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Proc. Nadl. Acad. Sci. USA Vol. 88, pp.

9675-9679, November 1991

Biochemistry

Positive DNA supercoiling generates a chromatin conformation characteristic of highly active genes
(transcription/torsional stress/nucleosome/2-Itm minichromosomes/yeast)

MYEONG-SOK LEE

AND

WILLIAM T. GARRARD*

Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235

Communicated by James Bonner, August 5, 1991

During transcription, positive DNA superABSTRACT coils generated ahead of RNA polymerase could theoretically uncoil the negative DNA supercoils associated with nucleosomes and thereby decondense the chromatin fiber in preparation for RNA polymerase passage. Here we exmine the effect of positive DNA supercoiling on the structure of yeast 2-pm minichromosomes. We utilized a conditional topoisomerase mutant expressing Escherchia coli topoisomerase I to convert the DNA supercoiling state from negative to positive in vivo. Minichromosomes containing positively supercoiled DNA exhibited a striking increase in DNase I sensitivity. They also displayed additional micrococcal nuclease cleavage sites but yielded nearly typical nucleosomal ladders after extensive digestion. Upon in vitro relaxation with eukaryotic topoisomerase I, the minichromosomes remained DNase I sensitive but were converted to negative DNA supercoiling with a slightly increased linking number compared to typical minichromosomes, thus indicating the presence of bound histones. Therefore, positive DNA supercoiling provides a mechanism for generating, but is not required for maintaining, a conformation in chromatin characteristic of highly transcribed genes.

transcription complex. Here we have utilized an in vivo system to examine this model by studying the effects of positive DNA supercoiling on the structure of yeast 2-,um minichromosomes. We demonstrate that positive DNA supercoils generate a chromatin conformation similar to that exhibited by highly transcribed genes and that these supercoils are not required to maintain this conformation.

Mechanisms that alter the conformation of chromosomes in living cells are of considerable interest as they likely determine the efficiencies of transcription, replication, recombination, mutagenesis, and DNA repair. Gene activation is accompanied by alterations in the conformation of chromatin, which for highly transcribed genes are manifested by increased sensitivity to cleavage by DNase I (1), a partial disruption of nucleosomal structure as revealed by micrococcal nuclease (MNase) digestion (2), and morphological decondensation (3) (for reviews, see refs. 4 and 5). Particularly intriguing is that these characteristics often extend considerable distances beyond the 5' and 3' boundaries of transcription units (refs. 6 and 7 and references therein), making it unlikely that such alterations are caused by direct contact with traversing RNA polymerase molecules. One possible way to spread alterations along a chromatin fiber is through torsional stress generated during transcription. Experimental evidence supports the hypothesis that the template rotates relative to traversing RNA polymerase molecules (8), leading to twin domains of DNA supercoiling: positive supercoiling downstream and negative supercoiling upstream of the transcription complex (9). Although DNA topoisomerases relax these structures, it seems likely that this relaxation may kinetically lag behind supercoil generation (8). We have proposed (10) that positive DNA supercoiling may play an important role in chromatin decondensation and pave the way for RNA polymerase by uncoiling the negatively supercoiled nucleosomal DNA downstream of the
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact.
9675

MATERIALS AND METHODS Yeast Strain and Growth Conditions. Two 1-liter cultures of Saccharomyces cerevisiae RS192 (Mata ade2 ura3-1 his311,15 trpl-J leu2-3,112 canl-100 top J-8::LEU2 top2-1) transformed with YepTopA-pGAL (8) were grown in 2% (wt/vol) galactose/1% yeast extract/2% (wt/vol) peptone. Where indicated, galactose was replaced with glucose. For most experiments, one culture was inoculated with 3 x 106 cells per ml and grown at 240C for 16 h to an A6N of -0.50 (5 x 107 cells per ml) and the other was inoculated with 5.4 x 106 cells per ml, grown for 13 h at 240C to an A6N of -0.50, and then shifted to 370C for 3 h. To obtain a sample with a mixture of DNA topoisomers, cells were shifted to 370C for only 1.5 h. Analysis of DNA Supercoiling States. Washed cells were incubated with Oxalyticase (Enzogenetics, Corvallis, OR) as described (10) except that the 370C cell sample was also incubated at 370C to inhibit potential residual topoisomerase II activity (11). Spheroplasts were collected by centrifugation (5000 x g for 5 min at 40() and lysed by suspension to a final DNA concentration of 100 ,ug/ml in ice-cold nuclease digestion buffer [40 mM HepesNaOH, pH 7.5/1 mM MgCl2/0.2 mM CaC12/1 mM phenylmethylsulfonyl fluoride/chymostatin (0.5 Ag/ml)/aprotinin (1.7 jug/ml)/trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (Sigma; 7.2 ug/ml)/ phosphoramidon (1.1 ,g/ml)/leupeptin (2.0,ug/ml)/antipain (2.0 ,ug/ml)/pepstatin (2.0 tug/ml)/2 mM benzamidine]. For DNA purification, 400 ,ul of 50 mM TrisHHCl, pH 7.0/20 mM EDTA was added to each 100-ulI sample. After gentle mixing, 50 ,ul of 10%o (wt/vol) SDS was added and samples were incubated at 650C for 30 min, extracted with phenol/ chloroform, and ethanol-precipitated. Suspended pellets were treated with RNase A and extracted as before. Twodimensional chloroquine/agarose gels (12) were loaded with 5 Ag of DNA samples. After transfer to Zeta-Probe membranes (Bio-Rad) (13), filters were prehybridized for 4 h at 650C and hybridized for 15 h at 450C with a 32P-end-labeled 2-pum 40-mer probe (5'-GGTAGGATCGATCCACTTGTATATTTGGATGAATTTTTGA-3'). Filters were washed once at room temperature for 10 min and for three 30-min periods at 530C with 2x standard saline citrate/20 mM sodium phosphate, pH 7.0/2% SDS. Autoradiograms were exposed with intensifying screens at -700C. Where indicated, stripped filters were rehybridized with a pBR322 probe
Abbreviation: MNase, micrococcal nuclease. *To whom reprint requests should be addressed.

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Biochemistry: Lee and Garrard

Proc. Natl. Acad. Sci. USA 88 (1991)

(5'-AAGAATTCTCATGTTTGACAGCTTATCATCGATAAGCTTT-3').
Nuclease Digestion, Chromatin Analysis, and Topoisomer-

ase I Treatment. Lysates were digested at 370C for 10 min with DNase I at 0.14 or 0.32 units/ml or MNase (Worthington) at 0.55 or 1.2 units/ml (footprinting) or MNase at 5.8, 12.7, 28, or 62 units/ml (structural analysis). Naked DNA was digested with 30-times less enzyme. Footprinting was by indirect end-labeling from the EcoRI site and structural analysis was by published techniques (10) with the above 2-,um probe and hybridization conditions. For relaxation, spheroplasts were lysed in nuclease digestion buffer (1 mM EDTA), and samples were diluted with an equal volume of nuclease digestion buffer (50 mM NaCl/1 mM EDTA) and centrifuged to remove whole cells (1000 x g for 3 min). For a 200-/l sample (5 ug of DNA), 1 pug of relaxed or negatively supercoiled form I pBR322 DNA was added followed by incubation for 1.5 min at 370C, addition of purified chicken topoisomerase I (1000 units in 10 ILI) (14), and 8.5 min of further incubation. When required, an equal volume of digestion buffer (5 mM MgC12) was added and samples were digested for 7 min at 370C with DNase I at 1.1 or 2.5 units/ml.

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RESULTS
Generation of Positively Supercoiled DNA in vivo Within Yeast 2-jam Minichromosomes. To study the effect of positive DNA supercoiling on chromatin structure, we utilized a top] top2-ts yeast strain, expressing Escherichia coli topoisomerase I, in which the supercoiled state of DNA can be manipulated in vivo by altering the growth conditions (8). At the permissive temperature of 240C, DNA is organized into topologically constrained negative supercoils because of the histones and a functional topoisomerase II (8, 11). However, when the topoisomerase II is inactivated at the nonpermissive temperature of 370C, positive supercoils slowly accumulate due to preferential relaxation of transient negative DNA supercoils by E. coli topoisomerase I (8). We selected for study the endogenous yeast multicopy 2-pum plasmid since these 6.3-kilobase (kb) circular molecules exist in the nucleus as minichromosomes (15) with a welldefined typical nucleosomal structure (16-18). In agreement with Giaever and Wang (8), two-dimensional gel electrophoresis revealed that the majority of the 2-pum minichromosomes contained positively supercoiled DNA when isolated from the mutant cells after they had been shifted to 370C (Fig. 1B, spot dl). In contrast, 2-p~m circles contained negative supercoils as expected when a functional topoisomerase II was present at 240C (Fig. 1A, spot a1) (11). These gel-mobility assignments were confirmed utilizing a standard containing a mixture of topological forms (Fig. 1C), in which we could visualize at least 15 positively supercoiled intermediate species (e.g., Fig. 1C and data not shown), yielding a minimum of one positive supercoil for every -0.4 kb of 2-pum DNA for molecules migrating in the d, position. Positive DNA Supercoiling Exposes Nuclease Cleavage Sites in Chromatin. We first employed DNase I footprinting in whole-cell lysates to study the effect of positive DNA supercoiling on the structure of the rarely transcribed REP2 gene region within 2-pum minichromosomes (19, 20). As shown in Fig. 2A, lanes a and b, the REP2 locus in minichromosomes containing negatively supercoiled DNA was flanked both 5' and 3' by hypersensitive sites (solid bars). The coding region was relatively resistant to DNase I and exhibited seven footprints with the intervening cutting sites following nucleosomal periodicity (open circles). In striking contrast, the entire REP2 coding region exhibited high DNase I sensitivity in minichromosomes containing positively supercoiled DNA (Fig. 2A, lanes d and e). This increased sensitivity extended both upstream into the FLP gene coding region and down-

FIG. 1. Supercoiling state of 2-Am DNA. A yeast top] top2-ts mutant, expressing E. coli topoisomerase I, was grown at 240C (A) or shifted to 370C (B) for 3 h. DNA was purified, separated by two-dimensional electrophoresis, blotted to a filter, and hybridized with a 2-Am probe. C depicts a mixture of topoisomers. Indicated are negatively supercoiled single molecules and interlocked rings containing two and three molecules (a,, a2, and a3, respectively), nicked circles (b), linear molecules (c), positively supercoiled single molecules and interlocked rings of two molecules (d1 and d2, respectively), and relaxed covalently closed circles (e). D, dimension.

stream into distal regions and was very similar to the cutting pattern exhibited by naked DNA (Fig. 2A, compare lanes d and e with lane c). However, the nuclease-hypersensitive sites flanking the REP2 locus were not disrupted by positive DNA supercoiling (Fig. 2A, lanes d and e, solid bars). The overall enhanced DNase I sensitivity was not correlated with elevated REP2 gene transcription as assessed by Northern blot analysis (data not shown) nor was it caused by heat shock, since control experiments revealed that the REP2 locus was not DNase I-sensitive in mutant cells shifted to 370C in the absence of E. coli topoisomerase I expression (see Fig. 5, lanes a and b). Furthermore, naked DNA samples were digested at similar rates, whether they were positively

conclude that positive DNA supercoiling increases the DNase I sensitivity of the chromatin. We also studied the pattern of MNase cleavage within the REP2 locus of the corresponding samples. As with the DNase I digests, the coding region exhibited seven footprints with the intervening MNase cutting sites following nucleosomal periodicity in minichromosomes containing negatively supercoiled DNA (Fig. 2B, lanes a and b, open circles; see also ref. 18). Minichromosomes containing positively supercoiled DNA exhibited these cutting sites but in addition displayed all sites that were present in digests of naked DNA (Fig. 2B, compare lanes d and e with lane c). Thus with the DNase I footprinting results, we conclude that positive DNA supercoiling exposes nuclease cleavage sites in chromatin. Minihromosomes Containing Positively Supercoid DNA Exhibit a Nearly Typcal Nucleosomal Structure. Nearly typical nucleosomal ladders were generated upon extensive MNase digestion of minichromosomes containing positively supercoiled DNA. These exhibited an oligomeric DNA repeat length of 168 + 5 base pairs (Fig. 3A, lanes e-h), a value similar to that measured for the negatively supercoiled counterparts (Fig. 3A, lanes a-d; see also refs. 16 and 17). Those minichromosomes containing positively supercoiled DNA were digested -2 times faster and their nucleosomal ladders exhibited a higher interband background (Fig. 3A). Similar patterns were observed when bulk genomic DNA was probed on the same filters (Fig. 3B). Thus, genomes that have been topologically converted from negative to positive DNA supercoiling exhibit a nearly typical nucleosomal organization upon MNase digestion.

or negatively supercoiled (data not shown).

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Biochemistry: Lee and Garrard


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Proc. Natl. Acad. Sci. USA 88 (1991)

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structure ofthe REP2 locus. Cultures were grown at 240C (lanes a and b) or shifted to 370C (lanes d and e) as described in Fig. 1. Lysate samples were each digested with two concentrations ofeither DNase

FIG. 3. Effect of positive DNA supercoiling on nucleosomal structure. Cultures were grown at 240C or shifted to 370C as described in Fig. 1. Lysates were digested with various concentrations of MNase (equal concentrations were in lanes a and e, lanes b and f, lanes c and g, and lanes d and h). DNA was purified, separated by electrophoresis, blotted to a filter, and hybridized with 2-gm DNA (A) or bulk DNA (B) probes. 1-8N represent the oligonucleosomal length of DNA fragments.

I (A) or MNase (B) (equal concentrations were in lanes a and d and lanes b and d). DNA was purified, cleaved with EcoRl, separated by electrophoresis, blotted to a filter, and hybridized with a 2-/Am probe that abutted the EcoRl site. Lanes c (D) contain the corresponding nuclease digests of naked DNA processed similarly. Calibration on the left is absolute DNA length (kb) and in the center is nucleotide map position. The open vertical arrows illustrate open reading frames, solid vertical bars depict DNase I hypersensitive regions, and open circles refer to cleavage sites with a nucleosomal periodicity. Because two isomers of 2-Am DNA exist (19), the chromatin structure above 2.4 kb is a mixture.

pBR322 had been relaxed (Fig. 4 C and D) or negatively supercoiled (data not shown). Thus, no evidence was obtained for an increase in free histones as a consequence of positive DNA supercoiling or for stripping histones off chromatin containing positively supercoiled DNA by exposure to negatively supercoiled naked DNA [the histone transfer model in transcription (21)]. Thus we conclude that positive DNA supercoils are not topologically constrained and infer

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Relaxation Converts Chromatin Containing Positively Supercoiled DNA to a Negatively Supercoiled State. We determined whether minichromosomes could be converted from positive to negative DNA supercoiling by treatment of lysates in vitro with eukaryotic topoisomerase I (after preincubating samples with a 10-fold excess of naked pBR322 DNA relative to 2-gm DNA to mop up any free histones). As shown in Fig. 4, this treatment indeed converted the minichromosomes containing positive DNA supercoils (Fig. 4A) to negative DNA supercoiling (Fig. 4B). These results provide no evidence for the presence of naked DNA in a fraction of the original population of 2-gm molecules. The higher resolution electrophoresis shown in Fig. 4E revealed that these relaxed minichromosomes (lane 2) possessed a somewhat broader topoisomer distribution with a slightly higher linking number as compared to the control (relaxed typical minichromosomes) (lane 3). [Similar results were obtained in the absence of added pBR322 (data not shown).] Simultaneously monitoring the extent of chromatin assembly onto the added naked pBR322 DNA molecules revealed that lysates prepared from control cells or lysates containing positively supercoiled DNA introduced on average only about four or five negative supercoils into pBR322 DNA molecules, whether the input

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FIG. 4. Relaxation of positive DNA supercoils by eukaryotic topoisomerase I. (A and B) Lysates of a culture shifted to 370C as described in Fig. 1, preincubated with relaxed pBR322 DNA, were further incubated with (B) or without (A) eukaryotic topoisomerase I. Samples were analyzed for 2-Am DNA as in Fig. 1. (C and D) Cultures were grown at 240C (C) or shifted to 370C (D) as described in Fig. 1. Lysates preincubated with relaxed pBR322 DNA circles were incubated further with eukaryotic topoisomerase I. Samples were processed as above, but filters were hybridized with a pBR322 probe. (E) Samples in A-C (lanes 1-3, respectively, in E) were resolved on a chloroquine/agarose gel (11), blotted to a filter, and hybridized with a 2-,um probe. Indicated are nicked circles (n), positively (+) or negatively (-) supercoiled circles, diagonal lines (dashes), negatively supercoiled topoisomers (0, -2, -4, -6), linear molecules (1), and an unknown DNA band seen in some experiments (*) (lanes 1 and 2).

9678

Biochemistry: Lee and Garrard

Proc. Natl. Acad. Sci. USA 88 (1991)

that positive DNA supercoiling does not significantly displace histones from chromatin. Positive DNA Supercoiling Is Not Required to Maintain DNase I Sensitivity. To determine whether positive torsional stress was responsible for the heightened DNase I sensitivity of 2-Eum minichromosomes (Fig. 2), we repeated the DNase I footprinting experiments before and after topoisomerase I relaxation. Fig. 5 shows that such relaxation did not reverse the DNase I sensitivity within or surrounding the REP2 locus (compare lanes c and d with lanes e and f). Two-dimensional electrophoresis confirmed that relaxation was complete (data not shown). Furthermore, as a control for potential heatshock effects, we topoisomerase-treated a lysate from cells that had been grown in glucose medium at 24TC and shifted in parallel to 370C and found that the REP2 locus did not become DNase I-sensitive (Fig. 5, lanes a and b) [these growth conditions repress expression of E. coli topoisomerase I (8)]. Thus, we can conclude that, although positive torsional stress may be responsible for generating DNase I sensitivity, such stress is not required for its maintenance.

DISCUSSION
Chromatin Containing Positively Supercoiled DNA Possesses Conformationally Altered Nucleosomes. Conformationally altered nucleosomes appear to represent the underlying GLU Kb STOPO 4.0
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structure of chromatin containing positively supercoiled DNA. Footprinting experiments demonstrated nuclease cutting within regions normally exhibiting a nucleosomal structure (Figs. 2 and 5). However, nearly typical nucleosomal ladders were generated after extensive MNase digestion (Fig. 3). These minichromosomes apparently still had nearly normal amounts of histones but contained positive torsional stress since relaxation with eukaryotic topoisomerase I generated minichromosomes with a similar (but not identical) linking number compared to typical minichromosomes (Fig. 4). The positive stress was not located exclusively in linker regions since nucleosomal footprints were disrupted (Figs. 2 and 5). In addition, growth conditions leading to positive DNA supercoiling did not result in an apparent increase in the intracellular free histone pool nor did added naked DNA preferentially strip off histones from the chromatin (Fig. 4). The conformationally altered nucleosomes may correspond to the unfolded or split nucleosomal structures associated with DNase I sensitivity in chromatin (10, 22). In some experiments, we have detected several split nucleosome footprints along the REP2 locus (data not shown); presumably imprecise positioning along the sequence has prevented a clearer demonstration of these structures. Perhaps the positive torsional stress achieved in the genetically engineered yeast system is so great that nucleosomes become flipped inside out. Utilizing in vitro reconstitution, Clark and Felsenfeld (21) concluded that nearly typical nucleosomes can be formed on positively supercoiled DNA. The findings from our in vivo study are not necessarily in disagreement. We agree that such chromatin still possesses histones, is capable of being relaxed by topoisomerase I to a negatively supercoiled state, and generates nearly typical nucleosomes upon MNase digestion. Although many important technical differences exist between our studies including the histone source (11, 23), Clark and Felsenfeld (21) could have missed the altered nucleosome conformation detected here because they did not perform

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footprinting experiments. Positive DNA Supercoiling Provides a Mechanism for Spreading a Chromatin Conformation Characteristic of ighly Active Genes. The structure of 2-,um minichromosomes containing positively supercoiled DNA resembles that of highly transcriptionally active genes: both are preferentially sensitive to DNase I digestion and contain a nucleosomal structure more prone to internal nuclease cleavage relative to that of inactive sequences (Figs. 2, 3, and 5; refs. 1-7 and 10). Positive torsional stress, therefore, may be a primary directive for establishing these features and could signal a series of stabilizing interactions mediated by posttranslational modifications and/or by other proteins. Once generated, the altered chromatin conformation appears to be "memorized," since relaxation with topoisomerase I did not reverse DNase I sensitivity (Fig. 5). Furthermore, heightened nuclease sensitivity ofthe active chicken globin gene locus, which appears
to be associated with torsional stress, cannot be fully reversed by DNA nicking (24) nor can that associated with the

I.

FIG. 5. Effect of positive torsional stress on the DNase I sensitivity of the REP2 locus. Cultures grown at 240C in glucose (lanes a and b) or galactose (lanes c-f) were shifted to 370C as described in Fig. 1. Lysate samples were incubated with (lanes a, b, e, and f) or without (lanes c and d) eukaryotic topoisomerase I and then digested with two concentrations of DNase I (equal concentration were in lanes a, c, and e and in lanes b, d, and I). Sample processing and I figure labels are as in Fig. 2.

yeast HSP82 gene (10). Typical 2-,um minichromosomes are estimated to contain -32 nucleosomes (16). The slightly increased linking number of the relaxed minichromosomes [Fig. 4E; AL 4] may not necessarily reflect '12% fewer nucleosomes but instead could be due to alterations in nucleosome conformation at the level of DNA writhe and/or twist, possibly caused by the loss of an H2A-H2B dimer (see refs. 21 and 25). Similar changes in linking number per nucleosome can be caused by histone acetylation (26). Several experimentally testable models can be considered on how positive torsional stress could alter chromatin structure. Fig. 6A schematically illustrates the consequences of the twin domain model of DNA supercoiling of Liu and Wang (9) at the level of chromatin as we have proposed (10). The

Biochemistry: Lee and Garrard

Proc. Natl. Acad. Sci. USA 88 (1991)

9679

-V-

molecules

are

transcriptionally active in vivo. Moreover,

contrary to previous views (33), recent studies reveal that nucleosomes are potent inhibitors of transcriptional elonga-

tion in vitro (34). Thus, positive DNA supercoiling may be required to facilitate the process of elongation by decondensing chromatin.
We are indebted to Drs. L. A. Freeman, D. S. Gross, P. K. Pfaffie, and K. W. Trevorrow for a critical review of this manuscript. We are particularly grateful to Drs. V. Jackson, R. Sternglanz, and J. C. Wang for their generous gifts of topoisomerase I, yeast strains, and recombinant plasmids. This investigation was supported by Grants GM22201, GM29935, and GM31689 from the National Institutes of Health and Grant 1-823 from the Robert A. Welch Foundation.
C

FIG. 6. Chromatin structural changes caused by transient DNA supercoiling. The schematic diagrams depict chromosomal loops with anchored ends, composed of polynucleosomal chains that are being traversed from left to right (arrow) by RNA polymerase that initiated at the triangular site labeled I. (A) Twin domains of DNA supercoiling (9) lead to positive supercoils (+) downstream of the transcription complex, which conformationally unfold or split nucleosomes, and negative supercoils (-) upstream, which tightly pack the structure (10). (B) Selective relaxation of negative supercoils spreads positive torsional stress throughout the loop. (C) Conformational alterations in nucleosomes initially created by positive stress downstream of the transcription complex are not fully reversed by subsequent negative stress after traversal by RNA polymerase.

positive supercoils downstream of traversing RNA polymerwould uncoil the negatively supercoiled DNA about histone octamers leading to nucleosome unfolding or splitting (10, 22). In addition, the 30-nm chromatin fiber, which consists of a nucleosome superhelix [even in yeast (27)], may also be negatively supercoiled (28) and positive supercoils could also uncoil these structures. The model shown in Fig. 6A, however, cannot account for the observation that preferential DNase I sensitivity sometimes extends upstream of transcription units (e.g., ref. 6). As shown in Fig. 6B, a net positive stress and accompanying DNase I sensitivity could be generated throughout an entire chromosomal loop if negative DNA supercoils are preferentially relaxed. Alternatively, positive supercoils might create an altered chromatin structure that is not fully reversed by negative stress (e.g., Fig. 5), leading to heightened DNase I sensitivity throughout and downstream of the transcribed gene region but not upstream of the initiation site (Fig. 6C). Net superhelical stress apparently can even be negative if positive supercoils are preferentially relaxed (29). Implications of Positive DNA Supercoiling for Transcription. As described above, positive DNA supercoiling downstream of the transcription complex is believed to decondense the chromatin fiber and pave the way for RNA polymerase passage (Fig. 6 and ref. 10). (The process of traversal is a separate topological problem requiring unwinding of the DNA double helix.) Significantly, the results of in vitro experiments are consistent with the possibility that transcription-induced positive stress alters nucleosomes (14) and previous electron microscopic studies have visualized chromatin decondensation downstream of traversing RNA polymerase (3). Furthermore, torsional stress appears to be required for transcription to occur in living cells, since circular, but not linearized (30, 31) or nicked (32), DNA
ase

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