A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex.

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.

Microdissected tumor cuboids: a microscale cancer model that retains a complex tumor microenvironment.

Present cancer disease models - typically based on cell cultures and animal models that lack the human tumor microenvironment (TME) - are extremely poor predictors of human disease outcomes. Microscale cancer models that combine the micromanipulation of tissues and fluids offer the exciting possibility of miniaturizing the drug testing workflow, enabling inexpensive, more efficient tests of high clinical biomimicry that maximize the use of scarce human tissue and minimize animal testing. Critically, these microscale models allow for precisely addressing the impact of the structural features of the heterogeneous TME to properly target and understand the contributions of these unique zones to therapeutic response. We have recently developed a precision slicing method that yields large numbers of cuboidal micro-tissues ("cuboids", ∼ (400 µm) 3 ) from a single tumor biopsy. Here we evaluate cuboids from syngeneic mouse tumor models and human tumors, which contain native immune cells, as models for drug and immunotherapy evaluation. We characterize relevant TME parameters, such as their cellular architecture (immune cells and vasculature), cytokine secretion, proteomics profiles, and their response to drug panels in multi-well arrays. Despite the cutting procedure and the time spent in culture (up to 7 days), the cuboids display strong functional responses such as cytokine and drug responses. Overall, our results suggest that cuboids make an excellent model for applications that require the TME, such as immunotherapy drug evaluations, including for clinical trials and personalized oncology approaches.

Efficient and economic protein labeling for NMR in mammalian expression systems: Application to a preT-cell and T-cell receptor protein.

Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the ß subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 µM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.