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Smad and AML Proteins Synergistically Confer Transforming Growth Factor β1 Responsiveness to Human Germ-line IgA Genes

Smad and AML Proteins Synergistically Confer Transforming Growth Factor β1 Responsiveness to... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 5, Issue of February 4, pp. 3552–3560, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Smad and AML Proteins Synergistically Confer Transforming b1 Responsiveness to Human Germ-line IgA Genes* Growth Factor (Received for publication, August 9, 1999, and in revised form, October 8, 1999) Evangelia Pardali‡§¶, Xiao-Qi Xie‡§, Panagiotis Tsapogas‡, Susumu Itohi, Konstantinos Arvanitidis‡, Carl-Henrik Heldini, Peter ten Dijkei, Thomas Grundstro ¨ m‡, and Paschalis Sideras‡** ‡‡ From the ‡Division of Tumor Biology, Department of Cell and Molecular Biology, Umeå University, S-901 87 Umeå, the iLudwig Institute for Cancer Research, Box 595, Biomedical Center, S-751 24 Uppsala, and the **Department of Inflammation Pharmacology, ASTRAZENECA R&D LUND, S-22100 Lund, Sweden Transcription of germ-line immunoglobulin heavy positive regulator of IgA production (2– 4). There are two IgA isotypes in the human system, IgA1 and IgA2, encoded by two chain genes conditions them to participate in isotype b1 switch recombination. Transforming growth factor- different genes, one in each of the two duplication units that b1) stimulates promoter elements located up- (TGF- compose the human immunoglobulin heavy chain locus (5). stream of the IgA1 and IgA2 switch regions, designated Both genes contain, upstream of their switch regions, almost a1 and Ia2, and contributes to the development of IgA identical TGF-b1 responsive promoter/enhancer elements des- responses. We demonstrate that intracellular Smad pro- ignated intron (I) a1 and Ia2, respectively (6, 7). According to a1 promoter by TGF-b. teins mediate activation of the I the “accessibility model” for isotype class switching (8, 9) tran- b type 1 receptor (ALK-5), activin type IB receptor TGF- scribed germ line IgA genes become “accessible” to the Ig class a1 (ALK-4), and the “orphan” ALK-7 trans-activate the I switch recombination machinery and thus are preferentially promoter, thus raising the possibility that other mem- rearranged and expressed. The importance of the I region pro- b superfamily can also modulate IgA bers of the TGF- moter/enhancer elements for proper guiding of a given immu- synthesis. Smads physically interact with the AML fam- noglobulin heavy chain locus toward a certain class has been ily of transcription factors and cooperate with them to repeatedly illustrated by elegant gene targeting experiments a1 promoter. The Ia1 element provides a activate the I where disruption of I region elements prevented rearrange- canape ´ of interspersed high and low affinity sites for ment of the targeted alleles (10, 11). Smad and AML factors, some of which are indispensable Genomic segments encompassing approximately 130 base b responsiveness. While AMLzSmad complexes for TGF- pairs upstream of the human and mouse Ia transcription ini- are formed in the cytoplasm of DG75 and K562 cells tiation sites contain all the information necessary for expres- b receptor activation, constitutively, only after TGF- sion and TGF-b responsiveness in transient expression assays novel Smad3zSmad4zAML complexes are detected in nu- a1 promoter-derived (6, 7, 12). A portion of the above sequences, containing a di- clear extracts by EMSA with I rectly repeated motif (referred to as TbRE) for TGF-b respon- probes. Considering the wide range of biological phe- nomena that AMLs and Smads regulate, the physical/ sive element (12) or DRE for direct repeat element (13) could functional interplay between them has implications that transfer TGF-b responsiveness to an unrelated promoter. We extend beyond the regulation of class switching to IgA. have recently shown that the DRE element contains functional binding sites for transcription factors of the acute myeloid leukemia (AML) family (14), and that AML proteins cooperate Production of the appropriate antibody isotypes during a with transcription factors of the Ets family to activate the Ia1 humoral immune response is an important prerequisite for the promoter. The AML proteins, also known as core-binding fac- development of protective humoral immunity. Distinct isotypic tors (CBF) (15), polyoma enhancer binding proteins (PEBPs) profiles are shaped by differentially regulating immunoglobu- (16), or Runt domain transcription factors bind to DNA as a/b lin heavy chain class-switching-associated DNA rearrange- subunit heterodimers (15, 17). Three a chains are encoded by ments. Such events juxtapose selectively an expressed heavy the AML1 (CBFA2/PEBP2aB), AML2 (CBFA3/PEBP2aC), chain variable region (VDJ) to a new downstream constant and AML3 (CBFA1/PEBP2aA) genes and one b subunit is region (C ), deleting the DNA in between containing the ear- H encoded by the PEBP2b (CBFb) gene. The a subunits contain lier expressed IgM and IgD genes (1). Experimental evidence the DNA binding runt homology domains, as well as sequences has implicated transforming growth factor-b1 (TGF-b1) as a necessary for association with the b subunit and trans-activa- tion, whereas the b subunit does not bind DNA but increases the strength of the DNA binding of the AML a subunits * This work was supported in part by grants from the Swedish Cancer (15–17). Foundation (Cancerfonden), Swedish Medical Research Council (MFR), Umeå Biotechnology Fund, and the Petrus and Augusta Hedlunds Studies conducted during the last few years have demon- Foundation. The costs of publication of this article were defrayed in part strated that a family of molecules known as Smad proteins by the payment of page charges. This article must therefore be hereby mediate intracellular signaling for the TGF-b superfamily of marked “advertisement” in accordance with 18 U.S.C. Section 1734 secreted polypeptides, which includes the TGF-bs, the activins, solely to indicate this fact. § Contributed equally to the results of this work. and the bone morphogenic proteins (BMPs) (18 –20). Smads are Supported by a scholarship from the “Alexander S. Onnasis” Public composed of an N-terminal Mad homology domain-1 and a Benefit Foundation. ‡‡ To whom correspondence should be addressed. Tel.: 46-90- 7852528; Fax: 46-90-771420; E-mail: [email protected]. oma enhancer-binding protein; BMP, bone morphogenic protein; ALK, The abbreviations used are: TGF-b, transforming growth factor-b; activin type II receptor like kinase; PCR, polymerase chain reaction; AML, acute myeloid leukemia; CBF, core binding factor; PEBP, poly- EMSA, electromobility shift assay; GST, glutathione S-transferase. 3552 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. AML/Smad Cooperativity 3553 San Francisco, CA). All GST-Smad fusion proteins were made by a C-terminal MH2 domain connected with a proline-rich linker PCR-mediated approach and subcloned into the pGEX4T-1 vectors. region. They are classified into three groups; receptor-regu- Full-length Smad1, Smad3, Smad4, and in vivo phosphorylated Smad3 lated (R-Smad), common-mediator (Co-Smad), and inhibitory produced in baculovirus-infected cells were generously provided by Dr. (I-Smad) groups (18 –21). The R-Smads, i.e. Smad1, 2, 3, 5, and Allen Coner (Ophidian Pharmaceuticals, Madison, WI) and Dr. M. 8, interact transiently with activated receptor complexes and Hoffman (McArdle Laboratory, University of Wisconsin-Madison, WU). as a result become phosphorylated at their extreme C termini The mouse AML1b, AML3/PEBP2aA1, and PEBP2b1 cDNAs cloned in the Rous sarcoma virus driven expression vector pBJ9V have been (22, 23). Smad2 and Smad3 interact with and thus mediate previously described (14). signaling from the activin type IB receptor (ActR-1B/ALK-4), Flag-AML1b(1– 451) mouse cDNA was generated by PCR with Pfu the TGF-b type I receptor (TbR-I/ALK-5), and orphan ALK-7. polymerase and cloned as BamHI-XhoI insert in cytomegalovirus Smad1 and Smad5 interact with and mediate signaling from driven expression vector pCDNA-3. Deletion constructs Flag-AML(1– the orphan ALK-1, the ActR-I/ALK-2, BMPR-IA/ALK-3, and 411), Flag-AML(1–371), Flag-AML(1–331), Flag-AML(1–291), Flag- BMPR-IB/ALK-6 (18 –21). Following their phoshorylation the AML(1–243), and Flag-AML(1–177) were generated from Flag-AML(1– 451) by PCR and cloned as EcoRI-XbaI inserts in pCDNA-3. R-Smads form complexes with Co-Smads, i.e. Smad4, which AML1b(178 – 451) and AML1b(51– 451) were inserted into the EcoRI/ translocate into the nucleus and regulate transcription. The XhoI sites. A stop codon was added at the C terminus of all deletion I-Smads, i.e. Smad6 and Smad7, interfere with the BMP and variants. All the PCR-generated DNA segments were confirmed by TGF-b/activin-mediated activation of signal transducing DNA sequencing. Smads (18 –21). Transient transfections were performed as described previously (7) Here we demonstrate that the Ia1 promoter contains an using 2 mg of hCMV-b-gal plasmid (reference plasmid for normaliza- tion), 3 mg of reporter plasmid, and 1– 6 mg of each expression plasmid array of interspersed functional Smad and AML-binding sites as indicated. Where necessary, the empty expression vector pBJ9V was that mediate in a cooperative manner the TGF-b responsive- added to a total of 10 mg of expression plasmids. 10 3 10 cells were ness of this element. Our findings extend the repertoire of electroporated followed by incubation in a volume of 10 ml of media. Smad interacting transcription factors and provide the frame- Human TGF-b1 was added 0.5 h after electroporation where indicated. work for characterizing the signals that regulate the interplay The cells were harvested 20 h after electroporation. between AMLs and Smads in the context of the Ia1 promoter EMSA and DNase I Footprinting Analysis—EMSAs and footprinting analyses were performed as described previously (13, 14). and other promoter/enhancer elements that contain neighbor- Production and Purification of Recombinant Proteins—Glutathione ing AML- and Smad-binding sites. S-transferase (GST)-Smad fusion proteins were expressed in Esche- richia coli and partially purified by column chromatography according EXPERIMENTAL PROCEDURES to the instructions of the manufacturer (Amersham Pharmacia Bio- Cells and Reagents—The human malignant cell lines DG75 (an EBV- tech). Briefly, bacteria grown in 1 3 Luria broth medium were induced negative Burkitt’s lymphoma) and K562 (an early erythroleukemia) with 0.1 mM isopropyl-b-D-thiogalactopyranoside. After sonication the were cultured as described previously (7). Human TGF-b1(R&D GST fusions were isolated using glutathione-Sepharose 4B, washed 3 Systems, Minneapolis, MN) was used at 1 ng/ml. times, eluted, and dialyzed against phosphate-buffered saline supple- Rabbit antiserum was prepared against the synthetic peptide “DG- mented with 2 mM dithiothreitol and 0.5 nM phenylmethylsulfonyl PRPEPRRHRQKLDD” derived from a conserved region in the runt fluoride. Thereafter, the proteins were purified on heparin-Sepharose homology domain. The peptides were synthesized with an Applied Bio- columns following the instructions of the manufacturer (Amersham systems 430 A peptide synthesizer using t-butoxycarbonyl chemistry Pharmacia Biotech). and were purified by reverse-phase high performance liquid chroma- AML1b subcloned in the pSP64-poly(A) KSN vector was in vitro tography. The peptides were coupled to keyhole limpet hemocyanin transcribed and translated using the TnT-coupled reticulocyte lysate using glutaraldehyde. The coupled peptides were mixed with Freund’s system (Promega, Madison, WI) with SP6 polymerase. The proteins adjuvant and used to immunize rabbits. were purified by heparin-Sepharose chromatography as described pre- Ia1 Promoter Derivatives—A series of progressive deletions from the viously (31). upstream side of the human Ia1 promoter was generated by the polym- In Vivo AML/Smad Binding Assay—Fifteen hours after transfec- erase chain reaction (PCR) as described previously (14). The constructs tion, K562 or DG75 cells were lysed in 1 ml of lysis buffer (50 mM were: A (2351/179), B (2247/179), C (2142/179), D (299/179), and E Tris-HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, (267/179) (numbering according to Ref. 7). Mutations of the AML and 0.1% SDS, 20 mM sodium pyrophosphate, protease inhibitor mixture Smad sites in the promoter were created by the PCR based overlap (Roche Molecular Biochemicals, Germany)). The cell lysates were pre- extension technique (24) using the upper strand oligonucleotides shown cleaned with protein G-Sepharose beads and incubated with Flag M2- below and their complementary lower strand counterparts. The mu- agarose beads (Aldrich-Sigma) for2hat4 °C. After washing the im- tated nucleotides are underlined: wt, 59-GCCCCACCACAGCCAGAC- munoprecipitates with lysis buffer three times, immunoprecipitates CACAGGCCAGACATGAC-39; mAML ,59-GCCCCACCACAGCCAGA- and aliquots of cell lysates were separated by SDS-gel electrophoresis AAACAGGCCAGACATGAC-39; mSMAD ,59-GCCCCACCACAGCCA- using 10% polyacrylamide gels and transferred to a Hybond-C extra GACCACAGGCCAACTATGAC-39; mAML /mSMAD ,59-GCCCCACC- membrane (Amersham Pharmacia Biotech). The membrane was then D B ACAGCCAGAAAACAGGCCAACTATGAC-39. The same oligonucleot- probed with anti-Flag M5 or anti-Myc (9E10 monoclonal antibody; ides were used in electromobility shift assays (EMSA) as described Santa Cruz, CA) antibodies. Primary antibodies were detected with below. horseradish peroxidase-conjugated goat anti-mouse antibody (Amer- Plasmid Construction and Transfections—The mouse AML1b cDNA sham Pharmacia Biotech) and chemiluminescent substrate. was cloned into the pSP64-poly(A) KSN vector (a derivative of pSP64- In Vitro AML/Smad Binding Assays—Equal amounts of GST or poly(A) (Promega, Madison, WI) where the PstI site was replaced by GST-Smad proteins were coupled to CNBr-activated Sepharose 4B KpnI, SacII, and NotI sites between the HindIII and SalI sites). The (Amersham Pharmacia Biotech) according to the instructions of the sequence 59-GGTACCACC-39was introduced in front of the start codon manufacturer. The protein-coupled beads were incubated with S- of AML1b by PCR with Pfu polymerase and the modified AML1b cDNA labeled in vitro translated AML1b protein in binding buffer (20 mM was inserted into the KpnI/XhoI sites of pSP64-poly(A) KSN. HEPES, pH 8.0, 10% glycerol, 100 mM NaCl, 2 mM dithiothreitol, 0.05% Expression construct for Flag-Smad1 was provided by Dr. J. Wrana Triton X-100). After incubation for2hat4 °C,the beads were washed (Hospital of Sick Children Toronto, Ontario, Canada). Flag-Smad4 and with binding buffer. Bound proteins were eluted by boiling in SDS- Flag-Smad6 as well as Myc-Smad3 were provided by Dr. M. Kawabata polyacrylamide gel electrophoresis sample buffer, detected by SDS- (Cancer Institute, Tokyo, Japan). Flag-Smad2, Flag-Smad5, and Flag- polyacrylamide gel electrophoresis and autoradiography, and quanti- Smad7 were described previously (25–27). Wild-type and constitutively tated with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). active (ac) ALK-1, -2, -3, -4, -5, and -6 in the pcDNA3 vector have been described previously (28). ALK-7 cDNA (29) was provided by Dr. C. F. RESULTS Ibanez (Karolinska Institute, Stockholm, Sweden). Constructs with the Smad Proteins Regulate the TGF-b Responsiveness of the Ia1 Flag epitope at the C terminus of Smad3, Smad4, and Smad3 and 4 with Promoter Elements—Work over the past few years has demon- C-terminal truncations (3Dc and 4Dc) acting in a dominant negative manner (30) were provided by Dr. R. Derynck (University of California, strated that Smad proteins are major transcription regulators 3554 AML/Smad Cooperativity FIG.1. Smads trans-activate the Ia1 promoter. DG75 cells were FIG.2. Transactivation of the Ia1 promoter by constitutively co-transfected with the Ia1-luciferase reporter construct (2142/179) active type I receptors. Constitutively active type I receptors (muta- and 3 mg of the indicated N-terminal-Flagged Smad expression plas- tions: ALK1-Q201D, ALK2-Q207D, ALK3-Q233D, ALK4-T206D, mids, and thereafter incubated in the presence or absence of TGF-b1. ALK5-T204D, ALK5-T204D, ALK6-Q204D, and ALK7-T194D) as well Luciferase activity was measured 20 h after transfection. The results as wild-type receptors were co-transfected with the Ia1 reported con- shown represent mean values of three independent experiments 6 S.E. struct (2142/179) in DG75 cells and their capacity to stimulate the Ia1 Relative fold activation is calculated considering the activity of the Ia1 promoter was assayed 20 h later. Relative fold activation is calculated containing construct without any ligand or Smad stimulation as 1. 3Dc considering the activity of the Ia1 reported construct without and and 4Dc are C-terminal-Flagged Smad3 and Smad4 variants with their ligand or receptor stimulation as 1. MH2 domains truncated (29). (data not shown). Therefore, we also analyzed the capacity of of TGF-b responsive promoter/enhancer elements (18, 19). different constitutively active type I receptors to stimulate the However, TGF-b family members activate other signaling Ia1 driven reported construct. As shown in Fig. 2, expression of pathways as well (32, 33). In order to clarify whether the constitutively active ALK-1, ActR-I/ALK-2, BMPR-1A/ALK-3, TGF-b responsiveness of the Ia promoters is Smad-dependent or BMPR-1B/ALK-6, capable of activating Smad1 and Smad5 we co-transfected the Ia1 promoter-driven luciferase reporter did not affect the Ia1 promoter activity in DG75 cells. However, construct with expression constructs for different Smad pro- expression of constitutively active ActR-1B/ALK-4 or TbR-I/ teins in the Burkitt’s lymphoma B cell line DG75. This cell line ALK-5, involved in activin and TGF-b signaling, respectively, has been extensively used for analyzing Ia promoter activity (7, and the “orphan” ALK-7 (29), induced the Ia1 reporter in a 13). Smad3 increased significantly (;2.5-fold induction) the dose-dependent manner. ALK-4 and ALK-7 in particular could ligand-independent and ligand-dependent Ia1 promoter activ- reconstitute almost 80 –90% of the transcription activity ob- ity, whereas Smad2 and Smad4 increased it moderately (;1.4- tained by stimulating the cells with TGF-b1 (Fig. 2). Thus in fold induction). However, when Smad2, Smad3, and Smad4 addition to TGF-b, activins or even not as yet identified mem- were combined, a stronger transactivation was observed with bers of the TGF-b superfamily, can potentially activate tran- the highest response obtained when all three proteins were scription of the germ line IgA genes, by modulating the activity co-expressed (;10-fold induction for ligand-independent and of Smad3 and Smad4, and consequently participate in regula- ;5-fold induction for ligand-dependent transactivation). tion of IgA humoral responses. Smad1 and Smad5 transfected individually, together, or in Localization of the Smad Responsive Motifs in the Ia1 Pro- combination with Smad4, did not influence the Ia1 promoter moter—To identify the sequences mediating Smad responsive- (Fig. 1 and data not shown). BMP responsive Smad combina- ness in the Ia1 promoter, deletion mutants were constructed tions did not up-regulate the Ia1 promoter even when they and their responsiveness to TGF-b1 in the presence or absence were co-transfected with the constitutively active ALK-1, thus of expression plasmids for Smad proteins was studied in the B excluding the absence of the necessary receptors in the utilized cell line DG75. As shown in Fig. 3, deletion of sequences be- cell lines as the reason for the Ia1 unresponsiveness (data not tween 2142 and 299, encompassing the DRE element reduced shown). While the Smad variants that interfere with the TGF- dramatically the capacity of the promoter to respond to TGF-b1 b/activin signaling pathway, such as Smad7 and dominant in the presence or absence of exogenous Smad proteins. The negative variants of Smad3 (3Dc) or Smad4 (4Dc) (30) sup- small remaining Smad responsiveness is probably due to addi- pressed basal and TGF-b1 induced Ia1 activity, Smad6, if any- tional Smad-binding sites located downstream (see below, Fig. thing, induced a small increase in promoter activity (Fig. 1). In 4). conclusion, Smad3 and Smad4 and to some extend Smad2 are To define more specifically the Smad-interacting sequences major regulators of TGF-b responsiveness of the Ia1 promoter. of the Ia1 promoter, we produced recombinant Smad proteins Constitutively Activated TbR-I/ALK-5, ActR-IB/ALK4, and in E. coli and used the highly purified proteins in EMSA and Orphan ALK-7 Can Stimulate the Ia1 Promoter—Different DNase I footprinting analyses. EMSA demonstrated that members of the TGF-b/activin family can activate similar Smad1, Smad3, and Smad4 were able to bind to a double- Smads (18 –20). Therefore, we questioned the exclusive involve- stranded oligonucleotide spanning the DRE element (2131/ ment of TGF-b1 in the regulation of Ia1. We tested the effect of 296). Smad4 bound with the highest affinity since almost 10 TGF-b superfamily members to stimulate the Ia1 driven re- times more Smad1 and 4 times more Smad3 was required to porter construct. TGF-b1, TGF-b2, activin A, but not BMP2 produce the same level of retarded probe. Smad2 did not bind and BMP7, were able to induce a transcriptional response in even at 100-fold higher concentration (data not shown). Foot- K562 and DG75 cells (data not shown). Analysis of TGF-b printing analyses with recombinant Smad1, Smad3, and superfamily receptors on these cells showed the presence of Smad4 proteins demonstrated the presence of an array of pro- TGF-b and activin receptors but absence of BMP receptors tected segments within the Ia1 region (Fig. 4, A and B). Simi- AML/Smad Cooperativity 3555 FIG.3. Mapping of the Smad and TGF-b1 responsive portions of the Ia1 promoter. 59 Deletion mutants of the Ia1 promoter were subcloned in the luciferase reporter construct and their responsiveness FIG.4. Footprinting analysis of the Ia1 promoter region using in the presence or absence of TGF-b1 and/or a combination of Smads 3 recombinant Smad1, 2, 3, and 4. A, the indicated amounts of highly and4(3 mg of each) was analyzed in DG75 cells. The results shown purified recombinant GST-Smad fusion proteins were used in DNase I represent the mean value from three independent experiments 6 S.E. footprinting analyses of the Ia1 promoter region. A G/A sequencing The structure of the deletion mutants in relationship to the previously reaction was used to identify protected regions. The Smad-binding sites defined AML, CRE/ATF, and Ets sites, important for Ia1 promoter are indicated and named SMAD to SMAD . Hypersensitive sites are activity, is graphically illustrated. Relative fold activation is calculated A D indicated with asterisks. B, summary of footprinting profiles for Smad considering the activity of the Ia1 containing construct without any 1, 3, and 4 on both DNA strands. Hypersensitive sites are shown with ligand or Smad stimulation as 1. vertical solid arrows. The CAGA motifs are shown in bold letters and arrows indicate their orientation. larly to the EMSA analyses, Smad4 generated footprints at lower protein concentrations than Smad3, suggesting a higher Furthermore, phosphorylated Smad3 produced by co-express- affinity for the target DNA, and Smad1 bound less efficiently ing the activated TbR-I/ALK-5 kinase domain in insect cells than Smad3. Smad2 did not footprint even at the highest gave identical footprints as the non-phosphorylated and the concentration used, thus serving as a negative control. Inter- GST-Smad3 proteins, however, it required only one-fifth pro- estingly, the Smads differed from each other not only regarding tein concentration. Collectively, the footprinting analyses re- their affinity for DNA, but also in the way by which they veals differences in the way the various Smads interact with interacted with it. At optimal concentrations Smad1, Smad3, DNA, not only regarding the affinity of interaction, but also and Smad4 produced footprints around three areas (2127/ regarding the effect their binding has on the DNase I accessi- 2100, 295/265, and 251/220). However, the footprints were bility of the DNA, especially on neighboring sequences. similar, but not identical (Fig. 4, A and B, and footprints for Alignment of all the sequences protected by Smad proteins upper strand not shown). The footprints of Smad3 and Smad4 demonstrated the presence of the CAGAC(C/A) motif or its within the 2127/267 area were identical, but Smad4 foot- complementary (T/G)GTCTG in all of them (Fig. 4B). Recent printed a larger stretch in the 251/220 area. Smad1 protected structural and molecular studies have demonstrated that in- a limited stretch within the 2127/2100 segment and only the deed the AGAC sequence that is present within the above lower strand was weakly protected around the 251/220 area. motifs constitutes the core of Smad-binding sites (34 –38). Furthermore, while all three induced DNase I hypersensitivity Smad Proteins Co-operate with AML1 to Stimulate the Ia1 at position 288(G) in the upper strand (which lies within a Promoter—We have previously demonstrated that AML pro- putative CRE/ATF site), all three Smad molecules induced teins and their corresponding binding sites in the Ia1 promoter additional unique hypersensitive sites. Smad4, for example, are involved in its TGF-b1 responsiveness (14). In light of the was the only one that induced a hypersensitive site on the findings that Smad proteins integrate physically and function- upper strand next to the high affinity AML site, position ally with other transcription factors (20, 30, 39, 40) we inves- 2111(C), and another on the upper strand at position 229(G). tigated the interplay between Smad and AML proteins. For The recombinant Smads initially used in our studies were these experiments we utilized the K562 erythroleukemia cell synthesized as GST fusion proteins. Identical results were ob- line which expresses very low levels of AML proteins and tained using recombinant Smad3 and Smad4 proteins pro- displays very low TGF-b responsiveness as measured by the duced in baculovirus-infected insect cells (data not shown). Ia1 reporter construct (maximally 2-fold induction). As shown 3556 AML/Smad Cooperativity FIG.5. AML and Smad proteins synergistically activate the Ia1 promoter. K562 cells were co-transfected with either wild type Ia1-luciferase reporter construct (2142/179) or AML /SMAD mutants and combinations of Smad3/4 (3 mg of each) and AML1b (1 mg) followed D B by incubation in the presence or absence of TGF-b1. The results shown represent the mean value from three independent experiments 6 S.E. Relative fold induction is calculated considering the activity of the wild-type Ia1 construct without any ligand, AML1b, or Smad stimulation as 1. Inset, EMSA analysis using recombinant AML1b, and Smad3 proteins with wild-type or mutant DRE probes demonstrating that the mutations used in Fig. 5 affect binding of only the corresponding transcription factor (1, wild type probe; 2, AML mutant probe; 3, SMAD mutant probe; D B 4, AML /SMAD double mutant probe). D B in Fig. 5, overexpression of AML1b (the largest splice forms of were stimulated by TGF-b1 or co-transfected with the consti- the mouse AML1) or a combination of Smad3 and Smad4, tutively active type I receptor, this novel band increased sig- increased dramatically the TGF-b1 responsiveness of K562 nificantly. The novel complex was supershifted/inhibited with cells. When AML and Smad proteins were co-expressed, a antibodies against the AML1b protein (anti-HA), Smad3 (anti- synergistic effect on the Ia1 activity was observed. The syner- Myc), or Smad4 (anti-Flag), indicating that all these proteins gistic effect was clearer in the ligand independent responses can bind to the Ia1 DRE sequences in a receptor-dependent (open bars, Fig. 5). Better synergistic effect where obtained in manner. Thus, activation of the receptor induces the appear- AML1b/Smad3/Smad4-transfected TGF-b1-stimulated cells ance of novel complexes that contain Smad3, Smad4, and AML when the fetal calf serum in the culture medium was reduced proteins. from 5 to 1% instead (data not shown). Smad and AML Proteins Can Physically Interact in Vivo and The synergistic action of AML and Smad sites on the activity in Vitro—Recent studies have demonstrated that Smads regu- of the Ia1 promoter was illustrated more dramatically when late transcription through their ability to bind to DNA directly the highest affinity AML and the closely located high affinity and/or indirectly and to induce transcriptional responses Smad site were mutated (Fig. 5). EMSA analyses using wild- through cooperativity with other transcription factors (20, 30, type and mutated oligos demonstrated that mutations that 39 – 41). To investigate the molecular basis of AML/Smad syn- reduced binding of recombinant AML proteins to the AML ergy we expressed epitope-tagged AML1b and Smad3 in K562 site did not abrogate binding of recombinant Smad proteins in the and DG75 cells in the presence or absence of constitutively nearby SMAD active ALK-4 (Fig. 7). AML1b and Smad3 were co-immunopre- site and vice versa (Fig. 5, inset). However, mutation of either the AML or the SMAD sites abrogated the cipitated in both cells analyzed. The AML1b/Smad3 interaction D B was constitutive in both cell lines tested. Co-transfection of a positive effect that overexpression of AML1b and/or Smad3/ Smad4 had on the promoter activity, in the presence or absence PEBP2b expression vector did not improve the interaction, of TGF-b stimulation. Mutation of both these sites affected the probably due to the already existing adequate endogenous lev- Ia promoter even more. In addition to the above two sites, we els in DG75 and K562 (14). Fractionation of cytoplasmic from mutated the SMAD nuclear extracts showed the majority of complexes to be in the , SMAD , AML , and CRE/ATF sites in- A D U dividually or in combinations. In all cases the mutations de- cytoplasm (data not shown). creased the Ia1 promoter activity, indicating that all these sites To map the domains of Smad3 and AML1b involved in this even those with low affinity for the corresponding transcription protein-protein interaction, different epitope-tagged deriva- factors are necessary for optimal Ia1 function (data not shown). tives of either molecule (Fig. 8A) were expressed in DG75 cells Receptor-dependent Formation of Novel DNAzProtein Com- and their interaction was investigated by co-immunoprecipita- plexes Containing AML, Smad3, and Smad4 —To detect recep- tion. As shown in Fig. 8C, fragments containing the C-terminal tor dependent complex formation in EMSAs, we co-transfected MH2 domain of Smad3 exhibited the best AML1b binding DG75 cells with different combinations of epitope-tagged activity. AML1b, Smad3, Smad4 and constitutively active ActR-IB/ Similarly, different truncations of the AML1b molecule were ALK-4 receptor (Fig. 6). The constitutively active ALK-4 stim- tested for their capacity to interact with full-length Smad3. A ulates the same R-Smads as the constitutively active TbRI/ number of functional domains have been recently defined in ALK-5 and as shown in Fig. 2 it can trans-activate the Ia1 the AML molecule (42). Based on these mapping data a number promoter. Similarly transfected cells were also stimulated with of AML1b deletion variants were produced (Fig. 8A) and tested TGF-b1 (20 h after transfection for 30 min) instead of the for interaction with full-length Smad3 (Fig. 8B). A complex constitutively active receptor. Nuclear extracts were prepared pattern of interaction was observed since all the AML1b deriv- and analyzed in EMSA using a probe that contained the AML atives were able to interact with Smad3, albeit with different SMAD , AML , and SMAD sites (Fig. 6). Overexpression of efficiency. More specifically, N-terminal deletions up to amino A D B acid residue 411 did not affect binding. Deleting further amino tagged Smad3, Smad4, and AML1 resulted in the appearance of a weak, novel, lower mobility band. However, when the cells acids 371– 411 reduced significantly binding, without com- AML/Smad Cooperativity 3557 FIG.6. Detection of AML and Smad3/4 containing complexes induced by constitutively active ActR-1B/ALK-4 receptor. DG75 cells were co-transfected with the indicated combinations of expression vectors for AML1b, Smad3, Smad4, and constitutively active ALK-4 (caALK4). Nuclear extracts were prepared 20 h after transfection. TGF-b1 stimulation was done for 30 min before the termination of the culture and the preparation of the nuclear extracts. EMSA was performed with a probe containing the AML , Smad , AML , and Smad -binding sites (2131/296). U A D B Antibodies specific for the Myc, Flag, and HA epitopes recognizing the epitope-tagged Smad3, Smad4, and AML1b, respectively, were used to supershift the TGF-b1 or ALK4-induced complex. No nuclear extract was added in the first lane. Complex A represents binding of endogenous AML proteins, B represents the novel TGF-b1 or caALK-4 receptor induced complexes, C and D represent the anti-Smad3 and anti-Smad4 antibody produced supershifts. The anti-hemagglutinin antibody inhibited complex formation without producing a significant supershift. did not result in the appearance of unphysiological levels of AML1b protein and also to investigate whether TGF-b signal- ing could alter the levels of endogenous AML proteins, cells were harvested at different time points after transfection/ TGF-b1 stimulation and analyzed by Western blotting with an anti-AML antibody. This antibody was raised against a peptide that is conserved in all AML family members and thus can detect all of them. As shown in Fig. 10, exogenous AML1b was detected 1 h and reached maximal levels 7 h after transfection. The levels of exogenous AML1b protein even when they reached maximum were comparable with the levels of other endogenous AML variants. Since AML1b and Smad3 could be co-precipitated even when 10 times less expression vectors were transfected (data not shown), we conclude that the ob- served interaction is not due to unphysiologically high levels of expression. FIG.7. Interaction of AML1b with Smad3 in vivo. DG75 and K562 cells were transfected with the indicated combinations of epitope- It has recently been shown that TGF-b1 stimulates AML2 tagged AML1b and Smad3 with or without constitutively active ALK-4 mRNA synthesis in a mouse cell line (46). As shown in Fig. 10, expression vector (caALK4). The physical interaction between AML1b overexpression of AML1b, Smad3/4, and TGF-b1 stimulation, if and Smad3 was analyzed as described under “Experimental anything, resulted in reduction of some of the endogenous AML Procedures.” variants. This makes unlikely that the cooperation studied herein is due to an induction of AML gene expression by TGF-b pletely eliminating it. Deleting even further down to amino signaling. acid 291 did not alter the binding, however, a deletion reaching Collectively, the in vivo and in vitro binding data indicate down to amino acid 243 restored binding activity to the level that parts of the AML1b molecule spanning the RDH region even higher than the intact AML1b molecule. AML variants 6 and the area between amino acids 371 and 411 participate in and 9 containing either the N-terminal or C-terminal half of AML1b/Smad3 interaction, however, all these interactions tar- AML1b were able to interact with full-length or the MH2 get the MH2 domain of the Smad3 molecule. At least in the two variant of Smad3. No interaction was observed with the Mad cell lines analyzed, this interaction is constitutive and is not homology domain-1 portion of Smad3 (Fig. 8C). To make sure modulated by TGF-b1 signaling. that AML1b and Smad3 interact directly with each other and DISCUSSION not via an intermediate adapter molecule, we used S-labeled in vitro translated and purified AML1b and E. coli expressed The TGF-b Responsiveness of the Ia1 Promoter Is Regulated purified GST-Smad fusion protein immobilized on Sepharose by Direct Interaction of Smads with Specific Sequences within beads. As shown in Fig. 9, approximately 50 – 80% of the in It—TGF-b has been implicated as a major regulator of IgA vitro translated AML1b was retained on beads coupled with synthesis. One of the targets of TGF-b mediated signaling are recombinant Smad1, Smad2, Smad3, and Smad4, whereas only the Ia promoters, which are located upstream of the Sa regions 10% was bound to GST beads. Smad1 and Smad3 bound better and function as a regulator of the accessibility of the Ca germ than Smad2 and Smad4. Thus, purified Smads and AML1b can line loci to class switch recombinases. To clarify the molecular physically associate with each other in the absence of other mechanism(s) that skew immunoglobulin production toward cytoplasmic proteins. IgA, we have characterized the molecular components that To make sure that transfection with the expression vectors participate in the TGF-b1-regulated transcription through Ia1, 3558 AML/Smad Cooperativity FIG.9. Direct interaction between AML1b and Smad proteins in vitro. Equal amounts of S-labeled in vitro translated AML1b were incubated with either GST or GST-Smad1, 2, 3, and 4 coupled beads, and eluted AML1b was analyzed by polyacrylamide gel electrophoresis. Bars represent average of AML1b binding (presented as percentage of input protein eluted from the beads) in at least three independent experiments 6 S.E. the regulatory module of the human IgA1 locus. Our previous studies and the results presented in the current report demon- strate that the Ia1 promoter contains an array of interspersed binding sites for AML and Smad proteins whose cooperative activity underlines Ia1 germ line transcription. Mutation of AML and Smad sites, individually or in combinations, demon- strated that those of high affinity are indispensable for TGF-b responsiveness, whereas those of low affinity contribute to it, however, only when the high affinity sites are intact. The presence of multiple AML and Smad sites with different de- grees of importance in the Ia1 promoter, and possibly in other Smad-regulated promoters, might allow the promoter to func- tion as a “rheostat” responding gradually to signals of varying strength instead of responding in an all-or-none manner. This possibility is compatible with the finding that synthetic TGF-b responsive promoters respond better when they have several copies of Smad-binding sites. In an in vivo situation the varying parameter could be the strength of ligand induced signaling, or it could be the relative abundance of different Smads in the FIG.8. Mapping of AML1b and Smad3 domains involved in responding cells. their physical interaction. A, schematic representation of deletion Crystallographic studies have demonstrated that Smad3 mutants. Epitope-tagged deletion variants were produced as described under “Experimental Procedures” and were subcloned in the pCDNA3 binds DNA via an 11-amino acid b hairpin in the Mad homol- vector. B, full-length 6Myc-Smad3 expression vector was co-transfected ogy domain-1 domain that contacts the AGAC motif (38). with different variants of Flag-AML1b plasmids. Prior to immunopre- Smad2 cannot bind DNA despite its very high sequence simi- cipitation, an aliquot of the protein extracts was analyzed by Western larity to Smad3. This is probably due to a sequence insert in blot with anti-Myc antibodies to verify equal Smad3 expression in all transfections. After immunoprecipitation with anti-Flag antibodies the Smad2, immediately upstream of the DNA binding b-hairpin, precipitated material was analyzed by Western blot with anti-Myc which could potentially hinder Smad2-DNA interaction (38, antibodies to detect co-precipitated Smad3 protein and anti-Flag anti- 43). Interestingly, our footprinting experiments demonstrate body to verify equal precipitation of AML1b variants in all samples. C, that while Smad3 and Smad4, and less efficiently Smad1, bind full-length or deletion variants of Smad3 were co-expressed with full- length or deletion variants of AML1b. AML1b/Smad3 physical associa- to the same core sequence (CAGAC(C/A)), the way they impose tion was analyzed as in B. themselves on the DNA helix differs. They protect to different extent the two strands (Fig. 3, A and B) and more importantly, AML/Smad Cooperativity 3559 FIG. 10. Western blot analysis of AML family members. DG75 cells were transfected with AML1b-Smad3-Smad4 expression vectors or the control pBJ9V plasmid. Transfected cells were cultured in the presence or absence of TGF-b1 and cell lysates were prepared at the indicated time points. The anti-AML antibody was raised against a conserved amino acid stretch in the runt homology domain and thus detects all the AML proteins and their splice variants. Using commercially available monoclonal antibodies the lower molecular weight major species was recognized as AML1 and the two upper bands as AML2 (data not shown). A number of minor bands were recognized by antibodies against AML1, AML2, and AML3 and probably represent mixtures of different splice variants with similar electrophoretic mobility. the different Smads induce the appearance of different DNase alternative splicing of exons and possibly also alternative use of I-hypersensitive sites on the DNA template indicating that start codons for translation. Shi and Stavnezer (46) have re- they influence to different extents the access of neighboring cently presented evidence supporting the role of AML proteins DNA sequences to DNase I. If different Smads hinder to dif- for the regulation of the mouse Ia promoter. Since AML2 was ferent extent the accessibility of neighboring transcription fac- the only member of the family that was up-regulated by tor-binding sites, then the outcome of TGF-b signaling will TGF-b1 stimulation, they concluded that AML2 is the critical depend on which Smads will eventually bind to a given site. regulator of Ia transcription. Considering that AML1b is the The cooperative function of multiple Smad-binding sites and most efficient Ia transactivator among the AML isoforms (12, the differential influence that various Smads exert on neigh- 14), the quite rapid induction of germ line transcription (47– boring binding sites upon binding, could allow the Smad sig- 49), the fact that AML2 up-regulation occurs at very late stages naling system to transfer a whole spectrum of quantitatively of TGF-b signaling (46) and also the fact that in the DG75 and and qualitatively different instructions on a target promoter/ K562 cells we have analyzed in the present report TGF-b1 enhancer and be important in the context of the function of signaling does not induce AML2 up-regulation (Fig. 10), it is TGF-b family members as morphogens during development conceivable to either envision a dynamic process, during which (44). different isoforms of AML sequentially participate in the AML/ Recent findings including those of the present study have Smad interplay or to consider that the apparent differences are demonstrated that recombinant Smad proteins can bind di- cell line specific. Furthermore, it is possible to expect that rectly to certain DNA sequences that contain the AGAC core different AML variants will be more important in responses to motif. Truncation of the MH2 domain in Droshophila Mad different activating stimuli and in the context of different pro- protein increases the capacity of the Mad homology domain-1- moters. Furthermore, considering the quite significant diver- Linker portion to bind DNA (45). To make sure that the binding gence of AML proteins at their C termini, it is possible that activity in our preparations was not due to degradation prod- different domains of the AML and Smad molecules will differ- ucts mimicking the MH2 truncation we have carefully purified entially contribute in physical interaction of various AML/ full-length proteins. Removal of smaller size contaminants in- Smad combinations. creased the specific activity of the preparation instead of re- Is TGF-b1 the Exclusive Regulator of Ia Promoter Activity?— ducing it, suggesting that the full-length receptor unmodified Our finding that constitutively activated ALK-4, ALK-5, and Smads can bind DNA (data not shown). ALK-7 can stimulate the Ia1 promoter (Fig. 2) immediately Smads Cooperate with AML Proteins to Stimulate the Ia1 raises the possibility that Ia transcription can be achieved not Promoter—Our studies extend the list of transcription factors only by TGF-b1 but also by other members of the TGF-b su- that integrate functionally with the Smad proteins by adding perfamily that can interact with the above receptors. Indeed, the AML family of transcriptional regulators. Smad3 and TGF-b2 and activin A can also stimulate the Ia1 reporter AML1b physically interact in vitro and in vivo. Their interac- construct in DG75 cells (data not shown). TGF-b1 and TGF-b2 tion is constitutive and at least in the two cell lines used is not were more potent than activin A, however, this could be due to affected by receptor signaling, and appears to be of lower af- the high levels of TGF-b type III receptors and the low levels of finity than the interaction of TbR-I-phosphorylated Smad3 activin receptors these cells express (data not shown). We con- with Smad4. However, it is only after stimulation either by clude that other members of the TGF-b/activin family are po- TGF-b1 or the constitutively active type I receptors that higher tential regulators of IgA synthesis. order complexes containing AML1b, Smad3, and Smad4 are Consequences of the AML/Smad Interplay for Other Biolog- detected in nuclear extracts by EMSA (Fig. 6). It appears thus ical Systems—AML transcription factors bind to DNA sites in that despite the fact that these molecules form complexes con- the regulatory regions of a number of hematopoiesis specific stitutively, the effect of their interplay on Ia1 transcription can genes and play key roles in normal and pathological blood cell potentially be modulated at other levels, such as nuclear processes. Inactivation of either the AML1 gene or its associ- availability. ated factor PEBP2b results in death early in fetal development The AML family includes the AML1, AML2, and AML3 (day 12–14) and a complete block in fetal liver hematopoiesis genes. All of them give rise to a collection of polypeptides by (15, 50). Furthermore, the AML1/PEBP2b genes are the most 3560 AML/Smad Cooperativity 6859 – 6863 frequent targets of translocation in acute human leukemia (51). 17. Ogawa, E., Inuzuka. M., Maruyama, M., Satake, M., Naito Fujimoto, M., Ito, Smad proteins, as nuclear TGF-b effectors, regulate a large Y., and Shigesada, K. (1993) Virology 194, 314 –331 18. Heldin, C-H., Miyazono, K., and ten Dijke, P. (1997) Nature 390, 465– 471 range of biological processes. Furthermore, Smads play impor- 19. Massague ´ , J. (1998) Annu. Rev. Biochem. 67, 753–791 tant roles during processes leading to malignant transforma- 20. Derynck, R., Zhang, Y., and Feng, X.-H. (1998) Cell 95, 737–740 tion. Smad4 was originally identified as a putative tumor sup- 21. Hata, A., Shi, Y., and Massague ´ , J. (1998) Mol. Med. Today 4, 257–262 22. Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and pressor gene, frequently mutated in pancreatic carcinomas and Wrana, J. L. (1997) J. Biol. Chem. 272, 27678 –27685 occasionally in other types of tumors (18, 21). 23. Souchelnytskyi, S., Tamaki, K., Engstro ¨ m, U., Wernstedt, C., ten Dijke, P., and In vivo and in vitro studies have suggested that among the Heldin, C.-H. (1997) J. Biol. Chem. 272, 28107–28115 24. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) extracellular signals that can induce the osteoblastic pheno- Gene (Amst.) 77, 61– 68 type are the BMPs (52). Recent studies have demonstrated that 25. Nakao, A., Afrakhte, M., More ´ n, A., Nakayama, T., Christian, J. L., Heuchel, R., Itoh, S., Kawabata, M., Heldin, C.-H., and ten Dijke, P. (1997) Nature a splice form of CBFA1/AML3/PEBP2aA also plays a critical 389, 631– 635 role in osteoblast differentiation (53–55). One possible mecha- 26. Souchelnytskyi, S., Nakayama, T., Nakao, A., More ´ n, A., Heldin, C.-H., Chris- nism by which BMPs could mediate regulation of bone devel- tian, J. L., and ten Dijke, P. (1998) J. Biol. Chem. 273, 25364 –25370 27. Tamaki, K., Souchelnytskyi, S., Itoh, S., Nakao, A., Sampath, K., Heldin, opment is via the induction of CBFA1 synthesis. AML1b inter- C.-H., and ten Dijke, P. (1998) J. Cell. Physiol. 177, 355– 63 acts physically not only with the TGF-b/activin Smads but also 28. Nakao, A., Imamura, T., Souchelnytskyi, S., Kawabata, M., Ishisaki, A., Oeda, E., Tamaki, K., Hanai, J., Heldin, C.-H., Miyazono, K., and ten Dijke, P. with the BMP-activated Smad1 (Fig. 9). It is possible that the (1997) EMBO J. 16, 5353–5362 AML/Smad interplay involves many or all the members of the 29. Ryde ´ n, M., Imamura, T., Jornvall, H., Belluardo, N., Neveu, I., Trupp, M., two families, and that depending on the cell type and stimuli, Okadome, T., ten Dijke, P., and Ibanez, C. F. (1996) J. Biol. Chem. 271, 30603–30609 different combinations of AML and Smad proteins could be 30. Zhang, Y., Feng, X.-H., and Derynck, R. (1998) Nature 394, 909 –913 recruited to regulate transcription of selected genes. 31. Corneliussen, B., Holm, M., Waltersson, Y., Onions, J., Hallberg, B., Thornell, A., and Grundstro ¨ m, T. (1994) Nature 368, 760 –764 Considering the wide range of biological phenomena that 32. Hocevar, B. A., Brown, T. L., and Howe, P. H. (1999) EMBO J. 18, 1345–1356 AML and Smad proteins regulate and the potential oncogenic 33. Visser, J. A., and Themmen, A. P. (1998) Mol. Cell. Endocrinol. 146, 7–17 character of AML1 and tumor suppressor activity of some 34. Yingling, J. M., Datto, M. B., Wong, C., Frederick, J. P., Liberati, N. T., and Wang, X.-F. (1997) Mol. Cell. Biol. 17, 7019 –7028 Smads, the observed functional interplay between these tran- 35. Dennler, S. Itoh, S., Vivien, D., ten Dijke, P., Huet, S., and Gauthier, J. M. scription factors has implications not only for class switching to (1998) EMBO J. 17, 3091–3100 36. Jonk, L. J. C., Itoh, S., Heldin, C-H., ten Dijke, P., and Kruijer, W. (1998) IgA but also for other biological processes such as regulation of J. Biol. Chem. 273, 21145–21152 bone ossification as well as normal and pathological hemato- 37. Zawel, L., Dai, J. L., Buckhaults, P., Zhou, S., Kinzler, K. W., Vogelstein, B., poietic cell behavior. and Kern, S. E. (1998) Mol. Cell. 1, 611– 617 38. Shi, Y., Wang, Y. F., Jayaraman, L., Yang, H., Massague ´ , J., and Pavletich, N. P. (1998) Cell 94, 585–594 Acknowledgments—We thank Drs. M. Kawabata, R. Derynck, and J. 39. Chen, X., Weisberg, E., Fridmacher, V., Watanabe, M., Naco, G., and Whit- Wrana for epitope-tagged Smad expression vectors, C. F. Ibanez for man, M. (1997) Nature 389, 85– 89 ALK-7 cDNA, A. Coner and M. Hoffmann for recombinant Smad3 and 40. Hua, X., Liu, X., Ansari, D. O., and Lodish, H. F. (1998) Genes Dev. 12, phosphorylated Smad3 proteins, Y. Eto for activin A, T. K. Sampath for 3084 –3095 BMP-2 and OP-1, and B. Pratt for TGF-b2. 41. Labbe ´ , E., Silvestri, C., Hoodless, P. A., Wrana, J. L., and Attisano, L. (1998) Mol. Cell. 2, 109 –120 REFERENCES 42. Kanno, T., Kanno, Y., Chen, L. F., Ogawa, E., Kim, W. Y., and Ito, Y. (1998) Mol. Cell. Biol. 18, 2444 –2454 1. Honjo, T., and Kataoka, T. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2140 –2144 43. Yagi, K., Goto, D., Hamamoto, T., Takenoshita, S., Kato, M., and Miyazono, K. 2. Coffman, R., Lebman, D. A., and Shrader, B. (1989) J. Exp. Med. 170, (1999) J. Biol. Chem. 274, 703–709 1039 –1044 44. Wilson, P. A., Lagna, G., Suzuki, A., and Hemmati-Brivanlou, A. (1997) De- 3. Defrance, T., Vanbervliet, B., Briere, F., Durand, I., Rousset, F., and Banche- velopment 124, 3177–3184 reau, J. (1992) J. Exp. Med. 175, 671– 682 45. Kim, J., Johnson, K., Chen, H., Carroll, S., and Laughon, A. (1997) Nature 388, 4. Van Vlasselaer, P., Punnonen, J., and de Vries, J. E. (1992) J. Immunol. 148, 304 –308 2062–2067 46. Shi, M. J., and Stavnezer, J. (1998) J. Immunol. 161, 6751– 6760 5. Flanagan, J. G., and Rabbitts, T. H. (1982) Nature 300, 709 –714 47. Berton, M. T., Uhr, J. W., and Vitetta, E. S. (1989) Proc. Natl. Acad. Sci. 6. Nilsson, L., Islam, K. B., Olafsson, O., Zalcberg, I. Samakovlis, C., Ham- U. S. A. 86, 1819 –1833 marstrom, L., Smith, C. I. E., and Sideras, P. (1991) Int. Immunol. 3, 48. Lutzker, S., Rothman, P., Pollock, R., Coffman, R., and Alt, W. A. (1988) Cell 1107–1115 53, 177–184 7. Nilsson, L., and Sideras, P. (1993) Int. Immunol. 5, 271–282 8. Yancopoulos, G. D., DePinho, R. A., Zimmerman, K. A., Lutzker, S. G., Rosen- 49. Kitani, A., and Strober, W. (1993) J. Immunol. 151, 3478 –3488 50. Dzierzak, E., Medvinsky, A., and de Bruijn, M. (1998) Immunol. Today 19, berg, N., and Alt, F. W. (1986) EMBO J. 5, 3259 –3266 9. Stavnezer Nordgren, J., and Sirlin, S. (1986) EMBO J. 5, 95–102 228 –236 51. Look, A. T. (1997) Science 278, 1059 –1064 10. Snapper, C. M., Marcu, K. B., and Zelazowski, P. (1997) Immunity 6, 217–223 11. Stavnezer, J. (1996) Adv. Immunol. 61, 79 –146 52. Rodan, G. A., and Harada, S. (1997) Cell 89, 677– 680 53. Mundlos, S., Otto, F., Mundlos, C., Mulliken, J. B., Aylsworth, A. S., Albright, 12. Lin, Y. C., and Stavnezer, J. (1992) J. Immunol. 149, 2914 –2925 13. Nilsson, L., Grant, P., Larsson, I., Pettersson, S., and Sideras, P. (1995) Int. S., Lindhout, D., Cole, W. G., Henn, W., Knoll, J. H. M., Owen, M. J., Mertelsmann, R., Zabel, B. U., and Olsen, B. R. (1997) Cell 89, 773–779 Immunol. 7, 1191–1204 14. Xie, X-Q., Pardali, E., Holm, M., Sideras, P., and Grundstro ¨ m, T. (1999) Eur. 54. Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., Bronson, R. T., Gao, Y. H., Inada, M., Sato, M., Okamoto, R., J. Immunol. 29, 488 – 498 15. Wang, S., Wang, Q., Crute, B. E., Melnikova, I. N., Keller, S. R., and Speck, Kitamura, Y., Yoshiki, S., and Kishimoto, T. (1997) Cell 89, 755–764 55. Otto, F., Thornell, A., P., Crompton,, T., Denzel, A., Gilmour, K. C., Rosewell, N. A. (1993) Mol. Cell. Biol. 13, 3324 –3339 16. Ogawa, E., Maruyama, M., Kagoshima, H., Inuzuka, M., Lu, J., Satake, M., I. R., Stamp, G. W., Beddington, R. S., Mundlos, S., Olsen, B. R., Selby, P. B., and Owen, M. J. (1997) Cell 89, 765–771 Shigesada, K., and Ito, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Smad and AML Proteins Synergistically Confer Transforming Growth Factor β1 Responsiveness to Human Germ-line IgA Genes

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 5, Issue of February 4, pp. 3552–3560, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Smad and AML Proteins Synergistically Confer Transforming b1 Responsiveness to Human Germ-line IgA Genes* Growth Factor (Received for publication, August 9, 1999, and in revised form, October 8, 1999) Evangelia Pardali‡§¶, Xiao-Qi Xie‡§, Panagiotis Tsapogas‡, Susumu Itohi, Konstantinos Arvanitidis‡, Carl-Henrik Heldini, Peter ten Dijkei, Thomas Grundstro ¨ m‡, and Paschalis Sideras‡** ‡‡ From the ‡Division of Tumor Biology, Department of Cell and Molecular Biology, Umeå University, S-901 87 Umeå, the iLudwig Institute for Cancer Research, Box 595, Biomedical Center, S-751 24 Uppsala, and the **Department of Inflammation Pharmacology, ASTRAZENECA R&D LUND, S-22100 Lund, Sweden Transcription of germ-line immunoglobulin heavy positive regulator of IgA production (2– 4). There are two IgA isotypes in the human system, IgA1 and IgA2, encoded by two chain genes conditions them to participate in isotype b1 switch recombination. Transforming growth factor- different genes, one in each of the two duplication units that b1) stimulates promoter elements located up- (TGF- compose the human immunoglobulin heavy chain locus (5). stream of the IgA1 and IgA2 switch regions, designated Both genes contain, upstream of their switch regions, almost a1 and Ia2, and contributes to the development of IgA identical TGF-b1 responsive promoter/enhancer elements des- responses. We demonstrate that intracellular Smad pro- ignated intron (I) a1 and Ia2, respectively (6, 7). According to a1 promoter by TGF-b. teins mediate activation of the I the “accessibility model” for isotype class switching (8, 9) tran- b type 1 receptor (ALK-5), activin type IB receptor TGF- scribed germ line IgA genes become “accessible” to the Ig class a1 (ALK-4), and the “orphan” ALK-7 trans-activate the I switch recombination machinery and thus are preferentially promoter, thus raising the possibility that other mem- rearranged and expressed. The importance of the I region pro- b superfamily can also modulate IgA bers of the TGF- moter/enhancer elements for proper guiding of a given immu- synthesis. Smads physically interact with the AML fam- noglobulin heavy chain locus toward a certain class has been ily of transcription factors and cooperate with them to repeatedly illustrated by elegant gene targeting experiments a1 promoter. The Ia1 element provides a activate the I where disruption of I region elements prevented rearrange- canape ´ of interspersed high and low affinity sites for ment of the targeted alleles (10, 11). Smad and AML factors, some of which are indispensable Genomic segments encompassing approximately 130 base b responsiveness. While AMLzSmad complexes for TGF- pairs upstream of the human and mouse Ia transcription ini- are formed in the cytoplasm of DG75 and K562 cells tiation sites contain all the information necessary for expres- b receptor activation, constitutively, only after TGF- sion and TGF-b responsiveness in transient expression assays novel Smad3zSmad4zAML complexes are detected in nu- a1 promoter-derived (6, 7, 12). A portion of the above sequences, containing a di- clear extracts by EMSA with I rectly repeated motif (referred to as TbRE) for TGF-b respon- probes. Considering the wide range of biological phe- nomena that AMLs and Smads regulate, the physical/ sive element (12) or DRE for direct repeat element (13) could functional interplay between them has implications that transfer TGF-b responsiveness to an unrelated promoter. We extend beyond the regulation of class switching to IgA. have recently shown that the DRE element contains functional binding sites for transcription factors of the acute myeloid leukemia (AML) family (14), and that AML proteins cooperate Production of the appropriate antibody isotypes during a with transcription factors of the Ets family to activate the Ia1 humoral immune response is an important prerequisite for the promoter. The AML proteins, also known as core-binding fac- development of protective humoral immunity. Distinct isotypic tors (CBF) (15), polyoma enhancer binding proteins (PEBPs) profiles are shaped by differentially regulating immunoglobu- (16), or Runt domain transcription factors bind to DNA as a/b lin heavy chain class-switching-associated DNA rearrange- subunit heterodimers (15, 17). Three a chains are encoded by ments. Such events juxtapose selectively an expressed heavy the AML1 (CBFA2/PEBP2aB), AML2 (CBFA3/PEBP2aC), chain variable region (VDJ) to a new downstream constant and AML3 (CBFA1/PEBP2aA) genes and one b subunit is region (C ), deleting the DNA in between containing the ear- H encoded by the PEBP2b (CBFb) gene. The a subunits contain lier expressed IgM and IgD genes (1). Experimental evidence the DNA binding runt homology domains, as well as sequences has implicated transforming growth factor-b1 (TGF-b1) as a necessary for association with the b subunit and trans-activa- tion, whereas the b subunit does not bind DNA but increases the strength of the DNA binding of the AML a subunits * This work was supported in part by grants from the Swedish Cancer (15–17). Foundation (Cancerfonden), Swedish Medical Research Council (MFR), Umeå Biotechnology Fund, and the Petrus and Augusta Hedlunds Studies conducted during the last few years have demon- Foundation. The costs of publication of this article were defrayed in part strated that a family of molecules known as Smad proteins by the payment of page charges. This article must therefore be hereby mediate intracellular signaling for the TGF-b superfamily of marked “advertisement” in accordance with 18 U.S.C. Section 1734 secreted polypeptides, which includes the TGF-bs, the activins, solely to indicate this fact. § Contributed equally to the results of this work. and the bone morphogenic proteins (BMPs) (18 –20). Smads are Supported by a scholarship from the “Alexander S. Onnasis” Public composed of an N-terminal Mad homology domain-1 and a Benefit Foundation. ‡‡ To whom correspondence should be addressed. Tel.: 46-90- 7852528; Fax: 46-90-771420; E-mail: [email protected]. oma enhancer-binding protein; BMP, bone morphogenic protein; ALK, The abbreviations used are: TGF-b, transforming growth factor-b; activin type II receptor like kinase; PCR, polymerase chain reaction; AML, acute myeloid leukemia; CBF, core binding factor; PEBP, poly- EMSA, electromobility shift assay; GST, glutathione S-transferase. 3552 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. AML/Smad Cooperativity 3553 San Francisco, CA). All GST-Smad fusion proteins were made by a C-terminal MH2 domain connected with a proline-rich linker PCR-mediated approach and subcloned into the pGEX4T-1 vectors. region. They are classified into three groups; receptor-regu- Full-length Smad1, Smad3, Smad4, and in vivo phosphorylated Smad3 lated (R-Smad), common-mediator (Co-Smad), and inhibitory produced in baculovirus-infected cells were generously provided by Dr. (I-Smad) groups (18 –21). The R-Smads, i.e. Smad1, 2, 3, 5, and Allen Coner (Ophidian Pharmaceuticals, Madison, WI) and Dr. M. 8, interact transiently with activated receptor complexes and Hoffman (McArdle Laboratory, University of Wisconsin-Madison, WU). as a result become phosphorylated at their extreme C termini The mouse AML1b, AML3/PEBP2aA1, and PEBP2b1 cDNAs cloned in the Rous sarcoma virus driven expression vector pBJ9V have been (22, 23). Smad2 and Smad3 interact with and thus mediate previously described (14). signaling from the activin type IB receptor (ActR-1B/ALK-4), Flag-AML1b(1– 451) mouse cDNA was generated by PCR with Pfu the TGF-b type I receptor (TbR-I/ALK-5), and orphan ALK-7. polymerase and cloned as BamHI-XhoI insert in cytomegalovirus Smad1 and Smad5 interact with and mediate signaling from driven expression vector pCDNA-3. Deletion constructs Flag-AML(1– the orphan ALK-1, the ActR-I/ALK-2, BMPR-IA/ALK-3, and 411), Flag-AML(1–371), Flag-AML(1–331), Flag-AML(1–291), Flag- BMPR-IB/ALK-6 (18 –21). Following their phoshorylation the AML(1–243), and Flag-AML(1–177) were generated from Flag-AML(1– 451) by PCR and cloned as EcoRI-XbaI inserts in pCDNA-3. R-Smads form complexes with Co-Smads, i.e. Smad4, which AML1b(178 – 451) and AML1b(51– 451) were inserted into the EcoRI/ translocate into the nucleus and regulate transcription. The XhoI sites. A stop codon was added at the C terminus of all deletion I-Smads, i.e. Smad6 and Smad7, interfere with the BMP and variants. All the PCR-generated DNA segments were confirmed by TGF-b/activin-mediated activation of signal transducing DNA sequencing. Smads (18 –21). Transient transfections were performed as described previously (7) Here we demonstrate that the Ia1 promoter contains an using 2 mg of hCMV-b-gal plasmid (reference plasmid for normaliza- tion), 3 mg of reporter plasmid, and 1– 6 mg of each expression plasmid array of interspersed functional Smad and AML-binding sites as indicated. Where necessary, the empty expression vector pBJ9V was that mediate in a cooperative manner the TGF-b responsive- added to a total of 10 mg of expression plasmids. 10 3 10 cells were ness of this element. Our findings extend the repertoire of electroporated followed by incubation in a volume of 10 ml of media. Smad interacting transcription factors and provide the frame- Human TGF-b1 was added 0.5 h after electroporation where indicated. work for characterizing the signals that regulate the interplay The cells were harvested 20 h after electroporation. between AMLs and Smads in the context of the Ia1 promoter EMSA and DNase I Footprinting Analysis—EMSAs and footprinting analyses were performed as described previously (13, 14). and other promoter/enhancer elements that contain neighbor- Production and Purification of Recombinant Proteins—Glutathione ing AML- and Smad-binding sites. S-transferase (GST)-Smad fusion proteins were expressed in Esche- richia coli and partially purified by column chromatography according EXPERIMENTAL PROCEDURES to the instructions of the manufacturer (Amersham Pharmacia Bio- Cells and Reagents—The human malignant cell lines DG75 (an EBV- tech). Briefly, bacteria grown in 1 3 Luria broth medium were induced negative Burkitt’s lymphoma) and K562 (an early erythroleukemia) with 0.1 mM isopropyl-b-D-thiogalactopyranoside. After sonication the were cultured as described previously (7). Human TGF-b1(R&D GST fusions were isolated using glutathione-Sepharose 4B, washed 3 Systems, Minneapolis, MN) was used at 1 ng/ml. times, eluted, and dialyzed against phosphate-buffered saline supple- Rabbit antiserum was prepared against the synthetic peptide “DG- mented with 2 mM dithiothreitol and 0.5 nM phenylmethylsulfonyl PRPEPRRHRQKLDD” derived from a conserved region in the runt fluoride. Thereafter, the proteins were purified on heparin-Sepharose homology domain. The peptides were synthesized with an Applied Bio- columns following the instructions of the manufacturer (Amersham systems 430 A peptide synthesizer using t-butoxycarbonyl chemistry Pharmacia Biotech). and were purified by reverse-phase high performance liquid chroma- AML1b subcloned in the pSP64-poly(A) KSN vector was in vitro tography. The peptides were coupled to keyhole limpet hemocyanin transcribed and translated using the TnT-coupled reticulocyte lysate using glutaraldehyde. The coupled peptides were mixed with Freund’s system (Promega, Madison, WI) with SP6 polymerase. The proteins adjuvant and used to immunize rabbits. were purified by heparin-Sepharose chromatography as described pre- Ia1 Promoter Derivatives—A series of progressive deletions from the viously (31). upstream side of the human Ia1 promoter was generated by the polym- In Vivo AML/Smad Binding Assay—Fifteen hours after transfec- erase chain reaction (PCR) as described previously (14). The constructs tion, K562 or DG75 cells were lysed in 1 ml of lysis buffer (50 mM were: A (2351/179), B (2247/179), C (2142/179), D (299/179), and E Tris-HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, (267/179) (numbering according to Ref. 7). Mutations of the AML and 0.1% SDS, 20 mM sodium pyrophosphate, protease inhibitor mixture Smad sites in the promoter were created by the PCR based overlap (Roche Molecular Biochemicals, Germany)). The cell lysates were pre- extension technique (24) using the upper strand oligonucleotides shown cleaned with protein G-Sepharose beads and incubated with Flag M2- below and their complementary lower strand counterparts. The mu- agarose beads (Aldrich-Sigma) for2hat4 °C. After washing the im- tated nucleotides are underlined: wt, 59-GCCCCACCACAGCCAGAC- munoprecipitates with lysis buffer three times, immunoprecipitates CACAGGCCAGACATGAC-39; mAML ,59-GCCCCACCACAGCCAGA- and aliquots of cell lysates were separated by SDS-gel electrophoresis AAACAGGCCAGACATGAC-39; mSMAD ,59-GCCCCACCACAGCCA- using 10% polyacrylamide gels and transferred to a Hybond-C extra GACCACAGGCCAACTATGAC-39; mAML /mSMAD ,59-GCCCCACC- membrane (Amersham Pharmacia Biotech). The membrane was then D B ACAGCCAGAAAACAGGCCAACTATGAC-39. The same oligonucleot- probed with anti-Flag M5 or anti-Myc (9E10 monoclonal antibody; ides were used in electromobility shift assays (EMSA) as described Santa Cruz, CA) antibodies. Primary antibodies were detected with below. horseradish peroxidase-conjugated goat anti-mouse antibody (Amer- Plasmid Construction and Transfections—The mouse AML1b cDNA sham Pharmacia Biotech) and chemiluminescent substrate. was cloned into the pSP64-poly(A) KSN vector (a derivative of pSP64- In Vitro AML/Smad Binding Assays—Equal amounts of GST or poly(A) (Promega, Madison, WI) where the PstI site was replaced by GST-Smad proteins were coupled to CNBr-activated Sepharose 4B KpnI, SacII, and NotI sites between the HindIII and SalI sites). The (Amersham Pharmacia Biotech) according to the instructions of the sequence 59-GGTACCACC-39was introduced in front of the start codon manufacturer. The protein-coupled beads were incubated with S- of AML1b by PCR with Pfu polymerase and the modified AML1b cDNA labeled in vitro translated AML1b protein in binding buffer (20 mM was inserted into the KpnI/XhoI sites of pSP64-poly(A) KSN. HEPES, pH 8.0, 10% glycerol, 100 mM NaCl, 2 mM dithiothreitol, 0.05% Expression construct for Flag-Smad1 was provided by Dr. J. Wrana Triton X-100). After incubation for2hat4 °C,the beads were washed (Hospital of Sick Children Toronto, Ontario, Canada). Flag-Smad4 and with binding buffer. Bound proteins were eluted by boiling in SDS- Flag-Smad6 as well as Myc-Smad3 were provided by Dr. M. Kawabata polyacrylamide gel electrophoresis sample buffer, detected by SDS- (Cancer Institute, Tokyo, Japan). Flag-Smad2, Flag-Smad5, and Flag- polyacrylamide gel electrophoresis and autoradiography, and quanti- Smad7 were described previously (25–27). Wild-type and constitutively tated with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). active (ac) ALK-1, -2, -3, -4, -5, and -6 in the pcDNA3 vector have been described previously (28). ALK-7 cDNA (29) was provided by Dr. C. F. RESULTS Ibanez (Karolinska Institute, Stockholm, Sweden). Constructs with the Smad Proteins Regulate the TGF-b Responsiveness of the Ia1 Flag epitope at the C terminus of Smad3, Smad4, and Smad3 and 4 with Promoter Elements—Work over the past few years has demon- C-terminal truncations (3Dc and 4Dc) acting in a dominant negative manner (30) were provided by Dr. R. Derynck (University of California, strated that Smad proteins are major transcription regulators 3554 AML/Smad Cooperativity FIG.1. Smads trans-activate the Ia1 promoter. DG75 cells were FIG.2. Transactivation of the Ia1 promoter by constitutively co-transfected with the Ia1-luciferase reporter construct (2142/179) active type I receptors. Constitutively active type I receptors (muta- and 3 mg of the indicated N-terminal-Flagged Smad expression plas- tions: ALK1-Q201D, ALK2-Q207D, ALK3-Q233D, ALK4-T206D, mids, and thereafter incubated in the presence or absence of TGF-b1. ALK5-T204D, ALK5-T204D, ALK6-Q204D, and ALK7-T194D) as well Luciferase activity was measured 20 h after transfection. The results as wild-type receptors were co-transfected with the Ia1 reported con- shown represent mean values of three independent experiments 6 S.E. struct (2142/179) in DG75 cells and their capacity to stimulate the Ia1 Relative fold activation is calculated considering the activity of the Ia1 promoter was assayed 20 h later. Relative fold activation is calculated containing construct without any ligand or Smad stimulation as 1. 3Dc considering the activity of the Ia1 reported construct without and and 4Dc are C-terminal-Flagged Smad3 and Smad4 variants with their ligand or receptor stimulation as 1. MH2 domains truncated (29). (data not shown). Therefore, we also analyzed the capacity of of TGF-b responsive promoter/enhancer elements (18, 19). different constitutively active type I receptors to stimulate the However, TGF-b family members activate other signaling Ia1 driven reported construct. As shown in Fig. 2, expression of pathways as well (32, 33). In order to clarify whether the constitutively active ALK-1, ActR-I/ALK-2, BMPR-1A/ALK-3, TGF-b responsiveness of the Ia promoters is Smad-dependent or BMPR-1B/ALK-6, capable of activating Smad1 and Smad5 we co-transfected the Ia1 promoter-driven luciferase reporter did not affect the Ia1 promoter activity in DG75 cells. However, construct with expression constructs for different Smad pro- expression of constitutively active ActR-1B/ALK-4 or TbR-I/ teins in the Burkitt’s lymphoma B cell line DG75. This cell line ALK-5, involved in activin and TGF-b signaling, respectively, has been extensively used for analyzing Ia promoter activity (7, and the “orphan” ALK-7 (29), induced the Ia1 reporter in a 13). Smad3 increased significantly (;2.5-fold induction) the dose-dependent manner. ALK-4 and ALK-7 in particular could ligand-independent and ligand-dependent Ia1 promoter activ- reconstitute almost 80 –90% of the transcription activity ob- ity, whereas Smad2 and Smad4 increased it moderately (;1.4- tained by stimulating the cells with TGF-b1 (Fig. 2). Thus in fold induction). However, when Smad2, Smad3, and Smad4 addition to TGF-b, activins or even not as yet identified mem- were combined, a stronger transactivation was observed with bers of the TGF-b superfamily, can potentially activate tran- the highest response obtained when all three proteins were scription of the germ line IgA genes, by modulating the activity co-expressed (;10-fold induction for ligand-independent and of Smad3 and Smad4, and consequently participate in regula- ;5-fold induction for ligand-dependent transactivation). tion of IgA humoral responses. Smad1 and Smad5 transfected individually, together, or in Localization of the Smad Responsive Motifs in the Ia1 Pro- combination with Smad4, did not influence the Ia1 promoter moter—To identify the sequences mediating Smad responsive- (Fig. 1 and data not shown). BMP responsive Smad combina- ness in the Ia1 promoter, deletion mutants were constructed tions did not up-regulate the Ia1 promoter even when they and their responsiveness to TGF-b1 in the presence or absence were co-transfected with the constitutively active ALK-1, thus of expression plasmids for Smad proteins was studied in the B excluding the absence of the necessary receptors in the utilized cell line DG75. As shown in Fig. 3, deletion of sequences be- cell lines as the reason for the Ia1 unresponsiveness (data not tween 2142 and 299, encompassing the DRE element reduced shown). While the Smad variants that interfere with the TGF- dramatically the capacity of the promoter to respond to TGF-b1 b/activin signaling pathway, such as Smad7 and dominant in the presence or absence of exogenous Smad proteins. The negative variants of Smad3 (3Dc) or Smad4 (4Dc) (30) sup- small remaining Smad responsiveness is probably due to addi- pressed basal and TGF-b1 induced Ia1 activity, Smad6, if any- tional Smad-binding sites located downstream (see below, Fig. thing, induced a small increase in promoter activity (Fig. 1). In 4). conclusion, Smad3 and Smad4 and to some extend Smad2 are To define more specifically the Smad-interacting sequences major regulators of TGF-b responsiveness of the Ia1 promoter. of the Ia1 promoter, we produced recombinant Smad proteins Constitutively Activated TbR-I/ALK-5, ActR-IB/ALK4, and in E. coli and used the highly purified proteins in EMSA and Orphan ALK-7 Can Stimulate the Ia1 Promoter—Different DNase I footprinting analyses. EMSA demonstrated that members of the TGF-b/activin family can activate similar Smad1, Smad3, and Smad4 were able to bind to a double- Smads (18 –20). Therefore, we questioned the exclusive involve- stranded oligonucleotide spanning the DRE element (2131/ ment of TGF-b1 in the regulation of Ia1. We tested the effect of 296). Smad4 bound with the highest affinity since almost 10 TGF-b superfamily members to stimulate the Ia1 driven re- times more Smad1 and 4 times more Smad3 was required to porter construct. TGF-b1, TGF-b2, activin A, but not BMP2 produce the same level of retarded probe. Smad2 did not bind and BMP7, were able to induce a transcriptional response in even at 100-fold higher concentration (data not shown). Foot- K562 and DG75 cells (data not shown). Analysis of TGF-b printing analyses with recombinant Smad1, Smad3, and superfamily receptors on these cells showed the presence of Smad4 proteins demonstrated the presence of an array of pro- TGF-b and activin receptors but absence of BMP receptors tected segments within the Ia1 region (Fig. 4, A and B). Simi- AML/Smad Cooperativity 3555 FIG.3. Mapping of the Smad and TGF-b1 responsive portions of the Ia1 promoter. 59 Deletion mutants of the Ia1 promoter were subcloned in the luciferase reporter construct and their responsiveness FIG.4. Footprinting analysis of the Ia1 promoter region using in the presence or absence of TGF-b1 and/or a combination of Smads 3 recombinant Smad1, 2, 3, and 4. A, the indicated amounts of highly and4(3 mg of each) was analyzed in DG75 cells. The results shown purified recombinant GST-Smad fusion proteins were used in DNase I represent the mean value from three independent experiments 6 S.E. footprinting analyses of the Ia1 promoter region. A G/A sequencing The structure of the deletion mutants in relationship to the previously reaction was used to identify protected regions. The Smad-binding sites defined AML, CRE/ATF, and Ets sites, important for Ia1 promoter are indicated and named SMAD to SMAD . Hypersensitive sites are activity, is graphically illustrated. Relative fold activation is calculated A D indicated with asterisks. B, summary of footprinting profiles for Smad considering the activity of the Ia1 containing construct without any 1, 3, and 4 on both DNA strands. Hypersensitive sites are shown with ligand or Smad stimulation as 1. vertical solid arrows. The CAGA motifs are shown in bold letters and arrows indicate their orientation. larly to the EMSA analyses, Smad4 generated footprints at lower protein concentrations than Smad3, suggesting a higher Furthermore, phosphorylated Smad3 produced by co-express- affinity for the target DNA, and Smad1 bound less efficiently ing the activated TbR-I/ALK-5 kinase domain in insect cells than Smad3. Smad2 did not footprint even at the highest gave identical footprints as the non-phosphorylated and the concentration used, thus serving as a negative control. Inter- GST-Smad3 proteins, however, it required only one-fifth pro- estingly, the Smads differed from each other not only regarding tein concentration. Collectively, the footprinting analyses re- their affinity for DNA, but also in the way by which they veals differences in the way the various Smads interact with interacted with it. At optimal concentrations Smad1, Smad3, DNA, not only regarding the affinity of interaction, but also and Smad4 produced footprints around three areas (2127/ regarding the effect their binding has on the DNase I accessi- 2100, 295/265, and 251/220). However, the footprints were bility of the DNA, especially on neighboring sequences. similar, but not identical (Fig. 4, A and B, and footprints for Alignment of all the sequences protected by Smad proteins upper strand not shown). The footprints of Smad3 and Smad4 demonstrated the presence of the CAGAC(C/A) motif or its within the 2127/267 area were identical, but Smad4 foot- complementary (T/G)GTCTG in all of them (Fig. 4B). Recent printed a larger stretch in the 251/220 area. Smad1 protected structural and molecular studies have demonstrated that in- a limited stretch within the 2127/2100 segment and only the deed the AGAC sequence that is present within the above lower strand was weakly protected around the 251/220 area. motifs constitutes the core of Smad-binding sites (34 –38). Furthermore, while all three induced DNase I hypersensitivity Smad Proteins Co-operate with AML1 to Stimulate the Ia1 at position 288(G) in the upper strand (which lies within a Promoter—We have previously demonstrated that AML pro- putative CRE/ATF site), all three Smad molecules induced teins and their corresponding binding sites in the Ia1 promoter additional unique hypersensitive sites. Smad4, for example, are involved in its TGF-b1 responsiveness (14). In light of the was the only one that induced a hypersensitive site on the findings that Smad proteins integrate physically and function- upper strand next to the high affinity AML site, position ally with other transcription factors (20, 30, 39, 40) we inves- 2111(C), and another on the upper strand at position 229(G). tigated the interplay between Smad and AML proteins. For The recombinant Smads initially used in our studies were these experiments we utilized the K562 erythroleukemia cell synthesized as GST fusion proteins. Identical results were ob- line which expresses very low levels of AML proteins and tained using recombinant Smad3 and Smad4 proteins pro- displays very low TGF-b responsiveness as measured by the duced in baculovirus-infected insect cells (data not shown). Ia1 reporter construct (maximally 2-fold induction). As shown 3556 AML/Smad Cooperativity FIG.5. AML and Smad proteins synergistically activate the Ia1 promoter. K562 cells were co-transfected with either wild type Ia1-luciferase reporter construct (2142/179) or AML /SMAD mutants and combinations of Smad3/4 (3 mg of each) and AML1b (1 mg) followed D B by incubation in the presence or absence of TGF-b1. The results shown represent the mean value from three independent experiments 6 S.E. Relative fold induction is calculated considering the activity of the wild-type Ia1 construct without any ligand, AML1b, or Smad stimulation as 1. Inset, EMSA analysis using recombinant AML1b, and Smad3 proteins with wild-type or mutant DRE probes demonstrating that the mutations used in Fig. 5 affect binding of only the corresponding transcription factor (1, wild type probe; 2, AML mutant probe; 3, SMAD mutant probe; D B 4, AML /SMAD double mutant probe). D B in Fig. 5, overexpression of AML1b (the largest splice forms of were stimulated by TGF-b1 or co-transfected with the consti- the mouse AML1) or a combination of Smad3 and Smad4, tutively active type I receptor, this novel band increased sig- increased dramatically the TGF-b1 responsiveness of K562 nificantly. The novel complex was supershifted/inhibited with cells. When AML and Smad proteins were co-expressed, a antibodies against the AML1b protein (anti-HA), Smad3 (anti- synergistic effect on the Ia1 activity was observed. The syner- Myc), or Smad4 (anti-Flag), indicating that all these proteins gistic effect was clearer in the ligand independent responses can bind to the Ia1 DRE sequences in a receptor-dependent (open bars, Fig. 5). Better synergistic effect where obtained in manner. Thus, activation of the receptor induces the appear- AML1b/Smad3/Smad4-transfected TGF-b1-stimulated cells ance of novel complexes that contain Smad3, Smad4, and AML when the fetal calf serum in the culture medium was reduced proteins. from 5 to 1% instead (data not shown). Smad and AML Proteins Can Physically Interact in Vivo and The synergistic action of AML and Smad sites on the activity in Vitro—Recent studies have demonstrated that Smads regu- of the Ia1 promoter was illustrated more dramatically when late transcription through their ability to bind to DNA directly the highest affinity AML and the closely located high affinity and/or indirectly and to induce transcriptional responses Smad site were mutated (Fig. 5). EMSA analyses using wild- through cooperativity with other transcription factors (20, 30, type and mutated oligos demonstrated that mutations that 39 – 41). To investigate the molecular basis of AML/Smad syn- reduced binding of recombinant AML proteins to the AML ergy we expressed epitope-tagged AML1b and Smad3 in K562 site did not abrogate binding of recombinant Smad proteins in the and DG75 cells in the presence or absence of constitutively nearby SMAD active ALK-4 (Fig. 7). AML1b and Smad3 were co-immunopre- site and vice versa (Fig. 5, inset). However, mutation of either the AML or the SMAD sites abrogated the cipitated in both cells analyzed. The AML1b/Smad3 interaction D B was constitutive in both cell lines tested. Co-transfection of a positive effect that overexpression of AML1b and/or Smad3/ Smad4 had on the promoter activity, in the presence or absence PEBP2b expression vector did not improve the interaction, of TGF-b stimulation. Mutation of both these sites affected the probably due to the already existing adequate endogenous lev- Ia promoter even more. In addition to the above two sites, we els in DG75 and K562 (14). Fractionation of cytoplasmic from mutated the SMAD nuclear extracts showed the majority of complexes to be in the , SMAD , AML , and CRE/ATF sites in- A D U dividually or in combinations. In all cases the mutations de- cytoplasm (data not shown). creased the Ia1 promoter activity, indicating that all these sites To map the domains of Smad3 and AML1b involved in this even those with low affinity for the corresponding transcription protein-protein interaction, different epitope-tagged deriva- factors are necessary for optimal Ia1 function (data not shown). tives of either molecule (Fig. 8A) were expressed in DG75 cells Receptor-dependent Formation of Novel DNAzProtein Com- and their interaction was investigated by co-immunoprecipita- plexes Containing AML, Smad3, and Smad4 —To detect recep- tion. As shown in Fig. 8C, fragments containing the C-terminal tor dependent complex formation in EMSAs, we co-transfected MH2 domain of Smad3 exhibited the best AML1b binding DG75 cells with different combinations of epitope-tagged activity. AML1b, Smad3, Smad4 and constitutively active ActR-IB/ Similarly, different truncations of the AML1b molecule were ALK-4 receptor (Fig. 6). The constitutively active ALK-4 stim- tested for their capacity to interact with full-length Smad3. A ulates the same R-Smads as the constitutively active TbRI/ number of functional domains have been recently defined in ALK-5 and as shown in Fig. 2 it can trans-activate the Ia1 the AML molecule (42). Based on these mapping data a number promoter. Similarly transfected cells were also stimulated with of AML1b deletion variants were produced (Fig. 8A) and tested TGF-b1 (20 h after transfection for 30 min) instead of the for interaction with full-length Smad3 (Fig. 8B). A complex constitutively active receptor. Nuclear extracts were prepared pattern of interaction was observed since all the AML1b deriv- and analyzed in EMSA using a probe that contained the AML atives were able to interact with Smad3, albeit with different SMAD , AML , and SMAD sites (Fig. 6). Overexpression of efficiency. More specifically, N-terminal deletions up to amino A D B acid residue 411 did not affect binding. Deleting further amino tagged Smad3, Smad4, and AML1 resulted in the appearance of a weak, novel, lower mobility band. However, when the cells acids 371– 411 reduced significantly binding, without com- AML/Smad Cooperativity 3557 FIG.6. Detection of AML and Smad3/4 containing complexes induced by constitutively active ActR-1B/ALK-4 receptor. DG75 cells were co-transfected with the indicated combinations of expression vectors for AML1b, Smad3, Smad4, and constitutively active ALK-4 (caALK4). Nuclear extracts were prepared 20 h after transfection. TGF-b1 stimulation was done for 30 min before the termination of the culture and the preparation of the nuclear extracts. EMSA was performed with a probe containing the AML , Smad , AML , and Smad -binding sites (2131/296). U A D B Antibodies specific for the Myc, Flag, and HA epitopes recognizing the epitope-tagged Smad3, Smad4, and AML1b, respectively, were used to supershift the TGF-b1 or ALK4-induced complex. No nuclear extract was added in the first lane. Complex A represents binding of endogenous AML proteins, B represents the novel TGF-b1 or caALK-4 receptor induced complexes, C and D represent the anti-Smad3 and anti-Smad4 antibody produced supershifts. The anti-hemagglutinin antibody inhibited complex formation without producing a significant supershift. did not result in the appearance of unphysiological levels of AML1b protein and also to investigate whether TGF-b signal- ing could alter the levels of endogenous AML proteins, cells were harvested at different time points after transfection/ TGF-b1 stimulation and analyzed by Western blotting with an anti-AML antibody. This antibody was raised against a peptide that is conserved in all AML family members and thus can detect all of them. As shown in Fig. 10, exogenous AML1b was detected 1 h and reached maximal levels 7 h after transfection. The levels of exogenous AML1b protein even when they reached maximum were comparable with the levels of other endogenous AML variants. Since AML1b and Smad3 could be co-precipitated even when 10 times less expression vectors were transfected (data not shown), we conclude that the ob- served interaction is not due to unphysiologically high levels of expression. FIG.7. Interaction of AML1b with Smad3 in vivo. DG75 and K562 cells were transfected with the indicated combinations of epitope- It has recently been shown that TGF-b1 stimulates AML2 tagged AML1b and Smad3 with or without constitutively active ALK-4 mRNA synthesis in a mouse cell line (46). As shown in Fig. 10, expression vector (caALK4). The physical interaction between AML1b overexpression of AML1b, Smad3/4, and TGF-b1 stimulation, if and Smad3 was analyzed as described under “Experimental anything, resulted in reduction of some of the endogenous AML Procedures.” variants. This makes unlikely that the cooperation studied herein is due to an induction of AML gene expression by TGF-b pletely eliminating it. Deleting even further down to amino signaling. acid 291 did not alter the binding, however, a deletion reaching Collectively, the in vivo and in vitro binding data indicate down to amino acid 243 restored binding activity to the level that parts of the AML1b molecule spanning the RDH region even higher than the intact AML1b molecule. AML variants 6 and the area between amino acids 371 and 411 participate in and 9 containing either the N-terminal or C-terminal half of AML1b/Smad3 interaction, however, all these interactions tar- AML1b were able to interact with full-length or the MH2 get the MH2 domain of the Smad3 molecule. At least in the two variant of Smad3. No interaction was observed with the Mad cell lines analyzed, this interaction is constitutive and is not homology domain-1 portion of Smad3 (Fig. 8C). To make sure modulated by TGF-b1 signaling. that AML1b and Smad3 interact directly with each other and DISCUSSION not via an intermediate adapter molecule, we used S-labeled in vitro translated and purified AML1b and E. coli expressed The TGF-b Responsiveness of the Ia1 Promoter Is Regulated purified GST-Smad fusion protein immobilized on Sepharose by Direct Interaction of Smads with Specific Sequences within beads. As shown in Fig. 9, approximately 50 – 80% of the in It—TGF-b has been implicated as a major regulator of IgA vitro translated AML1b was retained on beads coupled with synthesis. One of the targets of TGF-b mediated signaling are recombinant Smad1, Smad2, Smad3, and Smad4, whereas only the Ia promoters, which are located upstream of the Sa regions 10% was bound to GST beads. Smad1 and Smad3 bound better and function as a regulator of the accessibility of the Ca germ than Smad2 and Smad4. Thus, purified Smads and AML1b can line loci to class switch recombinases. To clarify the molecular physically associate with each other in the absence of other mechanism(s) that skew immunoglobulin production toward cytoplasmic proteins. IgA, we have characterized the molecular components that To make sure that transfection with the expression vectors participate in the TGF-b1-regulated transcription through Ia1, 3558 AML/Smad Cooperativity FIG.9. Direct interaction between AML1b and Smad proteins in vitro. Equal amounts of S-labeled in vitro translated AML1b were incubated with either GST or GST-Smad1, 2, 3, and 4 coupled beads, and eluted AML1b was analyzed by polyacrylamide gel electrophoresis. Bars represent average of AML1b binding (presented as percentage of input protein eluted from the beads) in at least three independent experiments 6 S.E. the regulatory module of the human IgA1 locus. Our previous studies and the results presented in the current report demon- strate that the Ia1 promoter contains an array of interspersed binding sites for AML and Smad proteins whose cooperative activity underlines Ia1 germ line transcription. Mutation of AML and Smad sites, individually or in combinations, demon- strated that those of high affinity are indispensable for TGF-b responsiveness, whereas those of low affinity contribute to it, however, only when the high affinity sites are intact. The presence of multiple AML and Smad sites with different de- grees of importance in the Ia1 promoter, and possibly in other Smad-regulated promoters, might allow the promoter to func- tion as a “rheostat” responding gradually to signals of varying strength instead of responding in an all-or-none manner. This possibility is compatible with the finding that synthetic TGF-b responsive promoters respond better when they have several copies of Smad-binding sites. In an in vivo situation the varying parameter could be the strength of ligand induced signaling, or it could be the relative abundance of different Smads in the FIG.8. Mapping of AML1b and Smad3 domains involved in responding cells. their physical interaction. A, schematic representation of deletion Crystallographic studies have demonstrated that Smad3 mutants. Epitope-tagged deletion variants were produced as described under “Experimental Procedures” and were subcloned in the pCDNA3 binds DNA via an 11-amino acid b hairpin in the Mad homol- vector. B, full-length 6Myc-Smad3 expression vector was co-transfected ogy domain-1 domain that contacts the AGAC motif (38). with different variants of Flag-AML1b plasmids. Prior to immunopre- Smad2 cannot bind DNA despite its very high sequence simi- cipitation, an aliquot of the protein extracts was analyzed by Western larity to Smad3. This is probably due to a sequence insert in blot with anti-Myc antibodies to verify equal Smad3 expression in all transfections. After immunoprecipitation with anti-Flag antibodies the Smad2, immediately upstream of the DNA binding b-hairpin, precipitated material was analyzed by Western blot with anti-Myc which could potentially hinder Smad2-DNA interaction (38, antibodies to detect co-precipitated Smad3 protein and anti-Flag anti- 43). Interestingly, our footprinting experiments demonstrate body to verify equal precipitation of AML1b variants in all samples. C, that while Smad3 and Smad4, and less efficiently Smad1, bind full-length or deletion variants of Smad3 were co-expressed with full- length or deletion variants of AML1b. AML1b/Smad3 physical associa- to the same core sequence (CAGAC(C/A)), the way they impose tion was analyzed as in B. themselves on the DNA helix differs. They protect to different extent the two strands (Fig. 3, A and B) and more importantly, AML/Smad Cooperativity 3559 FIG. 10. Western blot analysis of AML family members. DG75 cells were transfected with AML1b-Smad3-Smad4 expression vectors or the control pBJ9V plasmid. Transfected cells were cultured in the presence or absence of TGF-b1 and cell lysates were prepared at the indicated time points. The anti-AML antibody was raised against a conserved amino acid stretch in the runt homology domain and thus detects all the AML proteins and their splice variants. Using commercially available monoclonal antibodies the lower molecular weight major species was recognized as AML1 and the two upper bands as AML2 (data not shown). A number of minor bands were recognized by antibodies against AML1, AML2, and AML3 and probably represent mixtures of different splice variants with similar electrophoretic mobility. the different Smads induce the appearance of different DNase alternative splicing of exons and possibly also alternative use of I-hypersensitive sites on the DNA template indicating that start codons for translation. Shi and Stavnezer (46) have re- they influence to different extents the access of neighboring cently presented evidence supporting the role of AML proteins DNA sequences to DNase I. If different Smads hinder to dif- for the regulation of the mouse Ia promoter. Since AML2 was ferent extent the accessibility of neighboring transcription fac- the only member of the family that was up-regulated by tor-binding sites, then the outcome of TGF-b signaling will TGF-b1 stimulation, they concluded that AML2 is the critical depend on which Smads will eventually bind to a given site. regulator of Ia transcription. Considering that AML1b is the The cooperative function of multiple Smad-binding sites and most efficient Ia transactivator among the AML isoforms (12, the differential influence that various Smads exert on neigh- 14), the quite rapid induction of germ line transcription (47– boring binding sites upon binding, could allow the Smad sig- 49), the fact that AML2 up-regulation occurs at very late stages naling system to transfer a whole spectrum of quantitatively of TGF-b signaling (46) and also the fact that in the DG75 and and qualitatively different instructions on a target promoter/ K562 cells we have analyzed in the present report TGF-b1 enhancer and be important in the context of the function of signaling does not induce AML2 up-regulation (Fig. 10), it is TGF-b family members as morphogens during development conceivable to either envision a dynamic process, during which (44). different isoforms of AML sequentially participate in the AML/ Recent findings including those of the present study have Smad interplay or to consider that the apparent differences are demonstrated that recombinant Smad proteins can bind di- cell line specific. Furthermore, it is possible to expect that rectly to certain DNA sequences that contain the AGAC core different AML variants will be more important in responses to motif. Truncation of the MH2 domain in Droshophila Mad different activating stimuli and in the context of different pro- protein increases the capacity of the Mad homology domain-1- moters. Furthermore, considering the quite significant diver- Linker portion to bind DNA (45). To make sure that the binding gence of AML proteins at their C termini, it is possible that activity in our preparations was not due to degradation prod- different domains of the AML and Smad molecules will differ- ucts mimicking the MH2 truncation we have carefully purified entially contribute in physical interaction of various AML/ full-length proteins. Removal of smaller size contaminants in- Smad combinations. creased the specific activity of the preparation instead of re- Is TGF-b1 the Exclusive Regulator of Ia Promoter Activity?— ducing it, suggesting that the full-length receptor unmodified Our finding that constitutively activated ALK-4, ALK-5, and Smads can bind DNA (data not shown). ALK-7 can stimulate the Ia1 promoter (Fig. 2) immediately Smads Cooperate with AML Proteins to Stimulate the Ia1 raises the possibility that Ia transcription can be achieved not Promoter—Our studies extend the list of transcription factors only by TGF-b1 but also by other members of the TGF-b su- that integrate functionally with the Smad proteins by adding perfamily that can interact with the above receptors. Indeed, the AML family of transcriptional regulators. Smad3 and TGF-b2 and activin A can also stimulate the Ia1 reporter AML1b physically interact in vitro and in vivo. Their interac- construct in DG75 cells (data not shown). TGF-b1 and TGF-b2 tion is constitutive and at least in the two cell lines used is not were more potent than activin A, however, this could be due to affected by receptor signaling, and appears to be of lower af- the high levels of TGF-b type III receptors and the low levels of finity than the interaction of TbR-I-phosphorylated Smad3 activin receptors these cells express (data not shown). We con- with Smad4. However, it is only after stimulation either by clude that other members of the TGF-b/activin family are po- TGF-b1 or the constitutively active type I receptors that higher tential regulators of IgA synthesis. order complexes containing AML1b, Smad3, and Smad4 are Consequences of the AML/Smad Interplay for Other Biolog- detected in nuclear extracts by EMSA (Fig. 6). It appears thus ical Systems—AML transcription factors bind to DNA sites in that despite the fact that these molecules form complexes con- the regulatory regions of a number of hematopoiesis specific stitutively, the effect of their interplay on Ia1 transcription can genes and play key roles in normal and pathological blood cell potentially be modulated at other levels, such as nuclear processes. Inactivation of either the AML1 gene or its associ- availability. ated factor PEBP2b results in death early in fetal development The AML family includes the AML1, AML2, and AML3 (day 12–14) and a complete block in fetal liver hematopoiesis genes. All of them give rise to a collection of polypeptides by (15, 50). Furthermore, the AML1/PEBP2b genes are the most 3560 AML/Smad Cooperativity 6859 – 6863 frequent targets of translocation in acute human leukemia (51). 17. Ogawa, E., Inuzuka. M., Maruyama, M., Satake, M., Naito Fujimoto, M., Ito, Smad proteins, as nuclear TGF-b effectors, regulate a large Y., and Shigesada, K. (1993) Virology 194, 314 –331 18. Heldin, C-H., Miyazono, K., and ten Dijke, P. (1997) Nature 390, 465– 471 range of biological processes. Furthermore, Smads play impor- 19. Massague ´ , J. (1998) Annu. Rev. Biochem. 67, 753–791 tant roles during processes leading to malignant transforma- 20. Derynck, R., Zhang, Y., and Feng, X.-H. (1998) Cell 95, 737–740 tion. Smad4 was originally identified as a putative tumor sup- 21. Hata, A., Shi, Y., and Massague ´ , J. (1998) Mol. Med. Today 4, 257–262 22. Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and pressor gene, frequently mutated in pancreatic carcinomas and Wrana, J. L. (1997) J. Biol. Chem. 272, 27678 –27685 occasionally in other types of tumors (18, 21). 23. Souchelnytskyi, S., Tamaki, K., Engstro ¨ m, U., Wernstedt, C., ten Dijke, P., and In vivo and in vitro studies have suggested that among the Heldin, C.-H. (1997) J. Biol. Chem. 272, 28107–28115 24. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) extracellular signals that can induce the osteoblastic pheno- Gene (Amst.) 77, 61– 68 type are the BMPs (52). Recent studies have demonstrated that 25. Nakao, A., Afrakhte, M., More ´ n, A., Nakayama, T., Christian, J. L., Heuchel, R., Itoh, S., Kawabata, M., Heldin, C.-H., and ten Dijke, P. (1997) Nature a splice form of CBFA1/AML3/PEBP2aA also plays a critical 389, 631– 635 role in osteoblast differentiation (53–55). One possible mecha- 26. Souchelnytskyi, S., Nakayama, T., Nakao, A., More ´ n, A., Heldin, C.-H., Chris- nism by which BMPs could mediate regulation of bone devel- tian, J. L., and ten Dijke, P. (1998) J. Biol. Chem. 273, 25364 –25370 27. Tamaki, K., Souchelnytskyi, S., Itoh, S., Nakao, A., Sampath, K., Heldin, opment is via the induction of CBFA1 synthesis. AML1b inter- C.-H., and ten Dijke, P. (1998) J. Cell. Physiol. 177, 355– 63 acts physically not only with the TGF-b/activin Smads but also 28. Nakao, A., Imamura, T., Souchelnytskyi, S., Kawabata, M., Ishisaki, A., Oeda, E., Tamaki, K., Hanai, J., Heldin, C.-H., Miyazono, K., and ten Dijke, P. with the BMP-activated Smad1 (Fig. 9). It is possible that the (1997) EMBO J. 16, 5353–5362 AML/Smad interplay involves many or all the members of the 29. Ryde ´ n, M., Imamura, T., Jornvall, H., Belluardo, N., Neveu, I., Trupp, M., two families, and that depending on the cell type and stimuli, Okadome, T., ten Dijke, P., and Ibanez, C. F. (1996) J. Biol. Chem. 271, 30603–30609 different combinations of AML and Smad proteins could be 30. Zhang, Y., Feng, X.-H., and Derynck, R. (1998) Nature 394, 909 –913 recruited to regulate transcription of selected genes. 31. Corneliussen, B., Holm, M., Waltersson, Y., Onions, J., Hallberg, B., Thornell, A., and Grundstro ¨ m, T. (1994) Nature 368, 760 –764 Considering the wide range of biological phenomena that 32. Hocevar, B. A., Brown, T. L., and Howe, P. H. (1999) EMBO J. 18, 1345–1356 AML and Smad proteins regulate and the potential oncogenic 33. Visser, J. A., and Themmen, A. P. (1998) Mol. Cell. Endocrinol. 146, 7–17 character of AML1 and tumor suppressor activity of some 34. Yingling, J. M., Datto, M. B., Wong, C., Frederick, J. P., Liberati, N. T., and Wang, X.-F. (1997) Mol. Cell. Biol. 17, 7019 –7028 Smads, the observed functional interplay between these tran- 35. Dennler, S. Itoh, S., Vivien, D., ten Dijke, P., Huet, S., and Gauthier, J. M. scription factors has implications not only for class switching to (1998) EMBO J. 17, 3091–3100 36. Jonk, L. J. C., Itoh, S., Heldin, C-H., ten Dijke, P., and Kruijer, W. (1998) IgA but also for other biological processes such as regulation of J. Biol. Chem. 273, 21145–21152 bone ossification as well as normal and pathological hemato- 37. Zawel, L., Dai, J. L., Buckhaults, P., Zhou, S., Kinzler, K. W., Vogelstein, B., poietic cell behavior. and Kern, S. E. (1998) Mol. Cell. 1, 611– 617 38. Shi, Y., Wang, Y. F., Jayaraman, L., Yang, H., Massague ´ , J., and Pavletich, N. P. (1998) Cell 94, 585–594 Acknowledgments—We thank Drs. M. Kawabata, R. Derynck, and J. 39. Chen, X., Weisberg, E., Fridmacher, V., Watanabe, M., Naco, G., and Whit- Wrana for epitope-tagged Smad expression vectors, C. F. Ibanez for man, M. (1997) Nature 389, 85– 89 ALK-7 cDNA, A. Coner and M. Hoffmann for recombinant Smad3 and 40. Hua, X., Liu, X., Ansari, D. O., and Lodish, H. F. (1998) Genes Dev. 12, phosphorylated Smad3 proteins, Y. Eto for activin A, T. K. Sampath for 3084 –3095 BMP-2 and OP-1, and B. Pratt for TGF-b2. 41. Labbe ´ , E., Silvestri, C., Hoodless, P. A., Wrana, J. L., and Attisano, L. (1998) Mol. Cell. 2, 109 –120 REFERENCES 42. Kanno, T., Kanno, Y., Chen, L. F., Ogawa, E., Kim, W. Y., and Ito, Y. (1998) Mol. Cell. Biol. 18, 2444 –2454 1. Honjo, T., and Kataoka, T. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2140 –2144 43. Yagi, K., Goto, D., Hamamoto, T., Takenoshita, S., Kato, M., and Miyazono, K. 2. Coffman, R., Lebman, D. A., and Shrader, B. (1989) J. Exp. Med. 170, (1999) J. Biol. Chem. 274, 703–709 1039 –1044 44. Wilson, P. A., Lagna, G., Suzuki, A., and Hemmati-Brivanlou, A. (1997) De- 3. Defrance, T., Vanbervliet, B., Briere, F., Durand, I., Rousset, F., and Banche- velopment 124, 3177–3184 reau, J. (1992) J. Exp. Med. 175, 671– 682 45. Kim, J., Johnson, K., Chen, H., Carroll, S., and Laughon, A. (1997) Nature 388, 4. Van Vlasselaer, P., Punnonen, J., and de Vries, J. E. (1992) J. Immunol. 148, 304 –308 2062–2067 46. Shi, M. J., and Stavnezer, J. (1998) J. Immunol. 161, 6751– 6760 5. Flanagan, J. G., and Rabbitts, T. H. (1982) Nature 300, 709 –714 47. Berton, M. T., Uhr, J. W., and Vitetta, E. S. (1989) Proc. Natl. Acad. Sci. 6. Nilsson, L., Islam, K. B., Olafsson, O., Zalcberg, I. Samakovlis, C., Ham- U. S. A. 86, 1819 –1833 marstrom, L., Smith, C. I. E., and Sideras, P. (1991) Int. Immunol. 3, 48. Lutzker, S., Rothman, P., Pollock, R., Coffman, R., and Alt, W. A. (1988) Cell 1107–1115 53, 177–184 7. Nilsson, L., and Sideras, P. (1993) Int. Immunol. 5, 271–282 8. Yancopoulos, G. D., DePinho, R. A., Zimmerman, K. A., Lutzker, S. G., Rosen- 49. Kitani, A., and Strober, W. (1993) J. Immunol. 151, 3478 –3488 50. Dzierzak, E., Medvinsky, A., and de Bruijn, M. (1998) Immunol. Today 19, berg, N., and Alt, F. W. (1986) EMBO J. 5, 3259 –3266 9. Stavnezer Nordgren, J., and Sirlin, S. (1986) EMBO J. 5, 95–102 228 –236 51. Look, A. T. (1997) Science 278, 1059 –1064 10. Snapper, C. M., Marcu, K. B., and Zelazowski, P. (1997) Immunity 6, 217–223 11. Stavnezer, J. (1996) Adv. Immunol. 61, 79 –146 52. Rodan, G. A., and Harada, S. (1997) Cell 89, 677– 680 53. Mundlos, S., Otto, F., Mundlos, C., Mulliken, J. B., Aylsworth, A. S., Albright, 12. Lin, Y. C., and Stavnezer, J. (1992) J. Immunol. 149, 2914 –2925 13. Nilsson, L., Grant, P., Larsson, I., Pettersson, S., and Sideras, P. (1995) Int. S., Lindhout, D., Cole, W. G., Henn, W., Knoll, J. H. M., Owen, M. J., Mertelsmann, R., Zabel, B. U., and Olsen, B. R. (1997) Cell 89, 773–779 Immunol. 7, 1191–1204 14. Xie, X-Q., Pardali, E., Holm, M., Sideras, P., and Grundstro ¨ m, T. (1999) Eur. 54. Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., Bronson, R. T., Gao, Y. H., Inada, M., Sato, M., Okamoto, R., J. Immunol. 29, 488 – 498 15. Wang, S., Wang, Q., Crute, B. E., Melnikova, I. N., Keller, S. R., and Speck, Kitamura, Y., Yoshiki, S., and Kishimoto, T. (1997) Cell 89, 755–764 55. Otto, F., Thornell, A., P., Crompton,, T., Denzel, A., Gilmour, K. C., Rosewell, N. A. (1993) Mol. Cell. Biol. 13, 3324 –3339 16. Ogawa, E., Maruyama, M., Kagoshima, H., Inuzuka, M., Lu, J., Satake, M., I. R., Stamp, G. W., Beddington, R. S., Mundlos, S., Olsen, B. R., Selby, P. B., and Owen, M. J. (1997) Cell 89, 765–771 Shigesada, K., and Ito, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,

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