From the Institute for Molecular and Cellular
Biology, University of Texas at Austin, Austin, Texas 78712-1075 and the ¶ Department of Pathology, University of Texas
Southwestern Medical Center, Dallas, Texas 75235
Received for publication, January 29, 2001, and in revised form, March 30, 2001
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ABSTRACT |
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Bright (B cell regulator
of IgH transcription) is a B cell-specific,
matrix associating region-binding protein that transactivates gene expression from the IgH intronic enhancer (Eµ). We show here that Bright has multiple contextual requirements to function as a
transcriptional activator. Bright cannot transactivate via out of
context, concatenated binding sites. Transactivation is maximal on
integrated substrates. Two of the three previously identified binding
sites in Eµ are required for full Bright transactivation. The Bright
DNA binding domain defined a new family, which includes SWI1, a
component of the SWI·SNF complex shown to have high mobility group-like DNA binding characteristics. Similar to one group of high mobility group box proteins, Bright distorts Eµ binding
site-containing DNA on binding, supporting the concept that it mediates
Eµ remodeling. Transfection studies further implicate Bright in
facilitating spatially separated promoter-enhancer interactions in both
transient and stable assays. Finally, we show that overexpression of
Bright leads to enhanced DNase I sensitivity of the endogenous Eµ
matrix associating regions. These data further suggest that Bright may contribute to increased gene expression by remodeling the
immunoglobulin locus during B cell development.
Transcriptional regulation of genes during development and
differentiation is tightly controlled through several mechanisms. The
tissue specificity conferred by the immunoglobulin heavy chain enhancer
(Eµ)1 has been studied
extensively both for understanding Ig regulation and as a model for
enhancer function (reviewed in Ref. 1). Eµ is a complex unit
containing binding sites for multiple transcription factors and can
functionally be broken down into two segments, the core and the
flanking matrix associating regions (MARs) (2-5). Most of the
previously identified factors bind to the enhancer core, and several
have been shown to have some B cell specificity in terms of expression
or ability to transactivate. However, no binding site in isolation can
confer all of the tissue-specific regulation seen in vivo.
Ultimately, this is the result of cumulative interactions of various
nuclear factors with both DNA and each other. The Eµ core segment
alone can increase transcription in transient systems (6, 7). In
vivo, however, the core alone is insufficient to drive
transcription or maintain tissue specificity. Transgenic studies have
demonstrated that high level tissue-specific expression is only seen
when the core is present in context of the MARs (8). This effect
requires the core, because MARs alone could not produce high level
expression. Although the MARs had previously been implicated in
negative regulation of the Ig locus in non-B cells (4, 9-12), this was
the first demonstration that the MARs were required for proper
expression in B cells.
Bright (B cell regulator of IgH
transcription) is the only B cell-specific transcription
factor shown to bind to, and transactivate via, the Eµ MARs (13).
Bright was first identified as a factor responsible for increased
expression of the immunoglobulin heavy chain gene following
antigen + interleukin 5 stimulation of B cells in culture (14,
15). The Bright binding complex has also recently been shown to contain
Btk, which is critical for the DNA binding complex (16). Bright binds
within the MARs of the IgH enhancer to distinct ATC motifs (P sites)
previously identified as binding sites for the Eµ negative regulator,
nuclear factor-µ negative regulator, and the MAR-binding protein,
SATB1 (11, 17). We have identified nuclear factor-µ negative
regulator as a previously characterized, lineage-nonrestricted
homeoprotein, Cux/CAAT displacement protein (18), that antagonizes
Bright binding and transactivation by direct competition for P sites. Developmentally, Bright expression is maximal in late stage B cells
(13, 19), a pattern opposite that of Cux/CAAT displacement protein
(18). Bright is found in the nuclear matrix and within matrix-associated PML nuclear bodies (13, 20), locations
consistent with a putative role in chromosomal organization. Although a
number of MAR binding factors have been cloned
(e.g. see Refs. 13, 17, and 21-29, and reviewed
in Ref. 30), Bright was the first shown to directly affect gene transcription.
MARs and attachment to the nuclear matrix can mediate specific
alterations in chromatin structure (31-34). Such a mechanism seemed
reasonable for Bright, based on features of its DNA binding. Highly
specific binding within the minor groove is achieved by virtue of two
domains (reviewed in Ref. 35), a self-association/tetramerization domain, termed REKLES for a heptapeptide conserved within this region among Bright orthologues, and a DNA binding region, termed ARID
for AT-rich interaction
domain. The Bright ARID defined a new family of DNA-binding
proteins, including SWI1, a component of the SWI·SNF complex that has
been shown to remodel chromatin (36) and p270, its apparent
mammalian orthologue (37). Components of human SWI·SNF appear to
be tightly associated with the nuclear matrix (38), suggesting that at
least a fraction of this complex could be involved in chromatin
organizational properties associated with MARs (reviewed in Ref. 39).
Like SWI·SNF, all ARID proteins bind AT-rich DNA, but only members
that contain both ARID and REKLES bind specifically to AT-rich MAR
motifs (35).
In this report we further characterize the mechanisms through which
Bright functions and the contextual requirements for Bright transactivation. We also show that Bright bends its DNA target on
binding. This, along with the observation that Bright overexpression induces increased DNase I hypersensitivity of the enhancer, provides a
rationale for how this protein may facilitate enhanced expression of
the immunoglobulin gene.
Constructs--
The derivation of Electrophoretic Mobility Shift Assay--
Specifics of binding
reactions were described previously (13). To assess binding to the four
P sites, the following contructs were used: the Transfections and Stable Lines--
Transfections of M12.4 and
J558L cells and analysis of CAT protein was done as described
previously (13). Stable transfectant lines were made by co-transfecting
the indicated CAT vector in a 3-fold excess to pBK-cytomegalovirus
(Stratagene). 48 h after transfection, cells were selected in
G418. Transient transfection of these cell with the Bright sense
or antisense constructs were done 3-5 weeks after the selection began.
Circular Permutation Distortion Assays--
The high affinity
Bright binding site (P2 × 3) and the circular permutation
plasmid have been described previously (22, 41). The P2 site concatamer
was cloned into the plasmid polylinker and confirmed by sequencing. A
second series utilized an ~500-bp fragment spanning the core octamer
and 3' MAR P3 site of Eµ. Circular permutated fragments were
generated by appropriate restriction digests. Mobility shift assay with
these fragments and in vitro-translated full-length or
truncated (amino acids 216-601) Bright protein were performed as
described above. The binding and functional activity of the truncated
Bright polypeptide were described previously (22). The distortion angle
was estimated by the method of Thompson and Landy (42). Briefly, the
relative mobilities of the fastest complex (µE) and the slowest
complex (µM) are determined. This ratio is then plotted on a graph of
µM/µE (abscissa) and distortion angle (ordinate) derived from
A-tract standards.
DNase I Digestion of Isolated Nuclei and Hypersensitive Site
Analysis--
Nuclei were isolated and treated with DNase I as
detailed previously (43). Nuclease digests were restricted with
BglII and analyzed on a 1.4% agarose gel in 1 × TAE
(40 mM Tris acetate, 1 mM EDTA). The DNA was
blotted onto a Bio-Rad Zeta probe nylon membrane by a modified
alkaline blotting protocol (43). The gel was blotted overnight in 0.4 M NaOH, 0.2 M NaCl. The membrane was then
neutralized in 50 mM Tris at pH 7.5 for 5 to 10 min, air
dried, and baked at 80 °C under a vacuum for 1 h.
Prehybridization was carried out from 2 h to overnight in 0.27 M NaCl, 15 mM sodium phosphate (pH 7.0), 1.5 mM EDTA, 0.5% BLOTTO dried milk powder, 1% SDS, 500 µg
of sonicated herring testis DNA per ml. Hybridization was carried out
overnight in the same buffer in the presence of at least 2.5 × 107 cpm of a radiolabeled DNA probe (specific activity, at
least 109 cpm/µg) generated by random primer synthesis
with a Decaprime DNA labeling kit (Ambion, Austin, TX). The DNA probe
used was a 300-bp XbaI-EcoRI restriction fragment
found just downstream of the Eµ 3' MAR (44). Autoradiograms were
calibrated with DNA standards 2.3, 2.0, 1.3, 1.1, and 0.87-kb-long by
constructing a plot of log DNA size versus mobility. The
sizes of the resulting hypersensitive fragments were interpolated from
the resulting linear fit.
Bright Does Not Transactivate from a Concatamerized Binding
Site--
In our first description of the Bright transcription factor
(13), we demonstrated that Bright could transactivate gene expression from a plasmid containing an IgH enhancer element (Eµ) upstream of a
reporter gene. To assess the ability of Bright to transactivate gene
expression from a binding site not in context of the Eµ enhancer, we
used reporter constructs containing concatamers of a binding site in
the S107 promoter (Bf150) or the Eµ P2 site in transient transfections. Expression constructs containing Bright in either the
sense or antisense orientation were co-transfected with reporter constructs driven by a thymidine kinase promoter and the additional elements as described in Fig. 1.
Concatamers of the P2 site, which gel shift analysis demonstrated is a
strong Bright binding site, could not increase CAT levels in either a B
cell or plasma cell line (Fig. 1). Similarly, a reporter construct with
the S107 MAR site concatamerized to eight repeats (Bf150 × 8) did
not show any significant increase in transcription when Bright was
co-transfected in the sense orientation.
Bright Requires Specific MAR Sequences for Transactivation
Function--
Despite the lack of Bright activity on a concatamerized
substrate, Bright clearly activated transcription from an Eµ element over the levels seen from Eµ alone (see Ref. 13 and Fig.
2). Bright binding sites were required
for this activity, because an Eµ that lacked the P sites ( Bright Mediates Promoter-Enhancer Interactions--
Knowing that
Bright could mediate transactivation from both the enhancer and the
S107 promoter (and possibly other Ig promoters, as well), we became
interested in determining whether these functions were independent or
whether these elements could function in concert. We constructed CAT
vectors that partially or completely recapitulated the immunoglobulin
locus promoter/enhancer arrangement. The S107 promoter fragment
contains two Bright binding sites, one of which functions as a MAR (15,
45). In a construct containing the promoter alone, Bright could not
transactivate in a transient assay (Fig.
4). This is in contrast to assays where
Eµ is placed 5' of the CAT gene and Bright effectively increased gene
expression. The ability of Bright to function through the IgH enhancer
is also seen when the enhancer is in the distal position. Strikingly, when both the promoter and enhancer are present in the same construct, the effects of Bright are synergistic, increasing transcription levels
more than 3-fold over that seen with Eµ alone in the distal position.
This Bright-mediated transactivation requires Bright binding, because a
construct with Bright Transactivates Integrated Targets by MAR
Interaction--
Because Bright binding sites have the potential to
act as MARs, we also studied these vectors in stably transfected cells to determine whether Bright can mediate MAR effects that would only be
detected from integrated targets. CAT constructs were stably
transfected into J558L cells and selected with neomycin for 21 days
before transient transfection with Bright sense or antisense
constructs. In contrast to results from the transient transfection
assay, Bright is able to transactivate from the promoter alone in the
stable system (Fig. 4). This supports a role for Bright as a
MAR-binding protein, because this phenomenon is only seen when the
promoter construct is integrated into the chromosome. A further
increase in S107 promoter-driven transcription is seen when Eµ is
present in the distal position. As in the transient studies, this
interaction is specific for Bright binding, because a construct with
Bright Mediates DNA Distortion--
The distance between
promoter-associated and enhancer-associated Bright sites that appear to
synergize in the constructs of Fig. 4 is about 2 kilobase pairs. We
assumed that Bright may affect DNA topology to facilitate these
interactions. We have previously shown that Bright binds DNA in the
minor groove (13). The class of high mobility group box proteins
typified by lymphoid enhancer-binding factor-1 and SRY bind DNA
in the minor groove and bend the double helix (41). To determine
whether Bright can also distort its DNA target on binding, we used the
circular permutation assay described by Giese et al. (41),
which measures DNA bending, as well as DNA flexibility caused by
changes in DNA structure such as melting of AT-rich regions. For this
assay, a series of equally sized fragments, differing only in the
position of a Bright binding site, were generated. If the DNA is
distorted during binding, then fragments bound near the center will
migrate through a gel at a slower rate than those bound near the ends.
In Fig. 5, a truncated Bright protein
(amino acids 216-601) with full binding activity distorts the circular
permutated fragments as assessed by differences in complex mobility.
The full-length Bright protein had an identical effect in this assay
(data not shown). The angle of induced distortion can be determined by
comparing the calculated ratio µM/µE to a plot of known A-tract
standards, where µM and µE are the relative mobilities of the
middle-bound (slowest migrating) and end-bound (fastest migrating)
fragments, respectively (42). For Bright, µM is calculated to be 0.41 and µE to 0.48 giving a ratio of 0.85. Based on A-tract standards in
4% polyacrylamide gels, this ratio corresponds to a distortion angle
of 80-90°.
Eµ Becomes DNase I Hypersensitive following Bright
Overexpression--
The ability of Bright to mediate specific
activation of integrated binding sites and to distort DNA suggested
that it may be involved with altering chromosomal architecture and
nucleosome-free regions of DNA. DNase I hypersensitive sites coincide
with nucleosome-free regions in chromatin. To test the ability of
Bright to alter the chromosomal organization of the endogenous IgH
locus, we stably transfected Bright into a murine mature B cell line
(WEHI 231) that produces low levels of endogenous Bright protein
(13). Following a 20-day culture in G418, we selected a clone that
expressed Bright at levels ~8-fold above that in the WEHI 231 parental lane and about twice that seen in two IgM-secreting
plasmacytomas (MOPC 104E and HNK-1; data not shown). Nuclease
sensitivity in mock-transfected WEHI 231 nuclei was limited to a 220-bp
region coinciding with the Eµ core (Fig.
6). In cells ectopically expressing
Bright, hypersensitivity was greater in magnitude and encompassed a
significantly larger (~500 bp) area that extended through the 5' MAR,
which contains the high affinity P2 binding site of Bright. A modest (2-3-fold) increase in µ transcription accompanied this effect (data
not shown) but is similar to the level of µ induction caused by
antigen + interleukin 5 stimulation (14, 15). A stronger and more
extended DNase I digestion pattern is observed (Fig. 6) in nuclei of
the plasmacytomas that transcribe the µ locus about 50-fold higher
than WEHI 231 (see Ref. 6 and data not shown). These results indicate
that the endogenous enhancer assumes a more extended chromatin
configuration as a direct or indirect consequence of ectopic Bright
overexpression.
Herrscher et al. (13) described Bright as a B
cell-specific transcription factor capable of transactivating
expression from the IgH enhancer (Eµ). In this report we have
characterized the contextual requirements of Bright transactivation to
further understand how it, and potentially other MAR binding factors,
can affect transcription levels. The data presented in this report
support several mechanisms for Bright-mediated transcriptional regulation.
Using transient transfection analysis we have demonstrated that context
is important for Bright transactivation. Bright was unable to
transactivate gene expression from a concatamerized binding site,
suggesting that it required interaction with specific factors to
function. Furthermore, Bright only acts through the P2 and P4 sites of
the Eµ MARs. This was initially surprising, because Bright binds the
P3 site as strongly as P2 and suggested spatial constraints for the
interactions of Bright with other factors. This suggested that Bright
might function to form tertiary structures of the enhancer DNA and
interact with additional DNA-binding proteins or adaptor molecules. In
support of this, we demonstrated that Bright distorts DNA. Studies with
the T cell receptor Synergy between promoter and enhancer transactivation in both the
transient and stable transfections suggest an additional level of
function for Bright. Because Bright exists in a tetrameric form, and
only two functional chains are required for Bright binding in a gel
shift assay (13), it is likely that one Bright molecule could bind two
sites. Indeed, these studies suggest that Bright could bring an
enhancer in apposition to the promoter and directly affect
transcriptional activation. This effect would be consistent with
studies that have implicated the IgH enhancer MARs in long range
(Ig heavy chain variable gene segment promoter-mediated) transcriptional activation (8, 44, 47). In comparing transgenic µ expression in lines generated from wild-type and MAR-deleted Eµ
constructs, no VH-initiated transcripts were detected from the MAR-deleted locus (47). Using a different approach, Artandi et al. (48) demonstrated that TFE3 proteins binding
in the Ig promoter and enhancer could cooperate when binding sites were placed proximal and distal of a CAT gene, presumably through
interaction of two dimers. Bright already exists as a tetramer and so
would not require any additional protein-protein interactions to carry out this function.
This study also provides functional evidence for the MAR binding
function of Bright. Transient transfections with the S107 promoter
fragment, which contains a MAR (45), demonstrated that Bright was
unable to transactivate from this site. In contrast, when this
construct was stably transfected, Bright was now able to affect
a 4-fold increase in transcription consistent with the concept that
MARs only have effects when they are integrated into the chromosome. We
previously demonstrated that Bright protein can be matrix-associated
(13). The fact that Bright is only capable of transactivating from the
S107 promoter only when it is integrated suggests that Bright can
function by modifying or mediating matrix attachment. One difference
between the S107 plasmid and the construct with Eµ in the proximal
position, which can mediate transactivation in a transient assay, may
be the availability of other interacting co-factors. This highlights
the context-dependent activity of Bright. Bright may
interact with some factors during a transient assay and allow
activation from Eµ, whereas matrix attachment is required for
transactivation from a substrate that may have limited DNA binding
factors associated with it for Bright interaction. In support of
interactions such as this, we have recently shown that Bright
associates with members of the Sp100 family, which co-localize with
Bright in nuclear domains and act as co-factors in transactivation
(20). Thus, Bright has multiple requirements for transactivation
activity, but the context-dependent activity may also
provide multiple mechanisms for Bright to activate gene transcription.
Ectopic overexpression of Bright revealed an altered pattern of
chromatin organization within the IgH enhancer in WEHI 231 B cell
nuclei. Consistent with previous studies (44, 47, 49), the pattern of
untransfected WEHI 231 nuclei is restricted to the Eµ core. The
assembly of this complex, as judged by in vivo dimethyl sulfate methylation patterns, has been shown to be
independent of the flanking MARs (47). Under conditions where Bright is expressed at high levels, DNase I hypersensitivity appears to extend
upstream, to include the high affinity P2 site-containing MAR, but not
downstream of the core. This third, highly extended configuration
extending across the 3' MAR is observed in the two IgM-secreting
plasmacytomas that transcribe µ at ~50-fold higher levels.
Similarly, the Ig Studies presented in this report suggest some novel mechanisms for the
regulation of immunoglobulin gene expression. They confirm that Bright
acts in a restricted manner by binding specific sites in the IgH
promoter and enhancer and by potentially interacting with other factors
within the enhancer core. It further provides some insight into the
mechanism of enhancer function and more specifically, how Bright may
play an important role in Ig gene expression. Further analysis of these
Bright new alternatives should yield a greater understanding of
long-standing questions regarding gene regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Eµ and
P Eµ mutants
was described previously (13). Eµ and
Eµ were cloned in the
XbaI site of the pBL-CAT2 vector. All
P mutants were
cloned in the SalI-BamHI sites of the CAT vector.
The hybrid SV40-MAR construct (40) was previously constructed. The
S107 promoter was isolated as a
BamHI-HaeIII fragment (covering nucleotides
550
to
40), blunt-ended, and cloned into the pBL-CAT2 vector. Vectors
containing elements distal of the cat gene were
constructed by first subcloning Eµ of the appropriate mutation into
pBluescript (Stratagene) and cloning a KpnI-SacI
fragment into the distal site of either pBL-CAT2 or pBL-CAT2 containing
the S107 promoter fragment.
P2 Eµ 5' MAR
isolates the P1 site, the
P1 Eµ 5' MAR isolates the P2 site, the
P4 Eµ 3' MAR isolates the P3 site, and the
P3 Eµ 3' MAR
isolates the P4 site. Briefly, these fragments were end-labeled (20,000 cpm/fmol), bound to in vitro-translated Bright protein in
the presence of increasing concentrations of poly d(I·C), and run on
a 4% nondenaturing polyacrylamide gel. Gels were dried and exposed to
x-ray film.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
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Fig. 1.
Bright does not transactivate from a
concatamerized binding site. M12.4 or J558L cells were transfected
with tk-CAT vectors containing concatamers of the Eµ P2 site or of
the high affinity Bf150 site from the S107 promoter as indicated. Cells
were co-transfected with an expression vector containing Bright in the
sense (filled bars) or antisense (open bars)
orientation. Results are expressed as -fold activation over tk-CAT
alone and are the average of at least four separate experiments.
Eµ)
did not mediate Bright transactivation (see Ref. 13 and Fig. 2). To
further examine the specificity for transactivation that Eµ ascribes
to Bright, we tested the effects of P site deletions. Because P2 is a
well characterized Bright binding site (13), we reasoned that it might
be capable of acting alone. Indeed, a construct that lacks P1, P3, and
P4 (
P1, P3, P4) was competent in mediating Bright transactivation (Fig. 2). However, a construct that lacked the P2 but had all other
sites intact (
P2) was still functional. The additional deletion of
the P4 site (
P2, P4) abrogated Bright-mediated function. That P4
could mediate Bright transactivation alone was verified using a P4-only
construct (
P1, P2, P3). Interestingly, there was a trend that the
P2-only and P4-only constructs were activated to a slightly lower
degree than the intact Eµ, though the difference was not
statistically significant. It seems possible that Bright can act
through both sites but that the activity seen in the intact Eµ may be
the combined effects of Bright binding to both sites. It was
unanticipated that Bright could not function through the P3 site,
because Bright also binds P3 very well (Fig.
3). This lack of function suggested that
competent Bright binding sites must be within a contextual arrangement
to allow them to mediate transactivation.
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Fig. 2.
Bright requires specific Eµ
P sites for function. J558L cells were transfected with
tk-CAT vectors containing wild-type or mutant Eµ elements upstream of
the tk promoter as indicated. Results are expressed as -fold
transactivation of cells transfected with the Bright sense construct
over transfections with the antisense construct. Eµ alone conferred a
10-20-fold increase in CAT over tk-CAT in the absence of Bright.
Results are the average of at least four separate transfections.
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Fig. 3.
Bright interacts with specific P sites in
Eµ, etc. Electrophoretic mobility shift
assays with 10 ng of in vitro-translated Bright and labeled
P Eµ MAR fragments are shown. A schematic of the four MAR
fragments used as probes (deletions isolate a single P site per probe)
are shown above three corresponding lanes that
contain 1, 2.5, and 5 µg of poly d(I·C) in the binding
reaction.
Eµ in the distal position could not mediate the
Bright effect (Fig. 4).
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Fig. 4.
Bright mediates promoter-enhancer and matrix
interactions. J558L cells were transfected either transiently or
for stable integration with tk-CAT vectors containing the S107 promoter
fragment upstream of the CAT gene and/or Eµ or Eµ downstream of
the CAT gene as indicated. Results are expressed as -fold activation as
described in Fig. 2 and are the average of four separate transfections.
N.D., not determined.
Eµ in the distal position does not transactivate beyond what is
seen with promoter alone (Fig. 4).
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Fig. 5.
Bright distorts DNA on binding.
A, schematic of probes used to create the circular
permutation of a Bright binding site (P2 × 3). Digestion of a
tandem sequence with the indicated restriction enzymes generates a
series of equal sized fragments (485 bp) that differ only in the
location of the binding site. Ba, BamHI;
Ea, EaeI; RV, EcoRv; Rs, RsaI;
Hd, HindIII; RI, EcoRI. B,
electrophoretic mobility shift assay with 10 ng of in
vitro-translated truncated Bright protein (amino acids 216-601),
1 µg poly d(I·C), and labeled circular permutated probes. Total
migration distance (start-end) and relative mobilities for the slower
middle-bound complex (µM) and the faster end-bound complex (µE) are
shown.
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Fig. 6.
Overexpression of Bright is accompanied by
enhanced and extended DNase hypersensitivity of
Eµ. The strategy for indirect end labeling
was described under "Experimental Procedures." Hypersensitive
sites, mapped by reference to a 1.6-kb BglII restriction
fragment spanning Eµ, were detected by using an upstream
XbaI-EcoRI 220-bp subfragment as a hybridization
probe. Nuclei, isolated from the indicated cell lines, were digested
with (from left to right in each
panel) 0, 0.5, 1.0, or 2.0 µg/ml DNase I. DNA was
purified, cut with BglII, and analyzed by Southern blotting.
Molecular size markers are indicated to the left.
Hypersensitive positions, mapped by subtracting the fragment size from
the parental BglII fragment, are indicated on the blots and
superimposed onto a vertical schematic of Eµ.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain enhancer have shown the
requirement for DNA bending and distortion to remodel DNA so that
transcription factors whose binding sites are spatially distant can
interact (46). It is possible that Bright plays a similar role in the
induced immunoglobulin expression of late stage B cells.
3' enhancer assumes three states of DNase I
detectable accessibility, which correlate strictly with stage of B cell
development (50). That the Bright overexpressing cells may have begun
to transition from mature to activated is consistent with the increased
Eµ accessibility and the slightly increased levels of µ transcription observed here and with the appearance of active
Bright·MAR binding complexes both in normal B cell populations and in
B cell lines observed previously (13, 19). Based on its SWI1
similarities, nuclear matrix residence, and MAR bending
properties, it is tempting to consider a direct role for Bright in this
remodeling. However, both known classes of chromatin remodeling
enzymes, SWI·SNF and the histone acetyltransferases, exist as large
multicomponent, ATP-hydrolyzing complexes (reviewed in Ref. 36). We
have no evidence for or against participation of Bright as a B
cell-restricted member or recruiter of either. However, MARs do confer
local regions of histone acetylation (51). In a different target gene
system, Cux has been shown to form a complex with histone deacetylase
that leads to gene inactivation (52). Bright could mediate derepressive
chromatin remodeling indirectly through its successful competition with
Cux/histone deacetylase. In a similar logic, Bright, along with
related chromatin remodeling proteins, would then be in a position to
clear out regions carrying the cis-acting regulatory
elements of the core, contributing to the accessibility of conventional
DNA binding transactivators to promoter and enhancer elements.
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ACKNOWLEDGEMENTS |
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We thank Chhaya Das for technical assistance and Utpal Das for preparation of this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI18016 and CA31534 (to P. W. T.) and GM50329 (to R. H. S.).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.
§ Present address: Dept. of Immunology and Microbiology and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202.
To whom correspondence should be addressed: Inst. for
Molecular and Cellular Biology, University of Texas at Austin, 100 W 24th St., ESB-534, Austin, TX 78712-1095. Tel.: 512-475-7705; Fax:
512-475-7707; E-mail: philtucker@mail.utexas.edu.
Published, JBC Papers in Press, April 6, 2001, DOI 10.1074/jbc.M100836200
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ABBREVIATIONS |
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The abbreviations used are: Eµ, immunoglobulin heavy chain intronic enhancer; MAR(s), matrix attachment/associating region(s); P site, Bright/nuclear factor-µ negative regulator binding site in Eµ; CAT, chloramphenicol acetyltransferase; bp, base pair(s); tk, thymidine kinase.
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REFERENCES |
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1. | Ernst, P., and Smale, S. T. (1995) Immunity 2, 427-438[Medline] [Order article via Infotrieve] |
2. | Adams, J. M., Harris, A. W., Pinkert, C. A., Corcoran, L. M., Alexander, W. S., Cory, S., Palmiter, R. D., and Brinster, R. L. (1985) Nature 318, 533-541[Medline] [Order article via Infotrieve] |
3. | Banerji, J., Olson, L., and Schaffner, W. (1983) Cell 33, 729-740[Medline] [Order article via Infotrieve] |
4. |
Cockerill, P. N.,
Yuen, M.-H.,
and Garrard, W. T
(1987)
J. Biol. Chem.
262,
5394-5397 |
5. | Gillies, S. D., Morrison, S. L., Oi, V. T., and Tonegawa, S. (1983) Cell 33, 717-728[Medline] [Order article via Infotrieve] |
6. | Kiledjian, M., Su, L. K., and Kadesch, T. (1988) Mol. Cell. Biol. 8, 145-152[Medline] [Order article via Infotrieve] |
7. | Lenardo, M., Pierce, J. W., and Baltimore, D. (1987) Science 236, 1573-1577[Medline] [Order article via Infotrieve] |
8. | Forrester, W. C., van Genderen, C., Jenuwein, T., and Grosschedl, R. (1994) Science 265, 1221-1225[Medline] [Order article via Infotrieve] |
9. | Genetta, T., Ruezinsky, D., and Kadesch, T. (1994) Mol. Cell. Biol. 14, 6153-6163[Abstract] |
10. | Imler, J.-L., Lemaire, C., Wasylyk, C., and Wasylyk, B. (1987) Mol. Cell. Biol. 7, 2558-2567[Medline] [Order article via Infotrieve] |
11. | Scheuermann, R. H., and Chen, U. (1989) Genes Dev. 3, 1255-1266[Abstract] |
12. | Weinberger, J., Jat, P. S., and Sharp, P. A. (1988) Mol. Cell Biol. 8, 988-992[Medline] [Order article via Infotrieve] |
13. | Herrscher, R. F., Kaplan, M. H., Lelsz, D. L., Das, C., Scheuermann, R., and Tucker, P. W. (1995) Genes Dev. 9, 3067-3082[Abstract] |
14. |
Webb, C. F.,
Das, C.,
Coffman, R. L.,
and Tucker, P. W.
(1989)
J. Immunol.
143,
3934-3939 |
15. | Webb, C. F., Das, C., Eaton, S., Calame, K., and Tucker, P. W. (1991) Mol. Cell. Biol. 11, 5197-5205[Medline] [Order article via Infotrieve] |
16. |
Webb, C. F.,
Yamashita, Y.,
Ayers, N.,
Evetts, S.,
Paulin, Y.,
Conley, M. E.,
and Smith, E. A.
(2000)
J. Immunol.
165,
6956-6965 |
17. | Dickinson, L. A., Joh, T., Kohwi, Y., and Kohwi-Shigematsu, T. (1992) Cell 70, 631-645[Medline] [Order article via Infotrieve] |
18. |
Wang, Z.,
Goldstein, A.,
Zong, R.-T.,
Lin, D.,
Neufeld, J. E.,
Scheuermann, R. H.,
and Tucker, P. W.
(1999)
Mol. Cell. Biol.
19,
284-295 |
19. |
Webb, C. F.,
Smith, F. A.,
Medina, K. L.,
Buchanan, K. L.,
Smithson, G.,
and Dou, S.
(1998)
J. Immunol.
160,
4747-4754 |
20. |
Zong, R.-T.,
Das, C.,
and Tucker, P. W.
(2000)
EMBO J.
19,
4123-4133 |
21. | Adachi, Y., Kas, E., and Laemmli, U. K. (1989) EMBO J. 8, 3997-4006[Abstract] |
22. | Hofmann, J. F.-X., Laroche, T., Brand, A. H., and Gasser, S. M. (1989) Cell 57, 725-737[Medline] [Order article via Infotrieve] |
23. | Izaurralde, E., Kas, E., and Laemmli, U. K. (1989) J. Mol. Biol. 210, 573-585[Medline] [Order article via Infotrieve] |
24. | Luderas, M. E., de Graaf, A., Mattia, E., den Blaauwen, J. L., Grande, M. A., de Jong, L., and van Driel, R. (1992) Cell 70, 949-959[Medline] [Order article via Infotrieve] |
25. | Nakagomi, K., Kohwi, Y., Dickinson, L. A., and Kohwi-Shigematsu, T. (1994) Mol. Cell. Biol. 14, 1852-1860[Abstract] |
26. | Romig, H., Fackelmayer, F. O., Renz, A., Ramsperger, U., and Richter, A. (1992) EMBO J. 11, 3431-3440[Abstract] |
27. |
Tsutsui, K.,
Okada, S.,
Watarai, S.,
Seki, S.,
Yasuda, T.,
and Shohmori, T.
(1993)
J. Biol. Chem.
268,
12886-12894 |
28. | Zhao, K., Kas, E., Gonzalez, E., and Laemmli, U. K. (1993) EMBO J. 12, 3237-3247[Abstract] |
29. |
Zong, R.-T.,
and Scheuermann, R. H.
(1995)
J. Biol. Chem.
270,
24010-24018 |
30. | Scheuermann, R. H., and Garrard, W. T. (1999) Crit. Rev. Eukaryotic Gene Expression 9, 295-310[Medline] [Order article via Infotrieve] |
31. | Käs, E., Polijak, L., Adachi, Y., and Laemmli, U. K. (1993) EMBO J. 12, 115-126[Abstract] |
32. |
Pemov, A.,
Bavykin, S.,
and Hamlin, J. L.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14757-14762 |
33. | Singh, L., Panicker, S. G., Nagaraj, R., and Majumdar, K. C. (1994) Nucleic Acids Res. 22, 2289-2295[Abstract] |
34. | Stein, G. S., van Wijnen, A. J., Stein, J. L., Lian, J. B., Pockwinse, S., and McNeil, S. (1998) J. Cell. Biochem. 70, 200-212[CrossRef][Medline] [Order article via Infotrieve] |
35. | Webb, C., Zong, R.-T., Lin, D., Wang, Z., Kaplan, M., Paulin, Y., Smith, E., Probst, L., Bryant, J., Goldstein, A., Scheuermann, R., and Tucker, P. (1999) Cold Spring Harbor Symp. Quant. Biol. 64, 109-118[Medline] [Order article via Infotrieve] |
36. | Peterson, C. L. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, 545-552[Medline] [Order article via Infotrieve] |
37. | Dallas, P. B., Cheney, I. W., Liao, D.-W., Bowrin, V., Byam, W., Pacchione, S., Kobayashi, R., Yaciuk, P., and Moran, E. (1998) Mol. Biol. Cell 18, 3596-3603 |
38. |
Reyes, J. C.,
Muchardt, C.,
and Yaniv, M.
(1997)
J. Cell Biol.
137,
263-274 |
39. | Schnitzer, G. R., Sif, S., and Kingston, R. E. (1988) Cold Spring Harbor Symp. Quant. Biol. 63, 535-543 |
40. | Ariizumi, K., Ghosh, M. R., and Tucker, P. W. (1993) Mol. Cell. Biol. 13, 5629-5636[Abstract] |
41. | Giese, K., Cox, J., and Grosschedl, R. (1992) Cell 69, 185-195[Medline] [Order article via Infotrieve] |
42. | Thompson, J. F., and Landy, A. (1988) Nucleic Acids Res. 16, 9687-9705[Abstract] |
43. |
Blasquez, V. C.,
Hale, M. A.,
Trevorrow, K. W.,
and Garrard, W. T.
(1992)
J. Biol. Chem.
267,
23888-23893 |
44. | Jenuwein, T., Forrester, W. C., Fernandez-Herrero, L. A., Laible, G., Dull, M., and Grosschedl, R. (1997) Nature 385, 269-281[CrossRef][Medline] [Order article via Infotrieve] |
45. | Webb, C. F., Das, C., Eneff, K. L., and Tucker, P. W. (1991) Mol. Cell. Biol. 11, 5206-5211[Medline] [Order article via Infotrieve] |
46. | Giese, K., Kingsley, C., Kirshner, J. R., and Grosschedl, R. (1995) Genes Dev. 9, 995-1008[Abstract] |
47. | Fernandez, L. A., Winkler, M., Forrester, W., and Grosschedl, R. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, 515-524[Medline] [Order article via Infotrieve] |
48. | Artandi, S. E., Cooper, C., Shrivastava, A., and Calame, K. (1994) Mol. Cell. Biol. 14, 7704-7716[Abstract] |
49. | Ephrussi, A., Church, G., Tonegawa, S., and Gilbert, W. (1985) Science 227, 134-0138[Medline] [Order article via Infotrieve] |
50. | Roque, M. C., Smith, P. A., and Blasquez, V. C. (1996) Mol. Cell. Biol. 16, 3138-3155[Abstract] |
51. |
Fernandez, L. A.,
Winkler, M.,
and Grosschedl, R.
(2001)
Mol. Cell. Biol.
21,
196-208 |
52. |
Li, S.,
Moy, L.,
Pittman, N.,
Shue, G.,
Aufiero, B.,
Neufeld, E. J.,
LeLeiko, N. S.,
and Walsh, M. J.
(1999)
J. Biol. Chem.
274,
7803-7815 |