Requiem Protein Links RelB/p52 and the Brm-type SWI/SNF Complex in a Noncanonical NF-κB Pathway

Abstract

The SWI/SNF chromatin remodeling complex plays pivotal roles in mammalian transcriptional regulation. In this study, we identify the human requiem protein (REQ/DPF2) as an adaptor molecule that links the NF-B and SWI/SNF chromatin remodeling factor. Through in vitro binding experiments, REQ was found to bind to several SWI/SNF complex subunits and also to the p52 NF-B subunit through its nuclear localization signal containing the N-terminal region. REQ, together with Brm, a catalytic subunit of the SWI/SNF complex, enhances the NF-Bdependent transcriptional activation that principally involves the RelB/p52 dimer. Both REQ and Brm were further found to be required for the induction of the endogenous BLC (CXCL13) gene in response to lymphotoxin stimulation, an inducer of the noncanonical NF-B pathway. Upon lymphotoxin treatment, REQ and Brm form a larger complex with RelB/p52 and are recruited to the BLC promoter in a ligand-dependent manner. Moreover, a REQ knockdown efficiently suppresses anchorageindependent growth in several cell lines in which the noncanonical NF-B pathway was constitutively activated. From these results, we conclude that REQ functions as an efficient adaptor protein between the SWI/SNF complex and RelB/p52 and plays important roles in noncanonical NF-B transcriptional activation and its associated oncogenic activity.The SWI/SNF (switch/sucrose-nonfermentable) chromatin remodeling complex has important functional roles in the epigenetic regulation of many organisms and regulates a wide variety of genes (1, 2). In mammals, this complex is an assembly of about nine polypeptides and contains a single molecule of either Brm or BRG1 but not both (3). These two proteins are the catalytic subunits that together with noncatalytic subunits, such as BAF170, BAF155, Ini1, BAF60a, and ␤-actin, drive the remodeling of nucleosomes via their ATP-dependent helicase activity (4). Evidence has now accumulated that the Brm-type and BRG1-type SWI/SNF complexes regulate a set of target promoters that do not fully overlap. Indeed, Brm and BRG1 show clear differences in their biological activities. For example, Brm-type, but not BRG1-type, SWI/SNF complexes are essential for the maintenance of gene expression driven by the long terminal repeats of murine leukemia virus (5, 6) and human immunodeficiency virus (7). Moreover, in gastrointestinal cells, Brm but not BRG1 can transactivate Cdx2-dependent villin expression, even though both proteins can interact with Cdx2 (8). Overall, the SWI/SNF complexes interact with various proteins, including transcriptional regulators, through the many specific and varied associations with their subunits. We previously demonstrated that among all of the dimers formed between Fos and Jun family proteins, which compose the representative transcription factor AP-1, the c-Fos/c-Jun heterodimer most efficiently recruits SWI/SNF complexes to AP-1-binding sites through its specific binding activity to the BAF60a SWI/SNF subunit (9).Like AP-1, the NF-B 3 is an important transcription facto...

Non-canonical NF-κB signaling pathway

“…Another possibility is the involvement of chromatin remodeling factors in the specific control of non-canonical NF-κB. In this regard, a recent study suggests that RelB/p52 is linked to the SWI/SNF chromatin remodeling complex via an adaptor protein, requiem [91]. In response to lymphotoxin stimulation, requiem forms a large complex with the SWI/SNF catalytic subunit, Brm, and RelB/p52, which is recruited to the promoter of the BLC gene.…”

Double Plant Homeodomain (PHD) Finger Proteins DPF3a and -3b Are Required as Transcriptional Co-activators in SWI/SNF Complex-dependent Activation of NF-κB RelA/p50 Heterodimer

“…Compared with the Luc activity of cells transfected with CV-1, the introduction of RelA/p50, RelB/p52, and c-Rel/p50 increased these reporter expression levels by 42-, 6-, and 3-fold, respectively (Fig. 1C, lanes 13,19, and 7 compared with lane 1). These results indicated that NF-B-MinP-Luc-A3 has very low basal NF-B activity and that none of the candidates have any effects alone under these conditions, even if expressed at high levels.…”

“…For TNF-␣ stimulation, the cells were treated with 10 ng/ml TNF-␣ (R&D Systems, Minneapolis, MN). Vesicular stomatitis virus G protein (VSV-G)-pseudotyped retro-or lentiviral vectors were prepared as described previously (17,19). For transduction, cells were incubated with the virus vector stocks in the presence of 8 g/ml Sequabrene TM (Sigma).…”

“…The target proteins were eluted from these columns with His tag Elution Buffer (20 mM Tris-HCl (pH 8.0), 600 mM NaCl, 1 mM MgCl 2 , 10% glycerol, 0.1% Nonidet P-40, 250 mM imidazole, and phosphatase inhibitors) and GST Elution Buffer (100 mM Tris-HCl (pH 8.0), 12 mM NaCl, 20 mM glutathione, and phosphatase inhibitors), respectively. In vitro transcription and translation were then performed as described previously (19). In vitro synthesized proteins were incubated with respective GST fusion proteins for 2 h at 4°C on a rotating platform.…”

“…We have recently shown that DPF2 (REQ/BAF45d) functions as an efficient adaptor protein between the SWI/SNF complex and RelB/p52 and plays important roles in NF-B transcriptional activation at the most downstream part of the NF-B noncanonical pathway (19). DPF2 belongs to the d4 family of proteins, the members of which are characterized by an N-terminal 2/3 domain containing a nuclear localization signal, a central C2H2-type Krüppel-like zinc finger motif, and a C-terminal d4 domain consisting of a tandem repeat of the PHD zinc finger (20 -22).…”