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Cell Growth & Differentiation Vol. 12, 457-463, September 2001
© 2001 American Association for Cancer Research

The Smad Transcriptional Corepressor TGIF Recruits mSin31

David Wotton2, Paul S. Knoepfler, Carol D. Laherty, Robert N. Eisenman and Joan Massagué

Department of Biochemistry and Molecular Genetics and Center for Cell Signaling, 800577 HSC, University of Virginia, Charlottesville, Virginia 22908 [D. W.]; Division of Basis Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 [P. S. K., C. D. L., R. N. E.]; Cell Biology Program, Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [J. M.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The homeodomain protein TG-interacting factor (TGIF) represses transcription by histone deacetylase-dependent and -independent means. Heterozygous mutations in human TGIF result in holoprosencephaly, a severe genetic disorder affecting craniofacial development, suggesting that TGIF is critical for normal development. After transforming growth factorß (TGFß) stimulation, Smad proteins enter the nucleus and form transcriptional activation complexes or interact with TGIF, which functions as a corepressor. The relative levels of Smad corepressors and coactivators present within the cell may determine the outcome of a TGFß response. We show that TGIF interacts directly with the paired amphipathic {alpha}-helix 2 domain of the mSin3 corepressor, and TGIF recruits mSin3 to a TGFß-activated Smad complex. The mSin3 interaction domain of TGIF has been shown to be essential for repression of a TGFß transcriptional response. Thus, TGIF represents a targeting component of the mSin3 corepressor complex.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
TGIF3 (1 , 2) is a member of the TALE superfamily of HD proteins (3) . Members of this family are characterized by the presence of an extra loop between helices one and two of the HD. This insertion does not affect DNA binding by TALE proteins but can play an important role in protein-protein interactions (4, 5, 6, 7, 8, 9) . Outside their HDs, members of this family are highly divergent at the primary sequence level, have been shown to activate or repress transcription, and play roles in diverse biological processes (8 , 10, 11, 12) . TGIF was first identified by its ability to bind to the retinoid-responsive element of the cellular retinol binding protein gene (1) . Although the precise physiological role that TGIF plays in retinoid-responsive transcription is unclear, it has been suggested to repress transcription by competing with retinoid receptors for binding to DNA. Recent evidence suggests that TGIF competes for DNA binding with the TALE HD protein, Meis2, and represses transcription of the dopamine 1A receptor gene (13) .

TGIF contains at least two separable transcriptional RDs, which act by recruiting transcriptional corepressors (14) . We have recently shown that a conserved motif at the NH2 terminus of TGIF recruits the corepressor CtBP (15) . This CtBP-interaction motif lies within a region of TGIF capable of repressing transcription autonomously. The COOH-terminal half of TGIF is also able to repress transcription, and this repression appears to be dependent on its ability to recruit HDACs. HDACs are able to interact with TGIF via the central region of the protein. However, it appears that the extreme COOH-terminal region of TGIF may also contribute to repression by HDACs (14) . Thus, TGIF may act to recruit multiple transcriptional corepressors to specific target genes.

In response to TGFß signaling, Smad2 and Smad3 are phosphorylated directly by the TGFß receptor, then complexed with Smad4, and translocated to the nucleus (16 , 17) . Once in the nucleus, activated Smad complexes bind to specific genes via interactions with other transcription factors and by direct Smad-DNA contacts (18, 19, 20, 21) . A DNA-bound Smad complex is able to participate in the activation of transcription, in part because of interactions with general coactivators. TGFß-activated Smads can interact with the coactivators, p300 and CREB-binding protein, both of which have histone acetyl transferase activity, suggesting that transcriptional activation by Smads is in part attributable to effects on the chromatin template (22, 23, 24, 25) . In addition to interacting with coactivators, a nuclear Smad complex can interact with specific transcriptional repressors (26, 27, 28) . Interaction of TGIF with TGFß-activated Smads prevents interaction with p300, thus reducing the ability of the Smad complex to activate transcription. In addition, TGIF brings with it transcriptional repressor functions, resulting in a switch from transcriptional activation to repression (26) . c-Ski, the cellular counterpart of the v-Ski oncoprotein, and the related protein SnoN have also been shown to act as Smad corepressors. Thus, c-Ski and SnoN compete with coactivators and are able to recruit a complex of general repressor proteins to the Smad complex (27, 28, 29) .

Recent evidence has demonstrated that mutations in the human TGIF gene result in HPE (30) , a severe genetic disorder affecting craniofacial development (31 , 32) . The primary defect in HPE is a failure of the brain to divide laterally, and this is often accompanied by facial anomalies, including cyclopia and proboscosis. Mutations in TGIF found in HPE are generally deletions of a single allele, although some missense mutations which result in partial loss of function have also been identified (30) . It is not yet clear whether the TGIF mutations which cause HPE are attributable to defects in the TGFß pathway or in other TGFß-independent pathways, possibly including retinoid-responsive transcription. However, transcriptional regulation by TGIF clearly plays an important role during development, and it appears that relatively small changes in the level of TGIF can have dramatic effects on brain development (30) .

The Sin3A and B proteins are corepressors which have been shown to be required for repression by a growing number of transcriptional regulators (33 , 34) . Both the Saccharomyces cerevisiae Sin3p and the mammalian mSin3 proteins mediate transcriptional repression by HDACs (35, 36, 37, 38, 39) . In addition to a region which interacts with HDACs, Sin3 proteins contain four paired amphipathic helices, termed PAH1–4 (40) . These PAH domains are protein-interaction motifs which target mSin3 to specific transcriptional regulators (41, 42, 43, 44, 45) or accessory proteins, such as SAP30 (46) . Transcriptional repression by Mad1 and other functionally related basic helix-loop-helix zipper proteins requires interaction with mSin3 and is dependent on the recruitment of HDACs to DNA elements bound by Mad dimerized with Max (41, 42, 43) . Thus, the interaction of mSin3 with these basic helix-loop-helix zipper proteins provides a paradigm for the recruitment of a general corepressor complex to a specific DNA element, via interactions with sequence-specific DNA binding proteins. The corepressors N-CoR and SMRT interact with mSin3 via PAH1 and PAH3, and this interaction is required for HDAC-dependent repression of transcription by nuclear hormone receptors in the absence of ligand (39 , 47 , 48) . In this case, the mSin3/HDAC complex is brought to a DNA binding protein indirectly, via interactions with another corepressor. In addition, to the recruitment of HDACs via mSin3, proteins such as YY1, LEF1, and Rb family members interact directly with HDACs (49, 50, 51, 52, 53) .

Here, we demonstrate that TGIF interacts with mSin3 via its extreme COOH-terminal RD. Thus, it appears that TGIF is a targeting component of a complex of general corepressors, including an HDAC and mSin3. In addition, we show that in response to TGFß, TGIF can recruit mSin3 to an activated Smad complex.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
TGIF Interacts with mSin3.
The PAH2 domain of mSin3 interacts with a number of transcriptional repressors, including the Mad family, Mnt, REST, and MNFß (42, 43, 44 , 54 , 55) . These interactions result in recruitment of the mSin3 complex to genes regulated by these proteins. To identify other transcriptional regulators which interact with the PAH2 domain of mSin3, we screened a yeast two hybrid murine T-cell library with a LexA DNA binding domain fusion to this region of mSin3. Of 30 positive clones, 2 contained in-frame fusions of the VP16 activation domain to a region of TGIF encoding amino acids 56–272 (Fig. 1A)Citation , suggesting that TGIF is a PAH2-interacting protein.



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Fig. 1. Identification of TGIF as a mSin3-interacting protein. A, mSin3 and TGIF are shown schematically, together with the LexA-PAH2 fusion used as a bait in the two hybrid screen and the VP16 activation domain fusions to TGIF isolated. B, TGIF interacts directly with the PAH2 domain of mSin3. His 6-tagged bacterially expressed TGIF was incubated with glutathione agarose alone (Lane 4; beads) or in the presence of purified GST or GST-PAH2 as indicated (Lanes 5 and 6). After extensive washing, bound proteins were separated by SDS-PAGE and visualized by Coomassie blue staining. A portion of each of the input proteins is shown (Lanes 1–3), and the GST-PAH2-bound TGIF is indicated.

 
To determine whether TGIF interacts directly with mSin3, both proteins were expressed and purified from bacteria. Purified histidine-tagged bacterial TGIF was incubated with glutathione agarose or with glutathione agarose, which had been prebound with either GST alone or GST-PAH2. As shown in Fig. 1BCitation , TGIF bound specifically to GST-PAH2 but not to GST alone or to glutathione agarose in the absence of any GST protein. Thus, it appears that TGIF interacts directly with the PAH2 domain of mSin3 in vitro.

To investigate whether TGIF and mSin3 interact in vivo at endogenous levels of expression, we precipitated proteins from A549 cell lysates using antisera specific for TGIF or mSin3. Immunocomplexes were analyzed by Western blotting for the presence of both TGIF and mSin3, as well as HDAC1. As shown in Fig. 2Citation , TGIF and HDAC1 were visible in complexes precipitated using a mSin3-specific antiserum, whereas no TGIF or HDAC reactive bands were present in complexes precipitated using the preimmune serum. Similarly, both mSin3 and HDAC1 coprecipitated with TGIF, suggesting that endogenous TGIF, mSin3, and HDAC1 interact in vivo.



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Fig. 2. Interaction of endogenous TGIF and mSin3. Protein complexes were precipitated from human lung A549 cells using antisera specific for TGIF or mSin3 or with the appropriate preimmune sera. Proteins were visualized by Western blotting for TGIF, mSin3, and HDAC1. The positions of molecular weight markers (kDa) are shown.

 
mSin3 Interacts with the COOH-terminal RD of TGIF.
We have identified previously at least two independent RDs within TGIF (Ref. 14 ; Fig. 3CCitation ) and demonstrated that TGIF interacts with the corepressor CtBP via the NH2-terminal RD-1 (15) . TGIF interacts with HDAC1, 2, and 3, and repression by the COOH-terminal half of TGIF is dependent on HDAC activity (14) . To determine whether any of these previously identified RDs within TGIF was responsible for the interaction with mSin3, we analyzed interactions between mSin3 and a series of TGIF deletion mutants. Myc-Sin3 was clearly detectable in Flag immunoprecipitates from cells coexpressing Flag-TGIF(1–262). However, removal of the COOH-terminal 80 amino acids, including RD-2b, resulted in a dramatic decrease in this interaction (Fig. 3A)Citation . In contrast, removal of the HD or the NH2-terminal RD (constructs 1–42:104–262, 108–262, and 138–272) did not affect interaction with mSin3. Deletion of RD-2a (construct 1–147:177–262) led to a slight decrease in the interaction of TGIF with mSin3 (Fig. 3A)Citation .



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Fig. 3. mSin3 interacts with RD-2b of TGIF. A, COS-1 cells were transfected with the indicated Flag-tagged TGIF expression constructs, together with myc-Sin3. Proteins were precipitated on Flag-agarose and analyzed for the presence of myc-Sin3 (arrow). Expression levels of transfected proteins were assessed by Western blotting a portion of the lysate (below). B, COS-1 cells were transfected and analyzed as in A, except that TGIF was tagged with two HA epitopes, and proteins were precipitated using an HA-specific antibody. C, the TGIF deletion constructs used in A and B are shown schematically. Diagonal stripes, RD-2a, and RD-2b. Vertical stripes within RD-1, the CtBP-interaction motif. The positions of the Flag (FL) and HA epitopes are shown. The interaction of each construct with mSin3 (from A and B) is shown. +++, strong interaction; ++, slight decrease in the interaction; +, barely detectable interaction; -, no detectable interaction.

 
We next tested interactions of mSin3 with HA-tagged TGIF mutants with deletions in both RD-2a and RD-2b. As shown in Fig. 3BCitation , deletion of RD-2b dramatically reduced interaction with mSin3 (constructs 1–230 and 1–192). However, when both RD-2a and RD-2b were deleted, no detectable interaction was observed (construct 1–147:177–230). Together, these results suggest that the major site for interaction of mSin3 with TGIF is within RD-2b, between amino acids 230 and 262 of TGIF (Fig. 3C)Citation . However, we cannot rule out a contribution of RD-2a, perhaps via interactions bridged by HDACs.

TGIF Recruits mSin3 to TGFß-activated Smads.
In response to TGFß, signals Smad2 and Smad3 move into the nucleus, where, in complex with Smad4, they are able to interact with general coactivators, such as p300/CREB-binding protein, or with specific corepressors, including TGIF (21 , 26) . To determine whether TGIF could recruit mSin3 to a TGFß-activated Smad complex, COS-1 cells were cotransfected with expression vectors encoding Smad2, HA-tagged TGIF, and Myc epitope-tagged mSin3. Complexes were precipitated using a Myc-specific antibody from control cells or from cells treated with TGFß for 1 h before cell lysis. As shown in Fig. 4Citation , TGIF coprecipitated with mSin3 in the presence or absence of TGFß, and Smad2 was also clearly detectable in immunocomplexes from cells treated with TGFß. Importantly, the inclusion of Smad2 into the TGIF-mSin3 complex was dependent not only on the addition of TGFß but also on coexpression of TGIF. Thus, it appears that the formation of Smad-TGIF complexes in response to TGFß results in the recruitment of mSin3 into these complexes by TGIF.



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Fig. 4. TGIF recruits mSin3 to Smad2. COS-1 cells were transfected with expression vectors encoding Smad2, myc-Sin3, and HA-TGIF or a control vector. TGFß was added for 1 h before cell lysis, as indicated. Proteins were precipitated via the myc-epitope present on mSin3 and analyzed by Western blotting using a Smad2-specific antiserum. mSin3-associated Smad2 is indicated by an arrow. A portion of the lysate was analyzed for expression of transfected proteins (below).

 
To determine whether overexpression of mSin3 could enhance repression of a TGFß transcriptional response by TGIF, we tested repression of the A3-lux reporter, which contains three copies of the activin response element from the Xenopus mix.2 gene (56) . The activin response element is bound by winged helix transcription factors, such as FAST2 (57 , 58) . When FAST2 is expressed in mammalian cells, luciferase activity from the A3-lux reporter is strongly induced by TGFß. Mink lung L17 cells were cotransfected with A3-lux, a FAST2 expression vector, and various combinations of TGIF and mSin3 expression constructs. As shown in Fig. 5Citation , the TGFß-induced activity of the A3-lux reporter was not significantly reduced by expression of mSin3 alone. TGIF repressed activity to <40% of the control, and in the presence of both TGIF and mSin3 together, this repression was slightly enhanced. The relatively small increase in TGIF-mediated repression on coexpression of mSin3 suggests that for this response, mSin3 levels may not be limiting.



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Fig. 5. Effects of TGIF and mSin3 expression on TGFß-activated transcription. L17 cells were cotransfected with the A3-lux reporter and a FAST2 expression construct, together with TGIF (+, 25 ng; ++, 50 ng/well) or mSin3 (+, 100 ng/well) expression vectors as indicated. TGFß was added after 24 h, and luciferase activity was assayed after an additional 18 h. Activity is shown as the mean ±SD of triplicate transfections.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
TGIF is a transcriptional repressor which interacts with TGFß-activated Smads, thereby regulating the transcriptional output of the TGFß signaling pathway (26) . In addition, TGIF can repress transcription when bound directly to DNA, independent of TGFß signaling (14) . Certain retinoid-responsive elements have been suggested to be targets for repression by TGIF, and TGIF can bind to an element in the promoter of the dopamine 1A receptor gene, competing for binding with Meis2 (1 , 13) . Thus, TGIF appears to act in multiple transcriptional regulatory pathways, either as a DNA binding repressor or as a corepressor in association with other DNA binding proteins (Fig. 6)Citation .



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Fig. 6. TGIF-corepressor interactions. The possible protein interactions with TGIF are shown. TGIF can be recruited to DNA either via interactions with an activated Smad complex or by direct DNA binding to its cognate site (CTGTCAA) as indicated by the arrows.

 
We have demonstrated previously that TGIF interacts with HDACs and that repression by the COOH-terminal half of TGIF is impaired by inhibitors of HDAC activity (14) . In addition, TGIF can recruit HDACs to a TGFß-activated Smad complex, and HDAC activity is required for full repression of a TGFß response by TGIF (26) . Within the COOH-terminal half of TGIF, it appears that there are two sub-domains, which can repress transcription in isolation.

Here we demonstrate that TGIF interacts with mSin3 primarily via the COOH-terminal RD-2b. We have shown recently that the TGIF-related protein, TGIF2, is a transcriptional repressor, which interacts with HDAC1. In addition, the RD-2b region of TGIF is conserved within TGIF2 and is a major determinant of repression by TGIF2. Thus, it is likely that TGIF2 will also interact with mSin3.

HDACs can be recruited directly by specific transcriptional repressors, such as YY1, Blimp-1, and LEF1 (49, 50, 51, 52, 53 , 59) . In these cases, there is no evidence for the presence of other corepressor proteins. HDACs can also bind corepressors, such as Sin3 and Mi2, which are required to mediate the interaction between certain DNA binding repressors and HDACs (39 , 41, 42, 43, 44 , 47 , 48) . Our results demonstrate that TGIF interacts directly with mSin3 and may also bind HDAC1 via its COOH-terminal deacetylase-dependent RD (Fig. 6)Citation . Similarly, the repressor proteins, Bcl-6 and PLZF, have been shown to interact with both HDAC and with SMRT/N-CoR (60 , 61) .

Because deletion of the RD-2b region of TGIF results in a loss of repression of a TGFß transcriptional response (26) , we believe that interaction of TGIF with mSin3 is functionally relevant. Thus, recruitment of mSin3-containing complexes to an activated Smad complex by TGIF may be an important way in which TGIF represses TGFß-activated transcription. However, we cannot rule out that separate TGIF corepressor complexes may exist, containing TGIF and HDAC; TGIF, HDAC, and mSin3; or other as yet unidentified components of the repression machinery. These complexes may play different roles in regulating TGFß-activated transcription. Because it appears that the Smad and HDAC interacting regions of TGIF may overlap, mSin3 may play a specific role in the recruitment of HDACs to a Smad complex by TGIF. In this context, it is of interest that RD-2b is critical for full repression of a TGFß response but appears to be dispensable for repression by TGIF when bound directly to DNA (14 , 26) .

In addition to TGIF, the SnoN and c-Ski oncoproteins have been demonstrated to be components of the Smad repression complex (27, 28, 29 , 62) . c-Ski is part of the HDAC/mSin3/N-CoR complex and has been shown to interact with both mSin3 and N-CoR (63) . Interaction of c-Ski with Smad complexes results in the recruitment at least of N-CoR to the Smad complex (28) . Because, as we have shown, TGIF also interacts with components of this corepressor complex, it is in principle possible that TGIF and Ski/Sno reside within the same corepressor complex, both acting to target this complex to TGFß-responsive Smads. However, because Smad2 and 3 associate with c-Ski/Sno proteins in the absence of TGFß (29 , 62) , whereas the mSin3 interaction via TGIF is stimulated by TGFß (Fig. 4)Citation , we postulate that the Sno/Ski and TGIF complexes form at different stages of the TGFß transcriptional response. Indeed, TGFß stimulation leads to rapid degradation of Ski/SnoN. One possibility is that the Ski/Sno complexes act to repress basal level transcription by Smads, whereas the TGIF-HDAC-mSin3 complexes act to limit the extent of the Smad response after TGFß treatment (26) . It is also possible that TGIF plays a specific role in the down-regulation of expression of certain genes in response to TGFß signaling (64) . In addition to HDAC and mSin3 associations, TGIF also interacts with CtBP via its NH2-terminal RD (15) , perhaps indicating the existence of an additional TGIF-containing corepressor complex.

In summary, TGIF appears to be able to interact with general corepressor proteins and can target these activities to DNA, either via interaction with other DNA binding proteins or by binding directly to DNA itself (Fig. 6)Citation .


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Yeast Two Hybrid Screening.
PAH2 was used as bait to screen a T-cell lymphoma library as described previously (41) . We obtained 30 positive clones, of which approximately one-third were Mad family members and two were derived from TGIF.

Bacterial Fusion Proteins.
TGIF (amino acids 35–272) was expressed as a six-histidine fusion from within pQE30 (Qiagen) and purified on cobalt agarose. GST-PAH2 and GST were purified on glutathione agarose, eluted with 10 mM glutathione, and dialyzed into PBS to remove free glutathione. Interaction assays were carried out by binding GST or GST-PAH2 to glutathione agarose, which had been preblocked with 5% BSA. After extensive washing, His6-TGIF was added, and binding allowed to proceed at 4°C for 60 min in PBS with 1% N-P40. Bound proteins were collected by centrifugation, washed four times with PBS/1% N-P40, separated by SDS-PAGE, and visualized by Coomassie blue staining.

Cell Culture and Transfection.
COS-1 cells were grown in DME with high glucose supplemented with 10% fetal bovine serum and transfected using LipofectAMINE (Life Technologies, Inc.), according to the manufacturer’s instructions. A549 cells were cultured in DME with high glucose and 10% fetal bovine serum. L17 cells were transfected in six-well plates using DEAE-dextran as described previously. TGFß (R & D Systems) was added to a final concentration of 100 pM as indicated.

Reporter Assays.
Cells were transfected with firefly and renilla (pCMV-RL; Promega) luciferase reporters, and luciferase activity was assayed after 40–48 h. Firefly luciferase was assayed with a luciferase assay kit (Promega), and Renilla was assayed with 0.09 µM coelenterazine (Biosynth) in 25 mM Tris (pH 7.5) and 100 mM NaCl. Luciferase activities were measured using a Berthold LB 953 luminometer.

Plasmids.
TGIF expression constructs were created by PCR or using internal restriction enzyme sites within modified pCMV5 vectors, which contained either a single FLAG epitope tag or two HA epitopes. Full-length, untagged Smad2 was expressed from within pCMV5. Myc-epitope tagged mSin3 was generated by cloning mSin3A into pCS2 with a Myc epitope tag. The A3-lux reporter and FAST2 expression vector are as described previously (58) .

Immunoprecipitation and Western Blotting.
COS-1 cells were harvested 36 h after transfection and lysed by sonication in PBS with 1% N-P40 and protease inhibitors (Complete Mini protease inhibitor cocktail; Roche). After removal of cell debris, lysates were precleared with protein A-Sepharose (Pierce), and protein complexes were precipitated with either anti-Flag-agarose (Sigma Chemical Co.) or an HA-specific monoclonal antibody (12CA5; Roche) and a mixture of protein A-Sepharose and protein G-Sepharose (Pierce). Immunocomplexes were washed four times in PBS and 1% NP40 and analyzed by SDS-PAGE. Proteins were transferred to Immobilon-P, and membranes were incubated with the appropriate antibodies (Flag M2, Sigma Chemical Co.; anti-HA [12CA5], Roche; anti-myc [9E10], Sigma Chemical Co.; anti-Smad2, Upstate Biotechnology), followed by horseradish peroxidase-conjugated goat antimouse immunoglobulin or goat antirabbit immunoglobulin (Pierce). Proteins were visualized using enhanced chemiluminescence (Amersham). A549 cells were lysed by sonication in PBS with 1% N-P40, and protein complexes were precipitated using polyclonal rabbit antisera specific for TGIF or mSin3 or with the appropriate preimmune sera, together with protein A-Sepharose (Pierce). Proteins were visualized by SDS-PAGE, and Western blotting was performed as above, using rabbit antisera directed against TGIF (SM79), mSin3 (J260), or HDAC1 (Upstate Biotechnology) and goat antirabbit immunoglobulin (Pierce).


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grant HD39926 (to D. W.) from the National Institute of Child Health and Human Development, Grant ROICA57138 (to R. N. E.) from the NIH, and an NIH grant to Memorial Sloan-Kettering Cancer Center. J. M. is an investigator of the Howard Hughes Medical Institute. P. S. K. is a fellow of the Jane Coffin Childs Fund for Medical Research. Back

2 To whom requests for reprints should addressed, at Department of Biochemistry and Molecular Genetics and Center for Cell Signaling, 800577 HSC, University of Virginia, Charlottesville, VA 22908. Phone: (434) 243-6752; Fax: (434) 924-1236; E-mail: dw2p{at}virginia.edu Back

3 The abbreviations used are: TGIF, TG-interacting factor; TALE, three amino acid loop extension; HDAC, histone deacetylases; TGF, transforming growth factor; HPE, holoprosencephaly; PAH, paired amphipathic {alpha}-helix; GST, glutathione S-transferase; RD, repression domain; HD, homeodomain; HA, hemagglutinin. Back

Received for publication 11/30/00. Revision received 7/17/01. Accepted for publication 8/ 2/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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