Supplemental Data

Full Text
Duration of Nuclear NF-kappaB Action Regulated by Reversible Acetylation
Lin-feng Chen, Wolfgang Fischle, Eric Verdin, and Warner C. Greene

Supplementary Material

Supplemental Figure 1. TSA abrogates HDAC3-mediated inhibition of TNF-alpha-induced kappaB-luciferase activity. 293T cells were cotransfected with an expression plasmid DNA encoding HDAC3 and the kappaB-luciferase reporter plasmid DNA. 16 hours later, the cells were treated with medium or medium containing the indicated amounts of TSA for 1 hour and then stimulated with TNF-alpha for 5 hours before measurement of kappaB-luciferase activity (19). Note the inhibition of the TNF-alpha response by HDAC3 and dose-related blockade of this inhibition by TSA.

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Supplemental Figure 2. RelA is acetylated by p300 and CBP but not by P/CAF. 293T cells were cotransfected with the indicated amounts of expression plasmid DNA encoding T7-RelA, p300, CBP, and P/CAF. 20 hours later, cell lysates were prepared and T7- RelA was sequentially immunoprecipitated (19) with anti-T7 antibodies and immunoblotted with anti-acetylated lysine antibodies (Cell Signaling). Note the dose-dependent acetylation of RelA produced by p300 (lanes 3 and 4) and CBP (lanes 5 and 6) but not by P/CAF (lanes 7 and 8) although this latter protein was effectively expressed. Levels of T7-RelA present in each of the lysates are shown in the lower panel.

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Supplemental Figure 3. (A) To map determinants in HDAC3 that are required for its assembly with RelA, various NH2- and COOH-terminal deletion mutants of HDAC3 were prepared and tested in coimmunoprecipitation assays (left and middle panel). Deletion of the NH2-terminal 45 amino acids of HDAC3 impairs its assembly with RelA. Expression plasmid DNA encoding the deletion mutants of HDAC3-Flag (1.0 mug) depicted in the left panel and T7-RelA (1.0 mug) were cotransfected into COS7 cells. Whole-cell lysates were prepared 30 hours later and immunoprecipitated with anti-T7 antibodies followed by immunoblotting of the immunoprecipitates with anti-Flag antibodies (19). Expression levels for the various HDAC3-Flag proteins and T7-RelA are shown in the lower two panels. Note that wild-type HDAC3 and the HDAC3 1-406 mutant assemble with RelA (lanes 3 and 6 of upper panel) while the HDAC3 46 to 428 and 121 to 428 deletion mutants fail to coimmunoprecipitate with RelA (lanes 4 and 5 of upper panel). In functional kappaB-luciferase assays, activation of this reporter by RelA (right panel, lane 2) was blocked by wild-type HDAC3 but not by the two N-terminal HDAC3 deletion mutants that failed to bind to RelA. (B) The N-terminal 180 amino acids of RelA are sufficient to support binding to HDAC3. Using the mammalian two-hybrid system (20), expression plasmids encoding wild-type RelA or various deletions of RelA fused to VP16 (1.0 mug) depicted in the left panel, were cotransfected into 293T cells with plasmids encoding the GAL4 DNA binding domain fused to HDAC3 (0.5 mug) and a GAL4 enhancer luciferase reporter (0.1 mug) (pFR-luc) (Stratagene) by FuGENE 6. Luciferase activity was measured 40 hours after transfection (19). Note that the 1 to 180 fragment of RelA supports interaction with HDAC3 comparably to wild-type RelA (lane 4) while two similarly sized fragments of Rel A from the mid- and COOH-terminal regions of the protein fail to stimulate luciferase activity.

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Supplementary Information on Experimental Protocols: Luciferase reporter assays. 293T cells were seeded into either 24 well (1 mult 105 cells per well) or 6 well (4 mult 105 cells per well) plates and cultured for 10 to 12 hours in DMEM-10%FCS before transfection. 0.1 mug of kappaB-luciferase reporter plasmid DNA (Stratagene) was transfected alone or cotransfected with expression plasmids encoding FLAG-HDACs (0.5 or 1.0 mug of DNA) using FuGENE 6 (Roche). 16 to 24 hours after transfection, cells were stimulated with TNF-alpha (10 to 20 ng/ml). For treatment with TSA, TSA was added 1 hour before adding TNF-alpha. 5 hours after TNF-alpha stimulation, cells were lysed with 5 mult passive lysis buffer (Promega). In each experiment, cells were also cotransfected with EF1alpha-Renilla luciferase reporter plasmids to permit normalization for differences in transfection efficiency occurring in the individual cultures. Firefly and Renilla luciferase activity were assayed using the dual luciferase assay system according to manufacturer's instructions (Promega).

Immunoprecipitation and immunoblotting analyses. Transfected COS-7 cells or 293T cells cultured in 6 well plates were lysed in 300 mul of lysis buffer (50 mM Hepes pH 7.4, 250 nM NaCl, 1% NP-40, 1 mM EDTA). Lysates from 2 or 3 wells were immunoprecipitated for 2 hours at 4°C using 20 mul of a slurry containing 50% anti-T7-conjugated agarose beads (Novagen). These immunoprecipitates were washed three times in lysis buffer. Immunoprecipitated proteins were separated by SDS-PAGE (10%), transferred to nitrocellulose transfer membranes and immunoblotted with various antibodies followed by visualization of the immunoreactive proteins by ECL (Amersham).

In vivo assay of acetylation. COS7 cells seeded in 100 mm dishes were transfected with T7-RelA (5 mug) in combination with HDAC1 (10 mug), HDAC3 (10 mug) or control plasmid DNA (10 mug) using FuGENE 6 (Roche). Thirty hours after transfection, the cells were pretreated with cycloheximide (CHX) (25 mug/ml) for 1 hour and then transferred to the same DMEM medium containing 1 mCi/ml [3H]-sodium acetate (Amersham), 25 mug/ml CHX for 1 hour at 37°C before lysis. Cell lysates were immunoprecipitated with anti-T7 antibodies conjugated to agarose (Novagen). Immunoprecipitated proteins were analyzed by SDS-PAGE (10%) followed by autoradiography for 4 to 6 weeks. For assay of the acetylation of the endogenous RelA, HeLa cells were stimulated with medium or TNF-alpha (20 ng/ml) for 30 min and radiolabeled for 1 hour with Na-[3H]-acetate (1 mCi/ml) in the presence of cycloheximide (25 mug/ml). Whole cell lysates were prepared and immunoprecipitated with anti-RelA conjugated agarose beads (SC-109AC, Santa Cruz) to evaluate the potential acetylation of RelA.