Cell division- and DNA replication-free reprogramming of somatic nuclei for embryonic transcription.

Nuclear transfer systems represent the efficient means to reprogram a cell and in theory provide a basis for investigating the development of endangered species. However, conventional nuclear transfer using oocytes of laboratory animals does not allow reprogramming of cross-species nuclei owing to defects in cell divisions and activation of embryonic genes. Here, we show that somatic nuclei transferred into mouse four-cell embryos arrested at the G2/M phase undergo reprogramming toward the embryonic state. Remarkably, genome-wide transcriptional reprogramming is induced within a day, and ZFP281 is important for this replication-free reprogramming. This system further enables transcriptional reprogramming of cells from Oryx dammah, now extinct in the wild. Thus, our findings indicate that arrested mouse embryos are competent to induce intra- and cross-species reprogramming. The direct induction of embryonic transcripts from diverse genomes paves a unique approach for identifying mechanisms of transcriptional reprogramming and genome activation from a diverse range of species.

A method for reverse interactome analysis: High-resolution mapping of interdomain interaction network in Dam1 complex and its specific disorganization based on the interaction domain expression.

An experimental methodology that facilitates functional analysis of numerous protein-protein interactions, which have been found in genome-wide interactome researches, has long been awaited. We propose herein an antagonistic inhibition-based approach. The antagonizing polypeptide is generated in the course of interaction domain mapping based on yeast 2-hybrid (Y2H) screening coupled with in vitro convergence of the Y2H-selected fragments, which is performed in a formatted procedure. Using the coupled methodology, we first performed a high-resolution mapping of an interdomain interaction network within budding yeast's Dam1 complex. Dam1 complex is a kinetochore protein complex composed of 10 essential subunits including Spc34p and Spc19p. The high-resolution mapping revealed the overall network structure within the complex for the first time: Dam1 components form into two separated subnetworks on N-terminal scaffolding domains of Spc34p and Spc19p, and the coiled-coil interaction in their C-terminal domains connects the subnetworks. Secondly, we show that the domain fragments converged in the high-resolution mapping acted as potent inhibitors for the endogenous interactions when episomally overexpressed. The in vivo Dam1 interaction targeting with the fragments conferred a similar phenotype on the host cells; a critical and irreversible damage, which was accompanied with disturbed budding and chromosome mis-segregation as a result of disorganized spindle. These phenotypes were strongly related to the cellular function of the Dam1 complex. The results and approach we demonstrated herein not only shed light on the Dam1 molecular architecture but also pave the road to reverse-interactome analysis and discoveries of novel drugs that target disease-related protein-protein interactions.

Quantitative Y2H screening: cloning and signal peptide engineering of a fungal secretory LacA gene and its application to yeast two-hybrid system as a quantitative reporter.

A quantitative protein/peptide screening system amenable to high-throughput screening has been developed by furnishing conventional yeast two-hybrid (Y2H) system with an engineered fungal secretory beta-galactosidase gene (designated LacA3). We describe the molecular cloning and signal peptide-optimization of the original fungal LacA gene of which extracellular expression was initially toxic to the host cell. The engineered LacA, LacA3, showed less toxicity, resulting in improved cultural properties of the host. The release of the enzyme to the medium was constant to the cell density under a certain induction condition and independent of the growth phase. The released enzyme kept the wild type properties, was highly glycosylated, stable in a wide pH range and high temperature, and had an acidic pH optimum. In the Y2H system with the novel reporter in combination with the conventional Y2H reporters, the yeast colonies are visibly stained in blue, white or red in the growth context, according to the interaction intensity. The clones with the more stable interactions are easily found as colonies with the larger blue halos, due to the increased extracellular LacA3 expression. A quantitative, high-throughput Y2H screening of cDNA library based on the novel reporter was demonstrated. An application of the novel Y2H system to directed evolution of a peptide fragment was also exemplified.