PMC:2222968 / 23457-27615 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2222968","sourcedb":"PMC","sourceid":"2222968","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2222968","text":"GATA3 Represses the FOXP3 Promoter\nTo investigate the molecular mechanism of GATA3-mediated repression of human FOXP3, the human FOXP3 promoter was studied and a palindromic binding site for GATA3 was discovered. The GATA-binding site is located 303 bp upstream from the transcription start site (TSS) [24]. This site is highly conserved between humans, mice, and rats (Figure S4) and may therefore play an important role in FOXP3 regulation. The functional relevance of this site was studied using a FOXP3-promoter construct [24]. We transfected human primary CD4+ T cells, in vitro differentiated Th2 cells, and Jurkat cells (Jurkat cells are known to constitutively express GATA3 [25,26]), and we measured FOXP3 promoter activity. The promoter was not active in the GATA3-expressing cell line Jurkat or in the in vitro-differentiated Th2 cells, whereas the construct was active in the CD4 cells, which express a lower amount of GATA3 (Figure 8A). Overexpression of GATA3 in naive T cells diminished luciferase activity of the FOXP3 promoter compared with the control vector (Figure 8B). To further address the function of the GATA3 site, we inserted a site-specific mutation deleting the GATA3-binding site. This mutation increased luciferase activity by 3-fold in memory CD4+CD45RO+ T cells, whereas no difference was observed in naive (GATA3–) CD4+CD45RA+ T cells, revealing a repressor activity of GATA3 on the FOXP3 promoter (Figure 8C). Furthermore GATA3 binds directly to the FOXP3 promoter as investigated by pull-down assay. HEK cells were transiently transfected with GATA3 or a control vector, and increasing amounts of lysates were incubated with oligonucleotides containing the GATA3 site of the FOXP3 promoter or a control oligonucleotide with a mutated GATA3-binding site. After the pull-down, GATA3 binding was detected by Western blot. Similarly, GATA3-expressing Th2 cells and iTreg cells were subjected to this approach. Only HEK cells overexpressing GATA3 and Th2 cells showed GATA3-binding activity (Figure 8D and 8E). These experiments demonstrated that GATA3 binds the palindromic FOXP3 promoter. To gain insights into the in vivo situation, we performed a chromatin immunoprecipitation (ChIP) using an anti-GATA3 antibody and showed that GATA3 binds to the FOXP3 promoter region in Th2 cells, but not in iTreg cells (Figure 7F). Taken together these data demonstrate that the GATA3-binding to the FOXP3 promoter is repressing FOXP3 expression.\nFigure 8 GATA3 Represses the Human FOXP3 Promoter\n(A) Jurkat, Th2 cells, and human primary CD4 cells were transfected with an empty vector (pGL3 basic) or a vector containing the putative FOXP3 promoter region fused to the luciferase reporter gene. Bars show the mean ± SD of arbitrary light units normalized for renilla luciferase of four independent experiments; samples were measured in triplicates.\n(B) Naive CD4 T cells were transfected with the FOXP3 promoter reporter construct together with a GATA3 expression vector or an empty vector. Bars show the mean ± SD of three independent experiments.\n(C) Naive (left panel) or memory (right panel) CD4 T cells were transfected with wild-type or a GATA3 mutated 511-FOXP3 promoter reporter construct and activated with PMA and ionomycin. Bars show the mean ± SD of arbitrary light units normalized for renilla luciferase of eight independent experiments; samples were measured in triplicates.\n(D) Nuclear extracts were prepared from HEK cells transfected with GATA3 or an empty vector, (E) Th1, Th2, or iTreg cells and binding factors precipitated using biotinylated oligonucleotides. The oligonucleotides–transcription factor complexes were separated on a SDS-PAGE gel. The amounts of GATA3 protein in the precipitates were assessed by immunoblotting with anti-GATA3 mAb. Total nuclear extracts were also run as controls. Data are representative of three different experiments.\n(F) iTreg or Th2 cells were analyzed by ChIP for GATA3 binding to the FOXP3 promoter. The “input” represents PCR amplification of the total sample, which was not subjected to any precipitation. Results are representative of three independent experiments.\n\nD","divisions":[{"label":"Title","span":{"begin":0,"end":34}},{"label":"Figure 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