Project description:The protein kinase ERK2 is recurrently mutated in human squamous cell carcinomas and other tumors. ERK2 mutations cluster in an essential docking recruitment site that interacts with short linear motifs found within intrinsically disordered regions of ERK substrates and regulators. Cancer-associated mutations do not disrupt ERK2 docking interactions altogether but selectively inhibit some interactions while sparing others. However, the full scope of disrupted or maintained interactions remains unknown, limiting our understanding of how these mutations contribute to cancer. We recently defined the docking interactome of wild-type ERK2 by screening a yeast two-hybrid library of proteomic short linear motifs. Here, we apply this approach to the two most recurrent cancer-associated mutants. We find that most sequences binding to WT ERK2 also interact with both mutant forms. Analysis of differentially interacting sequences revealed that ERK2 mutants selectively lose the ability to bind sequences conforming to a specific motif. We solved the co-crystal structure of ERK2 in complex with a peptide fragment of ISG20, a screening hit that binds exclusively to the WT kinase. This co-crystal structure demonstrates the mechanism by which cancer hotspot mutations at Glu81, Arg135, Asp321, and Glu322 selectively impact peptide binding. Finally, we found that cancer-associated ERK2 mutations had a decreased ability to phosphorylate GEF-H1/ARHGEF2, a known ERK substrate harboring a WT-selective docking motif. Collectively, our studies provide a structural rationale for how a broad set of interactions are disrupted by ERK2 hotspot mutations, suggesting mechanisms for pathway rewiring in cancers harboring these mutations.
Project description:Cell state-specific gene expression programs emerge from the interplay between cis-regulatory elements (CREs), such as enhancers, and transcription factors (TFs). Massively parallel reporter assays (MPRAs) have enabled large-scale dissection of CRE function, but bulk approaches cannot resolve cell state specificity on continuous trajectories of cellular differentiation, and existing single-cell MPRAs are not readily applicable to primary cell differentiation models. Here, we developed a single-cell lentiviral Massively Parallel Reporter Assay (sc-lentiMPRA) that overcomes these limitations and enables parallel quantification of enhancer activity and cellular transcriptome. Applying sc-lentiMPRA in blood stem differentiation, we profiled the activity and specificity of ~160 fully synthetic enhancers with controlled motif composition and affinities across ~190,000 single cells. Focusing on Trp53 and Cebpa, we show that enhancers with high and low affinity motifs differ qualitatively and quantitatively in their responses to TF expression gradients. For Trp53, low-affinity motifs exhibited near-linear correlation with TF expression, whereas high-affinity motifs showed reduced sensitivity to TF levels and a potential contribution of cofactor availability. In contrast, Cebpa-associated enhancers displayed non-linear behaviors. Together, sc-lentiMPRA establishes a powerful framework for systematically relating enhancer architecture and TF expression to regulatory output at single-cell resolution during cellular differentiation.
Project description:Specific interactions between proteins and their binding partners are fundamental to life processes. Differential protein footprinting using a new and efficient, photo-activated aryltrifluromethylcarbene probe together with mass spectrometry has been employed to identify protein-ligand and protein-protein interaction sites. In a model protein-small molecule system, the location of a penta-N-acetylchitopentaose carbohydrate substrate was accurately mapped to the binding cleft of lysozyme. As a fine detail, footprinting revealed disruption of an intramolecular hydrogen-bond network within lysozyme, which is known to be associated with substrate binding. In a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5, and a diubiquitin substrate were located to different functional domains thus clarifying uncertainties from an X-ray crystal structure of USP5. The much improved properties of this bespoke diazirine probe make differential carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.
Project description:Cell state-specific gene expression programs emerge from the interplay between cis-regulatory elements (CREs), such as enhancers, and transcription factors (TFs). Massively parallel reporter assays (MPRAs) have enabled large-scale dissection of CRE function, but bulk approaches cannot resolve cell state specificity on continuous trajectories of cellular differentiation, and existing single-cell MPRAs are not readily applicable to primary cell differentiation models. Here, we developed a single-cell lentiviral Massively Parallel Reporter Assay (sc-lentiMPRA) that overcomes these limitations and enables parallel quantification of enhancer activity and cellular transcriptome. Applying sc-lentiMPRA in blood stem differentiation, we profiled the activity and specificity of ~160 fully synthetic enhancers with controlled motif composition and affinities across ~190,000 single cells. Focusing on Trp53 and Cebpa, we show that enhancers with high and low affinity motifs differ qualitatively and quantitatively in their responses to TF expression gradients. For Trp53, low-affinity motifs exhibited near-linear correlation with TF expression, whereas high-affinity motifs showed reduced sensitivity to TF levels and a potential contribution of cofactor availability. In contrast, Cebpa-associated enhancers displayed non-linear behaviors. Together, sc-lentiMPRA establishes a powerful framework for systematically relating enhancer architecture and TF expression to regulatory output at single-cell resolution during cellular differentiation.