From the Department of Molecular Pathology, Windeyer
Institute of Medical Sciences, University College London,
Cleveland Street, London W1P 6DB, United Kingdom and the
§ Department of Medicine, Stanford University Medical
Center, Stanford, California 94305-5112
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The BCL-2 protein is able to protect neuronal and other cell types from apoptotic programmed cell death and plays a key role in regulating the rate of apoptosis during development of the nervous system. We have previously demonstrated that the Brn-3a POU domain transcription factor protects sensory neurons from apoptotic programmed cell death induced by nerve growth factor withdrawal. We report here that Bcl-2 transcription is predominantly initiated from the Bcl-2 P2 promoter in both the ND7 neuronal cell line and primary dorsal root ganglion neurons, in contrast to the predominant use of the Bcl-2 P1 promoter in other cell types. Moreover, Bcl-2 transcription initiated from the P2 region increases in ND7 cells stably overexpressing Brn-3a, resulting in enhanced BCL-2 protein levels. Similarly, the Bcl-2 P2 promoter is directly activated by Brn-3a in co-transfection assays in both ND7 cells and dorsal root ganglion neurons. Analysis of the Bcl-2 regulatory sequence revealed a binding site for Brn-3a that is required for maximal activation by Brn-3a both in transfected cells and during differentiation of ND7 cells. Together these data identify Brn-3a as the first transcription factor regulating Bcl-2 activity specifically in neuronal cells and indicate that the anti-apoptotic effect of Brn-3a is likely to be mediated, at least in part, via the up-regulation of Bcl-2 expression.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The survival of specific sets of neurons during embryonic and postnatal remodeling of the nervous system is thought to be regulated by the presence of neurotrophic factors. In the absence of these trophic signals, neurons die by apoptotic programmed cell death (1, 2). An increasing number of genes have been shown to coordinately regulate apoptosis in mammalian cells, with several lines of evidence implicating the members of the Bcl-2 family as regulators of neuronal survival (3, 4).
The bcl-2 gene was originally isolated at the chromosomal breakpoint in t(14;18) bearing follicular B cell lymphoma (5), the translocation of bcl-2 to the immunoglobulin heavy chain locus leading to the deregulation of Bcl-2 expression. Bcl-2 is a membrane-bound 26-kDa protein that is located in several subcellular locations including the outer mitochondrial, outer nuclear, and endoplasmic reticulum membranes (6, 7). Although the function of the Bcl-2 family of proteins remains to be elucidated, recent evidence suggests that Bcl-2 may act to target the protein kinase Raf-1 to the mitochondrial membrane (8), whereas Bcl-xL is able to form ion channels in lipid membranes (9). Bcl-2-related proteins have been demonstrated to regulate cell survival in response to a variety of apoptotic stimuli, including growth factor withdrawal and genotoxic damage, in a number of cell types (10). The homodimeric form of Bcl-2 has been shown to enhance the survival of both central and peripheral neurons in culture and has been shown to counter the effects of a variety of apoptotic stimuli including neurotrophic factor removal (3). Evidence suggests that the susceptibility of the cell to apoptotic programmed cell death in neuronal cells can be affected by the ratio of the levels of Bcl-2 to Bax (11) and is, in the case of neuronal cells, regulated by the levels of various neurotrophic factors (11, 12). In vivo, Bcl-2 mRNA transcripts and protein are detectable within the murine nervous system as early as embryonic day 10. Although levels peak during the periods in which remodeling of the nervous system occurs, the continued expression into adulthood suggests a role for Bcl-2 in the maintenance of the nervous system (Ref. 13 and references therein).
Whereas Bcl-2 is regulated in both a tissue- and temporal-specific
manner, little is known about the regulatory mechanisms governing its
transcription. Two promoter regions have been identified in the
5'-regulatory region of the bcl-2 gene. P1, the predominant promoter in B cells, is a TATA-less, GC-rich region containing multiple
initiation sites and several SP1 and 1 recognition elements (14-16). In contrast, the second promoter, P2, located approximately 1.3 kilobase pairs downstream of P1, contains a CCAAT box, an octamer
motif (ATGCAAAG), and a TATA element. This promoter is responsible for
the production of only a small percentage of the Bcl-2 transcripts in
the cell types, such as B cells (15), so far studied in detail. The
relative activity of the P1 and P2 promoters in neuronal cells has not
been characterized. Although transcriptional regulators of Bcl-2
identified to date include the p53 tumor suppressor gene product (17,
18), the products of the cellular and viral myb genes (19,
20), and the product of the Wilms tumor gene wt1 (21, 22),
none of these have been shown to play a role in the regulation of Bcl-2
expression in neuronal cells. Hence Bcl-2 promoter usage in neuronal
cells, together with the identity of the transcription factors which regulate its expression in these cells, remains uncharacterized.
The three members of the Brn-3 family of type IV POU domain transcription factors, Brn-3a (23-25), Brn-3b (25), and Brn-3c (24, 26), are expressed in distinct but overlapping sets of neurons in the developing and adult nervous systems (26, 27) suggesting that, like the highly homologous nematode Unc-86 POU factor, they may play a key role in the regulation of gene expression in neuronal cells. Such a function has been confirmed by knock-out studies that demonstrate that the various members of the Brn-3 family are critical for a number of aspects of central nervous system development (28-30). Brn-3a and Brn-3c are generally activating factors, whereas Brn-3b generally represses promoters that are activated by Brn-3a (31). Interestingly, our recent studies have implicated a role for Brn-3a in the development of the mature process bearing phenotype of neurons that occurs during differentiation of these cells. First, the levels of Brn-3a expression increase upon the differentiation of neuronal cells whereas levels of Brn-3b decrease (32-34). More importantly, however, is the observation that the overexpression of Brn-3a promotes neurite outgrowth in these cells (34), whereas antisense treatment inhibits process formation (35). Furthermore, we have recently demonstrated that Brn-3a is capable of protecting cells of neuronal origin, as well as primary cultures of sensory neurons, against apoptosis induced by either serum withdrawal (in the neuronally derived ND7 cell line) or by withdrawal of nerve growth factor (in primary cultures of trigeminal and dorsal root ganglion neurons).1 In view of the known role of Bcl-2 in promoting neuronal cell survival, we have therefore investigated promoter usage in neuronal cells and have tested the effect of Brn-3a on the two Bcl-2 promoters.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of Stable Cell Lines-- The ND7-derived cell lines overexpressing Brn-3a were generated by stable transfection of ND7 cells with cDNA clones under the control of the glucocorticoid-inducible mouse mammary tumor virus promoter in the vector PJ5 (36) and have been described previously (34, 37). The cells were grown in L15 medium containing 10% fetal calf serum supplemented with G418 to a final concentration of 800 µg/ml to maintain expression of the transgene. Treatment with dexamethasone at a final concentration of 1 µM was used to induce expression of the mouse mammary tumor virus promoter.
Protein Isolation and Western Blot Analysis-- Protein was isolated from cells by freeze-thaw extraction in protein buffer (20 mM Hepes, 0.45 M NaCl, 25% glycerol, 0.2 mM EDTA containing 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM dithiothreitol) and submitted to SDS-polyacrylamide gel electrophoresis. Gels were transferred to nitrocellulose filters by Western blotting, and antigens were probed with the primary antibodies to Bcl-2, Bcl-xL/S, Bax, Bad, and Mcl-1 and detected using ECL Western blotting reagent (Amersham Pharmacia Biotech). Replica gels were stained with Coomassie, and filters were stripped and reprobed with a control antibody (pGp9.5) to ensure equal loading.
Nuclear Run-on Assays--
Assays using human BCL-2 promoter
target sequences were performed essentially as described by Young and
Korsmeyer (14). Briefly, isolated nuclei were incubated in a reaction
mixture containing 250 µCi of [-32P]GTP (3000 Ci/mmol), 2.5 mg/ml phosphocreatine, and 50 µg/ml creatine
phosphokinase at 37 °C for 30 min. Following its isolation, radiolabeled run-on RNA from these reactions was purified over Sephadex
G-50 and recovered by ethanol precipitation.
Plasmid Constructs--
A full-length construct of the human
BCL-2 gene 5' (P1) promoter region cloned upstream of the
luciferase reporter gene has been described previously (16). In order
to generate a full-length construct of the 3' (P2) promoter a
HindIII restriction site was introduced at position 8
(relative to ATG) by polymerase chain reaction, prior to subcloning
upstream of the luciferase reporter gene. Deletions of both the 5' and
3' constructs were generated using the appropriate restriction sites
within the promoter sequences (see Fig. 2, A and
B).
Transient Transfection--
Sensory neuron-derived ND7 cells
(38) and baby hamster kidney-derived fibroblasts (BHK-21 cells) (39)
were routinely cultured in L15 medium containing 10% fetal calf serum
and Dulbecco's modified Eagle's medium containing 10% fetal calf
serum, respectively. Transient transfection was carried out according
to the method of Gorman (40). Routinely, 1 × 106
cells were transfected with 10 µg of the reporter plasmid and 10 µg
of expression vectors together with 2 µg of pCMV plasmid containing the Escherichia coli lacZ gene under the control
of the constitutive cytomegalovirus promoter, and cells were harvested 48 h later. The efficiency of transfection of each sample was determined using a chemiluminescent assay for
-galactosidase activity using a commercial kit (Galactolight Plus, Tropics), and these
values were used to subsequently equalize the values obtained from the
luciferase and chloramphenicol acetyltransferase assays.
Culture and Transfection of Primary Neurons--
Dorsal root and
trigeminal ganglia were dissected from newborn Sprague-Dawley rat pups
at postnatal day 1 (P1) and, following the removal of connective tissue
sheaths, were incubated in trypsin (0.1% (Worthington) in calcium and
magnesium-free Earle's balanced salt solution). Ganglia were washed
twice in phosphate-buffered saline, dissociated, and plated onto
sterile 35-mm dishes precoated with polyornithine (0.5 mg/ml) and
laminin (20 µg/ml). Cells were cultured in defined medium (41)
supplemented with recombinant nerve growth factor (NGF; Life
Technologies, Inc.) at a final concentration of 20 ng/ml. Expression
and reporter constructs (1 µg each), together with 1 µg of CMV
plasmid, were introduced to cultured cells by liposome-mediated
transfection. Control cultures were similarly transfected with 1 µg
of empty expression vector. The efficiency of transfection of each
sample was determined using a chemiluminescent assay for
-galactosidase activity using a commercial kit (Galactolight Plus,
Tropics), and these values were used to subsequently equalize the
values obtained from the luciferase and chloramphenicol
acetyltransferase assays.
Virus Construction and Growth-- The full-length Brn-3a cDNA was cloned downstream of the Rous sarcoma virus promotor, and in reverse orientation to the cDNA encoding green fluorescent protein (GFP) cloned under the control of the cytomegalovirus promoter. This expression cassette (pR20.5-Brn-3a) was subcloned into a shuttle vector so that it was flanked by herpes simplex virus 1 (HSV-1) UL43 gene sequences. The pR20.5-Brn-3a-UL43 shuttle vector was co-transfected into BHK cells together with HSV-1 strain 1764 DNA which contains an inactivating insertion in the VMW65 gene as well as deletion of both copies of the ICP34.5 gene (42). Recombinant virus was subsequently plaque-purified on the basis of the visualization of GFP under ultraviolet conditions. Western blot analysis was performed to confirm that high levels of Brn-3a protein were produced in infected cultures before a high titer stock was grown.
Control virus containing the bacterial lacZ gene under the Rous sarcoma virus promoter (that is, containing pR20.5-lacZ-UL43) was similarly generated. Dorsal root ganglion neurons from stage embryonic day 17 mouse and rat embryos were isolated and maintained in culture as described above on glass coverslips at a density of approximately 200 neurons per coverslip. Cultures were infected in duplicate with approximately 1 × 105 plaque-forming units/coverslip of Brn-3a or control virus for 60 min, washed, and then maintained in medium supplemented with NGF for 24 h. The efficiency of viral infection was determined by visualization of GFP (typically 100% of neurons were GFP-positive) before media were replaced with fresh media with or without the addition of NGF. Neuronal cell counts were then performed 24 h later.Luciferase and Chloramphenicol Acetyltransferase Assay-- Assays of luciferase activity were carried out using a commercially available kit (Promega) and a Turner luminometer, whereas assays of chloramphenicol acetyltransferase activity were carried out according to the method of Gorman (40). All samples were equalized for DNA uptake as above.
Electron Mobility Shift Assay--
Two pairs of complementary
oligonucleotides with the sequences
5'-GTCGACAGAGAATGAAGTAAGAGGACAGG-3' and
5'-GAATTCCTGTCCTCTTACTTCATTCTCTG-3' (from 1512 to
1488
of the Bcl-2 promoter), and
5'-GTCGAGTGCTGAAGATTGATGGGATCG-3' and
5'-GAATTCGATCCCATCAATCTTCAGCACTCT-3' (from
578 to
601 of
the Bcl-2 promoter), where highlighted nucleotides identify the Brn-3
binding motif were synthesized. 5'-GATC overhangs allowed radiolabeling
with T4 polynucleotide kinase. For the binding assay, 10 fmol of
[32P]ATP-labeled oligonucleotide probe was mixed with
in vitro translated Brn-3a in the presence of 20 mM Hepes, 5 mM MgCl2, 50 mM KCl, 0.5 mM dithiothreitol, 4% Ficoll, and
2 µg of poly(dI/dC) in a 20-µl reaction. Cold competitor DNA was
added at 100 molar excess at this stage as required. The binding
reaction was incubated on ice for 40 min prior to electrophoresis on a
6% polyacrylamide gel in 0.5× TBE (1× TBE = 100 mM
Tris/HCl, 100 mM boric acid, 2 mM EDTA, pH 8.3)
for 2.5 h at 150 V and 4 °C. DNA-protein complexes were
visualized by autoradiography of the dried gel.
PCR-mediated Point Mutagenesis--
Polymerase chain
reaction-mediated mutagenesis of the sequence CATCAATCTTC (584 to
594) was performed by combining two PCR products with overlapping
sequences using a method adapted from that of Higuchi (43). Briefly,
the 5'-product was generated using the primers MUT1
(5'-GAGCTCTTGAGATCTCCGGTTGGGATTC-3') and MUT2
(5'-CGATCCCATAAATATTCAGCACTCTCCA-3'),
whereas the more 3'-product was generated using the primers MUT3
(5'-TGCTGAATATTTATGGGATCGTTGCCTTA-3') and MUT4 (5'-AAGCTTACACCAAAAGTAACGGGGGCCAA-3') (bold bases indicated mutagenized nucleotides; underlined bases indicate overlapping sequences). The two products (174 and 597 bp) were purified using Hybaid Recovery (Hybaid), annealed, and used as template for a second
round of PCR using the two flanking primers (MUT1 and MUT4). The
resulting product was ligated into pGEM-T Easy vector (Promega) and
subcloned as a SacI/HindIII fragment into the
original luciferase reporter gene vector used in this study. This
generated a mutagenized construct identical to the deletion construct
containing Bcl-2 promoter sequence
746 to
8 apart from the two
targeted mutations converting CATCAATCTTC to CATAAATATTC.
Statistics-- Microstat software (Biosoft) was used for statistical analysis. Pairwise comparisons were performed using t test or Mann Whitney analysis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brn-3a Enhances Expression of Bcl-2 but Not Bad, Bax, or Bcl-x in ND7 Cells-- We have recently described the ability of transfected Brn-3a to confer protection against apoptosis in ND7 cells following serum removal, as well as in primary cultures of NGF-dependent sensory neurons following the removal of NGF from culture media.1 To confirm and extend these observations, we have generated a safe and efficient recombinant herpes simplex virus (HSV-1) vector that is capable of long term, high level co-expression of both Brn-3a and GFP in neuronal cells both in vitro and in vivo.3 Table I shows that virally delivered Brn-3a is capable of protecting embryonic mouse dorsal root ganglion neurons from apoptosis induced by the withdrawal of NGF from culture medium (Table I). Similar protection is afforded to cultures of trigeminal ganglion neurons (data not shown).
|
|
Bcl-2 Promoter Activity in Sensory Neurons-- The 5'-flanking sequence of the human BCL-2 gene contains two distinct promoters (P1 and P2) that are required for initiation of transcription (Fig. 2A). The P1 region is situated 1.3-1.5 kilobase pairs upstream of the open reading frame and is a GC-rich, TATA-less promoter containing several SP1, STAT, and B elements with multiple start sites (14, 15). The second promoter (P2) is located 1.3 kilobase pairs downstream of P1 and includes an octamer motif, as well as CCAAT and TATA elements. Although it is not known which of these two promoter regions is utilized in cells of neuronal origin, studies have demonstrated that in other cell types in which Bcl-2 promoter activity has been characterized (predominantly cells of hemopoietic lineage), transcription is predominantly initiated from the P1 region (14-16). We therefore wished to determine the extent of P1 and P2 promoter usage in cells of neuronal origin prior to analyzing the effect of Brn-3a on the Bcl-2 promoters.
|
The Bcl-2 Promoter Is Regulated by Brn-3a in the ND7 Cell
Line--
Luciferase reporter constructs containing either the
full-length Bcl-2 promoter region (that containing both P1 and P2
regions and spanning between 3934 to
8 bp relative to the ATG) or
the isolated P1 regions (spanning
3934 to
1280 bp) have previously been described (Ref. 16 and references therein). In order to generate a
full-length construct of the P2 promoter (spanning
746 to
8 bp) a
HindIII restriction site was introduced at position
8
(relative to ATG) by polymerase chain reaction, prior to subcloning upstream of the luciferase reporter gene. Co-transfection experiments into the parental ND7 cell line revealed that Brn-3a is capable of
strongly activating both the full-length P1/P2 construct (Fig. 3A) as well as the construct
containing the P2 region (Fig. 3C). Brn-3a only weakly
activated the construct containing the isolated P1 region (Fig.
3B), whereas the related members of the Brn-3 family, Brn-3b
and Brn-3c, had no significant effect on these constructs (Fig. 3,
A-C, and data not shown). Hence these results confirm that the enhanced transcription of the bcl-2 gene,
as well as the increase in BCL-2 protein levels, in cells
overexpressing Brn-3a is dependent upon the direct effect of Brn-3a on
the Bcl-2 P2 promoter.
|
The Bcl-2 Promoter Is Regulated by Brn-3 in Primary Neuronal Culture-- The ND7 cell line was originally established via the fusion of N18TG2 azaguanine-resistant neuroblastoma cells with neonatal dorsal root ganglion neurons (38). Therefore, in view of the ability of Brn-3a to activate the Bcl-2 promoter reporter gene constructs in ND7 cells, we wished to determine whether these observations were paralleled in primary neurons. We therefore performed co-transfection experiments in primary cultures of neurons isolated from dorsal root ganglia of adult rats (see "Materials and Methods"). In these experiments Brn-3a significantly activated reporter constructs containing both the full-length Bcl-2 promoter (that is P1 and P2) (Fig. 4A) as well as the construct containing the P2 region (Fig. 4C). Brn-3a, however, did not activate the isolated P1 region (Fig. 4B). The related factor Brn-3b had no significant effect on the activity of all three constructs (Fig. 4, A-C).
|
Mutation of a Putative Brn-3a Binding Element Reduces but Does Not
Abolish Brn-3a Responsiveness--
The 600 bp of regulatory sequence
immediately upstream of the Bcl-2 P2 promoter region contained in the
P2 construct spanning 746 to
8 bp (this construct will now be
referred to as clone
746WT) contains two repeat elements, CTTCTGCTTC
and CATCAATCTTC, similar in sequence to the internexin CTTCNNNCTTC
motif identified in the
-internexin gene promoter as a response
element mediating induction by Brn-3a (44). To test whether these
elements are capable of binding Brn-3a, we performed electromobility
shift assay on these sequences. Although the first of these two
sequences did not bind any Brn-3 family members, a synthetic
oligonucleotide containing the second of these two motifs (the sequence
5'-CATCAATCTTC-3' located at
584 bp to
594 bp of the Bcl-2
promoter) was able to bind Brn-3a (Fig.
5).
|
|
|
Oct-1 and Oct-2 Do Not Activate the Bcl-2 Promoter-- The specificity of these effects to the neuronally expressed Brn-3 family of POU factors was confirmed by co-transfection experiments using the Bcl-2 promoter construct in combination with constructs expressing the more distantly related POU domain transcription factors Oct-1, Oct-2.1, Oct-2.4, and Oct-2.5. No activation or repression of Bcl-2 promoter activity was observed upon co-transfection of these type II POU factors into either ND7 cells (Fig. 8), the BHK fibroblastic cell line, or primary cultures of neonatal rat dorsal root ganglion neurons (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
By having made an initial observation that BCL-2 protein levels are increased approximately 10-20-fold in ND7 cells stably overexpressing Brn-3a (Fig. 1), we wished to determine whether this increase in protein levels was due to alterations in the transcriptional activity of the bcl-2 gene in these cells. However, we first wished to determine the precise nature of Bcl-2 transcription in neuronal cells as this has yet to be documented in the literature. The data presented here comprise the first report of Bcl-2 promoter usage in cells of neuronal lineage and indicate that the two Bcl-2 promoter regions are differentially regulated in a cell type-specific manner.
Importantly, the number of transcripts initiated from the P2 region
(and to a lesser extent from the P1 region) increased significantly in
ND7 cells induced to overexpress Brn-3a (Fig. 2, lane 3).
This observation not only provides evidence that the increase in BCL-2
protein levels in the Brn-3a overexpressing cell lines is due to
transcriptional activation but also suggests that a different set of
transcription factors and enhancers are utilized in the regulation of
Bcl-2 expression in neurons. Co-transfection experiments confirmed that
Brn-3a (a transcription factor that is predominantly expressed in the
developing nervous system (27)) is capable of activating Bcl-2
expression via the enhancement of transcriptional initiation within the
P2 promoter (Figs. 3 and 4). In particular, the reporter construct
containing the P2 region and approximately 600 bp of upstream
regulatory sequence (8 to
746 bp in clone
746WT), which
significantly does not contain the P1 region, were activated by Brn-3a
(Figs. 3C and 4C), further emphasizing the
importance of the P2 promoter and the sequence immediately upstream of
this region in the regulation of Bcl-2 expression by Brn-3a in neuronal
cells. As the stages of neurogenesis are so marked, as is the
observation that Bcl-2 expression is dramatically down-regulated in
most neuronal populations following the completion of neonatal central
nervous system remodeling as neurons become postmitotic (45), it will
be interesting to investigate the relative promoter usage during the
stages of neurogenesis and into adulthood.
Whereas the Bcl-2 P2 promoter does contain the octamer motif ATGCAAAG
(which is bound by octamer binding POU domain factors) approximately
120 bp upstream of the ATG, members of the Brn-3 family, including
Brn-3a, show only a very weak affinity for this sequence.4 However, the P2
region contained two motifs similar to that previously identified in
the -internexin promoter as mediating a response to Brn-3a (44). The
most 3' of these elements specifically bound Brn-3a, and mutagenesis of
this site in the context of the
746- to
8-bp fragment (in clones
746WT and
746MUT) results in a reduction of the responsiveness to
transfected Brn-3a and ND7 differentiation by approximately 50-60%
but not a complete inhibition of the effect. Whereas this reduction is
reproducible and is statistically significant (p = <0.005 in a Mann Whitney test), it is thus likely that the action of
Brn-3a is not solely dependent on its binding through this particular
site. Interestingly, we have shown that the oligonucleotide containing
this site conferred a low responsiveness to Brn-3a onto a heterologous
promoter in co-transfection experiments but to a lesser extent than was
observed with larger fragments containing the Bcl-2 P2 promoter region
(data not shown) suggesting that this single site is involved in the
action of Brn-3a but that other adjacent sequences are also
necessary.
We have thus identified a site in the Bcl-2 promoter that is involved in its response to transfected Brn-3a. We are currently investigating the roles of adjacent sites within this region upstream of the P2 promoter in the regulation of the promoter by Brn-3a. Most importantly, mutation of this site that affects responsiveness to Brn-3a in transfection assays also reduces to a similar extent the induction of the Bcl-2 promoter during ND7 cell differentiation, suggesting that the rise in endogenous Brn-3a levels during the differentiation process plays a key role in this enhanced activity.
We have previously demonstrated that the coordinate expression of Brn-3a and Brn-3b plays a role in the regulation of process outgrowth during neuronal differentiation (34, 37). In the present study we have demonstrated that the Brn-3a POU domain transcription factor is capable of activating the bcl-2 gene promoter. Together with the observation that BCL-2 protein levels are significantly increased in neuronally derived ND7 stable cell lines overexpressing Brn-3a, these data suggest that this regulation of a factor known to promote cell survival is likely to be one pathway by which Brn-3a is able to confer neuronal cells with resistance to apoptosis. In particular, this phenomenon may explain our observations that Brn-3a overexpression is able to protect (i) the ND7 cell line from serum withdrawal-induced apoptotic programmed cell death and (ii) primary cultures of sensory neurons from the trigeminal and dorsal root ganglia of neonatal rat pups from apoptosis induced by withdrawal of nerve growth factor.1 Indeed, it is possible that this ability of Brn-3a to protect neurons from apoptosis via the regulation of bcl-2 gene expression may be one explanation as to why significant numbers of sensory neurons fail to survive the period of target field innervation in Brn-3a null mice (29, 30).
Thus, in conclusion, these data suggest that Brn-3a is an important regulator of the bcl-2 gene in neuronal cells and is the first factor shown to specifically regulate the proximal P2 promoter region which we have shown to be the predominant promoter in neuronal cells. Both Brn-3a and Bcl-2 are capable of promoting the differentiation of neuronally derived cell lines (34, 46), suggesting that the regulation of bcl-2 gene expression provides a possible pathway by which Brn-3a is able to both promote neurite outgrowth (34) and neuronal survival.1
![]() |
ACKNOWLEDGEMENT |
---|
We thank John Reed (La Jolla Cancer Research Foundation) for the gift of the bax promoter clone.
![]() |
FOOTNOTES |
---|
* This work was supported by the Medical Research Council, UK (to D. S. L.), the Cancer Research Campaign (to D. S. L.), and by the National Institutes of Health Grant CA56764 (to L. M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 0171 380 9343; Fax: 0171 387 3310; E-mail: d.latchman{at}ucl.ac.uk.
1 M. D. Smith, S. J. Dawson, L. M. Boxer, and D. S. Latchman, submitted for publication.
2 The abbreviations used are: bp, base pair(s); PCR, polymerase chain reaction; BHK, baby hamster kidney; GFP, green fluorescent protein; HSV-1, herpes simplex virus 1; NGF, nerve growth factor.
4 M. D. Smith and D. S. Latchman, unpublished observations.
3 M. D. Smith, E. A. Ensor, R. S. Coffin, and D. S. Latchman, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|