Lsr2 is a nucleoid-associated protein conserved throughout the actinobacteria, including the antibiotic-producing Streptomyces. Streptomyces species encode paralogous Lsr2 proteins (Lsr2 and Lsr2-like, or LsrL), and we show here that of the two, Lsr2 has greater functional significance. We found that Lsr2 binds AT-rich sequences throughout the chromosome, and broadly represses gene expression. Strikingly, specialized metabolic clusters were over-represented amongst its targets, and the cryptic nature of many of these clusters appears to stem from Lsr2-mediated repression. Manipulating Lsr2 activity in model species and uncharacterized isolates resulted in the production of new metabolites not seen in wild type strains. Our results suggest that the transcriptional silencing of biosynthetic clusters by Lsr2 may protect Streptomyces from the inappropriate expression of specialized metabolites, and provide global control over Streptomyces’ arsenal of signaling and antagonistic compounds.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

Background We identify and validate accurate diagnostic biomarkers for prostate cancer through a systematic evaluation of DNA methylation alterations. Materials and methods We assembled three early prostate cancer cohorts (total patients = 699) from which we collected and processed over 1300 prostatectomy tissue samples for DNA extraction. Using real‐time methylation‐specific PCR, we measured normalized methylation levels at 15 frequently methylated loci. After partitioning sample sets into independent training and validation cohorts, classifiers were developed using logistic regression, analyzed, and validated. Results In the training dataset, DNA methylation levels at 7 of 15 genomic loci (glutathione S‐transferase Pi 1 [GSTP1], CCDC181, hyaluronan, and proteoglycan link protein 3 [HAPLN3], GSTM2, growth arrest‐specific 6 [GAS6], RASSF1, and APC) showed large differences between cancer and benign samples. The best binary classifier was the GAS6/GSTP1/HAPLN3 logistic regression model, with an area under these curves of 0.97, which showed a sensitivity of 94%, and a specificity of 93% after external validation. Conclusion We created and validated a multigene model for the classification of benign and malignant prostate tissue. With false positive and negative rates below 7%, this three‐gene biomarker represents a promising basis for more accurate prostate cancer diagnosis.

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1Lsr2 is a nucleoid-associated protein conserved throughout the actinobacteria, including the antibiotic-2 producing Streptomyces. Streptomyces species encode paralogous Lsr2 proteins or 3 LsrL), and we show here that of the two, Lsr2 has greater functional significance. We found that Lsr2 4 binds AT-rich sequences throughout the chromosome, and broadly represses gene expression. 5 Strikingly, specialized metabolic clusters were over-represented amongst its targets, and the cryptic 6 nature of many of these clusters appears to stem from Lsr2-mediated repression. Manipulating Lsr2 7 activity in model species and uncharacterized isolates resulted in the production of new metabolites not 8 seen in wild type strains. Our results suggest that the transcriptional silencing of biosynthetic clusters by 9Lsr2 may protect Streptomyces from the inappropriate expression of specialized metabolites, and 10 provide global control over Streptomyces' arsenal of signalling and antagonistic compounds. 11 12 Interestingly, however, most clusters are poorly expressed under normal laboratory conditions, and in 54 many cases their associated metabolites remain uncharacterized. This is also the case for the 55 filamentous fungi, many of whom have a broad, untapped specialized metabolic repertoire, courtesy of 56 transcriptional silencing by histones (Pfannenstiel and Keller, 2019). Significant efforts are being made to 57 stimulate the production of these 'cryptic' metabolites in both bacteria and fungi, as they are widely 58 regarded as productive sources of new natural products (Craney et al., 2013;Ochi and Hosaka, 2013; 59 Scharf and Brakhage, 2013;Yoon and Nodwell, 2014; Daniel-Ivad et al., 2017;Onaka, 2017). 60We sought to investigate the role of the nucleoid-associated proteins Lsr2 and LsrL in gene 61 regulation in Streptomyces. We found that deleting lsr2 from the chromosome of Streptomyces 62 venezuelae had minor effects on S. venezuelae growth and development and major effects on 63 metabolism. In contrast, deleting lsrL had no detectable impact on development, and only a minor effect 64 on metabolism. Focussing on Lsr2, we determined that it bound AT-rich regions, generally repressed the 65 expression of prophage genes and other genes unique to S. venezuelae (presumably acquired by lateral 66 gene transfer), and suppressed antisense gene expression. The most profound effect of lsr2 deletion, 67 however, was the large-scale activation of specialized metabolic cluster gene expression. Lsr2 directly 68 repressed the transcription of many cryptic clusters in a way that is analogous to Lsr2-and H-NS-69 mediated repression of pathogenicity islands in other bacteria, and histone-mediated cluster silencing in 70 fungi. Unexpectedly, Lsr2 also controlled the expression of well-characterized and highly-conserved 71 clusters, suggesting that Lsr2 control has been broadly integrated into the regulatory cascades governing 72 specialized metabolism. Our results suggest that Lsr2 functions as a metabolic gatekeeper in the 73 strepto...