competition reveals processive PNAS PLUS by the SAGA HAT module

Alison E. Ringela, Anne M. Cieniewiczb,c, Sean D. Tavernab,c, and Cynthia Wolbergera,c,1

aDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205; bDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and cCenter for , Johns Hopkins University School of Medicine, Baltimore, MD 21205

Edited by Carl Wu, Howard Hughes Medical Institute, Ashburn, VA, and approved September 1, 2015 (received for review April 30, 2015) The Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex acetyltransferase (HAT) complexes also contain reader hyperacetylates histone tails in vivo in a manner that depends modules that recognize the H3K4me3 mark (16, 17). upon histone 3 4 trimethylation (H3K4me3), a histone mark The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is a enriched at promoters of actively transcribed genes. SAGA contains highly conserved transcriptional coactivator (18) that is involved in a separable subcomplex known as the histone acetyltransferase the transcription of nearly all yeast genes (19, 20) and mediates (HAT) module that contains the HAT, Gcn5, bound to Sgf29, Ada2, crosstalk between H3K4me3 and histone hyperacetylation. The and Ada3. Sgf29 contains a tandem Tudor domain that recognizes HAT activity of SAGA resides in a four-protein subcomplex H3K4me3-containing peptides and is required for histone hyper- known as the “HAT module” (21, 22), which contains the catalytic acetylation in vivo. However, the mechanism by which H3K4me3 subunit Gcn5 (23–25), Ada2, Ada3 (26, 27), and Sgf29 (21, 28). recognition leads to lysine hyperacetylation is unknown, as in The association of Gcn5 with the other HAT module subunits vitro studies show no effect of the H3K4me3 modification on modulates Gcn5 activity and specificity. Gcn5 on its own acetylates histone peptide acetylation by Gcn5. To determine how H3K4me3 free but not (22, 23, 25) and preferentially binding by Sgf29 leads to histone hyperacetylation by Gcn5, we modifies at K14 (25, 29). A complex containing Gcn5 used differential fluorescent labeling of histones to monitor acety- bound to Ada2 and Ada3 is more active overall and modifies lation of individual subpopulations of methylated and unmodified nucleosomal substrates (22, 25). Binding to Ada2/Ada3 also nucleosomes in a mixture. We find that the SAGA HAT module broadens the lysine specificity of Gcn5 to acetylate multiple lysine preferentially acetylates H3K4me3 nucleosomes in a mixture con- residues on the histone H3 tail (22, 25). Sgf29 contains a tandem taining excess unmodified nucleosomes and that this effect requires Tudor domain that binds to H3K4me3 (17, 30) and is required for the Tudor domain of Sgf29. The H3K4me3 mark promotes processive, maintaining wild-type levels of histone H3 acetylation in vivo multisite acetylation of histone H3 by Gcn5 that can account for the (30–32). However, in vitro studies comparing acetylation kinetics different acetylation patterns established by SAGA at promoters by the SAGA complex on peptide substrates in the presence or versus coding regions. Our results establish a model for Sgf29 absence of Sgf29 do not show a rate enhancement when the

function at gene promoters and define a mechanism governing H3K4me3 modification is present (30). Because these experi- BIOCHEMISTRY crosstalk between histone modifications. ments were limited in scope to peptide substrates and measured acetylation using relatively insensitive end-point assays (30), acetyltransferase | histone crosstalk | histone modifications | the full impact of H3K4me3 on HAT module activity may transcription | coactivator Significance he different patterns of histone posttranslational modifications Tdistributed across the genome are proposed to constitute a Crosstalk between histone modifications regulates transcription “” that orchestrates distinct transcriptional programs by establishing spatial and temporal relationships between his- by recruiting specific effector proteins (1–4). The phenome- tone marks. Despite discoveries of reader domains that physically non of histone code “crosstalk,” whereby one type of histone associate with -modifying , the mechanisms by modification directs the establishment of another, or through which recognition of one modification triggers other kinds of which several histone modifications are recognized in tandem, has modifications have remained elusive. Gcn5 is the catalytic subunit emerged as an important and widespread mechanism regulating of the Spt-Ada-Gcn5 acetyltransferase (SAGA) histone acetyl- chromatin-templated processes (5, 6). The multifunctional com- transferase (HAT) module, which also recognizes histone 3 lysine 4 plexes that activate transcription are thought to mediate crosstalk trimethylation (H3K4me3) through the tandem Tudor domain- through “reader” domains that recognize particular chromatin containing protein Sgf29. Although previous studies could not marks as well as through catalytic subunits that deposit or remove connect H3K4me3 recognition to differences in acetylation by histone modifications (5, 7, 8). As a result, distinct combinations of Gcn5, we report enhanced processivity by the HAT module on histone posttranslational modifications that correlate with tran- methylated substrates using a previously unpublished histone scriptional output cluster across the genome (9–11). High levels of color-coding assay. Our work defines a mechanism for histone histone 3 lysine 4 trimethylation (H3K4me3) and histone H3 crosstalk that may account for genome-wide patterns of Gcn5- hyperacetylation are present at the promoters of actively tran- mediated acetylation. scribed genes (9, 11, 12), and multiple lines of evidence support the Author contributions: A.E.R., S.D.T., and C.W. designed research; A.E.R. and A.M.C. per- existence of regulatory mechanisms coupling the two modifica- formed research; A.E.R., A.M.C., S.D.T., and C.W. analyzed data; and A.E.R. and C.W. tions. Studies using tandem mass spectrometry to sequence whole wrote the paper. histone tails have shown that H3K4me3 is highly correlated with The authors declare no conflict of interest. hyperacetylation of the same H3 tail (13, 14). Deleting the yeast This article is a PNAS Direct Submission. Set1 methyltransferase, which trimethylates H3K4, leads to dra- Freely available online through the PNAS open access option. matically lower levels of histone H3 tail acetylation overall (13, 15). 1To whom correspondence should be addressed. Email: [email protected]. These results are consistent with a role for H3K4 in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. triggering hyperacetylation of histone H3; indeed, a number of 1073/pnas.1508449112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1508449112 PNAS | Published online September 23, 2015 | E5461–E5470 Downloaded by guest on September 28, 2021 HAT module and variants lacking either the Sgf29 tandem Tudor domain or the Gcn5 bromodomain were expressed as complexes in Escherichia coli and purified to homogeneity (Fig. 1). We first confirmed that S. pombe Sgf29 interacts specifically with H3K4me3 peptides in a pull-down assay. As shown in Fig. 2A, streptavidin beads coated with H3K4me3 peptides efficiently re- tain Sgf29, whereas unmodified H3 peptides do not. To determine the effect of H3K4me3 on the kinetics of pep- tide acetylation by Gcn5, we measured initial acetylation rates by the HAT module at increasing concentrations of either un- modified or H3K4me3 peptides in the presence of saturating amounts of acetyl-CoA. Compared with unmodified peptides, Fig. 1. Domain architecture of HAT module complexes. (Left) SDS/PAGE gel the presence of the H3K4me3 modification increases kcat/Km by of purified S. pombe HAT module complexes containing different domain 3.1-fold (Fig. 2B and Table 1). This change in kcat/Km is caused truncations in which all four subunits are present in roughly equimolar solely by a decrease in Km from 250 ± 40 μMto77± 10 μM, with quantities. (Right) Location of the catalytic domain, tandem Tudor domain, no corresponding change in kcat (Fig. 2B and Table 1). Deletion and bromodomain within the HAT module. of the Sgf29 tandem Tudor domain (ΔTudor) eliminates the difference in acetylation kinetics on H3K4me3 versus un- be much greater than previously determined. Thus, although modified peptides (Fig. 2C and Table 1), as is consistent with a k K multiple lines of evidence suggest that H3K4me3 regulates role for H3K4me3 binding in the observed difference in cat/ m. histone acetylation by SAGA in yeast and mammalian cells (17, We also ruled out the possibility that the H3K4me3 modification k K D 30, 31, 33), in vitro studies have failed to explain how H3K4me3 affects cat or m for acetyl-CoA (Fig. 2 and Table 2); our recognition by Sgf29 gives rise to different patterns of histone acet- finding is consistent with the proposed Gcn5 reaction mecha- ylation. As a result, the underlying mechanism governing crosstalk nism, in which the cofactor binds before the peptide (34, 35). between H3K4 trimethylation and acetylation by SAGA remains To confirm that acetylation by Gcn5 is not directly affected by poorly understood. truncating the Sgf29 tandem Tudor domain, we compared kcat K Δ To elucidate the mechanism underlying crosstalk between HAT and m values of wild-type and Tudor HAT module complexes module binding to H3K4me3 and histone acetylation, we devel- for unmodified histone peptides and nucleosomes. Because the oped a histone color-coding assay for measuring acetylation rates kinetic parameters for the two complexes are nearly the same on distinct histone populations within a single mixture of meth- for peptides (Table 1) and nucleosomes (Table 3), differences ylated and unmodified nucleosomes. We find that the HAT observed with the ΔTudor HAT module must be related to module preferentially acetylates H3K4me3 nucleosomes in a H3K4me3 recognition. We note that the rate enhancement ob- mixture and that this preference depends on the Sgf29 tandem served resulting from H3K4me3 differs from the results of an Tudor domain. Compared with unmodified nucleosomes, acety- earlier study that had found no role for Sgf29 in governing lation by the HAT module is processive on nucleosomes con- SAGA activity on H3K4me3 versus unmodified peptides in vitro taining the H3K4me3 modification, thereby triggering histone H3 (30). However, the previously reported conclusions were drawn hyperacetylation. These data reveal the mechanism by which the from end-point assays (30), whereas the data presented here are Sgf29 tandem Tudor domain within the SAGA HAT module fa- fit from a full steady-state titration and therefore are more cilitates crosstalk between Gcn5 acetyltransferase activity and sensitive to differences in kinetic parameters. H3K4me3 recognition and provide an explanation for how SAGA can establish different patterns of acetylation at gene promoters H3K4me3 Stimulates HAT Module Activity on Nucleosomes. The ex- versus coding regions. periments described above and previously reported studies (30) used peptide substrates, leaving open the possibility that H3K4 Results trimethylation might have a greater impact on acetylation by the Effect of H3K4me3 on Histone Peptide Acetylation. To investigate SAGA HAT module in a nucleosomal context. We therefore how H3K4 trimethyl recognition may contribute to HAT module generated nucleosomes containing recombinant full-length his- activity on methylated versus unmethylated substrates, we used tone H3 that was homogeneously modified with H3K4me3. To recombinant HAT module from Schizosaccharomyces pombe, incorporate this modification, we performed -mediated li- which can be isolated as a stable four-protein complex con- gation using an engineered variant ofthebacterialtranspeptidase, taining Sgf29, Gcn5, Ada2, and Ada3. The full-length S. pombe Sortase A (F40 SrtA) (36, 37), which splices an N-terminal

Fig. 2. The presence of H3K4me3 increases overall acetylation by the HAT module on peptides. (A) Pull-down using biotinylated histone peptides containing H3K4 trimethylation and Sgf29. (B–D) Steady-state kinetic titrations using an enzyme-coupled assay comparing the activity of the wild-type HAT module on unmodified versus H3K4me3 peptides (B), the ΔTudor HAT module on unmodified versus H3K4me3 peptides (C), or the wild-type versus ΔTudor HAT module (D) using a constant amount of H3K4me3 peptide and varying the acetyl-CoA concentration.

E5462 | www.pnas.org/cgi/doi/10.1073/pnas.1508449112 Ringel et al. Downloaded by guest on September 28, 2021 Table 1. Summary of steady-state kinetic parameters fit from titrations in which the PNAS PLUS concentration of the histone H3 amino acids 1–21 peptide was varied and the acetyl-CoA concentration was held constant at 100 μM

−1 −1 −1 HAT module Histone H3 peptide amino acids 1–21 kcat,s Km, μM kcat/Km, μM ·s

Wild type Unmod 2.5 ± 0.1 250 ± 40 0.010 ± 0.002 ΔTudor Unmod 2.5 ± 0.1 210 ± 30 0.012 ± 0.002 Sgf29 T202A/Y205V Unmod 3.4 ± 0.2 320 ± 50 0.011 ± 0.002 ΔBromodomain Unmod 1.8 ± 0.07 380 ± 40 0.0047 ± 0.001 Wild type H3K4me3 2.4 ± 0.08 77 ± 10 0.031 ± 0.004 ΔTudor H3K4me3 2.5 ± 0.09 190 ± 20 0.013 ± 0.001

synthetic peptide onto recombinant histone H3 (Fig. S1A) to yield the presence of the methyl modification (Fig. S3). Therefore, fully native histone H3 uniformly trimethylated at H3K4 (Fig. H3K4me3 recognition does not change the intrinsic specificity of S1B). We confirmed the presence of the trimethyl modification Gcn5 for catalyzing H3K9 and H3K14 acetylation, instead using Western blotting with an antibody specific for the H3K4me3 stimulating overall acetylation of histone H3. In agreement with modification (Fig. S1C)aswellasMALDI-TOFmassspec- our results, deleting Sgf29 in yeast results in global reductions in trometry (Fig. S2). The modified histone H3 was reconstituted acetylation at multiple positions on histone H3 (30). with unmodified histones H2A, H2B, and H4 into nucleosome core particles, thus generating a pool of nucleosomes uniformly Differential Labeling Technique for Studying Mixtures of Post- trimethylated at H3K4. translationally Modified Substrates. Chromatin in vivo is hetero- To test whether H3K4 trimethylation affects acetylation by the geneous with respect to the set of posttranslational modifications HAT module in a nucleosomal context, we used a radioactive found on each nucleosome. For an enzyme complex such as filter-binding assay to measure steady-state rates of acetylation the HAT module, which contains multiple chromatin-binding as a function of nucleosome concentration. We find that the Km domains (16, 41), the ability to acetylate nucleosomes selectively for nucleosomes drops from 1.9 ± 0.3 μM for unmodified nu- with particular combinations of histone modifications may be as cleosomes to 0.41 ± 0.05 μM for H3K4me3 nucleosomes (Fig. 3A important as the overall enzymatic activity. Although multiple and Table 3), whereas kcat stays roughly the same, corresponding to biochemical assays yield bulk acetylation rates (42), there are no an ∼3.5-fold increase in the specificity constant kcat/Km (Table 3). reported assays for selectively monitoring the acetylation kinetics The Km for nucleosomes is almost two orders of magnitude lower of different types of substrates in the context of a mixture. We than that for peptides, indicating that the HAT module binds therefore devised an approach that combines differential fluo- far more tightly to nucleosomes than to peptides. Nonetheless, rescent labeling of histones with acid–urea gel electrophoresis to the 3.5-fold increase in kcat/Km caused by the H3K4me3 mod- quantitate HAT module activity on both modified and unmodified A ificationinanucleosomalcontext(Fig.3 and Table 3) is only nucleosomes within the same reaction mixture. BIOCHEMISTRY modestly greater than the 3.1-foldincreaseinspecificityob- To track histone-specific acetylation, we reconstituted nucle- served with peptides (Fig. 2B and Table 1). osomes whose histone H3 was color-coded according to whether Because radioactive filter-binding assays report on total acetyla- it contained the H3K4me3 modification. Methylated H3 was tion and do not distinguish differences among histones, we com- generated using F40 SrtA as described above; the fluorescent pared histone acetylation patterns on unmodified versus H3K4me3 label was incorporated by introducing a Q125C substitution in nucleosomes using acid–urea gels, which separate histones based on histone H3 that could be labeled with a dye of choice. Un- molecular weight as well as the number of acetylated lysine residues modified histone H3 was labeled with Alexa Fluor 647, and (38). At early time points the HAT module acetylates H3K4 H3K4 trimethylated histone H3 was labeled with Alexa Fluor trimethylated histone H3 in nucleosomes more quickly than 488 (Fig. S4A). After the SAGA HAT module was incubated unmodified nucleosomal histone H3 (Fig. 3B). Thirty seconds with the nucleosomes (Fig. S4B) and the reaction products were after the reaction is initiated, bands corresponding to three acet- resolved on acid–urea gels, the products corresponding to one or ylation sites are evident on the H3K4me3 histone, but only two the other color-coded histone could be visualized selectively on a acetylation sites are populated for unmodified histone H3 (Fig. laser scanner using excitation wavelengths and emission filters 3B). Although the HAT module also acetylates histone H4, the for one or the other fluorophore (Fig. S4C). In this way, acety- rate of histone H4 acetylation is insensitive to H3K4 trimethyla- lation of methylated versus unmethylated H3 can be quantitated tion (Fig. 3B). This pattern is consistent with a mechanism in cis, within a single mixture. whereby recognition of the methyl mark stimulates acetylation on the same histone tail. Sgf29 Mediates Selective Acetylation of H3K4me3 Nucleosomes in a To examine whether recognition of the H3K4me3 modifica- Heterogeneous Mixture. To see whether the HAT module pref- tion changes the intrinsic lysine specificity of the Gcn5 subunit, erentially acetylates nucleosomes containing H3K4 trimethyla- which preferentially catalyzes H3K14 acetylation in vitro (29, 39) tion, we used the color-coding method to assay histone H3 acetylation and H3K9 acetylation in vivo (20, 40), we probed the reaction in mixtures of methylated and unmodified nucleosomes. Because products using antibodies that recognize specific acetylated ly- sine residues. Antibodies to the H3K9ac and H3K14ac modifi- cations recognize five bands on acid–urea gels, corresponding to Table 2. Summary of steady-state kinetic parameters fit from titrations in which the concentration of the H3K4me3 amino one through five modification sites per histone, whereas anti- – μ bodies to H3K18ac and H3K23ac recognize only the top four acids 1 21 peptide was held at 400 M and the acetyl-CoA bands (Fig. S3). This pattern reveals that singly acetylated his- concentration was varied −1 −1 −1 tones are predominately modified at H3K9 or H3K14 on both HAT module Substrate kcat,s Km, μM kcat/Km, μM ·s unmodified and H3K4me3 substrates. Although H3K4 trimeth- ± ± ± ylation enhances the rate of H3K9 acetylation at early time Wild type Ac-CoA 1.7 0.1 3.4 0.7 0.50 0.1 ΔTudor Ac-CoA 1.8 ± 0.1 2.8 ± 0.3 0.64 ± 0.08 points, acetylation at all the other tested changes little in

Ringel et al. PNAS | Published online September 23, 2015 | E5463 Downloaded by guest on September 28, 2021 Table 3. Summary of steady-state kinetic parameters fit from acetylated at equal rates (Fig. S5C). Together, these results show titrations in which the concentration of the nucleosome was that when nucleosomes harboring different histone marks com- varied and the acetyl-CoA concentration was held constant pete for the HAT module active site, the interaction between the at 25 μM Sgf29 tandem Tudor domain and the H3K4me3 modification fa- −1 −1 −1 cilitates preferential acetylation of methylated nucleosomes. HAT module Nucleosome kcat,s Km, μM kcat/Km, μM ·s To rule out the possibility that H3K4me3 recognition acts in Wild type Unmod 1.3 ± 0.1 1.9 ± 0.3 0.68 ± 0.1 trans by stimulating overall HAT module activity, we assayed ΔTudor Unmod 1.9 ± 0.2 2.8 ± 0.5 0.68 ± 0.1 nucleosome acetylation in the presence of an added histone H3 Wild-type H3K4me3 1.0 ± 0.05 0.41 ± 0.05 2.4 ± 0.3 tail peptide containing the H3K4me3 modification. Peptide was added at a concentration 10-fold higher than the reported dis- sociation constant of methylated peptide for Sgf29 (30) and H3K4 trimethylation increases the kcat/Km for the HAT module by nucleosomes were held at a concentration matching the amount around fourfold (Fig. 3A and Table 3), the HAT module should of H3K4me3 substrate in our experiments using nucleosome acetylate H3K4me3 and unmodified nucleosomes at the same rate mixtures (Fig. 4). As shown in Fig. S6, the added peptide did not in mixtures containing a fourfold molar excess of unmodified increase HAT module activity on unmodified (Fig. S6 A and B) nucleosomes (43). However, in assays of SAGA HAT module or H3K4me3 nucleosomes (Fig. S6 C and D), ruling out trans activity using a 4:1 molar ratio of unmodified versus methylated stimulation of Gcn5 by H3K4me3 histone tails. To control for nucleosomes, we unexpectedly found that the HAT module fluorophore-specific effects on acetylation by the HAT module, preferentially acetylates the methylated nucleosomes despite we compared HAT module activity on otherwise unmodified the presence of a fourfold molar excess of unmodified nucle- nucleosomes containing histone H3 labeled with either Alexa osomes (Fig. 4A). An overlay of images corresponding to each Fluor 488 or Alexa Fluor 647. The HAT module acetylates these labeled substrate reveals that nucleosomes with the H3K4me3 two nucleosomes at the same rate in homogenous preparations modification are acetylated to completion within the first 5 (Fig. S7 A and B) and in mixtures containing varying proportions minutes, whereas unmodified nucleosomes still are not fully of the Alexa Fluor 488- or Alexa Fluor 647-labeled nucleosomes acetylated at the last time point (Fig. 4A). For the SAGA HAT (Fig. S7 C and D), indicating that the choice of fluorescent label module lacking the Tudor domain, the pattern of acetylation is does not influence the observed acetylation patterns. the same for both methylated and unmethylated nucleosomes (Fig. 4B). Because there is fourfold less H3K4me3 substrate in the Sgf29 Promotes Processive Acetylation by the SAGA HAT Module on reaction, this finding indicates that complexes lacking the tandem H3K4me3 Nucleosomes. What mechanism links H3K4me3 recog- Tudor domain have lost the ability to distinguish between meth- nition by Sgf29 at gene promoters to hyperacetylation by Gcn5? ylated and unmodified nucleosomes. To verify that deletion of the When provided with competing substrates, the HAT module Sgf29 tandem Tudor domain did not disrupt the overall integrity clearly modifies H3K4me3 nucleosomes first, even under con- and activity of the HAT module, we introduced point mutations in ditions in which one expects to see equal rates of acetylation the Tudor domain predicted to disrupt binding to a methylated (Fig. 4A and Table 3). We hypothesized that H3K4me3 binding peptide based on structures of the human and yeast Sgf29 tandem by Sgf29 might promote processive acetylation of methylated Tudor domains bound to H3K4me3 peptides (30). We mutated nucleosomes, with the HAT module acetylating multiple lysines T202 to alanine and Y205 to valine (T202A/Y205V), thereby before dissociating from the nucleosome. To test for processive eliminating two hydrogen bonds between Sgf29 and the H3 acetylation by Gcn5, we used the reaction scheme diagrammed backbone and disrupting the aromatic cage that surrounds the in Fig. 5A. We incubated the HAT module with labeled nucle- methyl lysine (Fig. S5A). In pull-down assays, we find that isolated osome for a short period and then added a 20-fold molar excess Sgf29 containing the T202A/Y205V substitutions binds more of unlabeled, unmodified competitor nucleosome. As controls, weakly to H3K4me3 peptides than does the wild-type protein (Fig. either a portion of the initial reaction was quenched with acetic S5B). When incorporated into the HAT module, the effect of acid, which halts all enzymatic activity, or buffer alone was added mutating Sgf29 mimics that of a full Tudor domain truncation, in (mock quench), which allows the reaction to proceed (Fig. 5A). The which mixtures of methylated and unmodified nucleosomes are resulting pattern of acetylation on fluorescently labeled histone H3

Fig. 3. The presence of H3K4me3 increases overall acetylation by the HAT module on nucleosomes. (A) Steady-state kinetic titrations comparing wild-type HAT module activity on unmodified versus H3K4me3 nucleosome core particles (NCPs) using a radioactive filter-binding assay. Replicates are colored with different shades of purple (wild-type) or green (H3K4me3). (B) HAT reaction time course on unmodified versus H3K4me3 NCPs resolved on an acid–urea gel and stained for total protein using SyproRuby stain.

E5464 | www.pnas.org/cgi/doi/10.1073/pnas.1508449112 Ringel et al. Downloaded by guest on September 28, 2021 PNAS PLUS

Fig. 4. The HAT module preferentially acetylates nucleosomes harboring H3K4me3 out of a mixture. (A) Fluorescent images of a HAT reaction containing a 4:1 BIOCHEMISTRY molar ratio of differentially labeled unmodified versus H3K4me3 nucleosomes resolved on an acid–urea gel. (Left) Merged image with unmodified nucleosomes colored green and H3K4me3 nucleosomes colored red. (Right) Individual images used to generate the merged image. (B) Fluorescent images of a ΔTudor HAT reaction containing a 4:1 molar ratio of differentially labeled unmodified versus H3K4me3 nucleosomes resolved on an acid–urea gel. (Left)Mergedimagewith unmodified nucleosomes colored green and H3K4me3 nucleosomes colored red. (Right) Individual images used to generate the merged image.

then was visualized using acid–urea gels. For a distributive (non- peptide has little effect on overall rates of acetylation by the HAT processive) enzyme, the acetylation pattern in the presence of module (Fig. S6D), HAT module processivity is reduced signifi- competitor should mimic the chemical quench, because the enzyme cantly. This behavior indicates that Sgf29 promotes processive should dissociate readily from the labeled substrate and acetylate acetylation by the SAGA HAT module on H3K4me3 nucleosomes. the unlabeled competitor instead. A processive enzyme should re- To probe whether processive acetylation of H3K4me3 nucle- main bound to the labeled substrate, even in the presence of added osomes depends on the interaction between the Sgf29 tandem competitor, thus allowing further acetylation of the labeled nucle- Tudor domain and the H3K4 trimethyl modification, we re- osome. As a result, the acetylation pattern for a processive enzyme peated the experiment using a HAT module lacking the Sgf29 in the presence of competitor should mimic the mock-quench. Tudor domain (ΔTudor). Unlike the intact HAT module, the When the SAGA HAT module was incubated with nucleo- ΔTudor complex can be competed off both unmodified (Fig. 5E) somes containing unmethylated histone H3 fluorescently labeled and H3K4me3 nucleosomes (Fig. 5F). These results, along with with Alexa Fluor 647, the addition of unlabeled nucleosomes the fact that the wild-type HAT module is efficiently competed competed for HAT module activity and prevented labeled histone off H3K4me3 nucleosomes in the presence of the H3K4me3 H3 from becoming fully acetylated (compare competitor and mock- peptide (Fig. 5D), are consistent with a role for the Sgf29 Tudor quenched samples in Fig. 5B). In contrast, the addition of unlabeled domain in engaging the methylated histone H3 tail. competitor nucleosomes had little effect on the acetylation of Because the Gcn5 bromodomain recognizes acetyl lysine (44, fluorescently labeled H3K4me3 nucleosomes, as can be seen by the 45) and regulates the lysine specificity of Gcn5 (39), we asked similarity in the intensity profiles of the competitor and mock- whether the bromodomain contributes to processive acetylation by quench samples (Fig. 5C). This pattern is characteristic of processive the HAT module. Truncating the bromodomain of Gcn5 while acetylation of H3K4me3 histone H3, presumably mediated by leaving the Sgf29 tandem Tudor domain intact (Fig. 1) causes a binding of the HAT module subunit, Sgf29, to the methylated twofold decrease in kcat/Km for peptide substrates compared with N-terminal tail of histone H3 via the Sgf29 tandem Tudor domain the wild-type HAT module (Table 1). As has been observed pre- (30). Consistent with the role of H3K4me3 binding in processivity, viously, deletion of the Gcn5 bromodomain changes the overall the addition of H3K4me3 peptide at the start of the reaction, at a pattern of acetylation catalyzed by the HAT module (39), partic- concentration that should saturate binding to the Sgf29 tandem ularly by delaying accumulation of the most highly acetylated states Tudor domain (30), abrogates processive acetylation by the HAT (Fig. S8 A and B). However, processive acetylation is clearly module (Fig. 5D). Even though the addition of the H3K4me3 impaired on H3K4me3 nucleosomes (compare competitor and

Ringel et al. PNAS | Published online September 23, 2015 | E5465 Downloaded by guest on September 28, 2021 Fig. 5. Sgf29 promotes processive acetylation against H3K4me3 nucleosomes. (A) Schematic of the competition assay to test for processive behavior. (B–H) Results from competition assays containing: the wild-type HAT module and unmodified nucleosomes (B), the wild-type HAT module and H3K4me3 nucleosomes (C), the wild-type HAT module, H3K4me3 nucleosomes, and H3K4me3 peptide (amino acids 1–8) added at the beginning of the reaction (D), the ΔTudor HAT module and unmodified nucleosomes (E), the ΔTudor HAT module and H3K4me3 nucleosomes (F), the Δbromodomain HAT module and un- modified nucleosomes (G), or the Δbromodomain HAT module and H3K4me3 nucleosomes (H). (Upper Row) Acid–urea gel of competition assay visualized using a Typhoon scanner. Background subtraction was performed using a sliding paraboloid in ImageJ. (Lower Row) Quantitation of acid–urea gel in ImageJ.

mock-quenched samples in Fig. 5 G and H), although not to the was not known. In this study we uncover a mechanism by which degree seen with the ΔTudor complex (Fig. 5 E and F). These the HAT subcomplex of the SAGA transcriptional coactivator results suggest that, although processivity is governed primarily by couples histone hyperacetylation to H3K4 trimethylation, a uni- binding of the Sgf29 tandem Tudor domain to H3K4me3 marks in versal mark associated with promoter regions of actively tran- nucleosomes, there is some contribution by the Gcn5 bromodo- scribed chromatin in organisms ranging from yeast (9, 47) to main, presumably through binding to acetylated lysine residues. humans (48). We show that H3K4 trimethylation modestly ac- Under the conditions used to monitor processive acetylation, in celerates acetylation by the HAT module on peptides and nucle- which the HAT module and labeled nucleosome are present osomes (Figs. 2B and 3A and Tables 1 and 3). Using differential equimolar ratios, we observe six acetylation events per histone tail histone labeling, we find that the HAT module is highly selective (Fig. 5 B–H;alsoseeFig. S10). However, we only observe five for H3K4me3 histones in mixtures of modified and unmodified acetylation events per histone in assays containing higher con- nucleosomes (Fig. 4A). This selectivity is a consequence of processive centrations of nucleosomes (Figs. 3B and 4 and Figs. S3, S5C, S6, acetylation of nucleosomes containing the H3K4me3 modifi- and S7). The catalytic efficiency of Gcn5 for the various lysine cation (Fig. 5 B and C), with the HAT module acetylating each residues on the H3 tail varies by two orders of magnitude (29). H3K4me3-containing nucleosome multiple times before dis- Thus, we attribute the extra acetylation site observed at low con- sociating. These data establish a paradigm for Sgf29 function at centrations of nucleosomes to the different specificity of Gcn5 for gene promoters and provide a mechanistic explanation for the each lysine and would anticipate six acetylation events per histone observation that acetylation by Gcn5 in vivo is governed by the at long time points in all our assays. presence of H3K4 trimethylation (13, 30). Processive, multisite acetylation on H3K4me3 nucleosomes also Discussion explains how the HAT module might establish different acetyla- Although crosstalk between histone modifications is a well-docu- tion patterns at promoters versus coding regions of transcribed mented feature of the histone code (4, 5), the underlying mech- genes. Although Gcn5 is broadly enriched across highly tran- anisms have remained obscure. Although studies in vivo have scribed genes in yeast (49), Gcn5-dependent acetylation patterns connected the presence of H3K4me3 to transcriptional activation differ between gene promoters and coding regions (20, 49). For by the SAGA complex (30, 31, 46), the way by which SAGA example, both H3K9 and H3K14 acetylation can be catalyzed by transduces H3K4me3 recognition into hyperacetylation by Gcn5 Gcn5 in vivo, but high-resolution ChIP-sequencing data show that

E5466 | www.pnas.org/cgi/doi/10.1073/pnas.1508449112 Ringel et al. Downloaded by guest on September 28, 2021 HAT module contains a number of other chromatin-interacting PNAS PLUS domains that are not sensitive to the H3K4me3 modification. With the potential for multiple histone-binding interactions that may further impact both binding and catalysis, there is not a simple correspondence between Sgf29-H3K4me3 affinity and Km for the HAT complex. Our results highlight the benefit of using defined, homoge- neous chromatin templates containing native modifications to study the mechanistic basis of crosstalk between histone modi- fications. For many chromatin-modifying complexes, the mech- anism by which reader domains translate the histone code into functional outcomes remains an open question that is difficult to address biochemically with recombinant unmodified histones or with heterogeneously modified chromatin isolated from cells. Our histone color-coding assay, combined with established methods for generating uniformly modified histones (36, 53), makes it possible to monitor acetylation on different pools of nucleosomes within the same reaction, thereby better approximating the heterogeneity of chromatin inside the cell. This differential labeling assay also makes it possible to observe substrate competition directly, rather than making inferences from independent measurements of spec- Fig. 6. Model depicting posttranslational modification-dependent acetylation ificity. In the present study, we compared activity on unmodified by SAGA. (Left) H3K4me3 enrichment at gene promoters stimulates processive, and H3K4me3 nucleosomes; however, the number of independent multisite acetylation by the SAGA HAT module, resulting in hyperacetylation of subpopulations that can be interrogated simultaneously using histone H3. (Right) Within gene bodies, Gcn5 catalyzes low levels of acetylation color-coding is limited only by the need to avoid significant spectral based on its intrinsic lysine specificity, preferentially modifying H3K14. overlap between the fluorophores used. This technique can be adapted to study tail-specific acetylation on asymmetrically modi- fied nucleosomes as well (54), for example those in which one copy H3K9 acetylation is narrowly limited to promoter regions (20), of histone H3 contains the H3K4me3 mark and the other copy is whereas H3K14 acetylation spreads across gene bodies as well unmodified. By incorporating more than one label into the same (49). How can the same enzyme generate different acetylation octamer, different histones within the same nucleosome could be patterns? Our results are consistent with a model in which H3K4 monitored independently. Our assay using fluorescent histone la- trimethylation, which is enriched at gene promoters compared beling, along with other recently developed techniques such as with coding regions (9, 50), promotes a pattern of acetylation in fluorescent labeling of nucleosomal DNA (55) and DNA-barcoded promoter regions different from that in gene bodies (Fig. 6). In the

nucleosome libraries (56), are particularly suited to addressing the BIOCHEMISTRY absence of H3K4 trimethylation, the HAT module likely acety- mechanistic basis of histone crosstalk. lates histones according to the intrinsic lysine specificity of Gcn5, which vastly favors modification at the H3K14 position (29, 39, Materials and Methods 51). At promoters, however, recognition of the H3K4me3 mark by Cloning, Expression, and Purification of Proteins. SAGA HAT module subunits Sgf29 promotes processive, multisite acetylation by the HAT were PCR-amplified from S. pombe genomic DNA using KOD polymerase module. The bromodomain of Gcn5 can bind to these newly (EMD Millipore) and were cloned into vectors for bacterial expression using acetylated residues, providing a second feedback mechanism that the In-Fusion Cloning kit (Clontech). Because S. pombe Sgf29 contains an promotes retention of the complex and, consequently, local intron, the two exons were PCR-amplified using primers containing a 15-bp hyperacetylation (Fig. 6). Together, the actions of the tandem internal overlap and were used together in a single In-Fusion reaction. The HAT Tudor domain and the bromodomain provide a mechanism by module subunits were cloned into three vectors, which were compatible for coexpression in E. coli. Ada3 was cloned into pET32a, The Gcn5/ Gcn5Δbro- which the reader functions of SAGA might fine-tune the activity modomain was cloned into the first multiple cloning site of CDFduet, Ada2 of Gcn5 along different genic regions, thus establishing diffuse was cloned into the second multiple cloning site of CDFduet, and Sgf29/ patterns of H3K14 acetylation punctuated by narrow regions of Sgf29ΔTudor was cloned into pRSF. Full-length Sgf29 for pull-down assays was hyperacetylation that colocalize with H3K4 trimethylation. These cloned into pET32a. Histone H3 from Xenopus laevis containing C110A/Q125C results also explain why depleting Sgf29, a noncatalytic subunit mutations and/or having the first 32 amino acids truncated (H3Δ32) for sortase containing a tandem Tudor domain that recognizes H3K4me3, reactions were generated using QuikChange site-directed mutagenesis reduces levels of H3K9, H3K14, and H3K18 acetylation in yeast (Agilent Technologies). and human cells (30) despite having little effect on overall SAGA Plasmids containing the wild-type HAT module and deletion constructs Δ activity (30). This model leaves open the possibility that other lacking the Sgf29 Tudor domain ( Tudor corresponding to Sgf29 amino acids 1–95) or the Gcn5 bromodomain (Δbromodomain corresponding to Gcn5 acetyltransferases, particularly those with H3K4me3-binding do- amino acids 1–340) were cotransformed into Rosetta2-(DE3) E. coli cells (EMD mains, also might contribute to acetylation at actively transcribed Millipore) and coexpressed as intact complexes bearing a hexahistidine tag on genes. For example, the yeast HAT complex NuA3 contains a the Ada3 subunit. Ada3 was expressed as a thioredoxin fusion in pET32a; Gcn5 noncatalytic subunit, Yng1, which binds to H3K4 trimethylation and Ada2 were provided on CDFduet; and Sgf29 was supplied on pRSF. Starter and enhances H3K14 acetylation by NuA3 on H3K4me3 peptides cultures cotransformed with all three plasmids were grown overnight at 37 °C in vitro (52). in Terrific broth (TB) containing 50 μg/mL carbenicillin, 31 μg/mL chloram- The effect of H3K4me3 on acetylation by Gcn5 is clearly more phenicol, 50 μg/mL streptomycin, and 25 μg/mL kanamycin. The saturated cultures then were diluted by 100-fold into 4–12 L of TB and grown at 37 °C complicated than simply providing additional binding energy for – methylated substrate. Binding of Sgf29 to methylated peptide until reaching an OD600 of 0.6 0.8. The flasks were transferred to an ice bath K for 45 min, and the shaker temperature was lowered to 16 °C. The cells were is not proportionally reflected in a lower m, as methylation β ∼ induced with the addition of 0.25 mM isopropyl -d-1-thiogalactopyranoside changes the affinity of isolated Sgf29 for peptides by 50-fold (30), overnight (16–18 h), harvested by centrifugation, and resuspended in lysis whereas we observe only a fourfold change in Km (Tables 1 and 3). buffer containing 40 mM Hepes, pH 7.6, 500 mM NaCl, 10% glycerol, 20 mM We note that, in addition to the Sg29 tandem Tudor domain, the imidazole, pH 8.0, and 5 mM β-mercaptoethanol (BME).

Ringel et al. PNAS | Published online September 23, 2015 | E5467 Downloaded by guest on September 28, 2021 To purify the HAT module, cell pellets were thawed, lysed using a Micro- which the globular and ligated histones gradually precipitated while the fluidizer (Microfluidics Corp.), and clarified by centrifugation at 32,000 × g.The sortase enzyme and histone peptide remained soluble. The soluble and in- soluble fraction was loaded onto a HisTrap HP column (GE Life Sciences) soluble portions of the sortase reaction were separated by spinning at equilibrated in lysis buffer. The HAT module was eluted with a 20–400 mM 21,000 × g in a microcentrifuge for 10 min at room temperature. The pellet imidazole gradient over 15 column volumes and dialyzed overnight at 4 °C was resuspended in five reaction volumes of buffer containing 7 M deion- into 20 mM Hepes, pH 7.6, 100 mM NaCl, 10% glycerol, and 5 mM BME in the ized urea, 20 mM Hepes, pH 7.5, 5 mM BME, and 0.5 mM EDTA disodium salt presence of 1 mg recombinant Tobacco Etch Virus (TEV) protease per 10 mg and was loaded onto a MonoS HR 5/5 column (GE Life Sciences) equilibrated purified protein to cleave the thioredoxin tag. Following dialysis, imidazole in the same buffer. To separate full-length histone from unligated substrate, was added to the cleavage reaction at a final concentration of 20 mM, and the column was developed with a 0.1–1 M NaCl gradient over 40 column then the mixture was run over the HisTrap HP column (GE Life Sciences), which volumes. Fractions containing the full-length H3K4me3 histone were pooled retained the hexahistidine-tagged thioredoxin while allowing the HAT mod- and dialyzed at 4 °C against three changes of MilliQ water containing ule to pass through. Solid ammonium sulfate was added to the flow-through 0.1 mM PMSF and 5 mM BME. Dialyzed histone was lyophilized and resus- over 10 min with stirring at 4 °C, to a final concentration of 0.7 M. Precipitated pended in a small amount of MilliQ water, and the concentration was de- protein was removed using a 0.4-μm filter, and the supernatant was applied to termined spectrophotometrically using the same extinction coefficient − − a Phenyl HP column (GE Life Sciences) equilibrated in buffer A containing (4040 M 1·cm 1) as for full-length H3. Equal amounts of purified H3K4me3 20 mM Hepes, pH 7.6, 200 mM NaCl, 0.7 M ammonium sulfate, 10% glycerol, histone H3 and full-length bacterially expressed histone H3 ranging from and 5 mM BME. The column was stepped to 30% buffer B containing 20 mM 0.5–10 μg were run on an SDS/PAGE gel and stained with Coomassie Brilliant Hepes, pH 7.6, 40 mM NaCl, 10% glycerol, and 5 mM BME. The column was Blue. To correct the extinction coefficient of purified H3K4me3 for the developed with a 30–100% gradient over five column volumes. Fractions presence of contaminants, band intensities were compared in ImageJ be- containing the HAT module were concentrated and loaded onto a HiPrep tween the samples to calculate a correction factor. Typically, the extinction 26/60 Sephacryl S-400 (GE Life Sciences) gel filtration column equilibrated coefficient for recombinant histone H3 overestimated the concentration of in 20 mM Hepes, pH 7.6, 100 mM NaCl, and 200 μM Tris(2-carboxyethyl) H3K4me3 by a factor of 1.1–1.5, and this correction factor was applied when phosphine (TCEP). The purified HAT module was concentrated to 4–10 mg/mL, calculating the H3K4me3 concentration at the octamer reconstitution step. flash-frozen in liquid nitrogen, and stored at −80 °C until use. Extinction co- The protein was lyophilized for long-term storage at −20 °C. efficients were calculated using the ProtParam tool from the ExPASy Bio- informatics Resource Portal (www.expasy.org). Fluorescent Labeling of Histones and Nucleosomes. Histone H3 C110A/Q125C Cells expressing Sgf29 were grown and lysed as described above for the HAT was used for fluorescent labeling with cysteine-reactive dyes because the module except that 50 μg/mL carbenicillin and 31 μg/mL chloramphenicol were endogenous cysteine in Xenopus histone H3 (C110) points to the interior of used as antibiotics. Clarified lysate was loaded onto a HisTrap HP column (GE the histone octamer and is not in a good position to accommodate a fluo- Life Sciences) equilibrated in buffer containing 20 mM Hepes, pH 7.6, 500 mM rescent dye, whereas Q125 is located on a solvent-exposed helix. Lyophilized NaCl, 20 mM imidazole, pH 8, and 5 mM BME. After washing with 10 column histones were resuspended and reduced in 6 M guanidine·HCl, 20 mM volumes of buffer, the proteins were eluted with 300 mM imidazole and then Hepes, pH 7.5, and 400 μM TCEP for 30–60 min at room temperature at a

were dialyzed overnight into 20 mM Hepes, pH 7.5, 100 mM NaCl, and 5 mM concentration of 4 mg/mL. Meanwhile, 1 mg of Alexa Fluor 488 C5 maleimide BME. The thioredoxin/hexahistidine tags were removed by adding 1 mg of TEV and 1 mg of Alexa Fluor 647 C2 maleimide (Life Technologies) were protease per 10 mg recombinant protein during dialysis. The dialyzed samples each dissolved in 100 μL of DMSO at final concentrations of 13.9 mM and were loaded onto the HisTrap HP column, and the tag was separated using a 7.7 mM, respectively. Histones bearing different posttranslational modifi- gradient of 0–200 mM imidazole over 10 column volumes. Fractions containing cations were labeled with unique colors; unmodified H3 was labeled with pure protein were dialyzed overnight at 4 °C against 20 mM Hepes, pH 7.6, Alexa Fluor 647, and H3K4me3 was labeled with Alexa Fluor 488. Unfolded/ 100 mM NaCl, and 200 μM TCEP. The proteins were concentrated to ∼5mg/mL, reduced histones were diluted to a concentration of 2 mg/mL with labeling flash-frozen in liquid nitrogen, and stored at −80 °C. buffer and were incubated with a fivefold molar excess dye for 2 h at room Sortase was purified as previously described (36). Purified protein was di- temperature with gentle agitation and protection from light. Reactions were alyzed overnight at 4 °C into 50 mM Hepes, pH 7.5, 150 mM NaCl, and 1 mM quenched with the addition of 1 mM BME and were combined immediately DTT, concentrated to 35–48 mg/mL, flash-frozen in liquid nitrogen, and stored with equimolar amounts of the other three histones to reconstitute histone at −80 °C. octamers as previously described (57). Refolded octamers were purified by gel Recombinant X. laevis histones, including the mutants used for differential filtration, which also removed excess dye that remained after dialysis. Nucle- labeling and the truncated version of histone H3 for enzyme-mediated liga- osomes were reconstituted with differentially labeled octamers as previously tion in which the first 32 amino acids were deleted (H3Δ32), were purified as described (57). Labeled nucleosomes were dialyzed into nucleosome storage previously described (57). DNA for recombinant mononucleosomes was puri- buffer [50 mM KCl, 10 mM Tris·HCl (pH 7.5), and 1 mM DTT], concentrated to fied from an EcoRV digest of pST55-16xNCP601, a kind gift from Song Tan >5 μM for storage at 4 °C, and were used within 6 wk. (Pennsylvania State University, State College, PA) as described previously (58). Nucleosomes were assembled using the salt gradient dialysis method Steady-State Kinetic Assays. Steady-state kinetic measurements using peptide (57) and then were dialyzed into low-salt buffer containing 10 mM substrates were done with a continuous spectrophotometric assay as pre- Tris·HCl, pH 7.5, 5 mM KCl, and 1 mM DTT for storage at 4 °C, where they viously described (42), with a few minor modifications. Briefly, each 50-μL

were used within 6 wk. reaction contained 5 mM MgCl2, 1 mM DTT, 0.2 mM thiamine pyrophos- + phate (Sigma), 0.2 mM NAD , 100 mM Hepes, pH 7.6, 50 mM NaCl, 2.5 mM Enzyme-Mediated Ligation of Methylated Peptide to Histone H3 with Sortase. pyruvate, 1 μL of 0.45 U/mg at 13 mg/mL pyruvate dehydrogenase (Sigma), – μ – – μ Peptide for enzyme-mediated ligation with the sequence H2N-ART(K-me3) 0.5 100 M acetyl-CoA, 25 50 nM HAT module, and 0 1,400 M histone H3 QTARKSTGGKAPRKQLATKAARKSAPATGGK-NH2 was purchased at >85% peptide containing the sequence H2N-ARTKQTARKSTGGKAPRKQLA-COOH purity (United Peptide). The peptide was solubilized in MilliQ water at a (purchased at >90% purity from United Peptide). Peptide concentrations concentration of 20 mM and was dialyzed against three changes of MilliQ were determined with a BCA assay using BSA as a standard (Thermo Scien- water at 4 °C using cellulose ester dialysis tubing with a molecular mass tific). For titrations in which the peptide concentration was varied, the cutoff of 100–500 kDa (Spectrum Labs). After dialysis, peptide concentra- acetyl-CoA concentration was held at 100 μM. For titrations in which the tions were determined with a bicinchoninic acid (BCA) assay using BSA as a acetyl-CoA concentration was varied, the peptide concentration was held at standard (Thermo Scientific) read in a POLARstar Omega plate reader (BMG 400 μM. All the reaction components, except for acetyl-CoA, were assembled Labtech), and then stored at −20 °C. Full-length histone H3 containing tri- in 384-well plates (Greiner Bio-One) and incubated at 30 °C for 5 min. Re- methylated H3K4 and either the wild-type H3 sequence or a C110A/Q125C actions were initiated by the addition of acetyl-CoA, and the absorbance at double mutant were prepared as previously described (36) with the fol- 340 nm was monitored continuously using a POLARstar Omega plate reader lowing modifications: Ligation reactions were run in assay buffer containing (BMG Labtech) for 5–60 min. Absorbance at 340 nm was converted into the molar ’ e = −1· −1 50 mM Hepes, pH 7.6, 150 mM NaCl, 10 mM CaCl2, and 1 mM DTT. Each concentration of NADH using Beer sLaw,assuming 340nm 6,220 M cm .To reaction contained 500 μM 35-mer H3 peptide, 90 μMH3Δ32, and 300 μM calculate initial rates, NADH production was plotted as a function of time and fit sortase enzyme. Because the globular form of histone H3 (missing the first to a line where initial velocity conditions were satisfied, typically within the first 32 amino acids) has poor solubility in water, lyophilized protein was first 3 min. A blank reaction containing the HAT module and acetyl-CoA but no dissolved at a concentration of 8–10 mM in solubilization buffer [6 M substrate was performed for each titration, and the rate was subtracted as guanidine·HCl, 20 mM Hepes (pH 7.6), and 10 mM DTT] before diluting it background from the other reactions. Initial rates were measured in triplicate, into the reaction. The reactions were incubated at 30 °C for 16–18 h, during normalized to the enzyme concentration, and plotted as a function of substrate

E5468 | www.pnas.org/cgi/doi/10.1073/pnas.1508449112 Ringel et al. Downloaded by guest on September 28, 2021 concentration. The resulting curve was fit to the Michaelis–Menten equation to the beads at a final concentration of 10 μM and allowed to bind at room PNAS PLUS using nonlinear least squares regression implemented in GraphPad Prism 5. temperature for 1 h. The beads were washed three times with complex Steady-state titrations with nucleosomal substrates were performed using binding buffer, eluted by boiling with 2× SDS/PAGE loading buffer, run on an a radioactive filter-binding assay (42). Nucleosome concentrations were de- SDS/PAGE gel, and stained with Coomassie brilliant blue. termined spectrophotometrically at 260 nm using the extinction coefficient e = −1· −1 of the 147-bp DNA fragment: 260nm 2,346,045 M cm . Briefly, samples Differential Labeling Assay. Assays using differentially labeled nucleosomes containing 100 mM Hepes, pH 7.6, 1 mM DTT, 0–10 μM unmodified NCP or were run as described in HAT Assays for Analysis by Acid–Urea Gel Electro- 0–1 μM H3K4me3 NCP, 50 mM NaCl, and 50 nM HAT module were incubated phoresis. Control reactions containing only one kind of labeled nucleosome at 30 °C for 5 min. Reactions were initiated with the addition of acetyl-CoA were performed at substrate concentrations of 0.2 μMand1μM. Nucleosome at 25 μM, containing a 3:1 molar ratio of unlabeled:titrated acetyl-CoA mixtures were prepared by combining 0.2 μMH3K4me3nucleosome/Alexa (PerkinElmer) and were quenched by spotting 25 μL onto P81 filter paper. Fluor 488 with 0.8 μM unmodified nucleosome/Alexa Fluor 647 and running Because proteins and peptides stick to P81 filter but free acetyl-CoA does the same time-course experiments. Quenched time points were resolved on not, acetylation was monitored by scintillation counting with the filter pa- 28-cm acid–urea gels and visualized using a Typhoon Imager (GE Life Sciences). per. At first, six time points were collected at 15-s intervals to determine the Reactions containing Alexa Fluor 488 were excited using the 473-nm laser and range in which product formation was linear with time. Under all conditions imaged using the 520BP40 filter, and reactions containing Alexa Fluor 647 tested, initial rate conditions were satisfied for the first minute of the re- were excited using the 635-nm laser and imaged using the 670BP30 filter. action. Subsequent experiments were performed by collecting three time Native gels containing mixtures of the labeled nucleosomes were imaged in points at 15, 30, and 45 s. An unwashed filter was reserved at each substrate the same way to ensure that there was no spectral overlap between the two concentration to convert counts per minute to a molar concentration of dyes (Fig. S9). Merged images were prepared using Adobe Photoshop. acetyl-CoA. Initial rates were determined by plotting product concentration as a function of time and fitting a line to the data, using the rate of acetyl- Processivity Assay. Three reactions were prepared for each combination of transfer in the absence of substrate as a reference. Each experiment was nucleosome type/HAT module complex containing 20 mM Hepes, pH 7.6, performed in duplicate, and kinetic parameters were fit as described for the 50 mM NaCl, 1 mM DTT, 50 μMacetyl-CoA,20μg/mL BSA, and 0.05 μMfluo- enzyme-coupled assay earlier in this paper. rescently labeled nucleosome in a total volume of 25–35 μL. Reactions con- taining peptide were performed with 50 μM H3K4me3 amino acids 1–8 – HAT Assays for Analysis by Acid Urea Gel Electrophoresis. Nucleosomes were peptide (ART-Kme3-QTAR), which does not contain any free lysine residues acetylated in buffer containing 20 mM Hepes, pH 7.6, 50 mM NaCl, 1 mM DTT, that can be acetylated by Gcn5. Reactions were incubated for 5 min at 30 °C, 50 μM acetyl-CoA, 20 μg/mL BSA, and 0.2 μMor1μM nucleosome core initiated with the addition of enzyme to a final concentration of 50 nM, and particle. Reactions were incubated in buffer at 30 °C for 5 min, initiated by mixed vigorously by pipetting up and down. After the reaction was allowed to the addition of 50 nM HAT module, quenched at different time points by proceed for 10–60 s, reactions were quenched by the addition of s one-fifth flash-freezing in liquid nitrogen, and then lyophilized for analysis by acid– volume acetic acid, mock-quenched with the addition of one-fifth volume urea gel electrophoresis. nucleosome storage buffer, or subjected to competition by the addition of a Acid–urea gels were assembled and run as previously described (38). His- one-fifth volume of unlabeled nucleosomes, bringing the concentration of the tones were visualized with either SYPRO Ruby protein stain (Life Technologies) unlabeled competitor to 1 μM. After the reactions were incubated for a total or by Western blotting. For Western blotting, proteins were transferred to of 1–10 min at 30 °C, the samples were flash-frozen in liquid nitrogen, ly- PVDF membrane as previously described (38). Membranes were blocked ophilized, resuspended in 10 μLofacid–urea sample buffer, and run on a overnight in 5% nonfat milk at 4 °C and were washed in TBS. Primary anti- 28-cm acid–urea gel (38). Gels were scanned using a Typhoon imager (GE Life bodies were diluted in 1% nonfat milk in TBS supplemented with 0.1% Tween Sciences) with the same excitation wavelengths/emission filter combinations as 20 (TBST) as follows: anti-H3 (Abcam ab1791, 1:25,000), anti-H3K9ac (Active

used in the differential labeling assay. ImageJ was used for background sub- BIOCHEMISTRY Motif 39917, 1:5,000), anti-H3K14ac (07-353, EMD Millipore, 1:5,000), anti- traction and to quantitate band intensities. To show that the concentration of H3K18ac (EMD Millipore 07-354, 1:7,500), anti-H3K23ac (EMD Millipore 07- unlabeled competitor used in the processivity assay acts as an effective chase 355, 1:5,000), or anti-H3K4me3 (Abcam ab8580, 1:5,000). Each primary anti- for methylated nucleosomes, we measured acetylation by the HAT module on body was applied for 1 h at room temperature followed by washing in TBST. reactions containing 50 nM H3K4me3 nucleosomes labeled with Alexa Fluor Goat anti-rabbit IgG-HRP secondary antibody (Amersham Biosciences) was μ diluted to 1:5,000 in 1% nonfat milk and TBST, applied for 1 h at room tem- 488, with 1 M unlabeled nucleosome added at the beginning of the reaction. perature, and washed in TBST. Blots were developed using Pierce ECL Western Reactions were monitored for the same length of time as the processivity assay Blotting Substrate (Thermo Scientific) and were exposed using film. using the wild-type HAT module with methylated nucleosomes. The addition of unlabeled nucleosomes effectively slows acetylation by the HAT module (Fig. S10B) compared with acetylation in the absence of competitor (Fig. S10A). Pull-Down Assays. To study the interaction between Sgf29 and the H3K4me3 modification, 25 μL of M-280 Streptavidin DynaBeads (Life Technologies) were washed into histone binding buffer [20 mM Hepes (pH 7.6), 50 mM NaCl, ACKNOWLEDGMENTS. We thank Greg Bowman (Johns Hopkins University) for providing the X. laevis histone expression plasmids, Song Tan (Pennsyl- 0.02% Tween-20, and 1% BSA] and then were incubated for 1 h at room μ ± vania State University) for providing the pST55-16xNCP601 plasmid, and Phil temperature with 1 g of biotinylated histone H3 21-mer peptide H3K4me3, Cole (Johns Hopkins University School of Medicine) for helpful discussions. which was generously provided by S.D.T. The beads were washed twice with This work was supported by National Institute of General Medical Sciences histone binding buffer and then twice in complex binding buffer [20 mM Grants R01GM098522 (to C.W.) and R01GM106024 (to S.D.T.) and by Na- Hepes (pH 7.6), 50 mM NaCl, 0.02% Tween 20]. Recombinant Sgf29 was added tional Science Foundation Grant DGE-1232825 (to A.M.C.).

1. Jenuwein T, Allis CD (2001) Translating the histone code. Science 293(5532):1074–1080. 11. Liu CL, et al. (2005) Single-nucleosome mapping of histone modifications in S. cerevisiae. 2. Strahl BD, Allis CD (2000) The language of covalent histone modifications. Nature PLoS Biol 3(10):e328. 403(6765):41–45. 12. Millar CB, Grunstein M (2006) Genome-wide patterns of histone modifications in 3. Turner BM (2000) Histone acetylation and an epigenetic code. BioEssays 22(9):836–845. yeast. Nat Rev Mol Cell Biol 7(9):657–666. 4. Fischle W, Wang Y, Allis CD (2003) Histone and chromatin cross-talk. Curr Opin Cell 13. Jiang L, et al. (2007) Global assessment of combinatorial post-translational modifica- Biol 15(2):172–183. tion of core histones in yeast using contemporary mass spectrometry. LYS4 trimeth- 5. Musselman CA, Lalonde ME, Côté J, Kutateladze TG (2012) Perceiving the epigenetic ylation correlates with degree of acetylation on the same H3 tail. J Biol Chem 282(38): landscape through histone readers. Nat Struct Mol Biol 19(12):1218–1227. 27923–27934. 6. Suganuma T, Workman JL (2008) Crosstalk among Histone Modifications. Cell 135(4): 14. Taverna SD, et al. (2007) Long-distance combinatorial linkage between methylation 604–607. and acetylation on histone H3 N termini. Proc Natl Acad Sci USA 104(7):2086–2091. 7. Lalonde ME, Cheng X, Côté J (2014) Histone target selection within chromatin: An 15. Noma K, Grewal SIS (2002) Histone H3 lysine 4 methylation is mediated by Set1 and exemplary case of teamwork. Genes Dev 28(10):1029–1041. promotes maintenance of active chromatin states in fission yeast. Proc Natl Acad Sci 8. Taverna SD, Li H, Ruthenburg AJ, Allis CD, Patel DJ (2007) How chromatin-binding USA 99(Suppl 4):16438–16445. modules interpret histone modifications: Lessons from professional pocket pickers. 16. Lee KK, Workman JL (2007) Histone acetyltransferase complexes: One size doesn’t fit Nat Struct Mol Biol 14(11):1025–1040. all. Nat Rev Mol Cell Biol 8(4):284–295. 9. Pokholok DK, et al. (2005) Genome-wide map of nucleosome acetylation and meth- 17. Vermeulen M, et al. (2010) Quantitative interaction proteomics and genome-wide ylation in yeast. Cell 122(4):517–527. profiling of epigenetic histone marks and their readers. Cell 142(6):967–980. 10. Kurdistani SK, Tavazoie S, Grunstein M (2004) Mapping global histone acetylation 18. Koutelou E, Hirsch CL, Dent SYR (2010) Multiple faces of the SAGA complex. Curr Opin patterns to gene expression. Cell 117(6):721–733. Cell Biol 22(3):374–382.

Ringel et al. PNAS | Published online September 23, 2015 | E5469 Downloaded by guest on September 28, 2021 19. Huisinga KL, Pugh BF (2004) A genome-wide housekeeping role for TFIID and a highly 38. Shechter D, Dormann HL, Allis CD, Hake SB (2007) Extraction, purification and analysis regulated stress-related role for SAGA in Saccharomyces cerevisiae. Mol Cell 13(4):573–585. of histones. Nat Protoc 2(6):1445–1457. 20. Bonnet J, et al. (2014) The SAGA coactivator complex acts on the whole transcribed 39. Cieniewicz AM, et al. (2014) The bromodomain of Gcn5 regulates site-specificity of genome and is required for RNA polymerase II transcription. Genes Dev 28(18): lysine acetylation on histone H3. Mol Cell Proteomics 13(11):2896–2910. 1999–2012. 40. Jin Q, et al. (2011) Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300- 21. Lee KK, et al. (2011) Combinatorial depletion analysis to assemble the network archi- mediated H3K18/27ac in nuclear receptor transactivation. EMBO J 30(2):249–262. tecture of the SAGA and ADA chromatin remodeling complexes. Mol Syst Biol 7:503. 41. Spedale G, Timmers HT, Pijnappel WW (2012) ATAC-king the complexity of SAGA 22. Balasubramanian R, Pray-Grant MG, Selleck W, Grant PA, Tan S (2002) Role of the during evolution. Genes Dev 26(6):527–541. Ada2 and Ada3 transcriptional coactivators in histone acetylation. J Biol Chem 42. Berndsen CE, Denu JM (2005) Assays for mechanistic investigations of protein/histone 277(10):7989–7995. acetyltransferases. Methods 36(4):321–331. 23. Grant PA, et al. (1997) Yeast Gcn5 functions in two multisubunit complexes to acet- 43. Cornish-Bowden A (1984) Enzyme specificity: Its meaning in the general case. J Theor ylate nucleosomal histones: Characterization of an Ada complex and the SAGA (Spt/ Biol 108(3):451–457. Ada) complex. Genes Dev 11(13):1640–1650. 44. Hudson BP, Martinez-Yamout MA, Dyson HJ, Wright PE (2000) Solution structure and 24. Saleh A, Lang V, Cook R, Brandl CJ (1997) Identification of native complexes con- acetyl-lysine binding activity of the GCN5 bromodomain. J Mol Biol 304(3):355–370. taining the yeast coactivator/repressor proteins NGG1/ADA3 and ADA2. J Biol Chem 45. Owen DJ, et al. (2000) The structural basis for the recognition of acetylated histone H4 272(9):5571–5578. – 25. Grant PA, et al. (1999) Expanded lysine acetylation specificity of Gcn5 in native by the bromodomain of histone acetyltransferase gcn5p. EMBO J 19(22):6141 6149. complexes. J Biol Chem 274(9):5895–5900. 46. Kurabe N, et al. (2007) Deregulated expression of a novel component of TFTC/STAGA 26. Horiuchi J, Silverman N, Marcus GA, Guarente L (1995) ADA3, a putative transcrip- histone acetyltransferase complexes, rat SGF29, in hepatocellular carcinoma: Possible tional adaptor, consists of two separable domains and interacts with ADA2 and GCN5 implication for the oncogenic potential of c-Myc. Oncogene 26(38):5626–5634. in a trimeric complex. Mol Cell Biol 15(3):1203–1209. 47. Santos-Rosa H, et al. (2002) Active genes are tri-methylated at K4 of histone H3. 27. Candau R, Berger SL (1996) Structural and functional analysis of yeast putative Nature 419(6905):407–411. adaptors. Evidence for an adaptor complex in vivo. J Biol Chem 271(9):5237–5245. 48. Barski A, et al. (2007) High-resolution profiling of histone in the human 28. Han Y, Luo J, Ranish J, Hahn S (2014) Architecture of the Saccharomyces cerevisiae genome. Cell 129(4):823–837. SAGA transcription coactivator complex. EMBO J 33(21):2534–2546. 49. Johnsson A, et al. (2009) HAT-HDAC interplay modulates global histone H3K14 29. Kuo YM, Andrews AJ (2013) Quantitating the specificity and selectivity of Gcn5- acetylation in gene-coding regions during stress. EMBO Rep 10(9):1009–1014. mediated acetylation of histone H3. PLoS One 8(2):e54896. 50. Wang Z, et al. (2008) Combinatorial patterns of histone and methylations 30. Bian C, et al. (2011) Sgf29 binds histone H3K4me2/3 and is required for SAGA complex in the human genome. Nat Genet 40(7):897–903. – recruitment and histone H3 acetylation. EMBO J 30(14):2829 2842. 51. Kuo MH, et al. (1996) Transcription-linked acetylation by Gcn5p of histones H3 and H4 31. Schram AW, et al. (2013) A dual role for SAGA-associated factor 29 (SGF29) in ER stress at specific lysines. Nature 383(6597):269–272. survival by coordination of both histone H3 acetylation and histone H3 lysine-4 tri- 52. Taverna SD, et al. (2006) Yng1 PHD finger binding to H3 trimethylated at K4 pro- methylation. PLoS One 8(7):e70035. motes NuA3 HAT activity at K14 of H3 and transcription at a subset of targeted ORFs. 32. Shukla A, Lahudkar S, Durairaj G, Bhaumik SR (2012) Sgf29p facilitates the re- Mol Cell 24(5):785–796. cruitment of TATA box binding protein but does not alter SAGA’s global structural 53. Chatterjee C, Muir TW (2010) Chemical approaches for studying histone modifica- integrity in vivo. Biochemistry 51(2):706–714. tions. J Biol Chem 285(15):11045–11050. 33. Clouaire T, Webb S, Bird A (2014) Cfp1 is required for gene expression-dependent H3K4 54. Li S, Shogren-Knaak MA (2008) Cross-talk between histone H3 tails produces co- trimethylation and H3K9 acetylation in embryonic stem cells. Genome Biol 15(9):451. – 34. Tanner KG, et al. (1999) Catalytic mechanism and function of invariant glutamic acid operative nucleosome acetylation. Proc Natl Acad Sci USA 105(47):18243 18248. 173 from the histone acetyltransferase GCN5 transcriptional coactivator. J Biol Chem 55. Ranjan A, et al. (2013) Nucleosome-free region dominates histone acetylation in – 274(26):18157–18160. targeting SWR1 to promoters for H2A.Z replacement. Cell 154(6):1232 1245. 35. Tanner KG, Langer MR, Kim Y, Denu JM (2000) Kinetic mechanism of the histone 56. Nguyen UT, et al. (2014) Accelerated chromatin biochemistry using DNA-barcoded acetyltransferase GCN5 from yeast. J Biol Chem 275(29):22048–22055. nucleosome libraries. Nat Methods 11(8):834–840. 36. Piotukh K, et al. (2011) Directed evolution of sortase A mutants with altered substrate 57. Luger K, Rechsteiner TJ, Richmond TJ (1999) Preparation of nucleosome core particle selectivity profiles. J Am Chem Soc 133(44):17536–17539. from recombinant histones. Methods Enzymol 304:3–19. 37. Popp MW-L, Ploegh HL (2011) Making and breaking peptide bonds: Protein engi- 58. Dyer PN, et al. (2004) Reconstitution of nucleosome core particles from recombinant neering using sortase. Angew Chem Int Ed Engl 50(22):5024–5032. histones and DNA. Methods Enzymol 375:23–44.

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