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2_test

{"project":"2_test","denotations":[{"id":"22187158-15203102-77584451","span":{"begin":2966,"end":2968},"obj":"15203102"},{"id":"22187158-10751368-77584452","span":{"begin":3084,"end":3086},"obj":"10751368"},{"id":"22187158-19596987-77584453","span":{"begin":3087,"end":3089},"obj":"19596987"},{"id":"22187158-8570619-77584454","span":{"begin":3152,"end":3154},"obj":"8570619"},{"id":"22187158-11602715-77584455","span":{"begin":3155,"end":3157},"obj":"11602715"},{"id":"22187158-10751368-77584456","span":{"begin":3176,"end":3178},"obj":"10751368"},{"id":"22187158-10964543-77584457","span":{"begin":3179,"end":3181},"obj":"10964543"},{"id":"22187158-20403075-77584458","span":{"begin":3182,"end":3184},"obj":"20403075"}],"text":"Tat activates the p65-dependent expression of NF-κB-responsive genes, occupies the NF-κB enhancers and promotes the p65 recruitment with IκB-α displacement\nWe analysed the action of Tat on the expression of NF-κB-responsive genes in vivo. To this end, HeLa cells were transfected with FLAG-Tat, FLAG-Tat C(22,25,27)A or FLAG-Tat R(49–57)A, and 48 h later the expression of a number of NF-κB-dependent genes was analysed by real-time PCR. Tat significantly increased the expression of MIP-1α, CSF3, LTA, NFKBIA and TLR2, while the mutants Tat C(22,25,27)A and Tat R(49–57)A were ineffective (Figure 5A–E). The Tat-dependent transactivation of the NF-κB-dependent genes was abolished when cells were transfected with siRNA p65, indicating that the Tat transcriptional activation was mediated by p65 (Figure 5A–E). Differently, the expression of the NF-κB-independent genes GAPDH and ACTB was unaffected by wild-type and Tat mutants (Figure 5F and G). These results were consistent with the requirement of both the cysteine-rich and arginine-rich domains of Tat to enhance the NF-κB activity, suggesting that the up-regulation of NF-κB-responsive genes by Tat might occur through physical interaction of the viral protein with p65 and IκB-α.\nFigure 5. Tat activates the p65-dependent expression of NF-κB-responsive genes by occupying the NF-κB enhancers and promoting the p65 recruitment with IκB-α displacement. HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, p3xFLAG-Tat C(22,25,27), p3xFLAG-Tat R(49–57) or empty vector (10 µg); for p65 RNA interference, cells (5 × 106) were transfected with p3xFLAG-Tat (10 µg) and siRNA p65 or siRNA control (200 pmol). Twenty-four-hour post-transfection, total RNA was extracted and analysed by real-time PCR to evaluate the expression of MIP-1α (A), CSF3 (B), LTA (C), NFKBIA (D), TLR2 (E), GAPDH (F), ACTB (G) genes. (H) HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, or empty vector (10 µg) and 48 h later ChIP was performed with anti-p65 antibody. Real-time PCR was performed with primers specific for the indicated promoters. (I) HeLa cells (5 × 106) were transfected with empty vector, or p3xFLAG-Tat (10 µg) in presence or absence of siRNA p65, or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-FLAG antibody. Real-time PCR was performed with primers specific for the indicated promoters. (J) HeLa cells (5 × 106) were transfected with empty vector, p3xFLAG-Tat (10 µg), or empty vector plus siRNA IκB-α or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-IκB-α antibody. Real-time PCR was performed with primers specific for the indicated promoters. Values (mean ± SE, n = 3) are shown. The asterisks indicate statistically significant differences compared to the control (empty vector) according to the Student's t-test (P \u003c 0.05).\nMIP-1α, which was the mostly activated gene by Tat, encodes for a chemokine that promotes the recruitment of pro-inflammatory cells (56). Consistently with our findings, MIP-1α expression was increased in glial cells and lymph nodes of AIDS patients (57,58); moreover, MIP-1α production was induced by HIV-1 infection (59,60) and Tat protein (57,61,62). The transcriptional regulation of MIP-1α has been poorly characterized. Jaspar-based analysis (http://jaspar.genereg.net/) predicted three putative NF-κB enhancers in the proximal promoter region of the MIP-1α gene: NF-κB1, −1023/−1014 nucleotides; NF-κB2, −661/−652 nucleotides; NF-κB3, +370/+379 nucleotides (Supplementary Figure S3A). To validate the predicted NF-κB binding sites of the MIP-1α promoter, we stimulated HeLa cells with TNF-α, a well-known NF-κB inducer, and measured the expression levels of MIP-1α by real-time PCR, and the p65 occupancy of the NF-κB sites by ChIP. TNF-α activated the expression of MIP-1α and induced the p65 recruitment to the NF-κB1 site of MIP-1α without affecting the occupancy of the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3B and S3C). Tat also promoted the recruitment of p65 to the NF-κB1 site of MIP-1α, while it did not affect the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3D). Altogether these results indicated that the NF-κB1 site was the only effective NF-κB enhancer of the MIP-1α promoter in response to TNF-α and Tat.\nSimilarly to MIP-1α, Tat increased the recruitment of p65 to the NF-κB enhancers of CSF3, LTA, NFKBIA and TLR2, while it was ineffective at the promoters of GAPDH and ACTB (Figure 5H). By ChIP, Tat bound to the NF-κB-responsive promoters, and not to the GAPDH and ACTB promoters, with loss of binding following p65 RNA interference (Figure 5I), suggesting that Tat occupancy occurred via p65 interaction. As additional findings, IκB-α was chromatin-immunoprecipitated at the NF-κB enhancers in absence of Tat, and it was significantly removed in presence of Tat, or following IκB-α RNA interference (Figure 5J), indicating that Tat displaced IκB-α from promoters."}

pmc-enju-pas

bionlp-st-ge-2016-coref

{"project":"bionlp-st-ge-2016-coref","denotations":[{"id":"T15820","span":{"begin":526,"end":537},"obj":"Anaphor"},{"id":"T15821","span":{"begin":538,"end":554},"obj":"Antecedent"},{"id":"T15822","span":{"begin":559,"end":572},"obj":"Antecedent"},{"id":"T15823","span":{"begin":1201,"end":1218},"obj":"Anaphor"},{"id":"T15824","span":{"begin":1153,"end":1156},"obj":"Antecedent"},{"id":"T15825","span":{"begin":2841,"end":2846},"obj":"Anaphor"},{"id":"T15826","span":{"begin":2833,"end":2839},"obj":"Antecedent"},{"id":"T15827","span":{"begin":4060,"end":4062},"obj":"Anaphor"},{"id":"T15828","span":{"begin":3983,"end":3986},"obj":"Antecedent"},{"id":"T15829","span":{"begin":4408,"end":4410},"obj":"Anaphor"},{"id":"T15830","span":{"begin":4311,"end":4314},"obj":"Antecedent"},{"id":"T15831","span":{"begin":4583,"end":4590},"obj":"Anaphor"},{"id":"T15832","span":{"begin":4488,"end":4493},"obj":"Antecedent"}],"relations":[{"id":"R12781","pred":"boundBy","subj":"T15820","obj":"T15821"},{"id":"R12782","pred":"boundBy","subj":"T15820","obj":"T15822"},{"id":"R12783","pred":"boundBy","subj":"T15823","obj":"T15824"},{"id":"R12784","pred":"boundBy","subj":"T15825","obj":"T15826"},{"id":"R12785","pred":"boundBy","subj":"T15827","obj":"T15828"},{"id":"R12786","pred":"boundBy","subj":"T15829","obj":"T15830"},{"id":"R12787","pred":"boundBy","subj":"T15831","obj":"T15832"}],"namespaces":[{"prefix":"_base","uri":"https://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"Tat activates the p65-dependent expression of NF-κB-responsive genes, occupies the NF-κB enhancers and promotes the p65 recruitment with IκB-α displacement\nWe analysed the action of Tat on the expression of NF-κB-responsive genes in vivo. To this end, HeLa cells were transfected with FLAG-Tat, FLAG-Tat C(22,25,27)A or FLAG-Tat R(49–57)A, and 48 h later the expression of a number of NF-κB-dependent genes was analysed by real-time PCR. Tat significantly increased the expression of MIP-1α, CSF3, LTA, NFKBIA and TLR2, while the mutants Tat C(22,25,27)A and Tat R(49–57)A were ineffective (Figure 5A–E). The Tat-dependent transactivation of the NF-κB-dependent genes was abolished when cells were transfected with siRNA p65, indicating that the Tat transcriptional activation was mediated by p65 (Figure 5A–E). Differently, the expression of the NF-κB-independent genes GAPDH and ACTB was unaffected by wild-type and Tat mutants (Figure 5F and G). These results were consistent with the requirement of both the cysteine-rich and arginine-rich domains of Tat to enhance the NF-κB activity, suggesting that the up-regulation of NF-κB-responsive genes by Tat might occur through physical interaction of the viral protein with p65 and IκB-α.\nFigure 5. Tat activates the p65-dependent expression of NF-κB-responsive genes by occupying the NF-κB enhancers and promoting the p65 recruitment with IκB-α displacement. HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, p3xFLAG-Tat C(22,25,27), p3xFLAG-Tat R(49–57) or empty vector (10 µg); for p65 RNA interference, cells (5 × 106) were transfected with p3xFLAG-Tat (10 µg) and siRNA p65 or siRNA control (200 pmol). Twenty-four-hour post-transfection, total RNA was extracted and analysed by real-time PCR to evaluate the expression of MIP-1α (A), CSF3 (B), LTA (C), NFKBIA (D), TLR2 (E), GAPDH (F), ACTB (G) genes. (H) HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, or empty vector (10 µg) and 48 h later ChIP was performed with anti-p65 antibody. Real-time PCR was performed with primers specific for the indicated promoters. (I) HeLa cells (5 × 106) were transfected with empty vector, or p3xFLAG-Tat (10 µg) in presence or absence of siRNA p65, or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-FLAG antibody. Real-time PCR was performed with primers specific for the indicated promoters. (J) HeLa cells (5 × 106) were transfected with empty vector, p3xFLAG-Tat (10 µg), or empty vector plus siRNA IκB-α or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-IκB-α antibody. Real-time PCR was performed with primers specific for the indicated promoters. Values (mean ± SE, n = 3) are shown. The asterisks indicate statistically significant differences compared to the control (empty vector) according to the Student's t-test (P \u003c 0.05).\nMIP-1α, which was the mostly activated gene by Tat, encodes for a chemokine that promotes the recruitment of pro-inflammatory cells (56). Consistently with our findings, MIP-1α expression was increased in glial cells and lymph nodes of AIDS patients (57,58); moreover, MIP-1α production was induced by HIV-1 infection (59,60) and Tat protein (57,61,62). The transcriptional regulation of MIP-1α has been poorly characterized. Jaspar-based analysis (http://jaspar.genereg.net/) predicted three putative NF-κB enhancers in the proximal promoter region of the MIP-1α gene: NF-κB1, −1023/−1014 nucleotides; NF-κB2, −661/−652 nucleotides; NF-κB3, +370/+379 nucleotides (Supplementary Figure S3A). To validate the predicted NF-κB binding sites of the MIP-1α promoter, we stimulated HeLa cells with TNF-α, a well-known NF-κB inducer, and measured the expression levels of MIP-1α by real-time PCR, and the p65 occupancy of the NF-κB sites by ChIP. TNF-α activated the expression of MIP-1α and induced the p65 recruitment to the NF-κB1 site of MIP-1α without affecting the occupancy of the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3B and S3C). Tat also promoted the recruitment of p65 to the NF-κB1 site of MIP-1α, while it did not affect the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3D). Altogether these results indicated that the NF-κB1 site was the only effective NF-κB enhancer of the MIP-1α promoter in response to TNF-α and Tat.\nSimilarly to MIP-1α, Tat increased the recruitment of p65 to the NF-κB enhancers of CSF3, LTA, NFKBIA and TLR2, while it was ineffective at the promoters of GAPDH and ACTB (Figure 5H). By ChIP, Tat bound to the NF-κB-responsive promoters, and not to the GAPDH and ACTB promoters, with loss of binding following p65 RNA interference (Figure 5I), suggesting that Tat occupancy occurred via p65 interaction. As additional findings, IκB-α was chromatin-immunoprecipitated at the NF-κB enhancers in absence of Tat, and it was significantly removed in presence of Tat, or following IκB-α RNA interference (Figure 5J), indicating that Tat displaced IκB-α from promoters."}

bionlp-st-ge-2016-spacy-parsed

UBERON-AE

{"project":"UBERON-AE","denotations":[{"id":"T15077","span":{"begin":3054,"end":3059},"obj":"http://purl.obolibrary.org/obo/UBERON_0002391"},{"id":"T15078","span":{"begin":3054,"end":3065},"obj":"http://purl.obolibrary.org/obo/UBERON_0000029"}],"text":"Tat activates the p65-dependent expression of NF-κB-responsive genes, occupies the NF-κB enhancers and promotes the p65 recruitment with IκB-α displacement\nWe analysed the action of Tat on the expression of NF-κB-responsive genes in vivo. To this end, HeLa cells were transfected with FLAG-Tat, FLAG-Tat C(22,25,27)A or FLAG-Tat R(49–57)A, and 48 h later the expression of a number of NF-κB-dependent genes was analysed by real-time PCR. Tat significantly increased the expression of MIP-1α, CSF3, LTA, NFKBIA and TLR2, while the mutants Tat C(22,25,27)A and Tat R(49–57)A were ineffective (Figure 5A–E). The Tat-dependent transactivation of the NF-κB-dependent genes was abolished when cells were transfected with siRNA p65, indicating that the Tat transcriptional activation was mediated by p65 (Figure 5A–E). Differently, the expression of the NF-κB-independent genes GAPDH and ACTB was unaffected by wild-type and Tat mutants (Figure 5F and G). These results were consistent with the requirement of both the cysteine-rich and arginine-rich domains of Tat to enhance the NF-κB activity, suggesting that the up-regulation of NF-κB-responsive genes by Tat might occur through physical interaction of the viral protein with p65 and IκB-α.\nFigure 5. Tat activates the p65-dependent expression of NF-κB-responsive genes by occupying the NF-κB enhancers and promoting the p65 recruitment with IκB-α displacement. HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, p3xFLAG-Tat C(22,25,27), p3xFLAG-Tat R(49–57) or empty vector (10 µg); for p65 RNA interference, cells (5 × 106) were transfected with p3xFLAG-Tat (10 µg) and siRNA p65 or siRNA control (200 pmol). Twenty-four-hour post-transfection, total RNA was extracted and analysed by real-time PCR to evaluate the expression of MIP-1α (A), CSF3 (B), LTA (C), NFKBIA (D), TLR2 (E), GAPDH (F), ACTB (G) genes. (H) HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, or empty vector (10 µg) and 48 h later ChIP was performed with anti-p65 antibody. Real-time PCR was performed with primers specific for the indicated promoters. (I) HeLa cells (5 × 106) were transfected with empty vector, or p3xFLAG-Tat (10 µg) in presence or absence of siRNA p65, or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-FLAG antibody. Real-time PCR was performed with primers specific for the indicated promoters. (J) HeLa cells (5 × 106) were transfected with empty vector, p3xFLAG-Tat (10 µg), or empty vector plus siRNA IκB-α or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-IκB-α antibody. Real-time PCR was performed with primers specific for the indicated promoters. Values (mean ± SE, n = 3) are shown. The asterisks indicate statistically significant differences compared to the control (empty vector) according to the Student's t-test (P \u003c 0.05).\nMIP-1α, which was the mostly activated gene by Tat, encodes for a chemokine that promotes the recruitment of pro-inflammatory cells (56). Consistently with our findings, MIP-1α expression was increased in glial cells and lymph nodes of AIDS patients (57,58); moreover, MIP-1α production was induced by HIV-1 infection (59,60) and Tat protein (57,61,62). The transcriptional regulation of MIP-1α has been poorly characterized. Jaspar-based analysis (http://jaspar.genereg.net/) predicted three putative NF-κB enhancers in the proximal promoter region of the MIP-1α gene: NF-κB1, −1023/−1014 nucleotides; NF-κB2, −661/−652 nucleotides; NF-κB3, +370/+379 nucleotides (Supplementary Figure S3A). To validate the predicted NF-κB binding sites of the MIP-1α promoter, we stimulated HeLa cells with TNF-α, a well-known NF-κB inducer, and measured the expression levels of MIP-1α by real-time PCR, and the p65 occupancy of the NF-κB sites by ChIP. TNF-α activated the expression of MIP-1α and induced the p65 recruitment to the NF-κB1 site of MIP-1α without affecting the occupancy of the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3B and S3C). Tat also promoted the recruitment of p65 to the NF-κB1 site of MIP-1α, while it did not affect the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3D). Altogether these results indicated that the NF-κB1 site was the only effective NF-κB enhancer of the MIP-1α promoter in response to TNF-α and Tat.\nSimilarly to MIP-1α, Tat increased the recruitment of p65 to the NF-κB enhancers of CSF3, LTA, NFKBIA and TLR2, while it was ineffective at the promoters of GAPDH and ACTB (Figure 5H). By ChIP, Tat bound to the NF-κB-responsive promoters, and not to the GAPDH and ACTB promoters, with loss of binding following p65 RNA interference (Figure 5I), suggesting that Tat occupancy occurred via p65 interaction. As additional findings, IκB-α was chromatin-immunoprecipitated at the NF-κB enhancers in absence of Tat, and it was significantly removed in presence of Tat, or following IκB-α RNA interference (Figure 5J), indicating that Tat displaced IκB-α from promoters."}

GO-BP

{"project":"GO-BP","denotations":[{"id":"T16611","span":{"begin":623,"end":638},"obj":"http://purl.obolibrary.org/obo/GO_2000144"},{"id":"T16612","span":{"begin":750,"end":765},"obj":"http://purl.obolibrary.org/obo/GO_0006351"},{"id":"T16613","span":{"begin":3191,"end":3206},"obj":"http://purl.obolibrary.org/obo/GO_0006351"},{"id":"T16614","span":{"begin":750,"end":789},"obj":"http://purl.obolibrary.org/obo/GO_1900622"},{"id":"T16615","span":{"begin":1113,"end":1123},"obj":"http://purl.obolibrary.org/obo/GO_0065007"},{"id":"T16616","span":{"begin":3207,"end":3217},"obj":"http://purl.obolibrary.org/obo/GO_0065007"},{"id":"T16617","span":{"begin":3773,"end":3788},"obj":"http://purl.obolibrary.org/obo/GO_1904469"},{"id":"T16618","span":{"begin":4263,"end":4278},"obj":"http://purl.obolibrary.org/obo/GO_0034612"},{"id":"T16619","span":{"begin":4605,"end":4621},"obj":"http://purl.obolibrary.org/obo/GO_0016246"},{"id":"T16620","span":{"begin":4872,"end":4888},"obj":"http://purl.obolibrary.org/obo/GO_0016246"},{"id":"T27981","span":{"begin":1545,"end":1561},"obj":"http://purl.obolibrary.org/obo/GO_0016246"}],"text":"Tat activates the p65-dependent expression of NF-κB-responsive genes, occupies the NF-κB enhancers and promotes the p65 recruitment with IκB-α displacement\nWe analysed the action of Tat on the expression of NF-κB-responsive genes in vivo. To this end, HeLa cells were transfected with FLAG-Tat, FLAG-Tat C(22,25,27)A or FLAG-Tat R(49–57)A, and 48 h later the expression of a number of NF-κB-dependent genes was analysed by real-time PCR. Tat significantly increased the expression of MIP-1α, CSF3, LTA, NFKBIA and TLR2, while the mutants Tat C(22,25,27)A and Tat R(49–57)A were ineffective (Figure 5A–E). The Tat-dependent transactivation of the NF-κB-dependent genes was abolished when cells were transfected with siRNA p65, indicating that the Tat transcriptional activation was mediated by p65 (Figure 5A–E). Differently, the expression of the NF-κB-independent genes GAPDH and ACTB was unaffected by wild-type and Tat mutants (Figure 5F and G). These results were consistent with the requirement of both the cysteine-rich and arginine-rich domains of Tat to enhance the NF-κB activity, suggesting that the up-regulation of NF-κB-responsive genes by Tat might occur through physical interaction of the viral protein with p65 and IκB-α.\nFigure 5. Tat activates the p65-dependent expression of NF-κB-responsive genes by occupying the NF-κB enhancers and promoting the p65 recruitment with IκB-α displacement. HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, p3xFLAG-Tat C(22,25,27), p3xFLAG-Tat R(49–57) or empty vector (10 µg); for p65 RNA interference, cells (5 × 106) were transfected with p3xFLAG-Tat (10 µg) and siRNA p65 or siRNA control (200 pmol). Twenty-four-hour post-transfection, total RNA was extracted and analysed by real-time PCR to evaluate the expression of MIP-1α (A), CSF3 (B), LTA (C), NFKBIA (D), TLR2 (E), GAPDH (F), ACTB (G) genes. (H) HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, or empty vector (10 µg) and 48 h later ChIP was performed with anti-p65 antibody. Real-time PCR was performed with primers specific for the indicated promoters. (I) HeLa cells (5 × 106) were transfected with empty vector, or p3xFLAG-Tat (10 µg) in presence or absence of siRNA p65, or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-FLAG antibody. Real-time PCR was performed with primers specific for the indicated promoters. (J) HeLa cells (5 × 106) were transfected with empty vector, p3xFLAG-Tat (10 µg), or empty vector plus siRNA IκB-α or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-IκB-α antibody. Real-time PCR was performed with primers specific for the indicated promoters. Values (mean ± SE, n = 3) are shown. The asterisks indicate statistically significant differences compared to the control (empty vector) according to the Student's t-test (P \u003c 0.05).\nMIP-1α, which was the mostly activated gene by Tat, encodes for a chemokine that promotes the recruitment of pro-inflammatory cells (56). Consistently with our findings, MIP-1α expression was increased in glial cells and lymph nodes of AIDS patients (57,58); moreover, MIP-1α production was induced by HIV-1 infection (59,60) and Tat protein (57,61,62). The transcriptional regulation of MIP-1α has been poorly characterized. Jaspar-based analysis (http://jaspar.genereg.net/) predicted three putative NF-κB enhancers in the proximal promoter region of the MIP-1α gene: NF-κB1, −1023/−1014 nucleotides; NF-κB2, −661/−652 nucleotides; NF-κB3, +370/+379 nucleotides (Supplementary Figure S3A). To validate the predicted NF-κB binding sites of the MIP-1α promoter, we stimulated HeLa cells with TNF-α, a well-known NF-κB inducer, and measured the expression levels of MIP-1α by real-time PCR, and the p65 occupancy of the NF-κB sites by ChIP. TNF-α activated the expression of MIP-1α and induced the p65 recruitment to the NF-κB1 site of MIP-1α without affecting the occupancy of the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3B and S3C). Tat also promoted the recruitment of p65 to the NF-κB1 site of MIP-1α, while it did not affect the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3D). Altogether these results indicated that the NF-κB1 site was the only effective NF-κB enhancer of the MIP-1α promoter in response to TNF-α and Tat.\nSimilarly to MIP-1α, Tat increased the recruitment of p65 to the NF-κB enhancers of CSF3, LTA, NFKBIA and TLR2, while it was ineffective at the promoters of GAPDH and ACTB (Figure 5H). By ChIP, Tat bound to the NF-κB-responsive promoters, and not to the GAPDH and ACTB promoters, with loss of binding following p65 RNA interference (Figure 5I), suggesting that Tat occupancy occurred via p65 interaction. As additional findings, IκB-α was chromatin-immunoprecipitated at the NF-κB enhancers in absence of Tat, and it was significantly removed in presence of Tat, or following IκB-α RNA interference (Figure 5J), indicating that Tat displaced IκB-α from promoters."}

GO-MF

{"project":"GO-MF","denotations":[{"id":"T16621","span":{"begin":3551,"end":3564},"obj":"http://purl.obolibrary.org/obo/GO_0051059"},{"id":"T16622","span":{"begin":3557,"end":3564},"obj":"http://purl.obolibrary.org/obo/GO_0005488"},{"id":"T16623","span":{"begin":4583,"end":4590},"obj":"http://purl.obolibrary.org/obo/GO_0005488"},{"id":"T27982","span":{"begin":1996,"end":2004},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T27983","span":{"begin":2281,"end":2289},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T27984","span":{"begin":2561,"end":2569},"obj":"http://purl.obolibrary.org/obo/GO_0003823"}],"text":"Tat activates the p65-dependent expression of NF-κB-responsive genes, occupies the NF-κB enhancers and promotes the p65 recruitment with IκB-α displacement\nWe analysed the action of Tat on the expression of NF-κB-responsive genes in vivo. To this end, HeLa cells were transfected with FLAG-Tat, FLAG-Tat C(22,25,27)A or FLAG-Tat R(49–57)A, and 48 h later the expression of a number of NF-κB-dependent genes was analysed by real-time PCR. Tat significantly increased the expression of MIP-1α, CSF3, LTA, NFKBIA and TLR2, while the mutants Tat C(22,25,27)A and Tat R(49–57)A were ineffective (Figure 5A–E). The Tat-dependent transactivation of the NF-κB-dependent genes was abolished when cells were transfected with siRNA p65, indicating that the Tat transcriptional activation was mediated by p65 (Figure 5A–E). Differently, the expression of the NF-κB-independent genes GAPDH and ACTB was unaffected by wild-type and Tat mutants (Figure 5F and G). These results were consistent with the requirement of both the cysteine-rich and arginine-rich domains of Tat to enhance the NF-κB activity, suggesting that the up-regulation of NF-κB-responsive genes by Tat might occur through physical interaction of the viral protein with p65 and IκB-α.\nFigure 5. Tat activates the p65-dependent expression of NF-κB-responsive genes by occupying the NF-κB enhancers and promoting the p65 recruitment with IκB-α displacement. HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, p3xFLAG-Tat C(22,25,27), p3xFLAG-Tat R(49–57) or empty vector (10 µg); for p65 RNA interference, cells (5 × 106) were transfected with p3xFLAG-Tat (10 µg) and siRNA p65 or siRNA control (200 pmol). Twenty-four-hour post-transfection, total RNA was extracted and analysed by real-time PCR to evaluate the expression of MIP-1α (A), CSF3 (B), LTA (C), NFKBIA (D), TLR2 (E), GAPDH (F), ACTB (G) genes. (H) HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, or empty vector (10 µg) and 48 h later ChIP was performed with anti-p65 antibody. Real-time PCR was performed with primers specific for the indicated promoters. (I) HeLa cells (5 × 106) were transfected with empty vector, or p3xFLAG-Tat (10 µg) in presence or absence of siRNA p65, or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-FLAG antibody. Real-time PCR was performed with primers specific for the indicated promoters. (J) HeLa cells (5 × 106) were transfected with empty vector, p3xFLAG-Tat (10 µg), or empty vector plus siRNA IκB-α or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-IκB-α antibody. Real-time PCR was performed with primers specific for the indicated promoters. Values (mean ± SE, n = 3) are shown. The asterisks indicate statistically significant differences compared to the control (empty vector) according to the Student's t-test (P \u003c 0.05).\nMIP-1α, which was the mostly activated gene by Tat, encodes for a chemokine that promotes the recruitment of pro-inflammatory cells (56). Consistently with our findings, MIP-1α expression was increased in glial cells and lymph nodes of AIDS patients (57,58); moreover, MIP-1α production was induced by HIV-1 infection (59,60) and Tat protein (57,61,62). The transcriptional regulation of MIP-1α has been poorly characterized. Jaspar-based analysis (http://jaspar.genereg.net/) predicted three putative NF-κB enhancers in the proximal promoter region of the MIP-1α gene: NF-κB1, −1023/−1014 nucleotides; NF-κB2, −661/−652 nucleotides; NF-κB3, +370/+379 nucleotides (Supplementary Figure S3A). To validate the predicted NF-κB binding sites of the MIP-1α promoter, we stimulated HeLa cells with TNF-α, a well-known NF-κB inducer, and measured the expression levels of MIP-1α by real-time PCR, and the p65 occupancy of the NF-κB sites by ChIP. TNF-α activated the expression of MIP-1α and induced the p65 recruitment to the NF-κB1 site of MIP-1α without affecting the occupancy of the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3B and S3C). Tat also promoted the recruitment of p65 to the NF-κB1 site of MIP-1α, while it did not affect the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3D). Altogether these results indicated that the NF-κB1 site was the only effective NF-κB enhancer of the MIP-1α promoter in response to TNF-α and Tat.\nSimilarly to MIP-1α, Tat increased the recruitment of p65 to the NF-κB enhancers of CSF3, LTA, NFKBIA and TLR2, while it was ineffective at the promoters of GAPDH and ACTB (Figure 5H). By ChIP, Tat bound to the NF-κB-responsive promoters, and not to the GAPDH and ACTB promoters, with loss of binding following p65 RNA interference (Figure 5I), suggesting that Tat occupancy occurred via p65 interaction. As additional findings, IκB-α was chromatin-immunoprecipitated at the NF-κB enhancers in absence of Tat, and it was significantly removed in presence of Tat, or following IκB-α RNA interference (Figure 5J), indicating that Tat displaced IκB-α from promoters."}

GO-CC

{"project":"GO-CC","denotations":[{"id":"T16624","span":{"begin":257,"end":262},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16625","span":{"begin":687,"end":692},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16626","span":{"begin":2959,"end":2964},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16627","span":{"begin":3044,"end":3049},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16628","span":{"begin":3614,"end":3619},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T16629","span":{"begin":4729,"end":4738},"obj":"http://purl.obolibrary.org/obo/GO_0000785"},{"id":"T27972","span":{"begin":1873,"end":1878},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T27973","span":{"begin":2094,"end":2099},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T27974","span":{"begin":2379,"end":2384},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T27975","span":{"begin":1996,"end":2004},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T27976","span":{"begin":2281,"end":2289},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T27977","span":{"begin":2561,"end":2569},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T27978","span":{"begin":1996,"end":2004},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T27979","span":{"begin":2281,"end":2289},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T27980","span":{"begin":2561,"end":2569},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T27970","span":{"begin":1415,"end":1420},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T27971","span":{"begin":1563,"end":1568},"obj":"http://purl.obolibrary.org/obo/GO_0005623"}],"text":"Tat activates the p65-dependent expression of NF-κB-responsive genes, occupies the NF-κB enhancers and promotes the p65 recruitment with IκB-α displacement\nWe analysed the action of Tat on the expression of NF-κB-responsive genes in vivo. To this end, HeLa cells were transfected with FLAG-Tat, FLAG-Tat C(22,25,27)A or FLAG-Tat R(49–57)A, and 48 h later the expression of a number of NF-κB-dependent genes was analysed by real-time PCR. Tat significantly increased the expression of MIP-1α, CSF3, LTA, NFKBIA and TLR2, while the mutants Tat C(22,25,27)A and Tat R(49–57)A were ineffective (Figure 5A–E). The Tat-dependent transactivation of the NF-κB-dependent genes was abolished when cells were transfected with siRNA p65, indicating that the Tat transcriptional activation was mediated by p65 (Figure 5A–E). Differently, the expression of the NF-κB-independent genes GAPDH and ACTB was unaffected by wild-type and Tat mutants (Figure 5F and G). These results were consistent with the requirement of both the cysteine-rich and arginine-rich domains of Tat to enhance the NF-κB activity, suggesting that the up-regulation of NF-κB-responsive genes by Tat might occur through physical interaction of the viral protein with p65 and IκB-α.\nFigure 5. Tat activates the p65-dependent expression of NF-κB-responsive genes by occupying the NF-κB enhancers and promoting the p65 recruitment with IκB-α displacement. HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, p3xFLAG-Tat C(22,25,27), p3xFLAG-Tat R(49–57) or empty vector (10 µg); for p65 RNA interference, cells (5 × 106) were transfected with p3xFLAG-Tat (10 µg) and siRNA p65 or siRNA control (200 pmol). Twenty-four-hour post-transfection, total RNA was extracted and analysed by real-time PCR to evaluate the expression of MIP-1α (A), CSF3 (B), LTA (C), NFKBIA (D), TLR2 (E), GAPDH (F), ACTB (G) genes. (H) HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, or empty vector (10 µg) and 48 h later ChIP was performed with anti-p65 antibody. Real-time PCR was performed with primers specific for the indicated promoters. (I) HeLa cells (5 × 106) were transfected with empty vector, or p3xFLAG-Tat (10 µg) in presence or absence of siRNA p65, or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-FLAG antibody. Real-time PCR was performed with primers specific for the indicated promoters. (J) HeLa cells (5 × 106) were transfected with empty vector, p3xFLAG-Tat (10 µg), or empty vector plus siRNA IκB-α or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-IκB-α antibody. Real-time PCR was performed with primers specific for the indicated promoters. Values (mean ± SE, n = 3) are shown. The asterisks indicate statistically significant differences compared to the control (empty vector) according to the Student's t-test (P \u003c 0.05).\nMIP-1α, which was the mostly activated gene by Tat, encodes for a chemokine that promotes the recruitment of pro-inflammatory cells (56). Consistently with our findings, MIP-1α expression was increased in glial cells and lymph nodes of AIDS patients (57,58); moreover, MIP-1α production was induced by HIV-1 infection (59,60) and Tat protein (57,61,62). The transcriptional regulation of MIP-1α has been poorly characterized. Jaspar-based analysis (http://jaspar.genereg.net/) predicted three putative NF-κB enhancers in the proximal promoter region of the MIP-1α gene: NF-κB1, −1023/−1014 nucleotides; NF-κB2, −661/−652 nucleotides; NF-κB3, +370/+379 nucleotides (Supplementary Figure S3A). To validate the predicted NF-κB binding sites of the MIP-1α promoter, we stimulated HeLa cells with TNF-α, a well-known NF-κB inducer, and measured the expression levels of MIP-1α by real-time PCR, and the p65 occupancy of the NF-κB sites by ChIP. TNF-α activated the expression of MIP-1α and induced the p65 recruitment to the NF-κB1 site of MIP-1α without affecting the occupancy of the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3B and S3C). Tat also promoted the recruitment of p65 to the NF-κB1 site of MIP-1α, while it did not affect the putative NF-κB2 and NF-κB3 sites (Supplementary Figure S3D). Altogether these results indicated that the NF-κB1 site was the only effective NF-κB enhancer of the MIP-1α promoter in response to TNF-α and Tat.\nSimilarly to MIP-1α, Tat increased the recruitment of p65 to the NF-κB enhancers of CSF3, LTA, NFKBIA and TLR2, while it was ineffective at the promoters of GAPDH and ACTB (Figure 5H). By ChIP, Tat bound to the NF-κB-responsive promoters, and not to the GAPDH and ACTB promoters, with loss of binding following p65 RNA interference (Figure 5I), suggesting that Tat occupancy occurred via p65 interaction. As additional findings, IκB-α was chromatin-immunoprecipitated at the NF-κB enhancers in absence of Tat, and it was significantly removed in presence of Tat, or following IκB-α RNA interference (Figure 5J), indicating that Tat displaced IκB-α from promoters."}

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events-check-again

bionlp-st-ge-2016-reference-tees

test2

bionlp-st-ge-2016-reference

bionlp-st-ge-2016-uniprot

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