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.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...

Activation of CDK4 Gene Expression in Human Breast Cancer Cells by the Brn-3b POU Family Transcription Factor

“…27 Our initial studies in breast cancer cells, identified the BRCA-1 gene as a target for transcriptional repression by Brn-3b. 16 Hence, although Brn-3b can interact with the oestrogen receptor and enhance its ability to activate its target genes, 11 it appeared that Brn-3b might achieve its effects on the growth of breast cancer cells 19 by inhibiting rather than activating gene expression.…”

“…18 Moreover, Brn-3b has also been shown to interact with the oestrogen receptor via a protein-protein interaction and to enhance its ability to activate its target genes. 11 These findings suggest therefore, that the overexpression of Brn-3b in human breast cancer cells may act to modulate the growth of these cells by regulating the expression of specific genes with growth regulatory properties. In agreement with this idea, we have shown that overexpression of Brn-3b in the MCF-7 human breast cancer cell line results in increased growth rate, higher saturation density and enhanced ability to form colonies in soft agar, compared to the control empty vector transfected cells.…”

“…Thus, the closely related POU family transcription factors Brn-3a, Brn-3b and Brn-3c were originally identified on the basis of their distinct but overlapping patterns of gene expression in the developing and adult nervous systems [3][4][5][6] and inactivation of the individual genes encoding these factors has been shown to produce defects in the development of specific parts of the nervous system. [7][8][9] However, expression of Brn-3a and Brn-3b has also been observed in nonneuronal cells notably, in the testis, 10 breast 11 and cervix. 4,12 This expression of Brn-3a in neuronal and some nonneuronal cell types is paralleled by their overexpression in tumors of neuronal and nonneuronal origin.…”

“…19 Moreover, although all these effects could be observed in the absence of oestrogen, cells overexpressing Brn-3b also demonstrated enhanced oestrogen responsiveness, 19 paralleling the effect of the protein-protein interaction between the oestrogen receptor and Brn-3b on the activation of the oestrogen receptors target genes. 11 Hence, Brn-3b is both overexpressed in human breast cancer samples 16 and can enhance the growth of breast cancer cells in culture. 19 Although these effects are likely to be dependent, at least in part, on the ability of Brn-3b to repress BRCA-1 gene expression 16 and to activate the oestrogen receptor, 11 we wished to identify further target genes by which Brn-3b might regulate the growth of human cancer cells.…”

“…11 Hence, Brn-3b is both overexpressed in human breast cancer samples 16 and can enhance the growth of breast cancer cells in culture. 19 Although these effects are likely to be dependent, at least in part, on the ability of Brn-3b to repress BRCA-1 gene expression 16 and to activate the oestrogen receptor, 11 we wished to identify further target genes by which Brn-3b might regulate the growth of human cancer cells. Here we report the result of such an analysis in which target genes were identified by screening an array of cancer-related genes and their altered expression in both MCF-7 cells, having different Brn-3b levels, and in human breast cancer samples was analysed.…”