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pmc-enju-pas

bionlp-st-ge-2016-spacy-parsed

GO-BP

{"project":"GO-BP","denotations":[{"id":"T7061","span":{"begin":43,"end":56},"obj":"http://purl.obolibrary.org/obo/GO_0006351"},{"id":"T7062","span":{"begin":195,"end":208},"obj":"http://purl.obolibrary.org/obo/GO_0006351"},{"id":"T30796","span":{"begin":2856,"end":2869},"obj":"http://purl.obolibrary.org/obo/GO_0006351"}],"text":"RUNX1 and RUNX3 bind to the FOXP3 promoter\nTranscription element search system analysis of the human FOXP3 promoter predicted 3 putative RUNX binding sites at 333, 287, and 53 bp upstream of the transcription start site (TSS). All three binding sites are conserved between human, mouse, and rat (Fig. S4). To verify the putative binding sites in the FOXP3 promoter, we transiently transfected HEK293T cells with RUNX1 and RUNX3. After the pull-down with oligonucleotides containing the wild-type binding sequences, but not mutant sequences, RUNX binding to the FOXP3 promoter oligonucleotides was detected by Western blot (Fig. 3 A). To confirm these results and test the ability of single binding site sequences to bind either RUNX1 or RUNX3, we used the promoter enzyme immunoassay. Cell lysates were obtained from HEK293T cells that had been transiently transfected with RUNX1 or RUNX3 expression vectors. The biotinylated FOXP3 promoter oligonucleotides were linked to a streptavidin-coated microtiter plate, and bound RUNX1 or RUNX3 was detected by using anti-RUNX antibodies and a peroxidase-labeled secondary antibody. We showed binding of RUNX1 and RUNX3 to the mixture of all three oligonucleotides containing the binding sites, whereas there was no binding detectable when a combination of the mutated oligonucleotides was used in the assay (Fig. 3 B). Although there was a similar and high degree of binding to the −333 and −287 sites, a lower degree of binding was detected when the oligonucleotide containing the −53 site in the Foxp3 promoter was used. This effect was observed both for binding to RUNX1 and RUNX3 (Fig. 3 B). The binding to the two single binding sites at −333 and −287 was comparable to the mixture of all three oligonucleotides. Chromatin immunoprecipitation (ChIP) assay results confirmed the binding of RUNX1 and RUNX3 complexes containing CBFβ to FOXP3 promoter during the differentiation of naive T cells toward T reg cells. Here, naive CD4+ T cells were cultured with IL-2, anti-CD2/3/28 mAb, and TGF-β as a Foxp3-inducing stimulation. Amplification of PCR products from the FOXP3 promoter region with the predicted RUNX binding sites showed that RUNX1, RUNX3, and CBFβ were immunoprecipitated together with the FOXP3 promoter (Fig. 3 C). Negative control primer targeting open reading frame-free intergenic DNA, IGX1A did not show any significant change in site occupancy.\nFigure 3. Binding of RUNX1 and RUNX3 proteins to the predicted binding sites in the FOXP3 promoter. (A) Mutated and wild-type oligonucleotides are shown. The predicted RUNX binding sites are accentuated (boxed and in green letters) and stars mark mutations introduced into the binding site of the control oligonucleotides. Nuclear extracts from HEK293T cells were incubated with biotinylated oligonucleotides. The precipitated oligonucleotide–transcription factor complexes were separated by SDS-PAGE and identified by Western blotting with anti-RUNX1 and anti-RUNX3 antibodies. A mixture of all three oligonucleotides with the predicted binding sites or with the inserted mutation into the predicted sites was used. Data shown are one representative of three independent experiments with similar results. (B) Promoter enzyme immunoassay using wild-type and mutated oligonucleotides within the FOXP3 promoter. Bars show mean ± SE of three independent experiments. (C) Chromatin immunoprecipitation assay results show binding of RUNX1 and RUNX3 complexes containing CBFβ to the human FOXP3 promoter in naive CD4+ T cells that were cultured with IL-2 together with anti-CD2/3/28 and TGF-β. There was no change in site occupancy in all immunoprecipitations when IGX1A negative control primers were used. The results are normalized to input and isotype control antibody. Bars show mean ± SE of three independent experiments. Statistical differences were verified by the paired Student's t test. *, P \u003c 0.05\n\nR"}

GO-MF

GO-CC

{"project":"GO-CC","denotations":[{"id":"T7090","span":{"begin":1116,"end":1124},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T7091","span":{"begin":1762,"end":1771},"obj":"http://purl.obolibrary.org/obo/GO_0000785"},{"id":"T30806","span":{"begin":2766,"end":2771},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T30807","span":{"begin":3527,"end":3532},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T30808","span":{"begin":2980,"end":2990},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T30809","span":{"begin":3770,"end":3778},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T30810","span":{"begin":2980,"end":2990},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T30811","span":{"begin":3770,"end":3778},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T30812","span":{"begin":3381,"end":3390},"obj":"http://purl.obolibrary.org/obo/GO_0000785"},{"id":"T7082","span":{"begin":401,"end":406},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T7083","span":{"begin":825,"end":830},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T7084","span":{"begin":1936,"end":1941},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T7085","span":{"begin":1955,"end":1960},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T7086","span":{"begin":1981,"end":1986},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T7087","span":{"begin":1070,"end":1080},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T7088","span":{"begin":1116,"end":1124},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T7089","span":{"begin":1070,"end":1080},"obj":"http://purl.obolibrary.org/obo/GO_0042571"}],"text":"RUNX1 and RUNX3 bind to the FOXP3 promoter\nTranscription element search system analysis of the human FOXP3 promoter predicted 3 putative RUNX binding sites at 333, 287, and 53 bp upstream of the transcription start site (TSS). All three binding sites are conserved between human, mouse, and rat (Fig. S4). To verify the putative binding sites in the FOXP3 promoter, we transiently transfected HEK293T cells with RUNX1 and RUNX3. After the pull-down with oligonucleotides containing the wild-type binding sequences, but not mutant sequences, RUNX binding to the FOXP3 promoter oligonucleotides was detected by Western blot (Fig. 3 A). To confirm these results and test the ability of single binding site sequences to bind either RUNX1 or RUNX3, we used the promoter enzyme immunoassay. Cell lysates were obtained from HEK293T cells that had been transiently transfected with RUNX1 or RUNX3 expression vectors. The biotinylated FOXP3 promoter oligonucleotides were linked to a streptavidin-coated microtiter plate, and bound RUNX1 or RUNX3 was detected by using anti-RUNX antibodies and a peroxidase-labeled secondary antibody. We showed binding of RUNX1 and RUNX3 to the mixture of all three oligonucleotides containing the binding sites, whereas there was no binding detectable when a combination of the mutated oligonucleotides was used in the assay (Fig. 3 B). Although there was a similar and high degree of binding to the −333 and −287 sites, a lower degree of binding was detected when the oligonucleotide containing the −53 site in the Foxp3 promoter was used. This effect was observed both for binding to RUNX1 and RUNX3 (Fig. 3 B). The binding to the two single binding sites at −333 and −287 was comparable to the mixture of all three oligonucleotides. Chromatin immunoprecipitation (ChIP) assay results confirmed the binding of RUNX1 and RUNX3 complexes containing CBFβ to FOXP3 promoter during the differentiation of naive T cells toward T reg cells. Here, naive CD4+ T cells were cultured with IL-2, anti-CD2/3/28 mAb, and TGF-β as a Foxp3-inducing stimulation. Amplification of PCR products from the FOXP3 promoter region with the predicted RUNX binding sites showed that RUNX1, RUNX3, and CBFβ were immunoprecipitated together with the FOXP3 promoter (Fig. 3 C). Negative control primer targeting open reading frame-free intergenic DNA, IGX1A did not show any significant change in site occupancy.\nFigure 3. Binding of RUNX1 and RUNX3 proteins to the predicted binding sites in the FOXP3 promoter. (A) Mutated and wild-type oligonucleotides are shown. The predicted RUNX binding sites are accentuated (boxed and in green letters) and stars mark mutations introduced into the binding site of the control oligonucleotides. Nuclear extracts from HEK293T cells were incubated with biotinylated oligonucleotides. The precipitated oligonucleotide–transcription factor complexes were separated by SDS-PAGE and identified by Western blotting with anti-RUNX1 and anti-RUNX3 antibodies. A mixture of all three oligonucleotides with the predicted binding sites or with the inserted mutation into the predicted sites was used. Data shown are one representative of three independent experiments with similar results. (B) Promoter enzyme immunoassay using wild-type and mutated oligonucleotides within the FOXP3 promoter. Bars show mean ± SE of three independent experiments. (C) Chromatin immunoprecipitation assay results show binding of RUNX1 and RUNX3 complexes containing CBFβ to the human FOXP3 promoter in naive CD4+ T cells that were cultured with IL-2 together with anti-CD2/3/28 and TGF-β. There was no change in site occupancy in all immunoprecipitations when IGX1A negative control primers were used. The results are normalized to input and isotype control antibody. Bars show mean ± SE of three independent experiments. Statistical differences were verified by the paired Student's t test. *, P \u003c 0.05\n\nR"}

sentences

events-check-again

bionlp-st-ge-2016-reference-tees

bionlp-st-ge-2016-reference

bionlp-st-ge-2016-uniprot

test2

PMC:2806624 / 11954-15872 JSONTXT

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PMC:2806624 / 11954-15872 JSON TXT