Degradation of promoter-bound p65/RelA is essential for the prompt termination of the nuclear factor kappaB response

Abstract

Transcription factors of the nuclear factor (NF)-kappaB/Rel family translocate into the nucleus upon degradation of the IkappaBs. Postinduction repression of NF-kappaB activity depends on NF-kappaB-regulated resynthesis of IkappaBalpha, which dissociates NF-kappaB from DNA and exports it to the cytosol. We found that after activation, p65/RelA is degraded by the proteasome in the nucleus and in a DNA binding-dependent manner. If proteasome activity is blocked, NF-kappaB is not promptly removed from some target genes in spite of IkappaBalpha resynthesis and sustained transcription occurs. These results indicate that proteasomal degradation of p65/RelA does not merely regulate its stability and abundance, but also actively promotes transcriptional termination.

Figure 1.

Termination of the NF-κB response in IκBα-deficient cells. (A) Kinetics of NF-κB down-regulation in WT and IκBα^−/− 3T3 cells. Cells were stimulated with TNF-α as indicated and analyzed for NF-κB binding activity by electrophoretic mobility shift assay (EMSA) using a canonical κB site as a probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′). Data were quantified with a phosphorimager. Kinetics of IκBα degradation and resynthesis are also shown. (B) Proteasome inhibition prevents NF-κB down-regulation in IκBα^−/− cells. After TNF-α washout, cells were incubated with β lactone and assayed by EMSA. An anti-p65 Western blot (W.B.) on cytoplasmic and nuclear extracts is shown. (C) Endogenous p65 is ubiquitinated after NF-κB activation. Extracts from IκBα^−/− cells were immunoprecipitated with an anti-p65 antibody and blotted with an anti-ubiquitin monoclonal antibody. H.C., heavy chains. (D) Reconstitution of p65 ubiquitination by cotransfection of FLAG-p65 and myc-ubiquitin expression vectors in HEK-293T cells. Blots obtained from whole cell extracts were probed with an anti-p65 polyclonal antibody and an anti-myc monoclonal antibody. (E) IκBα^−/− cells were stimulated with TNF for 15 min, washed, and incubated with 10 ng/ml LMB for an additional 3.15 h. EMSAs were performed on nuclear lysates. As a positive control for LMB effects, accumulation of IκB kinase complex α in the nuclear fraction of LMB-treated cells is shown.

Figure 2.

p65 ubiquitination requires sequence-specific binding to κB sites. (A) WT p65 and a p65 mutant bearing a double substitution in the Rel homology domain (23Y > A; 26E > D) were cloned with an NH₂-terminal GFP tag and transfected in HEK-293 cells alone or with a p50 expression vector. Total lysates were made and assayed by EMSA using a canonical κB site as a probe. The GFP tag allows easy discrimination between p65 homodimers and p65/p50 heterodimers (n.s., nonspecific). An anti-p65 immunoblot shows the expression of endogenous p65 and transfected GFP-p65 in total lysates. Expression of IκBα is also shown. (B) Association of WT and mutant GFP-p65 with p50 and IκBα. 293T cells were transfected as indicated. Total cell extracts were immunoprecipitated with either an anti-p50 or an anti-IκBα antibody and then blotted with an anti-p65 antibody. (C) The κB site binding-deficient p65 mutant is not efficiently polyubiquitinated. HEK-293T were cotransfected with the indicated expression vectors. Whole cell extracts were assayed for the appearance of high molecular weight ubiquitinated forms of p65 by anti-p65 immunoblotting. (D) p65 induces recruitment of proteasome components to target genes. HEK-293 cells were transfected with empty vector or a FLAG-p65 expression vector. ChIP assays with an anti-FLAG antibody, an antibody against Sug1, or a control antibody were performed. Recruitment of FLAG-p65 and Sug1 to the endogenous IκBα gene promoter is shown. (E) Anti-p65 and anti-Sug1 ChIP assays on HEK-293 cells stimulated with TNF-α. Immunoprecipitated DNA was amplified with primers spanning the IκBα promoter or a region immediately downstream of the IκBα gene.

Figure 3.

Proteasome inhibition in IκBα-deficient cells determines persistent promoter occupancy and increased NF-κB–dependent transcriptional activity. (A) Anti-p65 ChIP in TNF-α–stimulated IκBα^−/− cells. β lactone treatment after a pulse of TNF prolongs occupancy of all target genes tested and induces (B) increased and sustained transcription. (C) Proteasome inhibition increases κB site–directed transcription of a luciferase reporter in TNF-α–stimulated IκBα^−/− cells.

Figure 4.

Effects of proteasome inhibition on NF-κB response termination in IκBα-containing cells. (A) Proteasome inhibition impairs down-regulation of nuclear NF-κB activity (assayed by EMSA) in WT 3T3 cells. IκBα degradation and resynthesis is also shown. (B) Anti-p65 ChIP assay and mRNA analysis (C) in TNF-stimulated WT 3T3 cells treated with β lactone or vehicle as indicated. The effects of β lactone treatment on IP-10 and MIP-2 occupancy by p65 and on their transcriptional activity are shown. (D) Effects of proteasome inhibition on NF-κB response down-regulation and p65 occupancy of target genes (E) in NIH3T3 cells.