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. 2003 Jan;23(2):526–533. doi: 10.1128/MCB.23.2.526-533.2003

FIG.2.

FIG.2.

Identification of activator binding sites required for TNF-α gene regulation by M. tuberculosis. (A) J774 cells were transfected with 1 μg of a TNF-α luciferase reporter containing either 200 or 982 nt upstream of the transcription start site. Eight hours posttransfection, cells weretreated with H37Rv M. tuberculosis whole sonicate (10 μg/ml) for 16 h. All transfections included a control Renilla luciferase plasmid and were normalized to Renilla luciferase activity. Data are presented as means ± standard errors of the means (SEM) of three independent experiments. (B) The DNA sequence of the TNF-α promoter spanning nt −200 to −20 relative to the transcription start site is shown. Transcription factor binding sites are indicated, as are the locations of point mutations in the mutant constructs studied in panel C. (C) The CRE, Sp1, upstream Sp1, Egr-1, and Ets binding sites are required for M. tuberculosis induction of TNF-α. J774 cells were transfected with 1 μg of the −200 TNF-α luciferase reporter or with isogenic reporters containing the indicated mutations and treated with M. tuberculosis whole sonicate for 16 h as described above. We noted that the 3′M and −76 NFAT mutant abolished Ets binding to the adjacent −84-Ets/Elk site (reference 23 and data not shown). Data are presented as means ± SEM of three independent experiments. (D) Formaldehyde cross-linking and chromatin immunoprecipitation of J774 cells that were unstimulated (−) or treated with M. tuberculosis whole sonicate (+). Samples of sonicated and purified chromatin were immunoprecipitated with the indicated antibodies, and DNA isolated from immunoprecipitated material was amplified by PCR with primers specific for the TNF-α promoter. An increase in the relative amount of the TNF-α promoter-specific PCR product indicates binding of the protein to the endogenous amplified TNF-α promoter. Densitometry quantification of the induction ratios for the various transcription factors were 1.9 for ATF-2, 2.0 for c-Jun, 3.8 for Ets, 1.5 for Sp1, and 1.6 for Egr. Input DNA control lanes (lanes 12 and 13) and free primer (lane 1) are shown.