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Transcription factor IIS cooperates with the E3 ligase UBR5 to ubiquitinate the CDK9 subunit of the positive transcription elongation factor B.

Cojocaru M, Bouchard A, Cloutier P, Cooper JJ, Varzavand K, Price DH, Coulombe B - J. Biol. Chem. (2010)

Bottom Line: Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene.This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9.Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada.

ABSTRACT
Elongation of transcription by mammalian RNA polymerase II (RNAPII) is regulated by specific factors, including transcription factor IIS (TFIIS) and positive transcription elongation factor b (P-TEFb). We show that the E3 ubiquitin ligase UBR5 associates with the CDK9 subunit of positive transcription elongation factor b to mediate its polyubiquitination in human cells. TFIIS also binds UBR5 to stimulate CDK9 polyubiquitination. Co-localization of UBR5, CDK9, and TFIIS along specific regions of the γ fibrinogen (γFBG) gene indicates that a ternary complex involving these factors participates in the transcriptional regulation of this gene. In support of this notion, overexpression of TFIIS not only modifies the ubiquitination pattern of CDK9 in vivo but also increases the association of CDK9 with various regions of the γFBG gene. Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene. This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9. Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

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TFIIS interacts with the E3 ligase UBR5 and the P-TEFb kinase CDK9. A, high confidence interaction network for human TFIIS. The graph shows part of a network of high confidence interactions obtained using affinity purification (in vivo pulldown) coupled with mass spectrometry, with 77 TAP-tagged baits targeting many transcription and RNA processing factors (22). B, in vitro pulldown using TFIIS-GST. In vitro pulldown experiments used HEK 293 whole cell extracts with TFIIS-GST bound to glutathione-Sepharose beads or with control glutathione-Sepharose beads. The eluates and input (5%) were immunoblotted using antibodies directed against UBR5, CDK9, or the control protein CAND1. The NaCl concentration (m) in the elution buffer is indicated. C and D, immunoprecipitation using anti-CDK9 (C) and anti-UBR5 (D) antibodies. Immunoprecipitation experiments used HEK 293 whole cell extracts with an antibody raised against CDK9 (C) or UBR5 (D) bound to protein A beads or with mock protein A beads. The eluates and input (5%) were run on SDS gels and immunoblotted using antibodies against UBR5, TFIIS, CDK9, and Rpb1. WB, Western blotting; IP, immunoprecipitation; WCE, whole cell extract.
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Figure 1: TFIIS interacts with the E3 ligase UBR5 and the P-TEFb kinase CDK9. A, high confidence interaction network for human TFIIS. The graph shows part of a network of high confidence interactions obtained using affinity purification (in vivo pulldown) coupled with mass spectrometry, with 77 TAP-tagged baits targeting many transcription and RNA processing factors (22). B, in vitro pulldown using TFIIS-GST. In vitro pulldown experiments used HEK 293 whole cell extracts with TFIIS-GST bound to glutathione-Sepharose beads or with control glutathione-Sepharose beads. The eluates and input (5%) were immunoblotted using antibodies directed against UBR5, CDK9, or the control protein CAND1. The NaCl concentration (m) in the elution buffer is indicated. C and D, immunoprecipitation using anti-CDK9 (C) and anti-UBR5 (D) antibodies. Immunoprecipitation experiments used HEK 293 whole cell extracts with an antibody raised against CDK9 (C) or UBR5 (D) bound to protein A beads or with mock protein A beads. The eluates and input (5%) were run on SDS gels and immunoblotted using antibodies against UBR5, TFIIS, CDK9, and Rpb1. WB, Western blotting; IP, immunoprecipitation; WCE, whole cell extract.

Mentions: Human EcR 293 cells stably transfected were induced with 3 μm ponasterone A (Invitrogen) to express TAP-tagged versions of TFIIS and TFIIS.1. Whole cell extracts were subjected to TAP purification, and the eluates were run on SDS-PAGE gels, as described previously (20). Noninduced cells were used as TAP purification controls. After silver or Sypro Ruby (Bio-Rad) staining, the bands were excised and digested with trypsin. The tryptic peptides were purified and identified by tandem mass spectrometry, as described previously (21). To discriminate between specific and spurious interactions, we used a computational method that we have described previously (21, 22). This method uses an algorithm that assigns an interaction reliability score to each protein interaction based on parameters such as the strength of the MS score and the local topology of the network (e.g. number of common interaction partners and the identification of the interaction in reciprocal purifications). The graph in Fig. 1A shows the interaction partners of TFIIS with an interaction reliability score higher than 0.73. We estimate that above this threshold, the specificity is ∼88%, and the rate of false positives is lower than 12%. By setting this threshold value of interaction reliability, we evaluate that the sensitivity of our method to detect relevant interactions approaches 72%, which suggests a rate of 28% false negatives (see Ref. 22 for details on the computational method used to evaluate the confidence of our interaction networks).


Transcription factor IIS cooperates with the E3 ligase UBR5 to ubiquitinate the CDK9 subunit of the positive transcription elongation factor B.

Cojocaru M, Bouchard A, Cloutier P, Cooper JJ, Varzavand K, Price DH, Coulombe B - J. Biol. Chem. (2010)

TFIIS interacts with the E3 ligase UBR5 and the P-TEFb kinase CDK9. A, high confidence interaction network for human TFIIS. The graph shows part of a network of high confidence interactions obtained using affinity purification (in vivo pulldown) coupled with mass spectrometry, with 77 TAP-tagged baits targeting many transcription and RNA processing factors (22). B, in vitro pulldown using TFIIS-GST. In vitro pulldown experiments used HEK 293 whole cell extracts with TFIIS-GST bound to glutathione-Sepharose beads or with control glutathione-Sepharose beads. The eluates and input (5%) were immunoblotted using antibodies directed against UBR5, CDK9, or the control protein CAND1. The NaCl concentration (m) in the elution buffer is indicated. C and D, immunoprecipitation using anti-CDK9 (C) and anti-UBR5 (D) antibodies. Immunoprecipitation experiments used HEK 293 whole cell extracts with an antibody raised against CDK9 (C) or UBR5 (D) bound to protein A beads or with mock protein A beads. The eluates and input (5%) were run on SDS gels and immunoblotted using antibodies against UBR5, TFIIS, CDK9, and Rpb1. WB, Western blotting; IP, immunoprecipitation; WCE, whole cell extract.
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Related In: Results  -  Collection

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Figure 1: TFIIS interacts with the E3 ligase UBR5 and the P-TEFb kinase CDK9. A, high confidence interaction network for human TFIIS. The graph shows part of a network of high confidence interactions obtained using affinity purification (in vivo pulldown) coupled with mass spectrometry, with 77 TAP-tagged baits targeting many transcription and RNA processing factors (22). B, in vitro pulldown using TFIIS-GST. In vitro pulldown experiments used HEK 293 whole cell extracts with TFIIS-GST bound to glutathione-Sepharose beads or with control glutathione-Sepharose beads. The eluates and input (5%) were immunoblotted using antibodies directed against UBR5, CDK9, or the control protein CAND1. The NaCl concentration (m) in the elution buffer is indicated. C and D, immunoprecipitation using anti-CDK9 (C) and anti-UBR5 (D) antibodies. Immunoprecipitation experiments used HEK 293 whole cell extracts with an antibody raised against CDK9 (C) or UBR5 (D) bound to protein A beads or with mock protein A beads. The eluates and input (5%) were run on SDS gels and immunoblotted using antibodies against UBR5, TFIIS, CDK9, and Rpb1. WB, Western blotting; IP, immunoprecipitation; WCE, whole cell extract.
Mentions: Human EcR 293 cells stably transfected were induced with 3 μm ponasterone A (Invitrogen) to express TAP-tagged versions of TFIIS and TFIIS.1. Whole cell extracts were subjected to TAP purification, and the eluates were run on SDS-PAGE gels, as described previously (20). Noninduced cells were used as TAP purification controls. After silver or Sypro Ruby (Bio-Rad) staining, the bands were excised and digested with trypsin. The tryptic peptides were purified and identified by tandem mass spectrometry, as described previously (21). To discriminate between specific and spurious interactions, we used a computational method that we have described previously (21, 22). This method uses an algorithm that assigns an interaction reliability score to each protein interaction based on parameters such as the strength of the MS score and the local topology of the network (e.g. number of common interaction partners and the identification of the interaction in reciprocal purifications). The graph in Fig. 1A shows the interaction partners of TFIIS with an interaction reliability score higher than 0.73. We estimate that above this threshold, the specificity is ∼88%, and the rate of false positives is lower than 12%. By setting this threshold value of interaction reliability, we evaluate that the sensitivity of our method to detect relevant interactions approaches 72%, which suggests a rate of 28% false negatives (see Ref. 22 for details on the computational method used to evaluate the confidence of our interaction networks).

Bottom Line: Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene.This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9.Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada.

ABSTRACT
Elongation of transcription by mammalian RNA polymerase II (RNAPII) is regulated by specific factors, including transcription factor IIS (TFIIS) and positive transcription elongation factor b (P-TEFb). We show that the E3 ubiquitin ligase UBR5 associates with the CDK9 subunit of positive transcription elongation factor b to mediate its polyubiquitination in human cells. TFIIS also binds UBR5 to stimulate CDK9 polyubiquitination. Co-localization of UBR5, CDK9, and TFIIS along specific regions of the γ fibrinogen (γFBG) gene indicates that a ternary complex involving these factors participates in the transcriptional regulation of this gene. In support of this notion, overexpression of TFIIS not only modifies the ubiquitination pattern of CDK9 in vivo but also increases the association of CDK9 with various regions of the γFBG gene. Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene. This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9. Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

Show MeSH