SMAD BINDING ELEMENTS AND USES THEREOF

Abstract: The invention provides isolated nucleic acids including one or more Smad binding elements (SBEs) and Smad proteins and fragments thereof which bind to Smad binding elements. The invention also provides TGF beta response elements which confer TGF beta superfamily responsiveness to genes which have such elements in transcriptional control regions. Also provided are methods for using the Smad binding elements in methods of screening for compounds which bind the Smad binding element and in methods for modulating the binding of Smad proteins to Smad binding elements, e.g. to modulate TGF beta signal transduction. Further, the identification of a mammalian Smad binding sequence permits identification of the SBE binding determinants of Smad proteins.

Description: - 1 - SMAD BINDING ELEMENTS AND USES THEREOF Field of the Invention The invention relates to nucleic acid sequences which bind proteins and confer ligand- inducible transcriptional response. Background of the Invention The product of the JunB gene is a member of the AP-1 family of transcription factors that activate transcription by binding to TPA response elements (TRE) within the promoter of target genes. AP-1 components are immediate early gene products whose expression is rapidly induced byva variety of extra-cellular stimuli. JunB differs in biological properties from its homologs and appears to be a negative regulator of AP-1 function. This functional difference is due to a small number of amino acid changes between its DNA binding and dimerization motifs and the corresponding c-Jun sequences as well as to differences in phosphorylation status in response to mitogenic stimulation. The action of JunB as a negative regulator of TRE response elements corresponds with its induction by negative regulators of cell growth including TGFβ and related factors. In this respect, junB is among other known genes, e.g. cyclin dependent inhibitors (CDI) pi 5 and p21, and the plasminogen activator inhibitor (PAI-1) gene, that are known to be transcriptionally induced in response to TGFβ. However, the mechanism by which TGFβ and related factors transactivate these gene promoters has not been resolved. TGFβ initiates its action by binding to transmembrane receptors that possess serine/threonine kinase activity. Receptor activation involves binding of TGFβ to the type II receptor (TβR-II) which then recruits the type I receptor (TβR-I) in a tetrameric complex consisting of two ligand-bound type II and two type I receptors. This is followed by phosphorylation of the type I receptor by the type II receptor and phosphorylated type I receptor then further activates signal transmission leading to activation of gene transcription. Genetic studies in Drosophila and Caenorhabditis elegans have recently led to the identification of a conserved family of proteins termed Smads which play an important role in TGFβ receptor-downstream signal transduction. At present at least nine family members have been identified among vertebrates. Smads are 40-62 kDa proteins with N- and C-terminal homology domains (MH1 and MH2) connected by a proline-rich linker. Recent evidence suggests that in their inactive states the MH1 and MH2 domains interact and after - 2 - phosphorylation by receptor, Smad molecules undergo a conformational change that leads to heteromeric Smad complexes and translocation to the nucleus. Functional studies in Xenopus laevis have indicated different roles for Smads in receptor-downstream signalling. Smadl, Smad5 and presumably Smad9 associate with and are phosphorylated by BMP -mediated type I receptor activation, while Smad2 and Smad3 are phosphorylated after activation of TGFβR-I and activin type IB receptor (ActR-IB)-mediated activation, following phosphorylation, which occurs at a conserved SSXS motif at the extreme carboxy-terminus. These pathway-specific Smads heterodimerize with the common Smad4 to translocate to the nucleus to activate gene transcription. Phosphorylation of the pro line-linker by growth factor activated Erk-1 kinase activation was recently shown to play a role in Smad cytoplasmic-nuclear translocation and hence transcriptional activation. Smadό and Smad 7 are inhibitory Smads that have been shown to block Smad mediated signal transduction in a dominant-negative manner by stable receptor association. Support for a role of Smads as transcription factors has been obtained from a number of studies. The C-terminal domain of Smadl and Smad4 have transactivation activity when fused to the Gal4-DNA binding domain in a Gal4-reporter transactivation assay. Smad2 and Smad4 together with Fast 1 , a winged-helix DNA binding protein, form activin response factor (ARF) that bind to the activin response element of the Mix-2 promoter (Chen et al., Nature 383:691- 696, 1996; Chen et al., Nature 389:85-89, 1997). Smad3/4 overexpression transactivates the PAI- 1 and p3TP-lux promoters, which has been attributed to potentiation of AP 1 -dependent transcriptional activation. By contrast, Drosophila Mad domain binds a GC rich sequence in the Drosophila quadrant enhancer. These investigations have not been conclusive with regard to the role of Smads in transcriptional activation as well as the sequences with which they interact. Summary of the Invention The invention provides isolated nucleic acids including one or more Smad binding elements (SBEs), and Smad proteins and fragments thereof which bind to Smad binding elements. As described herein, binding of Smad proteins to SBEs results in transactivation of downstream nucleic acid sequences. The invention also provides TGFβ response elements which confer TGFβ superfamily responsiveness to genes which have such elements in transcriptional control regions; the SBEs surprisingly confer responsiveness via Smad binding to TGFβ, activin, and bone morphogenetic protein factors. The TGF-β superfamily members are - 3 - well known to those of ordinary skill in the art and include TGF-βs, activins, bone morphogenetic proteins (BMPs), Vgl, Mullerian inhibitory substance (MIS) and growth/differentiation factors (GDFs). Also provided are methods for using the Smad binding element in methods of screening for compounds which bind the Smad binding element and in methods for modulating the binding of Smad proteins to Smad binding elements to modulate TGFβ signal transduction. Further, the identification of a mammalian Smad binding sequence permits identification of the SBE binding determinants of Smad proteins by mutagenesis or crystallography. According to one aspect of the invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid includes a Smad binding element having a nucleotide sequence selected from the group consisting of CAGACA (SEQ ID NO:l), CAGACAG (SEQ ID NO:2), GAGACA (SEQ ID NO:3), GAGACAG (SEQ ID NO:4), CTGACA (SEQ ID NO:5), CTGACAG (SEQ ID NO:6), CAGACT (SEQ ID NO:7), CAGACTG (SEQ ID NO:8), CAGACAC (SEQ ID NO:9); and complements thereof, but preferably excludes known naturally occurring sequences which include a "wild-type" SBE sequence, such as the junB promoter. The activity of SEQ ID NOs:3-9 as SBEs is particularly unexpected because each of those sequences has a mutation relative to the "wild-type" j unB SBE sequence. In certain embodiments the isolated nucleic acid molecule includes at least two of the Smad binding elements, and preferably includes four or more of the Smad binding elements. In some embodiments the Smad binding elements are arranged as one or more inverted repeats. In preferred embodiments, the Smad binding elements are arranged in a non-natural arrangement, such as one which is not found in a naturally occurring nucleic acid known as of the effective filing date of this application, including arrangements which vary the sequence, spacing, inversion, flanking sequences, multiplicity and/or distance from promoter sequences of the Smad binding elements. In a preferred embodiment of the invention, the Smad binding elements include the nucleotide sequence CAGACAGTCTGTCTG (SEQ ID NO: 10). In other embodiments, the Smad binding element included in the isolated nucleic acid molecule is a fragment of the junB gene upstream region, wherein the nucleotide sequence of the nucleic acid molecule which flanks the Smad binding element is not derived ϊx vΑJunB. Preferred fragments of the junB gene upstream region include nucleotides -2813 through -2792, nucleotides -2908 through -2788, nucleotides -2908 through -2611, and nucleotides -3004 through -1534. In certain embodiments, the fragment of the junB gene upstream region is less than 50 nucleotides in length, preferably - 4 - including at least two Smad binding elements which more preferably are arranged as one or more inverted repeats. In certain embodiments, the foregoing isolated nucleic acid molecules are double stranded. According to another aspect of the invention, vectors which include the foregoing isolated nucleic acid molecules are provided. Preferably the isolated nucleic acid molecule is operably linked to a heterologous nucleic acid, preferably a nucleic acid which encodes a polypeptide. In preferred embodiments, the heterologous nucleic acid includes at least a portion of the coding sequence of a reporter gene which encodes a detectable nucleic acid and/or polypeptide. In certain embodiments, the operable linkage comprises a minimal promoter. In another aspect of the invention, host cells transformed or transfected with the foregoing nucleic acid molecules or vectors are provided. According to still other aspects of the invention, isolated polypeptides are provided which are fragments of Smad proteins which bind to Smad binding elements. In general such polypeptides include the Smad MH1 domain. Preferred isolated polypeptides include fragments of Smad proteins consisting of amino acids 1-279 of Smad3 or amino acids 1-321 of Smad4. In certain embodiments the foregoing isolated polypeptides are fusion proteins. SBE binding complexes of the foregoing polypeptides also are provided. According to yet another aspect of the invention, methods of screening for compounds which bind to a Smad binding element are provided. The method include the steps of contacting a nucleic acid comprising a Smad binding element with a test compound, and determining the binding of the test compound to the Smad binding element. In certain embodiments, the methods also include the steps of contacting a mutant Smad binding element with the test compound, determining the binding of the test compound to the mutant Smad binding element, and comparing the binding of the test compound to the Smad binding element and the mutant Smad binding element as a measure of the specific binding of the test compound to the Smad binding element. In some embodiments the mutant Smad binding element does not bind a Smad protein. In still another aspect of the invention, methods for identifying compounds that compete for binding of a Smad binding element with a Smad protein are provided. The methods include the steps of providing a nucleic acid comprising a Smad binding element, forming a complex comprising the nucleic acid and a Smad protein which binds the Smad binding element, contacting the complex with a test compound, and determining the binding of the Smad protein - 5 - to the nucleic acid as a measure of the displacement of the Smad protein by the test compound. In a further aspect, the invention provides methods of screening for compounds that modulate (i.e., increase or decrease) transcription mediated by Smad binding elements. The methods include providing a transcription system comprising a Smad protein which binds a Smad binding element and a nucleic acid molecule including a Smad binding element, contacting the transcription system with a test compound, and determining the transcription of the nucleic acid molecule as a measure of transcriptional modulation by the test compound. Increased transcription of the nucleic acid molecule in the presence of the test compound indicates that the test compound increases transcription mediated by the Smad binding element and decreased transcription indicates that the test compound decreases transcription mediated by the Smad binding element. In preferred embodiments the transcription system is a cell, preferably one which includes a Smad binding element reporter vector which is stably contained within the cell, such as a stably transfected cell line. In some embodiments the Smad protein which binds Smad binding element is Smad 1, Smad2, Smad3, Smad3ΔMH2, Smad4, Smad4ΔMH2, Smad5, a Smadl/Smad4 complex, a Smad2/Smad4 complex, a Smad3/Smad4 complex, or a Smad5/Smad4 complex. Preferably the nucleic acid molecule further comprises at least a portion of the coding sequence of a reporter gene, which encodes a detectable nucleic acid and/or polypeptide, operably linked to the Smad binding element. In the foregoing methods, the nucleic acid molecule preferably includes at least two Smad binding elements (preferably four or more) and more preferably the Smad binding elements are arranged as one or more inverted repeats. In some embodiments, the foregoing methods can be used to screen for compounds which regulate or modulate SBE-mediated ligand induced transcription. Such methods further include the steps of contacting the transcription system with a first ligand which induces transcription mediated by Smad binding elements, wherein the transcription system is responsive to the ligand. In these methods, a reduction in ligand induced transcription in the presence of the test compound indicates that the test compound interferes with the ligand induced transcription mediated by Smad binding elements and an increase in ligand induced transcription in the presence of the test compound indicates that the test compound enhances the ligand induced transcription mediated by Smad binding elements. The Smad binding elements described herein have the unexpected property of conferring transcriptional responsiveness to multiple TGFβ superfamily ligands, including at least TGFβ, activin and BMPs. Thus, in preferred embodiments, the methods described above can be used to - 6 - screen for and distinguish between compounds which selectively modulate transcription induced by a single TGFβ superfamily ligand (or other ligand which modulates transcription through SBEs) and compounds which modulate transcription induced by more than one such ligand. For these methods, the foregoing procedure is repeated at least once in the presence of a second ligand which induces transcription mediated by Smad binding elements. In these methods, a reduction or increase in transcription induced by the first ligand but not the second ligand indicates that the test compound selectively modulates the transcription mediated by Smad binding elements induced by the first ligand. A reduction or increase in transcription induced by the first ligand and the second ligand indicates that the test compound generally modulates the transcription mediated by Smad binding elements as induced by the first and the second ligands. The method can be repeated with several ligands in parallel assays to select for general and selective modulators of transcription. Preferred ligands include TGFβ, activin, OP-1, BMP-2, and GDF5. Likewise, the foregoing methods can be repeated with SBEs having various nucleotide sequences (e.g. SEQ ID NOs:l-10, and optionally including mutations of those sequences) to determine which sequences can bind Smads and/or are responsive or are non-responsive to any set of one or more TGFβ superfamily ligands. These sequences can then be included in nucleic acid molecules in accordance with the above-described procedures to identify compounds which modulate binding and/or transcription by certain Smads and/or TGFβ superfamily ligands. According to another aspect of the invention, methods for treating a subject having a condition characterized by an abnormally elevated level of Smad-mediated transcription mare provided. The methods include administering to a subject in need of such treatment an agent which decreases the level of binding of a Smad protein to an endogenous Smad binding element in an amount effective to reduce the abnormally elevated level of Smad-mediated transcription. In certain embodiments, the agent is an isolated nucleic acid molecule including a SBE, preferably which includes the nucleotide sequence CAGACA, an antisense nucleic acid which binds to a nucleic acid molecule encoding the Smad protein which binds a Smad binding element, or an antibody which binds to the Smad protein which binds a Smad binding element, the Smad binding element itself, or a complex of the Smad protein and the Smad binding element. According to still another aspect of the invention, methods for modulating transcription of genes which include a Smad binding element in a transcription control region are provided. - 7 - The methods include contacting a cell with an effective amount of an isolated nucleic acid molecule including a SBE, preferably which includes the nucleotide sequence CAGACA. In certain embodiments the nucleic acid includes at least two Smad binding elements, preferably arranged as one or more inverted repeats. In the foregoing methods, it is preferred that the isolated nucleic acid molecule is double stranded. In yet another aspect of the invention, methods are provided for modulating in a cell transcription of genes which include a Smad binding element in a transcription control region. The methods include contacting the cell with an effective amount of an agent which increases in the cell the level of a Smad protein which binds a Smad binding element or a fragment of the Smad protein which binds a Smad binding element. In some embodiments the agent is an expression vector which encodes the Smad protein which binds a Smad binding element or a fragment of the Smad protein which binds a Smad binding element. In other embodiments the agent is the Smad protein which binds a Smad binding element or a fragment of the Smad protein which binds a Smad binding element. Preferred Smad proteins in these methods include Smad3, Smad4 and fragments thereof. According to another aspect of the invention, methods are provided for modulating in a cell transcription of genes comprising a Smad binding element in a transcription control region. The methods include contacting the cell with an effective amount of an agent which decreases the level of a Smad protein which binds a Smad binding element in the cell. In certain embodiments, the agent is an antisense nucleic acid which binds to a nucleic acid molecule encoding the Smad protein which binds a Smad binding element, or an antibody which binds to the Smad protein which binds a Smad binding element. In the foregoing methods the Smad protein which binds a Smad binding element preferably is Smad 1, Smad2, Smad3, Smad3ΔMH2, Smad4, Smad4ΔMH2, Smad5, a Smadl/Smad4 complex, a Smad2/Smad4 complex, a Smad3/Smad4 complex, or a Smad5/Smad4 complex. In another aspect of the invention, methods are provided for modulating in a cell transcription of genes comprising a Smad binding element in a transcription control region. The methods include contacting the cell with an effective amount of an agent which modulates binding of a Smad protein to a Smad binding element, or which itself binds to a Smad binding element, wherein the agent modulates transcription. Agents may be identified in accordance with the methods disclosed herein According to a further aspect of the invention, methods of identifying a Smad protein - 8 - having altered binding to a Smad binding element nucleic acid sequence are provided. The methods include contacting a sample containing a Smad protein, the nonmutated form of which binds a Smad binding element in a first amount, with an isolated nucleic acid molecule comprising the Smad binding element, determining a second amount of binding of the Smad binding element by the Smad protein, and comparing the first amount and the second amount of binding of the Smad binding element. A difference between the first amount and the second amount indicates that the sample contains a mutant Smad protein with altered nucleic acid binding characteristics. In certain embodiments, the Smad binding element includes the nucleotide sequence CAGACA. For use in these methods, the Smad protein preferably is prepared by mutagenesis, more preferably by site-directed mutagenesis. According to another aspect of the invention, the use of isolated nucleic acid molecules which include a Smad binding element, including antisense nucleic acids, in the manufacture of a medicament is provided. The invention also embraces functional variants and equivalents of all of the molecules described above. These and other objects of the invention will be described in further detail in connection with the detailed description of the invention. Brief Description of the Drawings Fig. 1 shows that Smad3 and Smad4 activate the JunB promoter through a region that binds a TGFβ-induced protein. (A) The JunB promoter is activated by Smad3 and Smad4 between nucleotides -3004 and -1534. (B) The Smad3 and Smad4-responsive region within the JunB promoter is located in a 297 bp (nt -2908/-2611) fragment. Fig. 2 shows the identification of a 22 bp fragment containing a 7 bp inverted repeat as a Smad-responsive element in the JunB promoter. Fig. 3 shows that SBEs are essential TGFβ response elements. (A) TGFβ and Smad3 and Smad4 activate SBE containing reporter to an equal extent. (B) Activation of the TGFβ response element is inhibited by Smad7. Fig. 4 shows that the SBE is a responsive element for TGFβ superfamily. (A) The JunB promoter is activated by different pathway-specific Smad proteins. (B) Pathway-specific Smads differentially activate the SBE. (C) SBE is activated by TGFβ, activin and osteogenic protein- 1 (OP-1). (D) Activation of SBE is dependent on Smad4 protein. Detailed Description of the Invention A short sequence has been identified in the mouse JunB gene promoter, termed the Smad Binding Element (SBE), that mediates responsiveness to several members of the TGFβ ligand superfamily. The TGFβ superfamily members are well known to those of ordinary skill in the art and include TGFβs, activins, bone morphogenetic proteins (BMPs), Vgl, Mullerian inhibitory substance (MIS) and growth/differentiation factors (GDFs). It has been determined that Smad proteins, particularly Smad 3 and Smad 4, interact with specific nucleotide sequences in the upstream control region of the junB gene by investigation of the transcriptional regulation of the immediate early gene junB by TGFβ. Transient overexpression of Smad3 and Smad4 with various junB promoter constructs localized a region in the junB promoter that is transactivated and shows inducible binding of a Smad protein containing complex. Further characterization led to the identification of an inverted hexanucleotide repeated sequence to which the inducible DNA binding activity was localized and which unexpectedly creates a powerful TGFβ inducible enhancer when multimerized. Thus the invention provides nucleic acid molecules which include a Smad binding element. The minimal Smad binding element is the hexanucleotide CAGACA. One or more nucleotides can be added to one or both ends of the SBE without destroying the Smad binding characteristics of the nucleotide sequence. For example, one nucleotide can be added to the 3' end of the hexanucleotide to form the heptanucleotide sequence CAGACAG, which is shown below to be a SBE. The invention includes SBEs formed by the addition of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 75, and more nucleotides to one or both ends of the hexanucleotide CAGACA. For example, certain fragments of the junB transcriptional control region were found to effectively confer Smad transactivation upon a reporter construct. The Smad transactivation properties of the isolated junB transcriptional control region were not previously recognized, and were unexpected. These fragments range in size from 22 nucleotides to 1471 nucleotides and more. In preferred embodiments, the nucleotide sequence of the nucleic acid which flanks the Smad binding element is not derived from junB. In other embodiments where the sequence flanking the SBE is derived from junB, it is preferred that the fragment of the junB gene upstream region flanking region is less than 50 nucleotides in length in total. The Smad binding element in the junB gene was found to be a multimer of the hexanucleotide CAGACA, arranged as an inverted repeat (CAGACAGTCTGTCTG). As - 10 - described below, this inverted repeat multimer confers TGFβ responsiveness of transcription when the inverted repeat is included in a transcriptional control region. The junB SBE inverted repeat is thus a TGFβ response element. Additional experiments described herein demonstrate that other multimeric SBEs (including 2x and 4x direct repeat multimers) also provide TGFβ response element activity. Thus SBEs arranged as multimers are preferred when TGFβ response element activity is desired. SBE multimers are also preferred when it is desired to have Smad binding activity, as multimers have greater numbers of Smad binding sites per molecule. The number of SBEs in a multimer is not limited in the broadest embodiments of the invention. However, in certain embodiments of the invention it may preferred that the number of SBEs in a multimeric configuration is less than 50, more preferably less than 25, and still more preferably less than 10, and yet still more preferably less than 5. Limitation on the number of SBEs in a multimer may be of importance in those multimeric configurations which favor formation of stem-loop structures, such as inverted repeats. One of ordinary skill in the art can readily prepare and test a variety of multimeric configurations in accordance with the methods described herein. The Smad binding elements can be arranged in non-natural arrangements or configurations, such as an arrangement or configuration which is not found in a naturally occurring nucleic acid known as of the effective filing date of this application. Non-natural arrangements include arrangements which vary the sequence, spacing, inversion, flanking sequences, multiplicity and/or distance from promoter sequences of the Smad binding elements from those found in natural arrangements. "Sequence" means the nucleotide sequence of the Smad binding element itself. "Spacing" means the number of nucleotides between individual SBEs of a direct or inverted repeat, or the number of nucleotides between one SBE (or repeat) and a 5' or 3' SBE or repeat. For example, the SBE repeat set forth in SEQ ID NO: 10 contains a single nucleotide between the halves of the repeat, and thus has a spacing of one nucleotide. The number of nucleotides between the SBE repeat of SEQ ID NO: 10 in the JunB transcriptional control region and another SBE in that control region would be the other use of the term spacing with respect to SBEs. "Inversion" means the linear relation of SBE repeats to each other. For example, the SBE repeat set forth in SEQ ID NO: 10 is an inverted repeat; direct repeats are set forth in other sequences described herein. Thus, one can vary the inversion of SBE repeats, for example, by altering a direct repeat to an inverted repeat, and vice versa. "Flanking sequences" are the nucleotide sequences 5' and 3' of a natural SBE. "Multiplicity" means the number of - 11 - SBEs in a transcriptional control region or other nucleic acid, such as an oligonucleotide. "Distance from promoter sequences" means the number of nucleotides between a particular Smad binding element and a promoter sequence. One or more of the foregoing properties can be altered and tested by one of ordinary skill in the art, using routine experimentation, to prepare non-natural arrangements of SBEs. The Smad binding element sequences described above having a wild-type nucleotide sequence (e.g., SEQ ID NOs:l, 2, and 10) are preferred, but by no means the only functional SBE sequences. Unexpectedly, it was determined that certain variant SBEs having one or more mutations in the SBE nucleotide sequence were "functional variant SBEs"; that is, the variant SBEs retained the ability to bind Smad proteins, as shown by direct binding and competition experiments. Thus, functional variant SBEs which differ in one or more nucleotides from the wild-type SBE sequences provided above are also embraced by the invention. Exemplary functional variant SBEs which bind Smads (as shown by competition for binding of a "wild- type" nonmutant SBE, see the Examples) include gAGACA (SEQ ID NO:3), gAGACAG (SEQ ID NO:4), CtGACA (SEQ ID NO:5), CtGACAG (SEQ ID NO:6), CAGACt (SEQ ID NO:7), CAGACtG (SEQ ID NO:8) and CAGACAc (SEQ ID NO:9). The mutation in each variant SBE is indicated by a lower case letter. Variant SBEs in which nucleotides in the central GAC sequence of the CAGACA SBE were mutated ("non-functional variant SBEs") were found to have substantially decreased Smad binding characteristics as determined by a loss of the ability to compete for binding to Smad proteins with a wild type SBE in an electrophoretic mobility shift assay. Of course, such non-functional variant SBEs are useful as negative controls in the assays and methods described herein. As exemplified below, one of ordinary skill in the art can readily test a variant SBE to determine if retains the Smad binding property of wild type SBEs, i.e., that the variant SBE is a functional variant SBE. Additional variant SBEs can be made by standard methods in the art, including chemical synthesis of random nucleotide sequences, site directed mutagenesis, random mutagenesis, etc. The skilled artisan also will be familiar with methods of selecting additional SBE sequences by computer database searches using standard software packages which select sequences based on nucleotide homology. Preferably such a search is conducted on sequences derived from transcriptional control regions of genes. As used herein with respect to nucleic acids, the term "isolated" means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; - 12 - (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5' and 3 ' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art. In general, the Smad binding characteristics of a putative SBE can be determined experiments in which a Smad protein or complex of Smad proteins is contacted with the putative SBE under conditions which permit binding of the Smad protein to the SBE. The binding experiments can assay Smad-SBE binding directly, or indirectly, such as in competition experiments wherein the putative SBE is used as a competitor nucleic acid for a known SBE- Smad binding pair. These conditions are generally known to one of ordinary skill in the art of protein-nucleic acid binding and can be performed using routine experimentation. Certain preferred binding conditions are set forth in greater detail in the Examples below. Smad proteins bound in vitro by SBEs include Smad4 and derivatives of Smad3 and Smad4 in which the MH2 domain is deleted (Smad3ΔMH2 and Smad4ΔMH2). In cells, it is believed that other Smad proteins, including Smadl, Smad2, Smad3, and Smad5 bind to SBEs. It is also believed that complexes of the foregoing Smad proteins with Smad4 bind to SBEs in cells. As shown below, cells transfected with expression vectors encoding Smad proteins and Smad complexes exhibit increased transactivation of SBE-containing reporter constructs. The Smad binding elements are useful for binding Smad proteins. Thus SBEs can be used to isolate Smad proteins and complexes thereof, as well as to detect Smad proteins and complexes thereof. Smad binding elements, as a TGFβ superfamily response element, are also useful for regulating transcription of genes. When placed in a transcription control region, SBEs confer transcriptional responsiveness to ligands of the TGFβ superfamily in transcriptional systems having the appropriate ligand receptor and signal transduction apparatus. Receptors and signal transduction factors for TGFβ superfamily members are known in the art. Therefore, a detectable gene (e.g., a reporter gene having a detectable nucleic acid or protein gene product) - 13 - placed under the control of one or more Smad binding elements can be used to test the integrity of TGFβ superfamily signal transduction in a variety of cell types. Smad binding elements can further be used in nucleic acid hybridizations to identify related sequences and identify additional gene transcriptional control regions which include SBEs. Similarly, Smad binding element sequences can be incorporated into primers useful in polymerase chain reaction assays of SBEs in transcriptional control regions. Conditions, reagents and so forth for hybridization and polymerase chain reaction assays are well known in the art and it is within the skill of the person of ordinary skill in the art to modify such conditions to achieve the appropriate stringency of probe or primer hybridization to selectively isolate or identify SBE sequences. Examples of such conditions, reagents etc. are provided in compilations of molecular biology methods, including, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. As mentioned above, the invention embraces antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding a Smad polypeptide which binds to a Smad binding element, e.g., Smad3, Smad4 and derivatives thereof. Nucleic acid molecules which bind to SBEs and block binding of proteins thereto are also contemplated. This is desirable in virtually any medical condition wherein a reduction of Smad protein binding to a Smad binding element is desirable, e.g., to modulate TGF-β superfamily transcriptional response or reduce Smad transactivation of SBEs by reducing the amount of Smad. The antisense oligonucleotides also are useful in screening assays for identifying compounds which bind to or regulate binding to a Smad binding element, or which regulate TGF-β superfamily transcriptional response mediated by a Smad binding element. As used herein, the term "antisense oligonucleotide" or "antisense" describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA. The antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the - 14 - particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based upon known Smad gene sequences, or upon allelic or homologous genomic and/or cDNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention. In order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the target, although in certain cases modified oligonucleotides as short as 7 bases in length have been used successfully as antisense oligonucleotides (Wagner et al., Nature Biotechnol. 14:840-844, 1996). Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases. Although oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5' upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3'-untranslated regions may be targeted. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs. In addition, the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol. Neurobiol. 14(5):439-457, 1994) and at which proteins are not expected to bind. In one set of embodiments, the nucleic acids which contain a Smad binding element of the invention, or the antisense oligonucleotides of the invention may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5' end of one native nucleotide and the 3' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester intemucleoside linkage. These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors. In preferred embodiments, however, the nucleic acids of the invention (sense or antisense) also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from binding proteins or hybridizing to their target nucleic acids but which enhance their stability or targeting or which otherwise enhance their effectiveness. - 15 - The term "modified oligonucleotide" as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic intemucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide. Preferred synthetic intemucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides. The term "modified oligonucleotide" also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus modified oligonucleotides may include a 2'-O-alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. The present invention, thus, contemplates compositions containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding Smad polypeptides which bind a Smad binding element, optionally together with a carrier. Antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art. The compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The characteristics of the carrier will depend on the route of administration. Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art. As used herein, a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids and - 16 - virus genomes. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., luciferase, β-galactosidase and alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined. As used herein, a coding sequence and regulatory sequences are said to be "operably" joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' - 17 - non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Such 5' non-transcribed regulatory sequences especially will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include, in addition to one or more SBE sequences, other enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art. Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of a heterologous DNA (RNA). That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell. In certain embodiments the transcriptional elements include one or more Smad binding elements sufficient to confer Smad transactivation or TGFβ superfamily ligand responsiveness. Preferred systems for mRNA expression in mammalian cells are those such as pRc/CMV (available from Invitrogen, Carlsbad, CA) that contain a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences. Additionally, suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen), which contains an Epstein Barr vims (EBV) origin of replication, facilitating the maintenance of plasmid as a multicopy extrachromosomal element. Another expression vector is the pEF-BOS plasmid containing the promoter of polypeptide Elongation Factor lα, which stimulates efficiently transcription in vitro. The plasmid is described by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), and its use in transfection experiments is disclosed by, for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferred expression vector is an adenovirus, described by Stratford-Perricaudet (J Clin. Invest. 90:626-630, 1992), which is defective for El and E3 proteins. Still other expression vectors are described in the Examples below. Where the expression vector is a TGFβ superfamily inducible vector which contains one or more Smad binding elements, the vector will not contain a constitutive promoter, but may contain a minimal promoter. Alternatively, a Smad binding element containing expression - 18 - vector can contain a cloning site downstream of the Smad binding element for cloning of a promoter and a sequence to be expressed. This arrangement also permits testing of the ability of a Smad binding element to effectively regulate transcription from a given promoter. The invention also embraces kits, which allow the artisan to test compounds for their ability to bind a Smad binding element, to increase or reduce Smad binding to a Smad binding element, or to modulate transcription of nucleic acids which are controlled by a Smad binding element. Such kits include at least the previously discussed Smad binding element nucleic acid sequences. Other components may be added, as desired, as long as the previously mentioned sequences, which are required, are included. The invention also provides isolated polypeptides and complexes of polypeptides which bind a Smad binding element. The polypeptides include Smad proteins such as Smadl, Smad2, Smad3, Smad4 and Smad5, as well as Smad derivatives such as Smad proteins having a truncated C-terminus, e.g. a complete or partial deletion of the MH2 domain, such as the Smad3ΔMH2 and Smad4ΔMH2 proteins described below. The complexes of polypeptides include Smadl/Smad4 complex, a Smad2/Smad4 complex, a Smad3/Smad4 complex, and a Smad5/Smad4 complex, each component of which can be a wild-type Smad protein or a derivative thereof. Such polypeptides are useful, for example, in in vitro assays of SBE- regulated transcription. Such protein also are useful in therapeutic methods for modulating binding to a Smad binding element, or for modulating transcription regulated by a Smad binding element. The proteins also can be used in screening methods for identifying compounds which bind to a Smad binding element, or which modulate transcription regulated by a Smad binding element. The invention embraces variants of the Smad polypeptides described above. As used herein, a "variant" of a Smad polypeptide is a polypeptide which contains one or more modifications to the primary amino acid sequence of a Smad polypeptide. Modifications which create a Smad variant can be made to a Smad polypeptide 1) to reduce or eliminate an activity of a Smad polypeptide, such as binding to a SBE; 2) to enhance a property of a Smad polypeptide, such as protein stability in an expression system or the stability of protein-protein binding or such as binding to a SBE; or 3) to provide a novel activity or property to a Smad polypeptide, such as addition of an antigenic epitope or addition of a detectable moiety. Modifications to a Smad polypeptide are typically made to the nucleic acid which encodes the Smad polypeptide, and can include deletions, point mutations, truncations, amino acid substitutions and additions of - 19 - amino acids or non-amino acid moieties. Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part of the various Smad amino acid sequences. In general, variants include Smad polypeptides which are modified specifically to alter a feature of the polypeptide unrelated to its physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages. Similarly, certain amino acids can be changed to enhance expression of a Smad polypeptide by eliminating proteolysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present). Mutations of a nucleic acid which encode a Smad polypeptide preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such a hairpins or loops, which can be deleterious to expression of the variant polypeptide. Mutations can be made by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the polypeptide. Variant polypeptides are then expressed and tested for one or more activities such as binding to a Smad binding element to determine which mutation provides a variant polypeptide with the desired properties. Further mutations can be made to variants (or to non-variant Smad polypeptides) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli, are well known to those of ordinary skill in the art. Still other mutations can be made to the noncoding sequences of a Smad gene or cDNA clone to enhance expression of the polypeptide. The activity of variants of Smad polypeptides can be tested by cloning the gene encoding the variant Smad polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the variant Smad polypeptide, and testing for a functional capability of the Smad polypeptides as disclosed herein. For example, the variant Smad polypeptide can be tested for inhibition of TGFβ (and/or activin or BMP) signalling activity as disclosed in the Examples, or for inhibition of Smad transactivation from SBEs or for SBE binding as is also disclosed herein. Preparation of other variant polypeptides may favor testing of other activities, as will be known to one of ordinary skill in the art. The skilled artisan will also realize that conservative amino acid substitutions may be - 20 - made in Smad polypeptides to provide functionally equivalent variants of the foregoing polypeptides, i.e, variants which retain the functional capabilities of the Smad polypeptides. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution which does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al, eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Exemplary functionally equivalent variants of the Smad polypeptides include Smads having a deletion of the MH2 domain, such as Smad4ΔMH2. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Conservative amino-acid substitutions in the amino acid sequence of Smad polypeptides to produce functionally equivalent variants of Smad polypeptides (including fusion proteins) typically are made by alteration of the nucleic acid encoding Smad polypeptides. Such substitutions can be made by a variety of methods known to one of ordinary skill in the art. For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488- 492, 1985), or by chemical synthesis of a gene encoding a Smad polypeptide. Where amino acid substitutions are made to a small fragment of a Smad polypeptide, such as a SBE binding site, the substitutions can be made by directly synthesizing a peptide which includes the fragment. The activity of functionally equivalent fragments of Smad polypeptides can be tested by cloning the gene encoding the altered Smad polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the altered Smad polypeptide, and testing for a functional capability of the Smad polypeptides as disclosed herein. Peptides which are chemically synthesized can be tested directly for function, e.g., for binding to Smad binding elements. A variety of methodologies well known to the skilled practitioner can be utilized to obtain isolated Smad molecules. The polypeptide may be purified from cells which naturally produce the polypeptide by chromatographic means or immunological recognition. Alternatively, an expression vector may be introduced into cells to cause production of the - 21 - polypeptide. In another method, mRNA transcripts may be microinjected or otherwise introduced into cells to cause production of the encoded polypeptide. Translation of mRNA in cell-free extracts such as the reticulocyte lysate system also may be used to produce polypeptide. Those skilled in the art also can readily follow known methods for isolating Smad polypeptides. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography and immune-affinity chromatography. The invention also makes it possible isolate Smad protein complexes such as Smadl/Smad4, Smad2/Smad4, Smad3/Smad4 and Smad5/Smad4 by the binding of such proteins to a SBE as disclosed herein. The identification of this binding also permits one of skill in the art to block the binding of Smad proteins to endogenous Smad binding elements (e.g. in gene control regions of endogenous genes). For example, binding of such proteins can be affected by introducing into a biological system (e.g., a cell) in which the proteins bind an oligonucleotide including a Smad binding element in an amount sufficient to reduce or even block the binding of Smad proteins and endogenous SBEs. The identification of Smad binding to SBEs also enables one of skill in the art to identify Smad amino acid sequences which bind to such SBEs and prepare modified proteins, using standard recombinant DNA techniques, which can bind to SBEs. For example, when one desires to place a certain gene under the control of a particular transcription factor, one can prepare a fusion polypeptide of the transcription factor protein and the Smad SBE binding site. Inclusion of SBEs in the transcriptional control region of the desired gene will then confer control of the transcription of that gene by the modified transcription factor. The invention also provides, in certain embodiments, "dominant negative" Smad polypeptides which have mutated SBE binding sites. Such a dominant negative polypeptide interacts with the cellular machinery (such as another Smad protein in a Smad/Smad complex), thereby displacing an active protein from its interaction with the cellular machinery or competing with the active protein, thereby reducing the effect of the active protein. The end result of the expression of a dominant negative polypeptide in a cell is a reduction in function of active proteins. One of ordinary skill in the art can assess the potential for a dominant negative variant of a protein, and using standard mutagenesis techniques to create one or more dominant negative variant polypeptides. For example, given the teachings contained herein of Smad polypeptides which bind SBEs, one of ordinary skill in the art can modify the sequence of a Smad polypeptide by site-specific mutagenesis, scanning mutagenesis, partial gene - 22 - deletion or t ncation, and the like. See, e.g., U.S. Patent No. 5,580,723 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. The skilled artisan then can test the population of mutagenized polypeptides for diminution in a selected activity (e.g., Smad binding to SBEs, Smad transactivation of SBEs, TGFβ signalling activity) and/or for retention of such an activity. Other similar methods for creating and testing dominant negative variants of a protein will be apparent to one of ordinary skill in the art. The invention also involves the use of agents such as polypeptides which bind to Smad polypeptides and/or to complexes of Smad polypeptides and/or to SBEs to modulate SBE- mediated activities. The invention, therefore, embraces peptide binding agents which, for example, can be antibodies or fragments of antibodies having the ability to selectively bind to Smad polypeptides. Antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology. Agents also include antisense nucleic acid (described above) which are useful for reducing the expression of Smad proteins, thereby reducing SBE- mediated activities. Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W.R. (1986) The Experimental Foundations of Modem Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology. 7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab') 2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation. Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, - 23 - in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity. It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. Thus, for example, PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies. Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for the use of F(ab') 2 , Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab') 2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes the use of so-called single chain antibodies. Thus, the invention involves the use of polypeptides of numerous size and type that bind specifically to Smad polypeptides, and complexes of Smad polypeptides. These polypeptides may be derived also from sources other than antibody technology. For example, such polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries. Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptoids and non-peptide synthetic moieties (e.g., peptidomimetics). Such peptides and related molecules can readily be screened for the ability to reduce Smad-SBE - 24 - binding according to standard procedures, some of which are detailed herein. Phage display can be particularly effective in identifying binding peptides useful according to the invention. Briefly, one prepares a phage library (using e.g. ml 3, fd, or lambda phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures. The inserts may represent, for example, a completely degenerate or biased array. One then can select phage-bearing inserts which bind to a SBE containing nucleic acid, or to a Smad polypeptide, sufficiently to reduce Smad-SBE binding. This process can be repeated through several cycles of reselection of phage that bind to the SBE or Smad polypeptide. Repeated rounds lead to enrichment of phage bearing particular sequences. DNA sequence analysis can be conducted to identify the sequences of the expressed polypeptides. The minimal linear portion of the sequence that binds to the SBE or Smad polypeptide can be determined. One can repeat the procedure using a biased library containing inserts containing part or all of the minimal linear portion plus one or more additional degenerate residues upstream or downstream thereof. Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to the Smad polypeptides. Thus, the Smad binding element nucleotide sequences can be used to screen peptide libraries, including phage display libraries, to identify and select molecules which bind to SBEs. Other non-peptide compounds, including libraries of such compounds, can also be screened for SBE binding activity. Such molecules can be used, as described, for screening assays, for purification protocols, for interfering directly with the functioning of SBEs and for other purposes that will be apparent to those of ordinary skill in the art. A Smad binding element, or a multimers thereof, also can be used to isolate their native binding partners, including, e.g., the Smad proteins and Smad protein complexes. Isolation of such binding partners may be performed according to well-known methods. For example, isolated nucleic acids including one or more Smad binding elements can be attached to a substrate (e.g., chromatographic media, such as polystyrene beads, or a filter), and then a solution suspected of containing the Smad binding element binding proteins may be applied to the substrate. If a protein or protein complex which can bind a Smad binding element is present in the solution, then it will bind to the substrate-bound Smad binding element. The protein or protein complex then may be isolated. Other molecules which bind Smad binding elements, may be isolated by similar methods without undue experimentation. The invention further provides methods for modulating (reducing or increasing) TGFβ superfamily signal transduction in a cell. Such methods are useful in vitro for altering the TGF-β - 25 - signal transduction, for example, in testing compounds for potential to block abnormally elevated TGF-β signal transduction or increase deficient TGF-β signal transduction. In vivo, such methods are useful for modulating growth, e.g., to treat cancer and fibrosis. Increasing TGF-β signal transduction in a cell by, e.g., introducing a reporter gene under the control of one or more Smad binding elements (TGFβ response elements) in the cell, can be used to provide a model system for testing the effects of putative inhibitors of TGF-β signal transduction. Such methods also are useful in the treatment of conditions which result from excessive or deficient TGF-β signal transduction. TGF-β signal transduction can be measured by a variety of ways known to one of ordinary skill in the art, such as the reporter systems described in the Examples. Various modulators of Smad-SBE binding activity can be screened for effects on TGF-β signal transduction using the methods disclosed herein. The skilled artisan can first determine the modulation of a SBE activity, such as TGF-β signalling activity, and then apply such a modulator to a target cell or subject and assess the effect on the target cell or subject. For example, in screening for modulators of Smad-SBE binding useful in the treatment of cancer, cells in culture can be contacted with Smad-SBE binding modulators and the increase or decrease of growth or focus formation of the cells can be determined according to standard procedures. Smad-SBE binding activity modulators can be assessed for their effects on other TGF-β signal transduction downstream effects by similar methods in many cell types. Thus it can be of therapeutic benefit to administer an SBE-containing nucleic acid, or an antagonist of Smad-SBE binding, to modulate TGF-β superfamily activity in certain conditions characterized by abnormally elevated TGF-β superfamily activity. Specific examples of conditions involving abnormally elevated TGF-β superfamily activity include ossification of the posterior longitudinal ligament (Yonemori et al., Am. J. Pathol. 150:1335-1347, 1997); ossification of the ligament flavum (Hayashi et al., Bone 21 :23-30, 1997); liver fibrosis including cirrhosis and veno-occlusive disease; kidney fibrosis including glomerulonephritis, diabetic nephropathy, allograft rejection and HIV nephropathy; lung fibrosis including idiopathic fibrosis and autoimmune fibrosis; skin fibrosis including systemic sclerosis, keloids, hypertrophic bum scars and eosinophilia-myalgia syndrome; arterial fibrosis including vascular restenosis and atherosclerosis; central nervous system fibrosis including intraocular fibrosis; and other fibrotic diseases including rheumatoid arthritis and nasal polyposis. (see, e.g., Border and Noble, N. Engl. J. Med. 331 :1286-1292, 1994). An effective amount of an SBE-containing nucleic acid, or an agent which modulates - 26 - Smad-SBE binding or transcriptional effects thereof, is administered to treat the condition, which amount can be determined by one of ordinary skill in the art by routine experimentation. For example, to determine an effective amount of an SBE-containing nucleic acid for treating fibrosis or ossification, an SBE-containing nucleic acid can be administered and the progress of the fibrosis or ossification monitored using standard medical diagnostic methods. An amount of an SBE-containing nucleic acid, or an antagonist of Smad-SBE binding which reduces the progression of the fibrosis or ossification, or even halts the progression of the fibrosis or ossification is an effective amount. The person of ordinary skill in the art will be familiar with such methods. Other conditions involving abnormally elevated TGF-β superfamily activity can be treated in a like manner, by administering SBE or a Smad-SBE binding antagonist, , to reduce TGF-β superfamily activity into normal ranges as needed. Smad-SBE binding antagonists include antibodies to Smad proteins, nucleic acids including SBE sequences and antisense Smad nucleic acids described above. When administered, the therapeutic compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal. When antibodies are used therapeutically, a preferred route of administration is by pulmonary aerosol. Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences. 18th edition, 1990, pp 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing antibody aerosols without resort to undue experimentation. When using antisense preparations of the invention, slow intravenous administration is preferred. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl - 27 - oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. The preparations of the invention are administered in effective amounts. An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, produces the desired response. For example, in the case of treating cancer, the desired response is inhibiting the progression of the cancer. In the case of treating ossification of the ligamentum flavum, the desired response is inhibiting the progression of the ossification. In the case of treating fibrosis, the desired response is inhibiting the progression of the fibrosis. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods or can be monitored according to diagnostic methods of the invention discussed herein. The invention also contemplates gene therapy. The procedure for performing ex vivo gene therapy is outlined in U.S. Patent 5,399,346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents. In general, it involves introduction in vitro of a functional copy of a gene into a cell(s) of a subject which contains a defective copy of the gene, and returning the genetically engineered cell(s) to the subject. The functional copy of the gene is under operable control of regulatory elements which permit expression of the gene in the genetically engineered cell(s). In the present invention, a functional gene need not be expressed; the gene therapy can be the product of the administration of nucleic acids, especially double stranded nucleic acids, which contain one or preferably more than one SBEs and thereby can act by binding endogenous SBE binding factors. Numerous transfection and transduction techniques as well as appropriate expression vectors are well known to those of ordinary skill in the art, some of which are described in PCT application WO95/00654. In vivo gene therapy using vectors such as adenovirus, retroviruses, herpes vims, and targeted liposomes, as well as naked DNA, also is contemplated according to the invention. The invention further provides efficient methods of identifying pharmacological agents or lead compounds for agents active at the level of SBE-Smad binding or a Smad binding element modulatable cellular function. In particular, such functions include TGFβ superfamily signal - 28 - transduction, Smad transactivation and formation of a SBE-Smad complex. Generally, the screening methods involve assaying for compounds which interfere with one of these activities. Such methods are adaptable to automated, high throughput screening of compounds. The target therapeutic indications for pharmacological agents detected by the screening methods are limited only in that the target cellular function be subject to modulation by alteration of the formation of a complex comprising a Smad polypeptide or fragment thereof and one or more natural Smad nucleic acid binding targets, such as the Smad binding elements described herein. Target indications include cellular processes modulated by TGF-β superfamily signal transduction following receptor-ligand binding. A wide variety of assays for pharmacological agents are provided, including, labeled in vitro protein-protein binding assays, protein-nucleic acid binding assays including electrophoretic mobility shift assays, immunoassays, in vitro transcription assays, cell-based assays such as two- or three-hybrid screens and expression assays, etc. For example, expression assays are used to rapidly examine the effect of transfected nucleic acids or applied compounds on the binding of Smad proteins to Smad binding elements. The transfected nucleic acids can encode, for example, combinatorial peptide libraries or antisense molecules. Convenient reagents for such assays, e.g., GAL4 fusion proteins, are known in the art, and examples are provided below. An exemplary cell-based assay involves transfecting a cell with a nucleic acid encoding a Smad polypeptide, such as Smad4, and a nucleic acid encoding a test peptide which potentially modulates Smad-SBE binding. The cell also contains a reporter gene operably linked to a gene expression regulatory region containing one or more Smad binding elements. A change in the activation of reporter gene transcription occurs when the test peptide modulates Smad-SBE binding such that the Smad-mediate transcription of the reporter gene is modulated. Agents which modulate Smad transactivation of the reporter gene are then detected through a change in the expression of reporter gene. Methods for determining changes in the expression of a reporter gene are known in the art and exemplified below. Likewise, assays in which non-nucleic acid test compounds are added to the cell are provided. The foregoing cell-based assays can be used to screen for compounds which modulate TGFβ superfamily signal transduction activity, in the presence of TGFβ superfamily ligands or in the absence of TGFβ superfamily ligand. Addition of ligand to the cell based assays will permit identification and selection of compounds which reduce TGFβ superfamily ligand stimulated signal transduction. Thus the cell based assays permit the selection of compounds - 29 - which modulate TGFβ superfamily signal transduction at any stage in the signal transduction pathway upstream of SBE-mediated transcriptional induction, including ligand binding, activation of receptors independent of ligand, prevention of Smad nuclear translocation, modulation of receptor-Smad interaction, upregulation of inhibitory Smad expression, etc. As shown herein, the SBE (e.g. as a reporter construct) is responsive to several members of the TGFβ superfamily, including TGFβ, activin and OP-1. Thus the assays can be carried out in parallel by stimulating with one, two, or more TGFβ superfamily ligands in individual assays, thereby permitting the identification of compounds which modulate the signal transduction induced by one or more of the ligands. Where only one ligand is affected, the compound is specific for that member of the TGFβ superfamily. Where more than one ligand is affected, the compound has a broader spectrum of activity, e.g., is a more general or less specific modulator of TGFβ superfamily signal transduction. A variety of functional variant SBEs can also be used in such assays to determine sequences of SBEs which are responsive to particular sets of one or more TGFβ superfamily ligands. Smad proteins and fragments used in the methods, when not produced by a transfected nucleic acid are added to an assay mixture as isolated polypeptides. Smad polypeptides preferably are produced recombinantly, although such polypeptides may be isolated from biological extracts. Recombinantly produced Smad polypeptides include chimeric proteins comprising a fusion of a Smad protein with another polypeptide, e.g., a polypeptide capable of providing or enhancing protein-protein binding, sequence specific nucleic acid binding (such as GAL4), enhancing stability of the Smad polypeptide under assay conditions, or providing a detectable moiety, such as green fluorescent protein or Flag epitope as provided in the examples below. The assay mixture is comprised of a natural intracellular Smad nucleic acid binding target such as a Smad binding element or multimer thereof capable of interacting with Smad. While natural Smad binding targets may be used, it is frequently preferred to use multimers or analogs (i.e., agents which mimic the Smad binding properties of the natural binding target for purposes of the assay) of the Smad binding element so long as the multimer or analog provides binding of the Smad protein measurable in the assay. The assay mixture also comprises a candidate pharmacological agent. Typically, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations. Typically, one of these concentrations serves as - 30 - a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection. Candidate agents encompass numerous chemical classes, although typically they are organic compounds. Preferably, the candidate pharmacological agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500. Candidate agents comprise functional chemical groups necessary for structural interactions with polypeptides and/or nucleic acids, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups. The candidate agents can comprise cyclic carbon or heterocyclic stmcture and/or aromatic or polyaromatic stmctures substituted with one or more of the above-identified functional groups. Candidate agents also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the agent is a nucleic acid, the agent typically is a DNA or RNA molecule, although modified nucleic acids as defined herein are also contemplated. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the agents. A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease, inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used. - 31 - The mixture of the foregoing assay materials is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the Smad polypeptide or complex thereof specifically binds a Smad binding element, a multimer thereof or analog thereof. Preferably, for cell based assays, the conditions permit transactivation of a reporter gene under the control of the Smad binding element. The order of addition of components, incubation temperature, time of incubation, and other perimeters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4°C and 40 °C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 10 hours. After incubation, the presence or absence of specific binding between the Smad polypeptide and one or more Smad binding elements is detected by any convenient method available to the user. For cell free binding type assays, a separation step is often used to separate bound from unbound components. The separation step may be accomplished in a variety of ways. Conveniently, at least one of the components is immobilized on a solid substrate, from which the unbound components may be easily separated. The solid substrate can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc. The substrate preferably is chosen to maximum signal to noise ratios, primarily to minimize background binding, as well as for ease of separation and cost. Separation may be effected for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent. The separation step preferably includes multiple rinses or washes. For example, when the solid substrate is a microtiter plate, the wells may be washed several times with a washing solution, which typically includes those components of the incubation mixture that do not participate in specific bindings such as salts, buffer, detergent, non-specific protein, etc. Where the solid substrate is a magnetic bead, the beads may be washed one or more times with a washing solution and isolated using a magnet. Detection may be effected in any convenient way for cell-based assays such as expression assays. The transcript resulting from a reporter gene transcription assay of Smad polypeptide interacting with a SBE typically encodes a directly or indirectly detectable product, e.g., β- galactosidase activity, luciferase activity, green fluorescent protein and the like. For cell free binding assays, one of the components usually comprises, or is coupled to, a detectable label. A - 32 - wide variety of labels can be used, such as those that provide direct detection (e.g., radioactivity, luminescence, optical or electron density, etc), or indirect detection (e.g., epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase, etc.). The label may be bound to a Smad binding element, or incorporated into the stmcture of the SBE. A variety of methods may be used to detect the label, depending on the nature of the label and other assay components. For example, the label may be detected while bound to the solid substrate or subsequent to separation from the solid substrate. Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, streptavidin-biotin conjugates, etc. Methods for detecting the labels are well known in the art. The invention provides agents which modulate directly or indirectly Smad-SBE binding, methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, Smad-SBE binding modulator pharmacological agents are useful in a variety of diagnostic and therapeutic applications, especially where disease or disease prognosis is associated with improper utilization of a pathway involving Smad proteins, e.g., TGF-β superfamily ligand induced signal transduction. Novel Smad- and SBE- specific binding agents include Smad- and SBE-specific antibodies and other natural intracellular binding agents identified with assays such as two hybrid screens, and non-natural intracellular binding agents identified in screens of chemical libraries, phage-display libraries and the like. Cell based assays include one, two and three hybrid screens, assays in which Smad- mediated transcription is inhibited or increased, etc. Cell free assays include SBE-protein binding assays, immunoassays, etc. Other assays useful for screening agents which bind SBE- containing nucleic acids or Smad polypeptides include fluorescence resonance energy transfer (FRET), and electrophoretic mobility shift analysis (EMSA). Various techniques may be employed for introducing nucleic acids of the invention into cells, depending on whether the nucleic acids are introduced in vitro or in vivo in a host. Such techniques include transfection of nucleic acid-CaPO 4 precipitates, transfection of nucleic acids associated with DEAE, transfection with a retrovims (or other vims) including the nucleic acid of interest, liposome mediated transfection, electroporation and the like. For certain uses, it is preferred to target the nucleic acid to particular cells. In such instances, a vehicle used for delivering a nucleic acid of the invention into a cell (e.g., a retrovims, or other vims; a liposome) can have a targeting molecule attached thereto. For example, a molecule such as an antibody - 33 - specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid delivery vehicle. For example, where liposomes are employed to deliver the nucleic acids of the invention, proteins which bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake. Such proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo intemalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like. Polymeric delivery systems also have been used successfully to deliver nucleic acids into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acids. Examples Experimental Procedures Construction of plasmids An 8 kb EcoRI fragment was isolated from the Balb/c mouse genomic JunB clone lambda 31 which has been described previously (de Groot et al., Nucleic Acids Res. 19:775-781, 1991). All sequences upstream from the initiator ATG were cloned in the pGL3-basic reporter vector (Promega). For cloning purposes the ATG was converted to ATC by PCR (pJBl). A deletion series was constmcted by restriction digestion of the pJBl plasmid with EcoRI-PinAI (ρJB2), Sad (pJB3), SacI-BssHII (pJB4), or SacI-SacII (pJB5) and subsequent self-ligation. A minimal promoter construct (pGL3ti) was made from pGL3-basic by inserting oligonucleotides carrying the Adenovims Major Late promoter TATA box (AdTu: gatctGGGGCTATAAAAGGGGGTAGGGGgagct; SEQ ID NO:l 1 and AdThcCCCCTACCCCCTTTTATAGCCCCa; SEQ ID NO: 12) and the mouse Terminal deoxynucleotidyl Transferase gene Initiator sequence (TiuxGCCCTCATTCTGGAGACAg; SEQ ID NO:13 and Til: gatccTGTCTCCAGAATGAGGGCgagct; SEQ ID NO:14) in the Bglll site. A deletion series (pJBl 1-17) was constmcted as follows: an Asp718-BseAI (-3004/1561) fragment from pJB3 was inserted into the Asp718-XmaCl sites from pGL3ti (pJBl 1), a T4 DNA polymerase blunted Sad-BamHI fragment from pJB3 was inserted in the Smal site of pGL3ti (pJB12), a BamHI-Bglll fragment from pJBl 1 was inserted into the Bglll site of pGL3ti (pJB13), pJB12 was restriction digested with Asp718-PvuII or PvuII-Bglll and subsequently blunted and self ligated (pJB14 and pJB15 respectively), pJB15 was restriction digested with - 34 - Asp718-MluNI and subsequently blunted and self ligated (pJB17), and pJBl 1 was digested with MluNI and Bglll and subsequently blunted and self ligated (pJB16). For fine-mapping of the Smad3 and Smad4 response/binding site in the -3004/-2616 region, pJB15 was used as template for PCR reactions with the Til oligonucleotide and the upper strands of the following oligonucleotides: A: CCAATGACGGACTTTATGTAAGCCACTTTAGAACAGTGTCTGGTCCATG; SEQ ID NO:15 GGTTACTGCCTGAAATACATTCGGTGAAATCTTGTCACAGACCAGGTAC; SEQ ID NO:16 B: CTGGTCCATGGTAAGAACCATGTAAGTGTTAGCTATTA; SEQ ID NO:17 GACCAGGTACCATTCTTGGTACATTCACAATCGATAAT; SEQ ID NO:18 C: TTAGCTATTAAAATAATAATTACTATTTCTCAGAC;SEQIDNO:19 AATCGATAATTTTATTATTAATGATAAAGAGTCTG; SEQ ID NO:20 D: TTTCTCAGACAGTCTGTCTGCCTGTCTTAAGTGTCT; SEQ ID NO:21 AAAGAGTCTGTCAGACAGACGGACAGAATTCACAGA; SEQ IDNO:22 G: TAATAATTACTATTTCTCAGACAGTCTGTCTGCC; SEQ ID NO:23 ATTATTAATGATAAAGAGTCTGTCAGACAGACGG; SEQ ID NO:24 The PCR products were phosphorylated, restriction digested with Sad and ligated into a Asp718 digested and blunted, Sad digested pGL3ti vector. The internal deletion series was constmcted as follows: a Pstl/PvuII fragment (-2762/-2611) from pJB12 was cloned into the Pstl and EcoRV sites of pBluescript SKII- (pSK-PsPv). Next, pJB15 was used as template for PCR reactions with the upper strand of oligonucleotide A and the lower strands of oligonucleotides B, C, D and G. The PCR products were cloned into the Smal site of pSK-PsPv. Also double stranded oligonucleotides A and D were ligated into pSK-PsPv. The resulting plasmids were restriction digested with Xbal and Sail and the fusion fragments were inserted into Nhel-Xhol opened pGL3ti, pGL3ti-PsPv and pGL3ti-AfPv was constmcted by digestion of pJB15 with Asp718-Psl or Asp718-AfII, blunting and self ligation. Reporter constructs carrying multimerized versions of Smad Binding Element I V5 were created by ligating 4 oligonucleotides into the Bglll site of ρGL3ti (inv5u:gatccTTTCTCAGACAGTCTGTCTGCa; SEQ ID NO:25 and inv51: gAAAGAGTCTGTCAGACAGACGtctag; SEQ ID NO:26). pGL3ti(DIR)4 was created by inserting one 4xCAGACA-WT oligonucleotide (upper strand: - 35 - gtaccCAGACAGTCAGACAGTCAGACAGTCAGACAGTc; SEQ ID NO:27, lower strand: gGTCTGTCAGTCTGTCAGTCTGTCAGTCTGTCAgagct; SEQ ID NO:28) into Asp718-XhoI opened pGL3ti. The expression plasmids for Flag-Smadl, Smad2, Myc-Smad3 and Smad4 have been described previously. pGL3ti was generated by the insertion of adenovims major late promoter in pGL3 vector (Promega, Madison, WI). pGL3ti-(CAGACAGACTGTCTG) 2 (pGL3ti-(INV)4) was constmcted using the double strand oligonucleotides and cloned into pGL3ti. All constmcts were verified by sequencing. Smad expression plasmids were constmcted as follows: pSG5-XMADl and pSG5- XMAD2 were created by inserting EcoRI fragments from pSP64TEN-DOTl and -2 into EcoRI opened pSG5; pSG5-hSmad3f was created by inserting a blunted BamHI-Sall fragment from pRK5-hSmad3f into blunted BamHI opened pSG5, and pSG5-hSmad4 was created by inserting a blunted BamHI-EcoRI fragment from pDPC-wt3 into blunted EcoRI-BamHI opened pSG5. Bacterial expression vectors encoding GST fusion proteins were generated by PCR and cloned in the pGEX4T-l (Pharmacia, Uppsala, Sweden). GST-SmadlΔMH2 (residues 1-269), GST-Smad2ΔMH2 (residues 1-272), GST-Smad3ΔMH2 (residues 1-229), GST-Smad4ΔMH2 (residues 1-321), and GST-Smad5ΔMH2 (residues 1-269) contained MH1 plus linker regions of Smadl, Smad2, Smad3, Smad4 and Smad5, respectively. GST-Smad2 was also constructed using pGEX4T-l vector. GST-Smadl, GST-Smad3 and GST-Smad4 were generously provided by Dr. R. Derynck. Cell Culture P19EC embryonal carcinoma cells were maintained in a 1 :1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium supplemented with 7.5% fetal bovine serum (Integro, Zaandam, The Netherlands). NIH3T3 embryonic fibroblast cells were maintained in DMEM supplemented with 10% Newborn Calf Serum (Life Technologies, Gaithersburg, MD). HaCaT human keratinocytes, HepG2 human hepatocellular carcinoma cells, MDA-MB468 breast cancer cells and MvlLu mink lung epithelial cells were grown in DMEM medium supplemented with 10% FCS. All media were further supplemented with 100 IU/ml penicillin, 100 g/ml streptomycin and lx DMEM non-essential amino acids. For transient transfection P19EC, HepG2 and MDA-MB488 cells were seeded 5 x IO 4 , 2 x 10 5 and 2.5 x 10 5 cells per well of a 6-well tissue culture cluster, respectively. MDA-MB468, P19Ec and HepG2 - 36 - cells were transiently transfected using the calcium phosphate co-precipitation method. In all transfections β-galactosidase expression plasmids served as internal controls. Cells were treated with TGFβl (R&D Systems, Minneapolis, MN and Abingdon, UK), Activin or OP-1 at 10, 20 or 100 ng/ml respectively. Luciferase activities were measured using the Luciferase Assay System following lysis in lx Reporter Lysis Buffer (Promega). β-galactosidase activity was quantified in 100 mM Na 2 HPO 4 /NaH 2 PO 4 , 1 raM MgCl 2 , 100 mM 2-mercapto-ethanol and 0.67 mg/ml o- Nitrophenyl-Galactopyranoside, pH 7.3. Preparation of GST fusion protein GST fusion protein expression was induced in logarithmically growing cultures of E coli, TGI, by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, and growing the bacteria at 30 °C for an additional 5 h. Bacteria suspended in 50 ml of cold phosphate-buffered saline (PBS) were sonicated, mixed with 1% Triton X-100 and centrifuged at 12,000g for 10 min. Subsequently, an extract containing GST fusion protein was mixed with 0.25% glutathione-Sepharose 4B beads (Pharmacia) and incubated at 4°C under the constant agitation for 12 h. The beads were washed four times with PBS containing 1% Triton X-100, and then eluted three times with 50 mM Tris (pH 8.0) including 10 mM glutathione. After the dialysis of eluates with PBS containing 2 mM DTT and 0.5 mM PMSF, the sample was stored at -70°C until used. Electrophoretic Mobility Shift Assays Nuclear extracts were prepared from MvlLu cells using a modified Dignam protocol as described previously (Jonk et al., Mechanisms of Development 47:81-97, 1994). Oligonucleotides and dephosphorylated restriction fragments were labeled with [α- 3 P-ATP] and T4 Polynucleotide Kinase. Oligonucleotides used in EMSA experiments were: A, B, C, D, G and inv5 (described above) and: WT: tcgagCAGACAGTCAGACAGTc; SEQ IDNO:29 cGTCTGTCTGTCTGTCAgagct; SEQ IDNO:30 Ml: tcgaggAGACAGTgAGACAGTc; SEQ IDNO:31 ccTCTGTCTcTCTGTCAgagct; SEQ IDNO:32 - 37 - M2: tcgagCtGACAGTCtGACAGTc; SEQ ID NO:33 cGaCTGTCTGaCTGTCAgagct; SEQ ID NO:34 M3: tcgagCAcACAGTCAcACAGTc; SEQ ID NO:35 cGTgTGTCTGTgTGTCAgagct; SEQ ID NO:36 M4: tcgagCAGtCAGTCAGtCAGTc; SEQ ID NO:37 cGTCaGTCTGTCaGTCAgagct; SEQ ID NO:38 M5: tcgagCAGAgAGTCAGAgAGTc;SEQIDNO:39 cGTCTcTCTGTCTcTCAgagct; SEQ ID NO:40 M6: tcgagCAGACtGTCAGACtGTc; SEQ ID NO:41 cGTCTGaCTGTCTGaCAgagct; SEQ ID NO:42 M7: tcgagCAGACAcTCAGACAcTc; SEQ ID NO:43 cGTCTGTgTGTCTGTgAgagct; SEQ ID NO:44 4XWT 5' -GTACCCAGACAGTCAGACAGTCAGACAGTCAGACAGTC;SEQIDNO:45 3' -GGTCTGTCAGTCTGTCAGTCTGTCAGTCTGTCAGAGCT; SEQ ID NO:46 4XMu 5' -GTACCCAcAgAGTCAcAgAGTCAcAgAGTCAcAgAGTC; SEQ ID NO:47 3' -GGTgTcTCAGTgTcTCAGTgTcTCAGTgTcTCAGAGCT; SEQ ID NO:48 Binding reactions contained 4 microgram nuclear extract, 100 mM KC1, 0.2 mM EDTA, 0.5 mM Dithiothreitol, 20% glycerol, 20 mM HEPES pH 7.9, 100 ng poly dl-dC and approximately 10000 cpm labeled probe and 500-fold molar excess of competitor oligonucleotide where appropriate. Protein binding was allowed to proceed for 30 minutes at room temperature. Then 5 microliters 20% Ficoll was added to the reactions and samples were immediately loaded onto 4.5 or 5% polyacrylamide gels containing 0.5 x TBE. Smad3 and Smad4 antibodies (542 and HPP, respectively (Nakao et al, EMBO J. 16:5353-5362, 1997)) were added undiluted at 0.5 microliter, after proteins were allowed to bind the probe for 15 minutes. Samples were then incubated 15 minutes before loading onto the gel. The binding reaction using GST fusion proteins was the following procedure: GST fusion proteins were mixed with the binding buffer consisting of 20% glycerol, 20 mM Hepes (pH 7.9), 30 mM KC1, 4 mM MgCl 2 , 0.1 mM EDTA, 0.8 mM sodium phosphate, 4 mM spermidine, 0.3 μg/μl poly (dLdC) (Pharmacia) and 0.25 nM 32 P-labeled probe at the final - 38 - volume of 20 μl. If necessary, 500-fold molar excess of the cold competitor was added in the reaction mixture. The mixture was incubated at 25 °C for 1 h and ran on a 5% non-denatured polyacrylamide gel with 0.5X TBE. Example 1 : Identification of a Smad responsive region in the JunB gene Previously, it has been shown that the immediate early gene JunB gene is a direct target for transcription activation in response to activation of signal transduction by TGFβ (de Groot and Kruijer, Biochem. Biophys. Res. Comm. 168:1074-1081, 1990). To investigate the mechanism of transactivation of the JunB promoter by TGFβ, a JunB-luciferase fusion gene was constructed, containing the JunB TATA box and transcriptional start site, the complete 5' untranslated region as well as approximately 6.4kb of promoter upstream sequences (pJBl, Fig. 1 A). Transient transfection of this constmct in NIH3T3 and HepG2 cells and treatment of the cells with TGFβ did not result in an increase in TGFβ transactivation over uninduced levels. However, a 3-4 fold activation over high basal levels was observed when the constmct was co- transfected with plasmids expressing the TGFβ-specific signal transducers Smad3 and Smad4 (Fig. 1 A). Deletion analysis showed that the Smad-responsive region is located between nucleotides -3009 and -1534. A nested set of restriction fragments was derived from this region and cloned in front of a heterologous minimal promoter fused to the luciferase gene. Co-transfection of P19EC cells with these constmcts and Smad3 and Smad4 expression plasmids allowed localization of a Smad responsive region to between nucleotides -2908 and -2611 (Fig. IB). After 40 hours, cells were lysed to measure luciferase activity. Luciferase activity was normalized using β-galactosidase activity. Data are shown with the mean ± SD of triplicates. TGFβ-treatment of cells results in the translocation of activated Smad complexes into the nucleus (Heldin et al., Nature 390:465-471, 1997) where these complexes are thought to activate transcription of TGFβ target genes. To investigate whether TGFβ induces binding of endogenous activated Smads to the Smad-responsive region, an end labeled fragment containing the 297 bp Smad-responsive region (nucleotides-2908 and -2611 of the JunB promoter) was incubated with nuclear extracts from human HaCaT keratinocytes, and mink MvlLu lung epithelial cells treated with or without 10 ng/ml TGFβ for 1 h. Complexes were resolved on a non-denaturing 4.5% polyacrylamide gel followed by autoradiography of the dried gel. TGFβ treated HaCaT and MvlLu cells contained an induced DNA binding activity that migrated with a - 39 - lower mobility than that of a constitutively expressed binding entity. These results suggest that TGFβ activates the endogenous JunB gene by induced binding of a nuclear factor to a JunB promoter distal element located between nucleotides -2908 to -2611. Furthermore, transactivation of this sequence corresponds with overexpression of Smad3 and Smad4 suggesting that Smads might be part of the induced complex. Having established that the 297 bp (-2908/-2611) region can be transactivated by overexpressed Smads and binds a TGFβ-induced protein(s), a minimal Smad-responsive region was defined next (Fig. 2). A nested series of deletion fragments derived from the 297 bp region (see Fig. 1) were generated by PCR using oligonucleotides A, B, C, D, and G and a downstream primer or restriction digestion with Aflll or Pstl. In addition, a series of internal deletion fragments were made by PCR and fused to the Pstl-PvuII (PsPv) fragment. The fragments were ligated in front of the heterologous minimal promoter of the pGL3ti reporter plasmid. The resulting plasmids with or without Smad3 and Smad4 expression plasmids were transfected into P19EC cells. Normalized luciferase activities are shown as the mean ± SD of triplicates. The 22 bp region of overlap between oligonucleotides G and D was defined as a Smad3 and Smad4- responsive region. The sequence of this region is shown below the schematic reporter constmcts. The arrows denote a 7bp inverted repeat. The region between nt -2788 and -2616 is not responsive to overexpression of Smad3 and Smad4. However, it appeared to be required for transactivation of upstream located sequences by Smad3 and Smad4. Extending this region with sequences located just upstream restored transactivation by Smad 3 and -4 (D-PvuII), while further incorporation of 5' sequences appeared to have a slight repressive effect except when sequences unique to the A oligo were included. This analysis was complemented by a series of deletions of sequences located between nt -2908 and -2762. Exclusion of sequences upstream from nt -2792 abrogated transactivation by Smad3 and Smad4. This analysis therefore defines the 22 basepair region between -2813 and -2792 as that minimally required for transactivation by Smad3 and Smad4. Example 2: Smad3 and Smad4 bind an inverted CAGACA repeat The location of the Smad binding element on the 297 bp Smad responsive region was more specifically determined by detailed binding competition analysis. A 120 bp subfragment (-2908/-2788) was found to carry the binding site for the TGFβ induced complex. An end labeled restriction fragment derived from the 120 bp Smad-responsive region was incubated with - 40 - nuclear extracts from mouse NIH-3T3 embryonic fibroblasts, human HaCaT keratinocytes, and mink MvlLu lung epithelial cells treated with or without 10 mg/ml TGFβ for 1 h. Protein binding was visualized as above. A TGFβ-induced complex was observed. This fragment (-2908/-2788) contains the minimal region required for transactivation by overexpressed Smad3 and Smad4 (-2813 to -2792). To investigate whether the 22 bp sequence identified above confers binding to the TGFβ induced complex, nuclear extracts were incubated with a labeled oligonucleotide probe carrying this 22 bp region and complexes were separated on a non-denaturing gel. Specifically, DNA carrying the 22 bp Smad-responsive element (inv+5) was labeled and incubated with nuclear extracts from MvlLu cells. Protein binding was visualized as above. The nuclear extract from cells treated with TGFβ possessed two inducible complexes (denoted as complexes I and II) which contained the labeled probe. This showed that the 22 bp region is capable of binding two TGFβ induced complexes. To identify the composition of these complexes, antisera directed against Smad3 or Smad4 were added to the binding reactions. Complex I contains proteins which were supershifted by the addition of Smad3 and Smad4 antibodies to the reaction mixtures. The Smad3 antisemm quantitatively supershifted the slowest migrating TGFβ induced complex, while the Smad4 antisemm was slightly less effective. Both antisera produced a supershifted band with extracts from uninduced cells. Possibly, these complexes were formed by stabilization by the antisera of the interaction of contaminating cytoplasmic Smads with the probe. These results show that the minimal region required for activation by overexpressed Smad proteins is identical to that binding Smad3 and Smad4 from TGFβ treated cells. Close inspection of the sequence revealed that it contains a perfect 7 bp inverted repeat (CAGACAGtCTGTCTG; SEQ ID NO: 10). To identify bases that are important for complex formation, a series of oligonucleotides each carrying a different substitution of one basepair of the 7 bp half site were tested for their efficiency as competitors. Formation of the TGFβ-induced complex I was inhibited by inclusion of oligonucleotides carrying wild type (i5 and WT) CAGACA repeats (SEQ ID NO: 1). Mutation of individual base pairs in the CAGACA repeat (Ml -6) resulted in different competition efficiencies. This analysis identified the central nucleotides GAC as most important for binding the TGFβ complex while the flanking nucleotides contribute less, but still significantly, to the binding. This repeat is referred to as the CAGACA repeat. - 41 - Example 3: Smad proteins directly bind to the Smad responsive region Recently, Drosophila melanogaster Mad protein has been shown to bind to DNA directly. To investigate whether Smad proteins directly bind the Smad-responsive fragment, full length and C-terminally tmncated (MH2 region deletion, ΔMH2) Smad 1-5 were produced as GST fusion proteins in E. coli and analyzed for their ability to bind the 120 bp MluNI/Aflll subfragment from the 297 bp region. The purified proteins were incubated with the labeled probe and complexes were resolved on a 5% non-denaturing polyacrylamide gel. Binding of full length fusion protein was only observed for GST-Smad4. However, C-terminally tmncated Smad3 and Smad4 strongly interacted with the probe, while binding of C-terminally truncated Smadl, Smad2, and Smad5 to the 120 bp fragment was below the threshold of detection. Next, end labeled double-stranded oligonucleotides A, B, C, D and G were tested for binding the C-terminally tmncated GST-Smad proteins. Complexes were resolved on a non- denaturing 5% polyacrylamide gel. GST-Smad3ΔMH2 and to a lesser extent GST-Smad4ΔMH2 bound oligos D and G while oligo A weakly interacted with GST-Smad3ΔMH2. These results identify the region of overlap between oligo D and G as the major binding site, and oligo A as minor binding site for bacterially expressed Smad3 and Smad4 proteins. When comparing the sequence of oligos A and D, a CAGACA repeat sequence in one copy in oligo A and as an inverted copy in oligo D were noted. Furthermore, this region coincides with that bound by the Smad3 and Smad4 containing complex from TGFβ-stimulated MvlLu cells, implying that TFGβ induces direct binding of Smad3 and Smad4 to regulatory sequences in the oligo A and D containing JunB promoter fragment. To compare the binding characteristics of bacterially produced Smads with those of the TGFβ-induced complex from MvlLu cells, gel shift experiments were performed with a (CAGACA)2 repeat containing probe. Bacterially produced GST-Smad3 and GST-Smad4 proteins were incubated with an end labeled (CAGACA)2 repeat containing oligonucleotide. Probe binding was challenged by inclusion of oligonucleotides carrying wild type (WT) and mutant (Ml-7) SBEs (500-fold molar excess). Complexes were resolved on a non-denaturing polyacrylamide gel. Order of efficiency of competition was found to be WT=M7>M6>M1>M2=M4>M3=M5). As expected, GST-Smad4 readily complexed with the probe while interaction with GST-Smad3 was not detectable. A stronger binding of GST-Smad3 MH2 deleted protein and GST-Smad4 (full length and MH2 deleted version) was observed with a (CAGACA)4 repeat containing probe. Binding was specifically competed with wild type - 42 - oligo, but not with mutated oligo probe, and no binding to mutated probe was observed. Next, complex formation was challenged by the addition of wild-type or mutant repeat oligos (WT and M1-M7) to the binding reaction with (CAGACA)2 repeat oligos. Bacterially produced GST-Smad3 and GST-Smad4 proteins were incubated with end labeled wild type (WT) or mutant SBE containing oligonucleotides. Complexes were resolved on a non-denaturing polyacrylamide gel. GST-Smad3 did not bind the probe. This analysis revealed that the bases required for formation of the TGFβ induced complex observed above are essentially the same as those used by the bacterially produced GST-Smad4 protein to bind the probe, as the order of binding potential of the oligonucleotide is identical to that found for efficiency in the binding competition (WT=M7>M6>M1>M2=M4>M3=M5). Complementary experiments in which GST-Smad proteins were incubated with labeled wild type or mutant oligonucleotides as probes yielded almost identical results. Example 4: The CAGACA repeat is a TGFβ response element The minimal region required for transactivation of the 297 bp cis-acting region by overexpressed Smad3 and Smad4 is by itself not sufficient to render a heterologous promoter activatable by TGFβ or Smad3 and Smad4. It therefore was reasoned that efficient activation by Smad proteins requires multimerization of CAGACA repeat containing region. The JunB SBE (INV5) and a direct repeat version thereof (DIR) were multimerized and cloned in pGL3ti. These reporter plasmids with or without Smad expression plasmids were transfected into HepG2 cells. The cells were treated overnight with or without 10 ng/ml TGFβ. The next day cells were lysed and reporter activity was measured. Normalized luciferase activity is expressed as the mean ± SD of triplicates. When co-transfected with Smad3 and Smad4 expressing plasmids, reporter plasmids carrying four copies of the inverted repeat were activated 7-fold in HepG2 cells (Fig. 3 A). A constmct containing four copies of a direct CAGACA repeat was activated 16-fold in this assay. However, when cells were transfected with reporter plasmids only and subsequently treated with TGFβ an equally strong response was observed. These results show that the inverted CAGACA repeat isolated from the JunB promoter as well as direct repeat- derivatives thereof are tme TGFβ response elements. The results also indicate that the relative orientation of the CAGACA repeat half sites is not important for efficient binding. Recently, Smad7 has been isolated and identified as a negative regulator of TGFβ superfamily receptor mediated signaling (Nakao et al, Nature 389:631-635, 1997). Smad7 acts - 43 - by binding to the docking site for receptor-activated Smads thereby preventing phosphorylation and thus signaling. To show that activation of the (CAGACA)4 ((INV)4) reporter by TGFβ is mediated by receptor-activated Smad proteins, the (INV)4 reporter plasmid was cotransfected into HepG2 cells along with increasing amounts of a Smad7 expressing plasmid (Fig. 3B). The transfected cells were treated with TGFβ. Normalized luciferase activities are shown as the mean ± SD of triplicates. In the absence of Smad7, TGFβ strongly induced reporter activity while co-transfection of the Smad7 constmct resulted in an 80% reduction of the maximum level. The results indicate that activation of endogenous Smad proteins by the type I TGFβ receptor is required for activation of the JunB promoter-derived CAGACA repeat in HepG2 cells. Example 5: The CAGACA repeat is a response element for other members of the TGFβ superfamily. Besides TGFβ, other members of the TGFβ superfamily such as activin and OP-1 have been shown to induce JunB mRNA expression. Co-transfection of the Smad3 -responsive (-3000/-2333) reporter with Smad4 and a receptor-activatable Smad protein (Smadl, Smad2, or Smad3) expressing plasmids showed that each receptor-activatable Smad induced reporter activity when co-transfected with Smad4 expressing plasmid (Fig. 4A). P19EC cells were transfected with the pJB12 plasmid with or without Smadl, Smad2, Smad3, and/or Smad4 expression plasmids as in Example 1. Normalized reporter activity is expressed as the mean ± SD of triplicates from a representative experiment. It was noted, however, that Smad2 transactivated the reporter constmct significantly less than Smad3. Both Smad2 and Smad3 are phosphorylated by the activated type I TGFβ receptor. Therefore it was next investigated whether TGFβ preferentially activated the CAGACA repeat ((INV)4) through Smad3 over Smad2 (Fig. 4B). HepG2 cells were transfected with the pGL3ti-(INV)4 plasmid and with the combinations of Smad2, Smad3, and/or Smad4 expression plasmids shown. The cells were incubated for 16 h with or without 10 ng/ml TGFβ. Normalized luciferase activities are shown as the mean ± SD of triplicates. Co-transfection of Smad2+Smad4 did not enhance TGFβ mediated induction of the reporter constmct while Smad3+Smad4 superactivated ligand induced reported activity. The JunB gene is activated by TGFβ, activin and BMP2. Besides Smad3, Smadl and Smad2 also activated the JunB promoter when co-transfected with Smad4, indicating that overexpression of Smads mimics the ligand mediated activation of the endogenous promoter. - 44 - As the DNA binding (MH1) domains of all Smad proteins are very similar, it was hypothesized that induction of JunB promoter activity by OP-1 and activin are mediated by a similar response element. Therefore HepG2 cells were transfected with the TGFβ-responsive CAGACA reporter constmct(pGL3ti(INV)4). The transfected cells were incubated for 16 h with or without 10 ng/ml TGFβ, 20 ng/ml activin or 100 ng/ml OP-1. Normalized luciferase activities are shown as the mean ± SD of triplicates. As shown in Fig. 4C, all three ligands activate the CAGACA (INV)4 reporter albeit with different efficiencies. To corroborate these findings, Smad4-negative MDA-MB468 cells were transfected with the CAGACA reporter plasmid with or without the Smad4 expression plasmid. Following transfection cells were incubated for 16 h with or without 10 ng/ml TGFβ, 20 ng/ml activin or 100 ng/ml OP-1 (Fig. 4D). Normalized luciferase activities are expressed as the mean ± SD of triplicates. In the absence of Smad4, the reporter constmct was virtually unresponsive to any of the ligands. Co-transfection of Smad4 resulted in a slight activation of the reporter. However, treatment of the Smad4 co-transfected cells with the various members of the TGFβ superfamily showed that all ligands strongly activated the CAGACA repeat, indicating that these ligands activate the JunB promoter through the same response element. The ligands that induce phosphorylation of Smadl, Smad2 and Smad5 at their C-terminus, including OP-1 and BMP2 and GDF5 (not shown), efficiently activated a CAGACA-reporter constmct. These results indicate that the CAGACA repeat (INN)4 can be activated by different members of the TGFβ superfamily, implying involvement of their cognate receptor activatable Smad proteins. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention. All of the references described herein are incorporated by reference. What is claimed is:

Claims: - 45 - CLAIMS 1. An isolated nucleic acid comprising a Smad binding element having a nucleotide sequence selected from the group consisting of CAGACA (SEQ ID NO:l), CAGACAG (SEQ ID NO:2), GAGACA (SEQ ID NO:3), GAGACAG (SEQ ID NO:4), CTGACA (SEQ ID NO:5), CTGACAG (SEQ ID NO:6), CAGACT (SEQ ID NO:7), CAGACTG (SEQ ID NO:8), CAGACAC (SEQ ID NO:9); and complements thereof, wherein the Smad binding element is arranged in a non-natural arrangement. 2. The isolated nucleic acid of claim 1 , wherein the isolated nucleic acid comprises at least two of the Smad binding elements. 3. The isolated nucleic acid of claim 2, comprising at least four of the Smad binding elements. 4. The isolated nucleic acid of claim 2, wherein the Smad binding elements are arranged as one or more inverted repeats. 5. The isolated nucleic acid of claim 4, wherein the Smad binding elements comprise the nucleotide sequence CAGACAGTCTGTCTG (SEQ ID NO: 10). 6. The isolated nucleic acid of claim 1, wherein the Smad binding element is a fragment of the junB gene upstream region, wherein the nucleotide sequence of the nucleic acid which flanks the Smad binding element is not derived from junB. 7. The isolated nucleic acid of claim 6, wherein the fragment of the junB gene upstream region is selected from the group consisting of nucleotides -2813 through -2792, nucleotides -2908 through -2788, nucleotides -2908 through -2611, and nucleotides -3004 through -1534. 8. The isolated nucleic acid of claim 6, wherein the fragment of the junB gene upstream region is less than 50 nucleotides in length. - 46 - 9. The isolated nucleic acid of claim 8, wherein the isolated nucleic acid comprises at least two Smad binding elements. 10. The isolated nucleic acid of claim 9, wherein the Smad binding elements are arranged as one or more inverted repeats. 11. The isolated nucleic acid of any of claims 1 - 10, wherein the isolated nucleic acid is double stranded. 12. A vector comprising the isolated nucleic acid of any of claims 1-11. 13. The vector of claim 12, wherein the isolated nucleic acid is operably linked to a reporter gene. 14. A host cell transformed or transfected with a nucleic acid selected from the group consisting of the isolated nucleic acid of claim 1 and the isolated nucleic acid of claim 8. 15. A host cell transformed or transfected with the vector of claim 12. 16. A host cell transformed or transfected with the vector of claim 13. 17. An isolated polypeptide comprising a fragment of Smad3 consisting of Smad3 amino acids 1-279. 18. The isolated polypeptide of claim 17, wherein the isolated polypeptide is a fusion protein. 19. An isolated polypeptide comprising a fragment of Smad4 consisting of Smad4 amino acids 1-321. 20. The isolated polypeptide of claim 19, wherein the isolated polypeptide is a fusion protein. 21. A method of screening for compounds which bind to a Smad binding element comprising - 47 - contacting a nucleic acid comprising a Smad binding element with a test compound, and determining the binding of the test compound to the Smad binding element. 22. The method of claim 21 , further comprising contacting a mutant Smad binding element with the test compound, determining the binding of the test compound to the mutant Smad binding element, and comparing the binding of the test compound to the Smad binding element and the mutant Smad binding element as a measure of the specific binding of the test compound to the Smad binding element. 23. The method of claim 22, wherein the mutant Smad binding element does not bind a Smad protein. 24. A method for identifying compounds that compete for binding of a Smad binding element with a Smad protein, comprising providing a nucleic acid comprising a Smad binding element, forming a complex comprising the nucleic acid and a Smad protein which binds the Smad binding element, contacting the complex with a test compound, and determining the binding of the Smad protein to the nucleic acid as a measure of the displacement of the Smad protein by the test compound. 25. A method of screening for compounds that modulate transcription mediated by Smad binding elements, comprising providing a transcription system comprising a Smad protein which binds a Smad binding element and a nucleic acid comprising a Smad binding element, contacting the transcription system with a test compound, and determining the transcription of the nucleic acid as a measure of transcriptional modulation by the test compound, wherein increased transcription indicates that the test compound increases transcription mediated by the Smad binding element and decreased transcription indicates that the test compound decreases transcription mediated by the Smad binding element. - 48 - 26. The method of claim 25, wherein the transcription system is a cell. 27. The method of claim 26, wherein the cell comprises a Smad binding element reporter vector which is stably contained within the cell. 28. The method of claim 25, wherein the Smad protein which binds Smad binding element is selected from the group consisting of Smad 1, Smad2, Smad3, Smad3ΔMH2, Smad4, Smad4ΔMH2, Smad5, a Smadl/Smad4 complex, a Smad2/Smad4 complex, a Smad3/Smad4 complex, and a Smad5/Smad4 complex. 29. The method of claim 25, wherein the nucleic acid further comprises a reporter gene operably linked to the Smad binding element. 30. The method of any of claims 21 -26, wherein the nucleic acid comprises at least two Smad binding elements. 31. The method of claim 30, wherein the Smad binding elements are arranged as one or more inverted repeats. 32. The method of claim 25, further comprising contacting the transcription system with a first ligand which induces transcription mediated by Smad binding elements, wherein a reduction in ligand induced transcription in the presence of the test compound indicates that the test compound interferes with the ligand induced transcription mediated by Smad binding elements and wherein an increase in ligand induced transcription in the presence of the test compound indicates that the test compound enhances the ligand induced transcription mediated by Smad binding elements. 33. The method of claim 32, further comprising repeating the method at least once in the presence of a second ligand which induces transcription mediated by Smad binding elements, wherein a reduction in transcription induced by the first ligand but not the second ligand indicates that the test compound interferes selectively with the transcription mediated by Smad binding elements induced by the first ligand, and wherein a reduction in transcription induced by - 49 - the first ligand and the second ligand indicates that the test compound interferes generally with the transcription mediated by Smad binding elements. 34. The method of claims 32 or claim 33, wherein the first or the second ligand which induces transcription mediated by Smad binding elements is selected from the group consisting of TGFβ, activin, OP-1, BMP-2, and GDF5. 35. A method for treating a subject having a condition characterized by an abnormally elevated level of Smad-mediated transcription, comprising administering to a subject in need of such treatment an agent which decreases the level of binding of a Smad protein to an endogenous Smad binding element in an amount effective to reduce the abnormally elevated level of Smad-mediated transcription. 36. The method of claim 35, wherein the agent is an isolated nucleic acid molecule comprising the nucleotide sequence CAGACA. 37. The method of claim 35, wherein the agent is an antisense nucleic acid which binds to a nucleic acid molecule encoding the Smad protein which binds a Smad binding element. 38. The method of claim 35, wherein the agent is an antibody which binds to the Smad protein which binds a Smad binding element. 39. A method for modulating transcription of genes comprising a Smad binding element in a transcription control region, comprising contacting a cell with an isolated nucleic acid comprising the nucleotide sequence CAGACA. 40. The method of claims 36 or claim 39, wherein the nucleic acid comprises at least two Smad binding elements. 41. The method of claim 40, wherein the Smad binding elements are arranged as one or more inverted repeats. - 50 - 42. The method of claim 36 or claim 39, wherein the isolated nucleic acid is double stranded. 43. A method for modulating in a cell transcription of genes comprising a Smad binding element in a transcription control region, comprising contacting the cell with an agent which increases in the cell the level of a Smad protein which binds a Smad binding element or a fragment of the Smad protein which binds a Smad binding element. 44. The method of claim 43, wherein the agent is an expression vector which encodes the Smad protein which binds a Smad binding element or a fragment of the Smad protein which binds a Smad binding element. 45. The method of claim 43, wherein the agent is the Smad protein which binds a Smad binding element or a fragment of the Smad protein which binds a Smad binding element. 46. A method for modulating in a cell transcription of genes comprising a Smad binding element in a transcription control region, comprising contacting the cell with an agent which decreases the level of a Smad protein which binds a Smad binding element in the cell. 47. The method of claim 46, wherein the agent is an antisense nucleic acid which binds to a nucleic acid encoding the Smad protein which binds a Smad binding element. 48. The method of claim 46, wherein the agent is an antibody which binds to the Smad protein which binds a Smad binding element. 49. The method of claim 43 or 46, wherein the Smad protein which binds a Smad binding element is selected from the group consisting of Smad 1, Smad2, Smad3, Smad3ΔMH2, Smad4, Smad4ΔMH2, Smad5, a Smadl/Smad4 complex, a Smad2/Smad4 complex, a Smad3/Smad4 complex, and a Smad5/Smad4 complex. 50. A method of identifying a Smad protein having altered binding to a Smad binding - 51 - element nucleic acid sequence, comprising contacting a sample containing a Smad protein, the nonmutated form of which binds a Smad binding element in a first amount, with an isolated nucleic acid molecule comprising the Smad binding element, determining a second amount of binding of the Smad binding element by the Smad protein, and comparing the first amount and the second amount of binding of the Smad binding element, wherein a difference between the first amount and the second amount indicates that the sample contains a mutant Smad protein with altered nucleic acid binding characteristics. 51. The method of claim 50, wherein the Smad binding element comprises the nucleotide sequence CAGACA. 52. The method of claim 50, wherein the Smad protein is prepared by mutagenesis. 53. The method of claim 52, wherein the Smad protein is prepared by site-directed mutagenesis.

Inventors: HELDIN CARL-HENRIK, ITOH SUSUMO, JONK LUIGI J C, KRUIJER WIEBE, TEN DIJKE PETER, HELDIN, CARL-HENRIK, ITOH, SUSUMO, JONK, LUIGI, J., C., KRUIJER, WIEBE, TEN DIJKE, PETER
Applicants: LUDWIG INST CANCER RES, LUDWIG INSTITUTE FOR CANCER RESEARCH
Application year: 1999
Main classification: C12N15
IPC codes: A61K38, A61K48, C07K14, C12N15