By
From the * Institute for Molecular Biology and Genetics and Department of Molecular Biology, Seoul
National University, Seoul 151-742, Korea; the Laboratory of Immunology, Korea Cancer Center
Hospital, Seoul 139-240, Korea; and the § Department of Molecular Biology, Dankook University,
Seoul 140-714, Korea
We isolated a new mouse gene that is highly expressed in thymocytes, testis, and brain. This gene, SRG3, showed a significant sequence homology to SWI3, a yeast transcriptional activator, and its human homolog BAF155. SRG3 encodes 1,100 amino acids and has 33-47% identity with SWI3 protein over three regions. The SRG3 protein contains an acidic NH2 terminus, a myb-like DNA binding domain, a leucine-zipper motif, and a proline- and glutamine-rich region at its COOH terminus. Rabbit antiserum raised against a COOH-terminal polypeptide of the SRG3 recognized a protein with an apparent molecular mass of 155 kD. The serum also detected a 170-kD protein that seems to be a mouse homologue of human BAF170. Immunoprecipitation of cell extract with the antiserum against the mouse SRG3 also brought down a 195-kD protein that could be recognized by an antiserum raised against human SWI2 protein. The results suggest that the SRG3 protein associates with a mouse SWI2. The SRG3 protein is expressed about three times higher in thymocytes than in peripheral lymphocytes. The expression of anti-sense RNA to SRG3 mRNA in a thymoma cell line, S49.1, reduced the expression level of the SRG3 protein, and decreased the apoptotic cell death induced by glucocorticoids. These results suggest that the SRG3 protein is involved in the glucocorticoid-induced apoptosis in the thymoma cell line. This implicates that the SRG3 may play an important regulatory role during T cell development in thymus.
Progenitor T cells arise in the bone marrow and migrate
to thymus, where they continue to develop. During
the T cell development, more than 95% of developing immature thymocytes die by apoptosis as a consequence of
negative selection or lack of positive selection (1). This apoptotic death targets mainly the cortical double-positive (CD4+CD8+) thymocytes. In thymocytes, apoptosis can be
triggered by several exogenous stimuli such as glucocorticoids (2), removal of growth factors (5, 6), exposure to
GCs, when complexed with an activated receptor, can
induce or inhibit the expression of specific genes, which
may be related to the induction of apoptosis. The transcriptional regulation of downstream genes by GCs requires not
only GR itself but several additional transcription factors
such as the SWI-SNF protein complex (16). For example, the rat GR, when expressed in yeast, requires SWI-SNF
proteins for transcriptional activation of GR-responsive genes and the GR-SWI3 complexes were coimmunoprecipitated in yeast extract (19, 20). In addition, antibodies
against SWI3 interfere with the ability of rat GR to activate
transcription in Drosophila melanogaster nuclear extracts (19).
SWI3 is a subunit of the SWI-SNF complex that seems
to facilitate transcriptional activation by antagonizing the
repressive actions of chromatin (21). The other subunits
of the SWI-SNF complex so far identified include the
SWI1 (ADR6), SWI2 (SNF2), SNF5, SNF6, SNF11, and
SWP73. The complex was initially identified in Saccharomyces cerevisiae (S. cerevisiae) as a positive regulator of HO, a gene
involved in mating type switching (24, 25), and SUC2, a
glucose-repressible gene that encodes the enzyme invertase (26, 27). These SWI gene products were subsequently
found to be required for the transcriptional activation of
many other genes (28). Such activities of the SWI-SNF
proteins are closely interconnected and they seem to function as components of a complex that associates with genespecific activators (31). The functional significance of
the SWI-SNF complex is reflected by the evolutionary conservation of these genes in higher eukaryotes. Several
higher eukaryotic homologues of SWI-SNF genes such as
Drosophila homeotic gene activator brm, hbrm (also known
as hSNF2 In this paper, we describe a newly isolated mouse gene,
the SWI3-related gene (SRG3), expressed in thymus and
encoding a protein that shows significant amino acid sequence homology to both yeast SWI3 and human BAF155
proteins. The SRG3 protein coimmunoprecipitates with a
mouse SWI2-like protein, suggesting their forming a protein complex in vivo. In addition, our data show that the
SRG3 is expressed at much higher level in thymus than in
peripheral lymphocytes. Because the GC is proposed to be
a regulatory molecule in thymocyte development in thymus, and the SWI-related proteins have an important role
in GC-mediated gene regulation, the high level expression of SWI3-related gene (SRG3) in thymocytes may imply
that SRG3 has a crucial role in thymocyte development as
a mediator of GC-induced transcriptional activation and
apoptotic cell death of thymocytes. As a first step of testing
this hypothesis, we analyzed the effect of downregulation
of SRG3 expression in a GC-sensitive thymoma cell line
on GC-induced apoptosis.
Mice and Cells.
C57BL/6J mice were maintained in the Institute for Molecular Biology and Genetics (Seoul National University,
Seoul, Korea). The yeast strain CY165 (MAT Isolation and Purification of Poly(A)+ RNA.
Total RNA was isolated by CsCl banding, as described by Chomczynski and Sacchi
(44). Intact thymi and spleens were collected from 3-5-wk-old
mice and used as sources for RNA. Poly(A)+ RNA was isolated
by oligo(dT)-cellulose chromatography (45).
Preparation of Subtractive Probe.
~10 µg of poly(A)+ RNA was
heated at 65°C for 2 min and annealed with 1 mg of magnetic
beads containing oligo (dT) (Dynabeads oligo [dT]25; Dynal, Inc.,
Great Neck, NY) for 30 min at room temperature (46). The annealed poly(A)+ RNA was separated in magnetic field, and used
as templates for the synthesis of the first-strand cDNA. The second-stranded cDNA was synthesized by random priming using
hexanucleotides and 200 µCi of [ DNA Sequencing and Computer Analysis.
To determine the
nucleotide sequences, the restriction fragments of the cloned
gene were subcloned into pBluscript (SK Separation of T and B Cell.
Single cell suspensions were prepared from intact spleens and lymph nodes of C57BL/6J mice.
After the red blood cells were removed, cells were resuspended in
PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM
KH2PO4, pH 7.3). To isolate T cells, single cell suspensions were
sequentially reacted with biotinylated H57-597 antibody, which
is specific to Overexpression and Purification of GST-fusion Protein.
For the construction of GST-fusion protein, COOH-terminal region of the
SRG3 gene was inserted into pGEX4T-2 vector in frame. DH5 Immunoprecipitation and Immunoblot Analysis.
The immunoprecipitation of the SRG3 and SWI2-like protein was performed by
a method described by Muchardt et al. (40) with some modifications. The single cell suspension of the mouse thymus was harvested in immunoprecipitation (IP) buffer (20 mM Hepes, pH
7.6, 10% glycerol, 25 mM MgCl2, 0.1 mM EDTA, 0.2% NP-40)
containing 0.1 M potassium acetate and 2.25 µg/ml pepstatin, 10 µg/ml leupeptin, 1 µg/ml soybean inhibitor, 2 mM PMSF, and
0.1 mM DTT. The cells were sonicated and debris were pelleted
by centrifugation. The extracts were precleared with protein
A-sepharose suspension and anti-SRG3 or anti-hSWI2 rabbit antiserum were added. After overnight incubation at 4°C, the extracts were incubated with protein A-sepharose suspension. The
beads were washed three times in IP buffer containing 0.6 M potassium acetate, and once with IP buffer without Hepes. The precipitate was eluted by boiling in SDS-PAGE loading buffer. For
immunoblot analysis, the proteins separated on SDS-PAGE were electrotransferred to nitrocellulose paper, and incubated in blocking solution (3% non fat dry milk, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl) with gentle agitation for 2 h. After the blot was incubated with the SRG3 antiserum or hSWI2 antiserum, the specific
bands were detected by treating the blot with anti-rabbit IgG conjugated with alkaline phosphatase in blocking solution (100 mM
Tris-HCl, pH 9.5, 100 mM NaCl, 10 mM MgCl2) containing
165 µg/ml BCIP and 330 µg/ml NBT.
Complementation of Yeast swi3 Preparation of the Glycerol Density Gradient Sedimentation Fraction
from Thymocytes Extract.
The thymocytes were prepared as a single cell suspension from thymi and homogenized in 700 µl extraction buffer (40 mM Hepes, pH 7.3, 200 mM NaCl, 0.5 mM DTT,
2 mM EDTA, and 2.25 µg/ml pepstatin, 10 µg/ml leupeptin,
1 µg/ml soybean inhibitor). After centrifugation at 12,000 rpm
for 20 min at 4°C, the supernatant containing 7.2 mg of total protein in 400 µl was layered on the top of linear 18-40% 10 ml
glycerol gradient cushion containing 40 mM Hepes, pH 7.3, 200 mM NaCl. After ultracentrifugation at 36,500 rpm in Beckman
SW41 rotor for 20 h at 4°C, the samples were collected from the
bottom of the tube by fractionation into 33 tubes. These fractionated samples were analyzed by immunoblotting with SRG3 or
hSWI2 antiserum.
Construction of a Plasmid Expressing Anti-sense RNA of the SRG3
Gene.
A 2.8 kb XbaI fragment spanning 60 bases of 5 Induction and Measurement of Apoptosis.
The apoptotic cell death
of thymoma cells were induced by treatment with 10 µM hydrocortisone (Sigma Chem. Co., St. Louis, MO) for 72 h at 37°C. After hydrocortisone treatment, the cells were harvested and lysed
in lysis buffer (10 mM EDTA, 50 mM Tris-HCl, pH 8.0, 0.5%
sodium lauryl sarcosine) containing the 100 µg/ml proteinase K
at 55°C for 2 h. The DNA was extracted with phenol/chloroform and precipitated with ethanol. After RNA was removed by
RNAse A treatment, the DNA was analyzed in agarose gel electrophoresis to visualize the fragmented DNA. The apoptotic cell
death was also measured by flow cytometry as described by Nicoletti et al. (52). In brief, the harvested cells were fixed with 70%
ethanol and stained with 50 µg/ml of propidium iodide (PI) and
10 µg/ml RNAse A for 30 min at 4°C. After washing with PBS
buffer, stained cells were analyzed using the FACStar® (Becton
Dickinson, Mountain View, CA) for the DNA content.
We made an attempt to isolate genes that
are specifically expressed in thymus but not in spleen by
subtractive hybridization. One of the clones isolated was
found to be expressed preferentially in thymus and was
found to have similar amino acid sequences to a part of
SWI3 protein of S. cerevisiae. The isolated gene has an open
reading frame of 3,300 bp encoding 1,100 amino acids
(Fig. 1). The homology search in the GenBank sequence
database of NCBI using the BLASTP program showed
amino acid sequence similarity to the SWI3 protein of S. cerevisiae (Fig. 2). The new gene was named as SRG3 to
emphasize its relatedness to SWI3 gene.
The NH2-terminal part of the SRG3 is highly acidic;
23% of 231 amino acids (211-441) are either aspartate or
glutamate. The COOH-terminal part has the leucine-zipper motif (53), and the proline- and glutamine-rich region.
The proline and glutamine residues make up 44% over 150 amino acids (952-1,100). In addition, the myb-like tryptophan repeat domain or SANT (SWI3, ADA2, N-CoR, and TFIIIB B Recently, the human homologues of yeast SWI3, BAF155,
and BAF170, were identified and designated as BRG1 associated factors (42). We found that the SRG3 protein has
very high amino acid sequence homology to BAF155 protein (Fig. 2). They show 91% identity in their complete
amino acid sequences, with the major deviations occurring at
the NH2 termini. Therefore, we conclude that the SRG3
protein is the murine counterpart of human BAF155 protein. The SRG3 protein also matches well with the BAF170,
another human SWI3 homologue; they are 60% identical
and 70% similar at the amino acid level. In addition, the
SRG3 protein has a considerable homology with the yeast
YFK7 protein, which is also known as SWI3b (42) (Fig. 2).
The major similarity between the SWI3 and its mammalian homologues at the amino acid level is found over three
regions (Fig. 2 B). The three regions of the SWI3 and
SRG3 proteins shows 33-47% identity and 61-64% similarity. The NH2-terminal part of the SWI3 is also extremely acidic, yet similarity with the SRG3 is still very
low. The leucine-zipper motifs in the region III of these proteins are different from SWI3 in that the third leucine
of the SWI3 protein is replaced by phenylalanine in SRG3
and BAF155 (Fig. 2 C). In the COOH-terminal region,
SWI3 lacks proline- and glutamine-rich regions found in
its mammalian homologues. The data suggests that these
proteins have similar protein structures and probably similar
biochemical functions. However, there is divergence in
structure and, probably, also in function between these
proteins.
To identify the
protein product of the SRG3 gene, polyclonal antibody
was produced against the SRG3-GST fusion protein. The COOH-terminal part of the SRG3 gene was inserted inframe into the pGEX4T-2 plasmid containing the glutathione-S-transferase (GST) gene. The overexpressed GST-
3C fusion protein with ~85 kD of molecular mass was
used to immunize rabbits through subcutaneous injection.
After primary and booster injections, polyclonal antiserum against GST-3C fusion protein was obtained. The antiserum was confirmed to recognize specifically the fusion protein (data not shown). To identify the SRG3 gene product,
immunoblot analysis was performed with crude extracts
prepared from thymus and lymph node. As shown in Fig.
3 A, two bands of ~155 and 170 kD were observed; the
155-kD protein is likely to be SRG3. This is supported by
the observations that the protein matches well to the size of
the BAF155 in human (42), that the antiserum immunoprecipitates the 155-kD protein (Fig. 3 B), and that the intensity of the 155-kD protein band was specifically reduced
when anti-sense RNA to the SRG3 is expressed in a cell
line (see Fig. 6 A). The antiserum also recognized a 170-kD
protein that seemed to be similar to the SRG3 protein in
its structure. Considering the results of human SWI3 homologues (42), it is likely that the 170-kD protein may be
the mouse counterpart of the human BAF170 protein. The SRG3 and BAF170 are quite similar to each other over regions I (86%), II (93%), and III (89%) (see Fig. 2 B), and
anti-SRG3 antiserum seems to recognize a murine BAF170like protein as well as SRG3. Interestingly, SRG3 is expressed at a three times higher level in thymus than in lymph
nodes; however, the 170-kD protein is expressed at similar
levels in both tissues.
We tested whether the SRG3 protein is associated with
other proteins such as a mouse SWI2-like protein. After
immunoprecipitation of cell extract with the antiserum followed by blotting with the same antiserum, the specific
band of 155 kD was observed (Fig. 3 B, bottom). We have
also employed the hSWI2 antiserum that recognized a protein with molecular mass ~195 kD in thymocyte extract
(Fig. 3 B). The 195-kD protein is similar to the size of
hSWI2 (43) and seems to be a mouse homologue of
hSWI2. When thymocyte extract was immunoprecipitated
with the SRG3 antiserum and blotted with the hSWI2 antiserum, a specific band corresponding to the mouse SWI2like protein was detected (Fig. 3 B, top). These results suggest that the SRG3 associates with a SWI2-like protein and
possibly with other SWI-SNF proteins.
To test the possibility that the mouse SRG3 gene
product complements the yeast swi3 Northern blot analysis of SRG3 gene
expression showed that the transcripts of this gene were
3.5 and 5 kb in size (Fig. 4 A). It seems that the 5.0-kb
mRNA encodes the SRG3 protein because the 3.5-kb
mRNA is not long enough to include the 3,300-base open reading frame, 5
As shown
in Fig. 3 A and Fig. 4, the level of SRG3 expression in thymus was much higher than the level in peripheral lymphocytes. Therefore, even though these proteins may form the
SWI-SNF complex together with other proteins in mouse
cells, it is also possible that the SRG3 protein exists independently of the SWI-SNF complex, especially in thymus.
It has also been suggested that the SWI-SNF complexes are
present as multiple forms in different tissues and cell lines
(42). To see whether SRG3 protein exists mostly as a SWI-
SNF complex, we fractionated the thymus extract according to the size of molecules by a glycerol density gradient.
Each fraction was run on a gel and immunoblotted with
the anti-SRG3 and the anti-hSWI2 antiserum. As shown
in Fig. 5, the mouse SWI2 protein was mostly fractionated
as a 2 MD complex as previously reported (42). However,
the SRG3 protein was detected in a broad range of molecular masses, from 150 kD to 2 MD. Interestingly, a major
portion of the protein was fractionated as 300 kD. The 170-kD protein was also fractionated similarly to SRG3
protein. These results suggest that a major portion of the
SRG3 protein (and the 170 kD protein) exists independently of the SWI-SNF complex and that they may play a
special role in developing thymocytes. This was similarly
reported for BAF155 and BAF170 proteins; some BAF155
and BAF170 are not associated with human SWI-SNF
complexes (42).
One of the roles played by the SRG3 or the SWI-SNF
complex in thymus may be mediating GR-induced transcriptional activation. One consequence of it is the induction of GC-mediated apoptosis in immature thymocytes.
Thus, we have tested whether the SRG3 is required for the
GC-mediated apoptosis in GC sensitive thymocyte cell line.
A plasmid expressing the SRG3 gene in anti-sense orientation under the control of CMV promoter (pRcASRG3)
was constructed. The pRcASRG3 construct was introduced
into a GC-sensitive thymoma cell line, S49.1, through DNA
transfection, and two clones, clone A and B, displaying reduced expression of SRG3 were selected. As shown in Fig.
6 A, clone A and B express ~50 and 30% SRG3 protein of
the vector transfectants, respectively. The DNA fragmentation induced by GC treatment was greatly reduced in these
clones and this effect was more dramatic in clone B, which
expressed lower level of SRG3 protein than clone A (Fig. 6 B).
The reduction in apoptotic cell death of the pRcASRG3
transfectants was also confirmed by FACS® analysis of the
DNA contents of the cells. After GC treatment, about 46%
of the vector transfectants were subdiploid and apoptotic (Fig. 6 C, c); however, only about 4% of the treated clone
B transfectants were apoptotic (Fig. 6 C, d). These results
suggest that the SRG3 protein is involved in the GC-induced
apoptosis of the thymoma cell line.
The subunit proteins of the SWI-SNF complex were
initially identified in S. cerevisiae as transcriptional activators
of a set of genes. The SWI-SNF proteins seem to facilitate
transcription by antagonizing the repressive actions of chromatin (21). The subunit proteins seem to function as
components of a complex that associate with gene-specific
activators. Mutations in any of the subunit genes resulted in
very similar phenotypes and the phenotype of multiple defective swi In yeast, the rat GR can activate transcription from a
promoter bearing the GR-responsive element in the presence of glucocorticoid (GC) (57, 58). It has been reported
that transcriptional activation by GR, which regulates the
expression of a network of genes in a tissue-specific manner, is dependent on SWI1, SWI2, SWI3, SWP73 gene
functions in yeast (19, 59). Furthermore, SWI3 was coimmunoprecipitated with GR in yeast extracts (19), suggesting that the two proteins interact directly upon activation
of GR. Our study showed that SRG3 was highly expressed
in developing thymocytes compared with mature peripheral T and B lymphocytes (Fig. 3). When the level of SRG3
protein was reduced to ~50-30% of normal level of thymoma cells by expressing anti-sense RNA to the gene, apoptosis induced by GC on these cells was significantly reduced
(Fig. 6). These results indicate that SRG3 is required for
GC-induced apoptosis in the thymoma cells and suggests
that SRG3 is an important factor for GC-mediated regulation of thymocyte development.
It has been hypothesized that GC might affect thymocyte development in a number of ways. Thymocytes respond to GC by apoptosis both in vitro and in vivo (2, 60).
In vivo, the immature CD4+CD8+ thymocyte population
is rapidly killed in the presence of GC, whereas both the
CD4-irradiation (7), and antigen binding involving the CD3/
TCR (8). The effect of glucocorticoids (GCs)1 is selective; the immature CD4+CD8+ thymocyte fraction is rapidly killed by GC treatment, whereas both the precursor
population (TCR
CD4
CD8
) and mature thymocytes
(CD4+ or CD8+) are relatively resistant (11). It was reported that GC is produced within the thymus (12), and
that transgenic expression of anti-sense RNA to glucocorticoid receptor (GR) significantly affects the thymocyte development (13). These results suggest that endogenous GC
produced in thymus may participate as an important regulatory molecule of normal thymic development (14, 15).
) and BRG1 (also known as hSNF2
) have been
identified (36). In addition, a human protein homologue of SNF5 (40, 41) and mouse BAF60 (42), which is
homologous to SWP73, have been identified. Recently, distinct complexes containing the BRG1 or hbrm that have
an in vitro activity similar to yeast SWI-SNF have been
purified from human cell lines (42, 43). From these complexes, the human BAF155 and BAF170 proteins that are
homologous to SWI3 protein were identified (42).
, swi3
:: trp1-
1,
HO-lacZ, ura3-52, leu2-
1, his3-
200, ade2-101, lys-801) cells,
and yCP50 plasmid containing the SWI3 gene were gifts from C. Peterson (University of Massachusetts, Worcester, MA). Yeast
cells were grown in synthetic minimal medium (0.67% Bactoyeast nitrogen base without amino acids; GIBCO BRL, Gaithersburg, MD) supplemented with leucine, histidine, adenine, and lysine to a mid-log phase. The mouse thymoma cell line, S49.1, was
purchased from American Type Culture Collection (ATCC,
Rockville, MD) and grown in DMEM supplemented with 10% fetal bovine serum.
-32P]dCTP (3,000 Ci/mmol).
To prepare a subtractive probe, 200 µg of the first-stranded
spleen cDNA conjugated with magnetic beads were mixed with
the labeled probe. After incubating at 55°C for 1 h, the labeled
probe DNAs hybridized to the first-stranded spleen cDNA were
removed using magnetic field, and the remaining subtractive probe was used for the screening of thymic cDNA library. The
cDNA library was obtained from M.M. Davis at Stanford University (Stanford, CA).
) vector (Stratagene
Inc., La Jolla, CA). Nested deletions were generated by the Erasea-Base system (Promega Corp., Madison, WI). The nucleotide
sequence was determined by dideoxy chain termination method
(47) using Sequenase 2.0 kit (United States Biochemical Corp.,
Cleveland, OH). Homology searches of the nucleotide and deduced amino acid sequences with sequences were performed at the National Center for Biotechnology Information, using the
BLAST network service (48).
TCR, and streptavidin-conjugated microbeads.
The B cells were reacted with microbead-conjugated goat anti-
mouse IgM. The cell-magnetic bead conjugates were separated by a magnetic cell sorter (MACS; Miltenyi Biotec, GmbH, Bergisch Gladbach, FRG) (49). The purity of isolated populations
were confirmed by FACS® (Becton Dickinson, Mountain View,
CA) analysis and Northern blot assay using TCF-1, a T cell-specific gene, as a probe.
cells harboring recombinant plasmids with GST-3C fusion were grown overnight and diluted to 1:200 in 200 ml of LB medium.
After incubation at 37°C for 2 h with vigorous shaking, the culture was treated with 1 mM IPTG and then incubated for 3 h to
induce expression of the fusion protein. Cells were harvested and
resuspended in a sample loading buffer (50 mM Tris-HCl, pH
6.8, 100 mM DTT, 4% SDS, 0.2% BPB, 20% glycerol), and
boiled for 2 min. These lysates were analyzed by electrophoresis
on polyacrylamide gel. The overexpressed protein was purified
by glutathion-sepharose 4B affinity chromatography as described
by Smith and Johnson (50). The polyclonal antiserum was prepared by immunizing New Zealand white rabbit with the purified fusion protein.
Mutant.
For complementation
study, the SWI3-SRG3 hybrid gene encoding the NH2-terminal
part of SWI3 and the COOH-terminal part of SRG3 was synthesized. The full-length SRG3 gene or the SWI3-SRG3 hybrid gene
were inserted into the pRS316GU vector containing the URA3
promoter and the URA3 gene as auxotrophic marker. The resulting constructs were used to transform CY165 yeast cells that harbor swi3
mutation and the HO-lacZ fusion gene construct. The
Ura+ cells were cultured in synthetic minimal media. Cells were
collected when OD595 of the culture reached 0.5.
-gal assay was
performed as described by Breeden and Nasmyth (51). The Cp15
construct that contains the SWI3 gene in yCP50 vector was also
used to transform CY165 cells as a positive control.
-untranslated region and 2772 bases of the SRG3 coding sequence was
inserted into the pRc/CMV vector (Invitrogen, San Diego, CA)
in the anti-sense orientation. The resulting plasmid was designated
as pRcASRG3. The plasmid construct, pRcASRG3, or pRc/CMV
was transfected into S49.1, a thymoma cell line (ATCC), by
electroporation. The transfected cells were selected and maintained with 1 mg/ml Geneticin (GIBCO BRL, Gaithersburg, MD)
in DMEM supplemented with 10% fetal bovine serum.
Cloning and Characterization of the Mouse SWI3-related
Gene (SRG3).
Fig. 1.
Amino acid sequence of the SRG3 gene predicted from
cDNA sequence. The predicted leucine-zipper motif is indicated by asterisks, and the myb-like tryptophan repeat is indicated by the closed triangles. The regions showing highest homology to the yeast SWI3 are underlined. These sequence data are available from EMBL/GenBank/DDBJ under accession number U85614.
[View Larger Version of this Image (61K GIF file)]
Fig. 2.
The comparison of amino acid sequences of the SRG3 with SWI3 and its human homologues, BAF155 and BAF170. The YFK7, another yeast homologue of SWI3, is also presented. The regions showing highest homology are shown by rectangles with distinctive fillings (A). The SRG3 and
human homologues of SWI3 protein contain the proline- and glutamine-rich domains that lack in the yeast SWI3. Amino acid comparisons of the three
regions (Region I, II, and III) are shown in B and C. The three homologous regions of SRG3 and SWI3 protein displayed 33-47% identity and 61-64%
similarity. The amino acids that are identical to consensus sequences are indicated as dots (C).
[View Larger Version of this Image (28K GIF file)]
) domain (54), which may be involved in
interaction with DNA, was also found in the middle part of
this protein (Fig. 2 A). Therefore, this gene product seems
to act as a transcriptional activator probably by interacting
with other proteins or by interacting with DNA.
Fig. 3.
Immunoblotting and immunoprecipitation of the SRG3
protein. The overexpressed COOH-terminal part of SRG3 gene in Escherichia coli system was used to immunize rabbits to produce the polyclonal antiserum. When thymus and lymph node extract were blotted with the
SRG3 antiserum, bands at 155 and 170 kD were detected (A). When the
extract was blotted with the hSWI2 antiserum, a band at 195 kD (B, top)
was detected. After immunoprecipitating the extract with the SRG3 antiserum, the precipitates were blotted with the SRG3 antiserum (B, bottom)
or the hSWI2 antiserum (B, top), displaying the 155- and 195-kD bands, respectively. Immunoprecipitation with preimmune serum and blotting with
the SRG3 and hSWI2 antiserum dose not show any band (B). TCL, total
cell lysate; IP:Pre, immunoprecipitation with the pre immune serum; IP:
anti-SRG3, immunoprecipitation with the SRG3 antiserum.
[View Larger Version of this Image (29K GIF file)]
Fig. 6.
Effect of SRG3 expression on GR mediated apoptosis. (A) Expression of the antisense RNA to SRG3 in thymoma
cell line, S49.1, reduced the level
of SRG3 protein. The pRcASRG3
plasmid expressing 2.9 kb of
XbaI fragment (bases 1-2829) of
SRG3 gene in anti-sense orientation under the control of CMV
promoter was transfected into
the S49.1 cells. The expression
of the SRG3 protein in transfected cells was detected by immunoblotting using the SRG3
antiserum. lane 1, S49.1; lane 2,
vector only; lane 3, pRcASRG3
transfectant, clone A; lane 4,
pRcASRG3 transfectant, clone
B. (B) Effects of SRG3 expression
on apoptotic cell death induced
by glucocorticoid treatment.
The 10 mM of hydrocortisone
was treated for 72 h (lanes 1-4)
and the DNAs of each cell were
electrophoresed on 2% agarose gel containing ethidium bromide. The DNA fragmentation was reduced as SRG3 expression level was reduced. Lane C, control (untreated S49.1); lane 1, S49.1; lane 2, vector only;
lane 3, pRcASRG3 transfectant, clone A; lane 4, pRcASRG3 transfectant clone B. (C) FACS® analysis of the DNA contents of the cells transfected with
vector only (a and c) and pRcASRG3 transfectant, clone B (b and d). The subdiploid peak (closed and open arrowheads) indicates apoptotic cells induced
by glucocorticoid treatment (c and d).
[View Larger Version of this Image (19K GIF file)]
Mutant.
mutation, the S. cerevisiae host strain CY165 (swi3
) containing the HO-lacZ
gene (25) was transformed with the pRS316GU vector containing the SRG3 full coding sequence or SWI3-SRG3 hybrid gene at the downstream of the URA3 promoter.
Cells transformed with the SWI3-SRG3 hybrid gene were
confirmed to express the hybrid protein by Western blotting using the SRG3 and SWI3 antisera (data not shown).
The growth rate of swi3
mutant was very slow (33) and
was not significantly changed after the transformation with
either the SRG3 or the SWI3-SRG3 hybrid. In addition,
expression of lacZ gene, controlled by the HO promoter
which requires the SWI-SNF protein complex for transcriptional activation, was not induced after the transformations of SRG3 or SWI3-SRG3 hybrid. The level of expression of lacZ gene in the SRG3 transformant was only
~22% of the mutant cells transformed with the yeast SWI3
(a positive control), whereas the LacZ expression level in
mutant cells transformed only with a vector plasmid was ~20% of the SWI3 transformed cells. The result was consistent in three independent experiments.
- and 3
-untranslated regions, and poly(A)
tail. At this point, it is not clear what the 3.5-kb mRNA
species encodes for. However, when a 1.5-kb PstI fragment
from the 3
-end of the SRG3 gene was used as a probe, only
the 5-kb transcript was detected (data not shown), suggesting the possibility that these two transcripts are different at
their 3
termini. The SRG3 gene is expressed at higher levels in thymus, brain, and testis than in other tissues (Fig. 4
A). Northern blot analysis with RNAs isolated from separated splenic T and B cell populations showed that the two
populations express similar level of SRG3. The separated
population was highly pure, as judged by the Northern blot
using TCF-1 gene, a T cell-specific gene (55, 56), as a
probe (Fig. 4 B) and by FACS® analysis (data not shown).
Interestingly, however, the level expressed in each peripheral lymphocyte population was only about 20% of that expressed in thymocytes (Fig. 4 B), as it was similarly shown
by Western blot analysis (see Fig. 3 A).
Fig. 4.
Northern blot analysis of SRG3 gene expression in different
organs (A) and cell types (B). The same amount (15 µg) of total RNAs
isolated from various tissues were analyzed by probing with a 1.8-kb HindIII fragment (bases 653-2361). (A) The lanes represent thymus (1), spleen
(2), brain (3), lymph nodes (4), testis (5), and lung (6). Two transcripts of
about 5 and 3.5 kb in size were expressed highly in thymus (lane 1), brain
(lane 3), and testis (lane 5). (B) Both T and B cell expressed the SRG3.
Lane 1, thymus; lane 2, T cells; lane 3, B cells. Normal T and B cells were
separated from spleen and lymph nodes by magnetic activated cell sorter (miniMACS). The purity of the separated population was tested by probing the RNA blot with TCF-1 (closed arrowhead). Both B and T cells expressed about the same levels of SRG3, as judged by the control -actin
probe (open arrowhead).
[View Larger Version of this Image (46K GIF file)]
Fig. 5.
Size fractionation of the SRG3 and SWI-SNF protein complexes. After total thymocytes extract was separated on glycerol density gradient sedimentation, the gradients were fractionated and immunoblotted with the SRG3 and hSWI2 antisera. The SRG3 protein was fractionated as separated complexes (300 kD) which are different from SWI2 complexes (2 MD). The blue-dextran (2 MD), thyroglobulin (669 kD),
-amylase (200 kD), and alcohol-dehydronase (150 kD) were used as
standard molecular mass size markers.
[View Larger Version of this Image (43K GIF file)]
or snf
mutants was identical to that of a single
swi
or snf
mutant (33). Besides, SWI-SNF proteins functioned interdependently in transcriptional activation (32).
In addition, the SWI-SNF proteins were copurified and
coimmunoprecipitated (31, 34) and were shown to be
components of a large multisubunit complex (34, 35). Recently, human SWI-SNF complexes were shown to be
present in multiple forms made up of 9-12 proteins and
several of them were purified using the BRG1 antiserum
(42, 43). There seems to exist several different forms of
SWI-SNF complexes in a single cell and in different differentiated cell types (42). Based on this, it has been suggested
that these complexes are involved in tissue-specific and developmental process-specific chromatin remodeling activity needed for the differentiation of a cell (42). The human
homologues of yeast SWI3, the BAF155 and BAF170, were
also identified and the two proteins seem to exist as core
components in the same protein complex (42). SRG3 identified in the present study is homologous to human
BAF155. The overall sequences are 94% similar to each
other and the protein products are quite similar in size.
The antiserum produced against SRG3 recognized an additional 170-kD protein, which seems to correspond to human BAF170, implying that there also exists two SWI3
homologues in mouse. In spite of this similarity, the expression patterns of BAF155 and SRG3 seem to be somewhat different. BAF155 is selectively expressed in muscle
and heart but expressed at relatively low levels in liver and
brain (42); however, SRG3 is expressed at much higher
level in brain than in liver (Fig. 4 A). The selective expression patterns of BAF155 and BAF170 in different tissues were quite similar to each other. However, mouse SRG3 is
expressed at higher level in thymus than in peripheral lymphoid tissues (Fig. 3); on the other hand, the 170-kD protein is expressed at similar levels in these tissues. Interestingly, some SRG3 proteins and the 170 kD proteins seem
to exist independently of the SWI-SNF complex (Fig. 5).
It is not yet conclusive whether some SRG3 and 170-kD
proteins exist as heterodimers or homodimers. These results suggest that even though the human and mouse homologues of yeast SWI3 are structurally and functionally
similar, they may play distinct roles in each species and in
different tissues.
CD8
precursor population expressing no TCR
and mature thymocytes (CD4+ or CD8+) cells expressing
high levels of TCR are relatively resistant. Using anti-CD3
monoclonal antibodies as a model for negative selection, it
has been found that pretreatment of mice with a GR antagonist, RU486, protects immature CD4+CD8+ thymocytes
from apoptosis (61). Furthermore, it has been reported that
radioresistent thymic epithelial cells constitutively produce GC (12). Thus, GC and GR may function as important
regulators in normal thymic differentiation (14, 15). At this
point, it is not clear whether GC-induced apoptosis of thymoma cells requires SRG3 as a component of the SWI-
SNF complex or as a separate entity. In yeast, transcriptional activation by GR is blocked by disrupting any one of
SWI1, SWI2, and SWI3 genes, suggesting the possibility
that the SWI-SNF complex is required for the GC sensitivity of thymocytes. However, it is noteworthy that SRG3 protein is expressed at a much higher level in thymocytes
than in peripheral T lymphocytes and that the major part of
SRG3 protein may exist independently of the SWI-SNF
complex in thymus. Furthermore, even though the antisense RNA expression reduced the level of SRG3 protein
in the transfected cells, there still remained ~50% (clone A)
or 30% (clone B) of normal level of SRG3 protein (Fig. 6).
Even in the case of the clone B transfectant, this is at least the similar level of protein found in peripheral T lymphocytes (Fig. 3). Therefore, it is likely that there still may be
enough SRG3 protein left to form SWI-SNF complexes in
the transfectants. These results suggest a possibility that
SRG3 protein may function independently of the SWI-
SNF complex in GC-mediated apoptosis. No matter how
SRG3 functions in GC-mediated apoptosis in thymoma cells, either as a component of SWI-SNF complex or as an
independent factor of the complex, our present results
show that SRG3 protein is required for the process and
possibly plays an important regulatory role during thymocyte development.
Address correspondence to Dr. Rho Hyun Seong, Institute for Molecular Biology and Genetics, Seoul National University, Kwanak-gu Shinlim-dong San 56-1 Bldg. 105, Seoul 151-742, Korea.
Received for publication 22 January 1997 and in revised form 17 March 1997.
1 Abbreviations used in this paper: GC, glucocorticoids; GR, glucocorticoid receptor; GST, glutathione-S-transferase; IP, immunoprecipitation; MACS, magnetic activated cell sorter; PI, propidium iodide.We thank Dr. M.M. Davis for the kind gift of mouse thymus cDNA library and Dr. C. Peterson for providing yeast strains used in this study.
This work was supported in part by the S.N.U.-Daewoo Research Fund (94-06-2068, 96-06-2078) and the Biotech 2000 project to R.H. Seong, and in part by grants from the Korea Science and Engineering Foundation, through the Research Center for Cell Differentiation, to S.D. Park and R.H. Seong.
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