The Brn-3a POU family transcription factor has been shown to strongly activate expression of the Bcl-2 protooncogene 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 ␣-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.The rate of apoptosis (programmed cell death) is controlled by the balance between proteins that activate processes resulting in such death and other proteins that act to inhibit these processes. Thus, for example, the Bcl-2 proto-oncogene was originally identified on the basis of its activation by chromosomal translocation in non-Hodgkin B cell lymphomas (1) and was subsequently shown to protect a wide variety of different cell types from programmed cell death or apoptosis (for review see Ref.2). Conversely, the p53 anti-oncogene protein, as well as inhibiting cellular proliferation, can also stimulate programmed cell death (for review see Ref.3).It is clear therefore that Bcl-2 and p53 represent proteins with opposite effects on the rate of apoptosis. Moreover, it appears that Bcl-2 can specifically inhibit p53-dependent apoptosis. Thus, although overexpression of p53 can induce apoptosis in different cell types, this is prevented by overexpression of Bcl-2 (4, 5). In this regard, it is of interest that high levels of p53 are associated with low levels of Bcl-2 and vice versa, both during normal rat development (6) and in different types of tumors (7-9).These findings suggest therefore that as well as being functionally antagonistic to one another, p53 and Bcl-2 may also be interlinked in terms of the processes regulating their expression. This possibility is supported by the finding that artificial overexpression of p53 in a murine leukemia cell line results in reduced Bcl-2 expression (10). Similarly, Bcl-2 levels are elevated in several tissues of knockout mice lacking functional p53 (10, 11). Evidently, these results raised the possibility that the p53 transcription factor may have an inhibitory effect on Bcl-2 gene transcription. In agreement with this it has been shown that p53 can inhibit ...

The BRCA-1 tumour supressor gene was identi®ed 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 ®rst 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.

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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.

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.Transcriptional regulation by the complex interaction of different classes of transcription factors allows a limited number of proteins to elicit diverse effects on gene expression, depending on the expression of other proteins, such as tissue-specific factors and signals which may influence their interactions (reviewed in references 39, 40, 59, and 68 and references therein). We were interested in looking at proteins which interact with the transcription factors Brn-3a and Brn-3b and modulate the regulation of gene expression by these proteins. These two proteins belong to the POU (Pit-Oct-Unc) family of transcription factors (21,25,26,42,66,70,73,76). Members of this class of transcription factors are defined on the basis of the common POU domain, which consists of two highly conserved regions, the POU-specific domain and the POU homeodomain, which are separated by a poorly conserved linker region. The POU domain acts as the DNA-binding domain which recognizes and binds specific DNA sequences present in target gene promoters but is also involved in protein-protein interactions (3,72,73). There are three known members of the Brn-3 family of transcription factors, namely, Brn-3a (also known as Brn-3.0) (21, 42, 65), Brn-3b (also called Brn-3.2) (42, 65, 70), and Brn-3c (also known as Brn-3.1) (21, 52), which are encoded by different genes (65, 77). Furthermore, different isoforms of Brn-3a and Brn-3b which result from alternative splicing of the genes encoding these two proteins have been identified (21,43,65,70).The Brn-3 proteins show restricted homology outside the conserved carboxyl-terminal POU domain and the amino-terminal POU IV box (21,65,70). Since the studies reported here were carried out with Brn-3a and Brn-3b, references to Brn-3 proteins will pertain to observations with Brn-3a or Brn-3b and not Brn-3c. Sequence differences between Brn-3a and Brn-3b proteins are paralleled by different effects on promoters which contain binding sites recog...