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J. Biol. Chem., Vol. 277, Issue 48, 46066-46072, November 29, 2002
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§,§¶, and
**
From the Departments of Pathology and
Biochemistry and Molecular Biology, University of Southern
California, Los Angeles, California 90089
Received for publication, July 29, 2002, and in revised form, September 17, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The p160 coactivator complex plays a critical
role in transcriptional activation by nuclear receptors and possibly
other classes of DNA-binding transcriptional activators. The complex
contains at least one of three p160 coactivators (SRC-1, GRIP1/TIF2, or pCIP/RAC3/ACTR/AIB1/TRAM1), a histone acetyltransferase such as CBP or
p300, and the histone methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1). Methylation of histone H3 and possibly other proteins in the transcription initiation complex by CARM1 occurs
along with acetylation of histones and other proteins by CBP and p300
to help remodel chromatin structure and recruit RNA polymerase II. Here
we show that other domains of CARM1 are required for the coactivator
function of CARM1 in addition to the methyltransferase activity. The
methyltransferase GRIP1, binding, and homo-oligomerization activities
all reside in the central region of CARM1, which is highly conserved
among the entire protein arginine methyltransferase family. In addition
to this conserved domain, the unique N- and C-terminal regions of
CARM1 were also required for enhancement of transcriptional activation
by nuclear receptors. While the N-terminal region has no known activity
at present, the C-terminal part of CARM1 contains an autonomous
activation domain, suggesting that it interacts with other
proteins that help to mediate CARM1 coactivator function.
Activation of transcription by DNA-binding transcriptional
activator proteins is mediated by coactivators, which locally remodel chromatin structure and recruit RNA polymerase II and its transcription initiation complex to the promoter. Members of the nuclear receptor (NR)1 family of
transcriptional activator proteins, which include the receptors for
steroid and thyroid hormones, retinoids, and vitamin D, as well as
so-called orphan receptors (1-3), recruit several different complexes
of coactivator proteins to their target gene promoters (4-8).
One coactivator complex, which plays a central role in mediating
transcriptional activation includes at least one of the three related
160-kDa proteins commonly referred to as p160 coactivators (SRC-1,
GRIP1/TIF2, and pCIP/RAC3/ACTR/AIB1/TRAM1). The p160 coactivators bind
directly and in a ligand-dependent manner to the C-terminal AF2 activation domains of NRs through three LXXLL motifs
(where L is a leucine and X, any amino acid) located
in the central part of the p160 polypeptide chain. The C-terminal
region of the p160 coactivators can also interact with the N-terminal
AF1 activation domains of some NRs (9-11). The p160 coactivators
contribute to transcriptional activation by bringing other associated
coactivator proteins with them to the promoter. The p160 coactivator
complex includes either of the two related proteins p300 and CBP, which bind to the AD1 activation domain of p160 coactivators (12-14) and
function as coactivators for many DNA-binding transcriptional activators, including NRs (15). Recent studies have confirmed hormone-dependent recruitment of p160 coactivators, CBP,
and p300 to promoters activated by NRs (16-19). CBP and p300
contribute to chromatin remodeling by acetylating histones, and also
acetylate other components of the transcription initiation complex (16, 20-22). CBP and p300 can also bind directly to basal transcription factors and may thereby help to assemble the transcription initiation complex (12). Thus multiple domains of CBP and p300 apparently contribute to chromatin remodeling and recruitment/activation of RNA
polymerase II.
The activation domain AD2, located at the C terminus of p160 factors,
binds CARM1, which belongs to a family of previously identified
arginine-specific protein methyltransferases (PRMTs) (23). CARM1
enhances nuclear receptor function in a p160-dependent manner in transient transfection assays. CARM1, p300/CBP, and a p160
coactivator can also form a ternary complex which functions synergistically to enhance NR function and requires the
methyltransferase activity of CARM1 to do so (24, 25). CARM1 methylates
histone H3 at Arg-17 and Arg-26 in vitro (26), and chromatin
immunoprecipitation studies indicate that CARM1 is specifically
recruited to steroid hormone-regulated promoters in vivo in
response to the hormone and methylates histone H3 as part of the
transcription initiation process (27, 28).
PRMTs are homodimeric or homo-oligomeric proteins (29-31), which
transfer the methyl group from
S-adenosyl-L-methionine (AdoMet) to the
guanidino group of arginines in protein substrates (32). The enzymatic
activity is supported by a catalytic core domain, which is highly
conserved among PRMT family members and contains the AdoMet and
arginine binding sites and a barrel-like domain (29). In addition to
the conserved core region, each methyltransferase has a unique
N-terminal region of variable size. However, CARM1 is the only member
of the family to harbor unique domains at its N and C termini. The
contribution of these additional domains to the function of the PRMTs
is unknown, although some studies suggest that they may be involved in
the specificity of protein substrate binding (29, 30, 33).
Previous studies indicate that while the methyltransferase activity of
CARM1 is required for coactivator function, other unspecified domains
also contribute (25). Furthermore, because the coactivator function of
CARM1 depends on p160 coactivators (23, 24), the p160 binding site of
CARM1 is also presumably required, but its location has not been
determined. To better understand which activities and domains of CARM1
contribute to its coactivator function, we defined the locations of the
methyltransferase, p160 binding, and homo-oligomerization domains, as
well as an autonomous activation domain, with respect to the conserved
central domain (amino acids 150-480) and the unique N- and C-terminal
regions of CARM1. By testing deletion mutants of CARM1 lacking various
domains, we identified multiple regions of CARM1, which are required
for its coactivator function along with the methyltransferase and GRIP1 binding activities.
Plasmids--
Proteins with N-terminal hemagglutinin A (HA)
epitope tags were expressed in transient mammalian cell transfections
and in vitro from vector pSG5.HA which has SV40 and T7
promoters (23). pSG5.HA GRIP1 (9) and pSG5.HA CARM1 (23) were
previously described, as were pHE0, encoding human estrogen receptor
(ER) Protein-Protein Interaction in Vitro--
To measure binding of
35S-labeled proteins synthesized in vitro to GST
fusion proteins on glutathione-Sepharose beads, GST pull-down assays
were performed as previously described (36) with the following
exceptions: binding was conducted overnight at 4 °C with 20 µl of
the in vitro synthesis reaction in a 150-µl total volume
of NETN buffer (0.1% Nonidet P-40, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0, 100 mM NaCl) containing
Complete protease inhibitor mixture (Roche Molecular Biochemicals).
Cell Culture and Transfection--
Transfections of CV-1 cells
(37) were performed in 12-well dishes as described previously (25) with
1 µg of total DNA per well. Where indicated, medium was supplemented
with 20 nM estradiol (E2) during the last 30 h of
growth. Luciferase and Immunoprecipitation of CARM1 from Transfected Cells and in Vitro
Methylation Assays--
COS7 cells (37) were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum. One
million cells were seeded into 10-cm diameter cell dishes and
transfected with Superfect (Qiagen) according to the manufacturer's
protocol with 5 µg of pSG5.HA plasmid encoding CARM1 wild type or
CARM1 mutants. After transfection, cells were grown in Dulbecco's
modified Eagle's medium with 10% fetal bovine serum for 40 h
before harvest. Cells were harvested in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.1%
Nonidet P-40, and Complete protease inhibitor mixture. Cell lysates
were frozen at Immunoblots--
Ten percent of the immunoprecipitated
methyltransferases from the transfected cells (see above) were analyzed
by SDS-PAGE on 12% gels. Immunoblotting was performed as described
previously (38) with rat monoclonal antibody 3F10 against the HA
epitope at 100 ng/ml as the primary antibody and horseradish
peroxidase-conjugated anti-rat immunoglobin G (Santa Cruz
Biotechnology) at 160 ng/ml (1:2,500 dilution) as the secondary antibody.
Domains of CARM1 Involved in Coactivator Activity--
To
determine which parts of CARM1 are required for its coactivator
function, N- and C-terminal truncations were made near the boundaries
between the conserved central domain of CARM1 and its unique N-terminal
(amino acids 1-150) and C-terminal (amino acids 480-608) regions
(Fig. 1A). The coactivator
activity of CARM1 and its mutants were tested with ER by transient
transfection in CV-1 cells under two different conditions: with
relatively high levels of transfected ER expression vector, where CARM1
cooperates with a p160 coactivator (23); and at very low levels of
transfected ER expression vector, where CARM1 functions synergistically
with a p160 coactivator and p300, such that all three of these
coactivators must be co-expressed with ER to achieve efficient
activation of an estrogen-dependent reporter gene (25). In
both cases, the activity observed was shown previously to be completely
dependent on the exposure of the cells to estradiol to activate ER.
At the higher level of ER vector (5 ng), GRIP1 expression enhanced
reporter gene activation by ligand-bound ER, and co-expression of
full-length CARM1 resulted in a further enhancement approximately in
proportion to the amount of CARM1 expression vector used (Fig. 1B). However, mutants lacking the N-terminal part of CARM1
(mutant 121-608) or the C-terminal part (mutants 3-460, 3-500,
3-580) had no effect on the reporter gene expression mediated by ER
and GRIP1, suggesting that both ends of CARM1 contribute to its
coactivator activity.
With a low concentration of ER expression vector (0.1 ng), p300 had no
effect on the transcriptional activity observed with ER and GRIP1, and
CARM1 (in the absence of p300) caused an enhancement of only 2-fold
(Fig. 1C). However as previously described (25), expression
of both CARM1 and p300 with GRIP1 resulted in a dramatic increase of
ER-dependent reporter gene activity. Deletions of the
unique N- or C-terminal part of CARM1 severely impaired the synergistic effect.
Thus the unique N- and C-terminal regions of CARM1 are required for
coactivator activity with ER under both tested conditions. The
expression level of each mutant was similar to that of full-length CARM1, with the exception of CARM1 (121-608) (Fig. 1A). The
lack of coactivator activity of the CARM1-(121-608) fragment could be
partly due to its lower expression. We also recognized that the
deletions may disrupt the protein structure and thereby impair the
function of domains that are still present in the mutant protein. To
test this possibility, we examined whether other known functions of
CARM1 remained intact in the various mutants. This line of experimentation also allowed us to assign specific functions of CARM1
to specific domains of the protein and thereby to explore the
mechanisms by which the unique N- and C-terminal regions and the
conserved central region of CARM1 contribute to the coactivator activity.
The GRIP1 Binding Domain of CARM1 Is Located in the Conserved
Central Region--
The lack of coactivator activity of the CARM1
mutants could be due to their inability to bind GRIP1. Binding between
GRIP1 and CARM1 is required for CARM1 coactivator function (24), and the GRIP1 binding domain of CARM1 has not been mapped. Full-length CARM1 and its deletion mutants were translated in vitro in
the presence of [35S]methionine and incubated with either
GST or GST-GRIP1 AD2 fusion protein (consisting of the GRIP1 C-terminal
amino acids 1122-1462), which were preloaded on glutathione-Sepharose
beads. None of the CARM1 proteins bound to GST, but full-length CARM1
and several CARM1 fragments bound GST-GRIP1 AD2 (Fig.
2A). Deletion of the N
terminus (amino acids 1-120) or the C terminus (amino acids 501-608
or 581-608) of CARM1 did not impair binding to GRIP1 AD2, showing that
these domains are not required for the in vitro interaction. The CARM1 mutant lacking amino acids 461-608 was still retained by
GST-GRIP1 AD2 but to a lesser extent. However, the CARM1 C-terminal region alone was not able to bind GRIP1 AD2. A mammalian two-hybrid assay confirmed these results (Fig. 2B). These results
localized the GRIP1 binding domain of CARM1 within the central
conserved domain (amino acids 121-460) and demonstrated that the
unique N- and C-terminal regions of CARM1 are neither necessary nor
sufficient for the interaction with GRIP1.
The Unique CARM1 Ends Are Not Required for the Methyltransferase
Activity--
Full-length CARM1 and its deletion mutants were
expressed in COS7 cells by transient transfection, isolated by
immunoprecipitation, and incubated with histone H3 in the presence of
[methyl-3H]AdoMet; methylated histone H3 was
detected by SDS-PAGE and fluorography (Fig.
3). The mutant lacking the N-terminal
part of CARM1 (amino acids 1-120) or the C-terminal region (amino
acids 501-608) still methylated histone H3 efficiently, showing that
these unique domains were not required for the enzymatic activity of
CARM1, at least in vitro. The lower activity of
CARM1-(121-608) was due to its lower expression level (Fig.
1A). However, the mutant lacking amino acids 461-608, which
includes a small portion of the central conserved domain, was inactive.
The unique C-terminal region (amino acids 461-608) by itself did not
exhibit any enzymatic activity. Thus, the methyltransferase activity
resides within amino acids 121-500.
The Conserved Central Part of CARM1 Also Contains Its
Homo-oligomerization Domain--
To localize the homo-oligomerization
domain, we tested the ability of the CARM1 deletion mutants to interact
with wild type CARM1. All mutants lacking N- and C-terminal amino acids
were retained by the bead-bound GST-CARM1 fusion protein, although CARM1-(3-460) bound very weakly compared with wild type CARM1 (Fig.
4A); thus the unique ends of
CARM1 are not required for its homo-oligomerization in
vitro. Furthermore, by itself the C-terminal fragment (amino
acids 461-608) did not bind to GST-CARM1. Mammalian two-hybrid assays
produced very similar results in vivo (Fig. 4B).
We, therefore, located the CARM1 homo-oligomerization domain in the
conserved central part of the protein along with the methyltransferase
and GRIP1 binding activities.
The Unique C-terminal Region of CARM1 Is a Transcriptional
Activation Domain--
Since the unique N- and C-terminal regions of
CARM1 were important for its coactivator function but played no role in
its methyltransferase, GRIP1-binding, or homo-oligomerization
activities, we tested whether these unique regions might contain an
autonomous activation function. CARM1 mutants fused to the Gal4 DBD
were tested for their ability to activate expression of a reporter gene
controlled by Gal4 response elements (Fig.
5). Fusion of full-length CARM1 to the
Gal4 DBD enhanced the reporter gene expression, indicating the presence
of an autonomous transactivation domain somewhere in the CARM1 protein.
A mutant deficient in methyltransferase activity (C1 VLD) (23) fused to
Gal4 DBD also increased the transcription driven by Gal4 response
elements. This suggests that the methyltransferase activity of CARM1 is
not necessary for the observed transcriptional activation activity. A
mutant constituted only by the N-terminal part of CARM1 (amino acids 3-126) had little or no ability to increase the reporter gene expression. The mutant 121-608 was almost as effective as full-length CARM1, thus showing that the N-terminal part was not involved in the
activity. Mutants lacking the C-terminal part of CARM1 (mutants 3-500;
3-580) were also active but to a lesser extent than full-length CARM1.
Moreover, deletion of residues 461-608 totally abolished the
autonomous transactivation activity of CARM1. Finally, the CARM1
fragments 461-608 and 501-608 exhibited activity ten times that of
wild type CARM1, indicating that CARM1 contains a strong autonomous
activation domain in its unique C-terminal region (Fig. 5, right
panel).
The C-terminal activation domain of CARM1 may contribute to coactivator
function through protein-protein interactions with some important
component of the transcription machinery. If so, overexpression of the
isolated C-terminal domain might inhibit the coactivator function of
full-length CARM1 by competing with CARM1 for the interaction with this
transcription machinery component. CARM1-C (amino acids 461-608)
strongly inhibited the coactivator effect of CARM1 on the
hormone-dependent, ER-mediated activation of reporter gene
expression (Fig. 6A,
upper panel). CARM1-C had little or no effect on the basal
ER activity observed in the absence of hormone. The specificity of the
inhibitory effect was also demonstrated by the fact that CARM1-C had no
effect on the expression of a RSV promoter-driven Multiple Functions of the Conserved Central Region of
CARM1--
CARM1 belongs to the PRMT family of arginine-specific
protein methyltransferases, which share a conserved core region of
about 330 amino acids that contains the methyltransferase activity. X-ray crystallography of mammalian PRMT3 and yeast Rmt1/Hmt1
demonstrated that the conserved region forms two separate structural
domains that combine to form the active enzyme (29, 39) (Fig.
7). The N-terminal part, which is the
most highly conserved in primary amino acid sequence among family
members, is composed of mixed
As expected from the three-dimensional structure and the high degree of
conservation among PRMT members, our studies located the
methyltransferase and homo-oligomerization activities of CARM1 approximately within the conserved region (amino acids 150-480 of
CARM1) (Figs. 3, 4, and 7). A C-terminal deletion to amino acid 460, which removed the last
While CARM1 shares homology with the PRMT family throughout the
methyltransferase domain, CARM1 has a unique set of protein substrates,
including histone H3 and p300/CBP (23, 40). In addition, CARM1 is the
only PRMT member tested to date which can cooperate synergistically
with p300, CBP, or p/CAF to enhance transcriptional activation by NRs
(25). Previous studies have shown that the coactivator function of
CARM1 depends on its methyltransferase activity (23, 25). Moreover,
chromatin immunoprecipitation assays demonstrated that steroid hormones
stimulate recruitment of CARM1 and methylation of histone H3 in a
CARM1-specific manner at promoters of stably integrated, steroid
hormone-responsive genes (27, 28). Thus, the unique transcriptional
coactivator function of CARM1 is at least partly due to its unique
methyltransferase substrate specificity.
Role of the Unique N- and C-terminal Regions of CARM1 in Its
Coactivator Function--
Deletion of the unique N- or C-terminal part
of CARM1 totally abolished its coactivator function (Fig. 1), but had
no effect on its ability to bind GRIP1 (Fig. 2), methylate histone H3
(Fig. 3), or form homo-oligomers (Fig. 4). Thus the unique N- and
C-terminal regions must contribute to the coactivator function of CARM1
through a novel mechanism not involving any of these three activities. To date little is known about the functions of the unique N termini of
PRMT family members; these unique N termini vary greatly in length as
well as sequence (29). It has been proposed that the relatively short N
terminus of mammalian PRMT1 and yeast Rmt1/Hmt1 may contribute to
methyltransferase substrate specificity by interacting with regions of
the substrate protein distinct from the sequence immediately
surrounding the target arginine residue (29, 30). In addition, deletion
of the relatively long unique N terminus of PRMT3 altered its substrate
specificity in vitro (33). However, mutants of CARM1 lacking
the unique N- or C-terminal regions appeared to be unaffected in their
ability to methylate histone H3 (Fig. 3) and several other protein
substrates that we tested.2
We also found that the N terminus of CARM1 did not contribute to the
autonomous transcriptional activation activity of CARM1 (Fig. 5). Thus
the mechanism by which the N-terminal region of CARM1 contributes to
coactivator function remains unclear.
While the unique C-terminal part of CARM1 was not required for GRIP1
binding, methyltransferase, or homo-oligomerization activities, it
contains a strong autonomous activation domain (Fig. 5), which presumably explains why this domain is necessary for the coactivator function of CARM1. CARM1 mutants lacking this domain were almost devoid
of a transcriptional activation activity when fused to Gal4 DBD,
indicating that the C terminus is responsible for most or all of the
autonomous transactivation activity observed in full-length CARM1. The
ineffective autonomous transcriptional activation activity associated
with the mutants lacking the C-terminal region indicates that the
methyltransferase activity per se is not sufficient for
transcriptional activation (Fig. 5). PRMT1, which lacks a unique
C-terminal domain and has a very short unique N-terminal domain, also
exhibits no autonomous activation activity when fused to Gal4 DBD (36).
Thus, although CARM1 methylates histone H3 and PRMT1 methylates histone
H4, the simple recruitment of a histone methyltransferase activity to
the promoter of a transient reporter gene is not sufficient to activate
transcription. This is also consistent with our findings that the
unique terminal domains of CARM1 are required in addition to the
methyltransferase activity for the coactivator function of CARM1.
As a model for CARM1 function, we propose that CARM1 is recruited to
the promoter through its interaction with the C-terminal domain of a
p160 coactivator. The autonomous activation activity of the CARM1
C-terminal domain collaborates with the methyltransferase activity of
the central domain and possibly an unknown activity in the unique
N-terminal domain, to mediate the coactivator function of CARM1. The
methyltransferase activity is responsible for the methylation of
histone H3, which presumably contributes to chromatin remodeling.
In addition, CARM1 may methylate other protein components of the
transcription machinery. We propose that the autonomous activation
activity of the C-terminal domain is due to its ability to interact
with other proteins in the transcription machinery. For example,
CARM1-C could interact with a component of the basal transcription
machinery and thereby help to recruit RNA polymerase II; or CARM1-C
could interact with another, currently unknown, coactivator and thereby
recruit or maintain the additional coactivator in the complex with
GRIP1, CARM1, and p300. The ability of the co-expressed C-terminal
fragment of CARM1 to inhibit the coactivator function of full-length
CARM1 (Fig. 6) supports our proposal that this region binds an
important factor that contributes to the transcriptional activation process.
Thus, once bound to the promoter, CARM1 contributes to the
transcriptional activation process through multiple downstream signaling mechanisms, i.e. through methylation of histones
and possibly other proteins and through protein-protein interactions mediated by the unique C-terminal and possibly N-terminal domains. The
use of multiple downstream signaling mechanisms by a single coactivator
is not unique to CARM1. The coactivators p300, CBP, and p/CAF have
multiple protein-protein interaction domains which also contribute to
their coactivator function in collaboration with their histone
acetyltransferase activities (12).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(34); the luciferase reporter plasmids MMTV(ERE)-LUC (23) and
GK1, which is controlled by Gal4 response elements (10); and the
-galactosidase (-gal) reporter plasmid RSV.-gal (35). CARM1
deletion mutants were constructed by inserting PCR-amplified CARM1
cDNA fragments flanked by a 5' EcoRI site and a 3'
BglII site into EcoRI and BglII sites
of pSG5.HA and into EcoRI and BamHI sites of pM
(to make Gal4 DBD fusions) or pVP16 (Clontech) (to
make VP16 fusions). pM.GRIP1 (encoding Gal4DBD-GRIP1) was described
previously (9), and pCMV.p300 was kindly provided by Dr. T.-P. Yao
(Duke University). Bacterial expression vectors for glutathione
S-transferase (GST) fused to GRIP1 AD2 (consisting of the
GRIP1 C-terminal amino acids 1121-1462) and CARM1 were described
previously (9, 23).
-galactosidase activities are shown as the
mean and range of variation of two transfected cell cultures.
80 °C, thawed on ice, and clarified by
centrifugation before incubation overnight at 4 °C with monoclonal
antibody (clone 3F10, Roche Molecular Biochemicals) against the HA tag.
Preblocked protein G-Sepharose (Amersham Biosciences) was then added
for 2 h at 4 °C. Immunoprecipitates were recovered by rapid
centrifugation, washed three times with NETN containing 0.1% Nonidet
P-40 and resuspended in HMT Buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.4 mM EDTA). In vitro
histone methylation assays were performed as follows: 3 µg of histone H3 (Roche Molecular Biochemicals) was incubated with immunoprecipitated methyltransferases and 7 µM
S-adenosyl-L-[methyl-3H]methionine
(specific activity 14.7 Ci/mmol) in 30 µl of HMT buffer for 1 h
at 30 °C. Reactions were stopped by addition of SDS loading buffer
and analyzed by 15% SDS-PAGE and fluorography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Coactivator function of CARM1 and its
deletion mutants with ER . A, schematic
representation of CARM1 deletion mutants is shown on the
left. The expression level of each protein is shown on the
right, as determined by immunoblot of transfected COS7 cell
extracts, using anti-HA tag antibodies. B, CV1 cells were
transiently transfected with MMTV(ERE)-LUC reporter plasmid (250 ng)
and expression vectors encoding ER (5 ng), GRIP1 (125 ng), and
increasing concentrations (100, 200, and 400 ng) of vectors encoding
full-length CARM1 or deletion mutants as indicated. Each mutant protein
is identified by the amino acids residues it contains. Transfected
cells were grown in culture medium with 20 nM estradiol,
and extracts of the harvested cells were tested for luciferase
activity. C, transfections were performed as in
B, but amounts of ER and coactivator expression vectors
were: ER, 0.1 ng; GRIP1, 125 ng; wild type or mutant CARM1, 250 ng; and
p300, 250 ng. The results presented are from a single experiment
representative of four independent experiments.
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Fig. 2.
Location of GRIP1 binding activity in
conserved central region of CARM1. A,
[35S]methionine-labeled CARM1 (wild type or mutant) was
incubated with either GST or GST-GRIP1AD2 fusion proteins preloaded on
glutathione-coupled beads. Bound proteins were eluted and analyzed by
SDS-PAGE and fluorography. The input lanes represent 10% of each
35S-labeled protein used in the binding assay.
B, CV1 cells were transiently transfected with 250 ng of GK1
reporter plasmid, 125 ng of a pM vector encoding Gal4DBD (left
lane) or Gal4DBD-GRIP1 fusion protein (all other
lanes), and varying concentrations (100, 200, and 400 ng) of
vectors encoding VP16 fused to CARM1 or its deletion mutants as
indicated. The luciferase activities shown are from a single experiment
which is representative of three independent experiments.
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Fig. 3.
Histone H3 methylation by wild type and
mutant CARM1. COS7 cells were transfected with vectors encoding
HA.CARM1 or its deletion mutants and the methyltransferase proteins
were immunoprecipitated from cell extracts with anti-HA antibodies.
Histone H3 was incubated for 1 h at 30 °C with the
immunoprecipitated CARM1 and
[methyl-3H]AdoMet. Methylated histone H3 was
detected by SDS-PAGE and fluorography. The results shown are from a
single experiment representative of three independent
experiments.
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Fig. 4.
Location of homo-oligomerization domain in
conserved central region of CARM1. A,
[35S]methionine-labeled CARM1 or its deletion mutants
were incubated with either GST or GST-CARM1 fusion protein preloaded on
glutathione-coupled beads. Bound proteins were eluted and analyzed as
in Fig. 2. The input lanes represent 10% of each
35S-labeled protein in the binding assay. B, CV1
cells were transiently transfected with 250 ng of GK1 reporter plasmid,
125 ng of pM vector encoding Gal4DBD or Gal4DBD fused to wild type or
mutant CARM1, and 125 ng of pVP16 vector encoding VP16-CARM1 as
indicated. Luciferase activity of the transfected cell extracts was
determined. The results presented are from a single experiment
representative of two independent experiments.
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Fig. 5.
The CARM1 C-terminal region contains an
autonomous activation domain. CV1 cells were transiently
transfected with 250 ng of GK1 reporter plasmid, 400 ng of pM vector
encoding Gal4DBD or increasing amounts (100, 200, and 400 ng) of
vectors encoding Gal4DBD fused to CARM1 or its deletion mutants.
Luciferase activity of the transfected cell extracts was determined.
Note the different scale on the y-axis of the right
panel. The results shown are from a single experiment
representative of three independent experiments.
-galactosidase
reporter gene, which was tested in a parallel experiment (lower
panel). CARM1-C also inhibited the autonomous transactivation
activity of full-length CARM1 fused to Gal4 DBD (Fig. 6B,
left panel). In contrast full-length CARM1, used as a
positive control, enhanced the activity of the Gal4-CARM1 fusion
protein, presumably through homo-oligomerization. In a mammalian
two-hybrid assay, the interaction of Gal4-CARM1 with VP16-CARM1 was
only slightly inhibited by CARM1-C (Fig. 6B, right
panel), consistent with our previous finding that CARM1-C cannot
bind to full-length CARM1 (Fig. 4). Thus, the negative effect of
CARM1-C on the autonomous activation activity of CARM1 is not caused by
a disruption of the homo-oligomer but could rather be because of a
competition of CARM1-C with CARM1 for the interaction with another
transcription factor that binds CARM1-C.
View larger version (17K):
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Fig. 6.
Dominant negative effect of CARM1-C on the
coactivator function of CARM1. A, CV1 cells were
transiently transfected with 250 ng of MMTV(ERE)-LUC reporter plasmid
(upper panel) or RSV. -gal reporter plasmid (lower
panel); expression vectors encoding ER (5 ng) and GRIP1 (125 ng);
and 400 ng of a pSG5.HA vector encoding full-length CARM1 or CARM1-C
(residues 461-608) or both. Transfected cells were grown in culture
medium with 20 nM estradiol (E2) or with vehicle
(C), and extracts of the harvested cells were tested for
luciferase or -galactosidase activity. B, CV1 cells were
transiently transfected with 250 ng of GK1 reporter plasmid, 250 ng of
pM vector encoding Gal4DBD or Gal4DBD fused to CARM1, 250 ng of pVP16
vector encoding VP16-CARM1 (right panel only), and 400 ng of
pSG5.HA empty vector (C) or pSG5.HA vector encoding CARM1 or
CARM1-C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helices and -strands. The
C-terminal part of the core forms an elongated 9-stranded -barrel
structure. Homodimerization is apparently required to form an active
enzyme. The dimer interface is formed by reciprocal contact between the
/ region of one monomer and a tri-helical arm extending from the
surface of the -barrel structure of the other monomer. The AdoMet
binding pocket is formed by the / region. The arginine residue of
the protein substrate binds in an acidic pocket containing two
glutamate residues, which interact directly with the two terminal amino
groups of the arginine side chain. The portions of the protein
substrate surrounding the target arginine residue are predicted to fit
in a groove between the / region and the -barrel structure
(29, 39).
View larger version (44K):
[in a new window]
Fig. 7.
CARM1 structural and functional domains.
Regions of CARM1, which are conserved or unique among the PRMT family
are indicate by shading. Structural and functional features determined
from x-ray crystallography studies of PRMT3 and Rmt1/Hmt1 (29, 39) have
been superimposed on the homologous regions of CARM1 in the diagram;
these features are labeled inside of the diagram or indicated below the
diagram by the boxes with vertical stripes. The
minimum fragments retaining specific functions as determined in the
current study are indicated by the hatched boxes. All
numbers refer to the amino acid residues of mouse CARM1.
-strand of the -barrel structure, eliminated the methyltransferase activity and thus demonstrated that
the entire conserved barrel structure is required for methyltransferase activity. The C-terminal -strand may contribute to structural integrity of the entire domain or could help to form the protein substrate-binding groove. The same deletion mutant retained
partial-to-full homo-oligomerization and GRIP1 binding activity (Figs.
2 and 4), indicating that structural integrity was not completely
disrupted. Our finding that the central conserved region of CARM1 also
contained the GRIP1 binding activity (Figs. 2 and 7) is consistent with previous findings that multiple members of the PRMT family can bind to
the C-terminal region of GRIP1 (25, 36). The GRIP1 binding activity of
CARM1 is undoubtedly required for the coactivator function of CARM1,
since we previously showed that the presence of GRIP1 and its
C-terminal CARM1-binding region are required for the coactivator
function of CARM1 (23, 24).
| |
ACKNOWLEDGEMENT |
|---|
We thank Daniel Gerke for technical assistance.
| |
FOOTNOTES |
|---|
* This work was supported by United States Public Health Service Grant DK55274 from the National Institutes of Health (to M. R. S.).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.
§ These authors contributed equally.
¶ Present address: Deltagen, Inc., 1003 Hamilton Court, Menlo Park, CA 94025-1422.
** To whom correspondence should be addressed: Dept. of Pathology, HMR 301, University of Southern California, 2011 Zonal Ave., Los Angeles, CA 90089-9092. Tel.: 323-442-1289; Fax: 323-442-3049; E-mail: stallcup@usc.edu.
Published, JBC Papers in Press, September 25, 2002, DOI 10.1074/jbc.M207623200
2 C. Teyssier and M. R. Stallcup, unpublished results.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
NR(s), nuclear receptor(s);
AD, activation domain;
AdoMet, S-adenosyl-L-methionine;
AF, activation
function;
CARM1, coactivator-associated arginine methyltransferase 1;
CARM1-C, CARM1 amino acids 461-608;
DBD, DNA binding domain;
CBP, CREB
(cAMP-response element-binding protein)-binding protein;
ER, estrogen
receptor ;
ERE, estrogen response element;
GRIP1, glucocorticoid
receptor interacting protein 1;
GST, glutathione
S-transferase;
HA, hemagglutinin A;
LUC, luciferase coding
region;
MMTV, mouse mammary tumor virus promoter;
PRMT, protein
arginine methyltransferase.
| |
REFERENCES |
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ABSTRACT
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RESULTS
DISCUSSION
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