Repression of TERRA Expression by Subtelomeric DNA Methylation Is Dependent on NRF1 Binding

International audienceChromosome ends are transcribed into long noncoding telomeric repeat-containing RNA (TERRA) from subtelomeric promoters. A class of TERRA promoters are associated with CpG islands embedded in repetitive DNA tracts. Cytosines in these subtelomeric CpG islands are frequently methylated in telomerase-positive cancer cells, and demethylation induced by depletion of DNA methyltransferases is associated with increased TERRA levels. However, the direct evidence and the underlying mechanism regulating TERRA expression through subtelomeric CpG islands methylation are still to establish. To analyze TERRA regulation by subtelomeric DNA methylation in human cell line (HeLa), we used an epigenetic engineering tool based on CRISPR-dCas9 (clustered regularly interspaced short palindromic repeats - dead CRISPR associated protein 9) associated with TET1 (ten-eleven 1 hydroxylase) to specifically demethylate subtelomeric CpG islands. This targeted demethylation caused an up-regulation of TERRA, and the enhanced TERRA production depended on the methyl-sensitive transcription factor NRF1 (nuclear respiratory factor 1). Since AMPK (AMP-activated protein kinase) is a well-known activator of NRF1, we treated cells with an AMPK inhibitor (compound C). Surprisingly, compound C treatment increased TERRA levels but did not inhibit AMPK activity in these experimental conditions. Altogether, our results provide new insight in the fine-tuning of TERRA at specific subtelomeric promoters and could allow identifying new regulators of TERRA

AP-TSS: A New Method for the Analysis of RNA Expression from Particular and Challenging Transcription Start Sites

Alternative promoter usage involved in the regulation of transcription, splicing, and translation contributes to proteome diversity and is involved in a large number of diseases, in particular, cancer. Epigenetic mechanisms and cis regulatory elements are involved in alternative promoter activity. Multiple transcript isoforms can be produced from a gene, due to the initiation of transcription at different transcription start sites (TSS). These transcripts may not have regions that allow discrimination during RT-qPCR, making quantification technically challenging. This study presents a general method for the relative quantification of a transcript synthesized from a particular TSS that we called AP-TSS (analysis of particular TSS). AP-TSS is based on the specific elongation of the cDNA of interest, followed by its quantification by qPCR. As proof of principle, AP-TSS was applied to two non-coding RNA: telomeric repeat-containing RNAs (TERRA) from a particular subtelomeric TSS, and Alu transcripts. The treatment of cells with a DNA methylation inhibitor was associated with a global increase of the total TERRA level, but the TERRA expression from the TSS of interest did not change in HT1080 cells, and only modestly increased in HeLa cells. This result suggests that TERRA upregulation induced by global demethylation of the genome is mainly due to activation from sites other than this particular TSS. For Alu RNA, the signal obtained by AP-TSS is specific for the RNA Polymerase III-dependent Alu transcript. In summary, our method provides a tool to study regulation of gene expression from a given transcription start site, in different conditions that could be applied to many genes. In particular, AP-TSS can be used to investigate the epigenetic regulation of alternative TSS usage that is of importance for the development of epigenetic-targeted therapies

Investigating the Effect of Mono- and Dimeric 360A G-Quadruplex Ligands on Telomere Stability by Single Telomere Length Analysis (STELA)

Telomeres are nucleoprotein structures that cap and protect the natural ends of chromosomes. Telomeric DNA G-rich strands can form G-quadruplex (or G4) structures. Ligands that bind to and stabilize G4 structures can lead to telomere dysfunctions by displacing shelterin proteins and/or by interfering with the replication of telomeres. We previously reported that two pyridine dicarboxamide G4 ligands, 360A and its dimeric analogue (360A)2A, were able to displace in vitro hRPA (a single-stranded DNA-binding protein of the replication machinery) from telomeric DNA by stabilizing the G4 structures. In this paper, we perform for the first time single telomere length analysis (STELA) to investigate the effect of G4 ligands on telomere length and stability. We used the unique ability of STELA to reveal the full spectrum of telomere lengths at a chromosome terminus in cancer cells treated with 360A and (360A)2A. Upon treatment with these ligands, we readily detected an increase of ultrashort telomeres, whose lengths are significantly shorter than the mean telomere length, and that could not have been detected by other methods

Strain- and sex-dependent circadian changes in abcc2 transporter expression: implications for irinotecan chronotolerance in mouse ileum.

ATP-binding cassette transporter abcc2 is involved in the cellular efflux of irinotecan. The drug is toxic for mouse ileum, where abcc2 is highly expressed. Here, we investigate whether circadian changes in local abcc2 expression participate in the circadian rhythm of irinotecan toxicity for ileum mucosa, and further assess whether genetic background or sex modify this relation.Ileum mucosa was obtained every 3-4 h for 24 h in male and female B6D2F(1) and B6CBAF(1) mice synchronized with light from Zeitgeber Time (ZT)0 to ZT12 alternating with 12 h of darkness. Irinotecan (50 mg/kg i.v. daily for 4 days) was administered at the sex- and strain-specific times corresponding to least (ZT11-15) or largest drug-induced body weight loss (ZT23-03-07). Abcc2 expression was determined with qRT-PCR for mRNA and with immunohistochemistry and confocal microscopy for protein. Histopathologic lesions were graded in ileum tissues obtained 2, 4 or 6 days after treatment. Two- to six-fold circadian changes were demonstrated for mRNA and protein mean expressions of abcc2 in mouse ileum (p<0.05). ZT12 corresponded to high mRNA and protein expressions, with circadian waveforms differing according to genetic background and sex. The proportion of mice spared from ileum lesions varied three-fold according to irinotecan timing, with best tolerability at ZT11-15 (p = 0.00003). Irinotecan was also best tolerated in males (p = 0.05) and in B6CBAF(1) (p = 0.0006).Strain- and sex-dependent circadian patterns in abcc2 expressions displayed robust relations with the chronotolerance of ileum mucosa for irinotecan. This finding has strong potential implications for improving the intestinal tolerability of anticancer drugs through circadian delivery

Confocal immunohistochemistry imaging of abcc2 protein expression in mouse ileum, according to circadian time, genetic background and sex.

<p>A) Selected examples in male (upper row) and female (lower row) B6D2F₁. B) <i>Id</i> in B6CBAF₁. C) Histogram depicting the changes in mean (±SEM) protein expression at four ZTs associated with low or high mRNA expressions in male and female B6D2F₁. D) <i>Id</i> in B6CBAF₁. NOTE: Three-way ANOVA confirmed statistically significant differences as a function of ZT (p<0.001), strain (p = 0.048). Statistically significant interactions were detected between ZT*strain (p = 0.001), ZT*sex (p = 0.035) and ZT*strain*sex (p = 0.004).</p

Circadian patterns of a<i>bcc2</i> mRNA expression in the ileum mucosa of mice, according to genetic background and sex.

<p>Mean (± SEM) as a function of Zeitgeber Time (ZT), with ZT0 as light onset. (A) male and female B6D2F₁; (B) male and female B6CBAF₁. Mean <i>abcc2</i> expression increased six-fold from ZT0 (trough) to ZT12 (peak) for B6D2F₁ males and ZT0 (trough) to ZT9 (peak) for B6D2F₁ females (ANOVA, p = 0.04; Cosinor, p = 0.0023 for B6D2F₁ males and ANOVA, p = 0.0008; Cosinor, p = 0.0023 for B6D2F₁ females). Similar three- to four-fold circadian variations were found in male and female B6CBAF₁, with highest values occurring from ZT9 to ZT15, and a trough at ZT0 (ANOVA, p = 0.004 in male; p = 0.003 in female). The rhythm was validated by cosinor for B6CBAF₁ (p = 0.00026 and p = 0.00012 in male and female, respectively).</p

Co-localization of abcc2 and β-catenin in the membrane of ileum mucosa cells.

<p>Double immunofluorescence staining of abcc2 (green) and β-catenin (red) in mouse ileum using confocal laser scanning microscopy. Far-red fluorescent DNA dye DRAQ5 (blue) for visualizing nuclei. Co-localization between abcc2 and β-catenin was shown as yellow. A) Original magnification 40× (scale bar, 20 µm). B) Higher magnification of selected area.</p

Circadian patterns of a<i>bcc2</i> mRNA expression in the ileum of male B6D2F₁ mice.

<p>Mean (± SEM) in mucosa and in serosa as a function of Zeitgeber Time (ZT), with ZT0 as light onset. Statistically significant differences according to ZT were validated for mucosa with ANOVA (p = 0.02). A statistically significant 24 h rhythm was confirmed for mucosa with cosinor (p = 0.0011).</p

Key research challenges to supporting farm transitions to agroecology in advanced economies. A review

International audienceIn response to the sustainability issues that agriculture faces in advanced economies, agroecology has gained increasing relevance in scientific, political, and social debates. This has promoted discussion about transitions to agroecology, which represents a significant advancement. Accordingly, it has become a growing field of research. We reviewed the literature on and in support of farm transitions to agroecology in advanced economies in order to identify key research challenges and suggest innovative research paths. Our findings can be summarized as follows: (1) Research that supports exploration and definition of desired futures, whether based on future-oriented modeling or expert-based foresight approaches, should more explicitly include the farm level. It should stimulate the creativity and design ability of farmers and other stakeholders, and also address issues of representation and power among them. (2) Research that creates awareness and assesses farms before, during or after transition requires more holistic and dynamic assessment frameworks. These frameworks need to be more flexible to adapt to the diversity of global and local challenges. Their assessment should explicitly include uncertainty due to the feedback loops and emergent properties of transitions. (3) Research that analyzes and supports farms during transition should focus more on the dynamics of change processes by valuing what happens on the farms. Research should especially give more credence to on-farm experiments conducted by farmers and develop new tools and methods (e.g., for strategic monitoring) to support these transitions. This is the first review of scientific studies of farm transitions to agroecology. Overall, the review indicates that these transitions challenge the system boundaries, temporal horizons, and sustainability dimensions that agricultural researchers usually consider. In this context, farm transitions to agroecology require changes in the current organization and funding of research in order to encourage longer term and more adaptive configurations. Keywords Transformation • Sustainable agriculture • Farm • Coupled innovations • Assessment • Design • Foresight Contents Lorène Prost and Guillaume Martin contributed equally

Key research challenges to supporting farm transitions to agroecology in advanced economies. A review

International audienceIn response to the sustainability issues that agriculture faces in advanced economies, agroecology has gained increasing relevance in scientific, political, and social debates. This has promoted discussion about transitions to agroecology, which represents a significant advancement. Accordingly, it has become a growing field of research. We reviewed the literature on and in support of farm transitions to agroecology in advanced economies in order to identify key research challenges and suggest innovative research paths. Our findings can be summarized as follows: (1) Research that supports exploration and definition of desired futures, whether based on future-oriented modeling or expert-based foresight approaches, should more explicitly include the farm level. It should stimulate the creativity and design ability of farmers and other stakeholders, and also address issues of representation and power among them. (2) Research that creates awareness and assesses farms before, during or after transition requires more holistic and dynamic assessment frameworks. These frameworks need to be more flexible to adapt to the diversity of global and local challenges. Their assessment should explicitly include uncertainty due to the feedback loops and emergent properties of transitions. (3) Research that analyzes and supports farms during transition should focus more on the dynamics of change processes by valuing what happens on the farms. Research should especially give more credence to on-farm experiments conducted by farmers and develop new tools and methods (e.g., for strategic monitoring) to support these transitions. This is the first review of scientific studies of farm transitions to agroecology. Overall, the review indicates that these transitions challenge the system boundaries, temporal horizons, and sustainability dimensions that agricultural researchers usually consider. In this context, farm transitions to agroecology require changes in the current organization and funding of research in order to encourage longer term and more adaptive configurations. Keywords Transformation • Sustainable agriculture • Farm • Coupled innovations • Assessment • Design • Foresight Contents Lorène Prost and Guillaume Martin contributed equally