NF-κB-Dependent Assembly of an Enhanceosome-Like Complex on the Promoter Region of Apoptosis Inhibitor Bfl-1/A1

The binding of NF-κB to DNA promotes the binding of AP-1, C/EBPβ, HMGI-C, and p300 to the −933/−773 bfl-1 probe. (A) Nuclear extracts from Jurkat T cells treated with PMA plus ionomycin (Iono), or with DMSO as control, were incubated with 160-bp probes derived from the −933/−773 region of bfl-1. The probes were either wild type (WT) or mutated at the NF-κB, AP-1, or C/EBPβ binding sites as indicated. The bracket highlights the large DNA-protein complex. (B) Supershift analysis of DNA-protein complexes formed on mutant AP-1 or C/EBPβ probes with antibodies (Ab) specific to each factor. Arrows point to the DNA-protein complex, and brackets indicate supershifted complexes. (C) Supershift analysis of the complex bound to a −933/−773 probe in which the AP-1 and C/EBPβ DNA sites were mutated simultaneously.

NF-κB nucleates formation of a DNA-protein complex containing c-Rel/p50, c-Jun, C/EBPβ, and HMGI-C on the −933/−773 bfl-1 probe. (A) Nuclear extracts from uninduced (+tet, lanes 1 to 6 and 13 to 18) or induced (−tet, lanes 7 to 12 and 19 to 24) HtTA-CCR43 cells were incubated with a BrdU- and ³²P-labeled wild-type −933/−773 bfl-1 probe (lanes 1 to 12) or a mutant probe in which the NF-κB site was inactivated (lanes 13 to 24). After UV cross-linking, DNA-protein complexes were immunoprecipitated (Ippt) with antibodies (Ab) specific for c-Rel, c-Jun, C/EBPβ, and HMGI-C or control normal rabbit IgG, followed by autoradiography. (B) Western blot analysis of endogenous c-Rel, c-Jun, and C/EBPβ proteins in nuclear extracts from HtTA-CCR43 cells uninduced (+tet) or induced to express c-Rel (−tet).

Phasing and orientation of the NF-κB dimer affect the stability of the complex on the −933/−773 bfl-1 probe. (A) Schematic diagram of −933/−773 bfl-1 positional mutants. Arrows indicate the orientation of the NF-κB dimer with respect to the rest of the 5′ bfl-1 regulatory region. (B) Nuclear extracts from Jurkat T cells stimulated with PMA plus ionomycin were incubated with wild-type (WT) or mutant −933/−773 bfl-1 probes (rκB, κB+6, and κB+10). Aliquots of the binding reaction mixtures were loaded onto a running nondenaturing gel at time zero and at intervals following addition of a 250-fold molar excess (250x) of an unlabeled competitor oligonucleotide. (C) Bound complexes were quantitated with ImageQuant software (Molecular Dynamics). Relative bound complex represents the ratio of DNA-bound complex remaining at indicated time points over the amount of bound complex at time zero. The data are representative of three independent experiments.

NF-κB, AP-1, and C/EBPβ synergistically activate transcription from the bfl-1 regulatory region with coactivator p300. (A) Jurkat T cells were transiently cotransfected with a −1374/+83bfl-1:luc reporter construct (1.33 μg) and expression vectors for c-Rel, c-Jun, c-Fos, and C/EBPβ (667 ng each), alone or together (3 TF). The total amount of transfected DNA was kept constant by addition of empty pcDNA3.1HisC vector. The average of three independent experiments is shown. (B) Transcriptional activity of the −933/−773 bfl-1 regulatory region. Jurkat T cells were transfected with 360 ng of −933/−773:−100/+83 bfl-1:luc or control −100/+83 bfl-1:luc reporter and 3.6 μg of pcDNA3 control or pcDNA3:IκBαM. Cells were treated with DMSO or stimulated with PMA plus ionomycin for 18 h prior to harvest. The results show the average fold activation of −933/−773:−100/+83 bfl-1:luc by treatment with PMA plus ionomycin from which background activity of the −100/+83 bfl-1:luc control was subtracted. (C) Cells were transfected with wild-type or mκB, mAP-1, or mC/EBPβ mutant −993/−773 bfl-1:CAT reporters (1.5 μg) and 1.5 μg each of CMV-c-Rel, CMV-c-Jun, and CMV-C/EBPβ DNA or 4.5 μg of an empty pcDNA3.1HisC vector as a control. CAT activity was assayed at 48 h posttransfection. (D) Jurkat T cells were cotransfected with a total of 4 μg of DNA containing −1374/+81 bfl-1:luc (730 ng) together with expression vectors for c-Rel, c-Jun, c-Fos, and C/EBPβ (360 ng each; TF mix), alone or together with p300 (1.8 μg). The average of three independent experiments is shown. (E) Assays were conducted essentially as in panel D, but with the −933/−773:−100/+83 bfl-1:luc reporter construct. The average of three independent experiments is shown.

ChIP analysis of in vivo complex formation on the 5′ regulatory region of bfl-1 upon T-cell activation. (A) Representative real-time PCR analysis of ChIP assays with an anti-c-Rel antibody (Ab). Accumulation of product in cells treated with PMA plus ionomycin or with DMSO as a control normalized to reporter fluorescence (R_N; y axis) is plotted as a function of cycle number (x axis). The threshold (dashed line) was calculated by multiplying the standard deviation of the R_N of the baseline (cycles 3 to 15) by 10. The average of three independent reactions is shown. (B) ChIP analysis of transcription factors c-Rel, p65/RelA, c-Jun, and C/EBPβ and of architectural factor HMGI-C binding to the bfl-1 locus in human Jurkat T cells stimulated with PMA plus ionomycin (P+I) for 2 or 18 h or with a DMSO control. Factor binding was evaluated by determining the difference in the average number of PCR cycles at which amplification of the bfl-1 regulatory region reached the threshold between chromatin samples immunoprecipitated with transcription factor-specific antibodies and the no-antibody control (2^−ΔCT). Standard deviations were calculated by using the formula s₁² + s₂² (ABI Prism 7700 Sequence Detection System, user bulletin 2, 1997. Applied Biosystems, Foster City, Calif.). (C) Control ChIP analysis showing p65/RelA recruitment to the 5′ regulatory region of the NF-κB target gene encoding IκBα in Jurkat T cells activated with PMA plus ionomycin and of c-Rel, c-Jun, and C/EBPβ recruitment to the upstream regulatory region of the B-cell-specific chemokine receptor gene BLR1 as a negative control. (D) ChIP analysis of coactivator p300, TAFII250, CBP, and p/CAF recruitment in Jurkat T cells treated with DMSO or following activation with PMA plus ionomycin. (E) ChIP analysis of SWI/SNF chromatin remodeling factor component BRG-1 and basal transcription factors TBP and TFIIB recruitment to the proximal bfl-1 promoter region in human Jurkat T cells stimulated with PMA plus ionomycin or with a DMSO control. (F) ChIP analysis of histone H3 and H4 acetylation on the bfl-1 regulatory region before and after Jurkat T-cell activation with PMA plus ionomycin (top panel). ChIP samples from unstimulated or PMA-plus-ionomycin-treated Jurkat T cells were immunoprecipitated with an antibody for hyperacetylated histone H3 and subjected to 25 rounds of standard nonquantitative PCR. The products were resolved on an agarose gel and revealed by ethidium bromide staining (bottom panel).