Recent studies emphasized a more extensive structural and functional diversification among chromatin-remodeling complexes than was previously appreciated. Here, we report the identification of two novel BRM-associated proteins, which allowed us to define two distinct Drosophila BRM chromatin-remodeling complexes: BAP and PBAP (Fig. ). BAP is the Drosophila counterpart of ySWI/SNF and human BAF-SWI/SNF-α, whereas PBAP is related to yeast RSC and human PBAF-SWI/SNF-β. BAP and PBAP share seven identical subunits, which include the central ATPase BRM, actin, and an actin-like protein (BAP55). The distinguishing subunits, unique for each subclass of remodeler, are OSA in the BAP complex and Polybromo and BAP170 in the PBAP complex. Determination of the genome-wide distributions of OSA and Polybromo on larval salivary gland polytene chromosomes revealed differential targeting of BAP and PBAP. Combined with the results of studies of yeast and mammalian cells, these results suggest evolutionarily conserved structural and functional differences between the SWI/SNF-BAF-BAP and RSC-PBAF-PBAP subfamilies of remodelers.
FIG. 8. Drosophila contains two distinct BRM complexes: BAP and PBAP. To determine the relationship between the BRM complex and the SWI/SNF-BAF or RSC-PBAF subclass of remodelers, we purified BRM and its associated factors and identified two novel subunits: (more ...)
Polybromo and BAP170 are both evolutionarily highly conserved proteins. Polybromo appears to be a homologue of the yeast Rsc1, Rsc2, and Rsc4 proteins and of human BAF180 (5
). Likewise, BAP170 is highly conserved among flies, C. elegans
, and mammals, a fact suggesting essential cellular functions. BAP170 contains a putative ARID region DNA binding motif like those in OSA, yeast Swi1p, and yeast Rsc9. Because OSA, but not BAP170, shows additional sequence homology to Swi1p outside the ARID region (10
), it seems likely that OSA represents the Swi1p orthologue in higher eukaryotes. Consequently, it is an attractive possibility that yeast Rsc9 is distantly related to the PBAP component BAP170. However, we could not identify additional regions of sequence homology between these two proteins outside the ARID region. Because of the very tight association with the fly PBAP complex, we suspect that the BAP170 homologues in other species will also be shown to form parts of PBAP-related complexes. Yeast RSC is much more abundant than ySWI/SNF (7
), whereas in human cells, BAF appears to be more abundant than PBAF (53
). We estimate from our purifications that BAP and PBAP are approximately equally abundant in Drosophila
embryos. Moreover, OSA and Polybromo bind comparable numbers of chromosomal sites on polytene chromosomes, suggesting a similar number of target genes for BAP and PBAP. Thus, BAP and PBAP may play equally broad roles in chromatin regulation in Drosophila
, suggesting that the SWI/SNF family of complexes has become more widely utilized throughout evolution.
Like its human and yeast counterparts, Polybromo harbors a multitude of putative protein-protein interaction domains: six bromodomains and two BAH domains. Bromodomains are conserved in eukaryotes and frequently are found in chromatin binding proteins and in nearly all nuclear histone acetyltransferases (32
). These domains have been shown to mediate the recognition of specific acetylated lysine residues in histone tails (17
). Therefore, it is tempting to speculate that the bromodomains in Polybromo play a critical role in targeting of the PBAP complex. BRM is the only other bromodomain-containing protein present in both the BAP and the PBAP complexes. Deletion of its bromodomain, however, affects neither BRM function nor chromatin binding (1
). Furthermore, it should be noted that not only PBAP but also BAP (lacking Polybromo) is found associated with open, hyperacetylated chromatin. Therefore, Polybromo by itself cannot be exclusively responsible for the recognition of hyperacetylated chromatin. Nevertheless, in light of the distinct chromosomal distributions of OSA and Polybromo, it seems likely that these proteins somehow direct BAP and PBAP to differentially modified chromatin domains. Future studies will be directed at discovering the mechanism of selective targeting of BAP and PBAP. Another potential interface with chromatin in Polybromo is formed by two conserved BAH domains. These motifs are also present in other chromatin-associated proteins and have been implicated in critical protein-protein interactions (8
). Furthermore, Polybromo has two potential DNA binding domains: a highly conserved HMG box (49
) and two, albeit poorly conserved, C2
-type zinc fingers (28
). An attractive hypothesis is that Polybromo acts as a specialized anchoring subunit. For example, its bromodomains could mediate histone tail recognition, the BAH domains might contact transcription factors, and the HMG domain and the putative zinc fingers might stabilize DNA binding.
Like Polybromo, BAP170 contains multiple conserved motifs, which may also play a role in PBAP targeting. First, there is an N-terminal ARID region. ARID regions have been implicated in sequence-specific as well as sequence-independent DNA binding (10
). Although OSA regulates gene expression in a promoter-selective manner, its ARID region does not mediate sequence-specific DNA binding (10
). It is possible that the ARID region acquires specificity through interactions with other cofactors. For instance, the ARID region in the human OSA homologue BAF250/p270 was recently implicated in the transcriptional coactivation of hormone receptors (21
). The highly conserved C terminus of BAP170 contains a second putative DNA binding domain: a double zinc finger motif comprising a canonical C2
zinc finger and another zinc finger in which the spacing between the two cysteine residues is larger (28
). Finally, BAP170 contains several consensus as well as variant LXXLL motifs. These motifs may mediate ligand-dependent binding to hormone receptors or interactions with coactivators such as the histone acetyltransferase CBP (12
). A functional characterization of the biochemical properties of Polybromo and BAP170 is expected to provide valuable insights into the mechanism of selective targeting of the PBAP chromatin-remodeling complex.
Immunolocalization on larval salivary gland polytene chromosomes revealed that OSA and Polybromo, the defining subunits of BAP and PBAP, display distinct, albeit overlapping, genome-wide distributions. Interestingly, the relative amounts of Polybromo and OSA on distinct sites are highly dissimilar. While on some locations Polybromo and OSA appear to be almost mutually exclusive, on other sites both are present. When OSA and Polybromo are colocalized, their relative abundances often appear quite distinct. Thus, it seems that on some locations, either Polybromo or OSA largely dictates the recruitment of PBAP or BAP, respectively. On sites where both complexes are present, they may be tethered through any of their subunits. Indeed, Collins et al. found that OSA is not required for BRM localization on chromatin per se (10
). A corollary of these localization studies is that some genes may be exclusively regulated by either BAP or PBAP. For other genes, both BRM complexes may be involved in regulation. Taken together, our findings support the notion that, like SWI/SNF-BAF and RSC-PBAF, BAP and PBAP have distinct regulatory functions.
We found that both PBAP and BAP complexes preferentially associate with regions of open, hyperacetylated chromatin. In contrast, the BRM antagonist PC displays an inverse pattern of chromosome binding, localizing predominantly at sites of closed, hypoacetylated chromatin devoid of BRM. These findings reinforce the close interrelationship between chromatin modulation by ATP-dependent remodelers and chromatin modulation by enzymes that catalyze covalent protein modifications (17
). Our results correlate very well with the findings of Armstrong and colleagues, who showed that BRM marks nearly all transcriptionally active chromatin on polytene chromosomes (1
). These researchers also established that BRM is required for most RNA Pol II transcription in salivary gland nuclei.
An important question raised by these studies is how the BRM complexes are targeted. Because we as well as Armstrong et al. (1
) failed to detect a direct association with Pol II, we prefer the idea that BRM remodelers are targeted by a combination of recruitment by sequence-specific DNA binding proteins and recognition of a local chromatin environment (4
). An example of the former mechanism is the selective BRM complex recruitment by the Zeste trxG transcriptional activator (24
). A number of different studies have provided evidence for stabilization of the association of a remodeler with chromatin by histone acetylation or methylation (3
). The bromodomain, BAH, SANT, and other conserved protein-protein interaction motifs in BAP and PBAP are prime candidates for mediating binding to specifically modified histones (32
). Finally, the various ARID, zinc finger, SANT/Myb repeat, and HMG box DNA binding motifs present in the BRM complexes may direct association with areas of open chromatin where DNA is more accessible (45
). SWI/SNF-type remodelers have been implicated in transcriptional repression (33
). However, the virtually exclusive association of BRM with open but not with silent chromatin suggests that a role in repression may be transient, for example, through facilitating repressor binding to target genes. It should be noted, however, that salivary gland cells are terminally differentiated and that dynamic processes may not be evident in polytene immunolocalization studies.
In summary, we have presented evidence that, like yeast and human cells, Drosophila cells contain a SWI/SNF-BAF- and RSC-PBAF-type remodeling complex. Intriguingly, Drosophila contains only a single Swi2p/Snf2p-related ATPase, BRM, which is present in both BAP and PBAP. In fact, OSA, Polybromo, and BAP170 are the only distinguishing subunits between BAP and PBAP. The identification of Polybromo and BAP170 described here hopefully will allow discovery of the mechanism of differential target gene selection by BAP and PBAP. We anticipate that such studies will reveal further regulatory diversification and selectivity among SWI/SNF-type remodelers and provide insight into the cross talk between chromatin modulation by ATP-dependent remodelers and chromatin modulation by enzymes that catalyze covalent histone modifications.