Transient expression of BRG1 in SW-13 cells potentiates the maximal induction of a group of IFN target genes by IFN-α.
The BAF complex elevates the basal-level expression of 80 genes in SW-13 cells, including a number of IFN-inducible genes (27
). Since chromatin remodeling is considered an important step in the transcriptional activation of a gene, we hypothesized that the presence of the chromatin-remodeling BAF complex may enhance the induction of these IFN target genes by IFN. RT-PCR analysis confirmed the conclusion from the cDNA microarray results that the basal-level expression of four IFN target genes including IFITM2, IFITM3, IFIT1, and GBP was augmented by the transient expression of BRG1 in SW-13 cells, while no difference in expression was observed for ISG15 and ISGF3G (Fig. , compare lanes 1 and 2). IFN-α alone induced only weakly the first four genes. However, the presence of BRG1 in SW-13 cells dramatically enhanced the induction of these genes by IFN-α (Fig. , compare lanes 5 and 6). In contrast, ISG15 and ISGF3G were induced strongly by IFN-α alone and no significant difference in induction was observed in the presence of BRG1.
FIG. 1. Reconstitution of the BAF complex potentiates the maximal induction of a group of IFN target genes by IFN-α. SW-13 cells transfected with BRG1 expression construct for 24 h were treated with 500 U of IFN-α/ml for 10 h. Total RNA was extracted (more ...)
To rule out the possibility that the IFN-α signaling pathway is defective in SW-13 cells, we examined the phosphorylation status of STAT1 and STAT2 upon IFN-α treatment. We found that IFN-α treatment efficiently induced the phosphorylation of STAT1 and STAT2 in both SW-13 cells and HeLa cells (data not shown). Therefore, the defect in the induction of IFITM2, IFITM3, IFIT1, and GBP by IFN-α in SW-13 cells might be caused by the absence of the functional BAF complex, suggesting that the maximal induction of these genes may require chromatin remodeling by the BAF complex.
Chromatin remodeling at the IFITM3 promoter is compromised in SW-13 cells upon IFN-α induction.
The IFITM2 (1-8D) and IFITM3 (1-8U) genes are located in the same chromosome locus and share significant homology in their coding and 5′ promoter regions (26
). They are highly induced by IFN-α in HeLa cells (12
). The observation that the maximal induction of these genes by IFN-α requires the presence of BRG1 suggests that the chromatin structure at their promoters may not be efficiently remodeled in the absence of a functional BAF complex upon IFN-α induction. We examined whether there is a nucleosome-like structure at the IFITM3 promoter in SW-13 cells by MNase digestion and LM-PCR. Nuclei isolated from exponentially growing cells were treated with MNase, which generates double-stranded cleavages in the linker region. To map the double-stranded cleavage sites, a unidirectional linker was added to the blunt ends generated by the double-stranded cleavages by MNase. The linker primer and an IFITM3 promoter-specific primer were then used to amplify the DNA and then to label it with a nested 32
P-labeled IFITM3 promoter-specific primer. The purified genomic DNA was treated the same way as the control. As shown in Fig. , about 160 bp of DNA from +60 to −100 relative to the transcription start site was protected from MNase digestion, suggesting the presence of a nucleosome-like structure. The same MNase digestion pattern was observed in HeLa cells (data not shown). In order to map more precisely the boundaries of the possible nucleosomal structure, the mononucleosome-sized DNA isolated from SW-13 nuclei digested with MNase was analyzed by LM-PCR with primers within the protected region. As indicated in Fig. , the labeled F1 primer (−35 to −12) detected a major band of 118 bases that included 25 bases of the universal linker DNA, which was ligated to the mononucleosomes, indicating that the 3′ boundary is at +60 relative to the transcription start site. By using the labeled R1 primer (+10 to −14), the 5′ boundary of the nucleosome was determined to be at −90 (Fig. , lane 2). Therefore, taken together, the data shown in Fig. indicate the existence of a positioned nucleosome from −90 to +60. The majority of the two ISREs (−102 to −72) in the promoter are located within the nucleosomal DNA.
FIG. 2. Chromatin remodeling at the IFITM3 promoter requires the activity of the BAF complex. (A) MNase digestion defines a nucleosome positioned over the IFITM3 promoter. The nuclei or genomic DNA isolated from SW-13 cells were digested with MNase. The double-stranded (more ...)
MNase digestion was not able to reveal any differences in the chromatin structure at the IFITM3 promoter between SW-13 cells and HeLa cells with or without IFN-α treatment (data not shown). We then used the restriction enzyme accessibility assay to monitor the chromatin-remodeling event at the IFITM3 promoter upon IFN-α treatment. There is a HincII site (+34) located in the nucleosome which is downstream of the two ISREs, as shown in Fig. . Nuclei from SW-13 cells and HeLa cells with or without IFN-α treatment were briefly digested with HincII. The genomic DNA was purified and digested completely with XmnI, which recognizes a site at −152. The DNA cleavage pattern was analyzed by LM-PCR. Cleavage at the HincII site would generate a fragment of 181 bp, and cleavage at the XmnI site would generate a fragment of 367 bp. The nucleosome remodeling at the promoter would increase HincII digestion and therefore generate more 181-bp fragment. As shown in Fig. , the HincII site became significantly more accessible after 2 h of treatment with IFN-α in HeLa cells (lanes 3 and 4). However, no significant changes were observed in SW-13 cells upon IFN-α stimulation (lanes 1 and 2).
In order to demonstrate that the defect in remodeling the IFITM3 promoter in SW-13 cells was caused by the absence of the BRG1 protein, we transiently transfected the cells with pMACS Kk and pBJ5-BRG1. Following treatment with IFN-α for 2 h after 24 h of transfection, the transfected and nontransfected cells were sorted with magnetic beads and processed for HincII accessibility as described in the legend to Fig. . The results showed that BRG1 expression alone slightly increased HincII digestion (Fig. , compare lanes 1 and 2) and that chromatin remodeling was fully restored in SW-13 cells expressing BRG1 protein upon treatment with IFN-α (Fig. , compare lanes 3 and 4). The control XmnI site at −152 was somehow inefficiently digested, and the band of 520 bp was derived from the XmnI site at −320.
These results indicate that the BAF complex is required for chromatin remodeling at the IFITM3 promoter upon induction with IFN-α.
Activation of the IFITM3 promoter by the BAF complex is dependent on chromatin formation.
In order to identify the DNA elements and the corresponding protein factors that mediate the activity of the BAF complex, we cloned 238 bp of the IFITM3 promoter sequence into the pGL3-basic vector and tested the promoter activity in SW-13 cells by cotransfection with the BRG1 expression vector. As shown in Fig. , cotransfection with BRG1 did not activate the promoter. Since the BAF complex is a chromatin-remodeling complex, the activation of the IFITM3 promoter by the complex might require the formation of proper chromatin structure. Therefore, we cloned the promoter into a pREP4-based episomal reporter vector that forms proper chromatin structure when transiently transfected into cells (27
). The IFITM3 promoter in the pREP4 vector was significantly activated by BRG1 (Fig. ). Interestingly, transient expression of the BRG1 homologue, hBRM, also efficiently activated the IFITM3 promoter in the pREP4 vector (data not shown).
FIG. 3. Synergistic activation of the IFITM3 promoter by BRG1 and IFN-α is dependent on the formation of proper chromatin structure. (A) Activation of the IFITM3 promoter by the BAF complex requires formation of proper chromatin structure. The IFITM3 (more ...)
Since activation of the IFITM3 promoter by the BAF complex is dependent upon the formation of proper chromatin structure, we hypothesized that the synergistic effect of the BAF complex and IFN-α on the induction of the IFITM3 gene might also require the proper chromatin structure. As shown in Fig. , the IFITM3 promoter in pGL3 vector was activated 6.2 times by IFN-α alone. The presence of BRG1 did not cause a significant increase in the activation (compare columns 3 and 4). However, the IFITM3 promoter in the pREP4 vector was significantly more activated by IFN-α in the presence of BRG1 (30 times) than in the absence of BRG1 (5 times) (Fig. , compare columns 3 and 4), indicating that the proper chromatin structure is a critical mediator of synergy.
Since the histone acetyltransferase activity of CBP/p300 is required for the induction of IFN-α/β target genes (3
), we tested the effect of inhibiting histone deacetylase activity by butyrate treatment on activation of the IFITM3 promoter. As shown for the pGL3 vector in Fig. , butyrate activated the promoter 1.8-fold (column 5). Both IFN-α and butyrate synergistically activated the promoter about 17-fold (column 7). No further activation was observed in the presence of BRG1 (compare columns 7 and 8). In contrast, the presence of BRG1 dramatically increased activation of the IFITM3 promoter in the pREP4 vector from 9-fold to 52-fold (columns 7 and 8 in Fig. ), consistent with the idea that histone acetylation stabilizes the binding of the SWI/SNF complex to the chromatin template (18
A 5′ deletion analysis of the promoter revealed that deletion to −173 slightly reduced the fold activation by BRG1 (Fig. ). A more significant decrease in the fold activation by BRG1 was observed when the promoter sequence was deleted to −152. Further deletion to −138 abolished activation by BRG1. Therefore, at least two major elements may be mediating the activity of the BAF complex, one located between −173 and −152 and the other between −152 and −138.
Sp1-binding site contributes to activation of the IFITM3 promoter by the BAF complex.
While examination of the DNA sequence between −173 and −152 did not reveal any apparent binding sites for known protein factors, the region between −152 and −138 contains one Sp1-binding motif (Fig. ). EMSA showed that the SW-13 nuclear extracts produced two reproducible shifts (labeled C1 and C2) of the 22-mer probe covering the Sp1-binding site from the IFITM3 promoter (Fig. , lane 2). The shifts were inhibited with a 100-fold excess of the unlabeled wild-type probe (lane 3) but not with the mutant-binding site (lanes 4). Both of the monoclonal and polyclonal antibodies against the Sp1 protein induced a very faint supershifted band and at the same time completely inhibited the upper shift, C1, while the C2 shift was only partially inhibited (Fig. , lanes 3 and 5). These data indicate that the C1 shift and part of the C2 shift were generated by Sp1 binding (Fig. , lanes 2 and 3).
FIG. 4. Sp1 contributes to the activation of the IFITM3 promoter by the BAF complex. (A) The sequence of the oligonucleotide probe surrounding the Sp1-binding site (underlined) from the wild-type IFITM3 promoter used for the EMSA is shown. The mutated bases used (more ...)
In order to determine if the Sp1-binding site contributes to the regulation of the IFITM3 promoter by the BAF complex, we tested the pREP4-TM3-luc construct with the same mutation as shown in Fig. in transfection experiments. As shown in Fig. , mutation of the Sp1-binding site reduced dramatically the activation of the IFITM3 promoter by the BAF complex while it caused a modest decrease in the basal-level activity of the promoter.
These results suggest that Sp1 binds to the IFITM3 promoter and augments activation by the BAF complex.
BAF complex and Sp1 are constitutively associated with the IFITM3 promoter in vivo.
Previous studies suggest that the chromatin-remodeling complexes are recruited to their target promoters upon induction (2
). In order to determine if the SWI/SNF-like BAF complex and Sp1 are recruited to the IFITM3 promoter upon induction by IFN-α, we performed the ChIP assay with HeLa cells. Chromatin fractions prepared from formaldehyde-cross-linked HeLa cells with or without prior IFN-α treatment were immunoprecipitated with antibodies against Sp1, BRG1 (which recognizes both BRG1 and hBRM), and STAT2. The immunoprecipitated DNA was reverse cross-linked and analyzed by multiplex PCR, with the DNA sequence downstream of the 3′ untranslated region of the IFITM2 gene as the negative control. As expected, STAT2 became bound to the promoter upon treatment with IFN-α, while no binding was detected in the absence of IFN-α treatment (Fig. ). However, antibodies against BRG1 and Sp1 enriched the promoter sequence relative to the control sequence in both the presence and absence of IFN-α treatment, indicating that the BAF complex and Sp1 are constitutively associated with the promoter in vivo. These data are consistent with the observation that transient expression of BRG1 in SW-13 cells remodeled and activated the IFITM3 promoter even in the absence of IFN-α treatment (Fig. , 2D, and 3).
FIG. 5. BAF complex and Sp1 are constitutively associated with the IFITM3 promoter in vivo. Chromatin was prepared from HeLa cells without (−) or with (+) IFN-α treatment for 30 min prior to formaldehyde cross-linking. The DNA was purified (more ...) Sp1 is bound to the IFITM3 promoter in the absence of the active BAF complex in SW-13 cells.
The Sp1-binding site in the IFITM3 promoter appears to be located outside of the positioned nucleosome (Fig. ). Therefore, Sp1 binding to the promoter might not require the activity of the BAF complex. To test this hypothesis, we performed a ChIP assay with SW-13 cells. As quantified by real-time PCR in Fig. , Sp1 antibodies modestly enriched the promoter sequence in the immunoprecipitates while no enrichment was observed with antibodies against BRG1, which does not have detectable levels of expression in the cells. Similar results were obtained with SW-13 cells treated with IFN-α (data not shown). These results suggest that Sp1 is constitutively associated with the IFITM3 promoter, even in the absence of the BAF complex.
FIG. 6. Sp1 interacts with the BAF complex in vivo. (A) Association of Sp1 with the IFITM3 promoter does not require the activity of the BAF complex. The immunoprecipitated chromatin from SW-13 cells with each antibody was analyzed, respectively, with the IFITM3 (more ...) Sp1 interacts directly with the BAF complex in vivo.
Since the Sp1-binding site contributed to activation of the promoter by the BAF complex (Fig. ) and the zinc finger DNA-binding domain of Sp1 has been shown to interact with BRG1 and BAF155 in vitro in a GST pull-down assay (20
), we decided to test the direct in vivo interaction between Sp1 and the BAF complex. While coimunoprecipitation from the nuclear extracts of Jurkat cells did not yield conclusive results, we used an in vivo cross-linking strategy to stabilize any possible interaction between them. Exponentially growing Jurkat cells were cross-linked briefly with the cell membrane permeable and reversible cross-linker DSP, followed by nuclear extract preparation as described previously (50
). The cross-linked nuclear extracts were immunoprecipitated with anti-Sp1 and anti-BRG1 antibodies in high-salt buffer, and the coimmunoprecipitated proteins were detected by Western blot analysis. As shown in Fig. , the Sp1 and BRG1 antibodies could each coprecipitate the other (lanes 4 and 8). The preimmune serum and anti-GATA1 antibodies did not pull down either the Sp1 or BRG1 proteins (lanes 2, 3, 6, and 7). Since the cross-linker, DSP, does not cross-link protein to DNA, which rules out the possibility that the coimmunoprecipitation of the BAF complex with Sp1 resulted from the BAF complex and Sp1 being cross-linked to the same DNA fragment, and the mild conditions we used favor the cross-linking of directly interacting proteins (50
), these data strongly suggest that Sp1 and the BAF complex interact directly in vivo.