Báo cáo khoa học: 5-Bromodeoxyuridine induces transcription of repressed genes with disruption of nucleosome positioning pptx

5-Bromodeoxyuridine induces transcription of repressed genes with disruption of nucleosome positioning Kensuke Miki 1 , Mitsuhiro Shimizu 2 , Michihiko Fujii 1 , Shinichi Takayama 1 , Mohammad Nazir Hossain 1 and Dai Ayusawa 1 1 Department of Genome System Science, Yokohama City University, Yokohama, Kanagawa, Japan 2 Department of Chemistry, Meisei University, Hino, Tokyo, Japan Introduction 5-Bromodeoxyuridine (BrdU), which is frequently used to measure DNA synthesis immunochemically in living cells, is also well known to modulate various biological functions when incorporated into DNA as 5-bromoura- cil instead of thymine. Previously we have found that BrdU very clearly induces a senescent-like phenomenon in every type of mammalian cell and also in yeast cells [1,2]. Historically, BrdU has been used as a modulator of cellular differentiation with cAMP and butyrate [3,4]. The latter two are found to target protein kinase A and histone deacetylase, respectively, leading to func- tional understanding of cell signaling and gene expres- sion. BrdU is thought to influence the expression of key genes involved in cellular differentiation. However, the molecular mechanism underlying the actions of BrdU still remains a mystery in spite of many efforts [5]. We have extensively characterized genes up- or down-regulated by the addition of BrdU in various cell Keywords AT-tract; 5-bromodeoxyuridine; BAR1; nucleosome positioning; transcriptional derepression Correspondence D. Ayusawa, Department of Genome System Science, Yokohama City University, Seto 22-2, Kanazawa-Ku, Yokohama, Kanagawa 236-0027, Japan Fax: +81 45 787 2193 Tel: +81 45 787 2193 E-mail: dayusawa@yokohama-cu.ac.jp (Received 5 January 2010, revised 2 August 2010, accepted 2 September 2010) doi:10.1111/j.1742-4658.2010.07868.x 5-Bromodeoxyuridine (BrdU) modulates the expression of particular genes associated with cellular differentiation and senescence when incorporated into DNA instead of thymidine (dThd). To date, a molecular mechanism for this phenomenon remains a mystery in spite of a large number of stud- ies. Recently, we have demonstrated that BrdU disrupts nucleosome posi- tioning on model plasmids mediated by specific AT-tracts in yeast cells. Here we constructed a cognate plasmid that can form an ordered array of nucleosomes determined by an a2 operator and contains the BAR1 gene as an expression marker gene to examine BAR1 expression in dThd-auxotro- phic MATa cells under various conditions. In medium containing dThd, BAR1 expression was completely repressed, associated with the formation of the stable array of nucleosomes. Insertion of AT-tracts into a site of the promoter region slightly increased BAR1 expression and slightly destabi- lized nucleosome positioning dependent on their sequence specificity. In medium containing BrdU, BAR1 expression was further enhanced, associ- ated with more marked disruption of nucleosome positioning on the pro- moter region. Disruption of nucleosome positioning seems to be sufficient for full expression of the marker gene if necessary transcription factors are supplied. Incorporation of 5-bromouracil into the plasmid did not weaken the binding of the a2 ⁄ Mcm1 repressor complex to its legitimate binding site, as revealed by an in vivo UV photofootprinting assay. These results suggest that BrdU increases transcription of repressed genes by disruption of nucleosome positioning around their promoters. Abbreviations BrdU, 5-bromodeoxyuridine; dThd, thymidine; MNase, micrococcal nuclease; TK, thymidine kinase. FEBS Journal 277 (2010) 4539–4548 ª 2010 The Authors Journal compilation ª 2010 FEBS 4539 lines using PCR-based cDNA subtractive hybridization and DNA microarray analysis [6,7]. Such BrdU- responsive genes behave similarly in normal human fibroblasts undergoing replicative senescence. BrdU decondenses particular regions of chromosomes after incorporation into DNA [8], suppresses position effect variegation [9] and restores expression of silenced genes [10]. Consistent with this, BrdU-responsive genes are located on particular regions of human chromo- somes, forming clusters on or nearby Giemsa-dark bands of human chromosomes [11,12]. AT-tract minor groove binders, such as distamycin A, netropsin, Hoe- chst 33258 and the AT-hook protein HMG-I, have all been shown to markedly potentiate the effects of BrdU [11,13]. On the basis of the above observations, we suggest that BrdU targets certain types of AT-rich sequence and alters the chromatin structure to induce particular genes. In eukaryotes, DNA fibers exist as regularly arrayed beads of nucleosomes. Nucleosomes restrict the acces- sibility of transcription factors to promoters and regu- latory sequences of genes. Thus, an alteration of nucleosome positioning is an essential step for the transition from a repressed state to an active state by aid of chromatin remodeling complexes [14–16]. In our previous studies, we addressed the effect of BrdU on nucleosome positioning in vivo using TALS plasmids [17], which have been successfully utilized to study nucleosome positioning in Saccharomyces cerevisiae. We have clearly shown that 5-bromouracil incorpo- rated into the plasmids disrupts nucleosome position- ing by inducing A-form-like DNA conformation in yeast cells [18]. In mammalian cells, histones were shown to bind more tightly to 5-bromouracil-substi- tuted DNA in vitro than normal DNA containing thy- mine [19,20]. In this study, we examined whether BrdU induces the expression of genes associated with the destabiliza- tion of nucleosome positioning with model plasmids containing the BAR1 gene as a marker gene. We showed that BrdU increases the transcription of repressed genes by disruption of nucleosome position- ing around their promoters. These data will facilitate the understanding of the role of BrdU and AT-tracts in the induction of particular genes. Results Construction of model plasmids In yeast MATa cells, the a2 ⁄ Mcm1 repressor binds to the a2 operator and acts to repress MATa cell-spe- cific genes such as BAR1, STE2 and STE6 [21–25]. Previously, our high-resolution mapping of micrococ- cal nuclease (MNase) cleavage sites has indicated that nucleosomes were well positioned around the pro- moter region of the genomic BAR1 gene [26] and such nucleosome positioning was required for its full repression [27]. Here, we constructed a plasmid pRS- BAR1 that contains the BAR1 gene as a model sys- tem (Fig. 1) to easily examine the role of BrdU in gene expression and nucleosome positioning in vivo. To further examine the effects of AT-tracts on nucle- osome positioning, we inserted them into a KpnI site adjacent to the TATA box. These plasmids were introduced into a thymidine (dThd)-auxotrophic Inserts at the Kpn Isite pRS001-BAR1 pRS801-BAR1 :T 3 CCT 6 CT 5 GCT 5 CT 7 :A 34 BAR1 -UTR -UTR pRS-BAR1 TATA 2op mRNA Kpn I +1130 ––219 66 +95 +255 I II III IV V 60 – 500 Xho I –––235 +420 –158 Eco RV +1667 Probe A Probe B 5′ 3′ BAR1 Fig. 1. Schematic representation of pRS-BAR1 and its derivatives. Plasmid pRS-BAR1 contains a genomic fragment spanning the BAR1 gene regulated by a2 ⁄ Mcm1 repressor. The a2 operator (a2 op), the TATA box (TATA) and the BAR1 coding sequence are denoted by hatched, filled and dotted boxes, respectively. The transcriptional initiation site is indicated by a bent arrow. The site of KpnI, to which AT-tracts are inserted, is indicated by a vertical line. The probes A and B are indicated by gray boxes. Positions of stable nucleosomes are shown by shadowed ellipses with numbers. Molecular mechanism of 5-bromodeoxyuridine K. Miki et al. 4540 FEBS Journal 277 (2010) 4539–4548 ª 2010 The Authors Journal compilation ª 2010 FEBS mutant of yeast cells to ensure quantitative incorpora- tion of BrdU into DNA [2]. Nucleosome positioning on pRS-BAR1 We first examined whether pRS-BAR1 forms a stable and precise array of nucleosomes in dThd medium using the indirect end-labeling method. DNA samples were completely digested with XhoI after partially digestion with MNase, and were subjected to Southern blot analysis with probe A. Five MNase cleavage sites indicated by *a, *b, *c, *d and *e were observed in the naked DNA sample, and these sites were protected in a chromatin sample of pRS-BAR1 (Fig. 2A, lanes 1 and 2). Also, the cleavage sites showed equal intervals (140–150 bp) in the chromatin sample (Fig. 2A, lane 1). These results showed that at least five stable nucle- osomes were precisely and stably formed from the a2 operator to the coding region of the BAR1 gene on pRS-BAR1 in vivo (Fig. 1, bottom panel). We then examined how BrdU affects nucleosome positioning on the pRS-BAR1 plasmid. When the MATa cells were cultured in BrdU medium, the two bands (*b and *c) that were protected in the chromatin sample prepared in dThd medium were evident in the chromatin sample prepared in BrdU medium (Fig. 2A, lanes 1 versus 3). Densitometrical measurement clearly showed these differences (Fig. 2B). Consistently, the band corresponding to the linker region between nucle- osomes II and III became broader in the same sample (Fig. 2A, lane 3). Because there was no significant dif- ference in MNase cleavage patterns between naked DNA samples prepared in dThd and BrdU medium, BrdU did not affect the specificity and sensitivity to MNase (Fig. 2A, lanes 2 versus 4). These results indi- cate that BrdU destabilized nucleosome positioning. We then examined the sequence in nucleosome II, and found the following: the AT-content of the sequence was 69%, whereas in other nucleosomes it was approx- imately 60%, and A n ,T n or (AT) n tracts (n ‡ 6) were found in six sites in nucleosome II, but not in nucleo- some III. Therefore, BrdU seemed to destabilize the positioning of nucleosome II through AT-rich sequences in nucleosome II, which then led to the destabilization of nucleosome III. The same results were obtained when nucleosome positioning was deter- mined from the EcoRV site with probe B (Fig. 1, S1, lanes 1–4). Effects of AT-tracts on nucleosome positioning We examined two derivatives of pRS-BAR1, pRS001- BAR1 and pRS801-BAR1, containing T 3 CCT 6 CT 5 GCT 5 CT 7 and A 34 , respectively, in a promoter region of the BAR1 gene (Fig. 1A). Insertion of these AT- tracts did not affect MNase cleavage patterns in naked DNA samples (Fig. 2A, even-numbered lanes). The MNase cleavage pattern in a chromatin sample of pRS001-BAR1 was similar to that in the chromatin sample of pRS-BAR1 in dThd medium, although the bands *c and *d were more evident in the former sam- ple (Fig. 2A, lanes 1 versus 5). This suggests that the positioning of nucleosomes I and II on pRS001-BAR1 was less stable than on pRS-BAR1 in dThd medium. The bands *b, *c and *e in the chromatin sample of pRS001-BAR1 were more marked in BrdU medium than in dThd medium (Fig. 2A, B, lanes 5 versus 7), although the MNase cleavage patterns in the naked DNA sample were similar between dThd and BrdU medium. Similar results were obtained when nucleo- some positioning was mapped in the opposite direction from the EcoRV site with probe B (Fig. S1, lanes 5–8). We next analyzed the nucleosome positioning on pRS801-BAR1. Interestingly, the MNase mapping of nucleosomes with probe A showed that the three bands *b to *d (especially band *c) were not protected in a chromatin sample of pRS801-BAR1 (Fig. 2A, lane 9), indicating that insertion of A 34 disrupts nucleosome positioning more remarkably than that of T 3 CCT 6 CT 5 GCT 5 CT 7 . In BrdU medium, all of the bands that corresponded to the linker regions were eliminated and the bands *a, *b and *e were more clearly detected than in dThd medium (Fig. 2B, lanes 9 versus 11). These results were also confirmed by indi- rect end-labeling with probe B (Fig. S1, lanes 9–12). Taken together, these observations indicate that nucleosome positioning on pRS001- and pRS801- BAR1 were modestly and almost completely disrupted, respectively, by BrdU. Effect of BrdU on BAR1 expression We measured the expression of the BAR1 gene on the model plasmids by Northern blot analysis. In dThd medium, the BAR1 mRNA level for pRS-BAR1 was 30 times less in the MATa cells than in the MATa cells (Fig. 3A). These results indicate that the episomal BAR1 gene is regulated similarly to the genomic BAR1 gene. In the MATa cells, the mRNA level was 2.4 times higher in BrdU medium than in dThd medium (Fig. 3B). Insertion of T 3 CCT 6 CT 5 GCT 5 CT 7 (pRS001- BAR1) slightly increased the mRNA level in dThd medium, but additionally increased it in BrdU medium (Fig. 3B). On the other hand, insertion of A 34 (pRS801-BAR1) significantly increased the mRNA level in dThd medium and markedly increased it in K. Miki et al. Molecular mechanism of 5-bromodeoxyuridine FEBS Journal 277 (2010) 4539–4548 ª 2010 The Authors Journal compilation ª 2010 FEBS 4541 a b c d a b c d 2op c d a b c d a b c e a b c e a b pRS-BAR1 CDCD dThd BrdU pRS001-BAR1 CD CD dThd BrdU pRS801-BAR1 CD C D dThd BrdU Lane 3 412 56 78 9 10 1112 M 2.0 1.5 1.0 0.5 (kb) e e e e d d I II III IV V A Lane 1 3 5 7 9 11 B 2 op IIIII IV VI Naked DNA e d c b a Fig. 2. Nucleosome positioning on pRS- BAR1 and its derivatives. Chromatin (indi- cated by C) and naked DNA (indicated by D) samples were prepared from cells transfect- ed with the plasmid indicated and cultured in dThd or BrdU medium as indicated. The samples were partially digested with 5UÆmL )1 (odd-numbered lanes) or 0.5 UÆmL )1 (even-numbered lanes) MNase, completely digested with XhoI, and sub- jected to the indirect end-labeling analysis with probe A as described in Materials and Methods. At least three independent analy- ses for each plasmid gave similar results. (A) Autoradiography of MNase cleavage pat- terns. DNA size markers (M), positions of nucleosomes I–V and a2 operator (a2 op) are shown to the left. Specific cleavage sites on naked DNA samples are marked with *a, *b, *c, *d and *e. Ellipses with dotted lines indicate nucleosomes whose positioning is unstable. Open stars on some lanes denote bands that changed in BrdU medium. The transcriptional start site and the KpnI site are indicated by a bent arrow and arrowheads, respectively. (B) Densito- metric profiles of autoradiography. The odd- numbered lanes in (A) were densitometrical- ly scanned. The vertical dotted lines denote the bands (*a, *b, *c, *d and *e) specific to naked DNA. The positions of stable nucleo- somes are shown at the top. Molecular mechanism of 5-bromodeoxyuridine K. Miki et al. 4542 FEBS Journal 277 (2010) 4539–4548 ª 2010 The Authors Journal compilation ª 2010 FEBS BrdU medium (Fig. 3B). We also examined the effect of BrdU on pYBT1 containing the thymidine kinase (TK) gene driven by the yeast constitutive ADH1 pro- moter as a control plasmid. BrdU did not significantly affect the expression of the gene (Fig. 3B). These results show that the levels of expression of the BAR1 gene are parallel to those of the disruption of nucleo- some positioning in dThd and BrdU medium. To confirm that our experimental conditions can induce specific genomic genes, we examined the expres- sion of some genomic genes having an AT-tract on their promoter regions. The DED1 gene, having T 3 CCT 6 CT 5 GCT 5 CT 7 , and the MAK16 gene, having T 24 , were significantly up-regulated by the addition of BrdU (Fig. 4), suggesting that the mechanism found in the episomal genes also operates in the genomic genes. Effect of BrdU on a2/Mcm1 repressor–operator complex We examined whether the a2 ⁄ Mcm1 repressor changes its binding to the a2 operator on pRS-BAR1 upon incorporation of 5-bromouracil by an in vivo UV photofootprinting assay. The a2 ⁄ Mcm1 operator has numerous thymine bases necessary for recognition by the repressor [28] and thus its binding to the repressor may be disturbed by substitution of 5-bromouracil. In the noncoding strand of a naked DNA sample, several thymine dimers were found around the a2 operator (Fig. 5A, lane 2), but three sites (marked with + in Fig. 5A) were protected in chromatin samples (Fig. 5A, lanes 1 and 3). Although slight differences in the thymine dimers formed were observed in the naked DNA samples containing thymine or 5-bromouracil (Fig. 5A, lanes 2 versus 5), the three thymine dimers were equally protected in the chromatin samples con- taining thymine or 5-bromouracil (Fig. 5A, lanes 4 and 6). Similar results were obtained with the coding strand (Fig. 5B). These results show that BrdU does not weaken the formation of the a2 ⁄ Mcm1 repressor– operator complex, excluding the possibility that the DED1 MAK16 ACT1 dThd BrdU Fig. 4. Northern blot analysis of genomic genes. Total RNA sam- ples prepared from the MATa cells cultured in dThd or BrdU med- ium were subject to Northern blot analysis with probes derived from the genes indicated. TB pRS-BAR1 0 2 4 6 TB pRS001-BAR1 ## TB pRS801-BAR1 ### Relative mRNA level TB BAR1 TK pYBT1 Relative BAR1 mRNA level 20 0 MAT MAT a pRS-BAR1 10 30 AB Fig. 3. Gene expression profiles of pRS-BAR1, its derivatives and a reference plasmid. (A) BAR1 mRNA levels in MATa and MATa cells. Total RNA and DNA samples were prepared from the cell type indicated in dThd medium and subjected to Northern and Southern blot analy- ses as described in Materials and Methods. BAR1 mRNA levels were expressed relative to actin mRNA levels after normalization by copy numbers of plasmids. (B) Effects of BrdU on BAR1 and TK mRNA levels. Total RNA and DNA samples were prepared from the MATa cells transfected with the plasmid indicated and cultured in dThd (T) or BrdU medium (B), and processed as in (A). ***P < 0.001 compared with the values in dThd medium. ## P < 0.01 and ### P < 0.001 compared with the values of pRS-BAR1 in dThd medium. Histograms represent means ± standard error. At least four independent analyses carried out for each plasmid gave similar results. K. Miki et al. Molecular mechanism of 5-bromodeoxyuridine FEBS Journal 277 (2010) 4539–4548 ª 2010 The Authors Journal compilation ª 2010 FEBS 4543 disruption of nucleosome positioning by BrdU is due to a decrease in the formation of the a2 ⁄ Mcm1 com- plex [18]. Effect of BrdU on pRS-BAR1 lacking the promoter activity We addressed whether the above changes in nucleo- some positioning are affected by the promoter activity of the BAR1 gene, because transcription factors can affect nucleosome positioning. We constructed a plas- mid, pRS-DTA ⁄ BAR1, in which the TATA box of BAR1 was disrupted (Fig. 6A) [23]. With pRS-DTA ⁄ BAR1 we were able to determine the change in nucleo- some positioning without the effect of the expression of BAR1 (Fig. 6B). Disruption of the TATA box caused the disappear- ance of band *d in naked samples of pRS-DTA ⁄ BAR1 prepared in dThd and BrdU medium. Band *d corre- sponds to the nuclease hypersensitive site located at the TATA box on pRS-BAR1 (Fig. 2A, lanes 2 and 4 versus Fig. 6C, lanes 2 and 4) as described in the pre- vious reports by Shimizu et al. [26] and Cooper et al. [23]. In dThd medium, the MNase cleavage pattern in a chromatin sample of pRS-DTA ⁄ BAR1 showed well- ordered nucleosome positioning (Fig. 6C, lane 1), which was identical to that on the chromatin sample of pRS-BAR1 (Fig. 2A, lane 1), except for the pres- ence of band *d. In BrdU medium, the bands corresponding to the linker regions between nucleosomes I–III were broader, and the two bands (*b and *c) that were pro- tected in the chromatin sample prepared in dThd med- ium became more evident (Fig. 6C, lane 3). When dThd CDC BrdU CDC + + + + + + + + + A dThd CDC BrdU CDC + + + + + + B 123 456 789 101112 3′-CGTACATTAATGGCATTTTCCTTTAATGTAC-5′ 3′-GTACATTAAAGGAAAATGCCATTAATGTACG-5′ Fig. 5. In vivo UV photofootprinting of a2 operator on pRS-BAR1. Intact cells (indicated by C) and naked DNA (indicated by D) were irradiated with UV at dosages of 250 mJ (lanes 1, 4, 7 and 10), 500 mJ (lanes 3, 6, 9 and 12) and 60 mJ (lanes 2, 5, 8 and 11). UV photoproducts were analyzed using primer extension mapping on the noncoding (A) and the coding (B) strands as described in Mate- rials and Methods. The a2 operator sequence is shown to the left of each panel. The thymine bases protected from UV damage are indicated by +. a b c e I II III IV V a b c e BrdU –137 –125 AC CAGTATAAAAGTG TATA pRS-BAR1 CAGTGGATCCGTG Bam H I pRS-ΔTA/BAR1 pRS-ΔTA/BAR1 dThd 2 op Lane 3 41 2 B ACT1 BAR1 BAR1 TA/ BAR1 pRS- C DC D Fig. 6. Nucleosome positioning on p0RS- DTA ⁄ BAR1. (A) Sequences of the TATA box and disrupted TATA box of the BAR1 pro- moter. (B) Northern blot analysis of the BAR1 gene. (C) Autoradiography of MNase cleavage patterns. Nucleosome positioning was analyzed as in Fig. 2. Molecular mechanism of 5-bromodeoxyuridine K. Miki et al. 4544 FEBS Journal 277 (2010) 4539–4548 ª 2010 The Authors Journal compilation ª 2010 FEBS cultured in dThd or BrdU medium, the overall band patterns were almost identical in the chromatin sam- ples between pRS-BAR1 and pRS-DTA ⁄ BAR1, except for the presence of band *d. These results indicate that BrdU changes nucleosome positioning in the absence of the transcription factors involved. In line with this observation, we have shown that incorporation of BrdU into DNA converts the DNA structure into an unusual conformation [18]. It is thus reasonable to suggest that such a structural change in DNA induced by BrdU affects nucleosome positioning and results in altered gene expression at particular regions. Discussion We were able to show a positive correlation between gene expression and the disruption of nucleosome posi- tioning in yeast cells harboring minichromosomes and cultured with BrdU as the only source of thymine. To validate this observation, BrdU must directly affect DNA structure, but not interactions between DNA and DNA-binding proteins, to induce a change in nucleosome positioning. In our model plasmids used, the binding of a2 ⁄ Mcm1 repressor to its legitimate binding site has a critical role in the formation of sta- bly ordered nucleosomes. As expected, BrdU did not significantly affect the formation of the a2 ⁄ Mcm1 repressor–operator complex [18]. In support of this, expression of the genomic STE2 gene regulated by the a2 ⁄ Mcm1 complex was not affected by the addition of BrdU (data not shown). In addition, some DNA-bind- ing proteins and enzymes examined to date cannot functionally distinguish between 5-bromouracil and thymine on DNA [29–32]. These results suggest that an unusual DNA conformation induced by BrdU is the primary cause of altered nucleosome positioning. In fact, we have demonstrated that the incorporation of 5-bromouracil into DNA reduces the bending of DNA [11] and converts to A-form-like DNA or a rigid DNA structure [18]. However, a possibility cannot be ruled out that a change in interactions between 5-bro- mouracil-substituted DNA and specific proteins may additionally affect nucleosome positioning. We showed here that levels of BAR1 expression are parallel to those of the destabilization of nucleosome positioning with the use of model plasmids. In our pre- vious study employing different minichromosomes [18], disruption of nucleosome positioning by BrdU was shown to depend on the length and sequence specificity of AT-tracts located at particular sites of minichromo- somes. In this study, destabilization of nucleosome positioning by BrdU did not require the presence of the promoter or expression of the BAR1 marker gene. As shown in the Results, AT-tracts alone can destabi- lize nucleosome positioning in dThd medium. How- ever, not all AT-tracts have the ability to induce the destabilization of nucleosome positioning or expression of the marker genes. The nucleosome disruption did not lead to full expression of the BAR1 gene. This can be explained by the absence of an activator function of Mcm1 in MATa cells. Mcm1 acts as an activator of a-cell-spe- cific genes in MATa cells, whereas it acts as a repressor in MATa cells. Taken together, nucleosome position- ing seems to be sufficient for full repression of genes [27]. Also, disruption of it seems to be sufficient for full derepression of genes if necessary transcription fac- tors are supplied. Can our findings obtained with the episomal genes apply to genomic loci? The episomal BAR1 gene was shown to behave similarly to the genomic BAR1 gene [27] when A 34 was inserted into their promoters. Simi- larly, the genomic DED1 gene was significantly up-reg- ulated by BrdU, as the episomal BAR1 gene has the AT-tract derived from the DED1 promoter. Further- more, the genomic MAK16 gene having T 24 on its pro- moter region was also up-regulated by BrdU. These results suggest that episomal and genomic genes behave similarly, and prove to be useful in studying gene regulation controlled by the higher-order struc- ture of chromatin. However, the presence of AT-tracts does not always affect the expression of their adjacent genes if the genes have a strong promoter. Their promoter regions seem to be reluctant to form a stable nucleosome structure. In these genes, BrdU does not seem to cause an additional change in the nucleosome structure sur- rounding the genes and increase their expression. For example, the expression of the TK gene driven by the yeast constitutive ADH1 promoter on pYBT1 was not affected by BrdU (Fig. 3B). In the case of TALS-GFP, EGFP expression is driven by the ADH1 promoter (Fig. S2). pOM801-GFP, having A 34 inserted in the ADH1 promoter region (Fig. S2), showed significantly increased promoter activity even in dThd medium (Fig. S4). However, BrdU did not further increase the expression of GFP (Fig. S4) or the state of the already opened nucleosome structure in pOM801-GFP (Fig. S3). These results support our hypothesis that BrdU induces the expression of genes through the dis- ruption of nucleosome positioning on their promoter regions. In mammalian cells, BrdU is thought to induce the expression of genes when AT-tracts are located adja- cent to their promoters, similar to yeast systems [11]. In contrast to yeast genes, most of the mammalian K. Miki et al. Molecular mechanism of 5-bromodeoxyuridine FEBS Journal 277 (2010) 4539–4548 ª 2010 The Authors Journal compilation ª 2010 FEBS 4545 genes are silenced during lifetime, embedded in con- densed chromatin. As described previously, BrdU can restore the expression of silenced genes [9,10], and BrdU-responsive genes are frequently located on inac- tive chromatin regions, such as AT-rich Giemsa-dark bands of human chromosomes [11,12]. In this context, BrdU seems to disrupt nucleosome positioning around AT-rich condensed chromatin and results in the induc- tion of the expression of silenced or repressed genes. Finally, the data of this study may lead to a new understanding of the molecular mechanism of BrdU. They may answer the new and old question of why BrdU modulates the expression of particular genes associated with cellular differentiation and senescence. Materials and methods Plasmids and yeast strains To construct BAR1 expression plasmid pRS-BAR1, the )500 to +2064 sequence containing the promoter with a KpnI site ()158 to )154) [27], a coding sequence and 300 bp of 3¢-UTR of the BAR1 gene, were cloned into the XhoI-SacI site of pRS424DKpnI in which one KpnI site was filled in. Plasmid pRS-BAR1 derivatives were constructed by inserting oligonucleotides into the KpnI site of pRS- BAR1 to yield pRS001-BAR1 (T 3 CCT 6 CT 5 GCT 5 CT 7 ) and pRS801-BAR1 (A 34 ). To disrupt a TATA box in pRS-BAR1, the sequence of the TATA box at )134 ( TATAAAA) was changed to a sequence (T GGATCC) that contained a BamHI site by amplifying a KpnI-BglII sequence of pRS-BAR1 with the following two primers: 5¢ -TATTGGTACCGTGTGTTTT TTGATAACAGT GGATCCGTG-3¢ and 5¢-GTGGAAGA TCTATGCTCATTATAAGTACTC-3¢. The amplified sequence was digested with KpnI and BglII, and cloned into the KpnI–BglII site of pRS-BAR1 to yield pRS-DTA ⁄ - BAR1, which lacks a functional TATA box. These plasmids were introduced into the yeast dThd- autotrophic strains YKH2 (MAT a ura2-52 trp1 his3 leu2 cdc21::LEU2 pYBT1) or YKH4 (MATa ura2-52 trp1 his3 leu2 cdc21::LEU2 pYBT1) established as described previ- ously [2]. Plasmid pYBT1 contains the herpes simplex virus TK gene driven by the yeast ADH1 promoter. Chromatin preparation and nuclease digestion Yeast cells harboring plasmids were selected in SC medium (2% glucose, 0.67% yeast nitrogen base without amino acids) supplemented with appropriate amino acids (except for tryptophan) and 1 mm dThd. Cells were grown in 30 mL of medium containing 1 mm dThd or BrdU at 30 °C for 15 h to an optimal density of 0.6–1.0 at 600 nm. Chro- matin and naked DNA samples were prepared according to the method of Balasubramanian & Morse [33]. Each sample was digested with MNase (Takara, Kyoto, Japan) at 37 °C for 10 min. The reactions were initiated by the addition of 0.15% Nonidet P-40, and halted by the addition of SDS and proteinase K. Samples were purified with a phe- nol ⁄ chloroform extraction and ethanol precipitation. Indirect end-labeling DNA samples were completely digested with XhoIor EcoRV together with RNase A, run on a 1.4% agarose gel and transferred on to a Nylon membrane (Biodyne B, Pall, Port Washington, NY, USA) followed by cross-linking with UV light (Stratalimker 2400, Stratagene, La Jolla, CA, USA). 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 a probe labeled with [a- 32 P] dCTP using a random-primed DNA labeling kit (Mega-prime, Amersham, Piscataway, NJ, USA). The XhoI–MspI fragment of pRS- BAR1 was used as a probe to detect nucleosomes in sam- ples digested with XhoI. Likewise, a sequence amplified with the primers 5¢- ATCTTATAATTATCGAGATCG- 3¢ and 5¢- AAGTGTTCCACTG TCTAGTTTG- 3¢ from pRS-BAR1 was used as a probe to detect nucleosomes in samples digested with EcoRV. After washing, the membrane was subjected to autora- diography and densitometric analysis using an image analyzer FLA-5000 (FUJIFILM, T okyo, Japan). Gene expression analysis Total RNA samples were prepared, subjected to electropho- resis on a 1% formaldehyde agarose gel and blotted on to a Nylon membrane as described previously [34]. To deter- mine copy numbers of plasmids in yeast cells, DNA sam- ples were prepared as described above. The DNA samples were digested with XhoI to linearize and run on a 1% aga- rose gel and blotted on to a Nylon membrane. The mem- brane was hybridized with appropriate 32 P-labeled BAR1, TK, DED1, MAK16 or ACT1 coding sequences as probes. After washing, the membrane was subjected to autoradiog- raphy followed by imaging analysis as described above. In vivo UV photofootprinting An in vivo UV photofootprinting assay was performed as described previously [35,36]. Yeast cells harboring a plasmid were grown in SC (Trp ) ) medium containing 1 mm dThd or BrdU at 30 °C for 15 h, irradiated with UV at 254 nm using a Stratalinker 2400, and DNA samples purified. As controls, DNA samples were purified from unirradiated cells and irra- diated similarly. Sites and levels of the formation of UV photoproducts were determined by primer extension map- ping, using the IR-Dye-800-labeled MS-9 primer ()387 to )358 of the BAR1 coding strand) and MS-10 primer ()87 to Molecular mechanism of 5-bromodeoxyuridine K. Miki et al. 4546 FEBS Journal 277 (2010) 4539–4548 ª 2010 The Authors Journal compilation ª 2010 FEBS )116 of the BAR1 noncoding strand) with a LI-COR 4000L DNA sequencer (LI-COR, Lincoln, NE, USA) [26,37]. Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. 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Schematic representation of TALS-GFP and its derivative. Fig. S3. Nucleosome positioning on TALS-GFP and pOM801-GFP. Fig. S4. Effects of BrdU on EGFP mRNA levels. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Molecular mechanism of 5-bromodeoxyuridine K. Miki et al. 4548 FEBS Journal 277 (2010) 4539–4548 ª 2010 The Authors Journal compilation ª 2010 FEBS . 5-Bromodeoxyuridine induces transcription of repressed genes with disruption of nucleosome positioning Kensuke Miki 1 , Mitsuhiro. marked disruption of nucleosome positioning on the pro- moter region. Disruption of nucleosome positioning seems to be sufficient for full expression of the