REQ and Brm specifically promote RelB/p52-dependent transcriptional activity. A, REQ and Brm activate RelB/p52-dependent luciferase activity. 293FT cells harboring the NF-κB reporter gene were transfected with NF-κB expression vectors (p50, p52, RelA/p50, RelB/p52, and c-Rel/p50) or the corresponding empty vector (pRK5) and cotransfected with expression vectors for Brm, BRG1, REQ, or the empty vector (pCAGF). 48 h after transfection, luciferase activity was determined. Values were defined as a ratio in comparison with the results from pRK5- and pCAGF-transfected cells. The columns represent the average values from triplicate experiments, and the bars indicate the standard deviation. B, protein levels generated from exogenous FLAG-protein expression vectors in the 293 FT cells used in A. The total protein was analyzed by Western blotting using anti-FLAG antibody. IB, immunoblot. C, effects of the knockdown of Brm, BRG-1, or REQ upon the expression of the endogenous ELC, BLC, and GAPDH genes in 293FT cells transiently transfected with RelA/p50 or RelB/p52. 293FT cells were transfected with expression vectors for RelA/p50, RelB/p52, or empty vector and with vectors expressing shBrm, shBRG1, shREQ#1, or shGFP (control). 3 days after transfection, the endogenous gene levels were analyzed by semi-quantitative RT-PCR. Similar transfection experiments were repeated two more times, and results of their expression levels are shown in supplemental Fig. S3. Densitometric analysis of these three gels was summarized in the right panel after the expression levels were normalized relative to those of “RelA/p50 and shGFP-expressing cells,” which were given a value of 1. The columns represent the average of triplicate, and the bars indicate the standard deviation (except for shREQ#2).

REQ and the Brm-type SWI/SNF complex transactivate the BLC gene after lymphotoxin treatment by forming a larger complex with RelB/p52 in a ligand-dependent manner. A, BLC activation after lymphotoxin treatment requires either REQ or Brm in HT-29 cells. HT-29 cells were stably transduced with shBrm, shBRG1, shREQ, or shGFP (control) expressing retroviral vectors. After a 6-h treatment with LT (100 ng/ml) or no LT (+LT and −LT, respectively), total RNAs were extracted to determine the expression levels of the endogenous BLC and GAPDH genes by semi-quantitative RT-PCR. Similar LT treatment experiments were repeated two times, and the results of expression levels are shown in supplemental Fig. S4. Densitometric analysis of these three gels was summarized in the right panel after the expression levels were normalized relative to those of LT-treated shGFP-expressing cells, which were given a value of 1. The columns represent the average of triplicate experiments, and the bars indicate the standard deviation (except for shREQ#2). B, Brm, BRG1 and REQ do not affect IL8 activation by TNF-α. HeLaS3 cells stably transduced with vectors expressing shBrm, shBrg1, shREQ#1, and shGFP (control) were treated with or without 10 ng/ml TNF-α (+TNF-α and −TNF-α, respectively). After 3 h, total RNAs were extracted to determine expression levels of the endogenous IL8, Brm, BRG1, REQ, and GAPDH genes by semi-quantitative RT-PCR. C, chromatin immunoprecipitation analysis of HT-29 cells treated with lymphotoxin (100 ng/ml) for 6 h or untreated. Fragmented genomic DNA was immunoprecipitated (IP) with anti-Brm, anti-BRG1, anti-REQ, or anti-RelB antibodies or normal rabbit IgG. After removal of the cross-links, DNA immunoprecipitates were used as PCR templates to detect the endogenous BLC, CD44, or GAPDH promoters. D, REQ and Brm interact with RelB/p52 in HT-29 cells. HT-29 cells stably transduced with a retrovirus vector expressing FLAG-REQ or nontransduced HT-29 cells were stimulated with 100 ng/ml lymphotoxin for 6 h or untreated. Nuclear fractions were then isolated and immunoprecipitated with anti-Brm, anti-BRG1, anti-REQ, and anti-RelB antibodies or normal rabbit IgG. Each immunoprecipitate was analyzed by immunoblotting (IB) using antibodies against FLAG, Brm, BRG1, BAF155, Ini1, RelB, or p100/p52.

Interactions between REQ and the SWI/SNF complexes in vivo and in vitro. A, Brm and BRG1 interact with REQ in vivo. Nuclear extracts from 293FT cells transiently expressing FLAG-Brm or FLAG-BRG1 were immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted (IB) using anti-REQ, anti-BAF155, and anti-FLAG antibodies, respectively. B, REQ interacts with Brm and BRG-1 in vivo. Nuclear extracts of HT-29 cells stably transduced with the pMXs-FLAG-REQ retroviral vector were immunoprecipitated with anti-FLAG antibody and analyzed by immunoblotting using anti-Brm, anti-BRG1, anti-BAF60a, anti-BAF155, anti-Ini1, and anti-FLAG antibodies, respectively. The immunoprecipitation assay was performed in the absence (EtBr−) or presence (EtBr+) of 50 μg/ml ethidium bromide. C, REQ interacts with SWI/SNF components in vitro. GST and GST-REQ proteins were mixed with the in vitro synthesized ³⁵S-proteins indicated. Binding proteins were eluted by boiling in SDS sample buffer, resolved by PAGE, and detected by autoradiography. Molecular mass markers (left) and the predicted position of each in vitro synthesized protein (arrowheads) are shown.

N-terminal region of REQ interacts with either SWI/SNF components or NF-κB p52 in vitro. A, REQ interacts with NF-κB p52 in vitro. RelA, RelB, c-Rel, p50, and p52 were each synthesized in vitro in the presence of [³⁵S]methionine, and GST pulldown assays were performed as described in Fig. 1C. B, structure of GST-REQ and GST fused to C-terminally truncated (CT1653744) and N-terminally truncated (NT1653745) REQ products. NLS, nuclear localization signal. C, Coomassie Brilliant Blue staining of the GST fusion proteins analyzed in D. D, an N-terminal region of REQ interacts with Brm, BRG-1, BAF60a, Ini1, and p52 in vitro. GST fusion proteins (B and C) were mixed with Brm, BRG-1, BAF60a, Ini1, or p52 synthesized in the presence of [³⁵S]methionine. Bound proteins were analyzed as described in Figs. 1C and 3A. Arrowheads indicate the predicted position of each protein synthesized in vitro.

REQ is required for the oncogenic activity induced by RelB/p52. A, immunoblotting (IB) analysis by anti-RelB, anti-p52/p100, and anti-actin (control) antibodies using whole cell extracts of several human tumor cell lines. HT-29 cells were treated without (−LT) or with (+LT) lymphotoxin for 6 h. B, REQ-knockdown in Panc-1 and H1299 cells does not affect their growth in monolayer cultures. Panc-1 and H1299 cells were stably transduced with shREQ#1-expressing retrovirus or empty control vectors. Each transduced population of cells was seeded into 96-well plates (1 × 10³ cells/well), and cellular growth was assayed using a Cell-Titer Glo assay kit. Cellular growth was measured as a ratio of the corresponding mock-infected cells at day 0. The values shown are the average of triplicate experiments, and the bars indicate the standard deviations. C, REQ is required for the anchorage-independent growth of Panc-1 and H1299 cells. Panc-1, H1299, and HT-29 cells stably transduced with shREQ#1 and shREQ#2 retroviral vectors as well as an empty vector were transferred into soft agar plates. After 3 weeks, the numbers of colonies were counted using at least three plates. The values were shown as a percentage of the colony numbers in the corresponding mock-infected cells. The columns represent the average values of two independent experiments, and the bars indicate the standard deviation.