Unit:
Department of BiologyLibrary of the School of Science
Author:
Liakos Anastasios
Dissertation committee:
Παναγιώτα Παπαζαφείρη, Αναπληρώτρια Καθηγήτρια Τμήματος Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Σπύρος Ευθυμιόπουλος, Καθηγητής Τμήματος Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Μαρία Φουστέρη, Ερευνήτρια Β', ΕΚΕΒΕ Φλέμιγκ
Διαμάντης Σίδερης, Αναπληρωτής Καθηγητής Τμήματος Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Ζωή Λυγερού, Καθηγήτρια Τμήματος Ιατρικής, Πανεπιστήμιο Πατρών
Σταύρος Ταραβήρας, Καθηγητής Τμήματος Ιατρικής, Πανεπιστήμιο Πατρών
Παντελής Χατζής, Ερευνητής Β', ΕΚΕΒΕ Φλέμιγκ
Original Title:
Μελέτη του ρόλου των επιγενετικών και επιδιορθωτικών μηχανισμών στη ρύθμιση της γονιδιακής έκφρασης
Translated title:
Studying the role of epigenetic and repair mechanisms in the regulation of gene expression
Summary:
The integrity of the DNA sequence is constantly challenged by a variety of genotoxic perturbations. Nucleotide Excision Repair (NER), via its two subpathways, plays a vital role in sensing and removing a large panel of helix-distorting DNA adducts induced by ultraviolet (UV) light, cigarette smoke, and other chemicals currently used in chemotherapy. Global Genome-NER (GG-NER, GGR) operates through the entire genome and is triggered stochastically by helix distortions, while Transcription Coupled-NER (TC-NER, TCR) is triggered by elongating Pol II molecules encountering DNA adducts and speeds-up damage excision and repair in expressed loci, revealing that transcription is a major driving force in safeguarding genomic stability.
High-throughput genome-wide methodologies have revealed that cells cope with DNA damage-induced stress through a multilayered transcription-driven response that involves global alteration of the gene expression program and leads to accelerated repair of transcription-blocking DNA lesions. Nonetheless, the associated molecular events ruling these processes remain elusive.
By focusing on normal human skin fibroblasts, we uncovered a surprising gain in chromatin accessibility at virtually all active Transcription Start Sites (TSSs) of mRNAs, PROMoter uPstream Transcripts (PROMPTs) and enhancers in response to UV irradiation. This phenomenon is accompanied by the maintenance of active histone marks (H3K27ac) and the lack of deposition of transcription silencing modifications (H3K27me3) at the respective transcribed loci. Importantly, we revealed the continuous and dynamic recruitment of pre-initiating RNA Polymerase II (Pol II-hypo) at active TSSs and its unrestrained transition into initiation at these regulatory loci, as further demonstrated by the increased synthesis of start-RNAs and the production of high levels of nascent RNAs (nRNAs) proximal to TSS sites, upon genotoxic stress. Our data highlight a dynamic synergy between increased accessibility of proximal and distal transcription regulatory regions and an unanticipated constant initiation process that is unleashed by the UV-triggered release of Pol II molecules from promoter-proximal pause sites. This mechanism secures gene expression accuracy and preserves the integrity of essentially all active transcription regulatory regions.
In parallel, we studied the transcription initiation dynamics upon UV stress in CS-B fibroblasts, a cellular model characterized by its inability to restore transcription upon exposure to UV stress. Surprisingly, we observed that H3K27ac is not significantly altered and de novo binding of Pol II-hypo is not inhibited at active TSSs, early after exposure to UV, showing that transcription initiation is not inhibited in CS-B cells at least at the early post recovery times. These results suggest that the inability to restore transcription in these cells derives from aberrations in the way cells handle transcription elongation blockage.
Moreover, by comparing non-irradiated normal and CS-B skin fibroblasts, we found that a group of genes implicated in developmental processes was differentially expressed between the two cell lines, while also being differentially targeted by H3K27me3. These results indicate that CSB protein could act as epigenetic regulator, providing possible links between the phenotype and the molecular basis of Cockayne Syndrome.
Main subject category:
Science
Keywords:
Transcription, Chromatin, Ultraviolet Irradiation, Nucleotide Excision Repair, RNA polymerase II, Cockayne Syndrome
Number of references:
203
