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Originally published In Press as doi:10.1074/jbc.M408679200 on December 13, 2004

J. Biol. Chem., Vol. 280, Issue 12, 11851-11858, March 25, 2005
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Brn-3a Transcription Factor Blocks p53-mediated Activation of Proapoptotic Target Genes Noxa and Bax in Vitro and in Vivo to Determine Cell Fate*

Chantelle D. Hudson{ddagger}, Peter J. Morris{ddagger}, David S. Latchman, and Vishwanie S. Budhram-Mahadeo§

From the Medical Molecular Biology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, United Kingdom

Received for publication, July 30, 2004 , and in revised form, December 10, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Brn-3a POU transcription factor is associated with survival and the differentiation of sensory neuronal cells during development. Brn-3a mediates its effects either by the direct regulation of target genes or indirectly upon interaction with proteins such as p53. Brn-3a differentially regulates p53-mediated gene expression and modifies its effect on cell fate. Here we show that, like Bax, Brn-3a antagonizes p53-mediated transcription of another proapoptotic target, Noxa, significantly reducing transactivation of the Noxa promoter by p53. This effect requires the p53 binding site, and electrophoretic mobility shift assay studies suggest that Brn-3a is associated with p53 when it is bound to its site in the Noxa promoter. The wild type but not the mutant promoter can be immunoprecipitated with Brn-3a in chromatin immunoprecipitation assays. Thus, Brn-3a may act by preventing the recruitment of cofactors required for p53 to transactivate this promoter. The co-expression of Brn-3a and p53 results in decreased endogenous Noxa protein in the neuronal cell line, ND7, suggesting a direct functional effect of this interaction. Moreover, there is a significant elevation of both proapoptotic Bax and Noxa proteins in sensory neuronal tissue taken from Brn-3a-/- embryos during development, compared with wild type controls. Striking changes occurred at embryonic day 14.5, a time that precedes a significant loss of specific neurons in the mutant embryos, but not at embryonic day 16.5 when Brn-3a-expressing cells are already lost by apoptosis. Therefore, the lack of antagonism by Brn-3a on activation of proapoptotic p53 target genes may contribute to the increased apoptosis seen in the Brn-3a-/- embryos. These results support a crucial role for Brn-3a in determining the pathway taken by p53 when co-expressed during development and thus in controlling the fate of these cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During neuronal development, transcription factors, which are expressed in a temporal/spatial manner or in response to specific signals, play a critical role in determining the fate of specific progenitor cells in terms of proliferation, survival and differentiation, or apoptosis of excess cells. This role is necessary to achieve the correct balance of specific neurons required for the normal innervation and function. The pit-1, oct-1/2, and unc-86 (POU) transcription factor Brn-3a protein (also referred to as POU4f1 and Brn-3.0) is essential for the survival and differentiation of specific populations of sensory neuronal cells (1, 2), but the mechanism by which this is achieved has yet to be elucidated fully.

Brn-3a is expressed in regions of the developing and adult central nervous system and peripheral nervous system (3, 4). During development, Brn-3a expression is initiated just before neurons exit the cell cycle in the peripheral nervous system and just after exiting the cell cycle in the central nervous system (5). This transcription factor is expressed in and acts as a marker of the earliest postmitotic sensory neurons to appear in primary cultures of neural crest cells (6). Brn-3a protein is required for the survival of the sensory neurons that express it, because Brn-3a KO1 mice, in which this transcription factor is deleted by homologous recombination, demonstrate an extensive loss of somatosensory neurons and die within postnatal day 1 (7).

An analysis of Brn-3a-/- embryos has revealed significant apoptosis of sensory neurons during late embryogenesis in the trigeminal ganglia (TG) and cervical dorsal root ganglia (DRG) as well as in the red nuclei of the midbrain and the inferior olivary nucleus and medial habenula (4, 7). It appears that the neurons that would normally express Brn-3a are specified, but they fail to survive at these sites in the knock-out mice. Studies carried out by Eng et al. (8) have demonstrated that mice lacking Brn-3a have defects in sensory axon growth and innervation of the target field. Thus, at embryonic day (E) 11.5, the neurons in TG and DRG condense as expected and grow toward their peripheral targets. However, by E13.5, the Brn-3a-null mice demonstrate abnormal axon bundles and migration as well as premature branching in the TG. By E15.5, there is extensive apoptotic death, resulting in the loss of cells that would normally express Brn-3a by E16.5 (8).

The requirement for Brn-3a in the survival of neuronal cells has also been shown using in vitro systems. Thus, the overexpression of Brn-3a in the neuronal cell line ND7 increases survival on the removal of neurotrophic factors compared with controls (9). This protective effect of high Brn-3a levels is also observed in cultured DRG and TG neurons (10), whereas a reduction of Brn-3a expression using an antisense approach results in apoptosis of these neurons even in the presence of neurotrophic factors (11). Moreover, using a model of neonatal sciatic nerve crush, we have shown that the overexpression of Brn-3a can also enhance neuronal survival in vivo (12).

This ability of Brn-3a to enhance neuronal survival and differentiation is associated with both direct and indirect transcriptional effects. Thus, Brn-3a can directly activate the promoters of genes that are associated with protection against apoptosis (Bcl-2 and Bcl-XL) and differentiation (e.g. in structural genes such as {alpha}-internexin (13), neurofilament (9), receptors such as neurotrophin receptor TrkA (14), and synaptic genes such as SNAP-25 (15) and synaptophysin (16)). In addition to direct effects, Brn-3a can also modify gene transcription indirectly by interaction with cellular proteins such as p53, thus effectively increasing the range of target genes regulated by this transcription factor.

Brn-3a associates with p53 via a direct protein-protein interaction (17), and this association is quite complex because it results in the differential regulation of their respective target genes. Thus, Brn-3a cooperates with p53 to activate the transcription of the gene promoter for the cyclin-dependent kinase inhibitor p21cip1/waf1, and in support of this, the co-expression of Brn-3a with p53 in the ND7 neuronal cell line results in an increase of endogenous levels of p21cip1/waf1 protein associated with cell cycle arrest of these cells (18). In contrast, Brn-3a antagonizes p53-mediated activation of the proapoptotic protein Bax and decreases endogenous Bax protein levels in ND7 cells upon co-expression of Brn-3a with p53 compared with the levels seen with p53 alone (18). Consequently, the co-expression of Brn-3a and p53 in this cell line is accompanied by increased survival and a higher proportion of cells in the G0/G1 phase of the cell cycle (18).

In light of the effect of Brn-3a on Bax expression, it is interesting that Brn-3a-/- embryos showed a significant loss of specific neurons as a consequence of increased apoptosis. Bax plays a role in naturally occurring apoptosis in many neurons during development, including sensory neurons in DRG and TG, which are known to express Brn-3a. In support of this, neuronal cells from Bax-/- mice demonstrate decreased apoptosis (in the DRG, TG, spinal ganglia, brainstem nuclear complex, and cerebellum). Moreover, neurons cultured from Bax-/- mice survive even in the absence of neurotrophic factors (19, 20). However, apoptosis is still observed in some sensory neurons of the Bax-/- mice; that is independent of Bax. This suggests that, although Bax can contribute to some apoptosis in sensory neurons, other proapoptotic factors are likely to contribute to the fate of these cells.

Noxa is another known proapoptotic p53 target gene that is induced by stresses that can activate p53 expression (21). Noxa encodes for a Bcl-2 homology 3 (BH3)-only member of the Bcl-2 family of proteins. When expressed, Noxa undergoes BH3 motif-dependent localization to mitochondria where, like Bax, it interacts with anti-apoptotic Bcl-2 family members with the resultant activation of caspase-9 and apoptosis (21). Ectopic expression of Noxa results in increased apoptosis, whereas blocking endogenous Noxa induction using antisense oligonucleotides results in the suppression of apoptosis in SAOS cells (21). Both Noxa and Bax expression are elevated upon induction of p53 in neuronal cells (22). It is possible that, like Bax, Noxa may also contribute to apoptosis during development, and the regulation of such proteins in neuronal cells by p53 may be important in controlling cell fate.

The antagonistic effect of Brn-3a on p53-mediated activation of the Bax is interesting because it suggests that in cells that normally express both proteins, Brn-3a may be important for controlling the expression of such proapoptotic factors by p53 and, hence, affect cell fate in terms of survival or apoptosis. As such, the loss of Brn-3a in these cells would result in increased proapoptotic p53 targets with consequent increased death. To test these hypotheses, we investigated the effect of Brn-3a on p53-mediated activation of the Noxa gene. We also analyzed the levels of the proapoptotic factors, Noxa and Bax, in sensory neurons of Brn-3a-/- mice to test whether the loss of Brn-3a but with the normal expression of wild type p53 would lead to increased levels that might contribute to the augmented death observed in Brn-3a-/- mutants. These results and the implications are discussed herein.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Expression Vectors—The p53 expression construct (in which cDNA encoding p53 was cloned into the eukaryotic pcDNA3 expression vector) was a kind gift of Dr. K. Vousden (National Cancer Institute, Frederick, MD). The Brn-3a expression vector containing the full-length Brn-3a cDNA under the control of the Moloney murine leukemia virus promoter in the pLTR expression vector was described previously (21). The Noxa promoter (329-bp SacII fragment) and {Delta}p53-Noxa-luc construct (286-bp, XhoI to SacII fragment), which were cloned into the Picagene luciferase reporter plasmid (21) were kind gifts from Dr. Tsukasa Shibue (University of Tokyo, Japan) and were described previously (21).

Antibodies—Anti-Bax antibody (BD Biosciences) was used at a 1:500 dilution; anti-Noxa antibody (Santa Cruz Biotechnology) was used at a 1:200 dilution; anti-actin antibody (catalog no. I 19, Calbiochem) was used at a 1:1500 dilution; anti-p53 antibody (catalog no. ab4060, Abcam) was used at a 1:1000 dilution; and anti-Brn-3a antibody (catalog no. MAB1585, Chemicon) was used at a 1:1000 dilution. Peroxidase-conjugated secondary antibodies (anti-rat, anti-mouse, anti-rabbit, and anti-goat) were obtained from DAKO (Cambridgeshire, UK) and used at dilutions of 1:3000 or 1:2000).

Cell Culture, Transfection, and Luciferase Assays—The ND7 cell line, which was obtained by immortalization of primary sensory neurons from dorsal root ganglia (23), was grown in full growth medium (L15 medium with 10% (v/v) fetal calf serum supplemented with D-glucose and glutamine) (23).

Transient transfections were carried out using the calcium phosphate method according to the method described by Gorman et al. (24), because we have previously achieved 30-40% transfection efficiency in these cells with this method. ND7 cells were plated at a density of 5 x 105 in 10-cm Petri dishes and incubated overnight (in 5% CO2 humidified incubator at 37 °C). 3 h prior to transfection, the medium was changed to Dulbecco's modified Eagle's medium with 10% fetal calf serum. Routinely, 5 µg of Noxa reporter plasmid (wild type or mutant) was co-transfected with 5 µg each of p53 or Brn-3a expression vector, either alone or together. Co-expression of the reporter construct with the empty expression vectors was used as a control. 0.5 µg of Renilla luciferase construct was included in all transfections to control for transfection efficiency between different plates. For luciferase assays, cells were harvested 48 h after transfection using the passive lysis buffer (Promega) and an assay for firefly and Renilla luciferase products, which were measured using the dual luciferase reporter assay system according to the manufacturer's protocol. Following adjustments for differences in transfection efficiencies using Renilla luciferase activity, values were expressed as a percentage of the vector control to show promoter activity on co-transfection with appropriate proteins.

Electrophoretic Mobility Shift Assay—100 ng of the double-stranded DNA sequence corresponding to the p53 binding site in the Noxa promoter (5'-AGGCTTGCCCCGGCAAGTTG-3') (21) was labeled with [{gamma}-32P]ATP using T4 kinase. To prepare the labeled probe for use in EMSA, unincorporated [{gamma}-32P]ATP label was removed using a Sephadex G-25 column. 2 ng of the probe was incubated in all of the reactions that included cellular extracts prepared from the neuroblastoma cell line IMR32, which expresses wild type p53, or SAOS2 cells, which lack p53. To test for the specificity and affinity of protein binding, different competitors were used. Specific competition was carried out using a 50x or 100x cold unlabeled p53 site. The oligonucleotide corresponding to the estrogen receptor element was used as a nonspecific competitor (17). An oligonucleotide known to bind the Brn-3a protein was used to test whether this protein was in the complex and whether binding to its site could compete for binding to the probe. 1 µl of p53 pAb (Oncogene), actin pAb (Santa Cruz Biotechnology), or Brn-3a monoclonal Ab was used.

Chromatin Immunoprecipitation Assay—To test whether Brn-3a could be immunoprecipitated when bound to p53 on the wild type Noxa promoter but not on the mutant promoter, in which the p53 site has been mutated, ChIP analysis was undertaken. This technique was carried out as described by Gascoyne et al. (25) but with some minor modifications. Briefly, ND7 cells were plated at a confluence of 0.5 x 105 in 6-well plates. The wild type and mutant Noxa promoters were co-transfected with Brn-3a and/or p53 expression vectors into these cells. After 48 h, cross-linking was performed by adding 270 µl of 37% formaldehyde/10 ml of the medium and incubating the cells for 15 min. Cells were washed, harvested in cold phosphate buffered saline, and then lysed in lysis buffer (1% SDS, 0.01 M EDTA, 0.05 M Tris, pH 8.0, and 0.01% protease inhibitor mixture). The cells were sonicated to shear the DNA and then subjected to centrifugation. A small volume of the supernatant was taken out for use as an input sample in a subsequent PCR analysis, and the remaining sample was divided and subjected to immunoprecipitation overnight using the appropriate antibody (either Brn-3a rabbit polyclonal Ab (Chemicon) or the secondary anti-rabbit antibody (DAKO)). Incubation with protein G-Sepharose beads allowed immobilization of the protein-antibody complex, which was washed thoroughly and then eluted from the beads. The cross-links between the protein and DNA were reversed by incubation at 65 °C for 4 h, and the proteins were digested using proteinase K. Following phenol chloroform extraction, the DNA was precipitated with ethanol, then resuspended in water, and used for PCR. The primers used in the PCR to amplify a 152-bp fragment of the promoter containing the Brn-3 site were NoxaF (5'-CTC GAG ACC TGC TCC ACT TC-3') and NoxaR (5'-CGC TGG AAT CCT CTC TGT TC-3'). Standard conditions were used for PCR amplification and included the use of 2.5 mM MgCl2 with the following cycling parameters: 1 cycle at 94 °C for 15 min followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. The reaction was completed by an incubation at 72 °C for 5 min. PCR products were resolved on a 2.5% agarose/Tris borate EDTA gel.

Brn-3a Knock-out Mice—Brn-3a knock-out mice were made as described by Xiang et al. (4) and were a kind gift from Dr. M. Xiang (Rutgers University, New Brunswick, NJ). Heterozygous mice, maintained in accordance with Home Office guidelines (UK), were used to set up timed mating, and pregnant females were killed at specified time points to obtain the required embryos. Tissues that were enriched for Brn-3a expression (hindbrain, trigeminal ganglia, spinal column, and dorsal root ganglion) were dissected and pooled from each animal. Care was taken to obtain a similar range of tissues from embryos. Tissues were frozen in liquid nitrogen for later preparation of proteins. The tail and limb buds were taken from each embryo for genotyping using the PCR protocol described previously (4). All experiments involving animals were carried out in accordance with Home Office guidelines and were covered by local and national ethical approval.

Analysis of Protein Levels—Total cellular proteins were prepared from either transfected cell lines or embryonic tissues. For this procedure, transfected cell lines were harvested into 2x Laemmli buffer and kept on ice. Following the addition of 5% {beta}-mercaptoethanol or 1 mM dithiothreitol, samples were heated to 95 °C for 5 min, and then cell debris was removed by centrifugation. To extract proteins from tissues, the appropriate tissues were ground in liquid nitrogen, homogenized in 2x Laemmli buffer using a glass homogenizer, and then processed as above. Variation in protein concentration was assessed using the Bradford assay or Coomassie Blue-stained PAGE, which allowed visualization of the protein integrity as well as an estimation of variation in protein concentration among different samples.

For Western blot analysis, 50-100 µg of each protein sample were subjected to polyacrylamide gel electrophoresis on a 15% gel at a constant 150 V. Resolved proteins were transferred onto a Hybond C membrane using a wet transfer method as described by Maniatis et al. (26). Filters were incubated with the appropriate antibody, and anti-actin antibody was used to equalize for protein loading. Peroxidase-conjugated secondary antibodies (anti-rat and anti-goat) were used at dilutions of 1:3000 or 1:2000, respectively. Signals were developed using the enhanced chemiluminescence systems (Amersham Biosciences or Pierce).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Regulation of p53-mediated Activation of Noxa Promoter by Brn-3a—Because Brn-3a could block p53-mediated activation of the Bax promoter but enhance expression of the cell cycle arrest-associated gene p21cip1/waf1, we examined whether Brn-3a might also modulate the effect of p53 on the promoter of another target gene, Noxa. Transient co-transfection studies were carried out in the neuronal cell line ND7. Initial studies were carried out using the wild type promoter, which contained the regulatory region of the Noxa gene (-183 to +158) (21) driving expression of the luciferase reporter gene. The wild type promoter was co-transfected with the Brn-3a or p53 expression vector alone or both together and compared with the empty expression vector, whereas the Renilla luciferase reporter was included to control for transfection efficiency. Values were expressed as a percentage of vector control-transfected cells, which were set at 100%.

As expected, co-transfection with p53 expression vectors resulted in strong transactivation of the Noxa promoter (Fig. 1A) with up to 8-fold increased activity compared with the empty vector control (*). Although Brn-3a alone did not significantly alter Noxa promoter activity, co-transfection of Brn-3a with p53 resulted in a significant decrease of the transactivation observed with p53 alone on this promoter (**). These effects resulted from the changes in the expression of Brn-3a and p53 proteins in the transfected cells. Therefore, similar to the Bax promoter, Brn-3a can block p53-mediated activation of the Noxa promoter. However, although Brn-3a alone could reduce endogenous activity of the Bax promoter (18), no such direct repressive effect was observed on the Noxa promoter.



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  		FIG. 1.
Brn-3a antagonizes p53-mediated activation of the Noxa promoter. A, luciferase assay following co-transfection experiments showed that Brn-3a alone had a minimal effect on Noxa promoter activity compared with vector only control. Co-expression with p53 alone resulted in a significant activation of the promoter (*). Although Brn-3a alone did not repress this promoter, co-transfection of Brn-3a with p53 resulted in a significant loss of p53-mediated transactivation of the Noxa promoter (**, p < 0.05). Promoter activities are expressed as a percentage of vector control and are adjusted with Renilla luciferase to control for the variation in transfection efficiency. The figures are the average ± S.E. of four independent experiments. Statistical analysis was carried out using Student's paired t test. B, the p53 binding site in the Noxa promoter is required for the effect on p53. Co-transfection assays using a modified Noxa promoter containing a mutated p53 site are shown. Although Brn-3a alone had a minimal effect on the mutant Noxa promoter compared with the vector only control, the stimulation of the promoter by p53 was lost. Co-transfection of Brn-3a with p53 did not significantly modify promoter activity. Promoter activities are expressed as a percentage of vector control and are adjusted with Renilla luciferase for the variation in transfection efficiency. The figures are the average ± S.E. of four independent experiments. C, expression of Brn-3a and p53 proteins when transfected alone or together into ND7 cells is shown. Changes are shown in the expression of Brn-3a and/or p53 proteins when the appropriate expression vectors were co-transfected with the Noxa promoter reporter construct into ND7 cells compared with the empty expression vector used as a control.

 
Repression of p53 Activation of the Noxa Promoter by Brn-3a Requires the p53 Consensus Binding Site—Although the Bax promoter contained two Brn-3 binding sites flanking the p53 site, which are required for its effect (27), no Brn-3a consensus site was observed in the sequence of the Noxa promoter used in these studies. However, because Brn-3a can still efficiently block p53-mediated transactivation of this promoter, we tested whether this effect resulted from the direct interaction of Brn-3a with p53 and hence was dependent on the p53 consensus site present in the promoter. Co-transfection studies were carried out using a reporter construct containing the Noxa promoter, in which just the p53 site was deleted ({Delta}p53-Noxa) (21). Although the basal promoter activity was still present and similar to the wild type Noxa promoter (mean basal activity of mutant {Delta}p53-Noxa, 7.46 ± 0.18 relative light units, compared with wild type Noxa, 6.31 ± 0.77 relative light units), p53 failed to transactivate this promoter, which lacked the p53 binding site (Fig. 1B). Brn-3a on its own had little effect on promoter activity, whereas the co-expression of Brn-3a and p53 had no effect compared with p53 only. The expression of Brn-3a and p53 proteins was clearly increased in the transfected cells, as shown by Western blot in Fig. 1C. Therefore, the altered effect observed with the mutant promoter was a result of the loss of the p53 consensus site, and hence failure to be bound by the p53 protein. Therefore, it appears that the association of Brn-3a with p53 antagonizes p53 activity on the Noxa promoter and that this effect is dependent on the p53 cognate DNA site being present on the promoter.

Effect of Brn-3a on p53 Protein Binding to Its Consensus DNA Site—We next tested whether Brn-3a acted by blocking p53 binding to its consensus site in the Noxa promoter or whether it is associated with p53 and might prevent the interaction of another co-factor required for the activation of the Noxa promoter. EMSA were carried out using cellular extracts obtained from neuroblastoma cells, which overexpress Brn-3a and are known to express wild type p53. As shown in Fig. 2A, the incubation of ND7 cell extract with a 32P-labeled oligonucleotide probe corresponding to the p53 binding site resulted in specific complexes (Fig. 2A, lane 1) that were competed by the addition of increasing amounts of the unlabeled p53 binding site (Fig. 2A, lanes 2 and 3) but not the nonspecific oligonucleotide (Fig. 2A, lane 4). The presence of two main retarded complexes would suggest that p53 associates with either itself or other cellular proteins to form complexes of different sizes. Furthermore, the addition of p53 pAb resulted in further retardation of the complexes (Fig. 2A, lanes 5 and 6 asterisks), but this was not seen on the addition of the pAb against the unrelated actin protein (Fig. 2A, lane 7), confirming that p53 protein is present in these complexes. Interestingly, the addition of an excess of the oligonucleotide that corresponded to the Brn-3a site resulted in a loss of the complex binding to the probe (Fig. 2A, lanes 8 and 9). This suggests that Brn-3a is associated with p53 in this complex and that the binding of Brn-3a to its DNA consensus site may compete with the labeled probe for binding of Brn-3a and the associated p53 protein. The addition of Brn-3a monoclonal Ab resulted in a supershift of the complex with one larger complex observed (Fig. 2A, lane 9). However, there also appears to be some competition when Brn-3a antibody is added, suggesting that the binding of this antibody to Brn-3a within the complex may prevent binding to the labeled DNA, perhaps by steric hindrance.



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  		FIG. 2.
Brn-3a does not prevent p53 from binding to its cognate sequence in the Noxa promoter. A, EMSA were carried out to test whether Brn-3a protein associates with p53 on the p53 DNA sequence element taken from the Noxa promoter. Incubation of the cellular extract containing both p53 and Brn-3a with the labeled probe comprising the p53 DNA binding site in the Noxa promoter resulted in the formation of two complexes (lane 1) that were competed specifically by the addition of increasing amounts of the unlabeled DNA sequence (50- and 100-fold molar excess, lanes 2 and 3, respectively) but not on addition of 100-fold molar excess of an unrelated nonspecific oligonucleotide (lane 4). The addition of 1 and 3 µl of p53 pAb resulted in a supershift of the complexes (*, lanes 5 and 6), but this was not seen on incubation of the unrelated actin pAb (lane 7). The addition of a Brn-3a consensus site also resulted in the competition of this complex (lanes 8 and 9), suggesting that Brn-3a is bound to p53 in this complex. Moreover, incubation with an antibody to Brn-3a resulted in the supershifted complex (lane 10). B, immunoprecipitation of Brn-3a on the wild type Noxa promoter but not on the mutant promoter. The ChIP assay shows that Brn-3a is associated with the wild type but not the mutant Noxa promoter in ND7 cells expressing Brn-3a and p53 proteins. Following immunoprecipitation of ND7 cells (transfected with either the wild type or mutant Noxa promoter) with Brn-3a antibody, PCR amplification was carried out using specific primers that flanked the p53 site on the Noxa promoter. PCR amplification of the positive control input (total chromatin extract before immunoprecipitation) is shown in the second lane. The wild type Noxa promoter immunoprecipitated with Brn-3a is shown in the third lane, whereas the negative control in which the secondary antibody was used to immunoprecipitate the wild type Noxa promoter is shown in the fourth lane. The fifth lane shows the input from cells transfected with mutant Noxa promoter, whereas the sixth lane shows immunoprecipitation with the Brn-3a antibody, which is similar to the background level seen with the control secondary antibody only in both wild type and control experiments.

 
These results confirm that Brn-3a is bound to p53 on the p53 consensus site found in the Noxa promoter. Moreover, it appears that Brn-3a does not prevent p53 binding to this sequence but may act by preventing the recruitment of co-factors such as apoptosis-stimulating protein of p53 (ASPP) proteins required for the stimulation of proapoptotic proteins such as Bax (28).

Brn-3a Is Immunoprecipitated with the Wild Type but Not Mutant {Delta}p53-Noxa Promoter in Chromatin Immunoprecipitation Assay—We next tested whether Brn-3a is associated with the wild type but not the mutant Noxa promoter in intact cells by undertaking chromatin immunoprecipitation (ChIP) assays. ND7 cells were transfected with the wild type Noxa promoter, with the intact p53 site or the {Delta}p53 mutant promoter containing mutated p53 site. Expression vectors encoding Brn-3a and p53 were also co-transfected because endogenous levels of both proteins are relatively low in normal proliferating ND7 cells. Primers were designed to amplify the 152-bp fragment of the promoter that encompassed the p53 binding site. As can be seen in Fig. 2B, this site can be amplified using cell extract containing the wild type promoter from which Brn-3a protein has been immunoprecipitated (Fig. 2A, lanes 2-4) but much lower levels were seen in the presence of the mutant promoter (5-7). Fig. 2A, lanes 2 and 5, indicates the input sample, whereas Fig. 2A, lanes 3 and 6, shows amplification following immunoprecipitation of cells transfected with the wild type and mutant {Delta}p53-Noxa promoters, respectively, with Brn-3a rabbit pAb. Fig. 2A, lanes 4 and 7, indicates the amplification from cells that were immunoprecipitated with only the control anti-rabbit secondary antibody. A significantly higher amplification of the Noxa promoter region was observed in cells transfected with the wild type promoter compared with those with the mutant promoter from which low levels of the promoter sequence were amplified. Fig. 2A, lane 1, indicates the 100-bp DNA ladder.

These results demonstrate that in the intact cell, Brn-3a is indeed associated with the region of the Noxa promoter that contains the p53 binding site. Failure of the mutant promoter to be immunoprecipitated with Brn-3a suggests that Brn-3a is associated with p53 bound to this site, so the loss of p53 binding to this site prevents the promoter from being immunoprecipitated with Brn-3a in the ChIP assays in these cells.

Brn-3a Reduces the Levels of Endogenous Noxa Expressed When Co-transfected with p53—We next tested whether the repression of p53 transactivation of the Noxa promoter by Brn-3a was reflected in changes in the levels of Noxa protein when these two proteins are co-expressed. For these studies we used ND7 cells that were transfected with either Brn-3a or p53 alone or both together (shown previously to result in changes in endogenous Bax and p21cip1/waf1 levels).

As shown in Fig. 3, Noxa protein levels were elevated significantly in cells expressing p53 only compared with control cells (p = 0.009), whereas Brn-3a alone had no effect on Noxa levels. However, co-expression of Brn-3a with p53 resulted in a significant decrease in Noxa levels compared with the expression of the p53 only cells (Student's t test, p = 0.002). These results support the finding that in the ND7 neuronal cells, Brn-3a can down-regulate the p53-mediated increase of Noxa as well as Bax and may thus contribute to the increased survival observed previously (18) on co-transfection of Brn-3a with p53 in these cells compared with cells expressing high levels of p53 alone. As described previously (29), Brn-3a increases the level of p53 but that its ability to antagonize p53-mediated transcription of proapoptotic proteins may prevent apoptosis by p53.



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  		FIG. 3.
Brn3a represses p53-mediated expression of endogenous Noxa protein. A, levels of Noxa protein in ND7 cells transfected with p53 and/or Brn-3a or with vector alone are shown. The control actin protein for each sample is shown for comparison. B, protein levels from three independent experiments were quantified by densitometry and equalized for protein loading using actin levels. Values were expressed as a proportion of control cells in which levels were set at 1. Statistical analysis of levels using Student's t test showed statistically significant differences in Noxa protein levels in extracts taken from cells transfected with p53 alone and control cells (*, p < 0.05) and cells transfected with p53 compared with Brn-3a and p53 together (**, p < 0.05).

 
Levels of Proapoptotic Proteins Bax and Noxa in Brn-3a-/- Mice—Knock-out mice lacking Brn-3a demonstrated a significant loss of specific neuronal cells as a result of apoptosis, which peaked at E15.5. To test whether the loss of Brn-3a protein resulted in increased levels of proapoptotic p53 target genes, we measured the levels of Bax and Noxa proteins in pooled neuronal tissues (midbrain, hindbrain, trigeminal ganglia, spinal cord, and DRG) dissected from each Brn-3a-/- embryo and compared them with similar tissue taken from wild type littermates. This approach allowed us to enrich for proteins from specific tissues that would normally express Brn-3a.

As shown in Fig. 4A, significantly increased levels of both Noxa and Bax proteins were observed in E14.5 Brn-3a-/- embryos. Quantification for protein levels obtained from a number of litters showed significant differences (p < 0.05) between the levels of both Bax (Fig. 4B, i) and Noxa (Fig. 4B, ii) in the knock-out mice compared with levels in wild type littermates. However, by E16.5, the levels of both Bax and Noxa proteins were similar in the wild type and knock-out littermates (Fig. 4C), with no significant differences evident on quantification (Fig. 4D, i and ii).



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  		FIG. 4.
Elevated levels of Bax and Noxa protein in Brn3a-/- embryos at E14.5 but not at E16.5. A, a representative Western blot analysis shows the expression of Bax and Noxa proteins in tissues taken from either Brn-3a-/- embryos or from wild type (WT) embryos at E14.5. Actin levels are shown for the same samples. B, quantification of Bax protein levels (i) or Noxa protein levels (ii) in E14.5 embryos was carried out by densitometry. Values were equalized for actin and plotted to show the differences in protein levels in age-matched wild type and Brn-3a knock-out embryos. Statistical significance was established by Student's t test. C, a representative Western blot analysis showing the expression of Bax and Noxa proteins in tissues taken from either Brn-3a-/- embryos or from wild type embryos at E16.5. Actin levels are shown for the same samples. D, quantification of Bax protein levels (i) or Noxa protein levels (ii) in E16.5 embryos was carried out by densitometry, and values were equalized for actin and plotted to show the differences in protein levels in age-matched wild type and Brn-3a knock-out embryos. Statistical significance was established by Student's t test.

 
Therefore, the loss of Brn-3a in mice with wild type p53 resulted in higher levels of proapoptotic proteins Bax and Noxa, at a time preceding maximal apoptosis in the Brn-3a-/- embryos, suggesting that Brn-3a may be important in regulating the expression of these proapoptotic target proteins during development.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The Brn-3a transcription factor is required for the survival and differentiation of sensory neurons during development. In particular, Brn-3a knock-out mice demonstrate significant loss of specific sensory neurons during development, and these mutants die soon after birth (4, 7). Intriguingly, cells that would normally express Brn-3a are specified but failed to survive because of a wave of apoptosis in later development. This suggests that the progenitor cells are present, but once they are committed to their developmental fate, Brn-3a is necessary for survival and differentiation (8).

It is known that Brn-3a can directly transactivate genes associated with neuronal survival during embryogenesis, such as Bcl-XL (12, 30) and Trk receptors associated with neuronal differentiation (14), as well as indirectly on association with proteins such as p53 (17, 18). However, the molecular mechanisms underlying these effects are still to be determined. Analysis of the effect of the loss of Brn-3a in a Bax null background demonstrates that many cells are rescued from apoptosis (14), suggesting that proapoptotic factors such as Bax do contribute to the apoptosis observed in these mice. Although Brn-3a is critical for the regulation of expression of Trk receptors (neurons in trigeminal ganglia of Brn-3a KO mice show no expression of TrkC receptors, whereas TrkA and TrkB are expressed but lost during later development), these do not appear to affect survival directly. For instance, in the surviving neurons seen in the Brn-3a/Bax mutants, TrkA expression is eventually lost, suggesting that this receptor is necessary for neuronal differentiation rather than the apoptosis observed in the Brn-3a-/- neurons (31). Thus, it is clear that apoptosis occurring in these sensory neurons on the loss of Brn-3a is associated with its ability to control the expression of anti-apoptotic as well as proapoptotic factors.

The co-expression and ability of Brn-3a to alter p53-mediated cell fate are very interesting, because it is the first protein shown to decrease apoptosis but increase cell cycle arrest by p53 protein. Critically, this interaction of Brn-3a with p53 results in the enhancement of p53-mediated transcription of the p21cip1/waf1 gene associated with G0/G1 arrest but the antagonism of p53-mediated stimulation on its proapoptotic target, Bax (18, 27). The ability of Brn-3a to differentially regulate p53-mediated transcription may present an important mechanism by which this transcription factor can help to determine survival and differentiation of neuronal cells.

In this study, we demonstrated that Brn-3a also blocks the transcriptional activity of p53 on another proapoptotic p53 target gene, Noxa, in a manner similar to that seen on the Bax promoter (18). Thus, Brn-3a antagonizes p53-mediated activation of the Noxa gene promoter in reporter assays. Functionally, this is reflected in the decrease of endogenous Noxa protein on co-expression of Brn-3a with p53 in ND7 cells, compared with levels seen with p53 alone. It is interesting that, although Brn-3a alone could mildly repress Bax promoter activity, this was not observed on the Noxa promoter used in this study. However, although the Bax promoter contains two consensus binding sites for Brn-3a, which flanks the p53 sites, no such Brn-3 sites were observed on the Noxa promoter tested in this study. In the Bax promoter, both Brn-3a binding sites are required for its effects on this promoter because a mutation of these sites resulted in a failure of Brn-3a to repress transcription either alone or via association with p53 (27). The lack of Brn-3a consensus binding site on the Noxa promoter may explain why Brn-3a did not directly repress promoter activity in these experiments. However, because Brn-3a could significantly antagonize p53-mediated stimulation of this promoter, this effect must arise from its ability to interact physically with p53. Because Brn-3a interacts with the DNA-binding domain of p53, we investigated the mechanism by which it could block transactivation by p53. We tested whether the association of Brn-3a with p53 prevented the p53 protein from binding to its consensus binding site on the Noxa promoter transcription or alternatively, whether the interaction of Brn-3a with p53 when it is bound to the promoter prevents the association of other proteins that are required for full p53 transactivation of this promoter. Electrophoretic mobility shift assay results show that Brn-3a is, in fact, associated with p53 when it is bound to its DNA-binding site in the Noxa promoter. This suggests that the interaction of Brn-3a with p53 may prevent the transactivation of the Noxa promoter, possibly by excluding binding of co-activators, e.g. ASPP required for the stimulation of proapoptotic gene promoters (28). This was strengthened by our finding that in ChIP assays, immunoprecipitation of Brn-3a in ND7 cells containing the wild type p53 site in the Noxa promoter resulted in the amplification of the promoter DNA, but this was not observed on the same promoter that lacks this p53 site.

It is now evident that cellular proteins co-expressed with p53 can alter the effect of this protein on determining cell fate by modifying target genes regulated by p53 (32, 33). For instance, in adverse conditions (e.g. on DNA damage, in the absence of serum/trophic factors, or in the presence of cytotoxic agents), the induction of p53 expression results in apoptosis as a consequence of activation of proapoptotic targets including Bax, Noxa, Bid, PUMA, and PIG3 (34). The co-expression of ASPP proteins (ASPP1/2), which interact with p53, increases the expression of proapoptotic genes such as Bax and PIG3 and are clearly important for determining the apoptotic pathway (28). In contrast, the co-expression of Brn-3a with p53 increases the expression of p21cip1/waf1 but antagonizes the expression of the p53 proapoptotic target Bax (18) as well as Noxa and so contributes to increased survival and cell cycle arrest. It is interesting to note that, as shown previously (29), Brn-3a increases the expression of p53 (as seen in Fig. 3). However, the ability of Brn-3a to prevent proapoptotic gene expression by p53 would prevent cell death associated with elevated p53 protein.

The ability of Brn-3a to differentially modulate the expression of p53 target genes appears to be achieved by different mechanisms. Thus, although Brn-3a binding sites are required for it to repress p53-mediated transcription on the Bax promoter (18), it can repress activity on the Noxa promoter by direct protein-protein interaction with p53. In contrast, on the p21cip1/waf1 promoter, Brn-3a appears to mediate its effect by recruitment to the basal transcriptional complex (27). These findings demonstrate the complexity of regulation of expression of genes associated with cell death/survival by Brn-3a.

In this regard, it is possible that during neuronal development, cells that co-express Brn-3a and p53 would survive because of the repression of proapoptotic factors, whereas cooperation on the p21cip1/waf1 promoter would enhance cell cycle arrest (18). Because Brn-3a increases the expression of neuronal proteins such as neurotrophic receptors (trkA and trkC), neurofilament, and {alpha}-internexin, this would enhance differentiation of surviving cells into mature neurons. In agreement with this hypothesis, we have shown that in a comparison of neuronal tissue taken from Brn-3a-/- with wild type mice, the loss of Brn-3a resulted in significant increases in the levels of both Bax and Noxa at specific times during development. Both Bax and Noxa proteins were elevated by E14.5 in Brn-3a-/- embryos compared with wild type littermates, a time that coincides with increased apoptosis in these mutants (8). By E16.5, when most neurons that would normally express Brn-3a are lost in the Brn-3a-/- embryos, the levels of Bax and Noxa proteins remained similar in the wild type embryos compared with mutants. Therefore in Brn-3a expressing cells such as sensory neurons, it is likely that the expression of Bax and Noxa are controlled by the ability of this transcription factor to alter p53-mediated gene transcription.

The increase in proapoptotic Bax and Noxa, in the absence of Brn-3a, may result from the lack of antagonism of p53-mediated transcription and contribute to the increased death of sensory neuronal cells, which would normally express Brn-3a. Bax plays a role in naturally occurring apoptosis in many sensory and sympathetic neurons during development (19, 20, 35, 36). Bax-mediated apoptosis is also clearly important for the loss of sensory neurons in Brn-3a KO embryos because crosses of Brn-3a KO with Bax KO lacking Bax resulted in the rescue of many of the sensory neurons that would normally express Brn-3a (14). However, there is still apoptosis in some sensory neurons that are independent of Bax, and it is likely that other proapoptotic factors contribute to the fate of these cells (20). The regulation of Noxa by p53 has been demonstrated previously (21), but its role in developmental apoptosis has not been studied. Like Bax, Noxa undergoes BH3 motif-dependent localization to the mitochondria, where it interacts with anti-apoptotic Bcl-2 family members with the resultant activation of caspase-9 and apoptosis (21). Both Noxa and Bax have been shown to be elevated on induction of p53 in neuronal cells (22).

Therefore, it is likely that during normal development, the interaction of Brn-3a and p53 will be important in determining the fate of neuronal cells that co-express these proteins. The ability of Brn-3a to differentially modulate the transcriptional activity of the p53 protein on promoters of its target gene associated with cell fate determination describes an alternate mechanism by which this transcription factor can control the fate of cells that express it and supports previous findings (32, 33) showing that Brn-3a is a critical regulator of fate neuronal cells.


   FOOTNOTES
 
* This work was supported by the Association for International Cancer Research (United Kingdom), the Biotechnology and Biological Sciences Research Council (United Kingdom), and the Neuroblastoma Society (United Kingdom) and by contributions from the Great Ormond Street Hospital Children's Charity (London, United Kingdom), the Terry Fox Foundation, and the Four Seasons Hotel. 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. Back

{ddagger} Both authors contributed equally to this work. Back

§ To whom correspondence should be addressed. Tel.: 44-20-7242-9789 (ext. 0761); Fax: 44-20-7905-2301; E-mail: v.mahadeo{at}ich.ucl.ac.uk.

1 The abbreviations used are: KO, knock-out; TG, trigeminal ganglia; DRG, dorsal root ganglia; BH, Bcl-2 homology; E, embryonic day; EMSA, electrophoretic mobility shift assay; pAb, polyclonal antibody; ChIP, chromatin immunoprecipitation; ASPP, apoptosis-stimulating protein of p53; SAOS, human osteogenic sarcoma. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Tsukasa Shibue for Noxa promoter constructs, Dr. K. Vousden for p53 expression vector, and Dr. M. Xiang for Brn-3a knock-out mice. We also thank J. Sinclair (Institute of Child Health, London, UK) for assistance with fluorescence-activated cell sorter analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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