Tài liệu Báo cáo khóa học: Trichostatin A reduces hormone-induced transcription of the MMTV promoter and has pleiotropic effects on its chromatin structure pptx

Trichostatin A reduces hormone-induced transcription of the MMTV promoter and has pleiotropic effects on its chromatin structure Carolina A ˚ strand 1, *, Tomas Klenka 1, *, O ¨ rjan Wrange 1 and Sergey Belikov 1,2 1 Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden; 2 D. I. Ivanovsky Institute of Virology, Moscow, Russia The deacetylase inhibitor trichostatin A (TSA) has long been used to study the relationship between gene transcrip- tion and the acetylation status of chromatin. We have used Xenopus laevis oocytes to study the effects of TSA on glucocorticoid receptor (GR)-dependent transcription and we have related these effects to changes in the chromatin structure of a reporter mouse mammary tumor virus (MMTV) promoter. We show that TSA induces a low level of constitutive transcription. This correlates with a change of acetylation pattern and a more open chromatin structure over the MMTV chromatin, and with specific acetylation and remodeling events in the promoter region. Specifically, a repositioning of initially randomly positioned nucleosomes along the distal MMTV long terminal repeat is seen. This nucleosome rearrangement is similar to the translational nucleosome positioning that occurs upon hormone activa- tion. We also note a reduced hormone response in the presence of TSA. TSA effects have for a long time been associated with transcriptional activation and chromatin opening through inhibition of the deacetylation of histones. However, our results and those of others show that TSA- induced changes in expression and chromatin structure can be quite different in different promoter contexts and, thus, the effects of TSA are more complex than previously believed. Keywords: MMTV promoter; chromatin structure; tran- scription; Xenopus oocytes; TSA. The role of the nucleosome as the fundamental unit of DNA packaging has long been accepted, but its purely structural role has been challenged by an increasing body of experi- mental data [1]. Recent evidence suggests that the organ- ization of promoters into nucleosome arrays provides an additional mechanism of gene regulation [2]. In this study, we have used a promoter from the 5¢-long terminal repeat (LTR) region of the mouse mammary tumor virus (MMTV) to correlate chromatin structure and gene activity. The MMTV-LTR contains potential regulatory elements which mediate transcription in the presence of glucocorti- coid ligands and in the presence of androgen, progesterone and their respective nuclear receptors (Fig. 1A) [3]. Six translationally positioned nucleosomes (A–F) cover this region [4], one of which, nucleosome B, covers the DNA segment around position )60 to )240. This segment contains four glucocorticoid response elements (GREs) [4–6]. This whole DNA segment shows increased hyper- sensitivity to DNase I upon binding of glucocorticoid receptor (GR) homodimers [4,7,8]. We have used the Xenopus oocyte system to reconstitute chromatin in vivo using single stranded DNA containing the MMTV promoter as a template. Single-stranded DNA reconstitutes chromatin more effectively than double-stran- ded DNA as the second-strand synthesis is coupled to chromatin assembly, and thus, seems to mimic the replica- tion coupled chromatin assembly occurring during S phase of the cell cycle [9]. While the ordered helical domains in the globular body of the core histones provide a structure for DNA to wrap around [10], the N-terminal histone tails have been shown to protrude through and around the DNA helix in a far less ordered manner [11]. They harbor positively charged lysine residues at conserved positions. These lysine residues have been shown to act as targets for post-translational modifi- cation [12]. Deletion of H3 and H4 N-terminal tails is a lethal event in yeast that significantly alters gene regulation, nucleosome assembly and spacing [13]. It is believed that reversible modifications of charged residues can alter chromatin structure by causing changes in the overall charge of the N-terminal tails, and hence their interactions with the negatively charged sugar–phosphate DNA back- bone, or with negatively charged regions located on adjacent nucleosomes [11]. An alternative view is that the various chemical modifications of specific amino acids in histones act as a code by serving as binding sites for various effector complexes. These complexes can modify the chromatin structure and hence the expression of a gene [14]. The relationship between the histone acetylation status of chromatin and transcription has been studied in many systems using a variety of promoter constructs and native Correspondence to S. Belikov, Department of Cell and Molecular Biology, Medical Nobel Institute, Box 285 Karolinska Institute, SE-171 77 Stockholm, Sweden. Fax: + 46 8 31 35 29, Tel.: + 46 8 52 48 73, E-mail: sergey.belikov@cmb.ki.se Abbreviations: ChIP, chromatin immunoprecipitation; DMS, dimethylsulphate methylation; GR, glucocorticoid receptor; GRE, glucocorticoid response element; HAT, histone acetyltransferase; HDAC, histone deacetylase; LTR, long terminal repeat; MNase, micrococcal nuclease; MPE, methidiumpropyl-EDTA–Fe(II); NaBu, sodium butyrate; TSA, trichostatin A; TA, triamcinolone acetonide. *Note: Both these authors contributed equally to this work. (Received 26 August 2003, revised 26 January 2004, accepted 30 January 2004) Eur. J. Biochem. 271, 1153–1162 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04019.x genes. The studies have revealed that both histone acetyl- transferases (HATs) and histone deacetylases (HDACs) play a vital role in gene regulation by either allowing transcription or establishing correct repression. Blocking of HDACs using inhibitors such as trichoststin A (TSA), sodium butyrate (NaBu) and trapoxin [15], has revealed a complicated picture of the exact role of HDACs in promoter function. TSA has been shown to relieve repres- sion by non ligand-bound TR/RXR protein bound to exogenous TRbA promoter constructs in Xenopus oocytes [16], as well as p53/mSin3A-repressed genes in the mam- malian cell lines [17]. Such effects are due to the inhibition of HDACs, targeted by specific DNA binding factors to transcriptionally silent regions as a part of large corepressor complexes [18]. The endogenous Xenopus H1 gene can be activated in cell lines by TSA, but only after the mid blastula transition, when histones become hyperacetylated in the presence of TSA and NaBu [9,19]. In this study, we analyze TSA-treated chromatin in Xenopus oocytes, and relate its structure to the function of an MMTV-LTR reporter construct. We show that TSA increases the acetylation of bulk histone H3 as well as H3 acetylation over the MMTV–LTR. Furthermore, TSA treatment causes a generally more open chromatin struc- ture, and increases DNA-accessibility to micrococcal nuc- lease (MNase) in the MMTV promoter. It also triggers a nucleosome repositioning in the distal part of the MMTV LTR, similar to the nucleosome rearrangement that occurs during hormone activation [6]. Our results, and the results of others, highlight the pleiotropic effects that TSA administration has on chromatin structure and on gene expression. Materials and methods DNA and plasmids Construction of the MMTV reporter and the plasmid for in vitro transcription of rat GR mRNA has been described previously [6]. Culture and injection of Xenopus oocytes Xenopus laevis oocytes were prepared and injected as described previously [20]. Transcription analysis Quantification of MMTV transcription by S1-nuclease and DNA analysis was performed as described previously [21], with one difference. A synthetic oligonucleotide identical to the lower strand of the )8/+64DNAsegmentofthe MMTV-LTR was labeled using [ 32 P]ATP[cP] (Amersham Biosciences) and T4-polynucleotide kinase, and used as probe. MMTV transcription was also quantified by primer extension using the following procedure. Homogenate equivalent to eight oocytes was first treated with 0.5 mgÆmL )1 proteinase K for 2 h at 37 °Candthen RNA extracted with Trizol (GibcoBRL) and chloroform according to the manufacturer’s instructions, precipitated with 0.7 vol. of isopropanol. One oocyte equivalent was used for primer extension. The primers were 32 P end-labeled oligonucleotides with the following sequences: 5¢-GC GGGAGTTTCACGCCACCAAGATCC-3¢ (MMTV, 10 pmol) and 5¢-GGCTTGGTGATGCCCTGGATGTTAT CC-3¢ (H4, loading control, 20 pmol). Primer extension was performed according to a protocol modified from [22]: dry RNA pellets were resuspended in 4 lLofeachprimer dilution and 2 lL5· First Strand buffer (GibcoBRL), primers were then annealed at 95 °C for 10 min, 55 °Cfor 25 min, 45 °C for 10 min. Extension was performed in 20 lLat45°Cwith1lL Superscript II (–RNaseH) RT (GibcoBRL), in 10 m M dithiothreitol, 0.5 m M dNTP for a further 40 min. Samples were diluted 1 : 1 (v/v) with denaturing loading buffer and run on 6% polyacrylamide sequencing gels: extension products were analyzed and quantified on a Fuji Bio-Imaging analyzer BAS-2500 using IMAGE GAUGE V3.3 software. Chromatin and protein–DNA analysis Micrococcal nuclease (MNase) digestion and in situ cleavage by methidiumpropyl-EDTA–Fe(II) (MPE) was performed as described previously [6] as was the supercoiling assay [23] that used a chloroquine concentration of 60 lgÆmL )1 . Radioactivity scans and quantifications were performed using a Fuji Bio-Imaging analyzer BAS-2500 with IMAGE GAUGE V3.3 software. Analysis of proteins extracted from Xenopus oocytes Nuclear proteins were isolated by physical separation of the germinal vesicle from the cytoplasm of injected oocytes using fine forceps in a pool of isolation buffer (20 m M Tris/ HCl, pH 7.5, 0.5 m M MgSO 4 ,140m M KCl). Both fractions were homogenized in the same buffer with 1% SDS, boiled for 5 min, and samples run on 10% or 15% SDS/PAGE gels (for GR and histone analysis, respectively) in Tris/ glycine buffer with 0.1% SDS [24]. Proteins were transferred onto poly(vinylidene difluoride) (PVDF) membranes (Mil- lipore) in Tris/glycine buffer containing 20% (v/v) methanol and 0.037% (w/v) SDS [24] at 20 V for 1 h. Filters were probed with antibodies to acetylated histones H3 and H4 (Upstate Biotechnology) and acetylated H3 (Upstate Bio- technology), and antibodies against specific modifications such as acetylated H3-K9 (Cell Signaling Technology) and H3-K14 (Abcam) and anti-H3 C-terminal (Abcam). Ana- lysis of GR was performed at 1 : 1000 dilution of primary antibody in Tris-buffered saline with 0.05% (v/v) Tween 20 (TTBS) and 5% (w/v) dried milk powder. Secondary antibody HRP conjugates were used at 1 : 1000 dilution in TTBS. Protein bands were visualized by chemiluminescence (GibcoBRL). Quantification was via IMAGE GAUGE V3.3 software. For an internal standard and loading control, the oocytes were incubated in oocyte medium also containing [ 35 S]methionine (Amersham Biosciences) for 5 h. After Western blotting, the filters were analyzed for radioactivity using a Fuji Bio-Imaging analyzer BAS-2500 as above. Chromatin immunoprecipitation Pools of oocytes were injected with 4.5 ng sspMMTV [6] and treated with or without TSA prior to fixation with 1% (v/v) 1154 C. A ˚ strand et al. (Eur. J. Biochem. 271) Ó FEBS 2004 formaldehyde for 10 min at ambient temperature. Chro- matin immunoprecipitation (ChIP) was performed according to a protocol described previously with some modifications [25]. Cells were washed and 12 nuclei per pool were dissected and collected in sonication buffer (20 m M Tris/HCl, pH 7.2, 60 m M KCl, 15 m M NaCl, 1 m M EDTA, 1 m M dithiothre- itol and 1· protease inhibitor cocktail (Sigma), 50 lLper nucleus). After sonication on ice (4· 20 s), samples were diluted with 1 vol. of buffer I (0.1% sodium deoxycholate, 1% Triton X-100, 2 m M EDTA, 50 m M Hepes pH 7.2, 150 m M NaCl, 1 m M dithithreitol and 1· protease inhibitor cocktail (Sigma) and centrifuged at 13 000 g,4°C, for 10 min to remove insoluble debris. Supernatant, equival- ent to one nucleus, was used for immunoprecipitation. Acetylated histones were immunoprecipitated for 4 h with anti-AcH3 (Upstate Biotechnology), H3 C-terminal and AcH3-K14 (Abcam) on protein A sepharose beads (Amer- sham) precoated with calf thymus DNA in 5% (w/v) dry milk. Complexes were washed (15 min wash) with buffers: buffer I described above; buffer II [0.1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, 2 m M EDTA, 50 m M Hepes, pH 7.2, 500 m M NaCl, 1 m M dithiothreitol and 1· protease inhibitor cocktail]; buffer III [0.25 M LiCl, 0.5% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 1 m M EDTA, 10 m M Tris/HCl, pH 8.0, 1 m M dithiothreitol and 1· protease inhibitor cocktail] and TE, pH 8.0 (1 m M dithiothreitol, 1· protease inhibitor cocktail). Bound mater- ial was eluted in elution buffer [0.5% (w/v) SDS, 0.1 M NaHCO 3 ,0.5lgÆlL )1 proteinase K]. Crosslinking was reversed at 65 °C overnight and DNA was purified by extraction with phenol/chloroform and isopropanol preci- pitation. PCR was performed in 21 cycles with primers covering the nucleosome B ()291/+42), the nucleo- some F ()1044/)732) and the M13 vector (2699/2990), and products were analyzed on a 6% (w/v) polyacrylamide sequencing gel. Radioactivity scans and quantifications were performed using a Fuji Bio-Imaging analyzer BAS-2500 using IMAGE GAUGE V3.3 software. Results Trichostatin A has pleiotropic effects on MMTV transcription Pools of oocytes were injected with 5 ng GR mRNA into the cytoplasm, followed by intranuclear injection of 1 ng of sspMMTV:M13 DNA. TSA (16 n M ) was added to some of the pools immediately after DNA injection to assemble chromatin in the presence of TSA, this is referred to as early TSA (E). Hormone induction of half of these pools was performed 18 h later by addition of the synthetic glucocor- ticoid hormone, triamcinolone acetonide (TA) at a concen- tration of 10 )6 M . TSA was added, at the same time, to a pool of injected oocytes, these are referred to as late TSA (L). Transcription was allowed to continue for 6 h. Oocytes were harvested and total RNA was extracted from all pools (Fig. 1B). S1 nuclease or primer extension analysis of the injected MMTV reporter transcripts was performed; the latter using a primer for endogenous histone H4 RNA as an internal control, followed by quantification on a phosphor- imager (eight experiments). Both assays generated identical results. Hormone induction of the MMTV promoter caused a dramatic increase in transcription over the basal level. This basal level was  0.5% of full induction (Fig. 1C, compare lanes 1, 2 and 3, 4). However, TSA alone tended to induce transcription at a weak level, also in the absence of hormone (Fig. 1C, compare lanes 1, 2 and 5, 6 and 9, 10). A further effect of TSA treatment was a significant reduction in hormone-induced transcription ( 50% of full induction when added early, compare lanes 3, 4 and 11, 12). Neither of these effects depended on the TSA concentration over the range used in these experiments, i.e. 16 n M and 64 n M (data not shown). This agrees with similar studies that used TSA to affect gene transcription [9,16,26]. A TSA concentration of 16 n M was used for subsequent experiments as this was enough to elicit a reproducible response. The TSA-induced, hormone-independent, or leaky, transcription was clearly seen only in the case of early TSA-treated oocyte pools (Fig. 1D), [1.74 ± 0.4% (E) vs. 0.60 ± 0.5% (L); n ¼ 8]. Similarly, the TSA-mediated effect of reducing the response to hormone was less evident when added late [52.5 ± 17.1% (E) of full hormone induction compared to 76.2 ± 21.6% (L) (n ¼ 8)]. We conclude that TSA causes a weak hormone-independent transcription of the MMTV promoter and partly inhibits hormone-inducible transcription. TSA affects acetylation levels of endogenous histone pools as well as histones in MMTV containing minichromosomes Treatment of oocytes with deacetylase inhibitors such as TSA may change the bulk acetylation pattern of histones, and in this way alter the structure of chromatin incorpor- ating them. To see whether the increased transcription leakage observed at early addition of TSA can be explained by an accumulation of acetylated forms of histones in a time-dependent manner, we looked at the pattern of TSA-induced histone acetylation. Using antibodies specific to acetylated forms of H3 and H4, we endeavored to look at the level of histone hyper- acetylation 12 h (E) and 18 h (L) before the harvest of noninjected oocytes. Nuclei isolated from nontreated oocytes showed significant levels of nuclear AcH4, which did not increase upon late addition nor on early addition of TSA (Fig. 2A). A striking response to TSA was seen in the levels of AcH3: almost no AcH3 was found in nontreated oocytes while oocytes treated with late TSA showed a significant increase in AcH3. This increase was even more pronounced when TSA was added early (Fig. 2A). We conclude that TSA induces a time-dependent increase in the level of AcH3. We also looked for specific histone modifications (Fig. 2B) and noted an increased acetylation of histone H3 lysine residues 14 and 9 upon early addition of TSA. To monitor the acetylation status of the MMTV promoter subjected to TSA treatment, we used a chromatin immunoprecipitation assay (ChIP) and evaluated the acetylation status of the so-called B- and nucleosome F [4] and compared these patterns with the M13 vector (Fig. 2C). As a control for the potential loss of histone–DNA contacts during treatment, an antibody against the carboxyterminal segment of histone H3, which is not subjected to any known modifications, was also included [27]. The ChIP analysis Ó FEBS 2004 TSA effects on transcription and chromatin structure (Eur. J. Biochem. 271) 1155 demonstrated TSA-dependent five- to 10-fold increase in histone H3 acetylation which involved both the MMTV promoter, the nucleosome B, the distal MMTV LTR, here presented by the nucleosome F, and the M13 vector DNA (Fig. 2C). We conclude that early TSA addition increases the acetylation status of bulk histones as well as the histones organizing the minichromosomes. Structural alterations in nucleosomal organization caused by TSA treatment Changes in the acetylation status of histones by TSA treatment may cause changes in the organization of chromatin. Such altered chromatin may no longer be able to repress transcription from inducible promoters and it may have less capacity to organize effective transcription in the induced state. We used several methods to look at chromatin structure and chromatin remodeling within the MMTV. Chromatin remodeling can be followed by in situ chromatin digestion with appropriate restriction enzymes [8,28]. A restriction enzyme accessibility assay utilizing a SacIorHinfI restriction site revealed a hormone-dependent remodeling of the chromatin in this region [6]. However, similar experiments failed to show any significant effect of TSA on SacIorHinfI accessibility (data not shown). We therefore used other approaches that were more sensitive to the small changes in the chromatin structure over a wide area of the nucleosome B: MNase digestion assay, topology assay and MPE chemical cleavage together with indirect end-labeling. Previous MNase experiments revealed changes in the canonical nucleosomal ladder over the nucleosome B region in response to hormone-dependent GR binding [6,20]. As seen in Fig. 3, increasing amounts of MNase reduced the nucleosome B region of the hormone-activated promoter to predominantly mono- and subnucleosomal fragments. Compare lanes 4–6 (nucleosome B probe) with lanes 1–3 (nucleosome B probe) and also Fig. 3B (left panel). This hormone-dependent appearance of a subnucleosome is specific for the nucleosome B area as it is not seen while reprobing the filter with the vector probe. Compare lanes 4–6 (nucleosome B probe) with lanes 4–6 (M13 vector probe). The nucleosome B is further affected by the early addition of TSA. In this case, the nucleosome B area is distinctly hypersensitive to MNase action, especially at high concentrations. Late addition of TSA, on the other hand, had virtually no effect (compare lanes 4–6 with 10–12 and 16–18, nucleosome B probe). Thus, the observed reduction in hormone response of the system in the presence of TSA correlates with detectable changes in the local chromatin architecture of the MMTV promoter. The TSA-induced leaky transcription also seems to correlate with a loss in chromatin structure regularity of the bulk chromatin; at lower MNase concentrations this is seen as increased Fig. 1. TSA decreases hormone-induced transcription of the MMTV promoter, and increases basal transcription in the absence of hormone. (A) The reporter DNA construct, the pMMTV:M13 used for injection with the primer used for primer extension analysis of DMS methyla- tion protection (solid black arrow), and the restriction enzyme cleavage sites that are referred to in the text. White boxes designate GRE hexanucleotide elements numbers I to IV, the black box shows the NF1 site, dark gray boxes show the Oct 1 sites, and light gray box shows the TATA sequence. The nucleosome B probe used in the MNase experiments is shown below. (B) Time-course of the oocyte injection experiment. Collagenased oocytes were allowed to recover for 18 h prior to injection of GR mRNA and DNA, TSA addition [ÔearlyÕ (E) or ÔlateÕ (L)] and hormone induction. RNA and DNA were extracted from pools of eight oocytes each. (C) Representative dena- turing acrylamide gel showing analysis in duplicate of the MMTV transcription in the presence of hormone and TSA. (D) Phosphor- imager analysis of MMTV transcription assayed by primer extension normalized to H4. The lower panel shows a smaller scale graph highlighting the increase in basal transcription. Error bars signify SD (n ¼ 8). 1156 C. A ˚ strand et al. (Eur. J. Biochem. 271) Ó FEBS 2004 smearing of the nucleosomal pattern both in the promoter and vector sequences (Fig. 3A, both panels, compare lanes 1, 7 and 13 and Fig. 3B, right panel). Topological changes in chromatin induced by TSA treatment and GR binding We have demonstrated previously that hormone-dependent activation of the MMTV promoter is associated with alterations in the chromatin structure that can be detected in a DNA topology assay as the loss of negative superhelical turns [20]. These alterations in DNA topology take place even if histones are not physically disrupted from the chromatin template [29]. Oocytes were injected with sspBSLSwt, a construct containing the same MMTV-TK fusion used in other experiments, but cloned into a Bluescript vector. The size of the injected DNA was smaller in these experiments, and the resolution of the topoisomers was thus improved. Following treatment, oocyte pools were extracted and the DNA resolved on an agarose gel containing 60 lgÆmL )1 chloro- quine to visualize any changes in superhelical density arising from TSA/hormone treatment. Treatment with TSA decreased the negative superhelicity by 1.5 superhelical turns, equal to 1.5 nucleosomes (Fig. 4 lanes/scans 1, 3 and 5). This indicates a more open conformation of the chromatin, which correlates with a loss of chromatin regularity and is consistent with increased smearing observed in the MNase-digested DNA. Interestingly, this phenomenon was as evident in the presence of ÔearlyÕ TSA as it was with ÔlateÕ TSA, suggesting that changes in the topology may occur quickly. The overall change in the topology caused by GR binding and the MMTV induction results in an overall loss of about 7 negative supercoils, an effect which was decreased by TSA treatment by two and one superhelical turns for ÔearlyÕ and ÔlateÕ TSA, respectively (Fig. 4 lanes/scans 2, 4 and 6). This indicates that in contrast to the uninduced promoter, TSA treatment during hormone activation leads to a less open chromatin structure. This observation agrees with the reduced hormone-dependent transcription from the MMTV promoter in the presence of TSA (Fig. 1D). TSA treatment causes nucleosome repositioning within the MMTV LTR For mapping of the translational nucleosome positioning along the MMTV LTR, we have used the chemical nuclease MPE, which has a strong preference for internucleosomal regions and shows significantly less sequence bias in cleaving DNA than MNase [6,30]. The MPE cleavage data suppor- ted the previous finding [6] that hormone induction causes a dramatic remodeling event within the MMTV LTR, resulting in hypercutting over the nucleosome B area, protection of the nucleosome C area and repositioning of initially randomly positioned nucleosomes (Fig. 5A and B, compare lanes 1, 2, and 3, 4 and corresponding scans). Quite unexpectedly, we observed a hormone-independent remodeling event within the MMTV-LTR after the addition of TSA. On early addition of TSA alone, the pattern of remodeling was seen over the region covered by nucleosomes C–F, which resembles the pattern obtained by hormone treatment in the absence of TSA (Fig. 5A and B, compare lanes 5, 6 to lanes 1, 2 and lanes 3, 4 and corresponding scans). This effect was detectable but less evident when TSA was added after chromatin assembly, i.e. late TSA treatment (compare lanes 5, 6 and 9, 10). No significant effects of TSA treatment were detected on nucleosome B. On the other hand, simultaneous addition of TA and TSA resulted in a digestion pattern indistin- guishable from that observed after treatment with TA alone Fig. 2. TSA treatment causes acetylation of bulk histones and acetyla- tion of histones in MMTV-containing minichromosomes. Pools of dis- sected nuclei from noninjected oocytes were analyzed by SDS/PAGE. (A) Western blot probed with anti-acetylated H3 (upper panel) and anti-acetylated H4 (lower panel). In vivo [ 35 S]methionine-labeled pro- teins in the nuclear extract were detected on the filter after blotting and were used as an internal standard. This showed that equal amounts of protein were loaded in each lane (not shown). (B) Western blot probed with antibodies against AcH3-K14, AcH3-K9 and H3 C-terminal. TSA was either not added (-), added early (E) or added late (L), according to the schedule in Fig. 1B. (C) Effects of TSA treatment on H3 acetylation at different regions of the MMTV promoter and vector sequences. The DNA-injected oocytes were treated (early) or not treated with TSA. The ChIP assay was performed as described in Materials and methods. Radioactively labeled PCR fragments from nucleosome B, nucleosome F and vector are shown to the left, and corresponding bars to the right. Bars represent the intensity of the bands, normalization was performed according to actual histone H3-DNA binding (H3 C-term) in TSA-treated and nontreated oocytes. Dark bars show TSA-treated cells. Ó FEBS 2004 TSA effects on transcription and chromatin structure (Eur. J. Biochem. 271) 1157 (Fig. 5A and B, compare lane 4 and 8, lower scan). We conclude that TSA can cause specific nucleosome rear- rangements in the distal MMTV-LTR similar to the hormone-induced rearrangements in this DNA segment. TSA treatment does not affect GR binding to the chromatin template Graphical calculation of the total GR expressed in an oocyte following injection of 5 ng GR mRNA, and com- parison to a standard dilution curve (Fig. 6A) allowed us to estimate an average of 67 ng of GR protein is present in each oocyte under the injection conditions used. This is equivalent to 0.76 pmol per oocyte (relative molecular mass of GR ¼ 87 500). We also analyzed the nuclear localization of GR in nuclei microdissected from TSA treated/untreated oocytes and found no difference in localization patterns between the oocyte pools (data not shown). To find out whether the reduced hormone response of the MMTV promoter after addition of TSA could be a result of compromised binding of GR to GREs in a hyperacetylated chromatin context, we analyzed GR–DNA interactions by dimethylsulphate (DMS) methylation [8], and the cleavage of DNA by alkali [31]. The method allows easy detection of DNA–protein interactions via the N7 position of guanines in the major groove and via the N3 position of adenines in the minor groove. The DMS cleavage pattern was devel- oped by primer extension (Fig. 6B). The pattern of the nonhormone-induced MMTV promoter is virtually identi- cal to that obtained for naked DNA (data not shown). Hence, there is no protein binding detected by the DMS methylation assay in the MMTV promoter in the noninduced state. Addition of hormone resulted in a drastic reduction in DMS methylation (protection) over the glucocorticoid response elements. In agreement with our previous results [20], we observed  40% DMS methylation of the corresponding guanines over the GREs 1–4 (Fig. 6B, compare lanes 1 and 2 and corresponding radioactivity scans). Addition of TSA to the oocyte media had virtually no effect on the DMS digestion pattern (Fig. 6B, compare lanes 1, 2 and 3, 4). This shows that TSA has no effect on GR-DNA binding. Discussion We have shown that TSA treatment of oocytes alters the MMTV transcription profile and causes changes in the bulk chromatin structure, as well as specific changes in the promoter region. To the best of our knowledge this is the first report to demonstrate a specific translational reposi- tioning of nucleosomes induced by TSA or any other HDAC inhibitor. The fact that we see effects more clearly when TSA is present during chromatin assembly (early addition) suggests that the deacetylation step of chromatin maturation is being blocked. Pools of newly synthesized histone H4 diacetylated at the evolutionarily conserved K5 and K12 residues are known to exist in a variety of organisms [32], and diAcH4 is also the main form of stored H4 in Xenopus oocytes [19]. Newly synthesized H3 has also been found in a diacetylated form in Drosophila and Tetrahymena, although this appears to be more transient, and the pattern of lysine residues acetylated in this manner is far less well conserved between species [32]. We were able to detect only tiny amounts of Fig. 3. Nucleosomal organization of the MMTV promoter. (A) Autoradiogram of a Southern blot of chromatin digested with increasing amounts of MNase, probed with vector M13 DNA (left) and reprobed with nucleosome B probe (right). Positions of bands corresponding to tri-, di-, mono- and subnucleosomal bands are indicated. (B) Phosphorimager profiles of individual lanes from (A), indicating changes in MNase digestion on treatment of oocytes with TA or TSA. Positions of tri-, di-, and the mono- nucleosomal bands are indicated, as is the hormone-induced subnucleosomal fragment. 1158 C. A ˚ strand et al. (Eur. J. Biochem. 271) Ó FEBS 2004 AcH3 in resting oocytes in our experiments, and the other researchers have not detected AcH3 in HeLa cells [32]. 2D PAGE analysis of Xenopus oocytestreatedwithNaBuor TSA have revealed little change in the overall H4 acetyla- tion, and inconclusive changes to AcH3 until the mid-blastula transition [19]. In agreement with this, our experiments did not show any change in the level of AcH4 following treatment with TSA, but we did see a distinct and time-dependent increase in the hyperacetylation of histone H3 as well as an increased acetylation at specific sites, i.e. lysines 14 and 9 (Fig. 2B). There are a number of reasons to suspect, apriori,that changes in the acetylation status of histones result in alterations in chromatin structure and DNA–protein inter- actions. We have used several methods to address this issue. We have not found any differences in GR binding to GREs with or without TSA (Fig. 6). At the same time, histone acetylation facilitates the binding of TFIIIA, GAL4 and USF to nucleosomal DNA in vitro [33–35]. The restriction enzyme accessibility assay, which is a sensitive method that is capable of detecting even subtle changes in chromatin structure, also failed to reveal any difference between chromatin treated with TSA and untreated chromatin (data not shown). Fig. 4. Changes in DNA topology upon hormone induction of the MMTV promoter and addition of TSA. Southern blot of pBSLSwt minichromosomes extracted from GR-injected oocyte pools treated with TSA and/or hormone, followed by separation on an agarose gel containing 60 lgÆmL )1 chloroquine to reveal the superhelical density; probed with radiolabeled DNA fragment )103/+431. One oocyte equivalent per lane. Phosphorimager profiles of scanned lanes showing the distributions of superhelical species are shown below. The circle indicates the most frequent topoisomer(s). Fig. 5. Nucleosome positioning analysis in situ by MPE digestion. (A)SouthernblotofMPE-cleavedDNAfromtreatedoocytepools, probed with radiolabeled 513 bp probe (EcoRV-SacI fragment, +425/ )108). The diagram to the left shows the positions of nucleosomes on the MMTV promoter [6], the transcriptional start site and the cleavage sites for SacI(S),HinfI (H) and BamHI (B). (B) Radioactivity scans from selected lanes. Ó FEBS 2004 TSA effects on transcription and chromatin structure (Eur. J. Biochem. 271) 1159 The structural changes over the whole minichromosome upon TSA treatment, as indicated by the increased acces- sibility of MNase, have been reported previously [16]. Our MNase digestion results, when coupled with the topology changes resulting from the loss of 1.5 superhelical turns of the DNA over the whole construct, suggest that the chromatin is indeed more relaxed in the absence of hormone. Interestingly, this phenomenon is as evident in the presence of ÔearlyÕ TSA as it is with ÔlateÕ, suggesting that changes in the topology may occur quickly, and that these changes may be a sensitive readout of histone hyperacety- lation. The MMTV-LTR specific structural changes in chromatin, detected by MPE, require a longer exposure to TSA to develop and they require exposure to TSA during second-strand synthesis and chromatin assembly. On the other hand, hormone activation results in an overall loss of about seven negative supercoils. This effect was decreased by TSA treatment by one or two superhelical turns following TSA treatment, which indicates that addition of TSA leads to formation of less open structure. This is in striking correlation with a reduced hormone response in TSA-treated oocytes. Our results are in good agreement with those previously published from studies in vitro [29] and in vivo [36] on the effects of TSA on DNA topology. However, changes in the DNA topology of minichromo- somes assembled with acetylated/nonacetylated histones were not significant in other studies [16,37]. Experimental data are consistent with an idea that in a hyperacetylating environment, the net charge over positively charged lysine residues on histone tails will be neutralized, thus altering histone–DNA [38] and, possibly, histone–histone [11] inter- actions. This alteration would eventually lead to a decrease in chromatin compaction [39]. Previously, we have shown that hormone induction in Xenopus oocytes results in the establishment of a specific nucleosome positioning pattern over initially randomly organized nucleosomes in the MMTV promoter [6]. After treatment of injected oocytes with TSA alone, an altered pattern of MPE cleavage over nucleosomes C and D is seen (Fig. 5), reflecting a partial, hormone-independent chroma- tin remodeling event. This mimics the situation occurring during hormone induction, where GR bound to the nucleosome B region of the MMTV promoter renders an array of six positioned nucleosomes (A–F). A TSA-induced chromatin remodeling event occurs in the HIV promoter. This promoter harbors a nucleosome positioned at the initiation site that is disrupted upon TNF-a-induced expression. Treatment with TSA results in a similar chromatin remodeling of the HIV promoter and in consti- tutive transcription [40]. However, we are not aware of any previous report of a specific translational positioning being induced by TSA. Our observations suggest that nucleosome repositioning during hormone induction [6] is not solely explained by GR binding to the GRE sequences within the nucleosome B region, but also depends on the subsequent histone acetylation in the surrounding area. Indeed, it was shown that histone H4 acetylation at Lys8 is responsible directly for the recruitment of the SWI/SNF complex to IFN-b gene during activation [41]. Another possibility is that the TSA-induced remodeling of the MMTV promoter that we have observed is triggered by acetylation of a transactive factor(s), which might lead to specific binding to the distal part of the MMTV-LTR and thereby direct the translational nucleosome positioning. The acetylation status of histones H3 and H4 associated with different parts of the MMTV promoter has been studied recently using ChIP [42–44]. Rather unexpectedly, it was shown that upon activation, promoter-proximal histones (the nucleosome B area) become deacetylated whereas the acetylation of both H3 and H4 of nuclesome F was increased [42]. Addition of TSA resulted in only an insignificant increase of the acetylation level of histone H4 in the nucleosome B region [43]. These conclusions were made assuming that the overall amount of histone–DNA cross- links induced by formaldehyde in the nucleosome B and F areas are the same. However, this might not be the case, given the strong remodeling of nucleosome B that occurs during transcription activation [6,7]. This remodeling might result in the partial loss of histone–DNA contacts in the nucleosome B area. Thus, the decrease of the acetylated Fig. 6. DMS methylation protection over the nucleosome B segment. (A)SDS/PAGEandWesternblotoftotaloocyteproteinextracts following injection of 5 ng GR mRNA, probed with GR polyclonal antibodies. 0.5 and 0.25 oocyte equivalent was compared to a standard curve of GR protein of known concentration purified from rat liver [56]. (B) DMS methylation protection over the nucleosome B segment in the presence/absence of TA and TSA. Oocytes in groups of five were treated with DMS, see Materials and methods. The methylation pat- tern was developed by primer (+42/+15) extension. Corresponding guanidine residues that are protected after hormone induction are indicated with arrows. Radioactivity scans of corresponding lanes are shown to the right. 1160 C. A ˚ strand et al. (Eur. J. Biochem. 271) Ó FEBS 2004 signal in ChIP experiments might indicate a loss of histone in the respective area [27]. Our results and the results of others [25] show an increase in acetylation status of histone H3 over the MMTV-LTR upon TSA treatment. This hyperacetylation was also seen in the vector sequences. Interestingly, our results show that a small but clearly detectable increase in the level of basal transcription occurs upon the TSA treatment. This shows that TSA can only insignificantly overcome the repressive nature of the chro- matin. We also discovered a reduction of  50% in the hormone response of the system in the presence of TSA, whereas previous studies on MMTV have reported aug- mentation of hormone induction by TSA [26,45,46]. However, addition of HDAC inhibitors resulted in the down-regulation of the MMTV transcription to various extents in several studies [43,47,48]. The inhibitory effect in our experiments was more evident in the case of early TSA treatment, suggesting that HDAC activity during chromatin maturation may not only help to establish sufficiently repressive chromatin, but may also be necessary for the formation of transcriptionally competent chromatin [49]. Deacetylase activity has for a long time been associated with transcriptional repression through the deacetylation of histones [50]. However, several studies have shown that HDACs are required for both transcriptional activation and repression [51]. One recent example of the down-regulation by HDAC inhibitors is that of the STAT 5 target genes [52], where transcription involves recruitment of HDAC1 [53]. Chromatin remodeling of these genes is not affected by TSA, but recruitment of the components of the basal transcription machinery is blocked [52]. Interestingly, GR is able to recruit HDAC activity and thereby deacetylate histone H4, and in this way also repress the expression of IL-1b -stimulated granulocyte-macrophage colony-stimula- ting factor [54]. One may speculate that a specific pattern of modified histone tails is required to recruit the basal transcription machinery, and that TSA can distort this pattern and thus reduce the hormone-induced transcrip- tional response [41,52]. To understand these events, it will be essential to map the detailed pattern of histone modifica- tions that occurs in the MMTV promoter during transcrip- tion activation, as has recently been done in the PHO5 promoter [27]. This work is in progress in our laboratory. HDAC inhibitors are exciting and promising anticancer drugs [55], not only for their ability to inhibit histone deacetylases but also due to their strong potency to induce growth arrest, to promote differentiation and to induce apoptosis. It is believed that they exert their effects via up-regulation of gene expression [55]. 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Materials and methods DNA and plasmids Construction of the