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Cutting Edge |
Locus Follows IL-7 Induction
*
Laboratory of Molecular Immunoregulation and
Science Applications International Corporation, National Cancer Institute, Frederick, MD 21702
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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locus. We have recently reported that IL-7 controls chromatin
accessibility for RAG-mediated cleavage. Inhibition of histone
deacetylase substituted for the IL-7 signal, indicating a role for
histone acetylation in altering chromatin accessibility. We found a
greatly reduced histone 3 and histone 4 acetylation level in
IL-7R
-/- thymocytes in comparison with
RAG-/- thymocytes or fetal thymocytes. Sterile
transcripts, indicating an open chromatin configuration, were
suppressed in IL-7R-/- and
IL-7-/-RAG-/- thymocytes. Moreover,
exogenously added IL-7 induced sterile transcripts from the TCR
constant region in cultured thymocytes from
IL-7-/-RAG-/- mice. This induction
correlated with increased histone acetylation at the J-promoter and
C-enhancer regulatory elements at the TCR locus. These results
suggest that IL-7 regulates chromatin accessibility for V(D)J
recombination by specifically altering histone acetylation within
the TCR locus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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and 
locus that abrogate recombination correlates with the level of
histone acetylation (4, 5). This suggests another close
relationship between the regulation of chromatin accessibility for
transcription as well as the process of V(D)J recombination.
IL-7 is required for normal lymphoid development and specifically for
V(D)J recombination at the TCR locus (reviewed in Refs.
6, 7, 8). Deletion of different components of the IL-7 signal
transduction pathway inhibits 
T cell development and leads to a
specific suppression of V(D)J recombination at the TCR locus
(9, 10, 11, 12). Although IL-7 provides survival functions for
pro-T cells, the defect in recombination cannot be corrected by
substituting the IL-7 signal with a survival signal. Thus a
bcl-2 transgene did not restore V(D)J recombination at the
TCR locus in IL-7R-/- mice
(12). We have previously reported that in the absence of
the IL-7R signal, T cell precursors fail to initiate cleavage
at the TCR locus and that this failure is due to an inaccessible
state of chromatin for RAG-mediated cleavage (13, 14). The
need for the IL-7R signal can be replaced by Trichostatin A, a
specific inhibitor of histone deacetylases, suggesting a role of
histone acetylation in the opening of chromatin structure induced by
IL-7. However, Trichostatin A treatment induces a global, rather than
locus-specific, acetylation of histones, and thus its mechanism of
mimicking the IL-7 effect on the TCR locus might have been indirect.
Thus, in the present study we address the question of how IL-7
signaling modifies chromatin to allow access for the V(D)J recombinase.
We examined whether IL-7 induces specifically histone acetylation
directly at regulatory sites within the TCR locus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Embryonic thymus were derived from day 15 gestation of C57BL/6
mice (Animal Production, Frederick, MD).
IL-7R-/- mice and
Rag-2-/- mice (The Jackson Laboratory, Bar
Harbor, ME) were 
4–8 wk of age. The generation of
IL-7-/-Rag-1-/- were
kindly provided by R. Murray (EOS Biotechnology, San Francisco,
CA) (9). Animal care was provided in accordance
with the procedures outlined in the "Guide for the Care and Use of
Laboratory Animals" (National Institutes of Health Publication No.
86-23, 1985).
Primers and probes
The locations of primers and probes within the TCR locus are
shown in Fig. 1
. The primers for
detection of gene rearrangement and sterile transcripts were derived
from sequences as previously published (13, 14). The
following oligonucleotides were used in the chromatin
immunoprecipitation (ChIP)2-PCR assay: 1)
upstream: S:5'-GGCAAGAAACAGAACAACTG-3';
AS:5'-GGACAATGTCTATTCTAGTC-3'; and probe:
5'-GTTCTGATTGGCTACTTTCC-3'. 2) VHsA: S5'-GCTCTCTCTCCCTTCTTTG-3';
AS5'-TGGTTGCAGACATGCTGAGTG-3'; and P5'-GGAAACACAAGACGTGACG-3'. 3)
J promoter: S5'-CCTCTTCTCAGAAATATATCCCC-3';
AS5'-TAATTTCCAGGAACTCACTCGTG-3'; and P5'-ACATTGGTGCTTGTGAGAAC-3'. 4)
Constant region enhancer: S5'-GTTGCTTCCTGGAAAATGGTTAAAG-3';
AS5'-CTAATAGCTGTGGTCTTTCGAAAG-3'; and
P5'-AGGAGCAGTTAAACCACAGC-3'. 5) Downstream:
S5'-AGGAGCTTCAGTTGAGGAAG-3'; AS5'-TAGAACCAAGACTAACAGCC-3'; and
P5'-CATGAGATCCAGCTGCAAGG3'. 6) Enhancer:
S5'-CAAAATACATGCCCAGCCA-3'; AS 5'CAGCAAAACTGATAACCCC-3'; and
P5'-AAGAGATAGCAGGGCTTCTG-3'. 7) Oct-2:
S5'-TGGAGGAGCTGGAACAGTTTG-3'; AS 5'GGACCTTGGCATCTTTGTTCC-3'; and
P5'-ATCAAGCTGGGCTTCACACAG-3'. 8) Pgk-2:
S5'-TGAAGTAGAGCAAGCCTGTG-3'; AS 5'AGACAGTGATGCTTGGAAGG-3';
P5'-TGTGGAGGAAGAAGGTAAGG3'.
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PCR and RT-PCR analysis
The PCR after ChIP was performed with Supermix (Life
Technologies, Rockville, MD) started with 94°C for 5 min, followed by
25 cycles: 1 min at 94°C, 2 min at 51°C, and 3 min at 72°C,
followed by a 10-min extension at 72°C. Total RNA was reverse
transcribed with oligo(dT) primer by using Sensiscript Reverse
Transcriptase (Qiagen, Valencia, CA) or a control reaction was
performed omitting reverse transcriptase. PCR was amplified in 50 µl
of HotStartTaq Master Mix reaction buffer (Qiagen) containing 0.2 mM of
each NTP, 0.2 µM of each primer, and 2.5 U of HotStartTaq polymerase.
The amplification for RT-PCR was started at 94°C for 15 min, followed
by 35 cycles: 0.5 min at 94°C, 0.5 min at 56°C, and 1 min at
72°C, followed by a 10-min extension at 72°C. (The annealing
temperature was 64°C for -actin primers.) PCR products were
separated by agarose gel electrophoresis and transferred to NYTRAN
membranes (Schleicher & Schuell, Keene, NH). Hybridization was
performed in Rapid-hyb buffer (Amersham Pharmacia Biotech, Piscataway,
NJ) according to the manufacturer’s protocol and visualized by
autoradiography.
ChIPs assay
The procedure of chromatin immunoprecipitation was modified from
the manufacturer’s protocol (acetyl-histone H3 or H4 ChIP assay kit;
Upstate Biotechnology, Lake Placid, NY) as follows. Thymocytes were
cross-linked with 1% formaldehyde for 12 min at 37°C and the
reaction was stopped by adding glycine to a final concentration of 125
mM for 5 min. The cells were washed twice in ice-cold PBS and lysed in
SDS lysis buffer. Sonication was performed on ice and the samples were
cooled on dry ice between the pulses, but care was taken not to freeze
the samples. The length of the DNA fragments averaged between 200
and 500 bp. After precleaning, 10% of each sample was saved as input
fraction. Immunoprecipitation was performed using specific Abs against
acetylated histone 3 (5 µg/ml) and histone 4 (4 µl/ml), or normal
rabbit IgG (5 µg/ml) as control. After incubation for 16 h at
4°C with agitation, the Ab/histone/DNA complex was captured by using
salmon sperm DNA/protein A-agarose slurry for 1 h rotating at
4°C. The supernatant of each sample was kept as unbound fraction. The
complex was eluted and the cross-linking was reversed. After
purification, the DNA concentration was determined and diluted in
three-fold series (20 ng, 6.7 ng, and 2.33 ng) for PCR amplification.
The blot result was imaged by PhosporImager and quantitated by
ImageQuant v5.0 software (Molecular Dynamics, Sunnyvale, CA). The
relative acetylated enrichment as shown in the figures was determined
from the ratio of bound:input fractions which was very similar to the
ratio formed by bound:unbound control fractions. The results shown in
the figures are expressed as means ± SE from three to five
independent ChIP experiments.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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locus in normal fetal
thymocytes
To determine whether IL-7 stimulation effects histone
acetylation at the TCR locus, we first determined the status of
acetylation in the presence of IL-7 during normal development. TCR
locus rearrangement occurs in thymocytes between days 14 and 16 of
murine gestation. During this period, sterile transcripts and
recombination intermediates are detected, indicating an open chromatin
configuration and an actively occurring recombination process,
respectively (14). Thus, fetal thymocytes were used as a
nuclear source for ChIP using specific Abs against acetylated histones.
The acetylation of N-terminal lysine residues of histone 3 as well as
histone 4, components of the mononucleosomes, are thought to result in
an open chromatin structure (2, 3, 15). Fragmentation of
chromatin after cross-linking allowed for specific analysis of small
regulatory sequences (average size 200–500 bp). The murine TCR1
locus extends 40–50 kbp comprising four variable region gene segments
and one joining and constant region segment (Fig. 1
). We first
investigated the status of histone 3 and histone 4 acetylation at
regulatory sites recently identified as transcriptional response
elements within the TCR locus: the DNase I hypersensitive site A
(V-HsA), an enhancer-like element between the V5 and V2 genes, the J
region promoter (J-promoter), or the constant-region enhancer
(C-enhancer) (12, 16, 17). In addition, the enhancer of
the TCR locus (E) was examined as a positive control. A
representative ChIP experiment (Fig. 2A) indicates specific
acetylation at histone 3 as well as histone 4 for all three regulatory
sites in comparison with the control immunoprecipitation. A critical
question is whether this demonstrated acetylation level found at the
TCR locus is specifically elevated in comparison with other sites in
the genome. To evaluate this question, the ratio was calculated between
the specifically precipitated material and the input fraction and
expressed as fold acetylation, as shown in Fig. 2B,
summarizing five experiments. This ratio indicates how much a specific
sequence is enriched in the precipitated fraction in comparison
with the whole genome (Fig. 2B). Thus the J-promoter, the
C-enhancer, and the HsA sites are 22-, 10-, and 13-fold enriched in the
acetylated H3 fraction and 35-, 15-, and 25-fold in the acetylated H4
fraction. This suggests a locus-specific enhancement of histone
acetylation at the TCR locus. In contrast, the tissue-specific
expressed genes Oct-2 (B cell-specific) and Pgk-2 (testis-specific) are
not enriched in the precipitated fraction, indicating no significant
enhancement for acetylation in comparison with the overall genome. To
determine the borders of hyperacetylation at the TCR locus, we
analyzed additional sequences that lack any known regulatory function.
Whereas the upstream site (1300 bp upstream of V5) still showed a
4-fold enrichment (2-fold over Oct-2), the downstream sequence (1700 bp
downstream of the C-enhancer) was indistinguishable in its H3 or H4
acetylation level from the control gene Oct-2. Thus the three known
regulatory sites of the TCR locus are hyperacetylated in comparison
with the global genome and at least two other tissue-specific genes.
Thus the acetylation is rather focused within the locus, tapering off
with increasing distance from the regulatory sites.
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locus in the absence of
IL-7R signaling
Next we addressed the question of whether hyperacetylation as
observed at the TCR locus during normal T cell development occurs in
response to IL-7R signaling. Recently we reported a defect in V(D)J
recombination and a reduced chromatin accessibility in
IL-7R-/- thymocytes compared with
those with RAG-/- thymocytes. Deletion of RAG-1
or RAG-2 genes leads to an abrogation of V(D)J recombination and an
early arrest of T cell development at a stage similar than that of day
15 fetal thymocytes. However, RAG-/- thymocytes
have detectable sterile transcripts and their chromatin shows normal
accessibility to RAG-mediated cleavage in vitro in contrast to
IL-7R-/- thymocytes (14). Thus
we compared the level of histone acetylation at the TCR locus
between IL-7R-/- thymocytes and
RAG-/- thymocytes. As shown in Fig. 3A, the level of histone
acetylation was greatly reduced in the absence of IL-7R signaling.
The enrichment for acetylation in RAG-/-
thymocytes was 20-, 26-, and 19-fold at J-promoter, C-enhancer, and
V-HsA sites for H3 and 22-, 21-, and 16-fold for H4, and thus
comparable with the hyperacetylation in fetal thymocytes (Fig. 3B). The slight differences between
RAG-/- and fetal thymocytes possibly reflect
distinct developmental stages (the pro-T3 stage for
RAG-/- thymocytes in contrast with the
pro-T1/T2 stage for d14 fetal thymocytes). In both controls all three
regulatory sites were markedly hyperacetylated. In contrast, the
chromatin derived from IL-7R-/- thymocytes
showed an overall histone acetylation level indistinguishable from the
control site within the Oct-2 gene. The reduction in acetylation was
12- to 22-fold for H3 and 6- to 10-fold for H4 comparing
IL-7R-/- with RAG-/-
acetylation levels. Thus the specific histone acetylation at the
regulatory sites within the TCR locus that is observed during normal
development is dependent on IL-7R signaling.
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IL-7 is also an important survival signal for T cell precursors,
although Bcl-2 cannot rescue the defective TCR rearrangement in
IL-7R defective mice (12). To determine whether IL-7
can directly effect accessibility at the TCR locus instead of
promoting accessibility indirectly through enhanced viability, we
examined the induction of sterile transcripts. Transcription of
unrearranged gene segments usually precedes V(D)J recombination and is
considered an indicator of accessible chromatin. Previously we have
reported that sterile transcripts are reduced in
IL-7R-/- thymocytes in contrast to
RAG-/- thymocytes (13) (Fig. 4A). We also detected a
suppression of sterile transcripts for V2 and V3 and the constant
region in IL-7-/-RAG-/-
thymocytes, indicating that at least part of the detrimental effect of
IL-7R deletion is due to IL-7 itself (rather than, for example,
TSLP, which also binds IL-7R). Recently it had been reported that
IL-7 can induce TCR transcripts within 5 h in
CD4+ T cells purified from adult thymus
(18). Similarly, we were able to induce constant region
transcripts in
IL-7-/-RAG-/-
thymocytes within 5 h of stimulation with IL-7 in culture (Fig. 4A). This rapid process suggests that IL-7 signaling is
directly able to induce opening of chromatin in thymic precursors,
rather than promoting cell survival or growth, indirectly affecting
chromatin. We could not observe V 2 and V3 transcripts in this
time period, although the reduced acetylation levels at the HsA site
would suggest a control by IL-7R signaling as well. However,
we observed reduced viability of
IL-7-/-RAG-/-
thymocytes over time in culture that was not restored by addition of
additional cytokines such as stem cell factor. It remains
possible that a different ligand of IL-7R, such as TSLP
(19), could provide long-term survival in culture or that
TSLP may specifically participate in the control of V2 and V3
transcription. Recently it was reported that the histone acetylation
levels at the recombination recognition sites at V2 and V3 are
high in fetal thymocytes, as reported in this work, but distinctly
regulated in adult thymocytes (being lower at V3) (20).
This suggests regulatory factors other than simply IL-7 alone being
involved in the control of a developmentally ordered rearrangement of
the variable gene segments.
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locus is induced in
IL-7-/-RAG-/- thymocytes by IL-7
The induction of constant region transcripts by IL-7 within 5
h supports the idea that IL-7 directly effects chromatin
accessibility at the TCR locus. To determine whether histone
acetylation contributes to the inducible opening of chromatin at the
TCR locus by IL-7 and to determine which regulatory sites are
involved in this induction, ChIP experiments were performed. Fig. 4B summarizes a quantitative analysis of three independent
ChIP experiments using thymocytes derived from
IL-7-/-RAG-/- mice and
cultured with IL-7 in vitro for 5 h or left untreated. The level
of H3 or H4 acetylation at the J-promoter or C-enhancer in untreated
IL-7-/-RAG-/-
thymocytes was undistinguishable from the level observed at the Oct-2
gene. This underacetylation corresponds with the low acetylation level
detected in IL-7R-/- thymocytes. Within
5 h of IL-7 stimulation histone acetylation levels changed at the
J-promoter and C-enhancer. Although the histone acetylation after
stimulation with IL-7 was much smaller than that observed in wild-type
thymocytes (see Fig. 2) the induction by IL-7 was three- to four-fold
for H3 and H4 above the untreated control (Fig. 4B). The
J-promoter and the C-enhancer are thought to drive sterile constant
region transcripts. Thus the induction of histone acetylation levels
correlated closely with the induction of sterile transcripts. These
results suggest that the IL-7 signal transduction pathway controls
TCR locus recombination by increasing acetylation of histones at
specific cis-acting elements.
The precise signal transduction pathway leading from the IL-7R to the
site of specific histone acetylation within the TCR locus is not yet
known, although the transcription factor Stat5 would be a
candidate. IL-7 can activate Stat5, and there is strong evidence that
Stat5 can induce histone acetylation. Moreover, Stat5 can bind to
sequences within the J-promoter (the C-enhancer and V-HsA element
contain potential binding sites) (12). The most compelling
evidence that Stat5 could mediate the observed acetylation effect comes
from the report that a transgene expressing Stat5 restores TCR
recombination in IL-7R deleted lymphoid precursor cells
(12). Stat5 has been demonstrated to associate with the
interacting protein Nmi and with the histone acetylases CBP/P300
(21). Thus activation of Stat5 by IL-7 could lead to
site-specific recruitment of histone acetylases at the TCR locus. In
contrast, knockout of Stat5a/b was not reported to be deficient in
T cells, implicating additional mediators.
How can site-specific histone acetylation regulate V(D)J recombination?
Previous work suggested that mononucleosomes could inhibit RAG-mediated
cleavage in vitro (22, 23). This inhibition is thought to
be caused by inhibiting access of the RAG dimer to the recombination
signal sequence as well as to reducing cleavage capacity of the
RAG recombinase (23). How can histone acetylation overcome
this suppression? Acetylation of histone tails may affect the structure
of the mononucleosome or participate in unfolding of higher chromatin
structure. Acetylated histone tails may also serve as flags for
recruitment of other chromatin remodeling complexes. ATP-dependent
complexes such as SWI/SNF complexes may disrupt chromatin structures by
sliding mononucleosomes along the DNA, thus providing better access for
the RAG recombinase. In support of this model, it was recently reported
that both acetylation and SWI/SNF-dependent chromatin remodeling act in
concert to promote RAG-mediated cleavage in vitro (23).
However, it is not yet known whether SWI/SNF-dependent
remodeling contributes in vivo to accessibility for V(D)J recombination
or whether other recombination-specific chromatin remodeling activities
exist. The use of IL-7 to induce specifically TCR rearrangement may
serve as a valuable in vivo model to study the molecular mechanism for
control of chromatin accessibility for V(D)J recombination.
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Acknowledgments |
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Footnotes |
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2 Abbreviation used in this paper: ChIP, chromatin immunoprecipitation.
Received for publication September 11, 2001. Accepted for publication October 2, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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gene enhancer during early T cell development: implications for the control of TCR- locus recombination. J. Exp. Med. 192:625.c-dependent cytokines IL-2, IL-7, and IL-15. Semin. Immunol. 12:465.[Medline]
T cell development and early thymocyte maturation in IL-7-/- mice. J. Immunol. 157:2366.[Abstract]
genes is blocked in interleukin-7 receptor-deficient mice. J. Exp. Med. 184:2423. gene rearrangement in interleukin-7 receptor knockout mice. Immunol. Lett. 57:9.[Medline]
locus by Stat5: implications for accessibility control by the IL-7 receptor. Immunity 11:213.[Medline]
gene rearrangement: role of receptor-associated chains and locus accessibility. J. Exp. Med. 188:2233. locus accessibility to the V(D)J recombinase. J. Exp. Med. 191:1045.2 gene in the murine T cell receptor locus cooperates with the 3' enhancer to act as a locus control region. J. Exp. Med. 190:669.1 in the murine T-cell receptor locus. Proc. Natl. Acad. Sci. USA 88:800./ T cells in interleukin 7 receptor-deficient mice is due to impaired expression of T cell receptor genes. J. Exp. Med. 190:973. gene recombination. J. Exp. Med. 193:873.-mediated signaling. Cell 96:121.[Medline]
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