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Estrogen receptor-α recruits P-TEFb to overcome transcriptional pausing in intron 1 of the MYB gene.

Mitra P, Pereira LA, Drabsch Y, Ramsay RG, Gonda TJ - Nucleic Acids Res. (2012)

Bottom Line: The MYB proto-oncogene is expressed in most estrogen receptor-positive (ERα(+)) breast tumors and cell lines.However, little is known, in general, about the factors involved in relieving transcriptional attenuation, or specifically how ERα coordinates such factors to promote transcriptional elongation.These data provide new insights into MYB regulation, and given the critical roles of MYB in tumorigenesis, suggest targeting MYB elongation as potential therapeutic strategy.

View Article: PubMed Central - PubMed

Affiliation: University of Queensland Diamantina Institute, Brisbane, Queensland 4102, Australia.

ABSTRACT
The MYB proto-oncogene is expressed in most estrogen receptor-positive (ERα(+)) breast tumors and cell lines. Expression of MYB is controlled, in breast cancer and other cell types, by a transcriptional pausing mechanism involving an attenuation site located ∼1.7 kb downstream from the transcription start site. In breast cancer cells, ligand-bound ERα binds close to, and drives transcription beyond this attenuation site, allowing synthesis of complete transcripts. However, little is known, in general, about the factors involved in relieving transcriptional attenuation, or specifically how ERα coordinates such factors to promote transcriptional elongation. Using cyclin dependent kinase 9 (CDK9) inhibitors, reporter gene assays and measurements of total and intronic MYB transcription, we show that functionally active CDK9 is required for estrogen-dependent transcriptional elongation. We further show by ChIP and co-immunoprecipitation studies that the P-TEFb complex (CDK9/CyclinT1) is recruited to the attenuation region by ligand-bound ERα, resulting in increased RNA polymerase II Ser-2 phosphorylation. These data provide new insights into MYB regulation, and given the critical roles of MYB in tumorigenesis, suggest targeting MYB elongation as potential therapeutic strategy.

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Related in: MedlinePlus

Recruitment of the P-TEFb complex to the MYB regulatory region is dependent on ligand-bound ERα. (A,B) MCF-7 cells were cultured in estrogen-free medium for 48 h, following which 10 nM estrogen or vehicle was added for a further 16 h. Extracts prepared from cells cultured in both conditions were used to perform ChIP assays using (A) anti-ERα antibody (1.0 µg), (B) anti-CyclinT1 (0.5 µg) and anti-CDK9 (0.5 µg) antibodies and corresponding control antibody (IgG) or no antibody (mock). The amount of binding was estimated by qPCR and expressed as percent input. (C,D) MCF-7 cells were incubated with 10 µM ICI 182780 for 16 h (ICI). Half of these cells were harvested and the remaining cells were incubated in fresh drug-free growth medium for 24 h (Recovery). An extract prepared from MCF-7 cells grown in regular growth medium (RGM) was used as control. Extracts of harvested cells were used for ChIP assays using (C) anti-ERα (1.0 µg), (D) anti-CyclinT1 (0.5 µg) or anti-CDK9 (0.5 µg) antibodies as indicated.
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gks286-F6: Recruitment of the P-TEFb complex to the MYB regulatory region is dependent on ligand-bound ERα. (A,B) MCF-7 cells were cultured in estrogen-free medium for 48 h, following which 10 nM estrogen or vehicle was added for a further 16 h. Extracts prepared from cells cultured in both conditions were used to perform ChIP assays using (A) anti-ERα antibody (1.0 µg), (B) anti-CyclinT1 (0.5 µg) and anti-CDK9 (0.5 µg) antibodies and corresponding control antibody (IgG) or no antibody (mock). The amount of binding was estimated by qPCR and expressed as percent input. (C,D) MCF-7 cells were incubated with 10 µM ICI 182780 for 16 h (ICI). Half of these cells were harvested and the remaining cells were incubated in fresh drug-free growth medium for 24 h (Recovery). An extract prepared from MCF-7 cells grown in regular growth medium (RGM) was used as control. Extracts of harvested cells were used for ChIP assays using (C) anti-ERα (1.0 µg), (D) anti-CyclinT1 (0.5 µg) or anti-CDK9 (0.5 µg) antibodies as indicated.

Mentions: Recruitment of the P-TEFb complex at the pausing site is one of the most critical steps in resuming transcription. In the case of HIV, the virus-encoded Tat protein recruits this complex to the TAR RNA sequence to allow transcription of full-length viral RNA (35). The P-TEFb complex is also recruited to cellular target genes by transcription factors, such as NFκB, MyoD, c-Myc or chromatin remodeling proteins such as Brd4, in part, by protein–protein interactions (25,36). In MCF-7 cells, published data from our laboratory (9) and global ChIP-on-chip or ChIP-seq analyses (37,38) have shown in vivo ERα binding in a region close to the SL-dT motif in the presence of estrogen. We were interested to know whether the P-TEFb complex is also recruited to this region and whether, given that P-TEFb forms a complex with ERα, the latter is required for its recruitment. To do this, we used ChIP followed by qPCR with primers that amplify 419 bp of MYB intron1 sequence including 181 bp upstream and 166 bp downstream from the 72 bp SL-dT region, as described previously (9). As a control for an estrogen-regulated gene, we used primers designed to amplify the promoter segment of the pS2 gene, which contains a well-defined ERα-binding site (39). As expected, qPCR analysis of ChIP samples generated using ERα-specific antibody detected binding (several fold above the control IgG) to the regulatory region of the MYB gene and to the pS2 promoter element (Figure 5A and B). Significantly, we also detected the presence of both CyclinT1 and CDK9 when antibodies to each of these proteins and the same MYB intron1 primers were used in ChIP assays. Somewhat surprisingly, we did not detect CDK9 or CyclinT1 association with the pS2 promoter, indicating a selective recruitment of these P-TEFb components to particular sites (Figure 5C). Since co-immunoprecipitation suggested complex formation of P-TEFb with ERα, we next wished to determine the role of ERα in the recruitment of the components of this complex to the MYB SL-dT region. Therefore, ChIP assays using antibodies against ERα, CyclinT1 and CDK9 were carried out on MCF-7 cells that had been incubated in estrogen-free medium for 48 h. Results presented in Figure 6A show negligible binding of ERα to the Myb-regulatory region and no significant accumulation of CDK9 or CyclinT1 under these conditions, but subsequent exposure to 10 nM estrogen for 16 h showed a significant recovery of the ERα binding to the MYB regulatory region. In parallel to ERα binding, a significant increase in the binding of CyclinT1 and CDK9 was observed following estrogen addition (Figure 6B). The response to estrogen in each case is actually quite rapid, as strong binding was also seen after 1 h (Supplementary Figure S2). To establish further the role of ERα in the recruitment of P-TEFb complex, we used the drug Fulvestrant/ICI182780 which not only blocks nuclear localization of this receptor, but also expedites its proteasome-mediated degradation (21). ChIP assays were performed using extracts prepared from MCF-7 treated with 100 nM Fulvestrant for 16 h (Figure 6C). A significant down-regulation (about 3- to 4-fold) of both ERα and P-TEFb binding was observed in the drug-treated sample. Note that Fulvestrant did not affect the total amounts of CDK9 and cyclinT1 in these cells (Supplementary Figure S3). We also performed a recovery experiment where cells were returned to Fulvestrant-free medium for 24 h after treatment as above. ChIP assay of this extract showed a significant re-accumulation of both ERα and P-TEFb complex binding (Figure 6D). Collectively, from the above results, we conclude that P-TEFb is recruited to the MYB-regulatory region as part of the complex formed with ligand-bound ERα.Figure 5.


Estrogen receptor-α recruits P-TEFb to overcome transcriptional pausing in intron 1 of the MYB gene.

Mitra P, Pereira LA, Drabsch Y, Ramsay RG, Gonda TJ - Nucleic Acids Res. (2012)

Recruitment of the P-TEFb complex to the MYB regulatory region is dependent on ligand-bound ERα. (A,B) MCF-7 cells were cultured in estrogen-free medium for 48 h, following which 10 nM estrogen or vehicle was added for a further 16 h. Extracts prepared from cells cultured in both conditions were used to perform ChIP assays using (A) anti-ERα antibody (1.0 µg), (B) anti-CyclinT1 (0.5 µg) and anti-CDK9 (0.5 µg) antibodies and corresponding control antibody (IgG) or no antibody (mock). The amount of binding was estimated by qPCR and expressed as percent input. (C,D) MCF-7 cells were incubated with 10 µM ICI 182780 for 16 h (ICI). Half of these cells were harvested and the remaining cells were incubated in fresh drug-free growth medium for 24 h (Recovery). An extract prepared from MCF-7 cells grown in regular growth medium (RGM) was used as control. Extracts of harvested cells were used for ChIP assays using (C) anti-ERα (1.0 µg), (D) anti-CyclinT1 (0.5 µg) or anti-CDK9 (0.5 µg) antibodies as indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3401469&req=5

gks286-F6: Recruitment of the P-TEFb complex to the MYB regulatory region is dependent on ligand-bound ERα. (A,B) MCF-7 cells were cultured in estrogen-free medium for 48 h, following which 10 nM estrogen or vehicle was added for a further 16 h. Extracts prepared from cells cultured in both conditions were used to perform ChIP assays using (A) anti-ERα antibody (1.0 µg), (B) anti-CyclinT1 (0.5 µg) and anti-CDK9 (0.5 µg) antibodies and corresponding control antibody (IgG) or no antibody (mock). The amount of binding was estimated by qPCR and expressed as percent input. (C,D) MCF-7 cells were incubated with 10 µM ICI 182780 for 16 h (ICI). Half of these cells were harvested and the remaining cells were incubated in fresh drug-free growth medium for 24 h (Recovery). An extract prepared from MCF-7 cells grown in regular growth medium (RGM) was used as control. Extracts of harvested cells were used for ChIP assays using (C) anti-ERα (1.0 µg), (D) anti-CyclinT1 (0.5 µg) or anti-CDK9 (0.5 µg) antibodies as indicated.
Mentions: Recruitment of the P-TEFb complex at the pausing site is one of the most critical steps in resuming transcription. In the case of HIV, the virus-encoded Tat protein recruits this complex to the TAR RNA sequence to allow transcription of full-length viral RNA (35). The P-TEFb complex is also recruited to cellular target genes by transcription factors, such as NFκB, MyoD, c-Myc or chromatin remodeling proteins such as Brd4, in part, by protein–protein interactions (25,36). In MCF-7 cells, published data from our laboratory (9) and global ChIP-on-chip or ChIP-seq analyses (37,38) have shown in vivo ERα binding in a region close to the SL-dT motif in the presence of estrogen. We were interested to know whether the P-TEFb complex is also recruited to this region and whether, given that P-TEFb forms a complex with ERα, the latter is required for its recruitment. To do this, we used ChIP followed by qPCR with primers that amplify 419 bp of MYB intron1 sequence including 181 bp upstream and 166 bp downstream from the 72 bp SL-dT region, as described previously (9). As a control for an estrogen-regulated gene, we used primers designed to amplify the promoter segment of the pS2 gene, which contains a well-defined ERα-binding site (39). As expected, qPCR analysis of ChIP samples generated using ERα-specific antibody detected binding (several fold above the control IgG) to the regulatory region of the MYB gene and to the pS2 promoter element (Figure 5A and B). Significantly, we also detected the presence of both CyclinT1 and CDK9 when antibodies to each of these proteins and the same MYB intron1 primers were used in ChIP assays. Somewhat surprisingly, we did not detect CDK9 or CyclinT1 association with the pS2 promoter, indicating a selective recruitment of these P-TEFb components to particular sites (Figure 5C). Since co-immunoprecipitation suggested complex formation of P-TEFb with ERα, we next wished to determine the role of ERα in the recruitment of the components of this complex to the MYB SL-dT region. Therefore, ChIP assays using antibodies against ERα, CyclinT1 and CDK9 were carried out on MCF-7 cells that had been incubated in estrogen-free medium for 48 h. Results presented in Figure 6A show negligible binding of ERα to the Myb-regulatory region and no significant accumulation of CDK9 or CyclinT1 under these conditions, but subsequent exposure to 10 nM estrogen for 16 h showed a significant recovery of the ERα binding to the MYB regulatory region. In parallel to ERα binding, a significant increase in the binding of CyclinT1 and CDK9 was observed following estrogen addition (Figure 6B). The response to estrogen in each case is actually quite rapid, as strong binding was also seen after 1 h (Supplementary Figure S2). To establish further the role of ERα in the recruitment of P-TEFb complex, we used the drug Fulvestrant/ICI182780 which not only blocks nuclear localization of this receptor, but also expedites its proteasome-mediated degradation (21). ChIP assays were performed using extracts prepared from MCF-7 treated with 100 nM Fulvestrant for 16 h (Figure 6C). A significant down-regulation (about 3- to 4-fold) of both ERα and P-TEFb binding was observed in the drug-treated sample. Note that Fulvestrant did not affect the total amounts of CDK9 and cyclinT1 in these cells (Supplementary Figure S3). We also performed a recovery experiment where cells were returned to Fulvestrant-free medium for 24 h after treatment as above. ChIP assay of this extract showed a significant re-accumulation of both ERα and P-TEFb complex binding (Figure 6D). Collectively, from the above results, we conclude that P-TEFb is recruited to the MYB-regulatory region as part of the complex formed with ligand-bound ERα.Figure 5.

Bottom Line: The MYB proto-oncogene is expressed in most estrogen receptor-positive (ERα(+)) breast tumors and cell lines.However, little is known, in general, about the factors involved in relieving transcriptional attenuation, or specifically how ERα coordinates such factors to promote transcriptional elongation.These data provide new insights into MYB regulation, and given the critical roles of MYB in tumorigenesis, suggest targeting MYB elongation as potential therapeutic strategy.

View Article: PubMed Central - PubMed

Affiliation: University of Queensland Diamantina Institute, Brisbane, Queensland 4102, Australia.

ABSTRACT
The MYB proto-oncogene is expressed in most estrogen receptor-positive (ERα(+)) breast tumors and cell lines. Expression of MYB is controlled, in breast cancer and other cell types, by a transcriptional pausing mechanism involving an attenuation site located ∼1.7 kb downstream from the transcription start site. In breast cancer cells, ligand-bound ERα binds close to, and drives transcription beyond this attenuation site, allowing synthesis of complete transcripts. However, little is known, in general, about the factors involved in relieving transcriptional attenuation, or specifically how ERα coordinates such factors to promote transcriptional elongation. Using cyclin dependent kinase 9 (CDK9) inhibitors, reporter gene assays and measurements of total and intronic MYB transcription, we show that functionally active CDK9 is required for estrogen-dependent transcriptional elongation. We further show by ChIP and co-immunoprecipitation studies that the P-TEFb complex (CDK9/CyclinT1) is recruited to the attenuation region by ligand-bound ERα, resulting in increased RNA polymerase II Ser-2 phosphorylation. These data provide new insights into MYB regulation, and given the critical roles of MYB in tumorigenesis, suggest targeting MYB elongation as potential therapeutic strategy.

Show MeSH
Related in: MedlinePlus