Physical and functional interactions of Arabidopsis ADA2 transcriptional coactivator proteins with the acetyltransferase GCN5 and with the cold-induced transcription factor CBF1

Biochimica et Biophysica Acta 1759 (2006) 69 – 79 http://www.elsevier.com/locate/bba

Physical and functional interactions of Arabidopsis ADA2 transcriptional coactivator proteins with the acetyltransferase GCN5 and with the cold-induced transcription factor CBF1 Yaopan Mao a,b,1 , Kanchan A. Pavangadkar a,b , Michael F. Thomashow b,c,d , Steven J. Triezenberg a,b,⁎ a

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA b Graduate Program in Genetics, Michigan State University, East Lansing, MI 48824-4320, USA c Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824-1325, USA d MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824-1312, USA Received 5 January 2006; received in revised form 21 February 2006; accepted 28 February 2006 Available online 27 March 2006

Abstract The Arabidopsis GCN5, ADA2a and ADA2b proteins are homologs of components of several yeast and animal transcriptional coactivator complexes. Previous work has implicated these plant coactivator proteins in the stimulation of cold-regulated gene expression by the transcriptional activator protein CBF1. Surprisingly, protein interaction studies demonstrate that the DNA-binding domain of CBF1 (and of a related protein, TINY), rather than its transcriptional activation domain, can bind directly to the Arabidopsis ADA2 proteins. The ADA2a and ADA2b proteins can also bind directly to GCN5 through their N-terminal regions (comparable to a region previously defined in yeast Ada2) and through previously unmapped regions in the middle of the ADA2 proteins, which bind to the HAT domain of GCN5. The ADA2 proteins enhance the ability of GCN5 to acetylate histones in vitro and enable GCN5 to acetylate nucleosomal histones. Moreover, GCN5 can acetylate the ADA2 proteins at a motif unique to the plant homologs and absent from fungal and animal homologs. We speculate that this modification may represent a novel autoregulatory mechanism for the plant SAGA-like transcriptional coactivator complexes. © 2006 Elsevier B.V. All rights reserved. Keywords: Chromatin; Histone; Nucleosome; Acetylation; Cold acclimation; SAGA

1. Introduction Expression of protein-coding genes in eukaryotes by RNA polymerase II is triggered by the actions of regulatory proteins known as transcriptional activators (reviewed in [1,2]). These proteins typically comprise both a DNA-binding domain, for directing the activator to the particular genes at which it is to work, and an activation domain that recruits other components of the transcriptional machinery to those target genes. Activation ⁎ Corresponding author. Department of Biochemistry and Molecular Biology, 510 Biochemistry Building, Michigan State University, East Lansing, MI 48824-1319, USA. Tel.: +1 517 353 7120; fax: +1 517 353 9334. E-mail address: [email protected] (S.J. Triezenberg). 1 Present address: Waksman Institute, Rutgers University, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA. 0167-4781/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbaexp.2006.02.006

domains can bind to several basal or general transcription factors including components of TFIID, TFIIH, TFIIA and TFIIB; these interactions can facilitate the assembly of the RNA polymerase II pre-initiation complex at a target gene promoter [3]. Activation domains can also interact with the Mediator protein complex, often considered to be a module of the RNA polymerase II holoenzyme (reviewed in [4–6]). Moreover, activation domains can interact with protein complexes known as coactivators including two classes of chromatin-modifying coactivators [7–10]. One class of coactivators includes enzymes that covalently modify the histone proteins in the core of nucleosomes, the basic unit of chromatin structure [11]. These modifications, including acetylation, phosphorylation, methylation, ubiquitinylation, and ADP-ribosylation, may influence the higher-order structure of chromatin directly and may serve as signals for interaction with additional proteins that influence

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gene expression. A second class of coactivators includes enzymes that use the energy of ATP hydrolysis to modify the position of nucleosomes along the DNA [12]. These remodeling enzymes, as they are known, may also mediate nucleosome deposition and transcriptional repression in addition to their role in transcriptional activation. One of the prototypical histone acetyltransferases that functions as a transcriptional coactivator is known as Gcn5. The gene regulatory role of Gcn5 was initially identified by genetic studies in the yeast Saccharomyces cerevisiae [13–16]. Its biochemical activity as a histone acetyltransferase (HAT) was first revealed in experiments using the protozoan Tetrahymena [17]. In the past decade, homologs of Gcn5 have been characterized throughout the eukaryotic realm including fungi, flies, mammals and plants. In yeast, Gcn5 is a component of several distinct protein complexes known as ADA, SAGA, SALSA and SLIK [18–20]. In each of those complexes, Gcn5 associates with several other common subunits such as the Ada2 and Ada3 proteins originally identified genetically as transcriptional coactivators [14,21,22]. Each complex also contains a set of proteins that are specific for that particular complex [23,24]. Comparable complexes have been characterized to some extent in flies and mammals [25–28]. The distinctive biological roles for these complexes are still being explored. Several lines of evidence suggest a central role for the Ada2 protein in the activity of the SAGA and related complexes in yeast. The yeast Ada2 protein can interact with transcriptional activator proteins [29–31], although other studies implicate another SAGA component, Tra1, in such interactions [32–34]. Some mutations of yeast Ada2 result in loss of Gcn5 protein from the SAGA complex and thus in impaired gene expression and cell growth [35–37]. The enzymatic efficiency and substrate specificity of the Gcn5 HAT activity are also influenced by the protein complexes, and particularly by the Ada2 and Ada3 proteins [22]. For instance, the recombinant Gcn5 protein alone can acetylate free histones but not nucleosomal histones, whereas the addition of Ada2 and Ada3 [22] or the presence of the intact SAGA protein complex [38,39] results in greater HAT activity on free histones and on nucleosomal histones. Despite their name, some HAT enzymes can acetylate other, non-histone proteins as well (including several transcription factors) with downstream regulatory effects (reviewed in [40,41]). A complementary question is whether the biochemical activities of HAT enzymes or their associated proteins can also be modulated by post-translational modifications; a recent report of Gcn5 sumoylation provides some initial support for this hypothesis [42]. Our laboratories are investigating the gene expression events underlying the response of plants to low temperatures, and in particular during the process known as cold acclimation whereby exposure to low non-freezing temperatures results in protection from subsequent exposure to freezing temperatures [43,44]. In Arabidopsis thaliana, a family of transcription factors known as CBF1, CBF2 and CBF3 (also designated DREB1b, DREB1c, and DREB1a, respectively) [45] are induced by low temperatures and then direct the expression of a set of cold-regulated genes [46–48]. As part of this analysis,

we have explored the roles of the Arabidopsis GCN5 homolog and two ADA2 homologs (ADA2a and ADA2b) in plant growth, development, and stress-induced gene activation [49,50]. Homozygous disruptions of GCN5 or of ADA2b (but not of ADA2a) resulted in a dwarfed growth pattern and an array of leaf and flower phenotypes. Cold-regulated gene expression was diminished but not abolished in gcn5-1 and ada2b-1 mutant plants. Moreover, the CBF1 transcription factor was shown to bind to the Arabidopsis ADA2 proteins by both yeast twohybrid and in vitro pull-down assays. These observations led to the hypothesis that GCN5-containing coactivator complexes may mediate transcriptional activation by CBF proteins during cold acclimation in Arabidopsis. In this report, we confirm that plant ADA2 proteins can interact directly with CBF1, albeit by an unusual mechanism; the DNA-binding domain of CBF1, rather than its activation domain, is both necessary and sufficient for this interaction. We mapped the regions of the Arabidopsis ADA2 and GCN5 proteins that are sufficient for their mutual interaction, finding regions of interaction not previously observed in similar studies of the yeast homologs. We show that the presence of ADA2 both enhances the catalytic efficiency and modulates the substrate specificity of GCN5. Moreover, we discovered that GCN5 can acetylate both ADA2a and ADA2b at an amino-acid motif (unique among the plant ADA2 homologs) that closely resembles the GCN5 target sequence in the amino-terminal tail of histone H3. We propose that the acetylation of ADA2 by GCN5 may reflect an autoregulatory event for HAT complexes in plants. 2. Materials and methods 2.1. AGI locus identification numbers The locus identification numbers for the Arabidopsis genes encoding the proteins examined in this study include At3g54610 (GCN5), At3g07740 (ADA2a), At4g16420 (ADA2b), At4g25490 (CBF1) and At5g25810 (TINY).

2.2. Yeast two-hybrid assay Yeast two-hybrid assays were performed using the Matchmaker GAL4 System 3 (Clontech). cDNA fragments encoding various portions of Arabidopsis GCN5, ADA2a or ADA2b were amplified by PCR and inserted in frame with the Gal4 DNA-binding domain in the bait vector pGBKT7, which has TRP1 and kanamycin resistance markers for yeast and bacterial selection, respectively. cDNAs encoding various portions of Arabidopsis ADA2a, ADA2b, CBF1 or TINY were inserted in frame with the Gal4 activation domain in the prey vector pGADT7 which carries LEU2 and ampicillin resistance markers. Portions of the resulting plasmids were sequenced to ensure fidelity of the amplified regions and cloning junctions. Yeast cells of strain AH109 (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATAADE2, URA3::MEL1UAS-MEL1 TATA-lacZ) ([51] and A. Holtz, unpublished) were transformed with various combinations of bait and prey plasmids and selected on solid synthetic dropout (SD) medium lacking leucine and tryptophan (Leu- Trp-). Transformants were then streaked onto solid SD medium (Ade- His- Leu- Trp-) containing X-α-gal and were allowed to grow for 3 to 4 days at 30°. In these conditions, positive interactions between prey and bait proteins result in the simultaneous expression of the reporter genes ADE2 and HIS3 (indicated by adenine and histidine prototrophy) and MEL1, whose protein product, α-galactosidase, catalyzes the conversion of X-α-gal (5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside) into an insoluble blue product.

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2.3. Expression and purification of recombinant proteins Bacterial expression of the Arabidopsis proteins CBF1, GCN5, ADA2a, and ADA2b (or various portions thereof) was accomplished using the GST-tagged expression vector pGEX-6P-1 (Amersham Biosciences) or the His6-tagged expression vector pET-28 (Novagen). All recombinant proteins were expressed in E. coli strain BL21(DE3) Codon Plus (Stratagene) grown in LB broth and induced at room temperature for 16–18 h using 60 to 90 μM isopropyl-beta-Dthiogalactopyranoside. The purification procedures were carried out at 4 °C unless indicated. The cells were lysed in buffer H-150 (40 mM HEPES, 150 mM KCl, 10% glycerol, 7 mM 2-mercaptoethanol) with protease inhibitor cocktail tablets (Roche) using a French pressure cell. To improve the solubility of the expressed fusion proteins, Triton X-100 was typically added to the lysates at a final concentration of 0.2% and the lysate was rocked at 4 °C for 30 min. After centrifugation at 20,000×g for 20 min at 4 °C , the cleared supernatant was recovered and stored at −80 °C. To purify GST-fusion proteins, the supernatant was incubated with an appropriate volume of glutathione-Sepharose beads (Amersham Biosciences) at 4 °C for 1 h. The beads were washed with buffer H150 containing 0.001% Triton X-100 and then washed once with buffer H-50 (containing 50 mM KCl). GST fusion proteins were eluted from the Sepharose beads with buffer H-50 and 20 mM glutathione or with a Tris-based buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM DTT, 20 mM glutathione). For some experiments, the GST tag was subsequently removed by digestion with PreScission protease (Amersham Biosciences) while the fusion proteins were bound to the Sepharose beads in buffer H-50 for 4 h at 4 °C. Expression of the His6-ADA2b recombinant protein was induced as described above, and cell lysates were prepared by French pressure cell using buffer A (20 mM HEPES, 25 mM KCl, 5% glycerol, 7 mM 2-mercaptoethanol). The lysate was incubated with Ni-NTA beads (Qiagen) in buffer A containing 20 mM imidazole. Beads were washed several times in buffer A containing 200 mM potassium acetate and then finally in buffer A with 200 mM potassium acetate and 40 mM imidazole. The His6-ADA2b protein was eluted from the NiNTA beads using 225 mM imidazole in buffer A. The eluted His6-ADA2b was either used directly or dialyzed against buffer H at 4 °C.

2.4. In vitro acetylation assays Protein acetylation assays were performed in reaction volumes ranging from 24 to 51 μl (depending on specific experiments) containing 40 mM HEPES pH 7.3 (or 50 mM Tris–Cl, pH 7.0 or 7.8), 50 mM KCl, 10% glycerol, 7 mM 2mercaptoethanol (or 1 mM dithiothreitol), and 0.05 μCi to 0.2 μCi [3H]-acetylCoenzymeA (3.3 Ci/mmol, NEN). Recombinant GCN5 protein was added to these reactions in amounts ranging from 0.25 μg to 6 μg (4 pmol to 100 pmol). In some cases, parallel reactions contained yeast SAGA complexes which were kindly provided by Dr. Shelley Berger (Wistar Institute). Acetylation of free histones was tested using 15 μg of calf thymus core histones (Sigma). For testing acetylation of nucleosomal histones, mono- and oligonucleosomes were purified from turkey erythrocyte nuclei by micrococcal nuclease digestion and gel filtration chromatography using a minor modification of an established method for chicken erythrocytes [52]. Some reactions contained GST-ADA2b fusion proteins (or GST as a negative control) in amounts ranging from 1.2 to 10 μg. Some reactions contained 6 mM sodium butyrate to inhibit histone deacetylases. Acetylation reactions proceeded for 5 to 30 min before being quenched with 12 μl of 2N HCl. In some experiments, three aliquots of each reaction were spotted onto Whatman P81 filter discs which were then washed in 50 mM NaHCO3 (pH 9.1) three times. Incorporated radioactivity was detected by scintillation counter using Ecolume liquid scintillation cocktail (ICN). In other experiments, reaction products were analyzed following electrophoresis in 12% or 15% SDS-polyacrylamide gels. Gels were first stained with Coomassie Blue to visualize all proteins and were then soaked in autoradiographic enhancer (NEN Life Science Products), vacuum dried and exposed to X-ray film for 3 days to detect radiolabeled proteins.

2.5. Mass spectrometry of acetylated Ada2b fragments Acetylation reactions were performed in 16 μl buffer (40 mM Tris–Cl pH 7.4, 50 mM KCl 10% glycerol, 7 mM 2-mercaptoethanol) containing 1–2 μg of recombinant ADA2b protein fragments, 0.5 μg GCN5, and 60 nM nonradio-

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active acetyl-CoenzymeA. The reaction was allowed to proceed for 30 min at 25 °C. The reaction products were resolved in a 12% SDS-polyacrylamide gel. A gel slice containing the ADA2b fragments was excised and digested with trypsin prior to separation of digestion products by reverse-phase HPLC. Matrix assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOFMS) were obtained using a Voyager-DE-STR mass spectrometer (PerSeptive Biosystems, Framingham, MA) in linear mode. Spectra were compared to the predicted tryptic fragments of ADA2 as obtained through Protein Prospector (P. Baker and K. Clauser, University of California, San Francisco; http://prospector. ucsf.edu). Fragments in which the acetylation site could not be unambiguously identified using MALDI-TOF-MS were further analyzed using a Q-ToF Ultima API LC-MS/MS coupled to a Waters CapLC capillary HPLC (Micromass, Manchester, UK). Data were analyzed using the MASCOT search program (Matrix Science; http://www.matrixscience.com).

2.6. Mutations of putative acetylation sites in ADA2a and ADA2b Oligonucleotide-directed mutagenesis was used to substitute arginine or alanine codons for lysine codons 257 or 265 in the ADA2a cDNA or lysine codon 215 in the ADA2b cDNA. After verification of the correct mutation, the altered gene fragments were inserted into plasmids expressing GST fusion proteins for in vitro analysis. The mutant ADA2b gene fragments were also incorporated into a plasmid, pKVA31 [50], for transformation and expression in plants. The plasmids expressing wildtype ADA2b or the K215R or K215A mutants were initially transformed into Agrobacterium tumefaciens strain GV3101, which was then used to transform Arabidopsis plants heterozygous for the ada2b-1 T-DNA disruption allele [50] using the floral dip method [53]. Transformed plants were selected on agar plates containing Gamborg's B-5 medium with Basta herbicide at 4 mg/L. Basta resistant seedlings were transplanted to soil and maintained on a 24-h light regimen. To identify the plants' genotypes, genomic DNA from rosette leaves was amplified by PCR using primers designed to detect the endogenous wildtype allele, the T-DNA disruption allele, and the transgenic allele. Expression of the ADA2b transgenes and the endogenous locus was assayed by Northern blot analysis using total RNA isolated from rosette leaves. About 5 μg of total RNA was electrophoresed in 1% agarose gels and transferred to Hybond N+ membrane (Amersham Biosciences) by capillary transfer. The RNA on the membrane was cross-linked by UV light and was then hybridized with a [32P]-labeled probe representing nucleotides 1–434 of the ADA2b cDNA. Hybridization was performed in PerfectHyb™ buffer (Sigma) at 68 °C overnight with 100 μg/ml sheared herring testis single strand DNA as non-specific blocker. The membrane was subsequently washed in 2× SSC, 0.1% SDS and 0.01% sodium pyrophosphate briefly at room temperature, and then in 0.5× SSC, 0.1% SDS and 0.01% sodium pyrophosphate at 65 °C for 30 min. The hybridization signals were detected using a PhosphorImager.

3. Results 3.1. Interaction of Arabidopsis ADA2 proteins with the DNA-binding domain of CBF1 The Arabidopsis CBF1 protein is a transcriptional activator that activates transcription of cold-regulated COR genes in response to low temperature [54]. The CBF1 protein includes an N-terminal DNA-binding domain (aa 1–115) representing the AP2 domain family [55] and a C-terminal transcriptional activation domain (aa 115–213; [56]) (Fig. 1A). We have previously shown that CBF1 can interact with the Arabidopsis transcriptional coactivator proteins ADA2a and ADA2b in a GST pull-down assay [49]. These results were consistent with the hypothesis that ADA2 might serve as a contact point through which transcriptional activators recruit HAT complexes to target promoters [31]. Yeast two-hybrid assays were used to further define which part of the CBF1 protein interacts with the

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the Gal4 activation domain. This fusion protein also interacted with the bait protein bearing ADA2b (Fig. 1D) and ADA2a (data not shown). Prey plasmids with no insert or with the GCN5 HAT domain were used as negative and positive controls, respectively, for interaction with ADA2b (Fig. 1D). Thus, the ability of AP2 DNA-binding domains to interact with the ADA2 coactivator proteins is not limited to CBF1. 3.2. Protein domains involved in the interaction between Arabidopsis ADA2 and GCN5 proteins

Fig. 1. The DNA-binding domain of CBF1 is sufficient for interaction with coactivator proteins ADA2a and ADA2b. (A) Schematic diagram of CBF1 and the various regions (a–f) tested for interaction with ADA2 and GCN5 proteins. a: amino acids (aa) 24–115, b: aa 48–115, c: aa 1–78, d: aa 96–213, e: aa 78– 213, and f: aa 48–78. (B, C) Yeast two-hybrid assays testing interactions of various regions of CBF1 (cloned into the prey plasmid pGADT7) with ADA2a or ADA2b (cloned into the bait plasmid pGBKT7). Yeast cells transformed with various combinations of plasmids were grown on selective solid medium (SD Trp–Leu–His–Ade–/X-α-gal) for 4 days at 30 °C. (D) Yeast two-hybrid assays testing the interactions of the DNA-binding domain (amino acids 1–95) of the Arabidopsis TINY protein or the GCN5 HAT domain (amino acids 203–384) cloned into pGADT7 with ADA2b cloned into pGBKT7.

ADA2 proteins. The ADA2a or ADA2b proteins were fused to the Gal4 DNA binding domain (as bait proteins), and various regions of CBF1 (Fig. 1A) were fused to the Gal4 activation domain (as prey proteins). The results shown in Fig. 1B and C indicate that the DNA-binding domain of CBF1, rather than its transcriptional activation domain, is sufficient for interaction with either ADA2a or ADA2b. Four different fragments of the DNA binding domain (fragments a, b, c and f in Fig. 1B and C) showed evidence of interaction with ADA2a and with ADA2b. The smallest of these, fragment f, encompasses the three βstrands within the AP2 domain that directly contact DNA [57]. In contrast, neither of the two fragments encompassing the transcriptional activation domain (fragments d and e) showed evidence of interaction. Immunoblots of yeast cells transformed with the various plasmids confirmed that the bait proteins bearing CBF1 fragments d and e were indeed expressed (data not shown), ruling out the trivial possibility that the lack of interaction was due to the failure of protein expression. Given this unexpected observation that the ADA2 proteins interacted with the DNA-binding domain rather than the activation domain of CBF1, we tested whether another AP2 family member might also interact with ADA2a and ADA2b. For this purpose we selected the TINY protein, which is 73% identical and 83% similar to CBF1 within the 60-residue AP2 DNA-binding domain. An N-terminal fragment of the TINY protein (aa. 1–95) was expressed as a prey protein fusion with

The Arabidopsis GCN5 protein interacts with both ADA2a and ADA2b in GST pull-down assays [49]. Yeast two-hybrid assays were used to confirm this interaction and to map the interacting regions of each protein. The Arabidopsis GCN5 protein has three distinguishable domains including a unique Nterminus (aa 1–200) the function for which is not yet known, a catalytic HAT domain (aa 203–384), and a bromodomain (aa 451–568) (Fig. 2). The Arabidopsis ADA2a and ADA2b proteins were also divided into three regions based on blocks of sequence similarity to the human and yeast homologs. These three regions included an N-terminal fragment (ADA2a aa 1– 267, ADA2b aa 1–225), a middle fragment (ADA2a aa 267– 418, ADA2b aa 225–377) and a C-terminal fragment (ADA2a aa 415–548, ADA2b aa 353–486). For yeast two-hybrid assays, the various domains of GCN5 were fused to the Gal4 DNA binding domain and the three individual domains of ADA2a or ADA2b were fused to the Gal4 activation domain. The results summarized in Fig. 2 reveal a complicated set of interactions of the ADA2 proteins with GCN5. Many of those interactions are similar for ADA2a and ADA2b. The full-length ADA2a and ADA2b fusion proteins bound to the full-length GCN5 fusion protein, consistent with the GST pull-down results reported previously [49]. The N-terminal fragments and the middle fragments of both ADA2a and ADA2b interacted with the full-length GCN5 protein, indicating that at least two distinct regions of the ADA2 proteins can bind to GCN5. The N-terminal portions of the ADA2 proteins did not interact with any of the smaller GCN5 fragments. In contrast, the middle regions of both ADA2a and ADA2b interacted with the HAT domain of GCN5. The C-terminal portions of ADA2a and ADA2b showed no evidence of interaction with the GCN5 protein; on the other hand, the bromodomain of GCN5 was not sufficient for interaction with any of the fragments of ADA2a or ADA2b. We conclude that the middle regions of ADA2a and ADA2b are sufficient for interaction with the HAT catalytic domain of GCN5. These similar patterns of interaction of GCN5 with various fragments of ADA2a or ADA2b suggest that sequences or structures common to the two ADA2 proteins are responsible for this interaction. However, several characteristics distinguish the ADA2a and ADA2b proteins in their associations with GCN5. The full-length ADA2a protein, but not the ADA2b protein, interacted with the N-terminal fragment of GCN5 (1– 250) but not with a shorter region of GCN5 (1–210). A shortened version of the HAT domain (aa 203–369) of the GCN5 protein interacted with the middle region of ADA2a, but

Y. Mao et al. / Biochimica et Biophysica Acta 1759 (2006) 69–79 Fig. 2. Mapping the interacting regions of Arabidopsis ADA2 and GCN5 proteins. Yeast two-hybrid assays were performed using various regions of ADA2a or ADA2b cloned into the prey plasmid pGADT7 and various regions of GCN5 cloned into the bait plasmid pGBKT7. Yeast cells transformed with various combinations of plasmids were scored as positive or negative for growth after 4 days on selective solid medium as described in Fig. 1. Shaded portions in the schematic diagrams of ADA2a, GCN5 and ADA2b indicate regions of sequence similarity across phylogenetic lines; HAT indicates the histone acetyltransferase catalytic domain and Br indicates the bromodomain of GCN5. Numbers refer to amino acid residues in the respective proteins or protein fragments.

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not with the corresponding portion of ADA2b. Moreover, the N-terminal fragment of ADA2b (aa 1–225), but not the corresponding region of ADA2a (1–267), interacted with a fragment of GCN5 (aa 369–568) that encompassed the bromodomain and the region between the HAT domain and the bromodomain (H–B region), although neither the bromodomain nor the H–B region alone was sufficient for this interaction. Thus, although in large measure the two ADA2 proteins interact with GCN5 in similar ways, these interactions differ in subtle ways that may have implications for biological activities including associations with other components of the corresponding HAT complexes. 3.3. Both ADA2a and ADA2b can enhance the HAT activity of GCN5 Other reports have suggested that the yeast Ada2 protein might serve both as a direct contact for transcriptional activator proteins [31] and as a stimulator of GCN5 HAT activity [22]. We tested whether the Arabidopsis ADA2 proteins affected the in vitro HAT activity of GCN5. HAT assays were performed with free core histones as substrates for recombinant GCN5 enzyme in the presence or absence of recombinant ADA2b fusion protein (GST-ADA2b) or the control GST protein. The results showed that ADA2b significantly enhanced GCN5 HAT activity, primarily on histone H3 (Fig. 3A). Quantitative assays showed approximately five-fold greater HAT activity in the presence of either GST-ADA2b or His-tagged ADA2b (Fig. 3B). In contrast to intact SAGA complexes, recombinant yeast Gcn5 effectively acetylates free histones but reacts poorly with nucleosomal histones as substrates [22]. To investigate whether the plant ADA2 protein can influence the HAT activity of Arabidopsis GCN5 on nucleosomal histones, HAT assays were performed with oligonucleosomes purified from turkey erythrocytes. When a mixture of free core histones was used as substrate, the HAT activity of 2 pmol of GCN5 protein was enhanced by the presence of 5 pmol of GST-ADA2b protein to a level comparable to that observed for an aliquot of intact yeast SAGA complex (Fig. 3C). Parallel assays showed that turkey nucleosomes were not effectively acetylated by GCN5 alone (lane 1), but were detectably acetylated in the presence of GSTADA2b (lane 2) albeit to a level far less than that achieved by the SAGA complex (lane 3). Addition of recombinant Histagged ADA2b showed a similar enhancement of nucleosomal acetylation, whereas GST alone had no effect (data not shown). We conclude that Arabidopsis ADA2 proteins can indeed stimulate the HAT activity of GCN5 on free histones and on nucleosomal histones, although additional components of the SAGA-related protein complexes may further enhance this effect. The yeast two-hybrid assays described in the previous section indicated that two different regions of the ADA2 proteins (the N-terminal and middle fragments) could bind to the full-length GCN5 protein. We tested whether these interacting regions could directly influence the enzymatic activity of GCN5. HAT assays were performed using free core

Fig. 3. ADA2b enhances the in vitro HAT enzymatic activity of GCN5. (A) Qualitative HAT assays were carried out using recombinant GCN5, [3H]-acetylCoA, free core histones and recombinant GST or GST-ADA2b proteins. Reaction aliquots were electrophoresed in an SDS-PAGE gel which was stained with Coomassie (left panel) prior to fluorography (right panel). (B) The HAT activity of recombinant GCN5 on free core histones in the presence of GST, GST-ADA2b, His6-ADA2b, or a His6 vector control extract was measured by liquid scintillation counting. (C) The HAT activity of recombinant GCN5 alone (lane 1) or with addition of GST-ADA2b (lane 2), or of an aliquot of purified yeast SAGA complex (lane 3), was tested on free core histones (left panel) or purified turkey oligonucleosomes (right panel). Reaction products were separated by SDS-PAGE prior to fluorography.

histones, recombinant GCN5, and GST fusion proteins bearing the various regions of ADA2a or ADA2b. As shown in Fig. 4A, both the N-terminal and the middle regions of ADA2a or ADA2b enhanced GCN5 HAT activity, whereas the C-terminal fragments of ADA2a (or ADA2b, data not shown) did not. Thus, the abilities of various regions of ADA2 proteins to interact with GCN5 correlate with their abilities to stimulate the GCN5 HAT activity. 3.4. Both ADA2a and ADA2b are acetylated by GCN5 A surprising observation in the HAT assays described above was that the ADA2b protein itself was acetylated by GCN5 (Fig. 3A, C). Additional assays confirmed that acetylation of ADA2b by GCN5 was independent of the presence of histones (data not shown). Given that acetylation of ADA2 proteins from other species has not been reported to date, several experiments

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Fig. 4. Two regions of ADA2a and ADA2b can enhance the in vitro HAT activity of GCN5. (A) The HAT activity of recombinant GCN5 on free core histones was measured in the presence of GST, GST-ADA2b-N (aa 1–225), GST-ADA2b-M (aa 225–377), GST-ADA2a-N (aa 1–267), GST-ADA2a-M (aa 267–418), or GST-ADA2a-C (415–548). Acetylated products were quantified by liquid scintillation counting (indicated as cpm observed ×10−3). (B) Reaction aliquots were separated by SDS-PAGE prior to staining by Coomassie to show that equivalent amounts of GST fusion proteins were used in each reaction.

were done to identify the acetylation site(s) in ADA2b. First, purified GST fusion proteins bearing the three regions of ADA2b were tested for acetylation by GCN5. The results show that only the N-terminal fragment (aa 1–225) was acetylated by GCN5 (Fig. 5A). The presence of the middle fragment of ADA2b enhanced the acetylation of the N-terminal fragment by GCN5 (Fig. 5A, fourth lane), similar to the enhancing effect on histone acetylation (Fig. 4). To further map the acetylation site, additional GST fusion proteins with various truncations in the N-terminal fragment of ADA2b were tested. Only two truncated versions (amino acids 1–225 and 28–225) could be acetylated (Fig. 5B), whereas a fragment representing amino acids 1–208 was not acetylated. This result indicated that the region between amino acids 208–225 was necessary for acetylation by GCN5. Examination of the peptide sequence in this region revealed a stretch of nine amino acids similar to that of the N-terminal tail of histone H3 (Fig. 5C). Lysine14 of histone H3 is the major site acetylated by GCN5, suggesting that the corresponding lysine (K215) of ADA2b might be the acetylation site. To test this prediction, purified GST fusion proteins with full-length ADA2b in which K215 was changed to either arginine or alanine were tested for acetylation by GCN5. The mutant ADA2b proteins were not acetylated by GCN5 (Fig. 6), indicating that K215 was necessary for acetylation. Finally, mass spectrometric analysis of recombinant ADA2bN(1–222) protein, acetylated in vitro by GCN5, confirmed that K215 was the acetylation site (data not shown). The N-terminal fragment of ADA2a (aa 1–272) was also acetylated by GCN5 in vitro, whereas the corresponding Nterminal region of yeast Ada2 (aa 1–176) was not acetylated by either GCN5 or yeast Gcn5 (data not shown). The sequence alignment (Fig. 5C) revealed that K257 in ADA2a might be the potential acetylation site. This hypothesis was supported by the observation that alanine and arginine substitutions for K257 abolished the acetylation of ADA2a by GCN5 (data not shown). The acetylation site sequence in ADA2a and ADA2b is also conserved in other plant ADA2 proteins such as that of rice and

Fig. 5. Mapping the sites of ADA2b acetylation by GCN5. Recombinant protein fragments of ADA2b or ADA2a were acetylated in vitro by recombinant GCN5 and [3H]-acetyl-CoA. Reaction products were separated by SDS-PAGE prior to fluorography (left panels) or Coomassie staining (right panels). (A) Protein fragments tested include Ada2b-N (aa 1–225), Ada2b-M (aa 225–377), Ada2bC (aa 353–486), and Ada2a-N (aa 1–267). The asterisk denotes a truncated product of the M fragment. (B) Recombinant protein fragments of ADA2b are designated by amino acid numbers. Arrowhead indicates recombinant ADA2bM polypeptide added to enhance GCN5 activity in each reaction. (C) Alignment of amino acid sequences of ADA2a (residues 249–265), ADA2b (residues 207– 223), and histone H3 (residues 6–22). Arrow indicates lysine residues acetylated by GCN5.

Fig. 6. Mutation of ADA2b Lys215 disrupts acetylation of ADA2b but not stimulation of GCN5 acetylation of histones. HAT assays were performed using recombinant GCN5, [3H]-acetyl-CoA, free core histones (left panels) or turkey nucleosomes (right panels), and either GST (G), GST-ADA2b (wildtype, designated K), GST-ADA2bK215R (R), or GST-ADA2bK215A (A). Reaction products were separated by SDS-PAGE prior to fluorography or Coomassie staining.

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maize (data not shown), but is not found in budding yeast Ada2 and metazoan ADA2 proteins, suggesting the sequence is unique to plant ADA2 proteins.

mutations may be masked by the gross overexpression of the respective transgenes. 4. Discussion

3.5. The biological functions of the ADA2b acetylation 4.1. Interaction of CBF1 with ADA2 proteins As described above, wild type ADA2b can enhance GCN5 HAT activity on both free core histones and nucleosomal histone H3. To investigate whether K215 was important for ADA2b to enhance GCN5 HAT activity, HAT assays were performed with purified recombinant mutants of ADA2b (K215R and K215A). The results showed that the mutant ADA2b proteins (K215R and K215A) enhanced the HAT activity of GCN5 as well as wild type ADA2b, on either free core histones or nucleosomal histones (Fig. 6). These results indicate that K215 is not essential for ADA2b to enhance GCN5 HAT activity in vitro. We have previously described an Arabidopsis mutant, ada2b1, bearing a T-DNA insertion in the fifth intron of the ADA2b gene. Plants homozygous for this disruption mutation showed pleiotropic phenotypes including dwarf stature and sterility [50]. Under laboratory growth conditions, the ada2b-1 phenotype can be suppressed by transgenic overexpression of wild type ADA2b cDNA. To investigate the importance of ADA2b acetylation in vivo, the K215R and K215A mutations of ADA2b were tested for their ability to suppress the ada2b-1 mutant phenotype. Plants heterozygous for the ada2b-1 allele were transformed with cDNAs encoding ADA2b (K215R or K215A) under the control of the cauliflower mosaic virus 35S promoter. Progeny plants were screened by PCR to identify plants individuals homozygous for the T-DNA disruption mutation and bearing at least one copy of the 35S:ADA2b transgene. Several lines were identified that showed wild-type growth characteristics rather than the dwarf phenotype of the ada2b-1 mutant (Fig. 7A). This result, on its surface, indicates that K215 and its acetylation are not essential for the biological activity of the ADA2b protein. However, this outcome must be interpreted with caution, since the expression levels of the transgenes were dramatically higher than the expression level of the endogenous ADA2b gene (Fig. 7B). Thus, subtle (hypomorphic) effects of the K215R or K215A

Chromatin-modifying coactivator protein complexes are typically recruited to particular genes by interaction with the transcriptional activation domains of specific DNA-binding proteins. In that regard, the interaction of the Arabidopsis ADA2a and ADA2b proteins with the transcription factor CBF1 is quite unusual. The results of our yeast two-hybrid analysis indicate that the DNA-binding domain of CBF1, rather than its activation domain, is necessary and sufficient for interacting with either ADA2a or ADA2b. Although this observation is an exception to the standard model in which activation domains recruit coactivators, other examples of DNA-binding domains interacting with coactivator proteins are known. For example, the DNA-binding domains of the transcription factors Sp1 and KLF5 can directly bind to the mammalian histone acetyltransferase p300 [58,59] and the Sp1 DNA-binding domain can also interact with the histone chaperone TAF-1 [60]. Our observation that the ADA2 proteins can interact with DNA-binding domains is not limited to CBF1, since the related DNA-binding domain from the Arabidopsis TINY protein was also able to interact with ADA2a and ADA2b. Thus, the ability to directly bind to coactivators may be a common feature of the approximately 140 members of the AP2 DNA-binding domain family in Arabidopsis. According to a tertiary structural model of the AP2 domain (based on NMR analysis of the Arabidopsis protein ERF1 [57]), a three β-strand bundle within the AP2 domain is most directly involved in contact with its cognate DNA motif. Our experiments indicate that the corresponding region of CBF1 was sufficient for interaction with ADA2 proteins. Future work will test whether CBF1 can simultaneously interact with DNA and with ADA2 proteins, and will explore whether the

Fig. 7. Overexpression of ADA2b acetylation site mutant cDNA does not prevent complementation of the ada2b-1 null phenotype. (A) Arabidopsis plants were transformed with T-DNA vectors encoding the wildtype cDNA or K215R or K215A mutants thereof. Seedlings of selected ada2b-1, wildtype (Ws ecotype), or transformed lines were germinated on agar medium and then transplanted to soil for 10 days prior to photography. White bars represent 1 cm. (B) Northern blot of total RNAs isolated from leaves of wildtype plants (wt) or ada2b-1 plants transformed with the wildtype (K) or K215R mutant (R) ADA2b cDNAs. Left panel: blot probed with radiolabeled ADA2b cDNA. Right panel: agarose gel stained with ethidium bromide, showing that comparable amounts of total RNA were loaded in each lane.

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same amino acids of CBF1 are required for both DNA-binding and ADA2 interaction. The Arabidopsis ADA2 proteins might not be the only components of their respective complexes that can interact with transcriptional activators. Although early experiments implicated the yeast Ada2 protein in directly contacting activators, subsequent experiments have shown that the Tra1 protein (the largest subunit of the yeast SAGA and NuA4 complexes) or its mammalian homolog TRAPP can also make direct contact with activators [32–34]. The Arabidopsis genome has two putative TRA1-related genes (At2g17930 and At4g36080) but no biochemical or genetic analysis of these genes or their products have yet been reported. 4.2. GCN5-ADA2 protein interactions Our results showing that the Arabidopsis ADA2 proteins can interact with the GCN5 protein confirm our previous report [49] and are generally consistent with the precedent established with the homologous yeast proteins [15,35– 37,61]. However, the details of those interactions seem to differ between plants and yeast. Co-immunoprecipitation and GST pulldown assays indicated that, in yeast, Gcn5 can interact with an N-terminal fragment of Ada2 (1–166) but not with the remainder of Ada2 (167–434), although this latter region was sufficiently ordered to interact with another SAGA component, Ada3 [35,37,61]. This yeast N-terminus corresponds to the N-terminus of Arabidopsis ADA2a (aa 1– 267) and ADA2b (1–225). The corresponding regions of the Arabidopsis ADA2a and Ada2b proteins are likewise sufficient to interact with the full-length GCN5 protein. However, in contrast to the yeast precedent, we observed that the middle regions of ADA2a and ADA2b are sufficient for interaction with either full-length GCN5 or the isolated HAT domain of GCN5. This suggests that the plant ADA2 and GCN5 proteins have an additional interaction surface that was not apparent in the yeast binary complexes. More detailed mutational analysis will be required to define explicitly the motifs and amino acids involved in these interactions. Considering the GCN5 regions involved, a fragment of yeast GCN5(1–261) which encompasses the HAT domain was not sufficient for interacting with yeast Ada2 [36]; at least 19 additional amino acids were required (extending to amino acid 280) for both ADA2 interaction and biological activity in vivo. Intriguingly, the HAT domain itself of the Arabidopsis GCN5 (203–369) was sufficient for interacting with the middle region of ADA2a but not of ADA2b. The interaction of ADA2b with GCN5 resembles the yeast case, in that an additional 15 residues C-terminal to the HAT domain (203–384) are required. This outcome suggests that the two ADA2 proteins of Arabidopsis differ somewhat in their interactions with GCN5. Two other differences between the ADA2a and ADA2b proteins also emerge from our analysis. ADA2a but not ADA2b was able to interact with an N-terminal fragment (aa 1–250) of GCN5, whereas ADA2b but not ADA2a was able to interact with a Cterminal fragment (aa 369–568) of GCN5. Whether these

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modest but reproducible differences in how ADA2a and ADA2b interact with GCN5 have functional consequences will await further purification and analysis of the corresponding protein complexes from plant cells. Recombinant Arabidopsis GCN5 was capable of acetylating free core histones but did not detectably acetylate nucleosomal histones in vitro (Figs. 3, 4). Addition of recombinant Arabidopsis ADA2 proteins stimulated the HAT activity of GCN5 (Figs. 3, 4) when either free histones or nucleosomes were available as substrate. This effect of ADA2 on GCN5 HAT activity is thus comparable to a previous report demonstrating that yeast Ada2 enhances the HAT activity of Gcn5 [22]. However, even in the presence of ADA2 proteins, the specific activity of recombinant Arabidopsis GCN5 on nucleosomal histones is markedly less than the activity of yeast Gcn5 when incorporated into an intact SAGA complex (Fig. 3C). Thus, it is likely that additional components of the SAGA complex affect the catalytic efficiency of GCN5. Evidence from analysis of the yeast SAGA complex suggests that the substrate specificity may also be different when Gcn5 is present in its native complex than when it is isolated as a distinct polypeptide [38]. Whether ADA2a and ADA2b have different effects on the catalytic efficiency or substrate specificity of Arabidopsis GCN5 will require purified protein complexes from plant cells; efforts in this direction are now underway. 4.3. Acetylation of ADA2 by GCN5 Several published reports indicate that enzymes designated as histone acetyltransferases can also modify other proteins, often with interesting and important regulatory influences [40,41]. We tested the hypothesis that the transcription factor CBF1 might be acetylated by GCN5, but found no evidence for such a modification (data not shown). We were surprised to find that the ADA2 proteins of Arabidopsis can be acetylated in vitro by GCN5. This observation suggests the intriguing possibility of an autoregulatory feedback mechanism for SAGA-like complexes. Such a mechanism would be novel to plants, since the acetylation site in the plant ADA2 homologs is not found in animal or fungal homologs. Moreover, when tested explicitly we observed no acetylation of yeast ADA2 by yeast or plant GCN5 enzymes (data not shown). We have not yet developed the necessary tools for definitive biochemical verification that the plant ADA2 proteins are acetylated in vivo. The genetic test of this hypothesis yielded ambivalent results; variant ADA2b proteins in which the acetylatable Lys residue was replaced with Ala or Arg were apparently able to complement the ada2b-1 T-DNA disruption allele. However, in this experiment the variant transgenes were expressed at levels far above those normally seen for the endogenous allele. Thus, the complementation result should be interpreted with caution, given the possibility that hypomorphic effects of these mutants might be masked by overexpression of the corresponding transgene. Alternatively, the effects of acetylation might be most important in specific biological signaling contexts such as particular developmental stages or responses to environmental stresses that we have not yet tested.

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Acknowledgements We thank Dr. Amy Hark and Dr. Kostas Vlachonasios for useful discussions during the course of this work, Travis Sportel for Western blots of yeast two-hybrid proteins, and Susanne Hoffmann-Benning, Rhonda Husain, and Brett Phinney of the MSU Proteomics and Mass Spectrometry core facilities for mass spectrometric analysis. Dr. Min-Hao Kuo and Nathan Lord provided helpful critique of the manuscript. This work was supported by grants from the National Science Foundation (MCB-9728462 and MCB-0240309) to SJT and MFT.

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