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

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.

Figure 1.

P-TEFb and an intact SL region of intron I are required for the transcriptional regulation of MYB reporter constructs by estrogen. (A) HEK293 cells are transfected (∼0.5 × 10⁶/well of six-well plate) with 1.0 µg of WT-MYB CAT (Left panel) or 1.0 µg ΔSL-MYB CAT construct (Right panel) along with 0.1 or 0.2 µg each of CyclinT1 and CDK9 and 10 ng pCMV β-actin-promoter-luciferase expression plasmid. After 24 h, cells were incubated with fresh medium supplemented with or without 10 nM estradiol for 16 h. (B) Transient transfections in HEK293 cells were carried out, as described above, in the presence of either WT P-TEFb (0.1 µg each of CyclinT1 and CDK9) or dominant negative CDK9 expression construct (0.1 µg) along with WT P-TEFb complex before adding estrogen to the medium. (C) After transient transfection, cells were either exposed to estrogen or to 25 µM DRB as indicated for 16 h. In each case, CAT activity was normalized to β-actin-promoter-Luc activity as an internal control. Experiments were done in triplicate and repeated at least thrice.

Figure 2.

Functionally active P-TEFb complex is selectively required for MYB expression. CDK9 inhibitors DRB and flavopiridol down-regulate the endogenous MYB gene expression. MCF-7 cells grown in regular growth medium (RGM) were treated with (A) DRB and (B) flavopiridol for 4 h. Cells were harvested and expression of MYB and cyclophilin A (Cyclo) was measured by qRT-PCR. (C) Over-expression of DN CDK9 inhibits endogenous MYB gene expression. MCF-7 cells were transfected with WT CDK9 or DN-CDK9 along with pCMV-GFP and treated with 10 nM estrogen. Cells were harvested 16 h after the hormone treatment and GFP-positive cells were isolated by FACS. RNA samples were isolated from the GFP-positive cells and used in qRT-PCR. In all experiments, relative expression of each gene was normalized by 18s Ribosomal RNA expression values as a control. Each experiment was done in duplicate and repeated at least twice.

Figure 3.

Synthesis of transcripts beyond the SL-region strongly correlates with the estrogen mediated stimulation or drug mediated down-regulation of endogenous MYB gene. Diagrammatic representation of human MYB (A) and cyclophilin A (B) genes. In the case of MYB, the position of the SL and poly dT sites are shown. Positions of intron based primers are indicated as Pre-I, Post-III and Post-IV. Full-length MYB transcripts are detected by MYB exon primer recognizes the sequence of exon8/9 region. Cyclophilin A full-length transcripts are detected by Cyclo Exon primers which recognize exon 5. (C) Ligand bound ERα relieves transcriptional attenuation of MYB gene expression. MCF-7 cells were kept 48 h in DMEM medium supplemented with CSS and incubated 2 h with 10 nM estrogen. The difference in gene expression in the presence of estrogen is shown as a fold change in comparison to the non-estrogen-treated cells. (D) DRB-mediated down-regulation of MYB transcription is associated with significant reduction in the synthesis of transcripts beyond the SL-dT region. MCF-7 cells were treated with 25 µM DRB for 4 h and synthesis of transcripts was estimated by qRT-PCR. Gene expression in the presence of drug was compared with the untreated control cells.

Figure 4.

Complex formation between CyclinT1, CDK9 and ERα. (A) Expression plasmids encoding CyclinT1, CDK9 and ERα were co-transfected into HEK293 cells and immunoprecipitated (IP) with the indicated primary antibodies and the resultant complexes were analyzed by western blotting (WB) using the indicated secondary antibodies. (B) and (C) Identification of complex formation by endogenous proteins. MCF-7 cells were grown in estrogen free (CSS), 10 nM estrogen added and in regular growth medium (control). Extracts, prepared from cells grown in the three different conditions, were used for immunoprecipitation (IP) with anti-CyclinT1 in (B) and anti-ERα in (C). Complexes were analyzed by WB using the antibodies indicated under each panel. In each case, 10% of the total cell extract was loaded for comparison and is labeled ‘In’ (input).

Figure 5.

ChIP assays detect ERα and P-TEFb components in the attenuation region of the MYB gene. (A,B) ERα binding to the MYB intron-I attenuation region and the pS2 promoter. In total, 0.25, 0.5 or 1.0 µg of anti-ERα antibody, no antibody (mock) or control IgG (1.0 µg) was added to MCF-7 cell extracts and ChIP assays were performed as described in ‘Materials and Methods’ section. (C) P-TEFb complex components are selectively recruited to the MYB intron-1 region. Anti-CyclinT1 and anti-CDK9 antibody (0.25 or 0.5 µg) or 0.5 µg of IgG was added to the MCF-7 extract for ChIP assays. The amount of DNA bound to ERα, CyclinT1 and CDK9 was detected by qPCR using MYB intron- and pS2 promoter-specific primers and the results are plotted as percentage of input.

Figure 6.

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.

Figure 7.

Estrogen stimulation selectively induces P-TEFb complex-driven Ser-2 phosphorylation of RNA Pol II in the MYB attenuation site. ChIP assays were performed with MCF-7 extract from cells grown either in the absence or presence of estrogen using anti-RNA Pol II antibody or anti-RNA Pol II CTD phospho Ser-2 antibody. The amount of total RNA Pol II and Ser-2 phospho-Pol II bound was detected by qPCR using MYB intron-1-specific primers (A) and pS2 promoter-specific primers (B), and expressed with respect to the input. These data are also plotted (C) to show the proportion of Ser-2 phosphorylated RNA Pol II compared with the total RNA Pol II present at the pS2 promoter and MYB intron-I pausing site in the presence or absence of estrogen.

Figure 8.

Model for estrogen-mediated relief of MYB transcriptional attenuation. Transcription is paused at the SL-dT region in the absence of estrogen in MCF-7 cells where Pol II is hypophosphorylated at Ser-2. Upon estrogen stimulation, the ERα–CyclinT1–CDK9 tripartite complex is recruited to the attenuation (SL-dT) region; this, in turn, phosphorylates the Ser-2 residue of RNA Pol II to allow resumption of elongation.