Abstract
SMAD proteins can mediate transforming growth factor β (TGF-β)-inducible transcriptional responses. Whereas SMAD can recognize specific DNA sequences, it is usually recruited to a promoter through interaction with a DNA-binding partner. In an effort to search for TGF-β-inducible genes, we used a subtractive screening method and identified human Smad7, which can antagonize TGF-β signaling and is rapidly up-regulated by TGF-β. In this report, we show that TGF-β can stabilize Smad7 mRNA and activate Smad7 transcription. The Smad7 promoter is the first TGF-β responsive promoter identified in vertebrates that contains the 8-bp palindromic SMAD-binding element (SBE), an optimal binding site previously identified by a PCR-based selection from random oligonucleotides by using recombinant Smad3 and Smad4. We demonstrate that on TGF-β treatment, endogenous SMAD complex can bind to a Smad7 promoter DNA as short as 14 or 16 bp that contains the 8-bp SBE in gel mobility shift and supershift assays. Our studies provide strong evidence that SMAD proteins can bind to a natural TGF-β responsive promoter independent of other sequencespecific transcription factors. We further show that, whereas recombinant Smad3 binds to the SBE, endogenous or even transfected Smad3 cannot bind to the SBE in the absence of Smad4. These findings have important implications in the identification of target genes of the TGF-β/SMAD signaling pathways.
The transforming growth factor β (TGF-β) family plays a fundamental role in cell regulation (1–7). TGF-β signals through two types of transmembrane serine/threonine kinase receptors. On activation, the type I receptor phosphorylates Smad2 and Smad3, which then form complexes with Smad4 and move into the nucleus to regulate transcription (1–11).
SMAD possesses flexible DNA-binding activities. By using recombinant Smad3 and Smad4 with random oligonucleotides by a PCR-based approach, Zawel et al. (12) identified an 8-bp palindromic sequence GTCTAGAC as the SMAD-binding element (SBE). Interestingly, Smad2 cannot bind to the SBE because of the interference by a stretch of aa that is present immediately before the DNA-binding hairpin (13, 14). A number of TGF-β/SMAD-regulated promoters, such as the PAI-1, collagenase I, c-Jun, IgA, and Jun B promoters, contain one or multiple copies of the sequence GTCT or AGAC, which can be bound by the Smad3–Smad4 complex (15–24). Tandem repeats of GTCT, AGAC, or the SBE can confer TGF-β inducibility to heterologous promoters (12, 16, 24, 25). In addition to the GTCT and AGAC elements, SMAD can also recognize a GC-rich sequence (26, 27).
Although SMAD can bind to DNA, SMAD is usually recruited to promoters through interaction with DNA-binding partners, which has been demonstrated for the Mix.2, goosecoid, and Xvent2 promoters through interaction with transcription factors that include FAST-1, FAST-2, mixer, milk, and OAZ (27–33). SMAD has also been shown to physically interact with other transcription factors, such as AP-1 (21, 34), TFE3 (20), PEBP2 (23), and ATF-2 (35), to activate transcription from a variety of promoters that include PAI-1, collagenase I, c-Jun, and IgA (15–23). Whether interactions with these transcription factors are necessary for SMAD to bind to the respective target promoters remains to be investigated in greater detail.
In addition to the above mechanism, SMAD has been suggested to be able to bind directly to certain promoters. For example, the Drosophila Mad is implicated to interact directly with a GC-rich sequence in the vestigial promoter (26). However, whether SMAD can indeed bind to a natural TGF-β responsive promoter independent of other transcription factors was not fully determined.
In this report, we characterized the regulation of Smad7, which is an antagonistic SMAD that can inhibit TGF-β family signaling pathways and is rapidly induced by TGF-β-like molecules and a number of other agents, such as epidermal growth factor, the phorbol ester phorbol 12-myristate 13-acetate, IFN-γ, and tumor necrosis factor-α (5, 36–43). Smad7 is the first natural TGF-β-responsive promoter in vertebrates that contains the palindromic SBE. We demonstrate that on TGF-β treatment, SMAD can bind to the SBE in the absence of other sequence-specific transcription factors.
Materials and Methods
Subtractive cDNA Cloning and Genomic Screening.
Poly(A)+ RNA (2 μg) from HaCaT cells that were untreated or treated with TGF-β for 1 h were used as driver and tester, respectively, in a PCR-based cDNA subtraction method (CLONTECH). Smad7 comprises about 3% of the positive clones identified in the screening. An ≈900-bp DNA fragment from the 5′ end of the human Smad7 cDNA was used as a probe to screen a human placenta genomic library (CLONTECH). From 3 × 105 plaques, 13 positive clones were identified, which were subjected to a further round of screening by using the 5′ end ≈200-bp DNA fragment as a probe, yielding five positive clones. Genomic DNA from these positive clones was subjected to restriction enzyme digestion, Southern blot analysis, automatic and manual sequencing, and reporter gene assay. These combined studies indicated that over 10-kb of the Smad7 genomic DNA was upstream of the coding region.
RNA Preparation and Northern Blot Analysis.
PolyA+ RNA was prepared from HaCaT cells by using the FAST track kit (Invitrogen). Poly(A)+ RNA (4 μg) was separated on a 1% formaldehyde-agarose gel, transferred to a nylon membrane, and hybridized with a random primered probe from the N-terminal domain (≈900 bp) of human Smad7 cDNA. The same blot was subsequently hybridized with a glyceraldehyde-3-phosphate dehydrogenase probe.
Primer Extension Analysis.
Poly(A)+ RNA (0.4 μg) and 0.1 pM 32P-labeled primer were annealed in a buffer containing 40 mM Pipes, 1 mM EDTA, 0.2% SDS, and 0.4% NaCl (pH 6.7) at 37°C overnight, followed by precipitation with isopropyl alcohol. The extension reaction was performed by using the Promega primer extension system/avian myeloblastosis virus reverse transcriptase kit at 42°C for 2 h. The products were analyzed on a denaturing 8% polyacrylamide gel.
Constructs and Luciferase Assays.
The BglII-HindIII (−3.2 kb to +641), BamHI-HindIII (−584 to +641), and KpnI-HindIII (−339 to +641) fragments of the Smad7 promoter region were fused to the pGL2 luciferase reporter gene (Promega). HaCaT and SW480.7 cells were transfected by using DEAE-dextran (125 μg/ml) for 3–5 h, treated with or without 500 pM TGF-β for 18–24 h. Luciferase activity was analyzed by using the luciferase assay system (Promega) in a Berthold Lumat LB 9507 luminometer.
Gel Mobility Shift and Supershift Assay.
SW480.7 cells were transfected with lipofectin and treated with TGF-β for 1 h. Whole-cell extracts from HaCaT and SW480.7 cells were prepared as described (30). DNA-binding assays were performed in a buffer containing 20 mM Hepes (pH 7.9), 0.1 μg/μl poly(dI-dC), 6 mM MgCl2, 30 mM KCl, 1 mM EDTA, 1 mM EGTA, 2 mM DTT, 12.5% glycerol, 1.5% polyvinyl alcohol, nonspecific double-stranded DNA and single-stranded DNA with 0.5 ng of 32P-labeled 16-bp probe. The wild-type SBE probe sequence is 5′-GGGTGTCTAGACGGCC-3′. The specific competitor sequence is 5′-AATCGTCTAGACATAT-3′. The nonspecific competitor sequence is 5′-GGGTTTTTAGACGGCC-3′. Antibody supershift assays were performed as described (30) with 1 μg of Flag or HA antibodies and 1 μl of undiluted preimmune or immune serum against SMAD. DNA–protein complexes were resolved on a 4% (40:1) polyacrylamide gels containing 1% glycerol.
Results
Identification of Smad7 from a Subtractive cDNA Cloning.
To understand TGF-β-inducible gene regulation, we searched for TGF-β-responsive genes by using a subtractive screening method, comparing mRNA transcripts from untreated versus TGF-β-treated human keratinocytes (HaCaT cells), which are highly responsive to TGF-β. From the screening, we identified Smad7 and other candidate TGF-β-responsive genes. Fig. 1A confirmed that Smad7 mRNA from HaCaT cells was significantly induced by TGF-β (≈10-fold) as determined by Northern blot analysis.
TGF-β Can Stabilize Smad7 RNA.
The effect of TGF-β on Smad7 expression could result from stabilization of Smad7 RNA and/or induction of Smad7 transcription. To examine whether TGF-β can stabilize Smad7 RNA, we analyzed Smad7 RNA levels in HaCaT cells that were treated with the transcription inhibitor actinomycin D in the absence versus presence of TGF-β. As shown in Fig. 1B, treatment with actinomycin D significantly reduced the level of Smad7 RNA. When TGF-β was present together with actinomycin D, the Smad7 RNA level was also decreased but at a slower rate. The half-life of Smad7 in HaCaT cells in the absence and presence of TGF-β is ≈40 and 90 min, respectively. Thus, TGF-β can stabilize Smad7 RNA.
TGF-β Significantly Increases the Smad7 Transcript from the Distal Promoter.
Because the RNA stabilization effect of TGF-β can account for only part of the full induction rate of Smad7, we asked whether TGF-β was able to increase transcription of the Smad7 gene. By using a 5′ end human Smad7 cDNA fragment as a probe, we screened a human placenta genomic library and isolated the Smad7 promoter. To determine the transcription start site(s) of the Smad7 promoter, we first compared the human Smad7 genomic sequence with the mouse and rat Smad7 cDNA sequences. Both mouse and rat Smad7 cDNAs contain a 1.4-kb GC-rich 5′ untranslated region, which is also highly conserved in the human Smad7 genomic sequence. We therefore synthesized a 22-bp Smad7 oligonucleotide that was conserved among the three species and was located at the very 5′ end of the 1.4-kb 5′ untranslated region of mouse and rat cDNAs. With this oligonucleotide, we performed primer extension analysis by using PolyA+ RNAs from HaCaT cells that were untreated or treated with TGF-β for 1 h. As shown in Fig. 2A, untreated cells contained one major transcription start site located 134 bp from the primer. Interestingly, TGF-β preferentially increased the Smad7 RNA transcript that was initiated 180 bp from the primer. We designate the distal transcription start site as +1, the distal promoter as P1, and the proximal promoter as P2 (Fig. 2B). The transcription start sites that we mapped are further upstream of the major transcription start site from the mouse Smad7 promoter (44). Examination of the Smad7 promoter sequence does not reveal a canonical TATA box. Future studies are required to determine how transcription is initiated from P2 and especially the P1 promoter.
SMAD Can Activate Transcription from the Smad7 Promoter.
We analyzed Smad7 promoter activity by fusing different promoter fragments with the pGL2 luciferase reporter gene (Promega). Transfection into HaCaT cells of reporter fusion genes containing 3.2 kb, 584 bp, or 339 bp of promoter sequences and 641 bp of 5′ untranslated region were sufficient to yield high basal luciferase activities, which were further increased five- to eightfold in the presence of TGF-β (Fig. 3A). Examination of the Smad7 promoter sequence revealed that it contained the palindromic SBE GTCTAGAC at position −210. To test whether the SBE was essential for TGF-β induction, we changed the GTCTAGAC sequence to GTCTAGCTAGAC so that four nucleotides were inserted after the GTCT sequence. This mutation significantly reduced the TGF-β inducibility of the Smad7 promoter (Fig. 3A).
To further confirm that SMAD proteins directly regulate Smad7 transcription, we analyzed the TGF-β inducibility of the Smad7 promoter in SW480.7 cells, which do not express Smad4 protein (45). As shown in Fig. 3B, the reporter gene that contained 339 bp of Smad7 promoter sequence was not induced by TGF-β in SW480.7 cells. Cotransfection with Smad2 did not increase its activity. Cotransfection with Smad3 led to a minimal activation in the presence of TGF-β. Cotransfection with Smad4 resulted in a small degree of activation in the presence of TGF-β, presumably because of Smad4 forming complexes with endogenous SMAD. Cotransfection with both Smad2 and Smad4 significantly activated the reporter gene. Importantly, cotransfection with both Smad3 and Smad4 resulted in maximal activation, indicating that the Smad3–Smad4 complex is optimal for transcriptional activity.
Endogenous SMAD Can Bind to the SBE on Its Own.
To define the sequence requirement for endogenous SMAD binding to the SBE, we prepared whole-cell extract from HaCaT cells that were untreated or treated with TGF-β for 1 h. These extracts were then subjected to gel mobility shift assays by using a series of DNA probes that all contained the SBE but spanned different lengths of the Smad7 promoter. Interestingly, a TGF-β-induced DNA–protein complex was detected with a DNA probe as short as 16 bp that contained the 8-bp SBE flanked by 4 bp on each side from the Smad7 promoter (Fig. 4A). In all of the experiments described below, we used the 16-bp probe. Longer probes from 20 to 45 bp gave rise to similar or identical patterns of TGF-β-inducible DNA–protein complexes (data not shown). A 14-bp probe with only 3 bp flanking sequences on each side was also analyzed in all of the gel shift assays and yielded the same results except that it had an ≈1.5- to 2-fold lower affinity than the 16-bp probe (data not shown).
To confirm the presence of SMAD in the TGF-β-induced DNA–protein complex, we performed supershift assays by using antibodies against SMAD. As shown in Fig. 4A, an antibody raised against the linker region of Smad2 that recognized both Smad2 and Smad3 (45) caused a supershift of the TGF-β-induced DNA–protein complex, whereas the preimmune serum had no specific effect. Similarly, an antibody against the Smad4 C-terminal domain (amino acids 319–552) (46), which had about 10-fold higher affinity for Smad4 than for other SMAD (data not shown), also resulted in a supershift. Thus, an endogenous SMAD complex containing Smad4 with Smad3 and/or Smad2 binds to the SBE.
The endogenous SMAD recognized the 8-bp SBE in the 16-bp probe. When all of the flanking nucleotides were changed but the palindromic SBE remained intact, the cold oligonucleotide competed for binding partially at a 25-fold excess and completely at a 100-fold excess (Fig. 4B). In contrast, when point mutations were introduced into the GTCTAGAC sequence so that only the AGAC half was intact, even a 1,000-fold excess of mutant oligonucleotide competed for binding only modestly.
To determine whether the appearance of the TGF-β-induced SMAD–DNA complex correlated with the Smad7 RNA level, we prepared extracts from HaCaT cells that were treated with TGF-β for different times and performed gel mobility shift assays. As shown in Fig. 4C, the TGF-β-inducible SMAD–DNA complex occurred as early as 15 min after TGF-β treatment, and the maximal level appeared after a 30–60-min treatment by TGF-β. This is in agreement with the Smad7 RNA level, which peaks at ≈1 h after TGF-β treatment (ref. 38, Fig. 1A).
Smad4 Is Essential for SBE Binding and the Smad3–Smad4 Complex Binds Optimally to the SBE.
In contrast to HaCaT cells, SW480.7 cells did not show TGF-β-inducible binding to the SBE (Fig. 5A), suggesting that Smad4 is critical for binding to the SBE. To further analyze the contribution of different SMAD in binding to the SBE, Smad2, Smad3, and Smad4 were cotransfected into SW480.7 cells either individually or in combination. As expected, transfected Smad2 did not show detectable binding to the probe (Fig. 5A). Interestingly, overexpression of Smad3 also had no detectable binding (Fig. 5A), in contrast to the reported SBE-binding activity of recombinant Smad3. Transfection of Smad3 into a Smad4-expressing cell line, such as COS cells, however, resulted in good SBE-binding activity (data not shown). Apparently, Smad3 requires complex formation with Smad4 for binding to the SBE in vivo. Transfection of Smad4 alone led to basal binding, which was increased in the presence of TGF-β, presumably as a result of forming a complex with endogenous SMAD. Smad2 and Smad4 together resulted in low-level binding activity, similar to transfection of Smad4 alone. In contrast, Smad3 and Smad4 together gave rise to very abundant DNA-binding (Fig. 5A). To examine whether the Smad2–Smad4 and Smad3–Smad4 complexes can indeed bind to the SBE probe, Flag-Smad2 or Flag-Smad3 were cotransfected with Smad4-HA into SW480.7 cells, treated with TGF-β, and analyzed in supershift assays with antibodies against the Flag and HA epitopes. As shown in Fig. 5B, Flag antibody readily caused a supershift of Flag-Smad3, whereas it barely supershifted Flag-Smad2, which was detected only after longer exposure. The HA antibody supershifted Smad4-HA in both cases. Thus, whereas both Smad3–Smad4 and Smad2–Smad4 complexes can bind to the SBE, the DNA-binding properties of the two complexes differ.
To confirm that recombinant SMAD can bind to the 16-bp SBE probe, we analyzed glutathione S-transferase (GST)-Smad1, GST-Smad2, GST-Smad3, and GST-Smad4 in a gel mobility shift assay. Consistent with previous reports, GST-Smad2 cannot bind to the probe. GST-Smad1 showed quite weak binding that was detected only after a longer exposure (data not shown). Both GST-Smad3 and GST-Smad4 can bind to the probe, and GST-Smad4 has a higher affinity for the probe than GST-Smad3 (Fig. 5C).
Discussion
Endogenous or Transfected Smad3 Cannot Bind to the SBE in the Absence of Smad4.
Whereas recombinant Smad3 can bind to the SBE, we have shown that endogenous or transfected Smad3 has no detectable SBE-binding activity in SW480.7 cells that do not express Smad4 by using either whole-cell extracts (Fig. 5A) or nuclear extracts (data not shown). Regarding nuclear extracts, we and others have previously shown that TGF-β can induce transfected or endogenous Smad3 to translocate into the nucleus in the absence of Smad4 in SW480.7 cells (30, 45). We have confirmed again that although Smad3 did not bind to the DNA, Smad3 was indeed translocated to the nucleus as described (30). Apparently, Smad4 plays a critical role in binding to the SBE in vivo. Whereas two recombinant Smad3 monomers each was able to bind to a half-site of the palindromic SBE and mutation of one half-site still allowed Smad3 binding to the other half-site (13), we found that recombinant Smad4 DNA-binding activity was dramatically decreased when half of the palindromic SBE was mutated (data not shown). Because the endogenous SMAD complex cannot bind to a half-site of the palindromic SBE (Fig. 4B), it behaves similarly to Smad4, not to Smad3. Smad4 is a tumor suppressor that is significantly mutated in pancreatic and colorectal carcinomas, and to a lesser extent, in several other types of cancers (1–2, 47). The central role of Smad4 in TGF-β-inducible transcriptional control may be causally linked with its tumor suppressor activity.
Endogenous SMAD Complex Can Bind to the SBE Independent of Other Sequence-Specific Transcription Factors.
Previous studies have provided evidence that SMAD can be recruited to a promoter through interaction with a DNA-binding partner (27–33). In this report, we have demonstrated that a TGF-β-inducible SMAD complex can bind on its own to a 14- to 16-bp DNA probe that contains the SBE of the Smad7 promoter. Whereas both Smad3–Smad4 and Smad2–Smad4 complexes can bind to the SBE, these two complexes have different properties. The Smad3–Smad4 complex binds to the SBE at a much higher affinity and/or in a much more stable manner than either Smad4 or Smad3 alone. In contrast, the Smad2–Smad4 complex binds to the SBE similarly to Smad4 alone. We speculate that under physiological conditions, the Smad3–Smad4 complex plays a major role in binding to the SBE. A recent report showed that Smad3 and Smad4 can bind to the SBE of the mouse Smad7 promoter by using longer DNA probes (282 bp and 33 bp) (44). Because a number of putative binding sites for other transcription factors, such as NF-InsE2, NF-InsE3, USF, c-myc, AP-1, and ATF/CREB, are present in close vicinity to the SBE of the Smad7 promoter, that study did not exclude the possibility that other factors are required for SMAD binding to the SBE.
The palindromic SBE represents an optimal binding site for SMAD, which is the basis of its being selected from a pool of random oligonucleotides. A recent study examined Smad3–Smad4 complex binding to two or three copies of abutting sequences GTCT and AGAC in different combinations (25). Interestingly, Smad3–Smad4 has little or no activity to bind to two or three copies of the GTCT sequence, or the AGAC sequence followed by one or two copies of the GTCT sequence. These observations further indicate that the SBE is a high-affinity binding site for SMAD. Smad7 promoter is the first natural TGF-β responsive promoter in vertebrates that contains the SBE. The high affinity nature of the SBE explains, at least in part, its ability to be bound by a TGF-β-inducible SMAD complex independent of other transcription factors.
We speculate that when a high-affinity binding site, such as the palindromic SBE, is present on a TGF-β responsive promoter, a SMAD complex can bind to DNA on its own, as in the case of the Smad7 promoter. However, in a number of promoters that contain only one copy of the GTCT or AGAC sequences, as in the case of the Mix.2 promoter, SMAD requires association with a DNA-binding partner, such as FAST-1, to be able to bind to DNA (28–31). For a promoter that contains more than one copy of the GTCT or AGAC sequences, whether a SMAD complex can bind to these sequences on its own may vary, depending on the promoter context. Our findings presented in this report help us understand the complex DNA-binding activities of SMAD and can assist the identification of the transcriptional targets of SMAD that are important in a variety of biological processes including embryonic development as well as tumorigenesis.
Acknowledgments
We thank P. ten Dijke and C.-H. Heldin for Smad7 cDNA, J. Massagué for reagents, I. Matsuura for GST-SMAD proteins, the Robert Wood Johnson Medical School DNA lab for automatic sequencing, and many colleagues on campus, especially A. Rabson, C. Abate-Shen, M. Xiang, C. Gelinas, and A. Shatkin for encouragement, support, and suggestions. This work was supported by start-up funds from the CABM/Rutgers University, a grant from the New Jersey Commission on Cancer Research, and by awards from the American Association for Cancer Research-National Foundation for Cancer Research, the Cancer Institute of New Jersey, the Pharmaceutical Research and Manufacturer of America Foundation, and the Burroughs Wellcome Fund (to F.L.).
Abbreviations
- TGF-β
transforming growth factor β
- SBE
SMAD-binding element
- GST
glutathione S-transferase
Footnotes
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.090099297.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.090099297
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