Human SWI/SNF-associated PRMT5 methylates histone H3 arginine 8 and negatively regulates expression of ST7 and NM23 tumor suppressor genes.

Abstract

Protein arginine methyltransferases (PRMTs) have been implicated in transcriptional activation and repression, but their role in controlling cell growth and proliferation remains obscure. We have recently shown that PRMT5 can interact with flag-tagged BRG1- and hBRM-based hSWI/SNF chromatin remodelers and that both complexes can specifically methylate histones H3 and H4. Here we report that PRMT5 can be found in association with endogenous hSWI/SNF complexes, which can methylate H3 and H4 N-terminal tails, and show that H3 arginine 8 and H4 arginine 3 are preferred sites of methylation by recombinant and hSWI/SNF-associated PRMT5. To elucidate the role played by PRMT5 in gene regulation, we have established a PRMT5 antisense cell line and determined by microarray analysis that more genes are derepressed when PRMT5 levels are reduced. Among the affected genes, we show that suppressor of tumorigenicity 7 (ST7) and nonmetastatic 23 (NM23) are direct targets of PRMT5-containing BRG1 and hBRM complexes. Furthermore, we demonstrate that expression of ST7 and NM23 is reduced in a cell line that overexpresses PRMT5 and that this decrease in expression correlates with H3R8 methylation, H3K9 deacetylation, and increased transformation of NIH 3T3 cells. These findings suggest that the BRG1- and hBRM-associated PRMT5 regulates cell growth and proliferation by controlling expression of genes involved in tumor suppression.

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MOLECULAR AND CELLULAR BIOLOGY, Nov. 2004, p. 9630–9645
0270-7306/04/$08.00?0DOI: 10.1128/MCB.24.21.9630–9645.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 21
Human SWI/SNF-Associated PRMT5 Methylates Histone H3 Arginine
8 and Negatively Regulates Expression of ST7 and NM23
Tumor Suppressor Genes†
Sharmistha Pal,1Sheethal N. Vishwanath,1Hediye Erdjument-Bromage,2
Paul Tempst,2and Saïd Sif1*
Department of Molecular and Cellular Biochemistry, College of Medicine and Public Health, The Ohio State University,
Columbus, Ohio,1and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York2
Received 29 June 2004/Returned for modification 15 July 2004/Accepted 28 July 2004
Protein arginine methyltransferases (PRMTs) have been implicated in transcriptional activation and re-
pression, but their role in controlling cell growth and proliferation remains obscure. We have recently shown
that PRMT5 can interact with flag-tagged BRG1- and hBRM-based hSWI/SNF chromatin remodelers and that
both complexes can specifically methylate histones H3 and H4. Here we report that PRMT5 can be found in
association with endogenous hSWI/SNF complexes, which can methylate H3 and H4 N-terminal tails, and show
that H3 arginine 8 and H4 arginine 3 are preferred sites of methylation by recombinant and hSWI/SNF-
associated PRMT5. To elucidate the role played by PRMT5 in gene regulation, we have established a PRMT5
antisense cell line and determined by microarray analysis that more genes are derepressed when PRMT5 levels
are reduced. Among the affected genes, we show that suppressor of tumorigenicity 7 (ST7) and nonmetastatic 23
(NM23) are direct targets of PRMT5-containing BRG1 and hBRM complexes. Furthermore, we demonstrate
that expression of ST7 and NM23 is reduced in a cell line that overexpresses PRMT5 and that this decrease
in expression correlates with H3R8 methylation, H3K9 deacetylation, and increased transformation of NIH
3T3 cells. These findings suggest that the BRG1- and hBRM-associated PRMT5 regulates cell growth and
proliferation by controlling expression of genes involved in tumor suppression.
During cell growth and proliferation several genes become
either repressed or activated. These variations in expression
often correlate with changes in chromatin structure, which can
be induced by a variety of enzymes that can disrupt nucleo-
somes in an ATP-dependent manner and/or covalently modify
nucleosomal histones (17, 29, 41, 58). Biochemical character-
ization of different members of the SWI2/SNF2 family of chro-
matin remodeling complexes revealed that there are complexes
that can catalyze both ATP-dependent nucleosome disruption
and histone deacetylation (48, 49, 55, 59). Unlike the nucleo-
some remodeling and deacetylase complex, human SWI/SNF
(hSWI/SNF) complexes can be purified either alone or in com-
bination with mSin3A/histone deacetylase, indicating that
there are different pools of BRG1- and hBRM-based hSWI/
SNF complexes (19, 27, 42). Recent work has also shown that
flag-tagged BRG1 and hBRM complexes include the type II
protein arginine methyltransferase 5 (PRMT5) and that these
complexes are involved in transcriptional repression of the
MYC/MAX/MAD target gene CAD (32). These studies and
work by various groups suggest that ATP-dependent chroma-
tin remodeling complexes can act in concert with various his-
tone-modifying enzymes to modulate chromatin structure (10,
29). Although PRMT5 has been implicated in transcriptional
repression of CYCLIN E and CAD, it is not clear whether it is
involved in regulating a broader spectrum of genes and wheth-
er it has any effects on cell growth and proliferation.
Histone methylation has been identified as an important
modification for both transcriptional activation and transcrip-
tional repression. Thus far, it appears that there are two types
of histone methyltransferases which include either a SET (Su-
var3-9, Enhancer of Zeste, Trithorax) domain found primarily
in proteins possessing lysine-specific methylase activity or an
arginine-specific catalytic domain characteristic of PRMTs (18,
45, 58). Differential methylation of conserved lysine residues in
histones H3 and H4 can lead to distinct transcriptional out-
comes. For example, methylation of H3 lysine 4 (H3K4) by
either SET7 or SET9 can activate transcription (30, 50), while
methylation of H3K9 by either SUV39H1 or G9a can induce
binding of heterochromatin protein 1 and promote transcrip-
tional silencing by triggering the formation of heterochromatin
(1, 22, 28, 31). Similarly, methylation of H3 arginine residues
by PRMT4 has been shown to be involved in transcriptional
activation, whereas methylation by PRMT5, which can target
both H3 and H4, has been implicated in CAD and CYCLIN E
transcriptional repression (4, 7, 32, 39). Currently, the histone
residues modified by PRMT5 are still not known.
PRMTs can be divided into type I PRMTs, which catalyze
monomethylation and asymmetric dimethylation of arginine
residues, and type II PRMTs, which catalyze the formation
of monomethylated and symmetrically dimethylated arginines
(57). Among the six PRMT family members, only PRMT5
behaves as a type II PRMT that can target histones (3, 32, 34).
Besides methylating and modulating the activity of proteins
involved in nuclear export and signal transduction, PRMT1
and PRMT4 have also been shown to methylate histones H3
* Corresponding author. Mailing address: Department of Molecular
and Cellular Biochemistry, Ohio State University College of Medicine,
Columbus, OH 43210. Phone: (614) 247-7445. Fax: (614) 292-4118.
E-mail: Sif.1@osu.edu.
† Supplemental material for this article may be found at http://mcb
.asm.org/.
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and H4 and activate transcription (4, 51). Although no PRMT2
substrates have been identified yet, it appears that PRMT2 can
potentiate estrogen receptor transcriptional activity and that
coactivation relies on the catalytic activity of PRMT2 (35).
Both PRMT3 and PRMT6 can methylate cellular proteins, but
their function in vivo remains unknown (8, 47).
PRMT5 was first identified in Schizosaccharomyces pombe as
a protein that interacts and positively regulates Shk1 kinase, a
member of the p21Cdc42/Rac-activated kinases (PAKs), which
play critical roles in RAS signaling (13). Deletion of ras1 in
fission yeast results in altered cell shape, and overexpression of
PRMT5 partially restores wild-type morphology, indicating
that PRMT5 is involved in RAS-induced cytoskeletal and mor-
phological control pathways. Moreover, prmt5-null mutants
grow slower and are less elongated than wild-type cells, and
reexpression of either S. pombe or human PRMT5 rescues cell
morphology, suggesting that PRMT5 is functionally conserved
(12). Yeast two-hybrid screens have shown that PRMT5 can
interact with a wide variety of proteins including Janus kinase
2 (Jak2), Orb6p kinase, and somatostatin receptors 1 and 4,
implying that type II PRMTs might be targeted by or regu-
late components of different signaling modules (34, 40, 53).
PRMT5 has also been shown to be part of a complex that can
bind and methylate SmD1 and SmD3, which are involved in
the biogenesis of spliceosomal U-rich snRNPs (9, 26). More
recently, PRMT1 and PRMT5 were shown to interact and
colocalize with the transcription elongation factor SPT5 on the
IkB? and interleukin-8 promoters (20). Both arginine methyl-
transferases modify SPT5 and reduce its association with RNA
polymerase II, suggesting that PRMT1 and PRMT5 might be
involved in regulating transcriptional elongation. Further evi-
dence in support of a role for PRMT5 in transcriptional re-
pression comes from recent findings which show that PRMT5
is associated with the promoter region of genes that are either
silent, such as interleukin-8, or have low basal activity, includ-
ing IkB?, CYCLIN E, and CAD (7, 20, 32).
Our understanding of the mechanisms by which PRMT5
affects gene expression and cell growth and proliferation is
very limited. We have previously reported the association of
PRMT5 with flag-tagged BRG1 and hBRM complexes and
shown that hSWI/SNF-associated PRMT5 can methylate hy-
poacetylated H3 and H4 more efficiently than the hyperacety-
lated forms. In this study, we provide further evidence to show
that PRMT5 copurifies with endogenous BRG1 and hBRM
complexes and demonstrate that immunopurified recombinant
and hSWI/SNF-associated PRMT5 target the same H3 and H4
arginine residues. We also show that PRMT5 has the ability to
stimulate cell growth and anchorage-independent growth by
methylating H3R8 and reducing expression of genes involved
in tumor suppression. These results indicate that the BRG1-
and hBRM-associated PRMT5 is an important regulator of
cell growth and proliferation.
MATERIALS AND METHODS
Plasmid constructions. Retroviral expression vectors for sense and antisense
(AS) PRMT5 were generated using pBS(KS?)/flag-tagged PRMT5, which was
previously described (32). Plasmid pBabe/Fl-PRMT5 was constructed by insert-
ing a 2-kbp BamHI-EcoRI fragment, which was excised out of pBS(KS?)/Fl-
PRMT5, into BamHI-EcoRI-linearized pBabe-puromycin. To generate the ret-
roviral vector for knocking down PRMT5 expression, pBabe/AS-PRMT5, a
PCR-amplified 1.1-kbp PRMT5 DNA fragment (?39 to ?1140), was inserted in
reverse orientation into pBabe-puromycin digested with BamHI and SalI. The
5? primer (5?-ACGCGTCGACGTGATTGGCTACTAGTATCAAGGAATC-
3?) was modified to include a SalI restriction site (underlined), while the 3?
primer(5?-CGCGGATCCCATATGTCTGAGATTCCAGATTGTC-3?)
cluded a BamHI restriction site (underlined). For coupled in vitro transcription
and translation of PRMT5, pBS(KS?)/Fl-PRMT5 was linearized with SacII to
delete the flag-tag epitope. A retroviral vector for expression of catalytically
inactive flag-tagged hBRM was constructed by excising a 5-kbp EcoRI DNA
fragment from pBS(KS?)/CehBRM-NTP (6) and inserting it into EcoRI-linear-
ized pBabe-puromycin.
Purification of endogenous and flag-tagged hSWI/SNF complexes, recombi-
nant flag-tagged wild-type and mutant PRMT5, and mass spectrometry. To
purify endogenous BRG1 and hBRM complexes, 300 mg of HeLa nuclear extract
was loaded onto a 30-ml BioRex 70 column preequilibrated with BC100 (20 mM
HEPES [pH 7.9], 100 mM KCl, 5 mM MgCl2, 20% glycerol, 1 mM EDTA, 0.25
mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). Next, bound proteins
were eluted with BC100 buffer supplemented with increasing amounts of KCl,
and the collected fractions were analyzed by Western blotting for the presence of
PRMT5, BRG1, and hBRM. Peak fractions (0.5 M and 0.8 M KCl) containing
PRMT5 and hSWI/SNF complexes were pooled and dialyzed against BC100
before they were loaded on two separate DE 52 columns. Both columns were
eluted as described for the BioRex 70 column, and the collected fractions were
analyzed by Western blotting for the presence of PRMT5-containing hSWI/SNF
complexes. The 0.3 M KCl fractions from both DE 52 columns, which contained
the majority of endogenous hSWI/SNF complexes, were used in immunoprecipi-
tation assays. Approximately 100 ?g of each pooled peak fraction was incubated
with 10 ?l of either preimmune or immune anti-PRMT5 antibodies at 4°C for
4 h. Next, protein A agarose beads preblocked in 0.5 mg of bovine serum albumin
(BSA)/ml overnight at 4°C were added to each reaction mixture. Samples were
incubated at 4°C for 8 to 10 h, and beads were collected and washed three times
with 1 ml of washing buffer (40 mM Tris-HCl [pH 8.0], 250 mM NaCl, 0.5%
NP-40). Bound proteins were separated on a sodium dodecyl sulfate (SDS)–8%
polyacrylamide gel and analyzed by Western blotting. Flag-tagged hSWI/SNF
complexes and Sf9-expressed flag-tagged wild-type and mutant PRMT5 were
purified as described previously (32, 42, 43). The gel-purified 70-kDa band in the
HeLa and INI1 fractions was identified by mass spectrometry as described
previously (32).
In vitro translation and immunoprecipitation assays. PRMT5 was synthesized
in vitro by incubating 2 ?g of linearized plasmid DNA with 25 ?l of TNT-coupled
rabbit reticulocyte lysate in the presence of [35S]methionine and cysteine (NEN)
according to the manufacturer’s instructions (Promega). In vitro-translated
PRMT5 was immunoprecipitated by incubating approximately 60 ? 103to 70 ?
103cpm with antibodies in a 250-?l reaction mixture containing immunoprecipi-
tation buffer (20 mM Tris-HCl [pH 7.4], 100 mM NaCl, 5 mM MgCl2, 1 mM
EDTA, 0.05% NP-40, 1% aprotinin). After a 1-h incubation on ice, 75 ?l of a
50% slurry of protein A agarose beads was added, and the reaction mixture was
incubated overnight at 4°C. Samples were washed as described for immunopu-
rified hSWI/SNF complexes, and bound proteins were analyzed by SDS-poly-
acrylamide gel electrophoresis and autoradiography.
Histone and peptide methylation assays. Methylation assays were performed
using either HeLa core histones (2.5 ?g), acetylated or nonacetylated histone
N-terminal peptides (4 ?g), and Sf9-expressed recombinant flag-tagged PRMT5
(250 ng) or hSWI/SNF-associated PRMT5 (400 ng) in a 25-?l reaction mixture
containing 15 mM HEPES (pH 7.9), 100 mM KCl, 5 mM MgCl2, 20% glycerol,
1 mM EDTA, 0.25 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride,
and 2.75 ?Ci of S-[3H]adenosylmethionine (SAM) (Amersham Pharmacia Bio-
tech., Inc.). After a 1.5-h incubation at 30°C, HeLa core histones were visualized
as described previously (32). When acetylated peptides were used, reaction
mixtures were supplemented with 40 mM sodium butyrate. Reaction mixtures
containing histone N-terminal peptides were spotted on Whatman P-81 filter
paper and washed five times with 10 ml of 0.1 M sodium carbonate buffer (pH
9.0) to remove unincorporated [3H]SAM. Methylated peptides were detected by
scintillation counting.
Cell culture, proliferation, and transformation assays. HeLa S3, NIH 3T3,
and flag-tagged dominant-negative BRG1 and hBRM cell lines were cultured in
Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. HeLa
S3 and flag-tagged INI1-11 cell lines were grown at the National Cell Culture
Center (Biovest International, Inc.). HeLa S3 cell lines that express dominant-
negative flag-tagged BRG1 were described previously (42). HeLa S3 cell lines
that express mutant flag-tagged hBRM were generated as described previously
(43). To generate cell lines that express Fl-PRMT5, AS-PRMT5, or MYC and
RAS, 5 ? 105NIH 3T3 cells were transfected for 5 h with 2 ?g of pBabe/Fl-
PRMT5, pBabe/AS-PRMT5, or pBabe-hygromycin/MYC and pBabe-hygromy-
in-
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cin/RAS by using Lipofectamine (Invitrogen, Inc.). Two days after transfection,
drug-resistant cells expressing either Fl-PRMT5, AS-PRMT5, or MYC/RAS
were selected for 2 weeks in the presence of either 3 ?g of puromycin/ml or 480
U of hygromycin/ml. For NIH 3T3 cells expressing AS-PRMT5, several individ-
ual colonies were isolated, expanded into cell lines, and analyzed by reverse
transcriptase PCR (RT-PCR) as well as Western blotting to assess endogenous
PRMT5 levels. After several clones were screened, NIH 3T3/AS-PRMT5 clone
15 was used in subsequent experiments because expression of endogenous
PRMT5 was reduced by more than 90% as quantitated by RT-PCR.
To measure the proliferation rate of different cell lines, 2 ? 105cells were
seeded into 6-cm-diameter plates and allowed to grow for 6 days. Proliferation of
each cell line was repeated three times with duplicate plates, and cells were
counted every 2 days. To measure bromodeoxyuridine (BrdU) incorporation, 106
cells were treated with 10 ?M BrdU for either 4.5 or 9 h according to the
instructions of the manufacturer (Becton Dickinson). Next, cells were incubated
with 1 ?l of fluorescein isothiocyanate-conjugated anti-BrdU antibody that was
diluted 1:50 in supplied 1? BD Perm/Wash buffer. Samples were then washed
twice with 1 ml of 1? BD Perm/Wash buffer and resuspended in 1 ml of staining
buffer containing 20 ?l of 7-amino-actinomycin D before cells were analyzed by
fluorescence-activated cell sorting (FACS) analysis. To determine if NIH 3T3
cells expressing AS-PRMT5 were undergoing apoptosis, 2 ? 105cells were
plated and allowed to grow for 4 days before the DNA content was examined by
FACS analysis with a FACScalibur flow cytometer (Becton Dickinson).
To assess the anchorage-dependent growth potential of NIH 3T3/Fl-PRMT5
and NIH 3T3/AS-PRMT5, 4 ? 103cells were plated in 10-cm-diameter plates.
After 7 days, colonies were visualized by fixing the cells in 10% buffered formalin
solution and staining them with 0.1% crystal violet. For analysis of anchorage-
independent growth, 2 ? 102cells were seeded into 6-cm-diameter plates in
Dulbecco’s modified Eagle’s medium containing 0.3% agar as described previ-
ously (11). Each cell line was tested for its ability to grow in soft agar by using
triplicate plates. Colonies were counted, and the average from three different
experiments is shown.
RT-PCR and microarray analyses. RNA was prepared from NIH 3T3, NIH
3T3/AS-PRMT5, NIH 3T3/Fl-PRMT5, HeLa S3, flag-tagged dominant-negative
BRG1, and flag-tagged dominant-negative hBRM cell lines by using Trizol re-
agent (Invitrogen, Inc.) according to the manufacturer’s instructions. Approxi-
mately 10 to 20 ?g of total RNA was used in a 20-?l reaction mixture containing
20 pmol of specific 3? primer, 3.5 mM MgCl2, 1 mM deoxynucleoside triphos-
phates, 1? Taq Pol buffer (Invitrogen, Inc.), 15 U of avian myeloblastosis virus
RT (Promega, Inc.), and 2.5 U of RNasin. Either 0.2 ?l or 2.0 ?l of the RT
reaction mixture was PCR amplified using specific primers in a 50-?l reaction
mixture containing 2.5 U of Taq polymerase (Invitrogen, Inc.) and 2 ?Ci of
[?-32P]dCTP. Amplified fragments were separated from nonspecific products by
electrophoresis on a 5% polyacrylamide gel and quantitated with a Molecular
Dynamics PhosphorImager. Specific primer pairs were used to amplify the fol-
lowing genes: NM23 (?103 to ?313), ST7 (?448 to ?717), RRM1 (?181 to
?591), CYCLIN B2 (?172 to ?489), CYCLIN E2 (?69 to ?409), NF-?B (?393
to ?762), GAS2 (?275 to ?595), CDC20 (?161 to ?540), CDK4 (?151 to
?542), MYT1l (?796 to ?1112), ALL1 (?186 to ?432), SERPIN E1 (?368 to
?798), PRMT5 (?1093 to ?1306), glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) (?178 to ?578), and ?-ACTIN (?140 to ?585).
Microarray analysis was performed using 10 ?g of total RNA isolated from
either puromycin-resistant NIH 3T3 or NIH 3T3/AS-PRMT5 cells. Affymetrix
MG-U74Av2 high-density expression array chips, which include 6,000 cDNA
clones and 8,000 expressed sequence tags, were used to identify genes whose
expression was altered when PRMT5 levels were reduced. The analysis was
performed by the Ohio State University Comprehensive Cancer Center microar-
ray facility (http://www.dnaarrays.org). Gene expression levels were estimated
from GeneChip PM probe intensities by means of an enhanced two-array version
of the Li-Wong PM-only algorithm (5). The enhanced algorithm (i) scales all PM
and MM probe intensities so as to minimize between-array differences in the
scaled MM probe intensity distributions, (ii) applies between-array variance
analysis to the scaled PM probe intensities in order to estimate PM-specific
sensitivities, (iii) estimates gene expression levels by regressing scaled PM probe
intensities on estimated PM probe sensitivities within each probe set, and (iv)
tests a probe-level general linear model within each probe set in order to esti-
mate the P values for between-array differential gene expression. The estimated
P values can be several orders of magnitude lower than 0.05, as required by the
Bonferroni correction, which applies when simultaneously testing thousands of
genes for significant differential expression (16).
Antibodies and Western blot analysis. Proteins were separated on an SDS–8
to 10% polyacrylamide gel, transferred onto nitrocellulose, and detected using
anti-hSWI/SNF subunits, anti-PRMT5, and anti-flag M2 rabbit polyclonal anti-
bodies, which have been described previously (32, 42, 43). To test anti-
H3(Me2)R8 antibodies, which were generated by Covance, Inc., methylated and
unmethylated H3 peptides were spotted on nitrocellulose before Western blots
were developed using ECL reagent (Pharmacia Amersham Biotech. Inc.). Anti-
MAD and anti-H3AcK9 were purchased from Santa Cruz Biotechnology and
Upstate Biotechnology, respectively.
Chromatin immunoprecipitation (ChIP) assay. Cross-linked chromatin was
prepared as described previously (32). Briefly, 90 to 95% confluent 10-cm-
diameter plates were treated with 1% formaldehyde for 10 min at room tem-
perature and harvested in 1 ml of lysis buffer (50 mM Tris-HCl [pH 8.1], 100 mM
NaCl, 5 mM EDTA, 0.5% SDS, protease inhibitors) after glycine was added to
a final concentration of 0.1 M. Chromatin was collected by centrifugation and
resuspended in 250 ?l of immunoprecipitation buffer (100 mM Tris-HCl [pH
8.6], 5 mM EDTA, 0.3% SDS, 1.7% Triton X-100, protease inhibitors) before it
was sonicated. Solubilized bulk chromatin, which contained DNA fragments that
varied in size from 0.25 to 1.5 kbp, was immunoprecipitated using specific
antibodies in the presence of 40 ?l of preblocked protein A beads (0.5 mg of
BSA/ml, 0.2 mg of salmon sperm DNA/ml) at 4°C for 14 h. Bound nucleoprotein
complexes were washed successively with 300 ?l of mixed micelle buffer (20 mM
Tris-HCl [pH 8.1], 100 mM NaCl, 5 mM EDTA, 5% [wt/vol] sucrose, 0.2%
Triton X-100, 0.2% SDS), buffer 250 (50 mM HEPES [pH 7.5], 250 mM NaCl,
1 mM EDTA, 0.1% deoxycholine, 0.2% Triton X-100), LiCl detergent buffer (10
mM Tris-HCl [pH 8.0], 250 mM LiCl, 1 mM EDTA, 0.5% deoxycholine, 0.25%
NP-40), and Tris-EDTA (pH 7.6). After phenol and chloroform extraction, DNA
was resuspended in 30 ?l of Tris-EDTA (pH 7.6). Specific primers were used to
amplify promoter sequences in a 50-?l reaction mixture containing 2 ?Ci of
[?-P32]dCTP.
RESULTS
PRMT5 can interact with endogenous BRG1- and hBRM-
based hSWI/SNF complexes. We have previously shown using
flag-tagged BRG1 and hBRM cell lines that PRMT5 can be
found in association with affinity-purified hSWI/SNF com-
plexes (32). To rule out the possibility that PRMT5 interacted
with hSWI/SNF complexes only when BRG1 and hBRM were
overexpressed, we analyzed its association with endogenous
hSWI/SNF complexes (Fig. 1). HeLa nuclear extracts were
fractionated on a BioRex 70 column, and the eluted proteins
were analyzed by Western blotting with anti-PRMT5, anti-
BRG1, and anti-hBRM antibodies. Previous work has shown
that BRG1- and hBRM-based hSWI/SNF complexes copurify
on phosphocellulose 11 (21). Consistent with these results, we
found that BRG1 and hBRM coelute at 0.5 M and 0.8 M KCl.
In addition, PRMT5 cofractionates with both chromatin re-
modeling ATPases. When both hSWI/SNF-enriched fractions
were further purified on a DE 52 column, PRMT5 coeluted
with BRG1 and hBRM at 0.3 M KCl, suggesting that it is in
complex with endogenous BRG1 and hBRM ATPases. To
demonstrate that PRMT5 is tightly associated with hSWI/SNF
complexes, we immunoprecipitated partially purified BRG1
and hBRM complexes with either preimmune or immune anti-
PRMT5 antibodies and tested for the presence of BRG1 and
hBRM by Western blotting. Both BRG1 and hBRM coimmu-
noprecipitated with PRMT5 in the presence of 0.3 M KCl,
indicating that PRMT5 is tightly associated with endogenous
BRG1- and hBRM-based hSWI/SNF complexes. Similar re-
sults were observed when flag-tagged INI1 nuclear extracts
were fractionated on BioRex 70, DE 52, Mono S, and anti-flag
M2 columns, further supporting the specific and tight associa-
tion of PRMT5 with BRG1 and hBRM complexes (data not
shown).
Although purification by conventional chromatography can
allow us to monitor association of PRMT5 with hSWI/SNF
complexes, the amount of active fractions purified by this
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method is very limiting. Therefore, we used the flag-tagged
INI1-11 cell line to purify PRMT5-containing BRG1 and
hBRM complexes (43). Fl-INI1 or HeLa nuclear extracts were
incubated with anti-flag M2 affinity gel, and after extensive
washing the retained proteins were eluted with flag peptide.
The collected fractions were then analyzed by silver staining
and Western blotting (Fig. 2A and B). In addition to the
previously identified hSWI/SNF subunits, PRMT5 was en-
riched in fractions containing BRG1 and hBRM but not in
control fractions purified using HeLa nuclear extract (Fig. 2B).
To rule out the possibility that PRMT5 interacted nonspecifi-
cally with flag antibody, we conducted immunoprecipitation
experiments in the absence of hSWI/SNF subunits. In vitro-
translated and35S-labeled PRMT5 was incubated with either
agarose beads, anti-flag M2 beads, or preimmune or immune
anti-PRMT5 antibodies. As expected, PRMT5 was immuno-
precipitated only in the presence of anti-PRMT5 antibodies,
indicating that PRMT5 does not cross-react with anti-flag
antibody (Fig. 2C). We have also identified PRMT5 by mass
spectrometry in the immunopurified Fl-hSWI/SNF complexes
but not in the HeLa fraction (data not shown). These results
are in complete agreement with our previous results, which
show that PRMT5 can interact with specific hSWI/SNF sub-
units (32), and confirm the association of PRMT5 with endog-
enous hSWI/SNF complexes.
hSWI/SNF-associated PRMT5 preferentially methylates H3
arginine 8 and H4 arginine 3 and is inhibited by either H3K9
or K14 acetylation. PRMT5 is a type II arginine methyltrans-
ferase that can target proteins involved in signal transduction,
transcriptional elongation, and splicing (9, 20, 26, 34). More
recently, PRMT5 has also been shown to methylate histones;
however, the identity of its target sites remains unknown. Us-
ing immunopurified flag-tagged hSWI/SNF (Fl-hSWI/SNF)
complexes, we examined whether PRMT5 could methylate
H1-depleted HeLa core histones in the presence of [3H]SAM
(Fig. 2C). BRG1- and hBRM-associated PRMT5 was able to
methylate histones H3 and H4 but not H2A and H2B. Simi-
larly, when Sf9-expressed and affinity-purified flag-tagged wild-
type PRMT5 (Fl-PRMT5) was incubated with the four core
histones, both H3 and H4 were methylated. In contrast, cata-
lytically inactive Fl-PRMT5(G367A/R368A) was unable to
methylate HeLa core histones. It is important to note based on
Western blot analysis and silver staining that seven- to nine-
fold-more recombinant Fl-PRMT5 was required to detect H3
and H4 methylation (Fig. 2D and data not shown), suggesting
that association with BRG1 and hBRM complexes enhances
PRMT5 methylase activity.
Most regulatory histone posttranslational modifications oc-
cur in the N-terminal tails of histones H3 and H4 (17, 45, 58).
To test whether hSWI/SNF-associated PRMT5 could methyl-
FIG. 1. PRMT5 coelutes with endogenous BRG1 and hBRM complexes. HeLa nuclear extracts (300 mg) were loaded on a 25-ml BioRex 70
column. After the flowthrough (FT) was collected, the column was washed with increasing salt concentrations as indicated. Fractions were collected
and analyzed by Western blotting with either anti-PRMT5, anti-BRG1, or anti-hBRM antibodies. Peak fractions from the BioRex 70 column
(fractions 4 to 28 for the 0.5 M KCl elution and fractions 4 to 12 for the 0.8 M KCl elution) were pooled and dialyzed against BC100 buffer.
Dialyzed proteins were loaded on two DE 52 columns and eluted as described for the BioRex 70 column. Peak fractions (0.3 M KCl) were pooled
and immunoprecipitated using either preimmune (PI) or immune (I) anti-PRMT5 antibodies. After extensive washing, proteins were analyzed by
Western blotting with the indicated antibodies.
VOL. 24, 2004 PRMT5 SUPPRESSES ST7 AND NM239633

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FIG. 2. PRMT5 coelutes with flag-tagged INI1 complexes. Either HeLa S3 or Fl-INI1 nuclear extract (180 mg) was incubated with anti-flag M2
affinity gel, and hSWI/SNF complexes were eluted with a 20-fold molar excess of flag peptide. Eluted complexes were analyzed by silver staining
(A) or by Western blotting (B) with specific antibodies. Western blot assays were performed using 20 ?g of Fl-INI1 nuclear extract (Input, lane
1), 15 ?l of eluted fraction with HeLa nuclear extract (Ctrl, lane 2), and 15 ?l of eluted flag-tagged hSWI/SNF (Fl-hSWI/SNF) complexes (lane
3). (C) PRMT5 does not cross-react with anti-flag antibodies. In vitro-translated and35S-labeled PRMT5 was incubated with either agarose beads
or anti-flag M2 antibody cross-linked to agarose beads. As a control, preimmune (PI) and immune (I) anti-PRMT5 antibodies were also included.
9634 PAL ET AL.MOL. CELL. BIOL.