Co-expression of POU4F2/Brn-3b with p53 may be important for controlling expression of pro-apoptotic genes in cardiomyocytes following ischaemic/hypoxic insults.

Cardiomyocyte death following ischaemic/hypoxic injury causes irreversible damage to cardiac function and contributes to chronic diseases such as heart failure. Understanding the mechanisms associated with myocyte loss under these conditions can help to identify strategies to minimise/abrogate such detrimental effects. The p53 protein can induce apoptosis or cell cycle arrest, but effects on cell fate depend on interactions with other regulators such as POU4F2/Brn-3b (Brn-3b), which co-operates with p53 to increase the expression of pro-apoptotic genes. In contrast, the related POU4F1/Brn-3a (Brn-3a) blocks p53-mediated apoptosis but co-operates with p53 to enhance cell cycle arrest. In this study, we showed that permanent coronary artery ligation in mouse hearts, which induced apoptotic markers, activated caspase-3 and -8 and necroptosis markers; RIP-1 and -3 also increased Brn-3b and Brn-3a expression. However, Brn-3a was only detected in uninjured myocardium but not at the site of injury, whereas Brn-3b showed generalised increase, including within the infarct zone. Conversely, p53 was detected in the infarct zone and in some cells adjacent to the site of injury but not in uninjured myocardium. Co-localisation studies showed Brn-3a co-expression with p53 in cardiomyocytes adjacent to the infarct zone, whereas Brn-3b was co-localised with p53 in the infarct zone only. Increased Brn-3b and p53 correlated with elevated expression of pro-apoptotic target genes, Bax, Noxa and PUMA, whereas cleaved caspase-3 confirmed the presence of apoptotic cells within this region of the injured heart. Similarly, simulated ischaemia/reoxygenation (sI/R) injury in neonatal rat ventricular cardiomyocytes (NRVM) and heart derived H9c2 myoblasts increased Brn-3b, p53 as well as apoptotic genes, and this was associated with enhanced apoptosis. Furthermore, targeted reduction of Brn-3b using shRNA caused reduction in pro-apoptotic Bax and Noxa proteins, even though p53 expression remained intact, suggesting that Brn-3b is important for controlling the fate of the myocardium in the injured heart.

Brn-3a/POU4F1 interacts with and differentially affects p73-mediated transcription.

The Brn-3a/POU4F1 POU transcription factor is critical for the survival and differentiation of specific sensory neurons during development or upon injury; by regulating expression of target genes, either directly or indirectly upon interaction with other proteins. In this study, we demonstrated the physical interaction of Brn-3a with different p73 isoforms and showed co-localization in sensory neurons arising from the neural crest. The biological effects of p73/ Brn-3a interaction depend on the particular p73 isoform, because co-expression of Brn-3a with TAp73 enhanced cell cycle arrest, whereas Brn-3a and DeltaNp73 cooperated to increase protection from apoptosis. Brn-3a antagonized TAp73 transactivation of pro-apoptotic Bax, but co-operated to increase transcription of the cell cycle regulator p21 CIP1/Waf1. The region 425-494 amino acids within the TAp73 C terminus were critical for Brn-3a to repress Bax transactivation, but not for cooperation on the p21 CIP1/Waf1 promoter. Our results suggest that co-factors binding to the p73 C terminus facilitate maximal activation on the Bax but not p21 CIP1/Waf1 promoter and that Brn-3a modulates this interaction. Thus, the physical interaction of Brn-3a with specific p73 isoforms will be critical for determining cell fate during neuronal development or in injured neurons expressing both factors.

Proliferation-associated Brn-3b transcription factor can activate cyclin D1 expression in neuroblastoma and breast cancer cells.

Brn-3b transcription factor enhances proliferation of neuroblastoma (NB) and breast cancer cell lines in vitro and increases the rate and size of in vivo tumour growth, whereas reducing Brn-3b slows growth, both in vitro and in vivo. Brn-3b is elevated in >65% of breast cancer biopsies, and here we demonstrate that Brn-3b is also elevated in NB tumours. We show a significant correlation between Brn-3b and cyclin D1 (CD1) in breast cancers and NB tumours and cell lines. Brn-3b directly transactivates the CD1 promoter in co-transfection experiments, whereas electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrate that Brn-3b protein binds to an octamer sequence located in the proximal CD1 promoter. Site-directed mutagenesis of this sequence resulted in loss of transactivation of the CD1 promoter by Brn-3b. Thus, Brn-3b may act to alter growth properties of breast cancer and NB cells by enhancing CD1 expression in these cells.

Differential regulation of different human papilloma virus variants by the POU family transcription factor Brn-3a.

The Brn-3a POU family transcription factor is overexpressed in human cervical carcinoma biopsies and is able to activate expression of the human papilloma virus type 16 (HPV-16) upstream regulatory region (URR), which drives the expression of the E6 and E7 oncoproteins. Inhibition of Brn-3a expression in human cervical cancer cells inhibits HPV gene expression and reduces cellular growth and anchorage independence in vitro as well as the ability to form tumours in vivo. Here, we show that Brn-3a differentially regulates different HPV-16 variants that have previously been shown to be associated with different risks of progression to cervical carcinoma. In human cervical material, Brn-3a levels correlate directly with HPV E6 levels in individuals infected with a high risk variant of HPV-16, whereas this is not the case for a low-risk variant. Moreover, the URRs of high- and intermediate-risk variants are activated by Brn-3a in transfection assays, whereas the URR of a low-risk variant is not. The change of one or two bases in a low-risk variant URR to their equivalent in a higher-risk URR can render the URR responsive to Brn-3a and vice versa. These results help explain why the specific interplay between viral and cellular factors necessary for the progression to cervical carcinoma only occurs in a minority of those infected with HPV-16.

Brn-3a neuronal transcription factor functional expression in human prostate cancer.

Neuroendocrine differentiation has been associated with prostate cancer (CaP). Brn-3a (short isoform) and Brn-3c, transcriptional controllers of neuronal differentiation, were readily detectable in human CaP both in vitro and in vivo. Brn-3a expression, but not Brn-3c, was significantly upregulated in >50% of tumours. Furthermore, overexpression of this transcription factor in vitro (i) potentiated CaP cell growth and (ii) regulated the expression of a neuronal gene, the Nav1.7 sodium channel, concomitantly upregulated in human CaP, in an isoform-specific manner. It is concluded that targeting Brn-3a could be a useful strategy for controlling the expression of multiple genes that promote CaP.

Coexpression of Brn-3a POU protein with p53 in a population of neuronal progenitor cells is associated with differentiation and protection against apoptosis.

The Brn-3a transcription factor is critical for survival and differentiation of sensory neurons derived from neural crest cells (NCC). Interaction of Brn-3a with p53 results in differential effects on target gene expression, which profoundly affects fate of neuronal cells. Here we demonstrate colocalization of p53 in a subset of Brn-3a-positive NCC-derived cells fated for the sensory neuronal lineage. The distinct morphology of Brn-3a/p53-coexpressing cells suggested a differentiated neuronal cell type, and this was confirmed by colocalization of p53 with differentiation marker NF-160. Functional effects of Brn-3a/p53 coexpression were analyzed in NCC cultured from Brn-3a -/- embryos, which showed significantly increased apoptosis upon induction of p53 compared with wild-type NCC, suggesting that Brn-3a modulates the p53-mediated fate of NCC that coexpress both factors. Thus, p53 is expressed in neuronal cells undergoing differentiation as well as apoptosis. Interaction with Brn-3a in sensory neurons may be critical for modulating p53-mediated gene expression and hence cell fate.

Distinct promoter elements mediate the co-operative effect of Brn-3a and p53 on the p21 promoter and their antagonism on the Bax promoter.

Although the promoters of both the Bax and p21 genes are activated by p53, they differ in the effect on this activation of the POU family transcription factor Brn-3a. Thus, Brn-3a inhibits activation of the Bax promoter by p53 but enhances the ability of p53 to activate the p21 promoter. We demonstrate that repression of p53-mediated activation of the Bax promoter involves a complex upstream sequence in which two Brn-3a response elements flank the p53 response element. In contrast, a minimal p21 promoter is activated by Brn-3a and such activation cannot be abolished without abolishing basal promoter activity. Moreover, synergistic activation by Brn-3a and p53 continues to be observed when the p53-binding sites in the p21 promoter are substituted by the Bax p53 site or by the region of the Bax promoter essential for Brn-3a-mediated repression, indicating that the p21 core promoter plays a central role in this response. The significance of these effects is discussed in terms of the different responses of the Bax and p21 promoters and the overlapping but distinct roles of Brn-3a and p53 in neuronal growth arrest and apoptosis.

A minimal Bcl-x promoter is activated by Brn-3a and repressed by p53.

The Brn-3a transcription factor stimulates the expression of the anti-apoptotic Bcl-2 and Bcl-x proteins and protects neuronal cells from apoptosis. Here we show that a minimal Bcl-x promoter is activated by Brn-3a and that this stimulation is prevented by the pro-apoptotic p53 protein. Both these effects are mediated via Bcl-x promoter sequences, which are indistinguishable from those required for minimal basal promoter activity. A newly described upstream Bcl-x promoter is also activated by Brn-3a with this activation being prevented by p53. Hence, Brn-3a-mediated activation of two distinct Bcl-x promoters and of the Bcl-2 promoter is blocked by p53 whereas this is not observed for Brn-3a activated promoters derived from genes not involved in inhibiting apoptosis. p53 therefore appears to specifically target the activation by Brn-3a of promoters derived from genes with an anti-apoptotic effect and this may be involved in the pro-apoptotic activity of p53.

The Brn-3b POU family transcription factor regulates the cellular growth, proliferation, and anchorage dependence of MCF7 human breast cancer cells.

The Brn-3b POU domain containing transcription factor is expressed in the developing sensory nervous system as well as in epithelial cells of the breast, cervix, and testes. Brn-3b functionally interacts with the estrogen receptor (ER) and in association with the ER, regulates transcription from estrogen responsive genes. In addition, Brn-3b expression is elevated in breast tumours compared to levels in normal mammary cells. To explore the role of Brn-3b in breast cancer, we established stable cell lines derived from the MCF7 human breast cancer cell line which had been transfected with Brn-3b sense or anti-sense constructs. The Brn-3b over-expressing cell lines exhibited increased growth rate, reached confluence at a higher saturation density, had higher proliferative activity, and an enhanced ability to form colonies in soft agar when compared to the control empty vector transfected cells. Likewise, the Brn-3b anti-sense cell lines showed reduced cellular growth and proliferation, reached confluence at a lower density, and exhibited a decreased ability to form colonies in soft agar when compared to the vector controls. Five to ten per cent of the Brn-3b over-expressing cells exhibited a severely altered morphology characterized by reduced adherence to tissue culture plastic, increased cell size, and a vacuolar cell shape. These results thus further indicate a role for the Brn-3b transcription factor in regulating mammary cell growth and suggest that its elevation in breast cancer is of functional significance.

The Brn-3a transcription factor plays a key role in regulating the growth of cervical cancer cells in vivo.

The cellular Brn-3a transcription factor is known to activate transcription of the genes encoding the human papilloma virus E6 and E7 proteins and is over-expressed in women with cervical neoplasia. We show that cervical cell lines with reduced Brn-3a expression show a greatly reduced ability to form tumours in nude mice compared to control cells and also show reduced expression of the HPV E6 and cellular Bcl-2 oncogenes. These effects are also observed in cervical cells over-expressing the related Brn-3b factor, which is known to antagonize activation of HPV gene expression by Brn-3a. These results demonstrate, for the first time, that inhibition of Brn-3a expression or enhanced Brn-3b expression can inhibit cervical cell-derived tumour growth in vivo as well as in vitro. Hence they establish Brn-3a as a key factor in cervical tumorigenesis and as a potential therapeutic target in human cervical neoplasia.

The closely related POU family transcription factors Brn-3a and Brn-3b are expressed in distinct cell types in the testis.

Although the Brn-3a and Brn-3b POU family transcription factors were originally identified in neuronal cells, their expression in some non neuronal cell types has previously been reported. Here we report that Brn-3a and Brn-3b are also expressed in the testis with expression of each factor being observed at distinct stages of germ cell development. Thus, Brn-3a is expressed in spermatogonia whereas Brn-3b expression is observed in post-meiotic spermatids. In agreement with this, Brn-3a expression is detectable much earlier than that of Brn-3b in testes derived from sexually immature postnatal animals. Similarly, Brn-3b expression is absent in knock out mice lacking a functional CREM transcription factor in which the later stages of germ cell development do not occur, whereas Brn-3a expression is observed at similar levels in the testes of these knock out mice. Interestingly, the cellular pattern of Brn-3a expression during germ cell development coincides with that of the BRCA-1 anti-oncogene. Consistent with the possibility that Brn-3a may regulate expression of BRCA-1 in the testis, we have shown that Brn-3a can strongly activate the BRCA-1 promoter in co-transfection experiments whereas Brn-3b does not have this effect. Hence, as observed in neuronal cells, Brn-3a and Brn-3b may play distinct and important functional roles in the regulation of gene expression during germ cell development.

The Brn-3b POU family transcription factor represses expression of the BRCA-1 anti-oncogene in breast cancer cells.

The BRCA-1 tumour supressor gene was identified on the basis of mutations which occur in familial breast cancer indicating that its inactivation can cause this disease. Although BRCA-1 does not appear to be mutated in sporadic breast cancer, its expression has been shown to be reduced in tumour material from such cases. We show here that mammary tumours which have reduced levels of BRCA-1 expression show enhanced expression of the Brn-3b POU family transcription factor at both the mRNA and protein levels. This elevated expression of Brn-3b is not found in normal mammary cells, benign tumours or in malignant tumour samples which do not exhibit reduced levels of BRCA-1. In contrast, no correlation was noted between BRCA-1 and expression of the related factor Brn-3a. Moreover, Brn-3b but not Brn-3a can strongly repress the BRCA-1 promoter approximately 20-fold in mammary tumour cells. To our knowledge, this is the first report of a transcription factor which regulates BRCA-1 expression. Thus, Brn-3b may play an important role in regulating expression of BRCA-1 in mammary tumours with enhanced expression of Brn-3b resulting in reduced BRCA-1 expression and thereby being potentially important in tumour development.

The Brn-3a transcription factor plays a critical role in regulating human papilloma virus gene expression and determining the growth characteristics of cervical cancer cells.

The Brn-3a POU family transcription factor has previously been shown to activate the human papilloma virus type 16 (HPV-16) promoter driving the expression of the E6- and E7-transforming proteins. Moreover, Brn-3a is overexpressed approximately 300-fold in cervical biopsies from women with cervical intra-epithelial neoplasia type 3 (CIN3) compared with normal cervical material. To test the role of Brn-3a in cervical neoplasia we have manipulated its expression in cervical carcinoma-derived cell lines with or without endogenous HPV genes. In HPV-expressing cells, reduction in Brn-3a expression specifically reduces HPV gene expression, growth rate, saturation density and anchorage-independent growth, whereas these effects are not observed when Brn-3a expression is reduced in cervical cells lacking HPV genomes. Together with our previous observations, these findings indicate a critical role for Brn-3a in regulating HPV gene expression and thereby in controlling the growth/transformation of cervical cells.

p53 suppresses the activation of the Bcl-2 promoter by the Brn-3a POU family transcription factor.

The Brn-3a POU family transcription factor has been shown to strongly activate expression of the Bcl-2 proto-oncogene and thereby protect neuronal cells from programmed cell death (apoptosis). This activation of the Bcl-2 promoter by Brn-3a is strongly inhibited by the p53 anti-oncogene protein. This inhibitory effect of p53 on Brn-3a-mediated transactivation is observed with nonoverlapping gene fragments containing either the Bcl-2 p1 or p2 promoters but is not observed with other Brn-3a-activated promoters such as in the gene encoding alpha-internexin or with an isolated Brn-3a binding site from the Bcl-2 promoter linked to a heterologous promoter. In contrast, p53 mutants, which are incapable of binding to DNA, do not affect Brn-3a-mediated activation of the Bcl-2 p1 and p2 promoters. Moreover, Brn-3a and p53 have been shown to bind to adjacent sites in the p2 promoter and to directly interact with one another, both in vitro and in vivo, with this interaction being mediated by the POU domain of Brn-3a and the DNA binding domain of p53. The significance of these effects is discussed in terms of the antagonistic effects of Bcl-2 and p53 on the rate of apoptosis and the overexpression of Brn-3a in specific tumor cell types.

POU transcription factors Brn-3a and Brn-3b interact with the estrogen receptor and differentially regulate transcriptional activity via an estrogen response element.

The estrogen receptor (ER) modulates transcription by forming complexes with other proteins and then binding to the estrogen response element (ERE). We have identified a novel interaction of this receptor with the POU transcription factors Brn-3a and Brn-3b which was independent of ligand binding. By pull-down assays and the yeast two-hybrid system, the POU domain of Brn-3a and Brn-3b was shown to interact with the DNA-binding domain of the ER. Brn-3-ER interactions also affect transcriptional activity of an ERE-containing promoter, such that in estradiol-stimulated cells, Brn-3b strongly activated the promoter via the ERE, while Brn-3a had a mild inhibitory effect. The POU domain of Brn-3b which interacts with the ER was sufficient to confer this activation potential, and the change of a single amino acid in the first helix of the POU homeodomain of Brn-3a to its equivalent in Brn-3b can change the mild repressive effect of Brn-3a to a stimulatory Brn-3b-like effect. These observations and their implications for transcriptional regulation by the ER are discussed.

The different activities of the two activation domains of the Brn-3a transcription factor are dependent on the context of the binding site.

The POU (Pit-Oct-Unc) family transcription factor Brn-3a contains two distinct activation domains, one at the N terminus of the molecule and one at the C terminus coincident with the DNA binding domain. These different activation domains have been shown previously to differ in their ability to activate an artificial test promoter containing a Brn-3a binding site and the naturally occurring alpha-internexin gene promoter. Here we identify the target site for Brn-3a in the alpha-internexin gene promoter and show that it can confer responsiveness to Brn-3a on a heterologous promoter. One of the single-stranded DNA sequences derived from either this novel Brn-3a binding site or from the previously characterized site in the test promoter are shown to bind Brn-3a preferentially compared with the complementary single strand or the corresponding double-stranded sequence. The pattern of responsiveness of these two sequences when cloned upstream of the same test promoter and co-transfected with constructs encoding various portions of Brn-3a indicates that the activity of the two Brn-3a activation domains is dependent upon differences in the context of the target sequence in each promoter rather than on differences in the target sequence itself.

The levels of the antagonistic POU family transcription factors Brn-3a and Brn-3b in neuronal cells are regulated in opposite directions by serum growth factors.

The Brn-3a and Brn-3b proteins are closely related POU family transcription factors with generally antagonistic effects on gene expression. We show that transfer of ND7 neuronal cells to medium containing either no foetal calf serum or low concentrations of serum results in a rise in Brn-3a mRNA levels and a fall in Brn-3b mRNA levels, although the precise serum dependence of these two effects differ. These effects can be reversed by addition of specific growth factors to the medium lacking serum, although not all growth factor treatments which suppress the rise in Brn-3a can reverse the fall in Brn-3b levels. These effects do not correlate with the effects of each treatment on cellular proliferation indicating that they are not simply a consequence of changes in proliferation. Interestingly however, treatments which produce a rise in Brn-3a levels also induce the outgrowth of neuritic processes. Hence the expression of a functionally antagonistic pair of POU factors is regulated in opposite directions by treatments with serum growth factors and this is likely to represent one means by which such growth factors modulate the gene expression patterns and ultimately the behaviour of neuronal cells.