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Molecular and Cellular Biology, May 2008, p. 2920-2929, Vol. 28, No. 9
0270-7306/08/$08.00+0     doi:10.1128/MCB.02217-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Transcriptional Coactivator SAYP Is a Trithorax Group Signature Subunit of the PBAP Chromatin Remodeling Complex{triangledown}

Gillian E. Chalkley,1,{dagger} Yuri M. Moshkin,1,{dagger} Karin Langenberg,1 Karel Bezstarosti,2 Andras Blastyak,3 Henrik Gyurkovics,3 Jeroen A. A. Demmers,2 and C. Peter Verrijzer1*

Department of Biochemistry, Center for Biomedical Genetics,1 Proteomics Center, Erasmus University Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands,2 Hungarian Academy of Sciences, Biological Research Center, Institute of Genetics, H-6701 Szeged, Hungary3

Received 14 December 2007/ Returned for modification 7 February 2008/ Accepted 19 February 2008


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ABSTRACT
 
SWI/SNF ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in eukaryotic gene expression control. BAP and PBAP are the fly representatives of the two evolutionarily conserved major subclasses of SWI/SNF remodelers. Both complexes share seven core subunits, including the Brahma ATPase, but differ in a few signature subunits; POLYBROMO and BAP170 specify PBAP, whereas OSA defines BAP. Here, we show that the transcriptional coactivator and PHD finger protein SAYP is a novel PBAP subunit. Biochemical analysis established that SAYP is tightly associated with PBAP but absent from BAP. SAYP, POLYBROMO, and BAP170 display an intimately overlapping distribution on larval salivary gland polytene chromosomes. Genome-wide expression analysis revealed that SAYP is critical for PBAP-dependent transcription. SAYP is required for normal development and interacts genetically with core- and PBAP-selective subunits. Genetic analysis suggested that, like BAP, PBAP also counteracts Polycomb silencing. SAYP appears to be a key architectural component required for the integrity and association of the PBAP-specific module. We conclude that SAYP is a signature subunit that plays a major role in the functional specificity of the PBAP holoenzyme.


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INTRODUCTION
 
Gene expression control is one of the most fundamental biological processes and, to a large extent, occurs at the transcriptional level. The transcription of a single protein-encoding eukaryotic gene involves a stunning plethora of regulating factors comprising some 100 or so distinct polypeptides (16, 23, 28). These can be classified as sequence-specific DNA-binding transcription factors that initiate the recruitment of positive or negative coregulatory complexes and the basal transcription machinery. Coactivators include a variety of proteins performing distinct functions during the transcription cycle such as the opening up of chromatin structure, mediating posttranslational histone modifications or bridging between activators and the basal transcription machinery. It has become clear that the diversity among gene-specific activators and repressors is complemented by functional specification among coregulatory complexes and even the core transcription machinery. One important class of coregulators is formed by the ATP-dependent chromatin-remodeling factors (remodelers).

Remodelers are large multisubunit complexes defined by the presence of an ATPase "engine" subunit (2, 3, 21, 24, 32). These proteins act like DNA translocases and use the energy derived from ATP hydrolysis to change the DNA-histone contacts, thus remodeling chromatin structure (4, 41). Based on the identity of their central ATPase, four major classes of remodelers have been recognized: SWI/SNF, ISWI, CHD/Mi2, and Ino80/Swr1 (17). Different remodelers are not exchangeable; rather, each executes unique biological functions. An early example of functional diversification was our finding that the Drosophila SWI/SNF class Brahma (BRM) remodelers, but not the ISWI remodelers, act as chromatin-specific coactivators for the transcription factor Zeste (13).

SWI/SNF class remodelers perform broad yet gene-selective transcription regulatory functions during development, cell cycle control, and tumor suppression. There are two major SWI/SNF subclasses, conserved evolutionarily from yeast to humans. The first subclass includes yeast SWI/SNF (ySWI/SNF), fly BAP, and mammalian BAF, whereas the second subfamily includes yeast RSC, fly PBAP, and mammalian PBAF (21). The corresponding multiprotein complexes are composed of highly related paralogs or identical subunits and a limited number of subclass-specific proteins. For example, Drosophila melanogaster BAP and PBAP share seven core subunits, but each is defined by unique signature subunits: the BAP-specific OSA and the PBAP-specific POLYBROMO and BAP170. Here, we will sometimes use the term SWI/SNF when making general statements that apply to both subcomplexes.

Previous structure-function dissection of fly SWI/SNF revealed that the common core subunits play architectural and enzymatic roles, whereas the signature subunits are key to the functional specificity of BAP and PBAP holoenzymes (22, 40). In particular, BRM and MOR are critical for the structural integrity of both BAP and PBAP (22). Regulation of the majority of target genes required the signature subunit OSA, PB, or BAP170, suggesting that SWI/SNF remodelers function mostly as holoenzymes (22). BAP and PBAP regulate distinct but overlapping transcriptional circuitries, acting either independently, similarly, or antagonistically. Likewise, BAP and PBAP direct convergent as well as distinct biological processes. BAP, but not PBAP, is required for cell cycle progression through mitosis. BAP mediates G2/M transition through direct regulation of the cell cycle regulator string/cdc25. OSA is required for targeting BAP to the string/cdc25 promoter (22).

The genes encoding BRM, MOR, and OSA were originally discovered in screens for dominant suppressors of Polycomb (Pc) mutations and therefore were classified as trithorax group (trxG) proteins (7, 8, 13-15, 25, 31, 33). The trxG of activators, together with their antagonists, the Pc group (PcG) of repressors, maintain correct expression of many developmental regulators. So far, no other core- or PBAP-selective subunits have been identified as trxG proteins (21, 25). Thus, whether PBAP, like BAP, acts as a trxG suppressor of Pc remains unclear.

SAYP is a chromatin-associated transcriptional coactivator that was originally identified as the enhancer of yellow, e(y)3, gene (30). SAYP contains two PHD fingers, an AT hook and a highly conserved SAY domain that is essential for transcription coactivation. Analysis of mutants revealed that SAYP is essential for oogenesis and early development (30). However, the molecular functioning of SAYP remained completely unclear.

Because distinct SWI/SNF subunits each provide unique functionalities, the complete determination of the BAP and PBAP composition is an important objective. Here, we purified PBAP and identified the coactivator SAYP as a novel subunit. We found that SAYP is an essential and distinctive subunit of PBAP, which is absent from BAP. We used a variety of genomic, biochemical, and genetic approaches to dissect the role of SAYP. We conclude that the transcriptional coactivator SAYP is a novel signature subunit that is essential for the functional specificity of the PBAP holoenzyme.


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MATERIALS AND METHODS
 
Antibodies and immunological procedures. Polyclonal antibodies were generated by immunizing guinea pigs with the Escherichia coli-expressed and purified fusion polypeptides, as follows: for SAYP, a glutathione S-transferase (GST) fusion polypeptide corresponding to SAYP amino acids (aa) 422 to 720; for BAP170, two His-tagged polypeptides spanning aa 192 to 314 and aa 1141 to 1272 of BAP170, respectively; for Snr1, a His-tagged polypeptide corresponding to Snr1 aa 245 to 370; for BAP111, two GST-fusion polypeptides of residues 230 to 430 and 466 to 646 of BAP111. Antigens were expressed, purified, and used for immunization as described previously (5, 10). Other antibodies have been described, including anti-OSA (34), anti-BRM and anti-ISWI polyclonal antibodies (13), anti-POLYBROMO and anti-MOR polyclonal antibodies (20), anti-SAYP rabbit polyclonal antibodies, used for polytene chromosome staining (30); anti-histone H3 antibodies were obtained from Abcam (ab1791). Immunoblotting experiments were performed using standard procedures (10). Immunolocalization of BRM complex subunits with Drosophila salivary gland polytene chromosomes was performed essentially as described previously (1, 20), using affinity-purified primary antibodies at the following dilutions: anti-POLYBROMO was diluted 1:50; anti-BAP170, 1:50; and anti-SAYP, 1:250. Slides were mounted in mounting medium containing 4',6'-diamidino-2-phenylindole counterstain (Vector Laboratories).

Protein purification and mass spectrometry. Embryo nuclear extracts were prepared from 0-h to 12-h-old Drosophila embryos (11), and immunopurification procedures were performed essentially as extensively described previously (5). Briefly, extracts were incubated with affinity-purified anti-BRM or anti-POLYBROMO antibodies coupled to protein A-Sepharose beads (catalog no. 17-0963-03; GE Healthcare) by dimethyl pimelimidate cross-linking (5, 10) or with anti-OSA antibodies coupled to protein G-Sepharose beads (catalog no. 17-0618-01; GE Healthcare). Anti-GST antibodies were used in a mock purification (data not shown) experiment. After 2 h at 4°C on a rotating wheel, the beads were then washed with HEMG buffer (25 mM HEPES-KOH [pH 7.6], 0.1 mM EDTA, 12.5 mM MgCl2, 10% glycerol) containing 200 mM KCl and 0.1% NP-40 and then extensively washed with HEMG-400 mM KCl, 0.1% NP-40 and then again with HEMG-200 mM KCl, 0.1% NP-40. Following a final quick wash with 200 mM KCl, the retained proteins were eluted with 100 mM Na-citrate buffer (pH 2.5). After they were neutralized, the eluted proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Coomassie staining. Polypeptides were identified by mass spectrometry on an LTQ-Orbitrap hybrid mass spectrometer (ThermoFischer) (38). Data analysis was performed using a Mascot search algorithm (version 2.1; MatrixScience) searching against the FlyBase database (http://flybase.bio.indiana.edu; version FB2007_03, released 1 November 2007). SAYP (CG12238) was identified by 11 unique peptides in the BRM immunopurification, covering 10.2% of the sequence and yielding a Mascot score of 737. In the POLYBROMO immunoprecipitation, SAYP was identified by 25 unique peptides covering 21.7% of the sequence with a mascot score of 2055. No SAYP peptides were found in the OSA purification. For immunodepletion (5), Drosophila nuclear extract was slowly diluted with HEMG buffer containing 100 mM KCl, to a final total protein concentration of 5.5 mg/ml, and then cleared by centrifugation. This extract was then incubated at 4°C with protein A beads and either anti-MOR antibodies or preimmune serum as mock control. After 1 h, the beads were removed by centrifugation. Fresh beads and either anti-MOR antibodies or preimmune serum were again added to the supernatant. This procedure was repeated three more times. The supernatants were then resolved on 8% SDS-PAGE gels and analyzed by immunoblotting.

RNA interference and genome-wide expression analysis. These experiments and analyses were performed as described (22). Briefly, Drosophila S2 cells were cultured in Schneider's medium (catalog no. 21720-024; Invitrogen) and treated with double-stranded RNA (dsRNA) for 4 days, as described previously (39). dsRNA for the SAYP subunit was synthesized by using an Ambion Megascript T7 kit according to the manufacturer's protocol, with the following oligonucleotides, 5'-TTAATACGACTCACTATAGGGAGAATGGTGATCGACGATTCG-3' and 5'-TTAATACGACTCACTATAGGGAGACTCGATTATGGATTGGGC-3'.

RNA samples from three completely independent biological SAYP knockdown experiments were prepared and hybridized with Affymetrix microarrays as described previously (22). The new SAYP expression profiles were compared with those of various (P)BAP subunits and ISWI (22), employing the algorithms for statistical analysis of the microarray data using R and Bioconductor, as described by Moshkin et al. (22). Details will be provided upon request.

Drosophila genetics. The osa, brm, mor, and snr1 fly strains were obtained from the Bloomington stock center (http://flystocks.bio.indiana.edu/). The Pc1 and Pc3 mutant alleles were provided by F. Karch (Geneva). The SAYPEMSl and SAYPu1 strains were a gift from Sofia Georgieva and were described previously (30). The polybromo33.2 mutant allele was obtained by P element insertion mapping at the 10- to 40-bp position upstream of the first ATG codon of the polybromo gene. The P(EP)bap170kim1 mutant allele (referred to as bap170kim1) was purchased from GenExel (P element insertion, 13193). bap170kim1 carries the P(EP) element insertion at a position 447 bp downstream from the transcription start site in the first intron of the gene. Precise excision of the P element rescues bap170kim1 reduced viability, and flies appear to be of the wild type. Information on gene structure and chromosomal location of genes used in this study is present at FlyBase (http://www.flybase.org/). All crosses were performed at 25°C and repeated several times.


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RESULTS
 
Identification of the coactivator SAYP as a novel PBAP subunit. One of the aims of this study was to determine whether there were still uncharacterized BRM-associated proteins remaining. To this end, we immunopurified BRM complexes from Drosophila embryo nuclear extracts. We used highly specific affinity-purified polyclonal antibodies directed against either BRM (recognizing BAP and PBAP) or POLYBROMO (PBAP selective) and monoclonal antibodies directed against OSA (BAP selective). Antibodies were coupled to beads and incubated with a nuclear extract. Following extensive washes with a buffer containing 400 mM KCl and 0.1% NP-40, protein complexes were eluted and resolved by SDS-PAGE, followed by Coomassie staining (Fig. 1A). Mass spectrometric analysis revealed the presence of SAYP in the BRM and POLYBROMO immunopurification, but not in the OSA immunopurification.


Figure 1
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  		FIG. 1. Identification of SAYP as a novel PBAP signature subunit. (A) Characterization of BRM complexes immunopurified (IP) with anti-BRM (common to BAP and PBAP), anti-POLYBROMO (PBAP-specific), and anti-OSA (BAP-specific) antibodies. Proteins retained on the beads after extensive washing were resolved by SDS-PAGE and visualized by Coomassie staining. Bands were excised and proteins were identified by Nanoflow liquid chromatography tandem mass spectrometry. SAYP (CG12238) was identified by 11 unique peptides in the BRM immunopurification covering 10.2% of the sequence and yielding a mascot score of 737. In the POLYBROMO immunoprecipitation, SAYP was identified by 25 unique peptides covering 21.7% of the sequence with a mascot score of 2055. No SAYP peptides were found in the OSA purification. The PBAP signature subunits, including SAYP, are indicated in red, whereas the positions of the OSA bands are indicated in green. (B) PBAP, but not BAP, coimmunoprecipitates with anti-SAYP antibodies. Immunoprecipitated material was resolved by SDS-PAGE and analyzed by Western immunoblotting using the indicated antibodies. (C) SAYP coimmunoprecipitates with core and PBAP signature subunits but not with OSA. Drosophila nuclear extract was incubated either with preimmune serum (Mock), anti-BRM, anti-MOR, anti-OSA, anti-POLYBROMO, or anti-BAP170 antibodies. Immunoprecipitated material was resolved by SDS-PAGE, followed by Western blotting with antibodies directed against the indicated BAP and PBAP subunits. (D) The majority of SAYP is stably associated with PBAP. Nuclear extract was immunodepleted with beads that were either coated with preimmune serum (Mock) or antibodies directed against MOR. The supernatants were then resolved by SDS-PAGE and analyzed by Western immunoblotting with the indicated antibodies. Whereas PBAP was no longer detectable in the MOR-depleted extract, ISWI remained unaffected. (E) A speculative model summarizing the PBAP and BAP subunit composition. PBAP-specific subunits POLYBROMO, BAP170, and SAYP are shown in red; the BAP-defining OSA is shown in green, and core subunits are shown in blue.

To test the notion that SAYP might be a novel PBAP subunit, we performed a series of stringent coimmunoprecipitation experiments. We used antibodies that were directed against the core subunits BRM and MOR, the BAP-selective OSA, and the PBAP-selective subunits POLYBROMO and BAP170. Immunoprecipitates purified from embryo nuclear extracts were washed extensively and analyzed by Western immunoblotting (Fig. 1C). SAYP selectively copurifies with the common core and PBAP-selective subunits but not with OSA. Conversely, the core subunits BAP170 and POLYBROMO copurify in an anti-SAYP immunoprecipitation, whereas OSA is completely absent (Fig. 1B). To determine whether SAYP is present outside the PBAP complex, we depleted the embryo nuclear extract by using an antibody directed against MOR (Fig. 1D). Inspection of the MOR-depleted extract showed that SAYP could no longer be detected, revealing that the majority of all SAYP is a stable component of PBAP. In contrast, ISWI, which is the core subunit of separate remodeling complexes, is not affected by the MOR depletion of the extract. Taken together, our results suggest that the transcription coactivator SAYP is a novel PBAP-selective subunit. Thus, in addition to the seven common core subunits, PBAP harbors three signature subunits: POLYBROMO, SAYP, and BAP170. BAP is defined by OSA, bound to the same seven-subunit core. Importantly, our coimmunoprecipitation experiments demonstrate that an association with the core by either the OSA or the PBAP module is strictly mutually exclusive. No hybrid complexes were detected. This observation suggests that the OSA and the PBAP modules target the same docking platform.

To study the genome-wide distribution of SAYP, we compared its distribution on larval salivary gland polytene chromosomes to that of POLYBROMO and BAP170 (Fig. 2). All three signature subunits colocalize on the majority of their chromosomal binding sites, as is evident from the largely yellow staining of the merged images. Previously, we showed that these sites also bind BRM and overall represent the interband regions of open, less condensed chromatin (20). The colocalization of POLYBROMO, SAYP, and BAP170 is emphasized by high-magnification microscopy (Fig. 2D, E, I, and J). At this moment, we are unable to determine if the very few bands that appear to be unique to one of the PBAP subunits are relevant or due to minor variations in antibody specificity. Although SAYP appears to be substoichiometric, as judged from the Coomassie-stained gel (Fig. 1A), it colocalizes with POLYBROMO and BAP170 on the vast majority of chromosomal sites. In general, our results thus far suggest that there are not various PBAP subcomplexes harboring a selection of signature subunits. Rather, POLYBROMO, SAYP, and BAP170 appear to form a stably associated PBAP-defining module that is integrally recruited to chromatin.


Figure 2
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  		FIG. 2. SAYP colocalizes with PBAP-specific subunits on Drosophila salivary gland polytene chromosomes. The distributions of SAYP, BAP170 (A to E), and POLYBROMO (F to J) on salivary gland polytene chromosomes were determined by immunostaining, with antibodies directed against each of these subunits. SAYP (red) colocalizes with BAP170 (green), which in turn colocalizes with POLYBROMO (red), as demonstrated by the predominantly yellow staining in the merged panels.

Genome-wide expression profiling reveals a close overlap between SAYP, POLYBROMO, and BAP170 transcription control. To investigate SAYP's role in gene expression control, we treated S2 cells with dsRNA directed against SAYP. Although there is a weak residual SAYP band, Western immunoblotting revealed a significantly reduced protein level (Fig. 3A). Next, we extracted RNA from cells depleted of SAYP, mock cells, and cells from which a selection of individual BAP and PBAP subunits or ISWI was depleted (22). For each subunit, we performed three strictly independent RNA interference (RNAi) knockdown experiments using distinct cell batches with different passage numbers. For mock-treated cells, we performed six independent experiments. RNA was extracted, labeled, and hybridized with Affymetrix Drosophila Genome 2 arrays. Expression indexes were calculated using a robust-multichip-average (RMA) algorithm (12). Genes that were expressed at very low levels were removed from the data set by using a minimum-covariance-determinant algorithm (29). Next, we applied one-way analysis of variance with each probe set to identify genes that changed significantly (P < 0.05) upon RNAi treatment. For these approximately 1,655 genes, we determined gene expression profiles by taking the ratios between average gene expression indexes obtained from specific RNAi- and mock-treated cells.


Figure 3
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  		FIG. 3. Expression profiling reveals a close functional relationship between SAYP and PBAP. (A) S2 cells were either mock treated or treated with dsRNA against SAYP. Whole-cell extracts were analyzed by Western immunoblotting using antibodies directed against SAYP. Histone H3 serves as a loading control. (B) Spearman's correlation matrix for the microarray expression profiles obtained after the knockdown of SAYP and selective BAP and PBAP subunits, as well as ISWI, was described previously (22). The heat map reflects the indicated R values. (C) Agglomerative hierarchical cluster analysis of the microarray expression profiles based on the Spearman R values. P values show probabilities for each cluster deduced from the bootstrap procedure. The PBAP (red), SWI/SNF core (blue), BAP (green), and ISWI clusters are indicated. (D) PCA analysis reveals the very tight clustering of SAYP with POLYBROMO and BAP170. Shown is a projection of OSA, BRM, MOR, SNR1, POLYBROMO, BAP170, and POLYBROMO expression profiles after RNAi-mediated depletion in a two-dimensional transcriptome space defined by PC1 and PC2, explaining 87% of the variance. (E) The heat map shows the agglomerative hierarchical clustering of genes with the highest absolute scores for PC1 and PC2. Changes in gene expression compared to the mock-treated cells are shown in red for up-regulated genes and in blue for down-regulated genes. White indicates no change.

We used an unbiased statistical analysis of the whole data set to compare the SAYP transcriptome to that of core, PBAP-, and BAP-selective subunits. Spearman's correlation analysis (Fig. 3B) and the derived hierarchical agglomerative clustering (Fig. 3C) showed the very strong correlation between SAYP and BAP170 and POLYBROMO. As we reported earlier, the transcription profiles of the core subunits, OSA, or PBAP-selective subunits form well-separated clusters. We also performed principal component analysis (PCA), a powerful mathematical procedure that helps to uncover relationships in complex data sets. PCA is a linear transformation that finds and projects original variables to the fewest principal components (PCs), accounting for most of the variance in the data set. About 87% of the variance in gene expression profiles obtained after depletion for selective BAP and PBAP subunits is explained by PC1 (59%) and PC2 (28%). As shown in Fig. 3D, PCA revealed that the SAYP profile correlated highly with that of POLYBROMO and BAP170 (Fig. 3D). The core subunits BRM, MOR, and SNR1 cluster closely together and are well separated from OSA and the PBAP signature subunits. The PBAP, core, and albeit to a lesser extent, the OSA transcriptomes correlate well with PC1. PC2 is made up largely of genes that are antagonistically regulated by BAP and PBAP.

To identify and visualize the genes that are coregulated or antagonistically regulated by BAP and PBAP, we selected the top 5% of genes at the right and left tails of the PC1 and PC2 value distributions. Hierarchical cluster analysis of the top-scoring genes revealed coordinate as well as antagonistic regulation by BAP and PBAP (Fig. 3E). In particular, with PC2 genes, BAP and PBAP elicit opposite transcription effects. Moreover, this analysis illustrates clearly that BAP and PBAP can have positive as well as negative effects on gene expression. The three PBAP signature subunits have highly similar effects on the expression of the majority of genes analyzed, strongly suggesting that they act as a functional unit. Like the genome-wide colocalization of all PBAP signature subunits, these results suggest that SAYP is largely a stoichiometric PBAP subunit. In summary, biochemical purification, genome-wide colocalization on polytene chromosomes, and genome-wide gene expression analysis identify SAYP as a novel PBAP subunit.

SAYP interacts genetically with PBAP during development and suppresses Pc silencing. So far, our results showed that SAYP, POLYBROMO, and BAP170 associate in PBAP and are corecruited to chromatin, and their transcriptomes strongly correlate with each other. Collectively, these findings suggest that the PBAP signature subunits share biological functions during development. To test this hypothesis, we obtained the bap170kim1 and polybromo33.2 P element insertion mutants (see Materials and Methods) and the previously described SAYP alleles generated either by ethyl methane sulfonate (EMS) mutagenesis, SAYPEMSl, or by the insertion of the Stalker mobile element, SAYPu1 (30). The SAYPEMSl allele is mid-embryonic lethal. However, an examination of homozygous females and hemizygous males for the weaker SAYPu1 hypomorphic allele revealed defects in leg development (Fig. 4A). These flies display a characteristic "bent-leg" phenotype with a moderate penetrance of about 20%, allowing us to assay for genetic interactions between SAYP and other fly SWI/SNF subunits.


Figure 4
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  		FIG. 4. SAYP interacts genetically with POLYBROMO and BAP170. (A) The saypu1 hemizygous males display a characteristic developmental leg abnormality, which we refer to as "bent-leg" (arrow). (B) The penetrance of the bent-leg phenotype is strongly enhanced by mutations in core subunits and PBAP-selective subunits but not by osa mutants. The bar graphs display the frequency of bent legs in male saypu1 hemizygous flies carrying additional mutations as indicated. n, number of animals inspected. The standard error of the mean is indicated and asterisks indicate a statistically significant increase in penetrance, as determined by t test (P < 0.05). It is important to note that the saypu1/Y; brmI21/+ genotype is lethal. (C) Flies homozygous for both the bap170kim1 and polybromo33.2 mutant alleles do not hatch from their pupal cases. Dissection revealed a severe leg developmental defect and microcephaly (arrows).

We obtained males hemizygous for the SAYPu1 mutant allele and heterozygous for one of the following mutations: osa308, osa00090, brmI21, brm2, mor1, and snr101319, as well as transheterozygous for both the bap170kim1 and polybromo33.2 mutant alleles. As shown in Fig. 4B, mutant alleles of genes encoding core (BRM and MOR) and PBAP signature subunits (POLYBROMO and BAP170) act as strong dominant enhancers of the penetrance of the SAYPu1 bent-leg phenotype. The combination of SAYPu1 and brmI21 mutant alleles causes lethality, as no adult males hemizygous for SAYPu1 and heterozygous for brmI21 could be recovered. In contrast, none of the mutant alleles for the BAP-specific subunit OSA affected the frequency of the SAYPu1 bent-leg phenotype. These observations suggest that SAYP interacts genetically with PBAP but not with BAP.

To expand our analysis of the developmental roles of PBAP, we also analyzed the BAP170 and polybromo mutants in more detail. Inspection of the bap170kim1 heterozygous stock revealed that this allele severely affects viability. Flies homozygous for the bap170kim1 mutant allele die primarily as pharate adults. However, we noticed that they also display the characteristic "bent-leg" morphogenesis defect observed for homo- and hemizygous SAYPu1 flies. In a balanced bap170kim1 heterozygous stock, homozygous bap170kim1 males make up less than 3% of all male progeny that is able to hatch, whereas no female escapers were found. Flies homozygous for the polybromo33.2 mutant allele are viable with no apparent phenotype. However, when it is combined with bap170kim1, the polybromo33.2 allele further reduces bap170kim1 viability and enhances the leg malformation. Flies homozygous for both the bap170kim1 and the polybromo33.2 mutations are not viable and die before or at the pharate adult stage. Interestingly, dissection of these pharate adults revealed a severe leg elongation defect and microcephaly (Fig. 4C). Together, our results strongly suggest that the PBAP-specific subunits control common developmental pathways. Noticeably, the leg malformation and microcephaly are reminiscent of phenotypes caused by mutations in the ecdysone signaling pathway (9, 18, 36, 37). These observations suggest a potential link between PBAP and ecdysone-controlled developmental transcription, suggesting a role for PBAP in hormone receptor function.

Genome-wide localization on polytene chromosomes and expression profiling revealed that BAP and PBAP execute independent, antagonistic but also common functions. We therefore wondered whether PBAP, like BAP, might play a role in the control of homeotic gene expression. Thus far, it has remained unclear whether PBAP could act as a suppressor of Pc expression. Fly strains carrying the Pc mutation Pc1 or Pc3 provide convenient genetic readouts with which to assay the effects of chromatin-associated proteins on epigenetic gene regulation. Males heterozygous for either the Pc1 or Pc3 mutant allele exhibit posterior-to-anterior leg transformation due to ectopic activation of the homeotic Scr gene. This homeotic transformation is readily visualized in males by the ectopic appearance of the sex combs, which are normally present only on the first legs (L1). Due to a failure to repress Scr in a Pc mutant background, additional sex combs appear on the second (L2 to L1) or both the second and the third pair of legs (L3 to L1) (Fig. 5A). The frequency of posterior-to-anterior leg transformations is reduced in transheterozygous males when either Pc1 or Pc3 is combined with both bap170kim1 and polybromo33.2 mutations (Fig. 5B). In particular, SAYPu1 hemizygous males display an almost complete suppression of Pc-induced leg transformations. Based on these results, we conclude that both BAP and PBAP can function as trxG suppressors of Pc mutations.


Figure 5
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  		FIG. 5. Sayp suppresses Pc homeotic leg transformations. (A) In males heterozygous for Pc1 or Pc3 mutant alleles, sex combs, normally found only on the first pair of legs, now also appear on the second pair (top panel, L2-L1) or third legs (bottom panel, L3-L1). (B) saypu1 is a strong suppressor of homeotic transformations caused by Pc1 and Pc3. Moreover, mutations in both polybromo and bap170 also suppress the frequency of Pc leg transformations. We quantified the frequency of males displaying L3-L1 and L2-L1 transformations (white), L2-L1 transformations without L3-L1 (light blue), and no transformations (dark blue). The various genotypes and number of animals inspected (n) are indicated. Changes in transformation frequencies were statistically significant according to the log-likelihood chi-square test (P < 0.05).

SAYP is essential for PBAP assembly. Previously, we found that there is an architectural hierarchy between Drosophila SWI/SNF subunits (22). Certain subunits, such as MOR, are strictly required for complex assembly, while others are not generally important for the incorporation of other subunits. Furthermore, our results suggested that those subunits that fail to assemble into a complex are unstable and are quickly targeted for degradation. Finally, we found that the removal of OSA, POLYBROMO, and BAP170 did not affect the integrity of the core complex. However, the core was absolutely required for the stability of the signature subunits.

Here, we tested the role of SAYP in the PBAP complex assembly. As shown in Fig. 3A, we can efficiently deplete Drosophila S2 cells of SAYP by treatment with specific dsRNA. Protein extracts for Western immunoblotting analysis were prepared from mock-treated cells and from cells depleted of either SAYP or ISWI (Fig. 6A). Loss of SAYP led to a strong reduction in the abundance of POLYBROMO and BAP170, whereas the core subunits MOR, BRM, Snr1, and BAP111 remained unchanged. As expected, ISWI levels were not influenced by SAYP depletion, and ISWI depletion did not affect BAP or PBAP. The importance of SAYP on POLYBROMO and BAP170 stability strongly suggest that SAYP is a near-stoichiometric subunit required for the stable association of the PBAP-selective module to the core complex. The relatively minor SAYP band in Fig. 1A is most likely due to weak Coomassie staining rather than to underrepresentation within PBAP.


Figure 6
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  		FIG. 6. SAYP is a key architectural component of PBAP. (A) S2 cells were either mock treated or treated with dsRNA directed against SAYP or ISWI. Next, whole-cell extracts were prepared, resolved by SDS-PAGE, and analyzed by Western immunoblotting using the indicated antibodies. Histone H3 serves as a loading control. (B) Similarly, MOR and SAYP were analyzed following RNAi-mediated depletion of BRM, MOR, POLYBROMO, and BAP170. (C) Summary of the architectural relationships between the PBAP-specific subunits. Arrows indicate that MOR is required for the integrity of the PBAP module. SAYP plays a major role in stabilizing BAP170 and POLYBROMO. The other way around, POLYBROMO and BAP170 are not required for SAYP stability. BAP170 does help stabilization of POLYBROMO (22). Finally, the loss of POLYBROMO does not affect either BAP170 or SAYP. The architectural relationships between several other BAP and PBAP subunits have been described previously (22).

Next, we tested the effect of the depletion of BRM, MOR, POLYBROMO, and BAP170 on SAYP (Fig. 6B). The RNAi-mediated knockdown of these subunits, with the exception of SAYP, have been described previously (22). RNAi-mediated reduction of BRM, which partially destabilizes the core complex, does not significantly affect SAYP amounts. MOR depletion, which causes disintegration of the core, BAP, and PBAP, leads to a significant reduction of SAYP levels. Interestingly, the two other PBAP-selective subunits are not required for SAYP stability. Thus, there seems to be a hierarchy in PBAP architecture, in which SAYP is critical for the stable assembly of the tripartite PBAP module and its linkage to the SWI/SNF core.


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DISCUSSION
 
An important step in uncovering the protein-protein and protein-DNA interaction networks controlling eukaryotic gene expression is the identification of complexes that act as functional units. Here, we found that the essential coactivator SAYP is a novel subunit of the PBAP complex. Genome-wide localization studies and a comparison of the transcriptomes controlled by various subunits revealed that SAYP is critically required for PBAP function. SAYP, POLYBROMO, and BAP170 form an integral PBAP module that associates with the SWI/SNF core. PBAP is essential for normal development and counteracts Pc silencing. Together with our earlier studies (22), the results presented here suggest that the signature subunits are particularly important for the functional specificity of the BAP or PBAP holoenzymes.

SAYP is a key PBAP signature subunit. SAYP was identified through a genetic screen for modifiers of the yellow expression gene, e(y)3, which turned out to encode for an evolutionarily conserved chromatin-associated transcriptional coactivator (30). Here, we showed that SAYP is a novel PBAP signature subunit. Recently, we purified the human homologue of SAYP as a unique subunit of PBAF (S. Giannakopoulos and C. P. Verrijzer, unpublished data). Moreover, we identified genes homologous to sayp in the genomes of sequenced metazoans but failed to identify clear homologues in, e.g., Neurospora spp., Saccharomyces cerevisiae, or Schizosaccharomyces pombe, suggesting that SAYP might be a metazoan-specific subunit.

Immunodepletion of a nuclear extract using antibodies directed against MOR led to the concomitant loss of SAYP in the unbound fraction. This result suggests that the majority of SAYP exists as part of the PBAP complex. Previous results from our studies and those of others suggested that SWI/SNF subunits that are not incorporated in a complex are unstable and degraded (6, 22). Moreover, we established that particular subunits, e.g., MOR, are essential for the architectural integrity of BAP and PBAP (22). Here, we extended this analysis and found that SAYP is required for the incorporation of BAP170 and POLYBROMO into PBAP. This relationship was not reciprocal: neither BAP170 nor POLYBROMO was required for SAYP assembly.

Whereas the BAP and PBAP signature modules require the core, removal of the PBAP signature subunits (as shown in this and previous studies) or OSA depletion (22) did not affect the core complex. However, in the absence of the signature subunits, the SWI/SNF core alone turned out to be largely dysfunctional in global gene expression control (22). This finding highlights the interesting issue that untargeted chromatin remodeling, which can be mediated efficiently by the SWI/SNF core (4, 26) by itself does not suffice for transcription control. Therefore, we suggested that SWI/SNF remodelers act as holoenzymes, in which the core subunits provide key architectural and enzymatic functions, but the signature subunits determine most of the transcriptional specificity (22).

Another interesting outcome from our analysis is that there is no evidence for BAP/PBAP hybrid complexes. Thus, the docking of OSA and the PBAP signature module appear to be mutually exclusive. Although there are likely to be additional sites of contact, SAYP appears to be particularly important for the stable association of POLYBROMO and BAP170 with the core complex. BAP170 stabilizes POLYBROMO binding (22). Most likely, there is a hierarchy of incorporation in which SAYP and OSA compete for an overlapping docking site within the core complex. The association of POLYBROMO and BAP170 is stabilized by SAYP but blocked by OSA, preventing the formation of hybrid complexes.

Developmental gene control by BAP and PBAP. Our results presented here and in earlier studies (22) demonstrate the value of epistasis analysis combined with whole-genome expression profiling for the structure-function analysis of multisubunit regulatory complexes. PCA of gene expression profiles after BAP- or PBAP-selective depletion revealed that BAP and PBAP regulate distinct but overlapping transcriptional circuitries, acting either together, independently, or antagonistically. Likewise, BAP and PBAP execute related but also distinct biological functions. Previously, we found that BAP, but not PBAP, controls cell cycle progression (22). BAP mediates G2/M transition through direct regulation of the cell cycle regulator string/cdc25. BAP recruitment to the stg/cdc25 promoter was critically dependent on OSA. In contrast, PBAP neither bound nor activated the stg/cdc25 promoter. Deciphering the role of SWI/SNF in cell proliferation and differentiation is of particular interest because of the association between SWI/SNF malfunction and human cancer (27). For example, the loss of the human homologue of SNR1, hSNF5, causes a defective cell cycle and the loss of ploidy control in malignant rhabdoid tumor cells (35).

Previously, the genes encoding the BAP-signature subunit OSA and two core subunits, BRM and MOR, were identified as trxG members because they act as dominant suppressors of Pc (7, 8, 13-15, 25, 31, 33). However, it remained unclear whether PBAP also antagonizes Pc silencing. Here, we found that mutations in the genes coding for the PBAP signature subunits suppress the leg transformations caused by Pc mutations. Thus, both BAP and PBAP can be classified as trxG transcriptional coactivator complexes.

In this study we also implicated PBAP in additional developmental control pathways. We found that mutations in genes encoding PBAP-specific subunits cause characteristic leg malformations and microcephaly, which are reminiscent of phenotypes caused by defective ecdysone signaling (9, 18, 36, 37). These observations suggest that PBAP might be involved in ecdysone-inducible gene regulation in vivo. Previous in vitro results suggested that human PBAF is selectively required for ligand-dependent transactivation by nuclear hormone receptors (19). Therefore, it will be interesting to investigate the role of BAP and PBAP in nuclear hormone receptors signaling during development.

In summary, BAP and PBAP are essential chromatin remodeling factors that perform cooperative and unique functions during development. Because distinct subunits appear to be dedicated to specific regulatory pathways, a complete structure-function analysis is required to gain insight into the roles of SWI/SNF remodelers in development and disease. Our study presented here identified the coactivator SAYP as novel PBAP subunit. We conclude that SAYP is a novel signature subunit that is essential for the functional specificity of the PBAP holoenzyme. Furthermore, our analysis of SWI/SNF remodelers suggested that they are dedicated to specific transcriptional pathways, rather than acting as true general factors. Our future studies will aim at dissecting the gene-selective functions of remodelers during development and disease.


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ACKNOWLEDGMENTS
 
We thank Tsung-Wei Kan for excellent technical assistance, S. Georgieva and Y. Shidlovskii for the gift of SAYP mutant flies, antibodies, and communication of results, W. van Ijcken for microarray hybridizations and E.-J. Rijkers for help with programming.

This work was supported by grants from The Netherlands Organization for Scientific Research (NWO) Chemical Sciences, no. 700.52.312 (to P.V.), and an EMBO long-term fellowship (Y.M.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry, Center for Biomedical Genetics, Erasmus University Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Phone: 31-10-408-7461. Fax: 31-71-527-6284. E-mail: c.verrijzer{at}erasmusmc.nl Back

{triangledown} Published ahead of print on 25 February 2008. Back

{dagger} These authors contributed equally to this work and should both be considered first authors. Back


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Molecular and Cellular Biology, May 2008, p. 2920-2929, Vol. 28, No. 9
0270-7306/08/$08.00+0     doi:10.1128/MCB.02217-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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