VDJ recombination of T cell receptor and immunoglobulin loci occurs in immature lymphoid
cells. Although the molecular mechanisms of DNA cleavage and ligation have become more
clear, it is not understood what controls which target loci undergo rearrangement. In interleukin 7 receptor (IL-7R)
/ murine thymocytes, it has been shown that rearrangement of the T
cell receptor (TCR)-
locus is virtually abrogated, whereas other rearranging loci are less severely affected. By examining different strains of mice with targeted mutations, we now observe that the signaling pathway leading from IL-7R to rearrangement of the TCR- locus
requires the c receptor chain and the c-associated Janus kinase Jak3. Production of sterile
transcripts from the TCR- locus, a process that generally precedes rearrangement of a locus,
was greatly repressed in IL-7R/ thymocytes. The repressed transcription was not due to a
lack in transcription factors since the three transcription factors known to regulate this locus
were readily detected in IL-7R/ thymocytes. Instead, the TCR- locus was shown to be
methylated in IL-7R/ thymocytes. Treatment of IL-7R/ precursor T cells with the
specific histone deacetylase inhibitor trichostatin A released the block of TCR- gene rearrangement. This data supports the model that IL-7R promotes TCR- gene rearrangement by
regulating accessibility of the locus via demethylation and histone acetylation of the locus.
Key words:
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Introduction |
Signals from the IL-7 receptor are critical for early stages
in development of several of the lymphoid lineages (1,
2). Thus, IL-7R/ mice produce very few /
T cells and
B cells, and the /
T cell lineage is even more severely repressed (3, 4). The IL-7R signal triggers at least two types
of responses in lymphoid precursor cells (for review see reference 5). One signal is for survival of pro-T cells and is associated with the level of bcl-2 in the cells (6); thus, the / T
cell deficiency in IL-7R/ mice can be partly ameliorated
by a bcl-2 transgene (7, 8). The second signal from IL-7 receptor promotes VDJ recombination at several loci, including TCR- (9, 10), IgH (11, 12), and the TCR- locus (13,
14); of these loci, rearrangement of the TCR- locus is the
most severely repressed in IL-7R/ mice.
VDJ recombination is a stringently regulated event that is
restricted (with few exceptions) to certain early stages in
lymphoid development. The mechanisms of this strict regulation are not fully understood. However, several types of
controls are identified or presumed. One level of control is
expression of the recombinase components, RAG1 and -2, which mediate cleavage of target gene segments (15). Thus,
expression of the RAG genes is restricted to early lymphoid
cells. On the other hand, religation of the target locus involves components that are not restricted to the lymphoid
lineage; these include Ku, p350 kinase catalytic subunit, XRCC4, and DNA ligase (for review see references 16, 17). IL-7 has been shown to promote the expression of RAG1
and -2 in pro-T cells (9, 18). Moreover, IL-7R/ mice
showed suppressed expression of the Rag genes in pro-T
cells, whereas the later stage (CD4+CD8+) expressed Rag
genes normally, indicating that after the pro-T cell stage expression of the Rag genes becomes IL-7R independent (10).
A second control of VDJ recombination governs whether
a locus is accessible to cleavage by the Rag proteins (for
review see references 5, 16). This control is necessary because the motifs recognized by the Rag proteins are similar
in all rearranging loci. Since different cell types rearrange
different loci, there is presumed to be a mechanism governing accessibility of the locus. For example, pro-T cells rearrange the TCR-, -, and - loci at about the same time,
do not fully rearrange the immunoglobulin loci, and the
TCR- locus is rearranged at a later stage. Little is understood of the process rendering a locus accessible to recombination. The enhancer of a locus, normally defined based
on its ability to promote transcription, also can be involved
in promoting rearrangement of a locus. This is thought to
account for the observation that a given locus generally
produces sterile transcripts before it undergoes rearrangement. Deletion of the respective enhancers abolishes rearrangement of the TCR- locus (19, 20), and greatly suppresses rearrangement of the IgH locus (21), the Ig
locus (22), and the TCR- locus (23). Transcription of a gene is not required for its rearrangement (24), so the enhancer is presumed to play a role in remodeling chromatin structure,
rendering nearby regions accessible to both recombination
and transcriptional machinery.
In this study, we investigated the signaling mechanism by
which IL-7R promotes rearrangement of the TCR- locus.
There are two ligands for the murine IL-7R chain, IL-7 and
thymic stromal-derived lymphopoietin (TSLP)1 (25). IL-7 signaling involves pairing of the IL-7R chain with the c chain
(26, 27), whereas TSLP signaling is thought to involve pairing of the IL-7R chain with a different chain (2); for this
reason we examined the role of c in signaling rearrangement of the TCR- locus. There are several tyrosine kinases activated by IL-7R (28); we examined the role of one of
these, the Janus kinase Jak3 (31, 32), which is associated with
c (33). In B cells, it has also been shown that phosphatidylinositol 3 (PI3) kinase is activated by IL-7 and is involved
in triggering proliferation; however, we have not found a requirement for PI3 kinase in IL-7 signaling in pro-T cells (6).
We also examined whether the TCR- locus produces sterile
transcripts and test for several transcription factors implicated
in activating the TCR- enhancer. We examined whether
IL-7R signals could induce demethylation of the TCR- locus, which could influence actetylation of chromatin. Finally,
we circumvented the need for the IL-7R signal by the use of
the specific deacetylase inhibitor trichostatin A (TSA), showing that it promotes TCR- gene rearrangement in vitro.
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Materials and Methods |
Mice.
Embryonic thymus was obtained by performing timed
breeding of C57BL/6 mice maintained at Animal Production
(Frederick, MD). IL-7R/ mice (1) and Rag2/ mice (36)
were produced in the NCI facility (Frederick, MD) from breeders purchased from The Jackson Laboratory (Bar Harbor, ME).
c/ mice (37) were bred in the National Institutes of Health
(NIH) facility (Bethesda, MD). Jak3/ mice (38) were bred at
the University of Massachusetts Medical Center (Worcester,
MA). Animal care was provided in accordance with the procedures outlined in the "Guide for the Care and Use of Laboratory
Animals" (NIH Publication No. 86-23, 1985).
PCR Analysis.
DNA was extracted (39) from adult thymi of
the indicated strain or fetal C57Bl/6 thymus from day 15 of gestation. In the case of fetal Rag2/ or IL-7R/, several thymi
were pooled, whereas all other thymi were individually treated.
Approximately 1 µg of DNA was used for each PCR reaction in
50 µl reaction buffer (PE Applied Biosystems, Inc., Foster City,
CA) with 0.2 mM of each NTP, 1 µM of each primer, and 1 U of
Taq polymerase. The PCR reaction was performed with a hot start: (94°C for 3 min) 94°C for 1 min, 55°C for 1 min, 72°C for 1 min for 30 cycles (extension, 10 min at 72°C). RNA was extracted with RNAzol according to the manufacturer's protocol
and reverse transcribed as described with oligo(dT) primers (9).
PCR was performed as for genomic DNA with the exemption
that the annealing time was 2 min at 55°C. PCR products were
separated on a 1.4% agarose gel, blotted, and pre-hybridized (Nycomed Amersham, Arlington Heights, IL) for 1 h before addition
of the -ATP 5' end-labeled probe. After 12-16 h of hybridization at 42°C, blots were washed according to the manufacturer's
protocol (Nycomed Amersham) and visualized by autoradiography. In Fig. 3 (see Results), the PCR product has not been blotted, but rather is the negative image of an original ethidium bromide-stained agarose gel. The location of primers within the
TCR- locus are shown in Fig. 1 (see Results). The primers for
detection of DNA gene rearrangement were derived from sequences as previously published (40). The primers for detection
of sterile transcripts have been published previously (41), as have
the primers for detection of GAPDH mRNA (42). The following
oligonucleotides were used (the bp number in parentheses indicates the precise position in the locus as reported in EMBL/GenBank/DDBJ under accession No. AF037352). No. 1, V2 leader
peptide sense (8,280 bp): 5'-CTGGGAATTCAACCTGGCAGATG-3'; No. 2, V2 sense (8,460 bp): 5'-CATGGGAAGTTGGAGCAACCTGAAATATC-3'; No. 3, V2 antisense (8,734 bp): 5'-GCTTCGTCTTCTTCCTCCAAGGAATA-3'; No. 4, V2-3a'antisense (8,846 bp): 5'-GCTAAGAAGGATGTGGGTTG-3', probe for V2 (8,586 bp): 5'-AACCAAGGTTTAGAGTTTCTATTA-3'; No. 5, V3 leader peptide sense (15,508 bp): 5'-CCAGCAGCCACTAAAATGTC-3'; No. 6, V3 sense
(15,678 bp): 5'-GACTCCTGGATATCTCAGGATCAGC-3';
No. 7, V3 antisense (15,947 bp): 5'-CGTGGCTTCATCCGATGTGACAAC-3'; No. 8, V3-3a' antisense (16,059 bp): 5'-TGGAGGATCCTTGGTGGGTTCA-3', probe for V3 (15,795 bp):
5'-CTGAAAGAAGGGGAGCCCCTGAGACG-3'; No. 9, J1
antisense (33,661 bp): 5'-CGGGATCCCAGAGGGAATTACTATGAGC-3'; No. 10, TCR- constant first exon sense (37,363 bp):
5'-AGGCTTGATGCAGACATTTCCCCC-3'; No. 11, TCR-
constant first exon antisense (37,657 bp): 5'-GATCTGCTCCTCCTTTGTTGTTCTC-3'; No. 12, TCR- constant second
exon antisense (39,154 bp): 5'-CTTGCCAGCAAGTTGTAGGCTTGG-3', probe for TCR- constant (37,504 bp): 5'-GATGGCAATACTATCCTGGACTCC-3'.
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Fig. 3.
Defective rearrangement of the TCR- locus in Jak3/ thymocytes. Thymocytes from Jak3/ mice were compared with normal adult
C57BL/6, fetal (day 15), and RAG2/ mice for rearrangements of the
TCR- locus as in Fig. 2. Control lanes represent a pool of five individuals,
whereas individual mice are shown from the Jak3/ and Jak3+/ strains.
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Fig. 2.
Defective rearrangement of the TCR- locus in IL-7R/
and c/ thymocytes. Thymocyte suspensions were prepared from normal adult C57BL/6 or fetal (day 15) mice or from mice deficient in IL-7R, Rag2, or c. DNA was extracted and PCR reactions were performed to detect the indicated rearrangements of the TCR- locus. The
control PCR reaction uses internal primers for V2 (which detects both
germ line and rearranged DNA). Each lane represents a pool of five individuals except for c/ and +/, for which three individuals are shown.
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Gel Mobility Shift Assay.
Nuclear extracts were prepared (43)
from ~107 fetal thymocytes (day 15 of gestation) from normal
C57BL/6 mice, or from adult mice (4-6-wk-old) of Rag2/ or
IL7R/ strains. The binding reaction was conducted at room
temperature for 20 min with 1 to 3 µg of protein in 25 µl of
binding buffer (25 mM hepes, pH 7.5, 10% glycerol, 1 µg
poly[d(I-C)], 50 mM NaCl, 0.05% NP-40, 1 mM dithiothreitol)
and 10,000 cpm of -[32P]ATP 5' end-labeled oligonucleotide. The
reaction mixture was separated on a 7% polyacrylamide gel at
4°C, dried, and visualized by autoradiography. The nuclear extracts were tested for equal binding to the control nucleotide of
the housekeeping factor Sp-1. For competitive inhibition of the
gel shift, 50-fold excess of the specific oligonucleotide or a nonspecific competitor was used (the nuclear factor [NF]2 site for binding to NF3 or NF4, and Sp-1 for binding to NF2). For inhibition with antibodies, the extracts were incubated with 1 µl of specific
antisera against Stat5a and Stat5b (R&D Systems, Minneapolis, MN)
or control rabbit serum at 8°C for 2 h before the addition of the
radiolabeled oligonucleotides. The following double-stranded oligonucleotides were used for detection of nuclear proteins that bind to
the murine TCR- enhancer (52). Nf3: 5'-CATAGGAGCAGTTAAACCACAGCCAG-3', 5'-CTGGCTGTGGTTTAACTGCTCCTATG-3'; Nf2: 5'-GTTGCTTCCTGGAAAATGGTTAAAG-3', 5'-CTTTAACCATTTTCCAGGAAGCAAC-3'; and Nf4: 5'-CTTTCGAAAGACCACAGCTATTAG-3', 5'-CTAATAGCTGTGGTCTTTCGAAAG-3'.
Assay for Methylation of DNA.
To analyze the methylation
status of the TCR- locus, 15 µg of DNA was digested overnight with MspI or HpaII (which cannot cleave at methylated
residues). The product was fractionated on a 0.8% agarose gel,
blotted, and then prehybridized overnight. The blot was hybridized with a 294-bp probe generated by PCR from the first exon
of the constant region of TCR-1 using primer Nos. 10 and 11 (as
described above; see Fig. 1). The probe was random primed, then
hybridized with the blot overnight in hybridization buffer containing 5× SSC, 10× Denhardt's solution, salmon sperm DNA
(0.5 mg/ml), and 1% SDS. Blots were then washed 10 min in 3×
SSC and 0.5% SDS at room temperature followed by washing 45 min in 0.1% SSC and 0.5% SDS at 60°C. The location of the
MspI/HpaII sites in the TCR- locus is shown in Fig. 6.
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Fig. 6.
Methylation of the TCR- locus in IL-7R/ and c/ thymocytes. Thymocyte cell suspensions were prepared from IL-7R/, c/,
RAG2/, or SCID mice. DNA was extracted and digested with MspI or
HpaII. Southern blot analysis was performed using a probe for the TCR-
constant region. Each lane represents a pool of two to five individuals except c/ representing one individual per lane. Position of MspI/HpaII
cleavage sites within the TCR- locus are indicated with an arrow. Position of the probe used for hybridization is indicated with a closed bar.
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Reconstitution and Culture of Thymic Lobes Containing IL-7R/
Thymocytes.
Embryonic thymic lobes were obtained from
C57Bl/6 embryos at day 14 of gestation. Lobes were depleted of
endogenous thymocytes by irradiation (30 Gy), then reconstituted
by placing in hanging drops of 35 µl of medium together with
IL-7R/ bone marrow cells (5 × 104/lobe) for 24 h. Reconstituted lobes were then washed and cultured in microtiter wells, one
lobe per well, in 50 µl of medium for 96 h together with the specific histone deacetylase inhibitor TSA (Wako Chemicals).
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Results |
Role of the c Chain.
It was previously shown that IL-7R/ thymocytes from two independent lines were defective in rearrangement of the TCR- locus (13, 14). A
second chain, c, serves as part of the receptor for IL-7 (26,
27), whereas it has been speculated that TSLP (25), a homologue of IL-7, signals through IL-7R together with a
different chain. We therefore tested whether c was required for rearrangement of the TCR- locus. Several rearrangements of this locus were examined using PCR,
which generates a product if the locus is rearranged, bringing two gene segments in sufficiently close proximity to
permit the polymerization reaction (Fig. 1). Using this
method, thymocytes from various strains of mice were analyzed for rearrangement of V3 and V2, as shown in Fig.
2, and V4 (data not shown), which showed the same pattern. As shown in Fig. 2, c/ thymocytes were as deficient as IL-7R/ thymocytes in the rearrangement of
this locus. Rag2/ thymocytes are shown as a control
since they are unable to initiate VDJ recombination at any
locus. We conclude that both IL-7R and c chains are essential parts of the receptor complex that signals the rearrangement of the TCR- locus.
Role of Jak3 Kinase.
Several kinases are activated by
IL-7R cross-linking, including those of the src (28, 30) and
Janus families (29). One of the Janus kinases, Jak3, was examined for a possible role in signaling TCR- locus rearrangement because Jak3 is physically associated with c and
has been shown to be essential for normal lymphoid development (38, 44, 45). As shown in Fig. 3, a deficiency in
TCR- rearrangement was noted in these Jak3/ mice
that was comparable to the deficiency observed in thymocytes deficient in either IL-7R or c chains. Thus,
Jak3 is an essential component of the signaling pathway
leading from IL-7R cross-linking to rearrangement of the
TCR- locus.
Role of Expression of the Rag Genes.
Expression of Rag
genes was previously shown to be sustained by IL-7 (9, 18,
46, 47) and it has been observed previously that transcripts
for RAG1 and -2 are deficient in IL-7R/ thymocytes
(10). We examined IL-7R/ thymocytes for expression
of Rag1 and -2 and observed considerable variability (data
not shown): different batches of thymocytes (pooled from
five individuals) showed levels ranging from barely detectable to normal levels for both RAG1 and -2. Some IL-7R/ mice have been shown to display a "leaky" phenotype in that they develop small numbers of CD4+CD8+
cells (2); this cell population has been shown to express Rag
messages in IL-7R/ thymocytes (10), whereas the same
study observed suppressed Rag expression in the pro-T cells
from the same mice. However, in our IL-7R/ mouse
colony, some pools of thymocytes from "non-leaky" mice (with minimal numbers of CD4+CD8+ cells) also expressed high levels of Rag messages, so we presume that
even among pro-T1 cells (CD44+CD25) there is variability among individual mice. Thus, the deficiency in rearrangement of the TCR- locus, which is observed in all our IL-7R/ mice, could not be explained purely by a deficiency in expression of Rag genes. Moreover, rearrangement of the TCR- locus is more repressed than the other
loci that undergo VDJ recombination (14), strongly suggesting that locus-specific influences are signaled by the IL-7R (5). We therefore sought additional mechanisms that
could be involved in the severe repression of rearrangement of the TCR- locus in these mice.
Production of Sterile Transcripts.
A mechanism that is
thought to control VDJ recombination is whether or not the
locus is accessible to the RAG proteins (for review see reference 16). One indicator that the chromatin is open around a
rearranging gene is the production of sterile transcripts emanating from that locus before its rearrangement, a phenomenon that has been reported for most of the loci that undergo
VDJ recombination (48, 49), including the TCR- locus
(50). Sterile transcripts from the TCR- locus were greatly
reduced in IL-7R/ thymocytes as shown in Fig. 4. Transcription from both constant and variable regions were affected, and several different regions of the locus were repressed to a similar degree. Positive controls for production
of sterile transcripts are day 15 embryonic thymus and
RAG2/ thymus, both of which contain a high proportion
of pro-T cells. The deficiency in sterile transcripts in IL-7R/ thymocytes suggests that signals from the IL-7R
may control the accessibility of the TCR- locus, which
would in turn affect its ability to be cleaved by the RAG
proteins. However, an alternative explanation is that the locus is open before the IL-7R signal, which then induces its
transcription, for example by inducing transcription factors
that bind the enhancer and upregulate expression of the
gene. We therefore performed additional assays to distinguish whether the transcriptional defect in IL-7R/ thymocytes was due to a lack of transcription factors or to inaccessibility of chromatin to those transcription factors.
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Fig. 4.
Defective expression of sterile TCR- transcripts and constant
region transcripts in IL-7R/ thymocytes. Thymocyte suspensions were
prepared from normal C57BL/6 mice (embryonic day 15), IL-7R/, or
RAG2/ mice. RNA was extracted and cDNA was synthesized. PCR
reactions were performed for unrearranged transcripts from the TCR-
locus, including the constant region and the indicated V regions. GAPDH
transcripts were analyzed as a positive control for RNA loading. Three
dilutions of each cDNA were tested to reflect quantitation.
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Role of Transcription Factors.
To test for defects in transcription factors in IL-7R/ thymocytes, we examined
nuclear proteins that bind to the enhancer regions which
are located 3' to three of the four constant regions of the
TCR- locus. The enhancer confers lymphoid-specific
transcription if coupled to a heterologous promoter (51,
52). Within the enhancer, three sites (NF2, 3, and 4) have
been identified based on footprinting studies. The NF2
site resembles the consensus motif for binding of STAT5,
which has been previously shown to be activated by IL-7
(29, 53). The proteins binding the NF3 site were shown
to contain c myb and core-binding factor (54). The components of the complex that bind the NF4 site have not been identified. In Fig. 5, nuclear extracts from IL-7R/
thymocytes are compared with those from RAG2/ thymocytes, since thymic development in both of these strains is arrested at pro-T cell stages. The IL-7R/ thymocytes
did not show a deficiency of complexes that bound any of
the three enhancer sites. In some nuclear extract preparations from IL-7R/ thymocytes, an additional band was
observed that migrated faster than the band in the control
RAG2/ extract; we do not know whether this smaller
complex is functionally significant, but suspect it represents
a degradation product. In any case, we observed no clear
deficiency in any complex in IL-7R/ thymocytes.
Since STAT5 is activated by IL-7, and since the NF2 site
resembles a STAT5 consensus site (see Materials and Methods for sequence), we tested whether the complex binding
to that site contained STAT5a or -b using antibodies directed against these proteins. No effect of these antibodies
was observed on the complexes, nor was complex formation blocked, nor was an increase in mass observed (as a
positive control for the antisera, they were shown to supershift the STAT5 complex induced in the YT line by IL-2 stimulation; data not shown). Hence, the complex that
binds the NF2 site does not appear to contain STAT5. In
conclusion, we found no evidence that IL-7R/ thymocytes lacked any of the nuclear proteins previously implicated in activating the TCR- enhancer. However,
some transcription factors can bind their motif in an "inactive" state, then phosphorylation allows them to interact
with the transcriptional machinery (AP-1 is an example of
such a transcription factor). Thus, it is possible that one of
these three transcriptional complexes, although it binds
DNA, is inactive in these thymocytes. It is also possible, since these sites were identified in lines of mature T cells, that pro-T cells use different sites to regulate transcription.
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Fig. 5.
Nuclear proteins
from IL-7R/ thymocytes
binding to regions of the TCR-
enhancer. Thymocyte cell suspensions were prepared from
normal C57BL/6 embryos (day
15 of gestation), IL-7R/, or
Rag2/ mice. Nuclear extracts
were prepared. Gel mobility shift
assays were performed using
labeled oligonucleotides from
three sites in the enhancer region
of the TCR- locus. To assess
specificity of binding, unlabeled
competitors were added in 10-fold excess to the labeled oligonucleotide (Specific denotes the
same sequence used in the labeled probe; Non-specific is an
unrelated sequence of the same
size). Antibodies were added to
determine whether the NF2-binding protein complex contained STAT5a or -b proteins.
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Methylation Status of the TCR- Locus.
To determine
whether signals from the IL-7R could control chromatin
structure of the TCR- locus, we examined the methylation of this gene. Methylation of the cytosine residues in
the sequence 5' CpG are often associated with silencing of
genes (for review see reference 55). This silencing is
thought to be based on the binding of proteins to these
methylated sites, thereby altering the access of transcriptional machinery (see below). Methylation is also able to
block VDJ recombination of minichromosomes after replication (56). We previously noted that during T cell development, the TCR- locus was demethylated just before
undergoing VDJ recombination (Muegge, K., unpublished
data). We therefore tested whether IL-7R signals controlled the methylation of the TCR- locus. This was indeed the case as shown by Southern blot analysis in Fig. 6. This assay is based on the properties of two restriction enzymes, MspI and HpaII, both of which cleave at the same
restriction site, whereas methylation of the cytosine in that
site interferes with cleavage by HpaII but not MspI. As
shown, HpaII was unable to cleave sites in the TCR- locus in thymocytes from IL-7R/ or c/, whereas thymocytes from RAG2/ or SCID mice were cleaved by
HpaII. Fig. 6 also illustrates the position of the methylation-sensitive cleavage sites within TCR- clusters 1 and 2 (the probe used for hybridization is 100% identical with
cluster 1 and 95% identical with cluster 2). One site in cluster 1 generating the 7.7-kb fragment is located between the joining region and the constant region, whereas the other
site is located within the first enhancer. This indicates that
signals from the IL-7R induce demethylation of the TCR-
locus, which may in turn keep the locus "open" for either
transcription (production of sterile transcripts) or rearrangement. We have also examined the methylation status of the
TCR- locus (data not shown) and found that, in distinction to the TCR- locus, it appears to be equally demethylated in IL-7R/ and Rag2/ thymocytes. Thus, the
requirement for IL-7R signals in rearrangement of the
TCR- locus is consistent with an effect on accessibility.
Treatment of T Cell Precursors with a Specific Inhibitor of Histone
Deacetylase Overcomes the Need for the IL-7R Signal.
Methylation of DNA is thought to obstruct accessibility by altering chromatin structure. The methyl-CpG binding protein
MeCP2 interacts specifically with methylated DNA and
mediates repression of transcription. It has been shown that
MeCP2 complexes with transcriptional repressors including mSin3A and histone deacetylases (57). The specific histone deacetylase inhibitor TSA can relieve repressed transcription, indicating that histone acetylation is an important
feature of accessible chromatin (57, 58). Thus, we tested
whether the need for IL-7R could be bypassed by enhancing histone acetylation with the use of TSA. Bone marrow
cells from IL-7R/ mice were allowed to reconstitute an
irradiated fetal thymus in a hanging drop culture. The reconstituted thymus was then treated for 96 h with different
concentrations of TSA. As shown in Fig. 7, TSA induced
TCR- gene rearrangement in IL-R/ thymocytes. Fetal thymus that had not been reconstituted with bone marrow cells showed no TCR- rearrangement in the presence of TSA (data not shown). These data are consistent
with a model that IL-7R signals control rearrangement of
the TCR- locus via histone acetylation.
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Fig. 7.
TSA restores rearrangement of the TCR- locus in IL-7R/
thymocytes. Bone marrow from IL-7R/ mice was used to reconstitute irradiated embryonic thymic lobes (from C57BL/6 mice) in hanging drops.
Reconstituted lobes were then cultured in the indicated concentrations of
TSA for 96 h and analyzed for rearrangement of the TCR- locus.
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Discussion |
IL-7R/ mice show a severe deficiency in rearrangement of the TCR- locus. In this study we examine the
mechanism by which IL-7R signals VDJ recombination
of the TCR- locus. Based on studies in knockout mice,
we observed that the c component of the receptor and the
c-associated kinase Jak3 are required to deliver the signal
from IL-7R to the TCR- locus. The production of sterile transcripts from the TCR- locus was greatly impaired in IL-7R/ thymocytes. This transcriptional defect could
either reflect a deficiency in transcription factors or an inaccessibility of the TCR- locus. The latter hypothesis is favored by three observations in IL-7R/ thymocytes: (a)
we readily detected the transcription factors binding to regions that have been shown to be important for expression of the TCR- genes; (b) the TCR- locus was methylated,
which may obstruct its accessibility to both transcriptional
and recombinational proteins; and (c) the need for IL-7R
signals could be overcome in vitro by the use of the specific
histone deacetylase inhibitor TSA.
The IL-7R effect on rearrangement of the TCR- locus is unlikely to be simply the result of the trophic effect
of promoting the survival of cells undergoing the rearrangement. First, the TCR- locus rearranges in pro-T
cells at about the same time as do the TCR- and - loci.
Thus, death of the pro-T cell would be expected to equally
repress rearrangement of all three loci, yet the latter two
loci show detectable rearrangements in IL-7R/ mice,
whereas rearrangement of the TCR- locus is almost abrogated. Even though pro-T2 cells are virtually absent in
these mice, and this is the stage in which TCR gene rearrangements normally begin, only rearrangement of the
TCR- locus is eliminated. Second, a bcl-2 transgene was
shown to promote development of the / lineage but not
the / lineage (7, 8), so although the IL-7R signal also
provides a trophic effect, there is no evidence that this is
sufficient to sustain TCR- locus rearrangement. On the
other hand, introducing a rearranged TCR- transgene
only partially restored / T cell development in c/
mice (59), reflecting the requirement for a second trophic signal from the IL-7 receptor.
The IL-7R chain is reported to be a component of the
receptor for TSLP, a homologue of IL-7. Knockout of IL-7
(60, 61) has a less severe phenotype than knockout of
IL-7R, in that more / T cells develop and / T cells are
at least detectable. This has led to the suggestion that TSLP
can partly compensate for the absence of IL-7 in IL-7
knockout mice, and implies that TSLP and IL-7 have similar
activities. There has also been some speculation that the receptor for TSLP does not use the c chain (2). Thus, if TSLP
were capable of inducing T cell development, it might be
independent of c and Jak3, which could explain why
knockout mice for c and Jak3 have a less severe phenotype,
more like IL-7- than IL-7R-deficient mice. In any case,
our data show that both IL-7R and c chains are required
for the signal to rearrange the TCR- locus. To test whether
receptors from another cytokine family could induce rearrangement of the TCR- locus, we have injected IL-7R/
mice with oncostatin M. However, we have not detected
rearrangement of the locus as of the writing of this paper.
To determine whether the TCR- enhancer is the target of IL-7R stimulation, we examined nuclear proteins
from IL-7R/ thymocytes. We did not find a deficiency
in DNA binding based on gel mobility shift assays. However, the binding of a transcription factor to DNA does not
necessarily indicate that it is transcriptionally active. Cross-linking of the IL-7R has been shown to activate the transcription factor STAT5, inducing its translocation to the
nucleus (29), and it has been shown that there is a deficiency in nuclear STAT5 in IL-7R/ thymocytes (62).
We have confirmed the latter finding that IL-7R/ thymocytes were deficient in nuclear proteins that bind a
STAT5 motif (data not shown). STAT5 seemed a likely
candidate for the relevant IL-7-induced transcription factor, since one of the enhancer sites resembles a consensus
binding site for STAT5. However, we observed that the
complex that actually binds this enhancer site in vivo does
not contain STAT5a or -b based on a failure of specific antibodies to block binding in vitro. These findings argue
against the STAT5 pathway being involved in rearrangement of the TCR- locus, which is also consistent with the
observation of the relatively normal T cell development
that occurs in STAT5a/b/ mice (63).
It has recently been suggested that the IL-7 effect on rearrangement of the IgH locus may be via the transcription
factor Pax5 (12) based on several findings: (a) Pax5 binds a
site in the IgH locus; (b) Pax5/ mice resemble IL-7R/
mice in that the IgH locus successfully rearranges D to J,
but fails to normally rearrange V to D; (c) IL-7R/ B
cells showed reduced expression of Pax5. However, we detected normal levels of Pax5 transcripts in pro-T cells from
IL-7R/ mice (data not shown), suggesting that its regulation is dependent on IL-7R in the B but not the T lineage, and arguing against it mediating the signal to rearrange the TCR- locus. That study parallels ours in noting
that IL-7R/ mice were defective in producing sterile
transcripts from the IgH locus and proposing that IL-7R
regulated accessibility of that locus. Since thymocytes normally perform DH to JH but not VH to DH rearrangements,
we tested whether TSA treatment would overcome the latter block. TSA did not induce VH to DH rearrangements in
normal thymocytes when used under similar conditions to
those in Fig. 7 (data not shown), indicating that histone
deacetylation is not sufficient to account for the suppression
of this rearrangement in the T cell lineage.
Our data favor the hypothesis that IL-7R signals an opening of the TCR- locus accompanied by locus-specific
demethylation, with one site being in the enhancer, which
could be a major controlling region. This idea is supported
by the observations that in cell lines, methylated minichromosomes show repressed VDJ recombination (56) and that
in transgenic mice, methylation of the transgene represses its
rearrangement (64). A number of molecular mechanisms have been suggested to participate in transcriptional repression by DNA methylation (for review see reference 55).
Methylated DNA binds proteins such as MeCP2 (which is
essential for embryogenesis), which can silence chromatin
over a considerable distance; for example MeCP2 separated
by 1,774 bp from a transcriptional start site reduced transcription >70% (65). MeCP2 is thought to mediate this repression by attracting histone deacetylase to the region (57).
Our data supports the idea that IL-7 controls the acetylation status of histones resulting in "active" chromatin. To determine how rapidly IL-7 induced demethylation of the TCR-
locus, it would be useful to treat IL-7/ mice with IL-7,
then monitor the methylation status of the locus. The alteration of chromatin by IL-7R signals may then allow access for transcription as well as cleavage by the Rag proteins. It remains to be determined which cis-acting elements in the
TCR- locus respond to the IL-7 receptor signal, how they
induce demethylation, whether this can cause specific histone acetylation of the TCR- locus, and whether this
causes a direct increase of locus accessibility.
Address correspondence to Kathrin Muegge, SAIC, FCRDC, National Cancer Institute, Bldg. 560, Rm. 31-45, Frederick, MD 21702-1201. Phone: 301-846-1386; Fax: 301-846-7077; E-mail: muegge{at}mail.ncifcrf.gov
We are grateful to R. Wiles for technical assistance and to J.J. Oppenheim for comments on the manuscript.
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Abstract
Introduction
