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5-Bromouracil disrupts nucleosome positioning by inducing A-form-like DNA conformation in yeast cells
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Biochemical and Biophysical Research Communications 368 (2008) 662–669 www.elsevier.com/locate/ybbrc
5-Bromouracil disrupts nucleosome positioning by inducing A-form-like DNA conformation in yeast cells Kensuke Miki a, Mitsuhiro Shimizu b, Michihiko Fujii a, Mohammad Nazir Hossain a, Dai Ayusawa a,* a
International Graduate School of Arts and Sciences, Yokohama City University, Seto 22-2, Kanazawa-Ku, Yokohama, Kanagawa 236-0027, Japan b Department of Chemistry, Meisei University, Hino, Tokyo 191-8506, Japan Received 3 January 2008 Available online 5 February 2008
Abstract 5-Bromodeoxyuridine (BrdU) modulates expression of particular genes associated with cellular differentiation and senescence. Our previous studies have suggested an involvement of chromatin structure in this phenomenon. Here, we examined the effect of 5-bromouracil on nucleosome positioning in vivo using TALS plasmid in yeast cells. This plasmid can stably and precisely be assembled nucleosomes aided by the a2 repressor complex bound to its a2 operator. Insertion of AT-rich sequences into a site near the operator destabilized nucleosome positioning dependent on their length and sequences. Addition of BrdU almost completely disrupted nucleosome positioning through specific AT-tracts. The effective AT-rich sequences migrated faster on polyacrylamide gel electrophoresis, and their mobility was further accelerated by substitution of thymine with 5-bromouracil. Since this property is indicative of a rigid conformation of DNA, our results suggest that 5-bromouracil disrupts nucleosome positioning by inducing A-form-like DNA. Ó 2008 Elsevier Inc. All rights reserved. Keywords: 5-Bromouracil; 5-Bromodeoxyuridine; TALS plasmid; Nucleosome positioning; AT-tract; A-form DNA
BrdU is normally incorporated into DNA as 5-bromouracil instead of thymine and has been used as a modulator of cellular differentiation with cAMP and butyrate [1,2]. The latter two are found to target protein kinase A and histone deacetylase, respectively, leading to understanding of cell signaling and gene expression. In spite of considerable efforts done to date, the molecular target or mechanism for BrdU still remains a mystery. We have found that BrdU very clearly induces a senescent-like phenomenon in every mammalian cell type and also in yeast cells [3,4]. A PCR-based cDNA subtractive hybridization and DNA microarray analysis have shown that BrdU-responsive genes form clusters on or nearby Giemsa-dark bands of human chromosomes [5,6]. These results are consistent with the facts that BrdU decondenses particular regions of chromosomes [7], suppresses position *
Corresponding author. Fax: +81 45 787 2193. E-mail address: [email protected] (D. Ayusawa).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.149
effect variegation [8], and induces expression of silenced genes [9]. Moreover, AT-tract minor groove binders are all shown to markedly potentiate the effects of BrdU [5,10]. Based on these observations, we suggest that BrdU targets certain types of AT-rich sequences and alter specific chromatin structure. In eukaryotes, DNA fibers exist as regularly arrayed beads of nucleosomes, and this restricts accessibility of transcription factors to regulatory sequences of genes. An alternation in nucleosome positioning is an essential step in transition from a repressed state to an active state by aid of chromatin remodeling complexes [11]. Recent studies in the yeast genome suggest that nucleosome positioning is largely determined by intrinsic DNA sequences [12,13]. For example, the periodic presence of AA or TT dinucleotides at approximately 10 bp intervals is implicated in nucleosome positioning [12]. Bent DNA facilitated by AA and TT dinucleotides is located at an average of 680 bp in the human b-globin locus [14]. Thus, bent DNA has been
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considered to be a ubiquitous signal for nucleosome positioning [15]. In our previous studies, substitution of thymine with 5-bromouracil in AT-rich scaffold/nuclear matrix attachment region (S/MAR) sequences are shown to reduce their bending and increase their interaction with the nuclear matrix proteins [5,16]. Based on above observations, we have suggested that 5-bromouracil changes DNA topology of particular AT-rich sequences, leading to a change in nucleosome positioning and then chromatin structure. In this report, we examined the effect of 5-bromouracil on nucleosome positioning with use of TALS plasmid [17], which have been successfully utilized to study nucleosome positioning in Saccharomyces cerevisiae. Moreover, various derivatives have been constructed by inserting AT-tracts into TALS plasmid [18]. Since this TALS system has no potential artifact for nucleosome positioning unlike in vitro reconstitution systems employing high concentrations of salts and urea, we can directly address the effect of 5-bromouracil on nucleosome positioning in vivo. Materials and methods Plasmids and yeast strains. TALS-pUC19 chimera plasmid and its derivative plasmids (pTS713-, pOM801-, pOM804-, and pTS711pBR322DEcoRI) were constructed as described previously [18]. Plasmids pKM001- and pKM002-pBR322DRI were constructed by inserting oligonucleotides into the SacI site of TALS-pBR322DRI to yield pKM001(T3CCT6CT5GCT5CT7) and pKM002-pBR322DRI ((T3CCT6CT5GCT5 CT7)2). The plasmids were digested with HindIII to eliminate pUC19 or pBR322 vector portions, self-ligated, and were introduced into yeast thymidine-autotrophic strain, YKH2 (MATa ura2-52 trp1 his3 leu2 cdc21::LEU2 pYBT1) expressing herpes simplex virus thymidine kinase as described previously [4]. Chromatin isolation and nuclease digestion. Transformants were selected in SC medium (2% glucose, 0.67% yeast nitrogen base without amino acids) supplemented with appropriate amino acids (except for tryptophan)
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and 1 mM dThd. Cells were grown in 30 ml of SC medium containing 1 mM dThd or BrdU at 30 °C for 15 h to an optimal density at 600 nm of 0.6–1.0. Cell pellets were spheroplasted by addition of 1/10 volume of 10 mg/ml Zymolyase 100T (Seikagaku Corporation, Tokyo) according to the supplier’s manual. Chromatin and naked DNA samples were prepared according to the method of Balasubramanian et al. [19] with a slight modification. Each sample was digested with Micrococcal nuclease (MNase) (TAKARA, Kyoto) at 37 °C for 10 min with serial dilution of MNase. The reactions were initiated by addition of 0.15% nonidet P-40, and halted by addition of SDS and proteinase K. Indirect end-labeling. After digestion of samples with MNase, DNA samples were completely digested with EcoRV together with RNaseA. Each sample was run on 1.5% agarose gel and transferred onto a nylon membrane (Biodyne B, Pall) followed by cross-linking with ultraviolet light (UV Crosslinker, Stratagene). The membrane was incubated at 65 °C for 16 h in hybridization solution [0.5 M Na–Pi, 1 mM ETDA, and 7% SDS] containing the EcoRV–HindIII fragment of TALS plasmid labeled with [a-32P]dCTP using random-primed DNA labeling kit (Mega-prime, Amersham). After washing, the membrane was subjected to autoradiography and densitometrical analysis using an image analyzer FLA-5000 (Fuji Photo Film). Electrophoretic analysis. DNA fragments containing AT-tracts at their central positions were amplified from TALS plasmid and its derivatives by PCR with the following primers: 50 -TTGATAATTAGCGTTGCCTC-30 and 50 -CACAGGAAACAGCTATGACC-30 . Then, thymine was substituted with 5-bromouracil by 2–5 rounds of PCR with BrdUTP instead of dTTP using the amplified fragments as a template. The resulting PCR products were electrophoresed on a 5% non-denaturing polyacrylamide gel at 4 and 52 °C. After electrophoresis, gels were stained with SYBR Green (TAKARA, Kyoto). Electrophoretic mobility was measured using ImageGauge software (Fuji Photo Film).
Results Construction of plasmids TALS plasmid contains a2 operator derived from the STE6 promoter, and can be assembled precise and stable 10 nucleosomes in yeast MATa cells (Fig. 1) [17,20]. The a2/Mcm1 repressor binds to a2 operator and acts as a phase determinant in MATa cells.
Fig. 1. Schematic diagram of TALS chromatin in yeast MATa cells and the inserts used in this study. TALS plasmid lacks the Escherichia coli vector backbone, contains a2 operator indicated by the hatched box, and forms 10 stable nucleosomes in vivo as illustrated by numbered gray circles. AT-rich sequences shown in the table were inserted into the SacI site (1460 map units).
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To examine a role of AT-tracts in nucleosome positioning, we inserted several AT-rich sequences into the SacI site located 75 bp upstream of the a2 operator (Fig. 1). The inserts include a long mixed sequence GC(GAGCTCGC)6, a short AT-tract A7TATA8 resembling a yeast ARS consensus sequence, a long poly(dA). poly(dT) sequence, which is often found in promoters [21] and shows rigidity [22,23], another type of long AT-tract T3CCT6CT5 GCT5CT7 derived from DED1 promoter [24], and a hetero-polymeric sequence (CA)17 showing high flexibility [25]. These plasmids were introduced into dThd-auxotrophic MATa cells to ensure quantitative incorporation of 5-bromouracil into DNA [4,26]. Nucleosome positioning on TALS plasmid We confirmed nucleosome positioning on TALS plasmid by digestion with MNase followed by the indirect end-labeling method. Four MNase cleavage sites (indicated by *a, *b, *c, and *d in Fig. 2A) were observed on the naked DNA samples. These sites were protected in the chromatin samples, and instead new cleavage sites appeared with equal intervals (140–150 bp) (Fig. 2A, lanes 1–4). When chromatin samples were digested with HindIII, bands corresponding to linker regions between nucleosomes V–VIII were detected (data not shown). These results indicate that nucleosomes are assembled precisely and stably on TALS plasmid in vivo as described previously [17,18]. Effects of 5-bromouracil on nucleosome positioning We examined the effects of 5-bromouracil on nucleosome positioning on TALS plasmid. When cells were cultured with BrdU for more than three generations, the band corresponding to the linker region between nucleosomes IV and V disappeared, and the band *a was evident in the chromatin sample (Fig. 2, lane 5). Densitometrical measurement clearly showed these differences (Fig. 2B). The other bands were clearly protected similarly to the sample prepared in dThd-medium (Fig. 2A, lanes 5–8). Since 5-bromouracil did not affect MNase cleavage patterns on naked DNA samples (Fig. 2B), 5-bromouracil disrupted nucleosomes IV and V adjacent to the a2 operator sequence. We further examined binding of a2/Mcm1 to a2 operator with an in vivo UV photofootprinting assay as described previously [18,27]. There was no detectable difference in the presence of a2/Mcm1 between dThd- and BrdU-medium (data not shown). These results suggest that the above changes are caused by the presence of 28 bp ATrich sequence just downstream of the a2 operator (Fig. 2A). Effects of AT-tracts on nucleosome positioning To search for a role of AT-tracts in nucleosome positioning, we examined TALS derivatives containing several AT-rich sequences at its SacI site (Fig. 1). First,
we examined pTS711 containing a mixed sequence GC(GAGCTCGC)6. This plasmid was assembled nucleosomes similarly to TALS plasmid in dThd-medium (Fig. 3, lane 1 vs. 5), indicating that DNA insertion per se did not affect nucleosome positioning and that nucleosomes are positioned independently of DNA sequences flanked by the a2 operator [20]. Addition of BrdU eliminated protection of band *a in the chromatin sample (Fig. 3, lanes 7 and 8) but the other regions were not affected. Thus, only nucleosomes IV and V were affected by addition of BrdU as in the case of TALS. Plasmids pTS713 containing A7TATA8 (Fig. 3, lanes 9–12) and pOM802 containing A5TATA4 (not shown) behaved similarly to pTS711 in dThd- or BrdU-medium. These results suggest that a long mixed sequence and short AT-tracts do not affect nucleosome positioning on TALS plasmid regardless of whether it contains thymine or 5-bromouracil. Plasmid pKM001 containing a long AT-tract of T3CCT6CT5GCT5CT7 showed stable nucleosome positioning in dThd-medium (Fig. 3, lanes 13 and 14). Addition of BrdU also prevented protection of the band *a as in the cases of TALS, pTS711, and pTS713 plasmids, but it made the bands *b and *c in the chromatin sample more evident (Fig. 3, lane 13 vs. 15). Similar results were obtained with pOM804 containing a hetero-polymeric sequence of (CA)17 (Fig. 3A, lanes 17–20). These results demonstrated that 5-bromouracil disrupts nucleosome positioning on the sequence containing not only a long AT-tract but also a purine–pyrimidine repeat. Plasmids pOM801 containing a long AT-tract of A34 and pKM002 containing a longer AT-tract of (T3CCT6CT5GCT5CT7)2 were weakly assembled nucleosomes around the site of their insertion even in dThd-medium (Fig. 3, lanes 21 and 25) as described previously [18]. Addition of BrdU almost completely prevented the protection of nucleosome-specific bands in their chromatin samples as exemplified by appearance of the band *b (Fig. 3, lanes 23 and 27). Moreover, the regions of nucleosomes I–III were broader than those in dThd-medium. These results showed that 5-bromouracil further disrupts unstable nucleosome positioning mediated by long AT-tracts. Electrophoretic analysis We undertook electrophoretic analysis with sequences (323–400 bp) containing the above AT-tracts at their centers (Fig. 4A and B). 5-Bromouracil was incorporated into the sequences by PCR with BrdUTP as a substrate. The control sequence derived from TALS behaved similarly at 4 °C and 52 °C regardless of whether it contained thymine or 5-bromouracil. The relative mobility (Rm = mobility at 4 °C/mobility at 52 °C) of this sequence was 0.98 (Fig. 4B). The sequence derived from pTS713 (A7TATA8) migrated slower at 4 °C than at 52 °C (Rm 1.07) when containing thymine, indicating that this sequence was bent. Upon substitution with 5-bromouracil, it migrated nor-
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Fig. 2. Nucleosome positioning on TALS plasmid. Chromatin (indicated by C) and naked DNA (indicated by D) samples were prepared from cells transfected with TALS plasmid and cultured with dThd or BrdU. The samples were digested with MNase at the concentrations of 2 U/ml (lanes 1 and 5), 5 U/ml (lanes 3 and 7), 0.2 U/ml (lanes 2 and 6), and 0.4 U/ml (lanes 4 and 8), and subjected to indirect end-labeling analysis as described in Materials and methods. Independent experiments gave similar results. (A) Autoradiography of MNase cleavage patterns detected with the EcoRV–HindIII fragment (385–615 map units of TALS in Fig. 1) used as a probe. DNA size marker (lane M) and the positions of nucleosomes I–VI and a2 operator are shown to the left. Specific cleavage sites on naked DNA samples are marked with *a, *b, *c, and *d. Open stars on some lanes denote the bands that changed in BrdU-medium. The intrinsic AT-rich sequence (underlined) just downstream of a2 operator is shown between the panels. (B) Densitometrical profiles of autoradiography. Lanes 3, 4, 7, and 8 in the above panels are densitometrically scanned. The positions of stable nucleosomes are shown on the top.
mally, indicating that 5-bromouracil canceled DNA bending in agreement with the previous report [5]. Similar
results were obtained with the sequence derived from pKM001 (T3CCT6CT5GCT5CT7).
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Fig. 3. Nucleosome positioning on TALS-plasmids having various inserts. TALS and its derivatives shown in Fig. 1 were analyzed as in Fig. 2. Chromatin and naked DNA samples were digested with 5 U/ml and 0.4 U/ml MNase, respectively. Independent experiments gave similar results.
On the other hand, the sequences derived from pOM801 (A34) and pKM002 ((T3CCT6CT5GCT5CT7)2) migrated faster at 4 °C than at 52 °C when containing thymine (Rm 0.96 and 0.97, respectively), suggesting that these sequences were not bent but adopt a non B-form conformation. Substitution of thymine with 5-bromouracil further accelerated their mobility at 4 °C. The sequence derived from pOM804 containing (CA)17 migrated slightly slower than the control sequence when containing thymine (Rm 1.0). Substitution with 5-bromouracil intermediately accelerated its mobility at 4 °C. The above results suggested that rapid migration of DNA (Rm values less than 0.98) rather than cancellation
of DNA bending is correlated with instability of nucleosome positioning in TALS system. Discussion Incorporation of 5-bromouracil was shown to clearly disrupt nucleosome positioning on the plasmids dependent on a specific AT-rich sequence inserted into a site near the anchor position of a2 operator. The mixed sequence GC(GAGCTCGC)6 and the short AT-tracts (A5TATA4 and A7TATA8) did not affect nucleosome positioning. In contrast, long AT-tracts such as T3CCT6CT5GCT5CT7,
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Fig. 4. Electrophoretic analysis of sequences derived from TALS and its derivatives. DNA fragments containing AT-tracts at their central positions were amplified from the plasmids by PCR, and run on polyacrylamide gel at as described in Materials and methods. Independent experiments gave similar results. (A) Lane 1, DNA amplified by PCR with dTTP; lanes 2 and 3, DNA amplified by PCR with BrdUTP for 2 and 5 cycles, respectively; lane M, size marker. (B) Relative mobility (mobility at 4 °C/52 °C) of lanes 1 and 3 in (A).
(CA)17, A34, and (T3CCT6CT5GCT5CT7)2 destabilized nucleosome positioning on TALS plasmid. Addition of BrdU almost completely disrupted nucleosomes around the site of their insertion. How does 5-bromouracil disrupt nucleosome positioning? Historically, the bromine atom at the 50 position of uracil has been thought to stabilize interaction between DNA and certain DNA-binding proteins due to its electronegative nature stronger than methyl residue [28], although most DNA-binding proteins cannot distinguish sequence specificity of DNA containing 5-bromouracil or thymine [29,30]. According to our recent studies [5], 5-bromouraicl is thought to induce a change in DNA topology possibly
due to increase in neighboring base–base interaction or stacking of base pairs. In the present study, we showed that the AT-tracts effective in disrupting nucleosome positioning migrated rapidly on gel electrophoresis upon substitution with 5-bromouracil. This property is indicative of an A-form-like structure [31]. Runs of consecutive purines are known to set up an A-like helix [32,33] because stacking interactions of purines are thought to provide the mechanical rigidity of DNA helix [34]. To date, rapid migration is reported for AT-rich fragments from yeast centromeres [35] and for sites of illegitimate recombination in mammalian cells [36]. Bovine satellite I DNA is also shown to migrate rapidly dependent on temperature and to take an A-form-like structure as determined by circular permuta-
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tion and circular dichroism spectra [37,38]. Based on these observations, bromine atom is thought to increase rigidity in DNA, resulting in stiff and rod-like DNA. In support of this, 5-bromouracil-substituted chromatins showed a blue shift and increased positive ellipticity in the 250–300 nm region of circular dichroism spectra [28], which is a part of the feature of A-form DNA. Taken together, we suggest that disruption of nucleosome positioning by 5-bromouracil is caused by appearance of an unusual DNA conformation such as an A-form-like structure. Biological and structural features of A-form DNA are only poorly understood. The DNA binding site for transcription factor IIIA, which binds to the internal control region of the Xenopus 5S RNA gene, is shown to have an overall or global structure with characteristics of A-form DNA [32,39]. Since the hydrophobicity of the DNA surface in the minor grooves is increased by B- to A-form transition, hydrophobic amino acids can interact extensively with A-form structure [40,41]. Moreover, cardiolipin, a chromatin-bound phospholipid, is shown to provide Aform DNA in the complex with RNA polymerase to elicit active chromatin [42,43]. In this respect, it is interesting to note that cardiolipin induces premature senescence in normal human fibroblasts [44]. Finally, the novel findings described here may lead to direct understanding of the role of BrdU in modification of chromatin structure. They may also answer the new and old question why BrdU modulates expression of particular genes associated with cellular differentiation and senescence. References [1] F.H. Wilt, M. Anderson, The action of 5-bromodeoxyuridine on differentiation, Dev. Biol. 28 (1972) 443–447. [2] H. Weintraub, Size of the BUdR sensitive targets for differentiation, Nat. New Biol. 244 (1973) 142–143. [3] E. Michishita, K. Nakabayashi, T. Suzuki, S.C. Kaul, H. Ogino, M. Fujii, Y. Mitsui, D. Ayusawa, 5-Bromodeoxyuridine induces senescence-like phenomena in mammalian cells regardless of cell type or species, J. Biochem. (Tokyo) 126 (1999) 1052–1059. [4] M. Fujii, H. Ito, T. Hasegawa, T. Suzuki, N. Adachi, D. Ayusawa, 5Bromo-20 -deoxyuridine efficiently suppresses division potential of the yeast Saccharomyces cerevisiae, Biosci. Biotechnol. Biochem. 66 (2002) 906–909. [5] T. Suzuki, E. Michishita, H. Ogino, M. Fujii, D. Ayusawa, Synergistic induction of the senescence-associated genes by 5-bromodeoxyuridine and AT-binding ligands in HeLa cells, Exp. Cell Res. 276 (2002) 174–184. [6] S. Minagawa, K. Nakabayashi, M. Fujii, S.W. Scherer, D. Ayusawa, Functional and chromosomal clustering of genes responsive to 5bromodeoxyuridine in human cells, Exp. Gerontol. 39 (2004) 1069– 1078. [7] A.F. Zakharov, L.I. Baranovskaya, A.I. Ibraimov, V.A. Benjusch, V.S. Demintseva, N.G. Oblapenko, Differential spiralization along mammalian mitotic chromosomes. II. 5-bromodeoxyuridine and 5bromodeoxycytidine-revealed differentiation in human chromosomes, Chromosoma 44 (1974) 343–359. [8] T. Suzuki, M. Yaginuma, T. Oishi, E. Michishita, H. Ogino, M. Fujii, D. Ayusawa, 5-Bromodeoxyuridine suppresses position effect variegation of transgenes in HeLa cells, Exp. Cell Res. 266 (2001) 53–63.
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5-Bromouracil disrupts nucleosome positioning by inducing A-form-like DNA conformation in yeast cells Kensuke Miki a, Mitsuhiro Shimizu b, Michihiko Fujii a, Mohammad Nazir Hossain a, Dai Ayusawa a,* a
International Graduate School of Arts and Sciences, Yokohama City University, Seto 22-2, Kanazawa-Ku, Yokohama, Kanagawa 236-0027, Japan b Department of Chemistry, Meisei University, Hino, Tokyo 191-8506, Japan Received 3 January 2008 Available online 5 February 2008
Abstract 5-Bromodeoxyuridine (BrdU) modulates expression of particular genes associated with cellular differentiation and senescence. Our previous studies have suggested an involvement of chromatin structure in this phenomenon. Here, we examined the effect of 5-bromouracil on nucleosome positioning in vivo using TALS plasmid in yeast cells. This plasmid can stably and precisely be assembled nucleosomes aided by the a2 repressor complex bound to its a2 operator. Insertion of AT-rich sequences into a site near the operator destabilized nucleosome positioning dependent on their length and sequences. Addition of BrdU almost completely disrupted nucleosome positioning through specific AT-tracts. The effective AT-rich sequences migrated faster on polyacrylamide gel electrophoresis, and their mobility was further accelerated by substitution of thymine with 5-bromouracil. Since this property is indicative of a rigid conformation of DNA, our results suggest that 5-bromouracil disrupts nucleosome positioning by inducing A-form-like DNA. Ó 2008 Elsevier Inc. All rights reserved. Keywords: 5-Bromouracil; 5-Bromodeoxyuridine; TALS plasmid; Nucleosome positioning; AT-tract; A-form DNA
BrdU is normally incorporated into DNA as 5-bromouracil instead of thymine and has been used as a modulator of cellular differentiation with cAMP and butyrate [1,2]. The latter two are found to target protein kinase A and histone deacetylase, respectively, leading to understanding of cell signaling and gene expression. In spite of considerable efforts done to date, the molecular target or mechanism for BrdU still remains a mystery. We have found that BrdU very clearly induces a senescent-like phenomenon in every mammalian cell type and also in yeast cells [3,4]. A PCR-based cDNA subtractive hybridization and DNA microarray analysis have shown that BrdU-responsive genes form clusters on or nearby Giemsa-dark bands of human chromosomes [5,6]. These results are consistent with the facts that BrdU decondenses particular regions of chromosomes [7], suppresses position *
Corresponding author. Fax: +81 45 787 2193. E-mail address: [email protected] (D. Ayusawa).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.149
effect variegation [8], and induces expression of silenced genes [9]. Moreover, AT-tract minor groove binders are all shown to markedly potentiate the effects of BrdU [5,10]. Based on these observations, we suggest that BrdU targets certain types of AT-rich sequences and alter specific chromatin structure. In eukaryotes, DNA fibers exist as regularly arrayed beads of nucleosomes, and this restricts accessibility of transcription factors to regulatory sequences of genes. An alternation in nucleosome positioning is an essential step in transition from a repressed state to an active state by aid of chromatin remodeling complexes [11]. Recent studies in the yeast genome suggest that nucleosome positioning is largely determined by intrinsic DNA sequences [12,13]. For example, the periodic presence of AA or TT dinucleotides at approximately 10 bp intervals is implicated in nucleosome positioning [12]. Bent DNA facilitated by AA and TT dinucleotides is located at an average of 680 bp in the human b-globin locus [14]. Thus, bent DNA has been
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considered to be a ubiquitous signal for nucleosome positioning [15]. In our previous studies, substitution of thymine with 5-bromouracil in AT-rich scaffold/nuclear matrix attachment region (S/MAR) sequences are shown to reduce their bending and increase their interaction with the nuclear matrix proteins [5,16]. Based on above observations, we have suggested that 5-bromouracil changes DNA topology of particular AT-rich sequences, leading to a change in nucleosome positioning and then chromatin structure. In this report, we examined the effect of 5-bromouracil on nucleosome positioning with use of TALS plasmid [17], which have been successfully utilized to study nucleosome positioning in Saccharomyces cerevisiae. Moreover, various derivatives have been constructed by inserting AT-tracts into TALS plasmid [18]. Since this TALS system has no potential artifact for nucleosome positioning unlike in vitro reconstitution systems employing high concentrations of salts and urea, we can directly address the effect of 5-bromouracil on nucleosome positioning in vivo. Materials and methods Plasmids and yeast strains. TALS-pUC19 chimera plasmid and its derivative plasmids (pTS713-, pOM801-, pOM804-, and pTS711pBR322DEcoRI) were constructed as described previously [18]. Plasmids pKM001- and pKM002-pBR322DRI were constructed by inserting oligonucleotides into the SacI site of TALS-pBR322DRI to yield pKM001(T3CCT6CT5GCT5CT7) and pKM002-pBR322DRI ((T3CCT6CT5GCT5 CT7)2). The plasmids were digested with HindIII to eliminate pUC19 or pBR322 vector portions, self-ligated, and were introduced into yeast thymidine-autotrophic strain, YKH2 (MATa ura2-52 trp1 his3 leu2 cdc21::LEU2 pYBT1) expressing herpes simplex virus thymidine kinase as described previously [4]. Chromatin isolation and nuclease digestion. Transformants were selected in SC medium (2% glucose, 0.67% yeast nitrogen base without amino acids) supplemented with appropriate amino acids (except for tryptophan)
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and 1 mM dThd. Cells were grown in 30 ml of SC medium containing 1 mM dThd or BrdU at 30 °C for 15 h to an optimal density at 600 nm of 0.6–1.0. Cell pellets were spheroplasted by addition of 1/10 volume of 10 mg/ml Zymolyase 100T (Seikagaku Corporation, Tokyo) according to the supplier’s manual. Chromatin and naked DNA samples were prepared according to the method of Balasubramanian et al. [19] with a slight modification. Each sample was digested with Micrococcal nuclease (MNase) (TAKARA, Kyoto) at 37 °C for 10 min with serial dilution of MNase. The reactions were initiated by addition of 0.15% nonidet P-40, and halted by addition of SDS and proteinase K. Indirect end-labeling. After digestion of samples with MNase, DNA samples were completely digested with EcoRV together with RNaseA. Each sample was run on 1.5% agarose gel and transferred onto a nylon membrane (Biodyne B, Pall) followed by cross-linking with ultraviolet light (UV Crosslinker, Stratagene). The membrane was incubated at 65 °C for 16 h in hybridization solution [0.5 M Na–Pi, 1 mM ETDA, and 7% SDS] containing the EcoRV–HindIII fragment of TALS plasmid labeled with [a-32P]dCTP using random-primed DNA labeling kit (Mega-prime, Amersham). After washing, the membrane was subjected to autoradiography and densitometrical analysis using an image analyzer FLA-5000 (Fuji Photo Film). Electrophoretic analysis. DNA fragments containing AT-tracts at their central positions were amplified from TALS plasmid and its derivatives by PCR with the following primers: 50 -TTGATAATTAGCGTTGCCTC-30 and 50 -CACAGGAAACAGCTATGACC-30 . Then, thymine was substituted with 5-bromouracil by 2–5 rounds of PCR with BrdUTP instead of dTTP using the amplified fragments as a template. The resulting PCR products were electrophoresed on a 5% non-denaturing polyacrylamide gel at 4 and 52 °C. After electrophoresis, gels were stained with SYBR Green (TAKARA, Kyoto). Electrophoretic mobility was measured using ImageGauge software (Fuji Photo Film).
Results Construction of plasmids TALS plasmid contains a2 operator derived from the STE6 promoter, and can be assembled precise and stable 10 nucleosomes in yeast MATa cells (Fig. 1) [17,20]. The a2/Mcm1 repressor binds to a2 operator and acts as a phase determinant in MATa cells.
Fig. 1. Schematic diagram of TALS chromatin in yeast MATa cells and the inserts used in this study. TALS plasmid lacks the Escherichia coli vector backbone, contains a2 operator indicated by the hatched box, and forms 10 stable nucleosomes in vivo as illustrated by numbered gray circles. AT-rich sequences shown in the table were inserted into the SacI site (1460 map units).
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To examine a role of AT-tracts in nucleosome positioning, we inserted several AT-rich sequences into the SacI site located 75 bp upstream of the a2 operator (Fig. 1). The inserts include a long mixed sequence GC(GAGCTCGC)6, a short AT-tract A7TATA8 resembling a yeast ARS consensus sequence, a long poly(dA). poly(dT) sequence, which is often found in promoters [21] and shows rigidity [22,23], another type of long AT-tract T3CCT6CT5 GCT5CT7 derived from DED1 promoter [24], and a hetero-polymeric sequence (CA)17 showing high flexibility [25]. These plasmids were introduced into dThd-auxotrophic MATa cells to ensure quantitative incorporation of 5-bromouracil into DNA [4,26]. Nucleosome positioning on TALS plasmid We confirmed nucleosome positioning on TALS plasmid by digestion with MNase followed by the indirect end-labeling method. Four MNase cleavage sites (indicated by *a, *b, *c, and *d in Fig. 2A) were observed on the naked DNA samples. These sites were protected in the chromatin samples, and instead new cleavage sites appeared with equal intervals (140–150 bp) (Fig. 2A, lanes 1–4). When chromatin samples were digested with HindIII, bands corresponding to linker regions between nucleosomes V–VIII were detected (data not shown). These results indicate that nucleosomes are assembled precisely and stably on TALS plasmid in vivo as described previously [17,18]. Effects of 5-bromouracil on nucleosome positioning We examined the effects of 5-bromouracil on nucleosome positioning on TALS plasmid. When cells were cultured with BrdU for more than three generations, the band corresponding to the linker region between nucleosomes IV and V disappeared, and the band *a was evident in the chromatin sample (Fig. 2, lane 5). Densitometrical measurement clearly showed these differences (Fig. 2B). The other bands were clearly protected similarly to the sample prepared in dThd-medium (Fig. 2A, lanes 5–8). Since 5-bromouracil did not affect MNase cleavage patterns on naked DNA samples (Fig. 2B), 5-bromouracil disrupted nucleosomes IV and V adjacent to the a2 operator sequence. We further examined binding of a2/Mcm1 to a2 operator with an in vivo UV photofootprinting assay as described previously [18,27]. There was no detectable difference in the presence of a2/Mcm1 between dThd- and BrdU-medium (data not shown). These results suggest that the above changes are caused by the presence of 28 bp ATrich sequence just downstream of the a2 operator (Fig. 2A). Effects of AT-tracts on nucleosome positioning To search for a role of AT-tracts in nucleosome positioning, we examined TALS derivatives containing several AT-rich sequences at its SacI site (Fig. 1). First,
we examined pTS711 containing a mixed sequence GC(GAGCTCGC)6. This plasmid was assembled nucleosomes similarly to TALS plasmid in dThd-medium (Fig. 3, lane 1 vs. 5), indicating that DNA insertion per se did not affect nucleosome positioning and that nucleosomes are positioned independently of DNA sequences flanked by the a2 operator [20]. Addition of BrdU eliminated protection of band *a in the chromatin sample (Fig. 3, lanes 7 and 8) but the other regions were not affected. Thus, only nucleosomes IV and V were affected by addition of BrdU as in the case of TALS. Plasmids pTS713 containing A7TATA8 (Fig. 3, lanes 9–12) and pOM802 containing A5TATA4 (not shown) behaved similarly to pTS711 in dThd- or BrdU-medium. These results suggest that a long mixed sequence and short AT-tracts do not affect nucleosome positioning on TALS plasmid regardless of whether it contains thymine or 5-bromouracil. Plasmid pKM001 containing a long AT-tract of T3CCT6CT5GCT5CT7 showed stable nucleosome positioning in dThd-medium (Fig. 3, lanes 13 and 14). Addition of BrdU also prevented protection of the band *a as in the cases of TALS, pTS711, and pTS713 plasmids, but it made the bands *b and *c in the chromatin sample more evident (Fig. 3, lane 13 vs. 15). Similar results were obtained with pOM804 containing a hetero-polymeric sequence of (CA)17 (Fig. 3A, lanes 17–20). These results demonstrated that 5-bromouracil disrupts nucleosome positioning on the sequence containing not only a long AT-tract but also a purine–pyrimidine repeat. Plasmids pOM801 containing a long AT-tract of A34 and pKM002 containing a longer AT-tract of (T3CCT6CT5GCT5CT7)2 were weakly assembled nucleosomes around the site of their insertion even in dThd-medium (Fig. 3, lanes 21 and 25) as described previously [18]. Addition of BrdU almost completely prevented the protection of nucleosome-specific bands in their chromatin samples as exemplified by appearance of the band *b (Fig. 3, lanes 23 and 27). Moreover, the regions of nucleosomes I–III were broader than those in dThd-medium. These results showed that 5-bromouracil further disrupts unstable nucleosome positioning mediated by long AT-tracts. Electrophoretic analysis We undertook electrophoretic analysis with sequences (323–400 bp) containing the above AT-tracts at their centers (Fig. 4A and B). 5-Bromouracil was incorporated into the sequences by PCR with BrdUTP as a substrate. The control sequence derived from TALS behaved similarly at 4 °C and 52 °C regardless of whether it contained thymine or 5-bromouracil. The relative mobility (Rm = mobility at 4 °C/mobility at 52 °C) of this sequence was 0.98 (Fig. 4B). The sequence derived from pTS713 (A7TATA8) migrated slower at 4 °C than at 52 °C (Rm 1.07) when containing thymine, indicating that this sequence was bent. Upon substitution with 5-bromouracil, it migrated nor-
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Fig. 2. Nucleosome positioning on TALS plasmid. Chromatin (indicated by C) and naked DNA (indicated by D) samples were prepared from cells transfected with TALS plasmid and cultured with dThd or BrdU. The samples were digested with MNase at the concentrations of 2 U/ml (lanes 1 and 5), 5 U/ml (lanes 3 and 7), 0.2 U/ml (lanes 2 and 6), and 0.4 U/ml (lanes 4 and 8), and subjected to indirect end-labeling analysis as described in Materials and methods. Independent experiments gave similar results. (A) Autoradiography of MNase cleavage patterns detected with the EcoRV–HindIII fragment (385–615 map units of TALS in Fig. 1) used as a probe. DNA size marker (lane M) and the positions of nucleosomes I–VI and a2 operator are shown to the left. Specific cleavage sites on naked DNA samples are marked with *a, *b, *c, and *d. Open stars on some lanes denote the bands that changed in BrdU-medium. The intrinsic AT-rich sequence (underlined) just downstream of a2 operator is shown between the panels. (B) Densitometrical profiles of autoradiography. Lanes 3, 4, 7, and 8 in the above panels are densitometrically scanned. The positions of stable nucleosomes are shown on the top.
mally, indicating that 5-bromouracil canceled DNA bending in agreement with the previous report [5]. Similar
results were obtained with the sequence derived from pKM001 (T3CCT6CT5GCT5CT7).
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Fig. 3. Nucleosome positioning on TALS-plasmids having various inserts. TALS and its derivatives shown in Fig. 1 were analyzed as in Fig. 2. Chromatin and naked DNA samples were digested with 5 U/ml and 0.4 U/ml MNase, respectively. Independent experiments gave similar results.
On the other hand, the sequences derived from pOM801 (A34) and pKM002 ((T3CCT6CT5GCT5CT7)2) migrated faster at 4 °C than at 52 °C when containing thymine (Rm 0.96 and 0.97, respectively), suggesting that these sequences were not bent but adopt a non B-form conformation. Substitution of thymine with 5-bromouracil further accelerated their mobility at 4 °C. The sequence derived from pOM804 containing (CA)17 migrated slightly slower than the control sequence when containing thymine (Rm 1.0). Substitution with 5-bromouracil intermediately accelerated its mobility at 4 °C. The above results suggested that rapid migration of DNA (Rm values less than 0.98) rather than cancellation
of DNA bending is correlated with instability of nucleosome positioning in TALS system. Discussion Incorporation of 5-bromouracil was shown to clearly disrupt nucleosome positioning on the plasmids dependent on a specific AT-rich sequence inserted into a site near the anchor position of a2 operator. The mixed sequence GC(GAGCTCGC)6 and the short AT-tracts (A5TATA4 and A7TATA8) did not affect nucleosome positioning. In contrast, long AT-tracts such as T3CCT6CT5GCT5CT7,
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Fig. 4. Electrophoretic analysis of sequences derived from TALS and its derivatives. DNA fragments containing AT-tracts at their central positions were amplified from the plasmids by PCR, and run on polyacrylamide gel at as described in Materials and methods. Independent experiments gave similar results. (A) Lane 1, DNA amplified by PCR with dTTP; lanes 2 and 3, DNA amplified by PCR with BrdUTP for 2 and 5 cycles, respectively; lane M, size marker. (B) Relative mobility (mobility at 4 °C/52 °C) of lanes 1 and 3 in (A).
(CA)17, A34, and (T3CCT6CT5GCT5CT7)2 destabilized nucleosome positioning on TALS plasmid. Addition of BrdU almost completely disrupted nucleosomes around the site of their insertion. How does 5-bromouracil disrupt nucleosome positioning? Historically, the bromine atom at the 50 position of uracil has been thought to stabilize interaction between DNA and certain DNA-binding proteins due to its electronegative nature stronger than methyl residue [28], although most DNA-binding proteins cannot distinguish sequence specificity of DNA containing 5-bromouracil or thymine [29,30]. According to our recent studies [5], 5-bromouraicl is thought to induce a change in DNA topology possibly
due to increase in neighboring base–base interaction or stacking of base pairs. In the present study, we showed that the AT-tracts effective in disrupting nucleosome positioning migrated rapidly on gel electrophoresis upon substitution with 5-bromouracil. This property is indicative of an A-form-like structure [31]. Runs of consecutive purines are known to set up an A-like helix [32,33] because stacking interactions of purines are thought to provide the mechanical rigidity of DNA helix [34]. To date, rapid migration is reported for AT-rich fragments from yeast centromeres [35] and for sites of illegitimate recombination in mammalian cells [36]. Bovine satellite I DNA is also shown to migrate rapidly dependent on temperature and to take an A-form-like structure as determined by circular permuta-
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tion and circular dichroism spectra [37,38]. Based on these observations, bromine atom is thought to increase rigidity in DNA, resulting in stiff and rod-like DNA. In support of this, 5-bromouracil-substituted chromatins showed a blue shift and increased positive ellipticity in the 250–300 nm region of circular dichroism spectra [28], which is a part of the feature of A-form DNA. Taken together, we suggest that disruption of nucleosome positioning by 5-bromouracil is caused by appearance of an unusual DNA conformation such as an A-form-like structure. Biological and structural features of A-form DNA are only poorly understood. The DNA binding site for transcription factor IIIA, which binds to the internal control region of the Xenopus 5S RNA gene, is shown to have an overall or global structure with characteristics of A-form DNA [32,39]. Since the hydrophobicity of the DNA surface in the minor grooves is increased by B- to A-form transition, hydrophobic amino acids can interact extensively with A-form structure [40,41]. Moreover, cardiolipin, a chromatin-bound phospholipid, is shown to provide Aform DNA in the complex with RNA polymerase to elicit active chromatin [42,43]. In this respect, it is interesting to note that cardiolipin induces premature senescence in normal human fibroblasts [44]. Finally, the novel findings described here may lead to direct understanding of the role of BrdU in modification of chromatin structure. They may also answer the new and old question why BrdU modulates expression of particular genes associated with cellular differentiation and senescence. References [1] F.H. Wilt, M. Anderson, The action of 5-bromodeoxyuridine on differentiation, Dev. Biol. 28 (1972) 443–447. [2] H. Weintraub, Size of the BUdR sensitive targets for differentiation, Nat. New Biol. 244 (1973) 142–143. [3] E. Michishita, K. Nakabayashi, T. Suzuki, S.C. Kaul, H. Ogino, M. Fujii, Y. Mitsui, D. Ayusawa, 5-Bromodeoxyuridine induces senescence-like phenomena in mammalian cells regardless of cell type or species, J. Biochem. (Tokyo) 126 (1999) 1052–1059. [4] M. Fujii, H. Ito, T. Hasegawa, T. Suzuki, N. Adachi, D. Ayusawa, 5Bromo-20 -deoxyuridine efficiently suppresses division potential of the yeast Saccharomyces cerevisiae, Biosci. Biotechnol. Biochem. 66 (2002) 906–909. [5] T. Suzuki, E. Michishita, H. Ogino, M. Fujii, D. Ayusawa, Synergistic induction of the senescence-associated genes by 5-bromodeoxyuridine and AT-binding ligands in HeLa cells, Exp. Cell Res. 276 (2002) 174–184. [6] S. Minagawa, K. Nakabayashi, M. Fujii, S.W. Scherer, D. Ayusawa, Functional and chromosomal clustering of genes responsive to 5bromodeoxyuridine in human cells, Exp. Gerontol. 39 (2004) 1069– 1078. [7] A.F. Zakharov, L.I. Baranovskaya, A.I. Ibraimov, V.A. Benjusch, V.S. Demintseva, N.G. Oblapenko, Differential spiralization along mammalian mitotic chromosomes. II. 5-bromodeoxyuridine and 5bromodeoxycytidine-revealed differentiation in human chromosomes, Chromosoma 44 (1974) 343–359. [8] T. Suzuki, M. Yaginuma, T. Oishi, E. Michishita, H. Ogino, M. Fujii, D. Ayusawa, 5-Bromodeoxyuridine suppresses position effect variegation of transgenes in HeLa cells, Exp. Cell Res. 266 (2001) 53–63.
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