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bionlp-st-ge-2016-reference-tees

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2_test

{"project":"2_test","denotations":[{"id":"16652169-10692237-98518410","span":{"begin":2003,"end":2005},"obj":"10692237"},{"id":"16652169-1909027-98518411","span":{"begin":2006,"end":2008},"obj":"1909027"}],"text":"Foxp3 Suppresses HIV-1 Gene Expression in Part through Blocking Activation of NF-κB\nIf Foxp3 functions as a repressor of NF-κB-dependent gene expression, then we hypothesized that Foxp3 overexpression could selectively down-regulate transcription from promoters previously shown to be responsive to NF-κB. To address this question, we examined the transcriptional activation of the HIV-1 LTR, which contains two tandem cis-acting NF-κB binding sites located between positions −102 and −81 with respect to the transcription initiation site [21]. NF-κB plays a crucial role in regulating gene expression directed from the HIV-1 LTR in CD4+ T cells [21]. Overexpression of full-length Foxp3, but not ΔFKH, in HEK 293T cells was able to inhibit basal activation of the HIV-1 LTR (Figure 3A), similar to what was previously demonstrated with the synthetic NF-κB reporter vector (Figure 2B). Furthermore, HIV-1 LTR activation was suppressed by full-length Foxp3 and ΔFKH in Jurkat T cells (Figure 3B). To demonstrate that Foxp3-mediated HIV-1 LTR repression was associated with interactions with NF-κB bound to the HIV-1 LTR, we compared basal activation of the HIV-1 LTR or an identical HIV-1 LTR lacking the NF-κB sites located between −102 and −81 (HIV-1 Δ-κB LTR) (Figure 3C). This mutant HIV-1 LTR construct exhibited reduced levels of transcription compared to the parental HIV-1 LTR in purified healthy donor CD4+ T cells (unpublished data). However, directly comparing the effect of Foxp3 overexpression on the activation of these two viral promoters demonstrated that Foxp3 was more capable of suppressing transcriptional activation of the HIV-1 LTR (Figure 3D) compared to the mutated HIV-1 LTR (Figure 3E). These results suggest that Foxp3 down-regulation of HIV-1 LTR activation was mediated at least in part by cis-acting NF-κB binding sites. Residual levels of inhibition of the HIV-1 Δ-κB LTR by Foxp3 may be due to NF-AT binding sites located upstream of the NF-κB sites within the HIV-1 LTR [22,23].\nFigure 3 Foxp3 Inhibits Basal Activation of the HIV-1 LTR\n(A and B) HEK 293T cells (5 × 105) (A) were seeded into six-well plates 1 d prior to transfection or Jurkat T cells (1 × 106) (B) were seeded into six-well plates on the day of transfection with an HIV-1 LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (1,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity.\n(C) Schematic representation of HIV-1 LTR luciferase reporter constructs pHIV-1 LTR-luc and pHIV-1 Δ-κB LTR-luc.\n(D and E) CD4+ T cells (2 × 106) from two healthy donors were nucleofected with either parental HIV-1 LTR (D) or HIV-1 LTR lacking NF-κB binding sites (at −102 to −81) (E) (1,000 ng), Foxp3 or control expression vectors (2,000 ng), and an internal reference plasmid (1,000 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity shown in A, B, D, and E was normalized to the internal reference control.\n\nT"}

pmc-enju-pas

bionlp-st-ge-2016-spacy-parsed

GO-BP

{"project":"GO-BP","denotations":[{"id":"T5639","span":{"begin":23,"end":38},"obj":"http://purl.obolibrary.org/obo/GO_0010467"},{"id":"T5640","span":{"begin":137,"end":152},"obj":"http://purl.obolibrary.org/obo/GO_0010467"},{"id":"T5641","span":{"begin":586,"end":601},"obj":"http://purl.obolibrary.org/obo/GO_0010467"},{"id":"T5642","span":{"begin":233,"end":246},"obj":"http://purl.obolibrary.org/obo/GO_0006351"},{"id":"T5643","span":{"begin":509,"end":522},"obj":"http://purl.obolibrary.org/obo/GO_0006351"},{"id":"T5644","span":{"begin":1335,"end":1348},"obj":"http://purl.obolibrary.org/obo/GO_0006351"},{"id":"T5645","span":{"begin":1609,"end":1624},"obj":"http://purl.obolibrary.org/obo/GO_0006351"},{"id":"T5646","span":{"begin":1750,"end":1760},"obj":"http://purl.obolibrary.org/obo/GO_0065007"}],"text":"Foxp3 Suppresses HIV-1 Gene Expression in Part through Blocking Activation of NF-κB\nIf Foxp3 functions as a repressor of NF-κB-dependent gene expression, then we hypothesized that Foxp3 overexpression could selectively down-regulate transcription from promoters previously shown to be responsive to NF-κB. To address this question, we examined the transcriptional activation of the HIV-1 LTR, which contains two tandem cis-acting NF-κB binding sites located between positions −102 and −81 with respect to the transcription initiation site [21]. NF-κB plays a crucial role in regulating gene expression directed from the HIV-1 LTR in CD4+ T cells [21]. Overexpression of full-length Foxp3, but not ΔFKH, in HEK 293T cells was able to inhibit basal activation of the HIV-1 LTR (Figure 3A), similar to what was previously demonstrated with the synthetic NF-κB reporter vector (Figure 2B). Furthermore, HIV-1 LTR activation was suppressed by full-length Foxp3 and ΔFKH in Jurkat T cells (Figure 3B). To demonstrate that Foxp3-mediated HIV-1 LTR repression was associated with interactions with NF-κB bound to the HIV-1 LTR, we compared basal activation of the HIV-1 LTR or an identical HIV-1 LTR lacking the NF-κB sites located between −102 and −81 (HIV-1 Δ-κB LTR) (Figure 3C). This mutant HIV-1 LTR construct exhibited reduced levels of transcription compared to the parental HIV-1 LTR in purified healthy donor CD4+ T cells (unpublished data). However, directly comparing the effect of Foxp3 overexpression on the activation of these two viral promoters demonstrated that Foxp3 was more capable of suppressing transcriptional activation of the HIV-1 LTR (Figure 3D) compared to the mutated HIV-1 LTR (Figure 3E). These results suggest that Foxp3 down-regulation of HIV-1 LTR activation was mediated at least in part by cis-acting NF-κB binding sites. Residual levels of inhibition of the HIV-1 Δ-κB LTR by Foxp3 may be due to NF-AT binding sites located upstream of the NF-κB sites within the HIV-1 LTR [22,23].\nFigure 3 Foxp3 Inhibits Basal Activation of the HIV-1 LTR\n(A and B) HEK 293T cells (5 × 105) (A) were seeded into six-well plates 1 d prior to transfection or Jurkat T cells (1 × 106) (B) were seeded into six-well plates on the day of transfection with an HIV-1 LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (1,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity.\n(C) Schematic representation of HIV-1 LTR luciferase reporter constructs pHIV-1 LTR-luc and pHIV-1 Δ-κB LTR-luc.\n(D and E) CD4+ T cells (2 × 106) from two healthy donors were nucleofected with either parental HIV-1 LTR (D) or HIV-1 LTR lacking NF-κB binding sites (at −102 to −81) (E) (1,000 ng), Foxp3 or control expression vectors (2,000 ng), and an internal reference plasmid (1,000 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity shown in A, B, D, and E was normalized to the internal reference control.\n\nT"}

GO-MF

{"project":"GO-MF","denotations":[{"id":"T6369","span":{"begin":430,"end":443},"obj":"http://purl.obolibrary.org/obo/GO_0051059"},{"id":"T6370","span":{"begin":1829,"end":1842},"obj":"http://purl.obolibrary.org/obo/GO_0051059"},{"id":"T6371","span":{"begin":436,"end":443},"obj":"http://purl.obolibrary.org/obo/GO_0005488"},{"id":"T6372","span":{"begin":1835,"end":1842},"obj":"http://purl.obolibrary.org/obo/GO_0005488"},{"id":"T6373","span":{"begin":1931,"end":1938},"obj":"http://purl.obolibrary.org/obo/GO_0005488"},{"id":"T6374","span":{"begin":1928,"end":1938},"obj":"http://purl.obolibrary.org/obo/GO_0003680"}],"text":"Foxp3 Suppresses HIV-1 Gene Expression in Part through Blocking Activation of NF-κB\nIf Foxp3 functions as a repressor of NF-κB-dependent gene expression, then we hypothesized that Foxp3 overexpression could selectively down-regulate transcription from promoters previously shown to be responsive to NF-κB. To address this question, we examined the transcriptional activation of the HIV-1 LTR, which contains two tandem cis-acting NF-κB binding sites located between positions −102 and −81 with respect to the transcription initiation site [21]. NF-κB plays a crucial role in regulating gene expression directed from the HIV-1 LTR in CD4+ T cells [21]. Overexpression of full-length Foxp3, but not ΔFKH, in HEK 293T cells was able to inhibit basal activation of the HIV-1 LTR (Figure 3A), similar to what was previously demonstrated with the synthetic NF-κB reporter vector (Figure 2B). Furthermore, HIV-1 LTR activation was suppressed by full-length Foxp3 and ΔFKH in Jurkat T cells (Figure 3B). To demonstrate that Foxp3-mediated HIV-1 LTR repression was associated with interactions with NF-κB bound to the HIV-1 LTR, we compared basal activation of the HIV-1 LTR or an identical HIV-1 LTR lacking the NF-κB sites located between −102 and −81 (HIV-1 Δ-κB LTR) (Figure 3C). This mutant HIV-1 LTR construct exhibited reduced levels of transcription compared to the parental HIV-1 LTR in purified healthy donor CD4+ T cells (unpublished data). However, directly comparing the effect of Foxp3 overexpression on the activation of these two viral promoters demonstrated that Foxp3 was more capable of suppressing transcriptional activation of the HIV-1 LTR (Figure 3D) compared to the mutated HIV-1 LTR (Figure 3E). These results suggest that Foxp3 down-regulation of HIV-1 LTR activation was mediated at least in part by cis-acting NF-κB binding sites. Residual levels of inhibition of the HIV-1 Δ-κB LTR by Foxp3 may be due to NF-AT binding sites located upstream of the NF-κB sites within the HIV-1 LTR [22,23].\nFigure 3 Foxp3 Inhibits Basal Activation of the HIV-1 LTR\n(A and B) HEK 293T cells (5 × 105) (A) were seeded into six-well plates 1 d prior to transfection or Jurkat T cells (1 × 106) (B) were seeded into six-well plates on the day of transfection with an HIV-1 LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (1,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity.\n(C) Schematic representation of HIV-1 LTR luciferase reporter constructs pHIV-1 LTR-luc and pHIV-1 Δ-κB LTR-luc.\n(D and E) CD4+ T cells (2 × 106) from two healthy donors were nucleofected with either parental HIV-1 LTR (D) or HIV-1 LTR lacking NF-κB binding sites (at −102 to −81) (E) (1,000 ng), Foxp3 or control expression vectors (2,000 ng), and an internal reference plasmid (1,000 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity shown in A, B, D, and E was normalized to the internal reference control.\n\nT"}

GO-CC

{"project":"GO-CC","denotations":[{"id":"T6375","span":{"begin":640,"end":645},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T6376","span":{"begin":715,"end":720},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T6377","span":{"begin":977,"end":982},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T6378","span":{"begin":1417,"end":1422},"obj":"http://purl.obolibrary.org/obo/GO_0005623"}],"text":"Foxp3 Suppresses HIV-1 Gene Expression in Part through Blocking Activation of NF-κB\nIf Foxp3 functions as a repressor of NF-κB-dependent gene expression, then we hypothesized that Foxp3 overexpression could selectively down-regulate transcription from promoters previously shown to be responsive to NF-κB. To address this question, we examined the transcriptional activation of the HIV-1 LTR, which contains two tandem cis-acting NF-κB binding sites located between positions −102 and −81 with respect to the transcription initiation site [21]. NF-κB plays a crucial role in regulating gene expression directed from the HIV-1 LTR in CD4+ T cells [21]. Overexpression of full-length Foxp3, but not ΔFKH, in HEK 293T cells was able to inhibit basal activation of the HIV-1 LTR (Figure 3A), similar to what was previously demonstrated with the synthetic NF-κB reporter vector (Figure 2B). Furthermore, HIV-1 LTR activation was suppressed by full-length Foxp3 and ΔFKH in Jurkat T cells (Figure 3B). To demonstrate that Foxp3-mediated HIV-1 LTR repression was associated with interactions with NF-κB bound to the HIV-1 LTR, we compared basal activation of the HIV-1 LTR or an identical HIV-1 LTR lacking the NF-κB sites located between −102 and −81 (HIV-1 Δ-κB LTR) (Figure 3C). This mutant HIV-1 LTR construct exhibited reduced levels of transcription compared to the parental HIV-1 LTR in purified healthy donor CD4+ T cells (unpublished data). However, directly comparing the effect of Foxp3 overexpression on the activation of these two viral promoters demonstrated that Foxp3 was more capable of suppressing transcriptional activation of the HIV-1 LTR (Figure 3D) compared to the mutated HIV-1 LTR (Figure 3E). These results suggest that Foxp3 down-regulation of HIV-1 LTR activation was mediated at least in part by cis-acting NF-κB binding sites. Residual levels of inhibition of the HIV-1 Δ-κB LTR by Foxp3 may be due to NF-AT binding sites located upstream of the NF-κB sites within the HIV-1 LTR [22,23].\nFigure 3 Foxp3 Inhibits Basal Activation of the HIV-1 LTR\n(A and B) HEK 293T cells (5 × 105) (A) were seeded into six-well plates 1 d prior to transfection or Jurkat T cells (1 × 106) (B) were seeded into six-well plates on the day of transfection with an HIV-1 LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (1,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity.\n(C) Schematic representation of HIV-1 LTR luciferase reporter constructs pHIV-1 LTR-luc and pHIV-1 Δ-κB LTR-luc.\n(D and E) CD4+ T cells (2 × 106) from two healthy donors were nucleofected with either parental HIV-1 LTR (D) or HIV-1 LTR lacking NF-κB binding sites (at −102 to −81) (E) (1,000 ng), Foxp3 or control expression vectors (2,000 ng), and an internal reference plasmid (1,000 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity shown in A, B, D, and E was normalized to the internal reference control.\n\nT"}

events-check-again

{"project":"events-check-again","denotations":[{"id":"T6480","span":{"begin":0,"end":5},"obj":"Protein"},{"id":"T6481","span":{"begin":87,"end":92},"obj":"Protein"},{"id":"T6482","span":{"begin":180,"end":185},"obj":"Protein"},{"id":"T6483","span":{"begin":186,"end":200},"obj":"Positive_regulation"},{"id":"T6484","span":{"begin":633,"end":636},"obj":"Protein"},{"id":"T6485","span":{"begin":652,"end":666},"obj":"Positive_regulation"},{"id":"T6486","span":{"begin":652,"end":666},"obj":"Positive_regulation"},{"id":"T6487","span":{"begin":682,"end":687},"obj":"Protein"},{"id":"T6488","span":{"begin":697,"end":701},"obj":"Protein"},{"id":"T6489","span":{"begin":950,"end":955},"obj":"Protein"},{"id":"T6490","span":{"begin":960,"end":964},"obj":"Protein"},{"id":"T6491","span":{"begin":1016,"end":1021},"obj":"Protein"},{"id":"T6492","span":{"begin":1072,"end":1084},"obj":"Binding"},{"id":"T6493","span":{"begin":1410,"end":1413},"obj":"Protein"},{"id":"T6494","span":{"begin":1485,"end":1490},"obj":"Protein"},{"id":"T6495","span":{"begin":1491,"end":1505},"obj":"Positive_regulation"},{"id":"T6496","span":{"begin":1571,"end":1576},"obj":"Protein"},{"id":"T6497","span":{"begin":1739,"end":1744},"obj":"Protein"},{"id":"T6498","span":{"begin":1905,"end":1910},"obj":"Protein"}],"relations":[{"id":"R5064","pred":"themeOf","subj":"T6482","obj":"T6483"},{"id":"R5065","pred":"themeOf","subj":"T6487","obj":"T6486"},{"id":"R5066","pred":"themeOf","subj":"T6488","obj":"T6485"},{"id":"R5067","pred":"themeOf","subj":"T6491","obj":"T6492"},{"id":"R5068","pred":"themeOf","subj":"T6494","obj":"T6495"}],"attributes":[{"id":"M94","pred":"Speculation","subj":"T6492","obj":"true"}],"text":"Foxp3 Suppresses HIV-1 Gene Expression in Part through Blocking Activation of NF-κB\nIf Foxp3 functions as a repressor of NF-κB-dependent gene expression, then we hypothesized that Foxp3 overexpression could selectively down-regulate transcription from promoters previously shown to be responsive to NF-κB. To address this question, we examined the transcriptional activation of the HIV-1 LTR, which contains two tandem cis-acting NF-κB binding sites located between positions −102 and −81 with respect to the transcription initiation site [21]. NF-κB plays a crucial role in regulating gene expression directed from the HIV-1 LTR in CD4+ T cells [21]. Overexpression of full-length Foxp3, but not ΔFKH, in HEK 293T cells was able to inhibit basal activation of the HIV-1 LTR (Figure 3A), similar to what was previously demonstrated with the synthetic NF-κB reporter vector (Figure 2B). Furthermore, HIV-1 LTR activation was suppressed by full-length Foxp3 and ΔFKH in Jurkat T cells (Figure 3B). To demonstrate that Foxp3-mediated HIV-1 LTR repression was associated with interactions with NF-κB bound to the HIV-1 LTR, we compared basal activation of the HIV-1 LTR or an identical HIV-1 LTR lacking the NF-κB sites located between −102 and −81 (HIV-1 Δ-κB LTR) (Figure 3C). This mutant HIV-1 LTR construct exhibited reduced levels of transcription compared to the parental HIV-1 LTR in purified healthy donor CD4+ T cells (unpublished data). However, directly comparing the effect of Foxp3 overexpression on the activation of these two viral promoters demonstrated that Foxp3 was more capable of suppressing transcriptional activation of the HIV-1 LTR (Figure 3D) compared to the mutated HIV-1 LTR (Figure 3E). These results suggest that Foxp3 down-regulation of HIV-1 LTR activation was mediated at least in part by cis-acting NF-κB binding sites. Residual levels of inhibition of the HIV-1 Δ-κB LTR by Foxp3 may be due to NF-AT binding sites located upstream of the NF-κB sites within the HIV-1 LTR [22,23].\nFigure 3 Foxp3 Inhibits Basal Activation of the HIV-1 LTR\n(A and B) HEK 293T cells (5 × 105) (A) were seeded into six-well plates 1 d prior to transfection or Jurkat T cells (1 × 106) (B) were seeded into six-well plates on the day of transfection with an HIV-1 LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (1,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity.\n(C) Schematic representation of HIV-1 LTR luciferase reporter constructs pHIV-1 LTR-luc and pHIV-1 Δ-κB LTR-luc.\n(D and E) CD4+ T cells (2 × 106) from two healthy donors were nucleofected with either parental HIV-1 LTR (D) or HIV-1 LTR lacking NF-κB binding sites (at −102 to −81) (E) (1,000 ng), Foxp3 or control expression vectors (2,000 ng), and an internal reference plasmid (1,000 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity shown in A, B, D, and E was normalized to the internal reference control.\n\nT"}

bionlp-st-ge-2016-reference

bionlp-st-ge-2016-uniprot

{"project":"bionlp-st-ge-2016-uniprot","denotations":[{"id":"T6343","span":{"begin":0,"end":5},"obj":"Q9BZS1"},{"id":"T6344","span":{"begin":87,"end":92},"obj":"Q9BZS1"},{"id":"T6345","span":{"begin":180,"end":185},"obj":"Q9BZS1"},{"id":"T6346","span":{"begin":633,"end":636},"obj":"P01730"},{"id":"T6347","span":{"begin":682,"end":687},"obj":"Q9BZS1"},{"id":"T6348","span":{"begin":697,"end":701},"obj":"P55316"},{"id":"T6349","span":{"begin":725,"end":729},"obj":"P00519"},{"id":"T6350","span":{"begin":950,"end":955},"obj":"Q9BZS1"},{"id":"T6351","span":{"begin":960,"end":964},"obj":"P55316"},{"id":"T6352","span":{"begin":1016,"end":1021},"obj":"Q9BZS1"},{"id":"T6353","span":{"begin":1410,"end":1413},"obj":"P01730"},{"id":"T6354","span":{"begin":1485,"end":1490},"obj":"Q9BZS1"},{"id":"T6355","span":{"begin":1571,"end":1576},"obj":"Q9BZS1"},{"id":"T6356","span":{"begin":1739,"end":1744},"obj":"Q9BZS1"},{"id":"T6357","span":{"begin":1905,"end":1910},"obj":"Q9BZS1"}],"namespaces":[{"prefix":"_base","uri":"http://www.uniprot.org/uniprot/"}],"text":"Foxp3 Suppresses HIV-1 Gene Expression in Part through Blocking Activation of NF-κB\nIf Foxp3 functions as a repressor of NF-κB-dependent gene expression, then we hypothesized that Foxp3 overexpression could selectively down-regulate transcription from promoters previously shown to be responsive to NF-κB. To address this question, we examined the transcriptional activation of the HIV-1 LTR, which contains two tandem cis-acting NF-κB binding sites located between positions −102 and −81 with respect to the transcription initiation site [21]. NF-κB plays a crucial role in regulating gene expression directed from the HIV-1 LTR in CD4+ T cells [21]. Overexpression of full-length Foxp3, but not ΔFKH, in HEK 293T cells was able to inhibit basal activation of the HIV-1 LTR (Figure 3A), similar to what was previously demonstrated with the synthetic NF-κB reporter vector (Figure 2B). Furthermore, HIV-1 LTR activation was suppressed by full-length Foxp3 and ΔFKH in Jurkat T cells (Figure 3B). To demonstrate that Foxp3-mediated HIV-1 LTR repression was associated with interactions with NF-κB bound to the HIV-1 LTR, we compared basal activation of the HIV-1 LTR or an identical HIV-1 LTR lacking the NF-κB sites located between −102 and −81 (HIV-1 Δ-κB LTR) (Figure 3C). This mutant HIV-1 LTR construct exhibited reduced levels of transcription compared to the parental HIV-1 LTR in purified healthy donor CD4+ T cells (unpublished data). However, directly comparing the effect of Foxp3 overexpression on the activation of these two viral promoters demonstrated that Foxp3 was more capable of suppressing transcriptional activation of the HIV-1 LTR (Figure 3D) compared to the mutated HIV-1 LTR (Figure 3E). These results suggest that Foxp3 down-regulation of HIV-1 LTR activation was mediated at least in part by cis-acting NF-κB binding sites. Residual levels of inhibition of the HIV-1 Δ-κB LTR by Foxp3 may be due to NF-AT binding sites located upstream of the NF-κB sites within the HIV-1 LTR [22,23].\nFigure 3 Foxp3 Inhibits Basal Activation of the HIV-1 LTR\n(A and B) HEK 293T cells (5 × 105) (A) were seeded into six-well plates 1 d prior to transfection or Jurkat T cells (1 × 106) (B) were seeded into six-well plates on the day of transfection with an HIV-1 LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (1,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity.\n(C) Schematic representation of HIV-1 LTR luciferase reporter constructs pHIV-1 LTR-luc and pHIV-1 Δ-κB LTR-luc.\n(D and E) CD4+ T cells (2 × 106) from two healthy donors were nucleofected with either parental HIV-1 LTR (D) or HIV-1 LTR lacking NF-κB binding sites (at −102 to −81) (E) (1,000 ng), Foxp3 or control expression vectors (2,000 ng), and an internal reference plasmid (1,000 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity shown in A, B, D, and E was normalized to the internal reference control.\n\nT"}

test2

{"project":"test2","denotations":[{"id":"T5589","span":{"begin":0,"end":5},"obj":"Protein"},{"id":"T5590","span":{"begin":87,"end":92},"obj":"Protein"},{"id":"T5591","span":{"begin":180,"end":185},"obj":"Protein"},{"id":"T5592","span":{"begin":186,"end":200},"obj":"Positive_regulation"},{"id":"T5593","span":{"begin":219,"end":224},"obj":"Negative_regulation"},{"id":"T5594","span":{"begin":224,"end":232},"obj":"Positive_regulation"},{"id":"T5595","span":{"begin":633,"end":636},"obj":"Protein"},{"id":"T5596","span":{"begin":652,"end":666},"obj":"Positive_regulation"},{"id":"T5597","span":{"begin":682,"end":687},"obj":"Protein"},{"id":"T5598","span":{"begin":697,"end":701},"obj":"Protein"},{"id":"T5599","span":{"begin":950,"end":955},"obj":"Protein"},{"id":"T5600","span":{"begin":960,"end":964},"obj":"Protein"},{"id":"T5601","span":{"begin":1016,"end":1021},"obj":"Protein"},{"id":"T5602","span":{"begin":1410,"end":1413},"obj":"Protein"},{"id":"T5603","span":{"begin":1475,"end":1481},"obj":"Regulation"},{"id":"T5604","span":{"begin":1485,"end":1490},"obj":"Protein"},{"id":"T5605","span":{"begin":1491,"end":1505},"obj":"Positive_regulation"},{"id":"T5606","span":{"begin":1571,"end":1576},"obj":"Protein"},{"id":"T5607","span":{"begin":1739,"end":1744},"obj":"Protein"},{"id":"T5608","span":{"begin":1905,"end":1910},"obj":"Protein"}],"relations":[{"id":"R4325","pred":"themeOf","subj":"T5591","obj":"T5592"},{"id":"R4326","pred":"themeOf","subj":"T5597","obj":"T5596"},{"id":"R4327","pred":"themeOf","subj":"T5598","obj":"T5596"},{"id":"R4328","pred":"themeOf","subj":"T5604","obj":"T5605"}],"attributes":[{"id":"M87","pred":"Speculation","subj":"T5592","obj":"true"}],"text":"Foxp3 Suppresses HIV-1 Gene Expression in Part through Blocking Activation of NF-κB\nIf Foxp3 functions as a repressor of NF-κB-dependent gene expression, then we hypothesized that Foxp3 overexpression could selectively down-regulate transcription from promoters previously shown to be responsive to NF-κB. To address this question, we examined the transcriptional activation of the HIV-1 LTR, which contains two tandem cis-acting NF-κB binding sites located between positions −102 and −81 with respect to the transcription initiation site [21]. NF-κB plays a crucial role in regulating gene expression directed from the HIV-1 LTR in CD4+ T cells [21]. Overexpression of full-length Foxp3, but not ΔFKH, in HEK 293T cells was able to inhibit basal activation of the HIV-1 LTR (Figure 3A), similar to what was previously demonstrated with the synthetic NF-κB reporter vector (Figure 2B). Furthermore, HIV-1 LTR activation was suppressed by full-length Foxp3 and ΔFKH in Jurkat T cells (Figure 3B). To demonstrate that Foxp3-mediated HIV-1 LTR repression was associated with interactions with NF-κB bound to the HIV-1 LTR, we compared basal activation of the HIV-1 LTR or an identical HIV-1 LTR lacking the NF-κB sites located between −102 and −81 (HIV-1 Δ-κB LTR) (Figure 3C). This mutant HIV-1 LTR construct exhibited reduced levels of transcription compared to the parental HIV-1 LTR in purified healthy donor CD4+ T cells (unpublished data). However, directly comparing the effect of Foxp3 overexpression on the activation of these two viral promoters demonstrated that Foxp3 was more capable of suppressing transcriptional activation of the HIV-1 LTR (Figure 3D) compared to the mutated HIV-1 LTR (Figure 3E). These results suggest that Foxp3 down-regulation of HIV-1 LTR activation was mediated at least in part by cis-acting NF-κB binding sites. Residual levels of inhibition of the HIV-1 Δ-κB LTR by Foxp3 may be due to NF-AT binding sites located upstream of the NF-κB sites within the HIV-1 LTR [22,23].\nFigure 3 Foxp3 Inhibits Basal Activation of the HIV-1 LTR\n(A and B) HEK 293T cells (5 × 105) (A) were seeded into six-well plates 1 d prior to transfection or Jurkat T cells (1 × 106) (B) were seeded into six-well plates on the day of transfection with an HIV-1 LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (1,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity.\n(C) Schematic representation of HIV-1 LTR luciferase reporter constructs pHIV-1 LTR-luc and pHIV-1 Δ-κB LTR-luc.\n(D and E) CD4+ T cells (2 × 106) from two healthy donors were nucleofected with either parental HIV-1 LTR (D) or HIV-1 LTR lacking NF-κB binding sites (at −102 to −81) (E) (1,000 ng), Foxp3 or control expression vectors (2,000 ng), and an internal reference plasmid (1,000 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity shown in A, B, D, and E was normalized to the internal reference control.\n\nT"}

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