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Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II (RNAPII) are necessary for DNA damage response (DDR) foci formation. We demonstrate that induction of DSBs results in the assembly of functional promoters that include a complete RNAPII pre-initiation complex (PIC), MED1 and CDK9. Absence or inactivation of these factors causes DDR foci reduction both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. We also show that dilncRNAs drive molecular crowding of DDR proteins such as 53BP1 into foci that exhibit liquid-liquid phase separation (LLPS) condensate properties. We propose that the assembly of DSB-induced transcriptional promoters drives RNA synthesis which stimulates phase separation of DDR factors in the shape of foci.

Telomeres are transcribed into noncoding telomeric repeat-containing RNAs (TERRA), which are essential for telomere maintenance. Deregulation of TERRA transcription impairs telomere metabolism and a role in tumorigenesis has been proposed. Head and neck cancer (HNC) is one of the most frequent cancers worldwide, with head and neck squamous cell carcinoma (HNSCC) being the predominant type. Since HNSCC patients are characterized by altered telomere maintenance, a dysfunction in telomere transcription can be hypothesized. In this prospective study, we compared TERRA levels in the tumor and matched normal tissue from 23 HNSCC patients. We then classified patients in two categories according to the level of TERRA expression in the tumor compared to the normal tissue: (1) lower expression in the tumor, (2) higher or similar expression in tumor. A significant proportion of patients in the first group died of the disease within less than 34 months postsurgery, while the majority of patients in the second group were alive and disease-free. Our results highlight a striking correlation between TERRA expression and tumor aggressiveness in HNSCC suggesting that TERRA levels may be proposed as a novel molecular prognostic marker for HNSCC.

DNA double-strand breaks (DSBs) are toxic DNA lesions, which, if not properly repaired, may lead to genomic instability, cell death and senescence. Damage-induced long non-coding RNAs (dilncRNAs) are transcribed from broken DNA ends and contribute to DNA damage response (DDR) signaling. Here we show that dilncRNAs play a role in DSB repair by homologous recombination (HR) by contributing to the recruitment of the HR proteins BRCA1, BRCA2, and RAD51, without affecting DNA-end resection. In S/G2-phase cells, dilncRNAs pair to the resected DNA ends and form DNA:RNA hybrids, which are recognized by BRCA1. We also show that BRCA2 directly interacts with RNase H2, mediates its localization to DSBs in the S/G2 cell-cycle phase, and controls DNA:RNA hybrid levels at DSBs. These results demonstrate that regulated DNA:RNA hybrid levels at DSBs contribute to HR-mediated repair., Long non-coding RNAs transcribed at DNA damaged sites can play part in DNA damage response. Here the authors reveal that damaged induced lncRNAs can form DNA:RNA hybrids at resected DNA-ends. These hybrids are involved in recruiting HR-mediated repair machinery which, in turn, controls their level at DSBs.

Until recently, DNA damage arising from physiological DNA metabolism was considered a detrimental by-product for cells. However, an increasing amount of evidence has shown that DNA damage could have a positive role in transcription activation. In particular, DNA damage has been detected in transcriptional elements following different stimuli. These physiological DNA breaks are thought to be instrumental for the correct expression of genomic loci through different mechanisms. In this regard, although a plethora of methods are available to precisely map transcribed regions and transcription start sites, commonly used techniques for mapping DNA breaks lack sufficient resolution and sensitivity to draw a robust correlation between DNA damage generation and transcription. Recently, however, several methods have been developed to map DNA damage at single-nucleotide resolution, thus providing a new set of tools to correlate DNA damage and transcription. Here, we review how DNA damage can positively regulate transcription initiation, the current techniques for mapping DNA breaks at high resolution, and how these techniques can benefit future studies of DNA damage and transcription.

The DNA damage response (DDR) preserves genomic integrity. Small non-coding RNAs termed DDRNAs are generated at DNA double-strand breaks (DSBs) and are critical for DDR activation. Here we show that active DDRNAs specifically localize to their damaged homologous genomic sites in a transcription-dependent manner. Following DNA damage, RNA polymerase II (RNAPII) binds to the MRE11-RAD50-NBS1 complex, is recruited to DSBs and synthesizes damage-induced long non-coding RNAs (dilncRNAs) from and towards DNA ends. DilncRNAs act both as DDRNA precursors and by recruiting DDRNAs through RNA-RNA pairing. Together, dilncRNAs and DDRNAs fuel DDR focus formation and associate with 53BP1. Accordingly, inhibition of RNAPII prevents DDRNA recruitment, DDR activation and DNA repair. Antisense oligonucleotides matching dilncRNAs and DDRNAs impair site-specific DDR focus formation and DNA repair. We propose that DDR signalling sites, in addition to sharing a common pool of proteins, individually host a unique set of site-specific RNAs necessary for DDR activation.

Centromeres are the sites of kinetochore assembly and spindle fiber attachment and consist of protein-DNA complexes in which the DNA component is typically characterized by the presence of extended arrays of tandem repeats called satellite DNA. Here, we describe the isolation and characterization of a 137-bp-long new satellite DNA sequence from the horse genome (EC137), which is also present, even if less abundant, in the domestic donkey, the Grevy's zebra and the Burchelli's zebra. We investigated the chromosomal distribution of the EC137 sequence in these 4 species. Moreover, we analyzed its architectural organization by high-resolution FISH. The position of this sequence with respect to the primary constriction and in relation to the 2 major horse satellite tandem repeats (37cen and 2PI) on horse chromosomes suggests that the new centromeric equine satellite is an accessory DNA element, presumably contributing to the organization of pericentromeric chromatin. FISH on combed DNA fibers reveals that the EC137 satellite is organized in relatively short stretches (2-8 kb) which are strictly intermingled within 37cen or 2PI arrays. This arrangement suggests that interchanges between satellite families are a frequent occurrence in the horse genome.

Summary The insertion of mitochondrial DNA in the nuclear genome generates numts, nuclearsequences of mitochondrial origin. In the horse reference genome, we identified 82 numtsand showed that the entire horse mitochondrial DNA is represented as numts without grossbias. Numts were inserted in the horse nuclear genome at random sites and were probablygenerated during the repair of DNA double-strand breaks. We then analysed 12 numt lociin 20 unrelated horses and found that null alleles, lacking the mitochondrial DNA insertion,were present at six of these loci. At some loci, the null allele is prevalent in the sampleanalysed, suggesting that, in the horse population, the number of numt loci may be higherthan 82 present in the reference genome. Contrary to humans, the insertion polymorphismof numts is extremely frequent in the horse population, supporting the hypothesis that thegenome of this species is in a rapidly evolving state.Keywords horse genome, insertion polymorphism, mitochondrial DNA insertion.

The longstanding dogma that telomeres, the heterochromatic extremities of linear eukaryotic chromosomes, are transcriptionally silent was overturned by the discovery that DNA-dependent RNA polymerase II (RNAPII) transcribes telomeric DNA into telomeric repeat-containing RNA (TERRA). Here, we show that CpG dinucleotide-rich DNA islands, shared among multiple human chromosome ends, promote transcription of TERRA molecules. TERRA promoters sustain cellular expression of reporter genes, are located immediately upstream of TERRA transcription start sites, and are bound by active RNAPII in vivo. Finally, the identified promoter CpG dinucleotides are methylated in vivo, and cytosine methylation negatively regulates TERRA abundance. The existence of subtelomeric promoters, driving TERRA transcription from independent chromosome ends, supports the idea that TERRA exerts fundamental functions in the context of telomere biology.

Telomeres are ribonucleoprotein structures at the end of chromosomes composed of telomeric DNA, specific-binding proteins, and noncoding RNA (TERRA). Despite their importance in preventing chromosome instability, little is known about the cross talk between these three elements during the formation of the germ line. Here, we provide evidence that both TERRA and the telomerase enzymatic subunit (TERT) are components of telomeres in mammalian germ cells. We found that TERRA colocalizes with telomeres during mammalian meiosis and that its expression progressively increases during spermatogenesis until the beginning of spermiogenesis. While both TERRA levels and distribution would be regulated in a gender-specific manner, telomere-TERT colocalization appears to be regulated based on species-specific characteristics of the telomeric structure. Moreover, we found that TERT localization at telomeres is maintained throughout spermatogenesis as a structural component without affecting telomere elongation. Our results represent the first evidence of colocalization between telomerase and telomeres during mammalian gametogenesis.

Mammalian telomeres are transcribed into long non-coding telomeric RNA molecules (TERRA) that seem to play a role in the maintenance of telomere stability. In human cells, CpG island promoters drive TERRA transcription and are regulated by methylation. It was suggested that the amount of TERRA may be related to telomere length. To test this hypothesis we measured telomere length and TERRA levels in single clones isolated from five human cell lines: HeLa (cervical carcinoma), BRC-230 (breast cancer), AKG and GK2 (gastric cancers) and GM847 (SV40 immortalized skin fibroblasts). We observed great clonal heterogeneity both in TRF (Terminal Restriction Fragment) length and in TERRA levels. However, these two parameters did not correlate with each other. Moreover, cell survival to γ-rays did not show a significant variation among the clones, suggesting that, in this cellular system, the intra-population variability in telomere length and TERRA levels does not influence sensitivity to ionizing radiation. This conclusion was supported by the observation that in a cell line in which telomeres were greatly elongated by the ectopic expression of telomerase, TERRA expression levels and radiation sensitivity were similar to the parental HeLa cell line.

We appreciated the commentary of Van Beneden et al. (1) on our article (2) because is giving us the opportunity to discuss the advantages and limitations of the various methods currently in use to measure TElomeric Repeat containing RNA (TERRA) cellular levels and to present new data supporting our previous conclusions. Which is the most appropriate method to analyze TERRA levels? With qRT-PCR, specific primer pairs are used to amplify reverse-transcribed fragments complementary to a portion of the subtelomeric region adjacent to the telomere; the number of transcripts containing subtelomeric fragments is measured while no information on the number of UUAGGG repeats within TERRA molecules is obtained. The qRT-PCR method has been extensively used by several groups, including ours; however, we can identify several limitations: (1) primers are constructed on subtelomeric sequences (3), thus, very short and possibly functionally irrelevant RNA molecules containing only a few UUAGGG repeats are detected together with molecules containing large numbers of repeats. However, the mechanisms of TERRA processing and the structure of physiologically relevant molecules have not been clarified yet. Most likely the function of TERRA is related to the UUAGGG repeats for the following reasons: (i) the subtelomeric tract contained in TERRA molecules is relatively short while the UUAGGG repeats can reach several kilobases; in particular, in TERRA molecules transcribed from the XqYq human subtelomere, the distance between the transcription start site and the first telomeric repeat is 257 nt (4, 5); (ii) UUAGGG oligonucleotides interact with several telomere associated proteins, including TRF1 and TRF2 (6). Using mass spectrometry, it was demonstrated that different members of the heterogeneous nuclear ribonucleoprotein family bind abundantly to TERRA repeats (7, 8) and, more recently, 115 proteins, specifically binding to UUAGGG repeats, were identified (9). (iii) The UUAGGG repeats of TERRA molecules are able to fold into G-quadruplex structures (10) that are required for the binding of TERRA to chromatin (11). (iv) TERRA repeats can inhibit the telomerase enzymatic activity in vitro (12) through base pairing with the telomeric repeat template but their role in the regulation of telomerase in vivo is more controversial (8, 13). (2) Due to the repetitive nature of subtelomeric sequences, it has been often impossible to design primers specific for single subtelomeres; indeed it should be kept in mind that most primer pairs used so far amplify fragments from more than one subtelomere (3, 14, 15). (3) Not all human subtelomeric sequences have been fully assembled (15, 16) and to specifically analyze their transcription remains a challenge; therefore, until we have specific primer sets for each subtelomere, quantification of TERRA molecules by qRT-PCR will not reflect the whole TERRA transcriptome. (4) TERRA promoters and putative promoter start sites have been identified only on 20 human subtelomeres (4) and the transcriptional regulation of the remaining subtelomeres still needs to be elucidated. (5) Quantification of TERRA expression using qRT-PCR on transformed heteroploid cell lines may be biased by variations in the number of chromosome ends recognized by each primer pair. (6) The contribution of each subtelomere to total TERRA is variable, depending on its transcription efficiency (2, 15). It is also important to point out that, since telomere length (17) and TERRA transcription vary from end to end, we should be able to measure both of them at single chromosome-end level to precisely define the relationship between these two parameters. Comparing average telomere length with the expression of a few subtelomeric regions may be misleading. Regarding northern blotting, as clearly shown by the Decottignies group (1, 14) the visualization of high molecular weight RNA molecules is favored by alkaline treatment of the gels. Using this approach, these authors observed the appearance of high molecular weight TERRA molecules in cell lines in which telomeres were artificially hyper-elongated by ectopic expression of telomerase holoenzyme; in parallel, measuring TERRA levels by qRT-PCR, they concluded that, in the cell lines with longer telomeres, TERRA expression was reduced to 50%. However, the comparison between the qRT-PCR and the northern blotting results is confusing [Figures 1B,E in Ref. (1)]. In the northern blots, the appearance of a high molecular weight (>5.3 kb) TERRA fraction in cells with long telomeres does not parallel a loss of the lower molecular weight molecules (