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Previous Article | Next Article 
Molecular and Cellular Biology, January 2007, p. 651-661, Vol. 27, No. 2
0270-7306/07/$08.00+0 doi:10.1128/MCB.01257-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
,
Lisette Mohrmann,1,
Wilfred F. J. van Ijcken,2 and
C. Peter Verrijzer1*
Department of Biochemistry, Center for Biomedical Genetics,1 Center for Biomics, Erasmus University Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands2
Received 11 July 2006/ Returned for modification 4 August 2006/ Accepted 26 October 2006
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ABSTRACT |
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INTRODUCTION |
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The SWI/SNF group of remodelers can be subdivided further into two distinct, evolutionarily conserved subclasses. One subfamily comprises yeast SWI/SNF (ySWI/SNF), fly BAP, and mammalian BAF, whereas the second subfamily includes yeast RSC, fly PBAP, and mammalian PBAF (35, 55). These two classes of SWI/SNF complexes are composed of highly related paralogs or identical subunits and a few subclass-specific subunits. For example, Drosophila BAP and PBAP share seven core subunits, but each is defined by unique signature subunits: the BAP-specific OSA and the PBAP-specific Polybromo (PB) and BAP170 (12, 27, 34, 42).
Work on budding yeast has established that, despite structural similarities between ySWI/SNF and RSC, each complex performs distinct cellular tasks (35, 55). ySWI/SNF is nonessential, is present in relatively small amounts, and is required for the expression of only a small portion of the yeast genome (9, 22, 49). In contrast, RSC is abundant and is essential for cell cycle progression through G2/M and for viability (1, 9, 10). RSC and ySWI/SNF also play distinct roles during DNA double-strand break repair (11). Chromatin immunolocalization studies revealed that RSC is generally recruited to RNA polymerase III promoters and to specific polymerase II promoters by transcriptional activators and repressors (14, 39). However, it is not yet clear whether the functional differences between yeast SWI/SNF subclasses can be translated directly to higher eukaryotes.
In flies and mammals, the functional distinction between the two subclasses of SWI/SNF remodelers is not well defined (35, 55). For example, in flies, both BAP and PBAP are essential for viability and appear to be more or less equally abundant. The central ATPase of both BAP and PBAP, BRM, facilitates the expression of a large portion of the fly genome (2). However, it is unclear whether this is due to a requirement for BAP or PBAP. Genomewide localization of OSA and PB on polytene chromosomes revealed that BAP and PBAP are recruited differentially, suggesting that they control distinct sets of target genes (34). The two highly related human SWI/SNF ATPases BRG1 and BRM determine functional specificity in transcription (25). The mammalian BAF- or PBAF-selective subunits have been implicated in transcription activation by specific nuclear hormone receptors (31, 40, 50) and for the expression of selective interferon-responsive genes (57). OSA, the fly BAP selective subunit, is required for repression of Wingless target genes (13). These findings suggest that the BAP/BAF and PBAP/PBAF complexes perform distinct functions in gene expression control. However, a comprehensive side-by-side analysis of gene control by both types of remodelers is lacking.
Typically, the core ATPase of the multisubunit remodelers suffices for chromatin remodeling in vitro (4, 19, 33). Therefore, we were intrigued by the roles of the remaining subunits in global transcription regulation in cells. To determine the contributions of shared and unique BAP and PBAP subunits to gene expression control, we performed a comprehensive RNA interference (RNAi) knockdown survey in Drosophila S2 cells. Epistasis analysis combined with whole-genome expression profiling suggested that SWI/SNF remodelers act mostly as holoenzymes. In cells, the core complex, without the BAP and PBAP signature subunits, appears to be dysfunctional for general transcription control. Depending on the target genes, BAP and PBAP may act coordinately or independently or antagonize each other. We dissected the pathway through which BAP, but not PBAP, controls cell cycle progression. Our results outline the functional roles of SWI/SNF complexes at the genomic level. They also demonstrate the power of integrating RNAi knockdown studies with statistical analysis of expression profiles for the structure-function analysis of multisubunit transcription-regulatory complexes.
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MATERIALS AND METHODS |
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RNA extraction, RT-qPCR, and Affymetrix microarray hybridization. RNA was extracted using the SV Total RNA Isolation System (Promega) and tested on an Agilent BioAnalyzer (Agilent). Samples with RNA integrity numbers of >8 were selected. Labeling, hybridization, washes, and staining of microarrays were performed according to Affymetrix specifications. Reverse transcription-quantitative PCR (RT-qPCR) was done with SYBR green I using a MyiQ single-color real-time PCR detection system and iQ SYBR green supermix (Bio-Rad) according to the manufacturer's protocols. First-strand cDNA was prepared with an iScript cDNA synthesis kit (Bio-Rad). Primers for RT-qPCR were selected using Beacon Designer software (Premier Biosoft). The RT-qPCR primers were as follows: CG11874, 5'-AGTGTTGCTCTGCCTAAGTGG-3' and 5'-CGGATGATGGTGCGGATTGG-3'; string (stg), 5'-CGTTATCTAAGTTTGGGTGTTATCG-3' and 5'-TGTGTCTGCGTCGTGTGC-3'.
Statistical analysis. Statistical analysis of the microarray data was performed using the R and Bioconductor free software (http://www.bioconductor.org). Gene expression indexes were calculated using the robust multichip average (RMA) algorithm implemented in the Bioconductor affy package (23). The multiple covariance determinant algorithm is implemented in the rrcov R package and was used for filtering the genes showing low expression indexes across all experiments (46). Calculation of bootstrap P values for hierarchical clustering was performed using the pvclust R package. Heat maps were plotted using the glplots R package. PCA was performed on scaled and centered data using the R "prcomp" function. Gene ontology (GO) analysis was done with the Bioconductor GOstats library. Distances between GO terms were determined using the "simUI" (union intersection) function. Further details of the statistical analysis and R scripts will be provided upon request.
Flow cytometric analysis. Cells were collected and fixed with 70% ethanol as described previously (7). After a minimum of 2 h on ice, the cells were washed with phosphate-buffered saline and resuspended in 400 µl phosphate-buffered saline containing 0.1% Triton X-100, 0.1 mg/ml propidium iodide, and 0.1 mg/ml RNase. The cells were incubated overnight and analyzed on a FACScan (Becton Dickinson).
ChIPs and polytene chromosome immunolocalization. Chromatin immunoprecipitation (ChIP) assays were performed according to published procedures (53). Cross-linked chromatin was prepared from S2 cells either treated with dsRNA or untreated and sheared by sonication to an average length of 0.7 kb. After immunoprecipitation, the recovered DNA was analyzed by qPCR with SYBR green I using the MyiQ single-color real-time PCR detection system (Bio-Rad). The following primers were used in qPCRs to amplify the stg/cdc25 promoter: 5'-CAGTGGCCTCCATAGAGCTG-3' and 5'-GTCCCGAGAAACGAGGAGA-3'. We also performed ChIP using anti-MOR antibodies on promoters of nontarget genes, such as mRpS17, mRpL16, and mRpL52, to estimate background levels (data not shown). Immunolocalization of OSA and PB on polytene chromosomes was performed as described previously (34). Chromosomes were stained with DAPI (4',6'-diamidino-2-phenylindole), and the 99A cytological region was mapped using the photographic maps of G. Leffevre, Jr. (32).
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RESULTS |
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RT-qPCR analysis revealed that changes in mRNA levels accounted only for the decreased levels of targeted proteins, not for the concomitant loss of other subunits (data not shown). Thus, a knockdown of the core subunits directly affects the stability and composition of both BAP and PBAP. Subunits that fail to assemble into a complex are apparently unstable and are quickly targeted for degradation. These results provide detailed insights into the architectural properties of BAP and PBAP subunits. A major finding was that MOR is particularly critical for the structural integrity of both complexes. On the other hand, RNAi knockdown of the signature subunits OSA (BAP), PB, and BAP170 (PBAP) resulted in the specific depletion of the respective complex. The architectural relationships between the distinct BAP and PBAP subunits are summarized in Fig. 1D.
BAP and PBAP structure-function analysis through expression profiling. To investigate the roles of BAP and PBAP in gene expression control, we extracted RNA from S2 cells treated with dsRNA against individual subunits. Labeled RNA was hybridized on Affymetrix Drosophila Genome 2 arrays (http://www.affymetrix.com/support/technical/datasheets/drosophila2_datasheet.pdf), containing 18,500 probe sets representing all known transcripts and variants. Expression indexes were calculated using the RMA algorithm (23). Examination of RMA expression indexes revealed a bimodal distribution with low values for a large portion of the probe sets (see Fig. S1 in the supplemental material). Prior to further analysis, we removed genes that were expressed at very low levels from the data set, using the minimum covariance determinant algorithm (46).
To assess the technical variability of microarray experiments, we hybridized each RNA sample from mock- and RNAi-treated cells twice. For each replica, we found a high correlation between expression indexes (r > 0.9; P < 0.001). We also performed RT-qPCR analysis on a selection of BAP and PBAP target genes. As a reference gene, we used CG11874, encoding mannosyl-oligosaccharide mannosidase, which remained unaffected in all of our microarray experiments. We found that changes in gene expression after RNAi treatment determined by either microarray or RT-qPCR analysis were highly correlated (r = 0.96; P < 0.001; n = 72). Together, these results demonstrate that gene expression profiles obtained from microarray experiments are highly reproducible and provide a valid representation of the effects of BAP or PBAP subunit depletion on the transcriptome.
Three independent RNAi knockdown experiments, followed by RNA extraction and microarray hybridization, were performed for each subunit. For mock-treated cells, we performed six independent experiments. Next, we applied one-way analysis of variance (ANOVA) on each probe set to identify genes that changed significantly (P < 0.05) upon RNAi treatment. This analysis identified 1,714 genes, for which we determined gene expression profiles by taking the ratios between average gene expression indexes obtained from RNAi- and mock-treated cells.
To gain insight into the relationship between the transcriptomes determined by individual BAP and PBAP subunits, we decided to use an unbiased statistical analysis of the whole data set. Spearman correlation analysis and the derived hierarchical agglomerative clustering revealed a clear separation between the transcriptomes dependent on either the core subunits, BAP, PBAP, or ISWI (Fig. 2A and B).
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The most notable feature of hierarchical clustering of the top-scoring PC2 genes is the strong antagonism between BAP and PBAP. Overall, genes activated by OSA were repressed by PB and BAP170. Conversely, OSA-repressed genes were stimulated by PBAP. The transcriptional consequence of core subunit depletion grouped with either BAP or PBAP, most likely reflecting the relative dependence of a gene on either complex (Fig. 3C, right). We suggest that in conjunction with the appropriate activators or repressors, BAP and PBAP can function as either selective coactivators or corepressors of transcription. In conclusion, our genomewide expression analysis uncovered both coordinate and antagonistic functions of BAP and PBAP in transcription control.
BAP- and PBAP-selective subunits determine holoenzyme function. Our results so far had established that the core subunits, but not the signature subunits, are essential for the structural integrity of BAP and PBAP. We also found that, depending on the target gene, BAP and PBAP might have similar effects, act independently, or antagonize each other. However, the function of the core complex itself in transcription control remained unclear. In other words, what activities could the core complex lacking the BAP- and PBAP signature subunits perform in the cell?
To address this question, we knocked down OSA, PB, and BAP170 simultaneously. Western immunoblotting confirmed that, although the BAP- and PBAP-defining subunits were depleted, the core complex remained (Fig. 4A). Strikingly, Spearman correlation analysis, hierarchical clustering, and PCA all revealed that the gene expression profile of cells lacking the signature subunits was very similar to that of cells depleted of the core subunits (Fig. 4B, C, and D). Genes with the highest absolute PC1 or PC2 values after depletion of core subunits were thus affected similarly by the triple OSA, PB, and BAP170 knockdown (data not shown). This observation implies that loss of the signature subunits impedes both the coordinate and antagonistic functions of BAP and PBAP. As we showed above (Fig. 1), depletion of the core subunits destabilizes both BAP and PBAP. Therefore, the effect of the triple knockdown, reducing BAP and PBAP to their common core, is largely similar to the total removal of the BAP and PBAP remodelers from the cell (Fig. 4E). The surprising implication of these findings is that, in the cell, the core SWI/SNF complex is defective for transcription control of the majority of target genes. We suggest that, for regulation of most genes, SWI/SNF remodelers act as holoenzymes, which require the signature subunits OSA or PB and BAP170 for global gene expression control.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BAP and PBAP function as holoenzymes in cells. Most chromatin remodelers exist as large multiprotein complexes that can be isolated as entities under stringent conditions (5, 24, 33, 37, 38, 51). Nevertheless, the central ATPase typically suffices for in vitro chromatin remodeling (4, 19, 33). In the case of human SWI/SNF, remodeling by the ATPase is stimulated by the mammalian orthologues of the MOR and SNR1 subunits (43). The human BRM and MOR homologs BRG1 and BAF155 are necessary and sufficient for EKLF-directed chromatin remodeling and transcription in vitro (26).
Here, we have investigated the functions of individual SWI/SNF subunits in cells. The core subunits, shared by BAP and PBAP, execute critical architectural and enzymatic functions. However, when stripped of OSA, PB, and BAP170, the core complex was defective in regulation of a significant portion of the target genes. Thus, the BAP- and PBAP-selective subunits are essential for global in vivo functionality of the complex. In other words, global gene expression analysis suggests that SWI/SNF remodelers predominantly act as holoenzymes. Examination of the BAP and PBAP expression profiles revealed that only a minority of SWI/SNF-regulated genes do not require an intact BAP or PBAP complex for their regulation. We suggest critical roles for OSA, PB, and BAP170 in tethering SWI/SNF to target genes. Consistent with this hypothesis, we showed that OSA is strictly required for BAP recruitment to the stg/cdc25 promoter. We propose that the core subunits provide both the structural framework and enzymatic activity, but that the BAP and PBAP signature subunits are required for regulation of the majority of SWI/SNF target genes.
Coordinate and antagonistic BAP and PBAP functions. Our determination of their genomewide distribution on polytene chromosomes revealed that BAP and PBAP display distinct but overlapping patterns of distribution (34). This localization study was the first suggestion that BAP or PBAP had differential effects on gene expression. However, it remained unclear whether BAP and PBAP had similar or opposing activities during transcription control of common targets. PCA of gene expression profiles after BAP- or PBAP-selective depletion revealed two major classes of regulated genes. The first comprised genes coordinately activated or repressed by both complexes. The second class was composed of genes that were antagonistically regulated by BAP and PBAP. Thus, it appears that BAP and PBAP help to integrate and balance the activities of transcriptional activators and repressors to establish appropriate transcription levels.
BAP, but not PBAP, is required for cell cycle progression. Evaluation of gene ontologies combined with expression profiling provides a valuable predictive tool for the dissection of biological pathways (3, 21). Using a statistical approach to GO analysis, we found that BAP is involved in the regulation of cell cycle progression. Indeed, depletion of BAP, but not PBAP, caused the accumulation of cells in the G2/M phase of the cell cycle and increased aneu- and polyploidization. A combination of statistical GO term analysis and PCA identified stg/cdc25 as a potential target, responsible for the BAP-dependent cell cycle phenotype. This hypothesis was confirmed experimentally by demonstrating that BAP directly binds and activates the stg/cdc25 promoter (Fig. 7B, D, and F) and by showing that a reduction of the stg/cdc25 level alone is sufficient to cause a G2/M arrest (Fig. 7C). In contrast, PBAP neither bound nor activated the stg/cdc25 promoter.
These results reveal a remarkable evolutionary switch between budding yeast and flies concerning the roles of RSC-PBAP class remodelers and ySWI/SNF-BAP in cell cycle control. In budding yeast, mutations in ySWI/SNF do not affect viability or the cell cycle, whereas RSC is essential for progression through G2/M and viability (1, 9, 10). In metazoans, BAP-BAF appear to be more abundant and perform more critical functions than their yeast counterparts (35, 55). Other studies of Drosophila have also implicated the BAP-selective OSA and several core subunits in cell cycle control (6, 48, 58). Finally, we note that insights into the role of SWI/SNF remodelers in cell cycle control will improve our understanding of certain human cancers (28, 44). For example, we found that loss of hSNF5, the human homologue of SNR1, causes a defective cell cycle and loss of ploidy control in malignant rhabdoid tumor cells (54).
In conclusion, we have used expression profiling following RNAi-mediated subunit depletion for a structure-function dissection of the BAP- and PBAP chromatin remodelers. We combined epistatic analysis and unbiased data-mining tools to explore the relations among gene expression profiles on the whole data set. We identified structural, as well as transcription-selective, functions executed by distinct subunits. Our results suggest that in general SWI/SNF remodelers act as holoenzymes, which require the BAP- or PBAP signature subunits for global transcription control. Statistical analysis of gene expression profiles and GO terms revealed that BAP and PBAP each control different biological processes. For example, BAP, but not PBAP, is required for cell cycle progression. These findings demonstrate the value of a statistical analysis of gene expression profiles for the dissection of complex biological processes controlled by multisubunit regulators.
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ACKNOWLEDGMENTS |
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This work was supported by grants from The Netherlands Organization for Scientific Research (NWO), the European Commission (to C.P.V.) and a long-term EMBO fellowship (to Y.M.M.).
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FOOTNOTES |
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Published ahead of print on 13 November 2006.
Supplemental material for this article can be found at http://mcb.asm.org.
Y.M.M. and L.M. contributed equally to this work.
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0.01). The heat map represents the average change in gene expression on a log2 scale within each GO cluster after RNAi treatment targeting individual BAP or PBAP subunits.
of 0.05. Groups a to c are represented by BAP, and group d by PB and BAP170. (C) Cell cycle distribution of S2 cells treated with dsRNA directed against stg/cdc25, as determined by FACS analysis. Cells with a DNA content of 4n or more are indicated. Quantification was based on gated cells. The ungated FACS profiles are also shown. (D) OSA is required for targeting BAP to the stg/cdc25 promoter. ChIP/qPCR quantification of changes in MOR binding to the stg/cdc25 promoter following dsRNA treatment compared to mock-treated cells is represented as ratios on a linear scale. Significant changes upon RNAi-mediated knockdown of individual BAP, but not PBAP, subunits were determined by one-way ANOVA [F(5,18) = 7.9; P < 0.001] on four biological replicates. The error bars represent 95% confidence intervals. The Fisher LSD test revealed two homogenous groups at an