From the Clinical Research Institute of
Montréal, Montréal, Québec H2W 1R7, Canada and the
** Departments of Pharmacology and Biochemistry and the
Molecular Biology Program, University of Montréal,
Montréal, Québec H3C 3J7, Canada
Received for publication, September 25, 2002, and in revised form, December 23, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The expression of the pT The development of Recently, the promoter and the enhancer sequences of the
pT The mechanism through which bHLH factors regulate pT Cell Lines, Cell Culture, and Mice--
The DN T-cell line
AD10.1 was cultured in IMDM (Invitrogen) containing 10% inactivated
fetal calf serum and 50 µM
E2A+/ FACS Analysis and Cell Sorting--
Thymi were removed from
newborn, 1-week, or 2-month-old mice. Single cell suspensions and
immunostaining were performed as previously described (9). Thymocytes
were stained with anti-CD4 (H129.19), anti-CD8 (53-6.7), anti-CD25
(7D4), anti-CD44 (IM7), anti-CD3 RNA Preparation, cDNA Synthesis, and PCR
Amplification--
Total RNA was prepared according to Chomczynski and
Sacchi's protocol (31), using tRNA as carrier for ethanol
precipitation. First-strand cDNA synthesis and specific PCR was
performed as described (9). cDNA samples were 2-fold diluted in 1×
PCR buffer, and 2 µl were added in the PCR mixture containing 1 µM of each specific 5' and 3' primers, 1 mM
dNTP, 1.5 mM MgCl2, and 1 unit of
TaqDNA polymerase (Invitrogen). Primer sequences are
available upon request. Twenty-eight (pT
SyberGreen quantitative PCR was performed on a MX4000 apparatus
(Stratagene) according to the manufacturer's instructions. One
microliter of the cDNA sample was added in the PCR mixture containing 0.5 µM (pT Constructs, Expression Vectors, and Transfection Assays--
The
pT Western Blot Assays--
Protein expression of E47 and HEB were
analyzed by Western blot in nuclear extract from wild-type bone marrow
and wild-type and CD3 Electrophoretic Mobility Shift Assays (EMSA)--
Nuclear
extracts were prepared from AD10.1, AD10.1-MSCV, or AD10.1-SCL cell
lines as previously described (20). We used 8 µg of nuclear extracts
per binding reaction containing 100 ng of poly(dI-dC) in 20 mM Hepes (pH 7.5), 50 mM KCl, 1 mM
EDTA, 1 mM dithiothreitol, 5% glycerol, and 10 µg of
bovine serum albumin. For competition assays, 1-100-fold molar excess
of unlabeled oligonucleotides were added, and the mixture was kept on
ice for 30 min before adding 50,000 cpm of double-stranded
oligonucleotide probe. Supershift assays were performed with 2 µg of
the following antibodies: anti-E2A mouse monoclonal antiserum YAE
(Santa Cruz Biotechnology Inc.), anti-HEB rabbit polyclonal antiserum
(Santa Cruz Biotechnology Inc.), anti-SCL mouse monoclonal antibodies
BTL73 and BTL136 (provided by Dr. D. Mathieu-Mahul, Institut de
Génétique Moléculaire, Montpellier), and a control
anti-Myc antibody. Protein-DNA complexes were resolved by 4% PAGE in
0.5× Tris borate-EDTA at 150 V, at 4 °C. All EMSA experiments were
performed with an excess of probe. Oligonucleotide sequences used in
EMSA experiments are available upon request.
ChIP Assays--
Chromatin immunoprecipitations were performed
essentially as described previously (19, 32, 33). Twenty million
AD10.1-MSCV, AD10.1-SCL, or CD3 E2A- and HEB-deficient Mice Express Decreased Level of pT E2A and HEB Gene Products Bind the pT Functional Importance of Two E-box Binding Sites within the pT
We have previously reported that ectopic SCL expression in immature
thymocytes represses the expression of the endogenous pT E2A and HEB Gene Products as Well as SCL-containing Complexes Bind
E-box Elements of the pT
The specificity and the relative affinities of E2A-HEB and
SCL-containing complexes for E-box binding sites were tested by competition assays using increasing amounts of unlabeled
double-stranded oligonucleotides (Fig. 6C). Both E2 and E3
competitors efficiently displaced E2A-HEB heterodimers and
SCL-containing complexes binding to DNA (lanes 3-6 and
13-16). In contrast, mutations within E3 (lanes
7-10) and E2 (data not shown) sequences disrupted DNA binding as
these competitors failed to displace E-protein complex formation. Interestingly, higher concentrations of E1 or E4 competitors are required to displace E2A-HEB heterodimer formation on either E3 (lanes 20-27) or E2 probes (data not shown). The relative
levels of binding of E2A-HEB or SCL-containing complexes to these E-box sequences were E3 > E2 > E1 > E4. Analysis of
dissociation constants indicates a 70-fold difference in binding
affinities between E3 and E4 sequences. Interestingly, both E3 and E2
sequences conform to E47 (38) and HEB (39) consensus sequences while E4
does not (Fig. 4C). These relative binding affinities are in
agreement with our transactivation assays showing that E1 has a weaker
contribution to enhancer activity as compared with E2 and E3, and that
E4 is a negative regulator (Fig. 4).
SCL-containing Complexes Bind the pT Relative Levels of E2A, HEB, and SCL Determines pT In the present study, we show that the bHLH factors E2A and HEB
drive pT Importance of E2A-HEB in Driving Stage-specific Activity of the
pT
At the molecular level, the regulatory elements of the pT
Here we demonstrate that in addition to c-Myb and the CSL-NICD complex,
E2A and HEB are critical determinants of pT
There is substantive controversy with regards to the contribution of
E-boxes to pT
Transient transactivation assays in heterologous cells indicate that
E47 can activate the pT SCL-E2A/HEB Complexes Occupy E-box Elements within the
pT
In summary, we unambiguously identify the pT
gene is required for effective selection, proliferation, and survival
of
T-cell receptor (
TCR)-expressing immature thymocytes.
Here, we have identified two phylogenetically conserved E-boxes within
the pT
enhancer sequence that are required for optimal
enhancer activity and for its stage-specific activity in immature T
cells. We have shown that the transcription factors E2A and HEB
associate with high affinity to these E-boxes. Moreover, we have
identified pT
as a direct target of E2A-HEB heterodimers in immature thymocytes because they specifically occupy the enhancer in vivo. In these cells, pT
mRNA levels are
determined by the presence of one or two functional E2A or
HEB alleles. Furthermore, E2A/HEB transcriptional activity
is repressed by heterodimerization with SCL, a transcription factor
that is turned off in differentiating thymocytes exactly at a stage
when pT
is up-regulated. Taken together, our observations
suggest that the dosage of E2A, HEB, and SCL determines pT
gene
expression in immature T cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
T cells from multipotent progenitors is
a complex and multistep process that is critically dependent on genetic
recombination and extracellular signals. Maturation of recent thymic
immigrants from bone marrow derived precursors is characterized by the
sequential expression of the
pre-TCR1 complex and of
several surface markers including CD4 and CD8 (1). Within the CD4/CD8
double-negative (DN) population, cell survival, cell proliferation, and
allelic exclusion as well as the subsequent transition to the more
mature CD4/CD8 double-positive (DP) stage (2, 3) are critically
dependent on pre-TCR signaling. The pre-TCR is formed by the
association of a correctly rearranged
TCR chain with the invariant
pT
chain and signaling molecules of the CD3 complex. Since
expression of the pre-TCR is critical for
T-cell
differentiation, dissecting the molecular program that drives the
expression of its components is of particular interest to understand
the transcriptional regulation of early T-cell development.
gene have been cloned (4) and partially characterized
(5-8). A 250-bp enhancer element located 4-kb upstream of the
initiation site is necessary and sufficient for specific expression of
the pT
gene in immature DN thymocytes in transgenic mice
(5). Several transcription factors have been shown to regulate enhancer activity, including c-Myb, and the activated form of Notch (5-7). We
have previously observed that the basic helix-loop-helix (bHLH) transcription factor, HEB, plays a critical role in the regulation of
pT
expression (9). The E2A and HEB genes
encode class I bHLH transcription factors, also called E-proteins
(E12-E47 and HEB, respectively), which bind specific DNA sequences
(E-box, CANNTG) as homo- or heterodimers. E-protein function is
essential for T- and B-cell development as revealed by gene-targeting
experiments or expression of dominant-negative molecules, such as Id
factors or the SCL oncogene (9-15). E2A- and
HEB-deficient mice, as well as Id or
SCL-LMO1 transgenic mice display a T-cell differentiation defect characterized by a partial differentiation block at the DN to DP
transition associated with thymic atrophy (9, 16-18). Our previous
study revealed that the partial differentiation block of SCL-LMO1
transgenic thymocytes is, at least in part, due to decreased pT
gene expression in immature DN thymocytes (9). SCL is a tissue-specific
bHLH transcription factor that forms heterodimers with E-proteins and
acts as a transcriptional activator or repressor, depending on the
cellular context (9, 18-23). SCL expression is detected in primitive
DN thymocytes, and it is normally shut off during T-cell
differentiation (9). Enforced SCL expression, in combination with its
nuclear partner LMO1, inhibits E-protein function during early
thymopoiesis (9) and subsequently leads to leukemogenesis (18,
24-26).
expression
remains to be documented. Here, we provide evidence that E2A-HEB
oligomers determine pT
mRNA levels in DN thymocytes in vivo, through direct binding to two conserved E-boxes of the
upstream enhancer. The effect is tightly dose-dependent and
is disrupted by heterodimerization with SCL.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol. The
parental cell line was retrovirally infected with MSCV empty or
MSCV-SCL-expressing vector, and stable transfectants were kept under
neomycin selection (1 mg/ml). For the detection of SCL protein, nuclear
extract were analyzed by Western blot using the anti-SCL mouse
monoclonal antibodies BTL73 and BTL136 (generously provided by Dr. D. Mathieu-Mahul, Institut de Génétique Moléculaire, Montpellier) and an anti-Sp1 rabbit polyclonal antibody (Geneka Biotechnology Inc., Montreal), as control for loading.
and HEB+/
mice
were kindly provided by Dr. Y. Zhuang (Duke University Medical Center,
Durham, NC) (27-29) and bred with C57Bl6/J mice for more than three
generations. Heterozygous animals were crossed to get homozygous
knockout mice, and all the litters were genotyped by PCR as described
(27, 28). CD3
/
mice (C57Bl6/J) were
kindly provided by Dr. B. Malissen (Centre d'Immunologie INSERM-CNRS
de Marseille-Luminy, Marseille) (30). Animals were maintained under
pathogen-free conditions according to institutional animal care and use guidelines.
(145-2C11), anti-
TCR
(H57-597), anti-TCR
(GL3) (PharMingen BD Biosciences, San Jose),
and/or anti-Thy1.2 (30-H12) (Sigma) antibodies. Four-color
immunofluorescence analyzes were performed on Moflo flow cytometer
(Cytomation, Denver) or FACStar flow cytometer (BD Biosciences, San
Jose) using dual laser excitation. When cell sorting was performed,
10,000 cells were collected in RNA lysis buffer.
, E2A, and HEB) or 22 (S16)
cycles of amplification were performed and 10 µl of each reaction
were loaded on a 1.2% agarose gel, transferred on nylon membranes
(Biodyne B, Pall Corporation, Ann Arbor) and hybridized with the
corresponding internal oligonucleotide probes. The hybridization
signals were analyzed on a PhosphorImager apparatus (Molecular
Dynamics, Amersham Biosciences).
, E2A, and HEB) or 1 µM (S16) of each specific 5' and 3' primers, 0.2 mM dNTP, 1.5 mM MgCl2, 6%
glycerol, 1 unit of TaqDNA polymerase (Invitrogen) in a
final volume of 20 µl. SyberGreen quantitative dye and Rox passive
dye (Molecular Probes, Eugene, OR) were added to the mixture. Forty
cycles of amplification were performed, followed by 38 cycles of
denaturation-annealing steps. Amplification plots and dissociation
curves were analyzed with the MX4000 (Stratagene) and Excel (Microsoft,
Redmond, WA) softwares.
enhancer element (0.25 kb
BstEII-MluNI fragment), a gift from Dr. P. Leder
(Harvard Medical School, Boston, MA) (4), was subcloned upstream of a
minimal TATA promoter into the pXpIII vector (19). Mutations of E-box
elements were performed by PCR, and all constructs were subsequently
sequenced. Transactivation assays were performed by electroporation as
previously described (9). CMV-
gal (Clontech BD
Biosciences) was added in all samples as an internal control for
transfection efficiency. Cells were harvested 36 h after
transfection, and luciferase activity was assayed using a Berthold
LB953 luminometer.
-galactosidase assays were performed using
o-nitrophenyl
-D-galactopyranoside as a substrate in 96-well flat-bottomed plates for 20 min, and optical density (OD) at 405 nm was measured. The results shown are the mean ± S.D. normalized for the
-galactosidase activity of one experiment performed in duplicate and representative of 3-5 experiments.
/
thymus using the
anti-E47 rabbit polyclonal antiserum N-649 (Santa Cruz Biotechnology
Inc.), the anti-HEB rabbit polyclonal antiserum (Santa Cruz
Biotechnology Inc.) and an anti-PTP-1D mouse monoclonal antibody (BD
Biosciences), as loading control. For the detection of SCL protein,
Western blot of nuclear extract from AD10.1-MSCV, AD10.1-SCL cell lines
were done using the anti-SCL mouse monoclonal antibodies BTL73 and
BTL136 (generously provided by Dr. D. Mathieu-Mahul, Institut de
Génétique Moléculaire, Montpellier) and an anti-Sp1 rabbit polyclonal antibody (Geneka Biotechnology Inc.), as control for loading.
/
primary
thymocytes cells were fixed by adding 1% formaldehyde to the culture
media for 10 min at room temperature. Formaldehyde was then quenched by
addition of 0.125 M glycine. Subsequent steps were
performed at 4 °C. Fixed cells were pelleted by centrifugation, washed twice in cold phosphate-buffered saline, then washed once in
Triton buffer for 15 min (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100) and
once in NaCl buffer for 15 min (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). Cells were pelleted, resuspended in RIPA buffer (10 mM
Tris-HCl, pH 8.0, 140 mM NaCl, 1 mM EDTA, 1%
Triton X-100, 0.1% SDS, 0.1% deoxycholate) and sonicated (7 × 10 s bursts) to make soluble chromatin ranging in size from 500 to
1000 bp. Cellular debris were removed by centrifugation (16,000 × g for 10 min), and protein concentrations were determined by
Bradford staining. Aliquots were reserved for isolation of input DNA,
while 1 mg of chromatin extract was incubated overnight at 4 °C with
specific antibodies: anti-E47 rabbit polyclonal antiserum N-649 (Santa
Cruz Biotechnology Inc.), anti-HEB rabbit polyclonal antiserum (Santa
Cruz Biotechnology Inc.), anti-SCL mouse monoclonal antibodies BTL73
and BTL136 (provided by Dr. D. Mathieu-Mahul, Institut de
Génétique Moléculaire, Montpellier), anti-rabbit IgG
(Sigma) and anti-HA mouse monoclonal antisera (Covance). DNA-protein
complexes were immunoprecipitated with pansorbin® cells
(Calbiochem, San Diego) for 30 min at 4 °C, then,
pansorbin® cells were sequentially washed twice with 1 ml
of RIPA buffer containing 500 mM of NaCl, twice with 1 ml
of LiCl buffer (10 mM Tris-HCl, pH 8.0, 250 mM
LiCl, 1 mM EDTA, 1% Nonidet P-40, 1% deoxycholate) and
twice with 1 ml of TE buffer. Chromatin samples were then eluted by
heating for 15 min at 65 °C in 300 µl of elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS).
After centrifugation, supernatants were diluted by addition of 300 µl
of TE buffer and heated overnight at 65 °C to reverse cross-links.
RNA and proteins were sequentially degraded by addition of 30 µg of
RNase A for 30 min at 37 °C, and 120 µg of proteinase K for 2-3 h
at 37 °C. DNA was phenol/chloroform-extracted and
ethanol-precipitated in the presence of 10 µg of tRNA as a carrier.
DNA samples were resuspended in 30 µl of water, serially diluted, and
30 cycles of amplification were performed using specific primers for
pT
enhancer, pT
promoter, and the
hypoxanthine phosphoribosyltransferase (HPRT) promoter. Oligonucleotide sequences are available upon request. One-fifth of PCR
products were loaded on a 1.2% agarose gel, transferred on Biodyne B
membrane (Pall Corporation, Ann Arbor), hybridized with internal
oligonucleotide probes, and analyzed on a PhosphorImager apparatus.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mRNA--
The essential role of E-proteins during T-cell
development has been revealed by gene-targeted disruption of the
E2A and HEB genes in mice (9;14-18;34;35). As
shown in Fig. 1B, E2A- and
HEB-deficient mice have an increased percentage of immature DN
thymocytes and a decreased number of total thymocytes indicating a
partial block of T-cell differentiation at the DN to DP transition
step, a critical checkpoint controlled by the pre-TCR. Differentiation
of TCR
lineage thymocytes were analyzed in
E2A+/
and HEB+/
mice
and wild-type littermates. We show that the percentage
TCR
+ thymocytes in the immature DN subsets of
E2A or HEB heterozygote mice is similar to that
of wild-type littermates (Fig. 1C). Thus, differentiation in
the
lineage is unaffected, despite reduced levels of both E2A
and HEB in heterozygous mice (Fig.
2A). We have previously
reported that HEB deficiency is associated with a decreased level of
pT
mRNA in DN thymocytes (9). Since E2A also controls the DN to
DP transition, we assess here the role of E2A in vivo in
regulating pT
expression, as compared with that of HEB. We therefore
used semiquantitative and real-time PCR to investigate pT
mRNA
level in E2A- and HEB-deficient mice, as well as in heterozygous and
wild-type littermates. Immature DN and DP thymocyte populations were
purified by flow cytometry according to their surface expression of
CD4, CD8,
TCR, and Thy1.2 markers. Within the DN compartment, we
observed a 3- and 5-fold decrease of pT
mRNA level in
E2A-deficient thymocytes as compared with wild-type controls by
semiquantitative RT-PCR and real-time PCR, respectively (Fig. 2,
C and D). Similarly, HEB-deficient DN thymocytes
show a 2-3-fold decrease in pT
, as described previously (9).
Interestingly, E2A and HEB heterozygous DN
thymocytes expressed, on average, intermediate levels of pT
mRNA
as compared with wild-type and null DN thymocytes, despite some
variations between animals. This decrease is in direct correlation with
the decreased E2A or HEB mRNA levels observed in total thymi of
heterozygous mice (Fig. 2A). Moreover, decreased pT
mRNA levels were not due to an increase in
T cells, since
the percentage of CD3
+TCR
+ cells in
wild-type and heterozygous mice is not significantly different. For
this reason, we observed the same decrease in pT
mRNA level,
even when TCR
+ cells were excluded from analysis
(Fig. 2E). Despite this lower level of pT
, T-cell
differentiation in heterozygous mice was not impaired as assessed by
flow cytometry analysis, but the total numbers of thymocytes were
slightly lower and were on average 60% (n = 4) of that
of wild-type littermates (Fig. 1B). The observation is in
agreement with the reduced E2A and HEB mRNA levels observed in
heterozygous mice (Fig. 2A) and reveals that E2A and HEB are haploinsufficient in the thymus. Finally, E2A deficiency did not affect
pT
levels in the more mature DP thymocytes, while HEB deficiency
consistently impaired its expression. Together, these results indicate
that E2A and HEB gene dosage plays an important role in regulating pT
expression specifically at the immature DN
stage and suggest that E2A and HEB might have overlapping as well as
distinct function during T-cell development.
View larger version (44K):
[in a new window]
Fig. 1.
Perturbation of T-cell development in
E2A and HEB knockout mice. A,
immunostaining of normal thymocytes reveals seven subsets according to
their degree of maturity. B, thymocytes from newborn
(HEB) or 1-week-old (E2A) mice were stained with
CD4, CD8, TCR, and Thy1.2 antibodies and sorted by flow cytometry.
C, thymocytes from newborn (E2A) or 1-week-old (HEB) mice
were stained with CD4, CD8, CD3
, TCR
, and Thy1.2 antibodies
and analyzed by flow cytometry. The percentages of
TCR
+/CD3
+ DN thymocytes in different
litters (
, X, newborn;
, 1-week old;
, 2-month-old)
are shown on the right. Representative FACS profiles for
wild-type (WT), heterozygous (+/
), and knockout (
/
)
littermates are shown, and the percentages of cells in each quadrant
are indicated. The total numbers of cells per thymus are indicated in
brackets.
View larger version (38K):
[in a new window]
Fig. 2.
Reduction of pT
expression in DN thymocytes of E2A and HEB
knockout mice. Quantification of E2A (A) and HEB
(B) gene expression in thymocytes of wild-type and
heterozygous littermate were performed by semiquantitative and
real-time RT-PCR using specific primers for E2A, HEB, and ribosomal S16
mRNA, the latter as control for the amounts of cDNA. mRNA
levels are shown as percentage of wild-type levels. C,
thymocytes were fractionated into two subsets according to their degree
of maturity; DN
(CD4
/CD8
/
TCR
/Thy1.2+)
and DP
(CD4+/CD8+/
TCR+/Thy1.2+).
Semiquantitative RT-PCR were performed on purified cell populations
using specific primers for pT
and S16 (internal control).
D, real-time RT-PCR were performed to quantify pT
gene
expression in DN and DP cells from wild-type,
E2A+/
and E2A
/
mice. pT
amplification curves were normalized for the amount of
cDNA using S16 as control. mRNA levels for heterozygous and
knockout mice are 60 and 20% of wild type at the DN stage and 60 and
110% at the DP stage. E, mRNA levels of pT
were
analyzed by semiquantitative RT-PCR as described above in thymocytes of
DN
(CD4
/CD8
/CD3
/Thy1.2+/TCR
)
and DP
(CD4+/CD8+/CD3
+/Thy1.2+/TCR
)
subset depleted of TCR
-positive cells of wild-type and
heterozygous mice for E2A and HEB. Results are
shown as percentages of wild-type levels.
Enhancer in Vivo in DN3
Thymocytes--
The expression of the pT
gene is
directed by a promoter and a 5' enhancer, both containing multiple
E-protein binding sites (E-box) (4, 5). Since E2A and HEB deficiency
leads to decreased pT
expression, we addressed the question of
whether these proteins associate with pT
regulatory
sequences in primary thymocytes in vivo. We therefore
performed ChIP using primary thymocytes. Within the DN subset,
thymocyte maturation can be followed according to the sequential
expression of CD44 and CD25 molecules (1), thus identifying 4 subpopulations (DN1 to DN4), as described in Fig. 1. Since the
pT
gene is expressed at the DN3 and DN4 stages, we used
thymocytes from CD3
/
mice that are
arrested at the DN3 stage because of the lack of a pre-TCR signal (30,
36, 37). E2A and HEB protein expression assessed by Western blotting
were higher in these thymocytes as compared with wild-type thymocytes,
which contain essentially cells at the DP stage, or to bone marrow
cells (Fig. 3C). Cross-linked chromatin extracts were prepared and subjected to immunoprecipitation using specific antibodies against E2A and HEB, as well as an
isotype-matched control antibody (rabbit IgG). After
immunoprecipitation, decross-linking, and purification, serial
dilutions of DNA templates were used for PCR amplification using
oligonucleotide primers flanking the pT
enhancer regions
(Fig. 3A). Since, gel shift assays revealed that HEB could
bind E-boxes located within the pT
promoter sequence (5,
8), we also used oligonucleotides flanking the promoter region (Fig.
3A). As shown in Fig. 3B, anti-E47 and anti-HEB
antibodies efficiently immunoprecipitated the pT
enhancer
sequence as well as the pT
promoter sequence, whereas
little or no background was observed with a control antibody. The
specificity was further confirmed by the absence of amplification of an
irrelevant promoter sequence, the HPRT promoter that is not
regulated by E2A or HEB.
View larger version (38K):
[in a new window]
Fig. 3.
E2A and HEB specifically associate with the
pT enhancer and promoter in primary thymocytes.
A, schematic diagram of the pT
locus;
arrows show the position and orientation of primers used for
ChIP assays. B, chromatin immunoprecipitation assays were
performed as described under "Experimental Procedures" using
cross-linked CD3
/
thymocytes nuclear extracts.
Anti-HA and RbIgG were used as isotype-matched controls for
immunoprecipitations. 5-fold serial dilutions of immunoprecipitated DNA
were used for amplification with specific primers for pT
enhancer and promoter regions. The HPRT promoter region was
amplified as a negative control. Input chromatin served as a positive
control for PCR amplification. The PCR products were analyzed by
agarose gel electrophoresis, transferred onto Biodyne B membrane, and
hybridized with an internal oligonucleotide. C, Western blot
of nuclear extract of wild-type and CD3
/
thymus, wild-type bone marrow, and AD10.1 cell lines was performed
using specific antibodies against E47, HEB, and PTP-1D, the latter as
control for loading.
Enhancer--
It has been previously reported that the
pT
enhancer element is essential for specific pT
expression in immature DN thymocytes (4, 5). This enhancer element
contains potential binding sites for different transcription factors
(YY1, ZBP-89, Sp1, c-Myb, and CSL) and particularly four E-boxes (Fig.
4A). We therefore cloned the
pT
enhancer sequence upstream of a minimal TATA promoter, and the luciferase reporter gene and confirmed that the enhancer is
active in the pT
+ "immature" T-cell line AD10.1 but
not in the "mature" T-cell line Jurkat nor in fibroblast cell lines
(data not shown). In order to determine the importance of E-boxes in
driving enhancer activity in the immature DN AD10.1 cell line, each of
the four E-box sites was mutated individually or in combination. As
shown in Fig. 4B, mutation of either E2 or E3 sites induced
a 3-4-fold decrease of pT
enhancer activity, while
simultaneous mutations of these two sites abolished enhancer activity.
The E1 site seems to play a weaker role in transcriptional activity
since its mutation modestly affected enhancer activity when E2 and E3
were intact. However, E1 mutation combined with E2 or E3 mutations
further decreased enhancer activity, revealing its potential role as a positive regulator when E2 or E3 were mutated. Interestingly, mutation
of the E4 site induced a 2-3-fold increase of enhancer activity,
either alone or in combination with E1 or E3 mutations, suggesting that
it negatively regulates enhancer activity. Sequence comparison
indicates that E2 and E3 sequences (CACCTG) match with the E47 and HEB
consensus sequences (Fig. 4C), while the E4 sequence (CACGTG) differs, suggesting that this site might bind a different factor endowed with repressive activity (Fig. 4C). Together,
these results indicate the importance of E-box binding sites for
optimal pT
enhancer activity.
View larger version (33K):
[in a new window]
Fig. 4.
The integrity of two E-boxes is critical for
pT enhancer activity. A, schematic
representation of the pT
enhancer showing its different
binding sites. B, point mutations of specific E-box sites
impair pT
enhancer activity. The AD10.1 DN T-cell line
was transfected with wild-type or mutant pT
enhancer
constructs as shown. Results are expressed as luciferase activity
relative to the minimal TATA promoter and represent the average ± S.D. of replicate determinations and are representative of
(n) independent experiments. Luciferase reporter activities
were normalized to that of an internal control (CMV-
gal).
C, sequences of the different E-box sites within the
pT
enhancer. Shown in bold are residues that
match the core E47 (38) or HEB (39) consensus.
gene and that HEB overexpression rescues pT
enhancer
activity (9). Using the real-time PCR technique, we show here that
ectopic SCL expression in AD10.1 cells (Fig.
5A) induced a 3-fold decrease of endogenous pT
mRNA level (Fig. 5B), and this
decrease was confirmed by semiquantitative RT-PCR (Fig. 5C)
(9). Furthermore, this lower mRNA level is in direct correlation
with a 3-4-fold decrease of the pT
enhancer activity as
measured by transient transfection assays (Fig. 5D). To test
whether SCL-mediated repression depends on E-protein function, we
transiently expressed reporter constructs containing wild-type or
mutated E-box sites together with the SCL expression vector. As shown
in Fig. 5D, SCL decreased pT
enhancer activity
to the same extent as mutations of either E2 or E3 binding sites.
Moreover, enforced SCL expression did not further decrease the activity
of E2- or E3-mutated constructs. On the opposite, SCL overexpression
was still able to repress enhancer activity when E4 was mutated.
Together, these results demonstrate that SCL-mediated repression of the
pT
enhancer activity requires the integrity of the two
E-box binding sites, E2 and E3, suggesting that SCL directly represses
E-protein function.
View larger version (28K):
[in a new window]
Fig. 5.
Repression of pT enhancer
activity by SCL is mediated by the E2 and E3 sites. A,
Western blot against SCL was performed using nuclear extract of
AD10.1-MSCV and AD10.1-SCL cell lines, the blots were revealed using a
monoclonal anti-SCL antibody, stripped, and further analyzed with a
polyclonal anti-Sp1 antibody, as control for loading. B,
real time RT-PCR was performed to quantify pT
mRNA levels in
AD10.1-MSCV and AD10.1-SCL cell lines. Amplification curves were
normalized for the amount of cDNA using S16 as control. pT
mRNA levels in AD10.1-SCL cell lines were 30% of that obtained in
the AD10.1-MSCV control. C, SCL expression in AD10.1-MSCV
and AD10.1-SCL cell lines was investigated by semiquantitative RT-PCR.
S16 mRNA amplification was used as control for the amount of
cDNA. D, the integrity of the two E-box sites is
critical for repression of pT
enhancer activity by SCL.
AD10.1 cells were transfected with wild-type or mutated enhancer
constructs, and the MSCV empty vector (open bars) or MSCV
SCL-expressing vector (solid bars). Enhancer activities were
calculated as in Fig. 4.
Enhancer in Vitro--
E-box mutations or
SCL-induced repression of the pT
enhancer activity
indicate the crucial role of E-proteins in regulating pT
gene expression. Since E2A or HEB occupy the pT
enhancer in DN thymocytes, we addressed the question of whether E2A
or HEB gene products directly bind E-boxes within the
pT
enhancer. We therefore performed EMSA using nuclear
extracts prepared from AD10.1 cells and specific oligonucleotide probes
that cover the E2 or E3 binding sites. Fig.
6A illustrates the binding of
a slowly migrating complex (C1) on both the E2 and E3 probes.
Supershift assays using specific antibodies identified the E2A and HEB
gene products as components of this C1 complex (Fig. 6B,
lanes 2-4 and 7-9), while an isotype-matched
control antibody did not affect the mobility of the E2 and E3 binding
complex (Fig. 6B, lanes 5 and 10). In
addition, when nuclear extracts from SCL-expressing AD10.1 cells were
used, we observed a faster migrating complex (C2, Fig.
6A) containing E2A-SCL and HEB-SCL heterodimers as revealed by supershift assays using specific antibodies against E2A, HEB, and
SCL (Fig. 6B, lanes 11-20). Together, these
results demonstrate that E2A-HEB heterodimers bind in vitro
the E2 and E3 sequences and suggest that SCL repression of E-protein
activity is mediated by DNA binding of E2A-SCL or HEB-SCL heterodimers
to the same sites on the pT
enhancer sequence.
View larger version (102K):
[in a new window]
Fig. 6.
E2A and HEB heterodimers and SCL-containing
complexes preferentially bind E2 and E3 sequence in
vitro. A, EMSA were done using
32P-labeled E2 or E3 probes and AD10.1 cell nuclear
extracts transfected or not with MSCV empty or MSCV-SCL-expressing
vector. B, supershift assays were done with AD10.1 and
AD10.1-SCL nuclear extracts. Where indicated, antibodies were included
in the samples before addition of the labeled probes. Arrows
point to the binding of E2A/HEB or SCL-containing complexes. A
monoclonal antibody against c-Myc was used as an isotype-matched
control. C, we used AD10.1-SCL nuclear extracts for
competition assays with a gradient of 1, 3, 10, and 100-fold molar
excess of unlabeled E3 wild-type or E3-mutated oligonucleotides
(lanes 3-10 and 19), or E1, E2, and E4 wild-type
oligonucleotides (lanes 13-16 and 20-27).
Arrows point to the different complexes formed on the E3
probe. Dissociation constants estimated by analysis of competition
curves are 300 nM (E1), 163 nM (E2), 13 nM (E3), and 720 nM (E4) for each E-box
sites.
Enhancer in
Situ--
To test whether SCL associates with pT
regulatory sequences in situ, we performed chromatin
immunoprecipitation assays (ChIP) on stable AD10.1 transfectants,
expressing either the empty MSCV vector or the MSCV-SCL-encoding vector
(Fig. 7). Using specific antibodies
against E47 (E2A), HEB, and SCL, as well as isotype-matched control
antibodies (anti-HA and rabbit IgG), we were able to show that in
addition to anti-E47 and anti-HEB, the anti-human SCL antibody
efficiently immunoprecipitated both the pT
enhancer, and
promoter sequences when chromatin extracts from SCL-expressing AD10.1
cells were used. Combined, our observations indicate that the
pT
enhancer and promoter are direct targets of E2A-HEB
heterodimers in immature T cells. Moreover, SCL-induced repression of
E2A-HEB transcriptional activity is not due to an Id-like titration of these factors, but rather is mediated by a DNA
binding-dependent mechanism.
View larger version (22K):
[in a new window]
Fig. 7.
SCL, E2A, and HEB specifically associate with
the pT enhancer and promoter in
situ. Chromatin immunoprecipitation assays were
performed as in Fig. 3 using cross-linked AD10.1-MSCV and AD10.1-SCL
nuclear extracts. Anti-HA and RbIgG were used as isotype and species
matched controls for immunoprecipitations.
Gene
Expression in Immature Thymocytes--
During thymocyte maturation,
T-cell commitment at the DN2 stage is marked by the initiation of pT
gene expression that reaches maximal levels at the DN3 stage (Fig.
8). Interestingly, pT
starts to be
expressed at the DN2 stage, coinciding with an elevation of both E2A
and HEB mRNA (Fig. 8). This elevation, also observed at the protein
level (40), is however not sufficient for optimal pT
gene expression
since both E2A and HEB remain constant at the DN3 stage while pT
levels abruptly increase. We therefore investigated SCL expression in
purified thymocyte populations together with E2A, HEB, and pT
mRNA levels using semiquantitative RT-PCR. As shown in Fig. 8, SCL
and pT
exhibit opposite expression patterns, i.e. SCL is
expressed at the DN1 and DN2 stages and is down regulated at the DN3
stage, coinciding exactly with an elevation in pT
gene expression.
Taken together, our observations suggest that the relative dosage
between E2A-HEB and SCL determines pT
gene expression in maturing
thymocytes.
View larger version (27K):
[in a new window]
Fig. 8.
SCL, E2A, and
HEB gene expressions determine pT
mRNA levels. A, thymocytes were fractionated
into eight subsets according to their degree of maturity, and mRNA
levels were investigated by semiquantitative RT-PCR (DN1 to DN4,
lanes 1-4; DP, lane 5; mature CD4+,
lane 6; and mature CD8+, lane 7; as
depicted in Fig. 1A). Specific primers for E2A, HEB, SCL,
pT
, and ribosomal S16 mRNA were used, the latter as control for
the amounts of cDNA. B, mRNA levels were assessed by
Southern blot hybridization with internal oligonucleotide probes.
Signals were normalized to S16 levels to account variations in cDNA
amounts.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
enhancer activity in immature thymocytes through high affinity binding to two conserved E-boxes, E2 and E3. Moreover, SCL inhibits E2A-HEB activity through binding to the same regulatory elements.
Enhancer--
During thymocyte differentiation, E47 increases at
the mRNA and protein levels, determined by flow cytometry analysis,
from the DN1 to DN4 stage, and start to decrease as the cells progress to the DP stage (9, 40). HEB mRNA also follows the same pattern (9). Our Western blot analysis of CD3
/
thymocytes (consisting of more than 90% DN3 cells) and wild-type thymocytes (more than 80% DP cells) suggests that E47 and HEB protein
levels decrease at the DP stage. This expression pattern is in
agreement with the stage-specific expression of the pT
gene in immature T cells, which is maximal at the DN3 and DN4 stages
and then decreases after
selection (41), and the finding that the
pT
gene is a target of transcription regulation by E2A and HEB (7-9). More importantly, this correlation suggests that pT
levels are determined by E2A/HEB levels. In the present study, we
provide direct evidence that E2A and HEB gene
dosage determines pT
mRNA levels.
gene have been identified and Reizis and Leder (4, 5) have shown that
the pT
upstream enhancer is necessary and sufficient to
drive stage and tissue-specific expression of the pT
gene in immature thymocytes. Indeed, this regulatory sequence is inactive in
transient transfection assays in non-T cells or in mature T-cell lines
that do not express the endogenous pT
gene (data not
shown) (4). Furthermore, reporter transgenes driven by the
pT
enhancer element are preferentially expressed in
immature thymocytes, in a pattern closely resembling that of the
endogenous pT
gene (5, 42). Analysis of the
pT
enhancer sequence revealed potential binding sites for
several transcription factors, including ZBP89, YY1, c-Myb, CSL, and
E-proteins. Previous reports have shown that pT
enhancer
activity depends on the integrity of binding sites for c-Myb and CSL,
the latter a downstream effector of the Notch1 pathway (5, 6). However,
mutation of either c-Myb or the CSL binding site did not abolish
enhancer activity in transgenic reporter experiments, suggesting that
these transcription factors, otherwise important for optimal and
stage-specific pT
expression, are not absolutely required for
enhancer activity.
enhancer activity. Moreover, 80% of enhancer activity in a DN cell line is
determined by two E-box binding sites, E2 and E3, which are conserved
between mouse and man (5). Finally, we show that E2A and HEB associate
with these E-boxes with high affinity in vitro and in
vivo, while E4 that is not conserved (5) has a low affinity for
these proteins. We therefore propose that E2A and HEB serve as
nucleation factors for the assembly of a multimeric complex into an
enhanceosome-like structure.
enhancer activity. Indeed, Reizis and Leder
(5) and Takeuchi et al. (8) suggested that mutations of
these E-boxes did not affect enhancer activity, while Petersson et al. (7) reported the opposite. This discrepancy may be
attributed to different cellular contexts used for transient
transactivation assays or alternatively, to the reporter vector itself
used in these experiments. Indeed, our previous work indicated that the promoterless pXpII vector, as well as other luciferase reporter vectors
(pGL3 basic), contains E-boxes within or near their multiple cloning
sites (19). The use of the modified pXpIII vector (19) where all
E-boxes in the vicinity of the multiple cloning site were mutated has
permitted us to reveal the functional importance of the conserved E-box
sites on pT
enhancer activity.
enhancer and promoter, the latter containing a tandem E-box site previously shown to be required for full
promoter activity (5, 8). However, a role for E2A in activating the
pT
gene in primary thymocytes has not been assessed.
Using two complementary approaches, we show by chromatin immunoprecipitation that E47 binds both pT
regulatory
sequences in vivo. Furthermore, the activity of a single
E2A allele is not sufficient for full pT
gene expression,
indicating that HEB does not compensate for E2A haploinsufficiency, at
least with regards to the transcriptional activity of the
pT
locus. Hence, E2A+/
mice
exhibit a 30% lower level of pT
mRNA in DN thymocytes, with
variable penetrance, and a modest decrease in thymocyte numbers. Interestingly, loss of one E2A allele in the context of the
HEB+/
genotype exacerbates T-cell
differentiation defect caused by HEB haploinsufficiency, resulting in
an increase in the DN and ISP populations (28). These results revealed
the importance of E2A and HEB in T-cell development (28). We show here
that both E2A and HEB bind the pT
regulatory sequences in
chromatin and that full E2A and HEB loci
activities are required for proper pT
expression. We therefore
conclude that the combined dosage of E2A and HEB controls pre-TCR
levels and, consequently, determines cell fate in the thymus.
Enhancer and Promoter--
SCL is a tissue-specific bHLH
transcription factor that heterodimerizes with E47 and HEB and binds
DNA at consensus E-box sequences (43, 44). Furthermore, SCL shows
an opposite expression profile to that of E proteins (9) and decreases
from the DN1 to DN3 stage, exactly when pT
mRNA increases. We
and others have previously shown that ectopic SCL expression in the
thymus, in combination with its nuclear partners LMO1 or LMO2,
down-regulates E-protein target genes such as the CD4 and
pT
genes (9, 18, 45). However, the molecular mechanism
through which SCL represses E-protein function in thymocytes remains to
be determined. SCL could either sequester E2A-HEB factors into
complexes that do not bind DNA in the same way as Id proteins, HLH
factors lacking a DNA binding domain. Alternatively, SCL-containing
complexes could bind DNA on E-protein target sites and prevent
E-protein transcriptional activity. Here we show that SCL associates
with E2A or HEB and binds in vitro the pT
E-box sites, E2 and E3, with the same affinity as E2A-HEB heterodimers
and that SCL occupies the pT
enhancer and promoter
sequences in vivo, as revealed by chromatin
immunoprecipitation assays. Together, these results suggest that the
repression induced by SCL is mediated by a DNA binding-dependent mechanism rather than a sequestration
effect. It remains to be determined whether SCL disrupts the formation of a transcription factor complex required for optimal enhancer activity or alternatively, whether SCL recruits new cofactors that
repress pT
gene transcription. In B cell development, E2A recruits a
chromatin remodeling complex at target DNA (46-48) and drives the
transcription of immunoglobulin genes (29, 34, 49-52). It is possible
that high levels of the SCL transgene are sufficient to form inactive
SCL-E2A or SCL-HEB heterodimers, hampering the formation of this
complex (the present study) (45). Alternatively, since SCL genetically
interacts with LMO1 or LMO2 to inhibit T-cell development at the DN-DP
transition point controlled by the pre-TCR, it is possible that SCL
recruits new cofactors that actively repress pT
gene transcription.
Further protein-protein interaction studies are warranted to
distinguish between these two possibilities.
enhancer and
promoter sequences as direct targets of E2A-HEB heterodimers in the
thymus, as well as direct targets of repression by the SCL oncogene.
Moreover, we show that pT
and SCL exhibit opposite expression
patterns in DN1 to DN3 subsets, indicating that SCL may regulate
E-protein activity during early thymocyte development. These results,
together with previous reports, suggest that the pT
enhancer is regulated by a complex combination of transcription factors
including c-Myb, CSL-NICD complex, and E-proteins. This unique
combination may determine the tissue and stage-specific expression of
an essential component of the pre-TCR, the pT
chain, required for
T-cell development.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Nathalie Tessier and Eric Massicotte (IRCM) for their assistance with cell sorting and Dr. Martine Raymond of the IRCM Molecular Biology Service for the use of the real-time PCR.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Canadian Institute for Health Research (CIHR) and the National Cancer Institute of Canada (NCIC) with funds from the Canadian Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a studentship from the Fonds de recherche sur la nature et les technologies.
¶ Recipient of a postdoctoral fellowship from the Leukemia Research Fund.
Recipient of a studentship from CIHR.
To whom correspondence should be addressed: Institut de
Recherches Cliniques de Montréal, 110, avenue des Pins Ouest,
Montréal, Québec H2W 1R7, Canada. Tel.: 514-987-5588; Fax:
514-987-5757; E-mail: hoangt@ircm.qc.ca.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M209870200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TCR, T-cell
receptor;
pT, pre-TCR
;
DN, double-negative;
DP, double-positive;
ISP, immature single positive;
bHLH, basic helix-loop-helix;
-gal,
-galactosidase;
HPRT, hypoxanthine phosphoribosyltransferase;
RbIgG, rabbit IgG;
ChIP, chromatin immunoprecipitation assay;
EMSA, electrophoretic mobility shift assay;
RIPA, radioimmune precipitation
assay buffer.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Godfrey, D. I., and Zlotnik, A. (1993) Immunol. Today 14, 547-553[CrossRef][Medline] [Order article via Infotrieve] |
2. | Chaffin, K. E., Beals, C. R., Wilkie, T. M., Forbush, K. A., Simon, M. I., and Perlmutter, R. M. (1990) EMBO J. 9, 3821-3829[Abstract] |
3. | von Boehmer, H., Aifantis, I., Feinberg, J., Lechner, O., Saint-Ruf, C., Walter, U., Buer, J., and Azogui, O. (1999) Curr. Opin. Immunol. 11, 135-142[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Reizis, B.,
and Leder, P.
(1999)
J. Exp. Med.
189,
1669-1678 |
5. |
Reizis, B.,
and Leder, P.
(2001)
J. Exp. Med.
194,
979-990 |
6. |
Reizis, B.,
and Leder, P.
(2002)
Genes Dev.
16,
295-300 |
7. | Petersson, K., Ivars, F., and Sigvardsson, M. (2002) Eur. J. Immunol. 32, 911-920[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Takeuchi, A.,
Yamasaki, S.,
Takase, K.,
Nakatsu, F.,
Arase, H.,
Onodera, M.,
and Saito, T.
(2001)
J. Immunol.
167,
2157-2163 |
9. | Herblot, S., Steff, A. M., Hugo, P., Aplan, P. D., and Hoang, T. (2000) Nat. Immunol. 1, 138-144[CrossRef][Medline] [Order article via Infotrieve] |
10. | Zhuang, Y., Soriano, P., and Weintraub, H. (1994) Cell 79, 875-884[Medline] [Order article via Infotrieve] |
11. | Bain, G., and Murre, C. (1998) Semin. Immunol. 10, 143-153[CrossRef][Medline] [Order article via Infotrieve] |
12. | Engel, I., and Murre, C. (2001) Nat. Rev. Immunol. 1, 193-199[CrossRef][Medline] [Order article via Infotrieve] |
13. | Quong, M. W., Romanow, W. J., and Murre, C. (2002) Annu. Rev. Immunol. 20, 301-322[CrossRef][Medline] [Order article via Infotrieve] |
14. | Bain, G., Engel, I., Robanus, M. E., te, R. H., Voland, J. R., Sharp, L. L., Chun, J., Huey, B., Pinkel, D., and Murre, C. (1997) Mol. Cell. Biol. 17, 4782-4791[Abstract] |
15. |
Barndt, R.,
Dai, M. F.,
and Zhuang, Y.
(1999)
J. Immunol.
163,
3331-3343 |
16. |
Blom, B.,
Heemskerk, M. H.,
Verschuren, M. C.,
van Dongen, J. J.,
Stegmann, A. P.,
Bakker, A. Q.,
Couwenberg, F.,
Res, P. C.,
and Spits, H.
(1999)
EMBO J.
18,
2793-2802 |
17. | Yan, W., Young, A. Z., Soares, V. C., Kelley, R., Benezra, R., and Zhuang, Y. (1997) Mol. Cell. Biol. 17, 7317-7327[Abstract] |
18. |
Chervinsky, D. S.,
Zhao, X. F.,
Lam, D. H.,
Ellsworth, M.,
Gross, K. W.,
and Aplan, P. D.
(1999)
Mol. Cell. Biol.
19,
5025-5035 |
19. |
Lecuyer, E.,
Herblot, S.,
Saint-Denis, M.,
Martin, R.,
Begley, C. G.,
Porcher, C.,
Orkin, S. H.,
and Hoang, T.
(2002)
Blood
100,
2430-2440 |
20. |
Wadman, I. A.,
Osada, H.,
Grutz, G. G.,
Agulnick, A. D.,
Westphal, H.,
Forster, A.,
and Rabbitts, T. H.
(1997)
EMBO J.
16,
3145-3157 |
21. | Hsu, H. L., Wadman, I., Tsan, J. T., and Baer, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5947-5951[Abstract] |
22. |
Park, S. T.,
and Sun, X. H.
(1998)
J. Biol. Chem.
273,
7030-7037 |
23. | Voronova, A. F., and Lee, F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5952-5956[Abstract] |
24. |
Aplan, P. D.,
Jones, C. A.,
Chervinsky, D. S.,
Zhao, X.,
Ellsworth, M.,
Wu, C.,
McGuire, E. A.,
and Gross, K. W.
(1997)
EMBO J.
16,
2408-2419 |
25. | Larson, R. C., Lavenir, I., Larson, T. A., Baer, R., Warren, A. J., Wadman, I., Nottage, K., and Rabbitts, T. H. (1996) EMBO J. 15, 1021-1027[Abstract] |
26. | Rabbitts, T. H., Axelson, H., Forster, A., Grutz, G., Lavenir, I., Larson, R., Osada, H., Valge-Archer, V., Wadman, I., and Warren, A. (1997) Leukemia 11 Suppl. 3, 271-272[Medline] [Order article via Infotrieve] |
27. | Zhuang, Y., Kim, C. G., Bartelmez, S., Cheng, P., Groudine, M., and Weintraub, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 12132-12136[Abstract] |
28. | Zhuang, Y., Cheng, P., and Weintraub, H. (1996) Mol. Cell. Biol. 16, 2898-2905[Abstract] |
29. | Bain, G., Maandag, E. C., Izon, D. J., Amsen, D., Kruisbeek, A. M., Weintraub, B. C., Krop, I., Schlissel, M. S., Feeney, A. J., and van Roon, M. (1994) Cell 79, 885-892[Medline] [Order article via Infotrieve] |
30. | Malissen, M., Gillet, A., Ardouin, L., Bouvier, G., Trucy, J., Ferrier, P., Vivier, E., and Malissen, B. (1995) EMBO J. 14, 4641-4653[Abstract] |
31. | Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve] |
32. | Orlando, V., Strutt, H., and Paro, R. (1997) Methods 11, 205-214[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Xu, D.,
Popov, N.,
Hou, M.,
Wang, Q.,
Bjorkholm, M.,
Gruber, A.,
Menkel, A. R.,
and Henriksson, M.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
3826-3831 |
34. |
Bain, G.,
Quong, M. W.,
Soloff, R. S.,
Hedrick, S. M.,
and Murre, C.
(1999)
J. Exp. Med.
190,
1605-1616 |
35. |
Pan, L.,
Hanrahan, J.,
Li, J.,
Hale, L. P.,
and Zhuang, Y.
(2002)
J. Immunol.
168,
3923-3932 |
36. | Wang, B., Simpson, S. J., Hollander, G. A., and Terhorst, C. (1997) Immunol. Rev. 157, 53-60[Medline] [Order article via Infotrieve] |
37. | Wang, N., Wang, B., Salio, M., Allen, D., She, J., and Terhorst, C. (1998) Int. Immunol. 10, 1777-1788[Abstract] |
38. | Blackwell, T. K., and Weintraub, H. (1990) Science 250, 1104-1110[Medline] [Order article via Infotrieve] |
39. | Hu, J. S., Olson, E. N., and Kingston, R. E. (1992) Mol. Cell. Biol. 12, 1031-1042[Abstract] |
40. |
Engel, I.,
Johns, C.,
Bain, G.,
Rivera, R. R.,
and Murre, C.
(2001)
J. Exp. Med.
194,
733-745 |
41. | Saint-Ruf, C., Ungewiss, K., Groettrup, M., Bruno, L., Fehling, H. J., and von Boehmer, H. (1994) Science 266, 1208-1212[Medline] [Order article via Infotrieve] |
42. | Gounari, F., Aifantis, I., Martin, C., Fehling, H. J., Hoeflinger, S., Leder, P., von Boehmer, H., and Reizis, B. (2002) Nat. Immunol. 3, 489-496[Medline] [Order article via Infotrieve] |
43. | Hsu, H. L., Cheng, J. T., Chen, Q., and Baer, R. (1991) Mol. Cell. Biol. 11, 3037-3042[Medline] [Order article via Infotrieve] |
44. | Hsu, H. L., Huang, L., Tsan, J. T., Funk, W., Wright, W. E., Hu, J. S., Kingston, R. E., and Baer, R. (1994) Mol. Cell. Biol. 14, 1256-1265[Abstract] |
45. |
Goardon, N.,
Schuh, A.,
Hajar, I.,
Ma, X.,
Jouault, H.,
Dzierzak, E.,
Romeo, P. H.,
and Maouche-Chretien, L.
(2002)
Blood
100,
491-500 |
46. | Eckner, R., Yao, T. P., Oldread, E., and Livingston, D. M. (1996) Genes Dev. 10, 2478-2490[Abstract] |
47. | Massari, M. E., Grant, P. A., Pray-Grant, M. G., Berger, S. L., Workman, J. L., and Murre, C. (1999) Mol Cell 4, 63-73[Medline] [Order article via Infotrieve] |
48. |
Massari, M. E.,
and Murre, C.
(2000)
Mol. Cell. Biol.
20,
429-440 |
49. | Sigvardsson, M., O'Riordan, M., and Grosschedl, R. (1997) Immunity. 7, 25-36[Medline] [Order article via Infotrieve] |
50. |
Liu, X.,
Prabhu, A.,
and Van Ness, B.
(1999)
J. Biol. Chem.
274,
3285-3293 |
51. |
Kee, B. L.,
and Murre, C.
(1998)
J. Exp. Med.
188,
699-713 |
52. | Goldfarb, A. N., Flores, J. P., and Lewandowska, K. (1996) Mol Immunol 33, 947-956[CrossRef][Medline] [Order article via Infotrieve] |