Eliciting the role of transcription in the maintenance of genome integrity
Genome integrity is essential for accurate gene expression and epigenetic inheritance. On the other hand, a prolonged transcriptional arrest can challenge genomic stability, contributing to genetic and epigenetic defects and the mechanisms of ageing and disease.
Our studies revealed a transcription-driven mechanism that is activated in response to genotoxic stress in human skin fibroblasts and which promotes genetic accuracy by ensuring fast and efficient repair of the actively transcribed genome. In particular, we found that, in response to UV-C irradiation, global de novo stress induced RNA Pol II elongation waves are simultaneously released from promoter-proximal pausing sites, in virtually all expressed genes, regardless of gene size or expression level. This mechanism results in enhanced Transcription-Coupled NER activity and in limited mutagenesis of genotoxic-agents exposed tissues, such as skin and lung, and could lower oncogenesis.
Lavigne MD, Konstantopoulos D, Ntakou-Zamplara KZ, Liakos A, Fousteri M. (2017). Global unleashing of transcription elongation waves in response to genotoxic stress restricts somatic mutation rate. Nature Communications 8(1): 2076. doi: 10.1038/s41467-017-02145-4.
In a follow-up study, we demonstrated that maintenance of active transcription initiation at genes and regulatory regions, such as PROMPTS and enhancers, during genotoxic stress recovery secures the integrity of the whole transcriptome.
Performing high throughput genome wide approaches and time-, inhibitor-resolved measurements and by developing robust integrative bioinformatics analyses we dissected transcription and chromatin dynamics and showed how human cells respond to significantly limit the potentially carcinogenic effects of DNA damaging stresses present in our daily environment (i.e.: UV irradiation, chemicals or cigarette smoke). In the same study, we also established precise maps of chromatin state and addressed in highly resolutive fashion the molecular details driving the unforeseen synergy between increased chromatin accessibility and post-damage transcription dynamics.
Liakos A, Konstantopoulos D, Lavigne MD, Fousteri M. (2020), ‘Continuous transcription initiation guarantees robust repair of all transcribed genes and regulatory regions’. Nature Communications 11 (1): 916. DOI: 10.1038/s41467-020-14566-9.
aniFOUND: Analysing the associated proteome and genomic landscape of the repaired nascent non-replicative chromatin
Exposure to environmental agents, such as UV irradiation, cigarette smoke and several chemotherapeutics currently in clinical use, induces bulky helix distorting DNA lesions that trigger a multi-layered cellular NER-DNA Damage Response (DDR) for their repair. Increased numbers of mutations due to an overwhelmed or defective NER are causatively linked to cutaneous melanoma carcinogenesis as well as basal and squamous cell carcinoma and certain lung cancers. Moreover, research on NER-DDR may aid in deepening our understanding and provide new strategies for treatment of Xeroderma Pigmentosum, Cockayne Syndrome, and Trichothiodystrophy, which are rare human disorders caused by NER defects.
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. In collaboration with Dr Martina Samiotaki and the Proteomics facility of BSRC Fleming, coupling of aniFOUND to mass spectrometry provided an in-depth view of the repaired chromatin-associated proteome and revealed previously unknown players involved in the NER-DDR and the restoration of damaged chromatin.
Establishment of a novel protocol for deep sequencing (NGS) of the repaired nascent DNA segments (aniFOUND-seq) and specific analysis pipelines, revealed their genome-wide distribution and resolved repair efficacy of the rather unexplored repeated genome; in particular, rDNA and telomeres. In summary, aniFOUND is a method that can delineate 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.
Georgios C Stefos, Eszter Szantai, Dimitris Konstantopoulos, Martina Samiotaki, Maria Fousteri, 2021. “aniFOUND: analysing the associated proteome and genomic landscape of the repaired nascent non-replicative chromatin”. Nucleic Acids Research, 49 (11): e64; gkab144, https://doi.org/10.1093/nar/gkab144
Investigation of the molecular paths, gene regulatory networks and cellular heterogeneity that underlie disease-driven biological processes by single cell (sc) Next Generation Sequencing (NGS) approaches.
In collaboration with Prof. G. Kolias and Dr M. Armaka laboratories (at BSRC ‘Alexander Fleming’) we employed scRNA-seq and scATAC-seq methodologies and integrative analyses, to generate and successfully resolve functional sc maps of synovial fibroblasts (SF) populations derived from naïve and hTNFtg mice (mice that overexpress human TNF, a murine model for Rheumatoid Arthritis). Our studies defined a dynamic SF landscape from health to arthritis highlighting the underlying molecular switches, spatiotemporal dynamics and gene regulatory networks and provide novel insights into the pathogenic remodeling of synovial microenvironment.
-Marietta Armaka#*, Dimitris Konstantopoulos#, Christos Tzaferis#, Matthieu D Lavigne#, Maria Sakkou#, Anastasios Liakos, Petros P Sfikakis, Meletios A Dimopoulos, Maria Fousteri*, George Kollias*, 2022. ‘Single-cell multimodal analysis identifies common regulatory programs in Synovial Fibroblasts of Rheumatoid Arthritis patients and modelled TNF-driven arthritis’. bioRxiv doi: https://doi.org/10.1101/2021.08.27.457747 and Genome Medicine, In Press.
# equally contributed, first authors