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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 and UBR5 overexpression affects the ubiquitination of CDK9 in vivo. A, HeLa cells transfected with a vector driving the expression of FLAG-tagged TFIIS or the empty vector, together with a construct driving the expression of ubiquitin-HA, were treated with the proteasome inhibitor lactacystin and lysed. The whole cell extract (5% input) (left panel) or eluates from anti-CDK9 immunoprecipitation with the C20 antibody (right panel) were run on SDS gels and immunoblotted with anti-ubiquitin or anti-FLAG antibodies (B) Immunodetection of CDK9 in the anti-CDK9 immunoprecipitate obtained as described in A. C, HeLa cells treated with lactacystin or mock-treated were lysed. The lysates were immunoprecipitated using anti-CDK9 antibody (C20) bound to protein A beads or with mock protein A beads. The eluates and the input (5%) were run on SDS gels and immunoblotted using anti-ubiquitin and anti-CDK9 antibodies. D, HepG2 cells stably transfected with constructs driving the expression of UBR5-FLAG or TFIIS-FLAG were subjected to in vivo ubiquitination as described above. Parental HepG2 cells treated in parallel were used as a control. The eluates of CDK9 immunoprecipitation (C20) (right panel) and the input (5%) (left panel) were immunoblotted using anti-ubiquitin, anti-CDK9, anti-FLAG, and anti-UBR5 antibodies. WB, Western blotting; IP, immunoprecipitation; WCE, whole cell extract.
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Figure 3: TFIIS and UBR5 overexpression affects the ubiquitination of CDK9 in vivo. A, HeLa cells transfected with a vector driving the expression of FLAG-tagged TFIIS or the empty vector, together with a construct driving the expression of ubiquitin-HA, were treated with the proteasome inhibitor lactacystin and lysed. The whole cell extract (5% input) (left panel) or eluates from anti-CDK9 immunoprecipitation with the C20 antibody (right panel) were run on SDS gels and immunoblotted with anti-ubiquitin or anti-FLAG antibodies (B) Immunodetection of CDK9 in the anti-CDK9 immunoprecipitate obtained as described in A. C, HeLa cells treated with lactacystin or mock-treated were lysed. The lysates were immunoprecipitated using anti-CDK9 antibody (C20) bound to protein A beads or with mock protein A beads. The eluates and the input (5%) were run on SDS gels and immunoblotted using anti-ubiquitin and anti-CDK9 antibodies. D, HepG2 cells stably transfected with constructs driving the expression of UBR5-FLAG or TFIIS-FLAG were subjected to in vivo ubiquitination as described above. Parental HepG2 cells treated in parallel were used as a control. The eluates of CDK9 immunoprecipitation (C20) (right panel) and the input (5%) (left panel) were immunoblotted using anti-ubiquitin, anti-CDK9, anti-FLAG, and anti-UBR5 antibodies. WB, Western blotting; IP, immunoprecipitation; WCE, whole cell extract.

Mentions: We next performed in vivo ubiquitination assays in extracts from cells in which either UBR5 or TFIIS were individually overexpressed or depleted. HeLa cells were co-transfected with constructs driving the expression of TFIIS carrying a FLAG epitope (TFIIS-FLAG) and an ubiquitin-HA peptide. Co-transfections of the empty vector and the ubiquitin-HA construct were used as controls. The cells were treated with the proteasome inhibitor lactacystin, and the whole cell extracts were incubated with an anti-CDK9 antibody. After CDK9 was isolated, the eluates were resolved on SDS-PAGE and immunoblotted with anti-ubiquitin, anti-CDK9, or anti-FLAG antibodies. The overexpression of TFIIS markedly increased the smear of high molecular weight proteins as compared with the controls (Fig. 3A, right panel). This effect was specific to CDK9 because no significant modification in the general ubiquitination pattern was observed in the whole cell extracts (Fig. 3A, left panel). Immunoblotting with an anti-CDK9 antibody shows that the high molecular weight species are polyubiquitinated CDK9 molecules (Fig. 3B). Moreover, these species appear specifically in the eluates from the CDK9 immunoprecipitates but not in the eluates from mock immunoprecipitation (Fig. 3C). Interestingly, proteasome inhibition by lactacystin does not affect the stability of the polyubiquitinated CDK9 species, whereas stabilization of the polyubiquitinated proteins is observed in the whole cell extracts using the same treatment (Fig. 3C).


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 and UBR5 overexpression affects the ubiquitination of CDK9 in vivo. A, HeLa cells transfected with a vector driving the expression of FLAG-tagged TFIIS or the empty vector, together with a construct driving the expression of ubiquitin-HA, were treated with the proteasome inhibitor lactacystin and lysed. The whole cell extract (5% input) (left panel) or eluates from anti-CDK9 immunoprecipitation with the C20 antibody (right panel) were run on SDS gels and immunoblotted with anti-ubiquitin or anti-FLAG antibodies (B) Immunodetection of CDK9 in the anti-CDK9 immunoprecipitate obtained as described in A. C, HeLa cells treated with lactacystin or mock-treated were lysed. The lysates were immunoprecipitated using anti-CDK9 antibody (C20) bound to protein A beads or with mock protein A beads. The eluates and the input (5%) were run on SDS gels and immunoblotted using anti-ubiquitin and anti-CDK9 antibodies. D, HepG2 cells stably transfected with constructs driving the expression of UBR5-FLAG or TFIIS-FLAG were subjected to in vivo ubiquitination as described above. Parental HepG2 cells treated in parallel were used as a control. The eluates of CDK9 immunoprecipitation (C20) (right panel) and the input (5%) (left panel) were immunoblotted using anti-ubiquitin, anti-CDK9, anti-FLAG, and anti-UBR5 antibodies. WB, Western blotting; IP, immunoprecipitation; WCE, whole cell extract.
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Figure 3: TFIIS and UBR5 overexpression affects the ubiquitination of CDK9 in vivo. A, HeLa cells transfected with a vector driving the expression of FLAG-tagged TFIIS or the empty vector, together with a construct driving the expression of ubiquitin-HA, were treated with the proteasome inhibitor lactacystin and lysed. The whole cell extract (5% input) (left panel) or eluates from anti-CDK9 immunoprecipitation with the C20 antibody (right panel) were run on SDS gels and immunoblotted with anti-ubiquitin or anti-FLAG antibodies (B) Immunodetection of CDK9 in the anti-CDK9 immunoprecipitate obtained as described in A. C, HeLa cells treated with lactacystin or mock-treated were lysed. The lysates were immunoprecipitated using anti-CDK9 antibody (C20) bound to protein A beads or with mock protein A beads. The eluates and the input (5%) were run on SDS gels and immunoblotted using anti-ubiquitin and anti-CDK9 antibodies. D, HepG2 cells stably transfected with constructs driving the expression of UBR5-FLAG or TFIIS-FLAG were subjected to in vivo ubiquitination as described above. Parental HepG2 cells treated in parallel were used as a control. The eluates of CDK9 immunoprecipitation (C20) (right panel) and the input (5%) (left panel) were immunoblotted using anti-ubiquitin, anti-CDK9, anti-FLAG, and anti-UBR5 antibodies. WB, Western blotting; IP, immunoprecipitation; WCE, whole cell extract.
Mentions: We next performed in vivo ubiquitination assays in extracts from cells in which either UBR5 or TFIIS were individually overexpressed or depleted. HeLa cells were co-transfected with constructs driving the expression of TFIIS carrying a FLAG epitope (TFIIS-FLAG) and an ubiquitin-HA peptide. Co-transfections of the empty vector and the ubiquitin-HA construct were used as controls. The cells were treated with the proteasome inhibitor lactacystin, and the whole cell extracts were incubated with an anti-CDK9 antibody. After CDK9 was isolated, the eluates were resolved on SDS-PAGE and immunoblotted with anti-ubiquitin, anti-CDK9, or anti-FLAG antibodies. The overexpression of TFIIS markedly increased the smear of high molecular weight proteins as compared with the controls (Fig. 3A, right panel). This effect was specific to CDK9 because no significant modification in the general ubiquitination pattern was observed in the whole cell extracts (Fig. 3A, left panel). Immunoblotting with an anti-CDK9 antibody shows that the high molecular weight species are polyubiquitinated CDK9 molecules (Fig. 3B). Moreover, these species appear specifically in the eluates from the CDK9 immunoprecipitates but not in the eluates from mock immunoprecipitation (Fig. 3C). Interestingly, proteasome inhibition by lactacystin does not affect the stability of the polyubiquitinated CDK9 species, whereas stabilization of the polyubiquitinated proteins is observed in the whole cell extracts using the same treatment (Fig. 3C).

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