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

Abstract

Background:The NF-B dimer, RelA/p50, often requires the SWI/SNF complex for its transactivation function, but its molecular mechanisms remain elusive. Results: The NF-B canonical pathway induced by TNF-␣ is DPF3a/b-and SWI/SNF-dependent for some promoters. Conclusion: DPF3a and DPF3b are effective linkers for the SWI/SNF complex and RelA/p50. Significance: The NF-B⅐DPF3a/b⅐SWI/SNF complex would be an effective platform for promoter-specific transactivation.

The Werner Protein Acts as a Coactivator of Nuclear Factor κB (NF-κB) on HIV-1 and Interleukin-8 (IL-8) Promoters

“…293FT-LTR-Luc-5 has been previously described in detail (27). The complete sequence encoding the N-terminal HA tag fused to WRN cDNA was inserted into the expression vector pcDNA3 (Life Technologies).…”

“…ChIP Assay-ChIP assays were performed as described previously (27). Briefly, lysed cells were treated with 1% formaldehyde to cross-link protein DNA complexes, and then lysates were sonicated on ice using ELESTAIN035SD (ELECON Science Corp., Chiba, Japan) to shear the DNA into small fragments with an average length less than 0.5 kb.…”

“…In Vitro Binding Experiments-GST pulldown assays were performed as described previously (27). [ 35 S]Methionine-labeled RelA or p50 constructs were translated using a reticulocyte lysate system in vitro (Promega) and incubated with GST alone or GST-His-tagged WRN derivative deletion constructs.…”

“…Immunoprecipitation Assay-Immunoprecipitation assays were performed as described previously (27). Briefly, cells were lysed with TNE buffer (10 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and protease inhibitors, and cell lysates were incubated overnight on a rotating platform at 4°C with respective antibodies (5 g each), which were previously bound to Dynabeads Protein G (Life Technologies).…”

“…RelA/p50 after TNF-␣ Stimulation-Using 293FT-LTR-Luc5 cells, we further examined whether WRN is co-transcriptionally activated with NF-B during HIV-1 transcription (27). The 293FT-LTR-Luc5 cell line harbors a copy of luciferase, under the control of an intact HIV-1 LTR with no Tat gene, stably integrated as an HIV-1 vector (Fig.…”