Gene expression is highly regulated at the step of transcription initiation, and transcription activators play a critical role in this process. RbpA, an actinobacterial transcription activator that is essential in Mycobacterium tuberculosis (Mtb), binds selectively to group 1 and certain group 2 σ-factors. To delineate the molecular mechanism of RbpA, we show that the Mtb RbpA σ-interacting domain (SID) and basic linker are sufficient for transcription activation. We also present the crystal structure of the Mtb RbpA-SID in complex with domain 2 of the housekeeping σ-factor, σ A . The structure explains the basis of σ-selectivity by RbpA, showing that RbpA interacts with conserved regions of σ A as well as the nonconserved region (NCR), which is present only in housekeeping σ-factors. Thus, the structure is the first, to our knowledge, to show a protein interacting with the NCR of a σ-factor. We confirm the basis of selectivity and the observed interactions using mutagenesis and functional studies. In addition, the structure allows for a model of the RbpA-SID in the context of a transcription initiation complex. Unexpectedly, the structural modeling suggests that RbpA contacts the promoter DNA, and we present in vivo and in vitro studies supporting this finding. Our combined data lead to a better understanding of the mechanism of RbpA function as a transcription activator.RbpA | X-ray crystallography | transcription | actinobacteria | mycobacteria

SummaryExtracellular signals are transduced to the cell nucleus by effectors that bind to enhancer complexes to operate transcriptional switches. For example, the Wnt enhanceosome is a multiprotein complex associated with Wnt-responsive enhancers through T cell factors (TCF) and kept silent by Groucho/TLE co-repressors. Wnt-activated β-catenin binds to TCF to overcome this repression, but how it achieves this is unknown. Here, we discover that this process depends on the HECT E3 ubiquitin ligase Hyd/UBR5, which is required for Wnt signal responses in Drosophila and human cell lines downstream of activated Armadillo/β-catenin. We identify Groucho/TLE as a functionally relevant substrate, whose ubiquitylation by UBR5 is induced by Wnt signaling and conferred by β-catenin. Inactivation of TLE by UBR5-dependent ubiquitylation also involves VCP/p97, an AAA ATPase regulating the folding of various cellular substrates including ubiquitylated chromatin proteins. Thus, Groucho/TLE ubiquitylation by Hyd/UBR5 is a key prerequisite that enables Armadillo/β-catenin to activate transcription.

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Cultured meat is an emerging technology with the potential to solve huge challenges related to the environmental, ethical, and health implications of conventional meat production. Establishing the basic science of cultured meat has been the primary focus of the last decade but it is now feasible that cultured meat products will enter the market within the next 3 to 4 years. This proximity to market introduction demands an evaluation of aspects of the cultured meat production process that have not yet been outlined or discussed in significant detail. For example, one technological approach for the production of cultured meat uses adult muscle stem cells, the limited proliferative capacity of which necessitates repeated collection of tissue samples via biopsies of living donor animals. The selection of donor animals and the details of biopsy processes must be optimized, as this is a key bottleneck in the cultured meat production process. The number of stem cells harvested from a biopsy, together with their proliferative capacity, determines a ‘multiplicity factor’ achieved by a cultured meat production process, thus dictating the reduction in number of animals required to produce a given quantity of meat. This article considers potential scenarios for these critical upstream steps, focusing on the production of cultured beef as an example. Considerations related to donor selection and details of the biopsy process are discussed in detail. The practicalities of various scenarios for cultured beef production, the health of donor animals, and regulatory issues associated with the safety of cultured meat for consumers are also considered. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Cultured meat is an emergent technology with the potential for significant environmental and animal welfare benefits. Accurate mimicry of traditional meat requires fat tissue; a key contributor to both the flavour and texture of meat. Here, we show that fibro-adipogenic progenitor cells (FAPs) are present in bovine muscle, and are transcriptionally and immunophenotypically distinct from satellite cells. These two cell types can be purified from a single muscle sample using a simple fluorescence-activated cell sorting (FACS) strategy. FAPs demonstrate high levels of adipogenic potential, as measured by gene expression changes and lipid accumulation, and can be proliferated for a large number of population doublings, demonstrating their suitability for a scalable cultured meat production process. Crucially, FAPs reach a mature level of adipogenic differentiation in three-dimensional, edible hydrogels. The resultant tissue accurately mimics traditional beef fat in terms of lipid profile and taste, and FAPs thus represent a promising candidate cell type for the production of cultured fat.