Regulation of pTalpha Gene Expression by a Dosage of E2A, HEB, and SCL*

Mathieu TremblayDagger §, Sabine HerblotDagger , Eric LécuyerDagger ||, and Trang HoangDagger **DaggerDagger

From the Dagger  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The expression of the pTalpha gene is required for effective selection, proliferation, and survival of beta  T-cell receptor (beta TCR)-expressing immature thymocytes. Here, we have identified two phylogenetically conserved E-boxes within the pTalpha 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 pTalpha as a direct target of E2A-HEB heterodimers in immature thymocytes because they specifically occupy the enhancer in vivo. In these cells, pTalpha 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 pTalpha is up-regulated. Taken together, our observations suggest that the dosage of E2A, HEB, and SCL determines pTalpha gene expression in immature T cells.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The development of alpha beta 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 beta  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 beta TCR chain with the invariant pTalpha chain and signaling molecules of the CD3 complex. Since expression of the pre-TCR is critical for alpha beta 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.

Recently, the promoter and the enhancer sequences of the pTalpha 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 pTalpha 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 pTalpha 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 pTalpha 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).

The mechanism through which bHLH factors regulate pTalpha expression remains to be documented. Here, we provide evidence that E2A-HEB oligomers determine pTalpha 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
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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 beta -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.

E2A+/- 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). CD3epsilon -/- 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.

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-CD3epsilon (145-2C11), anti-beta TCR (H57-597), anti-TCRgamma delta (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.

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 (pTalpha , 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).

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 (pTalpha , 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.

Constructs, Expression Vectors, and Transfection Assays-- The pTalpha 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-beta 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. beta -galactosidase assays were performed using o-nitrophenyl beta -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 beta -galactosidase activity of one experiment performed in duplicate and representative of 3-5 experiments.

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 CD3epsilon -/- 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.

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 CD3epsilon -/- 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 pTalpha enhancer, pTalpha 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.

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INTRODUCTION
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E2A- and HEB-deficient Mice Express Decreased Level of pTalpha 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 TCRgamma delta lineage thymocytes were analyzed in E2A+/- and HEB+/- mice and wild-type littermates. We show that the percentage TCRgamma delta + 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 gamma delta 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 pTalpha 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 pTalpha expression, as compared with that of HEB. We therefore used semiquantitative and real-time PCR to investigate pTalpha 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, beta TCR, and Thy1.2 markers. Within the DN compartment, we observed a 3- and 5-fold decrease of pTalpha 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 pTalpha , as described previously (9). Interestingly, E2A and HEB heterozygous DN thymocytes expressed, on average, intermediate levels of pTalpha 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 pTalpha mRNA levels were not due to an increase in gamma delta T cells, since the percentage of CD3epsilon +TCRgamma delta + cells in wild-type and heterozygous mice is not significantly different. For this reason, we observed the same decrease in pTalpha mRNA level, even when TCRgamma delta + cells were excluded from analysis (Fig. 2E). Despite this lower level of pTalpha , 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 pTalpha 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 pTalpha 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.


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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, beta 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, CD3epsilon , TCRgamma delta , and Thy1.2 antibodies and analyzed by flow cytometry. The percentages of TCRgamma delta +/CD3epsilon + DN thymocytes in different litters (diamond , X, newborn; triangle , 1-week old; black-diamond , 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.


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Fig. 2.   Reduction of pTalpha 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-/beta TCR-/Thy1.2+) and DP (CD4+/CD8+/beta TCR+/Thy1.2+). Semiquantitative RT-PCR were performed on purified cell populations using specific primers for pTalpha and S16 (internal control). D, real-time RT-PCR were performed to quantify pTalpha gene expression in DN and DP cells from wild-type, E2A+/- and E2A-/- mice. pTalpha 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 pTalpha were analyzed by semiquantitative RT-PCR as described above in thymocytes of DN (CD4-/CD8-/CD3epsilon -/Thy1.2+/TCRgamma delta -) and DP (CD4+/CD8+/CD3epsilon +/Thy1.2+/TCRgamma delta -) subset depleted of TCRgamma delta -positive cells of wild-type and heterozygous mice for E2A and HEB. Results are shown as percentages of wild-type levels.

E2A and HEB Gene Products Bind the pTalpha Enhancer in Vivo in DN3 Thymocytes-- The expression of the pTalpha 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 pTalpha expression, we addressed the question of whether these proteins associate with pTalpha 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 pTalpha gene is expressed at the DN3 and DN4 stages, we used thymocytes from CD3epsilon -/- 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 pTalpha enhancer regions (Fig. 3A). Since, gel shift assays revealed that HEB could bind E-boxes located within the pTalpha 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 pTalpha enhancer sequence as well as the pTalpha 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.


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Fig. 3.   E2A and HEB specifically associate with the pTalpha enhancer and promoter in primary thymocytes. A, schematic diagram of the pTalpha 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 CD3epsilon -/- 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 pTalpha 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 CD3epsilon -/- 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.

Functional Importance of Two E-box Binding Sites within the pTalpha Enhancer-- It has been previously reported that the pTalpha enhancer element is essential for specific pTalpha 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 pTalpha enhancer sequence upstream of a minimal TATA promoter, and the luciferase reporter gene and confirmed that the enhancer is active in the pTalpha + "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 pTalpha 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 pTalpha enhancer activity.


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Fig. 4.   The integrity of two E-boxes is critical for pTalpha enhancer activity. A, schematic representation of the pTalpha enhancer showing its different binding sites. B, point mutations of specific E-box sites impair pTalpha enhancer activity. The AD10.1 DN T-cell line was transfected with wild-type or mutant pTalpha 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-beta gal). C, sequences of the different E-box sites within the pTalpha enhancer. Shown in bold are residues that match the core E47 (38) or HEB (39) consensus.

We have previously reported that ectopic SCL expression in immature thymocytes represses the expression of the endogenous pTalpha gene and that HEB overexpression rescues pTalpha 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 pTalpha 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 pTalpha 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 pTalpha 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 pTalpha enhancer activity requires the integrity of the two E-box binding sites, E2 and E3, suggesting that SCL directly represses E-protein function.


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Fig. 5.   Repression of pTalpha 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 pTalpha 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. pTalpha 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 pTalpha 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.

E2A and HEB Gene Products as Well as SCL-containing Complexes Bind E-box Elements of the pTalpha Enhancer in Vitro-- E-box mutations or SCL-induced repression of the pTalpha enhancer activity indicate the crucial role of E-proteins in regulating pTalpha gene expression. Since E2A or HEB occupy the pTalpha enhancer in DN thymocytes, we addressed the question of whether E2A or HEB gene products directly bind E-boxes within the pTalpha 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 pTalpha enhancer sequence.


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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.

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 pTalpha Enhancer in Situ-- To test whether SCL associates with pTalpha 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 pTalpha enhancer, and promoter sequences when chromatin extracts from SCL-expressing AD10.1 cells were used. Combined, our observations indicate that the pTalpha 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.


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Fig. 7.   SCL, E2A, and HEB specifically associate with the pTalpha 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.

Relative Levels of E2A, HEB, and SCL Determines pTalpha Gene Expression in Immature Thymocytes-- During thymocyte maturation, T-cell commitment at the DN2 stage is marked by the initiation of pTalpha gene expression that reaches maximal levels at the DN3 stage (Fig. 8). Interestingly, pTalpha 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 pTalpha gene expression since both E2A and HEB remain constant at the DN3 stage while pTalpha levels abruptly increase. We therefore investigated SCL expression in purified thymocyte populations together with E2A, HEB, and pTalpha mRNA levels using semiquantitative RT-PCR. As shown in Fig. 8, SCL and pTalpha 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 pTalpha gene expression. Taken together, our observations suggest that the relative dosage between E2A-HEB and SCL determines pTalpha gene expression in maturing thymocytes.


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Fig. 8.   SCL, E2A, and HEB gene expressions determine pTalpha 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, pTalpha , 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

In the present study, we show that the bHLH factors E2A and HEB drive pTalpha 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.

Importance of E2A-HEB in Driving Stage-specific Activity of the pTalpha 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 CD3epsilon -/- 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 pTalpha gene in immature T cells, which is maximal at the DN3 and DN4 stages and then decreases after beta  selection (41), and the finding that the pTalpha gene is a target of transcription regulation by E2A and HEB (7-9). More importantly, this correlation suggests that pTalpha levels are determined by E2A/HEB levels. In the present study, we provide direct evidence that E2A and HEB gene dosage determines pTalpha mRNA levels.

At the molecular level, the regulatory elements of the pTalpha gene have been identified and Reizis and Leder (4, 5) have shown that the pTalpha upstream enhancer is necessary and sufficient to drive stage and tissue-specific expression of the pTalpha 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 pTalpha gene (data not shown) (4). Furthermore, reporter transgenes driven by the pTalpha enhancer element are preferentially expressed in immature thymocytes, in a pattern closely resembling that of the endogenous pTalpha gene (5, 42). Analysis of the pTalpha enhancer sequence revealed potential binding sites for several transcription factors, including ZBP89, YY1, c-Myb, CSL, and E-proteins. Previous reports have shown that pTalpha 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 pTalpha expression, are not absolutely required for enhancer activity.

Here we demonstrate that in addition to c-Myb and the CSL-NICD complex, E2A and HEB are critical determinants of pTalpha 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.

There is substantive controversy with regards to the contribution of E-boxes to pTalpha 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 pTalpha enhancer activity.

Transient transactivation assays in heterologous cells indicate that E47 can activate the pTalpha 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 pTalpha gene in primary thymocytes has not been assessed. Using two complementary approaches, we show by chromatin immunoprecipitation that E47 binds both pTalpha regulatory sequences in vivo. Furthermore, the activity of a single E2A allele is not sufficient for full pTalpha gene expression, indicating that HEB does not compensate for E2A haploinsufficiency, at least with regards to the transcriptional activity of the pTalpha locus. Hence, E2A+/- mice exhibit a 30% lower level of pTalpha 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 pTalpha regulatory sequences in chromatin and that full E2A and HEB loci activities are required for proper pTalpha expression. We therefore conclude that the combined dosage of E2A and HEB controls pre-TCR levels and, consequently, determines cell fate in the thymus.

SCL-E2A/HEB Complexes Occupy E-box Elements within the pTalpha 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 pTalpha 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 pTalpha 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 pTalpha E-box sites, E2 and E3, with the same affinity as E2A-HEB heterodimers and that SCL occupies the pTalpha 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 pTalpha 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 pTalpha gene transcription. Further protein-protein interaction studies are warranted to distinguish between these two possibilities.

In summary, we unambiguously identify the pTalpha 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 pTalpha 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 pTalpha 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 pTalpha chain, required for alpha beta 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.

Dagger Dagger 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; pTalpha , pre-TCRalpha ; DN, double-negative; DP, double-positive; ISP, immature single positive; bHLH, basic helix-loop-helix; beta -gal, beta -galactosidase; HPRT, hypoxanthine phosphoribosyltransferase; RbIgG, rabbit IgG; ChIP, chromatin immunoprecipitation assay; EMSA, electrophoretic mobility shift assay; RIPA, radioimmune precipitation assay buffer.

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