A Transient Histone Hyperacetylation Signal Marks Nucleosomes for Remodeling at the PHO8 Promoter In Vivo

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1 Molecular Cell, Vol. 7, , March, 2001, Copyright 2001 by Cell Press A Transient Histone Hyperacetylation Signal Marks Nucleosomes for Remodeling at the PHO8 Promoter In Vivo Hans Reinke, Philip D. Gregory, and Wolfram Hörz* Institut für Physiologische Chemie Universität München Schillerstrasse 44 D München Germany Summary Chromatin remodeling of the yeast PHO8 promoter requires the SAGA histone acetyltransferase complex. We report here that SAGA is necessary and sufficient to establish an activator-dependent hyperacetylation peak over the PHO8 promoter that is restricted to those nucleosomes that are remodeled upon activation. This local hyperacetylated state is observed upon activation in the absence of the SWI/SNF complex when the remodeling process is frozen subsequent to activator binding. Hyperacetylation is lost, however, if remodeling is permitted to go to completion. Thus, a transient histone hyperacetylation signal is shown to be a prerequisite for, and determinant of, the domain of nucleosome remodeling in vivo. Introduction quent to activator binding (Gregory et al., 1998; Ryan et al., 1998). The plethora of SWI/SNF paralogs found in yeast, fly, mouse, and human (reviewed in Peterson, 2000) suggests a general role for chromatin remodeling in transcriptional regulation in all eukaryotes. Histone acetyltransferases (HATs) comprise a second group of cofactors, which modulate chromatin structure. The yeast protein Gcn5, originally identified as a transcriptional coactivator (Berger et al., 1992; Georgakopoulos and Thireos, 1992), is the catalytic subunit of at least two multisubunit HAT complexes, SAGA (Grant et al., 1997) and ADA (Eberharter et al., 1999), functionally linking histone acetylation and transcription. Moreover, Gcn5-dependent promoter histone hyperacetylation is correlated with transcriptional activation at both the HIS3 and HO promoters (Kuo et al., 1998; Krebs et al., 1999). On the other hand, histone acetyltransferases are not limited to histones as sole substrate, since the Gcn5 homolog PCAF appears to acetylate additionally tran- scription factors and/or members of the basal transcrip- tion apparatus, potentially altering their transcription po- tential (Gu and Roeder, 1997; Imhof et al., 1997; Sartorelli et al., 1999; Martinez-Balbas et al., 2000). How histone acetylation regulates transactivation re- mains an open question. One model suggests that histone acetylation renders the nucleosome a more tractable substrate permissive to both transactivator binding (Lee et al., 1993; Vettese-Dadey et al., 1996) and assembly of the general transcription machinery onto nucleo- somal DNA (Ura et al., 1997). Accordingly, large domains of transcriptionally active chromatin have been found to contain hyperacetylated histones and to demonstrate increased sensitivity to DNaseI (Hebbes et al., 1994). Moreover, both higher order folding and PolIII transcription of nucleosomal arrays in vitro is strongly affected by the level of histone acetylation (Tse et al., 1998). Yet in vivo evidence for hyperacetylation per se modulating histone DNA interactions and/or higher order chromatin structure is lacking. Moreover, the remarkable conserva- tion of the lysine positions in the histone tails, as well as the specific functional consequences of individual acetylation patterns, would speak for an alternative model, which assigns histone acetylation the role of a regulatory signal that is deciphered by downstream DNA, the fundamental genetic information of all eukaryotes, is presented to the transcriptional apparatus within a highly compact framework of histone and nonhistone proteins termed chromatin. Packaging of DNA into nucleosomes, the repeating unit of chromatin, has been shown to repress transcription by preventing access of both transcription factors and the general transcrip- tional machinery to their targets within the promoters of genes. To cope with chromatin repression, different coactivators exist in the cell, which are necessary to antagonize this barrier to transcription and permit the proper and timely expression of genes (Workman and Kingston, 1998; Kingston and Narlikar, 1999). The yeast SWI/SNF complex is the prototype of one such class of coactivators. It directly alters nucleosomal architecture at a subset of promoter regions and is consequently required for the transcriptional activation of functioning activator complexes (Strahl and Allis, 2000). these genes, including HO and SUC2 (for reviews, see Distinguishing between these models is complicated by Sudarsanam and Winston, 2000; Vignali et al., 2000 and the fact that at present no data exist which correlate references therein). Although SWI/SNF has been shown chromatin remodeling, acetylation status, and transcripto facilitate binding of transcription factors to their re- tional activation at a single promoter. spective target sites on nucleosomal DNA (Burns and The PHO8 promoter undergoes a precisely defined Peterson, 1997), recent evidence has demonstrated re- chromatin transition upon induction by phosphate starcruitment of SWI/SNF to specific promoters through vation (Barbaric et al., 1992). Moreover, chromatin remodeling, but not activator binding, is dependent on transactivators (Kowenz and Leutz, 1999; Natarajan et al., 1999; Tsukiyama et al., 1999), the function of SWI/ both the SAGA and SWI/SNF complexes at PHO8 (Greg- SNF remodeling therefore occurring at a step subseline phosphatase (Kaneko et al., 1985), is transcription- ory et al., 1999). PHO8, which encodes a vacuolar alkaally coregulated with the PHO5 gene by a common and * To whom correspondence should be addressed ( hoerz@bio. med.uni-muenchen.de). well-understood signal transduction pathway (Lenburg Present address: Sangamo BioSciences, Inc., Richmond, California detailed understanding of the PHO8 promoter at and Oshea, 1996; Svaren and Hörz, 1997). Thus, our three

2 Molecular Cell 530 different levels its regulation, chromatin modulation, 14 and tetraacetylated H4. Both antibodies have been and transcriptional activation provides a unique opportunity successfully used for acetylation analyses of the Gcn5- to address directly the role of histone acetyla- dependent HIS3 and HO promoters (Kuo et al., 1998; tion in gene activation. Krebs et al., 1999). Here we report that SAGA establishes a transient hy- Acetylation levels at the activated PHO8 promoter are peracetylation peak over the PHO8 promoter limited to shown in Figure 1B. H3 acetylation is slightly but repro- precisely those nucleosomes that are remodeled upon ducibly reduced compared to the pho4 strain, whereas activation. This intermediate state can be detected under the adjacent 5 region remains unaltered. The drop in conditions in which promoter activation is frozen at promoter acetylation is also observed at PGK1 at both a step following binding of the activator Pho4 but prior histones H3 and H4 and is therefore not specific to the to chromatin remodeling. Local hyperacetylation is lost PHO8 promoter. No difference in H4 acetylation was when remodeling is permitted to occur and the promoter observed between the repressed ( pho4, closed chro- becomes transcriptionally active. Hyperacetylation is matin) and activated state (wt, open chromatin) at the therefore not required for the maintenance of the remodeled proximal PHO8 promoter and the region adjacent to state. Rather, our results suggest a role for histone PHO8 (Figure 1C), whereas acetylation of the upstream hyperacetylation as a signal for nucleosome remodeling promoter was also reduced upon activation to a similar at the PHO8 promoter. extent to H3 acetylation in this region. No signal was obtained from non-crosslinked cells, from strains harboring Results a promoter deletion, or from IP samples if the antibody had been omitted (data not shown). From these Remodeled Chromatin at the Activated PHO8 results, we conclude that an open chromatin structure Promoter Is Not Hyperacetylated does not correlate with histone hyperacetylation at the Activation of the PHO8 gene is accompanied by a pronounced PHO8 promoter. chromatin transition in the promoter region. Chromatin remodeling and subsequent transcriptional Phosphate Starvation in the Absence of SWI/SNF activation have been shown previously to be dependent Captures the PHO8 Promoter in a Hyperacetylated upon the histone acetyltransferase activity of Gcn5, im- and Nonremodeled State plicating a role for histone acetylation in chromatin remodeling The effectively identical acetylation levels of the PHO8 at this promoter (Gregory et al., 1999). To de- promoter under both repressed and activated condi- termine directly whether histones at the PHO8 promoter tions raise the possibility that histone acetylation is not become hyperacetylated upon gene activation, we em- involved in chromatin opening and transcriptional activation ployed chromatin immunoprecipitation (ChIP) followed at this promoter. However, the observation that by quantitative PCR analysis. chromatin remodeling is blocked at PHO8 in the absence Histone acetylation at the activated PHO8 promoter of Gcn5 suggests a crucial role for this factor in the was analyzed after 16 hr induction in phosphate-free activation process prior to chromatin remodeling. Thus, medium. Rather than using cells before induction for the conditions employed in Figure 1, under which chro- comparison, we compared the histone acetylation levels matin remodeling is complete, would necessarily be in the presence or absence of the transactivator Pho4, after the point of action of Gcn5. We therefore sought deletion of which renders the cell unable to respond conditions where Pho4 is bound but subsequent chromatin to the phosphate starvation signal and maintains the remodeling does not occur. Such conditions exist promoter in a permanently repressed state. This way, in a snf2 strain starved for phosphate. As previously pleiotropic effects due to the comparison of cells under shown, in this situation Pho4 is bound to the critical very different growth conditions were avoided. (Induc- UASp2 element of the promoter, but remodeling does tion of cells in phosphate-free medium leads to cell not take place (Gregory et al., 1999). This strategy offers cycle arrest.) Indeed, a global increase in H3 and H4 the possibility to analyze acetylation levels at the activated acetylation had been observed at the onset of S phase promoter and at the same time to avoid any possiacetylation (Krebs et al., 1999). ble effects on histone acetylation by the actual remodeling Acetylation levels were determined with separate event. primer pairs at the upstream and proximal PHO8 pro- A dramatic increase in histone H4 acetylation across moter in order to analyze all nucleosomes that are remodeled the PHO8 promoter is observed in the induced snf2 upon activation (Figure 1A). The adjacent 5 strain (Figure 2B). The upstream region of the promoter region of PHO8 (solid nucleosomes in the lower panel shows more than 3-fold and the proximal promoter of Figure 1A) is not part of the promoter and correspond- 2-fold higher acetylation levels compared to both the repressed ingly does not undergo any observable chromatin perturbation and fully activated states. The result of quantitaingly in the course of gene activation. This region tive PCR of DNA from immunoprecipitated chromatin was therefore also analyzed to distinguish between truly fragments from the snf2 and the wild-type strain are promoter specific and general effects on histone ace- shown in Figures 2C and 2D. Note the striking increase tylation. As further controls, we determined acetylation in H4 acetylation at the PHO8 promoter compared to levels at the constitutive PDA1 and PGK1 promoters, the adjacent 5 region that is only observed in the snf2 which do not respond to the presence or absence of mutant (Figure 2D, compare IP snf2 lane 11 to lanes Pho4. They behaved very similarly in all experiments; 13 and 15), but not in a wild-type strain (Figure 2D, therefore, data are only shown for PGK1. compare IP wt in lane 11 to lanes 13 and 15). In all cases Acetylation of histones H3 and H4 was determined immunoprecipitated DNA was compared to Input DNA using antibodies against H3 acetylated on lysines 9 and (only Input DNA from the snf2 strain is shown in Figures

3 Nucleosome Acetylation and Remodeling In Vivo 531 Figure 1. Histone Acetylation Levels at the PHO8 Promoter upon Chromatin Remodeling and Transcriptional Activation (A) Positions of the PCR fragments used for ChIP analysis of the PHO8 promoter are shown with respect to the nucleosomal organization of the repressed and activated promoter. Stable nucleosomes (filled circles), partially stable (shaded circles), and unstable nucleosomes (open circles) are indicated (Barbaric et al., 1992). (B) Strains CY338 ( pho4) and CY337 (wt) were phosphate starved for 16 hr and the chromatin fixed by formaldehyde treatment. Chromatin fragments were precipitated with an antibody specific for diacetylated histone H3. The amounts of coimmunoprecipitated DNA determined by quantitative PCR were normalized to the respective Input DNA and are shown in arbitrary units. (C) H4 acetylation in strains CY338 and CY337 was determined by ChIP analysis employing an antibody against tetraacetylated histone H4. 2C and 2D), which gave very similar signals for all chro- moter. However, in this strain the SWI/SNF complex mosomal regions examined (Figure 2D, Input lanes 9, cannot be assembled, since Snf2 is necessary for its 11, 13, and 15). Acetylation of histone H3 follows exactly structural integrity (Richmond and Peterson, 1996). the same pattern (Figure 2A), showing a strong peak of Thus, in the absence of the SWI/SNF complex, any com- acetylation at the PHO8 promoter in the absence of petition between SAGA and SWI/SNF for Pho4 could be Snf2. Compared to remodeled chromatin of the wildtype thrown out of balance, allowing SAGA to gain access strain, histone acetylation of the upstream and to Pho4 more effectively and thereby hyperacetylate the proximal PHO8 promoter is increased by a factor of two. promoter chromatin. This, however, would not reflect The slightly increased amount of amplified DNA from the wild-type situation, in which both complexes are the proximal PHO8 promoter compared to the upstream intact and recruitable to the PHO8 promoter via Pho4. promoter in the wild-type strain (Figure 2C, compare IP To rule out that the observed acetylation peak is only wt lanes 7 and 8 with lanes 5 and 6) was also observed due to abnormally high SAGA occupancy of the PHO8 with Input DNA from this strain (data not shown) and promoter, we investigated acetylation levels in a strain therefore is likely to reflect different primer efficiencies carrying the Snf2 point mutation K798A under phos- in this particular experiment. These results demonstrate phate starvation conditions. This mutant is unable to that activation of the PHO8 promoter in a snf2 strain bind or hydrolyze ATP, thus crippling the chromatin re- leads to an intermediate state in the course of chromatin modeling activity of the SWI/SNF complex (Laurent et opening, in which the promoter is strongly hyperacetylated. al., 1993; Côté et al., 1994). With respect to PHO8 remod- eling ability, there is no difference between the SNF2 null mutant and the SNF2 (K798A) allele, in both cases Hyperacetylation of the Induced PHO8 Promoter Is remodeling is blocked after Pho4 binding (Gregory et Also Observed in the Presence of a Structurally Intact al., 1999). SWI/SNF Complex Defective for Its ATPase Activity Figure 3A shows that in the K798A point mutant there In the previous experiment, a SNF2 null mutant was is a prominent peak of histone H3 acetylation across employed to obtain the conditions necessary to observe the PHO8 promoter compared to the wild-type strain. the transient hyperacetylation peak at the PHO8 pro- In fact, there is virtually no difference in acetylation levels

4 Molecular Cell 532 Figure 2. The PHO8 Promoter Is Highly Acetylated in a snf2 Strain upon Phosphate Starvation (A) Strains CY338 ( pho4), CY407 ( snf2), and CY337 (wt) were subjected to ChIP analysis after phosphate starvation. Acetylation levels of histone H3 (A) and H4 (B) were determined at PHO8 and the PGK1 promoter. (C and D) DNA quantification by ethidium bromide gel electrophoresis following PCR. DNA from the snf2 strain was purified from the wholecell extract before immunoprecipitation and subjected to quantitative PCR (Input). Antibodies against diacetylated H3 (C) and tetraacetylated H4 (D) were used for ChIP analyses of the snf2 (IP snf2) and wild-type (IP wt) strain. Each combination of IP DNA and Input DNA with each specific primer pair was analyzed using two different concentrations of the respective template DNA (2.5-fold dilution of template DNA in each even numbered lane) to confirm that the PCR reaction was in the linear range. between the SNF2 null and the point mutant at all regions to the phosphate starvation signal at the PHO8 promoter. examined. This result is clearly apparent from the quantification We therefore compared histone acetylation of of immunoprecipitated DNA from the snf2 and the PHO8 promoter in strains deleted for PHO4 that snf2k798a strain (Figure 3B, compare IP snf2 and IP either contained or lacked SWI/SNF to monitor for acti- snf2k798a in lanes 1, 3, 5, and 7). Likewise, H4 acetyla- vator independent effects on histone acetylation. tion levels are indistinguishable between the SNF2 null Figures 3C and 3D show that no significant differences and mutant strains at the proximal PHO8 promoter. Only in histone H3 acetylation between the pho4, snf2 double at the upstream promoter does H4 show a slight reduction and the pho4 single mutant are observed at the in acetylation in the mutant strain, which is, how- PHO8 promoter or PGK1 (Figures 3C and 3D). More ever, still significantly higher than in the wild-type strain importantly, there is almost no increase in H3 acetylation (data not shown). We conclude that the loss of an intact at the PHO8 promoter compared to the PHO8 5 region SWI/SNF complex is not the cause of the high acetylation when both SWI/SNF and Pho4 are absent from the cell levels at PHO8. Rather the absence of chromatin (compare Figure 3B, IP snf2 and snf2k798a lanes 3 remodeling allows the capture of an intermediate state and 5 with Figure 3D, IP pho4, snf2 lanes 3 and 5). in the activation process and permits the detection of The same result was obtained when histone H4 was histone hyperacetylation at the PHO8 promoter. analyzed (data not shown). We have previously shown that Pho4 is bound to the PHO8 promoter under activating Recruitment of Pho4 to the PHO8 Promoter conditions even when SWI/SNF is absent and the Is a Prerequisite for the Generation of promoter chromatin frozen in the inactive state. The new Hyperacetylated Promoter Chromatin results show that this promoter occupancy by Pho4 is in the Absence of SWI/SNF a prerequisite for histone hyperacetylation at PHO8. We wanted to address the question of whether the observed increase in histone acetylation upon starving The Absence of Gcn5 HAT Activity Also Prevents snf2 cells for phosphate was dependent on the presence Remodeling upon Phosphate Starvation of the Pho4 activator at the promoter. An answer yet Does Not Generate Hyperacetylated to this question was necessary to distinguish between Chromatin at the PHO8 Promoter two possibilities: (1) The increase in histone acetylation Next, we wanted to determine whether the observed might be a nonspecific response seen in the absence peak of acetylation at the PHO8 promoter requires the of SWI/SNF; or (2) It might be part of a specific response HAT activity of Gcn5. To address this question directly,

5 Nucleosome Acetylation and Remodeling In Vivo 533 Figure 3. Requirements for Histone H3 Hyperacetylation at the PHO8 Promoter (A) Strain CY397 (snf2k798a) carries a point mutation in Snf2, which abolishes the ATPase activity of SWI/SNF. Cells were induced by phosphate starvation, and acetylation of histone H3 was determined by ChIP. Acetylation levels at the PHO8 promoter and the PGK1 control promoter were compared to those of strains CY407 ( snf2) and CY337 (wt). (B) Quantification of IP DNA from strains CY397 and CY407 and Input DNA from strain CY397 on ethidium bromide gels following the quantitative PCR. (C) Histone acetylation in strains CY338 ( pho4) and CY408 ( pho4, snf2) was determined by ChIP analysis following phosphate starvation. (D) Quantification of IP DNA by ethidium bromide gel electrophoresis following PCR. Antibodies against diacetylated H3 were used for ChIP analyses of the pho4 (IP pho4) and pho4, snf2 (IP pho4, snf2) strain; only Input DNA from strain CY 338 is shown. To confirm that the PCR reaction was in the linear range, we used the protocol described in the legend to Figure 2. Rpd3 Is Not Involved in Restoring the Basal Deacetylated State at the Remodeled PHO8 Promoter Hyperacetylation of the PHO8 promoter is a transient event, since complete remodeling of the activated pro- moter results in the same acetylation level as found for the repressed state (Figures 1B and 1C). Therefore, we addressed the question of whether specifically the histone deacetylase Rpd3 is needed to erase this acetyla- tion peak and analyzed histone acetylation in a rpd3 mutant. In this mutant, PHO8 is fully activated and chro- matin at the PHO8 promoter is completely remodeled under inducing conditions (data not shown). Whereas H3 as well as H4 acetylation of the PGK1 promoter is slightly increased in the rpd3 strain compared to the wild type, there is no difference in acetylation levels at the PHO8 promoter. Importantly, there is no peak of acetylation over the PHO8 promoter (Figures 4C and 4D). We conclude that Rpd3 is not required to erase the acetylation peak at the PHO8 promoter when chromatin remodeling goes to completion. one would ideally want to determine acetylation levels in a snf2, gcn5 double mutant, but this mutant is not viable in the strain background employed. However, in the Gcn5 mutant PKM that completely lacks HAT activity (described in Gregory et al., 1998; Wang et al., 1998), chromatin remodeling of the PHO8 promoter is also blocked at a step subsequent to activator binding (Gregory et al., 1999) and in this respect resembles the intermediate activation state in a snf2 strain. Therefore, should any histone acetyltransferase different from Gcn5 be recruited to the PHO8 promoter, one would expect to find the promoter hyperacetylated even in the absence of Gcn5 HAT activity. The peak of histone H3 and H4 acetylation over the PHO8 promoter characteristic of the snf2 strain analyzed under activating conditions (Figures 4A and 4B, black bars) was completely absent in the Gcn5 HAT domain mutant (Figures 4A and 4B, gray bars). Across the PHO8 promoter and also at the adjacent upstream region, H3 and H4 acetylation was found to be at the same level or lower than in the pho4 mutant (Figures 4A and 4B, white bars). H3 acetylation at the PGK1 promoter was not affected by the absence of Gcn5 HAT activity (Figure 4A), whereas acetylation of histone H4 was significantly reduced (Figure 4B). Determination of the acetylation levels at the PDA1 promoter revealed no differences for H4 between the PKM, the wild-type, and the Pho4 mutant strain (data not shown). These results demonstrate that the observed drop of H4 acetylation at PGK1 and the PHO8 5 region was not due to a global reduction in H4 acetylation levels. The same result was obtained for a Gcn5 null mutant (data not shown). We conclude from these experiments that the observed peak of acetylation at the PHO8 promoter is completely dependent on Gcn5.

6 Molecular Cell 534 Figure 4. Effect of Gcn5 and Rpd3 upon the Acetylation of the PHO8 Promoter (A) H3 acetylation and (B) H4 acetylation of the PHO8 promoter were determined in the Gcn5 HAT domain mutant PKM (Gregory et al., 1998; Wang et al., 1998) and compared to the snf2 (CY407) and wild-type (CY337) strains. Histone acetylation of the PHO8 promoter in strain CY637 ( rpd3) is shown for histone H3 in (C) and for histone H4 in (D). Hyperacetylation of the PHO8 Promoter Is Limited to Nucleosomes that Are Remodeled upon Activation in a Wild-Type Strain We wanted to determine the actual boundaries of the acetylation peak at the PHO8 promoter with respect to the region where chromatin remodeling takes place. Therefore, we analyzed the hyperacetylated state of the PHO8 promoter in the absence of Snf2 and attempted to determine how far it extended in the upstream and downstream direction. As shown above, acetylation of the upstream promoter is increased by more than a factor of three in a snf2 strain. Acetylation does not increase at the 5 adjacent region, even though the primer pairs we used in this region determine histone acetylation at neighboring nucleosomes (Figures 1A and 5). To define the boundary of the acetylation peak toward the transcribed region of the PHO8 gene, we used a primer pair specific for the immediately adjacent PHO8 coding region that is separated by less than two nucleosomes from the proximal PHO8 promoter. Again, acetylation of histone H4, which is 2-fold increased at the proximal PHO8 promoter in the snf2 strain, is not affected at all over the PHO8 coding region and exactly at the same level as it is in the Pho4 mutant and the wildtype strain. The same sharp boundaries were obtained when histone H3 acetylation levels were analyzed across the PHO8 locus (data not shown). The fact that hyperacetylation does not extend toward nucleosomes in the PHO8 5 region or the transcribed region indicates that hyperacetylation is a highly localized event, specific to the nucleosomes that undergo the characteristic chromatin transition in the active promoter. Discussion SAGA Is Necessary and Sufficient for Hyperacetylation of the PHO8 Promoter Prior to Chromatin Remodeling Several studies have correlated acetylation of core histones with transcriptionally competent chromatin, suggesting a positive role for histone acetylation in gene transcription (Allfrey et al., 1964; for review, see Kuo and Allis, 1998). Moreover, transcriptional activation of a subset of yeast genes, including PHO8, is dependent upon Gcn5, the catalytic subunit of the histone acetyl- transferase complex SAGA. Indeed, Gcn5 is necessary but not sufficient for the opening of chromatin at the PHO8 promoter (Gregory et al., 1999). This result points to a role of Gcn5-dependent acetylation at a stage early in the activation process, preceding chromatin perturbation. Indeed, to capture Gcn5 acetylation at the pro- moter, we found it necessary to block the activation process subsequent to activator binding but before chromatin remodeling. The absence of SWI/SNF pre-

7 Nucleosome Acetylation and Remodeling In Vivo 535 Figure 5. Histone H4 Acetylation across the PHO8 Promoter The nucleosomal composition of the repressed PHO8 promoter is shown schematically (compare to Figure 1A); the area of chromatin destined for remodeling is indicated below. The black horizontal bars above show the positions of the PHO8 promoter regions analyzed and the relative degree of histone H4 acetylation levels in the strains indicated. Importantly, there is no detectable hyperacetylation at both the adjacent 5 region (nucleosomes 6, 7) nor the PHO8 coding region ( 2, 3) (Figure 5). Indeed, in agreement with the minimal contribution of UASp1 to remodeling and activation of PHO8 (Münsterkötter et al., 2000), the data summarized in Figure 5 suggest a distribution of acetylation centered around UASp2, spanning those nucleosomes that would be remodeled in the presence of both SAGA and SWI/SNF. This peak is observed in the absence of Snf2 and therefore in the absence of any nucleosome remodeling (Gregory et al., 1999) and consequently cannot be a product of the re- modeling event. Instead, our data strongly support a role for acetylation in determining the region of chromatin at the promoter that is to be remodeled in a SWI/SNFdependent manner. vents the nucleosomal remodeling normally observed at the PHO8 promoter under activated conditions (Figure 1 in Gregory et al., 1999); importantly, however, Pho4 is bound to UASp2 and potentially able to recruit any other cofactors necessary for remodeling (Gregory et al., 1999). Under these conditions, we observed a striking highly localized peak of H3 and H4 hyperacetylation over the PHO8 promoter demonstrating the recruitment of a histone acetyltransferase (Figure 2). Significantly, this peak of hyperacetylation could not be observed in a strain with a mutant Gcn5 protein lacking histone acetylase activity (Figure 4), even though chromatin remodeling is also blocked after Pho4 binding (Gregory et al., 1999). Furthermore, given the indistinguishable effects of Gcn5 (found in SAGA and Ada) and Spt7 (found in SAGA only) deletions on the activity of the PHO8 promoter in vivo (Gregory et al., 1999), SAGA is the best candidate for a HAT that is targeted to the PHO8 promoter acetylating histones at a stage prior to, and as a prerequisite for, nucleosome remodeling. Interestingly, we found the peak of H4 hyperacetylation to be as prominent as that of H3, yet SAGA has been shown to acetylate preferentially histone H3 in a nucleosomal context in vitro (Grant et al., 1997). One possibility is that another HAT with a substrate specificity biased toward histone H4 (e.g., NuA4) may be involved in acetylating the PHO8 promoter. However, its recruitment would necessarily be completely dependent upon recruitment of SAGA or of SAGA-dependent H3 acetylation. It is also possible that Gcn5 is directly responsible for both H3 and H4 acetylation, since its substrate specificity in vivo is not known. It should be mentioned that combined acetylation of both histones has been shown to be dependent on SAGA in vivo also at the HO promoter (Krebs et al., 1999). Localized Hyperacetylation Defines the Region of Chromatin Remodeling at the PHO8 Promoter The upstream and proximal PHO8 promoter primers detect a peak of acetylation in a snf2 strain upon phosphate starvation that corresponds to nucleosomes 5, 4, and 2, 1 ofthepho8 promoter, respectively. Histone Acetylation Per Se Does Not Generate Accessible Chromatin Previous findings suggest that histone acetylation can make chromatin a more amenable substrate, facilitating the access of both transactivators (Lee et al., 1993; Vettese-Dadey et al., 1996) and the transcription machinery (Ura et al., 1997; Nightingale et al., 1998) to promoter regions. Our results obtained from the PHO8 promoter point against a direct effect of acetylation on chromatin accessibility in vivo. First, we have shown that Pho4 is bound to UASp2 of the promoter in the absence of Gcn5 (Gregory et al., 1999). Thus, SAGA, and therefore ace- tylation, is not necessary for transcription factor binding at this stage. Furthermore, the most highly acetylated chromatin we have observed, namely that found under inducing conditions in the absence of SWI/SNF (Figure 2), corresponds to the least accessible structure we have measured (Gregory et al., 1999). For example, the PHO8 promoter chromatin of an induced snf2 strain is even less susceptible to nuclease cleavage than a repressed wild-type strain, resembling instead the accessibility profile of a strain deleted for the activator Pho4 (Barbaric et al., 1992; Gregory et al., 1999). Thus, although the acetylated domain spans rather accurately those nucleosomes that undergo remodeling, we con-

8 Molecular Cell 536 very similar to the repressed state or even lower. This was unexpected, since induction of the promoter results in an open chromatin structure and transcriptional activation (Gregory et al., 1999; Münsterkötter et al., 2000). Thus, although promoter hyperacetylation correlates with both the ability to, and the extent of, nucleosomal remodeling, this result demonstrates that histone hyperacetylation is not necessary for maintaining the PHO8 promoter in the remodeled and transcriptionally active state. A similarly transient peak was observed at HO (Cosma et al., 1999; Krebs et al., 1999) but not at the HIS3 promoter, since in the latter case hyperacetylation persisted following activation (Kuo et al., 1998). We have looked to see whether we could detect tran- sient hyperacetylation directly during PHO8 activation in SNF2 cells and found a minimal increase in promoter acetylation, unfortunately within the error range of these experiments. As expected, the final level was lower in agreement with the results shown for a wild-type strain after phosphate starvation in Figures 1B and 1C. This is the expected result if deacetylation in the course of remodeling occurs rapidly following the transient hyperacetylation once both processes are allowed to occur simultaneously, as is the case in wild-type cells. Indeed, acetylation turnover seems to be a rapid process, a figure of min reported for the half-life of H3 acetyla- tion in Chlamydomonas reinhardtii (Waterborg, 1998). Any model for the regulation of the PHO8 promoter has to take into account the strict requirements for Gcn5 and Snf2 for chromatin remodeling and transcriptional activation. The simplest model for the role of Gcn5 is the generation of a transient histone hyperacetylation peak quickly erased if Snf2 is present. However, this effect of Gcn5 does not exclude the possibility that ace- tylation of alternative substrates such as transcriptional cofactors or transactivators themselves, even of Pho4 directly, could contribute to the activation process. In- deed, acetylation of a number of such transcription as- sociated factors causing a change in the properties of the proteins involved has been well documented (Gu and Roeder, 1997; Imhof et al., 1997; Sartorelli et al., 1999; Martinez-Balbas et al., 2000). How is the acetylation signal erased in the presence of Snf2? An attractive idea would be that following gene activation the acetylation label is actively removed by a histone deacetylase (HDAC). Since Rpd3 has been shown to have effects on the PHO system (Vidal and Gaber, 1991; Rundlett et al., 1996), the involvement of this deacetylase seemed an attractive possibility. However, in a rpd3 mutant the activated PHO8 promoter was acetylated to a level comparable to that of a wild- type strain (Figure 4). Thus, Rpd3 is not required to reset the basal acetylation level at the PHO8 promoter. The number of other HDACs and the functional redundancy observed between these deacetylases in yeast (Rundlett et al., 1996) makes it seem possible that alternative HDACs are involved, or are able to complement, a Rpd3 deficiency. Assuming a relatively rapid half-life for acetylated residues in vivo, the eviction of the SAGA complex from the PHO8 promoter might be sufficient to explain the transient nature of the histone hyperacetylation. At the HO promoter, the peak of acetylation correlated rather precisely with the physical presence of the HAT Gcn5 clude that acetylation alone does not generate accessible chromatin. What Is the Role of Acetylation in the Remodeling Process? It has recently been proposed that histone acetylation might be a signal, marking a specific domain of chroma- tin for the action of the remodeling and/or transcription machinery (Strahl and Allis, 2000). Support for the con- cept of an acetylation signal in the remodeling process stems from the fact that Pho4 is bound to the promoter in the absence of Gcn5 and is highly likely to interact independently with the SWI/SNF complex, as has been shown for the acidic activator Gcn4 (Natarajan et al., 1999; Syntichaki et al., 2000). Indeed, the proposed in- teraction between Pho4 and SWI/SNF is evidenced by a limited region of chromatin perturbation detectable at the upstream PHO8 promoter in the absence of Gcn5 (Gregory et al., 1999). (For a side-by-side comparison of the four chromatin states at the PHO8 promoter rele- vant for this work, wt and P i, gcn5 and snf2 at P i, see Supplemental Figure 1 at org/cgi/content/full/7/3/529/dc1). Importantly, the up- stream perturbation seen in the gcn5 strain is not prop- agated to the proximal promoter. Thus, in the absence of an acetylated chromatin template, SWI/SNF-dependent remodeling is completely restricted to the vicinity of the Pho4 binding site. Furthermore, SAGA-dependent histone hyperacetylation labels precisely those nucleo- somes that are to be remodeled in the presence of SWI/ SNF. Combining these results, we conclude that at the PHO8 promoter histone hyperacetylation does not weaken histone DNA interactions, but rather provides a signal for the remodeling of chromatin by the SWI/SNF ATPase. Why should the absence of acetylation physically restrict remodeling by SWI/SNF? Recent evidence has indicated that the conserved bromodomain of PCAF preferentially binds to acetylated lysine residues of the histone tails (Dhalluin et al., 1999). To date, this is the only protein domain that is known to differentiate between acetylated and nonacetylated histone tails. It is worthy of mention that Snf2 also contains a bromodo- main and that this complex may therefore read the acetylation status of the promoter using this domain. Experiments are in progress to determine whether the Snf2 bromodomain is indeed required for the propagation of this activity into proximal promoter chromatin. Thus, in this model acetylation of promoter histones would define the region remodeled by SWI/SNF after its recruitment. However, it remains a possibility that promoter acetylation is necessary to stably recruit the general transcription machinery to the promoter. Under these circumstances a ternary complex of activator, SWI/SNF, and basal machinery would effect complete chromatin remodeling and activation. Hyperacetylation Is a Transient State in the Remodeling Process and Is Lost upon Complete Chromatin Remodeling Although a peak of acetylation could be demonstrated prior to the remodeling process, we found the histone acetylation levels of the activated PHO8 promoter to be

9 Nucleosome Acetylation and Remodeling In Vivo 537 on the promoter, whereas loss of hyperacetylation saw Antibodies against acetylated histones H3 and H4 were purchased a simultaneous loss of Gcn5 from the promoter (Cosma from Upstate. Immunoprecipitated DNA was analyzed by quantitative PCR using et al., 1999; Krebs et al., 1999). Moreover, since the the following primers: PGK1-A: 5 -TCAAGTCCAAATCTTGGACA presence of the SWI/SNF complex precludes the cap- GAC-3 ; PGK1-B: 5 -CTTTTCTTCTAACCAAGGGGGTG-3 ; PDA1-A: ture of the hyperacetylation peak at PHO8 (see Figures 5 -GACATTTACCCGGTTGAGTAAGG-3 ; PDA1-B: 5 -ATGGACTT 1 and 2), either the SWI/SNF-dependent remodeling or CAGACATGTCCGAGT-3 ; PHO8 5 region-a: 5 -TGAAGAATCCA simply the physical presence of an intact SWI/SNF com- AGGCTCTGAAAGC-3 ; PHO8 5 region-b: 5 -GGAAGAAACAGCT plex might have been responsible for the loss of histone TTGGTGACTGC-3 ; upstream PHO8 prom.-a: 5 -CCGTCGAATG GTATTGTGTAGAGC-3 ; upstream PHO8 prom.-b: 5 -GAGTGGAA acetylation. Indeed, recent work connecting SWI/SNF- CTGCTTGCGAATATGG-3 ; proximal PHO8 prom.-a: 5 -ATGTTT like remodeling activities with deacetylases in higher ATGTAGCCACTTGCTGGC-3 ; proximal PHO8 prom.-b: 5 -TGAA eukaryotes (Tong et al., 1998; Xue et al., 1998; Wade et GTACAAGTTAGCGAGCTACG-3 ; PHO8 coding region-a: 5 -CAA al., 1999; Zhang et al., 2000) provided a precedent for CATGTGAACTGAATGAAGCCG-3 ; PHO8 coding region-b: 5 -ACA such combined action. However, we observed an indisa CTTGACGAGCATTTTATCGGG-3 ; PCR products were dissolved on tinguishable peak of promoter acetylation from a strain 2.5% agarose gel, visualized with ethidium bromide, and quantified in which the intact SWI/SNF complex is unable to remodel with the ImageMaster system (Amersham Pharmacia Biotech). chromatin due to a point mutation within the Snf2 ATPase (Figure 3A). This result suggests that promoter Acknowledgments deacetylation requires more than only the physical pres- We would like to thank Craig Peterson, David Allis, and Michael ence of the SWI/SNF complex but also its functionality. Grunstein for strains/protocols, antibodies, and communicating These data lend support to models in which the ATPase methods, respectively; Shelley Berger for the Gcn5PKM mutant DNA; and Peter Becker and members of his laboratory for careful activity of the SWI/SNF complex is responsible for either reading of the manuscript. Excellent assistance was provided SAGA eviction or facilitating HDAC action at the proby Andrea Schmid. This work was supported by grants from the moter. Experiments are currently underway to differenti- European Commission Human Capital and Mobility Network ate between these possibilities. (ERBCHRXCT940447), the Deutsche Forschungsgemeinschaft Why might the cell deacetylate/reset an active promoter? (SFB190), and Fonds der Chemischen Industrie to W. H. Although in the regulation of gene expression the process of how to go from the off state to the on Received September 25, 2000; revised February 15, or open state is an essential step, switching back from the on to the off state is an equally important step References in the process of proper and timely gene regulation. In Allfrey, V.G., Faulkner, R., and Mirsky, A.E. (1964). Acetylation and this context, it is essential to release the machinery methylation of histones and their possible role in the regulation of from the chromatin prior to shutdown, especially since RNA synthesis. Proc. Natl. Acad. Sci. USA 51, histone hyperacetylation is not necessary for mainteweakly Barbaric, S., Fascher, K.D., and Hörz, W. (1992). Activation of the nance of the PHO8 promoter in the remodeled configuration regulated PHO8 promoter in S. cerevisiae: chromatin transi- tion and transcriptionally active state (Figure 1). 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