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First published online 10 February 2009
doi: 10.1242/jcs.037556
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Short Report |
Department of Cell Death and Proliferation, Institute for Biomedical Research of Barcelona, IIBB-CSIC-IDIBAPS, Barcelona, Spain
* Author for correspondence (e-mail: spfnqi{at}iibb.csic.es)
Accepted 10 November 2008
Summary
Murine Mash1 (Ascl1) is a member of the basic helix-loop-helix family of transcription factors and has been described to promote differentiation in some neural precursors. The process of differentiation is coordinated with a concomitant cell-cycle arrest, but the molecular mechanism of this process is unclear. Here, we describe for the very first time a direct regulation of an oncogene by a proneural gene. When expressed in proliferating cerebellar granular precursors, expression of the proneural gene encoding Mash1 promotes cell-cycle exit and differentiation, whereas expression of the oncogene MYCN has the opposite effect, promoting the proliferation of these cells in the absence of sonic hedgehog. Moreover, Mash1 overexpression neutralizes MYCN-induced proliferation. We now propose that the mechanism of antagonism between both molecules is based on opposite functions in the transcriptional regulation of the E-box motif, particularly in the E-boxes within the cyclin-D2 promoter, with MYCN acting as a transcriptional activator and Mash1 as a repressor. In agreement with this result, overexpression of cyclin D2 suppressed the anti-proliferative activity of Mash1.
Key words: MYCN, Mash1, Cyclin D2, Proneural, Oncogene, Cerebellar granular neuronal precursors
Introduction
During the development of the cerebellum, cerebellar granular neuronal precursors (CGNPs), which are located in the external granular layer (EGL), actively proliferate in response to the sonic hedgehog (Shh) protein produced by Purkinje cells. Concomitantly, CGNPs occupying the internal face of the EGL stop proliferating and migrate through the Purkinje-cell layer towards their final disposition in the internal granular layer (IGL) (Cajal, 1995
; Marti and Bovolenta, 2002).
Shh binds to its membrane receptor Patched (Ptc), activating Smoothened, which propagates the signal to the cytoplasm. Activated Smoothened reduces PKA activity through the inhibition of adenylate cyclase. Under these conditions, the transcription factor Gli2 and/or Gli3 (collectively referred to as Gli2/3) is translocated to the nucleus, promoting the transcription of Gli1 and MYCN, ensuring cell-cycle progression. In the absence of Shh, active PKA phosphorylates Gli2/3, which is cleaved by the proteasome so that it loses its transactivator domain. Gli2/3 then becomes a potent repressor of the pathway, shutting down the proliferative response (Dahmane and Altaba, 1999; Wechsler-Reya and Scott, 1999).
Shh activity can be counteracted through activating the PKA pathway with the pituitary adenylate-cyclase-activating polypeptide (PACAP) (Suh et al., 2001), with forskolin, with the cAMP analog dibutyril cAMP (DBA) or with the extracellular-matrix protein vitronectin (Pons et al., 2001). Shh can be also repressed through PKA-independent signals such as bone morphogenetic proteins (BMPs) (Alvarez-Rodriguez et al., 2007; Rios et al., 2004) or basic fibroblast growth factor (bFGF) (Fogarty et al., 2007).
MYCN belongs to the basic helix-loop-helix (bHLH) family of transcription factors and is one of the main downstream effectors of the Shh pathway (Hatton et al., 2006). MYCN-Max heterodimers are transcriptional activators through binding to E-box motifs (CANNTG). Different genes involved in cell-cycle regulation, such as that encoding cyclin D2, have E-box motifs in their promoters (Bouchard et al., 1999; Bouchard et al., 2001). Murine Mash1 (also known as Ascl1) is also a member of the bHLH family. The heterodimer Mash1-E12 (ELSPBP1) binds E-box motifs, acting as a repressor or activator depending on the availability of the Hes factors and on cellular context (Dambly-Chaudiere and Vervoort, 1998). Consistently, Mash1 has been shown to promote neuronal differentiation during retinal development and its expression has been reported to promote differentiation in some other neuronal precursors, whereas, in neuroblastoma cells, the level of Hash1 (the Mash1 homolog in humans) decreases concomitantly with progression of the differentiation process (Ichimiya et al., 2001; Tomita et al., 1996; Verma-Kurvari et al., 1996).
Differentiation of neuronal precursors is characterized by a loss of multipotency and, thus, an irreversible cell-cycle exit. In P19 embryonal carcinoma cells, the expression of some bHLH proteins (e.g. NeuroD2, Mash1) demonstrated a potent proneural activity when co-transfected together with their partner E12. Simultaneously to the differentiation program, an antiproliferative response was also induced through the upregulation of the cyclin kinase inhibitor p27kip1 (Farah et al., 2000). However, it is not clear whether this p27kip1 upregulation is a cause or consequence of the cell-cycle arrest.
In the context of CGNPs, a microarray transcriptional profile performed in our laboratory demonstrated that Mash1 expression is upregulated in the presence of certain anti-mitotic stimuli, such as BMP2 (ArrayExpress, accession number E-MEXP-1129); in addition, Mash1 is expressed in the IGL of the cerebellum during development (Schuller et al., 2006). These facts suggest a proneural role for Mash1 in the context of the CGNPs.
Here, we report that Mash1 ectopic expression in proliferating CGNPs induces cell-cycle exit and differentiation, opposing the Shh mitotic signal. We also show that MYCN-induced proliferation is suppressed when Mash1 is coexpressed. Moreover, MYCN and Mash1 display opposed transcriptional activities in the regulation of the E-box sequences in CGNPs: whereas MYCN is as an activator, Mash1 acts as a transcriptional repressor. Furthermore, we also found that the cyclin-D2 promoter, which contains two E-boxes, is also regulated by MYCN and Mash1 in an opposing manner. Consistently, expression of cyclin D2 can counteract the anti-proliferative effect of Mash1. Together, these data support a model in which the proneural factor Mash1 can directly antagonize the action of the oncogene MYCN by competing directly for the occupancy of the E-box sequences within the cyclin-D2 promoter, providing a new paradigm for the anti-mitotic activity of bHLH proneural genes.
Results and Discussion
Mash1 expression promotes cell-cycle exit and differentiation in CGNPs
To confirm the proneural activity of Mash1, CGNP cultures were electroporated with a bicistronic expression plasmid containing Mash1 plus GFP or empty vector (pCIG), and plated in the presence of Shh. Different parameters were measured at 24, 48 and 72 hours post-transfection, such as proliferation (BrdU incorporation; Fig. 1A), differentiation (β-tubulin-III staining; Fig. 1B) and apoptosis (TUNEL assay; Fig. 1C). In each case the percentages of proliferating, differentiated or apoptotic cells were calculated considering only the transfected population (GFP positive). Although, at longer periods of time (48 and 72 hours), Mash1 expression in CGNP cultures promoted a robust cell-cycle arrest and terminal differentiation, in the short-term (24 hours) it also protected against apoptosis. This anti-apoptotic activity is most probably due to the anti-proliferative activity itself, because non-cycling cells are more resistant to the stress induced by transfection. The anti-proliferative activity of Mash1 was observed as soon as 24 hours post-transfection, with reduction in BrdU incorporation of more than a half and almost no proliferation detected at 72 hours. The differentiated phenotype, however, was only evident at 48 hours after transfection. Thus, Mash1 expression was able to overcome the mitotic activity induced by Shh.
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; Oliver et al., 2003), which presents a poorer prognosis. The central role of MYCN on CGNP proliferation encouraged us to study the possible implication of MYCN in the mechanism used by Mash1 to counteract Shh-induced proliferation. CGNPs were co-electroporated with the indicated amounts of MYCN, Mash1 or empty vector and, after 48 hours, proliferation was measured through [3H]-thymine incorporation. Interestingly, Mash1 was able to suppress mitotic activity of MYCN in a dose-dependent manner (Fig. 2A). These data demonstrated a competition between MYCN and Mash1 to control CGNP proliferation.
To delve further into the nature of such competition, we next studied the expression of cyclin D2, a direct downstream gene of MYCN. Previous publications have demonstrated that MYCN binds directly to the E-box sequences within the cyclin-D2 promoter, increasing its expression (Bouchard et al., 1999). Moreover, cyclin D2 and MYCN show overlapping expression patterns in the EGL, and mice lacking the gene encoding cyclin D2 have reduced mitotic activity in the EGL. As expected, when expressed in cultured CGNPs, cyclin D2 promoted proliferation but, unlike MYCN, it was unaffected by Mash1 expression (Fig. 2B). Consistently, coexpression of MYCN and cyclin D2 could also suppress the anti-proliferative response to Mash1. These data strongly suggest that the mechanism used by Mash1 to antagonize MYCN-induced proliferation includes the regulation of the cyclin-D2 gene.
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; Johnson et al., 1992). Although MYCN always acts as an activator, the role of Mash1 is more ambiguous, acting as a repressor or an activator depending on the cellular context. It was conceivable, therefore, that MYCN and Mash1 could be competing for the E-box sequences within the cyclin-D2 promoter and, once bound, have opposite functions. In order to test this hypothesis, we studied the ability of MYCN to upregulate the cyclin-D2 promoter and also whether MYCN was affected by the presence of Mash1. As expected, MYCN expression consistently increased the activity of the cyclin-D2 promoter. However, when Mash1 was coexpressed together with MYCN, the repressor activity of Mash1 prevailed (Fig. 3A). In order to assess whether the competition between MYCN and Mash1 occurred at the E-box level, we tested the ability of Mash1 to repress MYCN activity in an artificial promoter containing five copies (5x) of a consensus E-box motif (CACGTG). Interestingly, Mash1 was able to suppress the activation induced by MYCN on the 5x E-box promoter (Fig. 3B). These data clearly demonstrate that MYCN and Mash1 have opposite roles in the regulation of the cyclin-D2 promoter and E-box sequences in CGNPs. Furthermore, we performed a chromatin immunoprecipitation (ChIP) analysis to study the binding of both MYCN and Mash1 to the cyclin-D2 promoter E boxes in non-transfected CGNP cultures (Fig. 3C). Both factors were found to associate to the 5' E-box of the cyclin-D2 promoter, whereas we failed to detect association using primers directed at the 3' E-box or the coding region (data not shown). Interestingly, the addition of BMP2 to cells growing in the presence of Shh clearly increased the amount of E-box–Mash1 complex; simultaneously, a moderate but consistent decrease of the MYCN bound to that sequence could be observed. Finally, and supporting all the previous data, western blot analysis of CGNPs that were transfected with MYCN alone or MYCN and Mash1 showed that endogenous cyclin-D2 protein levels were increased by MYCN expression and decreased to their basal levels when Mash1 was co-electroporated. The fact that MYCN protein levels were unaffected by the presence of Mash1 further supports the competition model (Fig. 3D). Interestingly, previous work from our laboratory demonstrated that one of the mechanisms used by BMP2 to promote cell-cycle exit in CGNPs is a downregulation of MYCN levels through an inhibition of MYCN mRNA transcription. In this case, repression of the MYCN promoter was mediated by the transcription factor TIEG-1, which is produced in response to BMP2 (Alvarez-Rodriguez et al., 2007). Now, these novel data suggest that BMP2 carries out two redundant regulatory actions on MYCN, one upstream, decreasing MYCN mRNA levels through TIEG-1, and another downstream, regulating MYCN transcriptional activity through Mash1. Concordantly, the levels of the neuronal differentiation marker β-tubulin III were increased in cells coexpressing MYCN and Mash1.
The present work describes for the very first time a competitive interaction between an oncogene (MYCN) and a proneural gene (Mash1) in the context of CGNP proliferation. Together, these data demonstrate that MYCN and Mash1 posses opposite activities in the control and regulation of proliferation, and suggest that the main mechanism for this counteraction is the differential regulation of the E-boxes within the cyclin-D2 promoter, with MYCN acting as an activator and Mash1 as a repressor (Fig. 3E). In accordance with its possible role as a tumor suppressor in CGNPs, Hash1 is not expressed in infratentorial primitive neuroectodermal tumors samples (PNET) such as medulloblastoma, whereas supratentorial PNET samples are usually positive for this bHLH factor (Rostomily et al., 1997). The present work presents a novel point of view of proneural genes in the control of proliferation and raises the issue of whether loss of these proneural genes is necessary for the development of some neural tumors such as medulloblastoma. Future work should be aimed at clarifying the role of other proneural genes in another cellular context, at determining the control and regulation of the proliferation, and at assessing the role of proneural bHLH factors as putative tumor suppressors.
Materials and Methods
CGNP cell culture and electroporation
Cerebellar cultures and plate coating were performed using a procedure described before (Rios et al., 2004). For transient-transfection experiments, cells were electroporated in suspension and plated into Neurobasal media supplemented with KCl 25 mM, B-27 and glutamine (Invitrogen, Carlsbad, CA). Depending on the experiment, the mitogen Shh was added or not as indicated (3 µg/ml). Electroporation was performed using the Microporator MP-100 (Digital Bio, Seoul, Korea) according to the manufacturer's instructions, with a single pulse of 1700 mV for 20 milliseconds.
Immunoblotting
For western blot analysis, cell cultures were lysed at 48 hours after electroporation in 1x SDS loading buffer (10% glycerol, 2% SDS, 100 mM DTT and 60 mM Tris-HCl, pH 6.8) and the DNA was disrupted by sonication. Samples were separated by SDS gel electrophoresis, transferred to nitrocellulose membranes, blocked with 8% non-fat dry milk in TTBS (150 mM NaCl; 0.05 Tween 20 and 20 mM Tris-HCl, pH 7.4) and probed with the different antibodies. The blots were developed using anti-rabbit- or anti-mouse-coupled peroxidase plus the ECL system and captured with Versadoc Imaging System from Bio-Rad (Bio-Rad, Hercules, CA).
Antibodies and chemicals
Monoclonal anti-MYCN was purchased from BD Biosciences (San Jose, CA). Monoclonal anti-β-tubulin-III was obtained from Sigma-Aldrich (Buchs, Switzerland). Monoclonal anti-BrdU was obtained from the Developmental Studies Hybridoma Bank (DSHB; IA). Anti-actin, anti-cyclin-D2 and anti-HASH (H56) were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). For immunocytochemical analysis, anti-GFP, the fluorochrome-conjugated secondary antibodies anti-rabbit Alexa-Fluor-488 and anti-mouse Alexa-Fluor-594, and streptavidin–Alexa-Fluor-488 were purchased from Molecular Probes (Invitrogen, Carlsbad, CA). For immunoblotting, anti-mouse antibody, anti-rabbit antibody and streptavidin conjugated to horseradish peroxidase were obtained from Jackson ImmunoResearch (Suffolk, UK). The N-terminal fragment of Shh was produced as a histidine fusion protein in Escherichia coli and purified using a nickel column according to the manufacturer's instructions (QIAGEN, Germantown, MD).
Plasmid constructs
Mash1 cDNA was kindly provided by Rioichiro Kageyama (Institute for Virus Research, Kyoto University, Japan) and subcloned into the bicistronic mammalian expression plasmid pCIG, which includes an IRES-3xNLS-eGFP to track transgene expression. MYCN cDNA was provided by Ann Mary Kenney (Memorial Sloan-Kettering Cancer Center, NY) and was subcloned into pCIG, as was cDNA encoding cyclin D2, kindly gifted from Charles J. Sherr (St Jude Children's Research Hospital, Memphis, TN). The cyclin-D2 reporter plasmid enclosing the regions –1624 to +1 was obtained from Rene H. Medema (University Medical Center Utrecht, Utrecht, The Netherlands) and the CMV–Renilla-luciferase was purchased from Promega (Madison, WI). The 5x E-box reporter promoter was constructed within the pGL3 plasmid and includes five repetitions of the CACGTG motif and a minimal TATA box.
Immunocytochemistry and the proliferation assay
For the BrdU-incorporation assay, cells were pulsed with 24 ng/ml of 8-bromo-deoxyuridine (BrdU) 4 hours prior to fixation with 4% paraformaldehyde. Cells were then permeabilized with methanol for 5 minutes, washed twice with PBS and incubated for 10 minutes with DNAse I in a DNAse buffer (10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2, 0.1 mM CaCl2). Cells were then washed once with PBS and incubated overnight at 4°C with primary antibody. For TUNEL assay (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling), fixed cells were incubated for 1 hour at 37°C with TdT and a biotin-labeled nucleotide, washed, and developed with streptavidin–Alexa-Fluor-488. For TUNEL and β-tubulin-III staining, DNAse treatment was not performed. All images were captured with a Colorview digital camera and processed with Adobe Photoshop 6.0.
For the proliferation assay, electroporated cells were plated for 48 hours and then pulsed for 2 hours with [3H]-thymidine (1 µCi each well, from Amersham, Buckinghamshire, UK). Cells were then lysed in SDS 0.04% and proliferation measured with a Wallac scintillation counter (Wallac-Perkin Elmer, Quebec, Canada).
Reporter assay
The reporter plasmid containing the cyclin-D2 promoter and 5x E-box were co-electroporated together with the CMV-Renilla-luciferase vector and a fourfold excess of the indicated constructs. Cells were plated in the absence of Shh for 48 hours and then lysed with the Passive Lysis Buffer (Promega, Madison, WI). Lysates were collected and measured with an Orion II Microplate Luminometer from Berthold using the reagents previously described (Dyer et al., 2000). Luciferase data were normalized to the Renilla-luciferase values, and results were plotted and expressed in arbitrary units as the mean and standard deviation of three different experiments.
ChIP analysis
The ChIP assay was performed as previously described (Valls et al., 2005). CGNPs were cultured in the presence of Shh for 24 hours and with Shh plus BMP2 for an additional 24 hours. One or three micrograms of antibody were used to immunoprecipitate the chromatin corresponding to 1 million cells. The sequence surrounding the 5' cyclin-D2 promoter E-box was amplified by PCR with the following set of primers: forward, 5'-GCAACTCACGCCATGCTATC-3'; reverse, 5'-CTGTATTCCACTTGGGGAGG-3'.
Footnotes
We thank all of the researchers who sent us the cDNAs detailed in the Materials and Methods section. Work in the S.P. laboratory is supported by the Spanish Ministry of Education grant BFU2005-01599.
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-actin were determined. Mash1 expression did not affect MYCN protein levels and was able to downregulate cyclin-D2 expression and raise β-tubulin-III levels. (E) A model for MYCN and Mash1 antagonism in cell-cycle control. In the presence of Shh, MYCN expression is upregulated. The complex formed with its partner, Max, binds to and activates the E-box sequences (CACGTG) that are present in the cyclin-D2 promoter, turning on the proliferative response. When a differentiator stimulus such as BMP2 is added, Mash1 expression increases. Mash1 then forms heterodimers with E12, displacing the MYCN-Max complex from the E-box sequences within the cyclin-D2 promoter, repressing cyclin-D2 transcription and shutting down proliferation.