Karen A. Lillycrop

The opposite and antagonistic effects of the closely related POU family transcription factors Brn-3a and Brn-3b on the activity of a target promoter are dependent on differences in the POU domain.

The Brn-3a, Brn-3b, and Brn-3c POU family transcription factors are closely related to one another and are members of the group IV subfamily of POU factors. Here we show that despite this close relationship, the factors have different effects on the activity of a target promoter: Brn-3a and Brn-3c stimulate the promoter whereas Brn-3b represses it. Moreover, Brn-3b can antagonize the stimulatory effect of Brn-3a on promoter activity and can also inhibit promoter activation by the Oct-2.1 POU factor. The difference in the transactivation activities of Brn-3a and Brn-3b is dependent upon the C-terminal region containing the POU domain of the two proteins, since exchange of this domain between the two factors converts Brn-3a into a repressor and Brn-3b into an activator.

Repression of a herpes simplex virus immediate-early promoter by the Oct-2 transcription factor is dependent on an inhibitory region at the N terminus of the protein.

The B-cell form of the Oct-2 transcription factor Oct 2.1 can activate the herpes simplex virus immediate-early gene 3 (IE3) promoter, whereas the neuronally expressed Oct 2.4 and 2.5 forms of the protein, which contain a different C terminus, can repress this promoter. Here we show that partial or full deletion of the C terminus of Oct 2.1 in the presence of an intact N terminus results in a protein which can strongly repress the IE3 promoter. In contrast, deletion of the entire N terminus or a short region within it leaving the C terminus intact results in a very strong activator. Deletion of both N and C termini leaving only the isolated POU domain generates only a very weak repressor. The N-terminal region defined in this way can repress a heterologous promoter when linked to the DNA-binding domain of the GAL4 factor, indicating that it can function as an independent inhibitory domain. These results indicate that a specific region within the N terminus common to Oct 2.1, 2.4, and 2.5 plays a critical role in the ability of neuronally expressed forms of Oct-2 to repress the IE3 promoter but can do so only when the C-terminal region of Oct 2.1 is altered or deleted.

The B-cell and neuronal forms of the octamer-binding protein Oct-2 differ in DNA-binding specificity and functional activity.

B lymphocytes contain an octamer-binding transcription factor, Oct-2, that is absent in most other cell types and plays a critical role in the B-cell-specific transcription of the immunoglobulin genes. A neuronal form of this protein has also been detected in brain and neuronal cell lines by using a DNA mobility shift assay, and an Oct-2 mRNA is observed in these cells by Northern (RNA) blotting and in situ hybridization. We show that the neuronal form of Oct-2 differs from that found in B cells with respect to both DNA-binding specificity and functional activity. In particular, whereas the B-cell protein activates octamer-containing promoters, the neuronal protein inhibits octamer-mediated gene expression. The possible role of the neuronal form of Oct-2 in the regulation of neuronal gene expression and its relationship to B-cell Oct-2 are discussed.

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.

Down regulation of the octamer binding protein Oct-1 during growth arrest and differentiation of a neuronal cell line

The octamer binding transcription/DNA replication factor Oct-1 is present in virtually all cell types including proliferating cell lines of neuronal origin but is not detectable in mature non-dividing neurons. Cell cycle arrest in G0/G1 and morphological differentiation of a neuronal cell line is accompanied by a decline in the level of Oct-1 DNA binding, although the level of DNA binding by another octamer binding protein, Oct-2 is unaltered. This effect is paralled by a decline in the level of the Oct-1 mRNA in the non-dividing cells. The decrease in Oct-1 levels occurs only with the production of a mature, non-dividing neuronal phenotype and not when the cells are arrested in late G1 and do not undergo morphological differentiation. The potential role of Oct-1 and other octamer binding proteins in gene regulation in neuronal cells and in their differentiation is discussed.

The inhibitory domain in the Oct-2 transcription factor represses gene activity in a cell type-specific and promoter-independent manner

The Oct-2 transcription factor contains an N-terminal inhibitory domain which can act to inhibit promoter activity when linked to either its corresponding DNA-binding POU domain or the heterologous DNA binding domain of the yeast transcription factor GAL4. This inhibitory effect is independent of the number of DNA binding sites or their context in the target promoter. In contrast the effect is cell type-specific and can be relieved by over-expression of the isolated inhibitory domain in the absence of a DNA binding domain. These results suggest that the inhibitory domain acts by decreasing the activity of the basal transcriptional complex but that it operates indirectly by recruiting a second cell type-specific factor to the promoter which then interacts with the basal complex decreasing its activity.

Regulated splicing of the Oct-2 transcription factor RNA in neuronal cells.

The primary RNA transcript derived from the gene encoding the Oct-2 transcription factor is alternatively spliced to yield a number of different mRNAs which encode different isoforms of this protein. The mRNAs encoding two such isoforms Oct-2c and mini Oct-2 were originally detected in neuronal cells. We show here that the mRNAs encoding these forms also occur in other tissues with the proportion of the mini Oct-2 mRNA being much higher in the spleen than in the brain. However, the levels of the mini Oct-2 mRNA increase in neuronal cell lines in response to differentiation-inducing stimuli and decrease upon exposure to growth factors. Hence the splicing of the Oct-2 transcript can be regulated in both a tissue specific manner and in neuronal cells in response to specific stimuli. The significance of this effect is discussed in terms of the differing ability of different forms of Oct-2 to activate or inhibit gene expression.

Cell cycle arrest and morphological differentiation can occur in the absence of apoptosis in a neuronal cell line.

Apoptotic cell death plays a critical role in the development of the nervous system. Although the apoptotic death of mature non-dividing neurons has been extensively studied, the mechanisms mediating the extensive cell death in areas of the developing brain where proliferating neuroblasts are differentiating into mature neurons have not been analyzed. We have previously shown that the cell cycle arrest of a proliferating neuronal cell line by transfer from medium containing 10% foetal calf serum (FCS) to serum-free medium results in the morphological differentiation of some cells and the death of others by apoptosis. Here we show that the effect of 10% FCS can be mimicked by medium containing either similar concentrations of newborn or adult bovine serum or 1% FCS all of which maintain cellular proliferation and inhibit differentiation and apoptosis. In contrast, the presence of 0.5% FCS in the medium effectively prevents apoptosis but does not allow cellular proliferation or inhibit morphological differentiation. Hence cell cycle arrest and differentiation can occur in the absence of apoptosis in cells of neuronal origin and the factors in serum responsible for modulating these processes are likely to be distinct.