Inhibition of Neuronal Process Outgrowth and Neuronal Specific Gene Activation by the Brn-3b Transcription Factor

Abstract

The differentiation of the ND7 neuronal cell line to a nondividing phenotype bearing numerous neurite processes is accompanied by a dramatic increase in the levels of the activating POU family transcription factor Brn-3a and a corresponding fall in the levels of the closely related inhibitory factor Brn-3b. We have previously shown that the artificial overexpression of Brn-3a in these cells can induce neurite outgrowth and the activation of genes encoding synaptic vesicle proteins in the absence of a differentiation-inducing stimulus. Here we show that overexpression of Brn-3b can reduce process outgrowth and synaptic vesicle gene expression following exposure to a stimulus which would normally induce differentiation. These inhibitory effects are abolished by altering a single amino acid in the POU homeodomain of Brn-3b to its equivalent in Brn-3a. The converse mutation in Brn-3a allows it to inhibit process outgrowth in response to a differentiation-inducing stimulus. Hence a single amino acid difference results in these closely related factors having opposite effects and allows the balance between them to regulate differentiation.The Brn-3a, Brn-3b, and Brn-3c transcription factors (also known as Brn-3.0, 3.2, and 3.1, respectively) (1, 2) are closely related members of the POU family of transcription factors which are encoded by three distinct genes (3-6) (for review of POU factors see Refs. 7 and 8). Among the mammalian POU factors, the Brn-3 proteins are the most closely related to the nematode POU factor Unc-86, which plays a critical role in neuronal development in the nematode (9, 10) suggesting that the Brn-3 factors may play a similar role in mammals. In agreement with this idea the three members of the Brn-3 family are expressed in distinct but overlapping populations of neuronal cells during development and in the adult organism (1-5, 11) with Brn-3a for example being expressed in the first differentiated neurons to appear in the midbrain, hindbrain, and spinal cord during development (12). Interestingly Brn-3a has been shown in co-transfection experiments to activate several promoters including those of the neuronally expressed genes encoding pro-opiomelancortin (1), ␣-internexin (13), and the presynaptic vesicle protein SNAP-25 (14) as well as a thymidine kinase (tk) 1 promoter containing an added synthetic binding site for the various forms of Brn-3 (tk-Oct) (15, 18). In contrast Brn-3b represses these promoters (13,15,18) and also inhibits their activation by Brn-3a. Similarly Brn-3c has only a weak activating effect on these promoters (13,15,18).When the proliferating ND7 cell line (obtained by fusing primary dorsal root ganglion neurons with C1300 neuroblastoma cells) (17) is induced to cease dividing and differentiate to a mature neuronal-like phenotype by treatment with cyclic AMP or removal of serum from the medium (18), the levels of Brn-3a rise dramatically, and those of Brn-3b fall (3, 15), while Brn-3c levels remain unchanged.2 This differentiation process is likely to require the induct...

A SNAP25 promoter variant is associated with early-onset bipolar disorder and a high expression level in brain

“…30,34 This protein is a member of the Pict-Oct-Unc (POU) transcription factor family that involved in the development of the mammalian nervous system and for which an overexpression results in a failure of SNAP25 activation and neurite outgrowth. 35 Thus, SNP4 might result in a modification in the binding affinity of a transcriptional factor, such as POU4F2, leading to the increase of SNAP25b expression level that we observed in prefrontal cortex of homozygous subjects for the 'C' allele of SNP4.…”

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

“…26 Brn-3b represses the expression of a variety of genes involved in neuronal differentiation including SNAP-25 24 and the neurofilaments 25 and thereby represses neuronal differentiation itself. 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.…”

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

“…Overexpression of Brn-3b in ND7 neuronal cells inhibits neuronal process outgrowth and synaptic vesicle gene expression and allows continued proliferation under conditions which would normally induce dierentiation (Smith et al, 1997). Moreover, in epithelial cells, Brn-3b can interact with the ER to potentiate the transcriptional eects of the ER, and the ER enhances estradiol-stimulated MCF7 cell prolifera- Figure 5 Photomicrographs (all taken at610 magni®cation) of MCF7 clonal cell lines (a ± c) grown in monolayer, and (d ± f) grown in soft agar.…”

“…Likewise, when ND7 cells and other neuronal cell lines are induced to dierentiate, the levels of Brn3b fall and the levels of Brn-3a rise (Smith and Latchman, 1996). Finally, arti®cial over-expression of Brn-3a induces neurite outgrowth in the ND7 cell line, and arti®cial over-expression of Brn-3b inhibits neuronal process outgrowth and neuronal speci®c gene activation, thus preventing the dierentiation of these cells (Smith et al, 1997). These results suggest that a role for Brn-3b may be to maintain these cells in a proliferative state.…”

“…Brn-3 proteins are associated with the balance between proliferation and dierentiation of the ND7 and other neuronal cell lines (Latchman, 1999). Speci®cally, while Brn-3a activates promoters associated with neuronal dierentiation, Brn-3b not only represses these same promoters, but also is able to negate the stimulatory eects of Brn-3a on these promoters (Smith et al, 1997). Likewise, when ND7 cells and other neuronal cell lines are induced to dierentiate, the levels of Brn3b fall and the levels of Brn-3a rise (Smith and Latchman, 1996).…”