Unit:
Faculty of MedicineLibrary of the School of Health Sciences
Author:
Foutadakis Spyros
Dissertation committee:
Βασίλειος Γοργούλης, Καθηγητής, Ιατρική Σχολή Αθηνών, ΕΚΠΑ
Δημήτρης Θάνος, Ερευνητής Α, Ίδρυμα Ιατροβιολογικών Ερευνών Ακαδημίας Αθηνών
Αθανάσιος Κοτσίνας, Επίκουρος Καθηγητής, Ιατρική Σχολή Αθηνών, ΕΚΠΑ
Γεώργιος Μόσιαλος, Καθηγητής, Τμήμα Βιολογίας, Αριστοτέλειο Πανεπιστήμιο Θεσσαλονίκης
Μενέλαος Μανουσάκης, Καθηγητής, Ιατρική Σχολή Αθηνών, ΕΚΠΑ
Απόστολος Κλινάκης, Ερευνητής Α, Ίδρυμα Ιατροβιολογικών Ερευνών Ακαδημίας Αθηνών
Ευάγγελος Ανδρεάκος, Ερευνητής Α, Ίδρυμα Ιατροβιολογικών Ερευνών Ακαδημίας Αθηνών
Original Title:
Συντονισμός της γονιδιακής ρύθμισης μετά από ιϊκή μόλυνση
Translated title:
Coordination of gene regulation following virus infection
Summary:
The main subject of the present Doctoral Thesis were the regulatory mechanisms that govern gene expression during the antiviral cellular response. In order to achieve this goal an array of modern genomics, molecular biology, biochemistry and computational biology methodologies were implemented. Our main goals were [1] to identify differentially expressed human genes [2] the enhancer elements that regulate those genes and [3] the detailed study of the genomic distribution of master transcription factors that orchestrate the antiviral cellular response. Our experimental system includes two well characterized cell types, HeLa (epithelial-like cells) and Namalwa (B-like cells). As the environmental stimulus of Virus infection we used Sendai Virus that was added to the culture media and experiments were conducted prior and 3 and 6 hours upon infection
Initially, we identified differentially expressed genes at the transcriptome-wide level during the antiviral cellular response in Namalwa and HeLa cells. An antiviral core of approximately 200 genes was induced in both cell types, enriched for immune processes such as the interferon pathway and the response to cytokines. In order to identify the accessible part of the genome and its reconfiguration during the antiviral cellular response, FAIRE-seq and DNaseI-seq methodologies were implemented. This led to the identification of hundreds of regions with Virus inducible chromatin accessibility which are preferentially found in the vicinity of Virus inducible genes according to RNA-seq experiments and bear motifs for master regulators of the immune response such as the IRF family. Next, we studied in detail the genomic redistribution of the immune master regulators IRF3 and p65 ChIP-seq and found an extensive, inducible colocalization of those factors at hundreds of genomic regions, a fact underscoring their potential synergy in regulating the antiviral cellular response. The majority of inducible binding sites were found hosted in chromatin regions that were accessible even prior to infection and carried additional markers characteristic of active enhancers such as CBP and Med1 binding and constitutive H3K27ac levels. Nevertheless, it is important to mention the presence of inducible IRF3 binding sites with or without p65 colocalisation in chromatin regions that were not accessible and did not carry active chromatin marks prior to infection. These regions gain significant accessibility upon infection and inducible binding of the coactivators Med1 and CBP as well as H3K27ac. So, by centering our analyzes around IRF3 we found that inducible gene expression is accompanied in several cases by an increase in chromatin accessibility, inducible binding of IRF3 with or without p65, inducible coactivator binding and inducible H3K27ac levels. Moreover, these regions are preferentially found in the proximity of virus inducible genes and bear motifs for crucial regulators such as IRF and Jun/Fos, further underscoring their potential role in regulating critical antiviral genes.
By using FAIRE-seq, DNaseI-seq and ChIP-seq methodologies putative inducible enhancers were identified based on structural chromatin characteristics such as increased chromatin accessibility and inducible transcription factor binding. Although these markers are correlated with enhancers based on a plethora of studies, their presence does not prove the enhancer activity of the underlying DNA sequence. In order to fill this gap and test the activity of the identified putative enhancers in vivo, we implemented and modified the unique ChIP-STARR-seq methodology, a massive scale reporter assay to simultaneously test the virus inducible enhancer activity of thousands of IRF3 and p65 binding sites. Strikingly, we found that only 10% of the approximately 3000 strongly inducible IRF3 sites are active inducible enhancers.
An interesting finding revealed by scanning the DNA sequences of IRF3-STARR-seq enhancers was that many of them contain serial repetitive IRF motifs. According to our knowledge our finding of Virus inducible enhancers that exclusively carry arrays of IRF binding sites has not been described before. By scanning representative genomes spanning the entire course of evolution using a consensus IRF motif derived from repetitive enhancers we discovered that IRF repetitive sequences are a universal characteristic of genomes as they are found in all examined species. Moreover, individual sequences with repetitive elements are found in Viral genomes such as those of Herpes Viruses.
Main subject category:
Health Sciences
Keywords:
Genomics, Gene regulation, Enhancers
Number of references:
153
