Maximal induction of a subset of interferon target genes requires the chromatin-remodeling activity of the BAF complex

Abstract

The mammalian SWI/SNF-like chromatin-remodeling BAF complex plays several important roles in controlling cell proliferation and differentiation. Interferons (IFNs) are key mediators of cellular antiviral and antiproliferative activities. In this report, we demonstrate that the BAF complex is required for the maximal induction of a subset of IFN target genes by alpha IFN (IFN-alpha). The BAF complex is constitutively associated with the IFITM3 promoter in vivo and facilitates the chromatin remodeling of the promoter upon IFN-alpha induction. Furthermore, we show that the ubiquitous transcription activator Sp1 interacts with the BAF complex in vivo and augments the BAF-mediated activation of the IFITM3 promoter. Sp1 binds constitutively to the IFITM3 promoter in the absence of the BAF complex, suggesting that it may recruit and/or stabilize the BAF complex binding to the IFITM3 promoter. Our results bring new mechanistic insights into the antiproliferative effects of the chromatin-remodeling BAF complex.

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 from the cells, reverse transcribed, and used for RT-PCR analysis with primers for the genes shown on the right. The samples in lanes 1 and 2 were amplified five more cycles than the samples in lanes 3 to 6 to visualize the difference caused by the presence of BRG1. β actin was used as a negative control.

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 cleavages in the nucleosomal linker region were detected by LM-PCR. The open oval on the right side of the lanes indicates the protected region of DNA. The filled oval represents the Sp1-binding site. The filled squares represent ISREs. “+1” and an arrow indicate the position relative to the transcription start site and direction of transcription, respectively. The HincII and XmnI restriction sites are also indicated. Chr, nuclei (28). (B) A map of the boundaries of the protected region is shown. The mononucleosome-sized DNA isolated from SW-13 nuclei digested with MNase was subjected to LM-PCR analysis (28). The nested ³²P-labeled primers are indicated F1 (−35 to −12) and R1 (+10 to −14). The arrowhead indicates the major band. The nucleosomal boundaries indicated by the open oval were determined after deduction of the DNA sequence (25 bases) that was ligated to the mononucleosomes as a universal linker (adaptor). (C) Chromatin remodeling at the IFITM3 promoter is defective in SW-13 cells. Nuclei isolated from SW-13 and HeLa cells with or without IFN-α treatment were briefly digested with HincII, followed by complete digestion of the purified genomic DNA with XmnI. The cleavage products were detected by LM-PCR. (D) Transient expression of BRG1 in SW-13 cells restored the chromatin remodeling at the IFITM3 promoter upon IFN-α stimulation. SW-13 cells were transiently transfected with pMACS K^k and pBJ5-BRG1 for 24 h. Following treatment with IFN-α for 2 h, the transfected and nontransfected cells were sorted by magnetic beads and processed for HincII accessibility as described for panel C. The digestion of the XmnI site at −152 was somehow incomplete. The band of 520 bp was derived from complete digestion of the XmnI site at −330. Size markers are indicated on the left of each panel.

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 promoter was cloned into the pGL3/basic or pREP4-luc reporter vector. The constructs were cotransfected with either pBJ5 or BRG1 into SW-13 cells. Luciferase activity was determined with the dual luciferase assay kit from Promega. The bar graph was derived from the average of two experiments, with the error bar indicating the range from two experiments. (B and C) The IFITM3 promoter in pGL3 vector (panel B) or pREP4 vector (panel C) was cotransfected with pBJ5 or BRG1 into SW-13 cells for 24 h, followed by treatment with 500 U/ml and/or 5 mM sodium butyrate for 12 h. The luciferase activity was analyzed as described for panel A. The fold activation is indicated above each column. (D) Deletion analysis of the IFITM3 promoter is illustrated. The pREP4-TM3-luc constructs with 5′ deletions were analyzed as described for panel A. The numbers below the graph represent the base pairs from the transcription start site. The numbers above the bars represent the fold difference in activation by BRG1 compared to that by the pBJ5 vector.

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 in the EMSA and in the reporter construct for transfection are indicated. (B) EMSA was performed with the wild-type probe labeled with ³²P, 1 μg of SW-13 nuclear extracts, and 500 ng of poly(dI-dC) nonspecific competitor as described previously (27). Where applicable, 100× excess of unlabeled oligonucleotide was used as competitor. TM3, wild-type oligonucleotide from the IFITM3 promoter; m-TM3, Sp1 site-mutated oligonucleotide from the IFITM3 promoter. (C) The EMSA reaction included 1 μg of Sp1 antibody. M, monoclonal antibody; P, polyclonal antibody; PreIM, normal rabbit serum; ∗, band not observed reproducibly. (D) The IFITM3 promoter was mutated as mut-Sp1 as described for panel A. The mutant construct was analyzed by luciferase assay as described in the legend to Fig. 3A.

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 from immunoprecipitates with the antibodies indicated above the panel and analyzed by multiplex PCR with IFITM3 promoter primers (−251 to +61) and a pair of negative control primers (IFITM2U/F2 and IFITM2U/R1) that recognized the DNA sequence downstream of the 3′ untranslated region of the IFITM2 gene. PCR was performed in the presence of 5 μCi of [α-³²P]dCTP, and the products were analyzed in 5% polyacrylamide gels as described previously (39). Chromatin input was diluted 3 times at each step.

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 promoter primers and the negative control primers by real-time PCR. The graph shows the relative enrichment of the IFITM3 promoter sequence after normalization to the negative control sequence. The error bar indicates the range from two experiments. (B) Sp1 interacts with the BAF complex in vivo. Nuclear extracts prepared from DSP-cross-linked cells were immunoprecipitated with the indicated antibodies. The immunoprecipitates were resolved by SDS-PAGE and blotted with BRG1 or Sp1 antibodies. One percent of the nuclear extracts used for IP was loaded in lanes 1 and 5 as input. The size of the band is indicated on the left of the panels.