Visualization of unconstrained negative supercoils of DNA on polytene chromosomes of Drosophila

Kuniharu Matsumoto1 and Susumu Hirose1,2,*

1 Department of Developmental Genetics, National Institute of Genetics, SOKENDAI, 1111 Yata, Mishima, Shizuokaken 411-8540, Japan
2 Department of Genetics, SOKENDAI, 1111 Yata, Mishima, Shizuokaken 411-8540, Japan

* Author for correspondence (e-mail: shirose{at}lab.nig.ac.jp)

Accepted 17 March 2004


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Bulk DNA within the eukaryotic genome is torsionarily relaxed. However, unconstrained negative supercoils of DNA have been detected in few local domains of the genome through preferential binding of psoralen. To make a genome-wide survey for such domains, we introduced biotinylated psoralen into Drosophila salivary glands and visualized it on polytene chromosomes with fluorescent streptavidin. We observed bright psoralen signals on many transcriptionally active interbands and puffs. Upon heat shock, the signals appeared on heat-shock puffs. The signals were resistant to RNase treatment but disappeared or became faint by previous nicking of DNA or inhibition of transcription with {alpha}-amanitin. These data show that transcription-coupled, unconstrained negative supercoils of DNA exist in approximately 150 loci within the interphase genome.

Key words: Drosophila, Polytene Chromosome, Psoralen, Supercoil, Transcription


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Recent studies have established that chromatin structure is important for transcriptional regulation. Starting from analyses of DNase l hypersensitivity (Elgin, 1981Go), lines of evidence have been accumulated for changes in the chromatin structure, such as DNA methylation (Bird and Wolffe, 1999Go), chemical modifications of histones (Jenuwein and Allis, 2001Go) and chromatin remodeling (Vignari et al., 2000Go; Narliker et al., 2002) during regulation of gene expression. However, besides transcription-driven supercoiling of DNA (Giaever and Wang, 1988Go), our knowledge on conformation of chromatin DNA is elusive because of the lack of proper probes for analyses of DNA topology in vivo (Freeman and Garrard, 1992Go).

Psoralen is a planar, aromatic compound that intercalates into DNA. On exposure to 365 nm light the intercalated psoralen mediates crosslinking of opposite DNA strands via the formation of covalent bonds at each end of the molecule (for a review, see Sinden et al., 1992; Ussery et al., 1992Go). It has been shown that the rate of psoralen photocrosslink to double-stranded DNA is linearly related to its level of negative superhelicity (Sinden et al., 1980Go). Detection of negative supercoiling in living cells can be accomplished by comparing rates of crosslinking in intact cells with those in cells where potential torsional tension has been relaxed by DNA strand nicking. In prokaryotes, measurements averaged globally across the Escheridia coli genome have detected unconstrained negative supercoiling with a superhelical density of –0.05 (Sinden et al., 1980Go). In eukaryotes, similar global assays on human HeLa and Drosophila Schneider cell lines have shown that bulk DNA within the genome is torsionarily relaxed (Sinden et al., 1980Go). However, it does not necessarily exclude a possibility that there are negatively supercoiled microdomains within the genome. Indeed Jupe et al. (Jupe et al., 1993Go) have shown the presence of unconstrained negative supercoils in the hsp70 and the 18S-ribosomal RNA genes of the Schneider cell line. They observed a high level of unconstrained negative supercoils within the hsp70 transcription units, whereas downstream regions of the divergent hsp70 genes at 87A do not contain a significant level of supercoiling. Quantitative analyses of psoralen photocrosslinking have also shown an increase in the level of negative supercoils in the coding region of hsp70 upon heat shock. Accessibility of psoralen has been also reported for rDNA of growing Dictyostelium discoideum cells (Sogo et al., 1984Go), and the dihydrofolate reductase gene (Ljungman and Hanawalt, 1992Go) and the hygromycin resistance transgenes (Kramer and Sinden, 1997Go) in cultured human cells. Because these psoralen-based studies on the conformation of chromatin DNA rely on Southern hybridization for detecting the crosslinks, only limited regions of the genome can be analyzed.

In the larval polytene chromosome of Drosophila, approximately 1000 chromatids are laid down in juxtaposition, building up horizontally amplified chromosomes visible under the light microscope. Alternating more and less tightly condensed regions in precise register give rise to an alternating pattern of bands and interbands. As perceived in the 1930s, these structures display an amplified interphase genome on which specific genetic loci can be mapped and the transcriptional state of genes can be observed as a puff. For example, puffs are induced on loci carrying heat-shock genes with a brief heat shock. In heat-shock puffs, the active state of the hsp70 gene continues for more than 10 minutes and a half of the transcriptional activity is maintained even after 1 hour (Kroeger and Rowe, 1992Go). In addition, nascent RNA can be labeled with ribonucleotide analogues on puffs (Chang et al., 2000Go). All these characters are advantageous for in situ analyses of DNA conformation and transcription.

In this work, we photocrosslinked polytene chromosomes in salivary glands of Drosophila with biotinylated psoralen and detected it with fluorescent streptavidin to achieve genome-wide survey for the negatively supercoiled domains of DNA. We anticipated that we would detect a small number of psoralen signals because so far, only two highly transcribed genes have been known to harbor unconstrained negative supercoils (Jupe et al., 1993Go). To our surprise, we observed many signals of psoralen on the polytene chromosomes. These signals were detected in many but not all interbands and puffs that are the sites of active transcription, and disappeared on previous nicking of chromatin DNA or inhibition of transcription. This is the first visualization of unconstrained negatively supercoiled domains of DNA within the interphase genome.


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Heat shock and X-ray irradiation
Drosophila melanogaster Oregon R was grown at 18°C. To obtain heat-shocked salivary glands, third-instar larvae were collected in a polypropylene tube and submerged in a 37°C water bath for 10 minutes. Salivary glands were dissected in a dissection buffer (10 mM HEPES-KOH pH 7.6, 5 mM MgCl2, 5 mM KCl, 130 mM NaCl, 1% polyethylene glycol 6000). When necessary the dissected salivary glands were X-ray irradiated for 60 minutes at a dose rate of about 3 Gy/minute (TORREX CABINET X-RAY SYSTEM Model TRX2800, Faxitron).

Staining of Drosophila polytene chromosomes
For uptake of biotinylated psoralen, 4-5 pairs of salivary glands were treated with 0.01% digitionin (Calbiochem) in 40 µl of dissection buffer for 10 minutes, then rinsed with dissection buffer without digitonin and soaked in dissection buffer containing 0.2 ng/ml biotinylated psoralen (Ambion) for 10 minutes. Then, the salivary glands were illuminated with a long wave (365 nm) UV lamp (UVP model UVL-21) for 10 minutes to crosslink the psoralen. For RNA labeling, 2 mM Br-UTP (Sigma) was added in dissection buffer during the digitonin treatment. To inhibit transcription by RNA polymerase II, 3 µg/ml of {alpha}-amanitin (Sigma) was added in dissection buffer during the digitonin treatment. The bulk of the Br-UTP incorporation, except in nucleoli, was abolished by the amanitin treatment. After the light exposure salivary glands were fixed with 40% acetic acid and squashed. For RNase treatment, the squashed samples were incubated with 100 µg/ml of RNase A (Sigma) for 3 hours in 10 mM sodium phosphate pH 7.0/150 mM NaCl. Br-UTP was detected with anti-BrU monoclonal antibody (Roche) and Rhodamin-labeled anti-mouse IgG antibody. Biotinylated psoralen was detected with Alexa Fluor 488-labeled streptavidin (Molecular Probes). DNA was stained with DAPI. Fluoroimages were analyzed with Carl Zeiss Axioplan 2 microscope and IP lab software.

Southern analysis of photocrosslinked DNA
Heat shock of larvae and dissection were done as described for polytene chromosome staining. Twenty pairs of salivary glands were soaked in dissection buffer containing 4,5',8-trimethyl psoralen (Sigma), then exposed to 365 nm light for photocrosslinking. Crosslinked DNA was isolated by Proteinase K treatment in lysis buffer (10 mM Tris-Cl pH 8.2, 100 mM EDTA, 0.5% SDS) for 4 hours at 55°C, followed by phenol/chloroform and chloroform extraction. Purified DNA was digested with restriction enzymes (TAKARA shuzo) that produce fragments containing the regions of interest. Following precipitation with isopropanol, DNA pellets were dissolved in glyoxal denaturation buffer (1 M glyoxal, 10 mM sodium phosphate pH 7.0, 50% dimethylsulfoxide) and denatured at 50°C for 1 hour. Glyoxylated non-crosslinked and crosslinked DNA fragments were then separated by electrophoresis on a 1% agarose gel in 10 mM sodium phosphate buffer (pH 7.0) at 3.6 volt/cm for 100 minutes. After electrophoresis, the gel was incubated with denaturing solution (0.5 M NaOH, 1.5 M NaCl) at 65°C for 100 minutes to reverse psoralen crosslink. DNA fragments were transferred to a nylon membrane (Hybond-N, Amersham) in 10xSSC and the membrane was hybridized with 32P-labeled probe DNA produced by random priming, then washed and exposed to a X-ray film. The hsp70 CR and DDS fragments were excised from a plasmid p56H8RIA (Moran et al., 1979Go).


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Southern analyses for detecting negative supercoiling of DNA in salivary glands of Drosophila
Jupe et al. (Jupe et al., 1993Go) have developed a protocol for quantification of psoralen crosslinking localized to specific regions in the Drosophila genome using cultured cells of the Schneider line SL2. To detect negative supercoils of DNA on polytene chromosomes, we first tested whether the protocol can be applied to a tissue like the salivary gland. Briefly, salivary glands were dissected from heat-shocked or non-heat-shocked larvae. The dissected glands were soaked in a buffer containing various concentration of psoralen, followed by exposure to 365 nm light for photocrosslinking. DNA was purified, restriction digested and denatured with glyoxal. Crosslinked and non-crosslinked DNA fractions were separated on a neutral gel, treated with alkali at 65°C to reverse crosslink, blotted onto a nylon membrane and detected by Southern hybridization using probes in the hsp70 locus at 87A (Fig. 1A, CR and DDS). We included the hot alkaline incubation of the gel containing DNA because it was essential for efficient and reproducible probe hybridization to the crosslinked fraction. Otherwise, rapid self annealing of the crosslinked DNA prevented hybridization with the DNA probe. We observed crosslinking of DNA in the hsp70 coding region (CR) in a psoralen concentration-dependent manner (Fig. 1B). The frequency of crosslinking increased on heat shock of the larvae (Fig. 1B). When the glands isolated from heat-shocked larvae were irradiated with 180 Gy of X-rays to introduce nicks in chromatin DNA and then incubated with psoralen, we were unable to detect a significant level of crosslinking (Fig. 1C, lane 3 vs lane 4). As a control, X-ray irradiation after the photocrosslinking step did not alter the result (Fig. 1C, lane 5 vs lane 2). This dose of X-rays is estimated to induce about one single-strand break per 30 kb of cellular DNA (Arnström and Edvardsson, 1974Go), suggesting that the target size of relaxation is greater than the size of the CR fragment (2 kb). Similar results were obtained with the glands isolated from non-heat-shocked larvae (data not shown). By contrast, the level of photocrosslinking was low in the hsp70 distal downstream sequence (DDS) and was not enhanced on heat shock of larvae (Fig. 1C, lanes 8 and 9). These results are in good agreement with those obtained using SL2 cells (Jupe et al., 1993Go), and indicated the presence of unconstrained negative supercoils in the coding region of hsp70 and further increase in their levels on heat shock. From these data, we conclude that the psoralen photocrosslinking protocol is applicable to the salivary gland.



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Fig. 1. Detection of negative supercoils of DNA on hsp70 in salivary gland. (A) Genomic organization of the hsp70 genes at 87A. Arrows denote the orientation of transcription. The hsp70 genes are flanked by specialized chromatin structures, scs and scs' (Udvardy et al., 1985Go). Restriction sites used for Southern analysis are indicated: X, XbaI; E, EcoRI; B, BglII. DNA fragments CR and DDS were used for hybridization probes. (B) Psoralen photocrosslinking to the hsp70 genes at 87A in salivary gland cells. Salivary glands from non-heat-shocked or heat-shocked larvae were incubated with indicated concentrations of psoralen. After photocrosslinking, genomic DNA was isolated, digested with XbaI, denatured, electrophoresed and analyzed by Southern hybridization using CR fragment as a probe. (C) Effect of previous nicking of DNA or inhibition of transcription on the psoralen photocrosslinking. Lanes 1-7: salivary glands from heat-shocked larvae were incubated with (lanes 2-7) or without (lane 1) psoralen and analyzed as in (B). Where indicated, the glands were irradiated with X-rays before (lane 3) or after (lane 5) photocrosslinking. The sample in lane 4 was mock treated without X-ray. The glands were treated with (lane 6) or without (lane 7) {alpha}-amanitin before incubation with psoralen. Lanes 8 and 9: salivary glands from non-heat-shocked (lane 8) or heat-shocked larvae (lane 9) were incubated with psoralen. Genomic DNA was digested with BglII and EcoRI, and analyzed as in (B) using DDS fragment as a probe.

 

Interestingly, crosslinking was barely detectable when the glands were incubated with {alpha}-amanitin before the photocrosslinking step, even though the glands were dissected from heat-shocked larvae (Fig. 1C, lane 6 vs lane 7). These results reveal that the negative supercoils are readily relaxed on inhibition of transcription by RNA polymerase II.

Visualization of unconstrained negative supercoils of DNA on polytene chromosomes
We then extended the psoralen crosslinking protocol to genome-wide survey of negative supercoils through visualization of biotinylated psoralen on salivary gland polytene chromosomes with fluorescent streptavidin. The biotinylated psoralen was less permeable to cells than free psoralen, and fluorescent signals on polytene chromosomes were hardly detectable when salivary glands were challenged directly (data not shown). Therefore, the glands were first treated with 0.01% digitonin. This mild treatment with the nonionic detergent has been successfully used for permeabilization of salivary gland cells and subsequent observation of puff formation and transcription on polytene chromosomes (Myohara and Okada, 1987Go). The digitonin-treated salivary glands were soaked in a buffer containing biotinylated psoralen and then exposed to 365 nm light, fixed and squashed. Biotinylated psoralen was detected with Alexa 488-labeled streptavidin. We observed many bright bands of psoralen signals over weak and rather homogeneous signals along polytene chromosomes (Fig. 2A). There were approximately 150 bright signals within the genome. Such signals were not detected when the glands were incubated with Alexa 488-labeled streptavidin directly without previous psoralen treatment (data not shown), or the exposure to 365 nm light was omitted (Fig. 2D). When compared with the image of DAPI, the bright signals were detected on many but not all interbands and puffs where DAPI signals were barely detectable (e.g. arrows in Fig. 2B,C), while the vague signals almost coincided with those of DAPI (Fig. 2B,C). Some bright signals were observed only on the side margins of interbands or puffs (e.g. arrowheads in Fig. 2B,C). These bright signals were not due to binding of psoralen to RNA because the signals were clearly seen after RNase treatment (Fig. 2F), whereas staining of RNA with YOYO dye was erased completely (data not shown). The psoralen signals on interbands and puffs disappeared or became faint when salivary glands were irradiated with 180 Gy of X-rays to introduce nicks in chromatin DNA before incubation with biotinylated psoralen, whereas the vague psoralen signals were left unaffected (Fig. 3A-C). Essentially similar disapperance of the bright psoralen signals was observed when salivary glands were treated with {alpha}-amanitin before photocrosslinking (Fig. 3D,E). As controls, mock-treated samples without X-ray or {alpha}-amanitin showed essentially the same patterns as in Fig. 2 (data not shown). These results indicate the presence of many local domains of DNA that harbor transcription-coupled, unconstrained negative supercoils within the interphase genome. The vague psoralen signals are most probably due to nonspecific binding of biotinylated psoralen to bulk relaxed DNA.



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Fig. 2. Visualization of psoralen signals on polytene chromosomes. (A) Biotinylated psoralen signals were detected with Alexa 488-labeled streptavidin (light green color). (B) DAPI signals (blue color). (C) Merged image. Arrows indicate representative interbands and puffs. In some loci, intense psoralen signals were observed only on the side margins of an interband or puff (arrowheads). (D) Biotinylated psoralen signals disappeared when the photo-crosslinking step was omitted. (E) DAPI image of (D). (F) Biotinylated psoralen signals were resistant to RNase treatment. (G) DAPI image of (F). Bars, 10 µm.

 


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Fig. 3. Bright psoralen signals disappear upon previous nicking of DNA or inhibition of transcription. Salivary glands were irradiated with X-rays for relaxation of DNA by nicking (A-C) or treated with {alpha}-amanitin for inhibition of transcription (D-F) before photocrosslinking with biotinylated psoralen. (A,D) Biotinylated psoralen. (B,E) DAPI. (C,F) Merged. Representative interbands and puffs are indicated by arrows. Bars, 10 µm.

 

Unconstrained negative supercoils of DNA on heat-shock puffs
Heat treatment of larvae induces puffs on heat-shock genes while transcription of other genes is declined. To examine whether these drastic changes in the mode of transcription affect the superhelical state of DNA within the genome, biotinylated psoralen was introduced into salivary glands dissected from heat-shocked larvae. An impressive signal of psoralen was observed on a heat-shock puff at 87B (Fig. 4, arrow 87B). The signals were detected on both side margins of a heat-shock puff at 87A (Fig. 4, arrow 87A). The psoralen signal was also observed on another heat-shock gene locus at 93D (Fig. 4, arrow 93D). Many psoralen signals detected on interbands of polytene chromosomes from non-heat-shocked larvae disappeared or became less prominent on polytene chromosomes from heat-shocked larvae (Fig. 4, arrowheads). Upon X-ray irradiation (Fig. 5A-C), or {alpha}-amanitin treatment of salivary glands (Fig. 5D-F) before photocrosslinking, the psoralen signals on heat-shock puffs disappeared or became faint, although heat-shock puffs were clearly seen after these treatments (Fig. 5, arrows). The X-ray irradiation did not significantly affect transcription on the heat-shock puffs as revealed by Br-UTP incorporation followed by its immunological detection (data not shown), suggesting that the disappearance of psoralen signals after the X-ray irradiation is not due to a secondary effect of transcription inhibition. Essentially the same results as shown in Fig. 4 were obtained from mock-treated samples without X-ray or {alpha}-amanitin (data not shown). These data show transcription-coupled, unconstrained negative supercoils on heat-shock puffs. The data also reveal that the superhelical state of DNA within the genome is not static but dynamic.



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Fig. 4. Distribution of psoralen signals on polytene chromosomes from heat-shocked larvae. (A) Biotinylated psoralen. (B) DAPI. (C) Merged. Major heat-shock puffs at 87A, 87B and 93D are indicated by arrows. Arrowheads represent interbands with biotinylated psoralen signals that became less prominent after heat shock. Bar, 10 µm.

 


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Fig. 5. Effect of previous nicking of DNA or inhibition of transcription on psoralen signals on heat-shock puffs. Salivary glands dissected from heat-shocked larvae were irradiated with X-ray (A-C) or treated with {alpha}-amanitin (D-F) and then photocrosslinked with biotinylated psoralen. (A,D) Biotinylated psoralen. (B,E) DAPI. (C,F) Merged. Arrows indicate heat-shock puffs at 87A and 87B. Bars, 10 µm.

 

Correlation between unconstrained negative supercoils and transcription
Judging from {alpha}-amamtin sensitivity, the generation of negative supercoils on interbands and puffs appears to be coupled with transcription. To confirm this, dissected salivary glands were incubated with Br-UTP to label nascent transcripts just before photocrosslinking (see Materials and Methods). The labeled RNA was detected with anti-BrU antibody in many interbands and puffs (Fig. 6D,E). On a merged image, all signals of psoralen were highlighted in interbands and puffs together with the nascent RNA (Fig. 6F). After heat shock, uptake of Br-UTP was impressive on the heat-shock puffs at 87A and 87B. A less-prominent signal was also observed on the heat-shock gene locus at 93D (Fig. 7). This result is consistent with the previous report that the C-terminal domain (CTD)-phosphorylated RNA polymerase II localizes exclusively on heat-shock puffs (O'Brien et al., 1994Go). Strong signals of psoralen were seen on the heat-shock puff at 87B and on both side margins of the heat-shock puff at 87A. For detailed inspection on the distribution of negative supercoils and transcripts, the signal intensities of psoralen, nascent RNA and DNA were quantified along the 87A and 87B puffs (Fig. 7B,C). The distribution of signals of psoralen and nascent RNA was not even within the 87A and 87B puffs, but showed a similar pattern to each other. DNA signals were extremely low on these puffs. When the ratios of signal intensities of psoralen to DNA were plotted, these exhibit a sharp peak at 87B puff and a broad peak at 87A puff (Fig. 7C, yellow curve). Collectively, these results establish a correlation between unconstrained negative supercoils and transcription.



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Fig. 6. Colocalization of bright psoralen signals with those of nascent RNA on polytene chromosomes. (A) Biotinylated psoralen. (B) DAPI. (C) Merged image of A and B. (D). Nascent RNA labeled with Br-UTP. (E) Merged image of (B) and (D). (F) Merged image of (A) and (D). Bar, 10 µm.

 


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Fig. 7. Colocalization of bright psoralen and nascent RNA signals on heat-shock puffs. (A) Comparison of psoralen and nascent RNA signals on polytene chromosomes dissected from heat-shocked larvae. (B,C) Detailed inspection on the heat-shock puffs at 87A and 87B. (C) Signal intensities of biotinylated psoralen (green), nascent RNA (pink) and DAPI (blue), and ratio of signal intensity of biotinylated psoralen to DNA (yellow) quantified along the region indicated in (B). Bar, 10 µm.

 


    Discussion
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 Materials and Methods
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 References
 
Unconstrained negative supercoils within interphase genome
In this study we detected bright signals of biotinylated psoralen on many transcriptionally active interbands and puffs along polytene chromosomes of the salivary gland. Upon heat shock, the signals on the interbands and endogenous puffs disappeared or diminished, and strong signals appeared on newly induced heat-shock puffs. The signals were resistant to RNase treatment but abolished by previous nicking of cellular DNA or inhibition of transcription with {alpha}-amanitin. These data indicate the presence of transcription-coupled, unconstrained negative supercoils in many loci within the interphase genome.

A priori, we analyzed the binding of biotinylated psoralen to polytene chromosomes in case there were other reasons for the observation. For example, psoralen could be more easily accessible to open chromatin than tightly packed chromatin. It is possible that the psoralen signals on interbands and puffs reflect its preferential binding to open chromatin. However, the bright psoralen signals disappeared upon nicking of chromatin DNA or {alpha}-amanitin treatment, although the interbands and puffs were clearly seen after these treatments. These results can not be explained by simple binding of psoralen to open chromatin. From the available data, we conclude that the bright psoralen signals on interbads and puffs represent unconstrained negative supercoils in chromatin DNA. This does not contradict the previous conclusion that bulk DNA of the eukaryote genome is relaxed (Sinden et al., 1980Go). Although we detected negative supercoils on many interbands and puffs, DNA contents of these loci are extremely low and account for a tiny portion compared with bulk DNA.

The negative supercoils should be in equilibrium with their generation and relaxation. The heterogeneity in the ratio of signal intensities of psoralen to nascent RNA on various interbands and puffs (Figs 6, 7) suggests that the equilibrium is not even throughout the genome. It has been shown that topoisomerase I is associated with transcribed regions of active genes (Fleischmann et al., 1984; Gilmour et al., 1986Go; Gilmour and Elgin, 1987Go). Interestingly, the level of RNA polymerase II present on the transcriptionally active heat-shock genes exceeds the level of topoisomerase I by twofold to fourfold, whereas twofold more topoisomerase I than RNA polymerase II occupies the modestly transcribed copia gene (Gilmour et al., 1986Go). The different ratios of topoisomerase I and RNA polymerase II on different genes would contribute to the heterogeneity in the equilibrium between generation and relaxation of negative supercoils.

Given that the difference in psoralen binding is about twofold greater for supercoiled DNA with a negative superhelicity of 0.05 (Sinden et al., 1980Go), why does our fluorescence system work so clearly? There may be two reasons for this. The first one is related to the accessibility of psoralen. Bulk chromatin DNA is tightly packaged and may be less accessible to psoralen compared with DNA in interbands and puffs. The second one is a threshold effect of fluorescence detection. Fluorescence is barely detectable under a microscope if the concentration of a fluorescent molecule is below certain threshold level. We have used the lowest concentration of biotinylated psoralen (0.2 ng/ml) at which we really detected the bright signals. When we dropped below it, the signals blended into background. At this concentration, the difference in the fluorescent signals between supercoiled and relaxed DNA may be exaggerated.

Mechanism of negative supercoiling of DNA
What is the mechanism underlying the generation of the negative supercoils that are coupled with transcription? The most potent mechanism is transcription-driven supercoiling of DNA (Liu and Wang, 1987Go). As transcription proceeds, a positively supercoiled domain is formed in front of the transcription machinery, and a negatively supercoiled domain is formed behind it. This type of supercoiling has been documented in eukaryote using a yeast mutant defective in topoisomerases (Giaever and Wang, 1988Go). According to the twin-supercoiled domain model, one could imagine accumulation of negative supercoils in the intergenic region of two oppositely oriented hsp70 transcription units at 87A (Fig. 1A). Consistent with the expectation, the ratios of signal intensities of psoralen to DNA formed a broad peak at 87A puff (Fig. 7C).

In addition, chromatin remodeling could release negative supercoils that are constrained by histone-DNA interactions. Indeed, SWI/SNF-type remodeling complexes have been shown to reduce the constrained superhelicity (Kwon et al., 1994Go; Gavin et al., 2001Go). Unconstrained negative superhelicity could be also released by transcription-induced displacement of a single H2A·H2B dimer from a nucleosome (Kireeva et al., 2002Go). The possibility is supported by a recent finding that FACT (facilitates chromatin transcription, a heterodimer of SSRP1 and SPT16) facilitates the displacement of H2A and H2B from nucleosomal DNA (Formosa et al., 2002Go; Belotserkovaskaya et al., 2003; Shimojima et al., 2003Go). Although the release of histone-DNA interactions in a single nucleosome has only a subtle effect on the superhelical state of DNA, the sum of these effects over nucleosome arrays would generate a detectable level of negative supercoils. However, the processes are at least one order of magnitude less efficient in generating supercoils per unit length of DNA than the transcription-driven supercoiling.

Finally, negative supercoils could be generated by the action of supercoiling factor (SCF) and topoisomerase II. SCF is a protein capable of introducing negative supercoils into DNA in conjuction with topoisomerase II (Ohta and Hirose, 1990Go). Drosophila SCF localizes to interbands and puffs on polytene chromosomes and hence, it is thought to be involved in the formation of transcriptionally active chromatin (Koyayashi et al., 1998). The idea is supported by recent genetic studies from this laboratory (H. Furuhashi and S.H., unpublished). All three mechanisms proposed for genration of negative supercoils are not mutually exclusive but could operate simultaneously.

Implication of negative supercoils in transcriptional regulation
The present study illuminates a dynamic nature of the superhelical state of DNA in many local domains of the eukaryotic genome. On the basis of the data we propose transcriptional regulation through conformation of chromatin DNA. For example, transcription activities from various promoters have been shown to change markedly by the degree of DNA supercoiling (Harland et al., 1983Go; Hirose and Suzuki, 1988Go; Mizutani et al., 1991Go; Schultz et al., 1992; Dunaway and Ostorander, 1993; Parvin and Sharp, 1993Go; Tabuchi et al., 1993Go). Dissection of transcription revealed that the binding of TATA element-binding protein (TBP) to TATA element is facilitated by negative supercoiling of DNA in most genes examined (Mizutani et al., 1991Go; Tabuchi et al., 1993Go). Therefore, transcription-coupled, unconstrained negative supercoils of DNA shown here, in turn, can affect transcription. Although relaxation of DNA by X-ray treatment did not significantly affect transcription under our conditions, it does not necessarily exclude the above idea because once TBP binds to the TATA element, subsequent relaxation of DNA will not reduce transcription until the TBP dissociates from the promoter. However, negative supercoils are not necessarily detectable in all transcriptionally active regions. Studies from other group have shown active genes without unconstrained negative supercoils (Kramer and Sinden, 1997Go; Kramer et al., 1999Go). It is possible that local DNA domains should be encompassed by some special structures such as chromatin boundaries to accumulate detectable levels of supercoils during transcription.

Formation of unusual DNA structures such as cruciform (Lilley, 1980Go; Panayatatos and Wells, 1981) and Z-form (Nordheim et al., 1982Go; Singleton et al., 1992) is significantly facilitated by negative supercoiling and hence, the observed negative supercoils can also affect conformation of DNA. Such unusual DNA structures are likely to affect transcription. Indeed, Z-DNA in a promoter region has been suggested to participate in transcriptional activation in collaboration with a chromatin remodeling complex BAF (Liu et al., 2001Go). Finally, it is possible that negative supercoiling of DNA has influence on higher order chromatin structure which, in turn, can affect transcription.


    Acknowledgments
 
We thank R. R. Sinden, J. C. Wang and A. Travers for helpful suggestions, and H. R. Drew for encouragement. This work was supported by Grants in aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology of Japan. K.M. was supported by a Center of Excellence Program of Japan.


    References
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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