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•3238 genes were differentially expressed after the exposure to tributyltin (TBT).•Transcriptomic point of departure occurred at concentrations 1/7 of the macroscopic ones.•Steroid, ...cholesterol and vitamin D3 related pathways were the most affected.•Lipid, general stress and DNA damage related pathways were altered at higher doses.•Usefulness of functional benchmark dose approach for risk assessment is highlighted.
Exposure to the antifouling tributyltin (TBT) has been related to imposex in mollusks and to obesogenicity, adipogenesis and masculinization in fish. To understand the underlying molecular mechanisms, we evaluated dose-response effects of TBT (1.7−56 nM) in zebrafish eleutheroembryos transcriptome exposed from 2 to 5 days post-fertilization. RNA-sequencing analysis identified 3238 differentially expressed transcripts in eleutheroembryos exposed to TBT. Benchmark dose analyses (BMD) showed that the point of departure (PoD) for transcriptomic effects (9.28 nM) was similar to the metabolomic PoD (11.5 nM) and about one order of magnitude lower than the morphometric PoD (67.9 nM) or the median lethal concentration (LC50: 93.6 nM). Functional analysis of BMD transcriptomic data identified steroid metabolism and cholesterol and vitamin D3 biosynthesis as the most sensitive pathways to TBT (<50% PoD). Conversely, transcripts related to general stress and DNA damage became affected only at doses above the PoD. Therefore, our results indicate that transcriptomes can act as early molecular indicators of pollutant exposure, and illustrates their usefulness for the mechanistic identification of the initial toxic events. As the estimated molecular PoDs are close to environmental levels, we concluded that TBT may represent a substantial risk in some natural environments.
Exposure to PFOS (perfluorooctanesulfonate) has been related to toxic effects on lipid metabolism, immunological response, and different endocrine systems. We present here a transcriptomic analysis ...of zebrafish embryos exposed to different concentrations of PFOS (0.03–1.0 mg/L) from 48 to 120 hpf. No major survival or morphological alterations (swimming bladder inflation, kyphosis, eye separation and size…) were observed below the 1.0 mg/L mark. Conversely, we observed significant increase in transcripts related to lipid transport and metabolism even at the lowest used concentration. In addition, we observed a general decrease on transcripts related to natural immunity and defense again infections, which adds to the recent concerns about PFOS as immunotoxicant, particularly in humans. Derived PoD (Point of Departure) values for transcriptional changes (0.011 mg/L) were about 200-fold lower than the corresponding PoD values for morphometric effects (2.53 mg/L), and close to levels observed in human blood serum or bird eggs. Our data suggest that currently applicable tolerable levels of PFOS in commercial goods should be re-evaluated, taking into account its potential effects on lipid metabolism and the immune system.
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•Zebrafish embryos show macroscopic alterations at PFOS concentrations >1 mg/L.•Transcriptomic changes occur at concentrations 1/10 to 1/100 the macroscopic LOEC.•Transcript analyses suggest alterations on lipid metabolism and the immune system.•Some of the observed changes occur at concentrations already detected in humans.•We propose tighter regulations limiting human and environmental exposure to PFOS.
DNA methylation is one of the main epigenetic mechanisms for the regulation of gene expression in eukaryotes. In the standard model, methylation in gene promoters has received the most attention ...since it is generally associated with transcriptional silencing. Nevertheless, recent studies in human tissues reveal that methylation of the region downstream of the transcription start site is highly informative of gene expression. Also, in some cell types and specific genes it has been found that methylation of the first intron, a gene feature typically rich in enhancers, is linked with gene expression. However, a genome-wide, tissue-independent, systematic comparative analysis of the relationship between DNA methylation in the first intron and gene expression across vertebrates has not been explored yet.
The most important findings of this study are: (1) using different tissues from a modern fish, we show a clear genome-wide, tissue-independent quasi-linear inverse relationship between DNA methylation of the first intron and gene expression. (2) This relationship is conserved across vertebrates, since it is also present in the genomes of a model pufferfish, a model frog and different human tissues. Among the gene features, tissues and species interrogated, the first intron's negative correlation with the gene expression was most consistent. (3) We identified more tissue-specific differentially methylated regions (tDMRs) in the first intron than in any other gene feature. These tDMRs have positive or negative correlation with gene expression, indicative of distinct mechanisms of tissue-specific regulation. (4) Lastly, we identified CpGs in transcription factor binding motifs, enriched in the first intron, the methylation of which tended to increase with the distance from the first exon-first intron boundary, with a concomitant decrease in gene expression.
Our integrative analysis clearly reveals the important and conserved role of the methylation level of the first intron and its inverse association with gene expression regardless of tissue and species. These findings not only contribute to our basic understanding of the epigenetic regulation of gene expression but also identify the first intron as an informative gene feature regarding the relationship between DNA methylation and gene expression where future studies should be focused.
The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using ...next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis. We compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, our results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserved.
Accumulation of tumor‐associated macrophages (TAMs) associates with malignant progression in cancer. However, the mechanisms that drive the pro‐tumor functions of TAMs are not fully understood. ZEB1 ...is best known for driving an epithelial‐to‐mesenchymal transition (EMT) in cancer cells to promote tumor progression. However, a role for ZEB1 in macrophages and TAMs has not been studied. Here we describe that TAMs require ZEB1 for their tumor‐promoting and chemotherapy resistance functions in a mouse model of ovarian cancer. Only TAMs that expressed full levels of Zeb1 accelerated tumor growth. Mechanistically, ZEB1 expression in TAMs induced their polarization toward an F4/80low pro‐tumor phenotype, including direct activation of Ccr2. In turn, expression of ZEB1 by TAMs induced Ccl2, Cd74, and a mesenchymal/stem‐like phenotype in cancer cells. In human ovarian carcinomas, TAM infiltration and CCR2 expression correlated with ZEB1 in tumor cells, where along with CCL2 and CD74 determined poorer prognosis. Importantly, ZEB1 in TAMs was a factor of poorer survival in human ovarian carcinomas. These data establish ZEB1 as a key factor in the tumor microenvironment and for maintaining TAMs’ tumor‐promoting functions.
Synopsis
Tumor‐associated macrophages (TAMs) require the expression of the EMT transcription factor ZEB1 for their cancer‐promoting functions.
ZEB1 activates a F4/80low phenotype in macrophages and inhibits their maturation into F4/80high macrophages.
ZEB1 is required for the activation of macrophages toward pro‐tumor F4/80low TAMs.
ZEB1 induces a Ccr2‐Mmp9‐Ccl2 positive loop between TAMs and cancer cells to enhance tumor progression.
Expression of ZEB1 in TAMs and cancer cells correlates with poorer prognosis in human ovarian carcinomas.
Tumor‐associated macrophages express the EMT‐associated transcription factor ZEB1, which drives their tumor‐promoting and chemotherapy‐resistance functions in mouse ovarian cancer models.
Histone H1 has seven variants in human somatic cells and contributes to chromatin compaction and transcriptional regulation. Knock-down (KD) of each H1 variant in breast cancer cells results in ...altered gene expression and proliferation differently in a variant specific manner with H1.2 and H1.4 KDs being most deleterious. Here we show combined depletion of H1.2 and H1.4 has a strong deleterious effect resulting in a strong interferon (IFN) response, as evidenced by an up-regulation of many IFN-stimulated genes (ISGs) not seen in individual nor in other combinations of H1 variant KDs. Although H1 participates to repress ISG promoters, IFN activation upon H1.2 and H1.4 KD is mainly generated through the activation of the IFN response by cytosolic nucleic acid receptors and IFN synthesis, and without changes in histone modifications at induced ISG promoters. H1.2 and H1.4 co-KD also promotes the appearance of accessibility sites genome wide and, particularly, at satellites and other repeats. The IFN response may be triggered by the expression of noncoding RNA generated from heterochromatic repeats or endogenous retroviruses upon H1 KD. In conclusion, redundant H1-mediated silencing of heterochromatin is important to maintain cell homeostasis and to avoid an unspecific IFN response.
New advances in high-throughput technologies have allowed for the massive analysis of genomic data, providing new opportunities for the characterization of the transcriptome architectures. Recent ...studies in pigs have employed RNA-Seq to explore the transcriptome of different tissues in a reduced number of animals. The main goal of this study was the identification of differentially-expressed genes in the liver of Iberian x Landrace crossbred pigs showing extreme phenotypes for intramuscular fatty acid composition using RNA-Seq.
The liver transcriptomes of two female groups (H and L) with phenotypically extreme intramuscular fatty acid composition were sequenced using RNA-Seq. A total of 146 and 180 unannotated protein-coding genes were identified in intergenic regions for the L and H groups, respectively. In addition, a range of 5.8 to 7.3% of repetitive elements was found, with SINEs being the most abundant elements. The expression in liver of 186 (L) and 270 (H) lncRNAs was also detected. The higher reproducibility of the RNA-Seq data was validated by RT-qPCR and porcine expression microarrays, therefore showing a strong correlation between RT-qPCR and RNA-Seq data (ranking from 0.79 to 0.96), as well as between microarrays and RNA-Seq (r=0.72). A differential expression analysis between H and L animals identified 55 genes differentially-expressed between groups. Pathways analysis revealed that these genes belong to biological functions, canonical pathways and three gene networks related to lipid and fatty acid metabolism. In concordance with the phenotypic classification, the pathways analysis inferred that linolenic and arachidonic acids metabolism was altered between extreme individuals. In addition, a connection was observed among the top three networks, hence suggesting that these genes are interconnected and play an important role in lipid and fatty acid metabolism.
In the present study RNA-Seq was used as a tool to explore the liver transcriptome of pigs with extreme phenotypes for intramuscular fatty acid composition. The differential gene expression analysis showed potential gene networks which affect lipid and fatty acid metabolism. These results may help in the design of selection strategies to improve the sensorial and nutritional quality of pork meat.
Cumulus cells provide an interesting biological material to perform analyses to understand the molecular clues determining oocyte competence. The objective of this study was to analyze the ...transcriptional differences between cumulus cells from oocytes exhibiting different developmental potentials following individual in vitro embryo production by RNA‐seq. Cumulus cells were allocated into three groups according to the developmental potential of the oocyte following fertilization: (1) oocytes developing to blastocysts (Bl+), (2) oocytes cleaving but arresting development before the blastocyst stage (Bl−), and (3) oocytes not cleaving (Cl−). RNAseq was performed on 4 (Cl−) or 5 samples (Bl+ and Bl−) of cumulus cells pooled from 10 cumulus‐oocyte complexes per group. A total of 49, 50, and 18 differentially expressed genes (DEGs) were detected in the comparisons Bl+ versus Bl−, Bl+ versus Cl− and Bl‐ versus Cl−, respectively, showing a fold change greater than 1.5 at an adjusted p value <0.05. Focussing on DEGs in cumulus cells from Bl+ group, 10 DEGs were common to both comparisons (10/49 from Bl+ vs. Bl−, 10/50 from Bl+ vs. Cl−). These DEGs correspond to 6 upregulated genes (HBE1, ITGA1, PAPPA, AKAP12, ITGA5, and SLC1A4), and 4 downregulated genes (GSTA1, PSMB8, FMOD, and SFRP4) in Bl+ compared to the other groups, from which 7 were validated by quantitative PCR (HBE1, ITGA1, PAPPA, AKAP12, ITGA5, PSMB8 and SFRP4). These genes are involved in critical biological functions such as integrin‐mediated cell adhesion, oxygen availability, IGF and Wnt signaling or PKA pathway, highlighting specific biological processes altered in incompetent in vitro maturation oocytes.
The article has compared the transcriptome of cumulus cells obtained from bovine cumulus‐oocyte‐complexes exhibiting different developmental competence: (1) unable to cleavage, (2) able to cleavage but unable to develop to blastocyst, or (3) able to develop to blastocyst.
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