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Two waves of nuclear factor kappaB recruitment to target promoters.

Saccani S, Pantano S, Natoli G - J. Exp. Med. (2001)

Bottom Line: Proinflammatory stimuli induce the rapid and transient translocation of nuclear factor (NF)-kappaB to the nucleus, where it activates transcription from several genes, including those encoding inflammatory cytokines and chemokines, adhesion molecules, and cytoprotective proteins.Using chromatin immunoprecipitation, we show that after an acute stimulation two distinct waves of NF-kappaB recruitment to target promoters occur: a fast recruitment to constitutively and immediately accessible (CIA) promoters and a late recruitment to promoters requiring stimulus-dependent modifications in chromatin structure to make NF-kappaB sites accessible (promoters with regulated and late accessibility [RLA]).Our results suggest that a mechanism of specificity in NF-kappaB-dependent transcriptional responses relies on the ability of individual stimuli to make RLA promoters accessible to NF-kappaB before its rapid extrusion from the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Biomedicine, CH6501 Bellinzona, Switzerland.

ABSTRACT
Proinflammatory stimuli induce the rapid and transient translocation of nuclear factor (NF)-kappaB to the nucleus, where it activates transcription from several genes, including those encoding inflammatory cytokines and chemokines, adhesion molecules, and cytoprotective proteins. Using chromatin immunoprecipitation, we show that after an acute stimulation two distinct waves of NF-kappaB recruitment to target promoters occur: a fast recruitment to constitutively and immediately accessible (CIA) promoters and a late recruitment to promoters requiring stimulus-dependent modifications in chromatin structure to make NF-kappaB sites accessible (promoters with regulated and late accessibility [RLA]). Our results suggest that a mechanism of specificity in NF-kappaB-dependent transcriptional responses relies on the ability of individual stimuli to make RLA promoters accessible to NF-kappaB before its rapid extrusion from the nucleus.

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A subset of NF-κB–dependent promoters is not constitutively accessible to NF-κB, but accessibility can be increased by adequate stimulation. (a) Raw 264.7 macrophages were transfected with a p65 expression vector or empty vector. Under these experimental conditions, transfected p65 is constitutively nuclear (and can activate transcription from naked cotransfected DNA). ChIP assay with an anti-p65 antibody shows that p65 is recruited only to MnSOD and MIP-2 promoters but not on the MCP-1 and RANTES promoters. Stimulation with LPS for 2 h is followed by NF-κB recruitment to the latter promoters. (b) IFN-γ pretreatment induces hyperacetylation of the MCP-1 promoter and makes it immediately accessible to NF-κB. Raw 264.7 cells were prestimulated with IFN-γ for 2 h and then stimulated with LPS for 20 or 40 min. ChIP assay was performed with anti-p65 antibody or anti-AcH4 serum. Control dilutions of IP supernatant in the anti-AcH4 ChIP are shown. Similar results were obtained with the RANTES promoter.
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Figure 4: A subset of NF-κB–dependent promoters is not constitutively accessible to NF-κB, but accessibility can be increased by adequate stimulation. (a) Raw 264.7 macrophages were transfected with a p65 expression vector or empty vector. Under these experimental conditions, transfected p65 is constitutively nuclear (and can activate transcription from naked cotransfected DNA). ChIP assay with an anti-p65 antibody shows that p65 is recruited only to MnSOD and MIP-2 promoters but not on the MCP-1 and RANTES promoters. Stimulation with LPS for 2 h is followed by NF-κB recruitment to the latter promoters. (b) IFN-γ pretreatment induces hyperacetylation of the MCP-1 promoter and makes it immediately accessible to NF-κB. Raw 264.7 cells were prestimulated with IFN-γ for 2 h and then stimulated with LPS for 20 or 40 min. ChIP assay was performed with anti-p65 antibody or anti-AcH4 serum. Control dilutions of IP supernatant in the anti-AcH4 ChIP are shown. Similar results were obtained with the RANTES promoter.

Mentions: To test if signal-induced modifications in chromatin structure are required for MCP-1, RANTES, and IL-6 promoters to become accessible to NF-κB, we transfected Raw 264.7 with a p65/RelA expression vector. Under these conditions, p65 exceeds endogenous IκBs levels and freely enters the nucleus. We found that although transfected p65 could effectively bind the MIP-2 promoter and the MnSOD enhancer in unstimulated cells, it was not recruited to either the MCP-1, RANTES, or IL-6 promoters (Fig. 4 a, and data not shown), thus indicating that these promoters have to undergo modifications to become accessible to NF-κB. Additional evidence that signal-induced modifications of these promoters facilitates NF-κB binding comes from the analysis of IFN-γ effects. IFN-γ enhances the response of macrophages to LPS, in part by activating transcription factors such as IFN regulatory factor (IRF)1 and signal transducer and activator of transcription (STAT)1, which cooperate with c-Jun, NF-κB, and other transcription factors on target promoters. We found that IFN-γ pretreatment allows NF-κB to be recruited to the MCP-1 promoter with a considerably faster kinetic than in nonprestimulated cells (20 min vs. 90 min; Fig. 4 b). Analysis of histone H4 acetylation shows that IFN-γ induces a significant increase in MCP-1 promoter acetylation levels (Fig. 4 b), possibly through stimulation of IRF-1 binding and activity. Similar results were obtained with the RANTES promoter (data not shown).


Two waves of nuclear factor kappaB recruitment to target promoters.

Saccani S, Pantano S, Natoli G - J. Exp. Med. (2001)

A subset of NF-κB–dependent promoters is not constitutively accessible to NF-κB, but accessibility can be increased by adequate stimulation. (a) Raw 264.7 macrophages were transfected with a p65 expression vector or empty vector. Under these experimental conditions, transfected p65 is constitutively nuclear (and can activate transcription from naked cotransfected DNA). ChIP assay with an anti-p65 antibody shows that p65 is recruited only to MnSOD and MIP-2 promoters but not on the MCP-1 and RANTES promoters. Stimulation with LPS for 2 h is followed by NF-κB recruitment to the latter promoters. (b) IFN-γ pretreatment induces hyperacetylation of the MCP-1 promoter and makes it immediately accessible to NF-κB. Raw 264.7 cells were prestimulated with IFN-γ for 2 h and then stimulated with LPS for 20 or 40 min. ChIP assay was performed with anti-p65 antibody or anti-AcH4 serum. Control dilutions of IP supernatant in the anti-AcH4 ChIP are shown. Similar results were obtained with the RANTES promoter.
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Figure 4: A subset of NF-κB–dependent promoters is not constitutively accessible to NF-κB, but accessibility can be increased by adequate stimulation. (a) Raw 264.7 macrophages were transfected with a p65 expression vector or empty vector. Under these experimental conditions, transfected p65 is constitutively nuclear (and can activate transcription from naked cotransfected DNA). ChIP assay with an anti-p65 antibody shows that p65 is recruited only to MnSOD and MIP-2 promoters but not on the MCP-1 and RANTES promoters. Stimulation with LPS for 2 h is followed by NF-κB recruitment to the latter promoters. (b) IFN-γ pretreatment induces hyperacetylation of the MCP-1 promoter and makes it immediately accessible to NF-κB. Raw 264.7 cells were prestimulated with IFN-γ for 2 h and then stimulated with LPS for 20 or 40 min. ChIP assay was performed with anti-p65 antibody or anti-AcH4 serum. Control dilutions of IP supernatant in the anti-AcH4 ChIP are shown. Similar results were obtained with the RANTES promoter.
Mentions: To test if signal-induced modifications in chromatin structure are required for MCP-1, RANTES, and IL-6 promoters to become accessible to NF-κB, we transfected Raw 264.7 with a p65/RelA expression vector. Under these conditions, p65 exceeds endogenous IκBs levels and freely enters the nucleus. We found that although transfected p65 could effectively bind the MIP-2 promoter and the MnSOD enhancer in unstimulated cells, it was not recruited to either the MCP-1, RANTES, or IL-6 promoters (Fig. 4 a, and data not shown), thus indicating that these promoters have to undergo modifications to become accessible to NF-κB. Additional evidence that signal-induced modifications of these promoters facilitates NF-κB binding comes from the analysis of IFN-γ effects. IFN-γ enhances the response of macrophages to LPS, in part by activating transcription factors such as IFN regulatory factor (IRF)1 and signal transducer and activator of transcription (STAT)1, which cooperate with c-Jun, NF-κB, and other transcription factors on target promoters. We found that IFN-γ pretreatment allows NF-κB to be recruited to the MCP-1 promoter with a considerably faster kinetic than in nonprestimulated cells (20 min vs. 90 min; Fig. 4 b). Analysis of histone H4 acetylation shows that IFN-γ induces a significant increase in MCP-1 promoter acetylation levels (Fig. 4 b), possibly through stimulation of IRF-1 binding and activity. Similar results were obtained with the RANTES promoter (data not shown).

Bottom Line: Proinflammatory stimuli induce the rapid and transient translocation of nuclear factor (NF)-kappaB to the nucleus, where it activates transcription from several genes, including those encoding inflammatory cytokines and chemokines, adhesion molecules, and cytoprotective proteins.Using chromatin immunoprecipitation, we show that after an acute stimulation two distinct waves of NF-kappaB recruitment to target promoters occur: a fast recruitment to constitutively and immediately accessible (CIA) promoters and a late recruitment to promoters requiring stimulus-dependent modifications in chromatin structure to make NF-kappaB sites accessible (promoters with regulated and late accessibility [RLA]).Our results suggest that a mechanism of specificity in NF-kappaB-dependent transcriptional responses relies on the ability of individual stimuli to make RLA promoters accessible to NF-kappaB before its rapid extrusion from the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Biomedicine, CH6501 Bellinzona, Switzerland.

ABSTRACT
Proinflammatory stimuli induce the rapid and transient translocation of nuclear factor (NF)-kappaB to the nucleus, where it activates transcription from several genes, including those encoding inflammatory cytokines and chemokines, adhesion molecules, and cytoprotective proteins. Using chromatin immunoprecipitation, we show that after an acute stimulation two distinct waves of NF-kappaB recruitment to target promoters occur: a fast recruitment to constitutively and immediately accessible (CIA) promoters and a late recruitment to promoters requiring stimulus-dependent modifications in chromatin structure to make NF-kappaB sites accessible (promoters with regulated and late accessibility [RLA]). Our results suggest that a mechanism of specificity in NF-kappaB-dependent transcriptional responses relies on the ability of individual stimuli to make RLA promoters accessible to NF-kappaB before its rapid extrusion from the nucleus.

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Related in: MedlinePlus