Enhanced frequency of transcription pre-initiation complexes assembly after exposure to UV irradiation results in increased repair activity and reduced probabilities for mutagenesis.

In addition to being essential for gene expression, transcription is crucial for the maintenance of genome integrity. Here, we undertook a systematic approach, to monitor the assembly kinetics of the pre-initiating RNA Polymerase (Pol) II at promoters at steady state and different stages during recovery from UV irradiation-stress, when pre-initiation and initiation steps have been suggested to be transiently shut down. Taking advantage of the reversible dissociation of pre-initiating Pol II after high salt treatment, we found that de novo recruitment of the available Pol II molecules at active promoters not only persists upon UV at all times tested but occurs significantly faster in the early phase of recovery (2 h) than in unexposed human fibroblasts at the majority of active genes. Our method unveiled groups of genes with significantly different pre-initiation complex (PIC) assembly dynamics after UV that present distinct rates of UV-related mutational signatures in melanoma tumours, providing functional relevance to the importance of keeping transcription initiation active during UV recovery. Our findings uncover novel mechanistic insights further detailing the multilayered transcriptional response to genotoxic stress and link PIC assembly dynamics after exposure to genotoxins with cancer mutational landscapes.

Loss of Kmt2c in vivo leads to EMT, mitochondrial dysfunction and improved response to lapatinib in breast cancer.

Deep sequencing of human tumours has uncovered a previously unappreciated role for epigenetic regulators in tumorigenesis. H3K4 methyltransferase KMT2C/MLL3 is mutated in several solid malignancies, including more than 10% of breast tumours. To study the tumour suppressor role of KMT2C in breast cancer, we generated mouse models of Erbb2/Neu, Myc or PIK3CA-driven tumorigenesis, in which the Kmt2c locus is knocked out specifically in the luminal lineage of mouse mammary glands using the Cre recombinase. Kmt2c knock out mice develop tumours earlier, irrespective of the oncogene, assigning a bona fide tumour suppressor role for KMT2C in mammary tumorigenesis. Loss of Kmt2c induces extensive epigenetic and transcriptional changes, which lead to increased ERK1/2 activity, extracellular matrix re-organization, epithelial-to-mesenchymal transition and mitochondrial dysfunction, the latter associated with increased reactive oxygen species production. Loss of Kmt2c renders the Erbb2/Neu-driven tumours more responsive to lapatinib. Publicly available clinical datasets revealed an association of low Kmt2c gene expression and better long-term outcome. Collectively, our findings solidify the role of KMT2C as a tumour suppressor in breast cancer and identify dependencies that could be therapeutically amenable.

MAP3K8 Regulates Cox-2-Mediated Prostaglandin E2 Production in the Lung and Suppresses Pulmonary Inflammation and Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by exuberant deposition of extracellular matrix components, leading to the deterioration of lung architecture and respiratory functions. Profibrotic mechanisms are controlled by multiple regulatory molecules, including MAPKs, in turn regulated by multiple phosphorylation cascades. MAP3K8 is an MAPK kinase kinase suggested to pleiotropically regulate multiple pathogenic pathways in the context of inflammation and cancer; however, a possible role in the pathogenesis of IPF has not been investigated. In this report, MAP3K8 mRNA levels were found decreased in the lungs of IPF patients and of mice upon bleomycin-induced pulmonary fibrosis. Ubiquitous genetic deletion of Map3k8 in mice exacerbated the modeled disease, whereas bone marrow transfer experiments indicated that although MAP3K8 regulatory functions are active in both hematopoietic and nonhematopoietic cells, Map3k8 in hematopoietic cells has a more dominant role. Macrophage-specific deletion of Map3k8 was further found to be sufficient for disease exacerbation thus confirming a major role for macrophages in pulmonary fibrotic responses and suggesting a main role for Map3k8 in the homeostasis of their effector functions in the lung. Map3k8 deficiency was further shown to be associated with decreased Cox-2 expression, followed by a decrease in PGE2 production in the lung; accordingly, exogenous administration of PGE2 reduced inflammation and reversed the exacerbated fibrotic profile of Map3k8 -/- mice. Therefore, MAP3K8 has a central role in the regulation of inflammatory responses and Cox-2-mediated PGE2 production in the lung, and the attenuation of its expression is integral to pulmonary fibrosis development.

aniFOUND: analysing the associated proteome and genomic landscape of the repaired nascent non-replicative chromatin.

Specific capture of chromatin fractions with distinct and well-defined features has emerged as both challenging and a key strategy towards a comprehensive understanding of genome biology. In this context, we developed aniFOUND (accelerated native isolation of factors on unscheduled nascent DNA), an antibody-free method, which can label, capture, map and characterise nascent chromatin fragments that are synthesized in response to specific cues outside S-phase. We used the 'unscheduled' DNA synthesis (UDS) that takes place during the repair of UV-induced DNA lesions and coupled the captured chromatin to high-throughput analytical technologies. By mass-spectrometry we identified several factors with no previously known role in UVC-DNA damage response (DDR) as well as known DDR proteins. We experimentally validated the repair-dependent recruitment of the chromatin remodeller RSF1 and the cohesin-loader NIPBL at sites of UVC-induced photolesions. Developing aniFOUND-seq, a protocol for mapping UDS activity with high resolution, allowed us to monitor the landscape of UVC repair-synthesis events genome wide. We further resolved repair efficacy of the rather unexplored repeated genome, in particular rDNA and telomeres. In summary, aniFOUND delineates the proteome composition and genomic landscape of chromatin loci with specific features by integrating state-of-the-art 'omics' technologies to promote a comprehensive view of their function.

Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α-dependent pathways.

In multiple sclerosis (MS), Th17 cells are critical drivers of autoimmune central nervous system (CNS) inflammation and demyelination. Th17 cells exhibit functional heterogeneity fostering both pathogenic and nonpathogenic, tissue-protective functions. Still, the factors that control Th17 pathogenicity remain incompletely defined. Here, using experimental autoimmune encephalomyelitis, an established mouse MS model, we report that therapeutic administration of activin-A ameliorates disease severity and alleviates CNS immunopathology and demyelination, associated with decreased activation of Th17 cells. In fact, activin-A signaling through activin-like kinase-4 receptor represses pathogenic transcriptional programs in Th17-polarized cells, while it enhances antiinflammatory gene modules. Whole-genome profiling and in vivo functional studies revealed that activation of the ATP-depleting CD39 and CD73 ectonucleotidases is essential for activin-A-induced suppression of the pathogenic signature and the encephalitogenic functions of Th17 cells. Mechanistically, the aryl hydrocarbon receptor, along with STAT3 and c-Maf, are recruited to promoter elements on Entpd1 and Nt5e (encoding CD39 and CD73, respectively) and other antiinflammatory genes, and control their expression in Th17 cells in response to activin-A. Notably, we show that activin-A negatively regulates the metabolic sensor, hypoxia-inducible factor-1α, and key inflammatory proteins linked to pathogenic Th17 cell states. Of translational relevance, we demonstrate that activin-A is induced in the CNS of individuals with MS and restrains human Th17 cell responses. These findings uncover activin-A as a critical controller of Th17 cell pathogenicity that can be targeted for the suppression of autoimmune CNS inflammation.

Activin-A co-opts IRF4 and AhR signaling to induce human regulatory T cells that restrain asthmatic responses.

Type 1 regulatory T (Tr1) cells play a pivotal role in restraining human T-cell responses toward environmental allergens and protecting against allergic diseases. Still, the precise molecular cues that underlie their transcriptional and functional specification remain elusive. Here, we show that the cytokine activin-A instructs the generation of CD4+ T cells that express the Tr1-cell-associated molecules IL-10, inducible T-Cell costimulator (ICOS), lymphocyte activation gene 3 protein (LAG-3), and CD49b, and exert strongly suppressive functions toward allergic responses induced by naive and in vivo-primed human T helper 2 cells. Moreover, mechanistic studies reveal that activin-A signaling induces the activation of the transcription factor interferon regulatory factor (IRF4), which, along with the environmental sensor aryl hydrocarbon receptor, forms a multipartite transcriptional complex that binds in IL-10 and ICOS promoter elements and controls gene expression in human CD4+ T cells. In fact, IRF4 silencing abrogates activin-A-driven IL10 and ICOS up-regulation and impairs the suppressive functions of human activin-A-induced Tr1-like (act-A-iTr1) cells. Importantly, using a humanized mouse model of allergic asthma, we demonstrate that adoptive transfer of human act-A-iTr1 cells, both in preventive and therapeutic protocols, confers significant protection against cardinal asthma manifestations, including pulmonary inflammation. Overall, our findings uncover an activin-A-induced IRF4-aryl hydrocarbon receptor (AhR)-dependent transcriptional network, which generates suppressive human Tr1 cells that may be harnessed for the control of allergic diseases.

Dendritic cells conditioned by activin A-induced regulatory T cells exhibit enhanced tolerogenic properties and protect against experimental asthma.

BACKGROUND: Previously, we demonstrated that regulatory T (Treg) cells induced by the cytokine activin-A suppress TH2-mediated allergic responses and linked airway disease. Still, the effects of activin-A-induced regulatory T (Act-A-iTreg) cells on the regulation of dendritic cell (DC)-driven allergic inflammation remain elusive. OBJECTIVE: Here we investigated whether Act-A-iTreg cells can modulate DC responses and endow them with enhanced tolerogenic functions. METHODS: Using adoptive cell transfer studies in mouse models of allergic airway disease, we examined the effects of Act-A-iTreg cells on DC phenotype, maturation status, and TH2 cell priming potential. Genome-wide gene expression profiling characterized the transcriptional networks induced in tolerogenic DCs by Act-A-iTreg cells. The ability of DCs conditioned by Act-A-iTreg cells (Act-A-iTreg cell-modified DCs) to protect against experimental asthma, and the mechanisms involved were also explored. RESULTS: Act-A-iTreg cell-modified DCs exhibited a significantly impaired capacity to uptake allergen and stimulate naive and TH2 effector responses on allergen stimulation in vivo accompanied by markedly attenuated inflammatory cytokine release in response to LPS. Gene-profiling studies revealed that Act-A-iTreg cells dampened crucial TH2-skewing transcriptional networks in DCs. Administration of Act-A-iTreg cell-modified DCs ameliorated cardinal asthma manifestations in preventive and therapeutic protocols through generation of strongly suppressive forkhead box P3+ Treg cells. Finally, programed death protein 1/programmed death ligand 1 signaling pathways were essential in potentiating the generation of DCs with tolerogenic properties by Act-A-iTreg cells. CONCLUSION: Our studies reveal that Act-A-iTreg cells instruct the generation of a highly effective immunoregulatory circuit encompassing tolerogenic DCs and forkhead box P3+ Treg cells that could be targeted for the design of novel immunotherapies for allergic disorders.

Mir221/222 drive synovial hyperplasia and arthritis by targeting cell cycle inhibitors and chromatin remodeling components.

miRNAs constitute fine-tuners of gene expression and are implicated in a variety of diseases spanning from inflammation to cancer. miRNA expression is deregulated in rheumatoid arthritis (RA); however, their specific role in key arthritogenic cells such as the synovial fibroblast (SF) remains elusive. Previous studies have shown that Mir221/222 expression is upregulated in RA SFs. Here, we demonstrate that TNF and IL-1ß but not IFN-γ activated Mir221/222 gene expression in murine SFs. SF-specific overexpression of Mir221/222 in huTNFtg mice led to further expansion of SFs and disease exacerbation, while its total ablation led to reduced SF expansion and attenuated disease. Mir221/222 overexpression altered the SF transcriptional profile igniting pathways involved in cell cycle and ECM (extracellular matrix) regulation. Validation of targets of Mir221/222 revealed cell cycle inhibitors Cdkn1b and Cdkn1c, as well as the epigenetic regulator Smarca1. Single-cell ATAC-seq data analysis revealed increased Mir221/222 gene activity in pathogenic SF subclusters and transcriptional regulation by Rela, Relb, Junb, Bach1, and Nfe2l2. Our results establish an SF-specific pathogenic role of Mir221/222 in arthritis and suggest that its therapeutic targeting in specific subpopulations could lead to novel fibroblast-targeted therapies.

Cockayne syndrome B protein is implicated in transcription and associated chromatin dynamics in homeostatic and genotoxic conditions.

The integrity of the actively transcribed genome against helix-distorting DNA lesions relies on a multilayered cellular response that enhances Transcription-Coupled Nucleotide Excision Repair (TC-NER). When defective, TC-NER is causatively associated with Cockayne-Syndrome (CS), a rare severe human progeroid disorder. Although the presence of unresolved transcription-blocking lesions is considered a driver of the aging process, the molecular features of the transcription-driven response to genotoxic stress in CS-B cells remain largely unknown. Here, an in-depth view of the transcriptional and associated chromatin dynamics that occur in CS-B cells illuminates the role of CSB therein. By employing high-throughput genome-wide approaches, we observed that absence of a functional CSB protein results in a delay in transcription progression, more positioned +1 nucleosomes, and less dynamic chromatin structure, compared to normal cells. We found that early after exposure to UV, CS-B cells released RNA polymerase II (RNAPII) from promoter-proximal pause sites into elongation. However, the magnitude of this response and the progression of RNAPII were reduced compared to normal counterparts. Notably, we detected increased post-UV retainment of unprocessed nascent RNA transcripts and chromatin-associated elongating RNAPII molecules. Contrary to the prevailing models, we found that transcription initiation is operational in CS-B fibroblasts early after UV and that chromatin accessibility showed a marginal increase. Our study provides robust evidence for the role of CSB in shaping the transcription and chromatin landscape both in homeostasis and in response to genotoxic insults, which is independent of its known role in TC-NER, and which may underlie major aspects of the CS phenotype.

Histone H2Bub dynamics in the 5' region of active genes are tightly linked to the UV-induced transcriptional response.

The timing and location of writing and erasing of histone modifications determine gene expression programs and are tightly controlled processes. One such modification is the monoubiquitination of histone H2B (H2Bub), whose precise level during transcription elongation is dynamically regulated by the synergistic action of RNF20/40 ubiquitin-ligase and the de-ubiquitinase (DUB) of the ATXN7L3-containing DUB modules. Here, we characterize the dynamics of H2Bub in transcription and explore its role in perspective with the recently updated model of UV damage-induced transcription reorganization. Employing integrative analysis of genome-wide high-throughput approaches, transcription inhibitors and ATXN7L3-DUB knockdown cells, we find that H2Bub levels and patterns depend on intron-exon architecture both in steady state and upon UV. Importantly, our analysis reveals a widespread redistribution of this histone mark, rather than a uniform loss as previously suggested, which closely mirrors the post-UV dynamics of elongating RNA Polymerase II (RNAPII) at transcribed loci. The observed effects are due to a direct inter-dependence on RNAPII local concentration and speed, and we show that deficient ATXN7L3-mediated DUB activity leads to increased elongation rates in both non-irradiated and irradiated conditions. Our data and the implementation of a high-resolution computational framework reveal that the H2Bub pattern follows that of RNAPII, both in the ATXNL3 knockdown and in response to UV guaranteeing faithful elongation speed, especially in the context of the transcription-driven DNA damage response.

SCALA: A complete solution for multimodal analysis of single-cell Next Generation Sequencing data.

Analysis and interpretation of high-throughput transcriptional and chromatin accessibility data at single-cell (sc) resolution are still open challenges in the biomedical field. The existence of countless bioinformatics tools, for the different analytical steps, increases the complexity of data interpretation and the difficulty to derive biological insights. In this article, we present SCALA, a bioinformatics tool for analysis and visualization of single-cell RNA sequencing (scRNA-seq) and Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq) datasets, enabling either independent or integrative analysis of the two modalities. SCALA combines standard types of analysis by integrating multiple software packages varying from quality control to the identification of distinct cell populations and cell states. Additional analysis options enable functional enrichment, cellular trajectory inference, ligand-receptor analysis, and regulatory network reconstruction. SCALA is fully parameterizable, presenting data in tabular format and producing publication-ready visualizations. The different available analysis modules can aid biomedical researchers in exploring, analyzing, and visualizing their data without any prior experience in coding. We demonstrate the functionality of SCALA through two use-cases related to TNF-driven arthritic mice, handling both scRNA-seq and scATAC-seq datasets. SCALA is developed in R, Shiny and JavaScript and is mainly available as a standalone version, while an online service of more limited capacity can be found at http://scala.pavlopouloslab.info or https://scala.fleming.gr.

Single-cell multimodal analysis identifies common regulatory programs in synovial fibroblasts of rheumatoid arthritis patients and modeled TNF-driven arthritis.

BACKGROUND: Synovial fibroblasts (SFs) are specialized cells of the synovium that provide nutrients and lubricants for the proper function of diarthrodial joints. Recent evidence appreciates the contribution of SF heterogeneity in arthritic pathologies. However, the normal SF profiles and the molecular networks that govern the transition from homeostatic to arthritic SF heterogeneity remain poorly defined. METHODS: We applied a combined analysis of single-cell (sc) transcriptomes and epigenomes (scRNA-seq and scATAC-seq) to SFs derived from naïve and hTNFtg mice (mice that overexpress human TNF, a murine model for rheumatoid arthritis), by employing the Seurat and ArchR packages. To identify the cellular differentiation lineages, we conducted velocity and trajectory analysis by combining state-of-the-art algorithms including scVelo, Slingshot, and PAGA. We integrated the transcriptomic and epigenomic data to infer gene regulatory networks using ArchR and custom-implemented algorithms. We performed a canonical correlation analysis-based integration of murine data with publicly available datasets from SFs of rheumatoid arthritis patients and sought to identify conserved gene regulatory networks by utilizing the SCENIC algorithm in the human arthritic scRNA-seq atlas. RESULTS: By comparing SFs from healthy and hTNFtg mice, we revealed seven homeostatic and two disease-specific subsets of SFs. In healthy synovium, SFs function towards chondro- and osteogenesis, tissue repair, and immune surveillance. The development of arthritis leads to shrinkage of homeostatic SFs and favors the emergence of SF profiles marked by Dkk3 and Lrrc15 expression, functioning towards enhanced inflammatory responses and matrix catabolic processes. Lineage inference analysis indicated that specific Thy1+ SFs at the root of trajectories lead to the intermediate Thy1+/Dkk3+/Lrrc15+ SF states and culminate in a destructive and inflammatory Thy1- SF identity. We further uncovered epigenetically primed gene programs driving the expansion of these arthritic SFs, regulated by NFkB and new candidates, such as Runx1. Cross-species analysis of human/mouse arthritic SF data determined conserved regulatory and transcriptional networks. CONCLUSIONS: We revealed a dynamic SF landscape from health to arthritis providing a functional genomic blueprint to understand the joint pathophysiology and highlight the fibroblast-oriented therapeutic targets for combating chronic inflammatory and destructive arthritic disease.

Continuous transcription initiation guarantees robust repair of all transcribed genes and regulatory regions.

Inhibition of transcription caused by DNA damage-impaired RNA polymerase II (Pol II) elongation conceals a local increase in de novo transcription, slowly progressing from Transcription Start Sites (TSSs) to gene ends. Although associated with accelerated repair of Pol II-encountered lesions and limited mutagenesis, it is still unclear how this mechanism is maintained during genotoxic stress-recovery. Here we uncover a widespread gain in chromatin accessibility and preservation of the active H3K27ac mark after UV-irradiation. The concomitant increase in Pol II escape from promoter-proximal pause (PPP) sites of most active genes, PROMPTs and enhancer RNAs favors unrestrained initiation, as evidenced by the synthesis of nascent RNAs including start RNAs. Accordingly, drug-inhibition of PPP-release replenishes levels of pre-initiating Pol II at TSSs after UV. Our data show that such continuous engagement of Pol II molecules ensures maximal transcription-driven repair throughout expressed genes and regulatory loci. Importantly, revealing this unanticipated regulatory layer of UV-response provides physiological relevant traction to the emerging concept that Pol II initiation rate is determined by pause-release dynamics.

Voltammetric fingerprinting of oils and its combination with chemometrics for the detection of extra virgin olive oil adulteration.

In the present work, two approaches for the voltammetric fingerprinting of oils and their combination with chemometrics were investigated in order to detect the adulteration of extra virgin olive oil with olive pomace oil as well as the most common seed oils, namely sunflower, soybean and corn oil. In particular, cyclic voltammograms of diluted extra virgin olive oils, regular (pure) olive oils (blends of refined olive oils with virgin olive oils), olive pomace oils and seed oils in presence of dichloromethane and 0.1 M of LiClO4 in EtOH as electrolyte were recorded at a glassy carbon working electrode. Cyclic voltammetry was also employed in methanolic extracts of olive and seed oils. Datapoints of cyclic voltammograms were exported and submitted to Principal Component Analysis (PCA), Partial Least Square- Discriminant Analysis (PLS-DA) and soft independent modeling of class analogy (SIMCA). In diluted oils, PLS-DA provided a clear discrimination between olive oils (extra virgin and regular) and olive pomace/seed oils, while SIMCA showed a clear discrimination of extra virgin olive oil in regard to all other samples. Using methanolic extracts and considering datapoints recorded between 0.6 and 1.3 V, PLS-DA provided more information, resulting in three clusters-extra virgin olive oils, regular olive oils and seed/olive pomace oils-while SIMCA showed inferior performance. For the quantification of extra virgin olive oil adulteration with olive pomace oil or seed oils, a model based on Partial Least Square (PLS) analysis was developed. Detection limit of adulteration in olive oil was found to be 2% (v/v) and the linearity range up to 33% (v/v). Validation and applicability of all models was proved using a suitable test set. In the case of PLS, synthetic oil mixtures with 4 known adulteration levels in the range of 4-26% were also employed as a blind test set.

Global unleashing of transcription elongation waves in response to genotoxic stress restricts somatic mutation rate.

Complex molecular responses preserve gene expression accuracy and genome integrity in the face of environmental perturbations. Here we report that, in response to UV irradiation, RNA polymerase II (RNAPII) molecules are dynamically and synchronously released from promoter-proximal regions into elongation to promote uniform and accelerated surveillance of the whole transcribed genome. The maximised influx of de novo released RNAPII correlates with increased damage-sensing, as confirmed by RNAPII progressive accumulation at dipyrimidine sites and by the average slow-down of elongation rates in gene bodies. In turn, this transcription elongation 'safe' mode guarantees efficient DNA repair regardless of damage location, gene size and transcription level. Accordingly, we detect low and homogenous rates of mutational signatures associated with UV exposure or cigarette smoke across all active genes. Our study reveals a novel advantage for transcription regulation at the promoter-proximal level and provides unanticipated insights into how active transcription shapes the mutagenic landscape of cancer genomes.