We tested the role of ATP-dependent remodeling in differentiation using primary T cells and BRG1 as a model system. We found BRG1 was required for Th2 differentiation. BRG1 was required during and after differentiation for Th2 cytokine transcription and during differentiation (but not after) for expression of the c-Maf and GATA3 mRNAs. BRG1 bound to the Th2 cytokine cluster, including the LCR, and also the GATA3 gene. Reduction in BRG1 expression affected IL-4 and IL-13 more strongly than IL-5, a finding reminiscent of the previously described phenotype of deletion of part of the LCR. BRG1 binding was regulated by the extent of differentiation and the choice of cell fate and cell activation. BRG1 was acting at least in part through the BAF complex. BRG1 was required for the Th2 cytokine cluster and GATA3 genes to adopt their typical chromatin structure. STAT6 bound the Th2 LCR, was required for NFAT and BRG1 binding to the LCR, and was associated with BRG1. NFAT activity was also required for BRG1 binding to the Th2 LCR.
We infer that at least part of the observed changes in gene expression are the result of BRG1 directly regulating the target genes rather than indirect (i.e., regulating a regulator of the target genes). First, we found that BRG1 bound to target loci with kinetics such that it is competent to regulate the cytokines and transcription factors. Second, reduction in BRG1 expression triggered both changes in gene expression and changes in chromatin structure at BRG1 binding sites, which is consistent with a direct, causal role for BRG1 remodeling in transcriptional regulation. Third, in mature effector cells BRG1 knockdown does not appear to regulate known regulators of Th2 cytokines. Fourth, BRG1 binding was observed specifically at known and putative regulatory regions. In differentiating cells BRG1 knockdown decreases mRNA encoding GATA3 and c-Maf, two transcription factors known to positively regulate Th2 cytokines. However, it does not strongly change the protein expression. Thus, in differentiating cells, GATA3 (and perhaps c-Maf) is a direct target of BRG1, whereas the Th2 cluster appears to be regulated by a combination of direct and perhaps indirect BRG1 effects. We propose the following simple model: BRG1 changes the chromatin structure of target loci, which allows enhancers and perhaps silencers to change their activity, which directly regulates gene expression.
We found that BRG1 plays a direct role in changing chromatin structure at the Th2 cytokine and transcription factor genes, which provides insight into the mechanism of a remodeling enzyme during choice of developmental fates. The change in DNase I hypersensitivity in the Th2 cytokine cluster have been carefully documented by several labs under various conditions. To our knowledge, this is the first report identifying an ATP-dependent chromatin remodeling enzyme that plays a role in their formation. Given the well-documented activity of BRG1 and SWI/SNF complexes in cell-free systems, we hypothesize that BRG1 is unfolding, sliding, or displacing nucleosomes to create DNase I HS; our experiments do not distinguish among these possibilities. DNase I hypersensitivity can reflect nucleosome-free regions, transcription factor binding, histone modifications, or nucleosome unfolding. At least part of this BRG1 activity appears to be through the BAF complex.
It is perhaps surprising that BRG1 is required in effector cells, since some of the Th2 cytokine locus DNase I HS, such as HSIV, are present in resting Th2 cells (1
). One might have predicted that the chromatin structure was mostly programmed during differentiation, and after it was set there would be little if any need for remodeling. However, we found that BRG1 is required for Th2 effector cell chromatin structure and that BRG1 is functionally important in these cells. We found that nuclease sensitivity at RHS7 is inducible after Th2 differentiation is complete, consistent with a prior report (29
); thus, the function of BRG1 may be to alter chromatin structure in effector cells. Our finding is also consistent with the recently described role of SWI/SNF in mediating the recall of transcriptional regulation memory in budding yeast (26
We found that BRG1 binding to the Th2 LCR is dependent on STAT6 and, furthermore, that BRG1 directly interacts with this transcription factor. Interestingly, a number of other STAT proteins have also been suggested to activate transcription and remodel chromatin, either directly or indirectly, through BRG1, suggesting that the BRG/STAT pathway is general (18
). In our system, we found that BRG1 recruitment to the Th2 LCR depends both on cytokine signals through STAT6, as well as signaling through the TCR via NFAT1. Since the differentiation of a Thp to a Th2 cell depends on these same signals, we suggest that BRG1 uses the mechanism of chromatin remodeling to integrate these signals. This is reminiscent of results reported for the IFN-γ promoter, where BRG1 binding in Th1 cells is dependent on both NFAT and STAT4 (64
). It is tempting to speculate that the recruitment of chromatin remodeling factors to STAT binding sites via STAT proteins is a general feature of cytokine-induced transcription, providing a direct connection between extracellular signals and epigenetic changes at the level of nucleosome remodeling.
Although our data suggest that the developmentally regulated and inducible recruitment of BRG1 to the Th2 locus is dependent on the activation of Th2 transcription factors, we cannot rule out a role for direct modification of BRG1-containing complexes or Th2 transcription factors by T-cell activation. Antigen receptor signaling has been shown to induce the rapid association of BRG complexes to chromatin, an activity that is dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) (65
). In addition, p38 mitogen-activated protein (MAP) kinase has been demonstrated to directly phosphorylate the BAF60 subunit of SWI-SNF resulting in the recruitment of SWI-SNF to a target promoter (52
). BRG1 itself is also capable of being phosphorylated by ERK1 in a cell cycle-dependent manner, resulting in the inactivation of BRG1 activity (51
). GATA3 has been found to be a target of MAP kinase cascades (12
). Given the established role for MAP kinases in Th differentiation (15
), future experiments will be needed to determine whether this signaling pathway plays a role in directing BRG1 recruitment to cytokine loci.
We do not yet know how BRG1 is activating transcription in this system. We found that BRG1 binds strongly to RHS7 in a Th2-specific manner, before DNase I hypersensitivity is established, and that BRG1 is required for DNase I HS at RHS7 in Th2 effector cells. Our finding that IL-4, IL-13, and IL-10 require BRG1 in Th2 effector cells, whereas IL-5 does not, is reminiscent of a previous finding that RHS7 is required in vitro under Th2 conditions for IL-4 and IL-13 mRNA production, but not for IL-5 (33
). It is unclear why IL-5 is strongly affected by BRG1 knockdown during differentiation but is unaffected after differentiation. We note that BRG1 binding is weak or absent at the IL-5 proximal regions that we have tested to date; perhaps another remodeling enzyme is regulating IL-5. It may be significant that we measure the strongest BRG1 binding at sites that are distant from the transcriptional start sites rather than the more frequently studied promoters. BRG1-mediated remodeling might be affording access to transcriptional activators or RNA polymerase, facilitating the action of other remodeling enzymes such as histone-modifying enzymes, or mediating changes in higher-order chromatin structure that permit long-range interactions in the locus.
We hypothesized that BRG1 would be important for the chromatin structure of at least some of the previously mapped DNase I HS. Unexpectedly, BRG1 binds to nearly every regulatory element we tested. Our assays are specific, since nonconserved regions in the Th2 locus (IL-13, kb -11; IL-5, kb -6) and a neuron-specific gene (NfM/Nef3, exon 1) lack BRG1 binding, histone acetylation, and DNase I HS. Moreover, BRG1 bound the IFN-γ promoter in Th1 cells but not Th2 cells. Although we favor a model wherein BRG1 is binding to specific regulatory elements, further experiments are in progress to determine whether BRG1 binds discrete elements or broad regions. We also detected BRG1 binding at sites that do not appear to have BRG1-dependent chromatin structure; we do not yet know whether BRG1 has no effect at these sites or is redundant with other remodeling enzymes, or whether the residual BRG1 following knockdown is sufficient to program the chromatin structure at these sites.
We have preferentially reduced BRG1 expression and revealed a role for the SWI/SNF component in Th2 differentiation, cytokine gene expression, and chromatin structure. The remaining Th2 function may be dependent on the residual BRG1 expression, BRM, or other remodeling enzymes; our experiments do not exclude a role for BRM. Our observation that reduced expression of BAF250a, another SWI/SNF component, has a similar effect as BRG1 provides independent confirmation of the role of SWI/SNF in these processes. This finding also detects a function for the BAF version of SWI/SNF; however, this does not exclude a role for P-BAF versions of SWI/SNF.
In a recent study examining the role of SATB1 in the Th2 locus, it was reported that BRG1 was present throughout the locus in a Th2 cell clone and that binding increased upon activation (11
). We extended these findings here by using primary cells to determine that BRG1 is not bound in naive cells and that binding occurs during Th2 differentiation. We found high BRG1 binding to the LCR and low binding to the IL-4 promoter and CNS1, whereas this pattern is reversed in the Th2 cell clone; this might reflect a difference between primary cells versus a T-cell clone maintained in vitro.
BRG1 is essential for embryonic development and is ubiquitously expressed. BRG1 has demonstrated roles in T-cell development, T-cell lineage choice, Th differentiation and function, and macrophage function. However, much remains to be discovered about how BRG1 helps to control these processes. We have found that in Th2 differentiation, BRG1 directly regulates targets at the transcriptional level by binding to proximal and distal regulatory regions and programming chromatin structure. We speculate this mechanism is likely to be recapitulated in other cell types throughout the body during development rather than being specific to T cells.