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* The Scripps Research Institute, La Jolla, CA 92037; and
Oklahoma Medical Research Foundation, Oklahoma City, OK 73104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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genes were analyzed extensively for the presence of MARs by in
vitro matrix-binding assay, and for interaction with the MAR-binding
proteins cut-like protein x/CCAAT-displacement protein (Cux/CDP), B
cell regulator of IgH transcription (Bright), and special AT-rich
sequence-binding protein (SATB1) by EMSA. Cux/CDP and SATB1 are
associated with repression, while Bright is an activator of Ig
transcription. Binding sites were identified in the vicinity of all
analyzed Ig V genes, and were also found flanking TCR V
genes. We
also show that the binding sites of the different factors do not always
occur at MAR sequences. MAR sequences were also found within the Ig V
loci at a much higher frequency than throughout the rest of the genome.
Overall, the frequency and location of binding sites relative to the
coding regions, and the strength of DNA-protein interaction showed much
heterogeneity. Thus, variations in factor binding and MAR activity
could potentially influence the extent of localized accessibility to
V(D)J recombination and thus could play a role in unequal rearrangement
of individual V genes. These sites could also contribute to effective
transcription of Ig genes in mature and/or activated B cells, bringing
both the promoter as well as the enhancer regions into close proximity
at the nuclear matrix. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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genes, while neighboring genes from other
V families were not induced (16, 17). This suggests
that accessibility for V(D)J recombination is controlled locally at the
level of gene families or even individual genes. Furthermore, these
data suggest that the transcription factors themselves may be involved
in targeting chromatin remodeling or gene-specific accessibility,
either through effects on germline transcription, or by recruiting
chromatin-modifying complexes (18). Additional evidence
that promoters or other 5' flanking regions are involved in controlling
accessibility of individual genes for recombination came from studies
by Baker et al. (19), in which they showed that switching
1 kb of promoter and 5' flanking DNA adjacent to two TCR V
genes led
to the reversal of the relative frequency of recombination of the two
genes in the adult thymus.
Although V gene coding region sequences are highly conserved
among members of the same family and to a lesser degree between
families, core promoter regions between families show little
conservation, and the remaining flanking regions of these genes exhibit
quite extensive sequence variability. Only the octamer and some TATA
box-like motifs are conserved among V gene promoters, while other
transcription factor binding sites may only be shared among members of
the same family (20, 21, 22). One such example is the human
VII family, in which most genes carry an early B cell transcription
factor binding site in their promoters, but this site is absent from
all other V promoters (23). The majority of V gene
promoters therefore bind combinations of transcription factors unique
to each individual gene family.
Besides transcription factor binding sites, Ig gene promoter regions
contain other regulatory elements, but to date their frequency and
distribution have been much less well characterized. For example, the
VHS107/V1 gene was shown to contain a matrix
attachment region (MAR) within 500 bp 5' of the transcription start
site, and two MAR-binding proteins, NF-µ negative regulator
(NF-µNR) and B cell regulator of IgH transcription (Bright),
also interact with this region (24, 25). MARs are AT-rich
regions located throughout the genome and have been shown to be
involved in organizing chromatin into topological loops by anchoring
DNA to the nonhistone proteins of the nuclear matrix (26, 27). Additional studies of MARs have shown that they are also
associated with more complex functions (reviewed in Refs.
28 and 29). For example, actively transcribed
genes have been shown to be associated with the nuclear matrix, and MAR
sequences have recognition sites for topoisomerase II
(27), which could result in the introduction of torsional
stress making nearby genes more accessible. The two MARs flanking the H
chain intronic enhancer Eµ have been shown to extend the local
accessibility induced by the core enhancer alone over a larger region
(30, 31), while deletion of the Ig -chain intronic
enhancer iE-associated MAR resulted in hyperrecombination of the
J genes closest to the deleted MAR (32). Furthermore,
mice lacking the iE-MAR showed premature onset of V-J
recombination and a decreased level of somatic hypermutation
(33). These observations suggest that MAR sequences may
contribute to changes in chromatin structure and accessibility and
therefore may be relevant for studies of control of V(D)J recombination
and Ig gene transcription.
Our preliminary observations that other MARs may be present in the Ig VH locus (34) and our sequencing of the upstream promoter region of another VH gene revealing a previously unknown AT-rich stretch led us to investigate whether extended areas surrounding Ig V gene coding regions would contain MAR sequences as a general feature, because this could certainly influence Ig functions. We also wanted to determine whether these flanking regions could be recognized by some of the MAR-binding proteins such as cut-like protein x (Cux)/CCAAT-displacement protein (CDP), Bright, and special AT-rich sequence-binding protein (SATB1), which differ in their expression pattern. Most of our investigation focused on the ubiquitously expressed Cux/CDP. The CCAAT-displacement protein Cux/CDP has been shown to be a component of NF-µNR (35), one of the proteins binding to the MAR in the V1 promoter and the Eµ enhancer region. Cux/CDP is a homeodomain protein that recognizes MARs and also regulates genes at many different stages of development and cell differentiation (36, 37, 38, 39). Cux/CDP can cause gene repression by competing with transcriptional activators for binding sites (40, 41), or through its recruitment of the histone deacetylase 1 (42). Furthermore, Cux/CDP binds to MAR DNA, but not to the nuclear matrix, thus preventing matrix association of the MAR DNA (37, 38). Because individual IgV genes contribute unequally to the Ab repertoire, we wanted to determine whether the occurrence and distribution of Cux/CDP binding sites and/or MARs in the vicinity of individual V genes would be related to the frequency with which a particular gene undergoes V(D)J recombination. It is possible that repression by Cux/CDP, possibly by histone deacetylase recruitment or prevention of matrix binding, could be one mechanism to restrict V gene accessibility.
We have extensively analyzed several kilobases of 5' and 3' flanking
region from 13 mouse and human IgH and Ig V region genes. Of these
genes, 70% showed Cux/CDP binding sites within 1 kb of the coding
region on either the 5' or the 3' side, while 50% had Cux/CDP
recognition sites on both sides. We have also analyzed flanking
sequences from three frequently recombining TCR V genes and here too
we identified Cux/CDP binding regions. Several fragments were also
tested for Bright binding and matrix attachment by in vitro MAR assay.
Previous studies had shown that the binding sites for SATB1, Cux/CDP
(NF-µNR), and/or Bright partially overlap within the MARs flanking
Eµ, CD8, and TCR intronic enhancer E (35, 37, 38).
However, the results from our analysis of Ig V gene flanking regions
presented in this study differ from these previous observations.
Specifically, our binding studies revealed that on several occasions
the protein recognition sites and MAR regions did not colocalize, but
rather were identified in different fragments. Overall, we detected
much heterogeneity in the frequency and location of binding sites and
the capability to bind to the nuclear matrix within the flanking
sequences of these genes, suggesting that variations in Cux/CDP and
Bright binding and MAR activity could possibly contribute to localized
control of accessibility and therefore nonrandom gene use during V(D)J
recombination. In addition, the presence of MARs and Bright binding
sites in the promoter-associated regions of most Ig V genes is also
likely to affect transcription of Ig genes in mature B cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EL4 cells were grown in RPMI medium supplemented with 10% FCS, 200 µM glutamine, 2-ME, and gentamicin sulfate. The B cell line, BCg3R-1d, was grown as previously described (25). Preparations of nuclear extracts were performed as previously described. For larger numbers of cells, the protocol by Dignam et al. (43) was used, while for smaller cell numbers nuclear extracts were prepared following Schreiber et al. (44). EL4 extracts were used for all EMSA experiments, because they are a rich source of Cux/CDP. Nuclear matrix preparations from EL4 and BCg3R-1d cells were isolated following the procedure by Cockerill and Garrard (27). Matrix preparations from EL4 and BCg3R-1d cells were used interchangeably throughout the in vitro matrix-binding assay with similar results. The final nuclear matrix preparations were resuspended in buffer at a concentration of 2 x 105 cell equivalents/µl.
Probes
All DNA fragments for which the genomic DNA sequence was
available were isolated by PCR using primers designed based on the
published sequence. The sizes of all PCR fragments were in the range of
300–600 bp. The VHS107/V1 upstream fragments
were isolated by restriction digestion and subcloning of genomic
fragments from a bacterial artificial chromosome clone spanning
the V1/V3 region (generously provided by R. Riblet, Torrey Pines
Institute for Molecular Studies, La Jolla, CA). All PCR fragments were
end labeled using [-32P]ATP and
polynucleotide kinase (New England Biolabs, Beverly, MA), according to
the manufacturer’s instructions. Restriction fragments were end
labeled by either using polynucleotide kinase in a nucleotide exchange
buffer (Life Technologies, Rockville, MD) or by fill-in reaction
of the overhanging ends using [
-32P]dCTP and
Klenow (New England Biolabs). As the negative control in the
matrix-binding assay, pBluescript was linearized by digesting with an
appropriate restriction enzyme and 32P labeled. A
total of 100–300 ng of each fragment, depending on the fragment size,
was labeled in 25 µl total reaction volume and separated from the
unincorporated nucleotides by purification using MicroSpin G-25 Spin
Columns (Amersham Pharmacia Biotech, Piscataway, NJ). The labeled
fragments were further purified by gel electrophoresis on 4%
polyacrylamide gels, containing 5% glycerol, in 1x TAE buffer
(40 mM Tris-acetate, 1 mM EDTA). The appropriate band was visualized by
autoradiography and excised from the gel, and the DNA was eluted from
the gel slice overnight using elution buffer (0.5 M
NH4OAc, 10 mM Mg(OAc)2, 1
mM EDTA, pH 8.0, 0.1% SDS). DNA was then precipitated, washed with
75% ethanol, air dried, and resuspended in an appropriate volume of
water. Probes were used at 5,000 cpm/µl for EMSA experiments and at
10,000 cpm/µl for matrix-binding assays.
EMSA
A total of 3 µl nuclear extract was preincubated with 1–2
µg poly(dIdC) in buffer D (20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M
KCl, 0.2 mM EDTA, 0.5 mM DTT) for 5 min at 37°C. For supershift
experiments, 1 µl 1/10 diluted anti-CDP guinea pig serum
(generously provided by E. Neufeld, Harvard Medical School, Boston, MA)
or, as negative control, 1 µl 1/10 diluted preimmune guinea pig serum
was added to the preincubation. A total of 5000 cpm probe was then
added to each reaction tube and the incubation continued for 10 min at
37°C, followed by 15 min at room temperature. Samples were loaded
onto 4% polyacrylamide gels, containing 5% glycerol in 1x
Tris-acetate-EDTA, and were run at 
120 V at 4°C. Gels were
dried and exposed to film overnight at -70°C. In vitro translated
Bright was prepared using a full-length Bright cDNA clone and
TNT rabbit reticulocyte lysates (Promega, Madison, WI), and 1
µl reaction mixture was used for binding.
SATB1 binding was assessed by incubating a constant amount of labeled
probe with increasing amounts of rSATB1 protein. Briefly, rSATB1 in the
range from 0.15 to 20 nM final concentration was preincubated with 1
µg poly(dIdC) in buffer C (10 mM HEPES, pH 7.9, 50 mM KCl, 2.5 mM
MgCl2, 1 mM DTT, 10% glycerol) supplemented with
2.5 mg/ml BSA in 20 µl reaction volume. After 5 min of preincubation
at room temperature, 5,000–10,000 cpm probe was added and incubated
for an additional 15 min at room temperature. Samples were loaded on 4
or 6% polyacrylamide gels with 5% glycerol in 1x TBE (90 mM
Tris-borate, 1 mM EDTA) and run at 120 V at 4°C. Gels were
dried and either exposed to film overnight at -70°C or exposed to a
phosphor imager overnight at room temperature.
Matrix-binding assay
For each binding reaction, nuclear matrix equivalent to 2 x 106 cells equaling 10 µl nuclear matrix preparation was used. Matrix preparations from EL4 and BCg3R-1d cells were used interchangeably throughout the in vitro matrix-binding assay with similar results. The nuclear matrix was washed three times in matrix wash buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM MgCl2, 0.25 M sucrose, 0.25 mg/ml BSA) with spinning between washes at 4000 rpm for 4 min at 4°C. The matrix was resuspended in 25 µl assay buffer (10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 0.25 M sucrose, 0.25 mg/ml BSA), and sonicated Escherichia coli DNA was added to a final concentration of 100 or 200 µg/ml reaction volume. Matrix was preincubated with the E. coli DNA for 30 min at room temperature on an orbital shaker before adding 20,000 cpm labeled probe and 20,000 cpm labeled pBluescript, followed by incubation 90 min at room temperature with shaking. Thereafter, 500 µl assay buffer was added to the reaction and mixed by inverting. The matrix was pelleted by spinning at 12,000 rpm for 1 min at 4°C, and the supernatant was saved for determination of the unbound fraction. The matrix was washed once more, the supernatant was discarded, and the matrix pellet was resuspended in 25 µl TE (10 mM Tris, 1 mM EDTA, pH 8.0), 0.5% SDS, and 400 µg/ml proteinase K, and incubated at 37°C overnight. After adding loading dye, the matrix-bound DNA was separated on agarose gels. The unbound DNA from the first wash was precipitated overnight and resuspended in 100 µl TE, 0.5% SDS, of which 25 µl, equivalent to one-fourth of the total amount, was loaded. To compare bound and unbound fractions with the amount of input DNA, 20,000 cpm of the same probe and 20,000 cpm pBluescript were diluted in 100 µl TE, 0.5% SDS, of which one-fourth was loaded. Samples were separated by electrophoresis on 0.8% vertical agarose gels, and gels were dried and exposed to film overnight at -70°C. The strong MAR 5' of the V1 gene was used as a positive control.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A region from -574 to -125 bp upstream of the
VHS107/V1 transcription start site had been shown
to harbor one nuclear MAR and to contain two binding sites of the
MAR-binding proteins Bright and Cux/CDP/NF-µNR (24, 25).
However, it had not been determined how far 5' the MAR extended, or
whether the repressor protein Cux/CDP could bind to this extended
promoter flanking region. To obtain additional sequence information, a
genomic clone was isolated from a 
-phage library, and the flanking
region was sequenced up to the EcoRI site 4.4 kb upstream
of the transcription start site. The sequence information was used to
design PCR primers, and the amplified PCR products were analyzed for
Cux/CDP binding by EMSA. Fig. 1
shows a
representative Cux/CDP-EMSA experiment for some of these fragments.
Cux/CDP-positive regions extended to 1 kb upstream of the
transcription start site, followed by a stretch of 1.8 kb that did not
have any Cux/CDP binding sites (Fig. 2).
Another 1.4 kb located directly 3' of the EcoRI site
contained two Cux/CDP binding regions separated by 700 bp of
Cux/CDP-negative sequence.
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18 kb
(20). The appearance of Cux/CDP binding sites not only in
the previously described MAR, but as far upstream as 4.4 kb 5' of the
V1 transcription start site, led us to ask whether Cux/CDP binding
sites are restricted to promoter-proximal flanking region or if the
more distal intervening sequence between V genes may carry additional
interspersed Cux/CDP binding sites. To address this question, the
region upstream of the EcoRI site was analyzed. DNA spanning
this region was isolated from a 10.5-kb BamHI genomic
fragment that had been subcloned from a bacterial artificial
chromosome clone carrying the V1 and V3 genes. This
BamHI fragment overlapped the already analyzed
EcoRI fragment by 2.5 kb. The remaining 8-kb
BamHI/EcoRI fragment was further subcloned and
subdivided into shorter regions for Cux/CDP-EMSA experiments, either by
restriction digest or by PCR. Fig. 2
shows that the 1.7-kb region
directly 5' of the EcoRI site was negative for Cux/CDP
binding, followed by 1.4 kb of Cux/CDP-positive fragments. The
remaining 4.9 kb up to the most 5' BamHI site did not
exhibit any Cux/CDP binding in EMSA experiments. Overall, our results
indicate that binding sites for the MAR-binding repressor protein
Cux/CDP occur not only near the promoter, but also extend as far as 7.5
kb from the transcription start site of V1. Because multiple Cux/CDP
binding sites were found in intervals of several kilobases upstream of
the V1 gene, we wanted to investigate whether sequences 3' of the RSS
might also contain binding sites. We therefore analyzed an area
extending 4 kb downstream of the RSS. Here too, we identified
Cux/CDP-binding fragments as shown on the map, indicating that the V1
coding region is flanked by Cux/CDP binding sites on both sides over an
extended region. CDP binding is not limited to MARs
Cux/CDP binds to many MARs (35, 37, 38). To determine
whether all Cux/CDP binding sites found in the V1 flanking sequences
were also located in MARs, we assayed these same regions for MAR
activity using an in vitro MAR assay. A representative experiment is
shown in Fig. 3. The location of
MAR-positive fragments in the VHS107/V1 gene is
illustrated in Fig. 2. In the region from the promoter up to the
EcoRI site, we found a good correlation between Cux/CDP
binding and matrix attachment; however, in the area from the
EcoRI to the 5' BamHI site, Cux/CDP binding and
matrix attachment did not correlate. Specifically, two Cux/CDP-binding
fragments were negative in the MAR assay, while two other
matrix-binding fragments did not bind Cux/CDP, indicating that matrix
attachment and Cux/CDP binding do not always occur within the same
region.
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Because Cux/CDP and Bright binding sites overlap
significantly in the intronic enhancer and promoter-proximal V1 MARs
(24, 25, 35), and because we identified multiple Cux/CDP
binding sites over a larger region surrounding the
VHS107/V1 gene, we asked whether additional
Bright binding sites might also exist distant from the core promoter.
Thus, several of the fragments identified as MARs were tested for
Bright binding. In addition to the previously described
promoter-proximal Bright binding sites, we identified one Bright
binding site 2.5 kb upstream of the transcription start site and
another one 300 bp downstream of the RSS. We also found a Bright
binding site 5.8 kb upstream of the V1 coding region that did not bind
Cux/CDP or have MAR activity. Furthermore, the 600-bp fragment
immediately 3' to the EcoRI site did not bind Bright,
although it is a MAR and bound Cux/CDP by EMSA. A few additional
fragments that were tested for Bright binding during our investigation
based on their Cux/CDP reactivity were also found to be negative and
are shown in Fig. 2. Thus, Bright binding sites were observed both 5'
and 3' of V1, but Bright-binding activity was not always correlated
with Cux/CDP binding or with MAR activity.
The A/T profile alone does not allow prediction of binding sites
One of the prominent characteristics of MARs is the increased
occurrence of (A + T) nucleotides clustered in longer AT-rich
stretches. DNA binding by Cux/CDP occurs through three cut repeats,
CR1, CR2, and CR3. CR1 binds to DNA with a consensus core sequence of
AATAAT, while CR2- and CR3-binding motifs contain a shorter ATA core
flanked by one or two additional A/T nucleotides (45). To
determine whether there might be a correlation between the occurrence
of Cux/CDP binding sites, matrix attachment, and the percentage of A/T
nucleotides, we analyzed our entire V1 sequence for its A/T profile. We
arbitrarily chose to test for the presence of five consecutive A/T
nucleotides in the sequence by using the "window" program from the
GCG package (Wisconsin Package Version 10.2; Genetics Computer Group,
Madison, WI), using the search parameter "WWWWW," with W
representing either an A or a T nucleotide. Although a high
W5 profile does not allow accurate prediction of
Cux/CDP or Bright binding or matrix attachment, long stretches with a
low W5 profile correlated with fragments that
were completely negative for MARs and MAR-binding proteins (Fig. 2 and
data not shown).
One of the challenges, however, of predicting MAR sequences or recognition sequences for proteins such as Cux/CDP or SATB1 is that there are no well-defined recognition sites, contrary to what is often found for enzymes or transcription factors. There are a limited number of computer programs that claim to predict potential MAR sequences. To investigate whether these programs and their approach to identify MARs would be more successful than our approach with the W5 profile, we analyzed our 5-kb V1 sequence using three of the MAR prediction computer programs: MARFinder (http://www.futuresoft.org/MAR-Wiz) (46), SMARTest (http://genomatix.gsf.de/cgi-bin/smartest_pd/smartest.pl), and ChrClass (ftp://ftp.bionet.nsc.ru/pub/biology/chrclass) (47). However, none of the three programs was capable of predicting our experimentally identified MARs by their algorithms.
LINE elements do not correlate with protein binding or matrix attachment
Previous studies of the mouse Ig H locus had revealed that the C
region carries many copies of repetitive sequences and that these may
represent LINE elements (48). An extensive analysis of the
entire murine Ig C region identified the presence of three distinct MAR
clusters (49). The same study also showed, based on
hybridization patterns, a very strong correlation between the presence
of MAR sequences and the occurrence of repetitive elements, most likely
LINE elements, within the same genomic fragments. Based on these
findings, we analyzed our sequence of VHS107/V1
for the presence of repetitive sequences using the computer program
RepeatMasker (http://repeatmasker.genome.washington.edu/)
(50). The positions of identified repetitive elements
within the entire sequence are indicated by black lines directly
underneath the W5 profile (Fig. 2). The position
of a "simple repeat" is further marked with an asterisk below the
line. In contrast to the findings for repetitive elements in the Ig C
locus, the identified LINE elements and simple repeats in the
VHS107/V1 flanking region did not correlate with
MAR sequences or Cux/CDP, Bright, and SATB1 binding sites.
Murine VH genes show differences in their Cux/CDP binding
The murine VH gene
VH81x is unique in being the most 3' functional
mouse VH gene, and is the most frequently
rearranged gene (51, 52). We sequenced the flanking
regions of VH81x to obtain more sequence
information than was currently available. The entire region, 4 kb in
total, was first analyzed by calculating the W5
profile (Fig. 4). The A/T-rich region
correlating with the spike in the W5 profile
within the intron between the 81x leader sequence and the coding region
was already known from the published sequence, and this sequence
contains a stretch of 40 bp that is mostly composed of T
nucleotides. Such homopolymeric stretches are very uncommon in the
regions we analyzed. However, we found that the 5' flanking region
beyond the published sequence also showed an increased A/T content. In
addition, we identified another A/T-rich segment starting 500 bp 3'
of the RSS with even greater concentration of A/T nucleotides. The
VH81x flanking regions were therefore analyzed
for Cux/CDP binding by EMSA. Surprisingly, only one segment located >1
kb upstream of the 81x coding region was identified as being Cux/CDP
positive. Because 81x rearranges with such high frequency, this result
was in accordance with our hypothesis that frequently rearranging genes
might have a paucity of flanking Cux/CDP sites.
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). These limited results
indicate that the Cux/CDP binding sites were not restricted to flanking
regions in the VHS107/V1 gene, but can be found
in the flanking regions of other mouse VH genes
too. The location and distance relative to the individual coding region
nonetheless varied among genes. Many human Ig VH genes are flanked by Cux/CDP binding sites
Our study of murine VH genes for binding sites of Cux/CDP and Bright was limited by the sequence information available for the mouse Ig loci. However, the human Ig H and L chain loci have recently been sequenced (53, 54, 55). This sequence information provided us with the opportunity to study more extensive flanking regions for the presence of repressor and/or activator binding sites, and the potential capability of these flanking regions to interact with the nuclear matrix.
V genes contribute to the Ab repertoire in a nonrandom fashion in both mice and humans (1, 2, 3, 4, 5). We therefore wished to investigate whether the presence, number, and relative location of Cux/CDP binding sites would correlate with the recombination frequency of individual genes to extend our observation with 81x. In addition, testing additional V gene flanking sequences for Cux/CDP binding would also address the question of whether clustering of repressor/MAR binding sites and their proximity to the promoter region are common among human VH genes.
The human VH locus contains 123 genes distributed
over 950 kb that belong to one of seven families (53).
We first chose to analyze three genes from the
VH3 family that rearrange at different levels:
VH3-23, which has been reported to be the single
most frequently rearranging VH3 gene;
VH3-33, which recombines at a moderate frequency;
and VH3-13, which contributes at a very low level
to the VH3 repertoire (1). The most
frequently rearranging VH3-23 gene was found to
be flanked by Cux/CDP binding sites both close to the promoter and
immediately 3' of the RSS (Fig. 5A).
VH3-33 showed Cux/CDP-reactive fragments 5' and
3' of the coding region, but at some distance from the regulatory
elements (Fig. 5A). However, we were unable to amplify one
AT-rich region within the 5' flank that might carry additional binding
sites. Finally, VH3-13, the gene recombining the
least frequently showed strong Cux/CDP binding close to and upstream of
the promoter, while the downstream Cux/CDP-reactive fragment was
located at a distance of >1 kb from the RSS (Fig. 5A). Two
fragments isolated from the VH3-23 flanking
region that were Cux/CDP positive also showed binding to the nuclear
matrix by in vitro MAR assay and were positive for binding of Bright.
Interestingly, the fragment directly 3' of the
VH3-23 RSS was positive in all three assays,
although it did not have a particularly high A/T content.
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B). Two more Cux/CDP-binding fragments were identified 2
kb upstream and 3.3 kb downstream, respectively, relative to the
VH4-34 coding region. However,
VH4-34 is surrounded by two other
VH genes at relatively close distances.
VH7-34.1 is located only 3.2 kb 5' of
VH4-34, while VH3-33.2 is
situated only 4 kb 3' of the VH4-34 RSS.
Therefore, the two distant Cux/CDP-positive fragments may be
functionally more closely associated with the neighboring genes than
with VH4-34. This is especially pronounced for
the most 3' Cux/CDP-binding fragment, which would have to be
characterized as promoter associated with regard to the next gene,
VH3-33.2. Three promoter-associated fragments of
VH4-28, a gene that does not rearrange, were also
tested for Cux/CDP binding. Two of the three fragments were positive in
Cux/CDP-EMSA experiments; however, the first 200 bp closest to the
VH4-28 coding region did not carry Cux/CDP
recognition sites.
We also asked whether Cux/CDP binding sites might be more prevalent
within certain regions of the human VH locus. We
therefore chose to analyze two VH genes situated
at opposite ends of the VH locus, namely
VH1-69, which is located almost 800 kb upstream
of the DH locus, and VH6-1,
the most 3' VH gene. Fig. 5C shows our
results obtained from Cux/CDP-EMSA experiments for these two genes in
correlation with their W5 profile. Several
fragments located upstream of the VH1-69 coding region were
positive for Cux/CDP binding, and their location correlated with spikes
or plateaus in the W5 profile (Fig. 5C). One fragment directly downstream of the RSS and another
fragment 4 kb 3' of the RSS also showed Cux/CDP binding, separated
by 3 kb that did not show any Cux/CDP reactivity. Surprisingly,
several of the fragments that did not bind Cux/CDP colocalized with
relatively high spikes in the W5 profile. We
therefore chose four fragments to assess their capability to bind to
the nuclear matrix and to bind the transcriptional activator Bright. As
shown in Fig. 5A, three of the four fragments carried MARs
and one fragment also bound Bright. The fragment that was MAR positive
and Bright positive also showed the highest A/T level in the
W5 profile. In contrast to the other human
VH genes, VH6-1, the gene
located most 3' in the locus, had the lowest overall
W5 profile of all genes analyzed (Fig. 5C), showing only two distinct regions of increased A/T
content. The only detected Cux/CDP-binding fragment in the 5' flanking
sequence was located 2 kb from the coding region, while the two
fragments immediately adjacent to the RSS also bound Cux/CDP. Because
VH6-1 showed a fairly good correlation between
the W5 profile and Cux/CDP binding, and because
the RSS-associated fragment bound strongly, we chose this particular
fragment for further examination. However, it did not bind the nuclear
matrix or Bright.
Thus, all human VH genes that we analyzed in this study showed Cux/CDP binding sites in their flanking regions. Furthermore, as seen in the murine VHS107/V1 gene, fragments flanking the human VH genes also showed extensive variability in the different assays, in that some fragments were positive in all three binding assays, while others were only positive for one or two and in different combinations. However, the flanking regions showed much heterogeneity, and no easily recognizable differences were detected between human VH genes that either recombine with very different frequencies or which are located far apart within the locus. Interestingly, the two VH genes with the fewest adjacent MARs and Cux/CDP binding sites are the most 3' VH genes in mice (81x) and humans (VH6-1).
CDP binding also occurs in Ig V genes
We also investigated whether a similar occurrence of Cux/CDP
binding sites would be found in the human V locus. We chose three
genes at different locations within the proximal arm of the locus and
from different families for analysis: V4-1 (B3), the most
J-proximal gene; V1-19 (L8), 150 kb upstream of the J
cluster; and V2-54 (A17), 400 kb from the J genes and close to
the 5' end of the proximal arm. All three genes rearrange fairly
frequently (3, 4). Fig. 6
shows the W5 profiles for each of these genes
together with the results obtained from the Cux/CDP-EMSA experiments.
All three V genes had segments that bound Cux/CDP on either side of
the coding region. The Cux/CDP-binding fragments in the VII gene A17
were at some distance from the coding region, while the VI and the
VIV gene showed Cux/CDP binding somewhat closer to the coding
region.
|
II genes showed a common increase in the W5
profile 2 kb downstream of the RSS that was observed for V2-54.
Similarly, several genes of the VI family exhibited a peak in the
W5 profile 2.3 kb 3' of the RSS seen in
V1-19 (data not shown). Interestingly, however, while this A/T-rich
fragment associated with the VI (L8) gene bound Cux/CDP strongly,
the similar fragment 3' of the VII (A17) gene was only weakly
positive.
The intron between the leader and the coding region of all VII genes
is 400 bp in length; the intron of the single VIV gene is 200
bp. Both introns are much larger than the short 50 bp intron
observed in VI genes. The W5 profile of the
VII (A17) and VIV (B3) gene showed a spike in the intronic
sequence. Surprisingly, when tested for Cux/CDP binding, only the
VIV intron was weakly positive, while the A17 intron fragment did
not bind Cux/CDP at all (Fig. 6).
TCR V genes also have Cux/CDP-binding fragments in close
proximity
The TCR and Ig loci share the same overall gene organization and
use the same enzymatic mechanisms to undergo V(D)J recombination, and
both exhibit allelic exclusion. We therefore asked whether TCR V region
genes would also contain Cux/CDP sites. We chose to analyze the regions
surrounding two commonly rearranged TCR V genes, namely V5.1 and
V8.2, that are in close proximity to each other and to another gene,
V8.3. These three genes are located within a genomic region of only
6 kb (Fig. 7), and only 5.6 kb upstream of the pseudo gene V5.3. The
W5 profile showed a higher A/T content in
sequences between V8.3 and V5.1 than between V5.1 and V8.2.
All of the fragments analyzed bound Cux/CDP, except for the fragments
located directly 5' of both the V5.1 and V8.2 coding region.
Furthermore, fragments directly 3' of the RSS of all three closely
located genes bound Cux/CDP, as was observed for many Ig V genes
(Fig. 7).
|
gene flanking regions
SATB1 is another MAR-binding protein whose expression, in contrast
to Cux/CDP and Bright, is limited to thymocytes (56). It
has been shown to bind to flanking regions of the intronic enhancers of
the Ig H-chain, Eµ, as well as the enhancer of the TCR locus, E
(38). Furthermore, SATB1 has been demonstrated to be
involved in the repression of many T cell-specific genes, in that
SATB1-null mice exhibit inappropriate ectopic up-regulation of several
genes (57). Because the MARs flanking the Ig and TCR
enhancers bound SATB1, we wanted to investigate whether the same
correlation would hold up for the promoter-associated MARs identified
in Ig V genes. We therefore screened the promoter region sequences of
several VH and V genes specifically for the
presence of ATC motifs, the preferred binding sites for SATB1, in
addition to our W5 analysis. Regions that were
identified as potential ATC sequences were then analyzed for SATB1
binding.
SATB1-EMSA experiments were performed in a semiquantitative way, in that a constant amount of 32P-labeled DNA fragment was incubated with increasing quantities of rSATB1 and the relative affinity was estimated based on DNA-SATB1 complex formation. Of the eight ATC fragments tested, only one was SATB1 negative, while the others showed binding to varying degrees. Interestingly, when the same or overlapping fragments had been tested for Cux/CDP binding, only three bound Cux/CDP, while four SATB1-positive fragments did not bind Cux/CDP. This indicates that competition between the two proteins is not the only mechanism of down-modulating Cux/CDP binding, and therefore proposes an even more complex regulation of Cux/CDP binding and associated V gene repression.
Low W5 profiles suggest absence of protein binding sites from the vicinity of mouse CH region exons
An earlier study by Cockerill (49), extensively
analyzing the murine Ig C region for the presence of MARs, revealed
three distinct MAR clusters located in the intergenic regions between
Ig CH region exons. However, all fragments that
facilitated binding to the nuclear matrix in the in vitro assay were
found at distances of at least several kilobases away from the nearest
exon. Our previous results had shown that in several instances, the
location of repressor/activator protein binding sites did not
colocalize within the same fragment that also bound to the nuclear
matrix. We therefore wanted to investigate whether potential protein
recognition sites could be found in closer proximity to the
CH region exons. Because our
W5 profile showed reasonable correlation in many
assayed fragments, specifically in that the absence of any noticeable
peaks in the W5 profile correlated with the
absence of binding sites, the profiles for several
CH region exons and their flanking sequence were
calculated. To further confirm this approach, the sequence surrounding
the Eµ enhancer was also evaluated (data not shown). Two peaks
present in the W5 profile and situated on either
side of the Eµ core enhancer sequence correlated with the well-known
MARs flanking the enhancer region. Unfortunately, the majority of the
mouse Ig CH region sequence, including the
reported MAR-containing regions in the CH locus,
is not yet publicly available, thus limiting our analysis to the few
publicly available genomic sequences. Our analysis showed an overall
very low W5 profile in the surrounding sequences
of Ig µ/Ig 
, Ig 3, and Ig . Only one relatively high peak
was found in the intron between the first and second Ig 
exon.
However, this sequence contains long stretches of poly(A) homopolymers.
Because a similar homopolymeric region had been found in the intron of
the 81x coding region, which did not bind Cux/CDP or SATB1, it was also
assumed that this homopolymer sequence would not bind to any repressor
and/or activator protein. Based on the absence of any
W5 predictable binding sites, we assumed that no
Cux/CDP, Bright, and SATB1 binding sites would be present in the
immediate vicinity of these CH region exons;
therefore, no fragment from the Ig CH locus was
analyzed experimentally.
In addition to the W5 profile, all five sequences were also analyzed for the presence of repetitive elements using RepeatMasker. Of all the identified repetitive elements, only two short stretches in the Ig µ(m) region were actual LINE elements, while all other segments were simple di-, tri-, or tetranucleotide repeats. These theoretical findings correlate well with the absence of MAR activity in the immediate flanking sequences of the Ig C region exons reported earlier (49).
|
Discussion |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
200 kb of the IgH C region
locus, reaching from the last DH gene to 20 kb
past the C region, had identified three clusters of MARs in addition
to the ones flanking the Eµ, but even here the distances between them
varied from 20 to over 70 kb (49), and therefore do not
occur at any higher frequency than throughout the rest of the genome.
Recently, a 100-kb region encompassing the CD8 gene was analyzed for
the presence of MARs, and four strong MARs, along with some weaker
ones, were observed. Three of the strong MARs were clustered around two
DNase-hypersensitive sites within a 9-kb region; two of them also
contained SATB1 binding sites. The fourth strong MAR was located 60
kb 5' of this region (59). In contrast to these findings,
we identified MARs and/or binding sites for MAR-binding proteins near
all Ig V genes analyzed. Moreover, the analyzed V genes were not
limited to a specific region within the loci and consequently cannot
represent a localized MAR cluster. The average distance between
neighboring VH genes in the human locus is less
than 8 kb (53), while the average distance between
neighboring V genes is even shorter, less than 4 kb
(55). Thus, based on our findings, the average distance
between MARs in the human Ig H and Ig V gene locus also can be
assumed to be less than 8 or even 4 kb, respectively, which is much
shorter than the previously reported minimum distance between adjacent
MARs of 20–30 kb (49, 58). Therefore, MARs in the Ig V
loci occur at a frequency much higher than throughout the rest of the
genome.
Ig V genes have been shown to contribute in a nonrandom frequency to
the Ab repertoire. Although there are naturally occurring variations
among the different RSSs, which can affect the frequency of
rearrangement of individual genes, there are clearly other factors in
addition to the RSS potency influencing V(D)J recombination and
therefore the contribution of the various genes to the peripheral
repertoire (5, 7, 8, 9). One other mechanism by which
nonrandom V(D)J recombination could occur is through differential and
localized accessibility of individual genes to the recombinase enzymes.
MAR sequences themselves may directly affect V gene accessibility. For
example, it has been shown that deletion of the iE MAR results in
premature V-J recombination (33). Local changes in
chromatin could be imposed through histone acetylation and DNA
demethylation, both of which have been associated with MARs (60, 61). For example, it has been shown that demethylation of the
Ig locus resulted in an increase in gene rearrangement, and this
demethylation has been linked to the iE-associated MAR sequences, as
well as the core enhancer. A recent study by Fernandez et al.
(62) also showed that histone acetylation of nucleosomes
distant from the Eµ was augmented by the Eµ-associated MAR
sequence, and histone acetylation has been tightly linked to
accessibility for V(D)J rearrangement (62). Matrix
attachment is also associated with transcription, and germline
transcription is strongly correlated with the onset of recombination
(51).
One of the MAR-binding proteins, Cux/CDP, had been reported to be frequently involved in the transcriptional repression of genes that are developmentally regulated (39). Cux/CDP is evolutionarily conserved, and it is expressed in a wide variety of tissues (39). In most reported cases, only single Cux/CDP binding sites were described in promoters. In this study, we demonstrate that Cux/CDP binding sites are present in fairly high number and located throughout the intergenic regions between VH coding sequences, presenting the possibility that Cux/CDP binding may be involved in keeping the chromatin and thereby the different V genes within the loci in an inaccessible state. We show that Cux/CDP binding does not always colocalize with sequences that can facilitate binding to the nuclear matrix. Cux/CDP-mediated repression could therefore be imposed by discrete mechanisms: 1) in cases in which Cux/CDP binding does colocalize with MARs, Cux/CDP binding could prevent these MAR regions from interacting with the nuclear matrix and/or matrix-associated proteins, such as Bright or topoisomerase II; 2) under the circumstance that Cux/CDP binding occurs at a region with no MAR activity or in which the nearest MAR is located at some distance, recruitment of histone deacetylase complexes by Cux/CDP might be responsible for keeping DNA in a closed inaccessible conformation.
Cux/CDP has been shown to be a component of NF-µNR (35),
a protein first isolated because of its capability to bind to the
Eµ-flanking MARs (63). This study also showed that
NF-µNR activity as detected by EMSA experiments was present in many
murine non-B lineage cell lines and in cell lines representing the
pro-B stage. However, NF-µNR activity was not detectable in any
µ-expressing pre-B or B cell lines. It was therefore proposed that
NF-µNR, and thus Cux/CDP, may function as a lineage-specific
repressor of the Eµ enhancer activity in non-B cells by binding to
the Eµ flanking regions, consequently repressing Ig V(D)J
recombination in T cells (63). The high number of Cux/CDP
binding sites that we have identified in this study near Ig V genes,
and their location not only associated with the promoter regions, but
also 3' of the analyzed genes, support the hypothesis that Cux/CDP may
be involved in accessibility control of recombining loci. However, if
Cux/CDP binding would be involved in specifically preventing
accessibility of Ig genes, but not TCR genes in T cells, one might
predict that we would find Cux/CDP binding sites associated only with
the Ig gene loci and not the TCR loci. However, we identified Cux/CDP
binding sites in the flanking sequence of all three TCR V
genes examined. This suggests that Cux/CDP-mediated repression may
contribute to both TCR and Ig loci inaccessibility. Recently,
Cux/CDP-deficient mice were generated. Although, as expected, in the
absence of the repressor Cux/CDP, Ig and TCR rearrangement occurs, the
mice were lymphopenic presumably due to misregulation of other genes
normally controlled by Cux/CDP (64).
Finding Cux/CDP binding sites surrounding genes of the TCR locus, however, does not affect its potential involvement in control of accessibility at the Ig locus because repression of Ig gene rearrangement only persists until the appropriate time point in B cell development. Although EMSAs on normal bone marrow B cell progenitors have not been done, analysis of murine pre-B cell lines and normal mouse splenic B cells would suggest that Cux/CDP-binding activity appears to be lost as lymphocytes pass through the stages of V(D)J rearrangement (Refs. 63, 65 and unpublished data). This might occur either by decreasing expression levels of Cux/CDP or by inactivating its DNA-binding capability as the cells differentiate. Several posttranslational modifications of Cux/CDP have been shown to influence its capability to bind to DNA, such as serine phosphorylation and/or acetylation of the cut homeodomain (66, 67). If Cux/CDP-binding activity does diminish as VH gene rearrangement progresses, it may contribute to the nonrandom gene accessibility throughout the IgH locus. In one potential model, a decrease in Cux/CDP concentration or its lowered affinity for binding sites would then remove Cux/CDP, and therefore Cux/CDP-mediated repression from V genes that are flanked by low affinity binding sites, while high affinity Cux/CDP recognition sequences would remain occupied. As a result, some V genes would become locally derepressed, while other genes even within the same locus could remain inaccessible.
In another model, repression could be modulated through the competition of Cux/CDP with other proteins for DNA binding. It has been demonstrated that several other MAR-binding proteins can specifically compete or interfere with Cux/CDP for MAR binding (35, 37, 38, 68). Interestingly, one of these proteins, Bright, functions as a transcriptional trans-activator (35), while another protein that can compete with Cux/CDP, SATB1, exhibits predominantly repressive properties (69, 70), although SATB1 displacement of Cux/CDP has also been associated with activation (37). Importantly, while Cux/CDP is expressed in many cell types, Bright expression is limited to two specific stages during B cell development (65) and SATB1 is expressed predominantly in thymocytes (56). Bright shares homology with mating type switching (SWI), a component of the sucrose nonfermenting/mating type switching complex involved in chromatin remodeling (71). Thus, because Bright outcompetes Cux/CDP for binding to the MAR sequences (35), expression of Bright in bone marrow progenitors and binding to its recognition sites could result in Bright-mediated chromatin remodeling. SATB1 has also been reported to interact directly with Cux/CDP in a DNA-independent fashion (70), resulting in inactivation of DNA binding in both proteins. It was therefore hypothesized that the ratio of the two proteins could contribute to modulation of their net function.
If competition between Cux/CDP and other proteins is an important factor in modulating Cux/CDP binding, one should frequently observe binding sites for Cux/CDP and the competing proteins within the same flanking fragment. To date, only a few MARs have been analyzed extensively (28, 37, 59, 68). In all these previous studies, it was found that the binding sites of MAR-binding proteins such as Cux/CDP, Bright, SATB1, and MAR-BP1 occurred within the same sequence that also facilitated direct binding of DNA to the nuclear matrix (35, 37, 38). It was therefore assumed that most of these proteins can be characterized as MAR binding. In this study, we analyzed a selected number of fragments for matrix attachment, Cux/CDP, Bright, and SATB1 binding. Surprisingly, and in contrast to expected results, we found several fragments that showed dissociation of binding any of these proteins and binding to nuclear matrix preparations. This indicated that the binding sites of the different repressors/activators not always overlap and/or colocalize. Moreover, this also ascribes additional properties to these proteins separate from their MAR-binding activity. Thus, individual V genes may be more or less likely to have Cux/CDP binding successfully competed out by binding of other MAR-binding proteins, depending upon whether their flanking DNA has sites for those proteins. It is also very likely that other MAR-binding proteins that were not addressed in this study may also interact with or displace Cux/CDP, or that they may directly affect matrix binding, which could influence Ig V gene accessibility and/or Ig transcription.
It is possible that the frequent occurrence of MARs and/or
Cux/CDP binding sites flanking recombining genes could potentially be
involved in preferentially targeting recombination to some genes and
not others. In the case of Cux/CDP, a simple hypothesis would be that
genes that exhibit a lower recombination frequency might be more
repressed by having more Cux/CDP sites in their vicinity, while more
frequently rearranging genes would have few binding sites. Although
this seemed to be true for 81x, which rearranges at very high frequency
and was almost deficient in Cux/CDP binding sites on either side, this
was not the general case. One therefore could hypothesize that a
differential impact of Cux/CDP on RAG accessibility would be most
profound if the proteins would be bound in close range to either the
RSS, and directly exclude RAG binding, or within the promoter regions.
The latter site may be important in influencing germline transcription
or transcription factor binding whose role in V(D)J recombination has
not yet been fully established (17, 51). We therefore
analyzed the presence of Cux/CDP-binding fragments 5' and 3' of the Ig
V gene coding regions. However, there was not a simple correlation
between clustering and/or relative location of Cux/CDP binding sites
and the frequency with which the corresponding V gene recombines, as
one would expect if only Cux/CDP occupancy influenced local
accessibility. Instead we found much heterogeneity in the number and
location of the individual flanking Cux/CDP binding sites. We also
detected heterogeneity in the location of recognition sites for other
MAR-binding proteins and for matrix binding as well as divergence in
the colocalization of these binding sequences. Thus, the affinity and
frequency with which Cux/CDP binds in the vicinity of individual V
genes, and its interaction with MARs and MAR-binding proteins could
result in a net chromatin conformation that has the potential for
influencing V(D)J recombination by altering localized accessibility to
the recombinase enzymes. The importance of these recognition sites for
the individual V gene segments is further strengthened by the predicted
absence of MAR-binding protein recognition sites from the murine Ig C
region locus. The number of C regions is much smaller than the number
of V region genes, and importantly, isotype switch recombination
requires completely different mechanisms of control than V(D)J
recombination. In addition, it has been hypothesized that an important
function of MARs in the Ig C region could be to separate the highly
recombining V, D, and J genes from the C regions, and to segregate the
early Ig µ- and -chains from the other isotype C regions
(49).
Because it is becoming progressively clearer that chromatin remodeling and histone acetylation are associated with the increase in accessibility to V(D)J recombination (10, 11, 12, 13, 14, 15), and because the Eµ MARs have been shown to promote histone acetylation over extended distances (62), the sites that we describe in this work may also play a role in chromatin changes augmenting V(D)J recombination in the V region loci. It has also recently been shown that the Ig locus is redirected during B cell development from the nuclear periphery to the center of the nucleus (72). The VH region in particular shows more nuclear peripheralization than the C region portion of the locus, leading to the suggestion that the more distal portion of the V gene locus, but not the C gene locus, may preferentially facilitate this subnuclear compartmentalization. It is thus also possible that these V gene-associated MARs could play a role in the subnuclear localization of the Ig loci during B cell development. Finally, the frequent occurrence of MARs, and binding sites for transcriptional activators such as Bright, could also affect Ig gene transcription at later stages of B cell development. Bright has been shown to activate Ig transcription in activated cells (65), so the presence of Bright sites in the 5' flank of many V genes could influence levels of Ig transcription. Mature B cells may use promoter-flanking and enhancer-flanking MARs to colocalize these regions of rearranged Ig H and L chain genes at the nuclear matrix, thus promoting Ig transcription. Thus, the occurrence of MARs, and binding sites for MAR-binding transcriptional activator and repressor proteins may be relevant for multiple aspects of B cell function.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Ann J. Feeney, Department of Immunology, The Scripps Research Institute, IMM-22, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: feeney{at}scripps.edu
3 Abbreviations used in this paper: RSS, recombination signal sequence; CDP, CCAAT-displacement protein; Cux, cut-like protein x; Bright, B cell regulator of IgH transcription; Eµ, Ig H chain intronic enhancer; iE, Ig -chain intronic enhancer; MAR, matrix attachment region; NF-µNR, NF-µ negative regulator; SATB1, special AT-rich sequence-binding protein; TE, tris-EDTA.
Received for publication December 17, 2001. Accepted for publication June 25, 2002.
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References |
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