From the Department of Pharmacology and Cancer
Biology, Duke University Medical Center, Durham, North Carolina, 27708 and ¶ Manitoba Institute of Cell Biology and Department of
Biochemistry and Molecular Biology, University of Manitoba,
Winnipeg, Manitoba, R3E OW3, Canada
Received for publication, November 29, 2000, and in revised form, March 7, 2001
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ABSTRACT |
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The Smads are a family of sequence-specific
DNA-binding proteins that modulate transcription in response to
transforming growth factor Transforming growth factor Studies of the Smad proteins have revealed much about the mechanism of
TGF Far less is known, however, about the mechanism through which TGF Given the established role of Smads as effectors of TGF Although these studies establish a physical link between the Smads and
HDAC1, the contribution of Smad-mediated recruitment of histone
deacetylase enzymatic activity to specific promoters has not been
evaluated. To address this issue, we show that the ability of Smad3 to
associate with endogenous HDAC enzymatic activity correlates with its
ability to repress transcription. Using random mutagenesis and
"reverse" two-hybrid analysis, a cluster of residues in the MH1
domain of Smad3 that are critical for the ability of Smad3 to associate
with HDAC enzymatic activity are identified. A mutation at one of these
residues does not affect the ability of Smad3 to activate transcription
or to bind TGIF, c-ski, or SnoN in vitro. Surprisingly, this
same mutation does abrogate the ability of Smad3 to repress
transcription. Together, these results indicate that the association of
Smad3 with histone deacetylase activity may require interactions with
an unidentified HDAC-associated factor(s) at its amino terminus and
that this association is critical for Smad3 to repress constitutive
transcription. Moreover, these findings suggest that Smad3 may have
distinct functional motifs associated with either the activation or
repression of transcription.
Cell Culture--
293T and Mv1Lu cells were maintained in
Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum, penicillin, and streptomycin.
Plasmids--
The 4xSBE-Lux reporter construct was made
by annealing primers 5'-GATCTAAGTCTAGACGGCAGTCTAGAC-3' and
5'-GATCGTCTAGACTGCCGTCTAGACTTA-3' and then concatamerizing them into
the BglII site of pGL2-T+I (34). HDAC1/pBSK was created by
subcloning a PCR product derived from pBJ5-HD1-F into the
BamHI site of pBSK. Primers used to amplify the HDAC1
cDNA were as follows: 5'-CGGGATCCCGATGGCGCAGACGCAGGGCAC-3' and
5'-CGGGATCCCGTCAGGCCAACTTGACCTCCTC-3'. HA-c-ski/pBSK and HA-SnoN/pBSK were constructed by subcloning the ClaI/XbaI
fragment of HA-c-ski/pCMV5b and HA-SnoN/pCMV5b into the
ClaI/XbaI sites in pBSK. TGIF/pCAN was
constructed by subcloning the EcoRI/XhoI
restriction fragment of pCMV-FLAG-TGIF into the
EcoRI/XhoI sites of pCAN (Onyx Pharmaceuticals). Partial EcoRI/PpuMI fragments of mutant Smad3
cDNAs in pGBT9 were subcloned into
EcoRI/PpuMI site of Smad3/pRK5. Transfer of the mutant cDNAs was confirmed by sequencing. The
SmaI/SalI fragments of mutant Smad3 cDNAs in
pRK5 were subcloned into pGEX-KG. Amino acids 1-147 encoding the DNA
binding domain of Gal4 were subcloned into the
HindIII-SalI site of Smad3/pRK5F (R. Derynck). pRK5Gal4 vector was constructed by replacing the
Smad3-FLAG sequence in Smad3/pRK5F with this Gal4 DNA sequence.
Smad3NL/pRK5Gal4, mutants of Smad3NL/pRK5Gal4, and Smad3C/pRK5Gal4 and
were created by PCR using the primers
5'-TCCTCCCCCGGGATGTCGTCCATCCTGCCTTTC-3' and 5'-CGCGGATCCCGGCTCGCAGTAGGTAACTGG-3' and
5'-TCCTCCCCCGGGATGGCCTTCTGGTGCTCCATC-3' and
5'-CGCGGATCCAGACACACTGGAACAGCGGAT-3' to amplify cDNA from the
appropriate Smad3/pRK5FLAG vectors. Each PCR product was subcloned into
the SmaI/BamHI site of pRK5Gal4.
Yeast Two Hybrid Screen--
A yeast two-hybrid screen of a
HaCaT cell cDNA library (CLONTECH) was
performed using Smad3 as bait according to the
CLONTECH Matchmaker protocol and as described (35).
Briefly, an Hf7c yeast line carrying Smad3 in the Gal4 DNA binding
(DBD) domain fusion vector, pGBT9 (CLONTECH) and
expressing DBD-Smad3 was transformed with the HaCaT cDNA library
fused to the Gal4 activation (AD) domain in pACT2
(CLONTECH). 5 × 106 transformants
were screened for their ability to grow in the absence of histidine. 5 mM 3-aminotriazole was included in the media to reduce
background HIS3 reporter activity. 242 transformants grew
under these conditions and were positive for lacZ reporter gene activity. Bait dependence of the interaction was established by
reintroducing fusion vectors for AD clone 349 with DBD alone, with
DBD-Smad3, or with the DBD fused to an unrelated protein, G12, into Hf7c parental yeast. Co-transformants were
re-screened for the HIS3 and lacZ reporter gene
activities according to the CLONTECH Matchmaker protocol.
PCR Mutagenesis--
The PCR mixture contained 100 ng of
Smad3/pGBT9 DNA, 1 µM 5' pGBT9 770 (5'-TCAGTGGAGACTGATATGCC-3'), 1 µM 3' pGBT9 1077 (5'-GCTATACCTGAGAAAGCAAC-3'), 1× PCR buffer (Life Technologies, Inc.),
all four dNTPs (each at 50 µM), 0.1% Triton X-100, 1.5 mM MgCl2, 1 µg/µl bovine serum albumin, and
5 units of Taq DNA polymerase in 100 µl (1 min at 94 °C, 1 min at 45 °C, and 2 min at 72 °C for 40 cycles). 100 µM MnCl2 was added after 10 cycles. The PCR
product was gel-purified.
Reverse Two-hybrid--
Performed according to the
protocol developed by Vidal et al. (see Ref. 36). The MaV103
strain of Saccharomyces cerevisiae carries the
SPAL10::URA3 marker. Yeast two-hybrid
protein-protein interactions confer sensitivity to 5-fluoorotic acid
(FOA) in yeast carrying this marker. A clonal line of MaV103 that
expressed AD-349 isolated from the yeast two hybrid screen was
transformed with 300 ng of the mutagenized Smad3 library and 150 ng of
BamHI-linearized pGBT9. Several thousand transformants were
first selected for growth in the presence of 0.1% FOA. These
FOA-resistant transformants were tested for Histone Deacetylase Assays--
293T cells (~5 × 106) were transfected with 12 µg of pCGN vector or
Smad/pRK5F constructs using a standard calcium phosphate protocol.
12 h post-transfection the cells were treated with 100 ng/ml
trichostatin A (Wako BioProducts). 36 h post-transfection, the
cells were lysed in LS (phosphate-buffered saline and 0.5% Nonidet
P-40), 1 mM phenylmethylsulfonyl fluoride containing
protease inhibitors. Lysates were immunoprecipitated with 1 µg
anti-FLAG M2 (Eastman Kodak Co.) and protein A/G-Sepharose (Amersham
Pharmacia Biotech) as described. Immunoprecipitated complexes were
washed 5 times with LS buffer containing 1 mM
phenylmethylsulfonyl fluoride and protease inhibitors and resuspended
in HD buffer (20 mM Tris (pH 8.0), 150 mM NaCl,
10% glycerol). Histone deacetylase assays were performed as described
(37). Briefly, immunoprecipitated complexes were incubated for 30 min
at 37 °C with 180 mg (150,000 dpm) of acid-soluble histones isolated
from [3H]acetate-labeled chicken erythrocytes. Released
[3H]acetate was extracted using ethyl acetate and
quantified by scintillation counting.
In Vitro Binding Studies--
Bacterially produced,
glutathione-Sepharose-bound glutathione S-transferase (GST)
and GST-Smad fusions were normalized for the amount of protein and
volume of glutathione-Sepharose. These GST protein preparations were
incubated with rabbit reticulocyte lysate in vitro
transcribed translated (TNT) products (Promega) as described (35). TNT
products were made using HDAC1/pBSK, HA-c-ski/pBSK, HA-SnoN/pBSK,
TGIF/pCAN Sin3A-pCS2+MT, and c-Jun/pBSK constructs as templates.
Reporter Assays--
Luciferase assays were performed as
previously described (38). LipofectAMINE (Life Technologies, Inc.)
transfections were performed using the manufacturer's protocol. All
transfections were normalized to The Smads Associate with HDAC1 in Yeast--
To elucidate
the mechanism of Smad-mediated transcriptional modulation of
TGF
The Hf7c parental line carries two reporter genes for protein
interaction, HIS3 and lacZ, each containing
multiple consensus Gal4 DNA binding sequences upstream of their
respective coding regions. Thus, growth on media lacking histidine and
elevated
In vitro binding experiments show that the Smad/HDAC1
interaction was not direct (Fig. 5C and data not shown) but
may have been mediated by highly conserved yeast homologs to components of the HDAC repressor complex. Conserved proteins in this complex include yeast homologs to Sin3A, HDAC1, HDAC2, RbAP46, RbAP48, SAP18,
and SAP30 (41-45). Indeed, yeast proteins have been attributed with
bridging between bait and prey in other yeast two-hybrid screens (46).
Consistent with this possibility, Smad2, Smad3, and Smad4 bound
directly to Sin3A in vitro (data not shown and Fig.
5C).
Smads Associate with Histone Deacetylase Enzymatic Activity in
Mammalian Cells--
These data support the findings of several
studies that describe interactions between the Smads and other
HDAC1-binding proteins, TGIF, c-ski, and SnoN. Taken together, these
results indicate that the Smads may make direct contact with several
members of the repressor complex, with the functional end point being
an association with histone deacetylase enzymatic activity.
To determine whether the Smads actually complex with an active
HDAC enzyme, we measured the amount of endogenous histone deacetylase activity associated with the Smads. The method used to show that the
transcriptional repressor Mad1 and the Sin3A and N-CoR/SMRT co-repressors associate with endogenous HDAC enzymatic activity was
employed (Fig. 2A) (47-50).
Carboxyl-terminal FLAG-tagged Smads were overexpressed in 293T cells
and immunoprecipitated with an antibody directed against the FLAG
epitope. These immunoprecipitated complexes were incubated with
[3H]acetate-labeled histones, and the amount of histone
deacetylase activity was recorded as the number of dpm released from
the reactions. Smad1, Smad2, Smad3, and Smad4 co-immunoprecipitated
with significant levels of histone deacetylase activity that could be
inhibited upon treatment with trichostatin A, a reversible inhibitor of histone deacetylases (51). Smad1 and Smad2 consistently associated with
less histone deacetylase activity than either Smad3 or Smad4. The
levels of HDAC activity bound to Smad3 and Smad4 were ~4-fold less
than that associated with the HDAC1 binding partner and transcriptional repressor, Sin3A. A FLAG immunoblot of the immunoprecipitations confirmed that trichostatin A had no effect on the amount of Smad protein immunoprecipitated from each sample (Fig.
2B).
Screen for Loss-of-function Mutations in Smad3--
We
reasoned that the identification of the amino acids in Smad3 required
for its association with histone deacetylase activity would provide us
with tools to dissect the role of histone deacetylases in Smad-directed
transcription. To identify specific residues in Smad3 involved in the
association between Smad3 and HDAC1, a reverse two-hybrid screen of a
randomly mutagenized Smad3 library was performed. The entire Smad3
cDNA was mutated using random PCR mutagenesis. These PCR products
were then introduced into a MaV103 yeast line expressing the AD-349
clone isolated in the screen for Smad3 binding partners (Fig. 1). The
MaV103 yeast strain carries the
SPAL10::URA3 marker in addition to the
Gal4::lacZ and
Gal4::HIS3 markers previously described
(36). Yeast two-hybrid protein-protein interactions confer sensitivity
to FOA in lines carrying this reporter. Several thousand transformants
were screened for their ability to grow in the presence of 0.1% FOA
and their inability to produce a blue color in the presence of X-gal.
From this set of positives, clones that expressed full-length DBD-Smad3 proteins to a level similar to the wild type Smad3 line were selected. In all, nine clones met the selection criteria. A map of these mutants
is depicted in Fig. 3. As a measure of
lacZ reporter gene activity, the level of
Confirming the validity of the screen, Characterization of Smad3 HDAC Activity Mutants--
Given that
the yeast two-hybrid interaction between Smad3 and HDAC1 may be
dependent upon yeast proteins, the HDAC1-containing complexes that
associate with Smad3 in our yeast two-hybrid interaction may be very
different from those that associate with Smad3 in mammalian cells. To
determine if these mutations have any effect on the association of
Smad3 with endogenous histone deacetylases in human cells, we measured
the level of histone deacetylase activity associated with individual
mutants in 293T cells (Fig.
4A). Approximately equal
amounts of wild type and mutant Smad3-FLAG fusion proteins were
immunoprecipitated, suggesting that each mutant is stable in mammalian
cells. In this assay, each mutant was significantly deficient in its
ability to associate with HDAC activity, demonstrating the importance
of these particular MH1 domain residues in the association of active
histone deacetylases with Smad3.
Similar assays using Smad3 deletion mutants confirmed the importance of
the MH1 domain for the coupling of HDAC activity with Smad3. Smad3NL,
Smad3C, and Smad3
Examination of the crystal structure of Smad3 MH1 domain revealed that
many of these mutations alter amino acids in the hydrophobic core of
the protein (52). Thus, some of these amino acid changes may lead to
gross conformational changes in the MH1 domain that block several Smad3
functions such as DNA binding, nuclear entry, Smad4 binding, and
transcriptional activation. To address these issues, the ability of
Smad3 and the Smad3 mutants to activate the 4xSBE reporter was assessed
(Fig. 5A). This reporter
contains Smad consensus DNA binding sites (SBEs) to which Smad3 and
Smad4 directly bind and activate transcription. In this assay, the
ability of mutant L38P to activate transcription was severely
inhibited. In contrast, F111S/W394R, Y125C, and Y127C activated
transcription to levels similar to those of wild type Smad3. Activation
of transcription by P95S and N123D/I298M was partially hindered.
Mutated amino acids that are accessible to the surface of the wild type
protein are noted (Fig. 5B). Taken together, these data
suggest that unlike other mutations isolated from the reverse
two-hybrid screen, the F111S and Y125C mutations might disrupt a
discreet binding surface for HDAC1-binding proteins without inhibiting
the DNA binding, nuclear entry, Smad4 cooperation, and transcriptional
activation functions of Smad3. Supporting this notion, the MH1 domains
of bacterially produced GST-Smad3NL-F111S and GST-Smad3NL-Y125C retain the ability to directly associate with the SBE by electrophoretic mobility shift assay (data not shown).
c-ski, SnoN, and TGIF have been shown to mediate the association of
specific Smads with HDAC1. The inability of the mutant clones derived
from the reverse two-hybrid screen to associate with HDAC activity in
mammalian cells may be the result of deficient association with these
co-repressor proteins. To address this issue, the ability of in
vitro TNT c-ski, SnoN, and TGIF to bind GST fusion proteins of
Smad3, Smad3F111S/W394R, and Smad3Y125C was assessed (Fig.
5C). The inability of GST-Smad3 to bind TNT-HDAC1 was
mentioned previously and is shown here as a negative control for the
binding assays. As a positive control, the results of in
vitro binding reactions between each Smad3 mutant and TNT- c-Jun
is shown (35, 53). c-Jun forms critical contacts with lysine 41 in the
MH1 domain of Smad3 (54). Although Smad3F111S/W394R bound TNT-c-Jun
with similar efficiency as wild type Smad3, consistently the
association of Smad3Y125C and TNT-c-Jun was slightly diminished. Therefore, at least under in vitro conditions, the Y125C
mutation may cause minor alterations to overall protein structure
rather than solely affecting a discreet surface. Consistent with this observation, the ability of Smad3Y125C to associate with other TNT
products was slightly diminished. In contrast, no consistent difference
between the affinities of Smad3 and Smad3YF111S/W394R for TNT-c-ski,
TNT-SnoN, and TNT-TGIF was observed, indicating that direct binding to
c-ski, SnoN, and TGIF is unaffected by this mutation.
Transcriptional Repression by Smad3 HDAC Activity Mutants Is
Deficient--
Based on evidence showing a connection between HDAC
activity and the repression of transcription, we postulated that the
ability of the Smads to associate with HDAC activity would allow them to repress constitutive transcription. To quantify Smad-mediated repression, a reporter construct containing GAL4 DNA binding sites upstream of the constitutively active SV40 promoter was employed (Fig.
6A). Previous reports show
that Gal4 fusions of the transcriptional repressors, Mad1 and Rb,
inhibit the expression of this same reporter (55, 56). In this assay,
Smad3 and Smad4 significantly repressed transcription when tethered to
DNA (Fig. 6, B and C, and data not shown).
Reporter activity was repressed ~8-fold further by Gal4-Smad3NL but
was unaffected by Gal4-Smad3C expression.
If the ability of Smad3NL to repress transcription was indeed dependent
upon its association with HDAC enzymatic activity, we predicted that
repression of reporter gene expression by Smad3 HDAC activity mutants
would be deficient. To address this, the effect of the NL domains of
the Smad3Y125C and Smad3F111S HDAC activity mutants on reporter gene
activity was assessed (Fig. 6D). Consistent with a role for
the Smad3-HDAC interaction in the repression of active transcription,
the ability of each mutant to repress reporter gene expression was
significantly inhibited compared with wild type Smad3NL. In contrast,
the expression level of each mutant was similar to wild type Smad3 NL
(Fig. 6E).
The ability of TGF The difference in HDAC activity isolated from Smad3 full-length and
Smad3NL immunoprecipitations suggests that the MH2 domain may inhibit
the association of histone deacetylases with full-length Smad3. A
previously characterized autoinhibitory interaction between the MH1 and
MH2 domains may be responsible for this effect. Activation of Smad3 by
TGF We have further dissected this endogenous HDAC·Smad3 complex by
identifying particular residues in Smad3 that are required for Smad3 to
either associate with the endogenous HDAC complex or affect the
enzymatic activity of the bound HDAC complex. Consistent with the Smad3
deletion analysis, all of the isolated mutants contain sequence
alterations in the MH1 domain of Smad3, revealing the importance of
this domain in the coupling of HDAC enzymatic activity with Smad3.
Amino acid residues Leu-38, Tyr-88, and Val-122 interact with each
other and constitute a portion of the MH1 domain hydrophobic core (52).
Asn-123 and Tyr-127 are also buried within the MH1 domain and are
conserved among Smad members. Mutations at these residues most likely
alter the shape of the entire MH1 globular domain and in this way alter
the association of Smad3 with the endogenous HDAC activity. Three
mutations in the MH1 domain, P95S, F111S, and Y125C, however, change
conserved surface residues. They are located on the side of the MH1
domain opposite the DNA binding surface and may define an area of
co-repressor protein contact. Pro-95 and Tyr-125 are a part of the
"double loop" region defined by crystallographic analysis (52).
This region, characterized by an extensive surface area that includes the L2 and L4 loops, has been proposed to contribute to macromolecular interactions. Phe-111 is located on an adjacent surface that is of
particular interest because it contains the hydrophobic Phe-111 residue
surrounded by several hydrophilic residues, Glu-110, Lys-117, Asp-118,
and Glu-119. The structural distinctiveness of this surface suggests it
may, indeed, contribute toward a protein-protein interface.
In vitro binding of TGIF, c-ski, or SnoN to the HDAC
activity mutant identified in these studies, Smad3F111S/W394R, was
essentially unaffected. It is possible that, TGIF, c-ski, and SnoN are
not present in these complexes, but they do make up and may be
essential for the formation of other Smad·HDAC complexes not isolated
or characterized in these assays. Supporting this possibility, our Smad3 HDAC activity mutants were based on an interaction between Smad3
and HDAC1 in S. cerevisiae, an organism with no homologues to TGIF, c-ski, or SnoN. Furthermore, previous studies show that the
Smad MH2 domain rather than the MH1 domain makes contact with c-ski and
SnoN (29-32, 63, 64). We demonstrate here, however, that Smad3NL, a
Smad3 fragment lacking the MH2 domain, associates with biologically
relevant levels of HDAC enzymatic activity.
Alternatively, TGIF, c-ski, and SnoN do participate in the HDAC
complexes isolated in the studies presented here, but their association
with Smad3 is not sufficient to mediate the interaction between Smad3
and HDAC activity when HDAC binding at the MH1 domain is blocked. In
this scenario, TGIF, c-ski, and SnoN association at the MH2 domain may
serve to stabilize the essential MH1 interaction. Competition with p300
for binding at the MH2 domain may have masked this role of TGIF, c-ski,
and SnoN in the HDAC activity assays reported here. Indeed, previous
reports show that p300/CBP competes with TGIF and c-ski for Smad
binding (32, 65). If p300/CBP does compete with TGIF, c-ski, and SnoN
in our studies, we predict that inhibition of p300 binding in these
assays may result in the co-immunoprecipitation of even more HDAC
activity with Smad3 and Smad3 In the transcriptional assay used here, the Smads are tethered to a
viral promoter that is constitutively transcribed. We show that Smad3
represses constitutive transcription from this promoter. Inconsistent
with previous reports that Smad-activated transcription is blocked by
Smad binding partners, transcriptional repression by Smad3 in this
assay was independent of TGF The ability of the Smad3 HDAC-activity mutants to repress transcription
was, however, partially retained. This may simply reflect the nature of
the SV40 promoter that is sensitive to HDAC-independent modes of
repression (56). Thus, a portion of the inhibitory effect of Smad3 on
reporter gene activity may be independent of HDACs, suggesting that in
addition to the recruitment of histone deacetylase activity to a
particular promoter, the Smads may repress constitutive transcription
through HDAC-independent mechanisms. Nevertheless, a significant
portion of Smad3-mediated transcriptional repression found here is
dependent upon the association of Smad3 with histone deacetylase
activity. The finding that the repression-deficient mutants,
Smad3F111S/W394R and Smad3Y125C, retain the ability to activate 4xSBE
transcription indicates that the activation and repressive effects of
Smad3 on transcription may exist as two separable functions of the
Smad3 protein. We envision a model where, under specific promoter
contexts, association of Smad3 and p300/CBP is favored and
transcription is activated. The competition between p300/CBP and
HDAC-associated repressors would determine the extent of
transcriptional activation. On other promoters, HDAC complexes
associate with Smad3 at the MH1 domain and repress transcription. This
mode of Smad3-mediated transcriptional repression may not involve the
competition between co-activators and co-repressors. Supporting this
model, loss of association with HDAC enzymatic activity was not found
to result in a hyperactivation of 4xSBE-mediated transcription by
Smad3F111S or Smad3Y125C. Instead, 4xSBE-mediated transcription is
activated similarly by wild type Smad3, and each repression-deficient
Smad3 mutant examined in this study.
Our data suggest that an interaction between the MH1 domain of Smad3
and an unknown but conserved HDAC-binding factor is required for the
coupling of Smad3 to histone deacetylase enzymatic activity. A
plausible candidate for this factor is the co-repressor Sin3A, which
has a highly conserved homologue in yeast and directly binds to HDAC1.
In vitro binding experiments with TNT-Sin3A, however, demonstrate that Smad3F111S/W394R and Smad3Y125C associate with Sin3A
as efficiently as wild type Smad3. Thus, the identity of this factor
remains unknown. Furthermore, whether Smad-associated histone
deacetylase activity is the effect of one specific HDAC complex or the
net effect of many distinct complexes, each containing a specific
combination of HDACs and co-repressor proteins, also remains unclear
(68). Recruitment of distinct HDAC complexes by the Smads may
facilitate promoter-specific transcriptional repression. Future studies
are required to further characterize the components of Smad·HDAC
complexes and any distinct functions they may have.
Taken together, these findings suggest that by targeting HDAC1 activity
to TGF (TGF
) by recruiting transcriptional
activators like the histone acetyltransferase, p300/CBP, or repressors
like the histone deacetylase, HDAC1, to TGF
target genes. The
association of Smads and HDAC1 is mediated in part by direct binding of
Smads to the HDAC1-associated proteins, TG-interacting factor,
c-ski, and SnoN. Although ectopic expression of these proteins inhibits Smad-activated transcription, the contribution of histone deacetylase enzymatic activity to transcriptional repression by TGF
is unknown. Here, the biological requirements for the interaction between Smads and
endogenous histone deacetylase activity are investigated. We identify
residues in Mad homology domain 1 of Smad3 that are required for
association with histone deacetylase activity. An amino acid change at
one of these critical residues does not disrupt the association of
Smad3 with c-ski, SnoN, and transforming growth-interacting factor but does abrogate the ability of Smad3 to repress transcription. These findings indicate that the association of Smad3 and histone deacetylase activity relies on additional protein mediators that make
contact with Smad3 at its amino terminus. Moreover, these data suggest
that the suppressive effect of Smad3 on transcription is dependent upon
its association with histone deacetylase enzymatic activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF
)1 is a pleiotropic
cytokine that elicits a diverse array of cellular responses in a
variety of cell types (1). The combined actions of these cellular
responses are likely to mediate the more global effects of TGF
,
including its role in development, wound healing, immune responses, and the pathogenesis of cancer (2). TGF
initiates downstream signaling inside the cell through a heteromeric receptor complex containing the
type I and type II TGF
receptors at the cell surface (3, 4). In its
activated state, the type I receptor directly binds and phosphorylates
specific members of the Smad protein family (5-8). Each member of this
protein family contains two highly conserved globular domains, an
amino-terminal Mad homology domain 1 (MH1) and a carboxyl-terminal Mad
homology domain 2 (MH2), connected by a less conserved proline-rich
linker sequence. Phosphorylated Smad2 and Smad3 associate with Smad4
and each other (9-13). These Smad oligomers enter the nucleus and
modulate the transcription of various TGF
-responsive genes (2,
14).
-mediated transcriptional modulation. By binding directly to Smad
DNA consensus sites and to other promoter-bound transcription factors,
the Smads activate the transcription of several genes in response to
TGF
. For instance, Smads directly bind to sites in the plasminogen
activator inhibitor-1 (PAI-1) promoter itself and to the transcription
factors, transcription factor meE3 and Sp1, on the promoter to activate
PAI-1 transcription (15-20).
represses the expression of target genes that maximally produce
transcript in the absence of ligand. This set of genes includes
osteocalcin (21-23), transin/stromelysin (24), and the proto-oncogene,
c-myc (25). TGF
-mediated transcriptional
repression of these genes is important for many cellular effects of
TGF
s. For example, TGF
-mediated inactivation of c-myc
transcription is essential for the anti-proliferative capacity of
TGF
in epithelial cells (25).
-mediated
transcriptional regulation, we postulate that the Smads may mediate
transcriptional repression of these genes. Although the molecular
mechanism underlying this transcriptional repression remains largely
unknown, the recent findings showing that Smads associate with several
components of the histone deacetylase 1 (HDAC1) transcriptional
repressor complex may provide a clue. It has been shown that
recruitment of this large multi-nucleoprotein complex to DNA by
site-specific DNA binding transcription factors induces localized
regions of deacetylated chromatin, resulting in the tight packing of
nucleosomes and the effective repression of transcription (for
review see Refs. 26 and 27)). Several studies describe direct
interactions between Smads and several HDAC1-associated proteins, TGIF,
c-ski, and SnoN (28-33). Ectopic expression of TGIF, c-ski, or SnoN
blocks the ability of TGF
to modulate transcription. Furthermore,
c-ski and TGIF were shown to compete with the co-activator, p300, for
binding at the MH2 domain of Smad3. Therefore, the reported inhibitory
effects of these HDAC1-associated proteins on TGF
-activated
transcription may result from competition between transcriptional
co-activators and co-repressors for Smad binding.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (
-gal)
activity using the
5-bromo-4-chloro-3-indolyl-
-D-galactoside (X-gal) filter
lift assay (CLONTECH MatchMaker protocol).
FOA-resistant lines with lacZ reporter activities less than
wild type Smad3 were tested for Smad3 protein expression by Western
analysis with anti-Gal4 DBD (Santa Cruz) monoclonal antibody. Clonal
lines that expressed full-length, DBD-Smad3 fusion proteins at levels
approximately equal to the wild type DBD-Smad3-expressing control line
were considered positives. The Smad3 cDNAs from these lines were
rescued and reintroduced with AD 349 into parental MaV103.
Co-transformants were selected on synthetic dropout without tryptophan
or leucine. Smad3 alleles with
-gal levels less than wild type Smad3
were sequenced.
-galactosidase activity by
co-transfection of 0.5 µg of a cytomegalovirus
-galactosidase
(CMV-
-gal) expression vector. Twelve hours after transfection, 10 ng/ml TGF
-1 in Dulbecco's modified Eagle's medium containing 0.1%
fetal bovine serum was added. Luciferase activity was measured 20-24 h
later. Chloramphenicol acetyltransferase assays were performed
according to a standard protocol (39, 40). 293T cells were transfected
for 7-12 h using calcium phosphate. 24 h later cells were assayed
for chloramphenicol acetyltransferase activity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-responsive genes, we set out to isolate transcription factors
that bind to the Smads. A yeast two-hybrid screen of a human
kerationcyte (HaCaT) cDNA library was performed using Smad3 as a
bait. A cDNA clone (AD-349) encoding amino acids 211-482 of the
histone deacetylase HDAC1 was isolated (Fig.
1A). Western analysis of yeast
lysates confirmed that the expected protein product was produced (data
not shown). To verify bait dependence of the interaction, AD-349 was
reintroduced into the parental yeast line Hf7c in combination with
expression vectors encoding the DBD alone or protein fusions of
DBD-Smad3 or DBD-Smad4. As a positive control for interaction, DBD-p53
and AD-T antigen were introduced into the parental yeast strain. As
negative controls for the interaction, either DBD-Smad3, DBD-Smad4, or
the AD empty expression vector were also introduced into Hf7c.
Transformants were selected on synthetic dropout media lacking
tryptophan and leucine.
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Fig. 1.
Smad3 and Smad4 associate with HDAC1 in
yeast. A, cDNA clone (349) encoding the
carboxyl-terminal portion of the HDAC1 cDNA was isolated in a yeast
two-hybrid screen using Smad3 as bait. A schematic depiction of clone
349 coding sequence compared with full-length HDAC1 is shown.
B, Smad3 and Smad4 associated with clone 349 in S. cerevisiae. Protein interaction of DBD fusion proteins with AD
fusion proteins activate the transcription of the HIS3 and
lacZ marker genes in the S. cerevisiae strain,
Hf7c. Here we show that the interaction of DBD-Smad3 or DBD-Smad4 with
AD-349 activates the HIS3 reporter. Yeast strain Hf7c was
transformed with the yeast expression vectors encoding DBD-Smad3 and
either AD or AD-349, DBD-Smad4, and either AD or AD-349 and DBD with
AD-349 according to the CLONTECH Matchmaker
protocol. As a positive control for interaction, DBD fused to amino
acids 72-390 of p53 (DBD-p53) and AD fused to amino acids
84-708 of SV40 large T antigen (AD-T antigen) were also
included. Transformants were isolated on synthetic dropout media that
lacks both tryptophan and leucine to select for cells carrying both a
DBD fusion vector and the AD fusion vector, respectively. These cells
were streaked onto synthetic dropout media that lacked tryptophan and
leucine and either contained (+histidine) or lacked
histidine ( histidine). 5 mM 3-aminotriazole
was included in the
histidine plate (see "Experimental
Procedures") WT, wild type.
-gal activity are indicative of fusion protein interaction.
As shown in Fig. 1B, both DBD-Smad3- and
DBD-Smad4-expressing cells survive in the absence of histidine only if
AD-349 is present, suggesting that Smad3 and Smad4 associate with amino
acids 211-482 of HDAC1. The inability of cells expressing DBD and
AD-349 to survive confirms the bait dependence of these interactions.
Similar results were found by
-gal activity assays (data not shown).
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Fig. 2.
Smads associate with endogenous histone
deacetylase activity. Histone deacetylase activity
co-immunoprecipitates with Smad complexes from transfected 293T cells.
A, 293T cells (~5 × 106) were
transfected with 12 µg of either CMV vector, the indicated Smad/pRK5F
construct, or Sin3A/pCS2+MT using calcium phosphate. 12 h
post-transfection the cells were treated with 100 ng/ml trichostatin A
(TSA) (Wako BioProducts). 36 h post-transfection, the
cells were lysed and immunoprecipitated with 1 µg of anti-FLAG M2
(Kodak) and protein A/G-Sepharose. The immunoprecipitated complexes
were incubated with [3H]acetate-labeled histones purified
from chicken erythrocytes as described under "Experimental
Procedures," and the number of dpm released from the
immunoprecipitations was used as a measurement of histone deacetylase
activity. Nonenzymatic release of label was subtracted to obtain the
reported values. Values reflect the average of triplicate reactions
from one immunoprecipitation, and error bars indicate the
S.D. of those reactions. Results are representative of two experiments.
B, anti-FLAG M2 (Kodak) immunoblot analysis of the
immunoprecipitated complexes in A to confirm that uniform
amounts of each Smad were immunoprecipitated.
-gal activity
produced by yeast expressing AD-349 and each Smad3 mutant is shown.
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Fig. 3.
Protein sequences of mutant Smad3
alleles. A domain map of Smad3 is drawn, indicating the two highly
conserved regions of the Smad proteins, MH1, and MH2, linked by a less
conserved, proline-rich linker sequence. The entire Smad3 cDNA
sequence was mutagenized. Smad3 mutants were screened for their
inability to associate with AD-349. The location of each mutation in
the Smad3 sequence is noted by the wild type residue, codon number, and
the mutant residue. Note that two alleles contain double mutations,
F111S/W394R and N123D/I298M indicated by * and #, respectively. In
addition, residue Tyr-127 was mutated in two different clones. The
interaction of each mutant Smad3 allele with AD-349 was quantified as a
function of GAL1::lacZ reporter gene
expression as determined by -gal activity assays using chlorophenol
red-
-D-galactopyranoside as substrate
(CLONTECH MatchMaker protocol). Values for the
AD-349/DBD-Smad3 wild type interaction were standardized to 100, and
reporter activity for each interaction is shown as a percentage of this
standard. Reporter activities for AD-349+DBD alone and AD
alone+DBD-Smad3 are shown as negative controls for the interaction.
Reporter activity for AD-T antigen + DBD-p53 is shown as a positive
control, and reporter activity for AD+DBD is shown as a negative
control for the assay. Error indicates the S.D. of
-gal activities
from two different transformant colonies.
-gal activity for each Smad3
mutant allele was significantly reduced compared with that of wild
type. Although reporter gene activity for some of the mutations is
approximately half that of wild type Smad3, other mutant alleles,
particularly L38P and N123D/I298M, give
-gal activities comparable
with negative controls for the interaction. Notably, all of the clones
contain a missense mutation in the MH1 domain of Smad3, suggesting that
the MH1 domain may make contact with HDAC1-binding proteins. One
particular amino acid, Tyr-127, was mutated in two different clones,
highlighting its importance for the interaction. Two other mutants,
Smad3F111S/W394R and Smad3N123D/I298M, contain two missense mutations,
one in the MH1 domain and one in the MH2 domain. A subset of these
mutations displaying the lowest
-gal activities was subcloned into
mammalian expression vectors to be studied further.
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Fig. 4.
The association of Smad3 with endogenous
histone deacetylase activity requires specific residues in the MH1
domain. Smad3 mutant alleles were subcloned into the mammalian
expression vector, pRK5FLAG, and assayed for their ability to
co-immunoprecipitate with histone deacetylase activity as described
under "Experimental Procedures" and in Fig. 3. A, a
graph of the number of dpm released from each immunoprecipitation as a
measure of histone deacetylase activity. Nonenzymatic release of label
was subtracted to obtain the reported values. Values reflect the
average of triplicate reactions from one immunoprecipitation, and
error bars indicate the S.D. of those reactions. Results are
representative of two experiments. Anti-FLAG M2 (Kodak) immunoblot
analysis of the immuno-complexes is shown in the lower panel
to confirm that uniform amounts of each Smad were immunoprecipitated.
B, map of wild type Smad3 and deletion mutants used in C. C, 293T cells (~5 × 106) were
transfected with 12 µg of either CMV vector or the indicated
Smad3/pRK5F construct as in A. Values reflect the average of
triplicate reactions from one experiment, and error bars
indicate the S.D. of those reactions.
c were overexpressed, immunoprecipitated, and
analyzed for HDAC activity (Fig. 4, B and C).
Strikingly, Smad3NL associated with 3-fold more HDAC activity than
full-length Smad3. The level of HDAC activity associated with Smad3NL
was comparable with Sin3A-bound HDAC activity, confirming that Smad3 binds biologically relevant levels of HDAC activity through its MH1
domain. Smad3
c co-immunoprecipitated with levels of associated HDAC
activity similar to full-length Smad3. In contrast, Smad3C associated
with little or no HDAC enzymatic activity.
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Fig. 5.
Effects of the mutations on Smad3
function. A, quantification of the ability of mutant
Smad3 alleles to activate transcription from Smad DNA binding sites.
Either Smad3/pRK5F (0.5 µg), mutant Smad3/pRK5F (0.5 µg), or empty
CMV vector, pCMV5 (0.5 µg) was co-transfected with Smad4/pRK5F (0.5 µg) into Mv1Lu cells along with the 4xSBE-lux (1.0 µg) and
CMV- -gal (0.5 µg) reporters described under "Experimental
Procedures." Total transfected DNA was kept constant with pCMV5.
Luciferase values were normalized to
-galactosidase activity to
control for transfection efficiency, giving relative luciferase units
(RLUs). Error bars indicate the S.D. of duplicate
transfections. WT, wild type. B,
surface-accessible residues are noted. The F111S and Y125C
surface-accessible mutations activate transcription as wild type Smad3.
C, in vitro binding experiments of GST-Smad3 and
GST-Smad3 mutants with in vitro TNT-c-ski, TNT-SnoN,
TNT-TGIF, TNT-Sin3A, and TNT-c-Jun. 10% of the total volume of TNT
product added to each binding reaction is shown (Input), and
total GST protein used in each reaction is shown in the lower
panel by Coomassie staining.
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Fig. 6.
The ability of Smad3 mutants to repress
transcription is inhibited. A, map of the pSVEC
reporter used in B and D with functional domains
noted. B, reporter activity in the presence of Smad3 or
Smad3 deletion mutants. Approximately 2 × 106 293T
cells were co-transfected with pSVEC (1.0 µg) and CMV- -gal (0.5 µg) reporter constructs and 5 µg of either pRK5Gal4,
Smad3/pRK5Gal4, Smad3NL/pRK5Gal4, or Smad3C/pRK5Gal4 or as a positive
control, Gal4-Mad1, into 239T cells. 36 h post-transfection the
cells were lysed and normalized for transfection efficiency. Normalized
lysates were assayed for chloramphenicol acetyltransferase activity and
analyzed by Western blot (C). C, Western analysis
of normalized lysates in B using anti-Gal4DBD monoclonal
antibody (Santa Cruz, sc-510). D, reporter activity in the
presence of Smad3NL or Smad3NL HDAC1 binding mutants. 293T cells were
transfected as in B with pSVEC (1.0 µg) and CMV-
-gal
(0.5 µg) reporter constructs and 5 µg of pRK5Gal4,
Smad3NL/pRK5Gal4, or Smad3NL/pRK5Gal4 mutants. Lysates were processed
as in B. E, Western analysis of lysates
normalized for transfection efficiency using the sc-510 antibody as in
C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
to repress the expression of critical cell
cycle regulators like c-Myc indicates that transcriptional repression
by TGF
may contribute to TGF
-mediated inhibition of cellular
proliferation. Thus, understanding the mechanisms through which TGF
represses transcription may provide insight into the anti-proliferative
capacity of TGF
and the role of Smads in tumor suppression. To this
end, we show that Smad1, Smad2, Smad3, and Smad4 associate with
endogenous histone deacetylase activity. Thus, Smad·HDAC
complexes are enzymatically active and may couple histone deacetylase
activity with nuclear Smad activities like DNA binding. A mutant
lacking the MH2 domain, Smad3NL, associated with 3-fold more histone
deacetylase enzymatic activity than Smad3. The amount of
Smad3NL-associated activity is comparable with the level of HDAC
activity that co-immunoprecipitated with the transcriptional repressor
and direct HDAC1-binding protein, Sin3A. Thus, Smad3 associates with
levels of HDAC activity that are biologically significant in the
repression of transcription.
is thought to interrupt the binding of the MH1 and MH2 domains
to each other and may facilitate the association of Smads with
HDAC-binding proteins (57). Alternatively, histone acetyltransferase
activity of p300/CBP bound at the MH2 domain of Smad3 (58-62) may
effectively reverse the effects of co-immunoprecipitated HDACs,
resulting in reduced Smad-associated HDAC activity levels in the
presence of the MH2 domain of Smad3 and Smad3
c.
c. In contrast, a block of Smad3C/p300
association is not expected to elevate HDAC activity levels associated
with Smad3C since the MH1 domain residues essential for the interaction
between Smads and HDAC activity are not present in this particular mutant.
-activated or Smad-activated
transcription (28, 30-32, 64, 66). In the context of this assay,
transcriptional repression by Smad3F111S and Smad3Y125C was
significantly abrogated compared with wild type Smad3. Consistent with
these findings, Smad3C, which did not associate with HDAC activity, did
not repress transcription. Together, these data suggest that, in the
context of this assay, the HDAC enzymatic activity of the Smad3·HDAC
complex is required for the repression of constitutive transcription by Smad3.
-repressed genes, the Smads may directly mediate the
repression of transcription of target genes, like c-myc, in response to TGF
. Future in vivo studies of such TGF
target genes using the Smad3 HDAC activity mutants identified here will
elucidate the contribution of the Smad-HDAC interaction to the
transcriptional modulation of a particular target gene by TGF
.
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ACKNOWLEDGEMENTS |
---|
We thank R. Eisenman for helpful discussion, the Sin3A construct, and pSPMadN35Gal; H. Moses and M. Aakre for the murine c-myc promoter and technical assistance; S. Schreiber for pBJ5-HD1-F; D. Dean for pSVEC; R. Derynck for the Smad/pRK5F constructs; Y. Xiong and M. Nichols for the yeast two hybrid cDNA library; K. Luo for the c-ski and c-SnoN cDNAs; J. Massagué and D. Wotton for the TGIF cDNA; J. Nevins, S. Long, A. Means, J. Frederick, A. Borton, X. Shen, M. Datto for helpful discussion of the manuscript; M. Long and Y. Yu for technical assistance.
![]() |
FOOTNOTES |
---|
§ Supported by a National Science Foundation predoctoral fellowship. Current address: Massachusetts General Hospital, Boston, MA 02114.
Supported by a predoctoral fellowship from the Department of
Defense Breast Cancer Research Program.
** A recipient of the Medical Council Senior Scientist award.
To whom correspondence should be addressed: Dept. of
Pharmacology and Cancer Biology, Box 3813, Duke University Medical
Center, Durham, NC 27708. Tel.: 919-681-4861; Fax: 919-681-7152;
E-mail: wang0011@mc.duke.edu
Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.M010778200
This work was supported by National Institutes of Health Grant CA75368 (to X. W.) and a grant from the Medical Research Council of Canada (to J. R. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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ABBREVIATIONS |
---|
The abbreviations used are:
TGF, transforming growth factor
;
HDAC, histone deacetylase;
MH, Mad
homology;
TGIF, transforming growth-interacting factor;
DBD, Gal4 DNA binding domain;
AD, Gal4 activation domain;
SBE, Smad binding
element;
-gal,
-galactosidase;
X-gal, 5-bromo-4-chloro-3-indolyl-
-D-galactoside;
FOA, 5-fluoorotic acid;
PCR, polymerase chain reaction;
HA, hemagglutinin;
GST, glutathione S-transferase;
TNT, transcribed and
translated;
CMV, cytomegalovirus;
CBP, cAMP-response element-binding
protein (CREB)-binding protein.
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