription cess of transcription elongation via displacement of histones elongation through nucleosome cores in vitro. from DNA or via splitting or unfolding of the nucleosome (reviewed in ref. 7). Proposed mechanisms involve the open- Abbreviation: PMSF, phenylmethylsulfonyl fluoride. tPresent address: Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, Building 6, The publication costs ofthis article were defrayed in part by page charge Room BlA13, National Institutes of Health, Bethesda, MD 20892. payment. This article must therefore be hereby marked "advertisement" tPresent address: Department of Food Science and Technology, in accordance with 18 U.S.C. §1734 solely to indicate this fact. University of Califormia, Davis, CA 95616. 6203 Downloaded by guest on October 1, 2021 6204 Biochemistry: O'Neill et al. Proc. Natl. Acad. Sci. USA 90 (1993) MATERIALS AND METHODS tical to those used in double-label experiments. Transcription reactions were analyzed by DE-81 filter binding assays and Preparation of Histone Octamers. Nucleosome core parti- electrophoresis in 6% polyacrylamide gels containing 8.3 M cles and histone octamers were prepared from HeLa nuclei urea. Appropriate sample volumes were loaded onto gels for as described (4). Crosslinked histone octamers were prepared comparison on the basis of equal 32p incorporation. essentially as described by Stein et al. (19). Nucleosome core particles at 100 ug/ml were crosslinked in 100 mM sodium borate (pH 10) containing dimethyl suberimidate (Pierce) at RESULTS 10 mg/ml for 40 min at 25°C. This material was then dialyzed Crosslinking of Octamers and Nucleosome Core Reconsti- against 100 mM sodium borate (pH 10) at 4°C, concentrated, tution. Stein et al. (19) demonstrated that dimethyl suberim- and crosslinked a second time with dimethyl suberimidate as idate-crosslinked histone octamers can be used to reconsti- described above. The crosslinked particles were then dia- tute nucleosome core particles that have properties quite lyzed against 10mM Tris HCl, pH 6.85/5 mM EDTA/0.1 mM similar to those of native nucleosome cores as assessed by phenylmethylsulfonyl fluoride (PMSF) and refractionated on circular dichroism spectra, DNase I digestion, electron mi- 5-20% sucrose gradients. The purified particles were then croscopy, and DNA supercoiling assays. Nucleosome core extracted with 3 M NaCl/10 mM Tris HCl, pH 7.4, several particles assembled from crosslinked histone octamers were times to precipitate histones and to remove DNA before only slightly more stable to thermal denaturation than native storage in 10 mM Tris HCl, pH 7.4/0.5 mM EDTA/0.1 mM core particles in these studies. A modification of their pro- PMSF. The purity and integrity ofthe individual histones and cedure was used to produce extensively crosslinked histone the extent of histone crosslinking were determined by 18% octamers from HeLa nuclei. Crosslinked and control histone (wt/vol) polyacrylamide gel electrophoresis (20), followed by were as in 1. staining with Coomassie brilliant blue R. octamers analyzed by SDS/PAGE shown Fig. Nudeosome Core Reconstitution. Nucleosome cores were The crosslinked histone octamer migrates with an apparent reconstituted onto supercoiled plasmid DNA by using a molecular weight of 4100,000 daltons, with no detectable modification of the salt dilution method of Germond et al. contamination of lower molecular weight components. (21). In reconstitutions with control histone octamers, oc- In the current study, nucleosome cores were reconstituted tamers were mixed with 12.5 pg of supercoiled pT207-18 onto supercoiled closed circular pT207-18 DNA from donor DNA in 2 M NaCl/10 mM Tris HCl, pH 7.4/10 mM sodium crosslinked or control histone octamers by salt