It is well-established that the remodeling of chromatin structure is an essential process that has a profound effect on basic cellular functions including transcription, DNA recombination, repair, and replication (Fletcher and Hansen, 1996
; Fyodorov and Kadonaga, 2001
; Becker et al., 2002
; Elgin and Workman, 2002
). The mechanisms of gene activation are highly complex and because of this complexity most studies focus on individual events using biochemical or genetic approaches. Here our study provides an integrated kinetic view of gene activation events on a target gene in vivo involving hormone-dependent recruitment of chromatin- remodeling complexes, dynamic interaction of chromatin-remodeling complexes with a target promoter, chromatin remodeling, regulation of higher order chromatin structure, RNA pol II loading, and transcriptional activation. Our results demonstrate that individual gene regulatory events are coordinated in vivo by members of the SWI/SNF chromatin-remodeling complex thereby providing a mechanistic basis for BRG1 and BRM chromatin-remodeling complexes in the transcriptional process.
The 3134 (murine mammary adenocarcinoma) cell line contains 200 copies of the MMTV-LTR array stably integrated in a head-to-tail orientation at a single integration event near the centromere of chromosome 4 (Kramer et al., 1999
). The hormone responsiveness of the MMTV array is identical to that of a single copy MMTV promoter, thereby making it a useful model system to directly visualize gene expression events such as the recruitment of chromatin-remodeling complexes and nuclear receptors to a target promoter in real time (Fragoso et al., 1998
; Fletcher et al., 2002
). Belmont and colleagues (Memedula and Belmont, 2003
) have used an amplified gene array based on the lac operator/repressor system to analyze the sequential recruitment of chromatin-remodeling complexes by the acidic activator VP16 to a condensed chromatin locus. Tsukamoto et al. (2000)
and Janicki et al. (2004)
have used a modified lac operator/repressor artificial array to demonstrate the recruitment of a lac repressor-VP16 chimera that resulted in chromatin decondensation. However in these studies, the contribution of chromatin-remodeling complexes on chromatin decondensation were not directly investigated. Here we extended these studies by observing the in vivo functional link between local chromatin remodeling, higher order chromatin reorganization, and transcriptional activation using various approaches including quantitative in vivo microscopy, chromatin accessibility, and decondensation assays as well as photobleaching approaches. Importantly, we have determined for the first time that BRG1 and BRM chromatin-remodeling complexes have distinct kinetic properties on the MMTV array, and they dynamically associate with and dissociate from MMTV chromatin in a manner dependent on hormone and a functional ATPase domain.
Three subclasses of ATP-dependent chromatin-remodeling complexes have been identified in mammalian cells: SWI/SNF, ISWI, and Mi-2/CHD (Narlikar et al., 2002
). We find that the members of the SWI/SNF remodeling complex, BRG1 and BRM, are preferentially recruited to the MMTV promoter in a hormone-dependent manner. Under the same experimental conditions, we failed to detect any enrichment of the ISWI (Snf2h) chromatin-remodeling complex at the MMTV array (). Although, we cannot define the molecular basis of this specificity, the subunit composition of individual chromatin-remodeling complexes is likely a contributory factor (Hsiao et al., 2003
). We have confirmed the contribution of BRG1 and BRM ATPases in the transcriptional activation of the MMTV promoter by biochemical and imaging approaches in well defined genetic backgrounds. Using SW13 cells that are deficient in BRG1, BRM, and GR expression, we find that both BRG1 and BRM potentiated transcription by GR on a transiently introduced MMTV reporter template (). Furthermore, transactivation required a functional BRG1, BRM, and ATP hydrolysis because the ATPase-deficient forms of BRG1 and BRM failed to stimulate transcription under similar conditions. The introduction of ATPase-deficient remodeling complexes can also dramatically compromise transcription from the stably integrated MMTV repeat (). We have further confirmed our observations by siRNA-mediated silencing of endogenous BRG1 expression (). At this point, we are unable to ascertain if BRG1 and BRM make distinct contributions to MMTV activation. BRG1 and BRM may have unique functions in the transcriptional process; alternatively, GR might be able recruit either BRG1 or BRM via shared BAFs. The use of cell lines lacking BRG1 or BRM might provide some insight into the complex(es) that contributes to MMTV activation.
ATP-dependent chromatin-remodeling complexes and histone-modifying complexes dynamically modulate chromatin structure both at the nucleosome as well as at a higher order level (Vignali et al., 2000
; Jenuwein and Allis, 2001
). We explored the consequences of ATPase-deficient remodeling proteins on chromatin structure by using a restriction enzyme accessibility assay to assess the disruption of local chromatin structure. Our studies demonstrated the expected hormone-dependent increase in restriction enzyme cutting in control cells (from 9 to 20%) compared with an inhibition of this hormone-dependent increase in endonuclease cutting in cells expressing the dominant negative form of BRG1 (). Interestingly, our ChIP analysis showed that this reduction in chromatin remodeling in cells expressing BRG1-K-R was accompanied by a reduction RNA pol II loading and transcription. These experiments provide data that implicate chromatin remodeling by BRG1 as a necessary prerequisite for optimal transcription of the MMTV promoter.
We have also used quantitative DNA FISH analysis in conjunction with indirect immunofluorescence microscopy, to examine higher order chromatin reorganization events in vivo. We observed a large-scale chromatin decondensation, of the MMTV array, in response to hormone when wild-type BRG1 and BRM is expressed, as has been previously described (Mueller et al., 2001
). When BRG1-K-R or BRM-K-R was expressed, the hormone-dependent decondensation events were inhibited significantly by BRG1-K-R and less so by BRM-K-R, in keeping with the differential transcriptional effects of these remodeling-deficient proteins (). These findings suggest that chromatin remodeling mediated by BRG1 and BRM ATPases can lead to higher-order chromatin unfolding and reorganization and this, in turn, correlates well with increased transcription from the MMTV array.
The dynamics of BRG1 and BRM chromatin-remodeling complexes at a specific promoter and the modulation of their kinetic properties in response to environmental stimuli have never been demonstrated in native chromatin in living cells. In our study, we found that BRG1, BRM, and BRG1-K-R dynamically exchange at the MMTV promoter with distinct kinetic properties in a manner dependent on hormone and a functional ATPase domain. The dynamic exchange of remodeling proteins on the MMTV array are consistent with our in vitro results obtained from rapid UV laser cross-linking where purified SWI/SNF binds to and is displaced from purified MMTV chromatin (Fletcher et al., 2002
; Nagaich et al., 2004
). Because the FRAP recovery kinetics of chromatin proteins are directly related to their chromatin-binding properties (Lefebvre et al., 1991
; Fragoso et al., 1998
; Lever et al., 2000
; Kimura and Cook, 2001
; Hager et al., 2002
; Kimura et al., 2002
; Maruvada et al., 2003
; Phair et al., 2004
; Becker et al., 2005
; Chen et al., 2005
), we conclude that the remodeling proteins with the slowest exchange rate reside longest on the MMTV promoter and associate most strongly with MMTV chromatin. A comparison of the kinetic properties of chromatin-remodeling complexes revealed that BRG1 was more strongly associated with the MMTV array than BRM (D). Interestingly, the remodeler with the slowest exchange rate is the dominant negative BRG1 (BRG1-K-R). Molecular chaperones have been demonstrated to regulate the dynamic properties of GR and PR in the nucleus and recently the high mobility group box 1 protein, HMGB1 has been found to influence the residence time of GR in chromatin (Stavreva et al., 2004
; Wagner et al., 2004
; Elbi et al., 2004a
; Agresti et al., 2005
). Considering that the dominant negative BRG1 (BRG1-K-R) is simply a single amino acid change in the ATPase domain, our study reveals the importance of ATP hydrolysis in the dynamic properties of BRG1 and BRM. Further studies will be necessary for a complete understanding of the regulation of chromatin protein dynamics and its role in gene expression.