Báo cáo khoa học: The histone demethylase JARID1A regulates progesterone receptor expression pot

The histone demethylase JARID1A regulates progesterone receptor expression Antje Stratmann1,2 and Bernard Haendler1 Therapeutic Research Group Oncology, Bayer Schering Pharma AG, Berlin, Germany Institute of Chemistry and Biochemistry, Free University Berlin, Germany Keywords ChIP; estrogen; histone demethylation; JARID1; progesterone receptor Correspondence B Haendler, Therapeutic Research Group Oncology, Bayer Schering Pharma AG, Mullerstr 178, D-13342 Berlin, Germany ă Fax: +49 30 468 18069 Tel: +49 40 468 12669 E-mail: bernard.haendler@bayer.com (Received 12 October 2010, revised 21 January 2011, accepted 21 February 2011) doi:10.1111/j.1742-4658.2011.08058.x Transcriptional control of the progesterone receptor gene by estrogen is a complex mechanism It involves estrogen receptor a which uses several enzymes that locally modify histone tails as cofactors Using MCF-7 cells as a model, we found that Jumonji AT-rich interactive domain 1A (JARID1A; KDM5A ⁄ RBP2), an enzyme that removes the activating H3K4 di- and trimethylation marks, was involved in the fine-tuning of progesterone receptor gene expression Reduction of JARID1A led to enhanced progesterone receptor expression, at both the basal and estrogenstimulated levels Conversely, overexpression of JARID1A wild-type, but not the enzymatically inactive mutant, suppressed progesterone receptor promoter activity Chromatin immunoprecipitation experiments showed JARID1A to bind in a ligand-independent manner to a progesterone receptor gene upstream region that contains an estrogen response element halfsite as well as the CCGCCC sequence, which is potentially recognized by JARID1A Estrogen treatment led to RNA polymerase II recruitment to this region and to increased estrogen receptor a binding to the PR enhancer region In addition, elevation of H3K4 trimethylation was detected at the estrogen response element half-site region Reduction of JARID1A expression was followed by higher H3K4 trimethylation in this region Analysis of MDA-MB-231 cells, which not express the progesterone receptor, indicated that H3K4 trimethylation did not take place in the regulatory regions examined Taken together, the results underscore the importance of epigenetic modifications for regulation of progesterone receptor expression They suggest that H3K4 tri- and dimethylation play an important role and that JARID1A is the histone-demethylating enzyme responsible for removal of this mark Introduction The progesterone receptor (PR) plays a fundamental role in reproduction [1,2] and its expression is under estrogen control [3–5] Several motifs that mediate the estrogen response have been identified in the human PR gene, including estrogen response element (ERE) half-sites, Sp1- and AP-1-binding sites [3,6,7] Two PR isoforms arising from a single gene have been identified in humans [3,4,8] They differ in the length of their N-terminal domain and the extension found in PRB endows it with specific properties, possibly linked to the differential recruitment of cofactors [2,9] Both PR isoforms are expressed in the breast and several reports Abbreviations ChIP, chromatin immunoprecipitation; ER, estrogen receptor; ERE, estrogen response element; JARID1, Jumonji AT-rich interactive domain 1A; Jmj, Jumonji; PR, progesterone receptor; siRNA, small interfering RNA; tsp, transcription start point 1458 FEBS Journal 278 (2011) 1458–1469 ª 2011 The Authors Journal compilation ª 2011 FEBS A Stratmann and B Haendler indicate that the ratio between PRA and PRB is altered during tumorigenesis, such that PRA predominates [9–11] PR gene transcription is controlled by estrogen in a complex way and is mainly driven by estrogen receptor (ER) a [5] Recent studies indicate that ERa interacts with histone-modifying enzymes to locally remodel chromatin, thus leading to activation or repression of gene expression CARM1 and PRMT1, two enzymes involved in the methylation of arginine residues, represent important coactivators for several steroid receptors, including ERa [12] Methylating enzymes that target lysines of histone (H3) such as SMYD3 [13,14], MLL2 [15], G9a [16], RIZ1 [17], EZH2 [18] and NSD1 [19] have all been shown to be ERa coactivators Far less is known about the role of demethylating enzymes in modulating ER function The lysine-specific demethylase 1, which belongs to the amine oxydase class, controls the expression of a subset of ER-regulated genes by counteracting the repressive function of H3K9 methylases [20] A second group of histone demethylases is the Jumonji (Jmj) C domain-containing family of which the Jumonji AT-rich interactive domain (JARID1) proteins represent a subgroup that specifically removes dimethyl and trimethyl marks from H3K4 [21,22] Four members have been identified in humans and mice, based on the conservation of the JmjC, JmjN, ARID and plant homeodomain domains, and of a C5H2C zinc finger [21,22] JARID1A (KDM5A ⁄ RBP2) was originally described as a protein that binds to the retinoblastoma protein, leading to the promotion of cellular differentiation [23] More recently, its function as a histone-demethylating enzyme was evidenced [24,25] Unlike other histone-modifying enzymes, JARID1A recognizes a specific CCGCCC DNA sequence via its ARID domain, thus regulating the expression of numerous genes [26] A genome-wide analysis showed that two groups of genes are controlled by JARID1A, some of which are involved in cellular differentiation, whereas others are implicated in mitochondrial function and RNA ⁄ DNA metabolism [27] Very recently, JARID1A was found to be required for the establishment of a reversible drug-tolerant phenotype in cancer cell subpopulations [28] JARID1B is upregulated in breast and prostate cancer and may favour tumour progression [24,29,30] Microarray studies have identified  100 genes controlled by JARID1B, several of which are involved in the cell cycle and in signal transduction [31] JARID1C is encoded by the SMCX gene on chromosome X and escapes X inactivation [32] It plays an important role in brain function and was found to be mutated in families with X-linked mental retardation [32] JARID1D is encoded by the Y chromosome and Regulation of PR expression by JARID1A forms a complex with a protein involved in meiosis during spermatogenesis [33] Here, we analysed the role of the JARID1 family in transducing ERa function using the estrogen-controlled progesterone receptor (PR) gene as a model We found that in the breast cancer cell line MCF-7, JARID1A acted as a repressor of PR expression Chromatin immunoprecipitation (ChIP) analysis showed the presence of JARID1A in a region covering an ERE half-site located downstream of the PRB transcription start point (tsp) We furthermore found JARID1A to control H3K4 trimethylation at this location and this modification was much increased after estrogen treatment Conversely, H3K4 trimethylation was not observed in the PR-negative cell line MDA-MB-231 Taken together, our results strongly suggest that H3K4 methylation in the region around the ERE half-site is important for modulation of PR expression by ERa, and JARID1A is involved in the removal of this mark Results JARID1A is involved in the regulation of PR expression in MCF-7 cells In order to find out whether the JARID1 demethylases played a role in PR expression, two small interfering RNA (siRNA) pairs with selectivity for each JARID1 family member were obtained and validated MCF-7 breast cancer cells were treated with each siRNA pair and the respective JARID1 mRNA levels were determined at different time-points after transfection, using quantitative PCR for measurement A robust and specific reduction of expression was observed for JARID1A, -1B and -1C two days post treatment, when compared with cells treated with lipids only or transfected with unrelated siRNAs (Fig S1) The selectivity of the siRNAs was ascertained by determining the expression levels of the nontargeted family members and no significant change was observed (Fig S1) JARID1D is not expressed in MCF-7 cells because it is encoded by the Y chromosome, and was therefore not analysed Western blot analysis was performed after siRNA transfection to confirm that the reduction in transcript levels also impacted the respective protein levels A strong decrease in JARID1A, -1B and -1C protein levels was seen days after transfection (Fig 1) No change in the GAPDH levels was observed in the treated cells compared with the different controls used Next, we looked at the effects of JARID1 knockdown on the induction of PR expression by estrogen in FEBS Journal 278 (2011) 1458–1469 ª 2011 The Authors Journal compilation ª 2011 FEBS 1459 Regulation of PR expression by JARID1A A Stratmann and B Haendler Fig Identification of siRNAs specific for JARID1 family members MCF-7 cells were transfected with two siRNA pairs (#1 and #2) specific for each JARID1 family member, as indicated In the controls, cells were transfected with unrelated siRNAs (siCo), with lipid only or left untreated (untr) JARID1 protein levels were determined by western blot analysis days later GAPDH protein levels were determined as loading control MCF-7 cells As reported previously [34], we found that the predominant PR isoform expressed in these cells is PRB A time-course experiment showed that the greatest PR stimulation by nm estrogen took place at 16 and 24 h, as evidenced by quantitative PCR (Fig 2A) Next, MCF-7 cells were transfected with siRNAs directed against the different JARID1 family members Three days after transfection when the protein was depleted, the cells were treated or not with estrogen and total RNA was purified 24 h later Quantitative PCR and western blot analysis showed that knocking-down the expression of JARID1A led to a strong increase in PR mRNA and protein levels (Fig 2B–D) The effect was more pronounced in the absence of estradiol (Fig 2B,C) than in its presence (Fig 2B,D) Reducing the expression of JARID1B or -1C had far less effect on basal PR levels (Fig 2C) and no effect on estrogenstimulated PR levels (Fig 2D) No change in GAPDH levels was observed (Fig 2E,F) Because PR expression was mostly affected by JARID1A knockdown, the underlying mechanisms were studied in more detail Overexpression of JARID1A represses PR promoter activity We then analysed whether JARID1A enzymatic activity was involved in regulating the PR promoter Because estrogen selectively increases the levels of transcripts derived from promoter B in breast cancer cells [4], a reporter plasmid containing the PRB promoter region from )711 to +29 (see Fig 4A) placed upstream of the 1460 Luc reporter was devised This region does not contain a canonical ERE, but has previously been shown to be strongly induced by estrogen in cell-based transactivation assays [3–5] This was confirmed in our experiments in which we measured a dose-dependent induction in MCF-7 cells co-transfected with an ERa expression plasmid and treated with increasing estrogen concentrations for 24 h (Fig 3B) The impact of overexpressed JARID1A either as a wild-type form or as a mutated form in which the iron-binding site was modified by replacing histidine at position 483 with alanine, as described [24], was compared The corresponding mutation in JARID1B is known to destroy enzymatic activity [35] Overexpression of the wild-type and mutated JARID1A forms after transfection of the corresponding plasmids in MCF-7 cells was confirmed at the protein level by western blot analysis (Fig 3A) Either plasmid was cotransfected with the ERa expression vector and the reporter vector containing the PRB promoter into MCF-7 cells After h, the cells were treated with different estrogen concentrations for an additional 24 h Determination of Luc activity revealed that overexpressing JARID1A wild-type, but not the enzymatically inactive mutant, entirely prevented estrogen induction of the PRB promoter, even at the highest hormone concentration tested (Fig 3B) JARID1A binds to the PR regulatory regions and influences the H3K4 methylation status Genome-wide surveys indicate that ER-binding sites are often located far away from the tsp of estrogenregulated genes [36–40] The precise regions involved in the estrogen control of the PR gene have not all been characterized and multiple EREs exist, as shown in several studies [41,42] A half-palindromic ERE located 571 bp downstream of the PRB tsp and adjacent to two Sp1-binding sites has been identified (Fig 4A) Both the ERE half-site and the Sp1 elements are implicated in the estrogen responsiveness of this regulatory region [43] In addition, two ER-binding sites located 168 kb and 206 kb upstream of the PRB tsp were identified by whole-genome cartography [42] In order to find out whether JARID1A binds to these different regulatory sequences and whether JARID1A and the H3K4 methylation status played a role in estrogen response, ChIP was performed using primers specific for each region As a control, we used primers directed against an intron of the unrelated F-box and leucine-rich repeat protein 11 gene, a region which is known to contain ERE motifs that are not bound by the ER [42] We first took an antibody directed against H3 and obtained similar signals at all analysed regions FEBS Journal 278 (2011) 1458–1469 ª 2011 The Authors Journal compilation ª 2011 FEBS A Stratmann and B Haendler Regulation of PR expression by JARID1A Fig Effects of JARID1 knockdown on PR expression (A) MCF-7 cells were treated with nM 17b-estradiol for the indicated times, total RNA was prepared and the PR transcript levels were determined by quantitative PCR (B–F) MCF-7 cells were transfected with siRNAs specific for each JARID1 (siJ1A: JARID1A; siJ1B: JARID1B; siJ1C: JARID1C) In the controls, cells were transfected with unrelated siRNAs (siCo), with lipid only (lip) or left untreated (untr) On day post transfection, MCF-7 cells were additionally treated with vehicle or with nM 17b-estradiol for 24 h (B) PR protein levels were determined by western blot analysis days post transfection GAPDH protein levels were measured as loading control (C,D) PR transcript levels were determined by quantitative PCR days post transfection (C) MCF-7 cells were additionally treated with vehicle (D) MCF-7 cells were additionally treated with nM 17b-estradiol (E,F) GAPDH transcript levels were determined by quantitative PCR days post transfection (E) MCF-7 cells were additionally treated with vehicle (F) MCF-7 cells were additionally treated with nM 17b-estradiol The fold inductions compared with siCon are given The asterisk denotes a significant difference (P < 0.01) between siRNA-treated cells and the corresponding untreated control, as analysed by Student’s t-test (data not shown) RNA polymerase II (Pol II) occupancy was then determined It was not observed in the absence of hormone, but increased significantly at the ERE half-site after estrogen treatment (Fig 4B) Loading of ERa was not observed in the absence of estrogen Conversely, it was seen at enhancer after hormone treatment (Fig 4C) JARID1A binding was near control levels in the absence and presence of estrogen at enhancer region 1, and slightly more elevated at enhancer region (Fig 4D) Much higher levels were seen at the ERE half-site region, independent of the hormone treatment (Fig 4D) Interestingly, sequence analysis indicated that two JARID1A-binding sites were present in this region We then examined the H3K4 methylation status using antibodies specific for each methylation pattern Mono- and dimethyla- FEBS Journal 278 (2011) 1458–1469 ª 2011 The Authors Journal compilation ª 2011 FEBS 1461 Regulation of PR expression by JARID1A A Stratmann and B Haendler Fig Effects of JARID1A on PR stimulation by estrogen (A) MCF-7 cells were transfected with JARID1A wild-type (wt), mutant H483A (H483A) or empty vector (vector) Protein levels were analysed by western blot analysis days later (B) MCF-7 cells were cotransfected with a PRB promoter reporter vector and an ERa expression vector (control) Expression vectors for wild-type and mutant H483A JARID1A were additionally cotransfected to generate the indicated curves (wt and H483A, respectively) Five hours post transfection, stimulation was performed with 0–100 nM 17b-estradiol Luc activity was determined 24 h later The fold inductions compared with cells not treated with estrogen are given tion were visible at all locations, with no effect of the estrogen treatment (Fig 4E,F) Interestingly, monomethylation was highest at the enhancer region and dimethylation at the half-ERE region Trimethylation was only observed at the half-ERE region and estrogen treatment led to a significant increase of the signal (Fig 4G) Because the modification in H3K4 trimethylation did not parallel a change in JARID1A binding, we looked whether the cellular JARID1A levels affected H3K4 methylation at the PR gene upstream region MCF-7 cells were transfected with JARID1A-specific siRNAs and the PR regulatory regions analysed by ChIP, as before No effect on H3K4 monomethylation was observed (Fig 5A) Concerning H3K4 dimethylation, a significant increase was seen at all sites examined (Fig 5B) The most dramatic changes were observed for H3K4 trimethylation at the ERE half-site region Here, a significant increase was observed following JARID1A knockdown both in the non- and hormone-stimulated cells (Fig 5C) By contrast, no trimethylation of H3K4 was detected at the far upstream enhancers or 1462 To further characterize the importance of H3K4 methylation in the control of PR expression, we used the ERa-negative cell line MDA-MB-231 These cells not express PR [44] We compared JARID1A protein levels by western blot analysis and found comparable signals in MDA-MB-231 and MCF-7 cells (Fig S2A) We then wanted to find out whether the local H3K4 methylation patterns in the PR gene regulatory region differed between these two cell lines ChIP analysis indicated H3 levels to be comparable at all sites examined (data not shown) No Pol II occupancy was detected at any analysed site (Fig S2B) H3K4 mono- and dimethylation were seen at the halfERE region (Fig S2C,D) but no trimethylation was seen (Fig S2E) In order to determine whether JARID1A was responsible for the lack of histone trimethylation and therefore possibly involved in the silencing of the PR gene in MDA-MB-231 cells, binding of this demethylase to the PR regulatory regions was determined ChIP results showed that there was no occupancy (Fig S2F) Taken together these data further underline the importance of H3K4 methylation in controlling PR gene expression and indicate that JARID1A controls PR gene transcription only in the context of ERa expression Discussion The role of the H3K4 methylation status in controlling gene transcription is documented by several studies [40,41,45], but how far nuclear receptors use this histone mark for the regulation of downstream target genes has not been extensively analysed Here we studied the role of the H3K4 demethylating enzymes of the JARID1 family in modulating estrogen control of the human PR gene transcriptional activity Using the breast cancer cell line MCF-7 as a model, we found that JARID1A regulated expression of the PR gene and fine-tuned its control by ERa Reducing JARID1A expression by specific siRNAs dramatically enhanced PR expression at the basal and less so at the estrogen-stimulated levels Conversely, overexpressing the JARID1A wild-type enzyme, but not its inactive mutant, suppressed the activity of the PRB promoter in transient transfection experiments This prompted us to determine the role of H3 methylation in regulating PR expression and the potential binding of JARID1A to the PR regulatory regions The H3K4 methylation pattern of three PR regulatory regions possibly involved in the control by estrogen, namely enhancer 1, enhancer and the half-ERE region located 571 bp downstream of the PRB tsp was analysed ChIP experiments showed the overall H3K4 FEBS Journal 278 (2011) 1458–1469 ª 2011 The Authors Journal compilation ª 2011 FEBS A Stratmann and B Haendler Regulation of PR expression by JARID1A Fig ChIP analysis of PR regulatory regions in MCF-7 cells (A) Schematic representation of the PR gene upstream region The tsp for the PRB and PRA transcripts are indicated with arrows Distal ER-binding sites and a proximal half-ERE located 571 bp downstream of the PRB tsp are highlighted with black diamonds The respective DNA regions amplified with the primer pairs used in ChIP are shown above the sites with black bars The white box indicates the PRB promoter region that was subcloned upstream of the Luc reporter construct (B) ChIP assay of Pol II binding (C) ChIP assay of ERa binding (D) ChIP assay of JARID1A binding (E) ChIP assay of H3K4 monomethylation (F) ChIP assay of H3K4 dimethylation (G) ChIP assay of H3K4 trimethylation Chromatin isolated from MCF-7 cells treated for h with nM 17b-estradiol (black bars) or untreated (white bars) was immunoprecipitated with the appropriate antibodies and the bound DNA analysed by quantitative PCR amplification An IgG-specific antibody was used in control experiments (light and dark grey bars) The asterisk denotes a significant difference (P < 0.01) between hormone-treated and the corresponding untreated data sets, as analysed by Student’s t-test methylation pattern to fit well with recent genomewide studies reporting monomethylation in different regions of active genes, dimethylation preferentially at promoters and trimethylation exclusively at promoters [46,47] The changes we observed in the PR locus upon estrogen stimulation were an increase in Pol II binding to the half-ERE region and of ERa binding to enhancer Interestingly, the enhancer region amplified in FEBS Journal 278 (2011) 1458–1469 ª 2011 The Authors Journal compilation ª 2011 FEBS 1463 Regulation of PR expression by JARID1A A Stratmann and B Haendler Fig Influence of JARID1A levels on H3K4 methylation of the PR regulatory regions in MCF-7 cells JARID1A-specific siRNAs (mixed pair #1 and #2) were used for expression knockdown (light and dark grey bars) An unrelated siRNA pair was used in the control experiments (white and black bars) Four days post transfection, chromatin isolated from MCF-7 cells and treated for h with nM 17b-estradiol (black and dark grey bars) or left untreated (white and light grey bars) was immunoprecipitated with antibodies specific for H3K4 methylation Bound DNA was analysed by quantitative PCR amplification An IgG-specific antibody was used as negative control (striped bars) (A) ChIP assay of H3K4 monomethylation (B) ChIP assay of H3K4 dimethylation (C) ChIP assay of H3K4 trimethylation The asterisk denotes a significant difference (P < 0.01) between the unrelated siRNA control and the JARID1A siRNA, hormone-treated and untreated respectively, as analysed by Student’s t-test our ChIP experiments is directly adjacent to a motif recently found to represent a bona fide ERE [48], and therefore likely to be detected under our conditions In addition, an increase in H3K4 trimethylation of the half-ERE region was also associated with hormone stimulation This change in H3K4 trimethylation corroborates previous findings showing the presence of this mark in the 5¢ region of genes to be essential 1464 for gene activity [45,47] Concerning JARID1A, a hormone-independent binding to the half-ERE was evidenced Sequence analysis showed that this region also contains two DNA binding motifs (CCGCCC) known to be recognized by the ARID domain of JARID1A It should however be added that other DNA motifs are also bound by JARID1A [49] so that only a comprehensive mutational analysis will allow to determine which PR regulatory region is involved Despite the fact that JARID1A levels were not affected by estrogen treatment, this demethylase is likely to be involved in controlling the H3K4 methylation status of the PR gene, as shown by our knockdown ChIP experiments in MCF-7 cells, which led to an increase of di- and especially of trimethylation, mainly at the half-ERE region The requirement of the demethylase activity for the repressive effects of JARID1A on PR gene expression was furthermore underlined by the luciferase assay which showed that overexpression of JARID1A wild-type but not of the active site mutant resulted in suppression of PRB promoter activity Our results show that both JARID1A and ERa are involved in the regulation of the PR promoter activity but that their binding to the three regulatory regions examined did not follow the same pattern ERa binds to the enhancer region in a hormone-dependent manner and is recruited following estrogen stimulation By contrast, the binding of JARID1A to the region close to the tsp does not depend on the hormonal status However, the activity of JARID1A is influenced by the presence of the hormone-activated ERa A possible scenario is that in the absence of estrogen, JARID1A is needed to shut down PR gene expression and that following hormone treatment, long-range interactions between distantly spaced cis-acting elements take place, thus allowing crosstalk between ERa, JARID1A and the transcriptional machinery at the tsp Indeed numerous long-range chromatin interactions have been detected for ERa [37,50], including interactions with the promoter-bound transcription factor Sp1 [51] These interactions might lead to conformational modifications of JARID1A and changes in protein–protein interactions that regulate its enzymatic activity The analysis of MDA-MB-231 cells which not express PR or ERa revealed that Pol II did not bind to the regulatory regions examined and that only the half-ERE region was methylated at H3K4 Importantly however, no enrichment in trimethylation was observed, in line with the fact that PR is not expressed in these cells Previous data generated in MDA-MB-231 cells showed that DNA methylation of the PR promoter at a specific CpG island led to repression and FEBS Journal 278 (2011) 1458–1469 ª 2011 The Authors Journal compilation ª 2011 FEBS A Stratmann and B Haendler that reactivation can be achieved by the DNMT inhibitor 5-aza-2¢-deoxycytidine [44] However, when treating the cells with a combination of 5-aza-2¢deoxycytidine and antiestrogen, the unmethylated PR gene remains in the repressed state [44] Our data strongly suggest that absence of H3K4 trimethylation is an important factor preventing PR expression in MDA-MB-231 cells and indicate a possible role of JARID1A in the silencing process in this cell line Because no binding of JARID1A to the PR regulatory region could be detected, the role of this demethylase may be that of an early regulator responsible for the active removal of positive marks For the later maintenance of the silenced status, DNA methylation and the absence of ER might be sufficient Future experiments will show whether for the regulation of PR expression a cross-talk exists between H3K4 methylation and DNA methylation or histone acetylation, as recently reported for other genes [52–54] A recent study showed that the H3K4 methyltransferase SMYD3 directly interacts with ERa and stimulates its activity in presence of estrogen [13] Examination of different estrogen-regulated genes such as pS2, CTSD and GREB1 indicates that this is linked to elevated H3K4 di- and trimethylation at ERE-containing regulatory regions [13] It is thus possible that the respective levels of SMYD3 and JARID1A, as well as their interaction with liganded ERa, govern the local H3K4 methylation levels at estrogen-regulated genes, and thereby their expression levels Materials and methods Plasmid construction For the design of full-length human JARID1A cDNA (NM_001042603.1), digestion of pCMV-SPORT6-JARID1A (RZPD, IRAKp961B10255Q) was performed with RsrII and the 5¢-terminal 3257 bp were obtained The 3¢terminal 1868 bp were amplified from a cDNA brain pool (QUICK-CloneÔ cDNA, BD Biosciences Clontech, San Jose, CA, USA) using the forward and reverse primers 5¢-C ATTGTTACAGGTGCTGAGCC-3¢ and 5¢-CTAACTGGT CTCTTTAAGATCCTC-3¢, transferred into the pCRÒ2.1-T OPOÒ (Invitrogen, Carlsbad, CA, USA) and partially digested with RsrII The two resulting fragments were ligated using the Rapid DNA Ligation Kit (Roche Diagnostics GmbH, Mannheim, Germany), transferred into the pCRỊ2.1-TOP plasmid and PCR-amplified with the primers 5¢-ATATGTCGACAATGGCGGGCGTGGGG-3¢ and 5¢-CGATGCGGCCGCCTAACTGGTCTCTTTAAG ATCC-3¢ The resulting product was cloned between the SalI and NotI sites of the pCMV-HA expression vector Regulation of PR expression by JARID1A (BD Biosciences Clontech) The H483A inactivating mutation was introduced into pCMV-HA-JARID1A by site-directed mutagenesis using the Quik-Change kit (Stratagene, La Jolla, CA, USA) and the primers: 5¢-CTTCTCTT CTTTTTGCTGGGCCATTGAGGATCACTGGAG-3¢ and 5¢-CTCCAGTGATCCTCAATGGCCCAGCAAAAAGAA GAGAAG-3¢ The PRB promoter luciferase (Luc) reporter construct was generated by amplifying the )711 to +29 bp region from Human Genomic DNA (Clontech, Heidelberg, Germany) using the primers 5¢-GGATCCATTTTATAAG CTCAAAGATAATTAC-3¢ and 5¢-CTTACCCCGATTAG TGACAGCTGTGGAC-3¢ The amplified fragment was then transferred into the pCRÒ2.1-TOPOÒ and subcloned into the KpnI and XhoI sites of the pGL4.26[luc2 ⁄ minP ⁄ Hygro] vector (Promega, Madison, WI, USA) Insertion of the human ERa cDNA into the pSG5 plasmid (Stratagene) was performed using standard procedures All plasmids were verified by DNA sequencing of the inserts and flanking regions Cell culture Cell lines were obtained from the American Tissue Culture Collection (LGC Promochem, Wesel, Germany) MCF-7 cells were grown at 37 °C in a 5% CO2 atmosphere in phenol-red RPMI-1640 supplemented with 10% fetal bovine serum (both from Biochrom AG, Berlin, Germany), and 100 mL)1 penicillin and 100 lgỈmL)1 streptomycin (Gibco, Invitrogen, Karlsruhe, Germany) For estrogen stimulation experiments, cells were cultured in estrogen-free medium (phenol-red-free RPMI-1640) supplemented with 10% charcoal-stripped fetal bovine serum and mm l-glutamine (Gibco) for 2-3 days prior to treatment with nm estrogen This concentration was chosen based on previous experience 17b-estradiol (ZK5018) was synthesized inhouse and dissolved in ethanol MDA-MB-231 cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco) with 10% fetal bovine serum, mm l-glutamine, 1· nonessential amino acids (NEA; Biochrom AG), and 100 mL)1 penicillin and 100 lgỈmL)1 streptomycin (Gibco) Knockdown experiments MCF-7 cells were transfected with JARID1 siRNAs (StealthÔ siRNA, Invitrogen) at a final concentration of 40 nm by using LipofectamineÔ 2000 (Invitrogen) StealthÔ RNAi Negative Universal Control Medium (Invitrogen) was transfected in the control experiments Specific siRNAs sequences for each JARID1 family member were selected For JARID1A: si#1 5¢-ATACTAACCAGCCAC CCTAGAGCTC-3¢, si#2 5¢-CAGCCTCCATTTGCCTG TGAAGTAA-3¢; for JARID1B: si#1 5¢-CACGTATCCA GAGACTGAATGAATT-3¢, si#2 5¢-GCCTTCTTGTTTG CCTGCATCATGT-3¢; for JARID1C: si#1 5¢-AGGCCC AGACGAGAGTGAAACTGAA-3¢, si#2 5¢-CGGTTTCCC FEBS Journal 278 (2011) 1458–1469 ª 2011 The Authors Journal compilation ª 2011 FEBS 1465 Regulation of PR expression by JARID1A A Stratmann and B Haendler TGTCAGTGACAGTAAA-3¢ Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse-transcription performed using the SuperScriptÔ III First-Strand (Invitrogen) and random primers Specific transcript levels were determined by real-time PCR using the following assays from Applied Biosystems (Darmstadt, Germany): JARID1A Hs00231908_m1, JARID1B Hs00366783_m1, JARID1C Hs00188277_m1, PGR Hs00172183_m1 and GAPDH 4326317E Human cyclophilin A levels were determined as control using the primers hu Cyc 4326316E Protein extracts were prepared using the Mammalian Protein Extraction Reagent (M-Per, Pierce Biotechnology, Rockford, IL, USA) supplemented with a protease inhibitor mix (Roche) and benzonase (Merck, Darmstadt, Germany) They were then analysed by western blot (see below) Cell-based transactivation For the transactivation assays, MCF-7 cells were seeded into 96-well plates at a concentration of 8000 cellsỈ80 lL)1 per well Transfection was carried out 44 h later using FuGENEÒ HD Transfection Reagent (Roche Diagnostics GmbH) in Opti-MEM Expression plasmids for wild-type JARID1A or mutant form (30 ngỈwell)1) were cotransfected together with a Luc-based reporter vector harbouring the PRB promoter (40 ngỈwell)1) and a pSG5-based ERaexpressing plasmid (80 ngỈwell)1) Hormone induction was performed 4–5 h later by adding estrogen up to 100 nm Measurement of Luc activity was carried out after 24 h in a Victor multilabel counter (Perkin–Elmer, Wellesley, MA, USA), following the addition of 100 lL of SteadyLite Plus Reagent (Perkin–Elmer) For all points the average value of six wells treated in parallel was taken Western blot analysis Total protein extracts prepared from transfected cells (see above) were analysed by western blot First, the protein lysates were separated using NuPAGE gradient gels and then transferred onto polyvinylidene fluoride membranes (both from Invitrogen) The membranes were blocked and incubated overnight with the primary antibody The antibodies were G-20, sc-544 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for ERa, A300-897A (Bethyl Laboratories, Montgomery, TX, USA) for JARID1A, ab50958 (abcam, Cambridge, UK) for JARID1B, A301-035A (Bethyl Laboratories) for JARID1C, MS-298 Ab-8 (Thermo Scientific, Rugby, UK) for PR and MAb 6C5 (Advanced ImmunoChemical, Long Beach, CA, USA) for GAPDH After incubation with the appropriate secondary antibodies (rabbit NA934V or mouse NA931V antibody from GE Healthcare, Chalfont St Giles, UK), the membranes were developed using the Western Lightning chemiluminescence kit (Perkin–Elmer) 1466 Chromatin immunoprecipitation MCF-7 and MDA-MB-231 cells were grown in 150-mm dishes in estrogen-free medium for days They were then treated with nm estrogen for h and dual cross-linked at room temperature by a 45-min incubation in mm disuccinimidyl glutarate, followed by treatment with 1% formaldehyde for 10 The reaction was stopped by adding glycine to a final concentration of 125 nm After min, the cells were washed twice with cold NaCl ⁄ Pi, detached by scraping into cold NaCl ⁄ Pi and pelleted by centrifugation at °C for at 180 g Chromatin was isolated after cell lysis in mm Pipes pH 8, 85 mm KCl, 0.5% Nonidet P40, supplemented with protease inhibitors (Roche Diagnostics GmbH) This was followed by nuclear lysis using RIPA-buffer (10 mm Tris ⁄ HCl pH 8, mm EDTA, 0.5 mm EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mm NaCl and protease inhibitors) DNA ` was sheared using the BiorupterÔ (Diagenode, Liege, Belgium) by sonicating for · 10 with a 30-s interval at high level The samples were then centrifuged at 15 700 g for 10 and at °C In order to pre-clear the chromatin solution, 60 lL of DynabeadsÒ Protein A ⁄ G (Invitrogen) were added to the cell lysates and incubated for h while rotating at °C For immunoprecipitation, we used 3–4 lg antibody specific for ERa (HC-20X, sc-543X, Santa Cruz Biotechnology), for JARID1A (A300-897A, Bethyl Laboratories), for RNA Pol II (ab5408, abcam), for H3 (ab1791 abcam), for H3K4me1 (ab8895, abcam), for H3K4me2 (ab7766, abcam) or for H3K4me3 (ab1012, abcam) Rabbit IgG (PP64, Millipore, Bedford, MA, USA) was used as control The antibodies were first pre-bound to 40 lL DynabeadsÒ Protein A ⁄ G in NaCl ⁄ Pi by incubating them for 2-3 h while rotating at °C After removing an input sample, the cell lysate was added to the prebound antibodies and incubated overnight at °C Washing was performed with buffer I (20 mm Tris ⁄ HCl pH 8, mm EDTA, 150 mm NaCl, 1% Triton X-100, 0.1% SDS), buffer II (20 mm Tris ⁄ HCl pH 8, mm EDTA, 500 mm NaCl, 1% Triton X-100, 0.1% SDS) and buffer III (10 mm Tris ⁄ HCl pH 8, mm EDTA, 250 mm LiCl, 1% Nonidet-P40, 1% sodium deoxycholate) followed by two washes with TE (10 mm Tris ⁄ HCl pH 8, mm EDTA) The immunocomplexes were eluted with 0.1 m NaHCO3, 1% SDS and the cross-link was reverted at 65 °C overnight The eluates were sequentially treated for h with RNase H and then with proteinase K (both from Qiagen) The DNA was then purified using the DNeasy Blood and Tissue kit (Qiagen) The immunoprecipitated DNA was analysed by quantitative PCR using QuantiFastÔ SYBRÒ Green PCR Kit (Qiagen) The reaction was performed at a combined annealing ⁄ extension temperature of 60 °C for 40 cycles using primers designed to specifically amplify the sequence containing the +571 ERE half-site [3] and two regions covering ER-binding sites named enhancer and enhancer FEBS Journal 278 (2011) 1458–1469 ª 2011 The Authors Journal compilation ª 2011 FEBS A Stratmann and B Haendler located at )168 and )206 kb in the PR regulatory regions [42] Primers directed against an unrelated region located in intron of the F-box and leucine-rich repeat protein 11 gene (Chromosome 11, position 66 656 165 to 66 657 244; Control in Carroll et al [42]) were used as control This region contains ERE motifs which are, however, not bound by the ER [42] For the experiments in which JARID1A was immunoprecipitated, an expression plasmid containing the corresponding cDNA needed to be transfected, due to the low sensitivity of the JARID1A antibody Regulation of PR expression by JARID1A Statistical analysis All data represent three independent experiments using cells from separate cultures and are expressed as the mean ± sample SD The results between the siRNA treatment and control groups in Figs and 5, and between the hormone-treated and untreated groups in Fig were compared using the Student’s t-test A P-value < 0.01 was considered significant 10 Acknowledgements We are indebted to Karl Ziegelbauer and Dominik Mumberg (BSP, Berlin), and to Petra Knaus (Institute of Chemistry 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indicated Fig S2 ChIP analysis of PR regulatory regions in MDA-MB-231 cells ChIP analysis of PR regulatory regions in MDA-MB-231 cells This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 278 (2011) 1458–1469 ª 2011 The Authors Journal compilation ª 2011 FEBS 1469 ... trimethylation, mainly at the half-ERE region The requirement of the demethylase activity for the repressive effects of JARID1A on PR gene expression was furthermore underlined by the luciferase assay... stimulation By contrast, the binding of JARID1A to the region close to the tsp does not depend on the hormonal status However, the activity of JARID1A is influenced by the presence of the hormone-activated... that JARID1A regulated expression of the PR gene and fine-tuned its control by ERa Reducing JARID1A expression by specific siRNAs dramatically enhanced PR expression at the basal and less so at the