Activator-binding domains of the SWI ⁄ SNF chromatin remodeling complex characterized in vitro are required for its recruitment to promoters in vivo Monica E. Ferreira 1,2 , Philippe Prochasson 3, *, Kurt D. Berndt 1,2 , Jerry L. Workman 3 and Anthony P. H. Wright 1,2 1 School of Life Sciences, So ¨ derto ¨ rns Ho ¨ gskola, Huddinge, Sweden 2 Department of Biosciences and Medical Nutrition, Karolinska Institutet, Huddinge, Sweden 3 Stowers Institute for Medical Research, Kansas City, MO, USA ATP-dependent chromatin remodeling complexes are a group of enzymes that modulate transcriptional activa- tion, as well as other chromosomal processes, by con- trolling the accessibility of specific DNA sequences within chromatin. A large number of remodeling com- plexes have been identified based on similarities between their ATPase subunits. The SWI ⁄ SNF com- plex was the first remodeling complex to be discovered by studies in yeast but it is conserved in eukaryotes and has been intensively studied. The yeast SWI ⁄ SNF complex has an estimated molecular weight of just over 1 MDa and is composed of twelve different subunits, one of which is the single ATPase of the complex, Swi2 ⁄ Snf2 [1–6]. The SWI ⁄ SNF complex interacts nonspecifically with DNA through multiple interaction surfaces, using the energy of ATP-hydroly- sis to remodel chromatin both in cis by sliding histone octamers along the DNA molecule and in trans by nucleosome disassembly, evicting H2A ⁄ H2B dimers or entire histone octamers [7–13]. The inherent ability of SWI ⁄ SNF to influence accessi- bility of important target sequences is manifested by the subset of yeast genes that depend on SWI ⁄ SNF for normal expression during standard growth conditions on rich media [14,15]. Early functional studies indicated that SWI ⁄ SNF facilitates DNA binding of transcrip- Keywords chromatin remodeling; coactivator recruitment; SWI ⁄ SNF complex; transcriptional activation; yeast Correspondence A. P. H. Wright, School of Life Sciences, So ¨ derto ¨ rns Ho ¨ gskola, S-141 89 Huddinge, Sweden Fax: +46 8 6084510 Tel: +46 8 6084708 E-mail: anthony.wright@sh.se *Present address Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, KS, USA (Received 15 December 2008, revised 20 February 2009, accepted 23 February 2009) doi:10.1111/j.1742-4658.2009.06979.x Interaction between acidic activation domains and the activator-binding domains of Swi1 and Snf5 of the yeast SWI ⁄ SNF chromatin remodeling complex has previously been characterized in vitro. Although deletion of both activator-binding domains leads to phenotypes that differ from the wild-type, their relative importance for SWI ⁄ SNF recruitment to target genes has not been investigated. In the present study, we used chromatin immunoprecipitation assays to investigate the individual and collective importance of the activator-binding domains for SWI ⁄ SNF recruitment to genes within the GAL regulon in vivo. We also investigated the conse- quences of defective SWI ⁄ SNF recruitment for target gene activation. We demonstrate that deletion of both activator-binding domains essentially abolishes galactose-induced SWI ⁄ SNF recruitment and causes a reduction in transcriptional activation similar in magnitude to that associated with a complete loss of SWI ⁄ SNF activity. The activator-binding domains in Swi1 and Snf5 make approximately equal contributions to the recruitment of SWI ⁄ SNF to each of the genes studied. The requirement for SWI ⁄ SNF recruitment correlates with GAL genes that are highly and rapidly induced by galactose. Abbreviation FRET, fluorescence resonance energy transfer. FEBS Journal 276 (2009) 2557–2565 ª 2009 The Authors Journal compilation ª 2009 FEBS 2557 tional activators and general transcription factors to target genes [1,16–18]. However, the abundance of the SWI ⁄ SNF complex was found to be low [1], suggesting that remodeling of target promoters in vivo requires recruitment of SWI ⁄ SNF by interaction with specific transcription factors. Consistently, many activator- bound sequences in the yeast genome are located within nucleosome-free regions, such as linker regions, nucleo- some excluding sequences or regions where maintenance of the nucleosome-free state depends at least in part on chromatin binding factors [19–23]. By contrast, TATA boxes and transcription initiation sites are commonly found within positioned nucleosomes. Taken together, these findings suggest that the predominant coactivating role of SWI ⁄ SNF may be at steps downstream of activa- tor binding rather than as a facilitator of activator bind- ing, although these roles are not mutually exclusive, as exemplified by a recent in vivo study on activation of the PHO5 gene by the Pho4 activator [24]. Further support for activator-dependent recruitment of SWI ⁄ SNF comes from a number of studies demonstrating that the activa- tion domain of activators is required for SWI ⁄ SNF recruitment [12,25–29]. Although activator dependence of coactivator recruit- ment has been well established, fewer studies have provided evidence for a direct interaction between acti- vators and coactivators in vivo. Two main approaches have been adopted to identify direct targets. Using fluo- rescence resonance energy transfer (FRET) to measure in vivo interactions between proteins fused to derivatives of the green fluorescent protein, the activation domain of the transcriptional activator Gal4 was reported to interact directly with the Tra1 subunit of the histone acetyl transferase complex SAGA [30]. A photo-cross- linking strategy has independently identified Tra1 as a direct target of Gal4, and several other acidic activators [31]. Thus, the FRET and cross-linking approaches appear to cross-validate each other. A photo-cross- linking approach has identified the Swi1, Snf5 and Swi2 ⁄ Snf2 subunits of the SWI ⁄ SNF complex as direct targets bound by several acidic activators [32]. Subse- quently, two partially redundant regions of Swi1 and Snf5, respectively, were shown to mediate interaction with transcriptional activators in vitro [33]. Recombi- nant activator interaction domains interact with activation domains in vitro using a two-step mechanism, where rapid ionic interaction with an unfolded activa- tion domain is followed by a slow entropy-driven step during which the activation domain folds [34]. A similar mechanism has been reported in another activator target interaction context and it has been suggested that the intrinsic conformational flexibility of the interaction mechanism may facilitate activator interactions with different coactivator targets [35]. Deletion of both acti- vator-binding domains does not disrupt the composi- tion, stability or catalytic properties of the SWI ⁄ SNF complex, but the phenotype of a mutant lacking both domains differs from the wild-type [33]. Thus, it is possible that the phenotypic defects are a result of the inability of this mutant SWI ⁄ SNF complex to interact with- and be recruited by- activator proteins in vivo. This conclusion would support the importance of acti- vator-dependent recruitment of SWI ⁄ SNF in relation to other possible recruitment mechanisms mediated via the nonspecific DNA binding domains in the complex, the acetyl-histone binding bromo-domain of Swi2⁄ Snf2 or protein interactions mediated by accessory subunits that could also potentially contribute to SWI ⁄ SNF recruit- ment in vivo [7,13,36,37]. The present study aimed to determine whether the activator-binding domains of the SWI ⁄ SNF complex are important for its recruitment to target genes in vivo, as well as for their transcriptional activation. We also investigated the relative significance of the two activator-binding domains for recruitment of SWI ⁄ SNF to different target genes. Results A subset of GAL genes require SWI/SNF activity for efficient activation by galactose To determine whether the activator-binding domains of the SWI ⁄ SNF complex are important for its recruit- ment to promoters in vivo and to determine whether the Swi1 and Snf5 domains are differentially important on different promoters, we required a group of genes that are activated by the same activator. We chose to study the GAL regulon, which is known to be regu- lated by the Gal4 activator protein. We first tested a group of known Gal4 target genes [38] to identify a group of genes showing robust induction under our conditions. Table 1 shows that several GAL genes are activated shortly after addition of galactose to cultures grown with raffinose as carbon source. Induction of PGM2, FUR4, MTH1 and PCL10 was not detected under our conditions. It is likely that these genes are induced at a later time after galactose addition. Using the identified set of galactose-induced genes, we next screened for those genes that require the SWI ⁄ SNF complex for induction by galactose. For this purpose, we used strain YPP33, in which the entire ORFs of SWI1 and SNF5 are both deleted, because deletion of these genes has been shown to disrupt the integrity of the SWI ⁄ SNF complex [39,40]. The most appropriate time for revealing SWI ⁄ SNF dependence was found to Mechanism of SWI ⁄ SNF recruitment to promoters M. E. Ferreira et al. 2558 FEBS Journal 276 (2009) 2557–2565 ª 2009 The Authors Journal compilation ª 2009 FEBS be 30 min after galactose addition to the cultures. Under these conditions GAL1, GAL10, GCY1, GAL2 and GAL7 showed a high degree of SWI ⁄ SNF depen- dence for induction by galactose (Fig. 1). GAL80 and GAL3 did not show significant SWI ⁄ SNF dependence, nor did PGM2, which was included as a control to represent those genes that were not induced at this time-point. We conclude that a subset of Gal4-induc- ible genes were induced under our conditions, and that a further subset of these genes were SWI ⁄ SNF depen- dent. The SWI⁄ SNF-dependent set of galactose- induced genes was studied further. Identification of galactose-induced genes that are direct targets of SWI ⁄ SNF Before we could test the significance of the Swi1 and Snf5 activator interaction domains, it was necessary to investigate whether the selected genes were direct tar- gets of the SWI ⁄ SNF complex. We therefore used a chromatin immunoprecipitation assay to determine whether the SWI ⁄ SNF complex is associated with the promoters of the selected genes under identical galac- tose induction conditions. A schematic of the investi- gated GAL promoter regions is shown in Fig. 2A. Figure 2B shows that SWI ⁄ SNF is recruited to the GAL1-10, GAL2 and GAL7 promoters within 30 min of galactose induction. It is noteworthy that the GAL1 and GAL10 genes are divergent genes regulated by a common regulatory region. SWI ⁄ SNF recruitment to GCY1 did not differ significantly from the ILS1 gene that is not regulated by SWI ⁄ SNF. The specificity of the Snf2 antibody used in this assay was demonstrated by the observation that only background levels of precipitation were observed using chromatin extracts lacking the Snf2 protein (Fig. 2C). Based on these observations, we conclude that GAL1, GAL10, GAL2 and GAL7 are direct targets bound by SWI ⁄ SNF under our experimental conditions. The activator-binding domains of Swi1 and Snf5 are required for promoter recruitment of the SWI ⁄ SNF complex We next studied the level of SWI ⁄ SNF recruitment to the GAL1-10, GAL2 and GAL7 regulatory regions in strains lacking either or both the Swi1 (residues 329– 655) and Snf5 (residues 2–327) activator-binding domains, 30 min after galactose induction. Quantifying recruitment in relation to background binding to the promoter of the SWI ⁄ SNF-independent ILS1 gene, we found that SWI ⁄ SNF recruitment to GAL1-10 and GAL2 is reduced by approximately 50% in strains lacking either the Swi1 or the Snf5 activator-binding domains (Fig. 3). In the strain lacking both activator- binding domains, the level of SWI ⁄ SNF recruitment is reduced to background levels. We have consistently observed that the activation domains are required for the recruitment of SWI ⁄ SNF to the GAL7 gene, but technical problems have thus far prevented acquisition of quantitative data. We conclude that the activator- binding domains identified in vitro are essential for SWI ⁄ SNF recruitment in vivo and that Swi1 and Snf5 Table 1. Wild-type induction 30 min post galactose addition: screening by real-time RT-PCR. Gene Fold induction GAL2 522 GAL1 > 1000 GAL7 > 1000 GAL10 197 PGM2 1.2 GAL80 4.6 GAL3 51 GCY1 1.8 FUR4 1.5 a MTH1 0.7 PCL10 2.4 a a Large clonal variation. Fig. 1. Identification of SWI ⁄ SNF dependent GAL genes. Normal- ized expression of galactose-induced genes 30 min after galactose addition in strain YPP33 (grey bars, swi1D, snf5D), in which the entire ORFs of SWI1 and SNF5 are deleted, relative to normalized expression in the wild-type strain (arbitrarily set to 1, black bars, Wt). PGM2 was included as a non-induced control. The expression levels of Gal4 target genes, quantified by real-time PCR after cDNA synthesis, were normalized against expression of a control gene, ILS1, and normalized expression levels were scaled to give a wild- type value of 1 for all tested genes. Error bars indicate the SD of mean values from three independent cultures. M. E. Ferreira et al. Mechanism of SWI ⁄ SNF recruitment to promoters FEBS Journal 276 (2009) 2557–2565 ª 2009 The Authors Journal compilation ª 2009 FEBS 2559 contribute similarly to its recruitment to GAL1-10 and GAL2. The activator-binding domains of Swi1 and Snf5 are required for galactose-induced expression of SWI ⁄ SNF-dependent genes It was next necessary to determine whether the defect in SWI ⁄ SNF recruitment resulting from the deletion of one or both activator-binding domains would lead to reduced galactose-induced expression of target genes. Figure 4 shows that induction of GAL1, GAL10, GAL2 and GAL7 is severely reduced in a strain lacking both the Swi1 and Snf5 activator-binding domains. Recruitment of the SWI ⁄ SNF complex via activator interactions with these activator-binding domains is thus critical for normal galactose induction of the tested genes. Interestingly, the strains lacking either the Fig. 3. The activator-binding domains of the SWI ⁄ SNF complex are required for its recruitment to promoters. Enrichment of the immu- noprecipitated GAL1-10 and GAL2 promoters under inducing condi- tions (galactose, 30 min) in the wild-type strain (black bars, Wt), strain YPP310 (light grey bars, DDABDswi1 + snf5) lacking both activator-binding domains (SWI1D329-655, SNF5D2-327), strain YPP211 (white bars, DABDsnf5) lacking the Snf5 activator-binding domain (SNF5D2-327) and strain YPP247 (dark grey bars, DAB- Dswi1) lacking the Swi1 activator-binding domain (SWI1D329-655). The level of SWI ⁄ SNF enrichment on the promoters is relative to the negative control promoter, ILS1. Error bars indicate the values obtained from two independent experiments. A B C Fig. 2. SWI ⁄ SNF is recruited to the GAL1-10, GAL7 and GAL2 pro- moters within 30 min of galactose induction. (A) Schematic of the investigated GAL promoter regions. Solid black lines indicate the promoter regions that were used as the input sequence for primer design using PRIMER3 software. The positions of primers used for detection in chromatin immunoprecipitation experiments are indicated by arrows. (B) Amount of promoter DNA, detected by real-time PCR, for different genes in wild-type cells grown under non-inducing conditions (grey bars, Raf) and inducing conditions (galactose 30 min, black bars, Gal) precipitated by anti-Snf2 serum, shown as a percentage of input (% IP). ILS1 is included as a SWI ⁄ SNF-independent control. The asterisk indicates the level of background with beads only. Error bars indicate the values obtained from two independent cultures. (C) Enrichment of the GAL1-10 promoter relative to a region of the ILS1 coding sequence under inducing conditions, using different amounts of Snf2 antibody on extracts from the SNF2 deletion strain 11586 (grey bars, snf2D) and the wild-type strain (black bars, Wt). Mechanism of SWI ⁄ SNF recruitment to promoters M. E. Ferreira et al. 2560 FEBS Journal 276 (2009) 2557–2565 ª 2009 The Authors Journal compilation ª 2009 FEBS Swi1 or Snf5 activator-binding domains showed little, if any, reduction in the level of galacose induction of the same genes. Thus, the small reduction in SWI ⁄ SNF recruitment observed in these mutants is not sufficient to cause a reduction in induced gene expression under the conditions studied. Discussion The galactose regulon in Saccharomyces cerevisiae is an appropriate system for studying the components of coactivators that are required for their recruitment. During growth on nonrepressing, non-inducing sugars such as raffinose, the Gal4 activator is bound to its DNA binding sites in target genes but its activity is repressed by its association with the Gal80 repressor protein. Upon addition of galactose to such cultures, the Gal3 protein antagonizes Gal80-mediated repres- sion and Gal4 is immediately able to recruit necessary factors to its target genes [41]. As shown in the present study, the SWI ⁄ SNF complex is rapidly recruited to a subset of Gal4 target genes within 30 min of galactose addition, and defects affecting the SWI ⁄ SNF complex caused reduced activation of these genes within the same time window. The activator-binding domains in the Swi1 and Snf5 subunits of the SWI ⁄ SNF complex are essential for recruitment of the SWI ⁄ SNF complex to these genes, as well for their subsequent activation. Our observation thus strongly supports the model pro- posing that the activator-binding domains, largely defined and characterized in vitro, are necessary for SWI ⁄ SNF recruitment in vivo. This is crucial for understanding the roles played by the different SWI ⁄ SNF subunits and, from the results obtained in the present study, we can now add that other regions of SWI ⁄ SNF cannot replace the recruiting function of the Swi1 and Snf5 activator-binding domains. The in vivo validation of the Swi1 and Snf5 activator-bind- ing domains is also of interest in relation to the two- step binding mechanism between Gal4 and Swi1 that has been characterized in vitro [34], and our results suggest that the coupled binding and folding mecha- nism is likely to be relevant in vivo. This binding mech- anism is assumed to be generally important for transcription factor interactions with other proteins and may be important for the function of the increas- ingly large group of proteins that have been shown to contain intrinsically unstructured regions. The intrinsic conformation flexibility that is inherent in this binding mechanism would help to explain how activator proteins are able to form specific interactions with the large range of structurally distinct factors that they recruit to target genes. The Swi1 and Snf5 activator-binding domains appear to work additively because deletion of one or the other domain individually reduces SWI ⁄ SNF recruitment to approximately 50% of that seen in wild-type cells. The existence of two activator-binding domains might be an adaptation to the fact that acti- vators generally bind DNA as dimers, with the result that two activation domains are available to recruit target factors via two independent interactions. Alter- natively, a larger number of activator-binding domains could contribute to recruitment by increasing the prob- ability of contact between activator and coactivator, leading to a faster on-rate during recruitment. The Fig. 4. The activator-binding domains of the SWI ⁄ SNF complex are required for activation of target promoters. Normalized expression of the GAL1, GAL10, GAL2 and GAL7 genes under inducing conditions (galactose, 30 min), in strain YPP310 (light grey bars, DDABDswi1 + snf5) lacking both activator-binding domains (SWI1D329-655, SNF5D2-327), strain YPP211 (white bars, DAB- Dsnf5) lacking the Snf5 activator-binding domain (SNF5D2-327) and strain YPP247 (dark grey bars, DABDswi1) lacking the Swi1 activa- tor-binding domain (SWI1D329-655), relative to the wild-type tested in parallel (black bars, Wt, level arbitrarily set to 1). GAL transcript levels were quantified by real-time PCR after cDNA synthesis and normalized against transcript levels of a control (ILS1) to obtain nor- malized GAL expression. Normalized GAL expression levels were subsequently scaled to give a wild-type value of 1 for all tested genes. Error bars indicate the SD of means obtained from three independent cultures. M. E. Ferreira et al. Mechanism of SWI ⁄ SNF recruitment to promoters FEBS Journal 276 (2009) 2557–2565 ª 2009 The Authors Journal compilation ª 2009 FEBS 2561 latter alternative is consistent with measurements showing rapid dynamics of activator and coactivator association and disassociation with chromatin in vivo [42,43]. For the genes investigated in the present study, we have not observed differences in the relative impor- tance of the Swi1 and Snf5 activator interaction domains during SWI ⁄ SNF recruitment to different genes. It remains possible that the two interaction domains play differential roles on other SWI ⁄ SNF- dependent genes or under different physiological conditions; for example, during mitosis when a larger number of genes become SWI ⁄ SNF dependent [44]. We have previously shown that such differences can exist as demonstrated by the observation that different subunits of the Tup-Ssn6 corepressor complex are differentially important for the repression of different target genes in fission yeast [45,46]. Swi1 and Snf5 are known to be required for the structural integrity of the SWI ⁄ SNF complex [39,40]. The defect in transcriptional activation of GAL genes in the mutant lacking both the Swi1 and Snf5 activator interaction domains is essentially the same as that observed in the absence of both subunits. Therefore, the two activation domains appear to be essential for SWI ⁄ SNF activity, at least for the genes that we have studied. This is expected if SWI ⁄ SNF recruitment by activator proteins is required for its activity. However, in mutants lacking only one of the activator-binding domains, the transcriptional activation levels were not affected under our conditions, despite the observed 50% reduction in SWI ⁄ SNF recruitment. One possible explanation for the lack of an exact correlation between the amount of recruited SWI ⁄ SNF and tran- scriptional activation could be that the wild-type SWI ⁄ SNF complex is recruited in excess, at least under the conditions investigated. Another possibility is that the reduced level of SWI ⁄ SNF recruitment does not affect transcriptional activation as a result of the com- pensatory action of some other factor. The SAGA complex, another Gal4-associated co-activator [30,47,48], is a potential candidate for such a factor because partial redundancy between SWI ⁄ SNF and Gcn5, the histone acetyl transferase of SAGA, has pre- viously been demonstrated in relation to activation of the SUC2 gene [49]. Among the collection of GAL genes screened in the present study, we identified both SWI ⁄ SNF-dependent genes as well as GAL genes that have little or no requirement for SWI ⁄ SNF. SWI ⁄ SNF dependence was associated with a class of highly inducible GAL genes (GAL1, GAL10, GAL2 and GAL7), whereas genes showing lower inducibility by galactose after 30 min (GAL3, GAL80 and PGM2) were not significantly dependent on SWI ⁄ SNF. Thus, the extent of SWI ⁄ SNF dependence may depend on the extent and ⁄ or rate of induction. GCY1 is an exception because it was strongly dependent on SWI ⁄ SNF, even though it is relatively mildly induced by galactose. The results obtained in the present study suggest that GCY1 is an indirect target of SWI ⁄ SNF because we were unable to detect an association of SWI ⁄ SNF with GCY1. However, other explanations are possible. For example, SWI ⁄ SNF might interact with regions of GCY1 that are not detected by the primers used. Unlike the other SWI ⁄ SNF-dependent genes, basal levels of GCY1 expression were also dependent on SWI ⁄ SNF (data not shown). It is therefore likely that a factor required for GCY1 expression in both unin- duced and induced conditions is SWI ⁄ SNF dependent. Although an association of SWI ⁄ SNF with the GAL1- 10 and GAL7 genes has been reported previously [50], albeit under different conditions than those reported here, SWI ⁄ SNF association with GAL2 is a novel find- ing of the present study. Furthermore, we demonstrate that SWI ⁄ SNF is important for the efficient activation of a number of GAL genes upon galactose induction of cultures grown with raffinose as carbon source, which comprise conditions where SWI ⁄ SNF has previ- ously been reported not to play a role [51]. This discrepancy may be explained by differences in assay conditions because the apparent dependence on SWI ⁄ SNF is lower at later times after galactose induc- tion under our conditions (data not shown). It is thus possible that SWI ⁄ SNF is predominantly required for the transition to induced expression levels rather than their maintenance under prolonged growth on galactose. Experimental procedures Yeast strains The yeast strains used in the present study are listed in Table 2. Genomic partial deletions of the SWI1 and SNF5 ORFs, corresponding to amino acid residues 329–655 of Table 2. Strains used in the present study. Strain Relevant genotype Reference W303 1A Wild-type [55] YPP33 swi1D::HIS, snf5D::HIS [33] YPP247 a SWI1D329-655 Present study YPP211 a SNF5D2-327 Present study YPP310 a SWI1D329-655, SNF5D2-327 Present study 11586 snf2D::KanMX4 [56] a Isogenic to W303 1A. Mechanism of SWI ⁄ SNF recruitment to promoters M. E. Ferreira et al. 2562 FEBS Journal 276 (2009) 2557–2565 ª 2009 The Authors Journal compilation ª 2009 FEBS Swi1 and 2–327 of Snf5, were made using the Cre-loxP system [52] and verified by PCR. Galactose induction experiments Yeast were pre-cultured in yeast-peptone-dextrose medium at 30 °C for approximately 24 h, washed and diluted in sterile-filtered YP 2% raffinose, supplemented with adenine, and grown for at least 16 h until a D 600 of approximately 0.4–0.7 was reached, at which point each culture was split in two and induced for 30 min at 30 °C by the addition of one-tenth of the culture volume of a 20% galactose solu- tion, or mock induced using an equal volume of sterile deionized water. Preparation of RNA Samples for total RNA extraction were harvested at room temperature by centrifugation, immediately frozen in liquid nitrogen and stored at )70 °C until purification. Total RNA was extracted using hot phenol extraction, followed by further purification using an RNA Easy mini kit (Qia- gen, Solna, Sweden). Total RNA was treated with amplifi- cation grade DNase I (Invitrogen, Lidingo ¨ , Sweden) according to manufacturer’s instructions, followed by another round of purification using an RNA Easy mini kit. The quality of RNA was checked by electrophoresis of total RNA, and by control RT-PCR, using 50 ng total RNA per 25 lL of reaction, 200 nm each of ARN1 specific primers and SuperScript III Platinum One-Step RT-PCR System with Platinum Taq (Invitrogen), followed by elec- trophoresis. RNA preparations were verified DNA-free by control reactions without reverse transcriptase. Chromatin immunoprecipitation The chromatin immunoprecipitation protocol was adapted from a previously described method [53], with several modi- fications. Samples were cross-linked for 1 h. Lysis was carried out using a bead-beater and lysis buffer contained 150 mm NaCl. Prior to immunoprecipitation, lysates were pre-cleared by 1 h incubation at 4 °C with Protein A Agarose ⁄ Salmon Sperm DNA (catalogue number 16-157; Millipore, Solna, Sweden), and the total protein concentra- tion of pre-cleared chromatin extracts determined using the Bradford assay. Chromatin equivalent to 25 lg of total protein was used in each IP reaction, incubated overnight at 4 °C with 5 lg of a polyclonal rabbit Snf2 antibody (catalogue number sc-33629; SDS Biosciences, Falkenberg, Sweden), followed by Protein A Agarose ⁄ Salmon Sperm DNA incubation for no more than 1 h. Beads were washed twice for 15 min in lysis buffer containing 150 m m NaCl, followed by a 15-min wash in lysis buffer containing 500 mm NaCl, two 15-min washes in deoxycholate buffer and, finally, a 5-min wash in TE buffer. Chromatin was eluted from washed beads by 1 h of incubation with elution buffer at 65 °C, repeated once, and cross-links were sub- sequently reversed by overnight incubation at 65 °C. After proteinase K treatment, samples were treated with RNase (Roche, Bromma, Sweden) and purified using PCR clean- up columns (Qiagen). The specificity of the antibody was tested with a non-isogenic Dsnf2 strain as a control, using 1, 5 and 10 lg of Snf2 antibody per reaction, and the routinely-used beads only as a control. Real-time PCR and RT-PCR Quantitative PCR was performed in duplicate 25-lL reac- tions using the MyiQ Single-Color Real-Time PCR Detec- tion System (Bio-Rad, Sundbyberg, Sweden), 200 nm of each primer and, unless otherwise stated, iQ SYBR Green Supermix (Bio-Rad). One-step quantitative RT-PCR was performed with SuperScript III Platinum SYBR Green One- Step qRT-PCR kit (Invitrogen) supplemented with 10 n m fluorescein (Invitrogen), using 50 ng of total RNA per reac- tion, and expression of ARN1 was used for normalization. For two-step qRT-PCR, cDNA was prepared using iScript cDNA Synthesis kit (Bio-Rad), containing oligo-dT and random primers. cDNA corresponding to 50 ng of input total RNA was used for each subsequent quantitative PCR reaction with specific primers, and expression of ILS1 was used for normalization. Unless otherwise stated, the ILS1 promoter region was used for normalization in chromatin immunoprecipitation experiments for comparison of wild- type and mutant strains. Primers used for quantification of transcripts and promoter regions were for the most part designed using primer3 software [54]. Primer sequences are available from the authors upon request. Acknowledgements We thank Professor Hans Ronne at Uppsala Univer- sity for sharing the SNF2 deletion strain. This work was supported by a grant to A.W. from the Swedish Research Council, and by a Leukemia and Lymphoma Society Special Fellowship to P.P. 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