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Proximity biotinylation is a commonly used method to identify the in vivo proximal proteome for proteins of interest. This technology typically relies on fusing a bait protein to a biotin ligase using overexpression or clustered regularly interspaced short palindromic repeats (CRISPR)-based tagging, thus prohibiting the use of such assays in cell types that are difficult to transfect or transduce. We recently developed an 'off-the-shelf' proximity biotinylation method that makes use of a recombinant enzyme consisting of the biotin ligase TurboID fused to the antibody-recognizing moiety Protein A. In this method, a bait-specific antibody and the ProteinA-Turbo enzyme are consecutively added to permeabilized fixed or unfixed cells. Following incubation, during which ProteinA-Turbo antibody-antigen complexes are formed, unbound molecules are washed away, after which bait-proximal biotinylation is triggered by the addition of exogenous biotin. Finally, biotinylated proteins are enriched from crude lysates using streptavidin beads followed by mass spectrometry-based protein identification. In principle, any scientist can perform this protocol within 3 days, although generating the proteomics data requires access to a high-end liquid chromatography-mass spectrometry setup. Data analysis and data visualization are relatively straightforward and can be performed using any type of software that converts raw mass spectrometry spectra files into identified and quantified proteins. The protocol has been optimized for nuclear targets but may also be adapted to other subcellular regions of interest.

Many enhancers play a crucial role in regulating gene expression by assembling regulatory factor (RF) clusters, also referred to as condensates. This process is essential for facilitating enhancer communication and establishing cellular identity. However, how DNA sequence and transcription factor (TF) binding instruct the formation of such high RF environments is still poorly understood. To address this, we developed a novel approach leveraging enhancer-centric chromatin accessibility quantitative trait loci (caQTLs) to nominate RF clusters genome-wide. By analyzing TF binding signatures within the context of caQTLs, we discovered a new class of TFs that specifically contributes to establishing cooperative environments. These “context-only” TFs bind promiscuously with cell type-specific pioneers, recruit coactivators, and, like super enhancers, render downstream gene expression sensitive to condensate-disrupting molecules. We further demonstrate that joint context-only and pioneer TF binding explains enhancer compatibility and provides a mechanistic rationale for how a loose TF syntax can still confer regulatory specificity.

Adipose tissue plasticity is orchestrated by molecularly and functionally diverse cells within the stromal vascular fraction (SVF). While several mouse and human adipose SVF cellular subpopulations have now been identified, we still lack an understanding of the cellular and functional variability of adipose stem and progenitor cell (ASPC) populations across human fat depots. To address this, we performed single-cell and bulk RNA-seq analyses of >30 Lin–SVF samples across four human adipose depots, revealing two ubiquitous hASPC subpopulations with distinct proliferative and adipogenic properties but also depot- and BMI-dependent proportions. Furthermore, we identified an omental-specific, highIGFBP2-expressing stromal population that transitions between mesothelial and mesenchymal cell states and inhibits hASPC adipogenesis through IGFBP2 secretion. Our analyses highlight the molecular and cellular uniqueness of different adipose niches while our discovery of an anti-adipogenic IGFBP2+ omental-specific population provides a new rationale for the biomedically relevant, limited adipogenic capacity of omental hASPCs.

Akkermansia muciniphila is considered a key constituent of a healthy gut microbiota. In inflammatory bowel disease (IBD), A. muciniphila has a reduced abundance while other, putative pathogenic, mucus colonizers bloom. We hypothesized that interbacterial competition may contribute to this observation. By screening the supernatants of a panel of enteric bacteria, we discovered that a previously uncharacterized Allobaculum species potently inhibits the growth of A. muciniphila. Mass spectrometry analysis identified a secreted Allobaculum sialidase as inhibitor of A. muciniphila growth. The sialidase targets sialic acids on casein O-glycans, thereby altering the accessibility of nutrients critical for A. muciniphila. The altered glycometabolic niche results in distorted A. muciniphila cell division and efficiently arrests its growth. The identification of a novel mechanism of A. muciniphila growth inhibition by a competing bacterial pathobiont may provide a rationale for interventions aimed at restoring and maintaining a healthy microbiota symbiosis in patients with intestinal disease.

Cell cycle transitions arise from collective changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs), but conceptual and mechanistic explanations for the abrupt cellular reorganisation that occurs upon mitotic entry are lacking. Specific interactions between distinct CDK-cyclin complexes and sequence motifs encoded in substrates might result in highly ordered phosphorylation1, while bistability in the mitotic CDK1 control network can trigger switch-like phosphorylation2. Yet the dynamics of mitotic phosphorylation has not been demonstrated in vivo, and the roles of most cell cycle-regulated phosphorylations are unclear. Here, we show evidence that switch-like phosphorylation of intrinsically disordered proteins (IDPs) by CDKs contributes to mitotic cellular reorganisation by controlling protein-protein interactions and phase separation. We studied protein phosphorylation in single Xenopus embryos throughout synchronous cell cycles, performed parallel assignment of cell cycle phases using egg extracts, and analysed dynamics of mitotic phosphorylation using quantitative targeted phosphoproteomics. This provided a high-resolution map of dynamic phosphosites from the egg to the 16-cell embryo and showed that mitotic phosphorylation occurs on entire protein complexes involved in diverse subcellular processes and is switch-like in vivo. Most cell cycle-regulated phosphosites occurred in CDK consensus motifs and located to intrinsically disordered regions. We found that substrates of CDKs and other cell cycle kinases are significantly more disordered than phosphoproteins in general, a principle conserved from yeast to humans, while around half are components of membraneless organelles (MLOs), whose assembly is thought to involve phase separation. Analytical modelling predicts modulation of homotypic IDP interactions by CDK-mediated phosphorylation, which was confirmed by biophysical and biochemical analysis of a model IDP, Ki-67. These results highlight the dynamic control of intrinsic disorder as a conserved hallmark of the cell cycle and suggest a mechanism for CDK-mediated mitotic cellular reorganisation.