Transcriptional activation by NF-kappaB requires multiple coactivators

Abstract

Nuclear factor-kappaB (NF-kappaB) plays a role in the transcriptional regulation of genes involved in inflammation and cell survival. In this report we demonstrate that NF-kappaB recruits a coactivator complex that has striking similarities to that recruited by nuclear receptors. Inactivation of either cyclic AMP response element binding protein (CREB)-binding protein (CBP), members of the p160 family of coactivators, or the CBP-associated factor (p/CAF) by nuclear antibody microinjection prevents NF-kappaB-dependent transactivation. Like nuclear receptor-dependent gene expression, NF-kappaB-dependent gene expression requires specific LXXLL motifs in one of the p160 family members, and enhancement of NF-kappaB activity requires the histone acetyltransferase (HAT) activity of p/CAF but not that of CBP. This coactivator complex is differentially recruited by members of the Rel family. The p50 homodimer fails to recruit coactivators, although the p50-p65 heterodimeric form of the transcription factor assembles the integrator complex. These findings provide new mechanistic insights into how this family of dimeric transcription factors has a differential effect on gene expression.

FIG. 1

Stimulation of p65-dependent transactivation requires multiple functional domains of CBP. (A) Schematic of CBP wild-type (wt) and mutant expression constructs used in the transient transfection assays. (B) Expression of CBP deletion mutants block p65 transactivation. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT, 250 ng of pcDNA-p65 (lanes 2 to 10), and either 1 or 10 μg of the indicated CBP expression construct. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The data are representative of three independent experiments performed in duplicate. (C) Schematic of E1A wild-type and mutant expression constructs used in the transient transfection assays. (D) Differential effects of mutated forms of E1A on p65-stimulated reporter gene expression. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT, 250 ng of pcDNA-p65, and the indicated concentrations of the wild-type E1A, E1Ad2-36, or E1AH3N expression construct. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The data are representative of three independent experiments performed in duplicate.

FIG. 2

Coactivator requirements for NF-κB-dependent gene expression. Plasmids consisting of a LacZ reporter under the transcriptional control of the E-selectin promoter were injected into the nuclei of Rat-1 cells in the presence of either preimmune IgG or affinity-purified antibodies to the indicated coactivators. The expression of the reporter plasmid was monitored by staining with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) and quantitated based on the percentage of injected cells that stained blue. (A) Effects of nuclear microinjection of anti-CBP, SRC-1/NCoA-1, and pCAF antibodies on p65-induced E-selectin-lacZ reporter gene expression. Photomicrographs of rhodamine-stained injected cells (top panel) and the corresponding phase-contrast pictures (lower panel) display typical results. (B) Coactivator requirements of NF-κB-dependent gene expression. Results were repeated in three separate experiments with more than 200 cells injected for each data point; data are expressed as means, and error bars represent standard errors of the means. (C) Effect of nuclear microinjection of anti-CBP, SRC-1/NCoA-1, and p/CAF antibodies on Sp1-LacZ and CMV-LacZ reporter constructs.

FIG. 3

Coexpression of CBP and SRC-1/NCoA-1, GRIP-1, TIF-2, or p/CAF enhances p65-mediated transcriptional activity. (A) SRC-1/NCoA-1 potentiates NF-κB-dependent gene expression. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT and 100 ng of pcDNA-p65 (lanes 2, 7, and 8) and either 3.25 μg (lanes 5, 9, and 11) or 6.5 μg (lanes 6, 10, and 12) of CMV-SRC-1/NCoA-1 and/or 3.25 μg (lanes 3, 7, and 11) or 6.5 μg (lanes 4, 8, and 12) of RSV-CBP. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon transfection of E-selectin CAT alone was set at one. The data are presented as means; error bars, standard deviations. (B) Western blot analysis of p65 levels in transfected cell extracts. A portion of each whole-cell extract was separated by SDS–10% PAGE, transferred to nitrocellulose, and probed with a rabbit anti-p65 antibody (Rockland) as described in Materials and Methods. Following incubation with a horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody, the bands were visualized by enhanced chemiluminescence (Amersham Life Science). (C) GRIP-1 and TIF-2 increase p65-dependent gene expression. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT and 100 ng of pcDNA-p65 (lanes 4 to 16) and 1, 2.5, 4, or 6.6 μg of simian virus 40 (SV40)-GRIP-1, SV40-TIF-2, or RSV-CBP. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon cotransfection of E-selectin CAT and p65 was set at one. Data are presented as means; error bars, standard deviations. (D) Western blot analysis of p65 levels in transfected cell extracts performed as described above and in Materials and Methods. (E) p/CAF potentiates p65-dependent transactivation and synergizes with CBP. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin–CAT and 100 ng of pcDNA-p65 (lanes 2 and 7 to 12) and either 3.25 μg (lanes 5, 9, and 11) or 6.5 μg (lanes 6, 10, and 12) of CMV-p/CAF and/or 3.25 μg (lanes 3, 7, and 11) or 6.5 μg (lanes 4, 8, and 12) of RSV-CBP. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon transfection of E-selectin CAT alone was set at one. Data are presented as means; error bars, standard deviations. (F) Western blot analysis of p65 levels in cell extracts performed as described above and in Materials and Methods.

FIG. 3

Coexpression of CBP and SRC-1/NCoA-1, GRIP-1, TIF-2, or p/CAF enhances p65-mediated transcriptional activity. (A) SRC-1/NCoA-1 potentiates NF-κB-dependent gene expression. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT and 100 ng of pcDNA-p65 (lanes 2, 7, and 8) and either 3.25 μg (lanes 5, 9, and 11) or 6.5 μg (lanes 6, 10, and 12) of CMV-SRC-1/NCoA-1 and/or 3.25 μg (lanes 3, 7, and 11) or 6.5 μg (lanes 4, 8, and 12) of RSV-CBP. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon transfection of E-selectin CAT alone was set at one. The data are presented as means; error bars, standard deviations. (B) Western blot analysis of p65 levels in transfected cell extracts. A portion of each whole-cell extract was separated by SDS–10% PAGE, transferred to nitrocellulose, and probed with a rabbit anti-p65 antibody (Rockland) as described in Materials and Methods. Following incubation with a horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody, the bands were visualized by enhanced chemiluminescence (Amersham Life Science). (C) GRIP-1 and TIF-2 increase p65-dependent gene expression. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT and 100 ng of pcDNA-p65 (lanes 4 to 16) and 1, 2.5, 4, or 6.6 μg of simian virus 40 (SV40)-GRIP-1, SV40-TIF-2, or RSV-CBP. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon cotransfection of E-selectin CAT and p65 was set at one. Data are presented as means; error bars, standard deviations. (D) Western blot analysis of p65 levels in transfected cell extracts performed as described above and in Materials and Methods. (E) p/CAF potentiates p65-dependent transactivation and synergizes with CBP. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin–CAT and 100 ng of pcDNA-p65 (lanes 2 and 7 to 12) and either 3.25 μg (lanes 5, 9, and 11) or 6.5 μg (lanes 6, 10, and 12) of CMV-p/CAF and/or 3.25 μg (lanes 3, 7, and 11) or 6.5 μg (lanes 4, 8, and 12) of RSV-CBP. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon transfection of E-selectin CAT alone was set at one. Data are presented as means; error bars, standard deviations. (F) Western blot analysis of p65 levels in cell extracts performed as described above and in Materials and Methods.

FIG. 4

NF-κB-dependent transactivation in vitro requires specific functional domains of SRC-1/NCoA-1. (A) Schematic representation of SRC-1/NCoA-1 expression plasmids used in the overexpression experiments. (B) The N terminus of SRC-1/NCoA-1 is required for potentiation of p65-dependent transactivation. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT, 100 ng of pcDNA-p65, and 0.5, 1, 4, or 8 μg of either wild-type CMV-SRC-1/NCoA-1 (WT), CMV-SRC-1-NR/CBP, or CMV-SRC-1 ΔN. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon cotransfection of E-selectin CAT and p65 was set at one. Data are presented as means; error bars, standard deviations.

FIG. 5

Coactivator LXXLL motif specificity in NF-κB-dependent gene expression. (A) The LXD2 and LXD4 domains of SRC-1/NCoA-1 are required for NF-κB-mediated transcription. Plasmids consisting of a LacZ reporter under the transcriptional control of the E-selectin promoter were injected into the nuclei of Rat-1 cells in the presence of either preimmune IgG or an affinity-purified antibody to SRC-1/NCoA-1. The expression of the reporter plasmid was monitored by X-Gal staining and quantitated based on the percentage of injected cells that stained blue. Rescue experiments were performed by coinjecting the indicated expression plasmids. (B) Coinjection of NCoA-2 expression plasmid does not rescue the inhibitory effect of the anti-SRC-1/NCoA-1 antibody on p65-dependent transcription. (C) Coinjection of NCoA-2 expression plasmid rescues the inhibitory effect of the anti-SRC-1/NCoA-1 antibody on RAR-dependent transcription. In all panels, data are means from three separate experiments; error bars represent standard errors of the means.

FIG. 6

NF-κB-dependent transactivation in vitro requires p/CAF HAT activity. (A) The HAT activity of p/CAF is required for potentiation of p65-dependent transactivation (upper panel). COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT and 100 ng of pcDNA-p65 and 0.5, 1, or 4 μg of either wild-type (WT) CMV-p/CAF or CMV-p/CAF (HAT⁻). Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon cotransfection of E-selectin CAT and p65 was set at one. Data are presented as means; error bars, standard deviations. Representative Western blot analyses of wild-type p/CAF (lower panel, lanes 2 to 4) and HAT⁻ p/CAF (lower panel, lanes 5 to 7) are shown. (B) The HAT activity of CBP/p300 is not required for potentiation of p65-dependent transactivation. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT, 100 ng of pcDNA-p65, and 0.5, 1, or 4 μg of either wild-type (WT) RSV-CBP or CMV-CBP (HAT⁻). Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon cotransfection of E-selectin CAT and p65 was set at one. Data are presented as the means; error bars, standard deviations. Representative Western blots of wild-type CBP (lower panel, lanes 2 to 6) and HAT⁻ CBP (lower panel, lanes 7 to 11) are shown. (C) HAT requirements for NF-κB-dependent gene expression. Plasmids consisting of a LacZ reporter under the transcriptional control of the E-selectin promoter were injected in the nuclei of Rat-1 cells in the presence of either an anti-CBP or an anti-p/CAF antibody. The expression of the reporter plasmid was monitored by X-Gal staining and quantitated based on the percentage of injected cells that stained blue. Rescue experiments were performed by coinjecting the indicated p/CAF or CBP expression plasmid.

FIG. 6

NF-κB-dependent transactivation in vitro requires p/CAF HAT activity. (A) The HAT activity of p/CAF is required for potentiation of p65-dependent transactivation (upper panel). COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT and 100 ng of pcDNA-p65 and 0.5, 1, or 4 μg of either wild-type (WT) CMV-p/CAF or CMV-p/CAF (HAT⁻). Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon cotransfection of E-selectin CAT and p65 was set at one. Data are presented as means; error bars, standard deviations. Representative Western blot analyses of wild-type p/CAF (lower panel, lanes 2 to 4) and HAT⁻ p/CAF (lower panel, lanes 5 to 7) are shown. (B) The HAT activity of CBP/p300 is not required for potentiation of p65-dependent transactivation. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT, 100 ng of pcDNA-p65, and 0.5, 1, or 4 μg of either wild-type (WT) RSV-CBP or CMV-CBP (HAT⁻). Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon cotransfection of E-selectin CAT and p65 was set at one. Data are presented as the means; error bars, standard deviations. Representative Western blots of wild-type CBP (lower panel, lanes 2 to 6) and HAT⁻ CBP (lower panel, lanes 7 to 11) are shown. (C) HAT requirements for NF-κB-dependent gene expression. Plasmids consisting of a LacZ reporter under the transcriptional control of the E-selectin promoter were injected in the nuclei of Rat-1 cells in the presence of either an anti-CBP or an anti-p/CAF antibody. The expression of the reporter plasmid was monitored by X-Gal staining and quantitated based on the percentage of injected cells that stained blue. Rescue experiments were performed by coinjecting the indicated p/CAF or CBP expression plasmid.

FIG. 7

CBP/p300, SRC-1/NCoA-1, and p/CAF are recruited to a DNA-bound p65-p50 heterodimer. (Left panel) Coactivators are recruited to a DNA-bound p65-p50 heterodimer. A biotinylated oligonucleotide containing two NF-κB binding sites was bound to streptavidin-paramagnetic beads and loaded with 0 μg (lane 2), 1 μg (lane 3), or 3 μg of His-p50–His-p65 (lane 4). After a wash, the DNA-bound heterodimers were incubated with 2 mg of K562 nuclear extracts and then washed, and bound proteins were identified by Western blotting as described in Materials and Methods. Lane 1 represents 200 μg, or 1/10 of the total K562 input. The antibodies used for Western blotting are indicated on the left. (Right panel) The p50 homodimer inhibits recruitment of CBP/p300, SRC-1/NCoA-1, and p/CAF. A biotinylated oligonucleotide containing two NF-κB binding sites was bound to streptavidin-paramagnetic beads and loaded with 1 or 3 μg of either p50-p65 (lanes 6 and 7) or the p50–p50 homodimer (lanes 8 and 9). After a wash, the DNA bound dimers were incubated with 2 mg of K562 nuclear extracts and then washed, and bound proteins were identified by Western blot analysis, as described in Materials and Methods. Lane 5 (Input) represents 200 μg, or 1/10, of the total K562 input.