RARα association to transcribed regions in AF9-or BRD4-independent genes.

<p>The response of TTNPB-inducible genes (FC>2 after 4 hours) in P19^wt was compared to that in P19^Af9(−) or P19^Brd4(−) in similar conditions. Genes losing their responsiveness to TTNPB (FC<1.2) in either the P19^Brd4(−) background (cluster B), the P19^Af9(−)background (cluster C) or both (cluster D) were identified by microarray data analysis. Genes maintaining an inducibility similar to that observed in P19^wt in either the P19^Af9(−) or the P19^Brd4(−) background were grouped in Cluster A. Genes in each cluster were searched for the occurrence of RAR binding sites on the basis of RAR ChIP-Seq data carried out in mouse ES cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064880#pone.0064880-Mahony1" target="_blank">[42]</a>. Three representative genes were selected from each cluster and their inducibility was validated by RT-QPCR in each condition (n = 3, left inset). RARα and RNApol II association to an upstream region (UR), RAR binding site (RAR BS), transcriptional start site region (TSS) and an exon (Exon) was assessed in independent, duplicate ChIP-PCR assays after a 4-hour challenge of P19^wt with TTNPB. Input lanes showed an equal loading but were omitted for space purposes.</p

RAR localizes to transcribed regions of the Rarβ2 gene in a P-TEFB-dependent manner.

<p>(A) Structure of the mouse <i>Rarβ2</i> promoter. pRARE: proximal RARE; dRARE: distal RARE. (B) Gene expression in P19 cells. P19 cells were treated for 48 hours with DMSO, 1 µM all trans RA (atRA), 250 µM cAMP or transfected with a HA-tag COUP-TFI expression vector. Expression of <i>Rarβ2, Top2β</i>, <i>Creb</i>, <i>Coup-TFI</i>, <i>Tcf19</i> and <i>Rplp0</i> were quantified by RT-QPCR. Basal expression levels were arbitrarily set to 1 and data are expressed as the mean±SEM (n = 3). *, p<0.05; **, p<0.01; ***, p<0.005. (C) RARα and phosphorylated RNApol II loading at the <i>Rplp0</i> and <i>Pou5f1/Oct4</i> promoters. P19 cells were treated as in (B) and ChIP assays were performed with indicated antibodies. (D) AF9 colocalizes to the <i>Rarβ2</i> promoter. P19 cells were treated for 4 hours with 1 µM atRA, and ChIP assays were carried out as described. The specific enrichment in <i>Rarβ2</i> promoter sequence is expressed after normalization to background values (<i>Myoglobin</i> gene). Data are expressed as the mean±SEM (n = 2). *, p<0.05; **, p<0.01; ***, p<0.005. (E) DRB inhibition of the <i>Rarβ2</i> gene transcription. P19 cells were treated with the indicated combination of atRA (1 µM) and varying doses of DRB (50 to 5000 nM) for 4 hours. <i>Rarβ2</i> mRNA was quantified by RT-QPCR. (F) A CDK9 dominant negative mutant inhibits <i>Rarβ2</i> gene expression. P19 were cotransfected with increasing amount of pCMV-lacZ (control), pCMV-HA-wtCDK9, or pCMV-HA-dnCDK9 expression vectors at the indicated ratio, then treated 24 hours with 1 µM atRA. Gene expression was quantified as above and data expressed as the mean±SEM (n = 4). *, p<0.05; **, p<0.01; ***, p<0.005. (G) RAR and phosphorylated RNApol II are detected at transcribed regions of the <i>Rarβ2</i> gene. P19 cells were treated as in (D) and ChIP/reChIP assays were performed. (H) P-TEFb inhibition prevents RAR association to <i>Rarβ2</i> elongated regions. P19 cells were treated for 4 hours with the indicated combination of TTNPB (1 µM) or flavopiridol (250 nM). ChIP assays (n = 2) were performed as in (D).</p

AF9 and BRD4 coactivate RARα in a ligand-independent manner.

<p>(A, B) P19 cells were transfected with the indicated amounts of AF9, sBRD4 or lBRD4 expression vectors for 24 hours with or without 1 µM atRA and <i>Rarβ2</i> gene expression was assayed by RT-QPCR. The basal expression level in non transfected, untreated cells was arbitrarily set to 1 and data were expressed as the mean±SEM (n = 5). *, p<0.05; **, p<0.01; ***, p<0.005. (C) <i>Af9</i> or <i>Brd4</i> knockdowns. (C, upper panel) AF9 or BRD4 expression was assayed by western blot analysis in P19wt, P19^Af9(−) and P19^Brd4(−). (C, lower panel) <i>Rarβ2</i> gene expression in AF9- or BRD4-depleted P19 cells. The time-dependent expression of <i>Rarβ2</i> upon stimulation with 1 µM TTNPB was quantified by RT-QPCR. (D) Exon-specific RT-QPCR assay of the <i>Rarβ2</i> mRNA. Cloned <i>mRarβ2</i> cDNA was used as a standard in PCR reaction, and used to select PCR primer sets displaying a similar efficiency (“Cloned cDNA”). <i>Rarβ2</i> mRNA from either P19^wt, P19^Af9(−) or P19^Brd4(−) was then formally quantified by Q-PCR. **, p<0.01, intra-sample comparison; ^§§, p<0.01, inter-sample comparison. (E) RAR associates to <i>Rarβ2</i> transcribed regions as a function of AF9 and BRD4 levels. P19^wt, P19^Af9(−) or P19^Brd4(−) were treated with 1 µM TTNPB for 1 hour and ChIP assays were performed. The specific enrichment in the different <i>Rarβ2</i> amplicons was assayed by Q-PCR and expressed normalized to background values (myoglobin gene). Data are expressed as the mean±SEM (n = 2). *, p<0.05; **, p<0.01; ***, p<0.005. (F) The AF-1 region of RAR confers DRB sensitivity to RA-induced transcription of the <i>Rarβ2</i> promoter. P19 cells were cotransfected as indicated with expression vectors coding for wtRXRα, wtRARα or ΔAF-1-RARα or ΔAF-2-RARα together with the mRARβ2-Luc reporter gene. Cells were treated 24 hours with 1 µM atRA and/or DRB and luciferase activity was quantified. Basal expression levels were arbitrarily set to 1 and data are expressed as the mean±SEM (n = 6). *, p<0.05; **, p<0.01; ***, p<0.005.</p

Time-dependent induction of gene expression upon RARα activation in P19^wt, P19^Af9⁽⁻⁾ or P19^Brd4⁽⁻⁾.

<p>Cells were treated with TTNPB for indicated times and gene expression patterns were monitored. Genes induced more than 2-fold and peaking at either 60 minutes, 120 minutes or 240 minutes in the P19^wt background were clusterized to define cluster I (peaking at 60 minutes), cluster II (peaking at 120 minutes) and cluster III (peaking at 240 minutes). Associated gene lists were used to generate entity lists in Genespring to follow the expression of these genes in the P19^Af9(−) or P19^Brd4(−) background. Expression at different times in distinct cellular backgrounds is displayed as a heatmap.</p

AF9 or BRD4 are required for RAR interaction with exonic regions.

<p>Two representative genes from clusters defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064880#pone-0064880-g005" target="_blank">Figure 5</a> were selected. The loading of AF9 and BRD4 to the TSS was assayed by ChIP-QPCR (n = 2) and results normalized to background values (<i>Myoglobin</i> gene) are represented in left insets (bar graphs). The association of RAR to an upstream region (UR), RAR binding site (RAR BS), transcriptional start site region (TSS) and an exon (Exon) was assessed in independent, duplicate ChIP-PCR assays after a 4-hour challenge of P19^wt, P19^Brd4(−) or P19^Af9(−) cells with TTNPB. Data are expressed as the mean±SEM (n = 2). *, p<0.05; **, p<0.01.</p

The N-terminus of RARα interacts with nuclear proteins.

<p>(A) A nucleus-targeted RARα AF-1 domain acts as a dominant negative receptor. HeLa cells were cotransfected with expression vectors coding for wild type (wt) RXRα, wtRARα, GFP-NLS and GFP-NLS-AF-1 at the indicated ratio together with a m<i>Rarβ2</i> promoter-driven reporter gene (mRARβ2-Luc). Cells were treated overnight with 1 µM atRA and luciferase activity was quantified. Basal expression levels were arbitrarily set to 1 and data are expressed as the mean±SEM (n = 3). *, p<0.05; **, p<0.01; ***, p<0.005. (Right panel) Confocal laser microscopy of transfected HeLa cells. (B) The RARα AF-1 domain is transcriptionally active. HeLa cells were transfected with mRARβ2-Luc and expression vectors coding for wtRXRα, wtRARα, N–terminally ΔAF-1-RARα) or C-terminally truncated ΔAF-2-RARα) RARα. Cell treatment, luciferase assays and calculations are as in (A). (C, D) Isolation and identification of proteins interacting with the AF-1 transactivation motif of RARα. AF-1 fused to GST (GST-AF-1) or GST alone (GST) were immobilized on a matrix and incubated with HeLa nuclear extracts (+HeLa) or buffer alone (Mock). Numbers indicate bands that were subjected to mass spectrometry analysis. (D) The table indicates the name, protein abbreviation, the UniProtKB/TrEMBL entry, percentage of peptide coverage in two representative purifications, and the predicted molecular mass. (E) Target validation by GST pulldown assays. Various domains of RARα were expressed as fusion proteins to GST (left panel) and used as baits for ³⁵S-labeled shortBRD4 (sBRD4), AF9, PAK6 and NAP1L2. CB: Coomassie Blue staining of RAR derivatives adsorbed on glutathione-Sepharose. (F) Interaction of RARα with BRD4 or AF9. FLIM-based FRET fluorescence assays were performed to determine the lifetime of the donor (GFP) in the indicated conditions.</p

Gene expression level in response to RARα activation in wild type, AF9- and BRD4-deficient backgrounds.

<p>(A) Genes exhibiting a fold-change above 1.2 fold in TTNPB-treated P19^wt cells were clusterized according to a functional gene ontology classification. Representative functional clusters from the top 10 hits are shown. The basal level in non treated P19^wt was arbitrarily set to 1 and is depicted by black boxes. Numbers indicate the fold change ratio of individual genes relative to untreated wt P19 (red: upregulation; green: downregulation; black, no change). (B) Gene expression levels of known atRA-target genes. RA-target genes were selected from the literature and their expression levels were extracted from microarray data. Results are represented as in (A).</p

Additional file 1: Figure S1. of The AF-1-deficient estrogen receptor ERα46 isoform is frequently expressed in human breast tumors

Clinical parameters of the breast tumor samples. IDC invasive ductal carcinoma, ILC invasive lobular carcinoma. Figure S2. Expression of Flag-ERα36 in transiently transfected MDA-MB231 as detected by immunocytochemistry using an anti-Flag antibody. Figure S3. Analysis of potential correlation between the clinical parameters of the breast tumor samples and the expression of ERα46 isoform. A significant P value (indicated in red) was only found between ERα46 expression and HER-2 positive breast tumors. IDC invasive ductal carcinoma, ILC invasive Lobular Carcinoma. Figure S4. Results of the proteomic analysis of the ERα46 protein detected in tumor samples. A) Western blot with the SP1 antibody obtained after immunoprecipitation of ERα with HC20 antibody in two human tumors overexpressing the putative ERα46 isoform. B and C) Sequence coverage obtained from the peptides identified by proteomic analysis shown in bold red on ERα66 and on ERα46 isoforms M (methionine): putative translational start sites generating the ERα46 isoform. Figure S5. The stress-induced increase in LucF activity is reproducible in MCF7 cells (A) and is not due to the generation of mono-cistronic LucF transcripts via an internal promoter or cryptic splicing as observed in MDA-Lenti-AB exposed to two siRNAs-lucR (B and C). Figure S6. Modulation profiles of the interaction of ERα46 (red) and ERα66 (blue) with coregulators in A) Apo proteins and B) in response to E2 binding. C) Profile of EC50 values of 4-OH-tamoxifen- (red) and fulvestrant- (blue) induced modulation of ERα46 and ERα66 coregulators interaction when use in antagonists mode with 6.3 nM E2. Figure S7. List of primers used in the expression profiling of target genes. Figure S8. Fold-changes (FC) in gene expression± SEM in MDA-ERα46 and MDA-ERα66 cells in response to 4-h treatment with E2 (10–8 M). P values were determined by Mann-Whitney test. Significant P values are indicated in red. (PPTX 939 kb