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ARTICLE Co-dependent recruitment of Ino80p and Snf2p is required for yeast CUP1 activation Roshini N. Wimalarathna, Po Yun Pan, and Chang-Hui Shen

Abstract: In yeast, Ace1p-dependent induction of CUP1 is responsible for protecting cells from copper toxicity. Although the mechanism of yeast CUP1 induction has been studied intensively, it is still uncertain which chromatin remodelers are involved in CUP1 transcriptional activation. Here, we show that yeast cells are inviable in the presence of copper when either chromatin remodeler, Ino80p or Snf2p, is not present. This inviability is due to the lack of CUP1 expression in ino80⌬ and snf2⌬ cells. Subsequently, we observe that both Ino80p and Snf2p are present at the promoter and they are responsible for recruiting chromatin remodeling activity to the CUP1 promoter under induced conditions. These results suggest that they directly participate in CUP1 transcriptional activation. Furthermore, the codependent recruitment of both INO80 and SWI/SNF depends on the presence of the transcriptional activator, Ace1p. We also demonstrate that both remodelers are required to recruit RNA polymerase II and targeted histone acetylation, indicating that remodelers are recruited to the CUP1 promoter before RNA polymerase II and histone acetylases. These observations provide evidence for the mechanism of CUP1 induction. As such, we propose a model that describes novel insight into the order of events in CUP1 activation. Key words: CUP1, INO80, SWI/SNF, chromatin remodeling, copper homeostasis. Résumé : Chez la levure, l’induction de CUP1 dépendante de Ace1p permet de protéger les cellules de la toxicité du cuivre. Bien que le mécanisme d’induction de CUP1 chez la levure ait été étudié intensivement, il semble incertain que des protéines de remodelage de la chromatine soient impliquées dans l’activation transcriptionnelle de CUP1. Nous montrons ici que les levures ne sont pas viables en présence de cuivre lorsque les protéines de remodelage de la chromatine Ino80p et Snf2p sont absentes. Cette non viabilité est due a` l’absence d’expression de CUP1 chez les cellules ino80D et snf2D. Nous avons subséquemment observé que Ino80p et Snf2p sont toutes deux présentes dans la région du promoteur et qu’elles sont responsables de mobiliser l’activité de remodelage de la chromatine dans la région du promoteur de CUP1 sous des conditions induites. Ces résultats suggéraient qu’elles participent directement a` l’activation transcriptionnelle de CUP1. De plus, le recrutement co-dépendant de INO80 et SWI/SNF dépend de la présence de l’activateur transcriptionnel Ace1p. Nous avons aussi démontré que les deux protéines de remodelage sont requises au recrutement de l’ARN polymérase II et a` l’acétylation ciblée des histones, indiquant que ces protéines de remodelage sont recrutées sur le promoteur de CUP1 avant l’ARN polymérase II et les acétylases d’histones. Ces observations nous renseignent sur le mécanisme d’induction de CUP1. Ainsi, nous proposons un nouveau modèle qui décrit l’ordre des événements qui mènent a` l’activation de CUP1. [Traduit par la Rédaction] Mots-clés : CUP1, INO80, SWI/SNF, remodelage de la chromatine, homéostasie du cuivre.

Introduction It has long been agreed that chromatin structure can play a decisive role in gene regulation. Furthermore, trans-acting factors including transcriptional activators, coactivators, and transcription machinery, also contribute to gene expression. The role of these trans-acting factors in gene activation has been defined through intense study, but the mechanistic links among these trans-acting factors, as well as their functional implications are not yet fully understood (Hahn and Young 2011; Luger et al. 2012; Rando and Winston 2012). As such, establishing a detailed description of the gene activation process through the study of these trans-acting factors’ functional connections and implications can better illustrate the mechanism of gene expression. In Saccharomyces cerevisiae, Ace1p-dependent induction of the CUP1 gene is responsible for protecting cells from the toxic effects

of copper (Thiele 1988; Welch et al. 1989). In the presence of copper, copper ions bind to the N-terminal domain of Ace1p, which activates CUP1 transcription through its C-terminal acidic activation domain. When enough metallothionein is produced, it binds free copper in the cytoplasm and leads to the departure of Ace1p from the nucleus, which shuts off CUP1 transcription (Thiele and Hamer 1986; Fürst et al. 1988; Buchman et al. 1989; Huibregtse et al. 1989; Zhou et al. 1992). This is part of the copper homeostasis mechanisms in eukaryotic cells. It has been shown that the loss of copper homeostasis in humans often leads to diseases, including Menkes disease and Wilson disease (Askwith and Kaplan 1998; Shim and Harris 2003). As such, the study of copper homeostasis at the molecular levels can shed light on such disorders. Since the regulation of copper homeostatic genes is conserved throughout eukaryotes, S. cerevisiae is an excellent model organism for the

Received 15 October 2013. Revision received 17 November 2013. Accepted 22 November 2013. R.N. Wimalarathna. Department of Biology, College of Staten Island, City University of New York, 2800 Victory Blvd., Staten Island, NY 10314, USA; PhD Program in Biology, The Graduate Center, City University of New York, 365 Fifth Avenue, NY 10016, USA. P.Y. Pan. Department of Biology, College of Staten Island, City University of New York, 2800 Victory Blvd., Staten Island, NY 10314, USA. C.-H. Shen. Department of Biology, College of Staten Island, City University of New York, 2800 Victory Blvd., Staten Island, NY 10314, USA; PhD Program in Biology, The Graduate Center, City University of New York, 365 Fifth Avenue, NY 10016, USA; Institute for Macromolecular Assemblies, City University of New York, 2800 Victory Blvd, Staten Island, NY 10314, USA. Corresponding author: Chang-Hui Shen (e-mail: [email protected]). Biochem. Cell Biol. 92: 69–75 (2014) dx.doi.org/10.1139/bcb-2013-0097

Published at www.nrcresearchpress.com/bcb on 27 November 2013.

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study of copper homeostasis to gain insight into mammalian copper metabolism. Previously, we have shown that the induction of CUP1 with copper results in chromatin remodeling, and that this remodeling activity is recruited by the transcriptional activator, Ace1p (Shen et al. 2001; Wimalarathna et al. 2012). Induction of CUP1 with copper also results in targeted acetylation of both H3 and H4 at the CUP1 promoter. This targeted acetylation requires the presence of a putative acetylase, Spt10p (Shen et al. 2002). Targeted acetylation of H3 and H4 either occurs when the TATA-binding protein binds to the TATA box or at a later stage in initiation. As such, the CUP1 activation process is clear to some extent. However, it is still not known which chromatin remodelers are involved in CUP1 induction and when these remodelers are recruited to the promoter during activation. Here, biochemical analyses were used to identify which chromatin remodelers are involved in CUP1 activation. We demonstrated that yeast cells are sensitive to the presence of copper in the absence of Ino80p and Snf2p. Subsequently, chromatin immunoprecipitation (ChIP) coupled with real-time PCR (qPCR) analysis was employed to show that both Ino80p and Snf2p are directly involved in regulating CUP1 induction, and that they are recruited to the CUP1 promoter in an Ace1p-dependent manner. Both remodelers are required in the recruitment of chromatin remodeling activity, histone acetylation, and RNA polymerase II (Pol II). These observations provide direct evidence for the role of Ino80p and Snf2p in CUP1 induction.

Materials and methods Yeast strains and growth conditions WT (MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0), ino80⌬ (MATa ino80⌬:: trp1 his3⌬200 leu2⌬0 met15⌬0 trp1⌬63 ura3⌬0), isw1⌬ (MATa ade2-1 can1-100 HIS3 leu2-3112 trp1-1 ura3-1 RAD5+ SWI2-3FLAG-KanMX isw1⌬::ADE2), rsc3⌬ (MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0 rsc3⌬:: URA3), snf2⌬ (MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0 snf2⌬), WT (BJ) (MATa, ura3-52, trp1, lys2-801, leu2⌬1, his3⌬200, pep4::HIS3, prb1⌬1.6R, can1 GAL cir+), and ace1⌬ (BJ) (MATa, ura3-52, trp1, lys2-801, leu2⌬1, his3⌬200, pep4::HIS3, prb1⌬1.6R, can1 GAL cir+ ace1⌬URA3) were used in this study. Yeast cells were grown at 30 °C in synthetic complete media (SC) media containing 2% glucose (w/v) without copper, except ace1⌬ (BJ) and rsc3⌬ which were grown in SC-ura (SC medium lacking uracil and copper), ino80⌬ which was grown in SCtrp (SC medium lacking tryptophan and copper), and isw1⌬ which was grown in SC-ade (SC medium lacking adenine and copper). When the optical density (OD) reached ⬃0.9–1.1, copper was added to a final concentration of 1.5 mmol/L, as required for inducing conditions, and cells were incubated for 1 h at 30 °C. Total RNA preparation and qRT-PCR analysis The total RNA preparation and qRT-PCR analysis was performed as described previously (Ford et al. 2007, 2008; Esposito et al. 2010; Wimalarathna et al. 2011, 2012; Konarzewska et al. 2012) and is described in the Supplementary data.1 The real-time PCR primers are also described in the Supplementary data.1 All experiments were repeated twice, and in each experiment, PCR reactions were done in triplicate in a 7500 sequence detection system (Applied Biosystems). Target DNA sequence quantities were estimated as described previously (Wimalarathna et al. 2012). Briefly, target DNA sequence quantities were estimated from the threshold amplification cycle number (CT) using Sequence Detection System software (Applied Biosystems). Each DNA quantity was normalized to the ACT1 DNA quantity by taking the difference between each gene’s CT and ACT1’s CT value, which is a ⌬CT value. Each

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normalized mRNA level was calculated with the following formula: 2(−⌬CT). ChIP and real-time PCR analysis The preparation of cross-linked chromatin, immunoprecipitation procedures, and real-time PCR analyses were performed as described previously (Ford et al. 2008; Esposito et al. 2010; Konarzewska et al. 2012) and are described in the Supplementary data.1 All antibodies used in this study are also listed in the Supplementary data.1

Results Ace1p-dependent CUP1 expression requires Ino80p and Snf2p It has been shown that CUP1 is expressed in the presence of copper sulfate during the logarithmic phase of growth (Shen et al. 2001; Shen et al. 2002). Furthermore, CUP1 expression requires Ace1p-dependent recruitment of chromatin remodeling activities and transcription machinery (Wimalarathna et al. 2012). To examine how chromatin remodeling complexes, INO80 and SWI/SNF, are involved in CUP1 expression, WT and chromatin remodeler knockout strains were grown in the absence of copper. When the OD reached 0.2, copper was added to a final concentration of 3 mmol/L as is required for toxicity in yeast cells, 1.5 mmol/L as is required for CUP1 induction, or 0 mmol/L as uninduced conditions. Aliquots of culture were removed for OD measurements following copper addition. The results show that the WT cells grew well in the absence of copper (Fig. 1A). In the presence of 1.5 mmol/L copper, the WT cells also grew well but with a doubling time about 40% longer. In the presence of 3 mmol/L copper, the WT cells were inviable. Both ino80⌬ cells and snf2⌬ cells grew well in the absence of copper, but showed severe growth defects in the presence of 1.5 mmol/L and 3 mmol/L copper (Fig. 1A). Two other remodeler knockout strains, isw1⌬ and rsc3⌬ cells, were also tested and the results showed that both of these mutants displayed similar growth patterns as the WT cells in all 3 growth conditions (Fig. 1A). These results demonstrated that both ino80⌬ and snf2⌬ cells are inviable in the presence of 1.5 mmol/L copper, whereas WT, isw1⌬, and rsc3⌬ cells are viable in the presence of 1.5 mmol/L copper, which suggests that both Ino80p and Snf2p may be required for cell growth in the presence of 1.5 mmol/L copper. To further examine whether the growth defects of both ino80⌬ and snf2⌬ cells in the presence of copper result from their influence in CUP1 expression at the transcription level, CUP1 mRNA levels were measured by qRT-PCR. The ratios of CUP1/ACT1 in WT cells were 6.2 ± 1.4 and 6.6 ± 0.6 after a 15 min and 30 min addition of 1.5 mmol/L copper, respectively (Fig. 1B). In the absence of copper, the ratios were about 1.3 ± 0.2 and 0.9 ± 0.1 after a 15 min and 30 min incubation, respectively. Thus, CUP1 is expressed in WT cells within 15 min under induced conditions. For both ino80⌬ and snf2⌬ cells, the ratios did not significantly increase in either uninduced or induced conditions (Fig. 1B). The ratios of CUP1/ACT1 were approximately 1 under both conditions for both mutants. For both isw1⌬ and rsc3⌬ cells, the ratios of CUP1/ACT1 were similar to WT cells under both un-induced and induced conditions (Fig. 1B). Therefore, the deletion of INO80 and SNF2 led to an inability to express CUP1 at the transcriptional level under induced conditions. To exclude the possibility that the lack of CUP1 expression was due to a variation in the transcriptional activator’s expression, ACE1 expression levels were also examined. Our results showed no significant difference in ACE1 expression between WT cells and all knockout mutants (Fig. 1C). As such, these results suggested that both INO80 and SNF2 regulate CUP1 expression at the transcriptional level.

Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/bcb-2013-0097. Published by NRC Research Press

Wimalarathna et al.

Fig. 1. Both Ino80p and Snf2p are required for Ace1-regulated CUP1 induction. (A) Growth of yeast cells in the presence or absence of copper. Yeast cell growth was monitored by A600. WT: (Œ) 0 mmol/L copper, (●) 1.5 mmol/L copper, (●) 3 mmol/L copper; ino80⌬: (〫) 0 mmol/L copper, (⽧) 1.5 mmol/L copper, (⽧) 3 mmol/L copper; isw1⌬: (*) 0 mmol/L copper, ( ) 1.5 mmol/L copper, () 3 mmol/L copper; rsc3⌬: (+) 0 mmol/L copper, (µ) 1.5 mmol/L copper, (–) 3 mmol/L copper; snf2⌬: (o) 0 mmol/L copper, (Œ) 1.5 mmol/L copper, (Œ) 3 mmol/L copper. The expression of (B) CUP1 and (C) ACE1 in WT, ino80⌬, isw1⌬, rsc3⌬, and snf2⌬ cells. The expression ratio for CUP1 mRNA to ACT1 mRNA after 15 and 30 min treatment and the expression ratio for ACE1 mRNA to ACT1 mRNA after 30 min treatment are graphed as mean ± standard deviation. UN: uninduced conditions (no copper was added); IN: induced conditions (copper was added to a final concentration of 1.5 mmol/L).

Co-dependent recruitment of INO80 and SNF2 to CUP1 promoter. Subsequently, we examined whether both INO80 and SNF2 directly participate in CUP1 expression. The IP signals of the Arp8p subunit of the INO80 (Arp8-IP) and of the Snf2p subunit of SWI/

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SNF (Snf2-IP) were examined at the CUP1 upstream activation sequences (UAS) and part of the coding region of the POL1 gene (POL1). The normalized relative IP ratio for the CUP1 promoter (UAS/POL1) represented the ratio of the CUP1 promoter IP DNA normalized to CUP1 input DNA to POL1 IP DNA normalized to POL1 input DNA. Results showed that both Arp8-IP and Snf2-IP levels were around 1 under un-induced conditions for WT (BJ) cells (Fig. 2A). The relative IP levels for both remodelers increased significantly under induced conditions, which were 2.16 ± 0.04 and 2.21 ± 0.24 for Arp8-IP and Snf2-IP, respectively. These results suggested that both INO80 and SWI/SNF were recruited to the CUP1 promoter under induced conditions. As it has been shown that Ace1p recruits chromatin remodeling activities to the CUP1 promoter under induced conditions (Shen et al. 2001; Wimalarathna et al. 2012), we wanted to investigate whether both INO80 and SWI/SNF are directly recruited by Ace1p. We examined the IP/INPUT ratio to determine the presence of Arp8p and Snf2p at the CUP1 promoter in both WT (BJ) and ace1⌬ (BJ) cells. Our results showed that the IP/INPUT ratios increased significantly for both Arp8p and Snf2p in WT (BJ) cells under induced conditions (Fig. 2B). However, we did not observe any increase of Arp8p and Snf2p levels in ace1⌬ (BJ) cells under induced conditions, which suggested that both chromatin remodelers were not recruited to the CUP1 promoter in ace1⌬ (BJ) cells. Therefore, both remodelers were recruited to the CUP1 promoter by the transcription activator, Ace1p, under induced conditions. To further confirm that both Ino80p and Snf2p directly participated in CUP1 induction, we examined whether the presence of chromatin remodeling activity at the CUP1 promoter is brought in by INO80 and SWI/SNF2. The analysis of nucleosome density through the IP of histones H3 and H4 levels were performed for WT, ino80⌬, and snf2⌬ cells. We evaluated the change of H3 and H4 levels from un-induced conditions to induced conditions by taking the ratios of induced IP/INPUT to un-induced IP/INPUT. The results showed that both H3 and H4 ratios dropped to 0.65 ± 0.04 and 0.51 ± 0.13 in WT cells, respectively (Fig. 2C), which suggested that both H3 and H4 density decreased dramatically after induction. The decreasing nucleosome density was an indication of the chromatin remodeling activity. Such a decrease was not observed in ino80⌬ and snf2⌬ cells, which has ratios of approximately 1.0 for both H3 and H4. These results suggested that the remodeling activities were indeed brought in by both Ino80p and Snf2p. As such, our findings provided direct evidence for the participation of Ino80p and Snf2p in CUP1 chromatin remodeling during activation. Since both INO80 and SWI/SNF are involved in CUP1 induction, and both remodelers are recruited by Ace1p, we then wanted to determine the recruitment dependence for both remodelers. We investigated the presence of Arp8p at the CUP1 promoter in snf2⌬ cells and of Snf2p at the CUP1 promoter in ino80⌬ cells. Our results showed that the relative Arp8p IP ratios were about 1.0 under both un-induced and induced conditions in snf2⌬ cells (Fig. 2D). This pattern is completely different from what we observed in WT cells, in which the relative Arp8p IP ratios increased significantly under induced conditions (Fig. 2A). Therefore, INO80 was not present at the CUP1 promoter of snf2⌬ cells under both un-induced and induced conditions. This suggested that the recruitment of INO80 was affected in the absence of Snf2p. Likewise, the Snf2p IP ratios in ino80⌬ cells were also about 1.0 under both un-induced and induced conditions (Fig. 2D). This result was again different from the WT cells (Fig. 2A). As such, SWI/SNF was not present at the CUP1 promoter of ino80⌬ cells under both un-induced and induced conditions, which suggested that the recruitment of SWI/SNF depends on the presence of Ino80p. This indicates that they are mutually required for recruitment under induced conditions. This co-dependent relationship may suggest that both remodelers arPublished by NRC Research Press

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Fig. 2. Co-dependent recruitment of both INO80 and SWI/SNF to the CUP1 promoter during induction. (A) Real-time PCR analysis of the presence of INO80 and SWI/SNF at the CUP1 promoter and POL1 coding region. DNA was immunoprecipitated with antibodies against INO80 (␣-Arp8p) and SWI/SNF (␣-Snf2p) at the CUP1 upstream activation sequences (UAS) and part of the coding region of POL1 gene (POL1) for WT (BJ) cells. The relative IP ratios for UAS/POL1 are graphed as mean ± standard deviation. (B) Real-time PCR analysis of the presence of INO80 and SWI/SNF at the CUP1 promoter for both WT (BJ) and ace1⌬ (BJ) cells. The IP ratios for the IP/INPUT are graphed as mean ± standard deviation. (C) The change of histone occupancy at the CUP1 promoter under induced conditions. DNA was immunoprecipitated with antibodies against histone H3 (␣-H3) and histone H4 (␣-H4) at the CUP1 UAS for WT, ino80⌬ and snf2⌬ cells. The ratios for (induced IP/INPUT) to (uninduced IP/INPUT) are graphed as mean ± standard deviation. (D) Co-dependent recruitment of INO80 and SWI/SNF at the CUP1 promoter under induced conditions. UN: un-induced conditions (no copper was added); IN: induced conditions (copper was added to a final concentration of 1.5 mmol/L).

rive at the CUP1 promoter at the same time in an activatordependent manner. Both Ino80p and Snf2p are required to recruit transcription machinery and histone acetylation at CUP1 promoter It has been shown that the recruitment of Pol II is activatordependent, it does not require targeted histone acetylation during CUP1 induction (Shen et al. 2002; Wimalarathna et al. 2012). Since both chromatin remodelers, INO80 and SWI/SNF, are also required for CUP1 induction, it is crucial to determine whether Pol II recruitment requires the presence of Ino80p and Snf2p. By doing so, we can also determine the order of events in CUP1 induction. The IP signals of Pol II were measured at the CUP1 TATA sequences (TATA) and part of the coding region of the POL1 gene (POL) for WT, ino80⌬ and snf2⌬ cells. Results showed that the relative IP values of TATA/POL were 0.82 ± 0.18 and 2.76 ± 0.32 for un-induced and induced WT cells, respectively (Fig. 3A). These results suggested that Pol II was largely recruited to the CUP1 promoter under induced conditions. For ino80⌬ cells and snf2⌬ cells, the relative IP values of TATA/POL were 0.97 ± 0.36 and 1.21 ± 0.03 under induced conditions, respectively (Fig. 3A). These results suggest that Pol II is not recruited to the CUP1 TATA region in the absence of INO80

and SWI/SNF under inducing conditions. As such, the recruitment of Pol II requires both INO80 and SWI/SNF remodelers. It has been suggested that targeted acetylation of H3 and H4 occurred at very late stages in the initiation of CUP1 induction (Shen et al. 2002). As we demonstrated that both Ino80p and Snf2p are required to recruit the Pol II, it was subsequently necessary to examine whether both Ino80p and Snf2p are required for the recruitment of histone acetylation at the CUP1 promoter. We analyzed the change of histone acetylation levels from un-induced conditions to induced conditions by calculating ratios of induced IP/INPUT to un-induced IP/INPUT. We found that the acetylation levels of H3 and H4 increased dramatically under induced conditions for WT cells (Fig. 3B). However, such an increase was not observed in either ino80⌬ or snf2⌬ cells. These results suggest the absence of histone acetylation in both ino80⌬ and snf2⌬ under induced conditions. As such, targeted histone H3 and H4 acetylation requires the presence of the both Ino80p and Snf2p.

Discussion Ace1p-dependent CUP1 expression can ensure that sufficient copper is present in the cell to perform essential biochemical processes, yet prevents the accumulation of copper to toxic levels. Published by NRC Research Press

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Fig. 3. Both INO80 and SWI/SNF are required to recruit RNA polymerase II and histone acetylation in CUP1 induction. (A) Realtime PCR of DNA immunoprecipitated with antibodies against RNA polymerase II at the CUP1 TATA (TATA) and POL1 for WT, ino80⌬ and snf2⌬ cells. The relative IP ratios for Pol II occupancy are graphed as mean ± standard deviation. (B) The absence of either remodeler resulted in the loss of targeted histone acetylation at the CUP1 promoter under induced conditions. DNA immunoprecipitated with antibodies against acetylated histone H3 (␣-acH3) and acetylated histone H4 (␣-acH4) at the CUP1 UAS for WT, ino80⌬ and snf2⌬ cells. The ratios of (induced IP/INPUT) to (un-induced IP/INPUT) are graphed as mean ± standard deviation to reflect the change of acetylated histone levels during copper induction. UN: un-induced conditions (no copper was added); IN: induced conditions (copper was added to a final concentration of 1.5 mmol/L).

Such regulation of copper homeostatic genes is important to cell physiology and is conserved throughout eukaryotes. Studying the mechanism of CUP1 gene expression is thus critical to the understanding of copper homeostasis at the molecular level. In this study, we have demonstrated that both Ino80p and Snf2p are required for CUP1 transcriptional activation. Furthermore, they are both Ace1p-dependent and are recruited to the CUP1 promoter in a co-dependent fashion. Finally, we showed that both INO80 and SWI/SNF are required to recruit chromatin remodeling activity, Pol II, and histone acetylation to the CUP1 promoter. As such, the order of events in CUP1 expression has been elucidated. Previously, we have shown that the transcriptional activator, Ace1p, is required to recruit both chromatin remodeling activity and transcription machinery to the promoter in CUP1 induction (Shen et al. 2001, 2002; Wimalarathna et al. 2012). Although genewide chromatin remodeling was observed in CUP1 induction (Shen et al. 2001) and the swi1⌬ cells exhibited impaired CUP1 induction (Kuo et al. 2005), the exact remodelers involved in CUP1 induction have never been identified. Here, we showed that both Ino80p and Snf2p are required for CUP1 expression through growth experiments and mRNA analysis. Furthermore, they are both recruited to the CUP1 promoter under induced conditions (Fig. 2A). In the absence of either remodeler, remodeling activity was lost under induced conditions (Fig. 2C). They were also required to recruit the transcription machinery and histone acetylation (Fig. 3). Therefore, we have provided direct evidence to show these two remodelers are involved in CUP1 induction. In an attempt to determine the dependence of INO80 and SWI/ SNF in each other’s recruitment, we found that both INO80 and SWI/SNF are recruited in a co-dependent fashion (Fig. 2D). We observed the absence of Ino80p at the CUP1 promoter in snf2⌬ cells. Likewise, Snf2p was missing from the CUP1 promoter in ino80⌬ cells. As such, it is possible that both remodelers are recruited by the activator and arrive at the promoter concomitantly. If this is the case, both SWI/SNF and INO80 might need to form a complex for the recruitment and (or) remodeling purpose. If one is absent, then the other is unable to be recruited to the promoter alone. Another possible explanation for this mutual dependence could be that either remodeler can be recruited to the promoter separately. If one remodeler is not present, the other remodeler might interact only transiently with the CUP1 promoter and depart from the promoter without performing the remodeling activity. In either case, our results demonstrated that the histone density remained unchanged in the absence of either remodeler (Fig. 2C). Clearly, no chromatin remodeling activity was found at the promoter if either remodeler was absent. This suggests that even if either remodeler can be recruited to the promoter, it still cannot perform its remodeling activity in the absence of the other remodeler. Although the details of this mechanism still need to be further examined, this is a unique recruitment pattern and is different from other gene activation models. For example, the

recruitment of INO80 is independent of SWI/SNF in INO1 activation, but the recruitment of SWI/SNF depends on the presence of INO80 (Ford et al. 2008; Esposito et al. 2010). Another example is the ARG1 induction model, which demonstrated that the recruitment of one remodeler is not influenced by the absence of another remodeler in induction (Govind et al. 2005). Therefore, we have demonstrated a unique co-dependent recruitment of chromatin remodelers in CUP1 expression. We observed that INO80 and SWI/SNF were absent at the CUP1 promoter under un-induced conditions. However, both INO80 Published by NRC Research Press

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Fig. 4. A model for transcriptional activation of CUP1 chromatin. In the inactive state, the nucleosomes are immobile and the chromatin structure is organized into clusters of overlapping nucleosome positions separated by linker. In the intermediate state, copperactivated transcriptional activator Ace1p locates a binding site in the UAS region. Both INO80 and SWI/SNF are recruited to the CUP1 promoter and nucleosomes begin to slide back and forth, creating a dynamic chromatin structure over the entire gene. In the active state, both Pol II and TFIID are recruited to the TATA boxes. Subsequently, a histone acetylase is recruited and acetylates H3 and H4 in nucleosomes at the promoter region.

and SWI/SNF were largely recruited to the CUP1 promoter upon induction (Fig. 2A). This recruitment pattern depends on the presence of the transcriptional activator Ace1p (Shen et al. 2001, 2002; Wimalarathna et al. 2012), which is recruited to the promoter upon induction (Buchman et al. 1989; Huibregtse et al. 1989). Many studies have demonstrated similar recruitment dependence between activators and co-activators, including ARG1 (Govind et al. 2005) and GAL1 (Bryant and Ptashne 2003). These genes all require the recruitment of an activator upon induction, followed by the recruitment of transcriptional co-activators and transcription machinery. While we observed that co-activators were recruited upon induction, our observed patterns differed from other models, including INO1 and HIS3 induction (Brickner and Walter 2004; Dasgupta et al. 2005; Kim et al. 2006). It has been shown that chromatin remodelers are also activator-dependent in these cases, but the transcriptional activator and chromatin remodelers are present at the promoter under repressing conditions. Under de-repressing conditions, the repressor dissociates from the activator and the activator becomes functional. Subsequently, the activation process is initiated, and remodelers depart from the promoter (Brickner and Walter 2004; Dasgupta et al. 2005; Kim et al. 2006; Ford et al. 2008). These different activation patterns might arise from the different regulatory mechanism of gene expression. For example, the regulation of INO1 is controlled by the presence of the Opi1p repressor. INO1 is repressed in the presence

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of the repressor, and thus INO1 can only be induced in the absence of the repressor. As such, the activator and chromatin remodelers have to be in the promoter region all the time (Brickner and Walter 2004; Dasgupta et al. 2005; Ford et al. 2008). On the other hand, the regulation of the inducible CUP1 promoter depends on the copper-induced activator Ace1p. In this case, the activator will only become active in the presence of the copper but not in the presence of other trans-acting factors. Subsequently, active Ace1p is recruited to the CUP1 promoter, followed by the recruitment of remodelers. As such, it is reasonable to see the absence of remodelers at the CUP1 promoter under un-induced conditions and the presence of remodelers after induction. Previously, we have shown that targeted acetylation occurs either when the TATA-binding protein binds to the TATA box or at a later stage in CUP1 activation. Furthermore, the movement of nucleosomes occurring upon CUP1 induction is independent of targeted acetylation, which indirectly suggests that the recruitment of remodelers is prior to the recruitment of both transcription machinery and histone acetylation (Shen et al. 2002). Here, we observed that both Pol II and targeted histone acetylation required the presence of both Ino80p and Snf2p at the CUP1 promoter (Fig. 3). This observation provides direct evidence of recruitment dependence, and it proves that remodelers arrive at the CUP1 promoter prior to Pol II and histone acetylation. We now have a general picture of the CUP1 activation model (Fig. 4). Under un-induced conditions, CUP1 chromatin is organized into clusters of overlapping nucleosome positions separated by linkers (Shen et al. 2001). Upon induction, the transcription activator, Ace1p, becomes active and binds to the CUP1 promoter followed by the recruitment of both Ino80p and Snf2p (Thiele and Hamer 1986; Fürst et al. 1988; Buchman et al. 1989; Huibregtse et al. 1989; Zhou et al. 1992; Wimalarathna et al. 2012). The recruitment of both remodelers results in nucleosome repositioning (Shen et al. 2001). Subsequently, histone acetylases and transcription machinery are recruited to the promoter to complete the initiation process. It has been shown that histone acetylation occurs at or after the arrival of the transcription machinery (Shen et al. 2002). The late arrival of histone acetylase(s) suggest that histone acetylases do not play a critical role in transcription initiation (Choi et al. 2008; Kim et al. 2010; Konarzewska et al. 2012). It is possible that histone acetylases might be used for purposes more than just acetylating histones. Although there is no direct evidence, it is possible that the late arrival of histone acetylases may play a role in remodelers’ dissociation from the promoter (Ferreira et al. 2007; VanDemark et al. 2007; Choi et al. 2008; Kim et al. 2010; Konarzewska et al. 2012). Further analysis is necessary to uncover histone acetylases’ functional implications during transcriptional activation, which will provide key insight into the mechanism of CUP1 activation process and gain insight into the mechanism of copper homeostatic pathway.

Acknowledgements We are grateful to Michelle Esposito, Paulina Konarzewska, and Goldie Lazarus for helpful discussion and comments on the manuscript and to our laboratory colleagues for technical assistance. This work was supported by an NSF Grant (MCB-0919218) and PSC-CUNY awards (65351-0043) to C.-H.S.

References Askwith, C., and Kaplan, J. 1998. Iron and copper transport in yeast and its relevance to human disease. Trends Biochem. Sci. 23: 135–138. doi:10.1016/ S0968-0004(98)01192-X. PMID:9584616. Brickner, J.H., and Walter, P. 2004. Gene recruitment of the activated INO1 locus to the nuclear membrane. PLoS Biol. 2: e342. doi:10.1371/journal.pbio. 0020342. PMID:15455074. Bryant, G.O., and Ptashne, M. 2003. Independent recruitment in vivo by Gal4 of two complexes required for transcription. Mol. Cell, 11: 1301–1309. doi:10. 1016/S1097-2765(03)00144-8. PMID:12769853. Buchman, C., Skroch, P., Welch, J., Fogel, S., and Karin, M. 1989. The CUP2 gene Published by NRC Research Press

Wimalarathna et al.

product, regulator of yeast metallothionein expression, is a copper-activated DNA-binding protein. Mol. Cell. Biol. 9: 4091–4095. PMID:2674688. Choi, J.K., Grimes, D.E., Rowe, K.M., and Howe, L.J. 2008. Acetylation of Rsc4p by Gcn5p is essential in the absence of histone H3 acetylation. Mol. Cell. Biol. 28: 6967–6972. doi:10.1128/MCB.00570-08. PMID:18809572. Dasgupta, A., Juedes, S.A., Sprouse, R.O., and Auble, D.T. 2005. Mot1-mediated control of transcription complex assembly and activity. EMBO J. 24: 1717– 1729. doi:10.1038/sj.emboj.7600646. PMID:15861138. Esposito, M., Konarzewska, P., Odeyale, O., and Shen, C.-H. 2010. Gene-wide histone acetylation at the yeast INO1 requires the transcriptional activator Ino2p. Biochem. Biophys. Res. Commun. 391: 1285–1290. doi:10.1016/j.bbrc. 2009.12.063. PMID:20018175. Ferreira, R., Eberharter, A., Bonaldi, T., Chioda, M., Imhof, A., and Becker, P.B. 2007. Site-specific acetylation of ISWI by GCN5. BMC Mol. Biol. 8: 73. doi:10. 1186/1471-2199-8-73. PMID:17760996. Ford, J., Odeyale, O., Eskandar, A., Kouba, N., and Shen, C.-H. 2007. A SWI/SNFand INO80-dependent nucleosome movement at the INO1 promoter. Biochem. Biophys. Res. Commun. 361: 974–979. doi:10.1016/j.bbrc.2007.07. 109. PMID:17681272. Ford, J., Odeyale, O., and Shen, C.-H. 2008. Activator-dependent recruitment of SWI/SNF and INO80 during INO1 activation. Biochem. Biophys. Res. Commun. 373: 602–606. doi:10.1016/j.bbrc.2008.06.079. PMID:18593569. Fürst, P., Hu, S., Hackett, R., and Hamer, D. 1988. Copper activates metallothionein gene transcription by altering the conformation of a specific DNA binding protein. Cell, 55: 705–717. doi:10.1016/0092-8674(88)90229-2. PMID:3052856. Govind, C.K., Yoon, S., Qiu, H., Govind, S., and Hinnebusch, A.G. 2005. Simultaneous recruitment of coactivators by Gcn4p stimulates multiple steps of transcription in vivo. Mol. Cell. Biol. 13: 5626–5638. doi:10.1128/MCB.25.13. 5626-5638.2005. PMID:15964818. Hahn, S., and Young, E.T. 2011. Transcriptional regulation in Saccharomyces cerevisiae: transcription factor regulation and function, mechanisms of initiation, and roles of activators and coactivators. Genetics, 189: 705–736. doi: 10.1534/genetics.111.127019. PMID:22084422. Huibregtse, J.M., Engelke, D.R., and Thiele, D.J. 1989. Copper-induced binding of cellular factors to yeast metallothionein upstream activation sequences. Proc. Natl. Acad. Sci. U.S.A. 86: 65–69. doi:10.1073/pnas.86.1.65. PMID:2643107. Kim, J.H., Saraf, A., Florens, L., Washburn, M., and Workman, J.L. 2010. Gcn5 regulates the dissociation of SWI/SNF from chromatin by acetylation of Swi2/ Snf2. Genes Dev. 24: 2766–2771. doi:10.1101/gad.1979710. PMID:21159817. Kim, Y., McLaughlin, N., Lindstrom, K., Tsukiyama, T., and Clark, D.J. 2006. Activation of Saccharomyces cerevisiae HIS3 results in Gcn4p-dependent, SWI/ SNF-dependent mobilization of nucleosomes over the entire gene. Mol. Cell. Biol. 26: 8607–8622. doi:10.1128/MCB.00678-06. PMID:16982689. Konarzewska, P., Esposito, M., and Shen, C.-H. 2012. INO1 induction requires chromatin remodelers Ino80p and Snf2p but not the histone acetylases.

75

Biochem. Biophys. Res. Commun. 418: 483–488. doi:10.1016/j.bbrc.2012.01. 044. PMID:22281492. Kuo, H.C., Moore, J.D., and Krebs, J.E. 2005. Histone H2A and Spt10 cooperate to regulate induction and autoregulation of the CUP1 metallothionein. J. Biol. Chem. 280: 104–111. doi:10.1074/jbc.M411437200. PMID:15501826. Luger, K., Dechassa, M.L., and Tremethick, D.J. 2012. New insights into nucleosome and chromatin structure: an ordered state or a disordered affair? Nat. Rev. Mol. Cell Biol. 13: 436–447. doi:10.1038/nrm3382. PMID:22722606. Rando, O.J., and Winston, F. 2012. Chromatin and transcription in yeast. Genetics, 190, 351–387. doi:10.1534/genetics.111.132266. PMID:22345607. Shen, C.-H., Leblanc, B.P., Alfieri, J.A., and Clark, D.J. 2001. Remodeling of yeast CUP1 chromatin involves activator-dependent repositioning of nucleosomes over the entire gene and flanking sequences. Mol. Cell. Biol. 21: 534–547. doi:10.1128/MCB.21.2.534-547.2001. PMID:11134341. Shen, C.-H., Leblanc, B.P., Neal, C., Akhaven, R., and Clark, D.J. 2002. Targeted histone acetylation at the yeast CUP1 promoter requires the transcriptional activator, the TATA boxes, and the putative histone acetylase encoded by SPT10. Mol. Cell. Biol. 22: 6406–6416. doi:10.1128/MCB.22.18.6406-6416.2002. PMID:12192040. Shim, H., and Harris, Z.L. 2003. Genetic defects in copper metabolism. J. Nutr. 133: 1527S–1531S. PMID:12730458. Thiele, D.J. 1988. ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene. Mol. Cell. Biol. 8: 2745–2752. doi:10.1128/MCB.8.7.2745. PMID:3043194. Thiele, D.J., and Hamer, D.H. 1986. Tandemly duplicated upstream control sequences mediate copper-induced transcription of the Saccharomyces cerevisiae copper-metallothionein gene. Mol. Cell. Biol. 6: 1158–1163. doi:10.1128/MCB. 6.4.1158. PMID:3537699. VanDemark, A.P., Kasten, M.M., Ferris, E., Heroux, A., Hill, C.P., and Cairns, B.R. 2007. Autoregulation of the RSC4 tandem bromodomain by GCN5 acetylation. Mol. Cell, 27: 817–828. doi:10.1016/j.molcel.2007.08.018. PMID:17803945. Welch, J., Fogel, S., Buchman, C., and Karin, M. 1989. The CUP2 gene product regulates the expression of the CUP1 gene, coding for yeast metallothionein. EMBO J. 8: 255–260. PMID:2653812. Wimalarathna, R.N., Tsai, C.-H., and Shen, C.-H. 2011. Transcriptional regulation of genes involved in yeast phospholipid biosynthesis. J. Microbiol. 49: 265– 273. doi:10.1007/s12275-011-1130-1. PMID:21538248. Wimalarathna, R.N., Pan, P.Y., and Shen, C.-H. 2012. Chromatin repositioning activity and transcription machinery are both recruited by Ace1p in yeast CUP1. Biochem. Biophys. Res. Commun. 422: 658–663. doi:10.1016/j.bbrc.2012. 05.047. PMID:22609398. Zhou, P., Szczypka, M.S., Sosinowski, T., and Thiele, D.J. 1992. Expression of a yeast metallothionein gene family is activated by a single metalloregulatory transcription factor. Mol. Cell. Biol. 12: 3766–3775. PMID:1508182.

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Co-dependent recruitment of Ino80p and Snf2p is required for yeast CUP1 activation.

In yeast, Ace1p-dependent induction of CUP1 is responsible for protecting cells from copper toxicity. Although the mechanism of yeast CUP1 induction h...
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