TGFβ1 enhances MAD1 expression and stimulates promoter-bound Pol II phosphorylation: basic functions of C/EBP, SP and SMAD3 transcription factors
© Hein et al; licensee BioMed Central Ltd. 2011
Received: 10 September 2010
Accepted: 23 February 2011
Published: 23 February 2011
The MAD1 protein, a member of the MYC/MAX/MAD network of transcriptional regulators, controls cell proliferation, differentiation and apoptosis. MAD1 functions as a transcriptional repressor, one direct target gene being the tumor suppressor PTEN. Repression of this gene is critical to mediate the anti-apoptotic function of MAD1. Under certain conditions it also antagonizes the functions of the oncoprotein MYC. Previous studies have demonstrated that MAD1 expression is controlled by different cytokines and growth factors. Moreover we have recently demonstrated that the MAD1 promoter is controlled by the cytokine granulocyte colony-stimulating factor (G-CSF) through the activation of STAT3, MAP kinases and C/EBP transcription factors.
We observed that in addition to G-CSF, the cytokine transforming growth factor β (TGFβ1) rapidly induced the expression of MAD1 mRNA and protein in promyelocytic tumor cells. Moreover we found that C/EBP and SP transcription factors cooperated in regulating the expression of MAD1. This cooperativity was dependent on the respective binding sites in the proximal promoter, with the CCAAT boxes being bound by C/EBPα/β heterodimers. Both C/EBP and SP transcription factors bound constitutively to DNA without obvious changes in response to TGFβ1. In addition SMAD3 stimulated the MAD1 reporter, cooperated with C/EBPα and was bound to the core promoter region. Thus SMAD3 appears to be a potential link between TGFβ1 signaling and C/EBP regulated promoter activity. Moreover TGFβ1 stimulated the phosphorylation of polymerase II at serine 2 and its progression into the gene body, consistent with enhanced processivity.
Our findings suggest that C/EBP and SP factors provide a platform of transcription factors near the core promoter of the MAD1 gene that participate in mediating signal transduction events emanating from different cytokine receptors. SMAD3, a target of TGFβ1 signaling, appears to be functionally relevant. We suggest that a key event induced by TGFβ1 at the MAD1 promoter is the recruitment or activation of cofactors, possibly in complex with C/EBP, SP, and SMAD3 transcriptional regulators, that control polymerase activity.
The MYC/MAX/MAD network of transcriptional regulators is essential to control many aspects of cell physiology . MYC was originally identified as oncogene in several different chicken retroviruses. Subsequently the three human MYC genes, MYC, MYCN and MYCL were found deregulated in the large majority of human tumors . The potent capacity of MYC to transform cells has also been supported by a large number of studies in both primary cells and established cell lines and in animal models. Central to the ability to transform cells is MYC's function as transcriptional regulator in controlling the expression of a large number of target genes. This explains, at least in part, the broad biological activities associated of MYC [3, 4]. The functions of MYC in gene expression control depend largely on its interaction with MAX, the central component of the MYC/MAX/MAD network.
MAD proteins are alternative binding partners of MAX . Six different MAD proteins have been identified. MAD1-4 are highly related, while MNT and MGA are considerably larger multi-domain proteins. Similar to MYC, the MAD proteins are transcriptional regulators, with MAD1-4 primarily described as repressors. Unlike MYC proteins, the MADs have not been linked to human diseases, in particular they appear not to be tumor suppressors as one might have expected. For MAD1-4 the reason for their apparent lack to function as tumor suppressors may be in part due to their broad and overlapping expression pattern, suggesting that more than one MAD family member would need to be inactivated in tumors . In addition, MAD proteins, best studied for MAD1, have anti-apoptotic activity and thus may antagonize the pro-apoptotic functions of MYC proteins [6–8]. This activity of MAD proteins may be indispensable for tumor development. In support, one of the few MAD1 target genes that has been identified is the tumor suppressor gene PTEN. MAD1, which functions primarily as a transcriptional repressor by recruiting histone deacetylase-containing complexes [9–12], represses the PTEN promoter directly . This contributes to the anti-apoptotic phenotype elicited by MAD1. The analysis of granulocytes from mice lacking Mad1 revealed increased sensitivity to pro-apoptotic conditions , further supporting the view that MAD1 protects cells from different apoptotic stimuli.
In addition to the anti-apoptotic function, MAD1 has been suggested to control proliferation and differentiation antagonistically to MYC . Indeed the unscheduled expression of MAD1 interferes with cell proliferation and the lack of Mad1 results in a differentiation defect of granulocytes [6, 7, 13–15]. During the studies to elucidate the functions of MAD1 in proliferation and differentiation, it had been noted early on that the expression of the MAD1 gene is highly regulated, generally reciprocal to the regulation of MYC genes . Moreover MAD1 expression is directly downregulated by MYC (NH and BL, unpublished observations). In particular several differentiation inducing agents, including transforming growth factor β (TGFβ), retinoic acid, and granulocyte-colony stimulating factor (G-CSF), were identified as stimulators of MAD1 expression [16–20]. These findings led us to address the question how the MAD1 promoter is organized and how signals of these differentiation factors control gene expression. The MAD1 promoter contains a CpG island as part of a roughly 400 bp proximal promoter region highly conserved between humans and rodents . This region is responsive to G-CSF, integrating signals transduced from the G-CSF receptor by STAT3 and by the RAS-RAF-ERK pathway. This regulation of the MAD1 promoter by G-CSF is in agreement with the described role of this cytokine and of Mad1 in the control of granulocyte differentiation and survival .
Cytokines of the TGFβ family have broad activities in controlling cell physiology, including proliferation, differentiation and survival [21–23]. TGFβ signals through TGFβ type II and I receptors with Ser/Thr kinase activity, thereby activating SMAD proteins, in particular SMAD2 and 3 in combination with SMAD4. These proteins translocate to the cell nucleus and form complexes with additional molecules to control the expression of target genes . We have shown previously that the phorbol ester TPA and TGFβ activate the expression of MAD1 in U937 and in HaCaT keratinocytes, respectively [18, 19]. In both systems a substantial increase in mRNA expression was observed by 90 min, suggesting that the induction was direct. Different kinetics of MAD1 induction were observed in a clone of U937 promyelocytes that stably express a viral version of MYC (U937-myc6). In these cells a weak induction was observed in response to TGFβ by 8 hrs, possibly as a result of constitutive MYC expression . To understand in more detail how TGFβ1 regulates MAD1 gene expression, we addressed how this cytokine affects MAD1 promoter activity. It appears that TGFβ1 stimulates MAD1 through elements proximal to the core promoter.
Results and Discussion
Rapid activation of MAD1 by TGFβ
MAD1 has been demonstrated to interfere with cell proliferation in some cell types [7, 14]. Therefore we measured whether the induction of MAD1 by TGFβ1 affected the proliferation of U937 tumor cells. However the early TGFβ1-stimulated induction of MAD1 was not sufficient to block U937 proliferation (Figure 1D), similar to the observations made in U937-myc6 cells . Our findings suggest that tumor cells like U937 have the possibility to bypass at least transiently the repressive function of MAD1 in cell proliferation.
C/EBPα/β heterodimers bind constitutively to the MAD1 promoter
SP transcription factors bind to the MAD1 promoter independent of TGFβ signaling
C/EBP and SP transcription factors cooperate in stimulating the MAD1 promoter
SMAD3 interacts with and activates the MAD1 promoter dependent on C/EBP and SP binding sites
TGFβ1 stimulates Ser2 phosphorylation of Pol II
We observed that C/EBP and SP transcription factors bind constitutively to the proximal MAD1 promoter. In addition SMAD3, a factor typically activated by TGFβ signaling, also was found constitutively on the MAD1 promoter, despite the fact that no obvious binding sites for SMAD proteins are found. While the GC boxes are consensus binding sites for SP1, the proposed CCAAT boxes are deviating considerably from C/EBP consensus sequences. In fact, both elements that were identified functionally, represent only half sites . Consistent with this interpretation, these DNA elements do not bind efficiently C/EBP homodimers in EMSA experiments in vitro. Surprisingly substantial binding was only measurable with C/EBPα/β heterodimers in these EMSA experiments. Nevertheless both factors were able to stimulate MAD1 promoter reporter genes. We did however not observe a strong synergistic activation by the two proteins, possibly due to abundant endogenous C/EBP factors (data not shown). We suggest that C/EBP and SP transcription factors form a platform for incoming signals as exemplified by G-CSF  and possibly TGFβ1. In the case of G-CSF, STAT3 is recruited by C/EBPs, requiring MAPK signaling. Our new findings suggest that TGFβ1 signaling activates SMAD proteins and stimulates MAPK signaling. The activation of MAPK might be a common pathway that controls at least in part MAD1 expression. Consistent with this interpretation, SMAD3 cooperated with C/EBP proteins to activate MAD1 promoter reporter genes. The finding that SMAD3 was bound to the MAD1 promoter suggests that SMAD3 is directly recruited to the MAD1 promoter by binding to C/EBPs or C/EBP associated factors. Because the GC box was also relevant, we propose that a large transcription factor/cofactor complex interacts with the identified promoter proximal region, including SMAD3. However, we point out that we cannot exclude direct binding of SMAD3 to the MAD1 promoter. Although no obvious binding sites could be detected, SMAD binding sites are rather short and leave the possibility open that SMAD3 forms a dimeric or multimeric complex with other factors, in which SMAD3 might bind directly to DNA.
The signals that are integrated at the proximal MAD1 promoter translate into the activation of Pol II as measured by its progression into the gene body and the concomitant change in the phosphorylation of the C-terminal domain of Pol II. This is consistent with recent observations on many genes, which have provided evidence that Pol II phosphorylated at Ser-5 is located at the promoter in a preactivated or paused mode. The switch to Ser-2 phosphorylation, possibly by the recruitment and activation of the P-TEFb kinase CDK9, results in the activation and promoter clearance of Pol II . Thus this represents a situation as it is now becoming evident at many different promoters that are being studied in detail. It is worth noting that Pol II was found to be associated with the MAD1 promoter prior to stimulation with cytokines. Thus at least in U937 tumor cells, the MAD1 promoter is preoccupied by Pol II and thus allows for rapid activation by multiple signals. It will now be of interest to specifically dissect how different cytokines use the C/EBP-SP transcription factor platform to activate the paused Pol II.
Reporter gene construct and expression vectors
The cloning of MAD1 promoter reporter gene constructs has been reported previously . Descriptions of pEQ176-ß-galactosidase, pCB6+-C/EBPα, and pCB6+-C/EBPβ are found in [28, 35]; pCDNA3+-C/EBPε was obtained from A. Friedman ; pCL-neo-HA-SP1 and pCI-neo-HA-SP1-N (a dominant negative form of SP1) were provided by H. Rotheneder .
Cell culture and treatment
HEK293 (ATCC CRL-1573) and HeLa (ATCC CCL-2) cells were cultured in DMEM (Gibco) with 10% fetal calf serum (Gibco) and penicillin/streptomycin (Seromed). U937 (ATCC CRL-1593.2) promyelocytes were grown in RPMI 1640 (Gibco) with 10% fetal calf serum and penicillin/streptomycin. All cells were cultured at 37°C and 5% CO2. U937 cells were treated with TGFβ1 (Peprotec) at a concentration of 2.5 ng/ml and with 5 μM SB505124 (Sigma-Aldrich) as indicated. Proliferation and viability of U937 cells were analyzed using Trypan Blue staining and the CASY cell counting system (Innovatis).
Transient transfection and luciferase assay
Transient transfection of HEK293 and HeLa cells were performed using the calcium phosphate co-precipitation method as described previously . HeLa cell co-transfected with pSuper-sh-C/EBPβ were harvested 72 hours post-transfection. For luciferase assays HeLa cells were co-transfected overnight with a total amount of 3-5 μg plasmid DNA and cultured for 48 hrs under normal growth conditions prior to harvesting. Luciferase activity was measured using a bioluminator (ELISA-Reader Victor2). The relative luciferase activity was normalized to the β-galactosidase activity. All experiments were performed in duplicates or triplicates with at least three independent replicates.
The online program siDirect (genomics.jp/sidirect) was used to design shRNA oligonucleotides targeting the C/EBPβ mRNA and the resulting sequences were analyzed via the BLAST algorithm. The hybridized oligonucleotides were cloned into the pSuper vector (obtained from R. Bernards) linearised with Bgl II and Hin dIII .
RNA preparation and quantitative RT-PCR
The RNAeasy Mini Kit (Qiagen) was used for total RNA extraction, according to the manufacturer's instruction and residual genomic DNA was removed by DNase (Qiagen) digestion. 1 μg total RNA was reverse transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) and analyzed by quantitative real time PCR using a LightCycler (Roche). The real time PCR reactions were performed with the SYBRgreen Ready Mix (Qiagen) and the following primer pairs: MAD1 QantiTect primer assay (Qiagen) and β-GLUCURONIDASE-f 5'-CTCATTTGGAATTTTGCCGATT-3', β-GLUCURONIDASE-r 5'-CCGAGTGAAGATCCCCTTTTTA-3'. The relative quantification of MAD1 mRNA was calculated by the comparative CT method and normalized to β-GLUCURONIDASE using the Software RelQuant.
Chromatin immunoprecipitation (ChIP) assay and RE-ChIP assay
ChIP assays were performed as described previously . U937 cells were grown in a spinner flask to a maximal density of 106 cells/ml. Following TGFβ1 treatment 5-2.5 × 107 cells/ml per IP were harvested. For immunoprecipitation 2 μg of the following antibodies were used: H3ac (06-599, Upstate); H3K4me3 (8580-50, Abcam); Pol II N20 (SC-899, Santa Cruz Biotechnology); Pol II CTD phosphoserine 2 H5 (MMS-129R, Covance); Pol II CTD phosphoserine 5 H14 (MMS-134R, Covance), C/EBPα 14AA (SC-61, Santa Cruz Biotechnology); C/EBPβ C19 (SC-150, Santa Cruz Biotechnology), SP1 PEP2 (SC-59, Santa Cruz Biotechnology), SP1 (07-124, Upstate), Cytochrome C (SC-7159, Santa Cruz Biotechnology), SMAD3 (ab28379 Abcam). In addition SP1-specific antibodies were obtained from G. Suske . The following primer pairs were used for PCR analysis of the MAD1 gene:
For Re-ChIP assays the first immunoprecipitation was performed as above. Then the samples were washed once in ChIP RIPA buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% NP40, 0.1% DOC, 0.1% SDS, 0.5% aprotinin) and the protein-DNA complexes solubilized in release buffer (1% SDS, 10 mM DTT, TE-buffer pH 7.5). The beads were incubated at 37°C for 30 min. To the supernatant 4 volumes of RIPA-SDS (150 mM NaCl, 10 mM Tris pH 7.5, 1% NP40, 1% DOC, 0.5% aprotinin, 1 mM EDTA, 1 mM iodoacetamid) were added to perform the second immunoprecipitation.
Electrophoretic mobility shift assay (EMSA)
The following oligonucleotides were γ32P-ATP radiolableled and used in EMSAs:
CCAAT-b1 f: 5'AGCCCTCTCCCAATCGCACAAG3';
CCAAT-b1 r: 5'CTTGTGCGATTGGGAGAGGGCT-3';
NE-CCAAT f: 5'-TCGAGATGGGGCAATAT-3';
NE-CCAAT r: 5'-ATATTGCCCCATCTCGA-3';
GC box1 f: 5'-AAGTGTAGGGGCGGGGCATTCT-3';
GC box1 r: 5'-AGAATGCCCCGCCCCTACACTT-3'.
HEK293 whole cell extracts were prepared on ice in Frackelton-lysis buffer (10 mM Tris-HCL pH 7.05, 50 mM NaCl, 30 mM Na4P2O7, 50 mM NaF, 5 μM ZnCl2, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 100 μM Na3VO4, 150 μM benzamidin, 0.025 U/ml α-macroglobulin, 2.5 μg/ml leupeptin, 14 μg/ml aprotinin). Whole cell extracts were incubated with the radiolabeled oligonucleotides at 30°C for 30 min and then subjected to electrophoresis as described previously . In brief, for supershift assays antibodies or equivalent amounts of control antibodies or BSA were added and incubated on ice for 10 min, prior to oligonucleotide addition. The protein-DNA complexes were separated on a 4.5% polyacrylamide gel containing 7.5% glycerol in 0.25-fold TBE (20 mM Tris base, 20 mM boric acid, 0.5 mM EDTA, pH 8) at 20 V/cm for 4 h. Gels were fixed in 10% methanol, 10% acetic acid, and 80% water for 1 h, dried, and autoradiographed. The following antibodies were used in EMSAs: C/EBPα 14AA (SC-61, Santa Cruz Biotechnology); C/EBPβ C19 (SC-150, Santa Cruz Biotechnology); SP1 PEP2 (SC-59, Santa Cruz Biotechnology), SP1 (07-124, Upstate), SP3 D-20 (SC-644, Santa Cruz Biotechnology), Cytochrom C (SC-7159, Santa Cruz Biotechnology).
To generate highly concentrated U937 whole cell extracts (107 cells/preparation), U937 cells were lysed in 20 - 30 μl FT Lysis buffer (600 mM KCL, 20 mM Tris-Cl pH 7.8, 20% glycerol, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 14 μg/ml, aprotinin, 0.4 mg/ml, Pefabloc) by pipeting up and down as described previously . The freeze-thaw cycles in liquid nitrogen were repeated five times. The thawed lysates were incubated with 250 U Benzonase (Merck) at RT for 10 min. Whole cell extracts were resolved by SDS-Page and transferred onto nitrocellulose membranes, probed with MAD1 C19 (SC-222, Santa Cruz Biotechnology), α-Tubulin (B-5-1-2, T-5168, Sigma), or C/EBPβ C19 (SC-150, Santa Cruz Biotechnology) antibodies followed by horseradish peroxidase (HRP)-labeled secondary antibody. Detection was performed with the either chemiluminescence ECL kit (Pierce) or SuperSignal West Femto Maximum Sensitivity Substrate (Pierce).
List of abbreviations
Electrophoretic mobility shift assay
Granulocyte colony stimulationg factor
acetylated histone 3
trimethylated lysine 4 of histone 3
MYC-associated factor x
Myelocytomatosis viral oncogene
Phosphatase and tensin homologue deleted on chromosome ten
Mothers against decapentaplegic homolog 3
Specific protein dominate negative
Transforming growth factor
Transforming growth factor receptor
We thank R. Bernards, A. Friedman, H. Rotheneder, and G. Suske for providing reagents, K. Eckert, J. Lüscher-Firzlaff, and L.-G. Larsson for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft Grant SFB 542 projects B8 and C11 to BL.
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