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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 or UBR5 knockdown decreases the ubiquitination of CDK9 in vivo, and UBR5 is required for the effect of TFIIS on this ubiquitination event. A, HeLa cells were transfected with the indicated concentrations of siRNAs targeting TFIIS or control nontargeting siRNAs or mock transfected. The cells were treated with lactacystin and lysed, and the lysate was used in immunoprecipitation experiments with an anti-CDK9 antibody (C20). The eluates were run on SDS gels and immunoblotted with anti-ubiquitin or anti-CDK9 antibodies. B, comparison of knockdown efficiencies resulting from treatment with siRNAs targeting TFIIS or control nontargeting siRNAs. C, HeLa cells treated with the indicated concentrations of siRNAs targeting UBR5 or nontargeting siRNAs were assayed for in vivo ubiquitination (as in A). D, comparison of knockdown efficiencies resulting from treatment with siRNAs targeting UBR5 or control nontargeting siRNAs. E, HeLa cells treated with siRNAs targeting UBR5 or nontargeting siRNAs (50 nm) or mock-treated were transfected with a construct driving the expression of TFIIS-FLAG or with an empty vector. An in vivo ubiquitination assay (as in A) was performed with the transfected cells. The lysates were used for anti-CDK9 immunoprecipitation, and the eluates (left panel) or the input (5%) (right panel) were run on SDS gels and immunoblotted with anti-ubiquitin or anti-CDK9 antibodies. The expression of the FLAG-tagged protein was monitored by immunodetection with an anti-FLAG antibody. WB, Western blotting; IP, immunoprecipitation; WCE, whole cell extract.
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Figure 4: TFIIS or UBR5 knockdown decreases the ubiquitination of CDK9 in vivo, and UBR5 is required for the effect of TFIIS on this ubiquitination event. A, HeLa cells were transfected with the indicated concentrations of siRNAs targeting TFIIS or control nontargeting siRNAs or mock transfected. The cells were treated with lactacystin and lysed, and the lysate was used in immunoprecipitation experiments with an anti-CDK9 antibody (C20). The eluates were run on SDS gels and immunoblotted with anti-ubiquitin or anti-CDK9 antibodies. B, comparison of knockdown efficiencies resulting from treatment with siRNAs targeting TFIIS or control nontargeting siRNAs. C, HeLa cells treated with the indicated concentrations of siRNAs targeting UBR5 or nontargeting siRNAs were assayed for in vivo ubiquitination (as in A). D, comparison of knockdown efficiencies resulting from treatment with siRNAs targeting UBR5 or control nontargeting siRNAs. E, HeLa cells treated with siRNAs targeting UBR5 or nontargeting siRNAs (50 nm) or mock-treated were transfected with a construct driving the expression of TFIIS-FLAG or with an empty vector. An in vivo ubiquitination assay (as in A) was performed with the transfected cells. The lysates were used for anti-CDK9 immunoprecipitation, and the eluates (left panel) or the input (5%) (right panel) were run on SDS gels and immunoblotted with anti-ubiquitin or anti-CDK9 antibodies. The expression of the FLAG-tagged protein was monitored by immunodetection with an anti-FLAG antibody. WB, Western blotting; IP, immunoprecipitation; WCE, whole cell extract.

Mentions: To confirm the role of UBR5 and TFIIS in CDK9 ubiquitination, siRNA treatments were used to knock down each protein individually in HeLa cells (Fig. 4, B and D, respectively), and the whole cell extracts were subjected to CDK9 immunoprecipitation and in vivo ubiquitination assays, as described above. The knockdown of each of TFIIS or UBR5 protein reduced the smear of polyubiquitinated CDK9 species in a dose-dependent fashion compared with control nontargeting siRNAs (Fig. 4, A and C, respectively). Together, these results indicate that both UBR5 and TFIIS are involved in CDK9 ubiquitination in vivo.


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 or UBR5 knockdown decreases the ubiquitination of CDK9 in vivo, and UBR5 is required for the effect of TFIIS on this ubiquitination event. A, HeLa cells were transfected with the indicated concentrations of siRNAs targeting TFIIS or control nontargeting siRNAs or mock transfected. The cells were treated with lactacystin and lysed, and the lysate was used in immunoprecipitation experiments with an anti-CDK9 antibody (C20). The eluates were run on SDS gels and immunoblotted with anti-ubiquitin or anti-CDK9 antibodies. B, comparison of knockdown efficiencies resulting from treatment with siRNAs targeting TFIIS or control nontargeting siRNAs. C, HeLa cells treated with the indicated concentrations of siRNAs targeting UBR5 or nontargeting siRNAs were assayed for in vivo ubiquitination (as in A). D, comparison of knockdown efficiencies resulting from treatment with siRNAs targeting UBR5 or control nontargeting siRNAs. E, HeLa cells treated with siRNAs targeting UBR5 or nontargeting siRNAs (50 nm) or mock-treated were transfected with a construct driving the expression of TFIIS-FLAG or with an empty vector. An in vivo ubiquitination assay (as in A) was performed with the transfected cells. The lysates were used for anti-CDK9 immunoprecipitation, and the eluates (left panel) or the input (5%) (right panel) were run on SDS gels and immunoblotted with anti-ubiquitin or anti-CDK9 antibodies. The expression of the FLAG-tagged protein was monitored by immunodetection with an anti-FLAG antibody. WB, Western blotting; IP, immunoprecipitation; WCE, whole cell extract.
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Figure 4: TFIIS or UBR5 knockdown decreases the ubiquitination of CDK9 in vivo, and UBR5 is required for the effect of TFIIS on this ubiquitination event. A, HeLa cells were transfected with the indicated concentrations of siRNAs targeting TFIIS or control nontargeting siRNAs or mock transfected. The cells were treated with lactacystin and lysed, and the lysate was used in immunoprecipitation experiments with an anti-CDK9 antibody (C20). The eluates were run on SDS gels and immunoblotted with anti-ubiquitin or anti-CDK9 antibodies. B, comparison of knockdown efficiencies resulting from treatment with siRNAs targeting TFIIS or control nontargeting siRNAs. C, HeLa cells treated with the indicated concentrations of siRNAs targeting UBR5 or nontargeting siRNAs were assayed for in vivo ubiquitination (as in A). D, comparison of knockdown efficiencies resulting from treatment with siRNAs targeting UBR5 or control nontargeting siRNAs. E, HeLa cells treated with siRNAs targeting UBR5 or nontargeting siRNAs (50 nm) or mock-treated were transfected with a construct driving the expression of TFIIS-FLAG or with an empty vector. An in vivo ubiquitination assay (as in A) was performed with the transfected cells. The lysates were used for anti-CDK9 immunoprecipitation, and the eluates (left panel) or the input (5%) (right panel) were run on SDS gels and immunoblotted with anti-ubiquitin or anti-CDK9 antibodies. The expression of the FLAG-tagged protein was monitored by immunodetection with an anti-FLAG antibody. WB, Western blotting; IP, immunoprecipitation; WCE, whole cell extract.
Mentions: To confirm the role of UBR5 and TFIIS in CDK9 ubiquitination, siRNA treatments were used to knock down each protein individually in HeLa cells (Fig. 4, B and D, respectively), and the whole cell extracts were subjected to CDK9 immunoprecipitation and in vivo ubiquitination assays, as described above. The knockdown of each of TFIIS or UBR5 protein reduced the smear of polyubiquitinated CDK9 species in a dose-dependent fashion compared with control nontargeting siRNAs (Fig. 4, A and C, respectively). Together, these results indicate that both UBR5 and TFIIS are involved in CDK9 ubiquitination in vivo.

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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