From the Department of Pathology and the
¶ Department of Biochemistry and Molecular Biology, University of
Southern California, Los Angeles, California 90089
Received for publication, May 17, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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Nuclear receptors (NRs) activate gene
transcription by binding to specific enhancer elements and recruiting
coactivators of the p160 family to promoters of target genes. The p160
coactivators in turn enhance transcription by recruiting secondary
coactivators, including histone acetyltransferases such as CREB-binding
protein (CBP) and p300/CBP-associated factor (p/CAF), as well as the
recently identified protein methyltransferase, coactivator-associated
arginine methyltransferase 1 (CARM1). In the current study, protein
arginine methyltransferase 1 (PRMT1), another arginine-specific protein methyltransferase that shares a region of high homology with CARM1, was
also found to act as a coactivator for NRs. PRMT1, like CARM1, bound to
the C-terminal AD2 activation domain of p160 coactivators and thereby
enhanced the activity of NRs in transient transfection assays. The
shape of the graphs of reporter gene activity versus the
amounts of CARM1 or PRMT1 expression vector indicated a cooperative relationship between coactivator concentration and activity. Moreover, CARM1 and PRMT1 acted in a synergistic manner to enhance reporter gene
activation by both hormone-dependent and orphan NRs. The synergy was most evident at low levels of transfected NR expression vectors, where activation of reporter genes was almost completely dependent on the presence of NR and all three exogenously supplied coactivators, i.e. GRIP1, CARM1, and PRMT1. In contrast,
with the higher levels of NR expression vectors typically used in
transient transfection assays, NR activity was much less dependent on
the combination of coactivators, suggesting that target gene activation occurs by different mechanisms at high versus low cellular
concentrations of NR. Because multiple coactivators are presumably
required to mediate transcriptional activation of native genes in
vivo, the low-NR conditions may provide a more physiologically
relevant assay for coactivator function.
Nuclear receptors (NRs)1
belong to a large superfamily of transcriptional activator proteins
that include the receptors for steroid and thyroid hormones, retinoids,
and vitamin D (1-4); the family also includes so-called orphan
receptors for which an activating ligand is unknown or not required (5,
6). NRs contain a highly conserved DNA binding domain (DBD) in the central region of the polypeptide chain that binds specific enhancer elements in the promoters of target genes. Activation of transcription is directed by two transcriptional activation domains: AF-1, located in
the N-terminal end, and AF-2, located within the C-terminal hormone-binding domain (7-10). Transcriptional activation
involves two general mechanisms. Alteration of the chromatin structure around the promoter by ATP-dependent nucleosome remodeling
and histone modification facilitates the recruitment of the RNA
polymerase II transcription preinitiation complex (11-13). The NRs
also make direct or indirect physical contact with components of the
transcription preinitiation complex (14, 15). Activation is
accomplished with the help of complexes of coactivator proteins that
bind to the activation domains of the NRs (13, 16).
A growing list of potential coactivators has been defined primarily by
virtue of their ability to enhance the function of NRs in transient
transfection experiments (13, 16-19). Among the most well
characterized of NR coactivators are a family of three related 160-kDa
proteins called the p160 coactivators (SRC-1, GRIP1/TIF2, and
pCIP/RAC3/ACTR/AIB1/TRAM1). The p160 coactivators contain a
cluster of three conserved NR box motifs (LXXLL,
where L is leucine and X is any amino acid), located
approximately in the middle of the ~1400-amino acid polypeptide
chain. The NR boxes bind to NR AF-2 regions by fitting into a conserved
hydrophobic cleft on the surface of essentially all NRs that act as
transcriptional activators (20-22). In addition, the C terminus of the
p160 coactivators binds to the AF-1 regions of some but not all NRs
(23-25).
After binding to the activated NR through at least one of these two NR
interaction domains, the p160 coactivators propagate the activating
signal through at least two activation domains, AD1 and AD2, which act
by recruiting a number of secondary coactivator proteins. AD1, located
near amino acid 1000, binds p300 and CREB-binding protein (CBP), two
related proteins that bind to and serve as coactivators for a large
number of DNA-binding transcriptional activator proteins, including NRs
(26-28). CBP and p300 can acetylate histones and have been associated
with chromatin remodeling (29). In addition, their ability to acetylate
nonhistone proteins (30, 31) and the ability of even
acetyltransferase-negative mutants to act as coactivators in some
settings (28) suggest that they contribute to transcriptional
activation through multiple molecular mechanisms. For example, CBP and
p300 associate with p300/CBP-associated factor (p/CAF), another
coactivator with protein acetyltransferase activity (26). They also
interact with components of the basal transcription machinery and thus
may help to recruit the transcription preinitiation complex
to the promoter (32, 33).
We recently demonstrated that AD2, located at the C terminus of p160
proteins, can also transmit the activating signal received from NRs
(25). The action of AD2 is independent of AD1 and requires neither CBP
nor p300. We also identified a novel protein, coactivator-associated arginine methyltransferase 1 (CARM1), which binds to AD2 and acts as a
secondary coactivator for NRs, i.e. its effect on NR
function is totally dependent upon the presence of a primary
coactivator in the form of a p160 coactivator (34). By amino acid
sequence homology, CARM1 belongs to a family of previously identified
arginine-specific protein methyltransferases, which includes the
mammalian proteins, protein arginine methyltransferase 1 (PRMT1),
PRMT2, and PRMT3, and the yeast protein arginine methyltransferase 1 (RMT1) (34).
PRMT1 and CARM1 are very different in size, but they share a high
degree of sequence homology in the central region, which contains the
arginine-specific protein methyltransferase activity (34) (Fig.
1). The coactivator function of CARM1 and
the homology between CARM1 and PRMT1 led us to test whether PRMT1, like
CARM1, can bind to p160 coactivators and function as a coactivator for NRs. We also investigated the functional relationship between PRMT1 and
p160 coactivators and the possibility of synergistic enhancement of NR
function by p160 coactivators, CARM1, and PRMT1.
Plasmids--
Proteins with N-terminal hemagglutinin A (HA)
epitope tags were expressed in transient mammalian cell transfections
and in vitro from pSG5.HA, which has SV40 and T7 promoters
(34). The following proteins were expressed from previously described
pSG5.HA derivatives: GRIP1
Other previously described mammalian expression vectors were as
follows: pHE0 encoding estrogen receptor (ER) Protein-Protein Interaction in Vitro--
GST pull-down assays
were conducted essentially as described previously (42, 43). GST fusion
proteins were isolated from Escherichia coli BL21 (CARM1) or
DH5 Cell Culture and Transfection--
CV-1 cells (45) were
transfected in 6-well culture dishes, and cell extracts were assayed
for luciferase and Binding of PRMT1 to p160 Coactivators and NRs--
Because CARM1
binds specifically to the C-terminal region of p160 coactivator GRIP1
(34), we tested for a similar binding activity of the homologous
protein, PRMT1. When a GST-PRMT1 fusion protein was affixed to
glutathione-agarose beads, it specifically bound all three members of
the p160 coactivator family (GRIP1, SRC-1a, and ACTR), which were
synthesized in vitro in the presence of
[35S]methionine (Fig.
2A). In contrast, the p160
coactivators did not bind the control protein, GST. In similar GST
pull-down assays, PRMT1 specifically bound the C-terminal AD2 region of
GRIP1 (amino acids 1121-1462) (Fig. 2B), which was
previously shown to bind CARM1 (34). Also, like CARM1, PRMT1 failed to
bind an N-terminal fragment of GRIP1 (amino acids 5-765) containing
the NR box motifs or a central fragment (amino acids 730-1121)
containing NR box III and AD1 (Fig. 2B). The N-terminal and
central fragments thus served as negative controls.
GST pull-down assays also revealed specific protein-protein interaction
of GST-PRMT1 with three different NRs, i.e. ER Synergistic, p160-dependent Enhancement of NR Function
by PRMT1 and CARM1--
CARM1 was previously shown to function as a
secondary coactivator for NRs in transient transfection assays,
i.e. it enhanced NR function through its association with
the C-terminal AD2 region of p160 coactivators (34). Because PRMT1
shares extensive sequence homology with CARM1 and can bind to
the AD2 region of p160 coactivators and to NRs as well, we tested PRMT1
for coactivator function with ER, TR, and AR. CV-1 cells were
transiently transfected with an NR expression vector, a corresponding
luciferase reporter gene (driven by a mouse mammary tumor virus (MMTV)
promoter containing the natural hormone response elements for AR or
engineered enhancer elements for ER or TR), and expression vectors for
various coactivators. Transfected cells were grown with the appropriate
hormone for the NR used, and luciferase activity was determined from
the transfected cell extracts. In the presence of a hormone-activated
NR and GRIP1, PRMT1 enhanced reporter gene activity (Fig.
3). For example, transient expression of
AR caused hormone-dependent reporter gene activity (Fig.
3C, histograms 1 and 2), and GRIP1
enhanced that activity 10-fold (histogram 3). Cotransfection
of PRMT1 plasmid DNA caused a further 3-fold enhancement
(histogram 4). Similar results were seen with ER
When fixed amounts of reporter gene, NR vector, and p160 vector were
cotransfected with varying amounts of PRMT1 or CARM1 expression vector,
both CARM1 and PRMT1 enhanced reporter gene activity in a
dose-dependent manner (Fig.
4, A-C, graphs a and b) and had approximately equivalent coactivator effects on
NR function. The range of CARM1 and PRMT1 vectors tested produced upward curving graphs, i.e. the slopes of the dose-response
curves increased along with the amounts of CARM1 and PRMT1 vector used, suggesting a cooperative relationship between coactivator concentration and activity. When the reporter gene activity was replotted on a
logarithmic scale, the data fit straight lines, consistent with a
cooperative dose-response curve (Fig. 4E shows data for AR
replotted from Fig. 4C). Similar coactivator effects of
PRMT1 were also observed when other members of the p160 coactivator
family, SRC-1a or ACTR, were substituted for GRIP1 (Fig.
5, graphs a-c).
Because both PRMT1 and CARM1 acted as secondary coactivators for NRs,
we tested for additive or synergistic coactivator effects on NR
function when both proteins were expressed together with a p160
coactivator in the transient transfection assays. In the presence of
the reporter gene, NR, hormone, and GRIP1, varying doses of PRMT1
plasmid DNA were cotransfected with a fixed amount of CARM1 plasmid DNA
(0.5 µg) and vice versa (Fig. 4, graphs c and
d). The effects of combining CARM1 and PRMT1 were
synergistic. For example, in transfections with AR, adding 0.5 µg of
CARM1 DNA and 0.5 µg of PRMT1 DNA together (Fig. 4D, histogram
5) caused a synergistic enhancement of activity when compared with
the sum of their separate effects at the same doses (histograms
3 and 4). In other words, compared with the activity
observed with NR plus GRIP1 and no methyltransferase expression vectors
(histogram 2), the increase in activity achieved by the
simultaneous addition of CARM1 and PRMT1 (histogram 5 minus
histogram 2) was considerably greater than the sum of the
enhancements caused by adding CARM1 and PRMT1 in separate assays
(histogram 3 minus histogram 2 plus histogram 4 minus histogram 2).
Similar synergistic effects were observed for PRMT1 and CARM1 when
ER Synergy between CARM1 and PRMT1 Depends upon NR Levels--
The
coactivator effects of CARM1 and PRMT1, expressed separately or
together, were examined at different levels of AR by varying the amount
of transfected AR expression vector and fixing the amounts of all
coactivator expression vectors (Fig. 6).
At lower levels of AR expression vector (0.1 and 0.3 µg), the
addition of either CARM1 or PRMT1 without the other (graphs
c and d) produced a relatively small enhancement over
the activity observed with AR plus GRIP1 (graph b); however,
CARM1 and PRMT1 added together (graph e) had a strong
synergistic coactivator effect. In contrast, at higher levels of AR
expression vector (0.6 and 1.0 µg), CARM1 or PRMT1 each enhanced
reporter gene activity (graphs c and d) above the
level observed with AR plus GRIP1 (graph b), but
adding CARM1 and PRMT1 together produced no further increase
(graph e).
Because the degree of synergy increased at the lower levels of AR, we
extended the analysis to even lower levels (0.01 µg) of AR expression
vector. Under these conditions, the activity of hormone-activated AR
was extremely low (Fig. 7,
histogram 1) compared with the activity at higher levels of
AR expression vector (Fig. 6, graph a). Coexpression of
GRIP1 did not enhance the reporter gene activation observed with 0.01 µg of AR vector (Fig. 7, compare histograms 1 and
5). In the presence of GRIP1, coexpression of either CARM1
or PRMT1 produced a modest 2-5-fold enhancement (compare histograms 6 and 7 with 5) which was
GRIP1-dependent (compare histograms 6 and
7 with 2 and 3, respectively).
Simultaneous coexpression of all three coactivators (GRIP1, CARM1, and
PRMT1) produced a dramatic, synergistic, 35-fold enhancement of
reporter gene activity (histogram 8). This activity was
almost completely abolished by omission of any one of the three
coactivators (compare histogram 8 with histograms 4, 6, and 7) or AR (data not shown). Similar synergy among
these three coactivators and a similar dependence of synergy on low
levels of NR expression vector were observed with ER CARM1 and PRMT1 Coactivator Function Requires GRIP1 with a
Functional AD2 Domain--
The coactivator effects of CARM1 and PRMT1
on NRs required the presence of a p160 coactivator. The fact that CARM1
and PRMT1 both bind to the C-terminal AD2 region of GRIP1 suggests that this physical interaction is required for enhancement of NR function by
CARM1 and PRMT1. To test this hypothesis, GRIP1 deletion mutants lacking AD1, AD2, or both regions were tested with AR, CARM1, and PRMT1
in the transient transfection system.
As described above, wild type GRIP1 functioned in a highly synergistic
manner with CARM1 and PRMT1 when 0.01 µg of AR expression vector was
used (Fig. 7, histograms 1-8). The results observed with
GRIP1 Synergistic Action of CARM1 and PRMT1 on Orphan NRs--
AR, ER,
and TR represent Class I (AR and ER) and Class II (TR) NRs for which
known ligands are required to activate the receptor by inducing an NR
conformation that can interact with coactivators (13). A third class of
NRs are the orphan NRs, for which ligands are unknown or not required
for activation (5, 6). Orphan NRs that function as transcriptional
activators generally have AF-2 domains that share homology with those
of the hormone activated NRs and bind p160 coactivators, and the p160
coactivators can thus enhance transcriptional activation by the orphan
NRs (35, 39, 47). We therefore tested the ability of CARM1 and PRMT1 to
serve as synergistic secondary coactivators for one subfamily of orphan
NRs, which consists of ERR1, ERR2, and ERR3. As indicated by their
name, the ERRs are related to the ERs both in sequence and in function
(35, 48, 49). They do not bind estrogen, but they can bind to and
activate transcription from estrogen response elements and SF-1
(steroidogenic factor-1) response elements, and they also bind p160
coactivators in vitro and in vivo. All of these
activities occur in the absence of any added ligand, indicating that
the ERRs are constitutively active (i.e. ligand-independent) transcriptional activators.
Transient transfection experiments were performed with low amounts
(0.01 µg) of ERR expression vectors, since these conditions with the
hormone binding NRs were shown to optimize coactivator synergy (Figs. 6
and 7) and may give rise to NR concentrations closer to physiological
levels than those resulting from the use of higher levels (0.1-1 µg)
of NR expression vectors. In tests with GRIP1, CARM1, and PRMT1, these
conditions produced patterns of coactivator synergy for the ERRs (Fig.
8A) very similar to the
pattern observed for AR (Fig. 7). The reporter gene activity stimulated by each ERR, in the absence of coexpressed coactivators, was
very low (Fig. 8A, histograms 1) and was indistinguishable from the activity of the reporter gene in the absence of ERRs (compare
histograms 1 and 9). Coexpression of any single
coactivator with the ERR caused a negligible (less than 2-fold)
increase of reporter gene activity (histograms 2, 6, and
7). The addition of GRIP1 expression vector with either
CARM1 or PRMT1 expression vector caused a moderate 2-6-fold
enhancement of the low basal activity (histograms 3 and
4). As with the hormone-binding NRs, a combination of GRIP1,
CARM1, and PRMT1 vectors caused a dramatic enhancement of activity
(histogram 5). The activity in all of these cases was
entirely dependent on the presence of GRIP1 and ERR vectors
(histograms 6-11). Thus, at low ERR levels, GRIP1, CARM1, and PRMT1 together caused a dramatic and synergistic enhancement of reporter gene activity that was almost entirely dependent on the
presence of all three coactivators.
In the experiments performed with ERR3 (Fig. 8A), an
internal control for transfection efficiency was performed by including in the transfections a Synergistic Enhancement of GRIP1 Activity by PRMT1 and
CARM1--
Although PRMT1 bound NRs as well as GRIP1, the ability of
PRMT1 and CARM1 to enhance NR function depended on the presence of a
p160 coactivator. These results suggested that CARM1 and PRMT1 enhanced
NR function by enhancing or mediating the coactivator activity of
GRIP1. We therefore tested whether PRMT1 and/or CARM1 could directly
enhance GRIP1 activity in an assay with no NR involvement. When CV-1
cells were transiently transfected with GAL4 DBD fused to GRIP1, there
was weak activation of the GK1 luciferase reporter gene, which
contained Gal4 response elements; this activity was severalfold higher
than the activity observed with Gal4 DBD alone (Fig.
9B, histograms 1 and
2). Coexpression of PRMT1 with Gal4DBD-GRIP1 enhanced
reporter gene activity, although relatively high quantities of PRMT1
expression vector were required for a moderate effect (Fig. 9A,
lower graph). CARM1 and PRMT1 together caused a synergistic enhancement of GRIP1 activity, which depended on the dose of PRMT1 vector used (Fig. 9A, upper graph). For example, when used
separately, 0.5 µg of CARM1 vector and 0.5 µg of PRMT1 vector each
caused a 50% increase in reporter gene activity (Fig. 9B,
compare histograms 3 and 4 with
2). When expressed together, the same amounts of CARM1 and
PRMT1 vectors increased reporter gene activity more than 5-fold
(histogram 5). The synergistic enhancement of Gal4DBD-GRIP1 activity by PRMT1 and CARM1 was observed at all concentrations of
Gal4DBD-GRIP1 expression vector tested (Fig. 9C). When
Gal4DBD-GRIP1 was replaced by Gal4 DBD alone, the combination of CARM1
and PRMT1 caused a very small enhancement of activity, and the activity thus achieved was negligible compared with that observed with Gal4DBD-GRIP1 and the two methyltransferases (data not shown).
PRMT1 Had No Autonomous Activation Domain--
To begin examining
the mechanism of PRMT1 coactivator function, PRMT1 was fused to
Gal4-DBD. When this fusion protein was expressed in CV-1 cells, the
resulting activity of the GK1 reporter gene was no greater than that
observed with Gal4 DBD alone (Fig. 10,
histograms 1 and 3). In contrast, Gal4DBD-CARM1
had an activity almost 500 times higher than Gal4 DBD alone
(histogram 5). To test the functional integrity of the
Gal4DBD-PRMT1 expression vector, it was coexpressed with a VP16-PRMT1
fusion protein. This provided a two-hybrid test of the ability of PRMT1
to form homodimers or homo-oligomers, an ability that has been
demonstrated previously (50). Although VP16-PRMT1 and Gal4 DBD produced
no activity (histogram 2), the two PRMT1 fusion proteins
together produced a high reporter gene activity (histogram 4),
indicating that both fusion proteins produced PRMT1 proteins that can
form homo-oligomers.
Mechanism of PRMT1 Coactivator Function--
CARM1, a protein
methyltransferase, was recently identified as a new coactivator for NRs
(34). CARM1 bound to the C-terminal AD2 region of p160 coactivators and
enhanced NR function only in the presence of a p160 coactivator. These
findings suggested a model whereby p160 coactivators bind directly to
NRs and act as primary coactivators, whereas CARM1 is recruited
indirectly to the NR by binding to the C-terminal region of p160
coactivators and thus acts as a secondary coactivator. The evidence
presented here indicated that PRMT1, which shares sequence homology
with CARM1 and is also an arginine-specific protein methyltransferase, is also a coactivator for NRs and functions as a secondary NR coactivator in a manner similar to CARM1. Both PRMT1 and CARM1 enhanced
the function of ER, TR, AR, and ERRs, indicating their ability to act
as coactivators for a wide range of NRs, including hormone-activated as
well as hormone-independent NRs. Like CARM1, PRMT1 bound to the
C-terminal region of p160 coactivators, and its enhancement of NR
function depended on the presence of a p160 coactivator with an intact
AD2 region, which is the binding site for CARM1 and PRMT1. Thus,
although PRMT1 can bind directly to NRs in GST pull-down assays, this
binding interaction is obviously not sufficient for the functional
enhancement of NRs by PRMT1 and thus at most might play a subsidiary
role to the required binding between PRMT1 and p160 coactivator. The
dependence on NR and any required activating hormone demonstrated that
CARM1 and PRMT1 were not directly activating the reporter gene but were acting as coactivators; and the dependence on a p160 coactivator indicated roles for CARM1 and PRMT1 as secondary rather than primary coactivators. The coactivator effects of CARM1 and PRMT1 were lost when
the C-terminal AD2 region of GRIP1 was deleted but not when the AD1
region (the binding site for CBP and p300) was deleted, indicating that
the C-terminal region of GRIP1 is essential for CARM1 and PRMT1
coactivator function. These data support the conclusion that the
interaction of PRMT1 with the C-terminal region of p160 is the key
binding interaction that recruits PRMT1 to the promoter. Further
support for these conclusions was provided by the ability of PRMT1, as
well as CARM1, to enhance the function of GRIP1 fused to Gal4DBD in the
absence of NRs.
Activated NRs bound to enhancer elements of target genes stimulate
initiation of transcription with the help of coactivators. How PRMT1
and CARM1 pass along the activating signal from NRs and p160
coactivators to the transcription machinery still remains to be
determined. Both CARM1 and PRMT1 can methylate histones in
vitro (34), suggesting nucleosome remodeling as one possible mechanism for their action. Histone methylation could theoretically cooperate with other types of histone modification by coactivators, including acetylation and phosphorylation, to remodel nucleosomes and
thus help recruit a transcription preinitiation complex (51). Of
course, just as acetylation of nonhistone proteins by coactivators may
also play a role in transcriptional activation (29-31), so methylation
of nonhistone proteins by PRMT1 and CARM1 must also be considered. In
addition to histones, PRMT1 can methylate a number of proteins,
including heterogeneous nuclear ribonucleoprotein A1, nucleolin,
fibrillarin, and poly(A)-binding protein II (52-54), which play
diverse roles in RNA metabolism, including post-transcriptional steps
of gene regulation. An increase in luciferase activity in transient
transfection assays could result from coactivator effects on the
efficiency of transcriptional activation or any subsequent step in gene
expression required to produce a functional protein. Recent studies
provide some examples of possible post-transcriptional mechanisms by
which protein methyltransferase-type coactivators could act. In yeast
arginine methylation of some heterogeneous nuclear
ribonucleoproteins has been linked to their nuclear export efficiencies, and a genetic relationship was found between the function
of mRNA cap-binding protein 80 and an arginine-specific protein
methyltransferase (55).
The protein acetylation activities of CBP, p300, and p/CAF are required
for their coactivator function with some transcriptional activators but
not with others (28). Thus CBP, p300, and p/CAF use multiple mechanisms
and multiple domains to transmit activating signals to the
transcription machinery. By analogy, PRMT1 and CARM1 may use mechanisms
in addition to or even instead of protein methylation to enhance
reporter gene expression. CARM1 exhibited an autonomous transcriptional
activation activity when fused to Gal4 DBD (Fig. 10); this activity is
located in a domain separate from the methyltransferase region,
suggesting the possibility that regions other than the
methyltransferase domain of CARM1 may contribute to the transmission of
the activating signal.2 In
contrast, full-length PRMT1 fused to Gal4 DBD failed to exhibit any
transcriptional activation activity (Fig. 10). Thus, PRMT1 is
apparently not capable of activating transcription by itself when
recruited or tethered to the promoter. The transmission of an
activating signal by PRMT1, whether by the methyltransferase domain or
some other domain, apparently requires action by another protein,
either to activate PRMT1 or to act in cooperation with PRMT1. To
understand the specific mechanisms of each methyltransferase-type coactivator, it will be important to map their functional domains and
investigate possible downstream targets that bind to or are methylated
by these coactivators.
Mechanism of Coactivator Synergy--
Although CARM1 and PRMT1
both act as arginine-specific protein methyltransferases and as
secondary coactivators for NRs, they are unlikely to be redundant in
their functions because of their extensive structural and functional
differences. CARM1 and PRMT1 share extensive regions of amino acid
homology, primarily in a 125-amino acid sequence in the central region
of their polypeptide chains (34) (Fig.
1). A smaller subregion believed to be
the S-adenosylmethionine binding region is also homologous
to other S-adenosylmethionine dependent methyltransferases
(e.g. DNA-methyltransferases) (52). Aside from the 125-amino
acid homology region and one much smaller region of homology (Fig. 1),
there is no convincing sequence homology between extensive portions of
the N- and C-terminal regions of these two proteins. In addition, CARM1
(608 amino acids) is much longer than PRMT1 (353 amino acids) and thus
has extensive regions with no counterpart in PRMT1. CARM1 has an
autonomous activation domain, whereas PRMT1 does not (Fig. 10).
Although CARM1 and PRMT1, like other protein arginine
methyltransferases, both transfer methyl groups from
S-adenosylmethionine to specific arginine residues of
specific target proteins, their protein substrate specificities
(i.e. proteins that they methylate efficiently) are quite
different. The only known common substrate is histone H2A (34). In
contrast, CARM1 methylates histone H3, but PRMT1 does not; PRMT1 but
not CARM1 methylates heterogeneous nuclear ribonucleoprotein A1,
histone H4, and an arginine residue in a glycine-rich peptide
representing a region of fibrillarin (34, 52, 53). Thus, substantial
indirect evidence suggests that although CARM1 and PRMT1 share regions
of extensive homology, they also have extensive regions with no
homology and have distinct functions and modes of action. The fact that
their coactivator activities for NRs are synergistic also indicates
that they rely on different mechanisms for their coactivator activities.
One attractive hypothesis to explain the synergistic coactivator
functions between CARM1 and PRMT1 is their abilities to methylate different proteins in the coactivator complex, chromatin, or other components of the transcription machinery or RNA processing machinery. Their complementary abilities to methylate histones H3 and H4 suggest
one possible mechanism of synergy, but methylation of other proteins
could also play a role. Methylation of histones may cooperate with
acetylation and other types of covalent histone modifications to
remodel chromatin structure and/or provide a signal to facilitate
assembly of the active transcription initiation complex (51). Of
course, the synergistic coactivator function of CARM1 and PRMT1 could
involve other types of complementary mechanisms in addition to or
instead of protein methylation, such as interactions with other
components of the transcription or RNA processing machinery. Further
work will be required to characterize the domains of CARM1 and PRMT1
required for binding to p160 coactivators, for protein methylation, and
for transmitting the activating signal to the transcription machinery.
A cooperative relationship was observed between reporter gene activity
and the amount of CARM1 or PRMT1 expression vector used in
transfections (Fig. 4). Such a cooperative dose-response curve may
suggest that multimers of these methyltransferases function more
efficiently as coactivators than monomers or that the binding or action
of one CARM1 or PRMT1 molecule facilitates the binding or action of a
second CARM1 or PRMT1 molecule. Such a mechanism could account for the
cooperative curves observed as well as the synergy between CARM1 and
PRMT1. The precise mechanisms remain to be elucidated, but previously
published results and our preliminary data indicate that CARM1
and PRMT1 can form homo-oligomers (50) (Fig. 10 and
footnote 3). It should be stated that
although we have demonstrated a requirement for p160 and
methyltransferase-type coactivators in these assays, other coactivators
endogenous to CV-1 cells (such as CBP, p300, and p/CAF) may also be
necessary for the observed NR function.
Significance of Coactivator Synergy--
When low levels of NR
expression vectors were used in the transient transfection assays, a
remarkable degree of synergy was observed among the NRs and three
different coactivators (GRIP1, CARM1, and PRMT1) such that if any one
of these four components was not expressed, there was an almost
complete loss of reporter gene activity (Figs. 7-8). In other words,
the activity of low levels of NRs was almost undetectable unless GRIP1,
CARM1, and PRMT1 were all coexpressed with the NR. This extremely high
level of synergy was observed only when very low levels of NR
expression vectors were used. In contrast, at the higher levels of
transfected NR expression vectors more typically used in transient
transfection assays, the effects of the NRs were readily observable in
the absence of any coexpressed coactivators; p160 coactivators alone or
p160 coactivator combined with either CARM1 or PRMT1 caused moderate
enhancement of reporter gene activity (Fig. 6). However, the addition
of a third coactivator failed to cause further stimulation. How should
one interpret these dramatically different levels of requirement for
coactivators? Because transient transfections generally result in
higher than physiological levels of expression, the lower NR
concentrations achieved by transfecting very low amounts of NR
expression vector may be closer to the physiological levels of NR. Thus
it is possible that some cells expressing low endogenous levels of NRs
may require the presence of all three of these coactivators (GRIP1,
CARM1, and PRMT1), as well as other coactivators not tested for in
these studies, for NRs to activate their target genes efficiently. In
contrast, high levels of NRs appear capable of activating transcription
with less help from coactivators (i.e. with help from fewer
coactivators and/or lower levels of coactivators).
In this report, we have identified a new coactivator for NRs (PRMT1),
demonstrated synergy between p160 coactivators and two different
methyltransferase-type coactivators, determined specific physical
interactions between p160 and methyltransferase coactivators that
underlie this synergy, and defined conditions that make NR function
almost entirely dependent upon the presence of three different
coactivators (GRIP1, CARM1, and PRMT1). Although many putative
coactivators for NRs have been reported, most have been studied
separately from other coactivators. However, the current state of
knowledge suggests that many coactivators may need to cooperate to
mediate the process of transcriptional activation. Our finding of
conditions where multiple coactivators are required for NR function is
consistent with this prediction and furthermore provides a new set of
experimental conditions that may be important and valuable for
investigating coactivator function. Thus, for future coactivator
studies it may prove advantageous to use in parallel two sets of
experimental conditions: one with higher levels of NR expression
vector, where effects of individual coactivators and pairs of
coactivators can be observed; and a second set of conditions with lower
levels of NR expression vectors, where there is a high degree of
requirement for multiple coactivators. We suggest that the conditions
producing a requirement for multiple coactivators may more closely
resemble the physiological state. It will also be important to search
for other coactivator combinations that exhibit synergy and for other
experimental conditions that may allow a dependence on higher numbers
of coactivators.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Homology between PRMT1 and CARM1.
Regions of homology (black boxes) are indicated along with
the percentages of amino acids that are identical between PRMT1 and
CARM1 within each homology region.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
AD1 (full-length GRIP1 with amino acids
1057-1109 deleted) (25); ERR3 (35); GRIP1, SRC-1a, GRIP1-(5-765),
GRIP1-(730-1121), GRIP1-(1121-1462), GRIP1-(5-1121), and CARM1 (34).
Vector pSG5.HA-GRIP1
AD1/
AD2 was constructed using the Promega
Gene Editor kit to delete codons 1057-1109 from pSG5.HA-GRIP1
AD2, which encodes GRIP1-(5-1121). A polymerase chain
reaction-amplified cDNA fragment encoding full-length PRMT1 was
cloned into the EcoRI-BamHI sites of pSG5.HA. The
same EcoRI-BamHI fragment encoding PRMT1 was
cloned into vector pM (CLONTECH) for expression of
Gal4DBD-PRMT1 and into pVP16 (CLONTECH) for
expression of VP16-PRMT1. Vectors encoding Gal4DBD-GRIP1-(5-1462)
(full-length) and Gal4DBD-CARM1 were constructed by inserting an
EcoRI fragment encoding GRIP1 and an
EcoRI-BglII fragment encoding CARM1 into the
EcoRI and EcoRI-BamHI sites, respectively, of pM.
(36); pCMXhTR
1 encoding thyroid hormone receptor (TR)
1 (20); pSVAR0
(37) (for transient transfection) and pCMX.hAR (38) (for expression in vitro) for androgen receptor (AR); and pRShERR1 for
estrogen receptor-related protein (ERR) 1 and pRShERR2 for ERR2 (39). The luciferase reporter gene vectors were described previously: MMTV-LUC for AR, MMTV(ERE)-LUC for ER and ERRs, and MMTV(TRE)-LUC for
TR (40); GK1, controlled by Gal4 binding sites (24). The bacterial
expression vectors for recombinant glutathione S-transferase (GST) fused to PRMT1 (41) and to CARM1 (34) were also described previously.
(other proteins) after induction with 0.1 mM
isopropyl thio-
-D-galactoside for 5 h. Bacterial cells were harvested, resuspended in NETN buffer (100 mM
NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH8.0, and
containing 0.5% detergent, either Nonidet P-40 or IGEPAL CA-630). Cell
debris was removed by centrifugation (13,000 × g for
30 min at 4 °C), and GST fusion proteins were isolated by incubation
of the supernatant overnight at 4 °C with rotation with
glutathione-agarose beads (Sigma). Beads were washed by centrifugation
(500 × g for 5 min at 4 °C) once with NETN and then
twice with NETN containing 0.01% detergent. Proteins to be tested for
interaction with GST fusion proteins on the beads were synthesized by
transcription and translation in vitro in the presence of
[35S]methionine using the TNT-T7-coupled reticulocyte
lysate system (Promega). The binding assay was conducted by incubating
beads containing 1-2 µg of GST or GST fusion protein with slow
rotation for 60 min at 4 °C with 10 µl of the in vitro
synthesis reaction in a 100-µl total volume of NETN containing 0.01%
detergent. Beads were washed four times by centrifugation with NETN
containing 0.01% detergent. GST fusion protein and interacting
35S-labeled protein were eluted from beads with Laemmli
sample buffer (44) and analyzed by SDS-polyacrylamide gel
electrophoresis and autoradiography.
-galactosidase activity as described
previously (25). A Rous sarcoma virus (RSV)-
-galactosidase reporter
plasmid was used as the internal control to monitor for transfection
efficiency, and
-galactosidase activity was assayed in the cell
lysates as described previously (46). Data shown are the means and
standard deviations of results from three transfected cultures. When
hormone was required, transfected cells were grown in medium containing
charcoal-treated serum (Gemini BioProducts) and 20 nM
concentration of the appropriate hormone for each NR: estradiol for ER,
triiodothyronine for TR, or dihydrotestosterone for AR.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Binding of PRMT1 to GRIP1 and NRs.
A, in GST-pull down assays, 35S-labeled p160
coactivator, synthesized in vitro, was incubated with GST or
GST-PRMT1 recombinant protein bound to agarose beads. Bound proteins
were analyzed by SDS-polyacrylamide gel electrophoresis and
autoradiography. For comparison, 10% of the labeled protein initially
incubated with the agarose beads (10% input) is shown. B,
in GST pull-down assays performed as described in A, the
indicated 35S-labeled GRIP1 fragments were tested for
binding to GST and GST-PRMT1. C, in GST-pull down
assays performed as described in A, 35S-labeled
ER , TR
1, and AR were tested for binding to GST or GST-PRMT1 in
the presence or absence of the appropriate hormone for each NR. The
results presented are from a single experiment representative of three
separate experiments. M.M., molecular mass.
, TR
1, and AR (Fig. 2C). The binding was observed in the presence
or absence of a 20 nM concentration of the appropriate
hormone for each NR.
and
TR
1 (Fig. 3, A and B). Because PRMT1 bound directly to NRs as well as to p160 coactivators, we tested whether PRMT1 was able to serve as a direct or primary coactivator for NRs,
i.e. without exogenously expressed p160 coactivators. In the
absence of exogenous GRIP1, the activity observed with
hormone-activated NR (Fig. 3, A-C, histogram 2) was
enhanced only slightly or not at all by the addition of PRMT1
(histogram 5). In the presence of GRIP1, the enhancement of
NR function by PRMT1 was also dependent upon the presence of hormone
(compare histograms 6 and 8), indicating that
PRMT1 was acting as a coactivator for NRs rather than an independent
transcriptional activator. The fact that the activity of PRMT1 depended
on the presence of a p160 coactivator indicated that PRMT1, like CARM1,
was acting as a secondary coactivator.
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Fig. 3.
Dependence of PRMT1 coactivator function on
GRIP1 and the appropriate hormone. CV-1 cells were
transiently transfected with 0.25 µg of luciferase reporter plasmid
controlled by an appropriate hormone response element, expression
vectors for the indicated NR (1 ng for ER or TR, 0.1 µg for AR), 0.25 µg of pSG5.HA-GRIP1, 0.5 µg of pSG5.HA-PRMT1, and 0.5 µg of
pSG5.HA-CARM1 where indicated. Transfected cells were grown with the
appropriate hormone for each NR, except where indicated otherwise, and
cell extracts were assayed for luciferase activity, which is expressed
as relative light units (RLU). Each data point represents
the mean and S.D. from three transfected cultures. Results shown are
from a single experiment which is representative of 12 separate
experiments for ER, 4 experiments for TR, and 3 experiments for
AR.
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Fig. 4.
Synergistic enhancement of NR function by
CARM1 and PRMT1. A-C, transfections and reporter gene
assays were conducted as described in the legend for Fig. 3, with 0.25 µg of pSG5.HA-GRIP1 and varied or fixed (0.5 µg) amounts of
pSG5.HA-PRMT1 and/or pSG5.HA-CARM1 as indicated (graphs a d). Transfected cells were grown with the appropriate hormone
for each NR. Insets show the same graphs on a smaller scale.
All data for a given NR are from the same experiment, and data for
Figs. 3 and 4 are from the same experiment. D, selected data
from C are shown in histogram form. E, data from
C were replotted on a semilog scale, and straight
lines were fitted to graphs a-d.
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Fig. 5.
Synergistic activity of PRMT1 and CARM1 in
the presence of various p160 coactivators. CV-1 cells were
transfected with 0.25 µg of MMTV-LUC, 0.1 µg of pSVAR0,
0.25 µg of expression vector for the indicated p160 coactivator,
varied amounts of pSG5.HA-PRMT1, and (for graphs d and
e) 0.5 µg of pSG5.HA-CARM1. Data shown are from a single
experiment representative of two independent experiments.
or TR
1 was used instead of AR (Fig. 4, A and
B, graphs c and d), or when SRC-1a or
ACTR was used instead of GRIP1 as the p160 coactivator for AR (Fig. 5,
graphs d and e). The synergistic effects of CARM1
and PRMT1 were dependent upon the presence of p160 coactivator and
hormone (Fig. 3, compare histograms 7 and 8 with
6). Thus, an activated NR and a p160 coactivator were both required before CARM1 and PRMT1 could enhance reporter gene activity.
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Fig. 6.
Coactivator synergy depends upon NR
concentration. CV-1 cells were transfected with 0.25 µg of
MMTV-LUC, varied amounts of pSVAR0, 0.25 µg of pSG5.HA
GRIP1, and where indicated 0.5 µg of pSG5.HA-PRMT1 and/or 0.5 µg of
pSG5.HA-CARM1. The results shown are from a single experiment
representative of three independent experiments.
and TR
1
(data not shown). Thus, coactivator combinations had their largest
effects when NR was expressed at low levels, and under these
conditions, there was a very high degree of coactivator synergy.
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Fig. 7.
Synergy among GRIP1, PRMT1, and CARM1
coactivators depends on the AD2 region of GRIP1. CV-1 cells were
transfected with 0.25 µg MMTV-LUC, 10 ng of pSVAR0, 0.25 µg of the indicated wild type or mutant pSG5.HA-GRIP1 expression
vector, and where indicated 0.5 µg of pSG5.HA-PRMT1 and/or 0.5 µg
of pSG5.HA-CARM1. GRIP1
AD2, amino acids 5-1121; GRIP1
AD1, full-length GRIP1 (amino acids 5-1462) with amino acids
1057-1109 deleted. The results shown are from a single experiment
representative of two independent experiments.
AD1 and the two methyltransferases (histograms
13-16) closely resembled the activation patterns observed with
wild type GRIP1 (histograms 5-8). However, the activity
contributed by the combined methyltransferases was about 90% less when
coexpressed with the GRIP1
AD2 mutant (histograms 9-12)
or the GRIP1
AD1/
AD2 double mutant (histograms 17-20)
compared with their activity in the presence of wild type GRIP1.
Results with GRIP1 mutants lacking AD2 closely resembled the activity
levels seen when no GRIP1 vector was transfected (Fig. 7,
histograms 1-4). By immunoblot analysis, all three mutant
GRIP1 species were expressed at similar levels in COS cells (Ref. 25
and data not shown). Furthermore, at higher levels of AR and in the
absence of coexpressed coactivators, the GRIP1
AD2 mutant still
retained about half of the coactivator function of wild type GRIP1
(25), demonstrating that its loss of synergistic activity with CARM1
and PRMT1 was not due to a complete loss of function. However, even in
the absence of a GRIP1 AD2 domain, the combination of CARM1 and PRMT1
caused a small but reproducible enhancement of NR function (Fig. 7,
histograms 4, 12, and 20). Therefore, although a
small amount of coactivator activity from CARM1 and PRMT1 was
independent of exogenously expressed p160 coactivator, the great
majority of the CARM1 and PRMT1 coactivator activity required the
presence of a p160 protein with an intact AD2 domain. These results
support the conclusion that the binding of CARM1 and PRMT1 to the AD2
region is necessary and therefore presumably responsible for their
functional dependence on GRIP1.
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Fig. 8.
Synergy among GRIP1, PRMT1, and CARM1
coactivators with orphan NRs. CV-1 cells were transfected with 0.1 µg of RSV- -galactosidase reporter gene, 0.25 µg of MMTV(ERE)-LUC
reporter gene, 10 ng of the indicated ERR expression vector, and where
indicated 0.25 µg of pSG5.HA-GRIP1, 0.5 µg of pSG5.HA-CARM1, and/or
0.5 µg of pSG5.HA-PRMT1. Transfected cell extracts were assayed for
luciferase activity (A) and
-galactosidase activity
(B). The results shown are from a single experiment
representative of three independent experiments.
-galactosidase reporter gene driven by the
RSV promoter.
-Galactosidase activity was reproducibly enhanced 3-fold by the combination of CARM1 and PRMT1, whereas GRIP1 had little
if any effect on
-galactosidase activity (Fig. 8B).
Because the activity of the RSV and various other so-called
constitutive promoters is often enhanced somewhat by transcriptional
coactivators, they are not perfectly appropriate controls for
transfection efficiency in these experiments. Therefore, in this entire
study the luciferase activity of the NR-regulated reporter genes was
not normalized with the
-galactosidase activity. However, because
the effects of the coactivators on
-galactosidase activity was much
less than the effects on the NR-controlled luciferase activity (3-fold versus 33-fold in the ERR3 experiment),
-galactosidase
activity does serve as a valid control to show that differences in
transfection efficiency cannot explain the observed coactivator effects
on NR-dependent luciferase activity.
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Fig. 9.
Synergistic enhancement of Gal4DBD-GRIP1
activity by CARM1 and PRMT1. A, CV-1 cells were
transfected with 0.25 µg of GK-1 luciferase reporter gene, 0.5 µg
of pM.GRIP1, varied amounts of pSG5.HA-PRMT1, and where indicated 0.5 µg of pSG5.HA-CARM1. B, selected data from A
are replotted as a histogram. Where indicated, 0.5 µg of PRMT1 vector
was used. C, transfections were performed as described in
A, except that the amount of pM.GRIP1 DNA was varied, and
0.25 µg of pSG5.HA-PRMT1 and/or 0.25 µg of pSG5.HA-CARM1 were
included where indicated. In A and C, the symbol
X represents the activity of 0.5 µg of the pM vector
(encoding Gal4 DBD) in the absence of coactivators. Results shown in
A and C are from independent experiments.
Mtase, methyltransferase, i.e. CARM1 or
PRMT1.
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Fig. 10.
Transcriptional activation activity of CARM1
or PRMT1 fused to Gal4 DBD. CV-1 cells were transfected with 0.5 µg of GK-1 luciferase reporter gene and 0.5 µg of each of the
indicated expression vectors. The results presented are from a single
experiment representative of five independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the following colleagues at the
University of Southern California: Hung-Yi Wu for plasmid pSG5.ACTR;
Dr. Han Ma for plasmid pSG5.HA-GRIP1
AD1/
AD2; Shih-Ming Huang
for pSG5.HA and pGEX vectors encoding GRIP1 fragments; and
Gerhard Coetzee for critical comments on the manuscript. We
thank Drs. Harvey Herschman and Steve Clarke (University of California,
Los Angeles) for the vector encoding GST-PRMT1 and Drs. Beatrice
Darimont and Keith Yamamoto (University of California, San
Francisco) for pRSV-
-galactosidase.
![]() |
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.
§ Supported by a predoctoral traineeship from Grant AG00093 from the National Institutes of Health.
To whom correspondence should be addressed: Dept. of
Pathology, HMR 301, University of Southern California, 2011 Zonal Ave., Los Angeles, CA 90089. Tel.: 323-442-1289; Fax: 323-442-3049; E-mail:
stallcup@hsc.usc.edu.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M004228200
2 D. Chen and M. R. Stallcup, unpublished results.
3 S. S. Koh and M. R. Stallcup, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NR, nuclear
receptor;
ACTR, activator of thyroid and retinoic acid receptors;
AD, activation domain;
AR, androgen receptor;
CARM1, coactivator associated
arginine methyltransferase 1;
CBP, CREB (cAMP-response element-binding
protein)-binding protein;
DBD, DNA binding domain;
ER, estrogen
receptor ;
ERR1, estrogen receptor-related protein 1;
GRIP1, glucocorticoid receptor interacting protein 1;
GST, glutathione
S-transferase;
HA, hemagglutinin A;
MMTV, mouse mammary
tumor virus;
p/CAF, p300/CBP-associated factor;
PRMT1, protein arginine
methyltransferase 1;
RLU, relative light unit;
RSV, Rous sarcoma virus;
SRC-1, steroid receptor coactivator-1;
TR, thyroid hormone
receptor.
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REFERENCES |
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