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Unité de Génétique et Biochimie du Développement, Unité de Recherche Associée Centre National de la Recherche Scientifique 1960, Département d’Immunologie, Institut Pasteur, Paris, France; and
Anatomy Department, University of Birmingham Medical School, Edgbaston, Birmingham, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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L chain loci. In recombination-activating
gene-deficient pro-B cells, there is a reorganization of nucleosomal
structure over the H chain JH cluster and increased DNase I
sensitivity of VH and JH segments. The
pro-B/pre-B cell transition is marked by a decrease in the DNase I
sensitivity of VH segments and a reciprocal increase in the
nuclease sensitivity of V and J segments. In contrast,
JH segments remain DNase I sensitive, and their nucleosomal
organization is maintained in µ+ recombination-activating
gene-deficient pre-B cells. These results indicate that initiation of
rearrangement is associated with changes in the chromatin structure of
both V and J segments, whereas stopping recombination involves changes
in only V segment chromatin. We further find an increase in histone H4
acetylation at both the H and L chain loci at the pro-B cell stage.
Although histone H4 acetylation appears to be an early change
associated with B cell commitment, acetylation alone is not sufficient
to promote subsequent modifications in Ig
chromatin. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During B cell development, rearrangement at the Ig H chain locus
generally begins before L chain gene rearrangements in pro-B cell
precursors (4). H chain gene rearrangement occurs in two
steps, first joining of DH and
JH segments, which can take place on both
chromosomes, followed by VH to
DJH rearrangement. A productive
VDJH rearrangement at one allele and expression
of the µ H chain lead to down-regulation of RAG and inhibition of
further IgH gene rearrangement. The cells then undergo two to seven
rounds of division before differentiating to the pre-B cell stage, in
which RAG genes are up-regulated and V to J rearrangements at the L
chain loci are initiated. Despite the presence of an active
recombinase, IgH loci containing a DJH
rearrangement are not targeted for further V-DJH
recombination in pre-B cells. The ability of the recombination complex
to discriminate between IgH and Ig genes at the pro-B/pre-B cell
transition is essential for maintaining the monospecificity of the Ag
receptors in B cells.
How V(D)J recombination is regulated is not known. Available evidence indicates that rearrangement of all Ig and TCR genes is mediated by the same recombination trans-acting factors acting at conserved signal sequences (5). This suggests that regulating the accessibility of V region gene segments to the recombination complex may play an important role in the control of rearrangement. Several lines of evidence, including transfection and transgenic experiments, support the idea of differential accessibility of Ig and TCR genes in lymphoid precursors (reviewed in Ref. 6). However, it is not known what, at the molecular level, differentiates an accessible locus from one that is refractory to V(D)J recombination. In eukaryotic cells, DNA is associated with histones to form nucleosomes and higher order chromatin structures (7). This organization provides multiple levels at which the access of DNA-binding proteins to their target sequences can be restricted. It has become increasingly apparent that modulation of chromatin structure plays an important role in the regulation of transcription. A number of transcriptional coactivator complexes contains ATP-dependent nucleosome-remodeling factors and histone acetyltransferases (8, 9, 10), while numerous transcriptional repressors have been found to be associated with histone deacetylases and methyl-CpG-binding proteins (11, 12). Recent evidence from in vitro recombination studies has shown that packaging of DNA into chromatin also inhibits the initial stages of V(D)J recombination, namely RSS cleavage by the RAG proteins (13, 14). Furthermore, in contrast to naked DNA substrates, RAG-mediated cleavage of Ig and TCR gene segments within chromatin reflects the lineage and stage specificity of recombination (15). These in vitro results suggest that remodeling of chromatin structure may play an important role in the control of Ag receptor gene rearrangement.
A change in methylation status was one of the first epigenetic
modifications observed at the Ig loci. Demethylation of H and L chain
genes occurs during B cell development (16, 17, 18) and
appears to be associated with onset of V(D)J recombination at the
locus (19, 20). Similarly, DNase I analysis has shown that
functionally rearranged Ig genes are more sensitive to nuclease
digestion than transcriptionally inactive unrearranged loci (16, 21, 22). Although these early studies indicate that changes in
chromatin structure occur at Ig loci, it is unclear to what extent
these modifications precede or are a result of V(D)J recombination.
To address this question and to define the underlying changes in V and
J segment chromatin associated with the onset and inhibition of V(D)J
recombination, we have undertaken a detailed analysis of the chromatin
structure of Ig H and L chain genes. This study was conducted using B
cell precursors derived from RAG2-/- mice with
and without a functional IgH transgene (RAG2-/-
x Igµ). Inactivation of the RAG2 gene completely inhibits V(D)J
recombination; consequently, B cell development in
RAG2-/- mice is blocked at the pro-B cell
stage, in which Ig H chain gene rearrangement normally takes place
(23). Introduction of a functionally rearranged Igµ
transgene onto the RAG2-/- background promotes
B cell development to the pre-B cell stage (24). These B
cell precursors undergo appropriate developmentally regulated changes
in Ig gene structure, as assessed by RAG-mediated RSS cleavage of IgH
and gene segments and initiation of germline transcripts (15, 24). They therefore appear to be poised to initiate V(D)J
recombination, while maintaining their Ig genes in an unrearranged
configuration. The results of this study show dynamic changes in V and
J segment chromatin at the endogenous Ig loci during B cell
development, as assessed by histone H4 acetylation, DNase I
sensitivity, and nucleosomal organization. These modifications appear
to take place in successive stages, one of the earliest changes
observed being an increase in the level of histone H4 acetylation.
Furthermore, our results support the idea that alterations in V gene
chromatin play an important role in the control of Ig gene
rearrangement.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell lines used include the Abelson murine leukemia virus-transformed pro-B cell lines 63.12 (23), LM2 (24) derived from RAG2-/- mice, and the LC2 and HC8 pre-B cell lines (24) derived from RAG2-/- mice containing either low or high copy number, respectively, of a functional Igµ transgene (gift from F. Young (University of Rochester Medical School, Rochester, NY) and F. W. Alt (Harvard Medical School, Boston, MA)); the S194 plasma cell line (ATCC TIB-19); the YAC-1 T cell line (25); and the P815 mastocytoma cell line (ATCC TIB-64). FACS analysis showed that 63.12, LM2, LC2, and HC8 Abelson cell lines are all B220+, but only LC2 and HC8 are surface µ+ (data not shown).
For some experiments, cells were cultured in the presence of the histone deacetylase inhibitor trichostatin A (TSA; Sigma, St. Louis, MO) at either 100 ng/ml for 6 h or 5 ng/ml overnight, or with 10 µg/ml LPS (Sigma) for 24–72 h.
DNase I sensitivity analysis
DNase I digestion was performed on lysolecithin-permeabilized
cells, essentially as described (26). Following
permeabilization, 1.5 x 107 cells were
treated with 0.1–3.2 µg/ml DNase I (Worthington Biochemicals,
Lakewood, NJ) at 25°C for 5 min in 400 µl of 150 mM sucrose, 80 mM
KCl, 35 mM HEPES (pH 7.4), 5 mM
K2HPO4, 5 mM
MgCl2, 1 mM CaCl2, and
0.02% sodium azide. DNase I digestion was routinely controlled on a
1% agarose gel. Genomic DNA (20 µg) from the DNase I-treated cells
was digested with BamHI, then separated on a 0.7% agarose
gel in 1x TAE buffer (0.04 M Tris acetate, 0.001 M EDTA),
transferred to a nylon membrane (Positive Membrane; Appligene,
Strasbourg, France), and hybridized successively with the following
radiolabeled probes: JH, a 143-bp fragment
obtained by PCR amplification using the forward primer
5'-CTATGCTATGGACTACTGGGGT-3' and reverse primer
5'-GCTCCCTCAGGGCAAATATCC-3'; VHJ558, a 315-bp
PstI-EcoRI fragment (27); J, a
2.7-kb HindIII fragment; V11, a 430-bp
StyI-AccI fragment (28); V21, a
375-bp PstI-HincII fragment (28);
kidney androgen-regulated protein (KAP), a 550-bp cDNA
PstI-HindIII fragment (29). The
intensity of hybridization signals was quantified on a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
Micrococcal nuclease (MNase) digestion analysis
Cells were permeabilized as above, then treated with 0.5–10
U/ml MNase (Worthington Biochemicals) at 25°C for 5 min in 150 mM
sucrose, 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 2 mM
CaCl2, and 0.02% sodium azide (26).
Following MNase digestion, the extracted genomic DNA (20 µg) was
cleaved with MspI, separated on a 1.4% agarose gel in 1x
TAE buffer, then transferred to a nylon membrane (Positive Membrane;
Appligene), and hybridized with a radiolabeled 600-bp
BamHI-HindIII 5'JH fragment
(probe A, Fig. 4
A).
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Nuclear extracts of lymphoid cell lines and gel retardation
experiments were performed as described previously (30).
The mouse NF-B oligonucleotide used was
5'-GACAGAGGGGACTTTCCGAGAG-3'.
Chromatin immunoprecipitation
Chromatin immunoprecipitations were performed as previously
described using affinity-purified Abs to acetylated histone H4
(31). In brief, cells were grown overnight in medium
supplemented with [3H]thymidine (Amersham,
Arlington Heights, IL) at 0.1 µCi/ml before isolation of nuclei and
digestion with MNase (Pharmacia, Piscataway, NJ) to release chromatin.
Digestion conditions were adjusted so that the chromatin to be used for
immunoprecipitation was rich in mononucleosomes and the smaller
oligonucleosomes, typically 2- to 5-mers. Following chromatin
immunoprecipitation, DNA was isolated from the Ab-bound (i.e., highly
acetylated), unbound (i.e., underacetylated), and input chromatin
fractions, and analyzed by electrophoresis on 1.2% agarose gels. Equal
amounts of DNA from each fraction, based on
[3H]thymidine content, were serially diluted
and applied to nylon filters (Hybond N+;
Amersham) by slot blotting. Specific DNA sequences were detected by
hybridization with radiolabeled probes and quantified on a
PhosphorImager (Molecular Dynamics). 
-Tubulin and centric
heterochromatin (Het 266) probes are as described (31, 32).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although rearrangement of V segment genes appears to be the
regulated step for both H and L chain gene recombination
(5), little is known about the chromatin structure of
VH and V segments. The murine IgH and Ig V
loci each contain 100 or more V genes spread over >1 megabase
(33, 34). We therefore probed the chromatin structure of
VH and V segments by measuring general DNase I
sensitivity (35), since this technique allows the global
analysis of large, complex regions.
To compare DNase I sensitivity of Ig gene segments at different
developmental stages, we analyzed pro-B and pre-B cell lines derived
from RAG2-/- and
RAG2-/- x Igµ mice (23, 24), as
well as mature B and non-B cell lines. Cells were permeabilized with
lysolecithin and digested with increasing concentrations of DNase I.
Southern blots from extracted DNA were hybridized successively with
family-specific H and L chain V probes, as well as with probes for
JH and J. We analyzed gene segments of the
V11 and V21 family, which represent the most 5' and 3' V gene
clusters, respectively (34), and the
VHJ558 family, which is the largest murine
VH cluster (33). Relative
sensitivity to DNase I was assessed by measuring the loss of
hybridization signal for each probe. As an internal control, the blots
were also hybridized with a probe for the kidney-specific, KAP gene
(29). This probe detects a relatively DNase I-resistant
fragment as expected, which persists throughout the digestion in the
concentration range used with only a 2- to 5-fold loss in hybridization
signal intensity. Band intensities for the V and J segments were
quantified and plotted relative to the KAP gene. Results of two
representative Southern blots, obtained for the RAG2-deficient 63.12
pro-B and LC2 pre-B cell lines, are shown in Fig. 1A. The Ig V and J fragments analyzed vary in size. Since
large molecular mass fragments are more susceptible to random DNase I
digestion than smaller sized fragments, we compared, in Fig. 1B, the DNase I sensitivity of the same Ig gene fragments in
the different cell lines.
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genes are refractory to RAG-mediated cleavage (15),
the V and J segments are relatively DNase I resistant (Fig. 1
B). Similar results were obtained with thymocytes and the
YAC-1 T cell line, although a slightly greater DNase I sensitivity was
observed for the JH fragment (data not shown). In
contrast, in the 63.12 and LM2 pro-B cell lines, the
VH and JH segments are
significantly more sensitive to DNase I than in non-B lineage cells,
whereas the V genes and, to a lesser extent, the J segments
remain DNase I resistant. The relative DNase I sensitivity of the J
fragment may be due to the presence of a hypersensitive site, which
maps to the Ei enhancer element (36) and which we find
is already present at the pro-B stage. A different pattern is observed
with the LC2 and HC8 pre-B cell lines. In these cells, there is an
increase in the rate of loss of the V and J fragments, which
become more sensitive to DNase I than in pro-B cells, whereas the
VH genes become more resistant to DNase I
digestion (Fig. 1B). In contrast, the
JH segments remain DNase I sensitive in the pre-B
cells. Both IgH alleles can be visualized separately with the
JH probe due to restriction fragment length
polymorphism, and we find that they are both equally sensitive to DNase
I digestion in the RAG-deficient pro-B and pre-B cell lines. The
pro-B/pre-B cell transition, which corresponds to a stop in IgH and
activation of Ig rearrangements, is therefore marked by an increase
in the DNase I sensitivity of V and J segments and a reciprocal
decrease in the sensitivity of VH segments. In
mature B cell lines, no longer undergoing IgH or Ig gene
rearrangements, the J segments remain DNase I sensitive, whereas the
V, like the VH genes, become resistant. These
results suggest that initiation of V(D)J recombination at the Ig loci
is preceded by an increase in nuclease sensitivity of both V and J
segments, whereas stopping rearrangement only involves changes in V
segment chromatin.
LPS increases DNase I sensitivity of J, but not V segments
The above results show that DNase I sensitivity reflects lineage
and developmental accessibility of Ig gene segments to V(D)J
recombination factors. A model system for studying activation of
gene rearrangement is provided by the bacterial mitogen LPS. Treatment
of pro-B cells with LPS activates NF-B, a transcription factor that
binds to the intronic enhancer and induces both gene
transcription and rearrangement (37). We therefore
analyzed the effect of LPS treatment on the chromatin structure of V
and J segments in the 63.12 pro-B cell line. LPS stimulation of
63.12 cells, under conditions that induce transcriptionally active
p50/p65 NF-B heterodimers (Fig. 2A), increases the DNase I sensitivity of J segments to
that observed in pre-B cells (Fig. 2B). In contrast, no
change was observed in the nuclease sensitivity of V11 and V21
genes, indicating that LPS- induced modifications are restricted to the
J locus and do not totally mimic developmentally regulated
alterations in chromatin structure.
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The N-terminal domains of nucleosomal core histones are subject to
several posttranscriptional modifications, including acetylation at
specific lysine residues (8). Changes in histone
acetylation take place during physiological processes, such as
transcription and DNA replication. We have used a chromatin
immunoprecipitation approach to assay the level of H4 acetylation along
the H and L chain loci, to assess the role of histone acetylation
in the regulation of Ig gene accessibility to V(D)J recombination
factors.
Nuclei from 63.12 pro-B cells, HC8 pre-B cells, P815 mast cells, and
YAC-1 T cells were subjected to mild MNase digestion and chromatin
fragments immunoprecipitated with affinity-purified Abs to acetylated
H4 histones, as previously described (31). To examine
levels of acetylation at the Ig loci, slot blots containing DNA from
the Ab-bound (B) and unbound (U) fractions were hybridized successively
with H and L chain V and J probes (Fig. 3A). As control, blots were also hybridized with probes for
centric heterochromatin and the ubiquitously expressed -tubulin
gene. Hybridization was quantified by PhosphorImager analysis, and
levels of acetylation expressed as B/U ratio (Fig. 3B).
Consistent with previous results, we found that heterochromatin
sequences were underrepresented in the bound, acetylated fraction of
all the cell lines, whereas the -tubulin gene was constitutively
acetylated. B/U ratios equal or less than that of heterochromatin were
observed for all H and L chain V and J segments in P815 mast cells,
indicating that the Ig loci are underacetylated in these cells (Fig. 3B). In the B cell precursor lines, there is an increase in
the level of H4 acetylation for the Ig genes, which becomes comparable
or greater than that of -tubulin. This increase was observed not
only at the H chain locus, but also for L chain V and J segments in
63.12 pro-B cells. All the Ig segments remain hyperacetylated in HC8
pre-B cells. In contrast, the control KAP gene remains hypoacetylated
in pro-B and pre-B cells, indicating that the increase in acetylation
observed at the Ig loci is not due to a general increase in the level
of histone H4 acetylation in these cells. These results therefore
suggest that histone acetylation at the Ig loci occurs early in B cell
development and may be related to B cell commitment, rather than
stage-specific locus accessibility. Interestingly, although V and
J segments are underacetylated in YAC-1 T cells, the acetylation
level of H chain J segments approaches that of the -tubulin gene
(Fig. 3B) and may be related to the potential of T lineage
cells to undergo partial H chain D-JH
rearrangements.
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Selective positioning of the nucleosome core particle has been found to inhibit RSS cleavage by the RAG proteins in vitro (13, 14). Thus, regulating the position of the nucleosomes may be one of the ways of controlling the accessibility of recombination trans-acting factors to the Ig loci. We therefore performed nucleosome-mapping experiments using the MNase, which cleaves the DNA backbone in the linker region between nucleosomes. We concentrated on the JH locus, comparing the nucleosomal organization in non-B lineage cells and B cell precursors.
Permeabilized cells were digested with MNase, and the cleavage sites
were mapped with respect to the upstream MspI site by
Southern blot analysis using a probe hybridizing to the 5' end of the
JH fragment. Results for the P815 mast cell line
and the 63.12 pro-B and HC8 pre-B cells are shown in Fig. 4A. In all the cell lines, MNase digestion gave a discrete
banding pattern for the 1.9-kb MspI fragment containing the
JH segments, a result consistent with an ordered
nucleosomal structure. Control digestion of naked DNA showed that this
pattern was not due to the presence of preferential MNase-cutting
sites. These data therefore show that the JH
segments are covered by an ordered nucleosomal array in both lymphoid
and nonlymphoid cells. The 63.12 pro-B and HC8 pre-B cell lines
exhibited a similar digestion pattern. This profile was also observed
for the LM2 and LC2 B cell precursor lines, but was clearly different
from the pattern observed with P815 mast cells or thymic T cell
precursors (Fig. 4A and data not shown). The difference
between B cell precursor and non-B cells was confirmed using a 3'
hybridizing probe (probe B), whereas a similar MNase pattern was
observed with a probe for the KAP gene (Fig. 4B), providing
a control for the specificity of hybridization. Taken together, these
results provide evidence of a different nucleosomal structure at the
JH locus in B cell precursors, where the J
segments are accessible to RAG cleavage, and in P815 cells, where they
are not.
TSA treatment does not alter chromatin structure at the Ig loci in non-B cells
Our chromatin immunoprecipitation results show that the Ig loci
are underacetylated in non-B cells and become acetylated during B cell
development. To determine whether histone acetylation is sufficient to
induce the developmental changes in chromatin observed at the Ig loci,
we have used TSA, a specific inhibitor of histone deacetylase
(38). P815 mast cells were treated with either 100 ng/ml
TSA for 6 h or 5 ng/ml for 14 h, higher TSA concentrations or
longer incubation periods causing extensive cell death. This treatment
markedly increased the level of both bulk H4 acetylation and
acetylation at the JH and J loci (Fig. 5, A and B). However, no effect was observed on
nucleosomal organization at the JH locus. As
shown in Fig. 5C, the MNase digestion profile of TSA-treated
P815 cells resembled that of untreated cells and was different from
that of 63.12 pro-B cells. Similarly, no obvious effects of TSA
treatment on the DNase I sensitivity of the JH
locus were observed, nor did TSA modify the DNase I sensitivity of
JH or J segments in YAC-1 cells (data not
shown). These results suggest that histone acetylation alone cannot
induce the changes in chromatin structure occurring during B cell
development at the endogenous Ig loci.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L chain gene
rearrangement are preceded by a reorganization of V and J segment
chromatin structure.
The modifications of Ig gene chromatin structure appear to take place
in successive steps during B cell differentiation. One of the earliest
changes observed was an increase in the level of histone H4 acetylation
associated with the Ig loci in B cell precursors. We found that H and
L chain V and J segments carry hypoacetylated nucleosomes in
nonlymphoid cells, whereas all Ig gene segments become acetylated at
the pro-B cell stage and remain acetylated in pre-B cells. Since L
chain genes become acetylated in pro-B cells and yet are not rearranged
until the pre-B cell stage, acetylation of Ig genes does not seem to be
a developmental stage-specific modification, directly linked to onset
of V(D)J recombination. Rather, it appears to be a feature of loci,
which are capable of rearrangement in B lineage cells. In support of
this idea, we also find an increase in the level of acetylation of Ig
L chain J segments in both pro- and pre-B cell precursors (data not
shown). Furthermore, McMurry and Krangel (39) have
recently reported that histone H3 acetylation at the TCR
locus is
increased in T cell precursors. However, in this study, stage-specific
changes were observed, perhaps reflecting differences in H3 and H4
acetylation patterns (40). Histone H4 acetylation at the
Ig loci in pro- and pre-B cells closely resembles that observed for the
-globin locus. During erythrocyte development, H4 acetylation is not
only observed at the transcriptionally active -globin gene, but also
at the previously active and at the poised -globin genes, suggesting
that this modification is related to transcriptional competence rather
than transcriptional activity per se (41). Histone H4
acetylation may similarly be important in the generation of an open
chromatin conformation at the Ig loci in early B cell development, and
hence play a role in rendering Ig genes competent for V(D)J
recombination.
Additional stage-specific modifications in Ig chromatin structure
appear to take place during B cell development. The experiments
described in this work show an increase in the DNase I sensitivity of
VH and JH segments in pro-B
cells, whereas V and J segments at the recombinationally inactive
locus remain relatively resistant to nuclease digestion. Furthermore,
we find that the pro-B to pre-B cell transition is marked by an
increase in the sensitivity of V and J segments to DNase I, and a
reciprocal decrease in the nuclease sensitivity of
VH, but not JH gene
segments. These alterations in V and J segment chromatin coincide
directly with in vitro RSS cleavage observed in equivalent
RAG-deficient pro-B and pre-B cell nuclei (15). Thus,
VHJ558 and JH RSS are
cleaved by RAG in nuclei of pro-B cells, while J segments become
susceptible to RAG-mediated cleavage in µ+
pre-B cells. In pre-B cells, the JH segments are
also susceptible to RAG cleavage, which correlates with our findings of
continued sensitivity to DNase I at this stage. In mature B cells, no
longer undergoing V(D)J rearrangement, we observed that
VH and V genes are all DNase I resistant,
whereas J and JH segments remain accessible to
nuclease digestion. Again, this correlates with findings that in nuclei
from mature B cells, both J and JH segments
are susceptible to in vitro cutting in the presence of RAG, whereas
VHJ558 RSSs are not (15). These
results therefore indicate that changes in DNase I sensitivity occur
before rearrangement at the Ig loci and are highly predictive of gene
segment accessibility to V(D)J recombinase. Our results further show
that alterations in DNase I sensitivity of both V and J segments are
associated with induction of H chain rearrangement in pro-B cells and
L chain rearrangement in pre-B cells. The finding that once
activated, J segments remain in an active chromatin conformation
suggests that control of allelic exclusion at the H chain locus largely
depends on alterations in chromatin that result in selective
inaccessibility of VH segment genes. Taken
together, these results support the idea that alterations in V gene
chromatin play an important role in the control of V(D)J
recombination.
Demethylation of Ig H and L chain genes has previously been
reported to coincide with onset of rearrangement in B cell precursors
and to persist through the latter stages of B cell development
(16, 17, 19). As previously reported by Bergman and
colleagues (20), we find that demethylation of J
segments takes place in the absence of V(D)J recombination in bone
marrow pre-B cell precursors derived from
RAG2-/- x Igµ mice (data not shown). We have
extended these findings to the JH locus, which is
demethylated in both RAG-deficient pro-B and pre-B cell precursors.
These results strengthen the idea that demethylation occurs before
V(D)J recombination and is involved in derepression of Ig loci during B
cell development. The Abelson cell lines used in this study have, in
addition, allowed us to show that changes in histone H4 acetylation and
DNase I sensitivity do not require prior DNA demethylation. In the LC2
and HC8 Abelson-transformed pre-B cell lines, the J segments are not
demethylated (data not shown), probably due to sequestration of NF-B
in the cytoplasm (42, 43). Nevertheless, there is an
increase in histone H4 acetylation and DNase I sensitivity at the
locus in these cells. Furthermore, LPS treatment of pro-B cells causes
an increase in DNase I sensitivity, but not demethylation of J
segments, again indicating that methylation and nuclease sensitivity
can be uncoupled. These results are consistent with the idea that
demethylation may be a late step in the activation of the Ig loci.
Demethylation would therefore reflect prior chromatin changes and hence
a fully activated locus and may be important in targeting of
recombination factors to the individual Ig alleles, as originally
suggested by Mostoslavsky et al. (20).
It has recently been shown that packaging of RSSs into nucleosomes inhibits V(D)J cleavage in vitro (13, 14) as well as recombination of episomal substrates in vivo (44). This suggests that nonrearranging Ig loci might exist within a repressive nucleosomal array, and that activation of V(D)J recombination would require either removal or displacement of nucleosomes in B cell precursors. To address this question, we have investigated nucleosomal structure of endogenous H chain J segments. Our data show that JH segments are packaged within an ordered nucleosomal structure in both B cell precursors and nonlymphoid cells. However, as judged by MNase digestion profiles, there is a different nucleosomal organization over this region in nonlymphoid cells, where the IgH locus is refractory to V(D)J recombination, and in B cell precursors, where the JH segments are accessible to RAG-mediated cleavage. Higher resolution mapping will be required to determine the precise position of nucleosomes, but our results suggest that actively rearranging gene segments are packaged in a modified nucleosomal structure, rather than being totally devoid of nucleosomes. Interestingly, both pro-B and pre-B cells were found to have the same nucleosomal structure. This suggests that a reorganization of positioned nucleosomes over the JH segments occurs during early B cell development and is maintained at the pre-B cell stage, despite the fact that µ+ pre-B cells are submitted to heavy chain gene allelic exclusion. These results, along with those of DNase I sensitivity analysis, indicate that JH locus remains in an active chromatin conformation in pre-B cells and that inhibition of H chain rearrangement does not involve changes in JH segments.
The experiments described in this work provide a systematic analysis of Ig chromatin structure in a RAG-deficient background and clearly show that chromatin remodeling precedes V(D)J recombination at the endogenous Ig loci. To obtain sufficient numbers of cells to perform the chromatin assays, we used RAG-deficient Abelson pro-B and pre-B cell lines rather than bone marrow B cell precursors. Although the Abelson virus tyrosine kinase can interfere with signal transduction pathways, RAG-deficient Abelson cell lines have been used in prior studies and have been shown to undergo appropriate developmentally regulated changes in Ig structure, as assessed by RAG cleavage assays and initiation of germline transcription (15, 24, 45).We analyzed several different cell lines for each B cell stage and observed consistent results for cell lines at the same stage of development, as well as significant differences between the pro-B and pre-B cells. This strongly suggests that the changes observed are not due to the Abelson virus, but truly represent developmentally regulated modifications in chromatin.
The question then becomes, how is chromatin remodeling at the Ig loci
controlled? Targeting of chromatin-modifying complexes to specific
genes can occur through interactions with sequence-specific DNA-binding
proteins (8). Several transcription factors implicated in
the regulation of B cell development and Ig gene rearrangement have
recently been found to interact with remodeling complexes
(46, 47, 48, 49, 50, 51). A role of transcription factors in the targeting
of chromatin-modifying complexes is consistent with the finding that
cis-acting sequences that control transcription at the Ig
loci, notably enhancers, also mediate regulation of V(D)J recombination
(for review, see Ref. 3). To investigate the contribution
of transcription factors in chromatin remodeling at the Ig loci, we
treated pro-B cell lines with LPS, which causes a rapid increase in
nuclear NF-B and induction of gene rearrangement. We found that
LPS increases the DNase I sensitivity of J segments and therefore is
capable of causing alterations in gene chromatin. However, LPS
stimulation does not totally mimic developmentally regulated changes in
chromatin structure, since the J locus was not demethylated (data
not shown) and no change in V chromatin was observed. LPS therefore
promotes only limited changes in chromatin structure, probably via
binding of NF-B to the intronic enhancer, suggesting the
presence of additional regulatory elements associated with Ig V
segments. Similarly, Ferrier and coworkers (52) recently
reported that the TCR enhancer activity is confined to the D-J
region. An increase in histone acetylation is one of the earliest
changes observed at the Ig loci during B cell differentiation, raising
the question of whether histone acetylation is sufficient to induce
subsequent chromatin modifications. Previous experiments with the
deacetylase inhibitor, TSA, have shown that increasing histone
acetylation can relieve methylation-induced inhibition of gene activity
and remodel nucleosomes for transfected or microinjected constructs
(53). Furthermore, TSA treatment has recently been found
to increase V(D)J rearrangement of a premethylated episomal
recombination substrate and V-J rearrangements in a pre-B cell
line (44, 54). We, however, observed no obvious effects of
TSA on either nuclease sensitivity or reconfiguration of
JH-associated nucleosomes. In addition, TSA
treatment did not induce demethylation of JH and
J segments (data not shown). Histone acetylation alone is therefore
not sufficient to promote activation of the endogenous Ig loci. These
results are consistent with recent findings that endogenous methylated
genes cannot be activated by TSA treatment alone (55, 56).
Chromatin remodeling complexes may therefore act upstream of histone
acetylase-containing complexes at the Ig loci, as previously described
for the yeast HO promoter (57), or else nucleosome
remodeling and histone acetylation may occur by independent pathways.
Indeed, Kwon et al. (58) have shown that both the
mating-type switching/sucrose nonfermenting remodeling complex and
acetylation of histones are required to counter the repressive effects
of nucleosomes on V(D)J cleavage in vitro.
Our results suggest that in uncommitted lymphoid precursors or non-B
lineage cells, the Ig loci are highly methylated, nuclease insensitive,
and associated with hypoacetylated histones. This stably repressed
conformation may be due to targeting of histone deacetylase-containing
complexes, by lymphoid lineage-determining factors, such as Ikaros
family members (50, 51, 59). Repression may also be
generated or maintained by interaction of methyl-CpG-binding proteins
with the methylated Ig loci (11, 60). Commitment to the B
cell lineage is associated with histone acetylation and nucleosome
repositioning at the Ig loci, presumably due to recruitment of
ATP-dependent remodeling complexes and histone acetyltransferase
activity in early B cell precursors. The B lineage-determining
transcription factor, E2A, is a good candidate for recruitment of
histone acetyltransferases to the Ig loci since it binds to target
sites and activates V(D)J recombination at both H and L chain loci
(61) and has been shown to interact with the
Spt-Ada-Gen5-acetyltransferase and p300 acetyltransferase-containing
complexes (48, 49). Locus-specific activation, leading to
increased nuclease accessibility, probably requires the interaction of
a second set of site-specific DNA-binding proteins, such as NF-B,
which increases DNase I sensitivity of J locus. Demethylation may be
the final step in locus activation, allowing access of V(D)J
recombination factors to Ig gene segments. Future studies on the
interaction of chromatin-modifying complexes with Ig sequences both in
vivo and in vitro should provide a better understanding of the
molecular basis of the chromatin modifications at the Ig loci and hence
regulation of V(D)J recombination.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michele Goodhardt, Unité de Génétique et Biochimie du Développement, Unité de Recherche Associée, Centre National de la Recherche Scientifique 1960, Département d’Immunologie, Institut Pasteur, 75724 Paris, France. E-mail address: migood{at}pasteur.fr
3 Abbreviations used in this paper: RAG, recombination-activating gene; KAP, kidney androgen-regulated protein; MNase, micrococcal nuclease; RSS, recombination signal sequence; TSA, trichostatin A.
Accepted for publication May 15, 2001.
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