Different metazoan cell types are distinguished by extensive gene- and cell-specific transcription. This remarkable complexity of expression is not accomplished by a corresponding expansion in gene number (1
) or by cell-specific regulatory factors (51
). Instead, these selective and sophisticated patterns of transcription are generated by multifactor regulatory complexes comprised of different combinations of broadly expressed regulatory factors (4
). Although this concept of combinatorial regulation is long established (6
), we understand relatively little about how common factors assemble into regulatory complexes that differ in composition, geometry, and function. Indeed, even the total number of proteins associated in a given complex, and the dynamics of their interactions, have not been determined. However, it is apparent that mammalian transcriptional regulatory complexes, for example, may contain 50 to 100 or more different proteins that associate on demand at genomic response elements (15
In this study, we sought to determine whether as few as two proteins, GR and Brm, both residing in a select set of regulatory complexes, could serve as probes to functionally distinguish different roles of those complexes. The activities of GR and Brm can be monitored separately, since GR activates or represses transcription, whereas Brm remodels chromatin. Within a set of GR-activated and GR-repressed genes, we independently controlled the actions of the two proteins using glucocorticoid dependence of GR and knockdown of Brm, and we monitored the effects of GR and Brm on each other's occupancy and activity. At genes with GR-dependent Brm occupancy, we observed striking differences in the effects of Brm on GR occupancy and regulation (). Investigation of chromatin architecture at four such differentially controlled genes revealed distinct and unanticipated classes of chromatin remodeling. For example, in the absence of dex, Brm maintained either increased protection or increased accessibility, dex-dependent chromatin remodeling was Brm independent or Brm dependent, and the magnitude of Brm-dependent remodeling spanned a broad range. Overall, our analysis of four genes in A549 cells revealed four distinct patterns of transcriptional response, factor occupancy, and activity (). Thus, monitoring only two proteins within regulatory complexes is sufficient to identify functionally discrete assemblies.
At IGFBP1, an activated gene, Brm occupancy increased at the GBR in a GR-dependent manner, yet we cannot identify any activity associated with the recruitment of Brm to this regulatory region. Brm knockdown had no effect on GR occupancy, transcription, or chromatin remodeling, although Brm may remodel chromatin outside of the GBR and TSS regions that we analyzed. Previous studies have shown that increased GR and coregulator occupancy do not always correlate with their known activities (24
). In these cases, GR and coregulators may bind unproductively, or they may serve as scaffolds to recruit additional factors. Brm may be inactive at this site due to altered Swi/Snf subunit composition (34
) or posttranslational modifications of the Swi/Snf complex (5
). Since the two Swi/Snf ATPases possess distinct functions in certain settings (12
), future studies could determine whether Brg1-containing Swi/Snf complexes are responsible for the dex-dependent remodeling at IGFBP1. Alternatively, both Brm and Brg1 may participate in chromatin remodeling at this gene, but functional redundancy between these ATPases may allow Brg1-containing Swi/Snf complexes to compensate for Brm knockdown (10
). The choice between two ATPases with both redundant and distinct functions may increase the specificity and versatility of Swi/Snf-containing multifactor regulatory complexes. Preliminary ChIP assays indicate that Brg1 occupancy increases upon dex treatment at a subset of GBRs in tandem with Brm (see Fig. S8 in the supplemental material).
Interestingly, two genes with opposite transcriptional responses (MT2A and CCL2) displayed similar patterns of GR and Brm interdependence. Upon dex treatment, Brm occupancy and Brm-mediated chromatin remodeling increased at both genes. In the presence of Brm, GR occupancy increased, leading to the transcriptional activation or repression of MT2A or CCL2, respectively. Therefore, it appears that the regulatory complexes acting at these two genes similarly regulate GR and Brm occupancy as well as chromatin remodeling despite mediating opposite transcriptional responses. Perhaps differential recruitment of DNA-binding activators or repressors, due to Brm-mediated changes in the accessibility of their target sequences, dictates transcriptional activation or repression by stabilizing the associated chromatin conformation (22
). Alternatively, the overall composition of factors within the regulatory complex at the response element may specify the transcriptional output. For instance, differential recruitment of coregulators may produce an activating or repressing complex (24
), or differential recruitment of chromatin-modifying proteins may lead to activating or repressing histone marks (16
). Future experiments could identify differences within the MT2A and CCL2 regulatory complexes that might account for their opposite transcriptional outputs.
The reciprocal roles of GR in Brm occupancy and Brm in GR occupancy suggest positive feedback in which GR binds to chromatinized GBRs with low affinity, recruiting Brm-containing Swi/Snf complexes to these regulatory regions, causing local chromatin remodeling, which allows more GR, and in turn more Swi/Snf, to bind accessible GBRs. Even before dex treatment, Brm appears to associate and function at a low level throughout the genome. This basal level of Brm remodeling activity may be important for initial “pioneer” GR binding events to relatively inaccessible chromatin, as we observed GR-independent Brm-mediated chromatin remodeling at the GBR of CCL2. Consistent with previous studies, the GBRs of the genes we investigated appear to be in regions that are relatively accessible (18
). Although we examined Brm occupancy only at GBRs, others have suggested that Brm is also recruited to TSSs of GR-regulated genes in a dex-dependent manner (31
), possibly through looping interactions between regulatory complexes and the promoter (14
). These long-range interactions could produce the Brm-dependent remodeling that we observe around the TSSs.
In contrast to the similarities between repressed CCL2 and activated MT2A, CCL2 and GDF15 are both repressed genes, yet they have very different patterns of GR and Brm interdependence. Both genes showed a dex-dependent increase in Brm occupancy, leading to greater GR-mediated transcriptional repression. While robust Brm-mediated chromatin remodeling at CCL2 allowed increased GR occupancy, the mild remodeling observed at GDF15 was associated with decreased GR occupancy. The multifactor regulatory complexes acting at these two genes apparently dictate transcriptional repression through different mechanisms. At CCL2, changes in DNA accessibility regulate GR occupancy. However, GR occupancy at GDF15 appears fairly independent of chromatin remodeling, as the GBR is relatively accessible under all observed conditions. Instead, the Brm-dependent decrease in GR occupancy may reflect competition for binding with Brm or an unknown factor that is recruited by Brm. If Brm can indeed exclude a factor such as GR without remodeling chromatin, this would represent a fundamentally new mechanism of Brm action. Alternatively, local chromatin rearrangements not detectable by MNase digestion might account for the Brm-dependent decrease in GR occupancy.
Our investigation of two proteins present in functionally different regulatory complexes, each bearing perhaps 50 times as many components, revealed their distinctive roles and interdependencies in those complexes. In doing so, we laid the groundwork for an experimental system to monitor combinatorial assembly and function, in which we can independently modify and measure factor occupancy and activity. Since four genes showed four different patterns of transcriptional response and GR and Brm interdependence, we conclude that these two factors interact through multiple distinct mechanisms. Thus, our results underscore the power and specificity of combinatorial regulation.
By what mechanisms might factors engage different physical interactions that create distinctive regulatory complex geometries? One way to think about these results comes from the view of proteins as “mosaics” of potentially functional surfaces, each of which may be more “active” in some contexts than in others. For example, Rogatsky et al. (36
) found that GR activates the transcription of different genes in human U2OS cells by utilizing gene-specific patterns of functional surfaces. These differential patterns likely reflect, in part, allosteric effects on GR conformation driven by the precise DNA sequence with which GR interacts at each target gene (27
). However, there exist many contextual influences, including ligand chemistry, posttranslational modifications, and occupancy by other DNA-binding regulatory factors and non-DNA-binding coregulators. In turn, the functional surfaces so created or stabilized may confer enzymatic activities, serve as sites for posttranslational modification or interact with other factors, thereby greatly expanding combinatorial diversity. We demonstrate that profiling the interactions and functions of two factors is sufficient to separate regulatory complexes into distinct classes, implicating additional context-specific interactions. Overall, this strategy provides a general route toward the discovery of functional surfaces and components within regulatory complexes and showcases how combinatorial control produces remarkable regulatory specificity. Understanding the functional relationships between regulatory complex components will eventually enable prediction, rather than mere observation, of the diverse outputs of combinatorial regulation.