From the Division of Host-Parasite Interaction, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Received for publication, August 19, 2002, and in revised form, November 21, 2002
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ABSTRACT |
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The human adrenal carcinoma cell line,
SW13, has been reported to be deficient in both BRG1 and
Brm expression and therefore is considered to lack a
functional SWI/SNF complex. We found that the original cell line
of SW13 is composed of two subtypes, one that expresses neither BRG1
nor Brm (SW13(vim During development, differential gene expression generates the
cellular diversity required to establish pattern formation and
organogenesis. Although the cellular specificity involved in
development has been described well, the mechanisms that ensure long
term cellular memory, by which the developmental fate of a particular
cell is passed on to its descendents, are largely unknown. In both
Drosophila and mammals regulatory mechanisms that allow gene
expression patterns to be maintained have been shown to be supported by
the products of two classes of regulatory genes, the Polycomb group
(Pc-G)1 and the trithorax
group (trx-G) (1-3). The proteins required to maintain a repressed
state are contained in the Pc-G complex, whereas those required for
persistence of expression are in the trx-G complex. The Pc-G and trx-G
protein complexes counteract each other to repair previously
established chromosomal domains of specific genes throughout
development (3).
Insight into the role of the trx-G protein in transcriptional
regulation has come from studies of the Brahma gene
(belonging to trx-G) in Drosophila and
its mammalian homologues, Brm and BRG1. Brm and
BRG1 have DNA-dependent ATPase activity and are the
catalytic subunits of the mammalian SWI/SNF chromatin remodeling complex. This complex contains either Brm or BRG1, but not both (4),
and recent reports on the glucocorticoid receptor (5), c-Myc
(6), C/EBP The SW13 cell line derived from human adrenal adenocarcinoma was
reported to express neither BRG1 nor Brm (14, 15)
and has been used in many experiments because of this unique property (9, 14-20). In our previous analysis on AP-1 (21) (composed of Fos/Jun
dimers), we found that a SWI/SNF complex subunit, BAF60a, binds to
different Fos/Jun dimers with distinct affinities by interacting with
two interfaces for c-Fos and c-Jun, respectively, and further showed
that the SWI/SNF complex is a major determinant of the transactivation
potential of Fos/Jun dimers. In SW13 cells, which lack a functional
SWI/SNF complex, the transactivating activity of Fos/Jun dimers is kept
at basal levels when assayed by transient transfection experiments.
However, transactivation by the c-Fos/c-Jun heterodimer (having the
highest affinity to BAF60a) was specifically enhanced by cotransfecting
Brm or BRG1 into SW13 to recover the functional
SWI/SNF complex (9).
Considering that the SWI/SNF complex is composed of important trx-G
proteins, functional loss of the SWI/SNF complex in SW13 might affect
long term cellular memory, leading to an obvious question whether the
SW13 cells are epigenetically stable. We are especially interested in
previous observations (22) that SW13 cells express vimentin
in a mosaic pattern. There are two cellular subtypes in SW13 culture,
the subtype that does not express vimentin protein (designated as
SW13(vim Cell Lines--
Human tumor cell lines SW13 (adrenocortical
carcinoma), MDA-MB435 (breast ductal carcinoma), and PtG-S2
(prepackaging cell line for retrovirus vector production) were
maintained in high glucose Dulbecco's modified Eagle's medium
(Invitrogen) supplemented with 10% fetal calf serum and
incubated at 37 °C. CHAP31 (24) (gifts from the Japan Energy Corp.,
Saitama, Japan) at 2-8 nM, 50-200 nM
trichostatin A (TSA; Wako Pure Chemicals, Tokyo, Japan), 0.5-2
µM 5-azacytidine (Sigma), 0.5-2 µM
5-azadeoxycytidine (5-azadC; Sigma), or 100 nM
12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma) were added
to the culture medium.
Immunocytochemistry--
SW13 cells were fixed with 4%
paraformaldehyde, treated with anti-vimentin mouse monoclonal antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antibody binding was
visualized with Alexa Fluor 488 conjugated anti-mouse IgG antibody
(Molecular Probes Inc., Eugene, OR) for immunofluorescence microscopy
or detected with biotinated anti-mouse IgG antibody (Chemicon
International, Temecula, CA) using the VECTASTAIN ABC kit (Vector
Laboratories Inc., Burlingame, CA).
Isolation of SW13(vim Plasmid Constructions--
pCR2.1-BRG1(B), -Brm(F), - Retrovirus Vectors--
Vesicular stomatitis virus G protein
(VSV-G) pseudotyped, murine leukemia virus-based retrovirus
vectors were prepared using prepackaging cell line PtG-S2 (26) and
transduced as described previously (15). A control vector was produced
with pBabe-IRESpuro (27), and Brm or BRG1 viruses were produced with
pBabe-hBrm-IRESpuro and pBabe-hBRG1-IRESpuro (15), respectively.
RT-PCR--
Total RNA was prepared from cells with the
ISOGEN RNA isolation reagent (Wako). RT-PCR was performed within the
linear range by the Superscript one-step RT-PCR with platinum
Taq kit (Invitrogen). The band density was semi-quantified
by Densitometry (ATTO Printgraph) using the software ATTO
Densito graph3. The other primer pairs for BRG1,
Brm (15), collagenase, c-met, and
GAPDH (9) were described previously. The primer pairs
were as follows: for vimentin, 5'-gacaatgcgtctctggcacgtcttgaccttgaacgc-3' and
5'-gcatctggcgttccagggactcattggttc-3', and for CD44,
5'-cagacctgcccaatgcctttgatggacc-3', 5'-caaagccaaggccaagagggatgcc-3'. RNA was reverse-transcribed for 30 min at 50 °C. Amplification conditions are as follows: an initial denaturation at 94 °C for 3 min followed by 30 cycles (for BRG1, Brm,
vimentin, and CD44) at 94 °C for 30 s,
67 °C for 2 min, and 72 °C for 1 min or 30 cycles (for
collagenase and c-met) or 25 cycles (for
GAPDH) at 94 °C for 30 s, 56 °C for 1 min, and
72 °C for 1 min.
Western Blotting--
Whole cell extracts (20 or 40 µg) were
prepared under denaturing conditions, separated by electrophoresis on
8% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride
membranes, immunostained with anti-BRG1 rabbit polyclonal antibody
(Santa Cruz), anti-Brm goat polyclonal antibody (Santa Cruz), or
anti-vimentin mouse monoclonal antibody (Santa Cruz) and detected with
an ECL kit (Amersham Biosciences), as described previously (15).
Northern Blotting--
Total RNA was prepared from cells with
the ISOGEN RNA isolation regent (Wako). Cytoplasmic poly(A) RNA was
isolated from total RNA with an mRNA purification kit (Amersham
Biosciences). For Northern blotting, 20 µg of poly(A) RNA was
separated on 1% agarose/formamide gel and blotted onto Hybond N+
(Amersham Biosciences). Membranes were hybridized with either a
32P-labeled BRG1 or Brm probe. The
BRG1 probe was generated from a 5.25-kb
SalI-NotI fragment of pSVhSNF2 Nuclear Run-on Assay--
Equal numbers of nuclei (2 × 107) were isolated from SW13(vim Probe Preparation--
Digoxigenin-labeled probes for in
situ hybridization were synthesized from linearized plasmids using
the digoxigenin RNA labeling kit (Roche Molecular Biochemicals).
To synthesize the BRG1 and Brm antisense
riboprobes, pBluescriptSK(+)-BRG1(D) and pBluescriptSK(+)-Brm(H) were
linearized with EcoRV and ClaI, respectively, and
transcribed by T3 RNA polymerase. To synthesize the vimentin
antisense riboprobe, pBluescriptSK(+)-vimentin was linearized with
EcoRI and transcribed by T7 RNA polymerase.
In SituHybridization--
SW13 cells were cultured on
collagen-coated chamber slides (Nalge Nunc International, Naperville,
IL) and fixed with 4% paraformaldehyde, hybridized with
digoxigenin-labeled antisense RNA probes specific for the
BRG1, Brm, and vimentin genes and
treated with anti-digoxigenin-alkaline phosphatase conjugated Fab
fragments (Roche Molecular Biochemicals). The probes were detected by a
2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate fluorescent
detection set (Roche Molecular Biochemicals). The cellular nuclei were
counterstained with TOTO-1 (Molecular Probes).
SW13(vim
The vimentin gene has been reported to be one of the
representative genes that are under the control of transcription factor AP-1. Tandem AP-1 binding sites that mediate serum inducibility were
identified in its promoter region (28). We have reported previously
that transactivating activity of AP-1 depends on the presence of a
functional SWI/SNF complex and that AP-1 function in SW13 cells was
strongly attenuated, because the cells are deficient in the expression
of both catalytic subunits of the SWI/SNF complex. Considering these
prior observations, we hypothesized that the expression of
BRG1 and Brm might be recovered in SW13(vim+).
Therefore, we have determined the expression levels of BRG1
and Brm in both SW13(vim
By Northern blot analysis of poly(A) RNA prepared from SW13(vim
To examine whether the clear difference in the expression patterns
observed between the two subtypes of SW13 can be stably maintained in
the cloned culture, we monitored the expression of BRG1,
Brm, and vimentin mRNA at single cell
resolution (Fig. 2). After in
situ hybridization with specific probes (red), each monolayer culture was counterstained with TOTO-1 to detect cellular nuclei. The entire population of SW13(vim Several Genes under the Control of AP-1 Are Expressed Specifically
in SW13(vim+) Because of the Presence of Functional BRG1and
Brm--
We have reported previously that collagenase
(intracisternal collagenase, MMP1) and
c-met genes could be induced after DNA transfection of
expression vectors carrying the BRG1 or Brm gene, together with c-Fos and c-Jun expression vectors, into SW13 cells (9).
The CD44 gene is another representative gene that is under the control of AP-1 (29) and was recently reported to be induced by
either BRG1 or Brm supplied exogenously into SW13 cells (16, 17, 30).
We therefore examined the expression status of three other genes,
collagenase, c-met, and CD44, in
SW13(vim Introduction of a Physiological Level of Exogenous BRG1 or Brm in
SW13(vim SW13(vim
We next analyzed mRNA expression 3 days after the treatment with
CHAP31 or 5-azadC by in situ hybridization using the
BRG1, Brm, and vimentin probes,
respectively. As shown for BRG1 (Fig. 6), all the populations of SW13(vim
We next measured the mRNA levels of BRG1 and
Brm, as well as vimentin, collagenase,
c-met, and CD44 in SW13(vim
In the case of 5-azadC treatment, the induced BRG1 mRNA
level was close to the level in SW13(vim+), but the expression level of
Brm mRNA was much lower than that in SW13(vim+).
Expression levels of the four AP-1-driven genes were also significantly
lower compared with those in SW13(vim+) (Fig. 7). When these
5-azadC-treated cells were kept for 7 days in the absence of this DNA
methyltransferase inhibitor, expression of CD44 and
collagenase mRNAs became undetectable (Fig. 7,
+5-azadC/day7). These observations are reminiscent of the
previous report that cells do not express the CD44 gene even in the presence of BRG1 protein in Brm null mice (18). These results show that 5-azadC can not establish stable transition of
SW13(vim SW13(vim Expression of the BRG1 and Brm Genes Is Completely Suppressed by
Post-transcriptional Regulation Operating in SW13(vim
To examine how the HDAC inhibitor released the post-transcriptional
suppression observed in SW13(vim Co-culture with SW13(vim
To examine whether BRG1 and Brm mRNA
expression in SW13(vim+) might be affected in the presence of
SW13(vim
The next important point would be whether the reduction of this gene
expression observation in SW13(vim+) is reversible. To discriminate
these two possibilities, we isolated cellular clones by end-point
dilution from a co-culture containing SW13(vim+) at 50%, which reduced
vimentin mRNA to one-fourth during 4 days of co-culture
as described above. The 15 isolated clones were analyzed for their
expression levels of vimentin mRNA using RT-PCR. Among them,
seven cellular clones expressed vimentin mRNA at the same
level as the pure culture of SW13(vim+) #21, whereas the other eight
clones expressed no detectable vimentin mRNA, like the
original SW13(vim The human adrenal carcinoma cell line, SW13, has been reported to
be deficient in both BRG1 and Brm expression. It
has been used for many experiments to analyze the function of the
SWI/SNF complex, because the complex completely loses its catalytic
subunits. In this work, however, we present evidence that the original
cell line of SW13 is composed of two subtypes, one that does not
express BRG1 and Brm (SW13(vim We intended to liberate SW13(vim Because the run-on experiments indicated that the HDAC inhibitor did
not affect either the frequency of transcriptional initiation or the
rate of transcriptional elongation in SW13(vim We have shown previously that murine leukemia virus-based retrovirus
vector transgene transcription is rapidly suppressed in human tumor
cell lines lacking expression of Brm, such as C33A, SW13,
Saos-2, and G401, even though these vectors can successfully enter,
integrate, and initiate transcription (15). We further presented
evidence that retroviral gene transcription was maintained by the
counteraction between the trx-G protein complex (Brm-containing SWI/SNF
subfamily) and a Pc-G protein complex (containing YY1 and HDACs)
through a chromatin structure and histone acetylation in the promoter
region of provirus, 5'-long terminal repeat (15, 37). In these
experiments, we used SW13(vim At this moment, the molecular mechanisms involved in the
post-transcriptional regulation of BRG1 and Brm
remain largely unsolved. Sequence similarity between BRG1
and Brm might suggest that similar molecular mechanisms are
operating in the regulation of these two genes, whereas all the direct
target genes of post-transcriptional suppression might not be fully
described. Therefore, we can not exclude the possibility that the
retroviral gene silencing observed in SW13(vim It is also noteworthy that expression of the BRG1 and
Brm genes in SW13(vim+) is efficiently suppressed when
co-cultured with SW13(vim)) and the another, which does express both
(SW13(vim+)). The presence of BRG1 and Brm in SW13 correlates
completely with the cellular ability to express such genes as
vimentin, collagenase, c-met, and
CD44 that were under the control of a transcription factor,
AP-1, which was shown previously to require a functional SWI/SNF
complex for its transactivating activity. Transient treatment with
inhibitors of histone deacetylase induced a stable transition of
SW13(vim
) to a cell type indistinguishable from SW13(vim+),
suggesting that these two subtypes are epigenetically different. Run-on
analysis indicated that, unlike these four genes driven by AP-1,
transcription of the BRG1 and Brm genes in
SW13(vim
) are initiated at a frequency comparable with
SW13(vim+). In both SW13(vim
) and SW13(vim+) cells, the
BRG1 and Brm genes were transcribed through the
entire gene at a similar efficiency, indicating that their expression was completely suppressed at the post-transcriptional level in SW13(vim
) cells. We would like to propose that SW13 can spontaneously transition between two subtypes by switching expression of
BRG1 and Brm at the post-transcriptional level.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(7), estrogen receptor (8), AP-1 (9), and p53 (10)
support a model in which transcription factors recruit the SWI/SNF
complex to target genes (11, 12), providing mechanistic links between
epigenetic transcriptional regulation and chromatin remodeling
(13).
)), and the other, which does (designated as SW13(vim+))
(23). We examined the molecular basis for the presence of these two
subtypes and addressed whether this phenomenon was related to the
deficiency in BRG1 and Brm expression in this
cell line.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) and SW13(vim+) by End-point
Dilution--
As reported previously (22, 23), SW13 cells express
vimentin protein in a mosaic pattern when observed under
immunofluorescence microscope (Eclipus 600; Nikon). SW13 cells were
subcloned by end-point dilution using 96-well plastic plates (Corning
Glass Inc., Corning, NY). Fifty subclones were obtained and examined for human vimentin fibers by immunofluorescence microscopy; 31 subclones were completely negative for vimentin expression
(SW13(vim
)), whereas 19 subclones were positive (SW13(vim+)). Two
subclones of SW13(vim
) were selected and designated as SW13(vim
) #1
and #2, respectively, and two subclones of SW13(vim+) were selected and
designated as SW13(vim+) #21 and #22, respectively.
-actin,
-collagenase, and -c-Met were generated by inserting RT-PCR products
from the corresponding mRNA into pCR2.1 (Invitrogen), a TA
cloning vector designed to directly clone PCR products. The
RT-PCR primer pairs used were as follows: BRG1(B),
5'-atccccacccaggggcctggagggt-3' and 5'-ctcctgcaggatctccacagggtcg-3'; Brm(F), 5'-caggggaaaaggacgttgcctggcttgc-3' and
5'-ggctgcctgggcttgcttgtgctcccaaacc-3'.
-actin,
5'-agctcaccatggatgatgatatcgccgcgc-3' and
5'-gtccgcctagaagcatttgcggtggac-3'. Primer pairs for
collagenase and c-met were described previously (9). pCR2.1-BRG1(A) and -Brm(E) were generated by inserting PCR
fragments of the genomic sequences covering the promoter proximal region of the corresponding gene into pCR2.1. The PCR primer pairs used
were as follows: BRG1(A), 5'-gccgcggtgctgaggggga-3' and
5'-tgccacggcctcgcgacactgtg-3'. Brm(E),
5'-tttctgtactctgggtgactcagagaggg-3' and 5'-tgccggctgggcttcttgacc-3'. To
construct pBluescriptSK(+)-vimentin, a 1.1-kb RT-PCR fragment (primer
pairs 5'-cggaattcgcctcttctccgggagcc-3' and
5'-gcatctggcgttccagggatccattggttc-3') was digested by EcoRI
and HincII, and the 0.7-kb fragment generated was inserted
into the EcoRI-HincII site of pBluescriptSK(+).
The pBluescriptSK(+)-CD44 was constructed from a 0.76-kb RT-PCR
fragment (primer pair 5'-cagacctgcccaatgcctttgatggacc-3' and
5'-cggaattccgtgtcccagctccctgtaatgg-3') was digested by
HincII, and the resulting 0.42-kb fragment was inserted into
the HincII site of pBluescriptSK(+).
pBluescriptSK(+)-BRG1(C) was created by inserting the 0.85-kb
BamHI-DraI fragment of pSVhSNF2
(25) into the
BamHI-EcoRV site of pBluescriptSK(+).
pBluescriptSK(+)-BRG1(D) was created by inserting the 0.8-kb
NotI fragment of pSVhSNF2
into the NotI site
of pBluescriptSK(+). pBluescriptSK(+)-Brm(G) was generated by inserting
the 0.88-kb PshAI-SacI fragment of pSVhSNF2
(25) into the EcoRV-SacI site of
pBluescriptSK(+). pBluescriptSK(+)-Brm(H) was generated by inserting
the 0.78-kb ClaI- HindIII fragment of
pSVhSNF2
into the ClaI-HindIII site of
pBluescriptSK(+).
, and the
Brm probe was generated from a 5.25-kb HindIII
fragment of pSVhSNF2
. The probes were labeled with the RediprimeII
DNA labeling system (Amersham Biosciences).
) #1, SW13(vim+) #21,
MDA-MB435, and SW13(vim
) #1 treated with CHAP31. Transcriptional
elongation was carried out in the presence of 200 µCi of
[
-32P]UTP, and radiolabeled RNA was isolated from each
sample. They had almost the same total radioactivity and were
hybridized to Hybond N+ filters that were slot-blotted to such plasmid
DNA (1 or 5 µg) as pCR2.1-BRG1(A), -BRG1(B), -Brm(E), -Brm(F),
-collagenase, -c-met, and -
-actin and pBluescriptSK(+)-BRG1(C),
-BRG1(D), -Brm(G), -Brm(H), -vimentin, and -CD44. As the control
plasmids, pCR2.1 and pBluescriptSK(+) were also slot-blotted.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) Does Not Express Either BRG1 or Brm but SW13(vim+)
Expresses Both--
Subclones of SW13 cells, either expressing or not
expressing vimentin, were selected previously by limiting
dilution from the parent cell line SW13, on the basis of the presence
or absence of a vimentin filament detectable by immunofluorescence
microscopy. Using the same protocol, we have cloned two SW13(vim
)
subclones, #1, #2, and two SW13(vim+) subclones, #21, #22. For each
clone, the expression levels of vimentin were determined
both by semiquantitative RT-PCR of total RNA (Fig.
1A) and by Western blot
analysis of the total cellular lysates (Fig. 1B) and were
compared with those of a control cell line, MDA-MB435, that express
vimentin. vimentin mRNA and vimentin protein
were clearly detectable in SW13(vim+) #21 and #22, whereas they are
completely undetectable in SW13(vim
) #1 and #2. The generation times
of clone SW13(vim
) #1 and #2 and SW13(vim+) #21 and #22 cultures were
32-34 h, indicating that SW13(vim
) and SW13(vim+) had no significant
difference in their growth rates in monolayer culture.
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Fig. 1.
Comparison in gene expression patterns
between SW13(vim ) and SW13(vim+). A, expression
levels of the endogenous vimentin, BRG1, and
Brm genes, as well as a housekeeping gene, GAPDH,
were measured by semi-quantitative RT-PCR of total RNA isolated from
SW13(vim
) #1 and #2 and SW13(vim+) #21 and #22, as well as a control
human cell line, MDA-MB435. Expression levels in SW13(vim+) #21 were
taken as 1.0, and columns are shown as the average ratio
obtained by three independent experiments. The bars
represent the S.D. B, expression of vimentin, BRG1, and Brm
protein was analyzed by Western blot of total cellular lysates (20 µg
(for anti-BRG1 and vimentin) or 40 µg (for anti-Brm) per
lane) prepared from SW13(vim
) #1 and #2, SW13(vim+) #21
and #22, and MDA-MB435. Arrows indicated the position of
vimentin (55 kDa), BRG1 (190 kDa), and Brm (190 kDa). C,
expression levels of the corresponding mRNA were determined by
Northern blot of the poly(A) RNA (20 µg per lane).
Position of single strand RNA markers (RNA Ladder; Invitrogen) are
shown on the left.
) and SW13(vim+). SW13(vim
) #1
and #2 have no detectable levels of both BRG1 and Brm protein (Fig.
1B), as judged by Western blot and as has been reported for
the original cell line of SW13. When BRG1 and Brm
mRNA levels in these clones were assessed by RT-PCR, they were also
negative (Fig. 1A). In SW13(vim+) #21 and #22, however,
mRNA and the protein product of either BRG1 or
Brm genes were clearly detectable (Fig. 1, A and
B) like in MDA-MB435, which is competent for the SWI/SNF
complex (15). Despite the clear contrast in the gene expression
patterns of vimentin, BRG1, and Brm,
SW13(vim
) #1 and #2 and SW13(vim+) #21 and #22 expressed almost the
same mRNA levels of a housekeeping gene, GAPDH.
) #1
or SW13(vim+) #21, SW13(vim
) did not express either full-length
mRNA of BRG1 and Brm or any aberrant
transcript when detected by specific probes that covered entire coding
regions (Fig. 1C). These results are consistent with our
hypothesis that BRG1 and Brm are expressed in
SW13(vim+) and further suggest that this subtype of SW13 has a
functional SWI/SNF complex, which supports the expression of several
endogenous genes, including vimentin.
) #1 (or #2) failed to
express these three genes, whereas all the cells in SW13(vim+) #21 (or
#22) cultures expressed them at a significant level, as shown for
SW13(vim
) #1 and SW13(vim+) #21 (Fig. 2). These results indicate that
each subtype maintains its phenotypes stably after cloning by end-point
dilution (corresponding to about 20 cycles of cell division).
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Fig. 2.
Expression of vimentin,
BRG1, and Brm mRNA in cultures of
SW13(vim ) #1 and SW13(vim+) #21 detected by in situ
hybridization. The cellular nuclei were
counterstained with TOTO-1 (green) and superimposed
to the RNA probes stained with red fluorescence. Overlapping regions
were stained in yellow. The bar indicates 20 µm.
) #1 and #2 and SW13(vim+) #21 and #22. Like the
vimentin gene, expression of collagenase,
c-met, and CD44 genes were completely
undetectable in SW13(vim
) #1 and #2, whereas they are expressed in
SW13(vim+) #21 and #22 (Fig. 3) at a
significant level. These results clearly reveals that several genes
under the control of AP-1 are expressed specifically in SW13(vim+)
because of the presence of functional BRG1 and Brm. In none of these
biochemical and cytological analyses of the expression levels (see
Figs. 1-3) (data not shown) did we detect any significant differences
between cellular clones #1 and #2 or between #21 and #22. Therefore, we
used clones #1 and #21 for SW13(vim
) and SW13(vim+), respectively,
for further analyses.
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Fig. 3.
Expression pattern of
collagenase, c-met, and
CD44 mRNA in cellular clones of SW13(vim ) and
SW13(vim+). The same RNA samples as used for Fig. 1 were analyzed
for the expression levels of collagenase, c-met,
and CD44 by semiquantitative RT-PCR. Expression levels in
SW13(vim+) #21 were taken as 1.0, and each column shows the average
ratio obtained by three independent experiments. The bars
represent the S.D.
) Switches Expression of vimentin, collagenase, c-met, and
CD44 Genes--
Next we used retrovirus vectors to examine whether
SW13(vim
) can induce vimentin, collagenase,
c-met, and CD44 by exogenous expression of
BRG1 or Brm at a physiological level. Three days after infection, we detected clear induction of the mRNA of these four genes in SW13(vim
) at levels comparable with those observed in
SW13(vim+) (Fig. 4). These results
indicate that exogenous expression of the BRG1 or
Brm gene in SW13(vim
) switches the expression of several
genes, driven by such transcriptional factors as AP-1, in an all or
none manner. In SW13(vim
), endogenous expression of both the
c-fos and c-jun genes was readily detectable by
RT-PCR at levels similar to those in SW13(vim+), indicating that the amount of c-Fos/c-Jun dimer, which contributed most strongly to the
recruitment of the SWI/SNF complex by AP-1, would not be rate-limiting (data not shown).
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Fig. 4.
Patterns of mRNA expression in
SW13(vim ) transduced with BRG1 or Brm virus, compared with those in
SW13(vim+). SW13(vim
) #1 was transduced with control vector,
BRG1 virus, and Brm virus, respectively, and total RNA was extracted 3 days after transduction. RNA was also prepared from untransduced
SW13(vim
) #1 and SW13(vim+) #21 using the same protocol. For each
RNA, expression levels of endogenous vimentin,
collagenase, c-met, and CD44 were
measured by semi-quantitative RT-PCR. Expression levels in the
untransduced SW13(vim+) #21 were taken as 1.0, and each
column shows the average ratio obtained by three independent
experiments. The bars represent the S.D.
) Can Transition to a Cell Type Indistinguishable from
SW13(vim+) by Adding HDAC Inhibitors--
Complete loss of the
BRG1 and Brm genes in SW13(vim
) could represent
either genetic defects or epigenetical suppression. If the
BRG1 and Brm genes are not mutated at all in
SW13(vim
), and if their expression is epigenetically suppressed in
SW13(vim
), we might artificially relax the suppression of
BRG1 and Brm expression in SW13(vim
) cultures
by adding some reagents that modulate the epigenetical regulation. In
such a case, BRG1 and Brm induced in SW13(vim
) could reconstitute the
functional SWI/SNF complex, and the cells would recover the ability to
express vimentin. We screened several reagents for an
ability to efficiently induce vimentin expression by
detecting the presence of vimentin protein using immunocytochemical
staining. The reagents tested included a protein kinase C inhibitor
(TPA), HDAC inhibitors (CHAP31 and trichostatin A), and DNA
methyltransferase inhibitors (5-azacytidine and 5-azadeoxycytidine). We
also tested conditioned medium recovered from SW13(vim+)
overnight cultures, considering that SW13(vim+) might secrete some
inducing factors that trigger the transition. We found that HDAC
inhibitors CHAP31 (4-8 nM) and TSA (200 nM) caused efficient induction of vimentin protein in SW13(vim
) within 4 days (Fig. 5). Because 200 nM
TSA had cytotoxic effects on SW13(vim
) cells 1 day after the
treatment, we hereafter show only the results obtained by 4 nM CHAP31 as the HDAC inhibitor, but essentially the same
results were obtained by using 200 nM TSA instead. 5-azadC (0.5 µM) treatment also caused vimentin induction in a
lesser extent, but 5-azacytidine (2 µM) induced vimentin
only marginally. Neither TPA treatment nor exchanges with the
conditioned medium of SW13(vim+) had any effect (data not shown).
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Fig. 5.
Induction of vimentin
mRNA by the treatment with CHAP31 or TSA. SW13(vim ) #1
was treated with 2 nM (closed squares), 4 nM (closed triangles), and 8 nM
(closed circles) CHAP31 or 100 nM (open
triangles) and 200 nM (open circles) TSA.
The medium was changed every day, and at the time indicated, the total
RNA was purified from the culture. Expression levels of
vimentin were measured by RT-PCR using essentially the same
procedure as shown in Fig. 1. Expression levels in untreated SW13(vim+)
#21 were taken as 1.0.
)
treated with either CHAP31 or 5-azadC were induced at a similar level
of BRG1 mRNA to that observed in SW13(vim+). Similar
results were obtained when a Brm or vimentin
probe was used instead (data not shown), indicating that a clear
phenotypic transition occurred in the entire population by these
treatments.
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Fig. 6.
Induction of BRG1 mRNA
in a culture of SW13(vim ) treated with CHAP31 or 5-azadC for 3 days. The cellular nuclei were counterstained with TOTO-1
(green) and superimposed to the RNA probes stained
with red fluorescence. The bar indicates 20 µm.
) treated for 3 days
with CHAP31 or 5-azadC and compared them with mRNA levels in
untreated SW13(vim
) or SW13(vim+) (Fig.
7). In the case of CHAP31 treatment,
BRG1, Brm, vimentin,
collagenase, c-met, and CD44 mRNA
became detectable in SW13(vim
), and their expression levels were very
close to the levels in SW13(vim+) (Fig. 7,
+CHAP31/day0). Induction of BRG1, Brm, and
vimentin protein was also confirmed by Western blot analysis (data not
shown). After this 3-day treatment with CHAP31, the cells were
subsequently grown for 7 days in the absence of this HDAC inhibitor and
analyzed for the expression levels of the same series of genes (Fig. 7,
+CHAP31/day7). The expression levels were
unchanged after the subsequent growth, indicating that SW13(vim
)
stably altered its phenotype to a cell type that was indistinguishable
from SW13(vim+). After the SW13(vim
) culture was treated for 6 days
with 4 nM CHAP31, we found that the cellular phenotype was
stably maintained for at least 2 months when judged from the expression
patterns of these six genes (data not shown).
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Fig. 7.
Induction of BRG1 and
Brm mRNA, as well as vimentin,
collagenase, c-met, and
CD44 mRNA, in SW13(vim ) by treatment with CHAP31
or 5-azadC. SW13(vim
) #1 was treated with either CHAP31 (4 nM) or 5-azadC (0.5 µM) for 3 days. Total RNA
was prepared immediately after treatment (day 0) or after
subsequent growth for 7 days in the absence of the reagent (day
7). Expression levels in untreated SW13(vim+) #21 were taken as
1.0, and each column shows the average ratio obtained by
three independent experiments. The bars represent the
S.D.
) to SW13(vim+), although SW13(vim
) can transiently assume
SW13(vim+)-like phenotype after this treatment. Although underlying
mechanisms here are largely unclear at this moment, we think a balance
between BRG1 and Brm proteins would be important to maintain the
SW13(vim+) phenotype.
) Cells Initiate BRG1 or Brm Transcription at a Similar
Frequency as Do SW13(vim+) Cells--
Because the phenotypes of these
two subtypes of SW13, SW13(vim
) and SW13(vim+), are likely to be
regulated primarily by the BRG1 and Brm
expression, we are interested in the molecular mechanisms involved in
the lack of expression of these two genes in SW13(vim
). We prepared
cellular nuclei from SW13(vim
), SW13(vim+) and MDA-MB435 and their
transcriptional activity was measured by a nuclear run-on assay (Fig.
8). Surprisingly, nuclear extracts of
either SW13(vim
) or SW13(vim+) synthesized mRNA of
BRG1 and Brm to a similar extent. Because the DNA
probes used to monitor the BRG1 and Brm
transcripts (Fig. 9) include the
promoter-proximal region covering the first (5'-noncoding) exon and the
subsequent intron of these genes, these results indicate that
SW13(vim
) cells initiate BRG1 or Brm
transcription at a similar frequency as do SW13(vim+) and MDA-MB435
cells. However, no transcription of vimentin,
collagenase, c-met, and CD44 genes was
detected in the nuclear extracts of SW13(vim
) at all even by the
slots containing 5-µg DNA probes, whereas they were readily
detectable in SW13(vim+) by the slots of 1-µg DNA probes. These
results confirm that these four genes are regulated at the
transcriptional level in an all or none manner by the presence or
absence of functional SWI/SNF complex in the cells.
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Fig. 8.
Nuclear run-on transcription assay of several
genes using SW13(vim ), SW13(vim+), and MDA-MB435. Cellular
nuclei were prepared from SW13(vim
) #1, SW13(vim+) #21, and
MDA-MB435. After transcriptional elongation in the presence of
[
-32P]UTP, the nuclear extracts were hybridized to the
DNA plasmids slot-blotted on the filter (1 or 5 µg). Control plasmid
(control) is the empty vector pCR2.1, which was used for
cloning of most of the genes examined here. Another empty vector,
pBluescriptSK(+), which was used for cloning vimentin and
CD44, gave the same result.
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Fig. 9.
Genomic structure of the BRG1
and Brm genes and nuclear run-on transcription
assay using DNA probes covering several regions of these genes.
The genomic structures of the BRG1 and Brm genes
are schematically presented using the NCBI Genome data base under NCBI
accession numbers NT 011176 and NT 008413, respectively
(upper panel). Vertical lines indicate exons, and
the arrowhead shows the start codon of the corresponding
gene that locates in the second exon. The sequences covered by the DNA
probes used for the run-on assay are also shown (A-H).
A and E are originated from genomic sequences,
whereas B-D and F-H are derived from cDNA
as described under "Experimental Procedures." In nuclear run-on
assay, cellular nuclei prepared from the same set of the cell lines as
was shown in Fig. 8 were hybridized to 1 µg of the DNA probes. Signal
intensity of each band was quantified by an Image Analyzer (BAS 2000).
The ratios B/A, C/A, and
D/A (or the ratios F/E,
G/E, and H/E) are
indicative of relative levels of transcriptional elongation
versus initiation in the BRG1 (or Brm)
genes. Averages of these independent experiments are shown as
S.D.
) Cells--
A
growing list of genes transcribed by RNA polymerase II are regulated at
the level of transcriptional elongation in the promoter-proximal region
(31). These genes include such important genes as c-myc, hsp70, c-fos, and MAP kinase
phasphatase-1 (32-36). In the case of c-myc, quiescent
or differentiated cells reveal significant blocks to elongation during
transcription through the first exon or first intron sequences, whereas
RNA polymerase II in proliferating cells transcribes through the
c-myc gene much more efficiently (32-34). As shown in the
genomic structure of the BRG1 and Brm genes (Fig.
9), both genes possess a non-coding exon like the c-myc or
N-myc genes. We therefore tested whether blocks of
the transcriptional elongation of BRG1 and Brm
are operating in SW13(vim
). For detailed run-on analysis in these
genes, we prepared additional sets of DNA probes that detect exons
covering the entire BRG1 gene (probe B,
C, and D) and the entire Brm gene
(probe F, G, and H), respectively.
Relative band densities of probes B-D (or probes
F-H) to that of probe A (or probe E) were
determined (Fig. 9) for each cell. The results clearly indicated that
the relative densities were unchanged between SW13(vim
) and
SW13(vim+) and were close to those of MDA-MB435 cells, which produce
high levels of BRG1 and Brm proteins. Thus we do not detect any
specific blocks in transcriptional elongation in SW13(vim
) cells.
These results indicate that the loss of mature mRNA of
BRG1 and Brm in SW13(vim
) cells can not be
explained by premature transcriptional termination observed in such
genes as c-myc and N-myc but are rather caused by
the post-transcriptional suppression that is specifically operating in
SW13(vim
) cells.
), cellular nuclei were isolated from
a SW13(vim
) culture treated with CHAP31 for 3 days and analyzed by
the run-on assay. As shown in Fig. 9, the HDAC inhibitor did not
affected either the frequency of transcriptional initiation of
BRG1 (probe A) and Brm (probe
E) or the rate of transcriptional elongation of these genes
(probes B-D and F-H). From these results, it is
clear that the BRG1 and Brm genes are not the
direct targets of CHAP31. Therefore we think that the HDAC treatment
activates transcription of certain genes that are essential to
counteract to the post-transcriptional suppression operating in
SW13(vim+).
) Causes Suppression of BRG1, Brm, and
vimentin Expression in SW13(vim+)--
SW13 has been reported to be
deficient in both BRG1 and Brm expression, but,
as described above, we have presented evidence that the original cell
line of SW13 is composed of two subtypes, one that does not express
either BRG1 or Brm (SW13(vim
)) and the other subtype that does
express both of them at a significant level (SW13(vim+)). Because SW13
(vim+) clones were isolated frequently (19 of 50 clones described from
the original cell line of SW13; see "Experimental Procedures"),
this rather high frequency of SW13(vim+) isolation was unexpected,
because many groups, including ours, have failed to detect any
BRG1 and Brm expression in the original cell line
of SW13. Indeed, BRG1, Brm, and
vimentin mRNA was nondetectable when the original cell
line of SW13 that was used to isolate SW13(vim
) #1 and #2 and
SW13(vim+) #21 and #22 was analyzed using the same RT-PCR procedure as
shown in Fig. 1A.
) in cultures, we prepared a series of mixed cultures
composed of these two subclones. Four days after the initiation of
mixed cultures, RNA was prepared, and mRNA expression levels of
BRG1 and Brm were measured by RT-PCR. In a
parallel experiment, we prepared a series of mixtures of RNA isolated
from pure cultures of SW13(vim
) and SW13(vim+). In both
BRG1 and Brm, mRNA levels of the mixtures of
RNA samples increased linearly by elevating the percentage of the RNA
sample isolated from pure SW13(vim+) culture. mRNA levels in the
mixed culture were significantly lower than those of mixture of the RNA
samples at the corresponding percentage (Fig.
10). For example, when the equal cell
number of SW13(vim
) and SW13(vim+) were co-cultured (50% of
SW13(vim+)), the mRNA levels of BRG1, Brm,
and vimentin were about one-fourth of those of the mixture
of the two RNA samples. In the co-culture containing SW13(vim+) at
10%, none of these mRNA was detectable at all (Fig. 10). mRNA
levels of collagenase, c-met, and CD44
(data not shown) were also significantly reduced in the co-cultures.
The mRNA level of GAPDH was not affected at all by the
co-cultivation (Fig. 10). These results indicate that expression of the
BRG1 and Brm genes in SW13(vim+) are efficiently suppressed when co-cultured with SW13(vim
), suggesting that
SW13(vim
) can somehow transfer its suppressive effects on
SW13(vim+).
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Fig. 10.
Expression levels of BRG1,
Brm, vimentin, and GAPDH
mRNA in mixed cultures of SW13(vim ) and SW13(vim+).
SW13(vim+) #21 were co-cultured with SW13(vim
) #1 at various ratios,
disrupted for total RNA isolation 4 days after the initiation of
co-culture. Expression levels in SW13(vim+) #21 were taken as 1.0, and
each column shows the average ratio obtained by three
independent experiments (closed circles). As a control, a
series of mixtures between the RNA isolated from the pure cultures of
SW13(vim
) #1 and SW13(vim+) #21 were analyzed in the same manner
(open circles). The bars represent the S.D.
) #1. These results clearly indicate that SW13(vim+) cells transiently reduce expression of
vimentin gene after the co-culture but recover the full
expression level of vimentin upon re-cloning. This
phenomenon will explain why BRG1 and Brm
expression in SW13 were not detected previously.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)) and another that does express both
genes at a significant level (SW13(vim+)). Both subtypes maintain their own phenotypes after cloning by end-point dilution and subsequent cell
growth for more than one month. The presence of BRG1 and Brm in SW13
has an exact correlation to the cellular ability to express genes such
as vimentin, collagenase, c-met, and
CD44 (see Figs. 3 and 4). Transcription of these four genes
has been known to be dependent upon either BRG1/Brm or AP-1, a
transcription factor that requires the SWI/SNF complex for its
transactivating activity. The clear switching of these four genes in an
all or none manner implies that chromatin remodeling by a functional SWI/SNF complex is essential for their transcription under
physiological conditions.
) from suppression and screened
several reagents that induce the transition. Transient treatment with
inhibitors of HDAC such as CHAP31 and TSA, supports the induction of
BRG1 and Brm expression (see Figs. 6 and 7),
which probably triggers the subsequent expression of the four genes
driven by AP-1. These results indicate that, like SW13(vim+),
SW13(vim
) retains the functional BRG1 and Brm
genes, but their expression is especially suppressed. Cellular
transition is relatively stable and produced a cellular phenotype that
is indistinguishable from that of SW13(vim+) (Fig. 9). The transition
is maintained for at least 2 months in the absence of CHAP31. Our
nuclear run-on analysis indicated that, unlike the four genes driven by
AP-1, transcription of the BRG1 and Brm genes are
initiated and elongated through the entire gene in SW13(vim
) at a
similar efficiency to SW13(vim+). These results indicate that the loss
of mature mRNA of BRG1 and Brm in SW13(vim
)
cells can not be explained by premature transcriptional termination
observed in such genes as c-myc and N-myc but
caused by the post-transcriptional suppression. Some specific steps
during mRNA maturation from the primary transcript would be inhibited.
) cells (Fig. 9), it is
clear that the transcriptional initiation of the BRG1 and
Brm genes are not the direct target of CHAP31. Therefore we
think that the HDAC treatment activates transcription of certain genes
that are essential to counteract the post-transcriptional suppression.
From our observations, we propose that SW13 can transit between two
subtypes, either of which has distinct epigenetical stability, by
switching expression of the BRG1 and Brm genes at the post-transcriptional level. We further suggest that the transition could occur spontaneously in a monolayer culture as a rare event.
) as SW13, and the extent of the
retroviral silencing in SW13(vim
) was high when we measured it by the
"mosaic colony ratio" (81%) (15). When we measured the
mosaic colony ratio of the SW13(vim+), it was 43%,2 indicating
significant recovery from the gene silencing. Therefore like genes
driven by AP-1, maintenance of retroviral transcription is enhanced by
the presence of the Brm protein in SW13(vim+). The clear difference
between provirus and the genes under the control of AP-1 examined here
(vimentin, collagenase, c-met, and CD44) was that BRG1 had no effect to maintain retroviral
gene expression (15).
) was partly enhanced
by the post-transcriptional suppression mechanisms we detected.
) (Fig. 10). This observation explains why
expression of these two genes had been undetected in the original SW13
cell line until now. By culturing SW13(vim+) in conditioned media that are freshly prepared from SW13(vim
) every 12 h, we did not
detect any phenotypic change in SW13(vim+).2 The apparent
transfer of suppressive signals might involve cell-cell interaction or
paracrine secretion from SW13(vim
), but the molecular mechanisms
involved in it remain unsolved.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Japan Energy, Corp. for supplying CHAP31 and N. Hashimoto and K. Takeda for assistance in preparing this manuscript. We are grateful to Dr. S. Minoguchi for critical reading of this paper. SW13 cells were obtained from the Japanese Collection of Research Bioresources and from Dr. Y. Miyaji (Toho University). We are grateful to A. Watanabe for the preparation of retrovirus vectors.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science, and Culture of Japan.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.
To whom correspondence should be addressed. Tel.: 81-3-5449-5730;
Fax: 81-3-5449-5449; E-mail: iba@ims.u-tokyo.ac.jp.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M208458200
2 M. Yamamichi-Nishina, T. Ito, T. Mizutani, N. Yamamichi, H. Watanabe, and H. Iba, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Pc-G, Polycomb group; trx-G, trithorax group; CHAP, cyclic hydroxamic acid-containing peptide; TSA, trichostatin A; 5-azadC, 5-azadeoxycytidine; TPA, 12-O-tetradecanoylphorbol-13-acetate; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC, histone deacetylase.
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