(Received for publication, August 10, 1995)
From the
B cell-specific transcriptional promoter activity mediated by
the octamer motif requires the Oct1 or Oct2 protein and additional B
cell-restricted cofactors. One such cofactor, BOB.1/OBF.1, was recently
isolated from human B cells. Here, we describe the isolation and
detailed characterization of the murine homolog. Full-length cDNAs and
genomic clones were isolated, and the gene structure was determined.
Comparison of the deduced amino acids shows 88% sequence identity
between mouse and human BOB.1/OBF.1. The NH-terminal 126
amino acids of BOB.1/OBF.1 are both essential and sufficient for
interaction with the POU domains of either Oct1 or Oct2. This
protein-protein interaction does not require the simultaneous binding
of Oct proteins to DNA, and high resolution footprinting of the Oct-DNA
interaction reveals that binding of BOB.1/OBF.1 to Oct1 or Oct2 does
not alter the interaction with DNA. BOB.1/OBF.1 can efficiently
activate octamer-dependent promoters in fibroblasts; however, it fails
to stimulate octamer-dependent enhancer activity. Fusion of subdomains
of BOB.1/OBF.1 with the GAL4 DNA binding domain reveals that both
NH
- and COOH-terminal domains of BOB.1/OBF.1 contribute to
full transactivation function, the COOH-terminal domain is more
efficient in this transactivation assay. Consistent with the failure of
full-length BOB.1/OBF.1 to stimulate octamer-dependent enhancer
elements in non B cells, the GAL4 fusions likewise only stimulate from
a promoter-proximal position.
The octamer motif (ATGCAAAT) or its reverse complement was originally identified as a conserved element present in virtually all immunoglobulin promoters as well as in enhancer elements of immunoglobulin genes(1, 2) . In addition, it is also conserved in a variety of other genes specifically expressed in B cells(3, 4, 5) . The role of the octamer motif for mediating B cell-specific transcription was most convincingly demonstrated when it was shown that a single copy of this motif confers B cell specificity onto a minimal heterologous promoter element(6) . Likewise, multimerized octamer motifs efficiently functioned as B cell-specific enhancer elements(7) . However, functional octamer motifs are also conserved in the regulatory regions of a variety of genes, which show ubiquitous expression patterns(1, 8, 9) .
Several transcription factors could be identified in different cell types that specifically interacted with the octamer motif(10, 11) . All mammalian cell types express the Oct1 protein(12, 13) . B cells, in addition, express a second type of octamer transcription factors, the Oct2 proteins(14, 15, 16, 17, 18) . Expression of Oct2 is largely confined to the lymphoid lineage, and there it is expressed as a family of isoforms which arise by alternative splicing from a single transcription unit (19) . Oct1 and Oct2 belong to a growing family of transcription factors that all share a homologous DNA-binding domain, the POU domain(20, 21) . This POU domain is a bipartite DNA-binding domain consisting of a POU-specific and a POU-homeo subdomain. Both subdomains are required for efficient DNA binding(22, 23) , and recent crystallographic studies reveal that the two subdomains interact with opposite major grooves in the DNA double helix(24) . In addition to Oct1 and Oct2, many other transcription factors have been identified that share a POU domain (25) . Some of them, like Oct4 and Oct6, which are expressed in the germ line, also efficiently interact with the conserved octamer motif (11) .
The original hypothesis that
Oct2 is determining the B cell-specific functions of the octamer
motifs, whereas Oct1 would be responsible for its ubiquitous
activities(8, 25, 26) , was questioned by a
variety of observations. In vitro transcription experiments
failed to reveal a significant difference between Oct1 and Oct2
proteins(27, 28) . In some B cell T cell
hybrid cell lines, octamer-dependent transcriptional activity was
extinguished, although Oct2 expression was maintained(29) .
Moreover, a thorough investigation of gene expression in somatic cell
hybrids from myeloma
fibroblast cells showed a variable
expression of the Oct2 gene. (
)Finally, stably transfected
Oct2 did not activate octamer-dependent regulatory elements in NIH/3T3
fibroblasts, BW5147 T cells, or COS1 cells, whereas octamer-dependent
promoter activity was evident in B cells lacking the Oct2 transcription
factor(31, 32) . Likewise, expression of many genes
containing octamer motifs in their regulatory elements, like the
immunoglobulin genes, B29/Ig-
and CD20, was unaffected in B cells
from mice lacking Oct2 due to a mutation introduced by homologous
recombination into the endogenous oct2 gene(33, 34) . These observations argued in favor
of the existence of B cell-specific cofactors, which upon interaction
with Oct1 or Oct2, would determine the transactivation potential of
these transcription factors(31, 35) . Biochemical
fractionation of B cell-derived nuclear extracts revealed the presence
of an activity (OCA-B = octamer coactivator from B cells)
stimulating octamer-dependent immunoglobulin promoter
activity(27, 36) . Indeed, employing a yeast
one-hybrid screen, two groups recently reported the successful
isolation of cDNAs for such a B cell-specific octamer cofactor from
human cDNA libraries. The identified cDNAs encode the same protein
which was designated BOB.1 for B cell Oct-binding protein 1 (37) or OBF.1 for Oct binding factor 1(38) . Here we
describe the isolation and detailed functional characterization of the
murine homolog of BOB.1/OBF.1. We show that BOB.1/OBF.1 is an efficient
octamer coactivator that allows Oct1 and Oct2 to function on
promoter-proximal octamer motifs. However, this factor is unable to
mediate the activity of octamer motifs from distal enhancer positions.
For isolation
of a genomic clone, 2 10
cosmid clones were
screened with the same BOB.1/OBF.1 polymerase chain reaction fragment.
A single cosmid was obtained that contains the complete coding region
of murine BOB.1/OBF.1. The genomic map was determined by a combination
of restriction enzyme mapping, subcloning, and sequencing.
The resulting full-length, NH- and
COOH-terminal fragments were cloned into three types of vectors:
pMT/PKA (39) that allows expression as a fusion protein with a
hexa-His moiety at the amino terminus; a vector containing the GAL4
DNA-binding domain(41) ; and a derivative of this GAL4
expression vector that had the GAL4 DNA-binding domain removed allowing
expression of BOB.1/OBF.1 or the individual domains by themselves.
Conditions for antibody
coprecipitation have also been described before (39, 45) . 10 µl of Oct2-specific rabbit
antibodies coupled to 10 mg of protein A-Sepharose CL4B (Pharmacia)
were mixed with 200 µg of HeLa nuclear extract from cells infected
with recombinant Oct2 expressing or wild type vaccinia
viruses(28) . 20 µl of in vitro translated,
[S]methionine-labeled full-length BOB.1/OBF.1 or
the individual protein domains were added and incubated for 3 h at 4
°C in 500 µl of interaction buffer A (20 mM Tris/HCl,
pH 8.0, 0.1 M NaCl, 1 mM EDTA, 0.5% Nonidet P-40).
Reactions were washed four times for 15 min each with 1 ml of
interaction buffer A. Where indicated, 200 µg/ml ethidium bromide
were present throughout the interaction and washing procedure.
Using the sequence information of the human BOB.1/OBF.1 cDNA clone, we generated a probe encompassing the complete coding sequence of murine BOB.1/OBF.1 by reverse transcription-polymerase chain reaction and used this probe to screen a cDNA library from the murine S194 plasmacytoma cell line (see ``Materials and Methods'' for details). The largest cDNA isolates contained about 2550 nucleotides and included the complete putative coding sequence for the murine BOB.1/OBF.1 homolog with about 70 nucleotides of 5` leader sequence and 1700 nucleotides 3`-nontranslated sequence (Fig. 1A). Comparison of the deduced amino acid sequence of the murine BOB.1/OBF.1 clone with its human counterpart revealed a high degree of sequence conservation. The two clones are 88% identical at the amino acid level in the coding region. Interestingly, the high degree of conservation also extends in the 5` leader sequence. Only two nucleotide exchanges can be found in the 60 nucleotides preceding the AUG initiation codon. The cDNA clone was further used to isolate a cosmid clone containing the genomic locus of murine BOB.1/OBF.1. A combination of restriction enzyme mapping and sequence analysis revealed that the coding sequence of murine BOB.1/OBF.1 is split over 5 exons (Fig. 1B). The AUG initiation codon is localized in exon 1, which is separated from the remainder of the coding region by a large 17-kilobase pair intron. Exons 2, 3, and 4 are small exons of 131, 43, and 266 base pairs separated by introns of 492 and 82 base pairs, respectively. Exon 5 is just over 2000 base pairs long and encompasses the 3` end of the coding sequence as well as the complete 3`-noncoding sequences. The 3`-noncoding sequence contains multiple simple nucleotide repeats, and these repeats show some mouse strain polymorphism as they differ between the cDNAs isolated from S194 cells (BALB/c mouse strain) and the genomic cosmid clone derived from a 129 mouse strain library (data not shown). With the exception of the splice acceptor site of exon 3, which is divergent from the consensus sequence, all other exon-intron boundaries are in excellent agreement with consensus sequences known for mammalian exons and introns (Fig. 1C). At the 3` end of the mRNA a consensus poly(A) addition signal is conserved (Fig. 1A).
Figure 1: Sequence and organization of the murine BOB.1/OBF.1 gene. A, composite nucleotide and predicted peptide sequence of murine BOB.1/OBF.1. The complete nucleotide sequence of the longest cDNA isolate is shown. The first 15 nucleotides were derived from the genomic sequence and the primer extension data shown in Fig. 2. The complete peptide sequence of the murine clone is shown. Only those amino acids that differ in the human sequence are shown underneath the murine sequence. Arrows indicate the exon/intron boundaries. B, restriction map of the genomic locus. The five exons are shown as black boxes; their sizes are not drawn to scale. C, nucleotide sequences of the exon/intron boundaries. Exon sequences are shown as capital letters and intron sequences as lowercase letters. Sizes of the different introns are indicated.
Figure 2: Localization of the start site of transcription by primer extension analysis. A, schematic representation of the primer extension strategy. On top, a scheme of the 5` end of the cDNA is shown. The thin line represents the 5` leader sequence present in the longest cDNA isolate; the coding sequence is indicated by the open rectangle. Numbers above indicate the individual exons, and arrowheads mark the exon/intron boundaries. Below, the position of the two primers (P1 and P2) is given. Primer extension products are shown as dotted lines and their length as judged from B, is shown. B, primer extension experiment using primer P1 (lanes 1, 3, and 5) or primer P2 (lanes 2, 4, and 6) and either tRNA (lanes 1 and 2), WEHI231 RNA (lanes 3 and 4), or RNA from S194 cells (lanes 5 and 6). Extension products for P1 and P2 are indicated by arrows. The arrow with a question mark indicates a longer extension product specifically obtained with primer P2 (see text). The asterisk marks a prematurely terminated primer extension product seen in the reactions with P2. M, size marker (labeled pBR 322 DNA digested with MspI). C, comigration of P1 primer extension products with a sequencing ladder derived from sequencing the genomic clone with primer P1. Sequencing reactions (A, C, G, T) were loaded on the left half of the figure, the extension products for WEHI231, S194, and, as a negative control, tRNA, are shown. The arrowhead indicates the position of the P1-specific extension products.
Inspection of the 5` leader sequence of BOB.1/OBF.1 of the longest cDNA isolate showed that there is no in frame stop codon present in this sequence. We therefore determined the 5` end of the murine BOB.1/OBF.1 RNA by primer extension analysis to test whether there might be a longer upstream reading frame. Two primers were utilized for this analysis, one extending from -30 to -47 with respect to the AUG initiation codon (P1), the second one was localized in exon 2 at a position extending from +107 to +90, again with respect to the AUG codon (P2) (Fig. 2A). Primer extension analysis was performed on two different murine B cell RNAs, S194 and WEHI 231, representing plasmacytoma and mature B cell lines, respectively. The size of the extension product with primer P1 was 65 nucleotides for both cell lines, primer P2 gave rise to an extension product of 205 nucleotides (Fig. 2B). An additional extension product of roughly 350 nucleotides was observed with primer P2 (marked by a question mark in Fig. 2B). As no corresponding product could be seen with P1, the significance of this extension product remains elusive. These analyses localize the start site of transcription at a position about 15-20 nucleotides upstream of the end of the longest cDNA isolate. To identify the starting nucleotide of the RNA sequence, the primer extension reaction with the P1 primer was run on a sequence gel together with the sequence of the genomic region using the same primer (Fig. 2C), and the deduced sequence was included in Fig. 1A. Consistent with the presence of a single major initiation site, inspection of the 5` upstream putative promoter region revealed a sequence element fitting known TATA consensus motifs (TTTAAAAA) at a position -22 to -29 relative to the transcriptional initiation site (data not shown).
A hallmark of the human
BOB.1/OBF.1 protein is its interaction with the Oct1 and Oct2
transcription factors, which is thought to be a prerequisite for the
transactivation of octamer-dependent promoters. Likewise, in vitro translated murine BOB.1/OBF.1 protein interacted with both Oct1
and Oct2 and resulted in supershifts which were detectable in an EMSA
experiment (Fig. 3A). The identity of the individual
complexes was confirmed using antibodies specific for Oct1, Oct2, or
the BOB.1/OBF.1 protein (Fig. 3B). Inspection of the
intensities of the various complexes suggests that Oct1 and Oct2
interact with BOB.1/OBF.1 with similar affinities. In an effort to
functionally dissect the BOB.1/OBF.1 protein, we expressed the
NH- and COOH-terminal half of murine BOB.1/OBF.1
individually and tested the interaction with Oct proteins in an EMSA
experiment. Only the NH
-terminal domain was able to
interact with Oct proteins, resulting in an indicative supershift (Fig. 3C). In contrast, no supershift could be detected
with the isolated COOH-terminal domain of BOB.1/OBF.1.
Figure 3:
BOB.1/OBF.1 interacts with Oct1 and Oct2
in EMSA experiments. A, ternary complex formed on an octamer
containing probe by BOB.1/OBF.1 together with Oct1 and Oct2,
respectively. In lanes 1 and 2, nuclear extract from
HeLa cells infected with a recombinant vaccinia virus expressing murine
Oct2 was added(28) . Unprogrammed reticulocyte lysate (lane
1) or in vitro translated BOB.1/OBF.1 (lanes 2 and 3) were included. The position of the binary Oct1 and
Oct2 DNA complexes, as well as the ternary complexes containing the
BOB.1/OBF.1 protein, are indicated. Note that in vitro translated BOB.1/OBF.1 does not bind to the DNA by itself (lane 3). B, characterization of EMSA complexes with
specific antibodies. The left panel (lanes 1-4)
shows EMSA experiments with HeLa nuclear extracts expressing
recombinant Oct2 in the presence of unprogrammed reticulocyte lysate,
in the EMSA lanes shown on the right panel, 2 µl of in
vitro translated BOB.1 were added. Extracts were preincubated with
either preimmune serum (PI, lanes 1 and 5),
antibodies specific for the Oct1 protein (lanes 2 and 6), antibodies specific for Oct2 (lanes 3 and 7), or antibodies raised against recombinant BOB.1/OBF.1 (lanes 4 and 8). The position of the respective
binary and ternary complexes are indicated on the right side of the figure. C, the NH-terminal half of
BOB.1/OBF.1 is essential and sufficient for formation of the ternary
complex with Oct2. EMSA experiments with HeLa nuclear extract
containing recombinant Oct2 (lanes 1-4). Unprogrammed
reticulocyte lysate (lane 1) or lysates containing full-length
BOB.1/OBF.1 (lane 2), the NH
-terminal 126 amino
acids (lane 3), or the COOH-terminal 130 amino acids of
BOB.1/OBF.1 (lane 4) were added. In lane 5,
full-length BOB.1/OBF.1 was tested in the absence of HeLa nuclear
extract. The positions of the respective complexes are indicated. Due
to the overexpression of Oct2 as compared with Oct1, the ternary
complexes containing Oct1 cannot seen on this
exposure.
So far, all
experiments analyzing the interaction of BOB.1/OBF.1 and Oct1/Oct2 had
been performed as EMSA supershifts (37, 38) and
above). In these experiments, the POU domain of the octamer proteins
had been identified as the domain sufficient for interaction; however,
interaction of BOB.1/OBF.1 with other domains of the octamer proteins
could not be ruled out. We have therefore utilized a coprecipitation
assay to study the interaction of various Oct2 domains with
BOB.1/OBF.1. Unlabeled in vitro translated BOB./OBF.1 was
generated as fusion protein with an NH-terminal hexa-His
moiety. Individual domains of Oct2 fused to the GAL4 DNA binding domain
were in vitro translated as labeled proteins, mixed with
BOB.1/OBF.1, and precipitated with nickel-nitrilotriacetic
acid-agarose. Only the POU domain of Oct2, but not the NH
-
and COOH-terminal domains, nor the GAL4 DNA binding domain alone, was
coprecipitated efficiently together with BOB.1/OBF.1 (Fig. 4A).
Figure 4:
The POU domain of Oct2 and the
NH-terminal domain of BOB.1/OBF.1 physically interact with
each other. A, coprecipitation experiment with immobilized
BOB.1/OBF.1 and the individual domains of Oct2. Unprogrammed
reticulocyte lysate (lanes 1, 3, 5, and 7) or in
vitro translated, unlabeled BOB.1/OBF.1 (a fusion protein
containing an NH
-terminal hexa his moiety) (lanes 2, 4,
6, and 8) were mixed with in vitro translated,
[
S]methionine-labeled proteins. The following
labeled proteins were added. Lanes 1 and 2, the GAL4
DNA binding domain (amino acids 1-92); lanes 3 and 4, a fusion protein of the Oct2 POU domain fused to the GAL4
DNA binding domain; lanes 5 and 6, a fusion protein
containing amino acids 3-173 of Oct2 fused to the GAL4 DNA
binding domain; lanes 7 and 8, a fusion protein
containing the COOH-terminal amino acids 351-463 of Oct2; all
GAL4 fusion proteins have been described previously(41) . M,
C-labeled molecular weight marker. The
position of the respective full-length fusion proteins is indicated on
the right side of the figure. The lower molecular weight
proteins seen in the GAL4/POU and GAL4/N lanes most likely represent
internal initiation events initiating at amino acids 79 and/or 83 of
the GAL4 protein. B, coimmunoprecipitation experiment with
Oct2-specific antibodies. HeLa nuclear extract containing recombinant
Oct2 (lanes 4-9) or HeLa extract from mock-infected
cells (lanes 10-12) were mixed with in vitro translated, labeled BOB.1/OBF.1 proteins. Either full-length
BOB.1/OBF.1 (BOB.1-FL, lanes 1, 4, 7, and 10), the
NH
-terminal 126 amino acids of BOB.1/OBF.1 (BOB.1-N, lanes 2, 5, 8, and 11) or the COOH-terminal 130 amino
acids of BOB.1/OBF.1 (BOB.1-C, lanes 3, 6, 9, and 12)
were added. In lanes 7-9, 200 µl/ml ethidium bromide
(EtBr) was present throughout the immunoprecipitation and washing
procedure. Lanes 1-3 show 10% of the respective proteins
loaded directly onto the gel (load). The positions of the individual
proteins are indicated. M,
C-labeled molecular
weight standard.
The POU domain is a multifunctional
domain important both for specific DNA binding as well as for
protein-protein interactions(39, 45) . We wanted to
determine whether DNA binding of the POU domain was a prerequisite for
interaction with BOB.1/OBF.1. We therefore mixed labeled full-length
BOB.1/OBF.1 or the individual NH- and COOH-terminal domains
with nuclear extracts from HeLa cells containing ectopically expressed
Oct2 protein. We then performed coprecipitation experiments with
antibodies specific for Oct2 in the presence and absence of ethidium
bromide. As ethidium bromide intercalates into DNA and thereby
interferes with protein-DNA interactions, this method has been
established as a tool to discriminate between bona fide protein-protein interactions versus assembly of proteins
on the same piece of DNA(46) . Full-length BOB.1/OBF.1 as well
as the NH
-terminal domain efficiently coprecipitated with
the anti Oct2 antibody regardless of the presence or absence of
ethidium bromide (Fig. 4B). In contrast, the
COOH-terminal domain was not recovered in these coprecipitation
experiments regardless of the conditions. This result is in line with
the failure of the COOH-terminal domain to interact with Oct proteins
in the EMSA experiments (Fig. 3B) and suggests that
this domain does not make stable contact with the Oct proteins. No
BOB.1/OBF.1 proteins were coprecipitated when Oct2 protein was missing
from the nuclear extracts confirming the specificity of the assay (Fig. 4B).
The fact that Oct2 binding to DNA was not required for interaction with BOB.1/OBF.1 did not exclude the possibility that the interaction with BOB.1/OBF.1 would affect the Oct-DNA interaction. To analyze this possibility, we performed high-resolution in-gel chemical footprinting analyses of the binary Oct-DNA complex as well as the ternary BOB.1/OBF.1-Oct-DNA complex. Identical protection patterns were observed for both complexes, regardless of whether Oct1 or Oct2 containing complexes were analyzed (Fig. 5, A and B). Using slightly modified conditions for EMSA experiments, we had previously identified a B cell-specific complex migrating slower than the Oct1 complex, but containing the Oct1 protein(31) . This complex resembled the ternary BOB.1/OBF.1-Oct1 complex in several respects, such as migration behavior and the fact that no extra contacts were detectable by copper-orthophenanthroline footprinting(31) . We therefore investigated whether this complex contained the BOB.1/OBF.1 protein. Whereas the preimmune serum did not affect this ternary complex, the antibody raised against the recombinant BOB.1/OBF.1 protein completely abolished this EMSA complex, suggesting that indeed this complex represents the endogenous BOB.1/OBF.1-Oct1 complex in B cells (Fig. 5C). We do not know presently why this endogenous complex is more difficult to detect in EMSA experiments as compared with the one containing the recombinant BOB.1/OBF.1 protein.
Figure 5:
Analysis of ternary complexes. A,
chemical footprinting of the binary and ternary complexes containing
the Oct1 protein. In-gel footprinting was performed on the free probe (lanes 1, 2, 6, and 7), the binary Oct1-DNA complex (lanes 3 and 5), or the ternary complex containing
Oct1, the BOB.1/OBF.1 protein, and DNA (lane 4). The position
and orientation of the octamer motif in the probe are indicated on the right of the figure. B, same as A, but for
Oct2-containing complexes. C, analysis of endogenous
BOB.1/OBF.1-containing ternary complexes from B cells. EMSA experiments
with nuclear extracts from Namalwa cells and an octamer-containing
probe were performed under slightly modified gel shift conditions (see (31) ). Preimmune serum (lane 2) or 2 µl of the
BOB.1/OBF.1-specific antibody (lane 3) were included. The
positions of the respective complexes are indicated. The asterisk indicates a nonspecific complex that is present whenever preimmune
or BOB.1 antiserum is included.
We had
previously proposed that B cells contain two types of octamer
coactivators(35) . One that can interact with either Oct1 or
Oct2 to mediate octamer-dependent promoter activity, and a second one
that specifically interacts with the carboxyl terminus of Oct2 and is
involved in mediating octamer-dependent enhancer activity in B cells.
The observation that BOB.1/OBF.1 interacts with both Oct1 and Oct2
suggested that it would be the first type of transcriptional
coactivator, namely a specific promoter cofactor. To test this
hypothesis, a BOB.1/OBF.1 expression vector was cotransfected with an
octamer-dependent promoter reporter into NIH/3T3 fibroblasts. In the
absence of cotransfected BOB.1/OBF.1, the wild type octamer reporter
showed the same activity as a mutant version, bearing point mutations
in the octamer motifs (Fig. 6A). BOB.1/OBF.1 stimulated
the wild type reporter construct to a level of activity comparable with
the activity of this reporter in B cells (Fig. 6A and (28) ). Activation depended on the integrity of the octamer
motifs because the mutant reporter was not stimulated by BOB.1/OBF.1
cotransfection. When the NH- and COOH-terminal halves of
BOB.1/OBF.1 were tested individually, only the NH
-terminal
domain gave a low, but reproducible, activation of the wild type
octamer containing reporter (Fig. 6B). In contrast, the
COOH-terminal fragment failed to show any stimulation, consistent with
our previous observation that this domain does not interact
specifically with the octamer transcription factors.
Figure 6:
Cotransfection of BOB.1/OBF.1 activates
octamer-dependent promoters, but not enhancers in NIH/3T3 fibroblasts. A, BOB.1/OBF.1 cotransfection activates octamer-containing
promoters. Cotransfection with the indicated reporter plasmids and
either the parental expression vector (pSV) or a full-length
BOB.1/OBF.1 expression vector. The activity of the reporter plasmid
containing four copies of the mutant octamer motif upstream of the
TATA-box was arbitrarily set to 1. B, cotransfection of the
wild type octamer reporter with expression vectors for full-length
BOB.1/OBF.1, the NH-terminal, or the COOH-terminal domain
of BOB.1/OBF.1 as indicated. The activity of the reporter cotransfected
with the parental expression vector (pSV) was set to 1. C, cotransfection of the indicated reporter plasmids into
NIH/3T3 cells stably expressing Oct2. An expression vector for
full-length BOB.1/OBF.1 (or the parental expression vector) was
cotransfected where indicated. D, schematic outline of the
various expression and reporter plasmids used in the cotransfection
experiments described in this figure. The SV40 based expression vector
was derived from pSG5 (Stratagene). The different reporter plasmids
have been described previously(28, 31, 47) .
Briefly, 4
wt.TATA contains four copies of a synthetic octamer
motif upstream of the HSV-thymidine kinase TATA box (T). The
octamer motifs contain a single point mutation in 4
mut.TATA.
CL(ED) contains the chicken lysozyme promoter and six copies of a
50-base pair fragment derived from the murine heavy chain intronic
enhancer element comprising the E4 and Oct motifs. The octamer motif
contains several point mutations in the CL(Ed) construct. The same
hexameric wild type and mutant enhancer multimers were cloned upstream
of the minimal HSV-TATA region in
(ED)/(Ed).TATA.
A slightly different strategy had to be used in order to investigate whether BOB.1/OBF.1 would also stimulate transcription from a distal enhancer position, because we had previously shown that this activity is strictly Oct2-dependent(31, 47) . We therefore analyzed BOB.1/OBF.1 activity in NIH/3T3 cells that were stably transfected with an Oct2 expression vector(31) . These stably transfected fibroblasts express amounts of the Oct2 protein similar to typical B cell lines (31) . We first tested the stable transfectants in a BOB.1/OBF.1 cotransfection experiment with the octamer-dependent promoter reporters described before. Interestingly, the stimulation observed by BOB.1/OBF.1 cotransfection was comparable in the parental NIH/3T3 cells and the Oct2-positive NIH/3T3 cells (Fig. 6C). This result suggests that Oct1-BOB.1/OBF.1 complexes have similar transactivation potential as Oct2-BOB.1/OBF.1 complexes. To test activation from an enhancer position, reporter constructs bearing a multimerized octamer motif-containing fragment from the murine heavy chain enhancer at a position 3` of the luciferase gene driven by an upstream chicken lysozyme promoter was used(31, 47) . This reporter has been previously used to detect octamer-dependent enhancer activity in B cells. Interestingly, no stimulation of this reporter could be observed by cotransfection of full length BOB.1/OBF.1 (Fig. 6C). This result suggests that BOB.1/OBF.1 is in fact a promoter-specific cofactor and unable to mediate the B cell-specific octamer enhancer activities. This inability of BOB.1/OBF.1 to stimulate the enhancer reporter was not due to the fact that no functional ternary Oct-BOB.1/OBF.1 complexes can be formed on the multimerized heavy chain enhancer fragment used. When the same multimerized fragment was moved into the proximity of the TATA box, BOB.1/OBF.1 was again capable of activating this element (Fig. 6C).
The above interaction
domain mapping experiments and cotransfection experiments had revealed
that the NH-terminal domain of BOB.1/OBF.1 interacts with
the Oct transcription factors and contains a residual transactivation
function, yet transactivation by the full-length BOB.1/OBF.1 clone was
significantly more prominent. In order to map the potential
transactivation functions within the BOB.1/OBF.1 coactivator, we
generated fusion proteins with the GAL4 DNA-binding domain and either
full-length BOB.1/OBF.1 or the NH
- or COOH-terminal domain
of BOB.1/OBF.1 separately (Fig. 7C). These clones were
cotransfected with a reporter bearing multimerized binding sites for
the GAL4 transcription factors in a promoter proximal
position(41) . All three fusion proteins efficiently stimulated
this reporter. The COOH-terminal domain of BOB.1/OBF.1 showed about
3-fold higher activity than the NH
terminus, however, (Fig. 7A). This result suggests that the predominant
transactivation function of the BOB.1/OBF.1 protein resides in the
COOH-terminal 130 amino acids. Given the above described inability of
BOB.1/OBF.1 to stimulate octamer-dependent enhancer elements together
with Oct2 in fibroblasts, we wanted to determine whether the GAL4
fusion proteins would be able to stimulate transcription from a
distance. To this end, we utilized a reporter bearing the GAL4 binding
sites in a distal enhancer position and cotransfected this reporter
with the GAL4 fusion protein expression vectors. Fig. 7B shows that neither of the GAL4 fusions is capable of stimulating
this reporter, suggesting that failure to stimulate from a distance is
an intrinsic property of the transactivation domains of the BOB.1/OBF.1
protein.
Figure 7: BOB.1/OBF.1 only activates from a promoter-proximal position when tethered to DNA via GAL4 binding sites. A, activation of GAL4 promoters by BOB.1/OBF.1-GAL4 fusion proteins in S194 plasmacytoma cells. Reporter plasmid pG4.TATA was cotransfected with the indicated GAL4-BOB.1/OBF.1 fusion proteins. The activity of the reporter cotransfected with the GAL4 DNA-binding domain (GAL4(1-92)) was set to 1. B, similar experiment as in A but using the pCL(G8) reporter plasmid. C, structure of the expression plasmids and reporter plasmids used. GAL4(1-92) and all reporter plasmids were described previously(41) .
One of the hitherto unique features of B cell-specific transactivation mediated by the octamer transcription factors is their requirement for the presence of additional B cell-specific coactivators. Here, we describe the molecular analysis of one such coactivator that allows Oct1 and Oct2 to activate transcription from a promoter-proximal position in B cells.
We had previously shown that B cells contain two distinct types of coactivators. One responsible for the B cell-specific activity of octamer-containing promoters and a second type that confers activity on octamer regulatory elements from distal enhancer positions(31, 35, 47) . The results presented here unequivocally identify BOB.1/OBF.1 as a specific coactivator from promoter-proximal positions. This conclusion is supported by the following lines of evidence.
1) Whereas cotransfection of BOB.1/OBF.1 into NIH/3T3 fibroblasts was sufficient to fully activate octamer-dependent promoter elements, it did not result in enhancer activation, regardless of the presence or absence of Oct2.
2) Fusion proteins of the GAL4 DNA binding domain with full-length BOB.1/OBF.1 or individual domains of the coactivator efficiently stimulated reporters containing GAL4 binding sites in a promoter-proximal position but failed to activate when the binding sites were present in distal enhancer positions.
3) BOB.1/OBF.1 promiscuously interacts with both Oct1 and Oct2, whereas the putative enhancer cofactor specifically requires Oct2 for a functional interaction(31, 35, 47) . Furthermore, our previous experiments suggested that Oct1, if anything, reduced octamer-dependent enhancer activity(31, 47) .
4) Finally, we had previously identified the COOH-terminal transactivation domain of Oct2 as a prerequisite for stimulating octamer-dependent enhancer activity in B cells. This result suggested that the enhancer cofactor might specifically interact with this domain rather than the POU domain. In our protein-protein interaction analysis presented here, we failed to detect any evidence for specific interaction between BOB.1/OBF.1 and the COOH-terminal domain of Oct2. In summary, these results suggest that BOB.1/OBF.1 is the/one of the B cell-specific coactivator(s) responsible for the B cell-specific function of octamer-containing promoters. Furthermore, they suggest that additional, distinct cofactors exist in B cells that are required for mediating the octamer-specific functions from distal enhancer positions.
At present, the molecular mechanism responsible for the
observed coactivation by BOB.1/OBF.1 is largely obscure. When
transactivation properties of the NH- and COOH-terminal
transactivation domains of the Oct2 transcription factor were measured
in GAL4 fusion experiments, significant activity of the individual
domains could be scored, which were not significantly lower than the
transactivation observed for GAL4 fusion proteins containing
full-length BOB.1/OBF.1 or individual domains
thereof(41, 48) . A possible explanation for this
apparent paradox could be that due to an intramolecular masking
process, the transactivation domains of Oct1 and Oct2 might not be
accessible for interaction with general transcription factors, most
likely the transcription factor IID (TFIID) complex(49) . The
main function of the coactivator then would be to unfold and unmask
these transactivation domains due to its physical interaction with the
Oct1 and Oct2 transcription factors. Intramolecular masking of the
transactivation domain has been suggested for the MyoD transcription
factor(50) . A specific conformational change induced by
binding of MyoD to DNA is hypothesized to be responsible for the
release of the masked transactivation domain(50, 51) .
From our results it is unclear whether a similar masking/unmasking
process is responsible for the activation of the octamer transcription
factors in B cells. Clearly, a more complex situation than for MyoD has
to be envisaged, as an additional cofactor, namely BOB.1/OBF.1, is
required for the activation to take place. Interestingly, involvement
of additional cofactors could also not be ruled out in the MyoD
activation process(51) . Although our results demonstrate that
Oct2 and BOB.1/OBF.1 can interact off DNA, these experiments do not
exclude a role for DNA-binding in the activation process. In that
respect it is of interest to note that in our previous GAL4 fusion
experiments with different Oct2 domains we failed to detect
transcriptional activity for a fusion protein that contained the POU
domain fused to the GAL4 DNA-binding domain (the identical protein used
for the coprecipitation experiment in Fig. 4A) even in
B cells(41) . This result could be interpreted in two ways: (i)
a functional complex requires DNA binding of the POU domain and/or (ii)
the transactivation domains of the Oct proteins are essential for
BOB.1/OBF.1-mediated transactivation.
Significantly more work has been performed to elucidate another coactivation pathway where octamer proteins are involved. The Oct1 protein is a critical component for the activation of several viral promoters after infection of cells with herpes simplex virus. There, a complex between the viral VP16 protein and Oct1 on specific promoter motifs containing a TAATGARAT consensus is responsible for efficient coactivation(52, 53) . The functional complex formed on the DNA actually is composed of Oct1, VP16, as well as an additional cellular protein, HCF(54, 55, 56, 57, 58) . However, as Oct1 is the only DNA-binding component in this system, it was unclear how specific octamer motifs (the GARAT-containing motifs) were selected for VP16-mediated transactivation. A recent investigation of Oct1 binding to different octamer motifs by high resolution chemical footprinting suggested that the Oct1 POU domain adopts a specific conformation when binding to GARAT-containing motifs as compared with two other octamer motifs. It was suggested that this specific conformation would then be recognized by the viral coactivator(59) . However, the additional binding of the coactivator to Oct1 did not affect the POU domain contacts to DNA as measured by chemical footprinting. In agreement with these findings, we also failed to detect any alterations of Oct1 or Oct2 DNA contact upon binding of the BOB.1/OBF.1 cofactor. This does not exclude the possibility, however, that, upon interaction with the cofactor, the overall conformation of the Oct1 and Oct2 proteins is changed.
Could different conformation of Oct proteins on different octamer binding sides be responsible for the differences observed with respect to promoter and enhancer activation by BOB.1/OBF.1? This interpretation is highly unlikely for the following two reasons. First, supershifted EMSA complexes containing Oct1 or Oct2 plus the BOB.1/OBF.1 coactivator could be observed on the various binding sites used for the promoter and the enhancer reporter constructs (compare, for example, Fig. 3and 5). Furthermore, we have shown that the very same elements that failed to function at a distance, were efficiently activated by BOB.1/OBF.1 when placed in a promoter-proximal position (Fig. 6D). These results together with the evidence discussed above argue for an independent distinct enhancer coactivator.
The observation that BOB.1/OBF.1 only contacts the POU domain of the octamer transcription factors further supports the dual function of the POU domain in these proteins. In addition to being responsible for specific DNA-binding, its role in orchestrating protein-protein interaction is becoming more and more apparent over the last years. In addition to viral proteins such as the described VP16 herpes simplex virus coactivator or the adenovirus E1A protein(30) , interaction with cellular proteins has also been shown to be mediated by the POU domain of Oct1 and Oct2. We could previously show that the POU domains of Oct1 and Oct2 specifically interact with TBP, the TATA-binding protein component of transcription factor IID(45) . More recently, using the POU domain as probe in a protein-protein interaction screening protocol, we were able to isolate HMG2, an abundant non-histone nucleoprotein, as an interacting partner protein(39) . In contrast to the interaction with BOB.1/OBF.1 which has been described to be specific for Oct1 and Oct2(37, 38) , HMG2-POU domain interactions are more promiscuous as the Oct6 POU domain was also shown to interact with HMG2(39) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z54283[GenBank].