Genomic analyses are yielding a host of new information on the multiple genetic abnormalities associated with specific types of cancer. A comprehensive description of cancer-associated genetic abnormalities can improve our ability to classify tumors into clinically relevant subgroups, and, on occasion, identify mutant genes that drive the cancer phenotype (“drivers”). More often, though, the functional significance of cancer-associated mutations is difficult to discern. Genome-wide pooled shRNA screens enable global identification of the genes essential for cancer cell survival and proliferation, providing a “functional genomic” map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting ~16,000 genes and a newly developed, dynamic scoring approach, we identified essential gene profiles in 72 breast, pancreatic, and ovarian cancer cell lines. Integrating our results with current and future genomic data should facilitate the systematic identification of drivers, unanticipated synthetic lethal relationships, and functional vulnerabilities of these tumor types.

The ubiquitin system regulates virtually all aspects of cellular function. We report a method to target the myriad enzymes that govern ubiquitination of protein substrates. We used massively diverse combinatorial libraries of ubiquitin variants to develop inhibitors of four deubiquitinases (DUBs) and analyzed the DUB-inhibitor complexes with crystallography. We extended the selection strategy to the ubiquitin conjugating (E2) and ubiquitin ligase (E3) enzymes and found that ubiquitin variants can also enhance enzyme activity. Last, we showed that ubiquitin variants can bind selectively to ubiquitin-binding domains. Ubiquitin variants exhibit selective function in cells and thus enable orthogonal modulation of specific enzymatic steps in the ubiquitin system.

Highlights d Pdgfra-expressing mesenchymal cells from uninjured digits establish the blastema d Adult digit tip regeneration is distinct from embryonic digit development d The blastema state is environmentally determined d The regenerative environment enables mesenchymal lineage plasticity

Peripheral nerves provide a supportive growth environment for developing and regenerating axons and are essential for maintenance and repair of many non-neural tissues. This capacity has largely been ascribed to paracrine factors secreted by nerve-resident Schwann cells. Here, we used single-cell transcriptional profiling to identify ligands made by different injured rodent nerve cell types and have combined this with cell-surface mass spectrometry to computationally model potential paracrine interactions with peripheral neurons. These analyses show that peripheral nerves make many ligands predicted to act on peripheral and CNS neurons, including known and previously uncharacterized ligands. While Schwann cells are an important ligand source within injured nerves, more than half of the predicted ligands are made by nerve-resident mesenchymal cells, including the endoneurial cells most closely associated with peripheral axons. At least three of these mesenchymal ligands, ANGPT1, CCL11, and VEGFC, promote growth when locally applied on sympathetic axons. These data therefore identify an unexpected paracrine role for nerve mesenchymal cells and suggest that multiple cell types contribute to creating a highly pro-growth environment for peripheral axons.

The bone morphogenetic protein (BMP) and growth and differentiation factor (GDF) signaling pathways have well-established and essential roles within the developing skeleton in coordinating the formation of cartilaginous anlagen. However, the identification of bona fide targets that underlie the action of these signaling molecules in chondrogenesis has remained elusive. We have identified the gene for the retinoic acid (RA) synthesis enzyme Aldh1a2 as a principal target of BMP signaling; prochondrogenic BMPs or GDFs lead to attenuation of Aldh1a2 expression and, consequently, to reduced activation of the retinoid signaling pathway. Consistent with this, antagonism of retinoid signaling phenocopies BMP4 action, whereas RA inhibits the chondrogenic stimulatory activity of BMP4. BMP4 also down-regulates Aldh1a2 expression in organ culture and, consistent with this, Aldh1a2 is actively excluded from the developing cartilage anlagens. Collectively, these findings provide novel insights into BMP action and demonstrate that BMP signaling governs the fate of prechondrogenic mesenchyme, at least in part, through regulation of retinoid signaling.

Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N-and C-terminal tags and multiple promoters), is compatible with Gateway TM cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming. Molecular & Cellular Proteomics 9:811-823, 2010.The analysis of protein-protein interactions (PPIs) 1 and protein complexes is of central importance to biological research and facilitates our understanding of how molecular events drive phenotypic outcomes. Moreover, large scale protein interaction data can be used to generate protein interaction networks, which can then be used to predict disease genes and model biology in any living organism.A number of methods (e.g. yeast two-hybrid) have been developed to examine binary protein interactions in a systematic format and applied to model systems (1-8). However, affinity purification (AP) coupled with tandem MS has become the method of choice for the identification of protein complexes (9, 10). Large scale PPI studies using a high throughput and systematic AP-MS approach have been performed for Escherichia coli (11,12) and Saccharomyces cerevisiae (13-15). In fact, large scale efforts using AP-MS have connected an estimated 60% of the yeast proteome, demonstrating the power of coupling systematic biochemical purifications with mass spectrometry (13-16).AP-MS has also been used extensively for purification of mammalian protein complexes (17), but this has been mostly restricted to small scale studies and the use of either cell lines that are easy to transfect or highly validated antibodies against specific targets. For example, Glatter et al. (18) recently developed an integrated workflow where a high density interactome was developed for the protein phosphatase 2A complex. This workflow relies on "flip-in" technology to introduce transgenes into a common genomic site in HEK293 cells and, similar to work by other groups (19,20), utilizes an From the ‡Banting and Best

No abstract

SummaryIn the developing axial skeleton, sequential sonic hedgehog (SHH) and bone morphogenetic protein (BMP) signals are required for specification of a chondrogenic fate in presomitic tissue. A similar paradigm is thought to operate in the limb, but the signals involved are unclear. To investigate the nature of these signals, we examined BMP action in mesenchymal populations derived from the early murine limb bud (approximately embryonic day 10.5). These populations exhibited a graded response to BMPs, in which early limb mesenchymal cells (from the distal hind limb) displayed an anti-chondrogenic response, whereas BMPs promoted chondrogenesis in more mature cell populations (from the proximal fore limb). Under these conditions, multiple Gata genes were induced by BMPs and the extent of induction correlated with BMP anti-chondrogenic activity. A screen of limb-bud-expressed ligands revealed that prior short-term exposure to transforming growth factor 1 (TGF1) ameliorated the anti-chondrogenic response to BMP. Furthermore, brief activation of the TGF pathway was found to be necessary for subsequent induction of chondrogenesis by BMPs. Our findings indicate that, similar to axial skeletogenesis, induction of chondrogenesis in the appendicular skeleton is a two-step process. However, the programs differ in the transient signals driving chondrogenic responsiveness to BMPs, with SHH operating in the former and TGF activation in the latter.