RelB controls Rb phosphorylation, EZH2 expression and senescence through PSMA5 induced regulation of p21^WAF1 and p53 protein stability.

<p>(A) PSMA5 and ANAPC1 regulate p21^WAF1 and p53 protein stability. Western blot analysis of NHD fibroblasts treated with the indicated siRNAs. (B & C) RelB regulates PSMA5 expression. Whole cell protein lysates (B) or RNA (C) was prepared from NHD fibroblasts treated with the indicated siRNAs and western blot or Q-PCR analysis of PSMA5 expression was performed. (D) RelB regulates ANAPC1 expression. RNA was prepared from NHD fibroblasts treated with the indicated siRNAs and Q-PCR analysis of ANAPC1 was performed. (E) siRNA mediated knock down of PSMA5 results in loss of Rb phosphorylation. Western blot analysis of NHD fibroblasts treated with the indicated siRNAs. (F) siRNA mediated knock down of PSMA5 results in loss of EZH2 expression. RNA was prepared from NHD fibroblasts treated with the indicated siRNAs and Q-PCR analysis of EZH2 was performed. Psma5: (*** p≤0.001) Anapc1: (* p≤0.05). (G & H) siRNA mediated knock down of PSMA5 (G) or ANAPC1 (H) induces p53 dependent cellular senescence. NHD fibroblasts were transfected with the listed siRNAs and analyzed for senescence by β-galactosidase staining after 7 days.</p

NF-κB2 controls Rb phosphorylation, EZH2 expression and senescence through CDK4 and CDK6 regulation.

<p>(A & B) NF-κB2 regulates CDK4 & 6 expression. RNA was prepared from NHD fibroblasts treated with the indicated siRNAs and Q-PCR analysis of CDK4 (A) and CDK6 (B) expression was performed. (C) NF-κB2 regulates CDK4 expression. Western blot analysis of NHD fibroblasts treated with the indicated siRNAs. Note that this is a reprobing of blots used in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004642#pgen-1004642-g002" target="_blank">Fig. 2A</a> and the β-actin blot shown here is the same as in that figure. (D) siRNA mediated knock down of CDK4 and CDK6 results in loss of Rb phosphorylation and EZH2 expression. Western blot analysis of NHD fibroblasts treated with the indicated siRNAs. (E) siRNA mediated knock down of CDK4 induces cellular senescence. NHD fibroblasts were transfected with the listed siRNAs and analyzed for senescence by β-galactosidase staining after 7 days. (F) Re-expression of CDK partially recovers the effects of NF-κB2 siRNA depletion. 96 hours after the transfection of NHD fibroblasts treated with the indicated siRNAs, cells were further transfected with CDK4 and CDK6 expression plasmids. After an additional 24 hours, protein extracts were prepared and western blot analysis performed.</p

The alternative NF-κB pathway suppresses ROS production through the regulation of RacGAP.

<p>(A) Table summarizing the fold effect on RACGAP1 expression induced by transfection of the listed siRNAs in the microarray analysis. (B) NF-κB2, RelB and EZH2 regulate RACGAP1 expression in NHD fibroblasts. RNA was prepared from NHD fibroblasts treated with the indicated siRNAs and Q-PCR analysis of RACGAP1 expression was performed. (C) siRNA mediated knock-down of RACGAP1 induces ROS production. NHD fibroblasts were transfected with the siRNAs shown and analyzed for ROS production after 4 days. (D) ROS production induced by siRNA mediated knock down of NF-κB2 and RelB is dependent upon Rac1 and Cdc42. NHD fibroblasts were transfected with the siRNAs shown and analyzed for ROS production after 4 days.</p

EZH2 is a critical effector of an antagonistic cross-talk between NF-κB and p53.

<p>(A) Heatmap showing effects on gene expression of NHD fibroblasts depleted for EZH2, NF-κB2, RelB and p53. NHD fibroblasts were transfected in triplicate with the listed siRNAs. After 48 hours RNA was extracted for microarray analysis. Shown with a bar is the group of genes where EZH2, NF-κB2 and RelB antagonize p53 dependent gene expression. (B) A subset of genes is co-regulated by NF-κB2, RelB, EZH2 and p53 in NHD fibroblasts. (C) Graphical representation of the 975 p53 regulated genes whose expression changes >1.5 fold that are also regulated (>1.5×) by NF-κB2, RelB and Ezh2. (D–E) NF-κB2, RelB, EZH2 regulate DEK expression in NHD fibroblasts. RNA (D) and whole cell protein lysates (E) was prepared from NHD fibroblasts treated with the indicated siRNAs and Q-PCR or western blot analysis of DEK expression was performed. Note that (E) is a reprobing of blots used in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004642#pgen-1004642-g001" target="_blank">Fig. 1C</a> and the β-actin blot shown here is the same as in that figure. (F) siRNA mediated knock down of DEK induces cellular senescence. NHD fibroblasts were transfected with the siRNAs shown and analyzed for senescence after 7 days. (G) EZH2 and DEK alone can rescue senescence. Fibroblasts conditionally immortalized with temperature sensitive T antigen (described in Rovillain et al.) were shifted to the non-permissive temperature and subjected to a clonogenic assay with and without expression of the indicated genes. The panel shows an image of cells upon completion of the assay.</p

Summary of the results identified in this manuscript through which NF-κB2 and RelB regulate p53 dependent cellular senescence in primary human NHD fibroblasts.

<p>Depletion of NF-κB2 and RelB leads to a decrease in Rb phosphorylation. Unphosphorylated Rb represses E2F transcriptional activity and consequently inhibits EZH2 expression (which is negatively regulated by p53). However, this occurs through distinct pathways. Depletion of NF-κB2 leads to down regulation of CDK4 and CDK6, which are known to phosphorylate Rb directly. RelB depletion leads to an increase in p53 and p21 protein stability as a consequence of loss of expression of genes such as PSMA5 and ANAPC1. Other gene targets may be involved in these processes. NF-κB subunits also bind directly to the EZH2 promoter and this may also contribute towards its regulation. As a consequence of this pathway, down regulation of EZH2 results in numerous changes in gene expression, including p53 dependent repression of a number of gene targets. These include inhibition of RACGAP1 expression, resulting in Rac1/Cdc42 dependent induction of reactive oxygen species (ROS). Together with other changes, such as suppression of DEK and ultimately induction of p14^ARF and p16^INK4a, the ultimate consequence of NF-κB2 and RelB siRNA depletion and subsequent loss of EZH2 expression is p53 dependent cell senescence. Note, RACGAP1 activity as well as various inducers/suppressors or senescence may also be regulated by p53. Many of these genes may also be direct targets of NF-κB. Not shown is that oxidative stress is required to drive the basal level p53 activity seen in these cells.</p

EZH2 is an NF-κB regulated target gene.

<p>(A) The basal level of p53 protein in NHD fibroblasts is ROS dependent. NHD fibroblasts were grown under normoxia at 3% O2 for 7 days before western blot analysis. (B & C) siRNA mediated knock-down of NF-κB2 and RelB leads to a reduction in EZH2 mRNA and protein levels. RNA (B) or protein (C) was prepared from NHD fibroblasts treated with the indicated siRNAs 48 hours after transfection and Q-PCR or western blot analysis was performed to determine EZH2 expression. *** P≤0.001. (D & E) Lymphotoxin β receptor stimulation leads to induction of EZH2 expression. NHD fibroblasts were treated with LTβR agonist antibody for the times indicated and either Q-PCR (D) or western blot analysis (E) was performed to determine the expression of EZH2 (D) or EZH2, p52/p100, RelB and p53 (E). * P≤0.05.</p

CD40 stimulation leads to NF-κB activation and CLL induction in Chronic Lymphocytic Leukemia cells.

<p>(A) Analysis of EZH2 mRNA expression in CLL cells. RNA was prepared from CLL cells stimulated with CD40L/IL4 expressing mouse fibroblasts or with untransfected fibroblasts (NTL) and IL4 for the indicated times and Q-PCR analysis of EZH2 expression was performed. (B) Analysis of EZH2 protein level in CLL cells. Western blot analysis of nuclear extracts from CLL cells stimulated with CD40L/IL4 or untransfected fibroblasts (NTL) and IL4 for the indicated times. (C & D) EZH2 protein and RNA levels in CLL cells is NF-κB dependent. Whole cell protein lysates (C) and RNA (D) were prepared from CLL cells stimulated for 24 hours with CD40L/IL4 and treated with the IKKβ inhibitor TPCA-1 where indicated.</p

The alternative NF-κB pathway suppresses p53 mediated senescence in primary fibroblasts.

<p>(A & B) Senescence induced by siRNA knock down of NF-κB2 and RelB is ROS dependent. NHD fibroblasts were transfected with the siRNAs shown and treated, where indicated, 2 days later with the anti-oxidant N-Acetyl cysteine (NAC). 7 days after transfection cells analyzed for senescence by acidic β-galactosidase staining (A). An image of RelB siRNA transfected cells after staining is shown (B). (C) siRNA mediated knock down of NF-κB2 and RelB induces ROS production. NHD fibroblasts were transfected with the siRNAs shown and treated, where indicated, 2 days later with NAC. After 7 days they were incubated for 30 minutes with 5 mM DCF-DA and analyzed by FACs. The percentage of cells with higher than baseline ROS levels are shown. (D) siRNA knock down of NF-κB2, RelB and Bcl3 induce cellular senescence in a p53 dependent manner. NHD fibroblasts were transfected with the listed siRNAs and analyzed for senescence by β-galactosidase staining after 7 days. (E) Lymphotoxin β receptor stimulation represses basal level senescence in fibroblasts. NHD fibroblasts were treated with LTβR agonist antibody and after 7 days analyzed for senescence by β-galactosidase staining. * P≤0.05.</p

NF-κB2 and RelB regulate EZH2 in an Rb/E2F dependent manner.

<p>(A) siRNA mediated knock-down of NF-κB2 and RelB leads to a reduction of Rb- phosphorylation. Western blot analysis of whole cell lysates prepared from NHD fibroblasts 48 hours after transfection with the indicated siRNAs. (B) siRNA mediated knock down of RelB leads to accumulation of p53 and p21^WAF1 protein level. Western blot analysis of NHD fibroblasts treated with the indicated siRNAs. Whole cell lysates were prepared 48 hours after transfection. (C & D) NF-κB2 and RelB depletion does not affect p21^WAF1 or p53 mRNA levels. RNA was prepared from NHD fibroblasts treated with the indicated siRNAs and Q-PCR analysis of p21^WAF1 (C) or p53 (D) expression was performed.</p

NF-κB subunits bind the EZH2 promoter in fibroblasts and B-cells.

<p>(A & B) ChIP analysis of the EZH2 promoter was performed in NHD fibroblast cells using primers close to the core promoter of the EZH2 gene (A) (+596/+888) or an upstream control region (B) (−2802/−2600). Results shown are representative of a minimum of 3 separate experiments. * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001. (C) ChIP Seq data showing NF-κB subunit binding in the region of the EZH2 gene in the human EBV-transformed lymphoblastoid B-cell line (LCL) GM12878.</p