Label-Free Human Disease Characterization through Circulating Cell-Free DNA Analysis Using Raman Spectroscopy.

Circulating cell-free DNA (ccfDNA) is a liquid biopsy biomaterial attracting significant attention for the implementation of precision medicine diagnostics. Deeper knowledge related to its structure and biology would enable the development of such applications. In this study, we employed Raman spectroscopy to unravel the biomolecular profile of human ccfDNA in health and disease. We established reference Raman spectra of ccfDNA samples from healthy males and females with different conditions, including cancer and diabetes, extracting information about their chemical composition. Comparative observations showed a distinct spectral pattern in ccfDNA from breast cancer patients taking neoadjuvant therapy. Raman analysis of ccfDNA from healthy, prediabetic, and diabetic males uncovered some differences in their biomolecular fingerprints. We also studied ccfDNA released from human benign and cancer cell lines and compared it to their respective gDNA, confirming it mirrors its cellular origin. Overall, we explored for the first time Raman spectroscopy in the study of ccfDNA and provided spectra of samples from different sources. Our findings introduce Raman spectroscopy as a new approach to implementing liquid biopsy diagnostics worthy of further elaboration.

Nucleotide Excision Repair and Transcription-Associated Genome Instability.

Transcription is a potential threat to genome integrity, and transcription-associated DNA damage must be repaired for proper messenger RNA (mRNA) synthesis and for cells to transmit their genome intact into progeny. For a wide range of structurally diverse DNA lesions, cells employ the highly conserved nucleotide excision repair (NER) pathway to restore their genome back to its native form. Recent evidence suggests that NER factors function, in addition to the canonical DNA repair mechanism, in processes that facilitate mRNA synthesis or shape the 3D chromatin architecture. Here, these findings are critically discussed and a working model that explains the puzzling clinical heterogeneity of NER syndromes highlighting the relevance of physiological, transcription-associated DNA damage to mammalian development and disease is proposed.

Spatial proximity of homologous alleles and long noncoding RNAs regulate a switch in allelic gene expression.

Physiological processes rely on the regulation of total mRNA levels in a cell. In diploid organisms, the transcriptional activation of one or both alleles of a gene may involve trans-allelic interactions that provide a tight spatial and temporal level of gene expression regulation. The mechanisms underlying such interactions still remain poorly understood. Here, we demonstrate that lipopolysaccharide stimulation of murine macrophages rapidly resulted in the actin-mediated and transient homologous spatial proximity of Tnfα alleles, which was necessary for the mono- to biallelic switch in gene expression. We identified two new complementary long noncoding RNAs transcribed from the TNFα locus and showed that their knockdown had opposite effects in Tnfα spatial proximity and allelic expression. Moreover, the observed spatial proximity of Tnfα alleles depended on pyruvate kinase muscle isoform 2 (PKM2) and T-helper-inducing POZ-Krüppel-like factor (ThPOK). This study suggests a role for lncRNAs in the regulation of somatic homologous spatial proximity and allelic expression control necessary for fine-tuning mammalian immune responses.

The Nucleolus: In Genome Maintenance and Repair.

The nucleolus is the subnuclear membrane-less organelle where rRNA is transcribed and processed and ribosomal assembly occurs. During the last 20 years, however, the nucleolus has emerged as a multifunctional organelle, regulating processes that go well beyond its traditional role. Moreover, the unique organization of rDNA in tandem arrays and its unusually high transcription rates make it prone to unscheduled DNA recombination events and frequent RNA:DNA hybrids leading to DNA double strand breaks (DSBs). If not properly repaired, rDNA damage may contribute to premature disease onset and aging. Deregulation of ribosomal synthesis at any level from transcription and processing to ribosomal subunit assembly elicits a stress response and is also associated with disease onset. Here, we discuss how genome integrity is maintained within nucleoli and how such structures are functionally linked to nuclear DNA damage response and repair giving an emphasis on the newly emerging roles of the nucleolus in mammalian physiology and disease.

Looping forward: exploring R-loop processing and therapeutic potential.

Recently, there has been increasing interest in the complex relationship between transcription and genome stability, with specific attention directed toward the physiological significance of molecular structures known as R-loops. These structures arise when an RNA strand invades into the DNA duplex, and their formation is involved in a wide range of regulatory functions affecting gene expression, DNA repair processes or cell homeostasis. The persistent presence of R-loops, if not effectively removed, contributes to genome instability, underscoring the significance of the factors responsible for their resolution and modification. In this review, we provide a comprehensive overview of how R-loop processing can drive either a beneficial or a harmful outcome. Additionally, we explore the potential for manipulating such structures to devise rationalized therapeutic strategies targeting the aberrant accumulation of R-loops.

Transcription stress at telomeres leads to cytosolic DNA release and paracrine senescence.

Transcription stress has been linked to DNA damage -driven aging, yet the underlying mechanism remains unclear. Here, we demonstrate that Tcea1-/- cells, which harbor a TFIIS defect in transcription elongation, exhibit RNAPII stalling at oxidative DNA damage sites, impaired transcription, accumulation of R-loops, telomere uncapping, chromatin bridges, and genome instability, ultimately resulting in cellular senescence. We found that R-loops at telomeres causally contribute to the release of telomeric DNA fragments in the cytoplasm of Tcea1-/- cells and primary cells derived from naturally aged animals triggering a viral-like immune response. TFIIS-defective cells release extracellular vesicles laden with telomeric DNA fragments that target neighboring cells, which consequently undergo cellular senescence. Thus, transcription stress elicits paracrine signals leading to cellular senescence, promoting aging.

High levels of p110δ PI3K expression in solid tumor cells suppress PTEN activity, generating cellular sensitivity to p110δ inhibitors through PTEN activation.

Class IA PI3K isoforms have divergent, nonredundant cell biological roles. In untransformed cells and tissues, p110α and p110ß are ubiquitously expressed, whereas p110δ expression is highly enriched in leukocytes. High levels of p110δ expression have been documented in some solid tumor cell lines, but the functional role is unknown. This study aimed to elucidate the link between elevated expression of p110δ PI3K and cancer. We report that in breast and prostate cancer cells that contain leukocyte levels of p110δ, p110δ activity dampens the activity of the PTEN tumor suppressor. Indeed, inactivation of p110δ in these cells led to PTEN activation, suppression of Akt phosphorylation, and inhibition of cell proliferation, with inhibition of PTEN activity being able to counterbalance p110δ inactivation. Likewise, forced overexpression of p110δ in cells with low p110δ expression reduced PTEN activity, resulting in increased Akt phosphorylation. Our data indicate that the oncogenic potential of p110δ PI3K overexpression might at least partially act through PTEN inactivation, and that p110δ-selective PI3K inhibitors can have a dual antitumor mechanism, namely by directly inhibiting p110δ signaling and by a broader inhibition of class I PI3K activity through PTEN activation. These data may have important implications in the intervention of breast cancer.

The DREAM complex functions as conserved master regulator of somatic DNA-repair capacities.

The DNA-repair capacity in somatic cells is limited compared with that in germ cells. It has remained unknown whether not only lesion-type-specific, but overall repair capacities could be improved. Here we show that the DREAM repressor complex curbs the DNA-repair capacities in somatic tissues of Caenorhabditis elegans. Mutations in the DREAM complex induce germline-like expression patterns of multiple mechanisms of DNA repair in the soma. Consequently, DREAM mutants confer resistance to a wide range of DNA-damage types during development and aging. Similarly, inhibition of the DREAM complex in human cells boosts DNA-repair gene expression and resistance to distinct DNA-damage types. DREAM inhibition leads to decreased DNA damage and prevents photoreceptor loss in progeroid Ercc1-/- mice. We show that the DREAM complex transcriptionally represses essentially all DNA-repair systems and thus operates as a highly conserved master regulator of the somatic limitation of DNA-repair capacities.

XPF interacts with TOP2B for R-loop processing and DNA looping on actively transcribed genes.

Co-transcriptional RNA-DNA hybrids can not only cause DNA damage threatening genome integrity but also regulate gene activity in a mechanism that remains unclear. Here, we show that the nucleotide excision repair factor XPF interacts with the insulator binding protein CTCF and the cohesin subunits SMC1A and SMC3, leading to R-loop-dependent DNA looping upon transcription activation. To facilitate R-loop processing, XPF interacts and recruits with TOP2B on active gene promoters, leading to double-strand break accumulation and the activation of a DNA damage response. Abrogation of TOP2B leads to the diminished recruitment of XPF, CTCF, and the cohesin subunits to promoters of actively transcribed genes and R-loops and the concurrent impairment of CTCF-mediated DNA looping. Together, our findings disclose an essential role for XPF with TOP2B and the CTCF/cohesin complex in R-loop processing for transcription activation with important ramifications for DNA repair-deficient syndromes associated with transcription-associated DNA damage.

DNA damage, inflammation and aging: Insights from mice.

Persistent DNA lesions build up with aging triggering inflammation, the body's first line of immune defense strategy against foreign pathogens and irritants. Once established, DNA damage-driven inflammation takes on a momentum of its own, due to the amplification and feedback loops of the immune system leading to cellular malfunction, tissue degenerative changes and metabolic complications. Here, we discuss the use of murine models with inborn defects in genome maintenance and the DNA damage response for understanding how irreparable DNA lesions are functionally linked to innate immune signaling highlighting their relevance for developing novel therapeutic strategies against the premature onset of aging-associated diseases.

R-loops trigger the release of cytoplasmic ssDNAs leading to chronic inflammation upon DNA damage.

How DNA damage leads to chronic inflammation and tissue degeneration with aging remains to be fully resolved. Here, we show that DNA damage leads to cellular senescence, fibrosis, loss-of-tissue architecture, and chronic pancreatitis in mice with an inborn defect in the excision repair cross complementation group 1 (Ercc1) gene. We find that DNA damage-driven R-loops causally contribute to the active release and buildup of single-stranded DNAs (ssDNAs) in the cytoplasm of cells triggering a viral-like immune response in progeroid and naturally aged pancreata. To reduce the proinflammatory load, we developed an extracellular vesicle (EV)-based strategy to deliver recombinant S1 or ribonuclease H nucleases in inflamed Ercc1−/− pancreatic cells. Treatment of Ercc1−/− animals with the EV-delivered nuclease cargo eliminates DNA damage-induced R-loops and cytoplasmic ssDNAs alleviating chronic inflammation. Thus, DNA damage-driven ssDNAs causally contribute to tissue degeneration, Ercc1−/− paving the way for novel rationalized intervention strategies against age-related chronic inflammation.

The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing.

RNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.

Tissue-infiltrating macrophages mediate an exosome-based metabolic reprogramming upon DNA damage.

DNA damage and metabolic disorders are intimately linked with premature disease onset but the underlying mechanisms remain poorly understood. Here, we show that persistent DNA damage accumulation in tissue-infiltrating macrophages carrying an ERCC1-XPF DNA repair defect (Er1F/-) triggers Golgi dispersal, dilation of endoplasmic reticulum, autophagy and exosome biogenesis leading to the secretion of extracellular vesicles (EVs) in vivo and ex vivo. Macrophage-derived EVs accumulate in Er1F/- animal sera and are secreted in macrophage media after DNA damage. The Er1F/- EV cargo is taken up by recipient cells leading to an increase in insulin-independent glucose transporter levels, enhanced cellular glucose uptake, higher cellular oxygen consumption rate and greater tolerance to glucose challenge in mice. We find that high glucose in EV-targeted cells triggers pro-inflammatory stimuli via mTOR activation. This, in turn, establishes chronic inflammation and tissue pathology in mice with important ramifications for DNA repair-deficient, progeroid syndromes and aging.

Intron 4 a/b polymorphism of the endothelial nitric oxide synthase gene is associated with both type 1 and type 2 diabetes in a genetically homogeneous population.

Current classifications of diabetes distinguish between type 1 diabetes (T1D) and type 2 diabetes (T2D), however recent evidence highlights overlap between T1D and T2D. Earlier studies have suggested altered nitric oxide (NO) metabolism in both T1D and T2D. In the present case-control study, we investigated whether the endothelial NO synthase gene intron 4 a/b polymorphism is associated with T1D and T2D in the island of Crete, a well-defined area with genetically homogeneous population. Mutated allele "a" was more common in individuals with both T1D and T2D than in controls (odds ratio [OR] = 1.71, 95% confidence interval [CI] = 1.06-2.77, p = 0.013; and OR = 1.50, 95% CI = 0.930-2.42, p = 0.047, respectively). Mutated genotype (a/a or a/b) was more common in individuals with T1D than in nondiabetic individuals (OR = 1.93, 95% CI = 1.12-3.32, p = 0.008); this increased frequency was also observed for T2D, although not at a significant level (OR = 1.38, 95% CI = 0.802-2.37). No difference was found in the frequency of mutated allele a or mutated genotype (a/a or a/b) between T1D and T2D populations. In conclusion, our results indicate that allele a of the intron 4 endothelial NO synthase gene is associated with susceptibility to both T1D and T2D and may represent a common genetic factor for diabetes.

DNA damage-induced inflammation and nuclear architecture.

Nuclear architecture and the chromatin state affect most-if not all- DNA-dependent transactions, including the ability of cells to sense DNA lesions and restore damaged DNA back to its native form. Recent evidence points to functional links between DNA damage sensors, DNA repair mechanisms and the innate immune responses. The latter raises the question of how such seemingly disparate processes operate within the intrinsically complex nuclear landscape and the chromatin environment. Here, we discuss how DNA damage-induced immune responses operate within chromatin and the distinct sub-nuclear compartments highlighting their relevance to chronic inflammation.

Long non-coding RNA SeT and miR-155 regulate the Tnfα gene allelic expression profile.

It is becoming increasingly appreciated that the non-coding genome may have a great impact on the regulation of chromatin structure and gene expression. The innate immune response can be mediated upon lipopolysaccharide stimulation of macrophages which leads to immediate transcriptional activation of early responsive genes including tumor necrosis factor alpha (Tnfα). The functional role of non-coding RNAs, such as lncRNAs and microRNAs, on the transcriptional activation of proinflammatory genes and the subsequent regulation of the innate immune response is still lacking mechanistic insights. In this study we wanted to unravel the functional role of the lncRNA SeT, which is encoded from the murine Tnfα gene locus, and miR-155 on the transcriptional regulation of the Tnfα gene. We utilized genetically modified mice harboring either a deletion of the SeT promoter elements or the mature miR-155 and studied the response of macrophages to lipopolysaccharide (LPS) stimulation. We found that decreased expression of the lncRNA SeT in murine primary macrophages resulted in increased mortality of mice challenged with LPS, which was corroborated by increased Tnfα steady state mRNA levels and a higher frequency of biallelically expressing macrophages. On the contrary, miR-155 deletion resulted in reduced Tnfα mRNA levels supported by a lower frequency of biallelically expressing macrophages upon stimulation with LPS. In both cases, in the absence of either lncRNA SeT or miR-155 we observed a deregulation of the Tnfα allele homologous pairing, previously shown to regulate the switch from mono- to bi-allelic gene expression. Although lncRNA SeT was not found to be a direct target of miR-155 its stability was increased upon miR-155 deletion. This study suggests a role of the non-coding genome in mediating Tnfα mRNA dosage control based on the regulation of homologous pairing of gene alleles and their subsequent biallelic expression.