Eukaryotic centromeres are defined by the presence of nucleosomes containing the histone H3 variant, centromere protein A (CENP-A). Once incorporated at centromeres, CENP-A nucleosomes are remarkably stable, exhibiting no detectable loss or exchange over many cell cycles. It is currently unclear whether this stability is an intrinsic property of CENP-A containing chromatin or whether it arises from proteins that specifically associate with CENP-A chromatin. Two proteins, CENP-C and CENP-N, are known to bind CENP-A human nucleosomes directly. Here we test the hypothesis that CENP-C or CENP-N stabilize CENP-A nucleosomes in vitro and in living cells. We show that CENP-N stabilizes CENP-A nucleosomes alone and additively with CENP-C in vitro. However, removal of CENP-C and CENP-N from cells, or mutating CENP-A so that it no longer interacts with CENP-C or CENP-N, had no effect on centromeric CENP-A stability in vivo. Thus, the stability of CENP-A nucleosomes in chromatin does not arise solely from its interactions with CENP-C or CENP-N.

The identification of factors that define adipocyte precursor potential has important implications for obesity. Preadipocytes are fibroblastoid cells committed to becoming round lipid-laden adipocytes. In vitro, this differentiation process is facilitated by confluency, followed by adipogenic stimuli. During adipogenesis, a large number of cytostructural genes are repressed before adipocyte gene induction. Here we report that the transcriptional repressor transcription factor 7-like 1 (TCF7L1) binds and directly regulates the expression of cell structure genes. Depletion of TCF7L1 inhibits differentiation, because TCF7L1 indirectly induces the adipogenic transcription factor peroxisome proliferator-activated receptor γ in a manner that can be replaced by inhibition of myosin II activity. TCF7L1 is induced by cell contact in adipogenic cell lines, and ectopic expression of TCF7L1 alleviates the confluency requirement for adipocytic differentiation of precursor cells. In contrast, TCF7L1 is not induced during confluency of non-adipogenic fibroblasts, and, remarkably, forced expression of TCF7L1 is sufficient to commit nonadipogenic fibroblasts to an adipogenic fate. These results establish TCF7L1 as a transcriptional hub coordinating cell-cell contact with the transcriptional repression required for adipogenic competency.A dipose tissue is a highly specialized compartment of cells actively involved in maintaining global metabolic homeostasis through lipid synthesis and storage, adipokine secretion, and insulin responsiveness (1). Adipocytes compose the majority of cells in adipose tissue and play a critical role in normal physiology, but their dysfunction is also at the center of a diverse range of diseases, including obesity, diabetes, and lipodystrophies (2). Furthermore, primary preadipocytes and adipose-derived stem cells have shown promise in treating multiple conditions (3-5). Therefore, it is critical to understand the process by which spindly fibroblastic precursor cells undergo conversion into round lipid-laden fat cells.In vitro models of adipogenesis, such as the extensively studied committed preadipocyte cell line 3T3-L1 cells, have elucidated two major phases of adipogenesis: commitment and terminal differentiation (6, 7). Terminal differentiation is characterized by the induction of metabolic genes, many of which are the direct targets of the transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and C/CAAT-binding protein (C/ EBP) α and β (8-14). Recent efforts have focused on identifying committed preadipocyte populations in vivo (15, 16), as well as on determining molecular factors that define the committed preadipocytes phenotype. Zinc finger protein 423 (Zfp423) is a critical preadipocyte factor upstream of PPARγ that is not present in non-adipogenic fibroblasts (17). However, Zfp423 also has been identified as a regulator of neurologic development (18), suggesting that other factors also may be involved in specifying adipogenic competency and commitment of precursor cells up...

Cell surface receptors are critical for cell signaling and constitute a quarter of all human genes. Despite their importance and abundance, receptor interaction networks remain understudied because of difficulties associated with maintaining membrane proteins in their native conformation and their typically weak interactions. To overcome these challenges, we developed an extracellular vesicle-based method for membrane protein display that enables purification-free and high-throughput detection of receptor–ligand interactions in membranes. We demonstrate that this platform is broadly applicable to a variety of membrane proteins, enabling enhanced detection of extracellular interactions over a wide range of binding affinities. We were able to recapitulate and expand the interactome for prominent members of the B7 family of immunoregulatory proteins such as PD-L1/CD274 and B7-H3/CD276. Moreover, when applied to the orphan cancer-associated fibroblast protein, LRRC15, we identified a membrane-dependent interaction with the tumor stroma marker TEM1/CD248. Furthermore, this platform enabled profiling of cellular receptors for target-expressing as well as endogenous extracellular vesicles. Overall, this study presents a sensitive and easy to use screening platform that bypasses membrane protein purification and enables characterization of interactomes for any cell surface–expressed target of interest in its native state.

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Technological improvements in unbiased screening have accelerated drug target discovery. In particular, membrane-embedded and secreted proteins have gained attention because of their ability to orchestrate intercellular communication. Dysregulation of their extracellular protein–protein interactions (ePPIs) underlies the initiation and progression of many human diseases. Practically, ePPIs are also accessible for modulation by therapeutics since they operate outside of the plasma membrane. Therefore, it is unsurprising that while these proteins make up about 30% of human genes, they encompass the majority of drug targets approved by the FDA. Even so, most secreted and membrane proteins remain uncharacterized in terms of binding partners and cellular functions. To address this, a number of approaches have been developed to overcome challenges associated with membrane protein biology and ePPI discovery. This chapter will cover recent advances that use high-throughput methods to move towards the generation of a comprehensive network of ePPIs in humans for future targeted drug discovery.