Requirement for multiple domains of the protein arginine

Requirement for multiple domains of the protein arginine methyltransferase CARM1 in

its transcriptional coactivator function

CATHERINE TEYSSIER1,‡, DAGANG CHEN1,‡,# AND MICHAEL R.

STALLCUP1,2*

Departments of Pathology1 and of Biochemistry and Molecular Biology2, University of

Southern California, Los Angeles, California 90089.

Running title: Functional sub-domains of coactivator CARM1

‡ These authors contributed equally.

# Current address: Deltagen, Inc., 1003 Hamilton Court, Menlo Park, CA 94025-1422

*Corresponding author

Michael R. Stallcup

Department of Pathology, HMR 301

Univ. of Southern California

2011 Zonal Ave.

Los Angeles, CA 90089-9092

Teyssier et al 9-10-02

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Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 25, 2002 as Manuscript M207623200 by guest on A

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Phone: (323) 442-1289; Fax: (323) 442-3049

E-mail: [email protected]


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ABSTRACT

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 the 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-terminal

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.


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INTRODUCTION

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


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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 Arg17 and Arg26 in vitro (26), and chromatin immunoprecipitation

studies indicated 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 homo-dimeric 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-terminus

and C-terminus. The contribution of these additional domains to the function of the

PRMTs is unknown, although some studies suggested that they may be involved in the


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specificity of protein substrate binding (29,30,33).

Previous studies suggested 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-terminal 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.

MATERIALS AND METHODS

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) α (34); the

luciferase reporter plasmids MMTV(ERE)-LUC (20) and GK1, which is controlled by Gal4

response elements (10); and the β-galactosidase (β-gal) reporter plasmid RSV.β-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


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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).

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% NP-40, 1 mM EDTA, 20 mM Tris pH 8.0,

100 mM NaCl) containing Complete protease inhibitor cocktail (Roche).

Cell Culture and Transfection. Transfections of CV-1 cells (37) were perfomred 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 β-galactosidase activities are shown as the mean and range of

variation of two transfected cell cultures.

Immunoprecipitation of CARM1 from transfected cells and in vitro methylation

assays. COS7 cells (37) were maintained in DMEM supplemented with 10% fetal bovine

serum. One million cells were seeded into 10-cm diameter cell dishes and transfected

with Superfect (Qiagen) according to manufacturer’s protocol with 5 µg of pSG5.HA

plasmid encoding CARM1 wild type or CARM1 mutants. After transfection, cells were

grown in DMEM with 10% fetal bovine serum for 40 h before harvest. Cells were


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harvested in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.1%

NP40 and Complete protease inhibitor cocktail (Roche). Cell lysates were frozen at -80

°C, thawed on ice, and clarified by centrifugation before incubation overnight at 4 °C

with monoclonal antibody (clone 3F10, Roche) against HA tag. Pre-blocked protein G-

Sepharose (Amersham Pharmacia Biotech) was then added for 2 h at 4 °C.

Immunoprecipitates were recovered by rapid centrifugation, washed three times with

NETN containing 0.1% NP40 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) was incubated with immunoprecipitated

methyltransferases and 7 µM S-adenosyl-L-[3H-methyl]methionine (specific activity

14.7 Ci/mmol) in 30 µl 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.

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 (Roche) 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.

RESULTS

Domains of CARM1 involved in coactivator activity. To determine which parts of

CARM1 are required for its coactivator function, N-terminal and C-terminal truncations

were made near the boundaries between the conserved central domain of CARM1 and its


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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.


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Thus the unique N-terminal 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-terminal 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 to 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 to


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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 [3H-methyl]AdoMet; methylated histone H3 was detected by SDS-PAGE

and fluorography (Fig. 3). The mutant lacking the N-terminal part of CARM1 (1-120) or

the C-terminal region (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-

terminal 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


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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-terminal 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 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 to 608 totally


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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, over-expression 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 β-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

due to a disruption of the homo-oligomer but could rather be due to a competition of


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CARM1-C with CARM1 for the interaction with another transcription factor that binds

CARM1-C.

DISCUSSION

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 which

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 α-helices and β-strands. The C-terminal part of the core forms an

elongated nine-stranded β-barrel structure. Homo-dimerization is apparently required to

form an active enzyme. The dimer interface is formed by reciprocal contact between the

α/β region of one monomer and a tri-helical arm extending from the surface of the β-

barrel structure of the other monomer. The AdoMet binding pocket is formed by the α/β

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 α/β region and the β-barrel structure

(29,39).

As expected from the three-dimensional structure and the high degree of

conservation among PRMT members, our studies located the methyltransferase and


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homo-oligomerization activities of CARM1 approximately within the conserved region

(amino acids 150-480 of CARM1) (Figs. 3, 4, & 7). A C-terminal deletion to amino acid

460, which removed the last β-strand of the β-barrel structure, eliminated the

methyltransferase activity and thus demonstrated that the entire conserved barrel structure

is required for methyltransferase activity. The C-terminal β-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 & 4), indicating that structural integrity was not

completely disrupted. Our finding that the central conserved region of CARM1 also

contained the GRIP1 binding activity (Fig. 2 & 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).

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


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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-terminal and C-terminal regions of CARM1 in its coactivator

function. Deletion of the unique N-terminal 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-terminal 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-

terminal 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 (our unpublished

results). 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 the GRIP1


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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


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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).

ACKNOWLEDGMENTS.

We thank Daniel Gerke for technical assistance. This work was supported by United

States Public Health Service Grant DK55274 from the National Institutes of Health to

M.R.S.


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FOOTNOTES

1 The abbreviations used are: AD, activation domain; AdoMet, S-adenosyl-L-

methionine; AF, activation function; β-gal, β-galactosidase; 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 α; 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; NR(s), nuclear receptor(s); PRMT, protein arginine methyltransferase; RLU,

relative lights units.

FIGURE LEGENDS

FIG. 1. Coactivator function of CARM1 and its deletion mutants with ERα. A, 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


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ng; wild type or mutant CARM1, 250 ng; and p300, 250 ng. The results presented are

from a single experiment representative of 4 independent experiments.

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-hand 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.

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 [3H-methyl]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. 2A. 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.

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-hand panel. The results shown are from a single experiment representative of three

independent experiments.

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.β-gal reporter plasmid (lower panel); expression vectors


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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 β-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.

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 boxes with diagonal

stripes. All numbers refer to the amino acid residues of mouse CARM1.


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Catherine Teyssier, Dagang Chen and Michael R. Stallcupin its transcriptional coactivator function

Requirement for multiple domains of the protein arginine methyltransferase CARM1

published online September 25, 2002J. Biol. Chem.

10.1074/jbc.M207623200Access the most updated version of this article at doi:

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Requirement for multiple domains of the protein arginine