Many transcription factors contain proline- or glutamine-rich activation domains. Here it is shown that simple homopolymeric stretches of these amino acids can activate transcription when fused to the DNA binding domain of GAL4 factor. In vitro, activity increased with polymer length, whereas in cell transfection assays maximal activity was achieved by 10 to 30 glutamines or about 10 prolines. Similar results were obtained when glutamine stretches were placed within a [GAL4]-VP16 chimeric protein. Because these stretches are encoded by rapidly evolving triplet repeats (microsatellites), they may be the main cause for modulation of transcription factor activity and thus result in subtle or overt genomic effects.

Rho-like GTP binding proteins play an essential role in regulating cell growth and actin polymerization. These molecular switches are positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP. Using the interaction-trap assay to identify candidate proteins that bind the cytoplasmic region of the LAR transmembrane protein tyrosine phosphatase (PT-Pase), we isolated a cDNA encoding a 2861-amino acid protein termed Trio that contains three enzyme domains: two functional GEF domains and a protein serine/threonine kinase (PSK) domain. One of the Trio GEF domains (Trio GEF-D1) has rac-specific GEF activity, while the other Trio GEF domain (Trio GEF-D2) has rho-specific activity. The C-terminal PSK domain is adjacent to an Ig-like domain and is most similar to calcium/calmodulin-dependent kinases, such as smooth muscle myosin light chain kinase which similarly contains associated Ig-like domains. Near the N terminus, Trio has four spectrin-like repeats that may play a role in intracellular targeting. Northern blot analysis indicates that Trio has a broad tissue distribution. Trio appears to be phosphorylated only on serine residues, suggesting that Trio is not a LAR substrate, but rather that it forms a complex with LAR. As the LAR PTPase localizes to the ends of focal adhesions, we propose that LAR and the Trio GEF/PSK may orchestrate cell-matrix and cytoskeletal rearrangements necessary for cell migration.

Dbl-homology guanine nucleotide exchange factors (DH-GEFs) regulate actin cytoskeletal reorganization, cell adhesion, and gene transcription via activation of Rho GTPases. However, little is known about the physiological role of mammalian DH-GEFs during development. The DH-GEF family member Trio is of particular interest because it is a multifunctional protein possessing two GEF domains, as well as a protein serine/threonine kinase domain, and trio-like genes in Caenorhabditis elegans and Drosophila were shown to function in neural migration and axon guidance. To determine the role of Trio during mammalian development, we generated a mouse trio loss-of-function mutation ( trio ^−/− ). Trio function is essential during late embryonic development as genotype analysis indicated that trio ^−/− embryos died between embryonic day (E)-15.5 and birth, or shortly thereafter. In the trio ^−/− embryos, primary skeletal myofibers were relatively normal at E14.5, but by E18.5 highly unusual spherical myofibers accumulated. Trio deficiency may cause a defect in secondary myogenesis, as the appearance of the abnormal trio ^−/− skeletal myofibers temporally coincided with the onset of secondary myogenesis, and smaller secondary myofibers located adjacent to the primary myofibers were absent. The proliferation of trio ^−/− secondary myoblasts appeared normal, suggesting that Trio may regulate secondary myoblast alignment or fusion. trio ^−/− embryos also displayed aberrant organization in several regions within the brain, including the hippocampal formation and olfactory bulb. We thus conclude that Trio is essential for late embryonic development, and that Trio functions in fetal skeletal muscle formation and in the organization of neural tissues.

In most animal phyla from insects to mammals, there is a clear division of somatic and germ line cells. This is however not the case in plants and some animal phyla including tunicates, flatworms and the basal phylum Cnidaria, where germ stem cells arise de novo from somatic cells. Piwi-like genes represent essential stem cell genes in diverse multicellular organisms. The cnidarian Piwihomolog Cniwiwas cloned from Podocoryne carnea, a hydrozoan with a full life cycle. CniwiRNA is present in all developmental stages with highest levels in the egg and the medusa. In the adult medusa, Cniwi expression is prominent in the gonads where it likely functions as a germ stem cell gene. The gene is also expressed, albeit at low levels, in differentiated somatic cells like the striated muscle of the medusa. Isolated striated muscle cells can be induced to transdifferentiate into smooth muscle cells which proliferate and differentiate into nerve cells. Cniwi expression is upregulated transiently after induction of transdifferentiation and again when the emerging smooth muscle cells proliferate and differentiate. The continuous low-level expression of an inducible stem cell gene in differentiated somatic cells may underlie the ability to form medusa buds from polyp cells and explain the extraordinary transdifferentation and regeneration potential of Podocoryne carnea.