Nutritional inter-dependencies and a carbazole-dioxygenase are key elements of a bacterial consortium relying on a Sphingomonas for the degradation of the fungicide thiabendazole.

Thiabendazole (TBZ), is a persistent fungicide/anthelminthic and a serious environmental threat. We previously enriched a TBZ-degrading bacterial consortium and provided first evidence for a Sphingomonas involvement in TBZ transformation. Here, using a multi-omic approach combined with DNA-stable isotope probing (SIP) we verified the key degrading role of Sphingomonas and identify potential microbial interactions governing consortium functioning. SIP and amplicon sequencing analysis of the heavy and light DNA fraction of cultures grown on 13 C-labelled versus 12 C-TBZ showed that 66% of the 13 C-labelled TBZ was assimilated by Sphingomonas. Metagenomic analysis retrieved 18 metagenome-assembled genomes with the dominant belonging to Sphingomonas, Sinobacteriaceae, Bradyrhizobium, Filimonas and Hydrogenophaga. Meta-transcriptomics/-proteomics and non-target mass spectrometry suggested TBZ transformation by Sphingomonas via initial cleavage by a carbazole dioxygenase (car) to thiazole-4-carboxamidine (terminal compound) and catechol or a cleaved benzyl ring derivative, further transformed through an ortho-cleavage (cat) pathway. Microbial co-occurrence and gene expression networks suggested strong interactions between Sphingomonas and a Hydrogenophaga. The latter activated its cobalamin biosynthetic pathway and Sphingomonas its cobalamin salvage pathway to satisfy its B12 auxotrophy. Our findings indicate microbial interactions aligning with the 'black queen hypothesis' where Sphingomonas (detoxifier, B12 recipient) and Hydrogenophaga (B12 producer, enjoying detoxification) act as both helpers and beneficiaries.

PTEN interacts with the transcription machinery on chromatin and regulates RNA polymerase II-mediated transcription.

Regulation of RNA polymerase II (RNAPII)-mediated transcription controls cellular phenotypes such as cancer. Phosphatase and tensin homologue deleted on chromosome ten (PTEN), one of the most commonly altered tumor suppressors in cancer, affects transcription via its role in antagonizing the PI3K/AKT signaling pathway. Using co-immunoprecipitations and proximal ligation assays we provide evidence that PTEN interacts with AFF4, RNAPII, CDK9, cyclin T1, XPB and CDK7. Using ChIP-seq, we show that PTEN co-localizes with RNAPII and binds to chromatin in promoter and putative enhancer regions identified by histone modifications. Furthermore, we show that loss of PTEN affects RNAPII occupancy in gene bodies and further correlates with gene expression changes. Interestingly, PTEN binds to promoters and negatively regulates the expression of genes involved in transcription including AFF4 and POL2RA, which encodes a subunit of RNAPII. Loss of PTEN also increased cells' sensitivity to transcription inhibition via small molecules, which could provide a strategy to target PTEN-deficient cancers. Overall, our work describes a previously unappreciated role of nuclear PTEN, which by interacting with the transcription machinery in the context of chromatin exerts an additional layer of regulatory control on RNAPII-mediated transcription.

A natural polymorphism alters odour and DEET sensitivity in an insect odorant receptor.

Blood-feeding insects such as mosquitoes are efficient vectors of human infectious diseases because they are strongly attracted by body heat, carbon dioxide and odours produced by their vertebrate hosts. Insect repellents containing DEET (N,N-diethyl-meta-toluamide) are highly effective, but the mechanism by which this chemical wards off biting insects remains controversial despite decades of investigation. DEET seems to act both at close range as a contact chemorepellent, by affecting insect gustatory receptors, and at long range, by affecting the olfactory system. Two opposing mechanisms for the observed behavioural effects of DEET in the gas phase have been proposed: that DEET interferes with the olfactory system to block host odour recognition and that DEET actively repels insects by activating olfactory neurons that elicit avoidance behaviour. Here we show that DEET functions as a modulator of the odour-gated ion channel formed by the insect odorant receptor complex. The functional insect odorant receptor complex consists of a common co-receptor, ORCO (ref. 15) (formerly called OR83B; ref. 16), and one or more variable odorant receptor subunits that confer odour selectivity. DEET acts on this complex to potentiate or inhibit odour-evoked activity or to inhibit odour-evoked suppression of spontaneous activity. This modulation depends on the specific odorant receptor and the concentration and identity of the odour ligand. We identify a single amino-acid polymorphism in the second transmembrane domain of receptor OR59B in a Drosophila melanogaster strain from Brazil that renders OR59B insensitive to inhibition by the odour ligand and modulation by DEET. Our data indicate that natural variation can modify the sensitivity of an odour-specific insect odorant receptor to odour ligands and DEET. Furthermore, they support the hypothesis that DEET acts as a molecular 'confusant' that scrambles the insect odour code, and provide a compelling explanation for the broad-spectrum efficacy of DEET against multiple insect species.

Notch1 controls macrophage recruitment and Notch signaling is activated at sites of endothelial cell anastomosis during retinal angiogenesis in mice.

Notch is a critical regulator of angiogenesis, vascular differentiation, and vascular integrity. We investigated whether Notch signaling affects macrophage function during retinal angiogenesis in mice. Retinal macrophage recruitment and localization in mice with myeloid-specific loss of Notch1 was altered, as these macrophages failed to localize at the leading edge of the vascular plexus and at vascular branchpoints. Furthermore, these retinas were characterized by elongated endothelial cell sprouts that failed to anastomose with neighboring sprouts. Using Notch reporter mice, we demonstrate that retinal macrophages localize between Dll4-positive tip cells and at vascular branchpoints, and that these macrophages had activated Notch signaling. Taken together, these data demonstrate that Notch signaling in macrophages is important for their localization and interaction with endothelial cells during sprouting angiogenesis.

Loss of PBRM1 Alters Promoter Histone Modifications and Activates ALDH1A1 to Drive Renal Cell Carcinoma.

Subunits of SWI/SNF chromatin remodeling complexes are frequently mutated in human malignancies. The PBAF complex is composed of multiple subunits, including the tumor-suppressor protein PBRM1 (BAF180), as well as ARID2 (BAF200), that are unique to this SWI/SNF complex. PBRM1 is mutated in various cancers, with a high mutation frequency in clear cell renal cell carcinoma (ccRCC). Here, we integrate RNA-seq, histone modification ChIP-seq, and ATAC-seq data to show that loss of PBRM1 results in de novo gains in H3K4me3 peaks throughout the epigenome, including activation of a retinoic acid biosynthesis and signaling gene signature. We show that one such target gene, ALDH1A1, which regulates a key step in retinoic acid biosynthesis, is consistently upregulated with PBRM1 loss in ccRCC cell lines and primary tumors, as well as non-malignant cells. We further find that ALDH1A1 increases the tumorigenic potential of ccRCC cells. Using biochemical methods, we show that ARID2 remains bound to other PBAF subunits after loss of PBRM1 and is essential for increased ALDH1A1 after loss of PBRM1, whereas other core SWI/SNF components are dispensable, including the ATPase subunit BRG1. In total, this study uses global epigenomic approaches to uncover novel mechanisms of PBRM1 tumor suppression in ccRCC. IMPLICATIONS: This study implicates the SWI/SNF subunit and tumor-suppressor PBRM1 in the regulation of promoter histone modifications and retinoic acid biosynthesis and signaling pathways in ccRCC and functionally validates one such target gene, the aldehyde dehydrogenase ALDH1A1.

PTEN Regulates Glutamine Flux to Pyrimidine Synthesis and Sensitivity to Dihydroorotate Dehydrogenase Inhibition.

Metabolic changes induced by oncogenic drivers of cancer contribute to tumor growth and are attractive targets for cancer treatment. Here, we found that increased growth of PTEN-mutant cells was dependent on glutamine flux through the de novo pyrimidine synthesis pathway, which created sensitivity to the inhibition of dihydroorotate dehydrogenase, a rate-limiting enzyme for pyrimidine ring synthesis. S-phase PTEN-mutant cells showed increased numbers of replication forks, and inhibitors of dihydroorotate dehydrogenase led to chromosome breaks and cell death due to inadequate ATR activation and DNA damage at replication forks. Our findings indicate that enhanced glutamine flux generates vulnerability to dihydroorotate dehydrogenase inhibition, which then causes synthetic lethality in PTEN-deficient cells due to inherent defects in ATR activation. Inhibition of dihydroorotate dehydrogenase could thus be a promising therapy for patients with PTEN-mutant cancers.Significance: We have found a prospective targeted therapy for PTEN-deficient tumors, with efficacy in vitro and in vivo in tumors derived from different tissues. This is based upon the changes in glutamine metabolism, DNA replication, and DNA damage response which are consequences of inactivation of PTENCancer Discov; 7(4); 380-90. ©2017 AACR.See related article by Brown et al., p. 391This article is highlighted in the In This Issue feature, p. 339.

p53 Maintains Baseline Expression of Multiple Tumor Suppressor Genes.

TP53 is the most commonly mutated tumor suppressor gene and its mutation drives tumorigenesis. Using ChIP-seq for p53 in the absence of acute cell stress, we found that wild-type but not mutant p53 binds and activates numerous tumor suppressor genes, including PTEN, STK11(LKB1), miR-34a, KDM6A(UTX), FOXO1, PHLDA3, and TNFRSF10B through consensus binding sites in enhancers and promoters. Depletion of p53 reduced expression of these target genes, and analysis across 18 tumor types showed that mutation of TP53 associated with reduced expression of many of these genes. Regarding PTEN, p53 activated expression of a luciferase reporter gene containing the p53-consensus site in the PTEN enhancer, and homozygous deletion of this region in cells decreased PTEN expression and increased growth and transformation. These findings show that p53 maintains expression of a team of tumor suppressor genes that may together with the stress-induced targets mediate the ability of p53 to suppress cancer development. p53 mutations selected during tumor initiation and progression, thus, inactivate multiple tumor suppressor genes in parallel, which could account for the high frequency of p53 mutations in cancer.Implications: In this study, we investigate the activities of p53 under normal low-stress conditions and discover that p53 is capable of maintaining the expression of a group of important tumor suppressor genes at baseline, many of which are haploinsufficient, which could contribute to p53-mediated tumor suppression. Mol Cancer Res; 15(8); 1051-62. ©2017 AACR.

PTEN inhibits PREX2-catalyzed activation of RAC1 to restrain tumor cell invasion.

The tumor suppressor PTEN restrains cell migration and invasion by a mechanism that is independent of inhibition of the PI3K pathway and decreased activation of the kinase AKT. PREX2, a widely distributed GEF that activates the GTPase RAC1, binds to and inhibits PTEN. We used mouse embryonic fibroblasts and breast cancer cell lines to show that PTEN suppresses cell migration and invasion by blocking PREX2 activity. In addition to metabolizing the phosphoinositide PIP3, PTEN inhibited PREX2-induced invasion by a mechanism that required the tail domain of PTEN, but not its lipid phosphatase activity. Fluorescent nucleotide exchange assays revealed that PTEN inhibited the GEF activity of PREX2 toward RAC1. PREX2 is a frequently mutated GEF in cancer, and examination of human tumor data showed that PREX2 mutation was associated with high PTEN expression. Therefore, we tested whether cancer-derived somatic PREX2 mutants, which accelerate tumor formation of immortalized melanocytes, were inhibited by PTEN. The three stably expressed, somatic PREX2 cancer mutants that we tested were resistant to PTEN-mediated inhibition of invasion but retained the ability to inhibit the lipid phosphatase activity of PTEN. In vitro analysis showed that PTEN did not block the GEF activity of two PREX2 cancer mutants and had a reduced binding affinity for the third. Thus, PTEN antagonized migration and invasion by restraining PREX2 GEF activity, and PREX2 mutants are likely selected in cancer to escape PTEN-mediated inhibition of invasion.

A secreted PTEN phosphatase that enters cells to alter signaling and survival.

Phosphatase and tensin homolog on chromosome ten (PTEN) is a tumor suppressor and an antagonist of the phosphoinositide-3 kinase (PI3K) pathway. We identified a 576-amino acid translational variant of PTEN, termed PTEN-Long, that arises from an alternative translation start site 519 base pairs upstream of the ATG initiation sequence, adding 173 N-terminal amino acids to the normal PTEN open reading frame. PTEN-Long is a membrane-permeable lipid phosphatase that is secreted from cells and can enter other cells. As an exogenous agent, PTEN-Long antagonized PI3K signaling and induced tumor cell death in vitro and in vivo. By providing a means to restore a functional tumor-suppressor protein to tumor cells, PTEN-Long may have therapeutic uses.