Inducibility of genes in the TNF locus by mitogen stimulation. Inducibility of NFKBIL1, LTA, TNF and LTB is shown as the ratio of transcript abundance in unstimulated cells versus cells induced with a combination of PMA and ionomycin for 6 h. Transcript levels were assayed using gene-specific primers normalized to an ACTB control primer set by real time Q-PCR using the ΔΔC_t method. Mean ± SD of three replicates are shown.

Effects of sequences spanning or flanking DHS 44500 on reporter gene expression in Jurkat T cells. Jurkat cells were transiently transfected with different pGL3 reporter constructs, either pGL3-Basic or driven by the LTA or SV40 promoters as indicated. The effects of inserting different sequences corresponding to the DHS 44500 or flanking regions were investigated. The mean ± SD of luciferase expression values are shown (normalized by pRL-TK) for five independent transfection experiments, each performed in duplicate. Open bars show expression for unstimulated cells, black bars values following mitogen stimulation (PMA/ionomycin). *P 0.01 on paired t-testing (two-tailed) versus pGL3.LTAprom.

Sequence conservation analysis and DNase I hypersensitivity map of TNF locus. Data from quantitative chromatin profiling and southern blotting for GM121565, Jurkat, U937, HeLa and HEK293T cells is summarized in the context of genomic structure and DNA sequence conservation analysis between species. The presence or absence of DNase hypersensitive sites in different cell types is indicated by filled and open boxes, respectively. Positions of Sca I restriction sites used in Southern blotting analysis are also shown with genomic coordinates from NCBI build 35.

DNase I footprinting analysis of DHS 44500. DNA probes spanning DHS 44500 radiolabelled on either the forward (panel A) or reverse (panel B) strands were digested with DNase I either alone or in the presence of NEs from Jurkat T cells. Maxam Gilbert sequencing ladder (lanes 1 and 5); naked DNA (0.0075 U DNase I) (lanes 2 and 6); NEs from unstimulated cells (0.25 U DNase I) (open circle) (lanes 3 and 7) or cells induced with PMA and ionomycin for 2 h (0.25 U DNase I) (filled circle) (lanes 4 and 8). (C) Schematic showing sites of protection in context of human and murine sequences.

Tiling path analysis of histone modifications across the TNF locus. Histone modifications revealed by chromatin immunoprecipitation experiments using antibodies to diacetylated histone H3 (H3Ac), tetracetylated histone H4 (H4Ac) and tri-methylated histone K4 H3 (H3K4Me3) are shown for Jurkat T cells, either in the resting state or harvested 1, 2 or 6 h after induction with 125 nM ionomycin plus 200 nM PMA. The immunoprecipitated DNA was analysed using real time Q-PCR using 24 primer pairs at an average of 1.1 kb intervals across the region (31634558–31660002). On the y-axis, the fold difference in enrichment of each of the PCR amplicons tiled across the region is expressed relative to input DNA. Genomic location is shown on the x-axis.

USF1 and USF2 bind within DHS 44500. (A) EMSA experiments using radiolabelled probes corresponding to the putative USF binding site within DHS 44500 (lanes 1–3, 7–18) or to a USF binding site from the adenovirus major late promoter (lanes 4–6) were subjected to electrophoresis without NE (lanes 1, 4, 7 and 14), with NE from unstimulated cells (open circle) (lanes 2, 5) or cells induced with PMA and ionomycin for 2 h (filled circle) (lanes 3, 6, 8–13, 15–18). Competition experiments using 10× or 100× molar excess of unlabelled probe are shown (lanes 9–13); supershift experiments using antibodies as indicated (lanes 16–18). (B) Chromatin immunoprecipitation experiment using Jurkat T cells either unstimulated (open circle) or after stimulation with PMA and ionomycin (filled circle). Results of PCR amplification for primer pair at DHS 44500 (31644348–622) are shown for input controls (lanes 1 and 2); mock antibody immunoprecipitated controls (lanes 3 and 4); USF1 (lanes 5 and 6) and USF2 (lanes 7 and 8) immunoprecipitated DNA.

DNase hypersensitive sites in the TNF locus detected by Southern blotting for a range of cell types. Sca I restriction fragments were hybridized to radiolabelled probe and the pattern of cleavage analysed for nuclei incubated with increasing amounts of DNase I (20–160 U) at 37°C, or without exposure to DNase I incubated on ice (0*) or at 37°C (0). DNase hypersensitive sites are indicated by arrows with nomenclature following that used in analysis of quantitative chromatin profiling. LCLs (GM12156, QBL and a non-EBV transformed cell line BL41), Jurkat, U937, HeLa and HEK293T cells were analysed. (A) Southern blotting for region 5′ to LTA gene. A 10.4 kb Sca I restriction fragment (31639037–31649422) was hybridized to a labelled 247 bp probe (31649015–31649262). (B) Southern blotting for NFKBIL1 and 3′ region. A 6.92 kb Sca I restriction fragment (31632112–31639036) was hybridized to a labelled 266 bp probe (31637483–31637748). (C) Southern blotting in region of TNF. A 6.83 kb Sca I restriction fragment (31649420–31656247) hybridized to a labelled 245 bp probe (31655630–31655874). (D) Southern blotting in region of LTB. A 4.3 kb Sca I restriction fragment (31656244–31660544) was hybridized to a labelled 206 bp probe (31658807–31659012).

Quantitative chromatin profiling for a 34 kb region spanning the TNF locus and flanking genes in GM12156. This was carried out for four replicate DNase I digestions to give DNase I sensitivity measurements (ratios of copy number in treated compared to untreated samples) from 140 contiguous PCR amplicons designed to tile across the region (31634558–31668876). All samples were quantified in triplicate by Q-PCR. Relative DNase I sensitivity measurements are shown (DNase I-treated versus untreated) together with SNRs with genomic coordinates given along the x-axis. GM12156 was used, either resting (panel A) or after induction with 125 nM ionomycin plus 200 nM PMA for 30 min (panel B). Analysis revealed strong evidence for six hypersensitive sites (highlighted in light blue on figure): ‘DHS 35000’ 0.3 kb 3′ to exon 4 of NFKBIL1 in unstimulated cells (PCR amplicon coordinates 31634776–31635055); ‘DHS 36000’ 1.7 kb and 1.5 kb 3′ to exon 4 of NFKBIL1 in unstimulated cells (31636120–31636410) and stimulated cells (31635824–31636410) respectively; ‘DHS 44500’ 3.4 kb 5′ to LTA in unstimulated cells (31644348–31644622) and stimulated cells (31644348–31644807); ‘DHS 48000’ within 0.1 kb of the LTA transcriptional start site in unstimulated cells (31648007–31648254) extending up to 0.3 kb downstream in stimulated cells (31648007–31648393); ‘DHS 51250’ within 0.1 kb of the TNF transcriptional start site in stimulated cells (31651222–31651465) which was also present in unstimulated cells but to a lesser extent; ‘DHS 56700’ in exon 4 of LTB in unstimulated cells (31656528–31656800) and exon 4 and intron 3 of LTB in stimulated cells (31656528–31657053). In addition, six further sites are suggested with marginal evidence (shown highlighted in grey on figure).