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.

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.

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.

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.

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.

Nucleotide Excision Repair: From Neurodegeneration to Cancer.

DNA damage poses a constant threat to genome integrity taking a variety of shapes and arising by normal cellular metabolism or environmental insults. Human syndromes, characterized by increased cancer pre-disposition or early onset of age-related pathology and developmental abnormalities, often result from defective DNA damage responses and compromised genome integrity. Over the last decades intensive research worldwide has made important contributions to our understanding of the molecular mechanisms underlying genomic instability and has substantiated the importance of DNA repair in cancer prevention in the general population. In this chapter, we discuss Nucleotide Excision Repair pathway, the causative role of its components in disease-related pathology and recent technological achievements that decipher mutational landscapes and may facilitate pathological classification and personalized therapy.

Structural and regulatory elements of the interaction between amyloid-ß protein precursor and Homer3.

Amyloid-ß protein precursor (AßPP) metabolism and the accumulation of its derivative amyloid-ß (Aß) peptide in senile plaques have been considered key players in the development of Alzheimer's disease (AD). However, the mechanisms underlying the generation and the deposition of Aß are not clear but emphasis has been given in the role of AßPP protein interactions that regulate its processing and offer a means to manipulate Aß production. We have previously shown that AßPP interacts with members of the Homer protein family, which leads to inhibition of Aß generation. Herein, we studied the structural parameters of AßPP/Homer3 interaction by analyzing the sequences and domains that play a role in the formation of the complex. We found that the cytoplasmic tail of AßPP is necessary for the interaction. Regarding Homer3, we report that both the EVH1 protein interacting domain and the polymerization coiled coil domain are essential for the complex assembly. Importantly, phosphorylation of Homers at certain serine residues seems to enhance the interaction with AßPP, possibly underlying our recent work suggesting that calcium signaling also regulates the interaction. Our results show that the regulation of AßPP/Homer3 interaction might be critical in the context of Alzheimer's disease pathology as a novel target for regulating AßPP function and metabolism.