Identification of nerve growth factor-responsive element of the TCL1 promoter as a novel negative regulatory element

The serine/threonine kinase, Akt (Protein Kinase B) plays a central role in the regulation of intra-cellular cell survival. Recently, we demonstrated that the protooncogene TCL1, overexpressed in human T-cell prolymphocytic leukemia, is an Akt kinase co-activator.


The serine/threonine kinase, Akt (Protein Kinase B) plays a central role in the regulation of intra-cellular cell survival. Recently, we demonstrated that the protooncogene TCL1, overexpressed in human T-cell prolymphocytic leukemia, is an Akt kinase co-activator.
Tightly restricted TCL1 gene expression in early developmental cells suggested that the TCL1 gene is regulated at a transcriptional level. To characterize the TCL1 gene regulation, we cloned the 5'-promoter of the TCL1 gene located at human chromosome 14q32. The 5'-TCL1 promoter region contains a TATA box with cis-regulatory elements for Nur77/NGFI-B, (NBRE, Nerve Growth Factor Responsive element, CCAAGGTCA), NFκB, and FKHRL (Fork Head Transcription Factor). Nur77/ NGFI-B, an orphan receptor super family transcription factor implicated in T cell apoptosis, is a substrate for Akt. We hypothesized that TCL1 transactivity is regulated through Akt-induced phosphorylation of Nur77/NGFI-B in vivo.
In EMSA with CHIP assays, wild type Nur77, but not S350A mutant Nur77, could specifically bind to TCL1-NBRE. A luciferase assay demonstrated that TCL1-NBRE is required for inhibition of TCL1 transactivity upon NGF/PDGF stimulation, which activates Akt and phosphorylates Nur77. Using a CHIP assay with RT-PCR, NGF stimulation inhibited binding of endogenous Nur77 to TCL1-NBRE, in turn, suppressing TCL1 gene expression.
The results together establish that TCL1-NBRE is a novel negative regulatory element of Nur77/NGFI-B. To the best of our knowledge, TCL1-NBRE is the first direct target of Nur77 involving the regulation of intracellular cell death-survival. This Akt-induced inhibitory mechanism of TCL1 should play an important role in immunological and/or neuronal development in vivo.
Serine threonine kinase Akt, viral homologue of v-Akt, is an important intracellular regulatory molecule that controls apoptosis (1)(2)(3). Recently, we demonstrated that the protooncogene TCL1, whose function was previously unknown, is an Akt kinase co-activator that physically binds, and hence activates Akt (4,5). These studies together provided not only a molecular basis, but also therapeutic implications for human T-cell prolymphocytic leukemia (T-PLL) in which the TCL1 oncogene is over expressed due to a chromosomal translocation (6)(7)(8)(9)(10).
In T cell prolymphocytic leukemia, both TCL1 and TCL1b genes on human chromosome 14q23.1 are activated by translocations and inversions at 14q32.1, juxtaposing them to regulatory elements of T cell receptor genes. In addition to T-PLL, in human diseases, TCL1 is over expressed in EBV infected B cell lymphoma, ataxia-telangiectasia, teratoma, and/or AIDS related lymphoma (10)(11)(12)(13). In physiological conditions, however, TCL1 expression is tightly restricted to early developmental T cells (CD3 negative) as well as early B cells with negative IgM expression (14)(15)(16).
Three major isoforms of the human TCL1 oncogene are present in both the human and mouse genome, namely TCL1, TCL1b, and MTCP1 (9,13). Under physiological conditions, TCL1 and TCL1b are highly expressed at early developmental stages in several fetal tissues including thymus, kidney, lung (TCL1), and spleen (TCL1b). After birth, expression of all TCL1 family members is mainly restricted to lymphoid tissues, although TCL1b mRNA is also found in the placenta. It is notable that in mice both TCL1b and TCL1 mRNAs are abundant in oocytes and two-cell embryos, but rare in various adult tissues and lymphoid cell lines (14,17,18). Tightly restricted physiological expression of TCL1 family proteins suggested that TCL1 gene expression is regulated at a transcriptional level (14,16,19).
To clarify molecular regulation of TCL1 gene expression, we cloned 1123 base pairs of the 5' promoter sequence of TCL1 (including 931 from base pairs of the 5'-TCL1 promoter region) from human chromosome 14q32. Nucleotide sequence analysis revealed that a Nur77 (NGFI-B) binding site {NBRE, Nerve Growth Factor response element, CCAAGGTCA (30)} is located within the 5'-proximal promoter.
Despite intensive studies, a direct gene target of Nur77, which regulates the cell death survival machinery, is yet to be determined (21). Therefore, it is noteworthy that the cis-regulatory element of Nur77 (NBRE) is well conserved within the TCL1 5'-proximal promoter region of human, mouse, and rattus (21). These observations lead us to hypothesize that the TCL1 gene can be regulated through NBRE by Akt-induced phosphorylation of Nur77/NGFI-B in vivo.
By EMSA and luciferase reporter assays, we demonstrated that Nur77/NGFI-B bound to the NBRE site of the TCL1 promoter (TCL1-NBRE), and negatively regulated TCL1 gene expression by Akt stimulation. In PC12 cells, NGF stimulation induced endogenous Nur77 phosphorylation, diminished binding of Nur77 with the TCL1-NBRE, in turn, and suppressed TCL1 transactivation. The results together demonstrated that TCL1-NBRE is a novel functional target of Nur77/NGFI, which could play an important role for the restricted expression of the TCL1 gene in vivo.
Luciferase reporter assays were performed using Dual Luciferase kit (Promega). Twenty four hours after transfection, cells were treated with 20μM wortmannin (Calbiochem, #KY-12420) or 20μM PD98059 (Biomol, #EI-360) or DMSA for 30 minutes at 37 o C as indicated. Cells were then stimulated with 50 ng/ml PDGF for 15 minutes and luciferase reporter activities were measured. Statistical analysis was performed using student t-test and P< .05 was considered to be significant.
Nur77 is an orphan receptor super family transcription factor that is implicated in neuronal development and apoptosis (20). It is noteworthy that none of the molecular targets of Nur77/NGFI-B in cell death machinery have been identified in literature (21). Recognition sequences of NGFI-B orphan receptor super family transcription factors present within the 5' proximal TCL1 promoter were well conserved (underlined) in humans (CCAAGGTCA, -396 to -388 from ATG), the mouse (ACCTGGTCA, -336 to -328), and rat (ACGAGGTCA, from -402 to -394 from ATG)(32,33) (Fig. 1C). The observation led us to hypothesize that TCL1 gene expression is regulated via the Akt-Nur77 (NGFI-B)/NBRE-TCL1 functional regulatory loop.
A recent study demonstrated that Nur77, an orphan receptor super family transcription factor, is a substrate of Akt phosphorylation both in vitro and in vivo. Activated Akt could induce phosphorylation of Nur77, which decreased DNA binding activity of Nur77, and as a consequence, dissociated from DNA (27,28).
To examine the specific binding of Nur77 with TCL1 NBRE, we generated a recombinant fusion protein of DNA Binding Domain of Nur77 (GST-Nur77-DBD).

TCL1-NBRE is a novel regulatory element in TCL1 transactivation.
Nur77, an orphan receptor super family transcription factor, was a substrate for Akt (27,28). None of the target genes for Nur77 linked to cell death and survival machinery have been identified; therefore, it was striking that NBRE {cis-regulatory elements of Nur77, CCAAGGTCA, (19,20)} was well conserved within the TCL1 5'-proximal promoter of human, mouse, and rattus (see Fig. 1C). The observation together lead us to hypothesize that Akt-induced phosphorylation of Nur77, in turn, can regulate TCL1 transactivity.
We next elucidated endogenous Nur77 could bind to DNA complex with TCL1-NBRE and be functional in the regulation of TCL1 transactivity. PC12 cells (rat pheochrmocytoma cells) were chosen, since Nur77/NGFI was originally identified from PC12 cells (20), and NBRE within the TCL1 promoter sequences was well conserved in human and rattus (Fig. 1C). Using PC12 cells, in which Nur77 was endogenously expressed, EMSA was performed to examine the formation of endogenous Nur77 protein with TCL1-NBRE.
First we confirmed that NGF stimulation induced Akt phosphorylation at Serine 473 (Fig.  4A, third row, before and after NGF stimulation), threonine 308 (data not shown), and Nur77 in PC12 cells (Fig. 4A, top row, before and after NGF stimulation).
EMSA experiment demonstrated that endogenous Nur77 formed DNA-protein complexes with TCL1-NBRE in a serum starved condition. However, after NGF stimulation, which activated Akt-induced Nur77 phosphorylation (Fig. 4A), resulted in diminishing the formation of DNA-protein complexes of endogenous Nur77 with TCL1-NBRE (Fig. 4B, compare lane 7 vs. lane 8, without vs. with NGF stimulation, respectively). Relative intensities of the formation of DNA-protein complexes were shown on the right side of the panel (3.8 vs. 1.0, before and after NGF stimulation, respectively). The results were consistent when authentic NBRE probes (AAAAGGTCA) that were used in an EMSA experiment (data not shown).
Moreover, we attempted to clarify the functional relationship of the physical interaction of endogenous Nur77 with TCL1-NBRE in TCL1 transactivation. mRNA was isolated from PC12 cells (0, 1, and 4 hours after NGF-stimulation) for RT-PCR to quantitate TCL1 gene expression in the absence or presence of NGF stimulation, which is known to stimulate Akt. Consistent with luciferase reporter assays, in PC12 cells NGF stimulation decreased TCL1 expression detected by RT-PCR (Fig. 5C, lane 1 vs. 2, before vs. 4 hours after NGF stimulation, respectively) with G3PDH as an internal control (Fig. 5C, lane 4 vs. 5, before vs. 4 hours after NGF stimulation, respectively). Moreover, consistent with the inhibitory effect observed in RT-PCR, the protein levels of TCL1 expression also decreased after NGF stimulation in a time dependent manner (Fig. 5D, bottom row, 0, 10 min, 4 hrs, and 18 hrs after NGF stimulation, respectively), which correlated well with activated Akt (third row) and phosphorylated Nur77 (top row). The results demonstrated that NGF-induced activation of endogenous Nur77 by Akt, diminished Nur77 binding to TCL1-NBRE, in turn, suppressed endogenous TCL1 gene activation in PC12 cells. The results supported the notion that TCL1 transactivation is regulated via an Akt-Nur77-TCL1-NBRE negative feedback loop.

DISCUSSION
We have demonstrated that the protooncogene TCL1 physically interacts with Akt and functions as a co-activator of Akt, which enhances Akt kinase activity (4,5,10). Protooncogene TCL1 was originally identified from the chromosomal breakpoint in human T-cell prolymphocytic leukemia (T-PLL), a rare form of an adulthood chronic leukemia. In human T-PLL, the TCL1 gene is activated due to chromosomal translocations involving a T-cell receptor gene and either the 14q32.1 or the Xq28 regions (13,34).
In pathological conditions, TCL1 is over expressed in several human diseases including Burkitt's lymphoma, ataxia-telangiectasia (A-T), and AIDs related lymphomas (11,14,35). In contrast, the physiological expression of TCL1 is tightly restricted to early developmental cells (14,17). The observation suggested that the TCL1 gene is regulated at the transcriptional level (36,37). To clarify as to how the TCL1 gene is regulated, we cloned 931 base pairs of the 5'-promoter of human TCL1 located at chromosome 14q32. We found that the 5'-TCL1 proximal promoter region contained a TATA box with cis-regulatory elements of NBRE (Nerve Growth Factor Responsive element, CCAAGGTCA, from -340 to -332), NFκB (GCCCCGCCCC, from -121 to -110), and FKHRL (Fork Head Transcription Factor, CAAAATAAA, from -857 to -850). Differential expression between TCL1 and TCL1b are consistent in that the TCL1b gene, located adjacent to the TCL1 oncogene on human chromosome 14q32, contains no TATA box and GC rich sequences with YY1 with c-myc, typical for a housekeeping gene. Recent studies clarified that Sp1 sites around the TCL1 transcriptional start site play a regulatory role for gene activation of the TCL1 oncogene (19). In contrast to TCL1 and TCL1b, physiological expression of MTCP1, the third member of the TCL1 family oncogene, is still unclear. However, a recent study suggested that SEB treatment induced MTCP gene expression in murine T cells in vivo (38).
It was striking that the proximal 5'-promoter of TCL1, an Akt kinase co-activator, bears NBRE, the cis-regulatory element of Nur77/NGFI-B (Nerve Growth Factor I-B) that is phosphorylated by Akt. The NGFI-B gene was originally identified by differential hybridization that is rapidly induced in PC12 cells by NGF, which is required for the development and survival of sympathetic and neural crest-derived sensory neurons (20,21). Importantly, subsequent in vivo studies demonstrated that Nur77, originally identified in neuronal cells, plays a pivotal role in the TCR activation that induced cell death in developmental thymocytes (21,25,26). It is also noteworthy that in some circumstances, Nur77 is shown to translocalize to mitochondria to regulate cytochrome c release for cell death (39). The presence of cis-regulatory elements of Akt substrate Nur77/NGFI-B within the proximal TCL1 promoter prompted us to hypothesize that TCL1 gene expression can be regulated through the Akt-Nur77-TCL1 regulatory loop.
Nur77/NGF1-B bears the DNA-binding domain and the region comprising the ligand-binding domain near the COOH-terminus end. The DNA-binding domain of Nur77 recognizes the NBRE element (NGFI-B response element, TTTTAAAAGGTCATGC) (33). Despite the importance of in vivo T cell apoptosis, direct molecular targets of Nur77/NGFI-B, which bind, and hence regulate cell death machinery, have not been identified. In literature, the only molecular target of Nur77/NGFI-B, though not relevant to cell death machinery, was found in steroid 21-hydroxylase promoter sequences. Steroid 21-hydroxylase is one of a group of related cytochrome p-450 enzymes that is required for steroid hormone biosynthesis (40,41). In addition, Fas-L, (42), or NDG 1, 2 were also suggested to be upregulated by overexpressing Nur77 (43); however, it is of note that these effecter molecules were not directly targeted and/or regulated by Nur77/NGFI-B.
In EMSA and transfected cells, we demonstrated that wild type Nur77, but not S350A Nur77 (28,31), could bind to TCL1-NBRE. Consistently, in CHIP assays using 293 cells transfected with the TCL1-NBRE reporter construct of wild type Nur77, exogenous wild type Nur77, but not S350A mutant, could bind to TCL1-NBRE. The affinity of wild type Nur77 with TCL1 NBRE was fairly weak, as human TCL1-NBRE has first two nucleotide bases that were substituted from the core NBRE sequences (AAAAGGTCA to CCAAGGTCA) (33). Our preliminary observation suggested a dissociation constant of Nur77 with TCL1 NBRE as 5 μ M range (MN and MH unpublished observation).
Nur77/NGFI-B belongs to orphan nuclear receptor superfamily transcription factors with three subfamily members (Nur77, NOR1, and Nurr1) (20,22,23). Over 90 % homology in their DNA binding domains were conserved among the Nur77/NGFI-B family (21,24). Based on the highly homologous DNA binding domain of Nur77/NGFI-B family members, it is logical that other Nur77/NGFI-B family members (NOR1 and Nurr1) could also bind to TCL1-NBRE. In CHIP assays and EMSA using PC12 cells, we showed that all three Nur77/NGFI-B subfamily transcription factors could interact with TCL1-NBRE. The result suggested that three isoforms of Nur77/NGFI-B subfamily transcription factors may differentially regulate TCL1 gene expression in various cellular environments in vivo.
Moreover, given the conserved binding motif among the orphan receptor super family proteins, it is possible that other orphan receptor superfamily transcription factors (other than the Nur77/NGFI-B subfamily) may also physically and/or functionally regulate TCL1 gene expression in vivo.
Functionally, TCL1-NBRE is required to inhibit TCL1 transactivation upon NGF/PDGF stimulation in luciferase reporter assays. Introduction of mutation of NBRE showed no inhibitory effect upon NGF/PDGF stimulation. The observation is consistent in that the non-phosphorylated form of Nur77 could bind to TCL1-NBRE, hence activating TCL1 transactivity. Upon PDGF/NGF stimulation, Nur77 was phosphorylated and it decreased binding to TCL1-NBRE, hence translocated to cytosole (27,28). Consistently, phosphorylation of Nur77 could inhibit TCL1 transactivity both in luciferase reporter assays and in CHIP assays. The results together supported the notion that TCL1-NBRE plays a key inhibitory role in TCL1 gene regulation in vivo.
In addition to NBRE, we localized a cis-regulatory element of FKHRL at the 5'-TCL1 promoter region (from -857 to -850). Recent investigation revealed that Akt induced phosphorylation of FKHRL (31). The presence of FKHRL upstream of human TCL1-NBRE strongly suggested that both FKHRL and Nur77, both of which were the substrate of Akt, could play a role in NGF-induced TCL1 gene expression in vivo. Consistent with previous reports, in luciferase reporter assays using TCL1-PGL3, increasing the amount of FKHRL inhibited TCL1 transactivation as a dose responsive manner. Moreover, we showed that the introduction of a FKHRL mutation modestly, but reproducibly enhanced TCL1 promoter activity in luciferase reporter assays. However, introduction of double mutation of both NBRE and FKHRL compromised the enhancement of TCL1 transactivity in luciferase reporter assays. It is of note that in contrast to the NBRE mutation, introduction of FKHRL mutation within -931 TCL1 promoters retained inhibition after NGF stimulation in PC12 cells. The results suggested that both Nur77 and FKHRL, both of which are substrates for Akt, coordinately, but differentially, regulated TCL1 gene expression in vivo. Since Akt interacts with and phosphorylates over ten intracellular signaling molecules (3), it is likely that additional regulatory mechanisms may also play a role in TCL1 gene regulation, which negatively regulates TCL1 gene expression upon NGF (or PDGF) stimulation in vivo.
EMSA using either endogenous Nur77 (derived from PC12 cells) or exogenous Nur77 (overexpression experiment in 293 cells) showed binding ability to TCL1-NBRE. We demonstrated that endogenous Nur77 could bind to NBRE only in a serum starved condition, but dissociated after NGF stimulation, which is known to activate Akt kinase. It is reported that phosphorylation of Nur77 by MAP family kinase induces translocation to the cytosole from nucleus, which was correlated with diminishing Nur77 binding activity in PC12 cells (44,45). Therefore, other kinases might also phosphorylate other serine threonine residues of Nur77, which, in turn, suppresses TCL1 transactivation in a negative feedback manner.