Journal:

Article Title: A Human Immunodeficiency Virus Type 1 Tat-Like Arginine-Rich RNA-Binding Domain Is Essential for HEXIM1 To Inhibit RNA Polymerase II Transcription through 7SK snRNA-Mediated Inactivation of P-TEFb

doi: 10.1128/MCB.24.12.5094-5105.2004

Figure Lengend Snippet: The native NLS domain is required for HEXIM1 to bind to 7SK and P-TEFb and inhibit P-TEFb's kinase activity. (A) The NLS-substituted HEXIM1 mutants fail to bind to 7SK and P-TEFb in vivo. Anti-Flag immunoprecipitation (IP) was performed in NE of HeLa cells transfected with either an empty vector (lane 1) or constructs expressing the indicated Flag-tagged HEXIM1 proteins (lanes 2 to 9). The peptide-eluted immune complexes were analyzed by Western blotting (WB) for the presence of CDK9, CycT1, and HEXIM1 and by Northern blotting (NB) for 7SK. (B) The native NLS is required for HEXIM1 to inhibit the P-TEFb kinase in a 7SK-dependent manner. Kinase reaction mixtures contained the affinity-purified wild-type or mutant HEXIM1, P-TEFb, GST-CTD, and the indicated RNA fractions. Total RNA was extracted from HeLa NE, which had been subjected to deoxyoligonucleotide-directed RNase H cleavage in the presence of 7SK antisense or scrambled oligonucleotide. Lanes 1 to 8, phosphorylated GST-CTD as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Lanes 9 to 11, relative amounts of HEXIM1 proteins used in the kinase reactions as determined by anti-Flag Western blotting. Lanes 12 and 13, purity of wild-type fHXM1 affinity purified from transfected cells as indicated by silver staining. (C) The HEXIM1 NLS is required for fΔN to inhibit P-TEFb kinase. Kinase reactions containing the indicated HEXIM1 mutants were performed exactly as for panel B.

Article Snippet: The HEXIM1 complexes were affinity purified from NE by incubating at 4°C for 2 h with anti-Flag agarose beads (Sigma), followed by extensive washes with buffer D (20 mM HEPES-KOH [pH 7.9], 15% glycerol, 0.2 mM EDTA, 0.2% NP-40, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) containing 0.3 M KCl (D0.3 M) and elution with the Flag peptide dissolved in D0.1 M. The eluted materials were analyzed by Western blotting with anti-Flag (M2; Sigma), anti-CDK9, and anti-CycT1 antibodies (Santa Cruz Biotechnology) and Northern hybridization using the 32 P-labeled full-length 7SK antisense RNA as the probe.

Techniques: Activity Assay, In Vivo, Immunoprecipitation, Transfection, Plasmid Preparation, Construct, Expressing, Western Blot, Northern Blot, Affinity Purification, Mutagenesis, Polyacrylamide Gel Electrophoresis, Autoradiography, Silver Staining

Journal:

Article Title: A Human Immunodeficiency Virus Type 1 Tat-Like Arginine-Rich RNA-Binding Domain Is Essential for HEXIM1 To Inhibit RNA Polymerase II Transcription through 7SK snRNA-Mediated Inactivation of P-TEFb

doi: 10.1128/MCB.24.12.5094-5105.2004

Figure Lengend Snippet: Direct and selective binding of the isolated NLS domain of HEXIM1 to 7SK in vivo and in vitro. (A) Efficient in vivo binding of the isolated NLS of HEXIM1 to 7SK but not P-TEFb. Anti-Flag immunoprecipitation (IP) was performed in NE of HeLa cells transfected with either an empty vector or vectors expressing the indicated Flag-tagged proteins. The peptide-eluted immune complexes were examined by Western blotting (WB) with antibodies against CDK9, CycT1, and Flag and Northern blotting (NB) with antisense 7SK RNA as the probe. (B) The isolated NLS of HEXIM1 binds 7SK with slightly reduced selectivity. Results shown are from Northern analyses of the levels of 7SK, U1, and U2 snRNAs present in HeLa NE and the various immune complexes (αFlag IP) obtained in panel A. (C) Direct binding of the isolated HEXIM1 NLS to 7SK in the absence of other factors. The indicated GFP fusion proteins were affinity purified from transfected cells, stripped off their associated factors, and analyzed in a gel mobility shift assay for their abilities to interact with the antisense (lanes 1 to 3) and sense (lanes 4 to 8) 7SK RNA. Anti-Flag monoclonal antibody was also present in the reaction mixtures in lanes 7 and 8. The amounts of GFP fusion proteins used in the reactions were examined by Western blotting in lanes 9 and 10.

Article Snippet: The HEXIM1 complexes were affinity purified from NE by incubating at 4°C for 2 h with anti-Flag agarose beads (Sigma), followed by extensive washes with buffer D (20 mM HEPES-KOH [pH 7.9], 15% glycerol, 0.2 mM EDTA, 0.2% NP-40, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) containing 0.3 M KCl (D0.3 M) and elution with the Flag peptide dissolved in D0.1 M. The eluted materials were analyzed by Western blotting with anti-Flag (M2; Sigma), anti-CDK9, and anti-CycT1 antibodies (Santa Cruz Biotechnology) and Northern hybridization using the 32 P-labeled full-length 7SK antisense RNA as the probe.

Techniques: Binding Assay, Isolation, In Vivo, In Vitro, Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Western Blot, Northern Blot, Affinity Purification, Mobility Shift

Journal: Nucleic Acids Research

Article Title: HMGA1 recruits CTIP2-repressed P-TEFb to the HIV-1 and cellular target promoters

doi: 10.1093/nar/gku168

Figure Lengend Snippet: HMGA1 interacts with CTIP2 in an RNA-independent fashion, while the association with inactive P-TEFb is RNase-sensitive. ( A ) Immunopurification of HMGA1-FLAG from transiently transfected HEK293 cells in the presence or the absence of RNase A. Input and precipitation steps were monitored for endogenous CTIP2, Cdk9, CycT1, HEXIM1 and the 7SK-associated LARP7 in western blot analyses. Mock-transfected cells were used as a control. ( B ) Immunopurification of endogenous HMGA1 from microglial cells using a specific anti-HMGA1 antibody. Input and precipitation steps were monitored for endogenous HMGA1 and CTIP2. An immunopurification with boiled anti-HMGA1 IgG was used as a control.

Article Snippet: Antibodies used in western blot analyses were M2 anti-FLAG antibody (Sigma), anti-HMGA1 antibody (Abcam), anti-CTIP2 antibody (Abcam), anti-Cdk9 antibody (Pierce), anti-CycT1 antibody (Proteintech), anti-HEXIM1 antibody (Proteintech) and anti-LARP7 antibody (Proteintech) with the corresponding secondary horseradish peroxidase-coupled antibodies (Sigma) as recommended by the manufacturer.

Techniques: Immu-Puri, Transfection, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: HMGA1 recruits CTIP2-repressed P-TEFb to the HIV-1 and cellular target promoters

doi: 10.1093/nar/gku168

Figure Lengend Snippet: HMGA1 recruits CTIP2-inactivated P-TEFb to cellular target promoters. ( A ) Network of 7SK-dependent P-TEFb target genes affected by both knock-down of CTIP2 and HMGA1. Up-regulated genes are coloured in red, down-regulated genes are coloured in green. Functional connections are indicated as arrows. The inactive P-TEFb snRNP is highlighted in yellow (Cdk9, CycT1, HEXIM1, 7SK RNA). ( B ) The overexpression of CTIP2 (pCTIP2) was verified by western blot analyses and β-actin was used as a housekeeping gene. ( C ) Scatter plot comparing the expression changes of 7SK-dependent P-TEFb targets on CTIP2 knock-down and overexpression. Genes statistically significantly ( ) regulated in both conditions are highlighted in blue. ( D ) The overexpression of HMGA1 (pHMGA1) was verified by western blot analyses, and β-actin was used as a housekeeping gene. ( E ) As in (C), but comparing HMGA1 knock-down and overexpression. Genes statistically significantly ( ) regulated in both conditions are highlighted in red. ( F ) Representative visualization of the effects of dnCdk9 expression, 7SK overexpression, 7SK knock-down, CTIP2 knock-down, CTIP2 overexpression, HMGA1 knock-down and HMGA1 overexpression on the expression of the 7SK-dependent P-TEFb targets PARP12 and EPSTI1. ( G ) ChIP analyses targeting the EPSTI1 promoter from HEK293 cells transfected with CTIP2-FLAG in the presence or absence of a HMGA1 knock-down. ( H ) Same as in (G), but for the PARP12 promoter.

Article Snippet: Antibodies used in western blot analyses were M2 anti-FLAG antibody (Sigma), anti-HMGA1 antibody (Abcam), anti-CTIP2 antibody (Abcam), anti-Cdk9 antibody (Pierce), anti-CycT1 antibody (Proteintech), anti-HEXIM1 antibody (Proteintech) and anti-LARP7 antibody (Proteintech) with the corresponding secondary horseradish peroxidase-coupled antibodies (Sigma) as recommended by the manufacturer.

Techniques: Knockdown, Functional Assay, Over Expression, Western Blot, Expressing, Transfection