NFATp binds to multiple sites in HSS-9 and HSS+3. Shown is quantitative DNase I footprinting analysis using sense (A) and antisense (B) probes spanning region I (306 bp) of HSS-9 and a sense probe (C) spanning HSS+3 (307 bp) and increasing amounts (4 ng, 20 ng, or 100 ng) of recombinant NFATp or NF-κB p50/p65. Positions of the NFAT (gray boxes) and NFAT/NF-κB (dark gray boxes) binding sites (relative to the TNF transcription start site) are indicated.

3C assay. (A) Genomic DNA products of BstY I digestion (arrows), with areas of interest (HSS and gene promoters) labeled A–F and PCR primers numbered 1 and 2. (B and C) Relative cross-linking frequencies (±SEM) between the indicated fragments and fragment A (HSS-9) using primers A2-B2, A1-C2, A2-D2, A1-E1, and A2-F2 (B) or fragment C (TNF promoter) using primers C2-A1, C1-B1, C1-D1, C1-E2, and C1-F1 (C) with chromatin from unstimulated (−) or P+I-stimulated (+) cells or genomic DNA control (c). PCR products in B and C are from independent PCR plates, and PCR products within each of these two sets were amplified simultaneously; for each pair of genomic fragments, similar results were obtained by using the other possible primer pair combinations (SI Fig. 13B).

TNF/LT locus DH profiles in naive, Th1, and Th2 subsets of primary murine T lymphocytes. (A and B) Nuclei from naive T cells and Th1 and Th2 cells were digested with increasing amounts of DNase I. Purified DNA was subsequently digested with BglII (A) or XbaI (B) followed by resolution by agarose gel electrophoresis (molecular weight markers in kb), Southern blot analysis, hybridization with a probe corresponding to the first exon of TNF, and visualization by autoradiography. HSS found in T cells are indicated with arrows and designated by their approximate positions relative to the first TNF exon. Migration of digested genomic DNA fragments is consistently greater than expected relative to the marker, most likely because of a greater amount of total DNA (≈15 μg) than marker (10 pg). (C) Positions and directions of transcription of the TNF, LT-β, and LT-α genes in the ≈23-kb locus are shown by white arrows; exons are shown by numbered gray boxes; and the Southern blotting probe (186 bp) is shown by a black box. Positions of the parental BglII and XbaI fragments and the DNase I digestion products are indicated. (D) Sequence conservation between murine and human TNF/LT loci using PipMaker (www.bx.psu.edu) (32) as described in SI Fig. 6, showing strongly and moderately conserved noncoding regions (green and blue, respectively) and exons (red).

HSS-9 and HSS+3 bind NFATp in murine primary CD4⁺ T cells in vivo and function as enhancers of TNF transcription. (A–D) ChIP analysis in unstimulated (−) or anti-CD3/CD28-stimulated (+) mouse CD4⁺ T cells. Chromatin was immunoprecipitated with the indicated antibodies to transcription factors NFATp, NF-κB p50, and NF-κB p65 (A and C) or the acetylated forms of histones H3 and H4 (B and D) and amplified with primers specific for regions I and II of HSS-9, HSS+3, and the IL-2 promoter (SI Figs. 7 and 8). Nonimmune IgG antibody and 10 times the amount of the input DNA used for the specific antibodies (to detect background) were used for PCR controls for nonspecific binding. (E) 68-41 cells were transiently transfected with the indicated reporter constructs. After mock stimulation or P+I stimulation (P+Io) in the presence or absence of cyclosporin A (CsA) pretreatment, luciferase activity was normalized to Renilla luciferase activity. Error bars represent standard deviation from at least four independent experiments.

A model of intrachromosomal interactions at the TNF/LT locus in activated T cells. Simultaneous intrachromosomal interactions at the TNF/LT locus (TNF promoter–HSS+3, TNF promoter–HSS-9, and HSS+3–HSS-9) would both juxtapose NFAT-binding regulatory regions and selectively circularize the TNF gene (see SI Fig. 14).