JMJD6 specifically interacts with the methylated form of ERα.

<p>(<b>A</b>) <i>In vitro</i> synthesized and radiolabelled ERα protein was incubated with GST-JMJD6 and binding was analysed by autoradiography. Luciferase was used as a negative control. The lower panel shows the corresponding coomassie staining. (<b>B</b>) MCF-7 were treated with E₂ for the indicated times and the interaction of JMJD6 with ERα was analysed by immunoprecipitation. On the same extract methylated ERα (metERα) was studied by immunoprecipitation with the anti-metERα revealed with an anti-ERα. (<b>C</b>) PRMT1 is essential to JMJD6/ERα interaction. Lysates of MCF-7 cells transfected with control siRNAs or with specific PRMT1 siRNAs were treated with vehicle or E₂ (10⁻⁸ M), analysed for ERα methylation and immunoprecipitated with the anti-JMJD6 antibody before analysis with the indicated antibodies. The amount of ERα, JMJD6 and PRMT1 in the different samples was determined by western blot in the input. (<b>D</b>) JMJD6 interacts specifically with metERα. MCF-7 treated with E₂ as above were lysed and analysed for JMJD6/ERα interaction. In parallel, cell extracts were immunoprecipitated with metERα antibody. Unbound fractions were then used for a second immunoprecipitation using anti-JMJD6 antibody followed by western blotting with anti-ERα and anti-JMJD6 antibodies. The amount of ERα and JMJD6 in the different samples was determined by western blot in the input.</p

List of antibodies and their application.

<p>List of antibodies and their application.</p

Potential interacting proteins of JMJD6 identified by affinity purification and mass spectrometry.

<p>MMA: monomethylarginine. DMA: dimethylarginine. The bold characters highlight arginine methylated proteins.</p><p>*indicate proteins already identified as JMJD6 putative partners <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087982#pone.0087982-Webby1" target="_blank">[14]</a>.</p

JMJD6 Regulates ERα Methylation on Arginine

<div><p>ERα functions are tightly controlled by numerous post-translational modifications including arginine methylation, which is required to mediate the extranuclear functions of the receptor. We report that upon oestrogenic stimulation, JMJD6, the only arginine demethylase described so far, interacts with and regulates methylated ERα (metERα) function. Moreover, by combining the silencing of JMJD6 with demethylation assays, we show that metERα is a new substrate for JMJD6. We propose that the demethylase activity of JMJD6 is a decisive regulator of the rapid physiological responses to oestrogen.</p></div

JMJD6 knock-down increases MCF-7 cell proliferation and migration.

<p><b>(A)</b> JMJD6 expression has been studied in several human breast tumour cell lines by western blotting. GAPDH expression has also been determined to validate the quality of the samples loading. <b>(+)</b> and <b>(−)</b> indicates oestrogen receptor alpha expression in the different cell lines. <b>(B)</b> JMJD6 expression was analysed by western blotting to verify the efficiency of the ShRNAs. <b>(C)</b> Proliferation assays were performed on MCF-7 cells invalidated for JMJD6. Cell viability was measured for the indicated times after seeding. *p<0.05; **p<0.01. <b>(D)</b> Wound healing assays were performed in MCF-7 cells invalidated for JMJD6. <b>(E)</b> The percent of migration was determined as a mean of distances of the wound for the different experiments. Analysis was performed in three separate experiments. The data are represented as means +/- SEM from three replicates in each of the three independent experiments.</p

Role of JMJD6 in Breast Tumourigenesis

<div><p>Background</p><p>Protein arginine methylation is a common post translational modification that regulates protein properties. This modification is carried out by a family of nine arginine methyltransferases (PRMTs). Arginine methylation has already been linked to tumourigenesis as overexpression of these enzymes was associated with various cancers, notably in breast cancers. Since the Jumonji Domain Containing 6 protein (JMJD6) possesses an arginine demethylase activity able to remove the methyl mark, we wanted to assess its potential role in breast tumourigenesis.</p><p>Methods</p><p>The expression of the protein by tissue microarray immunohistochemical staining was performed on a cohort of 133 breast tumours. Using cell lines stably overexpressing or knocked down for JMJD6, we evaluated its role on cell proliferation, cell migration, colony formation and mice tumour xenografts.</p><p>Results</p><p>The analysis of JMJD6 expression in a cohort of breast tumour samples indicates that JMJD6 was highly expressed in aggressive breast tumours. Moreover, high expression of JMJD6 was associated with poor disease-free survival of patients in this cohort. JMJD6 silencing in breast tumoural cells promotes certain characteristics of tumourigenesis including proliferation, migration <i>in vitro</i>, and tumour growth <i>in vivo</i>. These effects are dependent on its demethylase activity as an enzymatic dead mutant lost these properties.</p><p>Conclusions</p><p>Although JMJD6 displays anti-tumoral properties in cell lines, its expression in breast tumours may be a marker of poor prognosis, suggesting that its function could be altered in breast cancer.</p></div

Multivariate analysis of DFS integrating JMJD6 expression.

<p>Final multivariate Cox modelisation of DFS adjusted on JMJD6 expression. Hazard ratio for high JMJD6 expression (>80% stained cells) relative to low JMJD6 expression (<80% stained cells) is presented, adjusted on tumour size.</p><p>Multivariate analysis of DFS integrating JMJD6 expression.</p

Knock-down of JMJD6 increases breast tumour xenograft growth.

<p>Orthotopic xenograft tumours were generated with MCF-7 breast tumour cells stably expressing either control shRNA or shRNA targeting the JMJD6 gene. <b>(A)</b> Tumour growth curve was performed measuring tumours twice per week with an electronic caliper. Values represent mean +/- SEM from 10 animals. * p<0.05, ** p<0.01 ***p<0.005 <b>(B)</b> Mice were dissected and tumours isolated at eight weeks following injection. Tumour mass <b>(B)</b> and volumes <b>(C)</b> were measured for each tumour. <b>(D)</b> Pictures of the tumours were taken from mice xenografted with MCF-7 control cells (a,b,c) and MCF-7 shJMJD6 cells (d,e,f). Panels a and d show the tumours in the intact mice, denoted by an asterisk. Panels b and e show the tumours before surgery. In panel e, the arrow indicates a secondary tumour. Panels c and f show the tumours removed after surgery.</p

JMJD6 expression in breast tumours.

<p><b>(A)</b> JMJD6 expression has been analysed by IHC in normal mammary gland (panel a) as well as in infiltrating breast cancer showing low staining (panel b) intermediate (panel c) or strong staining (panel d) (objective X 40). <b>(B)</b> Kaplan Meier estimates of DFS according to JMJD6 expression groups.</p

JMJD6 expression negatively affects MCF-7 anchorage-independent growth.

<p><b>(A)</b> MCF-7 knocked down for JMJD6 were assessed for anchorage-independent growth 25 days after plating. The lower panel shows a magnification of the colonies. <b>(B)</b> Quantification of anchorage independent growth. Colonies composed of more than 50 cells were counted in each well. The data are represented as means +/- SEM from two replicates in each of the three independent experiments. <b>(C)</b> The same experiments were performed on MCF-7 cells stably expressing JMJD6 (WT or Mut). The lower panel shows a magnification of the colonies. <b>(D)</b> The quantification of colonies was performed as in (B). The data are represented as means +/- SEM from two replicates in each of the three independent experiments.</p