Vishwanie Budhram-Mahadeo

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-2proto-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 α-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, bothin 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.

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.

Activation of CDK4 gene expression in human breast cancer cells by the Brn-3b POU family transcription factor.

The Brn-3b POU family transcription factor has previously been shown to be over-expressed in human breast cancer and to enhance the growth rate and anchorage independent growth of human breast cancer cells, acting at least in part by inhibiting the expression of the BRCA-1 anti-oncogene. Here we have used gene arrays to identify several other targets for Brn-3b in human breast cancer cells, including cyclin-dependent kinase 4 (CDK4). In particular, we show that levels of CDK4 mRNA and protein correlate with the levels of Brn-3b in breast cancer cell lines manipulated to express different levels of Brn-3b and in human breast cancer biopsies and that Brn-3b can activate the CDK4 promoter. The effect of Brn-3b on the growth regulatory CDK4 protein provides a further mechanism by which Brn-3b can regulate breast cancer cell growth and indicates that it can do this by activating as well as repressing specific target genes.

POU Transcription Factors Brn-3a and Brn-3b Interact with the Estrogen Receptor and Differentially Regulate Transcriptional Activity via an Estrogen Response Element

ABSTRACT 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.

Expression of the Brn-3b transcription factor correlates with expression of HSP-27 in breast cancer biopsies and is required for maximal activation of the HSP-27 promoter

In breast cancer, overexpression of the small heat shock protein, HSP-27, is associated with increased anchorage-independent growth, increased invasiveness, and resistance to chemotherapeutic drugs and is associated with poor prognosis and reduced disease-free survival. Therefore, factors that increase the expression of HSP-27 in breast cancer are likely to affect the prognosis and outcome of treatment. In this study, we show a strong correlation between elevated levels of the Brn-3b POU transcription factor and high levels of HSP-27 protein in manipulated MCF-7 breast cancer cells as well as in human breast biopsies. Conversely, HSP-27 is decreased on loss of Brn-3b. In cotransfection assays, Brn-3b can strongly transactivate the HSP-27 promoter, supporting a role for direct regulation of HSP-27 expression. Brn-3b also cooperates with the estrogen receptor (ER) to facilitate maximal stimulation of the HSP-27 promoter, with significantly enhanced activity of this promoter observed on coexpression of Brn-3b and ER compared with either alone. RNA interference and site-directed mutagenesis support the requirement for the Brn-3b binding site on the HSP-27 promoter, which facilitates maximal transactivation either alone or on interaction with the ER. Chromatin immunoprecipitation provides evidence for association of Brn-3b with the HSP-27 promoter in the intact cell. Thus, Brn-3b can, directly and indirectly (via interaction with the ER), activate HSP-27 expression, and this may represent one mechanism by which Brn-3b mediates its effects in breast cancer cells.

Brn-3b enhances the pro-apoptotic effects of p53 but not its induction of cell cycle arrest by cooperating in trans-activation of bax expression

The Brn-3a and Brn-3b transcription factor have opposite and antagonistic effects in neuroblastoma cells since Brn-3a is associated with differentiation whilst Brn-3b enhances proliferation in these cells. In this study, we demonstrate that like Brn-3a, Brn-3b physically interacts with p53. However, whereas Brn-3a repressed p53 mediated Bax expression but cooperated with p53 to increase p21cip1/waf1, this study demonstrated that co-expression of Brn-3b with p53 increases trans-activation of Bax promoter but not p21cip1/waf1. Consequently co-expression of Brn-3b with p53 resulted in enhanced apoptosis, which is in contrast to the increased survival and differentiation, when Brn-3a is co-expressed with p53. For Brn-3b to cooperate with p53 on the Bax promoter, it requires binding sites that flank p53 sites on this promoter. Furthermore, neurons from Brn-3b knock-out (KO) mice were resistant to apoptosis and this correlated with reduced Bax expression upon induction of p53 in neurons lacking Brn-3b compared with controls. Thus, the ability of Brn-3b to interact with p53 and modulate Bax expression may demonstrate an important mechanism that helps to determine the fate of cells when p53 is induced.

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.

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 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.

Regulation of Hsp27 expression and cell survival by the POU transcription factor Brn3a

The POU family transcription factor Brn3a is expressed in specific regions of the developing and adult nervous system. Both in vivo and in vitro studies demonstrate a critical role for Brn3a in the survival and specification of sensory neurons. In the sensory neuron-derived cell line ND7 Brn3a is associated with neurite outgrowth, cell cycle arrest, differentiation and protection against apoptosis. cAMP or serum withdrawal (SW) causes the upregulation of Brn3a expression in ND7 cells and correlates with the transactivation of target promoters associated with survival or differentiation. Hsp27 is a member of the small stress protein (sHsp) family and can confer resistance to a number of cytotoxic stresses, including heat, ischemia, anticancer drugs, oxidative stress and excitotoxicity. Hsp27 is highly expressed in neuronal cells and is shown to protect against neuronal cell apoptosis. Mehlen et al. reported an increase in Hsp27 expression and Hsp27-dependent protection against apoptosis during dopamine-induced, cAMP-dependent differentiation of rat olfactory neuroblasts. Since there are direct parallels between survival and differentiation in these two experimental systems, we hypothesised that Hsp27 may represent a downstream target of the Brn3a transcription factor during neuronal cell survival and differentiation. Investigation of the DNA sequences of the mouse and human Hsp27 promoters revealed an A–T-rich sequence TTGCCATTAATAG, which corresponded to a consensus Brn3a binding site and consisting of a core ATTAAT element intersecting two half oestrogen response elements (EREs) at 87 to 91 and 71 to 73. We subcloned the Hsp27 promoter region into the pGL2-luciferase reporter plasmid and co-transfected this into ND7 cells along with plasmids expressing full-length Brn3a(l), which contains the N-terminal transactivation and POU DNA binding domains, Brn3a(s) that lacks the N-terminal transactivation domain and the Brn3a POU domain in isolation. Co-transfection with the oestrogen receptor (ER) was also used as a positive control. As shown in Figure 1a, the wild-type promoter was activated 29-fold by full-length Brn3a(l), compared to a seven-fold activation by co-expression of the ER, but not by Brn3a(s) or the POU domain alone. This suggests that the N-terminal activation domain of Brn3a is required. The long (l) and short (s) isoforms of Brn3a result from alternate promoter usage. The 46 kDa Brn3a(l) contains an N-terminal activation domain not present in the 35 kDa Brn3a(s). Interestingly, while some promoters are activated by both Brn3a isoforms (i.e. SNAP25; synaptophysin) others require the N-terminal activation domain, particularly those involved in cell survival, such as Bcl-2 and Bcl-XL. 13 Site-directed mutagenesis of three nucleotides within the CATTAAT sequence to CGCCAAT within the context of the intact promoter abolished the ability of Brn3a(l) to transactivate the promoter (Figure 1b), but did not affect basal promoter activity. An enhanced activation of the promoter by ER in this case is consistent with the observation that Brn3a is able to interact with ER and represses its effect on an ERE-containing promoter. Furthermore, activation of the promoter by ER in the presence of the mutated site indicates that promoter activity is otherwise intact and that the CATTAAT site is not simply functioning as a TATA box. We investigated whether this effect of Brn3a was mediated by binding to the CATTAAT sequence within the proximal Hsp27 promoter by performing EMSAs using labelled oligonucleotides corresponding to the putative Brn3a binding site (50-TTG CCA TTA ATA GAG-30) and in vitro-translated (IVT) Brn3a. Incubation of labelled probe with IVT Brn3a resulted in two major bands that were specifically competed upon addition of unlabelled (‘cold’) oligonucleotide, but not by a nonspecific oligonucleotide or an oligonucleotide in which the Brn3a site was mutated (Figure 1c). Moreover, Brn3a failed to bind the labelled mutant probe. Functional analysis of whether Brn3a modified endogenous Hsp27 levels in ND7 cells showed that either SW alone or overexpression of Brn3a(l) clearly induced expression of Hsp27 (Figure 1e and f). This induction was further potentiated by overexpression of Brn3a followed by SW. Brn3a(l) also induced expression of the Brn3a(s) isoform as previously described. The effect of increased Brn3a and Hsp27 expression on survival and apoptosis following SW in ND7 cells was assessed by staining of the cells with annexin V and propidium iodide (PI) and subsequent FACS analysis. Annexin V staining (Figure 1e and f) showed that SW caused a substantial increase in the number of cells undergoing apoptosis (total annexin V positive cells mean7S.E.M: pLTR 12.974.0% versus pLTRþSW 65.872.8%, Pr0.01). Overexpression of Brn3a(l) significantly reduced the number of cells that were undergoing apoptosis, as indicated by a substantial reduction in both annexin V positive subpopulations (3a 21.473.1%, Pr0.01). This observation was supported by PI staining and cell cycle analysis. SW in (control) pLTR transfected cells resulted Cell Death and Differentiation (2004) 11, 1242–1244 & 2004 Nature Publishing Group All rights reserved 1350-9047/04