(Received for publication, August 5, 1994; and in revised form, October 5, 1994)
From the
The B cell-specific expression of immunoglobulin (Ig) genes is
controlled by the concerted action of variable (V) region promoters and
intronic or 3` enhancers, all of which are active in a
lymphoid-specific manner. A crucial highly conserved element of the V
region promoters is the octamer site -ATTTGCAT-, which can be bound by
ubiquitous (Oct-1) as well as B cell-specific (Oct-2) factors. Another
less conserved element found in many Ig promoters is pyrimidine-rich
and has been shown to be functionally important, in particular for
those Ig promoters that have only an imperfect octamer site. In this
study we have analyzed the factors binding specifically to the
pyrimidine-rich motif of the V19 promoter, a light chain gene
promoter with an imperfect octamer site. Using nuclear extracts
prepared from B cells, we detected two sets of specific complexes in
electrophoretic mobility shift experiments. One complex appears to be
ubiquitous but enriched in lymphoid cells and represents the binding of
a potentially novel factor with an apparent molecular mass of
50
kDa. The other complex was found only with extracts from pre-B or B
cells as well as from a macrophage cell line and appears to be caused
by the binding of PU.1, a factor of the Ets family. We show that on
this Ig promoter Oct factors (Oct-1 or Oct-2) and PU.1 can bind
concomitantly but without synergism. By transfection experiments in
non-B cells we demonstrate that PU.1 is indeed able to activate this
promoter in concert with Oct-2. Furthermore, we show that PU.1 can bind
with varying affinities to the pyrimidine-rich elements of several
other Ig promoters. These data suggest a more general role for PU.1 or
other members of the Ets family in the activation of Ig promoters.
Immunoglobulin (Ig) ()genes are preferentially
expressed in cells of the B-lymphoid lineage. This tissue specificity
is a property of the promoter and enhancer elements (1, 2, 3, 4) , both of which are
lymphoid-specific. A highly conserved octamer element 5`-ATTTGCAT-3`
that is present in Ig light chain promoters and, in the inverted
orientation, in Ig heavy chain promoters (5) is essential for
efficient Ig promoter activity both in vivo and in
vitro(6, 7, 8, 9) . The octamer
motif, located 50-70 bp upstream of the transcriptional start
site, serves as a binding site for the lymphoid-specific transcription
factor Oct-2 (10, 11, 12, 13, 14) and
the ubiquitous factor
Oct-1(15, 16, 17, 18, 19, 20) ,
which are members of the POU family of homeodomain proteins. In
addition, some heavy chain promoters contain a so-called heptamer motif
5`-CTCATGA-3` that, in cooperation with the octamer motif, can also
bind octamer factors(21, 22) .
The B cell specificity of Ig promoters is largely caused by the octamer element(9) , although this same element is also implicated in the ubiquitous expression of small nuclear RNA genes(23) , the cell cycle regulation of the histone H2B gene(24) , and the VP16-dependent expression of herpesvirus intermediate early genes(25, 26, 27) . The ability of the octamer element to promote ubiquitous as well as B cell-specific gene expression was initially suggested to reside in its interaction with Oct-1 and Oct-2. The different cellular distribution of these two proteins led to the proposal that Oct-2 rather than Oct-1 is responsible for the B cell specificity of Ig promoters. The suggestion that Oct-2 specifically promotes Ig gene transcription has come into question, because from in vitro experiments with octamer-containing promoter constructs, it has been shown that Oct-1 and Oct-2 are essentially interchangeable(28) . Analysis of Oct-2 knockout mice has shown that Oct-2 is not required for the generation of Ig-bearing B cells but is crucial for their maturation to Ig-secreting cells, and this implies that Oct-2 plays an essential role only late in B cell differentiation(29) . Thus, it appears that both octamer factors can activate transcription from Ig promoters. Moreover, a B cell-restricted coactivator, OCA-B, which can interact with Oct-1 or Oct-2, has been suggested to be required for a high level of Ig promoter activity(30, 31) . In addition, a third less conserved pyrimidine-rich element is found upstream of the heptamer or the octamer motif in many Ig promoters and has been shown to be also important for full promoter activity. However, only little is known about the factors mediating Ig gene transcription from these pyrimidine-rich sequences(32, 33, 34) .
In
the studies reported here, we have analyzed the factors binding to the
pyrimidine-rich motif of a light chain promoter (V
19). By
electrophoretic mobility shift assays (EMSAs) with nuclear extracts
prepared from B cells and a V
19 promoter pyrimidine-rich motif we
detected two sets of specific complexes. We show that one of these
factors binding specifically to that site appears to be PU.1, a member
of the Ets family that is expressed in B cells and
macrophages(35) . PU.1 is identical to the Spi-1
proto-oncogene(36, 37) , which was isolated as the
site of Friend erythroleukemia virus integration in 95% of virally
induced tumors. In addition to PU.1, the Ets family contains several
proteins, for instance Ets-1, Ets-2, Elf-1, Elk-1, Erg, E74, Fli-1, and
PEA3 (38) that share a relatively conserved 80-90-amino
acid long DNA-binding domain that recognizes a purine-rich sequence
with the core motif
5`-GGA(A/T)-3`(35, 39, 40) . As with other
Ets proteins, however, very little is known about the cellular genes
that are regulated by PU.1.
The other factor recognizing
specifically the pyrimidine-rich motif has an apparent molecular mass
of 50 kDa and is ubiquitous but is enriched in lymphoid extracts
and might be a novel factor, perhaps also belonging to the Ets family.
Furthermore, we demonstrate that PU.1 is able to strongly activate
transcription of the V
19 light chain promoter through the
pyrimidine-rich motif and that maximal promoter activity was obtained
in cooperation with Oct-2 in transfected cells. Finally, by performing
binding studies with related pyrimidine-rich sequences derived from
light or heavy chain promoters, we show that PU.1 can also bind with
varying affinities to several of these motifs present in other Ig
promoters. Our data, thus, suggest that PU.1 as well as perhaps other
factors of the Ets family might be involved in the regulation of a
number of Ig promoters.
Lymphoid cells were grown to a density of 2 10
cells/ml in RPMI 1640 medium supplemented with 10% fetal calf
serum, and non-lymphoid cells were grown to confluency in
Dulbecco's modified Eagle's medium with 3% fetal calf serum
and 3% newborn calf serum. Nuclear extracts from different cell lines
were prepared as described previously(41) .
Figure 4:
PU.1 and Oct factors simultaneously bind
to the V19 promoter. A, EMSA was performed with a
fragment of the V
19 promoter, comprising both the
Py and the
octamer site, and 1 µl of RL PU.1 (lane 1), RL Oct-1 (lane 2), or RL Oct-2 (lane 3) alone or 1 µl of
RL PU.1 in combination with increasing amounts of RL Oct-1 (lane
4, 1 µl; lane 6, 2 µl), or Oct-2 (lane
5, 1 µl; lane 7, 5 µl). B, EMSA was
performed with 4 µg (lane 8) or 8 µg of BJA-B nuclear
extract (lane 9), and competition analysis was carried out
with 4 µg of BJA-B nuclear extract and a 1000-fold excess of an
unlabeled octamer motif (lane 10) or SV40 PU site (lane
11) as competitors. Lane 12, probe alone. The sequence of
the V
19 probe used containing the pyrimidine-rich and octamer
motif is represented from bp -97 to -39 at the bottom of
the figure. The two binding sequences are indicated in white on
black.
Figure 1:
A, nuclear factors bind to the
pyrimidine-rich motif of the V19 promoter. EMSA was performed with
a
Py probe covering the pyrimidine-rich site (bp -97 to
-74) of the V
19 promoter and 4 µg of nuclear extract
prepared from BJA-B cells, a human mature B cell line. Increasing
amounts (100, 500, 2500 fmol) of unlabeled wild type
Py (lanes
2-4) or mutated
Py (indicated with an asterisk (*))
oligonucleotide competitor (lanes 5-7) as indicated
above were added. Lane 8, probe alone. B, cell line
distribution of the V
19 promoter binding activities. 4 µg of
nuclear extract prepared from a number of non-lymphoid cell lines (lane 1, HeLa; lane 2, Cos7; lane 3, 3T3),
pre-B and B cells (lane 4, HAFTL; lane 5, BAF3; lane 6, 38B9; lane 7, PD31; lane 8, 70Z/3; lane 9, NFS5.3; lane 10, Nalm 6; lane 11,
Wehi 231; lane 12, Namalwa), T cells (lane 13, YAC1; lane 14, CEM; lane 15, Peer; lane 16,
BW5147; lane 17, Molt 4; lane 18, Jurkat), and
macrophages (lane 19, U937) was added to an EMSA using the
Py probe. For a description of the cell lines see ``Materials
and Methods.'' The two specific DNA-protein complexes are
designated C1 and C2 on the left.
Figure 2:
A, the C2 binding activity is competed
away by the SV40 PU box. Competition experiments were performed with
the Py probe and 4 µg of BJA-B nuclear extract using a
1000-fold molar excess of different unlabeled competitor
oligonucleotides as indicated above each lane. B, the
C2 factor binds to the SV40 PU box. 4 µg of nuclear extracts
derived from BJA-B cells (lanes 2-4), U937 macrophages (lanes 6-8), and HeLa cells (lanes 10-12)
was tested for binding to the radiolabeled
Py (lanes 2, 6, and 10),
Py* (lanes 3, 7,
and 11), or SV PU probe (lanes 4, 8, and 12) in an EMSA. C, the complex C2 comigrates with in vitro-translated PU.1. For EMSA, the
Py or SV40 PU
probe was incubated with either 4 µg of nuclear extract from BJA-B
cells (lanes 2 and 5) or 1 µl of in
vitro-translated PU.1 protein (lanes 3 and 6)
prepared from rabbit reticulocyte lysate (RL) as indicated above
each lane. Lanes 1 and 4, unprogrammed
reticulocyte lysate. D, identification of the C2 binding
activity as PU.1. 4 µg of nuclear extract from BJA-B cells (lanes 1-4) or 1 µl of in vitro-translated
PU.1 (lanes 5 and 6) was used in an EMSA. Lanes 1 and 2 contain the
Py probe, and lanes 3-6 contain the SV40 PU box. Rabbit polyclonal antibody against a
peptide consisting of amino acids 251-271 of PU.1 (lanes
2, 4, and 6) was added to the EMSA reaction as
indicated above each lane. S refers to the
supershifted bands. The positions of the PU.1/C2 complex and its
supershift are indicated with an arrow.
Figure 3:
Identification of the contact sites for
PU.1 and C1 by methylation interference and copper-phenanthroline
footprinting analysis. A and B, the partially
methylated, radiolabeled Py probe was incubated with nuclear
extract (BJA-B, lanes 2-5) or in
vitro-translated PU.1 protein (RL PU.1; lanes 6 and 7). Free and bound probes were separated on a 4%
nondenaturating polyacrylamide gel, isolated, and cleaved with sodium
hydroxide, as described under ``Materials and Methods.''
Cleavage products of both free (lanes 2 and 6) and
bound probe (lanes 3-5 and 7) were analyzed on
a 12% polyacrylamide, 8 M urea gel along with a G + A
piperidine cleavage reaction of the probe (lane 1). The
sequences of the top and bottom strands are aligned with the
gels. The pyrimidine-rich motif (top strand) and the
corresponding purine-rich sequence (bottom strand) are
indicated in white on black. The nucleotides whose methylation
strongly or partially interfered with protein binding are indicated by black or open triangles, respectively. C,
for copper-phenanthroline footprinting, the
Py probe used for
methylation interference was incubated with BJA-B nuclear extract. Free
and bound probes were separated by electrophoresis and digested with
copper-phenanthroline while they were still embedded in the gel matrix.
Digested products of both free (top strand, lane 3; bottom strand, lane 6) and bound probes (top
strand, lanes 1 and 2; bottom strand, lanes 4 and 5) were analyzed as described in A and B.
Figure 5:
The pyrimidine-rich motif and the
divergent octamer site are both required for optimal V19 promoter
activity. A, schematic representation of the reporter plasmids
used. All constructs are based on the OVEC reporter plasmid and contain
various factor binding sites in front of the
-globin TATA box; in
addition, these reporters contain an SV40 enhancer 3` of the globin
gene (black box). The Sp1 construct containing the binding
site for the ubiquitous transcription factor Sp1 derived from the
metallothionein gene promoter was used as a positive control. The
Py/octa construct containing the pyrimidine-rich and octamer motif
(bp -101 to -41) was derived from the V
19 promoter,
and the mutated motifs are indicated with an asterisk (*). The SV40 PU
inv construct contains the SV40 purine sequence in inverse orientation
(with the corresponding pyrimidine-rich sequence on the upper strand). B, RNase protection analysis was performed with RNA from S194
B cells transiently transfected with the plasmid OVEC-Ref alone in the
absence of a reporter plasmid (lane 1) or with the plasmid
OVEC-Ref together with the Sp1 (lane 2),
Py/octa (lane 3),
Py*/octa (lane 4),
Py/octa* (lane 5),
Py*/octa* (lane 6), SV40 PU inv (lane 7), SV40 PU (lane 8), or
Py reporter
plasmid (lane 9). Lanes M and Y, marker and
control hybridization with yeast RNA, respectively. Ref indicates the position of the reference signal produced by the
plasmid OVEC-Ref, and Test indicates the position of the
correctly initiated RNA derived from the reporter plasmids. rt denotes the position of ``readthrough'' transcripts. C, relative activities of the various reporter constructs in
S194 and Namalwa B cells were determined by PhosphorImager
quantification of representative experiments. The signals derived from
the reference transcripts were used to normalize the variability in
transfection effficiency. Transfection with the plasmid OVEC-Ref alone
in the absence of a reporter plasmid (S194, lane 1) or with
the plasmid OVEC-Ref together with the promoterless OVEC parental
plasmid (Namalwa, lane 1), Sp1 (lane 2),
Py/octa (lane 3),
Py*/octa (lane 4),
Py/octa* (lane 5),
Py*/octa* (lane 6), SV40 PU inv (lane 7), SV40 PU (lane 8), or
Py reporter
plasmid (lane 9). The quantified data correspond to the gel
represented in B for S194 B cells and the average of three
independent experiments for Namalwa B cells. The activities are shown
relative to the activity of
Py*/octa* reporter plasmid, which was
arbitrarily set to 1.0.
Figure 6:
PU.1 can activate transcription in concert
with Oct-2. A, RNase protection analysis was performed with
RNA from HeLa cells transiently transfected with the plasmid OVEC-Ref
together with the promoterless OVEC parental plasmid (lanes 1 and 2), Py/octa (lanes 3-6),
Py*/octa (lanes 7 and 8),
Py/octa* (lanes 9 and 10),
Py*/octa* (lanes 11 and 12), SV40 PU inv (lanes 13 and 14),
SV40 PU (lanes 15 and 16), or
Py reporter
plasmid (lanes 17 and 18).The OVEC constructs (as
described in Fig. 5) were cotransfected with or without PU.1
and/or Oct-2 expression plasmids as indicated above each lane. B, relative transactivations of the various reporter
constructs by PU.1 and/or Oct-2 in HeLa cells were determined by
PhosphorImager quantification of representative experiments. The
signals derived from the reference transcripts were used to normalize
the variability in transfection efficiency. Lanes 1-18,
as described in A. The quantified data correspond to the gel
represented in A and were verified in several independent
experiments. The activities are shown relative to the activity of the
Py*/octa* reporter plasmid, which was arbitrarily set to
1.0.
Figure 7: PU.1 binds to the pyrimidine-rich motifs of several other Ig promoters. Pyrimidine-rich motifs derived from various Ig promoters were tested for binding to 4 µg of BJA-B nuclear extract (odd numbered lanes) or RL PU.1 protein (lane 2, 1 µl; even numbered lanes 4-16, 2 µl), as indicated above each lane in an EMSA.
The most conserved element of Ig promoters is the octamer site(1, 5) , and numerous studies have shown its importance for Ig expression (6, 7, 8, 9) . The nuclear factors recognizing this motif, the ubiquitous Oct-1 and the lymphoid-specific Oct-2, have been cloned and extensively studied(12, 20) .
In addition, other, less
conserved elements also contribute to the B cell-specific activity of
Ig promoters. One such element is the pyrimidine-rich
motif(33, 34) , a relatively loosely conserved element
found in many Ig promoters. In this case, very little is known about
the factors that mediate its activity. We have begun our studies by
looking at the factors interacting specifically with the
pyrimidine-rich motif of the V19 promoter, a light chain gene
having a promoter with an imperfect octamer site. Our results show that
two nuclear factors are able to bind to the pyrimidine-rich site (the
Py site); one of these factors is a lymphoid-enriched but
ubiquitous protein with an apparent molecular mass of
50 kDa. This
protein appears to be a novel factor and might correspond to the
protein
Y, described by Atchison et al.(32) .
Multiple evidences support the conclusion that the second factor is
PU.1, a B cell- and macrophage-specific member of the Ets
family(35) . First, the complex C2 was competed away
efficiently by the SV40 PU box, which contains a strong PU.1 binding
site. Second, in vitro-translated PU.1 formed a complex
indistinguishable from complex C2 in electrophoretic mobility shift
assays. Finally, PU.1-specific antibodies recognized the complex C2 in
the same manner as in vitro-translated PU.1 protein (Fig. 2). Like other members of the Ets oncoprotein family, PU.1
is a transcription factor and binds to a purine-rich sequence that
contains a central core with the sequence 5`-GGAA-3`. However, only
little is known about which cellular genes are regulated by PU.1. Here,
we show that one of its roles is to regulate, probably in concert with
Oct-2 (or Oct-1), a critical event in B cell immune response, the
expression of the V
19 light chain gene. The evidence is based on
the following observations. First, PU.1 recognizes the pyrimidine-rich
motif in the V
19 light chain promoter and binds to this element
with high affinity (Fig. 2C). Second, a mutation of the
Py motif, which abolishes in vitro PU.1 binding, reduces
severalfold the activity of a reporter construct in transfected B cells (Fig. 5, A and B). Third, coexpression of PU.1
in HeLa cells, which lack PU.1, transactivates efficiently reporter
constructs containing the
Py motif (Fig. 6, A and B).
By performing binding studies with related
pyrimidine-rich sequences derived from other light or heavy chain Ig
promoters, we found that PU.1 not only shows binding affinity to the
V19 site but also to several such motifs present in other Ig
promoters (Fig. 7, Table 1). PU.1 expression is limited to
B cells and macrophages, and several other Ets family members, like
Ets-1, Elf-1, Elk-1, Fli-1, and Erg, are also predominantly expressed
in lymphoid
cells(48, 59, 60, 61, 62) .
It is interesting to note that two other mouse V
genes, V
Ser
and V
TNP, contain the same divergent octamer and pyrimidine-rich
elements, which are identically located as in the V
19
promoter(32) . Thus, these observations suggest that PU.1 and
other Ets-related factors might indeed play a critical role in tissue-
and development-specific regulation of a number of Ig genes and provide
new insights into the function of these factors in lymphoid cells. In
support of this, a number of groups have recently shown that
Ets-related factors contribute to Ig expression. Evidence was provided
that the expression of PU.1 and Ets-1 together is sufficient to
activate the µA and µB elements of the Ig µ enhancer core
in nonlymphoid cells(63) . Similar results were reported by
Rivera et al.(64) , who have shown that Erg-3 and
Fli-1 can activate a reporter construct containing a multimer of
µE2-
binding sites of the Ig µ enhancer, synergistically
with the helix-loop-helix protein E12. Furthermore, it has been shown
that PU.1 recruits the binding of a second B cell-restricted nuclear
factor to an adjacent DNA site in the Ig
3` enhancer (65, 66) and in the Ig
2-4 enhancer (67) and that this interaction is required for efficient
activity of these two enhancers. However, unlike in the Ig
3` and
the Ig
2-4 enhancer system, PU.1 binding does not appear to
assist the binding of Oct factors to the nearby octamer site in the
V
19 promoter since we were not able to detect a synergistic
binding between these factors in our assay (Fig. 4A and
data not shown). Thus, our data demonstrate that PU.1 not only has a
function in the activation of several Ig enhancers but is also likely
to be involved in the regulation of some Ig promoters. In addition, a
further important role for PU.1 in B cells was recently established in
the regulation of another B cell-specific gene, the Ig J-chain gene. In
this case, the element recognized by PU.1 is somewhat divergent from
the GGAA consensus that has generally been regarded to be the core of
the PU.1 recognition motif(68) . Finally, PU.1 has not only
been shown to be an important regulator of B cell target genes; it also
has a role in the regulation of macrophage gene expression including
the myeloid-specific CD11b and scavenger receptor
genes(69, 70) .
The other factor recognizing the
V19 pyrimidine-rich motif is ubiquitous but enriched in lymphoid
extracts (Fig. 1B). The identity of this factor is
still unclear. Southwestern experiments and UV cross-linking to a
BrdU-substituted
Py probe (data not shown) have indicated that
this factor has an apparent molecular mass of
50 kDa. In addition,
competition experiments with known binding sites for Ets-1, Ets-2, or
Elf-1 (Fig. 2A) and lack of reaction of a broad
specificity anti-Ets antibody (data not shown) have shown that this
50 kDa protein appears not to be Ets-1, Ets-2, or Elf-1 but rather
to be a novel factor. However, comparison of the methylation
interference and copper-phenanthroline footprint pattern of the C1
factor with that of PU.1 revealed that both proteins were able to bind
the
Py motif in a highly similar if not identical way and that the
protein binding contacts were limited to the
Py site (Fig. 3). Essential for the
Py site is the GGAA motif,
which is the characteristic recognition sequence for the Ets family
members (38, 71, 72) on the opposite strand.
It has been shown that the lymphoid-specific Ets-1 factor also binds
purine-rich sequences in their inverse orientation in the stromelysin
promoter(73) , the T cell receptor
enhancer(50) ,
and the GATA-1 promoter(74) . These observations suggest that
another factor of the large Ets family might correspond to the C1
complex. Potential candidates for this
50-kDa protein might be
Elk-1 (60 kDa), Fli-1 (51 kDa), and Erg (52 kDa), which correspond
roughly to the predicted size of the C1 factor and also display a more
or less lymphoid-specific expression pattern(38) .
Previously, Atchison et al.(32) demonstrated that
the Py motif of the V
19 promoter serves as a strong binding
site for a novel lymphoid-specific nuclear factor, which they called
Y. This
Py binding activity might be identical to our C1
factor, since electrophoretic mobility and cell line distribution
appear to be similar. Surprisingly, these authors did not observe PU.1
binding to the
Py motif in their assay, and they also had
considerable difficulty detecting Oct-2 complexes. It is possible that
the minor V
19-specific band, which they speculated to be a complex
with partially degraded
Y factor, was in fact the PU.1-specific
complex. A possible reason for the difference in results might be the
different preparation of nuclear extracts.
In the case of the
V19 promoter, both sites, the divergent octamer motif,
5`-CTTTGCAT-3`, and the
Py motif have a strong effect on promoter
activity. A mutation of the octamer motif that no longer binds the Oct
factors reduced the promoter activity to 20% in S194 B cells and to 45%
in HeLa cells cotransfected with PU.1 and Oct-2. A mutation of the
Py motif that abolishes its factor-binding capability reduced the
promoter activity to 27% in S194 B cells (Fig. 5, B and C) and to 20% in HeLa cells cotransfected with PU.1 and Oct-2 (Fig. 6, A and B). The mutation of the
Py
motif has a more severe effect on promoter activity in HeLa cells than
the mutation of the octamer motif. Apparently, cotransfected Oct-2 and
endogenous Oct-1 do not suffice to efficiently activate transcription
from the octamer motif of the
Py*/octa reporter construct in HeLa
cells, and this could reflect the absence in the transfected cells of
an additional B cell-specific factor that can interact with Oct-1 or
Oct-2 and is required for maximal octamer-dependent promoter activity.
Evidence for such a factor has been provided by previous
studies(30, 31) , which have shown that a B
cell-restricted coactivator, OCA-B, stimulates transcription from an
IgH promoter in conjunction with either Oct-1 or Oct-2. Moreover,
Atchison et al.(32) have shown that the divergent
octamer motif of the V
19 promoter binds Oct factors only poorly
and that the flanking DNA sequences do not supply the additional
contacts required to produce a strong Oct factor binding. Thus, these
data demonstrate that the pyrimidine-rich element and the divergent
octamer motif are both required for optimal V
19 promoter activity
and that interaction of these motifs with their cognate factors is
essential for tissue-specific expression of this gene. Moreover, no
synergism between PU.1 and Oct factors could be observed in binding
activity (Fig. 4A and data not shown), and only a weak
synergism could be detected in transactivation (Fig. 6). This
type of interaction contrasts with the proposed interplay between the
heptamer and octamer motifs in V heavy chain promoters, which appears
to involve cooperative binding of the Oct
factors(21, 52, 53) .
In conclusion, we
have demonstrated that the Ets-related protein PU.1 binds to the
pyrimidine-rich motif of V19 promoter and is able to activate,
probably in concert with Oct-2 (or Oct-1) this promoter. Since PU.1 is
expressed in B lymphocytes, it is likely that it is at least in part
responsible for the maintenance of the cell type-specific function of
this Ig promoter. How PU.1 contributes to the stage-specific activation
of this Ig light chain gene is not clear. Northern analysis revealed
that the level of PU.1 mRNA remains constant during B cell development,
and analyses of nuclear levels of PU.1 protein showed similar
results(63, 68, 75) . The concerted action of
PU.1 and Oct-2 or Oct-1 as well as other factors acting through the
promoter and enhancer elements will be required to account for
developmental stage-specific regulation of Ig expression. In the
future, it will be interesting to determine whether PU.1 is also
involved in the regulation of other Ig promoters. Moreover, further
characterization and study of the C1 factor will be required to
determine its identity and its role in transcription of the V
19
gene. Finally, it will be interesting to determine the influence of
other Ets family members on transcription of this gene.