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Chromatin Associated with TCR Allelic Exclusion1
Department of Immunology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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rearrangement and
allelic exclusion, we analyzed TCR chromatin structure in double
negative (DN) thymocytes, which are permissive for TCR
recombination, and in double positive (DP) thymocytes, which are
postallelic exclusion and nonpermissive for V to DJ
recombination. Histone acetylation mapping and DNase I sensitivity
studies indicate V and DJ segments to be hyperacetylated and
accessible in DN thymocytes. However, they are separated from each
other by hypoacetylated and inaccessible trypsinogen chromatin. The
transition from DN to DP is accompanied by selective down-regulation of
V acetylation and accessibility. The level of DP acetylation and
accessibility is minimal for five of six V segments studied but
remains substantial for one. Hence, the observed changes in V
chromatin structure appear sufficient to account for allelic exclusion
of many V segments. They may contribute to, but not by themselves
fully account for, allelic exclusion of others. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Ig
) initiates subsequently, at the pre-B
cell stage, and occurs in a single step (V to
J or V to J). In similar
fashion, V(D)J recombination occurs at two distinct stages in the
development of 
T lineage cells. TCR locus rearrangement
initiates in CD4-CD8-
double negative (DN)3
thymocytes and occurs in two steps, first D to
J and then V to DJ. TCR
locus rearrangement initiates subsequently in
CD4+CD8+ double
positive (DP) thymocytes and occurs in a single step (V
to J).
A striking regulatory feature of V(D)J recombination is the phenomenon
of allelic exclusion, which restricts precursor lymphocytes to the
production of a single, functional Ag receptor gene at a given locus.
Allelic exclusion functions in highly analogous fashion at the IgH and
TCR loci. In each case, the presence of a functional VDJ
rearrangement on one allele inhibits the V to DJ step of rearrangement
on the second allele by a feedback mechanism (7, 8, 9). For
TCR , the feedback signal depends on the assembly of a TCR
polypeptide into a pre-TCR complex with pre-T and CD3, and on the
activity of the nonreceptor protein tyrosine kinase lck
(10, 11). Similarly, for IgH this signal depends on the
assembly of a membrane Igµ polypeptide into a pre-B cell receptor
(BCR) complex (12, 13). Pre-TCR and pre-BCR signaling also
induces critical developmental transitions during T and B lymphocyte
development (from DN to DP and pro-B to pre-B, respectively), as well
as the proliferative expansion of developing lymphocytes (14, 15). How pre-TCR- and pre-BCR-derived signals impact the process
of V(D)J recombination to inhibit V to DJ rearrangement at the TCR
and IgH loci is not well understood.
V(D)J recombination is initiated by the recombinase-activating gene
(RAG)-1 and RAG-2 proteins, which introduce double-strand breaks at
recombination signal sequences (RSSs) flanking Ig and TCR gene segments
(1, 2). One level at which V(D)J recombination can be
regulated is by developmental activation and inactivation of RAG gene
expression. For example, developmental inactivation of RAG gene
expression can account for the termination of V to
J rearrangement upon transition of DP thymocytes to the
single positive stage, and for termination of Ig L chain rearrangement
on transition of pre-B cells to the immature B cell stage (6, 16). However, other developmental changes in V(D)J recombination
can occur in the face of ongoing RAG gene expression and recombinase
activity. The inhibition of V to DJ and
VH to DJH rearrangement
that characterizes allelic exclusion falls into this category. Although
pre-TCR and pre-BCR signaling down-regulates RAG expression in
late-stage DN thymocytes and pro-B cells, respectively, allelic
exclusion is enforced despite the subsequent up-regulation of RAG
expression that is associated with V to J
rearrangement in DP thymocytes, and with V to
J and V to J rearrangement in
pre-B cells (6, 16). In general, developmental changes in
recombinase targeting have been attributed to developmental changes in
locus accessibility to the recombinase, with chromatin structure under
the control of specific promoters and enhancers (3, 4, 5, 6, 17, 18, 19). However, data addressing the role of chromatin
structure in the process of allelic exclusion have been limited
(20).
The TCR locus spans roughly 700 kb (21). At the 3' end
are two D-J-C clusters, as well
as a single, inverted V gene segment. The majority of
V segments are scattered across a region that extends from
250 to 500 kb upstream of the
D-J-C clusters. This
V domain is flanked on the 5' side and, remarkably, on the
3' side as well, by extended arrays of trypsinogen genes and gene
fragments. Moreover, one V segment is located upstream of
the 5' trypsinogen cluster, 
650 kb away from the
D-J-C clusters. To date, V(D)J
recombination at the TCR locus has been shown to depend on two
cis-elements. E, situated downstream of
C2, is required for all D to J
and V to DJ rearrangement events (22, 23), whereas a germline promoter (PD1), situated
upstream of D1, is required specifically for rearrangement
events involving the D1-J1 cluster (24, 25). Thus, E and PD1 cooperate to
promote rearrangement of D1 and J1 segments,
but the extent to which this regulation occurs at the level of
accessibility, vs at a later step in the reaction, is not fully
resolved (24, 25, 26, 27).
TCR allelic exclusion appears not to involve a developmental
transition involving D-J-C
chromatin and the activities of E and PD1.
First, signal ends associated with D to J
rearrangement are present even in DP thymocytes, suggesting that these
segments maintain accessibility to the recombinase at this stage
(24). Second, in RAG-/- thymocytes
induced to differentiate to the DP stage,
D-J-C chromatin maintains the
germline transcription and CpG hypomethylation that characterizes the
DN compartment (28). These results leave open the
possibility that V chromatin is regulated independently of
E, and that allelic exclusion is regulated at the level of
V chromatin. Indeed, recent data indicate that
accessibility and transcription of upstream and downstream
V segments are maintained in DN thymocytes of E-/-
mice (27). However, only limited data speak to the status
of V segments in DP thymocytes: levels of germline
transcripts for several upstream V segments (but not the
downstream V) were found to decline on transition from DN
to DP (28, 29). Thus, the notion that TCR allelic
exclusion is associated with a change in the structure of
V chromatin remains largely untested. In this study we
present a detailed comparison of TCR locus chromatin structure both
before and subsequent to allelic exclusion signals that addresses this
issue in some detail.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chromatin immunoprecipitation (CHIP) analysis was performed as
described (30), using rabbit antisera to diacetylated
histone H3 (AcH3), tetraacetylated histone H4 (Upstate
Biotechnology, Lake Placid, NY), and control rabbit IgG (Sigma-Aldrich,
St. Louis, MO). Serial 3-fold dilutions of nucleosomal DNA (20, 6.7,
and 2.2 ng) isolated from Ab bound and unbound fractions were amplified
by 25 cycles of PCR (45 s at 94°C, 1 min at 51°C, 2 min at 72°C)
in a 25-µl reaction. PCR products were electrophoresed through 1.5%
agarose gels, transferred to nylon, and detected by hybridization with
DNA fragments labeled by random priming. PCR primers used to amplify
mononucleosomal DNA or to produce probes for hybridization are listed
in Table I
. TCR locus primers were
designed with reference to GenBank files MMAE000663, MMAE000664, and
MMAE000665. Primers for analysis of Oct-2 were described
previously (30). Blots were washed in 1x SSC and 0.5%
SDS for 20 min at 23°C with one change. A single serial dilution
series produced from each DNA fraction was used for all PCR included in
a single experiment. Quantification was accomplished using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Raw acetylation
values were derived by determining the displacement between titration
curves for the bound fraction of the AcH3 immunoprecipitation and
unbound fraction of the control immunoprecipitation. Several factors
insured that PCR signals were specific. First, there is typically only
50–70% sequence homology among V coding segments and
<50% sequence homology in promoter and 3' flank regions. Second,
repetitive elements in noncoding regions were avoided. Third, our
detection strategy involved multiple steps, each imparting a degree of
specificity: an initial PCR step to generate products from
mononucleosomes, a second PCR step using one primer from the first step
and one new primer to generate an overlapping probe, and, finally,
detection of PCR products by hybridization with radiolabeled PCR
probe.
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Thymocytes were permeabilized and treated with DNase I as
described (31, 32). Purified DNA (4 µg) was restriction
digested, electrophoresed through 0.7% agarose, transferred to nylon,
and assayed by hybridization with 32P-labeled
probes generated by random priming. DNA fragments
DJ (MMAE00665 153,405–153,996), T4T5
(MMAE00663 92,771–93,461), V11 (MMAE00664
23,621–24,290), V12 (MMAE00664 14,657–15,446), and
V13 (MMAE00664 7,916–8,370) were produced by PCR.
Hybridization was quantified using a PhosphorImager.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Acetylation is structural modification of the amino-terminal tails
of core histones that is associated with an open, nuclease-sensitive
chromatin configuration (33, 34, 35, 36, 37). A variety of approaches
have recently established a very tight relationship between histone
hyperacetylation and accessibility for V(D)J recombination (18, 19, 27, 30, 38, 39, 40, 41, 42). Based on these results, we used acetylation
mapping in the current study to evaluate changes in the structure of
TCR locus chromatin that might be associated with TCR allelic
exclusion.
We used a CHIP assay to measure the acetylation status of TCR locus
chromatin before and after allelic exclusion. As a source of
nonallelically excluded chromatin we used thymocytes of
RAG-/- mice (43). These thymocytes
are primarily DNIII, a stage in which V to DJ
rearrangement is normally permitted. However, an allelic exclusion
signal cannot be generated in these mice due to the lack of TCR
rearrangement and pre-TCR expression on the
RAG-/- background. As a source of allelically
excluded chromatin we used thymocytes of RAG-/-
mice complemented with a rearranged TCR transgene
(44). These thymocytes are almost exclusively DP and would
already have received an allelic exclusion signal from the TCR
-containing pre-TCR at the DN stage. The lack of V(D)J recombination
on the RAG-/- and
RAG-/- x TCR backgrounds ensures that
endogenous TCR gene segments will be uniformly in germline
configuration and therefore directly comparable in both chromatin
preparations. Mononucleosomes prepared from
RAG-/- and RAG-/- x
TCR chromatin were immunoprecipitated with anti-AcH3 or a
control serum, and PCR was used to assess the representation of
particular segments of TCR locus chromatin in equivalent quantities
of DNA isolated from the Ab bound and unbound fractions.
Structure of TCR locus chromatin in DN thymocytes
Because the DNIII compartment is permissive for V to
DJ rearrangement, both V
chromatin and DJ chromatin were expected to
be in an open or accessible configuration in this compartment. To assay
V chromatin, we initially analyzed a series of sites
spanning 40 kb of the V locus, including five functional
V segments, that is situated 400 kb upstream of
E (Fig. 1). Within this
region, we assayed the conserved decamer sequence (45, 46)
in several V promoters (V12P,
V11P, V9P), the RSS elements flanking
V segments (V13R, V12R,
V11R, V9R, V6R), and several
intergenic sites located between V segments. To assay
DJ chromatin we analyzed a site in
PD1 (47, 48), just upstream of the
D1 gene segment. Acetylation at these sites was compared
with that of Oct-2 as a negative control not expressed in
thymocytes (30), and to CD3
as a positive control
expressed at high levels in thymocytes. All TCR locus sites were
chosen so as to exclude the TCR transgene from analysis.
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gene and a hypoacetylated
Oct-2 gene. With respect to the TCR locus, all sites
assayed in both V and DJ
chromatin displayed levels of acetylation significantly elevated over
that of Oct-2 (Fig. 2). Hence,
both V and DJ chromatin display
characteristics of active chromatin in DN thymocytes, a finding
consistent with a permissive V(D)J recombination phenotype and with the
results of previous studies (27, 28). A striking feature
of V chromatin is the dramatic variation in H3 acetylation
across the region analyzed. Acetylation at individual V
segments varies widely, with acetylation at V11 10-fold
higher than at V12. Moreover, acetylation oscillates along
V chromatin, with levels locally elevated over
V promoters and RSSs and reduced, although still above
background, at positions between V segments.
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segments, we analyzed a series of sites that extended
upstream and downstream of V9 but nevertheless remained
quite distant from flanking V segments (Fig. 3A). We found acetylation to
be elevated over a relatively broad region extending from at least 1 kb
upstream of the promoter to 1 kb downstream of the RSS.
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locus chromatin structure in the
DN compartment, we analyzed acetylation within the trypsinogen regions
that flank the V cluster on both its 5' and 3' ends (Figs. 1
and 3B). T20 in the 3' trypsinogen region is known to
encode preprotrypsin and to be expressed in pancreatic acinar cells
(49), but we are unaware of expression data for other
murine trypsinogen genes. Our analysis revealed the majority of sites
assayed in both trypsinogen clusters to display H3 acetylation
essentially equivalent to that of the negative control Oct-2
gene. The only exception was the T1 gene at the distal end of the 5'
cluster, which displayed H3 acetylation that was slightly above
background. Nevertheless, acetylation at T1 was clearly lower than at
any site assayed in either V or
DJ chromatin. Thus, we conclude that in
contrast to the V and DJ
regions, the 5' and 3' trypsinogen clusters have characteristics of
inactive chromatin. Consistent with this, we failed to detect thymic
expression of T1 and T4 by Northern blot (data not shown).
V2 is isolated from all other V segments at
the extreme 5' end of the TCR locus, upstream of the 5' trypsinogen
cluster (Fig. 1). It was therefore of interest to characterize the
structure of V2 chromatin. As for V segments
in the main cluster, V2 was found to be hyperacetylated in
DN thymocytes (Fig. 4A). We
did not analyze V14, which is isolated at the 3' end of
the locus, because we could not distinguish endogenous V14
from a copy contained in the TCR transgene used to induce the DN to
DP transition.
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locus chromatin structure in DN and DP
thymocytes
To determine whether a change in TCR locus chromatin structure
accompanies allelic exclusion, we compared H3 acetylation along
V and DJ chromatin in DN and DP
thymocytes (Fig. 2). We found the control Oct-2 gene to be
equivalently hypoacetylated in the DN and DP compartments, confirming
the DN and DP samples to be comparable. CD3 acetylation was 3-fold
higher in DP thymocytes than in DN thymocytes. With respect to the TCR
locus, we found acetylation at PD1 to be nearly twice
as high in the DP as the DN compartment. Thus
DJ chromatin appears to be active in both
thymocyte populations, consistent with inferences drawn from previous
studies (24, 28). However, quite different results were
obtained for V chromatin. At all points assayed, including
both V segments and sites between V segments,
H3 acetylation was lower in the DP compartment than in the DN
compartment. This drop was in the 82–92% range for most
V segments but was as little as 54% for V11.
Importantly, these reductions occur despite elevated acetylation of
PD1 in the same samples. Hence, there is a striking and
highly selective change in the structure of V chromatin
that accompanies allelic exclusion. Similar results were obtained when
many of the same sites were analyzed in independent experiments (Fig. 4A and data not shown).
Comparison of TCR locus chromatin structure in thymocytes and
non-T cells
Although acetylation at V11 was reduced in DP as
compared with DN thymocytes, acetylation in the DP compartment was
still substantial (Figs. 2 and 4A). To evaluate the
significance of this observation, we compared acetylation in DN and DP
cells to that in non-T cells at selected sites across the locus (Fig. 4). As a source of non-T cells, we examined splenocytes of either
C-/-C
-/- mice (Fig. 4A) or
C-/- mice (Fig. 4B), both of which lack
T cells (50, 51). These splenocytes consist
primarily of B cells and macrophages and should retain the entire
V locus in germline configuration. To insure equivalence
of the DN, DP, and non-T cell samples, we analyzed acetylation at a
site in -actin (data not shown) and normalized the acetylation
values for TCR locus chromatin on the basis of the -actin data.
The validity of this approach was confirmed by the equivalent levels of
histone H3 (Fig. 4A) and H4 (Fig. 4B) acetylation
calculated for sites within trypsinogen chromatin, which appears to be
inactive in all three cell populations.
Analysis of histone H3 (Fig. 4A) and H4 (Fig. 4B)
acetylation in non-T cells revealed the TCR locus acetylation
profiles to be largely T cell-specific. Strikingly, the residual H3
acetylation at V11 in DP cells was found to be still
elevated with respect to non-T cells. A similar result was obtained for
V9, although the level of residual DP acetylation was much
lower in this case. Analysis of H4 acetylation yielded a similar
picture. At two sites associated with V segments and two
intergenic sites, H4 acetylation was substantially reduced in DP
relative to DN thymocytes. However, the reductions in H4 acetylation
were not as great as those for H3. Moreover, for V13 and
V9, H4 acetylation in the DP compartment was somewhat
elevated in DP cells as compared with non-T cells. We conclude that
V11, in particular, appears to be in an intermediate
rather than a fully repressed state in DP thymocytes. Several other
V segments may display low-level activity as
well.
One exception to the T cell specificity of the TCR locus
acetylation patterns was V2. As for most other
V segments, H3 acetylation at this site was dramatically
down-regulated on transition from DN to DP. However, V2
acetylation was slightly higher in non-T cells than in DP thymocytes
(Fig. 4A). Perhaps consistent with this, recent data
indicated that V2 displays promiscuous activity in other
lineages, as V2 germline transcripts were identified in NK
cells and in a myeloid-enriched population of bone marrow cells
(52).
DNase I sensitivity of TCR locus chromatin in DN and DP
thymocytes
To understand whether the above documented structural transition
in V chromatin reflects a functionally relevant difference
in V segment accessibility, we directly probed the
accessibility of TCR locus chromatin by measuring its general
sensitivity to DNase I digestion (Fig. 5, A–F). Thymocytes were mildly permeabilized with
detergent and incubated briefly with increasing concentrations of DNase
I, following which genomic DNA was purified, digested with restriction
enzymes, and analyzed by Southern blot. To minimize artifactual
differences in sensitivity to DNase I digestion, we chose to compare
fragments that avoided known promoters and thereby avoided potential
hypersensitive sites, that were of similar size and therefore provided
similar targets for DNase I digestion, and that were analyzed on a
single blot. Digest and probe combinations were chosen so as to exclude
the TCR transgene from analysis.
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J, and V12 chromatin in DP
thymocytes was obtained by probing simultaneously for all three
sequences (Fig. 5A). Trypsinogen chromatin was revealed to
be relatively resistant to DNase I digestion and
DJ chromatin relatively sensitive, consistent
with their quite distinct acetylation profiles in the DP compartment.
By this criterion, trypsinogen chromatin is inaccessible and
DJ chromatin is accessible in DP thymocytes.
Because V12 chromatin displays DNase I sensitivity similar
to that of trypsinogen chromatin and distinct from that of
DJ chromatin, it appears to be inaccessible
in DP thymocytes as well.
For additional comparative analyses that eliminated DNA loading as a
variable, hybridization signals for trypsinogen chromatin were
normalized to 100% at all DNase I concentrations, and residual
hybridization signals for other fragments were expressed relative to
the trypsinogen signal at each point (Fig. 5, B–F). In comparing DN to DP thymocytes, we found
that DP thymocytes routinely require less DNase I than DN thymocytes to
produce equivalent digests of bulk genomic DNA (data not shown). This
may result from increased intrinsic fragility and permeability of DP
thymocytes to DNase I or increased sensitivity to detergent
permeabilization. Concentrations of DNase I that produced equivalent
digests of bulk DNA revealed DJ chromatin to
have roughly similar sensitivity to DNase I digestion, relative to
trypsinogen chromatin, in the two compartments (Fig. 5, compare
B to C and D to E).
However, relative to trypsinogen and DJ
chromatin, the DNase I sensitivity of sites within V
chromatin was found to vary between DN and DP thymocytes.
V12 and V13 display sensitivities in the DN
compartment that are nearly equivalent to that of
DJ chromatin, but they display sensitivities
in the DP compartment that are much more like that of trypsinogen
chromatin (Fig. 5, B and C). Thus, both
V segments are highly accessible in DN thymocytes, with
V12 accessibility slightly less than that of
V13. V12 is converted to an inaccessible
configuration in DP thymocytes, whereas V13 displays
residual, albeit much diminished, accessibility.
V11 is notable for its unusually high level of acetylation
in DP thymocytes (Figs. 2 and 4). DNase I analysis revealed
V11 to be highly accessible in DN thymocytes and to
display only a modest reduction in accessibility on transition to DP
(Fig. 5, D and E). Strikingly, although
V11 is significantly more accessible than trypsinogen
chromatin in DP thymocytes, it is as inaccessible as trypsinogen
chromatin in non T cells (Fig. 5, E and F).
Overall, the conclusions drawn from DNase I sensitivity analysis of
V11, V12, and V13 match well
with the conclusions drawn from acetylation mapping.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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locus rearrangement and
allelic exclusion, we have analyzed the structure of TCR locus
chromatin in DN thymocytes, a stage that is permissive for TCR
recombination, and in DP thymocytes, a stage which is postallelic
exclusion and nonpermissive for V to
DJ recombination. Our results are summarized
schematically in Fig. 6. We find that
both V segments and DJ segments
reside in accessible chromatin in the DN compartment but that
accessible chromatin domains are separated from each other by large
stretches of inaccessible trypsinogen chromatin. The transition to DP
occurs without any reduction in accessibility of
DJ chromatin but is accompanied by conversion
of V chromatin to a less-accessible configuration. Of
note, this conversion is not uniform for all V segments.
Thus, V12 and V13 undergo rather dramatic
changes in accessibility as defined by DNase I sensitivity. Based on
the observed parallels between the acetylation and accessibility
transitions of these V segments, we predict that
V2, V6, and V9, which were only
studied at the level of acetylation, undergo dramatic changes in
accessibility as well. In contrast, the accessibility change at
V11 is rather mild, and significant V11
accessibility is maintained in DP thymocytes. Thus we document changes
in V chromatin structure that appear sufficient to account
for allelic exclusion of several V segments and that may
contribute to, but may not by themselves fully account for, allelic
exclusion of V11.
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chromatin structure is controlled. Although
E and PD1 are critical regulators of
DJC chromatin, these elements
appear not to play significant roles as regulators of V
chromatin (24, 25, 27). V chromatin could
theoretically be regulated by the promoters of individual
V segments, by some form of global regulation that affects
the entire V cluster or by interactions between individual
V promoters and a global regulator. Two aspects of the
acetylation pattern across the V cluster are worth
emphasizing in this regard. First, because acetylation in DN thymocytes
is elevated over the level observed for inactive chromatin at both
V segments and intergenic sites, it can be deduced that
there is a global change in structure that affects the entire
V domain. Second, because acetylation is locally elevated
over V segments and at intermediate levels between
V segments, there is evidence of local influence that is
superimposed on the global influence. In fact, the pattern of
acetylation across V chromatin is reminiscent of that
across the human -globin locus (37). In this case,
acetylation was found to be induced relatively evenly across the locus
under the control of an undefined global regulator and to be further
elevated over individual transcription units in association with locus
control region-dependent promoter activation. As in the
-globin locus, the compound acetylation pattern detected in our
experiments seems compatible with the effects of both local and long
distance regulators.
That locally elevated acetylation over individual
V segments might reflect V promoter function
begs the question of the relationship between acetylation levels and
transcriptional activity. We note that although CD3 and
V11 are both heavily acetylated in DN thymocytes, CD3
transcripts are detectable by Northern blot, whereas V11
transcripts are not (data not shown). Moreover, although we find
substantial acetylation of V6 in DN thymocytes,
V6 transcripts were not detected by RT-PCR
(52). Although suggestive that acetylation and
transcription do not correlate, an important caveat is that these
approaches measure steady state levels of V transcripts
rather than rates of V transcription. Nevertheless,
discordance between acetylation and transcription is certainly
possible, because recruitment of histone acetyltransferases to
promoters can precede, and can be experimentally segregated from,
transcriptional activity per se (53, 54). Additional work
will be required to clarify this issue.
An important finding of ours is the down-regulated but
nevertheless heterogeneous nature of V accessibility in
the DP compartment. All V segments analyzed displayed
reduced accessibility in the DP as compared with the DN compartment.
Nevertheless, whereas most analyzed V segments (i.e.,
V12, V13, and, we predict, V2,
V6, and V9 as well) become either
inaccessible or nearly so, V11 clearly displays residual
accessibility, at least as defined by sensitivity to DNase I digestion.
Thus, whereas the changes in chromatin structure at V2,
V6, V9, V12, and
V13 have the potential to account for allelic exclusion of
these gene segments, it is unclear whether the change at
V11 is sufficient to account for its allelic exclusion. We
considered the possibility that allelic exclusion might not be complete
for V11, but V11 was shown to be efficiently
allelically excluded in previous work (11).
An important issue with respect to V11 is how accurately
measures of general accessibility within chromatin (i.e., acetylation,
DNase I sensitivity) predict the frequency of RAG cleavage. Although
acetylation seems to correlate well with accessibility for V(D)J
recombination in numerous instances, another chromatin modification
(55, 56) could be most relevant for accessibility to RAG.
In addition, RSS positioning with respect to the underlying nucleosome
can significantly impact RAG access and cleavage (41, 57).
We note as well that accessibility is clearly not the only factor
governing RAG cleavage in chromatin. Sequence variation among
natural RSS heptamer and nonamers (58, 59), spacer regions
(60), and coding flanks (61, 62, 63, 64) can
significantly impact rearrangement frequencies as well. It is notable
that V12, which is inaccessible in the DP compartment,
carries a nonamer (GCAAAAACA) that diverges from the consensus
(ACAAAAACC) at only the end positions. In contrast, V13,
which displays low DP accessibility, and V11, which
displays substantial DP accessibility, carry more divergent nonamers
(GCACAAAGC and GCAAGAAAC, respectively), with substitutions at
positions known to significantly inhibit recombination in test
substrates (65, 66). Similarly, V12 has a
permissive C nucleotide immediately 5' of the heptamer, whereas
V13 has a T nucleotide that in some studies appears to
inhibit recombination (61, 64). Thus, chromatin
accessibility, as defined here, may integrate with factors like RSS
positioning with respect to nucleosomes and with variation in RSS or
flanking sequences to equalize V segments as recombination
substrates in DP cells. In this manner, V11 could have a
frequency of RAG cleavage in DP cells that is essentially equivalent to
that of V12, despite the difference in general chromatin
accessibility.
As a cautionary note, we point out that the fully germline TCR
locus alleles analyzed in our study are not identical to the
DJ rearranged alleles that are the
physiological substrates for allelic exclusion. Fully down-regulated
V accessibility could conceivably require prior
D to J rearrangement, a possibility we cannot
exclude at the present time.
The notion that TCR allelic exclusion is enforced primarily at the
level of V segment accessibility would be consistent with prior
analyses of the IgH locus, which demonstrated VH
RSSs to serve as substrates for exogenously introduced RAG in isolated
nuclei of pro-B cells but not mature B cells (20), and
demonstrated VH segments to display greater sensitivity to
DNase I digestion in pro-B cells as compared with pre-B or mature T
cells (67). Taken together, these studies support the
existence of a general mechanism for the allelic exclusion process.
Nevertheless, given the ambiguous data for V11, we cannot
exclude the possibility thatallelic exclusion is controlled,
at least in part, by a non-chromatinbased mechanism that
inhibits V to DJ rearrangement
on an allele in which V and DJ
segments retain some degree of accessibility to RAG. How this might
occur is unclear, but one possibility is suggested by recent data
demonstrating that V rearrangement can be mediated by the
12-bp RSS 5' of D1, but not those associated with
J segments (68, 69). This raises the
possibility of a coupling factor for V to
DJ rearrangement that might itself be
developmentally regulated. In summary, we have demonstrated clear
structural and functional changes in V chromatin in
thymocytes undergoing TCR allelic exclusion. Additional studies
will be required to carefully evaluate the relative roles of
chromatin-based and non-chromatin-based regulation in this process.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael S. Krangel, Department of Immunology, Duke University Medical Center, PO Box 3010, Durham, NC 27710. E-mail address: krang001{at}mc.duke.edu
3 Abbreviations used in this paper: DN, double negative; DP, double positive; AcH3, anti-diacetylated histone H3; BCR, B cell receptor; CHIP, chromatin immunoprecipitation; RAG, recombinase-activating gene; RSS, recombination signal sequence; B, bound; U, unbound.
Received for publication November 9, 2001. Accepted for publication December 21, 2001.
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, Gene segments; 
, RSSs; •,
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