Nucleolar detention of NONO shields DNA double-strand breaks from aberrant transcripts.

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.

Alpha-synuclein regulates nucleolar DNA double-strand break repair in melanoma.

Although an increased risk of the skin cancer melanoma in people with Parkinson's Disease (PD) has been shown in multiple studies, the mechanisms involved are poorly understood, but increased expression of the PD-associated protein alpha-synuclein (αSyn) in melanoma cells may be important. Our previous work suggests that αSyn can facilitate DNA double-strand break (DSB) repair, promoting genomic stability. We now show that αSyn is preferentially enriched within the nucleolus in the SK-MEL28 melanoma cell line, where it colocalizes with DNA damage markers and DSBs. Inducing DSBs specifically within nucleolar ribosomal DNA (rDNA) increases αSyn levels near sites of damage. αSyn knockout increases DNA damage within the nucleolus at baseline, after specific rDNA DSB induction, and prolongs the rate of recovery from this induced damage. αSyn is important downstream of ATM signaling to facilitate 53BP1 recruitment to DSBs, reducing micronuclei formation and promoting cellular proliferation, migration, and invasion.

The association between the susceptibility to Botrytis cinerea and the levels of volatile and non-volatile metabolites in red ripe strawberry genotypes.

The relations between physical and chemical characteristics (e.g., color, firmness, volatile and non-volatile metabolites) of red ripe strawberry fruit and the natural spoilage caused by Botrytis cinerea were investigated. The spoilage rates differed between genotypes, and this was highly correlated over two successive years. Among seventeen genotypes, a more intense red coloration of the fruit skin was associated with a lower spoilage rate (r = -0.63). Additionally, weakly negative correlations were found between the levels of anthocyanins, ascorbic acid, malic acid and spoilage rates. No clear correlations were found between spoilage rates and soluble sugars, most volatiles, firmness and dry weight percentage. High levels of two volatile compounds, ethyl butanoate (r = 0.55) and 1-hexanol (r = 0.61), were correlated to high spoilage rates. These characteristics may assist strawberry breeders in selecting for genotypes with reduced susceptibility to B. cinerea.

Lack of Blue Light Regulation of Antioxidants and Chilling Tolerance in Basil.

Blue light, measuring from 400 to 500 nm, is generally assumed to increase the content of antioxidants in plants independent of the species. Blue light stimulates the biosynthesis of phenolic compounds such as flavonoids and their subclass anthocyanins from the phenylpropanoid pathway. Flavonoids, anthocyanins, and phenolic acids are strong reactive oxygen species (ROS) scavengers and may lessen the symptoms of abiotic stresses such as chilling. We tested the hypothesis that a high percentage of blue light induces the accumulation of antioxidants and that this effect depends on the photosynthetic photon flux density (PPFD, 400-700 nm). The effect may be more pronounced at a lower PPFD. We investigated the changes in primary and secondary metabolites of basil in response to the percentage of blue light (9, 33, 65, and 100%) applied either as a 5-day End-Of-Production (EOP) treatment or continuous throughout the growth cycle in the green cv. Dolly. We also studied if the response to the percentage of blue light (9 or 90%) was dependent on the total PPFD (100 or 300 µmol m-2 s-1 PPFD) when applied as a 5-day EOP treatment in the green cv. Dolly and the purple cv. Rosie. For both green and purple basil, it was found that the percentage of blue light had little effect on the levels of antioxidants (rosmarinic acid, total ascorbic acid, total flavonoids, and total anthocyanins) at harvest and no interactive effect with PPFD was found. Antioxidants generally decreased during postharvest storage, wherein the decrease was more pronounced at 4 than at 12°C. Chilling injury, as judged from a decrease in F v /F m values and from the occurrence of black necrotic areas, was not affected by the percentage of blue light. Particularly, chilling tolerance in the purple cultivar was increased in plants grown under higher PPFD. This may be related to the increased levels of soluble sugar and starch in leaves from high PPFD treated plants.

High light intensity at End-Of-Production improves the nutritional value of basil but does not affect postharvest chilling tolerance.

Basil suffers from chilling injury (CI) when stored at temperatures below 10-12 °C which seems related to the imbalance between reactive oxygen species (ROS) and antioxidants. We hypothesized that increased light intensity applied shortly before harvest (EOP, End-Of-Production) increases nutritional value i.e. carbohydrates and antioxidants and could improve the chilling tolerance. Two basil cultivars were grown in a vertical farming set-up at a light intensity of 150 µmol m-2 s-1. During the last 5 days of growth, EOP light treatments ranging from 50 to 600 µmol m-2 s-1 were applied. After harvest the leaves were stored at 4 or 12 °C in darkness. Higher EOP light intensity increased the antioxidant (total ascorbic acid, rosmarinic acid) and carbohydrate contents at harvest. During storage antioxidants decreased more rapidly at 4 than at 12 °C. However, increased EOP light intensity did not alleviate chilling symptoms suggesting a minor role of antioxidants studied against chilling stress.

A recurrent chromosomal inversion suffices for driving escape from oncogene-induced senescence via subTAD reorganization.

Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence.

Far-red radiation stimulates dry mass partitioning to fruits by increasing fruit sink strength in tomato.

Far-red (FR) light promotes fruit growth by increasing dry mass partitioning to fruits, but the mechanism behind this is unknown. We hypothesise that it is due to an increased fruit sink strength as FR radiation enhances sugar transportation and metabolism. Tomato plants were grown with or without 50-80 µmol m-2  s-1 of FR radiation added to a common background 150-170 µmol m-2  s-1 red + blue light-emitting diode lighting. Potential fruit growth, achieved by pruning each truss to one remaining fruit, was measured to quantify fruit sink strength. Model simulation was conducted to test whether the measured fruit sink strength quantitatively explained the FR effect on dry mass partitioning. Starch, sucrose, fructose and glucose content were measured. Expression levels of key genes involved in sugar transportation and metabolism were determined. FR radiation increased fruit sink strength by 38%, which, in model simulation, led to an increased dry mass partitioned to fruits that quantitatively agreed very well with measured partitioning. FR radiation increased fruit sugar concentration and upregulated the expression of genes associated with both sugar transportation and metabolism. This is the first study to demonstrate that FR radiation stimulates dry mass partitioning to fruits mainly by increasing fruit sink strength via simultaneous upregulation of sugar transportation and metabolism.

Response of Basil Growth and Morphology to Light Intensity and Spectrum in a Vertical Farm.

Vertical farming is becoming increasingly popular for production of leafy vegetables and herbs, with basil (Ocimum basilicum L.) as one of the most popular herbs. In basil most research has focused on increasing secondary metabolites with light spectra. However, knowledge about the effect of light intensity (photosynthetic photon flux density, PPFD) and spectra on growth and morphology is key for optimizing quality at harvest. The impact of PPFD and spectrum on plant growth and development is species dependent and currently few studies in basil are available. Understanding the response to End-Of-Production (EOP) light of growth and morphology is important for successful vertical farming. We performed a comprehensive series of experiments, where the effects of EOP PPFD, fraction of blue and their interaction on the growth and morphology were analyzed in two green and one purple basil cultivar. In addition, the impact of different EOP intensities and duration of far-red were investigated. We found that increasing the PPFD increased fresh mass, dry matter content and plant height in all three cultivars. The responses were linear or quadratic depending on the cultivar. A high fraction of blue (>90%) increased plant height and decreased the dry mass partitioning to the leaves. The only interaction found between the fraction of blue and overall PPFD was on plant height in the green cultivar whereas other growth parameters and morphology responded stronger to PPFD than to the fraction of blue light. Plant dry matter production was increased with the addition of far-red. Far-red EOP intensity treatments enhanced the fraction of dry mass partitioned to the leaves, whereas a prolonged far-red treatment enhanced partitioning to the stem. Both plant fresh mass and dry matter content were improved by applying high PPFD shortly before harvest. Light spectra were found to be of less importance than PPFD with respect to plant dry matter content. Light use efficiency (LUE) based on fresh mass decreased with increasing PPFD whereas LUE based on dry mass increased with increasing PPFD, when given as EOP treatments. The overall physiological mechanisms of the light intensity and spectral effects are discussed.

Double-strand breaks in ribosomal RNA genes activate a distinct signaling and chromatin response to facilitate nucleolar restructuring and repair.

The nucleolus is a nuclear sub-domain containing the most highly transcribed genes in the genome. Hundreds of human ribosomal RNA (rRNA) genes, located in the nucleolus, rely on constant maintenance. DNA double-strand breaks (DSBs) in rRNA genes activate the ATM kinase, repress rRNA transcription and induce nucleolar cap formation. Yet how ribosomal-DNA (rDNA) lesions are detected and processed remains elusive. Here, we use CRISPR/Cas9-mediated induction of DSBs and report a chromatin response unique to rDNA depending on ATM-phosphorylation of the nucleolar protein TCOF1 and recruitment of the MRE11-RAD50-NBS1 (MRN) complex via the NBS1-subunit. NBS1- and MRE11-depleted cells fail to suppress rRNA transcription and to translocate rDNA into nucleolar caps. Furthermore, the DNA damage response (DDR) kinase ATR operates downstream of the ATM-TCOF1-MRN interplay and is required to fully suppress rRNA transcription and complete DSB-induced nucleolar restructuring. Unexpectedly, we find that DSBs in rDNA neither activate checkpoint kinases CHK1/CHK2 nor halt cell-cycle progression, yet the nucleolar-DDR protects against genomic aberrations and cell death. Our data highlight the concept of a specialized nucleolar DNA damage response (n-DDR) with a distinct protein composition, spatial organization and checkpoint communication. The n-DDR maintains integrity of ribosomal RNA genes, with implications for cell physiology and disease.

The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage.

Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks.

A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure.

The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation. Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks. Interestingly, RNF8-mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin-ligase activity of RNF8, but involved a non-canonical interaction with the forkhead-associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling-assisted ubiquitylation, which involves the cooperation between CHD4 and RNF8 to create a local chromatin environment that is permissive to the assembly of checkpoint and repair machineries at DNA lesions.

The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator.

Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a 'head-to-tail' dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage.