- Open Access
MK3 controls Polycomb target gene expression via negative feedback on ERK
- Peggy Prickaerts1, 3,
- Hanneke EC Niessen†1,
- Emmanuèle Mouchel-Vielh†3,
- Vivian EH Dahlmans1,
- Guus GH van den Akker1,
- Claudia Geijselaers1,
- Michiel E Adriaens2,
- Frank Spaapen1,
- Yoshihiro Takihara4,
- Ulf R Rapp5,
- Frédérique Peronnet3 and
- Jan Willem Voncken1Email author
© Prickaerts et al.; licensee BioMed Central Ltd. 2012
Received: 4 April 2012
Accepted: 11 July 2012
Published: 7 August 2012
Gene-environment interactions are mediated by epigenetic mechanisms. Polycomb Group proteins constitute part of an epigenetic cellular transcriptional memory system that is subject to dynamic modulation during differentiation. Molecular insight in processes that control dynamic chromatin association and dissociation of Polycomb repressive complexes during and beyond development is limited. We recently showed that MK3 interacts with Polycomb repressive complex 1 (PRC1). The functional relevance of this interaction, however, remained poorly understood. MK3 is activated downstream of mitogen- and stress-activated protein kinases (M/SAPKs), all of which fulfill crucial roles during development. We here use activation of the immediate-early response gene ATF3, a bona fide PRC1 target gene, as a model to study how MK3 and its effector kinases MAPK/ERK and SAPK/P38 are involved in regulation of PRC1-dependent ATF3 transcription.
Our current data show that mitogenic signaling through ERK, P38 and MK3 regulates ATF3 expression by PRC1/chromatin dissociation and epigenetic modulation. Mitogenic stimulation results in transient P38-dependent H3S28 phosphorylation and ERK-driven PRC1/chromatin dissociation at PRC1 targets. H3S28 phosphorylation by itself appears not sufficient to induce PRC1/chromatin dissociation, nor ATF3 transcription, as inhibition of MEK/ERK signaling blocks BMI1/chromatin dissociation and ATF3 expression, despite induced H3S28 phosphorylation. In addition, we establish that concomitant loss of local H3K27me3 promoter marking is not required for ATF3 activation. We identify pERK as a novel signaling-induced binding partner of PRC1, and provide evidence that MK3 controls ATF3 expression in cultured cells via negative regulatory feedback on M/SAPKs. Dramatically increased ectopic wing vein formation in the absence of Drosophila MK in a Drosophila ERK gain-of-function wing vein patterning model, supports the existence of MK-mediated negative feedback regulation on pERK.
We here identify and characterize important actors in a PRC1-dependent epigenetic signal/response mechanism, some of which appear to be nonspecific global responses, whereas others provide modular specificity. Our findings provide novel insight into a Polycomb-mediated epigenetic mechanism that dynamically controls gene transcription and support a direct link between PRC1 and cellular responses to changes in the microenvironment.
Mitogen- and stress-activated protein kinase (M/SAPK) signaling pathways relay environment-to-gene information and enable physiologically appropriate cellular responses . MK3 is an interaction partner of extracellular signal-regulated kinase (ERK) and P38 and is targeted by all three M/SAPK signaling cascades ; these phosphorylation cascades induce multiple responses, among which is altered gene transcription [3, 4].
We previously demonstrated that mitogen-activated protein kinase-activated protein kinase 3 (MK3/3pK/MAPKAPK3) binds Polycomb repressive complex 1 (PRC1), via the self-association motif (SAM)-domain of PHC . Polycomb Group and Trithorax Group proteins maintain transcriptionally repressed and activated epigenetic states respectively and as such are part of an important cellular transcriptional memory system. Although PRC1-mediated transcriptional repression has long been considered a stable repressive state, increasing evidence indicates that PRC1 repression is a dynamic process [6, 7]. Genome-wide chromatin association studies have revealed changes in Polycomb/chromatin distribution and transcriptional reprogramming during lineage commitment and differentiation [8–10]. As M/SAPKs and MKs also play important roles in differentiation, development and cell proliferation, the physical association of PRC1 and MK3 suggests a functionally relevant connection. We have previously established that PRC1/chromatin dissociation correlates with their phosphorylation status during cell cycle progression . Subsequently, we showed that acute mitogenic and stress signaling also results in PRC1/chromatin dissociation . The molecular mechanisms that underlie this dynamic relocation of PRC1 and the exact role of MK3 in signaling to chromatin remained unknown. We here hypothesized that M/SAPK-MK signaling imposes a molecular mechanism by which cells regulate PRC1/chromatin association and PRC1 target gene expression in response to environmental cues. We used the previously identified PRC1 target gene ATF3 to study PRC1-mediated transcriptional regulation . ATF3 is an immediate-early response gene (IEG) ; relevantly, ERK and MK have been implicated in IEG activation . Therefore, mitogen-induced ATF3 activation represents a suitable model to study the biological relevance of ERK/MK3/PRC1 signaling. Our current data establish that ATF3 expression is dynamically controlled by MAPK/MK/PRC1, and identify important players in an epigenetic switch-module in response to changes in the cellular microenvironment.
Results and Discussion
M/SAPK signaling controls PRC1 target gene transcription
Transcription correlates with PRC1 dissociation, not loss of H3K27me3
Consistent with a global mitogen-induced increase of H3S28ph, ChIP analysis shows increased H3S28ph enrichment at PRC1 targets as well as non-targets; this epigenetic change occurs independent of transcriptional induction (Figure 2B, Additional file 2: Figure S2A). Remarkably, the unaltered H3K27me3 occupation at the ATF3 promoter suggests that transcriptional activation of PRC1 target genes occurs without concomitant loss of H3K27me3 (Figure 2A). Thus, although PcG-NB/chromatin dissociation and H3S28ph correlate well [5, 24, 25], H3S28ph by itself does not determine transcriptional status. In addition, our findings argue that local maintenance of H3K27me3 does not obstruct transcriptional reactivation of PCR1 targets. The recently reported presence of H3K27me3 marks on active promoters supports this notion . Whether or not the H3K27me3 mark needs to be removed at reactivated genes remains contradictory at this point [24–26], and may, besides on models and tools used, depend on the subgenic location of epigenetic modifications (possibly in conjunction with other modifications). Consistent with this idea, repressive H3K27me3 marking correlates best with transcription at loci that display ‘blanketing’-type H3K27me3 enrichment, that is, gene body-wide (downstream of the transcription start site (TSS)) . It is conceivable that H3K27me3 promoter marking defines a specific class of response factors, which are dynamically controlled by rapid removal of PRC1 complexes. Thus far, our H3S28ph analyses suggest that H3S28ph, like CBX8 dissociation, is a global event; despite being essential for PRC1 target gene activation, it is clearly not the sole determining factor and as such is unlikely to specify regulation only at PRC1 target genes. Co-occurrence of methylphosphoryl modifications on adjacent histone lysines and serines was proposed to act as a binary epigenetic switch mechanism, by affecting chromobox-domain binding to histone-trimethyl marks [24, 27–29]; Interestingly, also EZH2/chromatin dissociation at muscle-specific promoters during myogenesis appears driven by local H3K27 methyl/H3S28 phosphoryl modification [30, 31]. The simultaneous detection of local H3S10ph and HP1 binding, however, seemingly challenges the strictness of the methylphosphoryl switch concept . Irrespective of the exact mechanism, our combined molecular epigenetic analyses suggest PRC1 target gene activation does not require loss of H3K27me3 and point to chromatin dissociation of multiple PRC1 proteins as an important event in transcription initiation.
MK3 negatively regulates ERK signaling
Consistent with our IF analyses (compare Figure 1C, Additional file 1: Figure S1B), H3S28 shows rapid and transitory phosphorylation kinetics in response to mitogens (Figure 3E). Relevantly, global H3S28ph is increased and sustained in MKi cells, whereas it is blunted in MK3OE cells (Figure 3)E in line with altered PRC1 target gene expression under these respective conditions (compare Figure 3A,D). This data demonstrates that H3S28 phosphorylation kinetics respond to MK3 activity, and support a regulatory role for MK3 in epigenetic modulation of cell responses to environmental stimuli.
To gain further insight into the molecular mechanisms by which MK3 controls M/SAPK activity, we studied MEK, an ERK effector kinase. MEKpT292 and MEKpS298 were examined, as these two phosphoresidues are part of a mechanism that controls MEK activity . Like pERK, MEKpT292 is also sustained in MK-deficient cells, suggesting that MK3 controls phosphatase activity directed toward upstream kinases (Figure 3)F. Dual-specificity phosphatases (DUSPs) play important roles in feedback loops on phosphorylation cascades . DUSP2 and DUSP5 induction meet the criteria of being dependent on mitogenic signaling and on MK, as MK3OE and MKi, enhance and decrease DUSP mRNA induction, respectively (Figure 3G). To validate the involvement of DUSPs in the negative regulatory network involving MK3, we used induction of EGR1 as a read-out for mitogen-induced ERK activity . MKi cells display a stronger EGR1 induction (Additional file 4: Figure S4A), consistent with loss of negative regulation via ERK. To validate a role for phosphatase activity in regulation of the ERK/EGR1 response, cells were pretreated with orthovanadate, a generic DUSPs inhibitor (DUSPi). Enhanced EGR1 expression in the presence of DUSPi resembles that observed in MKi cells and supports a negative regulatory role for DUSPs in the MK3/ERK/EGR1 response (Additional file 4: Figure S4B); in line with reduced negative feedback in DUSPi cells, both pERK and pP38 are increased in response to mitogenic stimulation. Relevantly, also signaling-induced H3S28ph levels are enhanced significantly in the presence of DUSPi, in agreement with a functional role for DUSPs in epigenetic regulation (Additional file 4: Figure S4B). Relevantly, the reduced ERK phosphorylation by MK3OE is partially reversed by DUSP inhibition, suggesting that DUSP activity acts as part of a relay system in the regulatory feedback of MK3 to MEK/ERK (Additional file 4: Figure S4C). Combined, the above findings support a negative regulatory role for MKs in MEK/ERK signaling, at least in part, via induction and/or activation of phosphatases.
Cell culture, viral infections
Human U2-OS osteosarcoma cells and TIG3 primary human fibroblasts expressing the murine ecotropic receptor were kindly provided by Dr. D. Shvarts (Utrecht Medical Center, Utrecht, The Netherlands) and Dr. D. Peeper (Netherlands Cancer Institute (NKI), Amsterdam, The Netherlands), respectively. Expression vectors encoding the murine ecotropic receptor were a courtesy of Dr. R. Bernards (NKI, Amsterdam, The Netherlands). All human cell lines were cultured under standard conditions in DMEM containing 10% fetal calf serum (FCS). Retroviral expression vectors were used to maximize percentages of expressing cells and to minimize integration effects. Production of infectious viral particles was carried out as described previously . Briefly, ecotropic retroviral supernatants were produced by transfection of producer cells using calcium-phosphate co-precipitation. Forty to forty-eight hours post-transfection, the supernatants were harvested, filtered and stored at −80°C until further use. Viral titers were sufficiently high to achieve near 100% infection. Cells were transduced with retrovirus in the presence of 4 μg/ml polybrene (Sigma-Aldrich, St. Louis, MO, USA) at around 25% confluency for six to eight hours and then allowed to recover for forty-eight hours on fresh medium before selection pressure was applied. Transduced cells were grown for ± 1 week on 1 to 4 μg/ml puromycin (Sigma) preceding experiments. Expression of all plasmid constructs was verified by immunoblotting.
Cells were serum-starved at 0.1% FCS for 48 hours. Mitogenic stimulation was achieved by supplementing 15% FCS/100 ng/ml tetradecanoylphorbol acetate (TPA; Sigma) for 45 minutes or as indicated. Cells were pretreated with kinase inhibitors: 30 minutes 10 μM MEK inhibitor (U0126; Promega, Madison, WI, USA), 30 minutes 10 μM MK inhibitor (MK2a Inhibitor; Calbiochem/Merck, Darmstadt, Germany), 30 minutes 20 μM p38 inhibitor (SB202190; Calbiochem), 30 minutes 25 μM JNK inhibitor (SP600125; Biomol, Plymouth Meeting, PA, USA), 30 minutes 85 μM etoposide (ETP; Calbiochem). MK2a was confirmed to inhibit MK3 (data not shown). Cells were preincubated with sodium orthovanadate for 30 minutes prior to mitogenic stimulation at indicated concentration; control cells were incubated with solvent. Sodium selenite (Sigma) -treated cells (90 minutes 0.2 mM) were used as a positive control. Drosophila S2 cells were cultured at 25°C in Schneider medium with 10% FCS. The day before transfection, 5.106 cells were transferred in medium with 0.1% FCS. For transfection, 2 μg of plasmid DNA were mixed with Effecten™ transfection reagent (Qiagen, Hilden, Germany) according to manufacturer’s instructions (1/10 DNA-Effecten™ ratio). Forty-eight hours later, cells were either untreated, stimulated with 10% FCS/100 ng/ml TPA or 0.5 mM sodium meta-arsenite for two hours at 25°C. For immunoprecipitation (IP) experiments, plasmids Act::Myc-Ph and Act::EGFP-MAPKAP were co-transfected into S2 Drosophila cells grown in 0.1% FCS. IPs were performed as described (Mouchel-Vielh et al., 2011); IP antibodies are listed in Additional file 6: Table S1.
Retroviral vectors (pBMN-LZRS.ires.GFP, pBMN-LZRS.ires.NEO) expressing murine BMI1-2Py or GST-MK3 have been described elsewhere [5, 11]. Retroviral systems were used as published [51, 52]. RNA-interfering MK3 sequences were cloned into stable shRNA vectors ; targeting sequences: Additional file 7: Table S2.
The dMK2 cDNA was amplified from clone SD05481 (Drosophila Genomics Resource Center; for oligonucleotides: see Additional file 7: Table S2). The resulting amplicon was introduced into pENTR/D-TOPO™ (Invitrogen, Carlsbad, CA, USA), then transferred through LR-recombination into T. Murphy’s vectors pAWG (https://dgrc.cgb.indiana.edu/vectors). Similarly, a cDNA corresponding to the C-terminal part of Drosophila Polyhomeotic-proximal gene (Ph or Ph-p) was amplified, introduced in pENTR/D-TOPO™, and subsequently transferred in pAMW (for oligonucleotides: see Additional file 7: Table S2) . Ph sequences in the Act::myc-Ph construct endoded the 346 C-terminal amino acids of the Ph-proximal protein (GenPept qualifiers: NP_476871.2 GI:24639272) and included the SAM domain through which MK3 and PHC1/2 interact .
Chromatin immunoprecipitation (ChIP) assays
ChIPs on primary human fibroblasts were performed and analyzed essentially as described . Cells were fixed for 10 minutes in 1% formaldehyde/phosphate buffered saline (PBS) and stopped by 5 minutes incubation in glycine (final concentration: 0.125 M). Fixed cells were washed twice with PBS and harvested in SDS buffer (50 mM Tris at pH 8.1, 100 mM NaCl, 0.5% SDS, and 5 mM EDTA), supplemented with protease inhibitors (aprotinin, antipain and leupeptin all at 5 μg/ml and 1 mM PMSF). Cells were pelleted by centrifugation, and suspended in IP buffer (100 mM Tris at pH 8.6, 100 mM NaCl, 0.3% SDS, 1.7% Triton X-100, and 5 mM EDTA), containing protease inhibitors. Cells were disrupted by sonication, yielding genomic DNA fragments with a bulk size of 200 to 500 bp. For each IP, 1 ml of lysate was precleared by adding 35 μl of blocked protein A beads (Protein A-Sepharose/CL-4B (GE Healthcare, Piscataway, NJ, USA); 0.5 mg/ml fatty acid-free BSA (Sigma); and 0.2 mg/ml herring sperm DNA in TE buffer), followed by centrifugation. 10 μl aliquots of precleared suspension were put aside as input DNA and kept at 4°C. Samples were immunoprecipitated overnight at 4°C. HA antiserum was used as negative control. Immune complexes were recovered by adding 40 μl of blocked protein A or G beads (GE Healthcare) and incubated for 4 hours at 4°C. Beads were washed three times in 1 ml of mixed micelle buffer (20 mM Tris at pH 8.1, 150 mM NaCl, 0.2% SDS, 1% Triton X-100, 5 mM EDTA, and 5% w/v sucrose), twice in 1 ml of buffer 500 (50 mM HEPES at pH 7.5, 1% Triton X-100, 1 mM EDTA, and 0.1% w/v sodium deoxycholate), twice in 1 ml of LiCl detergent wash buffer (10 mM Tris at pH 8.0, 1 mM EDTA, 0.5% sodium deoxycholate, 0.5% NP-40, and 250 mM LiCl), and once in 1 ml TE buffer. Immune complexes were eluted from beads in 250 μl elution buffer (1% SDS and 0.1 M NaHCO3) for 2 hours at 65°C (continuous shaking at 1000 rpm), and after centrifugation, supernatants were collected. 250 μl elution buffer was added to input DNA samples and these were processed in parallel with eluted samples. Cross-links were reversed overnight at 65°C, followed by 2 hours digestion with RNase A at 37°C and 2 hours proteinase K (0.2 μg/μl) at 55°C. DNA fragments were recovered using QIAquick PCR purification columns, according to manufacturer’s instructions (Qiagen, Hilden, Germany). Samples were eluted in 75 μl EB buffer and then diluted 1:5 in TE buffer. Immunoprecipitated DNA was quantified by real-time PCR (for ChIP antisera, primers: see Additional file 6: Tables S1, Additional file 7: Table S2). Each experiment was performed in triplicate. Results of one representative experiment are shown.
RNA isolation, cDNA synthesis, real-time (RT) PCR analysis
For RT-PCR analysis, total RNA was isolated using Tri Reagent (Sigma) according to the manufacturer’s protocol. Quantity and quality of the RNA were determined by 260/280 nm and 260/230 nm absorbance measurements, respectively, using the Nanodrop (Witec AG, Luzern, Switzerland). Total RNA (1 μg) for each sample/replicate was converted into first strand cDNA using the iScript™ cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Gene expression was determined by RT-PCR using the MyiQ™ thermal cycler (Bio-Rad) in combination with the IQ5 version 2.1 software (Bio-Rad). RT-PCR was performed on 25 ng of cDNA using the qPCR iQ™ Custom SYBR™ Green Supermix with fluorescein (Bio-Rad) and 300nM primer in 96-well plates (Bio-Rad). For each primer pair a standard curve was generated with a serial dilution of a cDNA pool. RT-PCR data was analyzed according to the relative standard curve method. All values were normalized to either beta-Actin (Additional file 2: Figure S2) or cyclophillin A (Figures 1,3, Additional file 1: Figure S1, Additional file 3: Figure S3). The control condition is used as a reference. Primer sets for the selected genes were developed with Primer Express version 2.0 (Applied Biosystems, Foster City, CA, USA) using default settings (Additional file 7: Table S2).
Cells were grown on 6- or 12-well culture plates (Greiner Bio-One, Alphen aan de Rijn, The Netherlands) to ± 60 to 80% confluency, pretreated when applicable, washed twice with PBS, and either fixed for 15 minutes in 2% formaldehyde/PBS at room temperature (RT) followed by a 15 minutes incubation in chilled 100% methanol (MetOH) at −20°C, or directly fixed in 100% MetOH for 15 minutes at −20°C. Fixed plates were stored at 4°C in 70% ethanol (EtOH) or directly washed three times with PBS and used for immunocytochemistry (IC). For detection of PRC1 proteins and histone modifications, cells were first permeabilized for 10 minutes at RT in 0.2% Triton X-100 (TrX) in PBS. After extensive washing in PBS/0.02% TrX, cells were incubated with primary antibody (Additional file 6: Table S1) for 1.5 to 2.5 hours in a prewarmed humidified chamber at 37°C, washed five times in PBS/0.02% TrX and incubated with fluorescently labeled secondary antibody for 60 minutes at 37°C. 4′-6-diamidino-2-phenylindole (DAPI) was co-incubated with secondary conjugated antibodies to counterstain cell nuclei. Plates were washed in PBS/0.02% TrX, rinsed in PBS and subsequently dehydrated: 1 minute in 70% EtOH, two times 1 minute in 100% EtOH and air-dried. Cells were mounted in Vectashield (Vector Laboratories, Inc. Burlingame, CA, USA), analyzed using a Nikon TE200 Eclipse fluorescence microscope and photographed using a Nikon DXM1200 digital camera in combination with NIS-Elements 3.0 imaging software. All antibodies were diluted in blocking buffer (1% BSA, 5% FCS, 5% normal goat serum (NGS), in PBS/0.02% TrX). Secondary antisera used were goat-anti-mouse Texas Red™ (TXRD; 1:100; Southern Biotech, Birmingham, LA, USA) and goat anti-rabbit fluorescein isothiocyanate (FITC; 1:100; Southern Biotech), to detect monoclonal and polyclonal primary antibodies respectively.
Protein isolation, differential extraction, immunoprecipitation (IP), immunoblotting (IB)
Protein extraction and immunoblotting (IB) were performed and analyzed as described previously with minor adjustments [2, 5]. For extraction, cells were washed twice with cold PBS and lysed in RIPA buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 0.1% SDS, 5 mM EDTA, 0.5% w/v sodium deoxycholate, and 1% NP-40) supplemented with protease and phosphatase inhibitors (5 mM benzamidine, 5 μg/ml antipain, 5 μg/ml leupeptin, 5 μg/μL aprotinin, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM pyrophosphate, 10 mM ß-glycerophosphate, 0.5 mM DTT, and 1 mM PMSF). Lysates were subjected to two freeze-thaw cycles in liquid nitrogen, followed by sonication on ice with a probe sonicator (Soniprep 150; MSE, London, UK) for 12 cycli (1 sec ON, 1 sec OFF) with amplitude 5. After 10 minutes centrifugation at 13200 rpm (4°C; Eppendorf centrifuge), the supernatant was transferred to a fresh tube and protein concentration was determined using a BCA protein assay kit (Pierce/Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s protocols on a Benchmark 550 microplate reader (Bio-Rad).
For differential extraction, cells were washed two times with cold PBS and scraped in lysis buffer (Tris HCl at pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA; supplemented with inhibitors). After 30 minutes incubation on ice, nuclei are collected in the pellet by centrifugation (8000 rpm; 4°C) the supernatant is the cytoplasmic fraction. Nuclei are washed in lysis buffer and suspended in ELB buffer and incubated on ice for 10 minutes. Nuclear soluble (supernatant) and chromatin-bound fractions (pellet) are separated by centrifugation (13200 rpm; 4°C). After an additional wash in ELB buffer, the pellets were suspended in ELB buffer and sonicated.
For IP, cells were either cross-linked followed by cell lysis, or cells were lysed directly in ELB buffer (50 mM HEPES at pH 7.0, 250 mM NaCl, 5 mM EDTA, and 0.1% NP-40) supplemented with 5 mM benzamidine, 5 μg/ml antipain, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 1 mM sodium vanadate, 10 mM sodium fluoride, 10 mM pyrophosphate, 10 mM ß-glycerophosphate, 0.5 mM DTT, and 1 mM PMSF. For cross-linking, cells were incubated in 1% formaldehyde (in PBS) for 10 minutes at RT, fixation was stopped by addition of 2 M glycin to a final concentration of 0.125 M. After 5 minutes incubation, cells were washed twice in cold PBS and lysed in ELB buffer. IP was carried out as described [11, 15]. Briefly, extracts were sonicated on ice, centrifuged for 10 minutes at 4°C at 13200 rpm and supernatants were transferred to a precooled tube; 10% of the supernatant was taken as input and stored at −80°C. Appropriate amount of antiserum (Additional file 6: Table S1) was added and tubes were rotated for 1 hour at 4°C on a spinning wheel. To precipitate immune complexes, washed protein G beads (Protein G Sepharose/4 Fast Flow; GE Healthcare) were added to the extracts and rotated for 3 to 4 hours at 4°C, followed by 3 minutes centrifugation at 3000 rpm at 4°C. Supernatant was collected as depleted fraction and stored at −80°C. Beads were washed four times in ELB buffer (with supplements) and stored dry at −80°C until IB analysis.
For IB, equal protein amounts were boiled in sample buffer for 5 minutes and loaded on 9 to 15% polyacrylamide gels. Following separation by SDS-PAGE, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare). Ponceau S (Sigma) staining was used to check protein transfer. PVDF membranes were blocked with 3.4% non-fat dry milk (Protifar; Nutricia, Zoetermeer, the Netherlands) in PBS containing 0.1% Tween 20 (pH 7.5) for 1 hour at RT, followed by an overnight incubation at 4°C with the primary antibody (Additional file 6: Table S1; anti-CBX4 ). After extensive washing with PBS/0.2% Tween 20, membranes were probed with corresponding horseradish peroxidase-conjugated secondary antibodies for 1 hour at RT: goat anti-rat (7077; 1:2,000; Cell Signaling, Danvers, MA, USA), rat anti-mouse (P0260; 1:5,000; DAKO, Glostrup, Denmark) and donkey anti-rabbit (711035-152; 1:15,000; Jackson Lab, Bar Harbor, ME, USA), to detect monoclonal rat, monoclonal mouse and rabbit polyclonal primary antibodies respectively. Signals were detected on autoradiograms using enhanced chemoluminescence (ECL; Pierce). Intensity of the bands was quantified with Quantity One software (Bio-Rad).
Drosophila melanogaster stocks and crosses were kept on standard media at 25°C. For crosses, five females were mated with five males; they were transferred each 48 hours in new tubes. The w 1118 line was used as control line. The UAS::rolled line was a gift from Dr. K. Moses (University of Cambridge, Cambridge, UK); the UAS:: -p38b was a gift from Dr. JM Gibert (University of Geneva, Geneva, Switzerland); UAS::rolled and UAS::D-p38b allow dERK and -p38b overexpression, respectively [38, 56]. The v3171 line (w 1118 ; MAPk-Ak2GD1597) that downregulates dMK2 by RNA interference, was purchased from the Vienna Drosophila RNAi Center (VDRC) ; it does not present any known off-target effects. Downregulation of dMK2 in third instar larvae was verified by real-time (RT) PCR using primers located outside the v3171 repeats (Additional file 5: Figure S5B) as described . Results were normalized against rp49 or Spt6 (Additional file 7: Table S2). Transgene overexpression was achieved using the wing-specific Gal4 transgenic driver scalloped sd 29.1 ( called sd::Gal4; BL-8609 line; Bloomington Drosophila Stock Center (BDSC), Bloomington, IN, USA)). All the transgenic lines display Gal4-independent mini-white expression that allows tracing of transgene transmission. dMK2-LOF and hMK3-GOF transgenic flies were also crossed to mutant Polycomb lines to study a potential interaction of MK and PcG in vivo. Neither hMK3- GOF nor dMK2- LOF induce a discernable sexcomb-phenotype by themselves or in combination with established Pc 1 and Scm D1 alleles in heterozygote crosses (data not shown). The most likely explanation for this lack of phenotype is that dMK2 may not play a role in anteroposterior (AP) patterning. In support of the latter, AP-axis abnormalities have not been reported in single or double MK2/3 knockout mice .
We thank S. Bloyer, A. Catling, J. Geraedts, K. Hansen, M. Inagaki, M. van Lohuizen, S. Ludwig, M. Mooij, A. Otten, M. Schepens, R. Sverdlov, T. van de Weijer, B. Wouters, the MAASTRO and MolGen departments for research materials, technical support and scientific discussions. Financial support: Dutch Science Organization (ZonMW-NWO): Research Support grant 908-02-040 (JWV); VIDI grant 016.046.362 (JWV), joint van Gogh grant (JWV, FP); tUL Grant (JWV); VSB Fonds scholarship (PP); Netherlands Genome Initiative (NGI) fellowship 050-72-422 (HN); UPMC and CNRS (FP).
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