A novel bioinformatic approach reveals cooperation between Cancer/Testis genes in basal-like breast tumors.

Breast cancer is the most prevalent type of cancer in women worldwide. Within breast tumors, the basal-like subtype has the worst prognosis, prompting the need for new tools to understand, detect, and treat these tumors. Certain germline-restricted genes show aberrant expression in tumors and are known as Cancer/Testis genes; their misexpression has diagnostic and therapeutic applications. Here we designed a new bioinformatic approach to examine Cancer/Testis gene misexpression in breast tumors. We identify several new markers in Luminal and HER-2 positive tumors, some of which predict response to chemotherapy. We then use machine learning to identify the two Cancer/Testis genes most associated with basal-like breast tumors: HORMAD1 and CT83. We show that these genes are expressed by tumor cells and not by the microenvironment, and that they are not expressed by normal breast progenitors; in other words, their activation occurs de novo. We find these genes are epigenetically repressed by DNA methylation, and that their activation upon DNA demethylation is irreversible, providing a memory of past epigenetic disturbances. Simultaneous expression of both genes in breast cells in vitro has a synergistic effect that increases stemness and activates a transcriptional profile also observed in double-positive tumors. Therefore, we reveal a functional cooperation between Cancer/Testis genes in basal breast tumors; these findings have consequences for the understanding, diagnosis, and therapy of the breast tumors with the worst outcomes.

Locus-level L1 DNA methylation profiling reveals the epigenetic and transcriptional interplay between L1s and their integration sites.

Long interspersed element 1 (L1) retrotransposons are implicated in human disease and evolution. Their global activity is repressed by DNA methylation, but deciphering the regulation of individual copies has been challenging. Here, we combine short- and long-read sequencing to unveil L1 methylation heterogeneity across cell types, families, and individual loci and elucidate key principles involved. We find that the youngest primate L1 families are specifically hypomethylated in pluripotent stem cells and the placenta but not in most tumors. Locally, intronic L1 methylation is intimately associated with gene transcription. Conversely, the L1 methylation state can propagate to the proximal region up to 300 bp. This phenomenon is accompanied by the binding of specific transcription factors, which drive the expression of L1 and chimeric transcripts. Finally, L1 hypomethylation alone is typically insufficient to trigger L1 expression due to redundant silencing pathways. Our results illuminate the epigenetic and transcriptional interplay between retrotransposons and their host genome.

Genome-Wide Young L1 Methylation Profiling by bs-ATLAS-seq.

By silencing L1 retrotransposons, DNA methylation protects mammalian genomes from potent endogenous mutagens. However, some loci can escape this repressive mechanism and become active, particularly in carcinomas. Alterations of L1 DNA methylation can also locally influence gene expression. Comprehensive measurement of L1 DNA methylation at the locus level remains challenging. Here, we present bs-ATLAS-seq, a genome-wide approach to locate full-length L1 elements in the human genome, and assess their methylation levels at single-base and single-locus resolutions. This strategy targets the youngest, and only retrotransposition-competent family, L1HS, but also detects a significant fraction of older elements (L1PA2 to L1PA8). Bs-ATLAS-seq evaluates methylation at the first 15 CpGs of L1 5' UTR, which corresponds to the first half of the sense promoter. It relies on random fragmentation of the genomic DNA, adapter ligation, bisulfite treatment and suppression PCR, and ends by asymmetrical paired-end sequencing. A dedicated pipeline provides the location of L1 elements and their methylation status, including for non-reference loci, as well as their single-molecule DNA profiles.

Epigenetic switch drives the conversion of fibroblasts into proinvasive cancer-associated fibroblasts.

Carcinoma-associated fibroblasts (CAF) mediate the onset of a proinvasive tumour microenvironment. The proinflammatory cytokine LIF reprograms fibroblasts into a proinvasive phenotype, which promotes extracellular matrix remodelling and collective invasion of cancer cells. Here we unveil that exposure to LIF initiates an epigenetic switch leading to the constitutive activation of JAK1/STAT3 signalling, which results in sustained proinvasive activity of CAF. Mechanistically, p300-histone acetyltransferase acetylates STAT3, which, in turn, upregulates and activates the DNMT3b DNA methyltransferase. DNMT3b methylates CpG sites of the SHP-1 phosphatase promoter, which abrogates SHP-1 expression, and results in constitutive phosphorylation of JAK1. Sustained JAK1/STAT3 signalling is maintained by DNA methyltransferase DNMT1. Consistently, in human lung and head and neck carcinomas, STAT3 acetylation and phosphorylation are inversely correlated with SHP-1 expression. Combined inhibition of DNMT activities and JAK signalling, in vitro and in vivo, results in long-term reversion of CAF-associated proinvasive activity and restoration of the wild-type fibroblast phenotype.

euL1db: the European database of L1HS retrotransposon insertions in humans.

Retrotransposons account for almost half of our genome. They are mobile genetics elements-also known as jumping genes--but only the L1HS subfamily of Long Interspersed Nuclear Elements (LINEs) has retained the ability to jump autonomously in modern humans. Their mobilization in germline--but also some somatic tissues--contributes to human genetic diversity and to diseases, such as cancer. Here, we present euL1db, the European database of L1HS retrotransposon insertions in humans (available at http://euL1db.unice.fr). euL1db provides a curated and comprehensive summary of L1HS insertion polymorphisms identified in healthy or pathological human samples and published in peer-reviewed journals. A key feature of euL1db is its sample--wise organization. Hence L1HS insertion polymorphisms are connected to samples, individuals, families and clinical conditions. The current version of euL1db centralizes results obtained in 32 studies. It contains >900 samples, >140,000 sample-wise insertions and almost 9000 distinct merged insertions. euL1db will help understanding the link between L1 retrotransposon insertion polymorphisms and phenotype or disease.

Mercury(II) removal from water by electrocoagulation using aluminium and iron electrodes.

In this work, electrocoagulation was used to evaluate the treatment of synthetic solutions containing mercury(II) of concentration 2 x 10(-5)M. The effects of the distance between the electrodes, current density, charge loading and initial pH on the removal efficiency were investigated, using aluminium and iron electrodes. Analysis of the filtrates resulting from the treatment was made by anodic redissolution in the differential pulse mode. The removal efficiency was above 99.9% when the distance between the electrodes was 3 cm, the current density ranging from 2.5 to 3.125 A dm(-2); for instance, 99.95% of the mercury(II) was eliminated when a charge loading of 9.33 and 15.55 F m(-3) were used for iron and aluminium respectively. In these conditions, by varying the pH of the mercury(II) solutions from 3 to 7, the removal efficiency remained higher than 99%. In addition, some experiments were carried out on a river water contaminated with mercury(II) ions, and the results obtained showed that the presence of organic matter do not influence the efficiency of the treatment. The elimination of mercury(II) ions is best performed with iron, where 15 min of electrolysis was sufficient to reach the highest removal compared to aluminium which required 25 min for the same result.

LRP1 controls intracellular cholesterol storage and fatty acid synthesis through modulation of Wnt signaling.

The low-density lipoprotein receptor-related protein LRP1 is a cell surface receptor with functions in diverse physiological pathways, including lipid metabolism. Here we show that LRP1-deficient fibroblasts accumulate high levels of intracellular cholesterol and cholesteryl-ester when stimulated for adipocyte differentiation. We demonstrate that LRP1 stimulates a canonical Wnt5a signaling pathway that prevents cholesterol accumulation. Moreover, we show that LRP1 is required for lipolysis and stimulates fatty acid synthesis independently of the noradrenergic pathway, through inhibition of GSK3beta and its previously unknown target acetyl-CoA carboxylase (ACC). As a result of ACC inhibition, mature LRP1-deficient adipocytes of adult mice are hypotrophic, and lower uptake of fatty acids into adipose tissue leads to their redistribution to the liver. These results establish LRP1 as a novel integrator of adipogenic differentiation and fat storage signals.

Heterocyclic bis-cations as starting hits for design of inhibitors of the bifunctional enzyme histidine-containing protein kinase/phosphatase from Bacillus subtilis.

The main mechanism of carbon catabolite repression/activation in low-guanine and low-cytosine Gram-positive bacteria seems to involve phosphorylation of HPr (histidine-containing protein) at Ser-46 by the ATP-dependent HPr kinase, which in Bacillus subtilis, Lactobacillus casei, and Staphylococcus xylosus also exhibits phosphatase activity and is thus a bifunctional enzyme (HPrK/P). Since deficiency of HPrK/P in S. xylosus, L. casei, and B. subtilis mutants leads to severe growth defects, inhibitors of the enzyme could form a new family of antibiotic drugs. The aim of the study was to screen an in-house chemical library for identification of hits as inhibitors of HPrK/P in B. subtilis and to further extract additional information of structural features from hit optimization using a radioactive in vitro assay. A symmetrical bis-cationic compound LPS 02-10-L-D09 (2a) with a 12-carbon alkyl linker bridging the two 2-aminobenzimidazole moieties was identified as a non-ATP mimetic compound exhibiting an EC(50) value of 10 microM in a kinase assay with HPr as substrate. The substance also inhibited the phosphatase activity of HPrK/P triggered by the addition of inorganic phosphate. Similar results were obtained with 2a and catabolite repression HPr, which, like HPr, can be phosphorylated at Ser-46 by HPrK/P and is involved in catabolite repression. Structure-activity relationship analysis indicated the importance in its structure of a substituted 2-aminobenzimidazole. This typical heterocycle is linked through a C12 alkyl chain to a second scaffold that can bear a cationic or a noncationic moiety but in all cases should present an aromatic ring in its vicinity.

Properties and regulation of the bifunctional enzyme HPr kinase/phosphatase in Bacillus subtilis.

The bifunctional allosteric enzyme HPr kinase/phosphatase (HPrK/P) from Bacillus subtilis is a key enzyme in the main mechanism of carbon catabolite repression/activation (i.e. a means for the bacteria to adapt rapidly to environmental changes in carbon sources). In this regulation system, the enzyme can phosphorylate and dephosphorylate two proteins, HPr/HPr(Ser(P)) and Crh/Crh(Ser(P)), sensing the metabolic state of the cell. To acquire further insight into the properties of HPrK/P, electrospray ionization mass spectrometry, dynamic light scattering, and BIACORE were used to determine the oligomeric state of the protein under native conditions, revealing that the enzyme exists as a hexamer at pH 6.8 and as a monomer and dimer at pH 9.5. Using an in vitro radioactive assay, the influence of divalent cations, pH, temperature, and different glycolytic intermediates on the activity as well as kinetic parameters were investigated. The presence of divalent cations was found to be essential for both opposing activities of the enzyme. Furthermore, pH values equal to the internal pH of vegetative cells seem to favor the kinase activity, whereas lower pH values increased the phosphatase activity. Among the glycolytic intermediates evaluated, fructose 1,6-diphosphate and fructose 2,6-diphosphate were found to be allosteric activators in the kinase assay, whereas high concentrations inhibited the phosphatase activity, except for fructose 1,6-diphosphate in the case of HPr(Ser(P)). Phosphatase activity was induced by inorganic phosphate as well as acetyl phosphate and glyceraldehyde 3-phosphate. Kinetic parameters indicate a preference for binding of HPr compared with Crh to the enzyme and supported a strong positive cooperativity. This work suggests that the oligomeric state of the enzyme is influenced by several effectors and is correlated to the kinase or phosphatase activity. The phosphatase activity is mainly supported by the hexameric form.