Pan-SMARCA/PB1 Bromodomain Inhibitors and Their Role in Regulating Adipogenesis.

Accessibility of the human genome is modulated by the ATP-driven SWI/SNF chromatin remodeling multiprotein complexes BAF (BRG1/BRM-associated factor) and PBAF (polybromo-associated BAF factor), which involves reading of acetylated histone tails by the bromodomain-containing proteins SMARCA2 (BRM), SMARCA4 (BRG1), and polybromo-1. Dysregulation of chromatin remodeling leads to aberrant cell proliferation and differentiation. Here, we have characterized a set of potent and cell-active bromodomain inhibitors with pan-selectivity for canonical family VIII bromodomains. Targeted SWI/SNF bromodomain inhibition blocked the expression of key genes during adipogenesis, including the transcription factors PPARγ and C/EBPα, and impaired the differentiation of 3T3-L1 murine fibroblasts into adipocytes. Our data highlight the role of SWI/SNF bromodomains in adipogenesis and provide a framework for the development of SWI/SNF bromodomain inhibitors for indirect targeting of key transcription factors regulating cell differentiation.

Promoter Activation in Δhfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct Bioactivity Testing.

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.

Cyclo(tetrahydroxybutyrate) production is sufficient to distinguish between Xenorhabdus and Photorhabdus isolates in Thailand.

Bacteria of the genera Photorhabdus and Xenorhabdus produce a plethora of natural products to support their similar symbiotic life cycles. For many of these compounds, the specific bioactivities are unknown. One common challenge in natural product research when trying to prioritize research efforts is the rediscovery of identical (or highly similar) compounds from different strains. Linking genome sequence to metabolite production can help in overcoming this problem. However, sequences are typically not available for entire collections of organisms. Here, we perform a comprehensive metabolic screening using HPLC-MS data associated with a 114-strain collection (58 Photorhabdus and 56 Xenorhabdus) across Thailand and explore the metabolic variation among the strains, matched with several abiotic factors. We utilize machine learning in order to rank the importance of individual metabolites in determining all given metadata. With this approach, we were able to prioritize metabolites in the context of natural product investigations, leading to the identification of previously unknown compounds. The top three highest ranking features were associated with Xenorhabdus and attributed to the same chemical entity, cyclo(tetrahydroxybutyrate). This work also addresses the need for prioritization in high-throughput metabolomic studies and demonstrates the viability of such an approach in future research.

Natural product diversity associated with the nematode symbionts Photorhabdus and Xenorhabdus.

Xenorhabdus and Photorhabdus species dedicate a large amount of resources to the production of specialized metabolites derived from non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS). Both bacteria undergo symbiosis with nematodes, which is followed by an insect pathogenic phase. So far, the molecular basis of this tripartite relationship and the exact roles that individual metabolites and metabolic pathways play have not been well understood. To close this gap, we have significantly expanded the database for comparative genomics studies in these bacteria. Clustering the genes encoded in the individual genomes into hierarchical orthologous groups reveals a high-resolution picture of functional evolution in this clade. It identifies groups of genes-many of which are involved in secondary metabolite production-that may account for the niche specificity of these bacteria. Photorhabdus and Xenorhabdus appear very similar at the DNA sequence level, which indicates their close evolutionary relationship. Yet, high-resolution mass spectrometry analyses reveal a huge chemical diversity in the two taxa. Molecular network reconstruction identified a large number of previously unidentified metabolite classes, including the xefoampeptides and tilivalline. Here, we apply genomic and metabolomic methods in a complementary manner to identify and elucidate additional classes of natural products. We also highlight the ability to rapidly and simultaneously identify potentially interesting bioactive products from NRPSs and PKSs, thereby augmenting the contribution of molecular biology techniques to the acceleration of natural product discovery.