Induction of Germline Transcription in the
Human TCR γ Locus by STAT5
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J Immunol 2001; 167:320-326; ;
doi: 10.4049/jimmunol.167.1.320
http://www.jimmunol.org/content/167/1/320
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References
Hai-Chon Lee, Sang-Kyu Ye, Tasuku Honjo and Koichi
Ikuta
Induction of Germline Transcription in the Human TCR␥
Locus by STAT51
Hai-Chon Lee, Sang-Kyu Ye, Tasuku Honjo, and Koichi Ikuta2
T
cell receptor and Ig variable region genes are assembled
during lymphocyte development from V, D, and J segments by V(D)J recombination. V(D)J recombination is
conducted by conserved recombination signal sequences and recombinase-activating gene-1 and -2 proteins. In developing T and
B cells, specific molecular mechanisms exist that target the common recombinase activity to appropriate TCR or Ig loci in a lineage-specific, developmentally ordered, and allelically excluded
manner. This recombinase targeting should occur at the level of the
substrate locus, leading to a concept known as recombinational
accessibility (1–3). Two kinds of cis chromatin elements are involved in controlling the accessibility. First, the enhancer elements
govern the general accessibility of each locus in V(D)J recombination. Targeted deletion of the respective enhancers abolishes rearrangements in the TCR␤ locus and greatly reduces rearrangements in the IgH, Ig␬, and TCR␣ loci (2, 3). Histone acetylation is
tightly correlated with V(D)J recombination in the TCR␣␦ and
TCR␤ loci and is proposed as a mechanism for coupling enhancer
activity to accessibility (4, 5). Second, the promoters for germline
transcription control the local accessibility. T early ␣ germline
transcription takes place in the 5⬘ region of the J␣ cluster in immature thymocytes. Targeted deletion of the T early ␣ promoter
results in severe impairment of the rearrangement of the 5⬘-most
J␣ segments (6). In another case, targeted deletion of the D␤1
germline promoter abolishes D␤1 germline transcription and reduces the rearrangement of the D␤1 gene segment (7). These reDepartment of Medical Chemistry, Graduate School of Medicine, Kyoto University,
Kyoto, Japan
Received for publication October 13, 2000. Accepted for publication April 30, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants-in-aid from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan.
2
Address correspondence and reprint requests to Dr. Koichi Ikuta, Department of
Medical Chemistry, Graduate School of Medicine, Kyoto University, Yoshida,
Sakyo-Ku, Kyoto 606-8501, Japan. E-mail address: [email protected]
Copyright © 2001 by The American Association of Immunologists
sults suggest a critical role for germline transcription in V(D)J
recombination of the Ag receptor genes.
IL-7 is an essential cytokine for early lymphocyte development
when V(D)J recombination takes place. IL-7 exerts its effect
through interaction with the IL-7R, consisting of a unique ␣-chain
(IL-7R␣)3 and the common cytokine receptor ␥-chain. Injection of
neutralizing Abs to IL-7 or IL-7R␣, or genetic ablation of IL-7,
IL-7R␣, or common cytokine receptor ␥-chain, leads to a block of
lymphocyte development. Although IL-7R␣-deficient mice have
small numbers of B cells and ␣␤ T cells in the periphery, they
totally lack ␥␦ T cells (8 –10). The IL-7R transmits two signals in
lymphocyte progenitors (11). One is for survival and proliferation.
For instance, IL-7R signaling induces the expression of Bcl-2 in T
cell precursors (12), and introduction of a bcl-2 transgene restores
␣␤ T cell development in IL-7R-deficient mice (13, 14). The
IL-7R also promotes the proliferation of lymphocyte precursors
through the activation of phosphatidylinositol 3 kinase (15, 16).
The other signal from the IL-7R is to promote V(D)J recombination in the IgH and TCR␥ loci. For example, IL-7R signaling induces germline transcription and DNA rearrangement in D-distal
V segments in pro-B cells (17). The V-J recombination and germline transcription of TCR␥ genes also are severely impaired in
IL-7R␣-deficient mice (18 –21).
IL-7 binding to IL-7R triggers the phosphorylation and activation of receptor-associated Jak1 and Jak3 tyrosine kinases (22).
After their activation, the Jak kinases phosphorylate the tyrosine
residue of IL-7R␣. STAT, as well as phosphatidylinositol 3 kinase,
are recruited to the tyrosine residue and subsequently phosphorylated and activated by the Jak kinases. IL-7R mainly activates
STAT5A and STAT5B, and to a lesser extent, STAT1 and STAT3.
The phosphorylated STAT proteins then form homo- and heterodimers through Src homology 2 domain-mediated interactions
and translocate into the nucleus. Dimerized STAT proteins then
bind to a consensus binding motif (TTCNNNGAA) and activate
3
Abbreviations used in this paper: IL-7R␣, IL-7R ␣-chain; hC␥1, human C␥1; E␥, 3⬘
enhancer of the TCR␥ locus.
0022-1767/01/$02.00
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TCR and Ig genes are assembled by V(D)J recombination during lymphocyte development. The enhancer and the germline
promoter control the accessibility of each locus for the common recombinase activity. In the mouse TCR␥ locus, STAT5 proteins
activated by the IL-7R interact with consensus motifs in 5ⴕ regions of J␥ segments and induce germline transcription. To evaluate
the role of STAT5 in controlling the accessibility of the TCR␥ locus, we characterized the germline transcription of human TCR␥
genes and compared it with mouse. We first demonstrated that J␥-C␥ germline transcripts are induced in a cytokine-dependent
human erythroleukemia cell line. STAT consensus motifs are present in 5ⴕ regions of J␥1.1 and J␥2.1 gene segments, and activated
STAT5 binds to these motifs. By using a reporter assay, we showed that the J␥1.1 germline promoter is transactivated by STAT5
and that mutations in any of the two STAT motifs abrogate this activity. Thus, this study demonstrates that STAT5 induces
germline transcription in the TCR␥ locus of both mouse and human and suggests the possibility that this mechanism may play
an essential role in controlling the TCR␥ locus accessibility. In addition, STAT motifs are conserved among 5ⴕ J␥ germline
promoters, 3ⴕ enhancers, and a locus control region-like element, HsA, in both mouse and human TCR␥ loci, indicating the
possibility that IL-7R/STAT5 signaling probably controls the locus-wide accessibility through these elements. The Journal of
Immunology, 2001, 167: 320 –326.
The Journal of Immunology
Materials and Methods
Cells
A mouse IL-3-dependent pro-B cell line, Ba/F3, was cultured as described
previously (21). A GM-CSF-dependent human erythroleukemia cell line,
TF-1 (26), was maintained in RPMI 1640 medium containing 10% FBS
and 5 ng/ml recombinant human GM-CSF (Genzyme, Cambridge, MA).
Northern blot and RT-PCR analyses
Total RNA was electrophoresed through 1% agarose gel and transferred to
a nylon membrane (Hybond-N⫹; Amersham Pharmacia, Arlington
Heights, IL). The membrane was hybridized sequentially with 32P-labeled
C␥ and GAPDH probes. The C␥ probe was a 530-bp human C␥1 (hC␥1)
fragment isolated by PCR with primers as follows: hC␥ 5⬘-1, 5⬘-CAACT
TGATGCAGATGTTTCC-3⬘; hC␥ 3⬘-3, 5⬘-TTGTGCCACCGTCTGT
TATG-3⬘. Northern blot was analyzed and radioactivity quantitated with a
Bio-image Analyzer (Fujix BAS1500; Fuji Film, Tokyo, Japan).
Oligo(dT)-primed cDNA from TF-1 cells cultured with or without GM-CSF
(for 18 h) was amplified with the primers. PCR was conducted for 30 cycles
consisting of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C. PCR products were
subcloned in pT7Blue T-Vector (Novagen, Madison, WI). Sequences of the
primer are as follows: 5⬘hJ␥P 5⬘-8, 5⬘-TCTGTTGGTGCTTTTCAAGAATTACTG-3⬘; hC␥ 3⬘-2, 5⬘-GGAAACATCTGCATCAAGTTG-3⬘.
Western blot analysis
Cells (1–2 ⫻ 106) were starved for 6 h, restimulated for 30 min, and lysed
for 30 min in 1 ml of ice-cold lysis buffer containing 0.2% Nonidet P-40.
After centrifugation, cell lysate was incubated with anti-STAT5 mAb (Santa Cruz Biotechnology, Santa Cruz, CA). The immune complex was incubated with protein G-Sepharose beads (Amersham Pharmacia), and the
immunoprecipitate was washed and eluted in 2⫻ SDS loading buffer. The
protein was subjected to 8% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Hybond-P; Amersham Pharmacia). The blot
was incubated with anti-STAT5 Ab or anti-phosphorylated STAT5 mAb
(Upstate Biotechnology, Lake Placid, NY), and visualized with HPR-conjugated rabbit anti-mouse IgG by ECL plus detection system (Amersham
Pharmacia).
EMSA
Three micrograms of nuclear extract was incubated on ice in 15 ␮l of
binding buffer (20 mM HEPES, pH 7.9, 60 mM NaCl, 2 mM EDTA, 1 mM
DTT, 20 ng/ml BSA, 10% glycerol) containing 2 ␮g of poly(dI:dC). Two
nanograms of end-labeled oligonucleotide probe was added to the extract
and incubated for 30 min at 20°C. For supershift assay the nuclear extract
was preincubated with 1 ␮g of anti-STAT5 mAb (Santa Cruz Biotechnology) for 1 h on ice. For competition assay, extract was preincubated with
10- or 50-fold molar excess of cold oligonucleotide for 5 min before the
addition of labeled oligonucleotide. The reactions then were resolved by
native 5% PAGE. A control mouse probe was described before (21). The
oligonucleotides used as probes were as follows: 5⬘hJ␥1.1 䡠 site3 䡠 S, 5⬘AGACAATTCTCTGAATACTTTTCC-3⬘ (probe A); 5⬘hJ␥1.1 䡠 site3 䡠 A, 5⬘GGAAAAGTATTCAGAGAATTGTCT-3⬘ (probe A); 5⬘hJ␥1.1 䡠 site2 䡠 S,
5⬘-AGACAATTCTCTGAATACTTTTCCTGGTAATTTAG-3⬘ (probe B);
5⬘hJ␥1.1 䡠 site2 䡠 A, 5⬘-CTAAATTACCAGGAAAAGTATTCAGAGAATT
GTCT-3⬘ (probe B); 5⬘hJ␥1.1 䡠 site4 䡠 S, 5⬘-GAATACTTTTCCTGGTAATT
TAG-3⬘ (probe C); 5⬘hJ␥1.1 䡠 site4 䡠 A, 5⬘-CTAAATTACCAGGAAAAG
TATTC-3⬘ (probe C). The mutated probe contained a mutated motif (TTC
NNNTCC) instead of the consensus motif (TTCNNNGAA) or the atypical
motif (TTCNNNGTA).
Isolation of human 5⬘ J␥ regions
The human 5⬘ J␥1.1 and 5⬘ J␥2.1 fragments were cloned by PCR. These
fragments are the 1.0-kb 5⬘ J␥1.1 and 1.3-kb 5⬘ J␥2.1 regions that cover the
sequences just before the first ATG of the germline transcripts. The 5⬘
J␥2.1 region contains an Alu repetitive sequence. Mutations were introduced in STAT consensus motifs by PCR-based mutagenesis with an LAPCR in vitro mutagenesis kit (Takara Shuzo, Kyoto, Japan). The 5⬘ end of
the germline J␥-C␥ transcript was determined by cloning and sequencing
its cDNA from TF-1 cells with a 5⬘-Full RACE kit (Takara). Sequences of
the primers for isolation of human 5⬘ J␥ regions are as follows: 5⬘hJ␥ 5⬘-1,
5⬘-CAGCTGCTAAGACTTCTGATGCTTC-3⬘; 5⬘hJ␥ 3⬘-1, 5⬘-AATTGCCT
G(T/C)TTTCTTCTAAGGCAG-3⬘.
Deletion of the Alu repetitive sequence from the 5⬘ J␥2.1 clone was done
as follows. The upstream and downstream DNA regions of the Alu sequence
were amplified with either 5⬘hJ␥P2 3⬘-1 or 5⬘hJ␥P2 5⬘-1, and universal primers. The PCR products were digested with appropriate restriction enzymes, and
assembled in pBluescript vector (Stratagene, La Jolla, CA). Sequence of the primers are as follows: 5⬘hJ␥P2 3⬘-1,5⬘-CTGATATCAAAGAGCAGGCTCAGT
GGGT-3⬘; 5⬘hJ␥P2 5⬘-1,5⬘-CCGATATCTTTCTTTTTTTGTTGTTGTT-3⬘.
Luciferase reporter gene transactivation assay
Transfection was done by electroporation as described previously (21). A
reporter construct with mouse 5⬘ J␥1 germline promoter was described
before (21). Ba/F3 cells were transiently transfected by electroporation
with 10 ␮g of luciferase reporter plasmids driven by the 1.0-kb 5⬘ J␥1.1 or
1.3-kb 5⬘ J␥2.1 fragment (pGL2; Promega, Madison, WI) and 1 ␮g of a
␤-galactosidase plasmid driven by the Rous sarcoma virus long-terminal
repeat promoter (pRSV-␤-gal) as well as 10 ␮g of a STAT5A vector
(pMX-STAT5A). The total amount of DNA was kept constant with pGL2basic or pMX vector. After a 12-h recovery period in the IL-3-containing
medium, the cells were incubated in RPMI 1640 medium supplemented
with 0.5% BSA for 12 h or stimulated with the cytokine for the last 6 h.
Cell lysates then were subjected to luciferase (luciferase assay system;
Promega) and ␤-galactosidase (Galato-Light; Tropix, Bedford, MA) assays
with a luminometer (Lumat LB9051; Berthold, Bad Wildbad, Germany).
Normalized luciferase activity (luciferase/␤-galactosidase ratio) then was
compared. In each experiment, samples were analyzed in triplicate, and
each experiment was repeated at least twice.
Comparison of DNA sequences between the human and mouse
TCR␥ locus
Nucleotide sequences of the TCR␥ locus were compared between human
(GenBank accession no. AF159056) and mouse (GenBank accession no.
AF037352) by GENETYX-MAC version 7.3 software (Software Development, Tokyo, Japan).
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the transcription of various target genes. STAT5 and STAT1 interact
with Nmi, an N-Myc interactor. Nmi enhances the association of
CBP/p300 transcriptional coactivator proteins with STAT5 and
STAT1 to augment IL-2- and IFN-␥-dependent transcription (23).
The IL-7R is required for the TCR␥ locus accessibility (24).
STAT5 proteins activated by the IL-7R interact with consensus
motifs in 5⬘ regions of J␥ segments and induce germline transcription (21). A constitutively active form of STAT5 induces germline
transcription, restores V-J recombination of TCR␥ genes, and rescues ␥␦ T cell development from IL-7R⫺/⫺ T cell precursors.
These results suggest that STAT5 controls the accessibility of the
TCR␥ locus by the induction of germline transcription. The transcriptional coactivators have intrinsic histone acetyltransferase activity and have been suggested to be involved in the accessibility
control of the transcriptional machinery (25). Thus, it is conceivable that these coactivators render the chromatin accessible not
only to the transcriptional machinery but also to the recombinational machinery. STAT5 may recruit the transcriptional coactivators to the J␥ regions and thereby regulate the recombinational
accessibility in the TCR loci, especially at the J␥ regions.
To evaluate the STAT5-induced germline transcription of the
TCR␥ locus, we characterized the germline transcription of human
TCR␥ genes and compared it with mouse. Cytokine stimulation
leads a human hemopoietic cell line to induce phosphorylation of
STAT5 and to augment germline transcription in the TCR␥ locus.
Activated STAT5 proteins interact with consensus motifs in 5⬘
regions of J␥ segments and induce germline transcription. These
results are basically the same with those in the mouse TCR␥ locus.
Thus, this study demonstrates that STAT5 induces germline transcription in the TCR␥ locus of both human and mouse and suggests the possibility that this mechanism may play an essential role
in controlling the accessibility of the TCR␥ locus. In addition,
STAT motifs are conserved among 5⬘ J␥ germline promoters, 3⬘
enhancer of the TCR␥ locus (E␥), and a locus control region-like
element, HsA, in both mouse and human TCR␥ loci, indicating the
possibility that IL-7R/STAT5 signaling probably controls the locus-wide accessibility through these elements. We will also discuss its evolutional implication in ␣␤ and ␥␦ T cell development.
321
322
Results
Cytokine stimulation augments germline transcription of the
human TCR␥ locus
FIGURE 1. Germline transcription in the human TCR␥ locus. A, The
schematic illustration of the human TCR␥ locus. Exons and pseudogenes
are depicted as f and 䡺, respectively. P represents a pseudogene. B, Induction of germline transcripts of TCR␥ genes in a human erythroleukemia
cell line. Total RNA was isolated from TF-1 cells cultured with (⫹) or
without (⫺) GM-CSF, and a Northern blot was hybridized sequentially
with C␥ and GAPDH probes. C, Phosphorylation of STAT5 by GM-CSF
stimulation. Total cellular extract was isolated from TF-1 cells with (⫹) or
without (⫺) cytokine stimulation and immunoprecipitated with antiSTAT5 Ab. Western blots were visualized sequentially with antiphosphorylated STAT5 and anti-STAT5 Abs.
STAT5 was tyrosine-phosphorylated after GM-CSF stimulation.
Before stimulation, STAT5 was not phosphorylated at all, even
though STAT5 molecules were detected before and after GM-CSF
stimulation. This result confirmed that STAT5 is activated by the
GM-CSF stimulation in TF-1 cells. Taken together, these results
indicated that germline transcription in the human TCR␥ locus is
induced in both cytokine-dependent and -independent manners in
TF-1 cells.
STAT consensus motifs are conserved in the 5⬘ regions of J␥1.1
and J␥2.1 gene segments
To elucidate the mechanism of TCR␥ germline transcription, we
characterized the promoter regions for the transcription. We
checked the sequence of 5⬘ regions of five J␥ gene segments (Fig.
2). A STAT consensus motif (TTCNNNGAA) was found at ⬃300
bp upstream of the J␥1.1 gene segment. By sequence homology,
we noticed a similar STAT consensus motif at ⬃600 bp upstream
of the J␥2.1 gene segment. An Alu repetitive sequence was inserted between the STAT motif and the J␥2.1 gene segment. We
also found second atypical STAT motif (TTCNNNGTA) at 5 bp
downstream of the first motifs in 5⬘ J␥1.1 and 5⬘ J␥2.1 regions.
We next determined transcription initiation sites of TCR␥ germline transcripts by rapid amplification of cDNA ends method. TF-1
cells transcribed two kinds of germline transcripts, J␥1.1-C␥1 and
FIGURE 2. STAT consensus motifs conserved in human 5⬘ J␥ regions.
A, STAT consensus motifs in two 5⬘ J␥ regions. STAT consensus motifs in
the 5⬘ regions of J␥1.1 and J␥2.1 are schematically compared. An Alu
repetitive sequence is inserted in the 5⬘ J␥2.1 region. B and C, DNA sequence of 5⬘ J␥1.1 (B) and 5⬘ J␥2.1 (C) regions. STAT consensus motifs,
the nonamer and heptamer recombination signals, and an Alu repetitive
sequence are boxed. The initiation sites of germline transcription, the coding regions of J␥1.1 and J␥2.1, and the region used for reporter constructs
are shown by arrows.
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We and others have reported previously that TCR␥ genes are frequently rearranged in the mouse and human thymus (18, 27–30).
These results suggested that V␥-J␥ recombination takes place in
the majority of ␣␤ T cells in the thymus. Germline transcription
from unrearranged alleles usually precedes V(D)J recombination
and is considered as an index of locus accessibility (1). To explore
the mechanism of accessibility control of the human TCR␥ locus
(Fig. 1A), we examined germline transcription in a human erythroleukemia cell line, TF-1, by Northern blot analysis (Fig. 1B).
TF-1 cells proliferate depending on erythropoietin, GM-CSF, or
IL-3 and have the TCR␥ locus in germline configuration (data not
shown). Because stimulation by these cytokines activates STAT5
(22), we had thought that TF-1 cells may induce germline transcripts of TCR␥ genes as we reported previously with a mouse
IL-3-dependent cell line, Ba/F3. A high level of germline transcripts was detected in TF-1 cells cultured with GM-CSF. When
deprived of GM-CSF, TF-1 cells slightly down-regulated the
germline transcripts, although they still expressed a significant
level of the transcripts. The levels of the TCR␥ germline transcripts were quantitated and normalized by those of GAPDH transcripts. By cytokine stimulation, the germline transcripts were increased by 33% (data not shown). This result indicated that GMCSF signaling augments germline transcription of TCR␥ genes,
even though the induction is not tightly controlled by the cytokine.
Next we examined the STAT5 activation in TF-1 cells cultured
with or without GM-CSF (Fig. 1C). GM-CSF receptor signaling
usually activates and phosphorylates JAK2 and STAT5 proteins.
STAT5 protein was immunoprecipitated with anti-STAT5 Ab and
immunoblotted with anti-phosphotyrosine or anti-STAT5 Ab.
HUMAN TCR␥ GERMLINE TRANSCRIPTION BY STAT5
The Journal of Immunology
323
J␥2.1-C␥2. The J␥1.1-C␥1 transcripts started from two sites of
⬃100 and 110 bp downstream of the first STAT motif in 5⬘ J␥1.1
region (Fig. 2B). This distance between the motifs and transcription initiation sites is typical of STAT5-dependent promoters. The
J␥2.1-C␥2 transcripts started from two sites of ⬃70 bp (from
within the Alu sequence) and 400 bp downstream of the first STAT
motif in 5⬘ J␥2.1 region (Fig. 2C). Either of them may have started
under the influence of promoter activity within the Alu sequence.
To distinguish the J␥1.1-C␥1 and J␥2.1-C␥2 transcripts, we designed PCR primers common to both, and amplified and subcloned
cDNA of TF-1 cells cultured with and without GM-CSF. DNA
sequences of randomly picked up clones were determined and
compared (Table I). The J␥1.1-C␥1 transcripts were the majority
of the germline transcripts in TF-1 cells before and after cytokine
stimulation. This result suggested that the insertion of Alu sequence and other changes probably diminished the promoter activity of the 5⬘ J␥2.1 region.
STAT5 binds to the consensus motifs in 5⬘ J␥ regions
STAT5 transactivates the germline promoters of the human
TCR␥ locus
Next we checked by luciferase reporter assay whether the binding
of STAT5 to the motifs results in the induction of transcription
(Fig. 4). We used Ba/F3 cells because they give a higher level of
transfection efficiency than other cell lines. A 1.0-kb and a 1.3-kb
fragment of 5⬘ J␥1.1 and 5⬘ J␥2.1 regions, respectively, were
joined to a luciferase reporter gene (Fig. 4A). These plasmid DNAs
Table I. DNA sequences of J␥-C␥ germline transcripts in TF-1 cell
linea
No. of DNA Sequences
Identified as
GM-CSF
⫺
⫹
Total No. of DNA
Sequences Examined
J␥1.1-C␥1
J␥2.1-C␥2
15
16
13
14
2
2
a
The J␥-C␥ germline transcripts were amplified by RT-PCR from TF-1 cells
cultured with and without GM-CSF. PCR products were subcloned, and DNA sequences of randomly isolated clones were determined.
FIGURE 3. STAT5 binds to consensus motifs conserved in human 5⬘ J␥
regions. A, Oligonucleotide probes for EMSA. Oligonucleotide probes carrying mutations in the motifs (TTCNNNTCC; m1 and m2) also were designed. B–D, Binding of STAT5 to the consensus motifs in 5⬘ J␥ regions.
Nuclear extracts of Ba/F3 cells stimulated with IL-3 were incubated with
labeled oligonucleotide probes for the STAT motifs in 5⬘ J␥1 region, and
subjected to EMSA. The DNA-protein complexes were confirmed to contain STAT5 by supershift assay with anti-STAT5 Ab. The specificity of
binding was proved by competition assay with excess amount (10- and
50-fold) of unlabeled oligonucleotide. Specific binding to the motif also
was confirmed with oligonucleotide probes carrying mutations in the
motifs.
were electroporated into Ba/F3 cells together with an expression
plasmid coding for STAT5A. The cells were starved for 6 h and
then restimulated with IL-3. Cytokine stimulation alone induced
the expression of control mouse 5⬘ J␥1 reporter (Fig. 4B, 5⬘mJ␥1).
With exogenous STAT5A this induction was augmented about
threefold. The human 5⬘ J␥1.1 promoter showed similar pattern of
STAT5-dependent induction, suggesting that STAT5 can transactivate this promoter. In contrast, the 5⬘ J␥2.1 promoter revealed
relatively higher levels of reporter expression even without exogenous STAT5. In addition, induction by cytokine stimulation was
less drastic. This result suggested that the 5⬘ J␥2.1 region has lost
STAT5-dependent promoter activity. To test whether the Alu sequence is responsible for the impaired activity, we next deleted the
Alu sequence from the 5⬘ J␥2.1 fragment and similarly tested its
promoter activity (Fig. 4B, 5⬘J␥2.1⌬Alu). The deletion of the Alu
sequence did not result in any significant change of the promoter
activity. This result suggested that the promoter activity of the 5⬘
J␥2.1 region was diminished mainly by changes other than insertion of the Alu sequence.
We next checked whether the conserved motifs are important
for this transactivation. Different sets of mutations were introduced
in the motifs of the 5⬘ J␥1.1 promoter, and these were tested for
Stat5- and IL-3-dependent transactivation in Ba/F3 cells by luciferase reporter assay (Fig. 4C). Single mutations in any of the two
motifs caused dramatic decreases in transcription mediated by exogenous Stat5A. Double mutations resulted in similar decrease to
single mutations. This result suggested that the interaction of
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The coincidence of the STAT consensus motifs in two different 5⬘
J␥ regions led us to the idea that STAT proteins, activated by
IL-7R signaling, bind to these motifs and induce germline transcription as in the mouse (21). To test this, we first analyzed the
binding of STAT proteins to the motifs by EMSA (Fig. 3). Because DNA sequences around the STAT consensus motifs are
highly conserved between 5⬘ J␥1.1 and 5⬘ J␥2.1 regions, we designed oligonucleotide probes to 5⬘ J␥1.1 region (Fig. 3A). IL-3
stimulation induced binding activity to the oligonucleotide probe
A corresponding to the typical motif in the 5⬘ J␥ region, in Ba/F3
cells, similarly to a control mouse probe (Fig. 3B). By incubation
with anti-STAT5 Ab, this activity showed a supershift. Mutation in
the motif deprived the binding capacity of the probe. To test
whether STAT5 also can bind to the second atypical motif, we next
conducted EMSA with the oligonucleotide probe B that covers two
STAT motifs (Fig. 3C). Strong binding activity was detected after
IL-3 stimulation and showed a supershift by anti-STAT5 Ab. Single mutation in the first typical motif did not affect the interaction.
Double mutations in the first and the second motifs completely
abrogated the binding capacity of the probe. The interaction of
STAT5 proteins with the second motif was unequivocally proved
by the oligonucleotide probe C, which covers only the second motif (Fig. 3D).
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HUMAN TCR␥ GERMLINE TRANSCRIPTION BY STAT5
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FIGURE 4. Transcriptional activation of 5⬘ J␥1 promoter by STAT5. A,
Schematic illustration of luciferase reporter constructs. The 1.0-kb 5⬘ J␥1.1
and 1.3-kb 5⬘ J␥2.1 fragments with or without mutated motifs and Alu
sequence deletion, respectively, were flanked by luciferase cDNA. B, Transcriptional activation of 5⬘ J␥1.1 and 5⬘ J␥2.1 fragments by STAT5. Ba/F3
cells were transfected with the mixture of the luciferase reporter plasmids,
STAT5A expression vector, and ␤-galactosidase control vector, and stimulated with or without IL-3. Luciferase activity in the whole cell lysate was
normalized with ␤-galactosidase activity. Data are the mean ⫾ SE of triplicate data points from a representative experiment. A construct with the
mouse 5⬘ J␥1 germline promoter (5⬘mJ␥1) was used as a control. C, Full
transactivation of 5⬘ J␥1.1 fragment requires two intact consensus motifs.
Promoter activity of the 5⬘ J␥1.1 fragments carrying single or double mutations in the motifs was determined as described in B.
STAT5 with a set of two intact motifs probably plays an essential
role to activate germline transcription from the 5⬘ J␥1.1 promoter.
Conserved STAT motifs in the human and mouse TCR␥ loci
Comparison of cis-control elements in the TCR␥ locus between
human and mouse revealed that STAT consensus motifs are conserved in 5⬘ J␥ germline promoters, 3⬘ enhancers, and a locus
control region-like element, HsA (Fig. 5). Human 5⬘ J␥1.1 and
mouse 5⬘ J␥1 germline promoters have relatively high sequence
homology (61% for ⬃600 bp; Fig. 5A). The first STAT motif in
human is conserved as the third motif in mouse. Other STAT motifs were not conserved. Homology search for other transcription
FIGURE 5. Comparison of cis-control elements in the human and mouse
TCR␥ loci. Human and mouse 5⬘ J␥ germline promoters (A), 3⬘ enhancers (B),
and HsA elements (C) were compared. (⫺), aligned identical bases and gaps
are indicated (.). Conserved motifs for transcription factor and the nonamer and
heptamer recombination signals are shown by boxes. Germline transcription
and J␥ genes are shown by arrows. The DNase footprints (NF-␥1 to NF-␥6)
of mouse 3⬘ enhancer are underlined in B. The consensus transcription factor
binding sites in mouse HsA are underlined in C.
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FIGURE 6. Conserved STAT motifs in the mouse
and human TCR␥ loci. Exons and pseudogenes are depicted as f and 䡺, respectively. STAT consensus motifs are conserved in 5⬘ J␥ germline promoters, 3⬘ enhancers (E), and HsA elements between mouse and
human. STAT5, activated by IL-7R signaling, may interact with the motifs in E␥ and HsA and control general accessibility of the locus. STAT5 binds to the motifs in the germline promoters, recruits transcriptional
coactivators, and acetylates histones, resulting in the induction of germline transcription and local accessibility.
Discussion
In this study, we first demonstrated that J␥-C␥ germline transcripts
are induced in a GM-CSF-dependent human erythroleukemia cell
line (Fig. 1). STAT consensus motifs are present in 5⬘ regions of
J␥1.1 and J␥2.1 gene segments (Fig. 2), and activated STAT5
binds to these motifs (Fig. 3). By a reporter gene assay, we showed
that the promoter of J␥1.1 germline transcription is transactivated
by STAT5 and that mutations in the STAT consensus motifs abrogate this activity. The 5⬘ J␥2.1 region have lost STAT5-dependent promoter activity, probably because of changes other than
insertion of the Alu sequence (Fig. 4). These results basically are
the same with those in the mouse TCR␥ locus (21). Thus, this
study demonstrates that STAT5 induces germline transcription in
the TCR␥ locus of both mouse and human, and suggests the possibility that this mechanism may play an essential role in controlling the accessibility of the TCR␥ locus.
STAT motifs are conserved among 5⬘ J␥ germline promoters,
E␥ elements, and a locus control region-like element, HsA, in both
mouse and human TCR␥ loci (Figs. 5 and 6). This evidence indicates that these cis-control elements altogether cooperate to regulate the accessibility of the TCR␥ locus through their possible
interaction with STAT5. First, it is probable that the E␥ elements
control the general accessibility of the locus, as has been described
for other TCR and Ig loci (2, 3). Second, the HsA element also is
likely to contribute to the locus-wide accessibility (32). Finally, as
characterized in the previous (21) and this study, STAT5 induces
germline transcription and regulates the local accessibility near J␥
gene segments by histone acetylation (S.K.Y. and K.I., unpublished data).
Enhancers and promoters can direct the hyperacetylation of core
histones because of histone acetyltransferase activity of transcriptional coactivators. Histone acetylation is tightly correlated with
V(D)J recombination in the TCR␣␦ and TCR␤ loci and is proposed as a mechanism for coupling enhancer activity to accessibility (4, 5). In the TCR␥ locus, histones are hyperacetylated at
transcriptionally active J␥ segments in normal but not in IL-7R␣deficient thymocytes precursors. Interestingly, the acetylation levels of E␥ region also are high in normal but low in IL-7R␣-deficient thymocytes (S.K.Y. and K.I., unpublished data). Interaction
of STAT5 with E␥ may recruit transcriptional coactivators and
induce histone acetylation. It will be interesting to elucidate the
role for STAT5 in locus-wide accessibility control of the TCR␥
locus through the E␥ and HsA elements.
The phylogenetic conservation of STAT consensus motifs in
three types of cis-control elements indicates that the TCR␥ locus is
under the strong influence of IL-7R and STAT5 signaling. In contrast, the STAT motifs are not conserved in enhancers and germline promoters of the other TCR loci. After entry into the thymus,
T cell precursors first proliferate by stimuli from c-kit and the
IL-7R. At this stage, they receive a signal from the IL-7R to induce
the rearrangement and transcription of the TCR␥ locus. This will
help them to commit and maintain themselves to the ␥␦ T cell
lineage. However, at later stages of ␣␤ T cell development, this
signal for ␥␦ T cells seems to be shut off. We speculate that preTCR signaling may cancel this IL-7R signal to facilitate the differentiation into the ␣␤ T cell lineage. During evolution of the
immune system, ␥␦ T cells may have emerged first with the simple
mechanism that IL-7 produced from epithelial cells of the skin and
intestine induces the V(D)J recombination and cell expansion. ␣␤
T cells probably evolved later with a more sophisticated system
where pre-TCR signaling invalidates the IL-7R signal enabling
positive and negative selection to operate on the basis of interactions between ␣␤ TCRs and self-MHC.
Acknowledgments
We thank Dr. T. Ikeda for human thymus; Dr. T. Kitamura for TF-1 line;
T. Taniuchi, M. Tanaka, Y. Tabuchi, T. Toyoshima, and Y. Doi for their
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factor binding sites failed to find any site in common between
human and mouse. The human and mouse E␥ also have high sequence homology (57% for ⬃750 bp; Fig. 5B). As previously reported (31), the region from NF-␥2 to NF-␥4 sites is highly conserved between human and mouse (74% for ⬃140 bp). In the
NF-␥2 site, a STAT consensus motif is conserved. A PEBP2 binding site also is conserved in the NF-␥3 site.
A previous report suggested that a DNase I hypersensitive site
between V␥5 and V␥2 genes serves as a locus control region in
concert with E␥ (32). Comparison between human and mouse V␥
regions demonstrated that human V␥1.8 and mouse V␥5 and human V␥4P and mouse V␥2 have sequence homology (data not
shown). Besides these, mouse HsA element have a homologous
region in human between V␥2 and V␥3P genes (59% for ⬃510 bp;
Figs. 5C and 6). Thus, the relative location of HsA also is conserved between human and mouse. A STAT motif and a p300
motif are conserved in human and mouse HsA. However, other
GATA-3, GAGA, LEF/TCF, E box, and myb motifs in the mouse
HsA are not conserved in human. A 2-kb DNA region including
mouse HsA also has 50 – 60% sequence homology to a DNA region between human V␥2 and V␥3P (data not shown). Thus, these
results demonstrated phylogenetic conservation of STAT consensus motifs in three types of cis-control elements in the TCR␥ locus.
HUMAN TCR␥ GERMLINE TRANSCRIPTION BY STAT5
excellent technical assistance; and Drs. S. Fujimoto and K. Kinoshita for
critically reading the manuscript.
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