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Transcriptional Regulation of CD4 Gene Expression by T Cell Factor-1/-Catenin Pathway

Zhaofeng Huang^*, Huimin Xie^*, Vassilio Ioannidis, Werner Held, Hans Clevers, Maureen S. Sadim^1,* and Zuoming Sun^2,*

^* Department of Microbiology & Immunology, College of Medicine, University of Illinois, Chicago, IL 60612; Ludwig Institute for Cancer Research, Lausanne Branch and University of Lausanne, Epalinges, Switzerland; and Hubrecht Laboratory, Center for Biomedical Genetics, Utrecht, The Netherlands

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-x_L. By both loss and gain of function studies, in this study we show additional function of TCF-1/-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4⁺CD8⁺ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1^–/– DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1^–/– cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with -catenin. Furthermore, forced expression of a stabilized -catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized -catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and -catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Precise regulation of gene expression determines the fate of a cell during development. A change in the expression of a single gene could result in converting a cell to a different fate. Therefore, great efforts have been spent on identifying cis-regulatory elements and trans factors controlling gene expression. By interacting with MHC class II molecule, CD4 directs the lineage development of Th cells in the thymus, and facilitates the activation of mature CD4 T cells (1, 2). Transcription regulation of CD4 gene expression is one of the best-studied models in T cell development (3). The earliest T cells in thymus are negative for both CD4 and CD8, and thus designated as double-negative (DN)³ cells. DN cells with successful rearrangement of TCR--chain are permitted to further differentiate into CD4⁺CD8⁺ double-positive (DP) cells. Productive rearrangement of TCR--chain results in expression of TCR- on the surface of DP cells. Differentiation of DP cells into CD4⁺ single-positive (SP) cells is determined by the engagement of TCR and CD4 with the same MHC class II molecule, whereas engagement of TCR and CD8 with MHC class I molecule allows the development of CD8⁺ SP cells (1).

Transcription of the CD4 gene is under the control of well-characterized cis-acting elements including both enhancers and silencers (3, 4). Expression of CD4 in DP and CD4 SP cells requires a basic CD4 promoter (Pr), a proximal enhancer (5, 6), and possibly a 3' enhancer that is only active in DP cells (4). However, a silencer overrides the enhancer activity and prevents CD4 expression in DN and CD8 SP cells (7, 8). Previous studies have focused on the silencer, and identified Runt domain transcription factors as well as BAF to be essential for silencer function in vivo (9, 10, 11). A basic CD4 Pr is not sufficient to drive CD4 expression. The proximal enhancer is required to stimulate basic Pr, to achieve physiological levels of CD4 expression in DP and SP T cells (5, 7). Several DNase I-hypersensitive regions including a T cell factor (TCF)/lymphoid enhancer factor (LEF) and a E12/HEB binding site have been identified within the enhancer region (5, 6), but their in vivo binding factors and functions are largely unknown.

Wnts are a family of secreted proteins that mediate communication between cells during development by binding to Frizzled receptors associated with the low-density lipoprotein receptor-related protein 5 and 6 (12, 13, 14). Wnt/-catenin pathway regulates multiple developmental processes ranging from regeneration of stem cells to organogenesis of kidney and reproductive systems (15, 16). TCF is the ultimate mediator of Wnt/-catenin signaling pathway (17). Wnt pathway has been shown to regulate early lymphopoiesis (18, 19). By gene ablation, TCF-1 has been shown to regulate thymocyte maturation partially via enhancing DP thymocyte survival (20). Deletion of TCF-1 leads to significantly reduced levels of antiapoptotic protein Bcl-x_L accompanied with increased apoptosis of DP cells (21, 22). The -catenin-stimulated TCF-1 activity is essential for thymocyte survival, because wild type (WT), but not a truncated version of TCF-1 lacking N-terminal 116 aas required for binding to -catenin, restored thymocyte survival in TCF-1-deficient mice (21). In addition, both loss and gain of function studies indicated the critical role of the Wnt pathway in the regulation of T cell development at the DN-DP transition stage. Deletion of -catenin leads to a developmental block at the DN-DP stage, whereas expression of a constitutively active -catenin allows development of SP T cells lacking surface TCR due to affecting at the similar DN-DP transition stage (23, 24). Consistently, similar phenotype was observed in mice deficient in adenomatous polyposis coli gene, a negative regulator of the -catenin pathway (25).

Mechanisms for -catenin-mediated activation of TCF have been extensively studied. Without Wnt signaling, -catenin is phosphorylated by glycogen synthase-3, and is targeted for ubiquitination and degradation by 26S proteosome (26). In the absence of -catenin, TCF associates with transcriptional repressor, GRG in mouse (Groucho in Drosophila, and transducin-like enhancer in human), and thus inhibits target gene expression (27). Activation of Wnt signaling leads to inactivation of glycogen synthase-3 and stabilization and accumulation of -catenin. Accumulated -catenin, a transcriptional coactivator, then replaces GRG to bind to TCF, resulting in activation of target gene expression. In this study, we provide evidence to indicate the role of -catenin pathway in the regulation of CD4 expression in DP cells.

	Materials and Methods

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Mice

Transgenic mice in FVB background expressing stabilized -catenin were generated as described previously (28), and C57/B6 TCF-1 null mice were obtained from Dr. H. Clevers (Hubrecht Laboratory, Center for Biomedical Genetics, Utrecht, The Netherlands). All mice were bred and housed under a specific pathogen-free condition in the animal facility of the University of Illinois at Chicago.

Plasmids

DNA fragment containing CD4 Pr and the proximal enhancer as described previously (5, 7) was cloned into pGL3 basic (Promega). Mutation of TCF binding site (from CAAAG to TTCAG) was conducted with a Quick Change Site-Directed Mutagenesis Kit (Stratagene; catalog no. 200524). Topflash (TOP) and fopflash (FOP) reporters were gifts from Dr. P. Howe (Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH). WT and stabilized -catenin expression plasmids were provided by Drs. F. McCormick and O. Tetsu (University of California, San Francisco, CA). TCF-1 and TCF expression plasmids were also provided by Dr. H. Clevers (Hubrecht Laboratory, Center for Biomedical Genetics, Utrecht, The Netherlands).

Flow cytometry and cell sorting

The following Abs were purchased from BD Pharmingen: biotin-anti-CD4 (catalog no. 553649), streptavidin-Cy5 (catalog no. 554062), and PE-anti-CD8 (catalog no. RM2204). FACS assays were performed by a FACSCalibur (BD Biosciences) and analyzed with CellQuest software (BD Biosciences).

Northern blot analysis and RT-PCR

For Northern blot analysis, total RNA was purified by TRIzol (Invitrogen Life Technologies). Twenty micrograms of total RNA was separated by electrophoresis on a 1.2% agarose gel. Separated RNA was then transferred to Hybond-N⁺ membrane (Amersham) and cross-linked using an UV chamber. CD4 and hypoxanthine guanine phosphoribosyl transferase (HPRT) cDNA fragments were amplified by a PCR with [³²P]dCTP. Hybridization of the membrane with ³²P-labeled probes was conducted by standard methods. Northern blot signals were collected by exposing to storage phosphor screen and detected by a phosphorimager (Storm 860; Molecular Dynamics). For RT-PCR analysis, total RNA was reverse transcribed with SuperScript II Rnase H-Reverse Transcriptase (Invitrogen Life Technologies). To normalize the cDNA content, primers specific for HPRT along with CD4 were used in PCR. The PCR products were resolved on a 1.4% agarose gel. Primers used for PCR were as follows: CD4, 5'-AGAGCCTGACCCTGACCTTG and 5'-GGAAACCCAGAAAGCCGAAG; and HPRT, 5'-GGTAGGCTGGCCTATAGGCT and 5'-GATACAGGCCAGACTTTGTTG.

Cell culture, transient transfection, and reporter assay

Jurkat cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 50 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. A total of 1 x 10⁷ cells in 0.4 ml of serum-free RPMI 1640 medium was transfected with 2 µg of reporter plasmid, 0.5 µg of Renilla luciferase control vector (pRL-TK; Promega), and various amounts of expression vector by electroporation (250 V and 950 µF). The total amount of transfected DNA was kept constant by adjusting the amount of a control plasmid. After electroporation, the cells were incubated at room temperature for 10 min and then transferred into 10 ml of growth medium. After incubation at 37°C with 5% CO₂ for 40–48 h, dual luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.

Chromatin immunoprecipitation (ChIP) assays

Rabbit polyclonal anti-TCF-1 Ab was purchased from Santa Cruz Biotechnology (catalog no. Sc-13025X). Salmon sperm DNA/protein G-agarose was purchased from Upstate Biotechnology (catalog no. 16-201). The ChIP procedure was performed according to the manufacturer’s instructions (Upstate Biotechnology). A total of (5 x 10⁶) cells was used for each assay. Input sample (5%) was used as a template for control PCR. All primers used in PCR analyses were described previously (29). Primer sequences were as follows: proximal enhancer 1 (PE-1), 5'-GGCCTAGATTTCCCTTCTGAG and 5'-TACTTTCTGTGACTTACAAAGGC; Pr, 5'-TAGTCTGGCCTTGAGCTTGTG and 5'-ACCCCCAGTTGTTGGGGAAG; locus control region (LCR), 5'-TAACAGAGCAGTCAGGTCTGC and 5'-CACAACTGGCCTATGAGAGTC; thymocyte enhancer (TE)-1, 5'-TGAAGTGAACTATAGCCGCGAC and 5'-TGACACAGGAAGGCAGAACTGC; and TE-2, 5'-CTTCAGGCCCTAACTGAAAGG and 5'-AGCTTATCTGGGGCGCTGATC.

Data used in Figs. 1g and 2i were produced in Dr. W. Held’s laboratory, whereas the other data were produced in Dr. Z. Sun’s laboratory. Although two laboratories independently observed differences in CD4 expression between WT and TCF-1-deficient mice, the fluorescent intensity of the flow cytometric analyses are not comparable between the data generated in two different laboratories.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. Down-regulation of CD4 in TCF-1–/– mice. a and b, Flow cytometric analysis of surface expression of CD4 and CD8. Thymocytes obtained from WT (a) and TCF-1–/– (TCF–/–; b) mice were stained with anti-CD4 Ab and anti-CD8 Ab. A flow cytometer was then used to analyze surface CD4 and CD8. Three rectangles indicate the corresponding gates for CD4+ SP, CD4+CD8+ DP, and CD8+ SP cells. c and d, Expression of CD4 (c) and CD8 (d) on DP cells was compared between WT (gray area) and TCF-1–/– (black solid line) mice. e and f, Expression of CD4 and CD8 on SP cells was compared between WT (gray) and TCF-1–/– (black solid line) mice. g, Transgenic Bcl-2 failed to restore CD4 levels on DP cells of TCF-1–/– mice. CD4 levels on DP thymocytes of WT (gray area), Bcl-2Tg (dotted line), TCF-1–/– (black solid line), and TCF-1–/–Bcl-2Tg (gray solid line) mice were compared by histogram overlay analysis. h, RT-PCR analysis of CD4 mRNA levels. RNA was purified from sorted CD4+CD8+ DP thymocytes of WT and TCF-1–/– mice. Specific primers for CD4 and HPRT were then used to perform PCR using two different concentrations (1 and 4) of RNA as templates. Two bands representing the PCR products of CD4 and HPRT are indicated. Data shown are representative of at least three independent experiments.

View larger version (43K): [in this window] [in a new window]   FIGURE 2. Up-regulation of CD4 in -CatTg mice. a–b, Flow cytometric analysis of surface expression of CD4 and CD8. Thymocytes obtained from WT (a) and -catenin transgenic (-CatTg; b) mice were stained with anti-CD4 Ab and anti-CD8 Ab. A flow cytometer was then used to analyze CD4 and CD8. Three rectangles indicate the corresponding gates for CD4+ SP, CD4+CD8+ DP, and CD8+ SP cells. c and d, Expression of CD4 (c) and CD8 (d) on DP cells was compared between WT (gray) and -CatTg (black) mice. e and f, Expression of CD4 and CD8 on SP cells was compared between WT (gray) and -CatTg (black) mice. g, Northern blot analysis of CD4 mRNA levels. Total RNA was purified from thymocytes of WT and -CatTg mice. Northern blot analyses were performed with CD4 cDNA as a probe. HPRT was used as a control for the equal amounts of loaded RNA. h, RT-PCR analysis of CD4 mRNA levels. RNA was purified from sorted CD4+CD8+ DP thymocytes of WT and -CatTg mice. Specific primers for CD4 and HPRT were then used to perform PCR using two different concentrations (1 and 4) of RNA as templates. Two bands representing the PCR products of CD4 and HPRT are indicated. Data shown are representative of at least three independent experiments. i, Restoration of CD4 levels on DP cells of TCF–/– mice by transgenic expression of p45 but not p33. CD4 levels on DP thymocytes of WT (gray area), TCF–/–p45Tg (dotted line), TCF-1–/– (black solid line), and TCF–/–p33Tg (gray solid line) mice were compared by histogram overlay analysis.

	Results

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We initially noticed that expression of a transgene driven by a CD4 Pr was significantly decreased in mice deficient in TCF-1 (data not shown). It is possible that activity of the CD4 Pr used to target transgene to T cell compartments was reduced in the absence of TCF-1. To determine whether the absence of TCF-1 also affected endogenous CD4 expression, we then examined surface expression of CD4 and CD8 on thymocytes by a flow cytometer (Fig. 1, a and b). Indeed, compared with the thymocytes obtained from WT littermates mean fluorescent intensity CD4 levels (387 vs 301) (Fig. 1c), but not CD8 levels, (1256 vs 1347) (Fig. 1d) were reduced on TCF-1^–/– DP cells, suggesting that TCF-1 is required for expression of physiological levels of CD4 on DP cells. Furthermore, no obvious difference was detected in CD4 (478 vs 447) as well as CD8 levels (1065 vs 1174) in SP cells between WT and TCF-1^–/– mice (Fig. 1, e and f). This result suggests that expression of CD4 on DP cells, but not SP cells, requires TCF-1.

DP cells of TCF-1^–/– mice undergo apoptosis due to reduced levels of antiapoptotic protein Bcl-x_L (21). To exclude the possibility that reduced CD4 levels on TCF-1^–/– DP cells result from apoptosis, we examined the CD4 levels on TCF-1^–/– DP cells that express an antiapoptotic protein Bcl-2 obtained from TCF-1^–/– Bcl-2^Tg mice described previously (21) (Fig. 1g) (see last paragraph in Materials and Methods). In agreement with the above results, CD4 levels on TCF-1^–/– DP cells were lower than that of the WT. No significant difference was detected in the CD4 levels of DP cells between WT and Bcl-2^Tg mice, suggesting that forced expression of Bcl-2 does not affect CD4 expression. Equivalent levels of CD4 were detected on TCF-1^–/– and TCF-1^–/– Bcl-2^Tg DP thymocytes, clearly demonstrated that transgenic Bcl-2, although rescued TCF-1^–/– DP from apoptosis as previously reported (21), failed to restore CD4 expression. These results suggest that lower CD4 levels on TCF-1^–/– DP cells is not linked to apoptosis.

To determine whether transcription of the CD4 gene is affected in the absence of TCF-1, we examined CD4 transcript levels in TCF-1^–/– DP thymocytes. Due to developmental defects, the TCF-1^–/– thymus contains fewer DP cells (50%) compared with WT (80–90%), and a significantly increased percentage of DN cells and immature SP cells (20, 21). To specifically detect CD4 transcripts in DP population, we sorted CD4⁺CD8⁺ DP cells from TCF-1^–/– and WT littermates. RNA was then purified from sorted DP cells and subjected to semiquantitative RT-PCR analyses with primers specific for CD4 (Fig. 1h). The band representing PCR product of CD4 transcripts was much weaker in TCF-1^–/– mice than that of the WT mice. These results suggest that TCF-1 is a positive transcriptional regulator for CD4 expression in vivo.

Stimulation of CD4 expression by transgenic expression of a stabilized -catenin that augments TCF-1 activity

By loss of function study, the above experiments demonstrated that TCF-1 is required to enhance CD4 expression. In this study, we use a gain of function study to examine the role of the TCF-1 pathway in the regulation of CD4 expression under the conditions that do not interfere with earlier T cell development. We have established transgenic mice (-Cat^Tg) that express TCF-1 coactivator, -catenin, in T cell compartments (28). Because the phosphorylated form of -catenin is usually degraded by a ubiquitination-dependent pathway (30), we chose a stabilized form of -catenin that has an internal deletion of 20 aas (29–48). Because the deleted 20 aa contain four critical phosphorylation sites (Ser⁴⁵, Thr⁴¹, Ser³⁷, and Ser³¹) that are targets for an ubiquitination-mediated degradation pathway, the transgenic -catenin is stabilized and resistant to degradation. Analyses of thymocytes obtained from -Cat^Tg and littermate WT mice indeed revealed specifically up-regulated surface CD4 levels (Fig. 2, a and b). CD4, but not CD8, levels were significantly increased in DP cells (365 vs 805) (Fig. 2, c–f). In contrast to deletion of TCF-1 that affected CD4 expression in DP but not SP cells (Fig. 1, c–f), stabilized -catenin enhanced CD4 expression in both DP and SP (405 vs 748) cells (Fig. 2, c and e). Expression of the transgene started at the DP stage, and development of DN cells was thus not affected in -Cat^Tg mice as shown previously (28), suggesting that enhancement of CD4 expression is not related to the function of TCF-1/-catenin pathway in earlier T cell development. Northern blot analyses of RNA from total thymocytes (Fig. 1g), as well as RT-PCR analyses of RNA purified from sorted CD4⁺CD8⁺ DP cells (Fig. 1h) clearly indicated that CD4 mRNA was also greatly increased in -Cat^Tg mice compared with that of the WT mice. These results suggest that enhanced TCF activity by a stabilized -catenin results in transcriptional activation of CD4 expression in vivo.

To determine whether -catenin is required for TCF-1-mediated up-regulation of CD4, we examined CD4 expression on DP cells of TCF-1^–/– mice that are reconstituted with a transgene encoding either WT TCF-1 (p45^Tg) or a truncated form of TCF-1 (p33^Tg) that lacks the domain required for binding to -catenin. TCF-1^–/–p45^Tg and TCF-1^–/–p33^Tg mice were generated previously by breeding p45^Tg and p33^Tg mice to TCF-1^–/– mice (21). CD4 levels on TCF-1^–/–p45^Tg DP cells were equivalent, if not higher, to that of the WT (Fig. 2i) (see last paragraph of Materials and Methods), indicating that forced expression of p45 restored CD4 expression. In contrast, CD4 levels on DP cells of TCF-1^–/–p33^Tg mice were as low as that detected on TCF-1^–/– mice, indicating that p33 failed to rescue CD4 expression. These results strongly suggest that binding to -catenin is required for TCF-1-mediated up-regulation of CD4.

CD4 vs CD8 lineage development in TCF-1^–/– and -Cat^Tg mice

CD4⁺CD8⁺ DP cells eventually differentiate into either CD4⁺ Th cells or CD8⁺ CTLs. Engagement of CD4 with MHC class II molecule is believed to facilitate the development of CD4⁺ Th cells, whereas engagement of CD8 with MHC class I molecule assists the development of CD8⁺ T cells (2, 31). Because CD4 levels are changed on DP cells of TCF-1^–/– and -Cat^Tg mice, we determined whether there is a corresponding change in the development of CD4 vs CD8 SP cells. Thymic cellularity and numbers of different subsets of thymocytes from each genotype of mice were listed in Table I. Consistent with the function of TCF-1/-catenin in supporting DP thymocyte survival (21, 28), there was a significantly decreased thymic cellularity in TCF-1^–/– mice (7.8 x 10⁶), whereas greatly increased thymic cellularity developed in -Cat^Tg mice (395 x 10⁶) compared with WT (148 x 10⁶). Therefore, the absolute numbers of each subset of thymocytes are not comparable between different genotypes of mice. Thus, we calculated the ratio of CD4 to CD8 SP cells based on the percentage of each subset of cells (Table I). Similar to other reported results, the ratio of CD4 to CD8 SP cells is 2 to 1 in WT mice. Corresponding to the higher CD4 levels, the CD4:CD8 ratio was increased to about 4 to 1 in -Cat^Tg mice, whereas the CD4:CD8 ratio was reduced to <1 (0.78) in TCF-1^–/– mice that express lower levels of CD4 on DP cells. Similar results were obtained when gated on CD3^high SP cells (data not shown). These data suggest that the TCF/-catenin pathway enhances CD4 lineage development, and such enhancement is correlated with the ability of TCF/-catenin to stimulate CD4 expression.

View this table: [in this window] [in a new window]   Table I. Regulation of CD4 vs. CD8 lineage development by TCF/-catenina

Requirement of a TCF binding site for TCF/-catenin-mediated stimulation of CD4 Pr activity

DNase I footprint analysis with T cell nuclear extracts identified a potential TCF binding site (7). However, mutation of this TCF site had limited effect on enhancer activity. Before examining the function of this potential TCF binding site, we first determined whether TCF-1 or -catenin was able to directly stimulate CD4 Pr. To monitor CD4 Pr activity, we used a CD4 reporter that contains a luciferase reporter gene driven by a basic CD4 Pr and a 339-bp proximal enhancer as described previously (7). The reporter along with different amounts of expression plasmids encoding -catenin or TCF-1 were introduced into Jurkat cells by electroporation. CD4 reporter activity was obviously stimulated by a stabilized -catenin that is the same as the one used to generate -Cat^Tg mice, and thus resistant to degradation. However, the WT -catenin that is susceptible to degradation had minimal effects on the CD4 reporter (Fig. 3a), suggesting that activation of the -catenin pathway stimulates CD4 Pr. Similarly, TCF-1 stimulated CD4 reporter in a dose-dependent manner. However, CD4 reporter was not stimulated by TCF, which contains only DNA-binding domain but not the domain responsible for binding to -catenin, and thus was not able to mediate transcriptional activation (28). Consistent with the results obtained from TCF-1^–/– and -Cat^Tg mice, the in vitro transfection analysis also demonstrated that the TCF/-catenin pathway enhances CD4 expression. Furthermore, TCF/-catenin can directly stimulate CD4 Pr activity.

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Critical role of the potential TCF binding site in TCF-1/-catenin-mediated activation of CD4. a, TCF-1 or stabilized -catenin stimulates CD4 reporter. By electroporation, 107 Jurkat cells were transfected with different amounts of expression plasmids for WT -catenin, stabilized -catenin, WT TCF-1, and TCF (deletion of -catenin-interacting domain). CD4 Pr activity was monitored with a luciferase reporter gene driven by a CD4 basic Pr and a CD4 proximal enhancer. Luciferase activity is indicated as the fold of stimulation relative to the activity obtained from cells transfected with reporter alone. b, The putative TCF binding site mediates the transcriptional activation of CD4 reporter by TCF-1 and stabilized -catenin. WT CD4 reporter () or the CD4 reporter containing mutant TCF binding site (, CD4-TCFm reporter) were transfected into Jurkat cells together with control plasmid (control) or expression plasmids encoding TCF-1 or stabilized -catenin. c, TCF-1 or stabilized -catenin stimulates TOP, but not FOP reporter. TOP is a positive control containing 3x WT TCF sites, whereas FOP is a negative control containing 3x mutant TCF binding sites. Luciferase activity is indicated as the fold of stimulation relative to the activity obtained from cells transfected TOP alone. The reporter activity with error bars denoting SD was averaged from at least three independent experiments.

In vivo binding of TCF-1 to CD4 enhancer

Previous in vitro studies showed that factors in T cell nuclear extracts were able to bandshift synthesized double-strand oligonucleotides containing the potential TCF binding site (7). However, it is not clear whether it is TCF-1 binding to the enhancer, and whether such binding takes place in vivo. We used ChIP analysis to determine whether TCF-1 binds to enhancer, because such analysis allows detection of in vivo protein-DNA interactions (32). Five pairs of primers were designed to cover different regulatory regions of CD4 locus (Fig. 4a). PCR products for PE-1 covered the region containing the potential TCF binding site (TCF). The other four primers covered the Pr, LCR, and the putative thymocyte enhancer (TE) region (TE-1 and TE-2). First, we used DNA templates that were not subjected to immunoprecipitation (input) to adjust conditions so that each pair of primers could successfully amplify a single PCR band of expected size (Fig. 4b). PCR were then performed using the anti-TCF-1 Ab or control Ab-immunoprecipitated DNA templates. In WT mice, anti-TCF-1 reproducibly gave an enrichment band with PE-1 primers compared with control Ab (top panel). However, the strong enrichment band was not observed in TCF-1^–/– mice. Anti-TCF-1 Ab did not give detectable signals with LCR, TE-1, and TE-2 primers. These results suggest that TCF-1 specifically interacts with the enhancer region containing the potential TCF binding site. Interestingly, an enrichment band was repeatedly detected using Pr primers, indicating a weak interaction of TCF-1 with CD4 basic Pr region. One of the common mechanisms for enhancer function is to stimulate transcriptional initiation complexes binding to the Pr region (33). Transcription factors binding to enhancer regions are believed to make contact with transcriptional initiation complexes, resulting in stimulating transcription. The observed binding of TCF-1 to CD4 Pr may reflect such an interaction between enhancer and Pr.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. In vivo binding of TCF-1 to CD4 proximal enhancer. a, Schematic representation of the CD4 locus. Exons for CD4 gene and ubiquitin isopeptidase T (ISOT) are shown in the upper half, whereas CD4 regulatory elements are indicated in the lower half. Five pairs of arrows indicate the positions of primers for ChIP assays: PE-1, CD4 Pr, LCR, TE-1, and TE-2. b, ChIP assays. Nuclei purified from formaldehyde-treated thymocytes of WT and TCF-1–/– mice were subject to sonication. A portion of the sonicated samples was used as a template (input) for positive controls of PCR. Anti-TCF-1 Ab or control Ab (C) was then added to sonicated samples to perform immunoprecipitation. Targeted sequences in the immunoprecipitated complexes were then identified by PCR using specific primers described in a. Data shown are representative of at least three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Expression of CD4 requires coordinated action of multiple cis-elements and trans-factors. The basic CD4 Pr has minimal activity. To achieve physiological levels of CD4 expression, CD4 enhancers are required to stimulate the basic Pr activity (3, 8). Overriding the enhancer activity by a silencer results in preventing CD4 expression in DN and SP CD8 cells. However, the silencer activity is inhibited in DP and SP CD4 cells, which allows CD4 enhancer to stimulate the basic CD4 Pr, resulting in expression of CD4. A 339-bp core enhancer fragment has been shown to be sufficient to direct expression of various reporter genes in T cells and thymocytes of transgenic mice (7, 8, 35, 36, 37). Three nuclear protein binding sites (CD4-1, CD4-2, and CD4-3) were identified by DNase I footprinting analyses of the core enhancer fragment (5). Both CD4-1 and CD4-3 binding sites contain E-box motifs, which are the binding sites for basic helix-loop-helix transcription factors. Two basic helix-loop-helix factors that are preferentially expressed in thymocytes, E12 and HEB, are heterodimerized to bind to the CD4-3 site. HEB has been shown to directly bind the CD4 enhancer to recruit Mi-2, another critical factor for up-regulating CD4 expression (29). The enhancer activity was abolished by a mutation that disrupted the binding of E12/HEB heterodimer in in vitro transfection analyses. However, deletion of either HEB or Mi-2 gene leads to reduced but not abolished CD4 expression (29, 38). HEB and Mi-2 therefore enhance, but are not absolutely required for, CD4 expression. These results suggest that other trans factors that bind to CD4-2 may also be required for optimal CD4 expression.

CD4-2 contains a putative TCF/LEF binding site (5), but previous studies did not identify CD4 as a target gene for -catenin pathway. Staal et al. (39) identified 30 potential target genes of -catenin using DN cells, but CD4 was not found as a target gene. CD4 is not expressed at all in DN cells because that silencer overrides the activity of the CD4 enhancer (3). We believe that activation of -catenin itself is not sufficient to override silencer activity. It is likely that TCF-1/-catenin is not easily accessible to its binding site on enhancer due to closed chromatin structure of CD4 locus at DN stage. In this study, we provide both in vitro and in vivo evidence to support that TCF/-catenin pathway stimulates CD4 expression in DP cells. First, our and previous in vitro transfection studies showed that mutation of the TCF/LEF binding site leads to 40–50% reduction of CD4 Pr activity (Fig. 3b) (5). Second, forced expression of TCF-1 and -catenin resulted in stimulation of CD4 Pr activity in a TCF binding site-dependent manner (Fig. 3a). Third, transgenic expression of a stabilized -catenin that enhances TCF-1 activity leads to up-regulation of CD4 in T cells. Fourth, deletion of TCF-1 gene resulted in reduced CD4 expression. Lastly, ChIP analyses indicated that TCF-1 physically binds to CD4 enhancer in vivo. However, similar to HEB that binds to the E-boxes of CD4 enhancer, TCF-1 is able to stimulate, but is not absolutely required for, CD4 expression, because deletion of TCF-1 only impaired but not abolished expression of CD4. Expression of CD4 is therefore dependent on synergistic action of TCF-1 and other trans-factors including HEB and Mi-2.

Previous studies have shown that CD4 proximal enhancer is active in both DP and CD4 SP T cells (7). Consistent with this notion, transgenic -catenin stimulates CD4 expression in both DP and CD4 SP cells (Fig. 2). However, deletion of TCF-1 only affected CD4 expression in DP, but not in CD4 SP cells (Fig. 1), suggesting that TCF-1 regulates CD4 expression specifically in DP cells. It is very likely that TCF-1 is replaced by other transcription factors in CD4 SP cells. Considering that stabilized -catenin still stimulates CD4 expression in SP cells, it is very likely that a developmental switch allows other members of the TCF family transcription factors to be recruited to the CD4 enhancer when DP cells differentiate into CD4 SP T cells. One of the possible mechanisms responsible for this switch may be the expression of TCF-1 itself, as followed by its peak expression in DP cells, and TCF-1 is down-regulated in SP cells (20). The fact that TCF-1 is differentially required for CD4 expression in DP and SP cells suggests that CD4 enhancer activity is differentially regulated during different stages of T cell development.

By binding to MHC II molecules, CD4 facilitates eventual development of CD4 SP T cells in the thymus. Consistent with this notion, decreased CD4 levels of thymocytes of TCF-1^–/– mice are associated with the reduced CD4:CD8 SP cell ratio. The decreased CD4:CD8 ratio could be due to other changes such as the developmental block at DN to DP stages observed in TCF-1^–/– mice (22). However, when TCF pathway was enhanced by transgenic expression of a stabilized -catenin, we observed a corresponding increased CD4:CD8 ratio that is associated with higher CD4 levels. Importantly, transgenic -catenin (-cat^Tg) starts expression at DP cells, and did not affect the DN to DP transition (28). It thus excludes the possibility that the skewed CD4:CD8 ratio and higher CD4 levels observed in -cat^Tg mice were due to effects on the development of earlier DN cells. Therefore, TCF/-catenin pathway appears to skew T cell development to CD4 lineage likely via up-regulation of CD4. In contrast to our results, Mulroy et al. (40) observed a relatively increased SP CD8 population in their transgenic mice expressing an active -catenin. We believe that the stabilized -catenin in their transgenic mice may not be sufficient to compete with endogenous -catenin, because the levels of transgenic -catenin in their mice were similar to that of the endogenous -catenin. Stabilized -catenin in our transgenic mice overcame the endogenous -catenin and enhanced thymocyte survival and CD4 expression. Due to lack of effects on both DN and DP cells, the effects of their transgene are only restricted to SP CD8 cells. We believe that the phenotype observed in their transgenic mice resulted from effects of transgene at the SP stage, whereas what we observed in our -catenin transgenic mice is due to the effects of the transgene at the DP stage.

The other reported function of TCF/-catenin pathway is to enhance thymocyte survival, because TCF-1^–/– thymocytes displayed increased apoptosis, and thymocytes obtained from our -cat^Tg mice resisted to spontaneous and glucocorticoid-induced apoptosis (21, 28). The ability of TCF/-catenin pathway to regulate Bcl-x_L is related to thymocyte survival, because forced expression of Bcl-2 rescued apoptosis of TCF-1^–/– thymocytes. However, Bcl-2 did not rescue surface CD4 levels in TCF-1^–/– mice. Thus, down-regulation of CD4 is not linked to the apoptosis observed in TCF-1^–/– mice. Furthermore, Bcl-2 did not rescue the reduced CD4:CD8 ratio in TCF-1^–/– mice either, suggesting that the reduced CD4:CD8 ratio is likely correlated to the lower CD4 levels, but not the apoptosis in TCF-1^–/– mice. Therefore, TCF/-catenin-regulated survival and CD4 expression are two separate events.

CD4 proximal enhancer augments CD4 Pr activity to achieve optimal CD4 expression. Understanding the mechanisms underlying this proximal enhancer-regulated CD4 expression requires identification of the trans-factors that bind to the enhancer and regulate its activity. In this study, we have identified TCF-1 as a trans-factor that regulates CD4 expression in vivo. However, other trans-factors such as HEB and Mi-2 are also required to stimulate CD4 expression. Elucidation of the relationship between TCF/-catenin pathway and other molecules in the regulation of CD4 expression will facilitate the understanding of T cell development and function.

	Acknowledgments

 
We thank Dr. Phil Howe for providing TOP and FOP reporters, Drs. Frank McCormick and Osamu Tetsu for -catenin expression plasmids, and Dr. Marian Waterman for expression plasmids of WT TCF and DN TCF. Transgenic mice were generated in the University of Illinois at Chicago transgenic core facility. We also thank Dr. Prasad Kanteti for critically reading the manuscript and providing helpful discussion.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Current address: Medicine-Hematology and Oncology, Northwestern University, 710 North Fairbanks, Olson 8370, Chicago, IL 60611.

² Address correspondence and reprint requests to Dr. Zuoming Sun, Department of Microbiology & Immunology, College of Medicine, University of Illinois at Chicago, 835 South Wolcott (M/C790), Chicago, IL 60612. E-mail address: zuoming{at}uic.edu

³ Abbreviations used in this paper: DN, double negative; DP, double positive; SP, single positive; Pr, promoter; TCF, T cell factor; LEF, lymphoid enhancer factor; WT, wild type; TOP, topflash; FOP, fopflash; HPRT, hypoxanthine guanine phosphoribosyl transferase; PE-1, proximal enhancer 1; LCR, locus control region; TE, thymocyte enhancer; ChIP, chromatin immunoprecipitation.

Received for publication October 4, 2005. Accepted for publication February 6, 2006.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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