Active Repression of Antiapoptotic Gene Expression by RelA(p65) NF-κB Kirsteen J Campbell, Sonia Rocha, Neil D Perkins  Molecular Cell  Volume 13, Issue 6, Pages 853-865 (March 2004) DOI: 10.1016/S1097-2765(04)00131-5

Figure 1 UV-C Treatment Induces NF-κB DNA Binding but Represses NF-κB-Dependent Transcription (A) UV-C induces NF-κB DNA binding. U-2 OS cells were stimulated with UV-C (40 J/m2) for the indicated times. Nuclear extracts were prepared and analyzed by EMSA using 32P labeled HIV-1 κB or Sp1 GC box oligonucleotides. (B) The UV-C-induced NF-κB complex consists of the RelA and p50 NF-κB subunits. EMSA analysis was performed as in (A) using nuclear extracts prepared from control, TNF-treated (30 min), and UV-C-treated (5 hr) U-2 OS cells. Antibodies to NF-κB subunits or 100× molar excess of unlabeled HIV-1 κB oligonucleotide were included as indicated. (C and D) UV-C represses NF-κB reporter plasmid activity. U-2 OS cells were transfected with 2 μg of NF-κB luciferase reporter plasmids (3x κB ConA luc., HIV-1 LTR luc.), ConA luciferase reporter plasmid without κB sites (ConA luc.), or HIV-1 LTR luciferase reporter plasmid with mutated κB sites (HIV-1 Δ κB LTR luc.). Cells were either unstimulated (control) or stimulated for 8 hr with UV-C or TNF 36 hr after transfection. (E) UV-C repression of NF-κB reporter plasmid activity is independent of p53 status. The p53 null Saos-2 or H1299 cell lines were transfected with the 3x κB ConA luciferase plasmid and treated as in (A). Molecular Cell 2004 13, 853-865DOI: (10.1016/S1097-2765(04)00131-5)

Figure 2 Daunorubicin Treatment Induces NF-κB DNA Binding but Represses NF-κB-Dependent Transcription (A) U-2 OS cells were stimulated with daunorubicin (1 μM) for the indicated times. Nuclear extracts were prepared and analyzed by EMSA using 32P labeled Bcl-xL κB or Sp1 GC box oligonucleotides. (B) Daunorubicin-induced NF-κB consists of RelA-containing complexes. EMSA analysis was performed as in (A) using nuclear extracts prepared from control and daunorubicin treated (3 hr) U-2 OS cells. Antibodies to NF-κB subunits or 100× molar excess of unlabeled HIV-1 κB oligonucleotide were included as indicated. (C) Daunorubicin and doxorubicin but not etoposide repress NF-κB-dependent transcription. U-2 OS cells were transfected with 2 μg of 3x κB ConA luc or ConA luc control and 36 hr later stimulated with 1 μM daunorubicin, 2 μM doxorubicin, or 40 μM etoposide as indicated for 8 hr prior to harvesting. (D) Daunorubicin repression of NF-κB reporter plasmid activity is independent of p53 status. The p53 null Saos-2 cells were transfected with the 3x κB ConA luciferase plasmid and treated as in (A). (E) UV-C and daunorubicin induce expression of p53-responsive promoters. U-2 OS cells were transfected as above, with 2 μg of p21WAF1 luciferase or Bax luciferase reporter plasmids. Cells were stimulated with UV-C and daunorubicin and subsequently harvested as indicated above and in Figure 1. (F) UV-C and daunorubicin induce IκBα degradation and serine 32/36 phosphorylation in U-2 OS cells. Whole-cell lysates were prepared from U-2 OS cells following no treatment or stimulation with 40 J/m2 UV-C or 1 μM daunorubicin for the times indicated. Western blot analysis was then performed with antibodies to IκBα, phospho serine 32/36 IκBα, RelA, and β actin. Where indicated, cells were also treated with the proteasome inhibitor MG132 (10 μM). Molecular Cell 2004 13, 853-865DOI: (10.1016/S1097-2765(04)00131-5)

Figure 3 UV-C and Daunorubicin Inhibit Expression of Endogenous NF-κB Target Genes (A) UV-C and daunorubicin inhibit mRNA expression of the antiapopototic NF-κB target genes X-IAP, Bcl-xL, and A20 in U-2 OS cells. Semiquantitative PCR analysis was performed, using primers specific to human X-IAP, Bcl-xL, A20, IκBα, Fas, or a GAPDH control, with 10 ng total RNA prepared from U-2 OS cells following no treatment or stimulation with TNF (10 ng/ml), 40 J/m2 UV-C, or 1 μM daunorubicin for the times indicated. (B) TNF induction of A20 and IκBα gene expression at earlier time points. Cells were stimulated as in (A) above with TNF (10 ng/ml) for the indicated times. A20 and IκBα expression was analyzed by semiquantitative PCR. (C) Downregulation of Bcl-xL and X-IAP is specific. U-2 OS cells were treated as in (A) above with UV-C for 6 hr, and semiquantitative PCR analysis was performed using primers to human Smac, survivin, Bcl-xL, X-IAP, and GAPDH. (D) UV-C and daunorubicin downregulation of NF-κB target genes is independent of p53. Total RNA was prepared from H1299 cells and analyzed by PCR as in (A). (E) UV-C and daunorubicin induce a time-dependent decrease in X-IAP and Bcl-xL protein levels in U-2 OS cells. Cells were stimulated with 40 J/m2 UV-C or 1 μM daunorubicin for the times indicated. Whole-cell lysates were prepared, and 30 μg of protein were analyzed by SDS-PAGE and Western blotting with antibodies specific for X-IAP, Bcl-xL, or β actin. (F) UV-C and daunorubicin utilize the κB site in the Bcl-xL promoter to mediate transcriptional repression. U-2 OS cells were transfected with 2 μg of Bcl-xL κB luciferase reporter plasmid (Bcl-xL luc.) or a Bcl-xL κB luciferase reporter plasmid with mutated κB sites (Bcl-xL Δ κB luc.) and treated as in Figures 1B and 2C. Results are normalized such that no change in luciferase activity has a value of 0. Molecular Cell 2004 13, 853-865DOI: (10.1016/S1097-2765(04)00131-5)

Figure 4 UV-C, Daunorubicin, and Doxorubicin Inhibit the RelA Transactivation Domain U-2 OS cells were transfected with 1.5 μg Gal4 E1B luciferase reporter plasmid and 500 pg of expression plasmids encoding the Gal4 DNA binding domain fused to amino acids 428–551 of RelA [Gal4-RelA(TAD)] (A); Gal4-RelA(TAD) with threonine 505 mutated to alanine [Gal4-RelA(TAD-T505A)] (A); and Gal4 RelA (TAD2), which contains amino acids 428–521 of RelA (A), Gal4 alone (B), Gal4 p53 (B), or Gal4 VP16 (C). As indicated, 24 hr after transfection, cells were stimulated for 16 hr with 40 μM etoposide, 1 μM daunorubicin, 40 J/m2 UV-C, or 2 μM doxorubicin. Molecular Cell 2004 13, 853-865DOI: (10.1016/S1097-2765(04)00131-5)

Figure 5 UV-C and Daunorubicin Are Dominant Inhibitors of TNF-Induced NF-κB Transcriptional Activity (A and B) UV-C and daunorubicin dominantly repress TNF-induced NF-κB transcriptional activity. U-2 OS (A) or p53 null Saos2 cells (B) were transfected with 2 μg of 3x κB ConA luciferase reporter plasmid. Cells were stimulated for 6 hr with UV-C or daunorubicin and 4 hr with TNF, 36 hr after transfection. (C) UV-C and daunorubicin dominantly repress TNF-induced endogenous Bcl-xL and X-IAP expression. U-2 OS cells were treated as above except that cells were harvested 2 hr following TNF stimulation and PCR analysis of Bcl-xL, X-IAP, and GAPDH expression was performed. (D and E) UV-C and daunorubicin do not alter TNFα-induced NF-κB DNA binding. U-2 OS cells were stimulated with 40 J/m2 UV-C, 0.1, 1, or 10 μM daunorubicin and TNF as indicated. Nuclear extracts were prepared and 5 μg were subjected to EMSA using a 32P labeled HIV-1 κB oligonucleotide. (F) UV-C and daunorubicin stimulation do not induce serine 536 phosphorylation or prevent TNF-induced phosphorylation. Nuclear extracts (20 μg) were resolved by SDS-PAGE and Western blotted with phospho-serine 536 RelA antibody. These membranes were then stripped and reprobed for RelA. (G) RelA is bound to the Bcl-xL and IκBα promoters following UV-C, daunorubicin, and TNF stimulation. U-2 OS cells were stimulated with either UV-C (5 hr), daunorubicin (3 hr), or TNF (30 min), and ChIP assays were performed using an antibody directed to RelA. RelA binding to the indicated promoters was detected by PCR. Molecular Cell 2004 13, 853-865DOI: (10.1016/S1097-2765(04)00131-5)

Figure 6 RelA Is Required for UV-C and Daunorubicin Inhibition of Bcl-xL Expression (A and B) UV-C and daunorubicin induce a time-dependent decrease in Bcl-xL protein levels in wild-type but not rela null MEFs. Cells were treated with 1 μM daunorubicin (A) or 40 J/m2 UV-C (B) for times indicated, whole-cell lysates were prepared, and 30 μg of protein was analyzed by SDS-PAGE and Western blot with antibodies against Bcl-xL or β actin. (C) UV-C and daunorubicin induce a time-dependent decrease in Bcl-xL mRNA levels in wild-type but not rela null MEFs. Cells were treated with 40 J/m2 UV-C or 1 μM daunorubicin for times indicated, and total RNA was prepared. Semiquantitative PCR was then performed using primers specific to mouse Bcl-xL and GAPDH control with 10 ng total RNA. (D) rela null MEFs lack detectable NF-κB DNA binding following UV-C stimulation. Equivalent concentrations of nuclear protein extracts from wild-type and rela null MEFs, stimulated for the indicated times with UV-C (40 J/m2) were analyzed for NF-κB DNA binding to a 32P labeled HIV-1 κB oligonucleotide by EMSA. (E) Effects of UV-C and daunorubicin on IκBα in MEFs. Whole-cell lysates were prepared from wild-type MEFs following no treatment or stimulation with 40 J/m2 UV-C, 1 μM daunorubicin, or 10 ng/ml TNF for the times indicated. Western blot analysis was then performed with antibodies to IκBα, phospho serine 32/36 IκBα, RelA, and β actin. Where indicated, cells were also treated with the proteasome inhibitor MG132 (10 μM). (F) UV-C and daunorubicin repression of Bcl-xL expression is independent of p53, Mdm2, and p19ARF status. Total RNA was prepared from wild-type or p53−/−/Mdm2−/−/ARF−/− MEFs following no treatment or stimulation with 40 J/m2 UV-C or 1 μM daunorubicin for 6 hr as indicated. Semiquantitative PCR was then performed using primers specific to mouse Bcl-xL and GAPDH control with 10 ng total RNA. (G) Knockdown of RelA by RNA interference prevents UV-C- and daunorubicin-induced repression of Bcl-xL and X-IAP mRNA levels. U-2 OS cells were transfected with a siRNA oligonucleotide targeting RelA or a scrambled control siRNA oligonucleotide and then treated with UV-C and daunorubicin for 6 hr. Cells were harvested and RelA, Bcl-xL, X-IAP, Fas, and GAPDH expression were analyzed by PCR. (H and I) rela null MEFs show increased resistance to UV-C-induced cell death. Wild-type or rela null MEFs were stimulated with 40 J/m2 UV-C (A) or 10 ng/ml TNF (B) 24 hr after subculture and harvested 24, 36, or 48 hr later. Cell death was assessed by trypan blue viability assay and counted in triplicate. The results from two separate experiments are shown. Molecular Cell 2004 13, 853-865DOI: (10.1016/S1097-2765(04)00131-5)

Figure 7 RelA-Mediated Repression of Bcl-xL Expression Requires Histone Deacetylase Activity (A) UV-C and daunorubicin induce RelA association with HDACS 1, 2, and 3. U-2 OS cells were stimulated with 40 J/m2 UV-C or 1 μM daunorubicin for 5 or 3 hr, respectively, to coincide with optimal NF-κB DNA binding activity. Whole-cell lysate (100 μg) was incubated with 1 μg of antibodies to HDAC1, HDAC2, HDAC3, or an IgG control. Following immunoprecipitation, protein was resolved by SDS-PAGE and Western blotted with an antibody specific to RelA. Shown is 10 μg of input extract. (B) TNF does not induce the association of RelA with HDAC1. U-2 OS cells were treated with 10 ng/ml TNF (1 hr) or 40 J/m2 UV-C (5 hr). Cells were harvested and immunoprecipitations performed as in (A) above. (C) Overexpression of HDAC1 represses transcription from the Bcl-xL promoter in a κB site-dependent manner. U-2 OS cells were transfected with 1 μg of HDAC1 (or pcDNA3 control) expression plasmid and 2 μg Bcl-xL luc or Bcl-xL Δ κB luc. (D) The histone deacetylase inhibitor TSA rescues UV-C- and daunorubicin-induced repression of Bcl-xL in MEFs. Cells were stimulated with 40 J/m2 UV-C, 1 μM daunorubicin, and/or TSA for 6 hr prior to harvesting. Semiquantitative PCR analysis was then performed, using primers specific to mouse Bcl-xL or a GAPDH control, with 10 ng total RNA. (E) UV-C and daunorubicin but not TNF stimulation induce histone H3 deacetylation at the Bcl-xL and IκBα promoters. U-2 OS cells were stimulated with either UV-C (5 hr), daunorubicin (3 hr), or TNF (30 min), and ChIP assays were performed using an antibody directed to acetylated histone H3 (lysines 9 and 14). The presence of acetylated histone H3 at the Bcl-xL and IκBα promoters was detected by PCR. Molecular Cell 2004 13, 853-865DOI: (10.1016/S1097-2765(04)00131-5)