Interferon-gamma-induced chromatin remodeling at the CIITA locus is BRG1 dependent - PubMed (original) (raw)
Interferon-gamma-induced chromatin remodeling at the CIITA locus is BRG1 dependent
Samantha G Pattenden et al. EMBO J. 2002.
Abstract
SWI/SNF regulates growth control, differentiation and tumor suppression, yet few direct targets of this chromatin-remodeling complex have been identified in mammalian cells. We report that SWI/SNF is required for interferon (IFN)-gamma induction of CIITA, the master regulator of major histocompatibility complex class II expression. Despite the presence of functional STAT1, IRF-1 and USF-1, activators implicated in CIITA expression, IFN-gamma did not induce CIITA in cells lacking BRG1 and hBRM, the ATPase subunits of SWI/SNF. Reconstitution with BRG1, but not an ATPase-deficient version of this protein (K798R), rescued CIITA induction, and enhanced the rate of induction of the IFN-gamma-responsive GBP-1 gene. Not ably, BRG1 inhibited the CIITA promoter in transient transfection assays, underscoring the importance of an appropriate chromosomal environment. Chromatin immunoprecipitation revealed that BRG1 interacts directly with the endogenous CIITA promoter in an IFN-gamma-inducible fashion, while in vivo DNase I footprinting and restriction enzyme accessibility assays showed that chromatin remodeling at this locus requires functional BRG1. These data provide the first link between a cytokine pathway and SWI/SNF, and suggest a novel role for this chromatin-remodeling complex in immune surveillance.
Figures
Fig. 1. IFN-γ does not induce MHC class II and CIITA in SW13 cells. (A) Lack of MHC class II induction in SW13 cells. Flow cytometry using a PE-conjugated antibody against human MHC class II DR antigen was performed on MTRB-1 control cells, or SW13 BRG1-deficient cells that were untreated (shaded curve), or exposed to IFN-γ for 24 h (solid line). The dotted line represents cells analyzed with an isotype control antibody. Untreated EBV-transformed B cells, which express MHC class II constitutively, were also analyzed. (B) Lack of CIITA induction. SW13 (lanes 1 and 2), MTRB-1 (lanes 3 and 4) or S4 (lanes 5 and 6) cells were left untreated (lanes 1, 3 and 5) or exposed to IFN-γ for 24 h (lanes 2, 4 and 6) and mRNA levels of CIITA, GBP-1 or β-actin assessed by RT–PCR. (C) Expression of BRG1. Western analysis of 50 µg of total cell lysate shows that BRG1 is expressed in control lines (MTRB-1 and S4), but not in SW13 cells.
Fig. 2. BRG1 rescues CIITA induction in SW13 cells. SW13 cells were left uninfected (lanes 1 and 2), or infected with AdtTa virus alone (lanes 3 and 4) or together with AdBRG1 (lanes 5–8) or AdK798R (lanes 9–12) in the absence (lanes 1–6) or presence of tetracyline (lanes 7–12). After 20 h, cells were exposed to no cytokine (lanes 1, 3, 5, 7, 9 and 11) or IFN-γ (lanes 2, 4, 6, 8, 10 and 12) for an additional 6, 12 or 24 h. RT–PCR was used to measure CIITA, GBP-1 or β-actin RNA levels. The experiment was performed three times. Representative samples are shown.
Fig. 3. The effect of BRG1 on the CIITA promoter is chromatin dependent. (A) STAT1, IRF-1 and USF-1 expression in SW13 cells. Cells were treated with IFN-γ for 0, 6, 12 or 24 h, and lysate analyzed by western blot using antibodies to STAT1, IRF-1 and USF-1. (B) IFN-γ activation of a transiently transfected CIITA reporter vector does not require BRG1. SW13 cells were transfected in duplicate with 0.8 µg of phCIITAPIV-LUC reporter plasmid (shown schematically) and increasing amounts (1, 3 and 5 µg) of a vector expressing BRG1 (pBJ5-BRG1). The empty expression vector pBJ5 was used to fill samples to ensure equimolar amounts of total plasmid. Samples were left untreated, or exposed to IFN-γ for 24 h. Baseline activity (relative activity = 1) is that obtained in untreated cells transfected with pBJ5. The results shown are the average and range of two experiments each performed in duplicate. (C) BRG1 has opposite effects on the endogenous versus transiently transfected CIITA promoter. SW13 cells were infected with AdtTA plus control AdGFP (lanes 1 and 2), AdBRG1 (lanes 3 and 4) or AdK798R (lanes 5 and 6) adenoviruses, transfected the next day with 1 µg of phCIITAPIV-LUC and then left untreated or exposed to IFN-γ for 24 h. Protein and RNA were prepared for luciferase (graph) and RT–PCR (lower panel) assays, respectively. The luciferase assays are the average and range of two experiments, each performed in duplicate. The RT–PCR is representative of two separate experiments (and reproduces the conclusions from Figure 2).
Fig. 4. IFN-γ-inducible BRG1 recruitment to the CIITA promoter. HeLa-Ini-11 cells were left untreated or exposed to IFN-γ for 24 h, formaldehyde cross-linked and the chromatin sonicated and used in ChIPs. Primers for CIITA promoter IV or the irrelevant PITX1 intron were used to amplify no template (lane 1), control human genomic DNA (lane 2), input DNA (lane 3) or DNA isolated following ChIP with no antibody (lane 4), anti-BRG1 (lane 5) or anti-GAL4 (lane 6). The CIITA primers amplify from –352 to +160 relative to the transcription start site.
Fig. 5. BRG1-dependent chromatin remodeling at the CIITA promoter. (A) DNase I accessibility requires BRG1. SW13 cells were infected with AdtTa plus AdBRG (lanes 2–7), or AdK798R (lanes 8–13). After 20 h, cells were left untreated (lanes 2–4 and 8–10) or exposed to IFN-γ for 24 h (lanes 5–7 and 11–13). Nuclei were harvested and treated with 12.8 (lanes 2, 5, 8 and 11), 25.6 (lanes 3, 6, 9 and 12) or 51.2 (lanes 4, 7, 10 and 13) ng/µl DNase I for 3 min at 37°C. DNA was isolated and analyzed by LM-PCR. (B) _Hae_III accessibility requires BRG1. Uninfected SW13 cells (lanes 2 and 3) or cells infected with AdtTa and AdBRG (lanes 4 and 5) were treated for 24 h with IFN-γ (lanes 2–5). Nuclei were isolated and incubated in vivo with _Hae_III (lanes 2 and 4) or no enzyme (lanes 3 and 5) at 37°C for 10 min (lanes 2 and 4). DNA was extracted, digested to completion in vitro with _Avr_II and analyzed by LM-PCR. The intensity of each _Hae_III band in lanes 2 and 4 was normalized to that of the full-length primer extension product to the _Avr_II site. The ratio of these values gave the fold increase in _Hae_III accessibility. Arrows on the left and right indicate the _Hae_III and _Avr_II sites, respectively. The marker in both (A) and (B) (lane 1) is _Msp_I-digested pBR322 DNA. The position of elements in the CIITA promoter is indicated to the right of each figure. Major (+1) and minor (+16) start sites of transcription are indicated.
References
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