From the Immunology Unit, Department of Cell and Molecular Biology, Lund University, P. O. Box 7031, S-220 07 Lund, Sweden
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ABSTRACT |
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A novel Ets protein was isolated by yeast
one-hybrid screening of a cDNA library made from
lipopolysaccharide-stimulated mouse splenic B cells, using the SP6 Proteins that are members of the Ets family of transcription
factors are involved in a variety of developmental and cellular responses (1). At present around 20 distinct proteins within the family
have been described, and all these bind to a similar, purine-rich DNA
sequence element (1, 2). Given the similarity of their binding sites,
and given that several of the proteins have overlapping pattern of
expression, it has been difficult to define the individual function of
each Ets protein. However, gene targeting experiments in mice have
shown that Ets proteins are not functionally redundant (3-6), but that
each Ets protein has a distinct function.
In the B lymphoid lineage, several of the Ets proteins are expressed;
among these are Ets-1 (7, 8), elf-1 (9), PU.1 (Spi-1) (10), and Spi-B
(11). However, none of these factors are restricted in their expression
to B cells only. Ets-1, elf-1, and Spi-B are also expressed in T cells
(3, 8, 9, 12), and PU.1 can be found in both lymphoid and myeloid cells
(10, 13). Still, all four factors have been implicated in
transcriptional regulation of B cell-specific genes. Hence, a binding
site for Ets-1 has been found in the Ig heavy chain intron enhancer
(14), while binding sites for elf-1 have been found in the Ig heavy chain 3' enhancer (15) and in a Spi-B is closely related to PU.1, and no clear difference between the
binding sequences of the two proteins has been defined (2). As the
expression pattern between PU.1 and Spi-B is overlapping within the B
cell lineage (3), it would not be surprising should the two proteins be
functionally redundant. However, this is not the case, since, although
PU.1 Here we describe the cloning and characterization of a novel Ets family
protein, Spi-C, which was identified in an yeast one-hybrid screen due
to its interaction with the SP6 Yeast Strains--
Four copies of a fragment containing the
pentadecamer element and the Yeast Library Construction and Screening--
BALB/c mouse
spleens were minced and set up at a lymphocyte concentration of 1 × 106 lymphocytes/ml in Iscove's modified Dulbecco's
medium with 5% fetal calf serum and 25 µg/ml LPS. After 72 h,
the activated cells were washed once in phosphate-buffered saline and
total RNA isolated using the acid phenol method. A total of
approximately 1 mg of RNA was isolated, and poly(A)+ RNA
was obtained by purification on an oligo(dT)-cellulose column (26).
Double-stranded cDNA was prepared, inserted into predigested Hybri-ZAP cDNA Library Screening and Sequencing--
The Hybri-ZAP In Vitro Translation, EMSA, and Methylation Interference
Analysis--
The full-length Spi-C clone was cloned between the
EcoRI and XhoI sites of the pcDNA3 vector
(Invitrogen), while the partial Spi-C cDNA obtained from the yeast
screening was cloned between the EcoRI and XhoI
sites in a modified pcDNA3 vector in which an OCT2 ATG region
(5'-AATTGCGGGCAGCATGGTTCATTCCAGCGAATT-3') had been inserted into the
EcoRI site. The cDNAs were in vitro
translated using Promega's coupled transcription/translation
reticulocyte system from the T7 promoter. The EMSAs were performed as
described (16), using between 1 and 0.1 µl of reticulocyte extract.
Competitions were performed using 10, 30, 100, and 300 times, or 30, 100, 300, and 1000 times molar excess of competitor; the sequences of
the competitors and probes can be found in Ref. 16. The methylation interference analyses were made as described (24) using protein translated from the yeast clone. After autoradiography of the wet gel
for 4 h, the free and bound probes were cut out, eluted in 1× TE
with 50 mM NaCl overnight, extracted with phenol, and ethanol-precipitated. The precipitated DNA was treated with piperidine as described (26) and subsequently separated on a 10% polyacrylamide gel electrophoresis, 1× TBE gel that was fixed, dried, and autoradiographed.
Recombinant Proteins, Antisera, and Site Selection--
The
full-length or carboxyl-terminal part of Spi-C cDNA were
amplified via PCR using Spi-C primers (full-length:
5'-ACGAGATCTATGACTTGTTGTATTGATC-3' against
5'-ACGGAATTCTCAGCTCTGGTAACTGG-3', carboxyl part: 5'-ACGA GATCTGAGGCTGTTCTCCAAAGA-3' against 5'-ACGGAATTCTCAGCTCTGG TAACTGG-3') and inserted between the BamHI and EcoRI sites of
the pGEX-2 vector (Amersham Pharmacia Biotech) after restriction with
BglII and EcoRI and verified by sequencing using
the pGEX 5' primer (Amersham Pharmacia Biotech). The proteins were
expressed and purified on glutathione-Sepharose according to the
manufacturer's instructions (Amersham Pharmacia Biotech), and the
carboxyl terminus-containing protein was further purified on a Q Column
(Beckman) and was then used to raise a polyclonal sera in rabbits
according to standard procedures. For the first round of site
selection, an N oligonucleotide (5'-GCCACTGCGAATTCTCT(N)20AATGGGATCCCGTCGCA-3') was
annealed to a [32P]polynucleotide
kinase-labeled oligonucleotide, and fill-in was performed
using Klenow enzyme. This oligonucleotide was then used in EMSA with
the recombinant, full-length glutathione S-transferase-Spi-C protein. A piece of the gel that corresponded to the mobility of the
Spi-C-DNA complex was cut out and eluted in 1× TBE overnight. The
eluted DNA was amplified for 15 cycles in PCR using 5' and 3' primers
(5' primer: 5'-GCCACTGCGAATTCTC-3', 3' primer: 5'-TGCGACGGGATCCCAT-3'), end-labeled, and purified by gel electrophoresis. The selection procedure was repeated twice more, and the final PCR products were cut
with EcoRI and BamHI, inserted into the
pGEM3Z vector (Promega), and sequenced.
RNA Extractions, Northern Blotting, and RT-PCR
Assays--
Northern blotting was performed using standard protocols,
and 2 µg of poly(A)+ RNA was loaded in each lane (26).
Isolation of cells was done either by cell sorting (FACS Vantage,
Beckton Dickinson) or by using magnetic MACS beads (Miltenyi Biotec).
RNAs were prepared from tissue using a Pharmacia poly(A) RNA kit (the B
cell lines), Qiaex spin columns (Qiagen; ES cells, lymph node and
liver), or RNAzol B (Tel-test, Inc.; the rest). The RT reactions were
performed using Superscript II (Life Technologies) according to the
manufacturer's instructions, and the primers and probe used were the
same as used in the library screen (see above). The amount of cDNA
in each reaction was titrated using HPRT primers (27). Primers used to
detect PU.1 expression in cell lines were described before (28). In
some cases different exposures from the same blots are shown, but the
HPRT exposure were always matched with the Ets probe exposure.
Transfection Constructs and Assay--
The 4× Southern Blotting and Chromosome Mapping--
Southern blotting
and the isolation of a genomic Spi-C clone was performed according to
standard protocols (26). The chromosome mapping using FISH was
performed by SeeDNA Biotech Inc., Toronto, Canada. Mouse chromosomes
were prepared according to the published procedure. Briefly,
lymphocytes were isolated from mouse spleen and cultured at 37 °C in
RPMI 1640 medium supplemented with 15% fetal calf serum, 3 µg/ml
concanavalin A, 10 µg/ml lipopolysaccharide, and 5 × 10 Cloning of Spi-C, a
To investigate the binding specificity of the Spi-C gene product, the
insert from the original yeast clone was transcribed and translated
in vitro and the protein product analyzed by band-shift using the bait sequence as a probe. As shown in Fig. 1C,
unlabeled probe competed efficiently for binding in this assay while a
competitor that contained the SP6 Spi-C Is a Member of the Ets Protein Family--
A full-length
cDNA clone of Spi-C was isolated from a
Fig. 2 (B and C) shows an analysis of the protein
sequence homology in the Ets domain between Spi-C, PU.1, Spi-B, elf-1,
and Ets-1. Outside the Ets domain, the amino acid sequence diverged quite extensively compared with the other Ets family proteins. With
regard to the Ets domain, that of Spi-C matched those of PU.1 and Spi-B
more closely than those of Ets-1 or elf-1, although all amino acids
that are universally conserved in Ets proteins (34) were also conserved
within the Ets domain of Spi-C (Fig. 2B). The amino acid
identity within the Ets domains of Spi-B and PU.1 was 73%, while it
was 59% or 63% when compared with Spi-C for PU.1 and Spi-B,
respectively (Fig. 2C). When the Spi-C DNA Interactions Are Indistinguishable from Those of
PU.1--
The interaction between Spi-C and the
To further investigate the relationship between Spi-C and PU.1 DNA
binding, we performed band-shift analysis using the Spi-C Is Preferentially Expressed in Mature B Cells--
To
characterize the expression pattern of Spi-C, we performed
semi-quantitative RT-PCRs from a panel of mouse tissues and purified
cell populations (Fig. 4A).
High expression was found in spleen and significant expression was also
detected in lymph node and bone marrow. Low expression could also be
detected in thymus, Peyer's patches, and liver, while no expression
was seen in heart, striated muscle, or kidney. We subsequently analyzed the Spi-C expression in purified cell populations. No Spi-C expression was detected in embryonic stem cells, double-negative or
double-positive thymocytes, or purified peripheral CD4+ or
CD8+ T cells. On the other hand, purified macrophages
expressed Spi-C RNA at low but detectable levels, and B220+
peripheral B lymphocytes at higher levels. Thus, Spi-C seems to be
preferentially expressed in the macrophage and B lymphoid lineage.
We next investigated the expression of Spi-C RNA in various cell lines,
as a complement to the tissue and polyclonal cell analysis discussed
above. For comparison, the expression of PU.1 RNA was also investigated
in these preparations while all samples had first been normalized using
HPRT expression (data not shown). Fig. 4B shows that the
macrophage cell line J774 showed low but significant Spi-C expression,
concordant with the observed expression in freshly isolated
macrophages. More interestingly, when cell lines representing various
stages of B lymphoid differentiation were analyzed for Spi-C
expression, only cell lines having a phenotype of mature B cells were
positive. The two plasmacytoma cell lines J558 and S194 were negative,
as were the pre-B cell lines 230-238, 18-81, and 70Z/3. The 70Z/3
cell line differentiates to a mature B cells upon addition of LPS (38),
but such induction did not induce Spi-C expression. Hence, it appears
as if Spi-C expression is temporally regulated during B lymphoid
differentiation and is preferentially expressed in mature,
non-secretory B cells.
We finally investigated the expression of Spi-C by Northern blotting
using poly(A)+ RNA from two plasmacytomas and from the B
cell lymphoma K46R (Fig. 4C). The Spi-C probe hybridized
with a single band migrating below the 18 S RNA marker in the K46R
lane, while no hybridization was detected in the lanes with
plasmacytoma RNA. All three lanes gave a similar hybridization signal
with the GADPH control probe. The size of the Spi-C transcript
correlates favorably with the isolated cDNA clone and the low
expression level with the RT-PCR data above.
Spi-C Can Transactivate a Spi-C Is Encoded by a Single-copy Gene Mapping to Chromosome 10, Region C--
We finally wanted to determine the genomic
representation of Spi-C and therefore performed a genomic Southern
blot. As shown in Fig. 6A,
digestion of genomic DNA with four different enzymes gave a single
strongly hybridizing band when probed with a Spi-C cDNA probe. With
the BamHI and XbaI digests, a weaker hybridizing band of higher mobility was also observed. For the XbaI
digest, this can be explained by the presence of an XbaI
site in the Spi-C coding region while a BamHI site is
present within the Spi-C
locus.2
We subsequently cloned the Spi-C gene and used a 12-kilobase pair
fragment containing the complete Spi-C locus for chromosomal mapping
using FISH (Fig. 6B). Under the conditions used, the FISH detection efficiency was 91% (among 100 checked mitotic figures, 91 of
them showed signals on one pair of chromosomes). DAPI banding was used
to identify the specific chromosome, and an assignment between signals
from the probe and mouse chromosome 10 was obtained. The detailed
position was further determined to region C based on the summary from
10 photos (Fig. 6C). Hence, Spi-C is encoded by a
single-copy gene mapping to chromosome 10, region C in the mouse genome.
The expression of several transcription factors has been
shown to be essential for B cell development (39, 40). Some of these
are obligate for the formation of several hematopoietic lineages, while
others seem to be selectively involved in B cell development. Examples
of transcription factors of both these types can be found within the
Ets family of transcription factors. For example, the phenotypes of
mice lacking either PU.1 or the closely related Spi-B, which have a
partially overlapping expression patterns within the B cell
compartment, differ significantly. In mice homozygous for an
inactivated PU.1 gene defects in the development of T and B
lymphocytes, granulocytes and monocytes were observed (5, 6). In mice
devoid of Spi-B, the phenotype was milder and only antigen receptor
signaling in B cells during immune responses seemed to be perturbed,
while the remaining hematopoietic system appeared to be normal
(23).
Here we describe a novel Ets family protein, Spi-C, that seems to be
preferentially expressed within the B cell compartment and has an
expression pattern distinct from that of Spi-B and PU.1. A low level of
Spi-C expression was detected in the macrophage cell line J774 and in
purified macrophages, but whether this is representative for the whole
macrophage lineage remains to be investigated. Furthermore, the Spi-C
RNA expression detected in liver and thymus could be due to the fact
that these tissues contained a low number of B cells that expressed
Spi-C. Upon very long exposures, a low level of Spi-C expression could
also be detected in heart and kidney RNA (data not shown). The
conclusion that Spi-C expression in some of these organs was derived
from contaminating B cells is strengthened by the finding that
purified, double-negative thymocytes as well as peripheral
CD4+ or CD8+ T cells did not express Spi-C,
while purified B220+ cells did. Lymphoid organs that
contain mature B cells like spleen, lymph node, and bone marrow did
express Spi-C RNA. As a comparison, PU.1 is expressed in cells of the
myeloid lineage (13) and both PU.1 and Spi-B are expressed in T
lymphocytes (3). Furthermore, Spi-C appears to be expressed during a
window of B cell differentiation representing peripheral, mature B
cells but neither early B cells nor plasma cells, a finding that also
distinguishes Spi-C expression from that of PU.1 and Spi-B. We did not
detect any Spi-C expression in embryonic stem cells using RT-PCR while
several expressed sequence tags can be found in the NCBI data base that
appears to be derived from Spi-C. Some of these have been cloned from a
cDNA library prepared from blastocysts, which might indicate an
additional role for Spi-C during early development. In conclusion, we
show evidence that Spi-C has an expression pattern distinct from that of other Ets proteins and we would like to propose that it has a unique
function during B lymphoid differentiation. The elucidation of that
putative function must await the targeted inactivation of the Spi-C
gene in the mouse germline.
Spi-C is related to Spi-B and PU.1 in the DNA binding Ets domain based
on amino acid sequence homology, while it showed no homology to other
Ets proteins outside this domain. Furthermore, PU.1 and Spi-B are more
closely related to each other than to Spi-C. Spi-C may therefore be
considered to represent a novel subgroup of Ets proteins, especially
considering the lower degree of amino acid sequence identity compared
with Spi-B and PU.1 within helix 1 of the Ets domain (1, 41). The DNA
binding characteristics of Spi-C were very similar to those of PU.1. In
methylation interference assays using the SP6 Spi-C was cloned using the SP6 Finally, the Spi-C locus was mapped to chromosome 10, region C in the
mouse. No homogeneous syntenic region with regard to the human genome
can be found, but several markers from this region map to human
chromosome 12q22-24 (46). Interestingly, several chromosomal
translocations in non-Hodgkin's lymphomas have been described in this
region (50). It should also be pointed out that, upon a data base
search, a sequence corresponding a human homolog of Spi-C was
identified on human chromosome 4p16.3 in the Huntington's disease
region (GenBank accession no. Z68163). However, this locus most likely
corresponds to a pseudogene, since it is a near perfect match of the
Spi-C cDNA with the exception of a deleted region that potentially
corresponds to a splice-intermediate. Furthermore, no synteny is
evident between mouse chromosome 10 and 4p16 (46). However, the
sequence of the human pseudogene would indicate that the Spi-C gene has
about a 70% homology between the mouse and the human genome. In
conclusion, our initial characterization indicates that Spi-C is an
important regulator of B lymphocyte development, but the elucidation of
its biological function must thus await further experimentation
in vitro and in vivo.
promoter
Y element as a bait. The novel Ets protein was most closely
related to PU.1 and Spi-B within the DNA binding Ets domain and was
therefore named Spi-C. However, Spi-C may represent a novel subgroup
within the Ets protein family, as it differed significantly from Spi-B
and PU.1 within helix 1 of the Ets domain. Spi-C was encoded by a
single-copy gene that was mapped to chromosome 10, region C. Spi-C
interacted with DNA similarly to PU.1 as judged by methylation
interference, band-shift and site selection analysis, and activated
transcription of a
Y element reporter gene upon co-transfection of
HeLa cells. Spi-C RNA was expressed in mature B lymphocytes and at
lower levels in macrophages. Furthermore, pre-B cell and plasma cell
lines were Spi-C-negative, suggesting that Spi-C might be a regulatory molecule during a specific phase of B lymphoid development.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
promoter (16). Binding sites for
PU.1 have been found in the Ig heavy chain intron enhancer (14), the Ig
3' enhancer (17),
promoters (18), the
2-4 enhancer (19),
and the mb-1 (Ig
) and B-29 (Ig
) genes (20, 21), as well as in the
Ig J-chain promoter (22). However, no target genes for PU.1 that can
explain the phenotype observed in PU.1
/
animals have
been identified.
/
mice have a severe distortion of hematopoietic
development (5, 6), Spi-B
/
mice form a normal myeloid
and lymphoid compartment with the exception of a defective B cell
receptor-mediated immune activation (23).
promoter
Y element. The protein
is mainly expressed in the peripheral B lymphoid compartment; it is not
expressed in early B cell precursors or in pre-B cell lines and appears
to be down-regulated as the B cell matures to the plasma cell stage.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Y core element (16, 24) were cloned
into the pHisi and pLacZi vectors (CLONTECH),
between the XbaI and EcoRI sites, and
SmaI and EcoRI sites, respectively. The vectors
were linearized by XhoI or NcoI digestions and
stably integrated into the YM 4271 yeast strain
(CLONTECH). The integration was verified by
Southern blotting of
PCR1-amplified DNA from
genomic yeast DNA using either Hisi- or LacZi-specific primers, using
one copy of the inserted fragment as a probe
(5'-TACTCTCAAACAGCTGTGTAATTTACTTTCC-3'). The EBF binding site yeast
strain was constructed using the same protocol with four copies of the
EBF binding CCCT element from the SP6
promoter (25). The obtained
yeast strains did not grow on histidine-deficient plates at a 15 mM concentration of 3-amino-1,2,4-triazole (3-AT).
arms and packaged as described for Stratagene's
two-hybrid system. A total of 3.2 × 106 independent
plaques were obtained with an average insert size of 1.1 kilobase
pairs. The amplified stock was converted to its phagemid form as
described by the manufacturer (Stratagene). 20 µg of phagemid was
transfected into the yeast strain and spread on SD agar plates minus
histidine and minus leucine, containing 15 mM 3-AT as
described (CLONTECH). Growing clones were tested in
a LacZi assay (CLONTECH), and positive clones were
grown, plasmids isolated, transformed into bacteria, and finally
retransformed into the screening yeast strain or the EBF control
strain. cDNA clones that remained positive through these procedures
were sequenced from the 5' end. A total of six E2A-containing, three
E2-2-containing, and one Spi-C-containing clones were identified in
the screen.
library was spread on 90-mm plates at 22,000 plaque-forming units/plate
and overlaid with sodium chloride magnesium to obtain 60 phage pools.
These were screened via PCR using two Spi-C-specific primers
(5'-GCAAACATTTCAAGACGCC-3' and 5'-CTGTACGGATTGGTGGAAGC-3'),
the products separated by electrophoresis and blotted onto
charged nylon membranes, which were probed with a polynucleotide kinase
end-labeled oligonucleotide (5'-CAGACCTGTATTTGGAAGGA-3') as described
by the manufacturer (Bio-Rad). Two positive pools were identified, and
these were subsequently screened according to standard procedures using
the original yeast Spi-C clone as probe and then excised as plasmids
according to the manufacturer's instructions (Stratagene). The longest
of the two clones was completely sequenced on each strand using the
Sanger dideoxy method (26) with a combination of internal primers and
restriction fragments cloned into pGEM 3Z (Promega). The original yeast
clone was sequenced in parallel, and no differences between the two
clones were detected in overlapping sequences.
Y reporter
construct was made by inserting an oligonucleotide containing four
copies of the
Y element and the TATA box from the SP6
promoter
between the HindIII and XhoI sites of the pGL3
luciferase reporter vector (Promega), and the TATA box only containing
was subsequently created by EcoRI digestion and re-ligation
to remove the
Y elements. To obtain a PU.1 expression vector, an
EcoRI/ApaI fragment containing the coding region
of PU.1 was inserted into the pcDNA3 vector (Invitrogen). HeLa
cells (2.5 × 106 cells/transfection) were transfected
in duplicate using LipofectAMINE (Life Technologies, Inc.). Each
transfection contained 0.2 µg of reporter plasmid and 0.3 µg of
expression plasmid, as indicated. All co-transfections were normalized
to contain 0.8 µg of DNA/transfection using the pcDNA3 plasmid.
The cells were harvested and the luciferase activity determined after
40 h of incubation, and the mean value ± standard deviation
from three experiments is shown.
5 M mercaptoethanol. After 44 h, the
cultures were treated with 0.18 mg/ml bromodeoxyuridine for an
additional 14 h. The synchronized cells were washed and recultured
at 37 °C for 4 h in
-minimal essential medium with thymidine
(2.5 µg/ml). Chromosome slides were made by conventional method as
used for human chromosome preparation (hypotonic treatment, fixation,
and air-drying). As a probe, a 12.5-kilobase pair genomic fragment
cloned from a I129 genomic library was used after biotinylation using a
Life Technologies BioNick labeling kit at 15 °C for 1 h (29).
FISH detection was performed as described (29, 30). Briefly, slides
were baked at 55 °C for 1 h, and after treatment with RNase A
the slides were denatured in 70% formamide in 2× SSC for 2 min at
70 °C, followed by dehydration with ethanol. Probes were denatured
at 75 °C for 5 min in a hybridization mix consisting of 50%
formamide, 10% dextran sulfate, and mouse cot I DNA, and prehybridized
for 15 min at 37 °C before being loaded on the denatured slides.
After overnight hybridization, slides were washed, detected, and
amplified as described (29). FISH signals and DAPI banding pattern were recorded separately by photography, and the assignment of the FISH
mapping data with chromosomal bands was achieved by superimposing FISH
signals with DAPI-banded chromosomes (30).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Y Interacting Protein--
Fig.
1A shows a schematic
illustration of the mouse SP6
promoter. This immunoglobulin
promoter contains several DNA elements that are poor activators of
transcription per se but that will stimulate
octamer-induced transcription (16, 24, 31). In this study we focused on
the octamer upstream region that contains two transcriptional control
elements, the bipartite pentadecamer element and the
Y element (16,
24, 32, 33). In an effort to identify the proteins that interact with
these DNA elements, we used the sequence shown in Fig. 1A as
bait in a yeast one-hybrid screen of a cDNA library made from
LPS-stimulated mouse B cells (Fig. 1B). We isolated several
E-box binding clones, all encoded by the E2A and E2-2 genes (data not
shown), and in addition a clone containing a novel DNA sequence that we
decided to call Spi-C. Also shown in Fig. 1B is a
confirmation of the specificity of the screening procedure, using a
control yeast strain that had an EBF binding site as bait. When using
the pentadecamer/
Y yeast strain, Spi-C could rescue yeast growth on
3-AT-containing media while an EBF-containing plasmid could not. The
opposite was true using the EBF control. Furthermore, in the absence of selection, the cDNA clones also activated the transcription of the
LacZ genes as expected; Spi-C was positive only in the yeast strain
used for screening, while EBF was positive only in the EBF bait strain.
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Fig. 1.
A, schematic drawing of the SP6 k
promoter and the sequence used as bait in the one-hybrid screening.
B, verification of specificity of the Spi-C expressing yeast
clone and an EBF-expressing yeast clone as a control in combination
with a yeast strain containing an EBF binding site as bait. The
transformed yeast strains were then tested for their ability to grow in
the presence and absence of 3-AT, or to activate the LacZ gene as
indicated. C, the Spi-C cDNA from the original yeast
clone was introduced into a vector containing the translational start
region from Oct-2 in frame with the cDNA. After transcription and
translation in vitro, Spi-C was tested for its ability to
interact with 5' region of the SP6 promoter in EMSA. The Spi-C
complex is indicated, and free probe is labeled F. The
interaction between Spi-C and the probe was mapped by competition with
the indicated competitors, and the result obtained is summarized in the
lower part of the panel.
promoter octamer and its
3'-flanking region did not. Further analysis of the region used as bait
showed that a competitor that contained an intact
Y element competed efficiently for binding, while competitors with mutations of the
Y
element or with a truncated
Y element did not. We concluded from
these experiments that Spi-C interacted specifically with the
Y
element in the SP6
promoter.
cDNA library made
from LPS-stimulated B cells using the insert from the yeast clone as a
probe. The full-length cDNA encompassed 1267 base pairs (accession
no. AF098863), and contained an open reading frame starting with an ATG
at position 144 and a stop codon at position 870. This would generate
an open reading frame of 242 amino acids, with a predicted molecular
mass of 28 kDa (Fig. 2A).
Homology searches using the protein sequence showed that the protein
had sequence similarity to the Ets proteins PU.1 and Spi-B in their DNA
binding domain, while no significant homology was detected outside of
this region. Hence, Spi-C appears to be a novel member of the Ets
transcription factor family.
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Fig. 2.
A, the deduced amino acid sequence Spi-C
with the Ets domain. B, an amino acid comparison between the
Ets domain of the indicated Ets proteins. Boldface
letters indicate amino acids conserved in all Ets proteins,
and helices 1-3 of the Ets domain are indicated. C, the
percentage of amino acid identity between Spi-B, PU.1, and Spi-C in the
Ets domain or indicated subregions. The analysis was performed by hand.
Identical amino acids in a given position were scored (X),
and the percentage of identity calculated as
X/N × 100, where N indicates the
total number of amino acids in the analyzed region.
helices within the Ets
domain were analyzed separately, the degree of identity between
PU.1/Spi-B and Spi-C was most pronounced in helix 3 (79%), while less
marked in helix 2 (54-62%) and in helix 1 (31%). This is in contrast
to the degree of identity between Spi-B and PU.1 that was very high
(77%) also in helix 1 and 2. Since helix 2 and 3 entail the amino
acids that contact DNA directly in PU.1 (35), the higher level of
identity between Spi-B/PU.1 and Spi-C is not surprising, given that
Spi-C binds a bona fide PU.1 binding site (18). Also, all
amino acid positions shown by mutagenesis to be critical for PU.1
binding to DNA were conserved between PU.1 and Spi-C (36). We conclude
from these results that Spi-C is more closely related to PU.1 and Spi-B
than to other Ets proteins, but more diverged from these two proteins
than those to each other. Given the low degree of identity in helix 1 of the Ets domain, Spi-C might be representing a novel subgroup within the Ets protein family.
Y element was
further characterized and compared with that of PU.1 (18). First, the binding of the two proteins were studied via methylation interference analysis. Methylation of the two central G nucleotides within the Ets
motif of the non-coding strand completely inhibited both Spi-C and PU.1
binding, while no specific interference could be detected by
methylation of the coding strand (Fig.
3A). Upon longer exposures,
detected partial interference by methylation of some of the A residues
could also be detected, identically for both proteins (data not
shown).
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Fig. 3.
A, methylation interference analyses was
performed using both strands of the 5' region of the SP6 promoter
with either in vitro translated PU.1 or Spi-C. Interfering
positions are marked with arrowheads, and below the
autoradiograph the sequence of the probe with interfering positions
marked is shown. B, competition of Spi-C or PU.1 binding to
the
Y element using competitors containing published PU.1 binding
sites.
Y element as a
probe, in vitro translated full-length Spi-C or PU.1 as
ligands and various defined PU.1 sites as competitors. The in
vitro translated, full-length Spi-C generated two complexes in the
band-shift and also two bands in SDS-polyacrylamide gel electrophoresis
(data not shown). The larger of these bands correlated in size with the
predicted value for full-length Spi-C (28 kDa), and we therefore assume
that the more abundant, faster migrating complex represents a partial
break-down product with an intact DNA binding domain. That the DNA
binding domain is not affected by the limited proteolysis is supported
by the fact that in vitro translation of the original yeast
clone generated only one product (Fig. 2C). As shown in Fig.
3B, Spi-C
Y binding could be competed by PU.1 binding
sites from the SV40 enhancer (11), the
3' enhancer (17), and the
J-chain promoter (22). Furthermore, the amount of competitor needed to
compete Spi-C binding was similar to that needed to compete PU.1
binding to the same probe. Finally, we performed a site-selection
experiment using recombinant Spi-C to ask the question whether any
novel Ets binding motifs could be revealed. As summarized in Table
I, 90% of the selected templates contained established PU.1 binding sites. The most common was a GGA
core motif (21/33 sequences), while 9 out of 33 sequences contained
instead the variant AGA core found in the J-chain promoter (22) and in
the macrophage colony-stimulating factor receptor (37). There was a
clear preference for an A in the -4, -1, and +1 positions, while a
preference for A or T could be seen in the -3 and +3 positions. We
conclude from these data that Spi-C and PU.1 bind to overlapping DNA
elements with similar selectivity.
Binding site preference for Spi-C
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Fig. 4.
A, semiquantitative RT-PCR analysis for
Spi-C and HPRT RNA expression in the indicated organs and purified cell
types. BM, bone marrow; DP, thymic
double-positive (CD4+ CD8+) T cells; ES, embryonic stem
cells. B, semiquantitative RT-PCR analysis for Spi-C and
PU.1 RNA expression in the indicated organs and cell lines.
C, Northern blot analysis of 2 µg of poly(A)+
RNA from the indicated cell lines probed with either a Spi-C or a GADPH
probe.
Y Reporter Gene Construct in HeLa
Cells--
We subsequently wanted to determine whether Spi-C was a
transcriptional activator as a first characterization of its function. To this end, 0.2 µg of a reporter construct, containing four copies of the
Y element from the SP6
promoter in front of the
luciferase gene, was co-transfected into HeLa cells together with 0.3 µg of an empty expression vector or a PU.1- or a Spi-C-containing expression vector (Fig. 5). All
transfections were adjusted to include 0.8 µg of total DNA using the
empty expression vector, except the control transfection with the
Y
reporter only. Co-transfection of the Spi-C expression vector induced
an almost 10-fold increase in luciferase activity compared with that
seen after co-transfection of the empty expression vector.
Co-transfection of the PU.1 expression vector induced a slightly higher
luciferase activity than that observed with Spi-C at the same
concentration, while the addition of PU.1 and Spi-C expression vectors
together resulted in luciferase activity at the same level as seen with
PU.1 alone. Transfection of the expression vectors alone or together
with a luciferase vector containing a TATA box only did not induce any
significant luciferase activity (data not shown). We conclude from
these experiments that Spi-C is a transcriptional activator of similar
efficacy as PU.1.
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Fig. 5.
Co-transfection in HeLa cells of a Y luciferase
reporter gene with expression vectors containing Spi-C or PU.1 as
indicated. The mean value and standard deviation from three experiments
is shown.
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Fig. 6.
A, Southern blot of E14 embryonic stem
cell DNA digested with the indicated restriction enzymes and probed
with a Spi-C probe. B, FISH mapping of the Spi-C locus in
mouse. The left panel shows the FISH signals on
the chromosome, while the right panel shows the
same mitotic figure stained with DAPI to identify mouse chromosome 10. C, diagram of FISH mapping results for probe Spi-C. Each
dot represents the double FISH signals detected on mouse
chromosome 10.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
promoter
Y
element, the same protection patterns were observed using either PU.1
or Spi-C. Furthermore, several PU.1 binding sites competed as
efficiently for Spi-C binding as for PU.1 binding, and a site selection
experiment selected defined PU.1 target sequences in >90% of the
templates. The only significant difference in binding site preference
between the two that could be noted was a clear preference for an A in
position -2 for Spi-C, while PU.1 selects a G in this position (2). Thus, it appears as if these two Ets proteins are alternative ligands
to the same recognition sequences in mature B cells, although a minor
divergence could be detected. This could be expected, given the
conservation of all residues in helix 2 and 3 of the Ets domain that
has been shown to interact with DNA either by structural analysis or
mutagenesis (34, 35). On the other hand, given the sequence divergence
in helix 1 of the Ets domain, some difference in sequence recognition
of motifs outside the Ets core could be envisioned. For example, amino
acids in the NH2 terminus of PU.1 helix 1 form
water-mediated or phosphate backbone interactions with DNA (34). It
should also be considered that Ets proteins have been described to
mainly exert their function as members of higher order protein
complexes (19, 42-44), which might qualitatively influence the
protein/DNA interaction. For example, the DNA interaction of GABP
is
of lower affinity than that observed for the GABP
/GABP
heterodimer, although only the former contains an Ets domain (45). It
has also been shown that the part of the Ets domain of GABP
that
interacts with GABP
is in helix 1 (44). It is thus tempting to
speculate that Spi-C might have a distinct preference from that of PU.1
and Spi-B with regard to protein/protein interactions and, as a
consequence, a distinct function with regard to gene regulation both
qualitatively and quantitatively. The identification of a selective
ligand for Spi-C is obviously an interesting topic for future studies,
as is the definition of its target genes.
promoter
Y element as bait. This
element is only found in some
promoters, and the role of Spi-C in
the regulation of
transcription is difficult to envision,
especially since Spi-C seems to be turned off in plasma cells. Rather,
the Ets motifs that have been shown to be critical for the control of
immunoglobulin expression should be interacting with PU.1 or other Ets
proteins at this late stage of B cell differentiation (14, 17-19, 22).
One obvious possibility is that the relevant target gene(s) for Spi-C
is not immunoglobulin but that our cloning approach rendered its
isolation fortuitously. On the other hand, we could show by
co-transfection that Spi-C contains a transcriptional activation domain
of similar efficiency as PU.1. Whether the potential of Spi-C as a
transcriptional activator, or its target sequence specificity, is
modified if proper adapter molecules (19) are present remains to be determined.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. Sigvardsson for critical reading of the manuscript and members of the Immunology Unit in Lund for reagents.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Cancer Society, Swedish Medical Research Council, Kock's Foundation, Crafoord Foundation, and Österlunds Foundation.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF098863.
Current address: Medical Research Council Laboratory of Molecular
Biology, Cambridge CB2 2QH, United Kingdom.
§ To whom correspondence should be addressed. Fax: 46-46-2224218; E-mail: Tomas.Leandersson{at}immuno.lu.se.
2 D. Liberg, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; LPS, lipoppolysaccharide; RT, reverse transcription; FISH, fluorescence in situ hybridization; 3-AT, 3-amino-1,2,4-triazole; EMSA, electrophoretic mobility shift assay; DAPI, 4',6-diamidino-2-phenyl-indole; GABP, GA-binding protein; EBF, early B cell factor.
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REFERENCES |
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