Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: HIV-1 Tat interacts directly with the human Ssu72 CTD phosphatase. ( A ) MudPIT analysis of affinity-purified HA-Tat101-associated protein complexes from a stable Tet-OFF/Tat-ON HeLa cell line. ( B ) Immunoblot analysis of anti-HA immunoprecipitates from HA-Tat HeLa stable cells with the indicated antibodies. ( C ) Immunoblot analysis of anti-Tat serum immunoprecipitates from 2D10 Jurkat cells before and after TNF-α treatment. ( D ) Recombinant S-tagged Ssu72 and S-tagged CycT1 (amino acids 1–303) were incubated with recombinant GST-Tat101. The recovered pull-down products (PD) were detected by immunoblot analysis with anti-GST antibody (to detect GST and GST-Tat proteins) and HRP-conjugated S-protein (to detect Ssu72 and Cyclin T1). ( E ) The pull-down assays were performed as in D for S-tagged Ssu72, with indicated GST-Tat fragments. The amounts of GST and GST-Tat proteins were monitored by Ponceau staining (PS). The regions of Tat binding to Ssu72 are shown in the schematic diagram at the bottom of the panel. ( F ) Recombinant full-length and truncated GST-Ssu72 were incubated with recombinant Flag-Tat101 (GST was cleaved after purification). The recovered pull-down products were detected by immunoblot with anti-Flag (to detect Flag-Tat101) and anti-GST (to detect GST and GST-Ssu72) antibodies. Arrowheads indicate GST-Ssu72 fusion proteins. ( G ) HA-Tat HeLa stable cells were transfected with Flag-tagged full-length or coiled-coil domain truncated (∆CC) Ssu72. The immunoprecipitation experiments were performed as in B . The region of Ssu72 binding to Tat is shown in the schematic diagram at the bottom of the panel. ( H ) Recombinant GST-CycT1 (amino acids 1–303) was incubated with recombinant S-tagged Ssu72 in the absence or presence of recombinant Flag-Tat101. The pull-down products were detected by immunoblot with the indicated antibodies.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: Affinity Purification, Western Blot, Recombinant, Incubation, Staining, Binding Assay, Purification, Transfection, Immunoprecipitation

Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: Ssu72 synergizes with Tat to activate HIV-1 transcription. ( A ) Luciferase activities were measured in extracts of HIV-1:Luc HeLa cells transfected with the indicated constructs. The protein expression was monitored by immunoblot analysis. ( B ) Luciferase activities were measured in extracts transfected with the indicated amounts of Ssu72 expression constructs in the presence or absence of Tat. The protein lysates were analyzed by immunoblot with the indicated antibodies. ( C ) Luciferase assays were performed as in B to compare the activity of wild type or catalytically inactive mutants (C12S and D143A) of Ssu72. ( D ) Immunoblot analysis of anti-HA immunoprecipitates from HA-Tat HeLa stable cells transfected with Flag-tagged Ssu72 (wild type, C12S, and D143A). ( E ) Luciferase assays were performed as in B to analyze the activity of full-length Ssu72 and the Ssu72∆CC mutant. ( F ) Wild-type Ssu72 or Ssu72 C12S expression constructs were transfected in the HIV-1:Luc HeLa cells in the absence or presence of Tat. Nuclear run-on analysis was performed as described in the Materials and Methods. Error bars represent the standard deviation obtained from three independent experiments.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: Luciferase, Transfection, Construct, Expressing, Western Blot, Activity Assay, Mutagenesis, Standard Deviation

Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: Tat stimulates Ssu72 CTD phosphatase activity in vitro. ( A ) TFIIH-phosphorylated GST-CTD was incubated with the indicated amounts of recombinant human GST-Ssu72 or catalytically inactive GST-Ssu72 (C12S) in the presence of GST or GST-Tat86. The phosphatase assay products were detected with the indicated antibodies by immunoblot analysis. ( B ) Kinetics of Tat-stimulated Ssu72 phosphatase activity were monitored for the indicated times. The intensity of the S5P bands was quantitated and plotted. The half-time for reduction of S5P levels in the absence or presence of GST-Tat86 was calculated as described in the Supplemental Material. ( C ) The phosphatase assays were performed as in A except that the indicated amounts of GST-Tat86 were included. ( D ) In vitro purified recombinant S-tagged Ssu72 was incubated with GST, wild-type GST-Tat86, or mutant GST-Tat86 proteins as indicated. The recovered pull-down products were subjected to immunoblot analysis with anti-GST (to detect GST or GST-Tat86) or anti-Ssu72 antibodies. The amounts of GST and GST-Tat86 proteins were monitored by Commassie staining. ( E ) The phosphatase assays were performed as in A for GST-Ssu72 except that the wild-type or mutant GST-Tat86 proteins were used, as indicated.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: Activity Assay, In Vitro, Incubation, Recombinant, Phosphatase Assay, Western Blot, Purification, Mutagenesis, Staining

Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: Ssu72 is essential for Tat:P-TEFb to phosphorylate the S5P-CTD in vitro. ( A ) The GST-CTD (52 repeats) substrate was incubated with indicated amounts of P-TEFb in the absence or presence of Tat (GST-Tat86). CTD phosphorylation was monitored by immunoblot with the indicated antibodies. ( B ) In vitro phosphorylation of the TFIIH:CDK7-phosphorylated GST-CTD substrate by recombinant Tat:P-TEFb complexes was monitored in the presence or absence of recombinant Ssu72, as indicated. The experimental procedure is outlined above the panel, and CTD phosphorylation was monitored by immunoblot with the indicated antibodies. Note that each reaction contained identical levels of the S5P-CTD substrate.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: In Vitro, Incubation, Phospho-proteomics, Western Blot, Recombinant

Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: Ssu72 promotes Tat transactivation and regulates S5P-CTD levels at the HIV-1 promoter in vivo. ( A ) Luciferase activities were measured in extracts of HIV-1:Luc HeLa cells transfected with different siRNAs against Ssu72, Cyclin T1, or AFF1 in the presence or absence of Tat101. The efficiency of siRNA-mediated knockdown was monitored by immunoblot. ( B ) HIV-1:Luc HeLa cells were transfected with siRNAs specific for Ssu72, RPAP2, or SCP1 in the presence or absence of Tat, as indicated. Luciferase assays were performed as in A . ( C ) HIV-1:Luc HeLa cells were first transfected with control or Ssu72 siRNAs for 24 h and then were transfected with 5 µg of EGFP or EGFP-Tat101 for another 24 h. The cell extracts were subjected to ChIP analysis with the indicated antibodies. Error bars represent the standard deviation obtained from three independent experiments. Protein lysates were monitored by immunoblot with the indicated antibodies.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: In Vivo, Luciferase, Transfection, Knockdown, Western Blot, Control, Standard Deviation

Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: Tat recruits Ssu72 to the integrated HIV-1 proviral promoter in 2D10 T cells. ( A ) Immunoblot analysis of Tat and cellular protein expression in TNF-α treated 2D10 cells for the indicated times. ( B ) Immunoblot analysis of protein expression of 2D10 cells transfected with Ssu72 or Cyclin T1 siRNAs with the indicated antibodies. ( C ) ChIP analysis of the recruitment of virus-encoded Tat and host cell factors to the integrated provirus in TNF-α-induced 2D10 cells. Schematic representation of the genomic organization of the lentiviral vector and the relative locations of the primers used for ChIP are shown at the top of the panel. ( D ) Model depicting three different steps regulated by Tat to facilitate the transition from promoter clearance to early elongation at the HIV-1 promoter. See the Discussion for details.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: Western Blot, Expressing, Transfection, Virus, Plasmid Preparation

Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: ChIP-seq and GRO-seq analysis of Ssu72 in hESCs. ( A , left ) Genomic distribution of Ssu72 ChIP-seq peaks. ( Right ) Metagene representation of the Ssu72 ChIP-seq profile. ( B ) The graph at the left shows a statistically significant (CC = 0.88; P -value < 1 × 10 −100 ) correlation between the S5P–RNAPII and Ssu72 ChIP-seq peaks around gene TSSs. The right panel shows a heat map analysis of the correlation of Ssu72 and S5P–RNAPII occupancy. ( C ) Ssu72 and S5P–RNAPII binding at the POU5F1 and NANOG genes, as captured from the Integrative Genomics Viewer genome browser. Images show visualization of WIG files. Gene diagrams are shown at the bottom , with scale bars above . ( D ) Immunoblot from H1 hESCs transfected with control (Ctrl) or Ssu72 siRNAs for 48 h. DDX39 antibody was used as a loading control. ( E ) GRO-seq read count covering 10 kb from the gene TTS in siCtrl and siSsu72 hESCs, as indicated. The read counts from both the sense and antisense strands are plotted, as indicated in the legend.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: ChIP-sequencing, Binding Assay, Western Blot, Transfection, Control