Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose a framework for binary protein-protein interaction (PPI) mapping based on optimally combining assays and/or assay versions to maximize detection of true positive interactions, while avoiding detection of random protein pairs. We have engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions, differing by protein expression systems and tagging configurations. The resulting union of N2H versions recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our findings should be applicable to systematic mapping of other biological landscapes.

The SRC Kinase Adaptor Phosphoprotein 2 (SKAP2) is a broadly expressed adaptor associated with the control of actin-polymerization, cell migration, and oncogenesis. After activation of different receptors at the cell surface, this dimeric protein serves as a platform for assembling other adaptors such as FYB and some SRC family kinase members, although these mechanisms are still poorly understood. The goal of this study is to map the SKAP2 interactome and characterize which domains or binding motifs are involved in these interactions. This is a prerequisite to finely analyze how these pathways are integrated in the cell machinery and to study their role in cancer and other human diseases when this network of interactions is perturbed. In this work, the domain and the binding motif of fourteen proteins interacting with SKAP2 were precisely defined and a new interactor, FAM102A was discovered. Herein, a fine-tuning between the binding of SRC kinases and their activation was identified. This last process, which depends on SKAP2 dimerization, indirectly affects the binding of FYB protein. Analysis of conformational changes associated with activation/inhibition of SRC family members, presently limited to their effect on kinase activity, is extended to their interactive network, which paves the way for therapeutic development.

We have sequenced to high levels of accuracy 714-kb and 233-kb regions of the mouse and bovine X-inactivation centers (Xic), respectively, centered on the Xist gene. This has provided the basis for a fully annotated comparative analysis of the mouse Xic with the 2.3-Mb orthologous region in human and has allowed a three-way species comparison of the core central region, including theXist gene. These comparisons have revealed conserved genes, both coding and noncoding, conserved CpG islands and, more surprisingly, conserved pseudogenes. The distribution of repeated elements, especially LINE repeats, in the mouse Xic region when compared to the rest of the genome does not support the hypothesis of a role for these repeat elements in the spreading of X inactivation. Interestingly, an asymmetric distribution of LINE elements on the two DNA strands was observed in the three species, not only within introns but also in intergenic regions. This feature is suggestive of important transcriptional activity within these intergenic regions. In silico prediction followed by experimental analysis has allowed four new genes, Cnbp2, Ftx, Jpx, and Ppnx, to be identified and novel, widespread, complex, and apparently noncoding transcriptional activity to be characterized in a region 5′ of Xist that was recently shown to attract histone modification early after the onset of X inactivation.[The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AJ421478, AJ421479, AJ421480, andAJ421481. Online supplemental data are available athttp://pbil.univ-lyon1.fr/datasets/Xic2002/data.html andwww.genome.org.]

After a pandemic wave in 2009 following their introduction in the human population, the H1N1pdm09 viruses replaced the previously circulating, pre-pandemic H1N1 virus and, along with H3N2 viruses, are now responsible for the seasonal influenza type A epidemics. So far, the evolutionary potential of influenza viruses has been mainly documented by consensus sequencing data. However, like other RNA viruses, influenza A viruses exist as a population of diverse, albeit related, viruses, or quasispecies. Interest in this quasispecies nature has increased with the development of next generation sequencing (NGS) technologies that allow a more in-depth study of the genetic variability. NGS deep sequencing methodologies were applied to determine the whole genome genetic heterogeneity of the three categories of influenza A viruses that circulated in humans between 2007 and 2012 in France, directly from clinical respiratory specimens. Mutation frequencies and single nucleotide polymorphisms were used for comparisons to address the level of natural intrinsic heterogeneity of influenza A viruses. Clear differences in single nucleotide polymorphism profiles between seasons for a given subtype also revealed the constant genetic drift that human influenza A virus quasispecies undergo.