Mouse Brn-3 family of POU transcription factors: a new aminoterminal domain is crucial for the oncogenic activity of Brn-3a

The class IV POU domain genes Brn-3a, -b and -c are differentially expressed during neural development and at least Brn-3a also in neuroectodermal tumors. In contrast to Brn-3b and Brn-3c, Brn-3a encodes two protein variants: Brn-3a(l) and Brn-3a(s). Brn-3a(s) lacks 84 aminoterminal residues but is otherwise identical to Brn-3a(l). Outside the well conserved carboxyterminal POU domains all three Brn-3 proteins (-a, -b and -c) diverge until the aminoterminal end where a new domain of about 100 amino acids is identified. This domain is conserved only between Brn-3 proteins and other class IV POU factors. Brn-3a(l) that contains the complete domain but not Brn-3a(s) that lacks 84 amino acids of it is able to tumorigenically transform primary fibroblasts. Brn-3b that lacks 40 amino acids of the new domain does itself not transform, but abolishes the oncogenic potential of Brn-3a(l) when transfected together. This demonstrates not only that Brn3-a(l) is a proto-oncogene and may well be causally involved in the generation of neuroectodermal tumors but also suggests that the intactness of the new aminoterminal domain described here is crucial for oncogenic activity. In addition, our data indicate that Brn-3b acts as an inhibitor of Brn-3a(l) activity.     

Cooperative function of POU proteins and SOX proteins in glial cells

Glial cells of the oligodendrocyte lineage express several highly related POU proteins including Tst-1/Oct6/SCIP and Brn-1. Tst-1/Oct6/SCIP, but not Brn-1 efficiently cooperated with Sox10, the only SRY box protein so far identified in oligodendrocytes. Here we show that, in addition to Sox10, cells of the oligodendrocyte lineage contain significant amounts of the related SRY box proteins Sox4 and Sox11. During development, Sox11 was strongly expressed in the central nervous system. It was first detected in neural precursors throughout the neuroepithelium. During later stages of neural development, Sox11 was additionally expressed in areas of the brain in which neurons undergo differentiation. In agreement with its expression in neural precursors, Sox11 levels in cells of the oligodendrocyte lineage were high in precursors and down-regulated during terminal differentiation. Outside the nervous system, expression of Sox11 was also detected in the developing limbs, face, and kidneys. Structure function analysis revealed that Sox11 has a strong intrinsic transactivation capacity which is mediated by a transactivation domain in its carboxyl-terminal part. In addition, Sox11 efficiently synergized with Brn-1. Synergy was dependent on binding of both proteins to adjacent DNA elements, and required the presence of the respective transactivation domain in each protein. Our data suggest the existence of a specific code in which POU proteins require specific Sox proteins to exhibit cooperative effects in glial cells.     

Differential expression of gap junctions in neurons and astrocytes derived from P19 embryonal carcinoma cells

The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell-cell and cell-neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes.     

The polycystic kidney disease-1 protein, polycystin-1, binds and activates heterotrimeric G-proteins in vitro

Analysis of the C-terminal cytosolic domain of human and mouse polycystin-1 has identified a number of conserved protein motifs, including a 20-amino-acid heterotrimeric G-protein activation sequence. A peptide specific for this sequence was synthesized and shown to activate purified bovine brain heterotrimeric Gi/Go in vitro. To test whether the C-terminal cytosolic domain of polycystin-1 stably binds G-proteins, GST-fusion constructs were used in pull-down and co-immunoprecipitation assays with purified bovine brain Gi/Go and rat brain lysates. This identified a 74-amino-acid minimal binding domain that includes the G-protein activation sequence. This region of polycystin-1, including the G-protein activation peptide and flanking amino acid sequences, is highly conserved in mouse, human, and puffer fish, lending further support to the functional importance of the minimal binding domain. These results suggest that polycystin-1 may function as a heterotrimeric G-protein coupled receptor.