BCL11B is a transcriptional regulator with an important role in T-cell development and leukaemogenesis. We demonstrated recently that BCL11B controls expression from the IL (interleukin)-2 promoter through direct binding to the US1 (upstream site 1). In the present study, we provide evidence that BCL11B also participates in the activation of IL-2 gene expression by enhancing NF-κB (nuclear factor κB) activity in the context of TCR (T-cell receptor)/CD28-triggered T-cell activation. Enhanced NF-κB activation is not a consequence of BCL11B binding to the NF-κB response elements or association with the NF-κB–DNA complexes, but rather the result of higher translocation of NF-κB to the nucleus caused by enhanced degradation of IκB (inhibitor of NF-κB). The enhanced IκB degradation in cells with increased levels of BCL11B was specific for T-cells activated through the TCR, but not for cells activated through TNFα (tumour necrosis factor α) or UV light, and was caused by increased activity of IκB kinase, as indicated by its increase in phosphorylation. As BCL11B is a transcription factor, we investigated whether the expression of genes upstream of IκB kinase in the TCR/CD28 signalling pathway was affected by increased BCL11B expression, and found that Cot (cancer Osaka thyroid oncogene) kinase mRNA levels were elevated. Cot kinase is known to promote enhanced IκB kinase activity, which results in the phosphorylation and degradation of IκB and activation of NF-κB. The implied involvement of Cot kinase in BCL11B-mediated NF-κB activation in response to TCR activation is supported by the fact that a Cot kinase dominant-negative mutant or Cot kinase siRNA (small interfering RNA) knockdown blocked BCL11B-mediated NF-κB activation. In support of our observations, in the present study we report that BCL11B enhances the expression of several other NF-κB target genes, in addition to IL-2. In addition, we provide evidence that BCL11B associates with intron 2 of the Cot kinase gene to regulate its expression.
BCL11B, also known as CTIP2 [CtBP (C-terminal binding protein)-interacting protein 2], is a C2H2 zinc-finger transcription factor with a critical role in T-cell and brain development [1–3]. BCL11B was initially identified as a co-repressor for COUP-TF (chicken ovalbumin upstream promoter-transcription factor) nuclear receptors  and shown later to bind directly to DNA and recruit the NuRD (nucleosome remodelling and deacetylase) complex to repress expression from targeted promoters [5,6]. In addition, we demonstrated previously that BCL11B participates in the transcriptional activation of IL-2 [IL (interleukin)-2] gene expression in response to TCR (T-cell receptor) activation by direct binding to the US1 (upstream site 1) in the IL-2 promoter . IL-2 is the first cytokine whose expression is induced immediately after T-cell activation through TCR/CD28 signalling . Briefly, calcineurin, a calcium/calmodulin-dependent serine/threonine phosphatase, and PKCθ (protein kinase Cθ) are activated. The primary target for calcineurin is the NFAT (nuclear factor of activated T-cells), which is dephosphorylated and translocated to the nucleus, where it binds to the IL-2 promoter . PKCθ is required for the activation of the transcription factors NF-κB (nuclear factor κB) and AP-1 (activator protein-1) (Fos/Jun) [10,11]. NF-κB activation requires a second co-stimulatory signal provided by the CD28 receptor .
In the present study, we demonstrate that BCL11B participates in the activation of IL-2 gene expression not only through binding to the US1, but also by enhancing NF-κB activation in the context of TCR/CD28-triggered T-cell activation. This process occurs without direct binding by BCL11B to the NF-κB response elements or association with NF-κB–DNA complexes, but rather occurs indirectly through regulation of Cot (cancer Osaka thyroid oncogene) kinase gene expression and the consequent higher activation of IκB (inhibitor of NF-κB) kinase. Cot/Tpl2 (tumour progression locus 2)/MAP3K8 (mitogen-activated protein kinase kinase kinase 8) is a kinase which has been implicated in NF-κB activation and IL-2 gene expression by regulation of the IKK (IκB kinase) complex in T-lymphocytes, downstream of the CD28 pathway [13–15]. Our results demonstrate that a DN (dominant negative) mutant of Cot kinase  and Cot kinase siRNA (small interfering RNA) knockdown inhibit BCL11B-mediated activation of NF-κB activity, supporting the idea that Cot kinase plays a role in BCL11B-mediated activation of NF-κB. We also report that BCL11B activates additional NF-κB target genes in response to T-cell activation.
MATERIALS AND METHODS
The pΔODLO 4xCD28RE-TRE-luciferase, AP-1-luciferase, NFAT-luciferase  and Cot kinase DN (Cot S400A/S413A)  plasmids were generously given by Dr Arthur Weiss (Department of Rheumatology, University of California San Francisco, San Francisco, CA, U.S.A.). The NF-κB consensus (pNF-κB-Luciferase) and Renilla (pRL-Luciferase) reporter vectors were purchased from Clontech. FLAG–BCL11B was cloned into pRevTRE (Clontech) to generate the pRevTRE-BCL11B plasmid. The MSCV–BCL11B plasmid has been described previously .
Antibodies and biochemicals
The polyclonal anti-BCL11B (B26-44) antibody has been described previously . Additional anti-BCL11B antibodies were purchased from Bethyl Laboratories. The mouse monoclonal anti-actin antibody was purchased from Sigma–Aldrich. The antihuman CD3 (OKT) and anti-human CD28 antibodies were from eBioscience. The antibodies against p65 (RelA) (F-6), p105/p50 (H-119), IκBα (C-21), IκBβ (C-20), IKKα/β (H-470) and Cot (M-20) were from Santa Cruz Biotechnology. The anti-(phospho-IκBα) and anti-(phospho-IKKα/β) antibodies were purchased from Cell Signaling Technology. PMA, ionomycin and MG132 were purchased from Sigma–Aldrich and used at the concentrations stated in the Figure legends. Recombinant human TNF (tumour necrosis factor) α was obtained from R&D Systems and used at a final concentration of 20 ng/ml.
Jurkat and HeLa cells were obtained from the A.T.C.C. Jurkat cells were grown in RPMI 1640 medium containing 2 mM L-glutamine, 10% (v/v) HI-FBS (heat-inactivated fetal bovine serum), 50 units/ml penicillin and 50 μg/ml streptomycin. HeLa cells were grown in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) HI-FBS, 50 units/ml penicillin and 50 μg/ml streptomycin. The generation of the Jurkat MSCV (murine stem cell virus) and MSCV–BCL11B cell lines was performed as described previously . In a similar manner, HeLa TetON cells (Clontech) were transduced with pRevTRE-BCL11B retroviruses to generate the inducible cell line HeLa TetON–BCL11B.
Transient transfections and luciferase reporter assays
Jurkat cells were transfected by electroporation as described previously [6,7]. pRevTRE-BCL11B HeLa TetON cells were seeded on 6-cm-diameter plates (106 cells/plate) and transfected the following day with 2 μg of the reporter plasmids using Lipofectamine™ (Invitrogen). The cells were maintained in medium supplemented with 50 μg/ml doxycyclin for 48 h before harvesting. For reporter assays, the cells were stimulated as indicated in the Figure legends and then harvested in Renilla Lysis Buffer (Promega). Luciferase activity was analysed as described previously .
Gene knockdown by siRNA
BCL11B- and Cot-specific and control non-targeting siRNAs were purchased from Santa Cruz Biotechnologies and Dharmacon respectively. Jurkat cells (0.5×106 cells) were electroporated as described previously . The following day, cells were treated with PMA and ionomycin and then harvested for reporter assays or RNA preparation as described previously .
Nuclear fractionation, RT-PCR (reverse transcription-PCR), quantitative RT-PCR and chromatin immunoprecipitation
EMSA (electrophoretic mobility-shift assay) and Western blotting
These methods were performed as described previously .
BCL11B regulates the IL-2 promoter through site(s) located between −210 and −190 nt, in addition to binding to the US1
Our previous results using populations of Jurkat cells stably transduced with retroviruses expressing GFP (green fluorescent protein) (MSCV) and FLAG–BCL11B–IRES–GFP (MSCV–BCL11B) demonstrated that BCL11B regulates IL-2 gene expression by binding directly to the US1 located between −243 and −210 nt in the IL-2 promoter . However, following deletion of the US1-containing region, although the activity of the reporter decreased considerably, BCL11B continued to activate expression of the reporter, suggesting that site(s) downstream of −210 nt may be implicated in BCL11B-mediated activation of the IL-2 promoter (Figure 1). Deletion of the region between −210 and −190 nt resulted in an approx. 3-fold reduction in BCL11B-mediated augmentation of the IL-2 promoter, bringing the levels of the reporter construct in MSCV–BCL11B and MSCV cells to an approximately equal level (Figure 1). These results suggest that BCL11B regulates the expression of IL-2 through site(s) located between −210 and −190 nt, in addition to the US1.
BCL11B regulates the expression of IL-2 through site(s) located between −210 and −190 nt in addition to US1
BCL11B activates transcription from the NF-κB sites in the context of TCR activation
The −210–−190 nt region of the IL-2 promoter does not contain any US1 or consensus BCL11B sites; however, it contains an NF-κB site, located upstream of −190 nt and designated as a distal NF-κB site (Figure 1A) [11,15]. To investigate whether BCL11B may regulate the −210 to −190 nt region through the NF-κB site, we conducted reporter assays with a construct containing the consensus NF-κB site. The results showed enhanced expression of the NF-κB reporter in MSCV–BCL11B Jurkat cells compared with MSCV, suggesting that BCL11B activates the expression driven by the NF-κB site (Figure 2A). Conversely, reporter constructs controlled by NFAT or AP-1 showed similar levels of activation in both MSCV–BCL11B and control cells (Figure 2B), indicating that BCL11B-mediated activation is specific for NF-κB.
BCL11B activates transcription from NF-κB sites, but not from AP-1 or NFAT sites
NF-κB also binds the CD28RE (CD28-responsive element)/TRE (PMA-responsive element) in the IL-2 promoter and participates in the co-stimulatory activation of IL-2 gene expression . The CD28RE/TRE site is located downstream of −190 nt (Figure 1A) and plays a critical role in CD28-mediated full activation of the IL-2 promoter . Its participation in the co-stimulatory activation of the IL-2 promoter can only be evaluated when the −210 nt IL-2 promoter is intact, as it requires the distal NF-κB site [11,15]. However, the activation of multimers of CD28RE/TRE can be evaluated in the context of a TATA-like promoter in T-lymphocytes, in response to activation through TCR/CD28 or PMA and ionomycin . We therefore tested the activity of a reporter controlled by the CD28RE/TRE composite element in the context of a TATA-like promoter and found that, in response to stimulation with PMA and ionomycin, the activity of this composite site was higher in MSCV–BCL11B cells compared with MSCV control cells (Figure 2C). We further investigated whether the enhanced activation of NF-κB mediated by BCL11B occurs also in conditions considered to be closer to physiological activation, through treatment with anti-CD3 and anti-CD28 antibodies. Previous observations demonstrated that activation of NF-κB requires co-stimulatory signals generated through CD28 . When TCR was stimulated alone through treatment with an anti-CD3 antibody, the level of expression of the reporter was minimal, as expected (Figure 2D). When cells were activated by both anti-CD3 and anti-CD28 antibodies, the luciferase activity was higher in MSCV–BCL11B Jurkat cells compared with control cells (Figure 2D). These results suggest that enhanced activation of NF-κB mediated by BCL11B requires CD28 co-stimulatory pathways.
Taken together, these results demonstrate that BCL11B augments transcription from NF-κB sites in the context of activation of T-cells through TCR/CD28.
BCL11B knockdown reduces the transcriptional activation controlled by NF-κB response elements
We had previously found that knockdown of BCL11B caused a significant decrease in IL-2 promoter activation . To demonstrate that endogenous BCL11B plays a role in the activation of transcription from NF-κB sites, we used siRNA knockdown to decrease the level of endogenous BCL11B (Figure 3A). Knockdown of BCL11B resulted in a reduction of CD28RE/TRE composite-element activity, demonstrating that endogenous BCL11B is involved in the regulation of NF-κB activation (Figure 3B).
Endogenous BCL11B is required for full activation of transcription from the NF-κB site
Augmentation of transcription from NF-κB sites by BCL11B is a consequence of increased nuclear levels of p50 and p65 in T-lymphocytes, activated through the TCR
BCL11B can regulate the NF-κB sites in the IL-2 promoter by binding directly to the NF-κB sites, through association with NF-κB or by increasing the activation of NF-κB. We first evaluated by EMSA whether BCL11B binds directly to the NF-κB sites and found that BCL11B did not bind either to the consensus site, distal NF-κB, nor to the response element/AP-1 sites (results not shown). We then used EMSA to determine whether BCL11B is present in the NF-κB–DNA complexes and co-operatively binds the NF-κB sites together with NF-κB. The results show that the p65 (RelA)/p50–DNA complex does not contain BCL11B, as it was not shifted by anti-BCL11B antibodies (Figure 4A). It should be noted that the anti-BCL11B antibodies are appropriate for EMSAs, as demonstrated previously . These results show that BCL11B neither binds NF-κB sites nor interacts with the NF-κB (p50/p65)–DNA complexes.
BCL11B enhances nuclear levels of p50 and p65, as well as binding to NF-κB consensus sites following T-cell activation
It has been demonstrated that NF-κB is translocated to the nucleus as a result of its activation . We therefore evaluated the nuclear levels of NF-κB in response to T-cell activation by PMA and ionomycin or anti-CD3 and anti-CD28 antibodies. At early time points of TCR activation, the p65(RelA)–p50 complex is the major NF-κB complex present in the nucleus  and, as a result of this, we evaluated the nuclear levels of p50 and p65 (RelA) and found that they were increased in MSCV–BCL11B Jurkat cells compared with control cells (Figure 4B). In addition, we observed that DNA–NF-κB complex formation was enhanced in MSCV–BCL11B cells compared with control cells (Figure 4C). These results show that the regulation of NF-κB activity by BCL11B is a consequence of higher levels of p50 and p65 in the nucleus of activated cells, and occurs without association of BCL11B with NF-κB binding sites or with NF-κB–DNA complexes.
BCL11B enhances IκB degradation in response to TCR, but not UV-mediated activation
We next wanted to determine the cause of enhanced NF-κB nuclear translocation in the presence of increased levels of BCL11B. Nuclear translocation of NF-κB is a direct consequence of IκB degradation . As enhanced expression of BCL11B leads to increased nuclear translocation of p65 and p50, we investigated whether IκB degradation was elevated in MSCV–BCL11B cells compared with control cells. Under resting conditions, the levels of IκBα and IκBβ were similar in both MSCV–BCL11B and MSCV cells (Figure 5A, top panel, 0 h). However, in response to activation with PMA and ionomycin, IκBα degradation occurred significantly faster in MSCV–BCL11B cells (Figure 5A, top panel, 15 and 45 min). Although degradation of IκBβ degradation occurred with slower kinetics, BCL11B also enhanced its degradation (Figure 5A, middle panel). Therefore these results suggest that the augmentation of NF-κB activity by BCL11B in cells activated by PMA and ionomycin occurs through enhanced degradation of IκB. Degradation of IκBα/β triggered by TCR/CD28 or PMA and ionomycin treatment is a three step process. First, IκB is phosphorylated by IKK. Secondly, phosphorylated IκB is ubiquitinated, and, thirdly, ubiquitinated IκB is degraded by the 26S proteasome . Since the level of IκB was reduced in MSCV–BCL11B cells compared with control cells, we wanted to investigate in which of these steps BCL11B is involved. It was demonstrated previously that UVC radiation triggers ubiquitin-dependent degradation of IκB through a process which is independent from IKK activation [21,22]. If BCL11B is implicated in the ubiquitination and/or degradation of IκBα/β, then its sustained expression would result in enhanced degradation of IκB, regardless of how the cells are activated, i.e. through TCR/CD28 or UV treatment. Our results indicate that IκBα/β degradation in response to UV radiation occurred at the same rate in both MSCV–BCL11B and control MSCV Jurkat cells (Figure 5B), suggesting therefore that BCL11B does not enhance the ubiquitination and/or proteosome-mediated degradation of IκBα/β proteins.
BCL11B enhances IκB phosphorylation and degradation, and IKK phosphorylation in a TCR-dependent manner
BCL11B enhances IκB and IKK phosphorylation in T-lymphocytes activated through TCR
We then tested whether phosphorylation of IκB is enhanced by BCL11B overexpression in conditions of TCR activation. In these experiments, the degradation of IκB was blocked by pre-treatment of the cells with the proteasome inhibitor MG132, and phosphorylated IκBα was analysed in MSCV–BCL11B and control cells after stimulation with PMA and ionomycin. The levels of phosphorylated IκBα were significantly higher in MSCV–BCL11B cells, suggesting that IKK may be more active in these cells (Figure 5C). To demonstrate directly that the enhanced phosphorylation of IκB is a consequence of increased activation of IKK, we tested the levels of phosphorylated IKK, known to be activated through phosphorylation following T-cell activation through TCR/CD28 or PMA and ionomycin treatments. The levels of phosphorylated IKKα/β were higher in MSCV–BCL11B cells compared with control cells (Figure 5D). In summary, these results demonstrate that enhanced degradation of IκB in MSCV–BCL11B cells is a consequence of enhanced phosphorylation, and consequently of activation of IKK.
BCL11B does not participate in the TNFα-mediated activation of NF-κB in T-lymphocytes
In addition to TCR/CD28 stimulation, IKK can be activated by TNFα, but the upstream components of the two pathways are different . We therefore investigated whether BCL11B enhances NF-κB activation by a mechanism specific for a TCR/CD28- or PMA- and ionomycin-activated-pathway, or by a common mechanism converging towards IKK activation. The results show that IκB degradation after TNFα treatment was similar in both MSCV–BCL11B and control cells (Figure 5E), contrary to what was observed when cells were activated by PMA and ionomycin (Figure 5A). These findings suggest that BCL11B acts specifically on TCR/CD28-mediated IKK activation.
Enhanced activation of NF-κB mediated by BCL11B occurs specifically in T-lymphocytes
To further demonstrate that enhanced activation of NF-κB mediated by BCL11B involves downstream components of the TCR/CD28 signalling pathway and occurs specifically in T-lymphocytes, we used a HeLa cell line in which we stably expressed ectopic BCL11B (Figure 5F, right-hand panel). We chose HeLa cells because they do not express endogenous BCL11B (Figure 5F, right-hand panel) and lack the TCR/CD28 signalling pathway. However, NF-κB can be activated by PMA and ionomycin treatment of these cells . We conducted reporter assays with the consensus NF-κB–luciferase construct. The results showed that the relative luciferase activity was the same regardless of the presence or absence of BCL11B (Figure 5F, left-hand panel).
These results collectively demonstrate that the enhanced activation of NF-κB mediated by BCL11B is specific for T-lymphocytes and involves the TCR/CD28 signalling pathway.
BCL11B up-regulates the levels of Cot kinase mRNA
The results presented above indicate that BCL11B enhances NF-κB activation specifically in T-lymphocytes, downstream of the TCR/CD28 signalling pathway and upstream of IKK. BCL11B is a transcription factor localized almost exclusively to the nucleus of Jurkat cells (results not shown). Therefore it is unlikely that BCL11B is involved directly in IKK activation. Rather, it is possible that BCL11B regulates the expression of genes involved in TCR/CD28-triggered IKK activation. To test this hypothesis, we investigated the expression levels of known genes implicated in TCR/CD28-mediated IKK activation in MSCV–BCL11B and MSCV control cells by quantitative RT-PCR, including IKKα, IKKβ, IKKγ, VAV, SLP-76 (SH2 domain-containing leucocyte protein of 76kDa), ZAP70 [ζ-chain (TCR)-associated protein kinase of 70 kDa], LAT (linker for activation of T-cells), LCK (lymphocyte-specific protein tyrosine kinase), PKCθ, calcineurin, CARMA1 [CARD11 (caspase recruitment domain family, member 11)], BCL10, MALT1 (mucosa-associated lymphoid tissue lymphoma translocation gene 1), PI3K (phosphoinositide 3-kinase), Akt, Cot, NIK (Nck-interacting kinase), PDK1 (phosphoinositide-dependent kinase 1), TRAF2 (where TRAF is TNF-receptor-associated factor), TRAF6, TAK1 (transforming growth factor-β-activated kinase), RIP2 (receptor-interacting protein 2), MLK3 (mixed-lineage kinase 3), β-TrCP1 (β-transducin repeat-containing protein 1), cullin1, RBX1 (ring-box 1), E2UbcH5 (E2 ubiquitin-conjugating enzyme UbcH5), RAC1, UBC13, Need 8 and caspase 8 (Table 1). From all of the genes tested, only Cot kinase mRNA levels were up-regulated in MSCV–BCL11B cells compared with control cells (Table 1 and Figure 6A). Cot kinase is known to be involved in the activation of IKK following T-lymphocyte stimulation through TCR/CD28 . It is notable that Cot kinase mRNA levels were already higher in cells overexpressing BCL11B even in the absence of TCR activation (Figure 6A).
|Gene||Ratio of relative expression level (MSCV–BCL11B/MSCV)|
|Gene||Ratio of relative expression level (MSCV–BCL11B/MSCV)|
Enhanced activation of NF-κB by BCL11B is mediated through Cot kinase gene expression
A Cot kinase DN mutant and knockdown of Cot kinase reduced BCL11B-mediated activation of NF-κB
To demonstrate further that BCL11B activates NF-κB through Cot kinase up-regulation, MSCV and MSCV–BCL11B Jurkat cells were transfected with a Cot kinase DN construct (Cot S400A/S413A)  or Cot kinase siRNA knockdown (Figure 6B). Expression of the Cot kinase DN mutant and knockdown of Cot kinase both blocked the enhanced activation mediated by BCL11B on the NF-κB-driven CD28RE/TRE reporter (Figure 6B, upper panel), demonstrating that Cot kinase is implicated in BCL11B-mediated activation of NF-κB. The levels of expression of the Cot kinase DN mutant were similar in both MSCV and MSCV–BCL11B Jurkat cell lines (Figure 6B, lower left-hand panel). Since the levels of endogenous Cot kinase are undetectable with the antibody used for the detection of the transfected mutant (results not shown), we used quantitative RT-PCR to detect the reduction in Cot kinase mRNA levels (Figure 6B, lower right-hand panel).
To demonstrate further that Cot kinase is responsible for BCL11B-mediated activation of NF-κB and further effects on downstream genes, such as IL-2, we knocked down Cot kinase and evaluated IL-2 mRNA levels in MSCV and MSCV–BCL11B cells. The reduction in the level of IL-2 mRNA in MSCV–BCL11B cells was more pronounced compared with MSCV cells (2.5- and 1.9-fold respectively) (Figure 6C), demonstrating the contribution of Cot kinase in the up-regulation of IL-2 mediated by BCL11B through NF-κB. The levels of IL-2 mRNA remained higher in MSCV–BCL11B cells compared with MSCV cells after knockdown of Cot kinase, supporting our previous observation that BCL11B also controls IL-2 gene expression directly .
Taken together, these results demonstrate that Cot kinase is responsible for the BCL11B-mediated activation of NF-κB, and for further downstream effects.
BCL11B modulates the expression of NF-κB-dependent genes in response to T-cell activation
NF-κB was demonstrated previously to regulate the expression of genes encoding cytokines, chemokines and several cytokine receptors, including IL-2 , TNFα , TNFβ , IFNγ (interferon γ) , IL-8 , lymphotoxin β , MIP1 (macrophage inflammatory protein 1) α/β , IL-2Rα (IL-2 receptor α)  and IL-7Rα (IL-7 receptor α) . As BCL11B enhances NF-κB activation in lymphocytes, we measured the mRNA levels for genes demonstrated previously to be regulated by NF-κB. Quantitative RT-PCR assays demonstrated that the expression of the NF-κB-dependent genes, but not the expression of IL-4, was up-regulated in MSCV–BCL11B cells (Table 2).
|Gene||Ratio of relative mRNA levels (MSCV–BCL11B/MSCV)|
|Gene||Ratio of relative mRNA levels (MSCV–BCL11B/MSCV)|
To demonstrate that endogenous BCL11B plays a role in the activation of the NF-κB-dependent genes, we knocked down BCL11B, which resulted in the reduction of mRNA levels of the majority of NF-κB-dependent genes (Table 3), demonstrating that endogenous BCL11B plays a role in regulation of their expression. As expected, Cot kinase and IL-2 mRNA levels were also down-regulated (Table 3).
|Gene||Ratio of relative mRNA levels (siRNA BCL11B/control)|
|Gene||Ratio of relative mRNA levels (siRNA BCL11B/control)|
BCL11B associates with intron 2 of the Cot kinase gene
We further investigated whether BCL11B associates with the Cot kinase promoter and found that there was no binding above background within the 519 bp upstream of the mRNA start site (Figure 7), which suggests that BCL11B is unlikely to bind to the promoter. It has been reported that the Cot kinase gene has nine exons, the first two of which correspond to the 5′-untranslated region [34,35]. Regulatory elements can be also located within intronic regions, often within the first introns. We therefore investigated whether BCL11B binds to intron 2, which precedes the first ATG start codon. Our results show that BCL11B bound within this intron to a region upstream of the 5′-splice site. Interestingly, the binding was enhanced in response to PMA and ionomycin treatment (Figure 7). These results suggest that BCL11B regulates the expression of the Cot kinase gene by associating with a region in intron 2. Our results also indicate that the Cot kinase gene intron 2 potentially contains regulatory elements responsive to PMA and ionomycin, in addition to those demonstrated previously to be present in the promoter.
Endogenous BCL11B binds to Cot kinase gene intron 2
Our previous studies demonstrated that BCL11B participates in IL-2 gene expression in response to T-cell activation through direct binding of the US1 in the IL-2 promoter . In the present study, we show that BCL11B also participates in the activation of IL-2 gene expression through an indirect mechanism, as a result of modulation of NF-κB activity, through enhanced Cot kinase gene expression. In the first mechanism, as a consequence of T-cell activation through TCR/CD28 stimulation or PMA and ionomycin treatment, BCL11B binds and directly activates the IL-2 promoter through the US1 . In the second, indirect, mechanism, BCL11B enhances NF-κB activity, which further binds and activates the IL-2 promoter. Interestingly, we found that BCL11B up-regulates Cot gene expression even before T-cell stimulation. Thus it is expected that NF-κB activation is enhanced ***immediately after T-cell stimulation in cells expressing ectopic BCL11B. Indeed, it is sufficient to stimulate the cells for only 1 h in order to detect stronger translocation of NF-κB into the nucleus and an increase in DNA–NF-κB complex formation. It is notable that at this early time point of activation, the p65 (RelA)–p50 complex is the major NF-κB complex present on the IL-2 promoter . Expression of a DN mutant of Cot kinase, as well as Cot kinase siRNA knockdown, inhibited BCL11B-mediated NF-κB activation, supporting the theory that Cot kinase plays a role in BCL11B-mediated NF-κB activation. Enhanced Cot kinase expression in cells with higher levels of BCL11B results in elevated IKK activity, and enhanced IκBα/β phosphorylation and degradation. In addition, our results suggest that BCL11B-mediated NF-κB activation involves pathways specific to T-lymphocytes and is triggered by TCR/CD28 signals or PMA and ionomycin, but not by TNFα or UV radiation.
The NF-κB family of transcription factors has a key role in co-ordinating expression of a wide variety of genes that control both innate and adaptive immune responses . NF-κB-activating signalling pathways converge into IKK complex activation [23,36]. IKK-mediated IκB phosphorylation triggers the subsequent proteolytic destruction of the inhibitory complex, thus allowing NF-κB to translocate to the nucleus and activate target genes . Activation of the NF-κB pathway by IL-1, lipopolysaccharide and TNFα has been well characterized. Much progress has been made previously in understanding the biochemical mechanisms involved in NF-κB activation triggered by the TCR/CD28 pathways (reviewed in [23,36]). TCR engagement leads to the activation of several transcription factors, including NFAT, AP-1 and NF-κB . Stimulation of the TCR complex alone efficiently activates NFAT, whereas activation of NF-κB requires co-stimulatory signals from CD28 . Several studies demonstrated that CD28 co-stimulation is required for potent NF-κB activation and sustained IL-2 expression in T-lymphocytes [12,38,39]. In the present study, we demonstrate that the enhanced activation of CD28RE/TRE response elements by BCL11B requires CD28 co-stimulatory signals. Interestingly, it has been demonstrated that Cot kinase is required for TCR/CD28-induced, but not TNFα-induced, NF-κB activation in lymphocytes [14,15], which supports our observations that BCL11B enhances IκBα/β degradation after TCR-, but not after TNFα-mediated stimulation of Jurkat cells.
In response to TCR activation, NF-κB controls the expression of genes encoding multiple cytokines, chemokines and cytokine receptors . In the present study, we show that BCL11B, in addition to participating in the activation of IL-2 gene expression, also plays a role in the activation of expression of other NF-κB target genes, including TNFα and TNFβ, lymphotoxin β and MIP1α, as well as IL-2Rα and IL-7Rα. In conclusion, the results presented here demonstrate that BCL11B enhances NF-κB activation through the up-regulation of Cot kinase expression and, consequently, by promoting TCR/CD28-triggered IKK activation.
In addition to the important role of NF-κB in the transcriptional control of genes encoding cytokines, chemokines and cytokine receptors in response to TCR/CD28-triggered T-lymphocyte activation (reviewed in ), several reports have demonstrated that NF-κB is constitutively activated in T-cell leukaemia, but the molecular mechanism involved remains poorly defined [40,41]. Also, Cot kinase was found to be constitutively activated in several human T-cell leukaemia cell lines , and BCL11B was recently identified as a potential oncogene for ATL (adult T-cell leukaemia/lymphoma) . Thus our results may also suggest an interesting functional link between the enhanced expression of BCL11B and the constitutive activation of NF-κB in the ATL cells.
We thank Dr Arthur Weiss (Department of Rheumatology, University of California San Francisco, San Francisco, CA, U.S.A.) for constructs and Dr Gary Nolan (Department of Microbiology and Immunology, Stanford University, Palo Alto, CA, U.S.A.) for Phoenix A and E packaging cells. We also thank Hong-Mei Chen and Jennifer Gecewicz for technical assistance, Adrian Avram for graphical presentation and Debbie Moran for secretarial assistance.
adult T-cell leukaemia/lymphoma
cancer Osaka thyroid oncogene
electrophoretic mobility-shift assay
green fluorescent protein
heat-inactivated fetal bovine serum
IL-2 receptor α
IL-7 receptor α
macrophage inflammatory protein 1
murine stem cell virus
nuclear factor of activated T-cells
nuclear factor κB
inhibitor of NF-κB
protein kinase Cθ
small interfering RNA
tumour necrosis factor
upstream site 1
This work was supported by the National Institutes of Health/National Institute of Allergy and Infectious Diseases [grant number R01AI078273]; and by the American Cancer Society [grant number ACS-RSG-04-265-01-MGO] awarded to D. A.